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Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
Nasa Nnc05 Ca90 C Final Report
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Nasa Nnc05 Ca90 C Final Report

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  • 1. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Executive SummaryThe overall goal of this program (through Phase III) is the development of an analyzer integrated into theInternational Space Station toilets capable of detecting key chemicals in urine to monitor and assess astronaut health.The analyzer will employ a novel metal-doped sol-gel material to simultaneously extract and quantify these keychemicals using surface-enhanced Raman spectroscopy (SERS). During the Phase I program feasibility wassuccessfully demonstrated by chemically extracting 3-methylhistidine, a muscle-loss indicator, and Raloxifene, abone-loss inhibitor from reconstituted urine and obtaining their SER spectra in 1-mm glass capillaries. During thePhase II program the following success were achieved: 1) 50 biochemicals (12 biomarkers, 13 drugs, and 25 potential interfering urine components) were successfully measured using these SERS-active capillaries. 2) The biomarkers and drugs were successfully detected at physiological concentrations (1 mg/L, and 0.01 mg/L, respectively). 3) Prototype micro-fluidic chips were fabricated that contained separation and the SERS-active materials. 4) 3-methylhistidine and Risedronate were separated from a real urine sample and detected at these required concentrations using the chip shown below (Figure E.1). 5) A design is offered that can be integrated into Hamilton Sundstrand’s ISS toilet that would allow automated extraction, measurement and analysis of each astronaut’s urine every day to monitor and assess health. 6) The success of this program has broad commercial value, considering that 55% of the US population over age 50 suffer from osteoporosis, and the micro-fluidic chips developed here can identify biomarkers indicative of bone loss as much as 1 year prior to standard X-ray measurements. This would allow administering bisphosphonate-based drugs to potentially avert fractures, which represent a $17 billion annual cost to the US healthcare system. A C 3 2 B a b 4 1 c d d 5 6 Top Views D E 350 500 750 1000 1250 1500 1850 350 500 750 1000 1250 1500 1850 Raman Shift, cm-1 Raman Shift, cm-1Figure E.1. Photographs of A) 1) urine sample, 2) syringe, 3) filter, 4) ion-retardation capillary, 5) transfer vial, 6)lab-on-a-chip containing a) flow-control syringe, b) ports, c) ion exchange resin loaded channel, and d) SERS-active sol-gel loaded channels. B) Chip mounted on XY plate reader. C) RTA Raman Analyzer. SERS of D) 1mg/L 3-methyl histidine and E) 0.01 mg/L Risedronate extracted from actual urine sample using this apparatusand detected in the d) channels. Complete analysis takes less than 10 minutes. This figure is proprietary. 2
  • 2. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 " On-Demand Urine Analyzer " Part 1: Table of ContentsProject Summary ..........................................................................................................................................................1Executive Summary.......................................................................................................................................................2Part 1: Table of Contents ..............................................................................................................................................3Part 2: Identification and Significance of the Innovation .............................................................................................3Part 3: Technical Objectives.......................................................................................................................................11Part 4: Work Plan (Phase II Results) ..........................................................................................................................13Part 5: Potential Applications .....................................................................................................................................53Part 6: Contacts...........................................................................................................................................................53Part 7: Future Technical Activities .............................................................................................................................55Part 8: Potential Customer and Commercialization Activities ...................................................................................55Part 9: Resources Status .............................................................................................................................................57Part 10: References.....................................................................................................................................................58 Part 2: Identification and Significance of the Innovation This part is reproduced verbatim from the Phase I proposal.This Small Business Innovation Research program will develop a novel surface-enhanced Raman (SER) sensor thatwill perform real-time chemical analysis of urine. It will provide key physiologic information to monitor astronauthealth and indicate appropriate preventative treatment. The Phase I program will demonstrate feasibility byestablishing the ability of sol-gel chemistry to both select key chemicals: amino acids, biomarkers, drugs, andmetabolites, and enhance their Raman signals. The Phase II program will design and build a prototype “On-Demand Urine Analyzer” for ground-based measurement. This will include interfacing the SER sensor between asampling system and a Raman instrument. The Phase II program will also design a low mass, low power version ofthis system (Figure 1) to be used on the International Space Station (ISS) and other vehicles employed duringextended space flight missions (e.g. Mars expedition). Toilet Data & Power Field Bus Urine Sample Separator System Power Supply To Solid Molecules Sol-Gel Waste Computer in Solution Matrix (circuit boards) Raman Filter Scattering Laser Fiber Optics Raman Metal Spectrometer Particle Sample Laser Delivery Pump Adsorbed Flow Cell with Molecules One of four Block Out Chemically Selective Chemically Selective To Water SER-Active Discs Reclaimation Valves SER-Active Discs SystemFigure 1. Block diagram of surface-enhanced Raman based urinalysis system. A pump or flush mechanism drawsthe sample from the process through a particle filter into a flow cell and back into the process. Four sol-gel coateddiscs in the side wall provide polar-positive, polar-negative, weakly polar-positive and weakly polar-negativechemical selectivity and SER-activity. The proposed system will weigh 7.6 kg, occupy a 20x12x10 cm space (0.1cubic foot), and require 20 watts (pump not included). Expanded view illustrates surface-enhanced Ramanscattering from one of the sol-gel coated discs. This Illustration is Confidential and Proprietary.2.1. The Problem or Opportunity - Extended weightlessness causes numerous deleterious changes in humanphysiology, including space motion sickness (SMS), cephalad fluid shifts, reduced immune response, and loss ofbone and muscle mass.1-6 The need to monitor and assess these effects is critical to developing exercise programs ordrug regimes that would maintain astronaut health.7 Many of these physiological changes are reflected in thechemical composition of urine.8-13 For example, 3-methylhistidine can be used to assess loss of muscle mass,hydroxyproline to assess bone loss, and uric acid to assess renal stone formation,11 while metabolites can beanalyzed to regulate dosage of anti-SMS drugs.14 According to the National Research Council Space Studies Board, 3
  • 3. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007"Analysis of in-flight specimens for markers of bone resorption and formation would offer a unique opportunity todetermine relative efficacy of these various exercise programs."15 The Board further states that current exerciseregimes are ineffective and physiological data is inadequate to properly develop methods to offset the maladies ofweightlessness. Furthermore, these physiological changes also influence metabolism of therapeutic drugs used byastronauts during space flight. Unfortunately, current earth based analytical laboratory methods that employ liquidor gas chromatography for separation and fluorescence or mass spectrometry for trace detection are labor intensive,slow, massive, and not cost-effective for operation in space, regardless of the type of bio-fluid sample analyzed.16-20Therefore, the ability to assess and rapidly diagnose the status of individual bio-fluid samples “on-demand” is acritical and necessary component for monitoring the health and future well being of the crew members duringextended flight missions in space.2.2. The Innovation - We at Real-Time Analyzers (RTA) believe a light-weight, real-time analyzer can bedeveloped based on surface-enhanced Raman spectroscopy (SERS) to provide detection and identification of severalkey chemicals (amino acids, biomarkers, drugs, and metabolites) in urine at relevant concentrations (from ng/mL tomicrog/mL) in under 5 minutes. This approach is based on the extreme sensitivity of the SERS technique, whichhas been demonstrated by detection of single molecules,21,22 and the ability to identify molecular structure of keyphysiological chemicals and drugs through the abundant vibration information provided by Raman spectroscopy.We proposed this concept in 2001 and received a high score, but not an award. Since that time we have madeseveral significant advances in our SER-active sol-gel technology, two of which address reviewer’s comments.First, the sol-gel process is highly reproducible, and we guarantee SER-activity at 20% RSD for our Simple SERSSample Vials, now sold commercially for 2 years (Figure 2). Second, we have successfully coated glass capillariesthat are capable of measuring analytes in a flowing solution, reversibly (Figure 3) and capable of performingchemical separations.23 Additional important aspects of the technology include: third, we have patented the sol-gelprocess (NASA sponsored) that incorporates silver and/or gold nanoparticles into a stable porous silica matrix.24,25,26 1008 cm-1 band intensity for BA 45 A 40.0-45.0 40 35.0-40.0 35 30.0-35.0 30 25.0-30.0 25 20.0-25.0 B 20 15.0-20.0 15 10.0-15.0 10 5.0-10.0 5 6 0 0.0-5.0 330 9 300 270 240 Height 210 12 180 150 along 120 90 60 15 vial (mm) 30 15o increments around vial 0Figure 2. Reproducible SER-intensity response for Figure 3. Reversible SER spectra of 30-sec “plug” ofbenzoic acid over entire surface of a Simple SERS benzoic acid flowing through a sol-gel coated capillary.Sample Vial. Average = 29.1± 4.26 (14.6%) for 240 A) Spectra from time 0 to 2.4 min (bottom to top) andpoints (10 sec per point). B) plot of corresponding 1000 cm-1 band intensity. Spectra are each 8-sec, using 100 mW of 785 nm.Fourth, virtually all solvents can be used, including aqueous solutions ranging in pH from 2 to 11. No specialreagents or conditions are required. Fifth, we have used these metal-doped sol-gels to measure SER spectra ofseveral hundred chemicals,27,28,29,30,31,32 with typical detection limits of 1 ng/mL using 100 mW of 785 nm and 3-minacquisition time. Sixth, we have measured creatinine, lactic acid, uric acid, and actual urine specimens by simplyadding the samples to vials and recording the SER spectra (Figure 4).33-35 Furthermore, in preparation of thisproposal, we measured 1 mg of 3-methylhistidine in 1 mL of water and estimate a current detection limit of 60microg/mL (0.35 mM, 6 ppm, Figure 5). It is worth noting that most drugs contain nitrogen functionalitiesindicative of SER activity, and we have been able to measure drugs and their metabolites (Figure 6).36 Althoughphysiological measurements require detection limits below 1 microg/mL for biomarkers and as low as 10 ng/mL fordrugs and their metabolites, we believe improvements in sol-gel chemistry and instrumentation would allow 4
  • 4. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information achieving these detection limits. Seventh, the choice of metal and alkoxide can be used to develop chemically selective sensors. We have successfully developed sol-gels that select for polar-positive, polar-negative, weakly polar-positive, and weakly polar-negative chemical species.37 This will allow discriminative enhancement of the biomarkers or drugs in favor of urine components. Finally, it is worth noting that RTA has developed an extremely rugged, compact Raman instrument that employs interferometry for absolute wavelength accuracy and an avalanche Si detector that improves sensitivity by ~100 times.38 And we are currently developing a hand portable, battery powered version of the system for the Navy, which will weigh 9.8 kg, occupy 13,500 cc (0.5 cubic foot), require 23.5 W, and will be capable of wireless communication.39 N COOH H NH 2 Nrelative intensity R relative intensity A A B B uric acid Wavenumbers (∆cm-1) Wavenumbers (∆cm-1) Figure 4. SERS of A) female and B) male urine Figure 5. A) Raman and B) SERS of 3-methylhistidine specimens. Both samples were pH of 6.5 and diluted (R= CH3). Conditions: Raman, pure solid, 500 mW and 3 to 50% with water. Bands attributed to uric acid occur min scan, SER, 6mM, 100 mW and 3 min. Labeled SER at 502, 650, 815, 1134, and 1616 cm-1. Conditions: bands have been observed for histidine (R = H) by 120 mW of 1064 nm, 50 scans at 8 cm-1. electrolytic SERS. Inset: molecular structure. 2.3. The Proposal - The overall objective of the proposed program (through Phase III) is the development of an analyzer integrated into the ISS toilets capable of immediate detection of key chemicals in urine to monitor and assess astronaut health (complete analysis within 5 minutes of flush). The focus of Phase I will be to establish the ability of the sol-gel chemistry to both select these key chemicals and enhance their Raman signals. These key chemicals, listed in Table 1, include: urine products, hormones, muscle loss, bone loss and stone forming indicators (biomarkers), drugs and their metabolites. This will be achieved in three tasks. The first task will employ combinatorial chemistry to synthesize 36 libraries of metal-doped sol-gel coated micro-plates varying in alkoxide composition (Si:Si = 1:0 or 1:1 v/v) to be screened for analyte specific SER activity in Task 2. This will be Urine Products (g/L):* Muscle Loss Indicators (<mg/L):** creatinine (1.4) 3-methyl histidine A glucose (0.1) glutamic acid (0.3) Bone Loss Indicators(<mg/L): hippuric acid (0.9) hydroxyproline hydroxyproline (0.9) deoxypyridinoline lactic acid (0.2) B nicotinic acid (0.25) Stone Formation Indicators (<mg/L): PABA (0.2) calcium oxalate uric acid (0.2) calcium phosphate thiamine (0.2) uric acid C histidine (0.2) pyridoxamine (0.1) Drugs (~microg/L): Hormones (g/L): alendronate - for Anti bone loss pregnanediol (0.9) scopolamine - for Anti motion sickness D pregnanetriol (2.2) D-penicilamine - for Anti stone formation raloxifene - for Anti bone loss estradiol lovastatin - for Anti bone loss Table 1. Partial list of chemicals in urine, muscle loss, Wavenumbers (∆cm-1) bone loss, and stone formation indicators, and Figure 6. SER spectra of A) amobarbital, B) barbital, administered drugs. C) phenobarbital, and D) secobarbital. Conditions: 1 *To be measured in Task 3. **Chemicals in italics to be measured in Task 2. mg/ml (analyte/methanol) in sol-gel coated sample vials, 80 mW of 1064 nm, 50 averaged scans. 5
  • 5. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Informationaccomplished by varying reactant concentrations delivered to 96 well micro-plates. Reactant variables include eightSi-alkoxide precursors (tetramethylorthosilicate (TMOS), tetraethylorthosilicate (TEOS), methyltrimethoxysilane(MTMOS), ethyltrimethoxysilane (ETMOS), methyltriethoxysilane (MTEOS), ethyltriethoxysilane (ETEOS),aminotrimethoxysilane (ATMOS), and aminotriethoxysilane (ATEOS)), and two types of metal particles (silver orgold). The second task will screen the sol-gel libraries with key physiological chemicals and drugs for SER activity.This will be accomplished by measuring the SER spectra of library subsets using the following four standardchemicals: p-aminobenzoic acid (PABA), aniline (AN), benzoic acid (BA), and phenyl acetylene (PA); and twelveinitial target chemicals: 3-methyl histidine, hydroxyproline, deoxypyridinoline, calcium phosphate, uric acid,alendronate, scopolamine, dextroamphetamine, raloxifene, lovastatin, acetaminophen, and acetylsalicylic acid. Thethird task will demonstrate the ability of the proposed sol-gel SERS plates to discriminatively detect and quantify thekey chemicals in a chemical matrix equivalent to a urine specimen. This will be accomplished by analyzing thechemicals in simulated urine samples prepared according to clinically defined formulations representing an averagehuman composition. An initial chemometrics urinalysis model will be developed for identifying, quantifying andcorrelating key components in urine of physiological interest with the Raman spectra.2.4. The Probability of Success - Dr. Frank Inscore, as the Principal Investigator, and Dr. Stuart Farquharson, asProgram Manager, and Dr. John Murren of Yale University as a Consultant have the required expertise to performthe proposed research. The PI has over eight years experience in designing Raman systems to make difficult anddemanding measurements requiring extreme sample preparation protocols and rigorous optical alignment proceduresfor collecting reproducible spectral data. This includes the implementation of continuous wave (CW), pulsed, andsolid-state lasers combined with dispersive instrumentation and multichannel detection employing a charge coupleddevice (CCD) for acquiring normal Raman and resonance enhanced Raman spectra of various inorganic-organicmodels of related metalloprotein enzymatic systems.40,41,42,43 The PI also has extensive experience in designing andcollecting Raman data for various sampling configurations, sample states and conditions (e.g., in vacuo and atcryogenic temperatures). The PI also has acquired considerable expertise and experience in the analysis andapplication of FT-Raman, SERS and metal-doped sol-gel chemistry at RTA that is relevant to the successfulcompletion of the proposed project. The PM has the experience and expertise in the analysis and application of FT-Raman.25,44,45,47 The PM also has considerable experience and expertise in designing new Raman analyzers formany applications including sensor design for Raman and surface-enhanced Raman spectroscopy applications.24-,46-52 This includes numerous sampling systems, and several Raman systems, including a state-of-the-art fiber optic FT-Raman spectrometer.52,53 He has designed, patented, and implemented several fiber optic probes for in-situ remotemonitoring in harsh physical and chemical environments.54 Dr. Farquharson also has extensive experience inperforming and managing large interdisciplinary experimental research projects. He has been the PrincipalInvestigator or Manager on contracts from DOD, DOE, NASA, NIH and NSF. Dr. John Murren is a Professor atYale University School of Medicine (Medical Oncology) and Director of the Lung Cancer Treatment Unit. Dr.Murren is very active in the evaluation of new chemotherapy drugs and drug combinations used for cancertreatment. He will provide guidance in our urinalysis experiments (e.g. likely interferents), and understanding ofdrug metabolic pathways.55 Finally, we presented a conceptual Urine Analyzer design to Hamilton Sundstrand,which was very well received. Based on their design review, and our previous working relationship, HamiltonSundstrand will support our Phase II research and Phase III commercialization efforts (see support letter).2.5. Background and Technical Approach - We at Real-Time Analyzers believe that a method based on surface-enhanced Raman spectroscopy can be developed to provide real-time detection and quantification of several keychemicals, biochemicals, and metabolites in urine to monitor astronaut health and indicate appropriate preventivetreatment. This is based on our SERS detection of several chemicals, such as creatinine, lactic acid, and uric acid inurine specimens, the DNA bases, amino acids, including 3-methylhistidine, and numerous drugs and theirmetabolites (see Figures 4 – 6).24 A background to this approach follows.Microgravity and Human Physiology - Extended weightlessness causes numerous deleterious changes in humanphysiology, including space motion sickness (SMS), cephalad fluid shifts, reduced immune response, and loss ofbone and muscle mass. The signs of SMS (nausea, dizziness) and fluid shifts (headaches, increased heart rate) areeasily detected, while changes in hormone and bone metabolism are not. Consequently, a more detailed analysis ofastronaut physiology is required to assess these effects. Many of these physiological changes are reflected in thechemical composition of urine. For example, 3-methylhistidine is a known product of muscle protein breakdownand is quantitatively excreted in urine,8-10 while the urinary concentration of hydroxyproline and deoxypyridinolinereleased during collagen breakdown show promise as indicators of bone turnover.11,12 Renal stone formation, 6
  • 6. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007associated with bone loss, can be evaluated by analyzing for calcium oxalate, calcium phosphate, uric acid, citrate,magnesium ammonium phosphate, and other stone-forming salts. Furthermore, the metabolic products of drugsadministered to relieve SMS (e.g. promethazine and scopolamine),56-58 reduce muscle loss (amino acid infusion59) orbone loss (alendronate, lovastatin,60 raloxifene61), or renal stone formation (e.g. D-penicilamine) can be analyzed toregulate dosage and adjust diet. Unfortunately, the approach used in previous missions (shuttle and Mir), astronautslogging their diet, collecting and sending urine samples back to earth for analysis, would not allow timely preventivemeasures. This wait may further jeopardize the health of astronauts performing physical labor associated with theconstruction of the International Space Station. In particular, a normal dose of an anti-SMS drug for one astronautmay metabolize as a high dose for another, causing drowsiness and making tasks difficult to perform and potentiallydangerous.7,62,63,64Earth based urinalysis employs multiple steps to separate the chemicals and perform the required detection. Thistypically includes particulate filtration, pH adjustment, and chromatographic separation (usually high performanceliquid chromatographic, HPLC), prior to introduction into a mass spectrometer (MS). Inclusion of standardsthroughout this process is required to ensure measurement accuracy. These methods are labor intensive and timeconsuming, and the massive instruments (e.g. MS) are inappropriate for the ISS. It should be noted, however, thatan effort to make this traditional approach to urinalysis practical for the ISS has been undertaken by a research teamat Johns Hopkins University headed by Dr. Potember.18,65 They have developed a rugged time-of-flight massspectrometer (TOFMS) to measure biomarkers. Virtues of the TOFMS technologies are that it is small (less than onecubic ft), lightweight (less than 5 kg), and requires low power (less than 50 watts). To introduce quantitativesamples into the TOFMS, without time consuming chromatographic columns (>30 minutes analysis times), theJohns Hopkins team has been investigating matrix-assisted laser desorption ionization (MALDI) sampling. WithMALDI sampling, a matrix standard must be used (and supplied), and high-powered ultraviolet pulse lasers arerequired. Unfortunately, these lasers are inefficient and typical power requirements are near 100 W.The ability to monitor and assess the effectiveness of therapeutic agents used by astronauts during space flight isalso problematic. Evidence exists that suggest the therapeutic effectiveness of some drugs, such asscopolamine/dextroamphetamine (a drug combination used to prevent motion sickness) and acetaminophen (a drugused frequently for pain relief by astronauts) may change in space. This is reflected by the fact that concentrationlevels of such drugs (and hence their pharmacokinetic behavior) measured in bio-fluid samples (blood-plasma,urine, and saliva) during the course of pre- and post-flight time by typical chromatographic, mass spectrometric andimmuno-assay techniques on earth are not invariant, and that these subsequent changes ultimately depend onmission length and individual physiological responses to space flight.According to the National Research Council Space Studies Board "Analysis of in-flight specimens for markers ofbone resorption and formation would offer a unique opportunity to determine relative efficacy of these variousexercise programs."15 The Board further states that the current exercise regimes are ineffective and physiologicaldata is inadequate to properly develop methods to offset the maladies of weightlessness. The proposed system willallow pre- and post-flight ground analysis at the end of Phase II and on-station analysis shortly thereafter (throughPhase II production by Hamilton Sundstrand).Raman Spectroscopy - Similar to an infrared spectrum, a Raman spectrum consists of a wavelength distribution ofbands specific to molecular vibrations corresponding to the sample being analyzed, which allows confidentidentification of chemicals and biochemicals (selectivity). For example, Figure 5 shows the Raman spectrum of 3-methyl histidine, which is slightly different from that of histidine. In practice, a laser is focused on the sample, theinelastically scattered radiation (Raman) is optically collected, and directed into a spectrometer, which provideswavelength dispersion, and a detector converts photon energy to electrical signal intensity. Historically, the verylow conversion of incident radiation to inelastic scattered radiation limited Raman spectroscopy to applications,which were difficult to perform by infrared spectroscopy, in particular, aqueous solutions. In addition to sensitivity,Raman spectroscopy has been limited by long-term instrument stability, fluorescence interference, and wavelengthreproducibility. These four limitations have been largely overcome in the past decade by several technologicaladvances, principally: air cooled stable diode lasers, notch filters, full spectrum detectors (i.e. no scanning), highquantum efficiency detectors, and associated fast electronics, data acquisition and analysis, which have made Ramanspectroscopy standard equipment in analytical laboratories,66 and allowed the development of portable systems. Anattractive advantage to this technique is that in many cases samples do not have to be extracted or prepared, and afiber optic probe can simply be aimed at a sample to perform chemical analysis. In this regard, Raman spectroscopy 7
  • 7. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007has been used to identify various chemicals (within glass cylinders). However, these measurements are bestperformed for pure or at least highly concentrated samples. Further improvements can be realized by using aFourier transform Raman spectrometer.67 These systems employ diode pumped Nd:YAG lasers that provideexcitation in the near infrared, virtually eliminating fluorescence interference associated with visible laser excitation.Another advantage of FT systems is high wavelength accuracy (Connes advantage)68afforded by the HeNewavelength reference laser. This allows reliable spectral subtraction and library matching,69 as well as continuous or"on-demand" monitoring. Spectral subtractions can be used to isolate contributions of trace chemicals in thepresence of much more concentrated interferent chemicals,69,69 and library matching can provide rapid chemicalidentification. The former may be very important, in that uric acid produces a significant SER spectrum that mightneed to be subtracted to observe other key chemicals. It also allows employing other urine components as internalconcentration standards, such as creatinine to quantify 3-methyl histidine. Here, however, spectral analysis will beaugmented by the chemical selectivity of the sol-gels to be developed (see below). Nevertheless, even with theseimprovements, the very low conversion of incident radiation to inelastic scattered radiation limits the sensitivity ofRaman spectroscopy and provides only moderate detection limits for normal Raman scattering. Relatively highlaser powers and long acquisition times are required to obtain a spectrum with a reasonable S/N, which in generalstill allow only modest detection limits to be estimated for normal Raman. For example, Figure 5 shows Ramanspectra of pure 3-methyl histidine. The detection limit for 3-methyl histidine in water or lactic acid is ~1% (Figure 5is a pure solid sample). Thus, normal Raman scattering would be capable of only moderate detection limits, such as100 mg/mL.Surface Enhanced Raman Spectroscopy - In 1974,70 it was discovered that when a molecule is in close proximityto a roughened silver electrode, the Raman signal was increased by as much as six orders of magnitude.49 Themechanism responsible for this large increase in scattering efficiency has been the subject of considerable research.71Briefly, the incident laser photons generate a surface plasmon field at the metal surface that provides an efficientpathway to transfer energy to the molecular vibrational modes, and produce Raman photons.71 This is possible onlyif: 1) the material is in the form of particles much smaller than the laser incident wavelength (Raleigh regime,surface imperfections of similar size also work) to couple the energy, 2) the material has the appropriate opticalproperties to couple the light (extinction), 3) the available free electrons, when excited, are confined by the particlesize forming surface modes or generating surface plasmons, and 4) the molecule has matching optical properties(absorption) to couple to the plasmon field.49,72 These very specific conditions, restrict SERS to the coinage metals,silver, gold, and copper with diameters between 5 and 200 nm.72,73SERS has been demonstrated for a number of inorganics, organics, and biochemicals, using three primary methodsdeveloped to produce SER active media: activated electrodes in electrolytic cells,48 activated silver and gold colloidreagents,74,75,76 and metal coated substrates.77,78,79,80,81 Unfortunately, these methods have not been reduced to aproduct because it has proven difficult to manufacture a surface by these methods that yields reproducibleenhancements or reversible adsorptions or both. Oxidation-reduction cycles are used to create surface features(roughness) on electrodes with the appropriate size to generate surface plasmons, but this roughness is difficult toreproduce from one measurement to the next.49,82 Reducing a metal salt solution can be used to produce a colloidcontaining metal particles capable of generating surface plasmons. The resultant particle size and aggregate size arestrongly influenced by the initial chemical concentrations, temperature, pH, and rate of mixing, and consequently arealso difficult to reproduce.75 Depositing one of the SER active metals onto a surface with the appropriate roughnesscan also be used to prepare a surface capable of supporting surface plasmons. The largest enhancements areobtained when the sample is dried onto the surface, in effect concentrating the analyte on the metal. This alsoresults in measurements that are difficult to reproduce. The relative merits and limitations of these methods haverecently been reviewed.83In an effort to overcome these limitations, we have been developing metal-doped sol-gels as an active SER medium.This medium should be capable of providing SER measurements that are reproducible, reversible, and quantitative,yet are not restricted to specific environments, such as electrolytes, solvents, or evaporated surfaces. 24 The generalconcept is shown Figure 1, where nanocomposite material has been coated on the inside walls of glass vials to yielda general use SER product: Simple SERS Sample Vials. We have 1) measured the SER spectrum of 1nanogram/milliliter of PABA in methanol (estimated detection limit of 10 pg/mL), and achieved a signal increase ofa factor of 107,25 2) have reproduced measurements from vial-to-vial with a standard deviation less than 10%, 3)have demonstrated reversibility using a flowing system (within 5 minutes at 1 ml/min flow through a coated NMRtube,25 and 4) we have successfully measured the amino acids, DNA bases, several drugs and their metabolites in 8
  • 8. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Informationboth aqueous and non-aqueous solutions using our Simple SERS Sample Vials. In particular, we measured 1 mg of3-methyl histidine in 1 mL of water and the signal-to-noise ratio (S/N) of 52 suggests a current detection limit of 60microg/mL (defined as S/N=3, Figure 5). Measurements at the required detection limits (<1 microg/mL, Table 1) isanticipated to be straightforward using a new hybrid FT-Raman spectrometer (increased sensitivity with 785 nmlaser and Si-APD) developed under another SBIR program (see Related R&D). However, we recently employed785 nm laser excitation and found near-equivalent SERS-enhancement compared to previous 1064 nm laserexcitation, even when taking the ν4 wavelength dependency of Raman scattering into account. This suggested thatour particle size, distribution, and/or aggregation were far from optimum. In recent years we have used our SimpleSERS Sample Vials to measure several hundred different chemicals. In general, we calculate enhancement factorsbetween 104 and 106. (The difference is due to the polarizability of the analyte and/or the extent of interaction withthe metal.) These values may prove insufficient for detecting certain drugs and/or metabolites present at very lowconcentration levels in the body fluids (e.g. 1ng/L to 1ng/mL). Except for highly polarizable molecules, wetypically measure LODs of 10 microg/L, suggesting that an improvement of 2 to 4-orders of magnitude is requiredto achieve the lower detection limits. Recently, we re-evaluated our metal-doped sol-gels in regards to optimumparticle size and aggregation. TEM measurements show that the silver particles are largely unnaggregated and small(5-20 nm). We are currently examining methods to increase particle size and aggregation (variations inconcentration and heating). According to theory improvements of 2 to 6 orders of magnitude can be expected.Such improvements in both the instrumentation and metal-doped sol-gel chemistry will be important forachieving the sensitivity required in this proposal.Sol-Gel Chemistry - The sol-gel process is a chemical route for the preparation of metal oxides and other inorganicmaterials such as glasses and ceramics.84,85 The sol-gel process involves the preparation of a sol of metal-alkoxideprecursors in a suitable solvent, which undergo a series of reactions including their initial hydrolysis followed bypoly-condensation to form a gel. Expulsion of the solvent from the gel by a drying process, results in a highlyporous xerogel consisting of the metal oxides and any other additives that may have been introduced during theprocess. Additional heating (fired) can be used to crystallize and/or densify the material. Typically, the sol-gelprocess involves a silicon alkoxide (such as tetramethyl orthosilicate), water and a solvent (methanol or ethanol),which are mixed thoroughly to achieve homogeneity on a molecular scale. The sol-gel matrixes offer severaladditional properties useful to the proposed ISS application: physical rigidity and high abrasion resistance,negligible swelling in aqueous solutions, chemical inertness, high photochemical and thermal stability, and excellentoptical transparency, and low intrinsic fluorescence.86 We have successfully developed metal-doped sol-gels thatcan coat a variety of substrate-surfaces to produce a wide range of sensor designs, including glass vials, multi-wellmicroplates (glass and polystyrene), and glass capillaries. We have used the latter to detect flowing samples as wellas to perform chemical separations.The Simple SERS Sample Vials are produced according to the following procedure. First, a silver amine precursorcomplex is prepared from a solution of ammonium hydroxide and silver nitrate. Second, the sol-gel solution isprepared from TMOS and methanol. Third, the amine complex and sol-gel solution are mixed, then the solution isspin-coated onto the inner walls of a glass vial, and dried. Fourth, the substrate is heated to form the xerogel. Thisstep defines the porosity (size and distribution) and silver particle size. Fifth, the silver ion is reduced to silver metalparticles (Ago) using dilute sodium borohydride. And sixth, the substrate is washed and dried prior to the addition ofa sample. Previously, we established that a volume ratio of 1:5:4, silver amine complex to TMOS to methanolheated at 120 oC for 2 hours yielded optimum SERS signals for PABA.25 It is worth noting that gold-doped sol-gelcoated vials have also been prepared in a similar fashion by using an aqueous solution of HAuCl4 (or NaAuCl4),nitric acid as a gellation catalyst, and a Si-alkoxide precursor (e.g. TMOS). These conditions were optimized usinga simplistic experimental design approach. Knowledge of sol-gel chemistry and surface-enhancement theory wasused to optimize chemistry and physical properties, while performing a minimum number of experiments.Nevertheless, the use of only PABA to maximize the sensitivity may have reduced the sensitivity to other analytes.For example, we estimate a surface enhancement of <105 for 3-methyle histidine. Unfortunately, tailoringsensitivity to every analyte of interest would be time consuming and tedious.Combinatorial Chemical Synthesis - To alleviate this problem, we are employing combinatorial chemistry tosystematically synthesize large numbers of well-defined sol-gel compositions (libraries) by combining the reactantsin all combinations.87 We are employing 96-well micro-plates to develop the chemistry. Initially two reservoirs (8and 12-cell) are filled with the two starting solutions (here TMOS and the amine complex) with incrementallyincreasing concentrations. Then multi-channel pipettes (8 and 12) are used to deliver microliter samples to each 9
  • 9. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 well fairly rapidly (by hand it takes ~4 minutes). Once completed, the plate is placed into an oven to cure the sol- gel. We are able to prepare as many as 20 plates per day or 1892 sol-gels with different SERS activity. The success of a library is determined by testing the activity of the sol-gel coated well. Since the number of wells quickly escalates using this procedure, testing of each well becomes impossible and high-throughput screening techniques are used. This is accomplished by selecting and testing an ordered subset of the 96 wells. The most active wells can be used to refine the chemistry and improve SERS activity. Using this approach, we have modified the alkoxide chemistry to obtain SERS active sol-gels that preferentially "solvate" polar or non-polar analytes. For example, the alkoxide precursor MTMOS has a higher -CH3 concentration (lower -OH) than TMOS, and consequently a higher affinity for non-polar chemicals (hydrophobic).86,88 Task I will focus on employing combinatorial chemistry to develop sol-gels that are selective and active (and stable) towards various key analytes to be screened for in Task II and Task III. During the past year we successfully developed our first chemically selective SER-active sol-gels, consisting of the four Libraries outlined in Table 2. Table 2. Summary of chemically selective surface-enhanced Raman active metal-doped sol-gels. Library 1 Ag + TMOS Selective for polar-negative species Library 2 Ag + (TMOS+MTMS) Selective for weakly polar-negative species Library 3 Au +TMOS Selective for polar-positive species Library 4 Au + TEOS Selective for weakly polar-positive species Figure 7 shows surface-enhanced Raman spectra of p-aminobenzoic acid (PABA) using Library 1 and 3, and phenyl acetylene (PA) using Library 2 and 4. For Libraries 1 and 3, the polar PABA passes through the polar sol-gel and is enhanced by either the silver or gold particles. For electropositive silver, the PABA anion (pKa = 4.8) interacts through the carboxylate group and COO- bands appear at 840 and 1405 cm-1. For electronegative gold, PABA interacts through the amine group and -NH2 bands appear at 1355 and 1585 cm-1. For Libraries 2 and 4, the non- polar PA passes through the non-polar sol-gel and is enhanced by either the silver or gold particles. For electropositive silver, PA interacts strongly through the cylindrical π cloud around the carbon-carbon triple bond and a -C≡C- doublet occurs near 2000 cm-1. For electronegative gold, this interaction is unlikely and only very weak bands occur near 2000 cm-1. The polar/non-polar selectivity of the polar-negative and weakly polar-negative sol- gels was tested by adding a 1:1 molar mixture of PABA and PA. The selective enhancement is quite good (Figure 8). The spectrum obtained using the polar sol-gel suggests 78% PABA and 22% PA reached or is active at the metal surface, while the spectrum obtained using the weakly polar sol-gel suggests a 9% PABA and 91% PA activity. The band peak intensities at 2000 cm-1 for PA and 1450 cm-1 for PABA were used for these calculations, and are expanded in Figure 8 for clarity. Silver-doped TMOS favored more rapid transit of the polar PABA than the non- polar PA. A B C CH H2N COOHrelative intensity relative intensity Wavenumber (∆cm-1) Wavenumber (∆cm-1) Figure 7. SER spectra of A) PABA using Libraries 1 (top) and 3 (polar-negative and polar-positive sol-gels), and B) PA using Libraries 2 (top) and 4 (weakly polar-negative and weakly polar-positive sol-gels). PABA is 1 mg/mL, PA is 1% v/v. Spectral conditions: 75 mw 1064 nm, 100 scans (1.5 min), 8 cm-1 resolution. The y-axis for all spectra represent intensity in arbitrary units. 10
  • 10. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information A Crelative intensity relative intensity B D Wavenumber (∆cm-1) Wavenumber (∆cm-1) Figure 8. SERS of 1:1 M/M of PABA and PA in A) polar-negative and C) weakly polar-negative sol-gels. The lower traces, compare the pure chemicals; B) 1 mg/ml PABA in polar-negative sol-gel and D) 1% PA in weakly polar-negative sol-gel, while the insets magnify the minority species for clarity (x5 in A and x10 in B). Spectral conditions as in Figure 5. Part 3: Technical Objectives The overall objective of the proposed program (through Phase III) is the development of an analyzer integrated into the International Space Station (ISS) toilets capable of immediate detection of key chemicals in urine to monitor and assess astronaut health. The following six comprehensive tasks have been designed to develop this proposed analyzer, as well as the required method of analysis with the following objectives and specific questions to be answered. Task 1 - Spectral Library Development. The overall objective of this task is to build a SER spectral library to allow rapid spectral matching (or functional group analysis) for identification of biochemical markers, drugs and their metabolites present in human urine. This will be accomplished by extending the Phase I measurements to include an in-depth analysis of 12 primary bio-indicators specific for assessing muscle/bone loss and renal stone formation, and 12 priority drugs (and their metabolites) that may be used to minimize or counter the adverse physiological affects associated with changes in the concentration levels of these biomarkers present in urine. Questions to be answered? Are all of the 24 chemicals SER-active? What is the preferred sol-gel for each bio- indicator and target drug? Are there potential interferants? How well does the spectral match software identify each? How well does it identify a chemical not in the library? Task 2 – Chemical Selectivity Development. The overall objective of this task is to refine the ability of the 4 basic SER-active sol-gels developed in Phase I to selectively extract biochemicals and drugs present in human urine, and enhance their Raman spectra. This will be accomplished by measuring reversibility of representative bio-indicators and drugs drawn through the 4 sol-gels. Questions to be answered? Which sol-gels provide irreversible adsorption of the bio-indicators and target drugs and SER-activity? What are the estimated LODs? Can the spectral deconvolution software identify each urine component (bio-indicators and drugs) on each sol-gel? Task 3. Component selection and testing. The overall goal of this task is to design a lab-on-a-chip that can be used to analyze chemical components present in human urine by SERS. This will be accomplished by designing a chip based on the Phase I results and the background information provided above. Questions to be answered: Are there components available to perform the desired extractions and separations? Are they effective in the context of our SER-active sol-gels, separately, and together? What is the best sequence of sol-gels? How universal is it for various urine components and drugs? Are the mixtures effectively separated and 11
  • 11. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007detected? What happens when other chemicals from Task 1 are analyzed? What are the detection limits?Task 4. Lab-on-a-chip fabrication (with Advanced Fuel Research as subcontractor). The overall goal of thistask is to build many microfluidic chips that can be used to test the preliminary lab-on-a-chip design. This will beaccomplished by fabricating poly(methyl methacrylate) chips. The process of Muck et al., slightly modified, will befollowed.89 The microfluidic chips will be prepared in a class 10 clean room at AFR, and all safety (HF) procedureswill be followed.Questions to be answered: Can AFR produce the test chips? Does the process need further modification? Doesthe interface perform as planned (no leaks)? Can the various channels be loaded, reduced? What size, length isbest? Can separation materials be introduced?Task 5. Define Analytical Figures of Merit. The aim of this task is to establish performance criteria for the lab-on-a-chip. This will be accomplished by measuring the analytical figures of merit for the analyzer, as outlined bythe FDA: sensitivity, reproducibility, linearity, accuracy, precision, resolution, and selectivity.Questions to be answered: What are the LODs for each of the biomarkers, drugs and their metabolites? What isthe reproducibility of the chips? Which channel design provides the best selectivity, reproducibility, and sensitivity?What is the best standardization method for quantitating target analyte concentrations?Task 6 - Prototype Design. The overall goal of this task is to design a prototype system to be used and tested byNASA in Phase III. This will be accomplished by redesigning the lab-on-a-chip for system integration andautonomous operation. No questions to be answered. 12
  • 12. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information Part 4: Work Plan (Phase II Results)Task 1 - Spectral Library Development. The overall objective of this task was to build a surface-enhanced Ramanspectral library to allow rapid spectral matching (or functional group analysis) for identification of biochemicalmarkers, drugs and their metabolites present in human urine. This was accomplished by measuring the SERS-activity of 25 analytes and 25 potential interferents using some 20 different chemically-selective sol-gels(Libraries).The proposed 24 analytes (12 biomarkers and 12 drugs) that were the focus of this program are listed in Table T1.1.Ten of the 12 biomarkers were commercially available (the two collagen bound bone-loss markers were not) and allwere SERS-active. Similarly, 10 of the 12 proposed drugs were commercially available, and all were SERS-active.Three additional drugs (secondary) were also measured, including the metabolites of acetaminophen and allopurinol.Table T1.1. List of biomarkers and associated drugs studied during Phase II. Biomarkers Drugs Muscle loss indicators: Deoxypyridinoline – Anti-bone loss: Anti-stone formation: Creatinine (CRE) collagen bound* Etidronate (ETI) Hydrochlorothiazide (HCT) 3-Methylhistidine (3-MeHIS) Clodronate (CLO) Allopurinol (ALLO) Bone loss indicators: Stone formation indicators: Pamidronate (PAM) Penicillamine (PEN) Hydroxyproline (HO-PRO) Calcium oxalate (CaOx) Alendronate (ALE) Anti-motion sickness: Hydroxylysine (HO-LYS) Calcium phosphate (CaP) Risedronate (RIS) Promethazine (PROM)** Pyridinoline (H-Pyd) Uric acid (UA) Ibandronate (IBA) Scopolamine (SCOP) ** Pyridinoline-collagen bound* Cystine (CYST) Raloxifene (RAL) Anti-inflammatory: Deoxypyridinoline (H-dPyd) Tiludronate*/Zoldronic acid* Acetaminophen (AM)*** ** Four analytes not available. Additional drugs measured.In the Phase I proposal we described 4 chemically-selective sol-gels (Libraries 1-4) to be used for this study. Duringthe Phase I program we modified the Library 1 and 2 chemistries to produce 4 additional sol-gels. During the PhaseII program, the number of chemistries was further expanded to some 20 available sol-gels that could be used forscreening chemical selectivity (see Quarterly Report 7). By examining many biomarkers and drugs using all 20libraries it was found that 6 proved most useful. L1, L2, and L4 correspond to the chemistries designated 1, 2, and 4while L3 corresponds to the chemistry designated 2d in the Phase I Final Report. We also developed and used twonew chemistries, which essentially are chemistry 1 (or L1) modified by the inclusion of polymers, eitherpoly(ethyleneglycol) or poly(dimethylsiloxane) (PEG and PDMS, respectively), designated L5 and L6. The relativeconcentrations of the precursors used to prepare these 6 libraries are summarized in Table T1.2.Table T1.2. Synthesis summary for SERS-active, chemically-selective, sol-gel chemical libraries (L1-L6).Sol-Gel Metal Precursor (A) Sol-Gel Precursor (B) A B Selective for: (µL) (µL) L1 5/1/10: 1N AgNO3/28%NH3OH/MeOH 1/5: TMOS/MTMS 100 120 mildly polar - negative L2 5/5/10: 1N AgNO3/28%NH3OH/MeOH MTMS 100 100 non-polar - negative L3 5/5/10: 1N AgNO3/28%NH3OH/MeOH 1/5/1:TMOS/MTMS/ODS 100 175 very non-polar-negative L4 4/1: 0.25N HAuCl4(aq)/70% HNO3 TMOS 100 100 very polar - positive L5 5/1/10: 1N AgNO3/28%NH3OH/MeOH 1/5: TMOS/MTMS 100 120 polar – negative (+ 10 µL PEG) L6 5/1/10: 1N AgNO3/28%NH3OH/MeOH 1/5: TMOS/MTMS 100 120 non-polar - negative (+ 10 µL PDMS)APTMS: aminopropyltrimethoxysilane, MTMS: methyltrimethoxysilane, PDMS: polydimethylsiloxane, PEG: polyethyleneglycol, ODS: octadecyltrimethoxysilane, TMOS: tetramethylorthosilicate.During the Phase I program, screening SERS-activity for the analytes using different sol-gels was initiallyperformed in 96-well microplates. It was found that better results were obtained using glass capillaries (1.1 mm 13
  • 13. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Informationouter diameter, 800 micron inner diameter) filled with metal-doped sol-gels. These capillaries operate in the active mode, in that the sample is forced to flow through the sol-gel. We therefore used these capillaries to test SER-activity for the analytes throughout the Phase II program. Furthermore, the capillaries became the basis of the PhaseII micro-chip sampling system. The basic design and use of the SER-active capillaries is shown in Figure T1.1, andare prepared as follows. The alkoxide and amine precursors are prepared according to Table T1.2, mixed, and thendrawn into the capillary by syringe. Typically 0.4 mL of solution coats a 4 cm length of capillary. The sol solutiongels in 5 minutes, and a more rigid structure is obtained after 24 hours. A solution of 0.1g/100mL NaBH4 is drawnthrough the capillary to reduce the metal. This is followed by a 0.035% HNO3 acid wash, and then the capillary isthen ready to be used.Figure T1.1. Photograph of sol-gel coated melting point capillaries attached to syringes, before (top) and afterreduction with sodium borohydride.Initial SERS-activity screening on the various sol-gels employed 1 mg samples in 1 mL HPLC water. The sampleswere drawn into the capillaries, which were mounted on an XY sample stage above a fiber optic probe coupled toRTA’s Industrial Raman Analyzer. Spectra were obtained using 80 or 100 mW of 785 nm excitation at the sampleand 1 minute acquisition time. Once the initial screening was performed, the samples were serially diluted over 4orders of magnitude to 0.01 mg/L to determine sensitivity. The required sensitivity is ~ 1 mg/L for the metabolitesand 10s of microg/L for the drugs. Tables T1.3 and T1.4 summarize the SERS-activity in terms of the lowestmeasured concentration (LMC) for the 10 biomarkers and 13 drugs, respectively. The best measurements wereultimately obtained by concentrating the sample using ion exchange resins (Task 3), and are included in the tablesfor comparison.Also, normal Raman spectra (NRS) of the analytes were acquired as neat liquids or pure solids in the same glasscapillaries using 300 mW at 785 nm for 5 minutes. The NRS and SERS are shown for the 10 urinary biomarkers inFigures T1.2-T1.11. This includes the two muscle loss indicators, CRE and 3-MeHIS, the quantifiable bone lossindicators H-Pyd and H-dPyd, the non-quantifiable bone loss indicators HO-PRO and HO-LYS, and the stoneformation indicators, UA, CaP, CaOx and CYST. During the Phase I program we focused on hydroxyproline as anindicator of bone loss, however, research into the chemical and metabolic reaction pathways associated withosteoporosis indicates that free and bound pyridinoline and deoxypyridinoline, associated with collagen cross-linking, are more quantitative indicators of bone loss. And they are present in urine. We were able to obtain the freeforms of pyridinoline and deoxypyridinoline from Quidel Corporation, but not their bound form. Analysis ofhydroxyproline is still important, and remained part of this studyTable T1.3. Summary of Biomarker screening results: SERS-response on select chemistries in mg/L. CRE 3-MeHis HO-PRO HO-LYS H-Pyd H-Dpd CaP CaOx UA CYST t L1 1 1 neg 1000 dnt dnt 500 neg 500 1000 L2 1000 10 neg neg neg dnt 500 500 500 neg L3 1000 1000 1000 neg 1.8 neg 1.8 500 500 500 neg L4 neg neg 1 f neg neg dnt neg neg neg neg L5 neg 1000 neg neg dnt neg 1.8 500 500 500 1000 L6 1000 1000 neg neg dnt dnt 500 neg 500 neg IEXLibrary L1sL3d L5 L1t L3 L5 L5 L2 L1 L1 LMC 0.1 0.001 dnt 0.01 0.018 1.8 1 1 0.05 0.1Peaks 1421 1563 1534 1397 1393 1372 925 894 633 613dnt=did not try, f = flow method, t = TMOS only, S = SPE, d = +PDMS 14
  • 14. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary InformationTable T1.4. Summary of Drug screening results: SERS-response on select chemistries in mg/L. CLO ETI PAM ALE IBA RIS RAL HCT ALLO PEN SCOP PROM AM L1 100 1000 1000 100 10t 1 10 1000 10 1000 neg 1000 1000 L2 1000 1000 1000 100 neg 100 100 neg 0.1 1000 neg 1000 1000 L3 1000 1000 1000 1000 neg 1000 1000 100 1000 1000 neg 1000 1 L4 neg neg neg neg neg neg neg 0.01 f neg 100 0.01 f 0.01 f 0.01f L5 neg 100 100 1000 neg 100 1000 1000 500 neg neg dnt dnt L6 100 1000 1000 1000 neg 1 1000 100 500 1000 neg dnt dnt IEXLibrary L1 L3s L1 L1 L1t L5 L3d L1 L5 L1 LMC 0.01 0.01 0.01 0.01 0.01 0.001 0.01 1 0.001 0.01 dnt dnt dnt Peaks 674 644 639 647 1010 1034 1593 678 721 1110 999 1025 1578dnt = did not try, f = flow method, t = TMOS only, S = SPE, d = +PDMS; Note: OxP (ALLO metabolite) LMC0.01 mg/L on L5; PAM, IBA, ALE LMC 0.01 mg/L on both IEX and SPE with L1 (L1t for IBA) N O A A N NH2 OH B N O B N NH2Fig. T1.2. A) NRS and B) SERS of Creatinine on L1. Fig.T1.3. A) NRS and B) SERS of 3-Methylhistidine on L1 Note: 1-methyl histidine was measured in Phase 1, see Fig T5.2) . OH A O H2N A HO O NH HO OH NH2 B BFig. T1.4. A) NRS and B) SERS of Hydroxy-proline, 0.01 Fig. T1.5. A) NRS and B) SERS of Hydroxy-lysine on L1tmg/L on gold L4. (with HCl wash, and 2nd reduction step. 15
  • 15. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 A NH2 HO NH2 O O NH2 HO OH HO OH O BFig. T1.6. SERS of H-Pyridinoline in 0.2M acetic acid A) 1.8 Fig. T1.7. SERS of H-deoxyPyridinoline 1.8 mg/L on L5mg/L on L3; and B) L3 (chem2c, reported in Phase I) with (conditions same as in Fig. T1.6).acetic acid (SERS) subtracted. - O O P O Ca+2 - O O- Ca+2 O- A O A - O- O P O- Ca+2 O- O Ca+2 B BFig. T1.8. A) NRS and B) SERS of Calcium phosphate on Fig. T1.9. A) NRS and B) SERS of Calcium oxalate on L2.L2. O NH2 A A S O HO S HO NH2 OH H N N B O B N HO N HFig. T1.10. A) NRS and B) SERS of Uric acid on L1. Fig. T1.11. A) NRS and B) SERS of L-Cystine on L1. 16
  • 16. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007The NRS and SERS are shown for the 13 drugs in Figures T1.12-T1.24. This includes the anti-bone loss drugsCLO, ETI, PAM, ALE, IBA and RIS), a selective estrogen receptor modulator, RAL, the anti-stone formation drugsHCT, PEN and ALLO, the anti-motion sickness drugs SCOP and PROM, and the anti-inflammation drug AM. O Cl HO P A A Cl HO P OH HO O O CH3 B HO P OH B HO P OH HO OFig. T1.12. A) NRS and B) SERS of Clodronate disodium on Fig. T1.13. A) NRS and B) SERS of Etidronate disodium onL1. L1. NH2 O HO NH2 A O A P OH HO P OH HO OH HO P OH HO P HO O O B BFig. T1.14. A) NRS and B) SERS of Pamidronate disodium on Fig. T1.15. A) NRS and B) SERS of Alendronate sodium onL1. L1. O HO P OH N CH3 CH3 A O N A HO P OH HO P OH HO HO OH P HO O O B BFig. T1.16. A) NRS and B) SERS of Ibandronate sodium on Fig. 16 T1.17. A) NRS and B) SERS of Risedronate sodiumL1t. on L1. 17
  • 17. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 O N O A HO A S OH B B H2N O O O S S NH O Cl N H CFig. T1.18. A) NRS and B) SERS of Raloxifene in MeOH on Fig. T1.19. A) NRS and SERS of Hydrochlorothiazide on B)L2. L3, and C) 0.01 mg/L on gold L4. OH A N A N N N H NH2 B B HS O H3C HO H3C C Fig. T1.20. A) NRS and B) SERS of Allopurinol on L2. Fig. T1.21. A) NRS and SERS of Penicillamine on B) L2, and C) 0.01 mg/L on gold L4. H3C N CH3 A A CH3 H O N O N O OH S B H B C CFig. T1.22. A) NRS and SERS of Promethazine HCl on B) Fig. T1.23. A) NRS and SERS of Scopolamine HCl on B)L2, and C) 0.01 mg/L on gold L4. gold L4 (chem3a, from Phase I) and C) 0.01 mg/L on gold L4. 18
  • 18. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 A A O B O O O NH O HN OH O O O O B C Fig. T1.24. Acetaminophen, A) NRS and SERS B) 0.1 Fig. T1.25. A) NR and B) SERS of AM-G; 0.1 mg/mL on mg/mL on L2, and C) L4 (gold). L2.Although the majority of primary target drugs in this study, such as the bis-phosphonate bone-loss drugs, aregenerally excreted unchanged in urine, other drugs metabolize, such as acetaminophen and allopurinol. The SERS(and NRS) of the metabolites of these two drugs, acetaminophen-glucuronide (AM-G) and oxipurinol (OxP), werealso measured (metabolites from other drugs were not readily available). As shown in Figures T1.25 and T1.25,acetaminophen and its metabolite produce different SER spectra. ALLO is a xanthine oxidase inhibitor that lowersthe level of uric acid in urine. Approximately 90% of ALLO is metabolized to OxP. ALLO is rapidly excreted inurine (T1/2 = 40 min), while OxP is excreted over a much longer period (T1/2 =14-30 hrs). The NRS and SERS forOxP are shown in Figure T1.26 (compare to ALLO Fig T1.20). OH A OH N N B N N N N N H HO N H C Allopurinol Oxypurinol Fig. T1.26. A) NRS and B) SERS of Allopurinol; C) NRS and D D) SERS of Oxypurinol; 0.5 mg/mL on L5.In addition to these 25 measured analytes, 25 additional chemicals that may be present in astronaut urine and couldbe potential interferents were measured (Table T1.5). This included five additional drugs, the pain reliever -acetylsalicylic acid (ASA, aspirin), representative sleeping aids – barbitol and phenobarbitol, and stimulants caffeineand Adderall (Figures T1.27-T1.30). The latter drug is a single entity amphetamine product combining the neutralsulfate salts of dextroamphetamine and amphetamine, with the dextro isomer of amphetamine saccharate and d,l-amphetamine aspartate monohydrate.Table T1.5. List of drugs, vitamins, and natural products of metabolism as interferents that may appear in urine. Drugs Vitamins/Supplements Natural metabolitesAcetylsalicylic acid (ASA) Vitamin A Lactic acid GlucoseBarbital Vitamin E Hippuric acid Gluconic acidPhenobarbital Thiamine Nicotinic acid CholesterolCaffeine Pyridoxamine Glutamic acid EstradiolAllderall Citric Acid Histidine Pregnane-diol 1-methylhistidine Theophyllene Cystine/cysteine Xanthene/Hypoxanthene 19
  • 19. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 2.600 2.400 2.200 A 2.000 1.800 A 1.600 1.400 O OH O 1.200 HN NH 1.000 O O O O 0.800 B B 0.600 0.400 0.200 0.000 350 500 750 1000 1250 1500 1750 1850Fig. T1.27. A) NRS and B) SERS of Acetylsalicylic acid. Fig. T1.28. A) NRS of Barbituric acid, and SERS of B)on L3 (Chem2c). Barbital, and C) Phenobarbital; 0.1 mg/mL on L3. A NH2 N N O B N N O Fig. T1.29. A) NRS and B) SERS of Caffeine on gold L4. Fig. T1.30. A) NRS and B) SERS of Adderall 18 (25 mg; active ingredient Dextroamphetamine). L2.In addition to drugs astronauts also take vitamins (and supplements) to help prevent the negative effects of lowgravity. For this reason we measured vitamins A and E, thiamine, pyridoxamine (B6), and citric acid, all ofwhich are potential interferents in urine (Figures T1.31- T1.38). A HO A OH O B B CFigure T1.31. A) NRS and SERS of Vitamin-A in MeOH on B) Figure T1.32. A) NRS and B) SERS of Vitamin-E on L5.gold L4 and C) silver L2. 20
  • 20. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 1.000 2.100 NH2 NH2 2.000 OH 0.900 Cl- 1.900 0.800 N N+ A 1.800 OH A 1.700 0.700 relative intensity relative intensity OH S 1.600 N N 0.600 1.500 1.400 0.500 1.300 0.400 1.200 0.300 B 1.100 B 1.000 0.200 0.900 0.800 0.100 0.700 0.000 0.600 350 500 750 1000 1250 1500 1750 1850 350 500 750 1000 1250 1500 1750 1850 Wavenumbers (∆cm-1) Wavenumbers (∆cm-1) Fig. T1.33. A) NRS and B) SERS of Thiamine on L2 Fig. T1.34. A) NRS and B) SERS of Pyridoxamine on (Chem2b). L1. 2.400 A 2.200 2.000 1.800 A 1.600 1.400 OH O 1.200 O OH OH 1.000 HO O OH B 0.800 O B HO 0.600 0.400 0.200 0.000 350 500 750 1000 1250 1500 1750 1850 Wavenumbers (∆cm-1) Figure T1.35. A) NRS and B) SERS of Citric acid on L1. Fig. T1.36. A) NRS and B) SERS of Lactic acid on L1. 3.500 4.500 O 3.250 4.000 3.000 NH 2.750 3.500 2.500 HO A Arelative intensity relative intensity 3.000 2.250 O 2.000 2.500 1.750 2.000 O 1.500 1.250 1.500 1.000 0.750 B 1.000 N OH B 0.500 0.500 0.250 0.000 0.000 350 500 750 1000 1250 1500 1750 1850 350 500 750 1000 1250 1500 1750 1850 Wavenumbers (∆cm-1) Wavenumbers (∆cm-1) Fig. T1.37. A) NRS and B) SERS of Hippuric acid on Fig. T1.38. A) NRS and B) SERS of Nicotinic acid on L2. L3 (Chem2c). During the Phase I program we also measured several natural occurring biochemicals that appear in urine as the result of various metabolic processes. This included lactic acid, hippuric acid, and nicotinic acid. In addition, all of the amino acids are present in urine to some extent, and we have measured all of their SER spectra. But since these measurements are the focus of another SBIR program, here we only report glutamic acid since it has a high urine concentration, cystine and cysteine (Figures T1.39 and T1.40), and histidine (Figure T1.41). Cystine, composed of two cysteine amino acids, is of interest because the drug, PEN solubilizes cystine by displacing one of the cysteines as part of the anti-stone forming process and thereby releasing a free cysteine that ends up in urine. Both cystine and cysteine have unique SERS features (Figures T1.40.B and T1.40.C). 21
  • 21. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 7.500 7.000 6.500 6.000 A 5.500 A relative intensityrelative intensity 5.000 4.500 4.000 O O B 3.500 3.000 2.500 HO OH 2.000 C 1.500 NH2 B 1.000 0.500 0.000 D 350 500 750 1000 1250 1500 1750 1850 Wavenumbers (∆cm-1) Wavenumbers (∆cm-1)Fig. T1.39. A) NRS and B) SERS of Glutamic acid on L1 Figure T1.40. A) NRS and B) SERS of Cystine on L1, and C)(Chem1b). NRS and D) SERS of Cysteine on L3 (chem2c). 0.800 O 0.750 H 0.700 N OH 0.650 NH2 A Arelative intensity 0.600 N 0.550 0.500 OH 0.450 N O 0.400 NH2 N 0.350 B B 0.300 0.250 0.200 350 500 750 1000 1250 1500 1750 1850 Wavenumbers (∆cm-1) Fig. T1.42. A) NRS and D) SERS of 1-MeHIS (Nτ); on L1tFig. T1.41. NRS and B) SERS of Histidine on L3 (Chem2c). with HCl wash.Histidine is included in this study since it is the base structure for 3-methyl histidine. For the same reason we alsomeasured 1-methyl histidine because there were some uncertainty reported in literature as to the position of themethyl group. Nevertheless, as can be seen in Figures T1.41 and T1.42, all three histidines produce unique spectra.The biochemical interferents studies are important health indicators in their own right, specifically glucose andcholesterol. Glucose is an important indicator of diabetes (although blood glucose better represents the condition),but its SER spectrum has been difficult to obtain (as indicated Phase I Final Report Figure 4.17). We madeadditional attempts to measure glucose using many of the sol-gel chemistries. A representative SER spectrum andthe NRS of a solution are shown in Figure T1.43. Only three peaks are observed with significant intensity, a weakpeak at 845 cm-1, a strong peak at 895 cm-1, and a broad peak at 1410 cm-1. These peaks are tentatively assigned to aC-C stretch, O-C-H bend, and a C-C-H bend, respectively, based on the NRS peak assignments. However, thepaucity of peaks suggested that only part of the molecule was being enhanced (due to surface proximity or selectiveplasmon field interaction) or it may be a molecular fragment produced by photo-degradation. To test the latter, wemeasured the oxidation product of glucose, gluconic acid, which can also be produced by photo-degradation. Ascan be seen, the SERS of gluconic acid is extremely similar to glucose (Figure T1.43). The only difference is theabsence of the weak 845 cm-1 peak. To further clarify the glucose SER spectrum, we also measured glucose using1064 nm laser excitation (as well as using reduced powers). As Figure T1.44 shows, this glucose SERS looksvirtually identical to gluconic acid. However, the fact that the spectrum is present at all powers (down to 20 mW),suggest that photo-oxidation is not occurring. Further measurements are required to clarify this point. 22
  • 22. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 OH A A O OH OH 895 1410 O 845 B OH OH HO OH OH B HO OH OH C Figure T1.43. A) NRS and B) and C) SERS of glucose. Figure T1.44. A) NRS and SERS of gluconic acid. Conditions: A) 1g/mL in HPLC water B) 1 mg/mL in HPLC Conditions: A) 50 wt% in water, 1 mg/mL in HPLC water on water on L1, 785 nm, and C) 48 mW of 1064 nm, 1-min. L1. Cholesterol is normally measured in blood since it is involved in plaque build-up on artery and vein walls, but in certain pathological conditions cholesterol crystals can be found in acidic to neutral urine. The SERS of cholesterol was obtained on both gold- and silver-doped sol-gels as shown in Figure T1.45. We also measured the hormones estradiol and pregnane-diol (Figures T1.46 and T1.47). It should be noted that pregnane-triol (as proposed for in Phase I), testosterone and spermine were also measured, but were found to be inactive on the initial sol-gel chemistries tried, and measurements in Phase II were not pursued. 2.400 A 2.200 A 2.000 relative intensity 1.800 1.600 OH 1.400 1.200 B H B 1.000 H H 0.800 OH 0.600 C 0.400 0.200 350 500 750 1000 1250 1500 1750 1850 Figure T1.45. A) NRS and SERS of Cholesterol on B) L4 Fig. T1.46. A) NRS and B) SERS of Estradiol on L2 (chem3b), and C) L2. (Chem 2b). 5.000 O 4.500 N N A 4.000 A O N N 3.500relative intensity 3.000 O B 2.500 O H H N 2.000 HN B H H N C 1.500 N O 1.000 0.500 D 0.000 350 500 750 1000 1250 1500 1750 1850 Wavenumbers (∆cm-1) Figure T1.48. A) NRS and B) SERS of Xanthine; C) NRS, Fig. T1.47. A) NRS and B) SERS of Pregnane-diol on and D) SERS of Hypoxanthine; on L2. L2. 23
  • 23. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007The enzyme, xanthine oxidase, catalyzes the oxidation of hypoxanthine to xanthine, which is further converted touric acid. Since allopurinol inhibits this process, it also influences the relative amounts of hypoxanthine, xanthine,and uric acid in urine. As Figures T1.48 and T1.49 shows, the xanthenes, and theophyllene (which is produced via asimilar metabolic process) produce unique SER spectra. O N N A HN O N Fig. T1.49. A) NRS and B) SERS of Theophyllene on L1. BAs part of this program, we evaluated the ability of four spectral library search routines to identify the SER spectraof the biomarkers and drugs. The Euclidean Distance Algorithm (EDA), the Absolute Value Algorithm (AVA), theLeast Squares Algorithm (LSA), and the Correlation Algorithm (CA) have been successfully applied to Ramanspectra, and we incorporated them into a LabVIEW program. These four different search algorithms aresummarized in Figure T1.50.Euclidean Distance Algorithm (EDA) Absolute Value Algorithm (AVA) Least Squares Algorithm (LSA) n n ∑ (Libi − Unkni) 2HQI 2⋅ 1 − Lib⋅ Unkn ∑ Lib − Unkn i i i=1 i=1 Lib⋅ Lib⋅ Unkn⋅ Unkn HQI HQI n nCorrelation Algorithm (CA) n nHQI 1− ( Lib ⋅ Unkn m m ) 2 ∑ Lib i ∑ Unkn i ( m m )( Lib ⋅ Lib ⋅ Unkn ⋅ Unkn m ) m Where: Lib Lib − i=n Unkn m Unkn − i=n m n n Figure T1.50. Equations for the spectral library search algorithms.In all cases, Lib is the library entry being searched and Unkn is the unknown sample spectrum. Each method scoresthe spectral match in terms of a hit quality index (HQI), where the best score or match is 0 (Lib = Unkn) and theworst score or match is at higher values. Initially, we tested the algorithms using a very limited library composed of7 biomarkers and 9 drugs. Spectra different from that used in the library were used for these tests (the HQI scoreswere presented in Table 3 of Quarterly Report #4). It was found that the best results were obtained by 1) subtractingthe glass background produced by the capillary from both the sample and the spectra in the library, and 2) taking thefirst derivative. Smoothing the data improved scores, but not significantly. In all cases the EDA and CA methodsresulted in a positive match (after this pretreatment). However, the CA method gave the best overall results foridentifying each chemical (low HQI score) as well as discriminating against chemicals (high HQI scores).This result proved correct even when we added all of the chemicals studied in this program (biomarkers, drugs,metabolites, and potential interferents) plus all of the basic amino and nucleic acids (a total of 102 SER-activechemicals). As an important test, the spectrum obtained for a 1 microg/L 3-MeHIS sample pre-concentrated usingan ion exchange column was examined using the Correlation Algorithm. As shown in Figure T1.51, thisbiomarker was correctly identified with a very low HQI of 0.069. Furthermore, the next closest match (theLabVIEW program ranks closest 10) is the structurally similar amino acid histidine (HIS), which nevertheless has a 24
  • 24. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007much higher HQI score of 0.434. This is not unexpected as distinct differences observed in the SERS (Fig. T1.52). OH A N O NH2 N OH N O NH2 N BFigure T1.51. Display of Spectral Library Search and Match Figure T1.52. SERS of A) 1 microg/L of 3-MeHIS assoftware. The program ranks the best matches (lowest the unknown sample, and B) 1 mg/mL of HIS from thescores) and overlays the unknown and matched spectra for spectral library.visual comparison (SERS reference library = 102 chemicals).In addition to these measurements, the search routines were challenged by using SERS of analytes at different pH,on different SERS-active sol-gels, and with or without an acid treatment (i.e. HCl wash). As demonstrated in PhaseI, the spectra of these chemicals are constant between pH 4 and 8, and only if the pH was outside this range was asample misidentified (see Report # 5). In general, the alkoxide chemistry of the sol-gels produces insignificantchanges in the SER spectra. In fact, only in a few cases does the acid wash or metal (i.e. silver vs gold) result insignificant spectral differences. For chemicals that had such spectral differences, proper identification occurred ifboth spectral versions were in the library.We also evaluated S-Quant, a classical least squares algorithm, to determine chemical composition of mixtures.This program first searches the library for the best spectral match, and then this spectrum is subtracted from theoriginal spectrum to produce a new spectrum, the residual spectrum. The search routine then uses this residualspectrum as the unknown. The process is repeated until the noise throughout the residual spectrum is uniform (nomore peaks). The program then creates a spectrum composed of each of the matched spectra. The contribution ofeach spectrum is varied (weighted) until the composite spectrum matches the measured spectrum, as defined by aresidual consisting of a flat baseline.We evaluated this program using a simple 50/50 mixture of Allopurinol and it’s metabolite Oxypurinol (both at 0.25mg/mL, Figure T1.53), since drugs and their metabolites may not be easily separated using ion exchange (or otherchromatography). It must be stated that these two chemicals can be identified by their unique non-overlappingpeaks at 717 and 651 cm-1, respectively. The results of the S-Quant program are shown in Figure 59D. Theunknown spectrum was fit with equal contributions of Allopurinol and Oxypurinol, at 56.0% and 55.8%respectively. The total is not 100%, because the program is attempting to fit the noise in the spectrum with 1-2% ofother chemicals (some negative) to achieve a flat baseline. In other words, the program is correctly indicating a50/50 mix of the two chemicals. Since all of the library spectra are 1 mg/mL concentrations, this program onlygives relative concentrations. Actual concentrations would require a calibration curve for each chemical. 25
  • 25. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 A D B CFigure T1.53. SERS of A) 50/50 mixture of Allopurinol and Oxipurinol (0.25 mg/mL each), and pure B) ALLOand C) OxP. D) S-Quant Results (part of LabView front panel, see Report #8).Questions to be answered? Are all of the 24 chemicals SERS-active? All of the chemicals that we were able toobtain were SERS-active. What is the preferred sol-gel for each bio-indicator and target drug? Six sol-gel librariesallowed measuring all of the target analytes as summarized in Tables 4.3 and 4.4. Are there potential interferants?Yes, such as histidine for 3-methyl histidine. How well does the spectral match software identify each? It hasproven exceptional, correctly identifying any of the target analytes in a library of over 100 chemicals that couldpotentially be in urine. How well does it identify a chemical not in the library? The software ranks all of thechemicals based on how well their spectra match the measured sample. If there is a close match (i.e. and HQI scoreless than 0.5) then the unknown is probably very similar in structure. If no score is below 0.75, then the chemical isnot in the library and likely rather different than any of the library chemicals.Task 2 – Chemical Selectivity Development. The overall objective of this task was to refine the ability of the 4basic SER-active sol-gels developed in Phase I to selectively extract biochemicals and drugs present in human urine,and enhance their Raman spectra to improve sensitivity. This was accomplished by measuring SERS-activity as afunction of flow time for the 25 analytes using the 6 chemically selective sol-gel libraries selected in Task 1.As stated, the Task 1 results indicated that 6 chemically selective sol-gels allowed measuring the 25 target analytesat 1 mg/mL in static measurements. During the Phase I program we determined that flowing the analytes throughthe sol-gel capillaries improved sensitivity if the sol-gel extracted the analyte, effectively increasing theconcentration. In this task, the biomarkers were prepared at 1 mg/L, while the drugs were prepared at 10 microg/Lto determine if this approach could achieve the required concentration ranges for analysis.The ability of the sol-gels to extract the target analytes was investigated by measuring the SERS signal as the sampleflowed through a capillary as a function of time. The apparatus is shown in Figure T2.1. In one configurationFigure T2.1A), a peristaltic pump (VWR model 54856-070, West Chester, PA) was used to cycle the sample (20mL) through the capillary at a rate of 1 to 2.5 mL/min until a signal was observed, then a 3-way valve was used toswitch the stream to a solvent, either water or methanol to flush the analyte out of the sol-gel. SER spectralcollection was initiated when the sample solution entered the capillary and spectra were collected continuously (20sec/spectrum) for ~5-30 minutes. Early on it was found that the sol-gels that yielded the most sensitive signals werethose in which the analyte was bound irreversibly. Consequently, the solvent flush was essentially unnecessary, andinstead, a syringe pump (Sage model 341B, Thermo Electron, Waltham, MA, Figure T2.1B) was used to monitor thetime required to generate a signal. Typically, a 50 mL syringe filled with a 20-50 mL sample was passed throughthe sol-gel filled capillary at a rate of 1 to 10 mL/min (typically 2 mL/min). It is worth noting that sol-gel plugs ofthe MTMS based chemistry (L2) could handle flow rates greater than 10 mL/min, while those using the TMOS andODS based chemistries (L1 and L3 respectively) became detached at rates greater than 2.5 mL/min. This limitationwas overcome to some degree by adding the polymer component (PEG or PDMS) to the L1 sol-gel to produce L5and L6. It also improved sensitivity. 26
  • 26. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 A B Sample Solvent 3-way valve XY Peristaltic Stage Pump Syringe Pump Laser Spot on Capillary Fig. T2.1. Photographs of apparatus used for flow measurements. A) Peristaltic pump with 3-way valve to flow sample until detection, then solvent to remove (20 mL vial reservoirs). B) Syringe pump for continuous flow to monitor signal increase due to chemical extraction by the sol-gel (10-50 mL sample in 50 mL syringe). The first representative biomarker tested (during Phase I) was 1-MeHis (mislabeled as 3-MeHis by the manufacture, see Fig T5.2). A 1 mg/L solution was prepared and flowed at 2 mL/min through a capillary containing a 2 cm plug of L1 (polar sol-gel). As can be seen in Figures T2.2 and T2.3, the spectral intensity (based on the 1564 cm-1 peak height) reaches 80% of the maximum in the first 20 seconds. A 10 microg/L sample was also measured, and although a signal was perceptible after just 5 minutes, it was somewhat unstable. Similar experiments were performed using 1 mg/L samples on the non-polar sol-gels L2 and L3, and in both cases, the 1564 cm-1 peak was detected, but at a lower intensity. 0.07 A 0.061564 cm-1 band intensity 0.05 B 0.04 0.03 0.02 C 0.01 0.00 D 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Time (min) Fig. T2.2. Concentrating 1-MeHIS on L1 as a function of flow. Conditions: initial concentration was 0.001mg/mL, Fig. T2.3. SERS of 1-MeHIS on L1 after flowing A) 100- and flow rate was 2 mL/min, spectra: 100 mW of 785 nm, sec (pt.5), B) 80-sec (pt.4), C) 60-sec (pt.3), D) 40-sec (pt.2), 20-sec each. as in Fig.4.61 CRE was also measured as it was flowed through an L1-filled capillary. The signal for a 1 mg/L samples slowly increased and became significant after 12 minutes (Figures T2.4 and T2.5). Next, the bone-loss indicator, HO-PRO was tested (we did not have sufficient quantities to test H-Pyd). Initially, this biomarker, active on L4-filled capillary (gold-doped) was difficult to measure. We found that a second reduction step dramatically improved the overall sensitivity, typically by a factor of 100. This double reduction allowed measuring 1 mg/L HO-PRO. (It also allowed detecting many of the drugs at 0.01 mg/L (10 microg/L) for HCT, PEN, SCOP, PROM and AM that were nominally active on gold.) Here a 1 mg/L HO-PRO sample was flowed through a doubly reduced L4 capillary. The sample signal increased rapidly within 1 minute, as shown in Figure T2.6. 27
  • 27. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 As a final example, uric acid was measured during flow through an L1 filled capillary. Here the signal increased slowly, but after 7 minutes, the laser spot on the capillary was moved and a significant signal appeared, indicating that the laser was degrading the sol-gel (Figure T2.7) 4.0 3.5 3.0643 cm-1 Peak Area 2.5 2.0 1.5 1.0 0.5 0.0 0 2 4 6 8 10 12 14 Time (min) Fig. T2.4. Concentrating CRE as a function of flow on L1 Fig. T2.5. SERS of CRE (pt. 29) after 10 minutes of flow. with HCl wash. Conditions: initial concentration was 0.001 Conditions as in Fig. 4.63. mg/mL in HPLC water and flow rate was 1 mL/min, spectra: 100 mW of 785 nm, 20-sec each. 350 500 1000 1250 1500750 1850 Raman Shift, cm-1 Fig. T2.6 SERS of HO-PRO on L4 A) after 4-min flow B) 20- Fig. T2.7. SERS of UA on L1 after 7 minutes of flow. sec flow. Conditions: 1 mg/L in HPLC water, flow rate 1.5 Conditions: 1 mg/L in HPLC water, flow rate 1 mL/min, 100 mL/min; spectra: 80 mW of 785 nm, 60-sec. mW of 785 nm, 20-sec. The first drug tested using flow to concentrate the analyte was raloxifene (Phase I). Initially, a 0.001 mg/mL sample was flowed at 2 ml/min through an L1 capillary, and the SERS appeared after 1 minute, reaching a maximum in just 2 minutes, as shown for the1166 cm-1 peak Figure T2.8 (baseline at 1200 cm-1 subtracted). Since the desired sensitivity for raloxifene is considerably lower (~ 10 microg/L) the flow experiments were repeated using 100 microg/L. Again, the SER spectrum is detected, but after longer flow times (Figure T2.9). 0.18 A 1165 cm-1 band intensity 1165 cm-1 band intensity 0.16 0.14 0.12 0.10 B 0.08 0.06 0.04 0.02 0.00 0 1 2 3 4 5 6 Time (min) Fig. T2.8. Concentrating RAL on L1 as a function of Fig.T2.9. SERS of RAL on L1 A) 1 mg/L after 2-min (see flow. Initial concentration was 1 mg/L, 100 mW of 785 Fig. T2.8), and B) 0.1 mg/L after 5-min. nm, 20-sec each. 28
  • 28. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 The ability to detect the other drugs at microg/L concentrations using the flow technique was also examined. The following drugs represent the variety of results. The SERS were measured for a 5 microg/L ALLO sample as it flowed through an L2 filled capillary. At this low concentration, more time (over 12-min) was required to obtain a detectable signal (721 cm-1 peak, Figures T2.10 and T2.11). In the case of RIS, 20-min were required for a 10 microg/L sample flowed through an L1 capillary (Figures T2.12 and T2.13). It was also noted, that moving the laser position along the capillary dramatically increased the signal intensity, suggesting that either a relatively inactive spot was originally chosen, or the laser caused sol-gel degradation. In the case of ETI, again ~12 minutes were required to detect the SERS, but shortly thereafter the signal began to decrease (Figures T2.14 and T2.15). This also suggests that the sol-gel or the analyte may be degrading. In all cases efforts continued to improve the uniformity and stability of the sol-gels, principally by the incorporation of the polymers into the alkoxides. 0.10 0.09721 cm-1 band intensity 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Time (min) Fig. T2.10 Concentrating ALLO as a function of flow on Fig. T2.11. SERS of ALLO (pt. 34) after 12 minutes of flow L2 Conditions: initial concentration was 0.000005 mg/mL in Conditions: 0.000005 mg/mL on L2 in HPLC water, flow HPLC water and flow rate was 2.5 mL/min, spectra: 100 rate of 2.5 mL/min, 100 mW at 785 nm, 20-sec. mW of 785 nm, 20-sec each. 1.601033 cm-1 band intensity 1.40 A 1.20 1.00 0.80 0.60 B 0.40 0.20 0.00 0.00 5.00 10.00 15.00 20.00 25.00 Time (min) Fig. T2.12. Concentrating RIS as a function of flow on L1. Fig. T2.13. SERS of RIS A) (pt. 53) after 20 minutes of Conditions: initial concentration was 0.00001 mg/mL in flow, and B) (pt. 63) on new spot. Conditions: 0.00001 HPLC water and flow rate was 2.5 mL/min, spectra: 100 mg/mL on L1 in HPLC water, flow rate of 2.5 mL/min, 100 mW of 785 nm, 20-sec each. mW at 785 nm, 20-sec. 29
  • 29. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information 0.18647 cm-1 band intensity 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Time (min) Fig. T2.14. Concentrating ETI as a function of flow on L1. Conditions: initial concentration was 0.00001 mg/mL in Fig. T2.15. SERS of ETI (pt. 32) after 10 minutes of flow HPLC water and flow rate was 2.5 mL/min, spectra: 100 Conditions: 0.00001 mg/mL on L1 in HPLC water, flow rate mW of 785 nm, 20-sec each. of 2.5 mL/min, 100 mW at 785 nm, 20-sec. As described in the proposal, flow could be mimicked by “pistoning” the sample back and forth through the sol-gel to concentrate the analyte. This would allow the use of a syringe to manually manipulate the sample, so that significant urine samples were not required. To perform these experiments, a syringe containing 100- 500 microL of sample was pushed back and forth through the sol-gel 5 times (10 passes) and a spectrum recorded. A typical example is shown for RIS in Figure T2.16. As can be seen, the LMC was improved by a factor of 10 compared to static measurements. In general, the pistoning approach allowed measuring samples to 1 mg/L, suggesting that it is a viable Fig. T2.16. SERS of RIS in an L1 capillary before and approach for biomarkers, but NOT for the drugs. after pistoning (10 passes) at 0.1 mg/L. Conditions: 100 mW at 785 nm, 20-sec. The results for this task are summarized in Tables T2.1 and T2.2. Note that most of the biomarkers could be detected at physiological concentrations by pistoning at 1 mg/L, while most of the drugs could be detected by flowing at 10 microg/L (indicated in red). Table T2.1. Summary of Biomarker flow and piston results (vs static): LMC in mg/L on sol-gel libraries. CRE 3-MeHis HO-PRO HO-LYS H-Pyd H-Dpd CaP CaOx UA CYST * Static 1.0 1.0 100 1000 18 dnt 500 500 500 1000 L1 L1 L4 L3 L3 L2 L2 L1 Piston 1.0 1.0 10.0 neg 1 1.8 dnt 1.0 5.0 5.0 neg 1 L1 L1 L4 L1 L3 L3 L6** L2 L1 Flow 1.0 0.01 1.0 neg 1 0.9 dnt 1.0 1.0 1.0 1.0 L1 L1 L4 L1 L3 L3 L6** L2 L1 * = sol-gel chemistry with TMOS only, ** = sol-gel chemistry 2a with PDMS Table T2.2. Summary of Drug flow and piston results (vs static): LMC in mg/L on sol-gel libraries. CLO ETI PAM ALE IBA RIS RAL HCT ALLO PEN SCOP PROM AM Static 100 100 100 100 10* 1 10 100 0.1 100 1.0 1.0 1.0 L6 L5 L5 L1 L1 L1 L6 L2 L4 L4 L4 L3 Piston neg1 neg1 neg1 1.0 neg1 0.1 neg1 neg1 0.01 neg1 1.0 1.0 1.0 L1 L1 L1 L6 L1 L1 L1 L3 L2 L2 L4 L4 L3 Flow 1.0 0.01 neg1 0.01 neg1 0.01 0.01 0.01 0.005 0.01 0.01 0.01 0.01 L2 L1 L1 L6 L1 L1 L1 L4 L2 L4 L4 L4 L4 * = sol-gel chemistry with TMOS only 30
  • 30. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary InformationQuestions to be answered? Which sol-gels provide irreversible adsorption of the bio-indicators and target drugsand SER-activity? This data is summarized in Tables T2.1 and T2.2. What are the estimated LODs? Instead ofcalculating LODs, we improved sensitivity sufficiently to measure the analytes at the required physiologicalconcentrations (again see the same tables). Can the spectral deconvolution software identify each urine component(bio-markers and drugs) on each sol-gel? Yes, see Task 1.Task 3. Component selection and testing. The overall goal of this task was to design a lab-on-a-chip that can beused to analyze chemical components present in human urine by SERS. This was accomplished by performing aseries of tests to determine the components and functionality required for the lab-on-a-chip.A preliminary design illustrating the required functionality that such a chip should have was presented and discussedin the Phase II proposal. That design and discussion are repeated here verbatim, followed by the experimentsperformed to select the components.1. A sample compartment will be used to accept a liquid sample 100ml Tank (see components in Fig. T3.1).2. The urine sample will be drawn through a micron filter to remove any entrained particles. Filter3. The sample will continue through an inorganic extraction material (ion retardation resin) that will remove inorganic salts. Ion Retardant4. The aqueous sample will be extracted with a polar solvent (dichloromethane). Neutral organic species (drugs) will be extracted into the organic phase. Note that this step will require Extract Aq/Org modifications for application in a microgravity environment. rg5. The organic phase will be directed into a series of SER-active Aq O sol-gels designed to selectively extract the target bio-indicators pH NaOH and drugs by type (Set 1). This is NOT a chromatographic style separation. A) A very-weakly-polar (essentially non-polar) a b silver-doped sol-gel will extract non-polar-negative drugs/ Cation Exchange biomarkers (NP-neg), but pass the other chemicals. B) A a b weakly-polar gold-doped sol-gel will extract weakly-polar- positive species (WP-pos), but pass other chemicals. C) A Set Set Set weakly-polar silver-doped sol-gel will extract weakly-polar- One Two Three negative species (WP-neg), but pass other chemicals. D) A negative-polar silver-doped sol-gel will extract polar-negative species (P-neg). As an example of selectivity, we have seen that Waste scopolamine and hydroxyproline are most active on electronegative gold, and that alendronate and 3-methyl-histidine are most active on electropositive silver (see Fig. T3.2). Fig. T3.1. Flow diagram used to define separation components. A D B C B C A D Figure T3.2. SERS 1mg/ml 100 mW, 1min, 785 nm A) Figure T3.3. SERS TMOS/MTMS 100 mW, 1-min, 785 nm A) ALE, Ag-TMOS/MTMS , B) 3-MeHIS, Ag-MTMS, C) Reconstituted urine, B) reconstituted urine doped with 0.001 HO-PRO, Au-TMOS, D) SCOP, Au-TMOS/MTMS. mg/mL of 3-MeHIS and RAL in a 50:50 mixture, C) RAL extracted from organic phase (dichloromethane) component 5, D) 3-MeHIS extracted off cationic exchange column (component 9). 31
  • 31. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information6. The aqueous phase will be directed into a chamber, where the pH will be adjusted with 1M HCl to a final pH of 1.0 to ensure complete protonation.7. This pH adjusted aqueous sample will enter into a cation exchange column. The protonated species will strongly adsorb onto the material, while the basic/neutral species will quickly pass through.8. These basic/neutral species will enter into a series of SER-active sol-gels designed to selectively extract the target chemicals by type (Set 2). Set 2 follows a similar sequential sol-gel configuration as described for Set 1 above.9. A NaOH gradient mobile phase from 0.1 M to 1.0 M will then be used to elute off the remaining species that are adsorbed on the column. Species elute off the column in order of increasing PKa/pI values (i.e. as pH increases due to increasing NaOH concentration, the protonated species will elute when the pH equals/exceeds their respective pKa/pI values. Each fraction is subsequently directed into the appropriate sol-gel (Set 3).10. The feasibility of this extraction, separation, and detection method to be incorporated into our proposed lab-on- a-chip is demonstrated in Figure T3.3, where a lyophilized male urine sample (Sigma-Aldrich) was reconstituted with distilled water and doped with 3-MeHIS and RAL.11. To optimize the sensitivity, the solution will be “pistoned” in and out of the sol-gel region numerous times. The Phase I data suggest that sensitivity can be improved by up to 1000 times. This coupled with 532 nm laser excitation suggest that sensitivity at the part-per-trillion level can be achieved (see below).The following basic experiments were performed to evaluate the use of a filter and the value of an organic solvent inthe above flow diagram. Since urine contains many large MW molecules and numerous salts, sometimes asparticulate mater, it is necessary to include a filter to remove these substances. A saturated solution of uric acid wasprepared and measured in a SERS-active capillary before and after filtration. A 0.2 µm pre-cut 13 mm Nylon 66membrane filter (in a filter holder) was used to remove the particulate matter (Figure T3.4). As can be seen thefiltrated sample yields a better quality spectrum (more pronounced peaks and less noise). It was also found that allof the samples could be passed through this filter without diminishing signal, including urine samples at various pH,as well as temperature (samples became turbid at room temperature). A B Before After C Nylon FilterFig. T3.4. A) Photograph of syringe and in-line Nylon 66 membrane particle filter and saturated uric acid solutionsbefore and after filtration. SERS of UA B) before and C) after filtration in L2-filled capillaries.The role of the organic phase was to extract non-polar drugs and possibly biomarkers into this phase for SERSmeasurements, with the goal of reducing the complexity of the spectra analysis. After reviewing the solubilityproperties of the 25 target analytes and performing a few experiments, it was realized that only Raloxifene fell intothis category (as shown in Figure T3.3). Since this drug also dissolves in water, the organic solvent extraction stepis not necessary and it was eliminated.The next series of experiments examined the chemical composition of urine (simulated, reconstituted, and real) interms of the Raman and SER spectra, how it and its major components change with pH, and how the proposed ionretardation and ion exchange filters can be used to remove unwanted urine components and separate the targetanalytes. The major components of urine: urea, creatinine, uric acid and lactic acid, and lyophilized urine werepurchased from Sigma-Aldrich. Each of these four urine components as pure solids produce distinctive Ramanspectra (Figure T3.5), but only the urea produces a perceptible Raman signal in either simulated (urea 20 mg/mL,creatinine 1.4 mg/mL, uric acid 0.15 mg/mL and lactic acid 0.2 mg/mL) or lyophilized urine (reconstituted as 1g/30 32
  • 32. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007mL water, pH = 5.67, Figure T3.6). In contrast, only uric acid and creatinine produce SER spectra at neutral pH(Figure T3.7). Urea does not appear to produce a SER spectrum under any condition, while lactic acid requires avery acid pH (see below). Furthermore, it is found that the SER spectra of simulated, reconstituted, and real urinesamples are dominated by uric acid with some contribution from creatinine (Figure T3.8). A A B B C C DFig.T3.5. NRS of Urine components A) Urea, B) Creatinine, Fig. T3.6. NRS A) Urea (20 mg/mL), B) Simulated urine (seeC) Uric acid, and D) Lactic acid. Conditions: pure solids, 290 text), and C) Reconstituted urine (10,000 mwt cutoff),mW at 785 nm, 10-min. Conditions: 290 mW at 785 nm, 10-min. O A N A H2N NH2 O N NH2 B B OH H N N O N OH HO N H C C OH D D OFig. T3.7. SERS of Urine components. A) Urea on L3, B) Fig. T3.8. SERS of A) Simulated urine on L1, B)Creatinine on L1, C) Uric acid on L1, and D) Lactic acid on Reconstituted urine on L3, C) Real male urine sample (fromL1, Conditions: 1 mg/mL (UA 0.5 mg/mL). Note: all appear to volunteer at RTA) on L1, and D) Reconstituted urine on goldbe inactive on gold L4 (not shown). L4. Conditions: as prepared in Fig. T3.6.As part of the Phase I study, the pH dependence of creatinine, uric acid and lactic acid were studied in detail.In each case, stock solutions of the analyte were prepared at 1 mg/mL and then pH adjusted using HNO3 or NaOH(no buffers, verified by pH electrode), and then for each pH a sample was drawn into a separate SER-active capillaryfor measurement. For creatinine, samples were prepared from pH 11 to 3, and their spectra measured (10.8, 7.5, 6.9,5.9, and 4.5 shown in Fig. T3.9A). The SER signal intensity was good at neutral pHs, but degraded at 4.5 and 10.8.It was noted that the peak at 614 cm-1 decreased with increasing pH value, while peaks at 675 and 1740 cm-1increased, and bands at 850, 930, and 1420 cm-1 stayed relatively constant. A plot of the 614, 675 and 1740 cm-1peak intensities divided by the 1420 cm-1 peak intensity as a function of pH is shown in Fig T3.9B. A plot of the pHdependence for each of the ion concentrations based on the pKa’s shows that the 614 cm-1 peak corresponds to thecation species, and the 675 and 1740 cm-1 peaks correspond to the neutral species. It is important to note that in thepH range of 5-9, which encompasses the urine pH range (5.5 to 7.5), the creatinine SERS spectrum does not change. 33
  • 33. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 0.01 Creatinine++ Creatinine+ Creatinine 10.8 1740 0.009 0.008 7.5 Concentration [M] 0.007 0.006 6.9 A B 0.005 pK1 =4.83 pK2 =9.20 614 5.9 0.004 0.003 4.5 0.002 0.001 0 0 2 4 6 8 10 12 14 Wavenumbers (∆cm-1) pH Figure T3.9. A) SER spectra of 1 mg/mL CRE at pHs indicated (100 mW 785 nm, 1-min, L3 (chem2c)). B) Plot of 614 ( ), 675 ( ), and 1740 cm-1 (■), normalized band intensities as a function of pH representing CRE++, CRE+ and CRE, respectively. Concentrations of CRE++, CRE+ and CRE based on pKa’s as a function of pH are Uric acid showed little pH dependence as shown for spectra collected at pHs of 9.10, 7.41, 6.05, 5.25, and 4.08 (Fig. T3.10). The only indication of a change in molecular species as the pH transitions the pKa of 5.4 is a slight change in the peak at 595 cm-1, which appears to be absent at pH of 4.08. It was noted that at basic pHs (near and above the other pKa of 10.3) the solubility of uric acid in water was higher. Lactic Acid samples were prepared from pH 11 to 3, and their spectra measured (9.7, 5.6, 4.4, and 2.9 shown in Fig. T3.11). Lactic acid is only SER-active in the neutral form below the pKa of 3.08 and consequently will not be observed in urine at relevant physiological pH’s. 9.10 9.7 7.41relative intensity 5.6 relative intensity 6.05 4.4 5.25 4.08 2.9 Wavenumbers (∆cm-1) Wavenumbers (∆cm-1) Fig. T3.10. UA SERS pH dependence on L3 Figure T3.11. SERS of 1 mg/mL LA on L3 (chem2c) at (chem2c), 0.5 mg/mL. 100 mW, 785 nm, 1-min. pH’s indicated; 100 mW, 785 nm, 1-min. This data suggests that lactic acid will not interfere with measurements of biomarkers or drugs at physiological pHs, but creatinine and uric acid will. Although it may be possible to remove their spectra contributions, this will be insufficient, since their nominally high concentrations in urine are likely to block the target analytes from reaching the SER-active metal. It is clear that these urine components must be removed from the sample in order to perform analysis of the target analytes. As proposed, an ion retardation resin was examined to determine if it would remove creatinine and uric acid. A sample of reconstituted urine at pH 5.7 was pH adjusted to 4.6, and 8.7 to produce 3 samples covering the normal pH range of urine. The SER spectra for the 3 samples were measured before and after passing through a 5 mL disposable glass pipette packed with ~1 mL volume of a ion retardation resin (AG 11A8 by BioRad, MA). At pH 4.6 the SER signal is diminished, while at pH 5.7, uric acid dominates the spectrum. At pH 8.7 the uric acid signal is slightly diminished with a trace of creatinine present. However, in all 3 cases, the ion retardation resin does not appear to change the spectra, and certainly does NOT remove the uric acid. 34
  • 34. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information A A B B C CFig.T3.12. SERS of Reconstituted urine on L2 before Fig. T3.13. SERS of Reconstituted urine on L2 after ion-filtration at pH A) 5.7, B) 4.6 and C) 8.7. Note: A) on L1 retardation step, for pH A) 5.7, B) 4.6, and C) 8.7. But here A) on L2.Although the focus of the last experiment was to determine if the ion exchange resin could remove creatinine anduric acid, it also was included as a necessary component to remove dissolved salts that the filter might pass,particularly NaCl and KCl. Such salts at high concentration can potentially alter the SERS response throughsurface-deactivation or particle aggregation. To test the effectiveness of the ion retardation resin to eliminate suchsalts, a 1 ml solution containing 0.5 mg/mL of SCOP and KSCN in water was measured on gold L4 (FigureT3.14B). KSCN was chosen since it produces a very intense SER peak at 2100 cm-1, while the other salts onlyproduce a Ag-Cl peak at ~ 230 cm-1, which is not observed due to a low-wavenumber cut-off filter in the Ramananalyzer. SCOP was included as a control, since it should appear in the sample spectrum even when passed throughthe resin. As shown (Figure T3.14A), the spectrum of the sample passed through the resin is dominated by SCOPand there is no evidence of SCN-, indicating that this ion had been effectively trapped. It is worth noting thatmeasurements using a silver-doped sol-gel yielded similar results.As an initial example of the value of the ion retardation resin, a 0.5 mg/mL sample of allopurinol in reconstitutedurine was measure before and after it was passed through the resin (Figure T3.15). As shown, the removal of saltsimproves the spectrum substantially. Although the ion retardation resin did not remove the creatinine or uric acid itis still an essential component for the proposed lab-on-a-chip, especially since high salt levels diminish theseparation capabilities of the ion-exchange resins (see below). A A B B CFig.T3.14. SERS of a 1:1 mixture of SCOP and SCN on L4. Fig. T3.15 SERS of 0.25 mg/mL ALLO A) in reconstitutedA) After and B) before passing through ion-retardation urine and B) after filtration and ion-retardation (pH 6.8), and C)column. On separate L4 capillaries. in pure water for comparison. All on L1.The next component tested was the ion exchange resin. Again, the first experiment examined the SER spectraobtained from urine at various pH to determine if uric acid could be removed as a function of pH. As above, a glasspipette was filled with a strong cation-exchange resin (model AG50W, Bio-Rad). A 1g lyophilized urine in 30 mLwater sample was prepared and pH adjusted to 1.0 using 1M HCl. 1 mL of this sample was passed through the resinto “load” the column. Next, NaOH was passed through the column, 1 mL at a time, each increasing in strength from 35
  • 35. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information0.1M to 1M. Samples were collected in vials then pH 1transferred to SER-active capillaries and measured(Figure T3.15, not a 96 well plate as stated in Quarterly 2Report #6). As the pH increased the SERS of uric acid 4became apparent from 4-6, while creatinine dominatedat pH 8. 5 6Next, the 25 target analytes were each separately loadedon this ion exchange resin and measured as a function of 8pH, by eluting samples using increasing 1 mL NaOH 12concentrations and measuring the SERS-activity. Intime it was found that the biomarkers could easily bemeasured at the require 1 mg/L, and most of the Fig. T3.15. SERS of reconstituted urine collected from an iondrugs could be measured at the required 10 exchange column collected as pH fractions of ~1, 2, 4, 5, 6, 8,microg/L. For example, 3-MeHis, RIS, CaP, Allo, and 12 measured on L1 capillaries, eluted off with NaOH. Noteand OXP were measured at 1 microg/mL (Figures UA and CRE contribution at pH 2-6, and 8 respectively.T3.16 and T3.17), HO-LYS and IBA at 10microg/mL (Figures T3.18), and even H-Pyd at 18 microg/mL (Figures T3.19). Note that oxypurinol was capturedon an anion exchange resin and eluted off using an acid (Figure T3.17C). A A B B CFig.T3.16. SERS of 0.001 mg/L A) 3-MeHIS and B) RIS Fig.T3.17. SERS of A) 1 mg/L CaP and B) 0.001 mg/L ALLOon L5; pre-concentrated with cation exchange resin (pH on L5; pre-concentrated with cation exchange resin (pH ~2)~2), and eluted off with 1M NaOH. and eluted off with 1M NaOH; C) OXP 0.001 mg/L on L3 pre- concentrated with anionic exchange resin (pH~10), and eluted off with 1M HCl. A BFig.T3.18. SERS of 0.018 mg/L H-Pyd on L3 pre-concentrated Fig.T3.19. SERS of A) 0.01 mg/L HO-LYS and B) 0.01 mg/Lwith cation exchange resin (pH ~2), and eluted off with NaOH IBA, both on L1t (HCl washed), pre-concentrated with cationbase gradient. exchange resin (pH ~2), and eluted off with 1M NaOH.Towards the development of the lab-on-a-chip, methods were developed to load the ion retardation and exchange 36
  • 36. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Informationresins into the 1-mm glass capillaries (Figure T3.20A). This was accomplished by first loading a ~0. 5 mm plug ofMTMS sol-gel (no metal doping) into the capillary and letting it gel to form a porous frit to hold the resins. Thenthe resins were dissolved in water and ~ 2 cm segments were loaded. Several experiments were performed todemonstrate that the resins within these capillaries function as designed. These capillaries, the SERS-activecapillaries, the filter and a syringe as the method of delivering and flowing sample formed the basis of the lab-on-a-chip components (Figure T3.20) and the first experiment to evaluate the design. B: ion-retardation resin D: filter E: syringe B A: MTMS Frit F: C urine sample C: ion-exchange resin G: SERS-active capillaryFig.T3.20. Photographs of A) MTMS frit, B) ion retardation resin, C) ion-exchange resin, D) filter in filter holder,E: syringe, F: urine sample, G: SERS-active capillary (L1).The ideal test sample would contain a biomarker and a drug associated with bone loss or muscle loss (stoneformation is less important). Unfortunately, such a pair could not be measured (the bone loss biomarkers H-Pyd andH-dPyd available only in acetic acid, cost too much to perform but a few measurements, and there are no acceptedpreventive muscle loss drugs). Consequently, the combination of 3-methylhistidine and Risedronate became thebest test case for a biomarker and drug that would realistically be found in an astronaut urine sample.An artificially doped urine sample was prepared by adding 1 mL of a 1/1 volume mixture of 3-methylhistidine andRisedronate, both at 1 mg/mL in HPLC water, to a 1 mL reconstituted urine sample (0.5 g in 15 mL HPLC water).This produced a urine sample containing 0.25 mg/mL of each analyte. This sample solution was vortexed andallowed to equilibrate at room temperature for 5 minutes. Two 10 microL samples were drawn into SERS-activecapillaries (L1) by syringe and measured, one before and one after passing the 2 mL urine sample through the nylonfilter. In this case and all others, Tygon tubing connected the syringe to the capillary. There was no noticeabledifference between the spectra (Fig.T3.21A and B). The entire urine sample was then drawn through a capillaryfilled with ion retardation resin, and again ~10 microL was drawn into a SERS-active capillary and measured.Peaks associated with both 3-methylhistidine and Risedronate are readily apparent, but so are peaks associated withuric acid (Fig.T3.21C). Nevertheless, these spectra again show that the ion-retardation resin substantially improvesthe SERS-response, presumably by removing dissolved salt ions. RIS E UA 3-MeHIS C B D AFig.T3.21. SERS of reconstituted urine sample doped with 3-methylhistidine and Risedronate, A) before and B)after filtration, and C) after passing through an ion retardation (IR) capillary, and after passing D) 0.1 M KOH (3-MeHIS) and E) 0.5 M KOH (RIS) through the ion exchange (IEX) capillary. Note that the analytes can be identifiedin C), but are unmistakable in D) and E). 37
  • 37. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary InformationNext, 1 mL of the artificially doped urine sample was pH adjusted to 2 by adding 1M HCl. Approximately 25microL of this sample was loaded onto an ion exchange resin (cation). Next, three 1 mL solutions of 0.1, 0.5, and1.0 M KOH were passed through the resin, while collecting the eluted sample, 2 drops each, into the wells of a 96-well micro-plate. Approximately 1 drop was drawn from each of the 30 filled wells into SERS-active capillaries andmeasured (somewhat time consuming). It was found that 3-methylhistidine was carried off the column by the 0.1 MKOH (wells 1-10, and Risedronate by the 0.5 M KOH (wells 12-20). This is consistent with the pKA’s for thesechemicals, 5.9 and ~8, respectively, since these base concentrations result in pHs of ~6 and 8, respectively.Although reasonable SER spectra were obtained, it is clear that uric acid is still present in the sample containing 3-methylhistidine. For this reason, a urine sample doped only with 3-methylhistidine was prepared and measuredusing various sol-gel libraries after following the above procedure. It was found that sol-gel chemistry L3 yielded aquality SER spectrum of this analyte with little contribution from uric acid (Figure T3.22). A A B C B DFig.T3.22. SERS of 1 mg/L 3-MeHIS separated from Fig.T3.23. SERS of A) 1 mg/L 3-MeHIS and B) 0.01 mg/Lreconstituted urine (see text) on A) L3 (chem2c), B) L2, C) L1 RIS separated from reconstituted urine (see text). Both onand D) L5. Note that uric acid is absent in A-C), but not D). L3 (chem2c). Note: separate urine sample for each analyte.Next a series of experiments were performed using urine samples artificially doped with 3-methylhistidine andRisedronate at physiologically relevant concentrations to determine the volume of sample required, the amount ofion retardation and exchange resins required, the volume of acid to prepare the ion exchange column, and thevolume of base to elute the analytes. It was found that the following conditions yielded very good results:a sample volume of 1 mL, 250 mg of each resin loaded into capillaries, 25 microL of 1M HCl to acidify the ionexchange resin, 0.5 mL of 0.1M NaOH for 3-methylhistidine or 0.5M NaOH for Risedronate, and collecting the first100 microL for SERS analysis. These conditions were used to successfully measure 1 mg/L 3-methylhistidine and0.01 mg/L Risedronate artificially added to reconstituted urine (separately, Figure T3.23)Finally, based on all of these experiments, a lab-on-a-chip would consist of the components as shown in FigureT3.24. Note that the use of a continuous base gradient and capillaries (or channels) would be used to measureadditional biomarkers and drugs. Figure T3.24. Illustration of the lab-on-a-chip components that will effectively separate 1 mg/L 3- methylhistidine and 0.01 mg/L Risedronate from urine. The analysis should take less than 10 minutes. Compare to Fig. T3.1. 38
  • 38. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007Questions to be answered: Are there components available to perform the desired extractions and separations?Yes, the evaluation results are summarized in Figure T3.24. Are they effective in the context of our SER-active sol-gels, separately, and together? Yes. What is the best sequence of sol-gels? L3 for both biomarkers and drugs. Howuniversal is it for various urine components and drugs? L5 is best for UA, while L3 (chem2c) excludes UA. Are themixtures effectively separated and detected? Yes, see Fig. T3.21. What happens when other chemicals from Task 1are analyzed? Method is amenable to all analytes; spectral deconvolution software provides confirmation. What arethe detection limits? See lowest measured concentrations in Tables T1.3 and T1.4, and T2.1 and T2.2., in general 1mg/L for biomarkers and 10 microg/L for drugs achieved.Task 4. Lab-on-a-chip fabrication (with Advanced Fuel Research as subcontractor). The overall goal of thistask was to build many microfluidic chips that can be used to test the preliminary lab-on-a-chip design. This wasaccomplished by fabricating two successful polymethylmethacrylate (PMMA) chip designs after makingconsiderable modifications to the process described by Muck et al.This task consisted of several parts, 1) preparing a silicon master chip (AFR subcontract), 2) producing PMMAchips, 3) incorporating SERS-active sol-gels and separation materials into the chip channels, and 4) performingSERS measurements using the chips. This task was substantially delayed as the initial design mask was incorrect,and the photo-resist identified by Muck could not be used as described (it is our opinion that much of the chemistrydescribed by Muck was intentionally miss-stated). Together, these circumstances caused more a 5 month delay indelivery of the first chips (May, 2006), and consequently we requested a 6 month extension.While we awaited the chips we focused on part 3) learning to load smaller and smaller channels with our SERS-active sol-gels. The initial test chip included a variety of channel widths for testing, including ranging from 100 to500 microns (see below). Consequently, we ordered glass and plastic capillary tubing ranging from 200 to 600microns. Initial measurements were performed on 600 ID glass capillaries, as these were not much smaller than the800 micron ID of our standard 1-mm capillaries, and the volume would be close to a 500 micron wide channel thatis 250 microns deep.Initial measurements were performed by loading the standard silver-doped sol-gel chemistries (L1 - L3) into thecapillaries. It was found that long plugs of 2 cm or more were very difficult to reduce, while short plugs of 0.5 cmdetached during reduction or sample loading. This was even true for L3, which incorporates octadecyltrimethoxysilane, which produces relatively open structures due to the long organic chain. For these reason, severalmodifications to the sol-gel chemistry were investigated. This included the incorporation examining internallycoated, instead of filled capillaries, incorporation of different alkoxides, such as PDMS, and modification of the sol-gel curing process.One way to overcome the restricted flow caused by the sol-gel plugs in the capillaries is to open the center of thecapillary so that flow would be unrestricted. This was accomplished by filling a capillary in the usual way (mixedprecursors drawn into the capillary by syringe), but then forcing the sol solution back out after 20 seconds, before itgels. This process was repeated 6 times to form a thin film of sol-gel on the inner wall of the capillary (FigureT4.1). This multi-layered approach also gives us flexibility in terms of coating thickness.These open tubular capillaries were prepared using a modified chemistry L1 (TMOS only). First two 1 mg/mLsamples, pyridine and acetylsalicylic acid, were measured in internally coated capillaries of standard diameter, andthen a 1 mg/mL 3-methyl histidine was measured in a internally coated 600 micron ID capillary (Figure T4.2).Not surprising, it was found that these coated capillaries were somewhat less sensitive, as the analyte has to diffusethrough the sol-gel to the SERS-active metal, as opposed to the standard capillaries, in which the analyte is forced tothe metal surface as it is introduced into the capillary. 39
  • 39. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 A A B B CFigure T4.1. Microscope photograph of a 600 micron Figure T4.2. SERS of A) PYR, B) ASA, C) 3-MeHISID capillary internally coated with a reduced silver- Conditions: A) and B) 800 micron ID, and C) 600 microndoped sol-gel. ID capillaries internally coated with L1. Conditions: 1 mg/mL in HPLC water, 100 mw of 785 nm, 1-min.To aid the development of methods to loading smaller andsmaller diameter capillaries (and ultimately chip channels), Gas Linean apparatus used to coat gas chromatography columnswas purchased (Rinse Kit FOR GC columns, Part No.23626, Sigma Aldrich, Figure T4.3). It consists of a Connectionreservoir for the sol-gel solution, a side arm for applying to capillary orgas pressure, a small slit to fit a capillary into the reservoir chip channelopening and a side support arm. To load capillaries, firstthe reservoir is filled with a sol solution, and one end of thecapillary is inserted into the reservoir opening and sealedwith a graphite ferrule. Gas pressure (40 psi) appliedthrough a side arm forces the sol solution from thereservoir through the column and into the capillaries. Reservoir Fig. T4.3. Apparatus for loading small capillaries and chip channels.The next method investigated to improve the flow through the sol-gels was the incorporation of polydimethylsiloxane (PDMS) in the alkoxide precursor. As with ODS this siloxane should yield more porous sol-gels. Asmentioned previously, it also appears to adhere to glass and plastic capillary walls very well. 600 micron capillarieswere filled with modified L1 and L3 in which PDMS was added (see L6 chemistry in Table T1.2, Figure T4.4). ThePDMS-L1 modified chemistry (designated L6) in a 600 micron ID capillary was used to measure 1 mg/mLRisedronate (Figure T4.5). A B A B C CFig. T4.4. 600 micron ID capillaries; A) L6, B) L1, C) L3 Fig. T4.5. SERS of RIS A) 1mg/mL, L1, B) 1mg/L L6, and C)(+PDMS). Sealed with rubber caps, cured 24 hours and 1mg/mL L6 600 micron ID capillary; 90 mW, 785 nm, 1-min.reduced by standard method. 40
  • 40. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007Another important requirement is that the sol-gels must bond to the PMMA channels of the chips. To test this idea,a length of 500 micron ID PTFE (poly(tetrafluoro ethylene)) tubing was filled with L2. Upon curing (~2 hrs), thesol-gel filled tubing was cut into segments (~5 cm each), which were reduced following the standard method. Thesol-gel did not detach during curing or upon flowing the reducing agent. In fact, one of these segments was reduced5 days later and as shown in Figure T4.6, it not only remained attached but exhibited good SERS activity for ALLO.It is anticipated that adherence will be even better in the PMMA channels due to the ability to form chemical bondsdirectly with the silanol groups of the sol-gel. A A B BFigure T4.6. A) SERS of 0.5 mg/mL Allopurinol in L2 sol-gel Figure T4.7. SERS of A) 1-MeHIS (Nt-MeHIS), 50 microg/Lloaded poly(tetrafluoro ethylene) tubing, and B) NRS of the urine and B) OxP, 5 microg/L water. Note: samples passedPTFE tubing, 300 mW at 785 nm, 5-min. Insets, PTFE tubing through sol-gel based ion exchange resin.with and without sol-gel.Another requirement for developing the SERS-active microchips is the ability to load the separation materials intothe chip channels. Although we know that we can use MTMS sol-gel frits to hold these materials, we alsoinvestigated the ability of designing ion exchange capability using our sol-gel chemistry. A sol-gel anion-exchangematerial was created by using N-octadecyldimethyl[3-(trimethoxysily)propyl]ammonium chloride (C-18 TMOS,100 microL) with TMOS (100 microL), and 95% triflouroacetic acid (200 microL) as a catalyst. Due to thepresence of a positively charged quaternary ammonium group in the C-18 TMOS, the resulting sol-gel coatingcarries a positive charge.This sol-gel anion-exchange resin was successfully incorporated into a 250 micron capillary. Two samples wereprepared to test the IEX capillary, 1-MeHIS (50 microg/L in urine) and OxP (5 microg/L in water.) First, a 100microL sample of 1-MeHIS at 50 microg/L doped in reconstituted urine was pH adjusted (15 microL of 1M KOH toimpart a negative charge) and passed through this capillary. The sample was successfully extracted and pre-concentrated. The sol-gel was washed with 500 microL of HPLC water to remove the sample matrix, followingwhich the extracted sample was eluted off the sol-gel coating by a 1M HCl solution (100 microL). The eluent wasthen drawn into a standard SERS-active capillary (L1) and measured. The same procedure was followed for theOxP. The SER spectrum for both analytes were weak, with 1-MeHIS dominated by uric acid peaks. Clearly theextraction efficiency of this ion exchange material requires further development. Since the MTMS frits work,attempt to develop a sol-gel based ion exchange resin was discontinued.As the chemical-selectivity and SER-activity for the standard sol-gel libraries in terms of the target analytes wasbeing developed, it became clear that these libraries yielded the desired results, and that the sol-gel process, not thechemistry, should be investigated. To this end, the relative precursor volumes and gelling conditions wereinvestigated (temperature, gel time, amount of oxygen, etc.). It was found that by maintaining a constanttemperature, the sol-gel cure process was more consistent and more uniform activity was obtained. This alsoallowed reducing the sol-gel earlier during the gelation with significantly less flow restriction. Consequently,measurements using smaller diameter glass capillaries filled with sol-gel chemistries L1 and L2 were repeated.Ultimately, it was found that L2 could readily be incorporated into capillaries with IDs of 250 microns, as well as a200 micron ID channel etched into a Si-wafer micro-chip (kindly supplied by Dr. Eric Wong, Jet PropulsionLaboratory). Figures T4.8 and T4.9 show the SER spectra obtained for 10 microg/L samples of etidronate (pre-concentrated on separation materials) obtained in these devices, respectively. 41
  • 41. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007Figure T4.8. SERS of 10 microg/L etidronate Figure T4.9. SERS of 10 microg/L etidronate obtained inobtained in a sol-gel filled (L2) 250 micron ID glass a sol-gel filled (L2) 200 micron ID U-channel in a Si-chip.capillary. Conditions: 40 mW of 785 nm, 20-sec. Conditions: 60 mW of 785 nm, 20-sec. Inset: Photo ofInset: glass capillary, the dark color is the protective chip, actual size.polymer coating.In a kick-off meeting between RTA and AFR, the development of the micro-fluids master was discussed, includingthe design. Although a 14-channel chip was proposed containing 2- and 4-SERS-active sol-gel segments insequence, it was clear from the measurements described above that flow would be very difficult, and so two simplerdesigns were developed. They contain most of the important features to develop the methods required to load theSERS-active sol-gels into the channels. The designs are for 2 inch diameter silicon wafers. The first design issimply 4-channels (each 250 micron width) suitable for practicing loading sol-gels and obtaining SERS. It also willserve to produce the 20 deliverable chips for Phase III measurements (each with a different sol-gel chemistry).The second design contains 6 straight channels and 2 T-channels with widths of width 100, 200, 300, 400, and 500microns. The mechanical drawings provided to Microtronics to fabricate the masks are shown in Figure T4.10. A BFig.T4.10. Drawing of initial microchip designs. The masks were developed on 4-inch x 4-inch x 0.09-inch sodalime glass plate (grey regions represent anti-reflective chrome coat). The following specifications were provided:A) all circular end holes 2.0 mm diameter on 0.25 mm horizontal strips; B) 1) all circular end holes on vertical andhorizontal strips 2.0 mm diameter, 2) vertical strips spaced 3.62 mm apart on centers, 3) width of vertical strips asfollows: a) 0.5, b) 0.4, c) 0.3, d) 0.4 mm including above horizontal connecting strip, e) 0.1 mm including abovehorizontal connecting strip, f) 0.2, g) 0.1, h) 0.05 mm.As previously stated, the lab-on-a chip masters were prepared following the method of Muck by Advanced Fuel 42
  • 42. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary InformationResearch (under subcontract). AFR employed a class 10 clean room to prepare the master and followed allnecessary safety (HF) procedures. The overall process is shown in Figure T4.11 and involves two basic phases: 1)standard photolithography for patterning the wafer with a protective photoresist coating and 2) wet chemical etchingto thin the silicon wafer and produce the raised ridges. 100 µmFigure T4.11. Illustration of chip fabrication process.89 Figure T4.12. Photograph of Master Chip (design #2) and microscope image (x110) of e channel.Two-inch diameter silicon (100) wafers (p-type, 1-10 ohm-cm, 500 µm thick) with a 1000 nm thick thermal oxidecoating were used. First the wafer was cleaned by soaking in an 80oC bath of NH4OH:H2O2:H2O (1:1:5) for 10minutes, and then rinsed with de-ionized water. Second, the wafer was spin-coated with a negative tone photoresist(SU-8 2, Microchem Corp.) at ~ 2000 rpm for 30 seconds to produce a ~ 2 µm coating (the photoresist indicated byMuck could not be used, and consequently all of the following parameters were refined by AFR). Third, the waferwas soft-baked on a programmable hotplate by ramping the temperature from 25 to 95oC and holding for twominutes. The wafer was then allowed to cool to room temperature. Fourth, the mask was manually aligned on thewafer using the major flat for guidance (A in Figure T4.11). Fifth, the wafer was exposed to 365 nm UV light (B inFigure T4.11). Sixth, a post-exposure bake was performed to complete the cross-linking of the exposed resistregions, using conditions identical to the preliminary soft-bake. Seventh, the unexposed resist was then removed bysubmersion in a stirred bath of SU-8 developer for two minutes followed by a 90 second rinse in isopropanol, and aspin-drying stage (C in Figure T4.11). Eighth, the wafer was hard-baked at ~ 165oC for 15 minutes and allowed tocool on the hotplate, completing the patterning phase of the processing. Ninth, the wafer was submersed in a roomtemperature bath of a buffered oxide etchant consisting of a blend of 40% ammonium fluoride and 49% hydrofluoricacid in a 7:1 volume ratio until the wafer is hydrophobic (~12 minutes, D in Figure T4.11). After a one minute rinsewith de-ionized water, the wafer is immediately submersed in a 30% (wt/wt) solution of aqueous KOH at 80oC withvigorous stirring (E in Figure T4.11).A photograph of the second chip design master is shown in Figure T4.12, along with a microscope image of thechannel f T-section. The latter shows well-defined corners, and reveals the tapered shape of the ridges owing to theanisotropic nature of the Si (100 and111 planes). The top of the ridge was measured at 99.6 µm, which comparesfavorably to the target value of 100 µm. Based on the width of the ridge base and assuming that the sidewall anglewith respect to the Si surface is 54.7 degrees [1], the height of the ridge is calculated to be ~ 83 µm. Thiscorresponds to a KOH etch rate of ~ 0.92 µm/min for the Si(100), which is in fair agreement with the predicted etchrate of ~1.3 µm/min for 30% KOH at a temperature of 80oC [2]. Thus far, the “tallest” micro-machined ridges wehave achieved on Si were measured to be ~ 138 µm (determined in an optical microscope) for a KOH etch period of150 minutes. 43
  • 43. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary InformationPart 3 of making the micro-fluidic chips was performed at RTA. Following the procedure of Muck, a mold wasprepared by gluing the chip Masters to 4x4” glass supports using epoxy. A Teflon circle was cut and placed aroundeach Master, and then filled with the PMMA monomers (F in Figure T4.11). Unfortunately, the prescribed PMMAsolution and curing conditions did not work. After investigating these conditions, it was found that a 10% benzoinmethyl ether concentration was required, the 20/80 w/v ratio of PMMA/MMA had to be procured under nitrogen (2-min at 60 °C), and UV cure required 5 hours (a UV lamp was purchased for this part; Dymax Blue Wave 200 model38905). Unfortunately, this cured acrylic bonds to the master chip, and it could not be separated without destroyingthe master. Eventually, the following procedure was developed. A 4 gm/2 drop solution of PMMA and benzoylperoxide (Electron Microscopy Sciences, Hatfield, PA)was mixed and allowed to pre-cure for 10 minutes. At thesame time a thin coat of Teflon spray (DuPont, Delaware) was applied to the master chip. The acrylic monomersolution was then poured onto the master contained in the Teflon ring. Cure was complete in 24 hours at 25 oC or 2hours at 75 oC. The acrylic chip separated from the master with a little amount of prying (Figure T4.13B).Once these chips were formed the ports at the end of the channels were drilled through using a 1 mm bit. Next,several methods were explored to bond the chips to glass substrates (2”x3” microscope slides were used), asopposed to another sheet of acrylic described by Muck (since this would produce a strong Raman background).Ultimately, it was found that coating the microscope slide with a thin layer of the same PMMA/ solution, clampingit to the chip, and allowing it to cure at room temperature for 4-5 hours resulted in a strong bond with good seals.Nevertheless, as can be seen in Figure T4.13C, we still have difficulty producing flat chips, and consequently onlypreliminary measurements were performed. A B CFig. T4.13. Photograph of A) Chip Design 1 Master, B) PMMA chip produced from master, and C) Chip Design 2attached to 2”x3” glass slide.While the above micro fluidic chip development was underway, alternative methods to fabricate credit card sizedlab-on-chips were explored. The focus was to develop a working chip that could be used to separate 3-methylhistidine and Risedronate from a urine sample and measure them at physiological concentrations. The followingbasic design was developed (Figure T4.14). It consists of a central channel in which the ion exchange resin could beloaded and two side channels containing SERS-active sol-gels to measure the two analytes. The chip wasmanufactured as follows. A 1.75” by 2.75” rectangle was cut out of a ¼” sheet of acrylic. Three 1 mm widechannels, 1 mm deep were milled into the sheet in a trident pattern. Four 1 mm holes were drilled through the sheetto allow introducing the resin, sol-gels, reducing agent, sample, acid and bases. As described above, the ¼” acrylicchip was bonded to a 2”x3” glass microscope slide using the PMMA/peroxide monomer solution, clamped andallowed to cure at room temperature. After 5 hours, a syringe and #22 needle were used to load the outside channelswith L2 sol-gel and a plug of MTMS in the central channel. The sol-gels were allowed to cure for 12 hours. Nextthe outside channels were reduced with sodium borohydride, again using a syringe with a #22 needle. The surfaceof the chip was then cleaned with isopropyl alcohol and the luer port connectors were attached (NanoPortAssemblies, Part No N-333, Upchurch Scientific, Oak Harbor, WA). The adhesive gasket is cured at 120 oC in 1hour (or 60 oC in 12 hours). The luer ports were not attached until after the channels are loaded and reduced, sincethey are prone to getting clogged by the sol-gel and could retain some of the reducing agent. These ports provided 44
  • 44. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Informationthe lab-on-a-chip to real world fluidic interface (as opposed to the Teflon block interface proposed). To obtainSERS from these chips, a 4”x 6.5” Plexiglas plate was machined to fit the standard 96-well plate reader and hold thechips (see Figure E.1). A B CFigure T4.14. Photograph of of A) machined Trident chip, B) chip attached to 2”x3” glass slide, and C) TridentChip with luer ports.Questions to be answered: Can AFR produce the test chips? AFR was subcontracted to make the chip masters,and had a number of set-backs, but eventually developed the process. Does the process need further modification?Thicker substrates are required. Does the interface perform as planned (no leaks)? Yes, commercial luer locks wereused that simplified the real-world interface. Can the various channels be loaded, reduced? Yes, see above. Whatsize, length is best? We were able to load 200 micron channels AND flow analyte. Can separation materials beintroduced? Yes.Task 5. Define Analytical Figures of Merit. The aim of this task was to establish performance criteria for the lab-on-a-chip. This was accomplished by measuring the analytical figures of merit for the analyzer, as outlined by theFDA: sensitivity, reproducibility, linearity, accuracy, precision, and selectivity.Although the focus of this task was to determine the analytical figures of merit for the micro-fluidic chips, thedifficulties encountered in manufacturing these chips, forced us to perform these tests on the SERS-activecapillaries. We believe that the chip performance would have the same linearity, accuracy, precision, andselectivity, and similar sensitivity, and reproducibility. Sensitivity may be reduced due to a smaller channel size ascompared to the laser focal spot (~365 micron diameter, i.e. fewer analyte molecules in the beam), while thereproducibility will depend on manufacturing. In the case of the former, changing optical components in the fiberoptic sample probe can address this difference. In the case of the latter, improved replication of the chips using aphotolithographic master, such as shown in Figs.T4.13-14, will result in reproducibility equivalent to the capillaries.We proposed to improve sensitivity by developing chemically-selective sol-gels, evaluating sample flow andpistioning, and exploring the use of shorter laser excitation wavelengths. The goal was limits-of-detection (LODsbased on a signal-to-noise ratio of 3) of 1 mg/L for the biomarkers, and 0.01 mg/L for the drugs. Based on Phase Iresults, LODs of 10 to 100 microg/L were predicted. In fact, as described in Task 2, not only were these LODsachieved by using an ion exchange resin, but the analytes were actually measured at these target concentrations (seelowest measured concentration, LMC). Nevertheless, measurements at even lower concentrations are alwaysdesirable.The use of shorter laser excitation wavelengths can further improve sensitivity by more closely matching theplasmon absorption maximum. As described in the proposal, the plasmon maximum for 15 nm silver particles is ~450 nm, while the maximum SERS response occurs at ~500 nm (Figure T5.1).90 Our silver particles are 30-80 nm indiameter, which shifts both of these maxima by ~100 nm, but also reduces the magnitude of the plasmon absorptionby ~ 10 (blue line in Figure T5.1). Nevertheless, shifting to laser excitation at 532 or 633 nm should increase the 45
  • 45. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 SER signal by at least 20 times, and as much as 100 times. Attempts to obtain SER spectra using 20 mW at 532 nm laser (1064 nm frequency doubled using a KDP crystal) and a recently purchased 17 mW at 633 nm HeNe laser (Model MRP170, THOR Labs) failed. This was even true for attempts to obtain normal Raman spectra of pure chemicals at the shorter wavelength. Closer examination of our Raman analyzer revealed that the spectral response of the system is essentially non-existent below 650 nm (red line in Figure T5.1). This is not due to the Si detector, but the inefficiency of the beam splitter, which was not taken into account when proposed. However, there was sufficient response at wavelengths longer than 725 nm when using 633 nm laser excitation (above 2000 cm-1 in the Raman spectrum). Unfortunately, our analytes do not produce any significant peaks above ~1700 cm-1. One of the few chemicals that produce a SERS peak above this wavenumber is cyanide (CN-) with a peak at 2140 cm-1. 6 10 100 15 nm Ag A 90 B 15 nm silverEnhancement Factor 5 50 nm Ag particles 80 10 Experiment (RTA) 70 60 4 10 50 40 532 633 785 Theory 30 C 103 20 10 2 10 0 800 600 650 700 750 800 400 500 600 700 Wavelength (nm) Figure T5.1. A) Theoretical SERS enhancement for RTA 50 nm silver particles (blue line) and Raman analyzer response (red line). SERS of B) 1 and C) 0.1 mg/L using 785 and 633 nm laser excitation, respectively. We measured 100 microg/L cyanide in a SERS-active vial using the 633 nm laser and a 1 mg/L cyanide sample using the 785 nm laser. The calculated enhancement was 34 times when including the sample concentration, signal intensity, laser power, laser wavelength (4th power dependence), and spectral response efficiency ((1mg/L/0.1 mg/L)x (0.07/0.4)x(84 mW/12 mW)x(785 nm/633 nm)4(100%/15%)). Although our Raman system is clearly not optimized for operation at this wavelength, significant improvements in sensitivity can be expected using a more efficient beam splitter. Measuring the SER signal intensity as a function of concentration allows determining linearity, multiple measurements allows determining precision, measuring a mixture allows determining accuracy, and measuring several of the samples on multiple capillaries allows determining repeatability. During the Phase I program, 1- methyl histidine (mislabeled 3-methyl histidine by the supplier, Figure T5.2) was measured over a concentration range of 0.001 to 1 g/L (pH adjusted to 7.5). For each concentration, 50 microL of the 1-MeHis solution was drawn into a SERS capillary (chem2c), and five spectra were collected at random spots along the capillary. The concentration was related to the height of the 1565 cm-1 band in the 1-MeHis SER spectrum. In each case, the largest 2 outliers were discarded (modified T-test). The results of the concentration curve are shown in Fig. T5.3. This procedure provided better than normal reproducibility for the SERS capillaries with relative standard deviation (RSD) of 10-15%. As can be seen, the concentration curve initially increases linearly from zero, but then “rolls- over” as the available silver surface area decreases and monolayer coverage is reached (at ~0.005 mg/mL). This SER response has been previously described in the literature and ascribed to a standard Langmuir-Blodgett isotherm equation. 46
  • 46. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information 0.9 OH N 0.8 O 0.7 NH2 N 0.6 Intensity 0.5 mg/mL Int.1565 OH 1 0.672 0.4 0.5 0.617 N 0.25 0.392 O 0.3 0.1 0.329 NH2 N 0.2 0.05 0.278 0.025 0.234 0.1 0.005 0.143 0.001 0.001 0 0 0.2 0.4 0.6 0.8 1 1.2 Concentration (mg/mL)Fig.T5.2. SERS of A) 1-methyl histidine andB) 3-methyl histidine. Conditions: 100 mW 785, 1- Fig. T5.3. Plot of the 1565 cm-1 peak height of 1-MeHismin 1 mg/mL on L1. as a function of concentration (20% error bars included). Conditions: 100 mW 785, 1-min, on L3 (chem2c).Although the Phase I measurement was a good start, it did not cover the relevant concentration range and it did notaccount for the use of an ion exchange resin to concentrate the sample. A stock solution (3-MeHIS) was prepared inHPLC water, and serially diluted to generate samples having concentrations of 5000, 1000, 500, 100, 50, and 10microg/L. Each sample was pH adjusted to 1 using 1M HCl, passed through an ion retardation column, pre-concentrated on an ion exchange column, eluted off with 0.1M NaOH, and the first 100 microL was loaded into aSERS-active capillary (L3, chem2c) and measured. Similar to the Phase I measurements, spectra were recorded at10 positions along the capillary (1.5 mm apart), but none were removed. The ten spots were averaged to produce asingle spectrum at each concentration and the 1364 cm-1 peak height (baseline at 1433 cm-1 subtracted) was used toprepare a calibration curve (Figure T5.4). The ten points were used to determine the RSD for each concentration,and it was found the RSD = ~20% for 5, 1, 0.5 microg/L, and 40% for the lower concentrations. A B 0.08 0.08 0.055 0.055 0.03 0.03 reversed 0.005 0 200 400 600 800 1000 0.005 350 500 750 1000 1250 1500 1850 0 1000 2000 3000 4000 5000 Raman Shift, cm-1 Concentration (microg/L)Figure T5.4. A) SERS of 3-methyl histidine at 1000, 500, 100, 50, and 10 microg/L. Conditions: pH=1, ionretardation and ion exchange, measured on L3 capillary, 100 mW of 785 nm, ten 1-min scans averaged. B) Plot of1364 cm-1 peak height (baseline at 1430 cm-1 subtracted). Inset of higher concentration. Red dot = 25 microg/L.Since it is clear that the capillaries do not produce the same signal intensity from spot to spot, a program was writtento scan a 15 mm length and continuously collect spectra. In 1 minute it makes 10 passes (5 each way), andeffectively averaged the spot-to-spot intensities. The success of this “Raster” approach is shown in Figure T5.5,where the identical set of capillaries was measured. Although the S/N was reduced (1 min vs 10 min per spectrum),the concentration curve becomes linear. Since repeat measurements were not performed, the precision (RSD) wasnot calculated for each concentration. However, the improved shape of the concentration curve suggests that theRSD has improved, and would likely be similar to our SERS-active vials at 15%. Nevertheless, the RSD for all ofthe concentrations combined allows calculating the reproducibility of the capillaries, which is 27%. If 10-minRaster scans were used, this value would also likely improve. 47
  • 47. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 0.125 A B 0.105 0.085 0.065 ] 0.045 0.025 0.005 350 500 750 1000 1250 1500 1850 0 200 400 600 800 1000 Raman Shift, cm-1 Concentration (microg/L)Figure T5.4. A) SERS of 3-methyl histidine at 1000, 500, 100, 50, and 10 microg/L. Conditions: pH=1, ionretardation and ion exchange, measured on L3 (chem2c) capillary, 100 mW of 785 nm, 1-min Raster scan. B) Plotof 1364 cm-1 peak height (baseline at 1430 cm-1 subtracted). Inset of higher concentration. Red dot = 25 microg/L.To test the accuracy of this method, a 25 microg/L 3-methylhistidine sample was independently prepared andmeasured, and the peak height plotted in the concentration curve (circle). Based on the concentration curve, themeasured peak height (0.036±0.01) corresponded to 150 microg/L, but is within the RSD.Risedronate concentration samples were also prepared, but over a lower concentration range (1000 to 1 microg/L).Again each sample was pH adjusted to 1, passed through an ion retardation column, pre-concentrated on an ionexchange column, eluted off with 0.8M NaOH, and the first 100 microL was loaded into a SERS-active capillary(L3, chem2c) and measured. Spectra collected using the Raster approach are plotted in Figure T5.5 along with themeasure peak height at 1032 cm-1 (baseline at 989 cm-1 subtracted). This time the data conformed more closely tothe Langmuir-Blodgett isotherm. Again, the entire data set was used to calculate a capillary-to-capillary RSD of~32%. Also, a 250 microg/L Risedronate sample was independently prepared, extracted following the proceduredescribed above, and measured to test the accuracy of the concentration curve. This time the peak height (0.022,green square) placed the concentration at ~300 microg/L, well within experimental error. As a further test of theability to use this calibration curve, a 10 microg/L Risedronate sample was prepared in reconstituted urine,extracted as above, and measured. The peak height (0.016, red dot) was very close to that measured for the pristine10 microg/L sample (0.017 peak height). 0.03 A B 0.025 0.02 0.015 0.01 0.005 350 600 800 1000 1250 0 200 400 600 800 1000 Raman Shift, cm-1 Concentration (microg/L)Figure T5.5. A) SERS of Risedronate at 1000, 500, 100, 50, 10 and 1 microg/L. Conditions: pH=1, ion retardationand ion exchange, measured on L3 (chem2c) capillary, 100 mW of 785 nm, 1-min Raster scan. B) Plot of 1032 cm-1 peak height (baseline at 989 cm-1 subtracted). Inset of higher concentration. Green square = 250 microg/L, red dot= 10 microg/L in urine.As proposed, concentration curves for several other biomarkers and drugs were prepared and measured. It wasfound that analytes that could be detected at very low concentrations (1 microg/L), followed the Langmuir-Blodgett 48
  • 48. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007at these lower concentrations and leveled off within 2 orders of magnitude, such as Risedronate. Conversely,analytes that could be detected only as low as 50 microg/L followed this curve at higher concentrations, leveling offat correspondingly higher concentrations. We attribute this response to the chemical-selectivity of the sol-gels andthe analyte-to-metal interactions, i.e. the greater the interaction, the greater the sensitivity, and the greater theselectivity the lower the surface saturation point.Questions to be answered: What are the LODs for each of the biomarkers, drugs and their metabolites? SeeTables T1.3 and T1.4, T2.1 and T2.2. What is the reproducibility of the chips? This was not determined, but islikely to be the same as the glass capillaries, at about 30%. Which channel design provides the best selectivity,reproducibility, and sensitivity? Since the chip manufacturing was substantially delayed, it was only determined thatwe could load and perform measurements in both glass and plastic channels at 800, 600, 500, 250, and 200 microns.What is the best standardization method for quantifying target analyte concentrations? It appears that the bestmethod would be to sue creatinine and uric acid as internal intensity references. Creatinine is a known constant forurine chemistry. Once the relative uric acid concentration is determined, all other chemicals could be referenced toit, since it appears in virtually all samples due to its 7 ionizable protons (7 pKas.)Task 6 - Prototype Design. The overall goal of this task was to design a prototype system to be used and tested byNASA in Phase III. This has been accomplished by redesigning the proposed compact disc (CD) style lab-on-a-chip based on the Task 3 and 4 data.The proposed analysis of urine from astronauts on long term space missions in a microgravity environment willemploy a multi-component analyzer that will be integrated into either Node 3 on the International Space Station(ISS) or on future manned flights. This ultra compact system will be placed into an integrated rack, and will beconnected to a water reclamation system currently being developed by Hamilton-Sundstrand. A 10 mL samplecontainer designated for each astronaut will collect urine samples over the course of 24 hrs. After 24 hrs a 1 mLsample is withdrawn for analysis, and the container subsequently flushed with water for the next days use. Thisanalyzer will consist of three major components, the sampling system, the lab-on-a-chip, and a Raman instrument.The final lab-on-a-chip design will depend upon the other two components.Employing a sequence similar to that of the operation outlined in Figure T3.24, the following very simpledesign is proposed (Figure T6.1). The system consists of four components, A) an injection cartridge, B) the lab-on-a-chip, C) pneumatic valve control manifold, and D) a fiber optic linked Raman probe. In many respects it willoperate like a compact disc player. The injection cartridge will contain up to five needles to deliver 1) a vacuum formicro-fluidics control, 2) the sample from the individual astronaut sample container (which will be pH adjusted to1 using acid from a reservoir), 3) a base gradient prepared by increasing the amount of NaOH mixed in with water(note reservoirs), 4) air from a bladder to allow fluid flow control when combined with the vacuum line, and 5)waste removal line. Again, like a CD player, six chips will be used, one per astronaut (based on expected ISScapacity). Each lab-on-a-chip will consist of a 12 cm diameter disc (CD-size) divided into 30 pie-shaped “slices”,12-degrees wide (12 mm across outer edge), one slice per sample. Thus, each lab-on-a-chip will provide 30measurements; 1 per day. (A practical first version using 36-degee slices can be made with the technologydeveloped in this program, see below). Each sample segment will contain five ports (6-10), each 1.5 mm diameter,2 mm deep to match the cartridge delivery needles. A circular layer of butadiene (septa material, 11) will be bondedto the disc to seal the ports prior to use. The sample acceptance port (7) will also contain a layer of Nylon 66 (orequivalent particle filter material, 12) melted into place. Valves (13) integrated into a second, lower layer of the lab-on-a-chip will be computer controlled through a pneumatic or magnetic manifold below the chip. Recently, a plug-and-play micro-fluidic device has been built that employs computer controlled diaphragm pumps to control flowthrough desired channels.91 The choice of valve control will be based on available technology at the time ofimplementing this design. The valves are placed to control flow through the various separating materials and directthe analytes to the appropriate SER-active sol-gel set. An ion retardation channel (14) will contain the requiredseparation materials to extract salts, while an ion exchange (cationic) column (15) will adsorb the protonatedanalytes. The remainder of the sample will be removed from the chip through port 10 to waste (back to waterreclamation). Then the NaOH gradient will be delivered through port 8 through the ion exchange resin andsequentially to 1 of 12 SER-active sol-gels (16). The first 3 sol-gels are for biomarkers (e.g. 3-methyl histidine), thenext 8 for drugs, and the last for creatinine. The sequence is based on their relative pKa’s (e.g. 3-MeHis = 5.9,Risendronate ~8.5). These channels will lead back toward the center of the disc where a central vacuum or pistonwill be used to move the 3 µL sample through the sol-gels for analysis. A fiber optic linked Raman probe will be 49
  • 49. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Informationfixed above the SER-active channels, such that the 100 micron illumination spot matches the channel. Actual Size A Base w Acid Vie D Water p To 1 2 3 4 5 B Side 12 cm C View A 11 D Top View Size x4 16 14 15 12 mm 11 Measurement Points 13 Side ViewFigure T6.1. Illustration of urine analyzer, composed of a A) sample delivery cartridge, B) lab-on-a-chip, C)pneumatic/magnetic valve control manifold, and D) Raman optic probe. This figure is proprietary.In operation, the cartridge head (A) will lower, the needles will pierce the seal, and 1 mL of urine, acidified with 25microL 1M HCl, will be loaded through Port 7, through the particle filter (12), and through both ion resins (14 and15), then out through the waste port (10). This volume will provide a significant amount of analytes adsorbed ontothe ion exchange resin, and does not influence the size of the channels. One the ion exchange resin is loaded, 120microL of NaOH gradient will enter through port 8 and flow through the exchange resin into the SERS-activechannels in sequence. Valves would be used to select and deliver 10 microL sample of increasing pH to eachchannel (e.g. top to bottom). The air line allows offsetting pressure drops. Based on Task 3, a 200 micron squarechannel - 20 cm long will work and we have successfully loaded a chip obtained from NASA (JPL) that has a 50
  • 50. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Informationserpentine channel of these dimensions and obtained a SERS spectrum of Risedronate separated from urine (off-chip, Figure T6.2). However, this channel dimension would require 36-degree “pie slices” per analysis, limitingeach CD chip to 10 analyses. This should be acceptable for a first version of this analyzer.At a flow rate of ~ 0.1 mL/min all of the channels would be loaded (we were able to flow 0.5 mL/min through the800 micron channels). Then the cartridge head separates from the lab-on-a-chip, and the chip rotates in a step-wisefashion, such that Raman spectra can be collected from each of the SER-active sol-gel segments. Onceaccomplished, the chip again rotates to align the cartridge of the ports of the next sample segment and awaits thenext sample. The total analysis for each sample is expected to take no more than 15 minutes. Analysis of thespectra to identify each biomarker or drug based on spectra matching will be accomplished in less than 1 second.All spectra could be sent to earth for further analysis, as well as for identifying unknowns. It should be realized thatthe pneumatic manifold and Raman probe are fixed, and the only motion is the rotation of the lab-on-a-chip and theup and down movement of the sample delivery cartridge. This device will require very little power. Furthermore,only 25 microL acid and 120 microL of base are required per analysis, or ~1.5 mL for a 10 sample CD disc, or 4.5mL for a 30 sample CD disc! The 1 mL urine sample is not included, since it is provided by the astronaut. A B C L3 ( Ag, very non-polar) L2 (Ag, non-polar) L5 (Ag, very polar) L4 (Au, very polar)Figure T6.2. A) Photograph of lab-on-chip provided by JPL for testing. Note the serpentine channels are 200 x 200microns, and the serpentine channels are ~ 20 cm long. B) SERS of 1 mg/L Risedronate measured in this chip.Drug extracted from real urine (off-chip) and loaded into this chip and measured. Conditions: 75 mW of 785 nm, 1-min. C) Photograph of 4-channel SERS-active test chip (representative of the deliverables).Mission Tests (Phase III). The overall objective of this task is to test the space-worthiness of the proposed lab-on-achip. This will be accomplished by supplying NASA with 20 of SER-active lab-on-a-chips for Phase III testing (adeliverable).Twenty 4-channel chips have been manufactured and filled with 4 SERS-active sol-gels (L2, L3, L4 and L5, FigureT6.2C). These tests will include shelf life (2 years/accelerated tests), temperature cycling between -80 and 40 oC,vibrational tests, shock, 20-g forces, micro-gravity, radiation resistance (3 krads), cross-contamination-elimination,etc. This may include shuttle or space station testing as appropriate. 51
  • 51. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007Performance Schedule - The Phase II research program was accomplished according to the following schedule,tasks, and milestones with the work load distributed as SE-150, ENG-200, RA-200 hours:The program was extended by 6-months. QUARTERS 1 2 3 4 5 6 7 8Task 1 - Develop Spectral Library. 1 2 3Task 2 - Develop Chemical Selectivity. 4 5 6Task 3 - Select and Test Components. 7 8 9 10 11Task 4 - Fabricate Lab-on-a-chip 12 13 14 15Task 5 - Define Analytical Figures of Merit. 16 17 18Task 6 - Design Prototype. 19 20Milestones1. Prepare 96-well SER-active sol-gel plates 11. Re-examine search routine capabilities2. Measure SERS of 24 chemicals 12. Finalize test chip design3. Test and refine library search algorithms 13. Fabricate test chips4. Identify unique bands and determine detection limits 14. Fabricate real-world interface5. Prepare sol-gel coated capillaries 15. Test fluid deliver, Raman collect, etc.6. Measure reversibility and prelim LODs 16. Measure sensitivity7. Purchase components/chromatography supplies 17. Determine reproducibility8. Prepare 4-zoned capillaries 18. Demonstrate selectivity9. Test separation methods 19. Perform field test10. Test interferents 20. Design test chips for NASAThis Final Report was delivered to NASA (June 1, 2007) summarizing the experimental and analytical results of theproject according to contract specifications. In addition, RTA mailed 20 lab-on-chips (see Figure T6.2c above) forcontinued testing in Phase III (see Mission Tests above). 52
  • 52. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Part 5: Potential ApplicationsThe overall goal of this program is to develop an analyzer to measure bio-indicators and prescribed drugs and theirmetabolites in urine to assess and monitor health of astronauts affected by microgravity conditions during extendedmissions in space. The analyzer will employ a lab-on-a-chip to extract and separate out chemical components fromurine samples and surface-enhanced Raman spectroscopy to detect and identify these indicators. The proposedanalyzer is immediately applicable to astronauts working in the International Space Station. For example,monitoring the concentration of pyridinoline may suggest increasing (or decreasing) the dosage of the anti-bone lossdrug zoledronate. The proposed analyzer will undoubtedly increase our understanding of the adverse effects ofnear-zero gravity, allow determining the efficacy of drugs used for treatment, and improve regulating dosage. Theanalyzer will have continued value through the life of the ISS and into developing a base on the moon.Finally, the knowledge gained will undoubtedly be critical to the strategy of traveling to Mars safely.The proposed analyzer will also have a substantial impact on the health of a significant portion of our population,the elderly, in its ability to measure bone-loss bio-indicators, as well as the drugs being used to treat osteoporosis.Osteoporosis, a condition correlated to enhanced bone fragility and increased risk of fracture. As of 2006,osteoporosis effects 55% of the US population over age 50. More than 50% of healthy American women aged 30-40 are likely to develop vertebral fractures as they age due to osteoporosis.92 Each year, more than 250,000 hip and500,000 vertebral fractures occur in postmenopausal women in the USA.93 In 2006, it was estimated that fracturesrepresent a $17 billion annual cost to the US healthcare system. The World Health Organization has declaredosteoporosis the 2nd largest medical problem, next to cardiovascular diseases.94 Unfortunately, treatment is at bestpartially successful once progressive bone weakening has started.The primary method of detecting osteoporosis is the measurement of mineral density and bone mass using dual-energy X-ray absorptiometry, which lacks sufficient image quality to adequately detect early stages. It provides astandard (adult) density (SD) score, SD = 0 = healthy, SD = -2.5 onset of osteoporosis. Recently, studies haveidentified pyridinoline and deoxy-pyridinoline as degradation products of type 1 collagen. Not only can these bone-loss indicators be detected in urine, their concentrations have been found to decrease in response to certain drugtherapies, suggesting their use in monitoring treatment efficacy.93 Two methods are currently being developed todetect these bio-indicators, high-performance liquid chromatography (HPLC) and immunoassay. HPLC methodsare slow (30-min per analysis), labor intensive, and require daily calibration, while immunoassays are notoriouslyinaccurate.The proposed lab-on-a-chip, coupled to a Raman analyzer, could be used by clinical labs that perform blood andurine analysis (e.g. Quest Diagostics). The family physician would simply check “test for osteoporosis” duringannual physicals for patients. One milliliter of the urine sample is injected into a disposable lab-on-a-chip ($25), itis placed on the Raman Analyzer ($75,000), and a “score” is reported (0 to -5 SD). The process takes 5 minutes.This compares favorably to performing x-rays (machine costs $300,000) and the x-ray cost (>$100 and 30 minanalysis time), which is not covered by most insurance. Furthermore, the patient does not have to make separateappointment. RTA plans to pursue a marketing agreement with Quest Diagnostics. Part 6: ContactsPrinciple Investigator: Dr. Frank E. Inscore (860-528-9806, x128, inscore@RTA.biz), a Research Scientist andManager of Raman Applications at RTA, will be the Principal Investigator of this program. Dr. Inscore received hisPh.D in May of 2000 under Prof. Martin L. Kirk at the University of New Mexico. His research was directedtowards elucidating the electronic structure of Mo and W complexes employing a combined spectroscopic andtheoretical approach that focused primarily on the use of Raman spectroscopy. The PI has considerable expertise inCCD dispersive systems, and in designing sampling configurations under a variety of conditions (cryogenic/anaerobic) employing Raman. The PI was previously the manager and key designer for the Raman Lab of Dr.Martin Kirk at the University of New Mexico as a graduate student (Research Assistant). As a Research Associateat the University of Arizona with Prof. John H. Enemark he examined simple and complicated inorganic systems asstructural and electronic models of metalloprotein active sites where he further employed his expertise in ResonanceRaman spectroscopy. The PI has over 12 years experience with vibrational/Raman spectroscopy, and has publishedpeer-reviewed articles regarding instrumental set-up and analysis of Raman and other spectroscopic datacollected.40,41,42,43 The PI has also presented relevant research at three national ACS meetings, a Gordon Research 53
  • 53. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007Conference, two SPIE presentations, and a Pittsburg conference. The PI has also gained extensive experience in sol-gel chemistry and surface-enhanced Raman spectroscopic applications at RTA. Since joining RTA, Dr. Inscore hasperformed critical development work of RTA’s SER-active capillaries. He has successfully used this technology tomeasure chemical warfare agents and their hydrolysis products, and trace pesticides residues. He was also a majorcontributor for detecting dipicolinic acid extracted from Bacillus anthracis spores on surfaces. This programrepresents Dr. Inscore’ first SBIR win. He is on 1 patent and 4 pending.Principal Investigator: Dr. Frank E. Inscore, Research Scientist, Real-Time Analyzers, Inc.Education University of New Mexico - Ph. D. Chemistry - 2000 University of New Mexico - B. S. Chemistry Major/Applied Mathematics Minor - 1993 Austin College - B. A. Biology - 1986Experience Real-Time Analyzers, 2003 – present Manager of Raman applications at RTA: responsible for Raman research and development. University of Arizona, 2000-2003 Post doctoral Research Associate for J.H. Enemark, University of Arizona. Managed group research program: responsible for synthesis/purification of 1st generation geometric and electronic structural models of metalloenzyme active sites, and subsequent characterization via NMR, FAB/ESI-MS, IR/Raman, XRD, XAS/EXAFS, UV-vis/NIR absorption, gas-phase/solution anionic PES, CW/pulsed-EPR spectroscopies, and DFT computations. Additional responsibilities included isolation/purification of various metalloproteins via chromatography, HPLC and FPLC. University of New Mexico, 1994-2000 Research Assistant for M.L. Kirk University of New Mexico. Built and managed Raman facilities: responsible for probing contributions to structure/function relationships in enzyme active sites using resonance Raman, VT-MCD/CD, and UV-vis/NIR absorption spectroscopy.Honors and Awards Research Award of Excellence 1998 &1999 DOE GAANN Fellowship 1994 -1996 Dean Uhl Award for Excellence in Chemistry 1994 Inducted Kappa Mu Epsilon National Mathematics Honorary 1993Program Manager: Dr. Stuart Farquharson (860-528-9806, x127, stu@rta.biz), President at Real-Time Analyzerswill be the Program Manager. He has extensive experience in designing and developing both infrared and Ramanspectrometers for multiple industrial applications.95 While employed by the Dow Chemical Company, he designedand integrated a Fourier transform infrared spectrometer into a polymer production plant, for continuous processcontrol. The PM designed and patented the required sample system, designed the optical interface, installed theinstrument, and assisted in designing user-friendly data analysis software to ensure customer satisfaction. The PMhas extensive experience in Raman and surface-enhanced Raman spectroscopic applications, as well as instrumentdesign, and has published 50 papers in scientific journals and holds 6 patents in the field. He has been a Chair orCo-chair at 10 SPIE Conferences. He has been an invited speaker at more than 20 conferences. In 2002 thisincludes the Pittsburgh Conference (New Orleans, LA, March), CPAC Summer Institute (Seattle, WA, June),Gordon Research Conference (Newport, RI, July), FACSS (Providence, RI, October), and Eastern AnalyticalConference (Somerset, NJ, November). Dr. Farquharson’s resume is available upon request, and is provided in thePhase I and II proposals.Senior Investigator: Mr. Chetan Shende (860-528-9806, x134, chetan@rta.biz), a senior chemist at Real-TimeAnalyzers, will be a senior investigator. He recently completed his MS in Analytical Chemistry from the Universityof South Florida, under the direction of Dr. Abdul Malik. His main research area was developing columntechnology for analytical micro-separations using sol-gel chemistry based systems. His MS thesis work involveddeveloping Open-Tubular columns for high resolution capillary gas chromatography (CGC), using polyethyleneglycols (PEG) as the stationary phase. Mr. Shende has expertise in sol-gel synthesis and coating capillary columns.His resume is available on request.NASA Technical Representative: Robert Hawersaat (216-433-8157, Robert.w.hawersaat@nasa.gov).Hamilton Sundstrand Contact: Patricia O’Donnell, Program Manager (860-654-5649,patricia.odonnell@HS.UTC.com) 54
  • 54. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Confidential Proprietary Information Part 7: Future Technical ActivitiesThe overall objective of the proposed program (through Phase III) is the development of an analyzer integrated intothe International Space Station (ISS) toilets capable of immediate detection of key chemicals in urine to monitor andassess astronaut health. During Phase I we initiated conversations with Hamilton Sundstrand (HS), and discussedthe design for their toilet and water reclamation systems to be integrated into Node 3. We also proposed to test ourchip performance using HS’s Raman system being developed for a Mars mission. Dr. Farquharson visited HS inPomona, CA and found that HS only built a single prototype that was unavailable for tests. However, RTA hasdeveloped a very rugged Raman analyzer, which may be suitable for space operation (~ 1/5th the weight of the HSprototype), and integration into their toilet system. This is still the desired goal of this program as HS’s Node 3Water Processor system is finally being integrated into the ISS (Shuttle Mission: 20A/STS-122).As described in Task 6 above, the analyzer will be comprised of three major components: a urine capture device, alab-on-a-chip, and a Raman analyzer. The entire analyzer will be sufficiently small to fit into a standard ISSintegrated rack (IR). In use, 1 ml urine sample will be extracted from water reclamation line exiting HS’ toilet andcollected into one of six sample containers (bladder), one per astronaut (ISS capacity). This will be performed 10times during a 24 hour period per astronaut for a total of 10 mL each. From these individual bladders, we expect todraw 1 mL per analysis per day. We will work with HS to develop a rinse cycle to flush the sample containers oncea day after each analysis. The lab-on-a-chip will be designed on a 12-cm disk (1 disk per astronaut, 10 or 30 days ofanalysis, versions 1 and 2) that will contain 10 or 30 identical pie “slices”, each section self-contained with fullextraction/separation capabilities and SERS-activity for detection. The disk will rotate once a day for a new urineanalysis. This approach will virtually eliminate the potential for cross-contamination. Multiple disks per astronautor higher lab-on-a-chip channel densities can be used for longer missions.Mission Tests (Phase III). The overall objective of this task is to test the space-worthiness of the proposed lab-on-achip. We have supplied NASA with 20 SERS-chips so that some can be 1) tested in space and 2) used to measureactual astronaut urine brought back to earth. Phase III commercialization tests will begin with comprehensiveanalysis of osteoporosis, including analysis of new bis-phosphonate drugs as they become available (e.g. withPfizer), and ultimately lead to preliminary clinical tests (possibly at the Nevada Cancer Institute.) Part 8: Potential Customer and Commercialization Activities8.1. The Company: Real-Time Analyzers, Inc. (RTA, www.rta.biz) - The mission of Real-Time Analyzers is todevelop, produce and market analyzers that detect, identify and quantify trace chemicals in real-time, inindustrial or field settings, either continuously or on-demand.Real-Time Analyzers is a spin-off company of Advanced Fuel Research launched September 1, 2001. RTA wasformed to consolidate and commercialize the considerable expertise in Raman and surface-enhanced Ramanspectroscopy developed at AFR into a focused product line. This approach of bringing high-technology productsto market was recently validated when AFR sold its first spin-off company, On-Line Technologies to MKSInstruments, Inc. for over $20 million.Dr. Stuart Farquharson has developed a formal Business Plan for RTA focusing on the Chemical ManufacturingIndustry (agricultural chemicals, fine chemicals, petroleum products, polymers, and pharmaceuticals). Dawnbreakerprovided the basic tools for developing this Business Plan (www.dawnbreaker.com through DOE sponsorship).Foresight is currently performing an osteoporosis market analysis. The Business Plan is a two phase growthstrategy, 1) design, develop, market and sell a portable fiber optic based FT-Raman instrument (Industrial RamanAnalyzer, IRA), and 2) develop a trace chemical analyzer (Surface-enhanced Raman Analyzer, SERA) forenvironmental, pharmaceutical, medical applications and homeland security. The IRA is being marketed to 1)Homeland Security, 2) the DOD, and 3) the Chemical Manufacturing Industry.RTA completed the first growth Phase as of June 2006 when it sold its first two analyzers! Since that time RTAhas sold 4 more analyzers, penetrating each of these 3 markets!Initially, Dr. Farquharson was the only full-time employee with 4 part-time employees occupying 600 sq. ft. Earlygrowth has been focusing on product and application development. In January 2002, two AFR employees, an 55
  • 55. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007instrument design engineer and a PhD chemist, transferred to RTA. In June 2002 RTA hired a mechanical engineer,an optics engineer, and a chemical engineer. In January 2003 RTA added two PhD Raman spectroscopists, an MSsol-gel chemist, and a full-time accountant. The engineers focus on developing the IRA, while the chemists focuson developing the SERA and applications. In January 2004 RTA added a product engineer with 12 years experienceproducing FT-infrared spectrometers. RTA currently has 7 full-time employees (all with shared and/or overlappingmanagement, technical and manufacturing responsibilities). RTA moved into new facilities in 2005 (5000 sq. ft. ofoffice, lab, and manufacturing space). RTA currently employs several consultants and subcontractors to meet theirR&D and manufacturing needs. RTA’s Revenues 2004-7. Revenues 2004 2005 2006 2007 Employees 9 8 8 9-12 Analyzer Sales $122,000 $300,000 Service Contracts $28,500 $2,500 $125,000 $400,000 Vial Sales $12,000 $12,000 $12,000 $12,000 SBIR Funding $582,000 $910,000 $717,000 $300,000 % Revenue SBIR 93.4% 98.1% 74.2% 29.6% Total Revenue $622,500 $927,500 $966,000 $1,012,0008.2. Markets. This program has provided the foundation for an osteoporosis product, the Urine-analySER. Thisproduct and market are part of RTA’s second growth phase, selling SERA products.Osteoporosis – When we submitted this proposal in 2005, there were 10 million men and women suffering fromosteoporosis in the USA. As of 2006, there are 44 million US patients! Each year, more than 250,000 hip and500,000 vertebral fractures occur in postmenopausal women in the USA.93 And more than 50% of healthyAmerican women aged 30-40 are likely to develop vertebral fractures as they age due to osteoporosis. Metabolicbone and joint diseases (osteo-arthritis, cancer, etc) account for an additional 12 million cases of accelerated boneloss per year.92 The World Health Organization has declared osteoporosis the 2nd largest medical problem, next tocardiovascular diseases.94 The estimated yearly cost associated with acute hospital care and rehabilitation wasestimated at $17 billion for 2006.93 Unfortunately, treatment is only partially successful once progressive boneweakening has started. Therefore, it is important to identify this condition at an early stage, so that treatmentfocuses on prevention instead of fixing fractures and attempting to reverse bone loss. Nevertheless, it should benoted that drugs developed to prevent, mitigate or even reverse bone loss are showing signs of success.The primary method of detecting osteoporosis is the measurement of mineral density and bone mass using dual-energy X-ray absorptiometry, which lacks sufficient image quality to adequately detect early stages. More accurateanalyses, such as bone biopsies, have been developed, but are undesirably invasive. Recently, studies haveidentified pyridinoline and deoxypyridinoline as degradation products of type 1 collagen. Not only can these bone-loss indicators be detected in urine, their concentrations have been found to decrease in response to certain drugtherapies, suggesting their use in monitoring treatment efficacy.92 Two methods are currently being developed todetect these bio-indicators, high-performance liquid chromatography (HPLC) and immunoassay. HPLC methodsare slow (30-min per analysis), labor intensive, require daily calibration, and high up front and maintenance costshave limited their market acceptance. Immunoassay kits (colorimetric microassay plate enzyme immunoassay) havebeen developed to measure total deoxypyridinoline in urine. However, the analysis requires complicated hydrolysis,incubation and assay steps totally 48 hrs prior, which must be followed precisely to obtain quantitative results.The Urine-analySER could be used to rapidly detect and quantify pyridinoline, deoxypyridinoline, and otherbiomarkers in urine and assess patient risk, stage, or response to treatment.8.3. Commercialization Strategy -The overall commercialization strategy is to bring Real-Time Analyzersproducts to market through strategic partners with the aid of leveraged investment. This strategic path tocommercialization involves the following specific steps: 1) Form a spin-off company to consolidate expertise anddevelop a focused product line (RTA has 4 scientists with over 40 years of Raman experience, and engineers withover 35 years experience designing spectrometers). 2) Develop a superior produce with substantial patent coverage 56
  • 56. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007(the superior performance of the IRA is covered by 4 patents, the SERS technology is covered by 3 patent, and 11more have been filed). 3) Leverage the protected technology to gain market access through strategic partners (seebelow). 4) Grow value of company so that acquisition is desirable.Strategic Partners - RTA has profiled more than 50 companies in its target markets and has identified severalpotential Strategic Partners. Initial meetings have been held to describe RTAs mission, technology, market plans,and strategic partnering. Relevant to this program, RTA has met with Hamilton Sundstrand and the Nevada CancerInstitute. RTA plans to initiate discussions with Quest Diagnostics, as well. In each case, RTA plans to sell thebeta-unit (based on successful on-site demonstration) and leverage investment against exclusive-use licenses.Developments in Strategic Partnerships with Hamilton Sundstrand and Nevada Cancer Institute, are worthelaborating. In addition, RTA has been actively pursuing matching state funds through Connecticut Innovations.Hamilton Sundstrand Sensor Systems (HSSS, Pomona, CA) is the Sensor Systems business unit of OrbitalSciences bought in 2001 by Hamilton Sundstrand, one of United Technologies Corporation six business units. HS,with annual sales of $3 billion, bought Sensor Systems to complete their portfolio of space-based atmospheric andspace station sensor systems.96 Included in this acquisition was Analect, a company with considerable knowledge ofchemical analyzers, and an understanding of Raman analyzers. Patricia O’Donnell, business developmentmanager, has been intimately involved in the development of both the HS toilet and water reclamation system.WE hope to collaborate on the development of the proposed analyzer for ISS Node 3 during Phase III.Nevada Cancer Institute (Las Vegas, NV) - NCI began operations in 2005 with the goal of becoming a recognizedworld-leader performing state-of-the-art research and implementing groundbreaking methods of prevention,detection and treatment of cancer. According to Director Dr. Vogelzang: "It will expand the tools available for thetreatment of cancer. That will be our first and primary thrust." NCI has agreed to assist RTA in the development ofa chemotherapy drug analyzer based on measurements of saliva using our SERA technology. The importance ofmeasuring bone-loss in urine due to chemotherapy is a natural extension of this technology, and arrangementswill be made to perform these critical clinical tests with NCI with the goal of obtaining FDA approval.Quest Diagnostics Incorporated (www.questdiagnostics.com), a $500 million per year revenue company, is thenations leading provider of diagnostic testing, information and services that patients and physicians need to makebetter healthcare decisions. QD performs diagnostic laboratory tests for approximately 145 million patients eachyear, and over 70% of all healthcare treatment decisions. QD has over 2,000 patient service centers. These servicecenters each represent one Urine-analySER sale.Connecticut Innovations (CI) is charged with growing the economy of Connecticut’s entrepreneurial technologythrough venture and other investments. RTA has had several meetings with CI over the past 3 years, and they arecurrently considering a $500,000 investment. Part 9: Resources StatusNo government equipment was required for the Phase II program. The following resources at RTA are available.RTA Industrial Raman Analyzer Production (second generation, 3 systems) - RTA latest FT-Raman system isextremely compact and portable. The entire system fits into a single 19” rack mountable box (19x20x7”). The mainfeatures are: 1) a diode laser (1 W at 785 nm, Process Instruments), 2) an interferometer with a quartz beam splitter(MKS), 3) a Si avalanche photodiode detector (RTA), 4) numerous fiber optic probes (designed in-house), and 5) adata station consisting of acquisition and analysis hardware and software (OLT, National Instruments, and aGateway Pentium II personal computer). The system measures Raman spectra from 785 to 1080 nm (0 to 3200∆cm-1).RTA Industrial Raman Analyzer Prototype (second generation, 1 system) - RTA’s initial compact FT-Ramansystem employs a Nd:YAG laser (800 mw at 1064 nm, Keopsis), a compact interferometer (MKS), and an InGaAsdetector (RTA). The system measures Raman spectra from 1064 to 1800 nm (0 to 3500 ∆cm-1). The entire systemalso fits into a single 19” box.RTA Raman Analyzer Research Grade - This system frequency doubles the high powered Spectra Physics laser to 57
  • 57. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007generate 20 mW of 532 nm laser excitation. This coupled with the second generation IRA that employs silicadetection will be used to increase the SER scattering efficiency from 50 to 100 times. This will provide importantdata regarding potential modifications to the Hamilton Sundstrand Raman system.General Facilities at RTA - RTA has 15 high-end, interconnected personnel and laptop computers, with Internetaccess. The systems are all also tied into a Snap Server for day-to-day back-up. RTA has an extensive wet labequipped with two fume hoods for safe preparation of the required samples. A dry box is also available forpreparation of oxygen sensitive chemicals. Also available to RTA are full service machine and electronic shops. Part 10: References1 Hughes-Fulford, M., “Review of the Biological Effects of Weightlessness on the Human Endocrine System,” Receptor, 3:3, 145-54 (Fall 1993).2 Kohl R.L. and S. MacDonald, “New Pharmacologic Approaches to the Prevention of Space/Motion Sickness,” J Clin Pharmacol,10, 934-46 (1991).3 Santy P.,M.Bungo, “Pharmacologic Considerations for Shuttle Astronauts,”J Clin Pharm, 31, 931-3 (1991).4 LeBlanc, A. and V.Schneider, “Can the Adult Skeleton Recover Lost Bone?” Exp Gerontol, 26, 189 (1991).5 Lane, H.W., A.D. LeBlanc, L. Putcha, and P.A. Whitson, “Nutrition and Human Physiological Adaptations to Space Flight,” Am J Clin Nutr, 58:5, 583-6 (Nov 1993).6 Tipton, C.M., J.E. Greenlead, and C.G.Jackson, “Neuroendocrine and Immune System Responses with Spaceflights,” Med Sci Sports Exerc, 28:8, 988-98 (Aug 1996).7 Wood C.D., J.E. Manno, B.R. Manno, H.M. Redetzki, M. Wood, W.A. Vekovius, “Side Effects of Antimotion Sickness Drugs,” Aviat Space Environ Med, 55:2, 113-6 (1984).8 Harris, C.I. and G. Milne, "The urinary excretion of N7-methylhistidine by cattle: validation of muscle protein breakdown," Br. J. Nuutr., 45, 411-422 (1981).9 Percy, S.D. and M.E. Murphy, "3-methylhistidine excretion as an index of muscle protein breakdown in birds in different states of malnutrition," Comp. Biochem. Physiol., 116A, 267-272 (1997).10 Rathmacher J.A. and S.L. Nissen "Development and application of a compartmental model of 3-methylhistidine metabolism in humans and domestic animals," Adv Exp Med Biol 445, 303-24 (1998).11 Brazier M, S. Kamel, M. Maamer, F. Agbomson, I. Elesper, M. Garabedian, G. Desmet, J.L. Sebert, "Markers of bone remodeling in the elderly subject: effects of vitamin D insufficiency and its correction," J Bone Miner Res,10, 1753-1761(1995).12 Krall, E.A., B. Dawson-Highes, K. Hirst, J.C. Gallagher, S.S. Sherman, G.J., and G Dalsky, "Bone mineral density and biochemical markers of bone turnover in healthy elderly men and women," J. Gerontol A Biol Sci Med Sci, 52, M61-67 (1997).13 Whitson, P.A., R.A. Pietrzyk, and C.Y. Pak, “Renal Risk Assessment During Space Shuttle Flights,” J Urol, 158:6, 2305-10 (Dec 1997).14 Taylor G, Houston JB, “Simultaneous Determination of Promethazine and Two of Its Circulating Metabolites by High-Performance Liquid Chromatography,” J Chromatogr, 230, 194-8 (1998).15 National Research Council Space Studies Board, A strategy for Research in Space Biology and Medicine in the New Century, Washington, DC, National Academy Press, 1998.16 Inoue, H., Y.Date, K.Kohashi, , H.Yoshitomi, , and Y.Tsuruta, “Determination of Total Hydroxyproline and Proline in Human Serum and Urine by HPLC with Fluorescence Detection,” Biol Pharm Bull, 19, 163-6 (1996).17 Strickland, P.T., Kang, D, Bowman, E.D., Fitzwilliam, A., Downing, T.E., Rothman, N., Groopman, J.D., and Weston, A., “Identification of 1-hydroxypyrene Glucuronide as a Major Pyrene Metabolite in Human Urine by Synchronous Fluorescence Spectroscopy and Gas Chromatrography-Mass Spectrometry,” Carcinogenesis, 15:3, 483-7 (Mar 1994).18 Potember, R.S., W.A. Bryden, M. Antoine, "Quantitative determination of 3-methylhistidine in urine by matrix- assisted laser desorption mass spectroscopy," STAIF- 2000, paper 106 (2000).19 Leelavathi DE, Dressler DE, Soffer EF, Yachetti SD, Knowles JA, “Determination of Promethazine in Human Plasma by Automated High-Performance Liquid Chromatography with Electrochemical Detection and by Gas Chromatography-Mass Spectrometry,” J Chromatogr, 339:11, 05-15 (1985).20 Finnigan product literature (www.finnigan.com), $99,950 for tandem mass spectrometer LCQDUO.21 Kneipp, K., Wang, Y., Dasari, R. R., and Feld, M. S., “Approach to Single-Molecule Detection Using Surface- Enhanced Resonance Raman Scattering (SERRS): A Study Using Rhodamine 6G on Colloidal Silver,” Applied 58
  • 58. "fi, sf" RTA P2004#65 NASA Topic No B3.05 CN: NNC05CA09C Phase II Final Report June 2, 2007 Spectroscopy, 49, 780-784 (1995).22 Nie, S. and Emory, S.R., “Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering,” Science, 275, 1102 (1997).23 Farquharson, S. and P. Maksymiuk, “Simultaneous chemical separation and surface-enhancement Raman spectral detection using silver-doped sol-gels”, Applied Spectroscopy, 57, 479-482 (2003).24 Lee, Y. H.; W. Smith, S. Farquharson, H.C. Kwon, M.R Shahriari, and P.M. Rainey,"Sol-Gel Chemical Sensors for Surface-Enhanced Raman Scattering Detection," SPIE, 3537, 252-260 (1998).25 Lee, Y.-H., S. Farquharson, And P. M. Rainey "Surface-enhanced Raman sensor for trace chemical detection in water," SPIE, 3857, 76-84 (1999).26 Farquharson, S., "Miniaturized Fiber Optic Chemical Sensor," STTR Final Phase II Report for NASA Grant Number NAS9-98024 (submitted February 2000).27 Farquharson, S., and W. W. Smith, W. H. Nelson and J. F. Sperry, ”Biological agent identification by nucleic acid base-pair analysis using surface-enhanced Raman spectroscopy,” SPIE, 3533, 207-214 (1998)28 Farquharson, S., and W. W. Smith, S. Elliott and J. F. Sperry, "Rapid biological agent identification by surface- enhanced Raman spectroscopy", SPIE, 3855, 110-116 (1999).29 Lee, Y.H. and S. Farquharson, "Rapid chemical agent identification by surface-enhanced Raman spectroscopy", SPIE, 4378, 21-26 (2001)30 Lee, Y.H., and S. Farquharson, "SERS sample vials based on sol-gel process for trace pesticide analysis", SPIE, 4206, 140-146 (2001)31 Farquharson, S., W.W. Smith, Y.H. Lee, S. Elliott and J. F. Sperry, "Detection of biological signatures: A comparison of electrolytic and metal-doped sol-gel surface-enhanced Raman media", SPIE, 4575, 62-72 (2002)32 Farquharson, S. and Lee, Y. H., "High Throughput Identification: Drug Analysis by Surface-Enhanced Raman Spectroscopy", D&MD Focus Reports, 4-7 (March 2001)33 Farquharson, S, ”Rapid Drug Assay by Surface-Enhanced Raman Spectroscopy,” SBIR Phase I Final Report for NIH, Grant No. 1 R43 GM54916-01 (4/14/1998).34 Farquharson, S., Y.H. Lee, H.C. Kwon, M.R. Shahriari, and P.M. Rainey, "Urinalysis by surface-enhanced Raman spectroscopy," STAIF- 2000, paper 105 (2000).35 Farquharson, S., and Y. H. Lee, "Trace drug analysis by surface-enhanced Raman spectroscopy," SPIE, 4200, 89-95 (2000).36 Wentrup-Byrne, E., Sarinas, S., and Fredericks, P.M., “Analytical Potential of Surface-Enhanced Fourier Transform Raman Spectroscopy on Silver Colloids,” Applied Spectroscopy, 47, 8, 1192-1197 (1993).37 Farquharson, S., P. Maksymiuk, K. Ong and S.D. Christesen, "Chemical agent identification by surface- enhanced Raman spectroscopy", SPIE, 4577, 166-173 (2002).38 Farquharson, S. PI, "High Sensitivity Raman Spectrometer" NSF CN DMI-0296116.39 Farquharson, F. PI, "A portable Raman instrument for fuel characterization", Navy, CN M6785-03-M-5043.40 Inscore, F. E., McNaughton, R., Westcott, B.L., Helton, M.E., Jones, R., Dhawan, I.K., Enemark, J.H., Kirk, M.L. “Spectroscopic Evidence for a Unique Bonding Interaction in Oxo-Molybdenum Dithiolate Complexes: Implications for σ Electron Transfer Pathways in the Pyranopterin Dithiolene Centers of Enzymes”, Inorg. Chem. 38, 1401-1410, (1999).41 Jones, R.M., Inscore, F.E., Hille, R., Kirk, M.L. “Freeze-Quench Magnetic Circular Dichroism Spectroscopic Study of the “Very Rapid” Intermediate in Xanthine Oxidase” Inorg. Chem. 38, 4963-4970, (1999).42 Wang, X.B., Inscore, F.E., Yang, X., Cooney, J.J.A., Enemark, J.H., Wang, L.S. “Probing the Electronic Structure of [MoOS4]- Centers Using Anionic Photoelectron Spectroscopy”, J. Am. Chem. Soc. 124, 10182- 10191, (2002).43 Joshi, H.K., Cooney, J.J.A., Inscore, F.E., Gruhn, N.E., Lichtenberger, D.L., Enemark, J.H. “Investigation of Metal-Dithiolate Fold Angle Effects: Implications for Molybdenum and Tungsten Enzymes”, Proc. Nat. Acad. Sci., 100, 3719-3724, (2003).44 Farquharson, S.; Lee, Y. H., and C. Nelson "Material for SERS and SERS sensors and method for preparing the same", U.S. Provisional Patent Number AFU-17-PROV (1999)45 Farquharson, S. and S. F. Simpson “Applications of Fiber Optic Raman Spectroscopy to Chemical Processes,” SPIE, 1796, 272-285 (1992).46 Woodruff, W.H., Farquharson, S., ”Time-Resolved Resonance Raman Spectroscopy of Hemoglobin Derivatives: Heme Structure Changes in 7 Nanoseconds,” Science, 201, 831-833 (1978).47 Farquharson, S., W. W. Smith, W. H. Nelson, and J. F. Sperry, “Biological agent identification by nucleic acid base-pair analysis using surface-enhanced Raman spectroscopy,” SPIE, 3533-37 (1998) 59
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