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Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
Sers Biofluids Fei Invited Talk Pittcon2010
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Sers Biofluids Fei Invited Talk Pittcon2010

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  • 1. Rapid Analysis of Drugs, Biomarkers andPathogens in Urine, Saliva and Nasal Mucus Using a Disposable Lab-on-a-chip Focus: SERS Applications in Bio-Fluids Frank Inscore, Chetan Shende Atanu Sengupta and Stuart Farquharson RTA NIH CN: 1R43CA94457-01 NSF CN: DMI-0215819 NASA CN: NNC05CA09C DoD Dr. John Murren Yale New Haven Hospital Dr. Eric Wong Jet Propulsion Laboratories Prof. Jay Sperry University of Rhode Island Dr. Steven Christesen ECBC UK Road Policing Technologies Home Office Scientific Development Branch (Dr. Helen Turner )www.RTA.bizBooth #2825 Pittcon 2010 Providing Chemical Information When & Where You Need It
  • 2. Challenge: Offset negative effects of weightlessness in spaceSolution: Monitor indicators and associated counter-drugs in urine Extended Weightlessness in Space Causes: • Muscle Loss, Bone Loss • Renal Stone Formation •Difficulty in Sleeping, Motion Sickness Minimize Effects with Drugs and Exercise NASA desires a light-weight On-Board Analyzer -that is non-invasive -with fast response -and has necessary sensitivity specificity selectivity Physiological Changes Reflected in Urine Composition: • Biomarkers (~1 mg/L)Drugs & Metabolites (~10 microg/L) Providing Chemical Information When & Where You Need It
  • 3. Challenge: Analysis of chemo-drugs and metabolites for dosage controlSolution: Monitor drugs and metabolites in saliva• Chemotherapy drugs also kills non-cancer cells• Dosage is critical (to little noneffective, to much kills patient)• No clinical trials to establish statistical based dosage• Current analysis requires large sample volume (10-20 ml blood and/or urine)• These methods are labor intensive and time consuming • Centrifugation to remove red blood cells • Extraction using organic solvents • Separation using chromatography • Detection with UV or Mass Spec• Standards are needed to ensure measurement accuracy•Consequently, measurements made on an “as needed” basis• Viable alternative, parent drug/ metabolites represented in saliva • Concentration ~10-50% of blood plasma • 1 microg/mL detection needed for chemo-drugs• Advantage, Non-invasive (no needles) • Issues of clotting and platelet count in blood of chemo-patients• Advantage, Saliva is 99.5% water • Interfering physiological chemicals 100X less Providing Chemical Information When & Where You Need It
  • 4. Challenge: Analysis for presence of prescription & illicit drugs of abuseSolution: Identify drugs and metabolites in saliva 2 scenarios: ER overdoses, road-side policing of impaired drivers Speed - Rapid diagnosis of Drugs in Saliva. •Saliva analysis will increase speed by eliminating time consuming extraction and separation steps. •Rapid diagnosis for drug overdose in ER can lead to appropriate medical care. Specificity - Correct Identification of abused drug. •Variety of overdose systems can lead to confusing diagnosis. •Explicit drug detection will lead to accurate treatment regime. Sensitivity - Low detection limit Required. •Drugs in Saliva need to be measured at ng/mL concentrations Providing Chemical Information When & Where You Need It
  • 5. The Biofluid Analysis Challenge Need non-invasive technique that meets following criteria: • Specificity – identify Bio-agents Biomarkers Drugs Metabolites • Uniquely, in the presence of of the biofluid matrix • With no False Positives (or False Negatives)! • Speed – Field Applications require rapid analysis • 10 minutes or less • Sensitivity – requirements to Monitor and Detect • Drugs Metabolites ~ 100 to 10 ng/mL (100-10 ppb) • Biomarkers ~ 1000 to 10 microg/L (1 ppm - 10 ppb) • Bioagents ~ 10-4 to 10-2 cfu/mL and 1 ng toxin/mLSelective detection of target analyte(s) at parts-per-billion and lower is challenging: even under ideal conditions, more so in a complex bio-matrix! Providing Chemical Information When & Where You Need It
  • 6. Approach: Surface-enhanced Raman Spectroscopy Raman, although weak effect, provides molecular specificity BUT, when a molecule is within a laser induced plasmon field, the efficiency of Raman scattering can hν Ag increase by 106 i.e. 1 million times! Sub part-per million detection becomes H N H N possible. N H N N H H Chemical contribution Plasmon Fieldcan provide additional 103 enhancement Surface-Enhanced Single Molecule Detection: requires 1012 -1014 Raman Photon Providing Chemical Information When & Where You Need It
  • 7. SERS-Active Substrates benzenethiol 10-3M 10-5M 10-8M (~10 ppb) LMC ~10-11M (~10 ppt) Providing Chemical Information When & Where You Need It
  • 8. Approach: RTA SERS Patented Sampling Systems (rapid response) 2001: Simple SERS Sample Vials Molecules Sol-Gel Matrix Raman in Solution Scattering Laser Adsorbed Molecules Metal Particle silver gold2003: SERS Microplate 2004: SERS-Active Capillary 1 10 More suited to extract and pre-concentrate Providing Chemical Information When & Where You Need It
  • 9. 2007: Functionalized Sol-Gel SERS Capillary (affords greater selectivity and sensitivity) PC OTC unreduced reduced Std SERS vials 2nd generation Providing Chemical Information When & Where You Need It
  • 10. RTA’s Portable Raman Analyzers785 nm Vials 1064 nm Advantages: • No sample preparation • Simple integration via fiber optics • Remote analysis, multi-component Microplates • Complete spectral coverage • Wavelength stability • Confident spectral subtraction • and library search/match • Real-time, On-demand analysis • Long term stability • Temperature and vibration immune 25 pounds Capillaries • Shock resistant water tight Lab-on-Chips Other Wavelengths 1532, 976, 633, 532 nm Providing Chemical Information When & Where You Need It
  • 11. Analysis: SERS of Urine Matrix and Components Simulated O urea OH H2N NH2 H N N O Lyophillized creatinine HO N N H uric acid O N Real N NH2 OH lactic acid OH OSimulated urine = 20 g/L urea, 3 g/L NaCl, 1.5 g/L KCl,1.4 g/L creatinine, 0.2 g/L lactic acid, 0.15 g/L uric acid•100 chemicals/biochemicals as potential interferents in urine were measured Uric acid: dominates urine SERS! Providing Chemical Information When & Where You Need It
  • 12. Analysis: Biomarker and Counter-Drug 3-MethylHistidine Risedronate NR OH O N N O HO P OH SERS HO NH2 P OH N HO O 0.125 0.03 0.105 0.025 0.085 0.02 0.065 0.045 0.015 0.025 0.01 0.005 0.005 0 200 400 600 800 1000 0 200 400 600 800 1000 Microgram/L Microgram/L100 mw 785 nm, 1-min Providing Chemical Information When & Where You Need It
  • 13. Analysis: doped reconstituted urine sample - multi-targets complicated Can we detect directly? Pre-cleanup necessary? Extract and separate? Urine Raloxifene (organic extract) 3-Methylhistidine (aqueous extract) Risedronate (aqueous extract) Scopolamine (directly on gold)With exception of drugs like Ral or Scop, further pre-treatment necessary LOC is ideally suited for urine analysis Providing Chemical Information When & Where You Need It
  • 14. Analysis: 3-Methylhistidine OH 3-MeHis N unknown O N NH2 best match Histidine OH N O N NH2• Unique vibrational features provide ability to identify and discriminate chemicals•RTA spectral search soft-ware identifies unknown chemical from library data base Providing Chemical Information When & Where You Need It
  • 15. Analysis: parent drug and metabolite Mixture OH N N Allopurinol N N H OH N Oxypurinol N N HO N H•Unique vibrational features provide ability to identify and discriminate•RTA quantification and chemometric soft-ware can define composition Providing Chemical Information When & Where You Need It
  • 16. Component selection: for urine sample pre-treatment 1) Filter particulates RIS 3-MeHIS UA 2) Exclude small inorganic ions 3-MeHIS RIS 3-MeHIS3) Refine SG selectivity, extract and separate Providing Chemical Information When & Where You Need It
  • 17. Analysis: Demonstrate sub microg/L detection levels1 microg/L (1 ng/mL) 3-MeHis Ibandronate HO P O OH N CH3 CH3 HO P OH HO Alendronate O O NH2 HO P OH HO P OH Pamidronate HO O O NH2 HO P OH HO P OH HO Clodronate O O Cl HO P Cl HO P OH O HO Etidronate CH3 O Risedronate HO P OH H-Deoxypyridinoline HO HO P O OH OH10 microg/L Calcium Phosphate Uric Acid N H N O Allopurinol N HO N H O O Calcium Oxalate O O - Hydrochlorothiazide H 2N S O S O Allopurinol - O O - Ca +2 O Cl N H NH O CaP - O P O- Ca+2 O - Ca+2 - O P O- NH2 O Ca+2 O O NH2 HS O Penicillamine H 3C HO S S Cystine HO Oxypurinol NH2 HO H3C HScopolamine O N O O OH H H3C CH3 N N CH3 Promethazine S Providing Chemical Information When & Where You Need It
  • 18. R&D: Optimize Lab-On-Chip Components Providing Chemical Information When & Where You Need It
  • 19. R&D: SERS Lab-On-Chip Different Lab-on-Chip designs used Providing Chemical Information When & Where You Need It
  • 20. R&D: SERS Lab-On-Chip Analysis of real human urine sample doped with 3- MeHis and Ris at 1 microg/L (1 ng/mL) in 10-min! Providing Chemical Information When & Where You Need It
  • 21. R&D: SERS Lab-On-ChipMultiplexed Sample Analysis on CD Style LOC Providing Chemical Information When & Where You Need It
  • 22. Base-line Analysis: Chemotherapy Drugs 5-Fluorouracil NR • LMC is 100-10 ng/mL SERS Dacarbazine 100 µg/mL mixture Dacarbazine 5-FU Doxorubicin • Incorrect dosage can be toxic or ineffective •Complex analysis: drug mixtures and metabolites Providing Chemical Information When & Where You Need It
  • 23. Base-line Analysis: OTC and Prescription Drugs H NR Aspirin O N O NH SERS Amobarbital O O SERS NH Secobarbital O N O H O HN NH Phenobarbital O O NR Diazepam on Ag SERS Acetaminophen Oxycodone on Au SERS • Lowest Measured Concentration (LMC) is 100 - 10 ng/mL • Overdose with Acetaminophen is common in elderly • Barbituates are one of the most abused of prescribed drugs Providing Chemical Information When & Where You Need It
  • 24. Base-line Analysis: Illicit Drugs of Abuse Cocaine NR 1.1 ROC Curve SERS Au-SG 1 0.9 0.8 Probability of Detection 0.7 0.6 0.5 0.4 0.3 SERS Ag-SG 0.2 0.1 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Probability of False Positive Conc. # of Mean Std Mean Std substrates peak hgt Deviation Deviation (α) Blank 10 0.002 0.0014 0.004 25 ppb 9 0.0113 0.006 50 ppb 10 0.0157 0.003• LMC and Reproducibility Conc. C K value Log C at LMC at modify SG chemistry 25 ppb 2.263 K=3.29 95 % 50 ppb 3.343 confidence -7.31583 48 ppb• Most ER reported overdose cases occur with cocaine•Other drugs measured - Amphetamine, Heroine, Morphine, LSD, THC Providing Chemical Information When & Where You Need It
  • 25. Analysis: Single Drug in Saliva Typical SERS Experiment 1. Mix sample with saliva 2. Suck sample into SER-active capillary (10-sec) 3. Measure SERS of sample (10-sec placement, 1-min scan) Providing Chemical Information When & Where You Need It
  • 26. Single Component Analysis: 5-FU in Saliva 50 microg/mL HCl 5-FU doped saliva pH adjusted 1 microg/mL weak acid• Filter improved SERS-response• Small ions can be excluded• Weak acid improved sensitivity Providing Chemical Information When & Where You Need It
  • 27. Proof of Concept: Multi-component analysis in saliva cis-platinum A B Manual C Manual A Flow B Switch Flow C oxypurinol Switch • drugs extracted from saliva of chemotherapy patient Providing Chemical Information When & Where You Need It
  • 28. Non-Invasive Pathogen Detection in Nasal Mucus Exosporium A Outer Core G Wall DNA B C Ribosomes Inner Core H Wall Cortex Core A B Spore Coat Manual C A Flow I Manual Flow B F Switch Internal intensity reference Switch C - O O - digesting reagent J 2+ O C N C O Ca 500 800 1000 1200 1400 1600 1800 (calcium dipicolinate) Raman Shift, cm-1 D Dipicolinic acid 25 ppm BC spores in mucus B. cereus E cysS-Ag 250 ppm B. subtilis No DPA signature! spectrum same as before adding BS Providing Chemical Information When & Where You Need It
  • 29. Summary: Non-invasive Urine Saliva Analysis • Small sample volume: < 100 microL • Rapid & simple analysis: < 10 minutes (total) • Specificity: detect various drugs biomarkers • Sensitivity: at required detection in urine, but in saliva needs improvement • Require 1 microg/mL to 10 nanog/mL • Future work: improve extraction efficiency Road-side Policing with UK HOSB LOC for Infectious Pathogens in Hospitals Providing Chemical Information When & Where You Need It

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