Breathsafe- a biosensor designed for detecting H1N1

BREATHSAFE® A Biosensor design for detecting H1N1 infections By Gayathri Vijayakumar NYU-POLY

BREATHSAFE® Purpose It is a breath analyzing biosensor for the immediate diagnosis of H1N1 infection in a patient. Analyte The analyte that is being detected by the biosensor are the H1N1 viral capsid proteins Hemagglutinin (H1) and Neuraminidase (N1). Sample The sample being analyzed are the respiratory secretions that the patient releases when he/she coughs or sneezes. Bioreceptor Ultra high affinity anti-hemagglutinin and anti-neuraminidase protein single chain antibody ( scFV ) fragments are used as the bioreceptors. Biosensor Platform – (Flourescent Detection of analyte through labeled Abs) Ultra thin, flat and transparent Glass slides are used. Immobilization of the antibodies and subsequent immunoassay reactions are carried out on it. Diagnosis is done through excitation of the fluorophores tagged to Secondary Abs and simultaneous detection through a CCD camera. Real Concentration range of Analyte Respiratory secretions can contain about 6.3 x 10 4 to 10 7 TCID 50 /ml (Tissue Culture Infectious Dose 50 ). [15]

BUSINESS/FINANCIAL RATIONAL: The sensor hardware will be in the shape of a small box as illustrated in slide 3. An ultra thin, flat and transparent Glass slides are used for carrying out the immunoassay diagnosis. It is reasonably cheap to manufacture as such it can be made disposable. It has to be changed after each use. The hardware will be a one time investment for the customer, but the membranes will have to be brought periodically, as such it will be a continuous revenue generating product for the manufacturer. scFV antibody fragments are used as bioreceptors. High affinity Ab fragments can be manufactured inexpensively using bacteriophages. Biotinylation of the bioreceptors to the glass slides are done which is inexpensive. It is suitable for disposable, one shot devices such as antibody-based assay technologies. Flu epidemics will always be a recurring problem as such this biosensor can be easily adapted to different strains.

Compartment containing secondary Abs Nozzle Vacuum Microfiltration/Ultrasonication Chamber scFV Fragment specific to H1 & N1 SCHEMATIC REPRESENTATION OF BREATHSAFE® Excitation Light Source (LASER) Wavelength Filters (isolation of emission photons from excitation photons) Wash buffer Detector (such as CCD) Analyzer Result Display Compartment where the unbound Abs and Ags are washed off to with the Wash Buffer. An outlet that can be used for changing the membrane after each diagnosis. Secondary Abs tagged to Alexa Fluor 488 N -maleimide (EMC)-functionalized glass substrates Condenser

Immobilization of the primary antibodies to the polystyrene membrane is done by means of Biotinylation which allows for controlled orientation and therefore greater sensitivity. Covalent conjugation of the antibodies to biotin does not inactivate the antibody and it is a relatively simple technique. In it, biotin is attached to the membrane, streptavidin is added, then the biotin conjugated antibodies are added. Aqueous Diels–Alder chemistry combined with a poly(ethylene glycol) (PEG) spacer was used for a chemical selective and biocompatible immobilization of Biotin on to the glass slide.[1] Linkers containing α, ω linear PEG conjugates were synthesized containing cyclopentadiene in the α position and Biotin in the ω position. Linkers were coupled to N -maleimide (EMC)-functionalized glass substrates , and surface immobilization of biomolecules was confirmed by confocal fluorescence imaging.[1] Single chain antibodies (shown in red) complexed with Hemagglutanin . (taken from PDB , Keyword 3 FKU) The Diels–Alder approach, which involves a diene and a dienophile not present in any biomolecule, allows for a chemoselective reaction without the need for protecting groups, and water has an extraordinary rate-accelerating effect on the reaction process. [1]

In the figure given below, the key heterofunctionalized PEG, cylcopentadiene tetra(ethylene glycol) acetic acid 2 was prepared in four steps from tetra(ethylene glycol) 1 . Next, the biotin–PEG–diene 6 was prepared by the coupling of carboxylic acid of 2 with biotinyl ethylamine ( 3 , Sigma) with the activation of isobutylchloroformate.[1] Incubation of EMC-slide with cyclopentadiene–PEG 4 –biotin 6 in water at room temperature for 12 h, followed by washing with deionized (di) water three times gave a biotin–PEG functionalized surface.[1] REFERENCE: Chemoselective immobilization of biomolecules through aqueous Diels–Alder and PEG chemistry Xue-Long Sun, a* LiuChun Yang, b and Elliot L. Chaikof b, , Pubmed Central, PMCID: PMC2703444. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703444/ [1] [1]

Biorecognition Interface Streptavidin Biotin Biotinylated scFV Excitation Light Source (Laser) Hemagglutnin Neuramindase Secondary Antibody tagged to AF 488 N -maleimide (EMC)-functionalized glass substrates PEG 4 Cyclopentadiene

Latest Biosensing technologies in the field of Influenza diagnosis SENSOR BIO-RECEPTOR ANALYSIS SURFACE IMMOBILIZATION METHOD ANALYTE LIMIT OF DETECTION DYNAMIC RANGE/SENSITIVITY POC I on-channel Switch Biosensor (ICSB)[1] Nucleocapsid specific MAbs/Fabs Electrical current change above a defined threshold level Gold film on polycarbonate slide SAMs on gold Nucleocapsid antigens 52% Yes Poly-silicon Nanowire FET [2] 5′-aminomodified complementary AIV captured DNA probe Change in charge density induces a change in electrical field N -type poly-Si nanowire-channels APTES + Glutaraldehyde + Capture DNA Viral DNA 60 target molecules at 1 fM 1 fM –10 pM Yes BIE [3] Mabs/scFv specific to H5 hemagglutinin protein High spatial resolution imaging ellipsometry(increase in gray scale value) Functionalized silicon wafers Covalent Immobilization with NHS and EDC Influenza A virus hemagglutinin 1:10 6 dilution in PBST 1:10–1:10 6 dilution in PBST No CdTe QDs-Proton Flux Sensor [4] H9 avian influenza virus antibody Capturing virus on the F 1 β-subunit of F 0 F 1 –ATPase increases QD fluorescence CdTe QDs labelled Chromatophores Biotin-Streptavidin Immobilization H9 Influenza A virus Yes

ICSB provides an objective readout within 10 min of specimen inoculation without the need for chemical or other pretreatments.[1] Despite the several advantages of poly-SiNW FET devices (( i) ultrasensitive and label-free, (ii) cost-effective, (iii) rapid, direct, turbid and light absorbing tolerant, and (iv) potential for developing a portable, robust, low-cost, and easy-to-handle electrical component suitable for field test and homecare use ), technology to mass produce NW FET devices, control their electronic properties and reduce the cost to a reasonable range will be the key aspects for the future applications of the devices in biomedical diagnoses.[2] Key advantage of BIE is its high-throughput allowing for multiplexed analysis, and quantitative, label-free and rapid testing.[3] Stability against photobleaching ,large molar extinction coefficients, high quantum yield, and large surface/volume ratios make QDs superior to organic fluorophores in detection sensitivity as well as in luminescent stability. Because of these unique features, QD Virus detectors have a wide range of applications. [4] SENSOR BIO-RECEPTOR ANALYSIS SURFACE IMMOBILIZATION METHOD ANALYTE LIMIT OF DETECTION DYNAMIC RANGE/SENSITIVITY POC Electrochemical Impedence Spectroscopy (EIS) [5] sulfhydryl-modified oligonucleotide probes Label-free impedance detection of oligonucleotide hybridization through change in electrical parameter of surface. 1mm Gold disk working electrodes SAMs on gold 120-nt base fragment of the influenza hemagglutinin gene sequence <200 fmol 3-10 nM DNA in 20 μ l of solution Yes QCM [6] MAbs Detects mass changes due to Ag-Ab interactions on the surface of the transducer Gold electrodes on quartz crystals Ab coupled through Protien A to surface Influenza A or B viral antigens 1 × 10 3 pfu/mL of sample (10 μ g/mL) 1 × 10 3 –1 × 10 8 pfu/mL Yes DNA Sensor [7] Complementary DNA strands Converting conventional hybridization signal of the DNA sequences into useful electrical signal carbon multi-walled nanotubes Covalent I mmoblization through Carbodiimide method Influenza virus DNA strands 0.5 nM of Target DNA 1-10 nM Yes

In case of EIS the major drawback is that interpretation of the data requires an iterative curve-fitting algorithm and user input that is not readily accommodated in a mobile device. Plus the analyte that is to be detected is the viral DNA, therefore the amino acid sequence has to be reverse translated into a DNA sequence for detection. [5] QCM devices are relatively simple and convenient to use and can detect rapid, real-time responses to binding events on the crystal surface, such as antigen–antibody interactions. Using antibody-functionalized nanoparticles as a mass amplification probe for a QCM sensor, resulted in significant enhancement of sensitivity. NP-QCM had the highest sensitivity and specificity in comparison with RT-PCR, shell vial assay, cell culture and ELISA . Maximum enhancement of the sensitivity was observed only in the range 10 3 –10 4 pfu/mL, suggesting that the adsorption of excess antibody–colloidal gold conjugate on the electrode surface leads to steric hindrance effects. QCM offers a number of potential advantages over existing techniques, which include obviating the need for labeling techniques to measure the binding reaction between virus and antibody, the use of short measurement times, operational simplicity, low cost, the opportunity to re-use the crystal sensors and the potential for online data collection. Nasal wash is used as the sample. [6] I personally think that QCM devices are the best sensors because of all the advantages listed above. Another observation that I made is that most of the label- based detection methods are lab assays that cannot be accomodated into a mobile device for field use as it usually requires a lot of reagents ( primary and secondary Abs, wash buffer, etc). I am satisfied with the overall design of my sensor which is based on the flourescent detection of H1N1 viral proteins. The sensor will have to have a software that controls the opening and closing of the valves of the Secondary Ab and Wash Buffer compartments. Plus since my sample are respiratory secretions, the viscosity of the sample may create a problem, so I plan to introduce a microfiltration or an ultrasonication chamber into the design for atomization of the secretions. Another reason why I am sticking to my design is that unlike label free detection systems(which are one time investments), my sensor has a disposible sensing element in it, which will be a continuous revenue generating product for the manufacturing company. Am also introducing a condenser into the vaccum to cool the exhaled breath of the patient into an aqueous phase.

Comprehensive Review of Rapid Diagnostic Tests and Biosensors for Influenza Bio-barcodes [14] ICSB [9] Nanogap Field-effect Transistor[13] SENSOR PRINCIPLE ICSB [9] Reduction of admittance of the membrane corresponds to the presence of respiratory-related viruses. Image Ellipsometry [10] Combination of high spatial resolution imaging ellipsometry with proteinchip microarray Optical Interferometric Waveguide Immuno assay [11] Based on change in refractive index that occurs on binding of analyte Nanobead-based Portable Microfluid Biosensor[12] Based on impedance change caused by capture of nanobead-H5 AI virus complex by immobilized MAbs Nanogap Field-effect Transistor[13] Based on a nanogap-embedded field-effect transistor Quantum Dot Barcode [14] This technology combines nanotechnology, microfluidics and antibody based detection to dramatically improve the sensitivity. Bio-barcodes [14] combines microfluidics, magnetic particles and gold nanoparticles encoded with antibodies and DNA to achieve extremely high sensitivity

Influenza Diagnostic Table [8] Procedure Influenza Types Detected Acceptable Specimens Time for Results Rapid result available Viral culture A and B NP swab 2 , throat swab, nasal wash, bronchial wash, nasal aspirate, sputum 3-10 days 3 No Immunofluorescence DFA Antibody Staining A and B NP swab 2 , nasal wash, bronchial wash, nasal aspirate, sputum 2-4 hours No RT-PCR 5 A and B NP swab 2 , throat swab, nasal wash, bronchial wash, nasal aspirate, sputum 2-4 hours No Serology A and B paired acute and convalescent serum samples 6 2 weeks or more No Enzyme Immuno Assay (EIA) A and B NP swab 2 , throat swab, nasal wash, bronchial wash 2 hours No Rapid Diagnostic Tests 3M ™ Rapid Detection Flu A+B Test 7,9 (3M) A and B NP 2 swab/aspirate; Nasal wash/aspirate 15 minutes Yes Directigen EZ Flu A+B 7,9 (Becton-Dickinson) A and B NP 2 wash/aspirate/swab; lower nasal swab; throat swab; bronchioalveolar lavage less than 15 minutes Yes BinaxNOW Influenza A&B 8,9 (Inverness) A and B Nasal wash/aspirate, NP swab 2 less than 15 minutes Yes OSOM ® Influenza A&B 9 (Genzyme) A and B Nasal swab less than 15 minutes Yes QuickVue Influenza Test 4,8 (Quidel) A and B NP swab 2 , nasal wash, nasal aspirate less than 15 minutes Yes QuickVue Influenza A+B Test 8,9 (Quidel) A and B NP swab 2 , nasal wash, nasal aspirate less than 15 minutes Yes SAS FluAlert 7,9 (SA Scientific) A and B Nasal wash/aspirate less than 15 minutes Yes TRU FLU 7,9 (Meridian Bioscience) A and B Nasal wash/swab, NP aspirate/swab 15 minutes Yes XPECT Flu A&B 7,9 (Remel) A and B Nasal wash, NP swab 2 , throat swab less than 15 minutes Yes

REFERNCE Rapid detection of influenza A virus in clinical samples using an ion channel switch biosensor, S.Y. Oh a , B. Cornell c , D. Smith d , G. Higgins a , b , C.J. Burrell a , b and T.W. Kok a , b , , Biosensors and Bioelectronics Volume 23, Issue 7 , 28 February 2008, Pages 1161-1165. Poly-silicon nanowire field-effect transistor for ultrasensitive and label-free detection of pathogenic avianinfluenza DNA, Chih-Heng Lin a , Cheng-Hsiung Hung b , Cheng-Yun Hsiao a , Horng-Chih Lin b , Fu-Hsiang Ko c and Yuh-Shyong Yang a , Biosensors and Bioelectronics Volume 24, Issue 10 , 15 June 2009, Pages 3019-3024. Detection of avian influenza virus subtype H5 using a biosensor based on imaging ellipsometry, Cai Qi a , c , e , 1 , Xin-Sheng Tian b , c , 1 , She Chen a , c , Jing-Hua Yan b , Zhen Cao d , Ke-Gong Tian d , George F. Gao b , , and Gang Jin a , Biosensors and Bioelectronics, doi:10.1016/j.bios.2009.10.030 Using cadmium telluride quantum dots as a proton flux sensor and applying to detect H9 avian influenza virus, Zhang Yun a , b , Deng Zhengtao c , Yue Jiachang a , , , Tang Fangqiong c and Wei Qun b , Analytical Biochemistry , Volume 364, Issue 2 , 15 May 2007, Pages 122-127. Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences, Andreas Kukol a , , , Peng Li b , Pedro Estrela b , Paul Ko-Ferrigno c , 1 and Piero Migliorato, Analytical Biochemistry , Volume 374, Issue 1 , 1 March 2008, Pages 143-153. The detection of influenza A and B viruses in clinical specimens using a quartz crystal microbalance, Thamara M. Peduru Hewa a , Gregory A. Tannock a , b , 1 , David E. Mainwaring a , Sally Harrison a and John V. Fecondo , Journal of Virological Methods , Volume 162, Issues 1-2 , December 2009, Pages 14-21. DNA sensor development based on multi-wall carbon nanotubes for label-free influenza virus (type A) detectio, Phuong Dinh Tam a , , , Nguyen Van Hieu b , , , Nguyen Duc Chien c , Anh-Tuan Le a and Mai Anh Tuan b , Journal of Immunological Methods, Volume 350, Issues 1-2 , 31 October 2009, Pages 118-124 http://www.cdc.gov/flu/professionals/diagnosis/labprocedures.htm#table

REFERNCE Patent application title: VIRAL NUCLEOPROTEIN DETECTION USING AN ION CHANNEL SWITCH BIOSENSOR, Manoj Kumar, Sang Kyu-Lee., International Patent Classification: C12Q 1/70(2006.01) : http://www.freepatentsonline.com/EP1869226.pdf Avian influenza virus detection with biosensor based on imaging ellipsometry, C. Qi1;3, X.S. Tian2, J.H. Yan2, F. Gao2, and G. Jin1, Institute of Mechanics, Chinese Academy of Sciences, #15, Beisihuan West Rd., Beijing 100080, P.R.China, Institute of Microbiology, Chinese Academy of Sciences, #13, Beiyitiao Zhongguancun, Beijing 100080, P. R. China. file:///C:/Users/Owner/Documents/Assignments/INFLUENZA%20BIOSENSORS/Lifeboat%20News%20%20The%20Blog%20»%20New%20field-deployable%20biosensor%20detects%20avian%20influenza%20virus%20in%20minutes%20instead%20of%20days.htm Varshney, M., and Y. Li. 2008. Review: Interdigitated array microelectrodes based impedance biosensors for detection of bacterial cells. Biosens. Bioelectron. (available on-line, October 17, 2008). Nanogap Field-Effect Transistor Biosensors for Electrical Detection of Avian Influenza, Bonsang Gu 1 , Tae Jung Park 2 , Jae-Hyuk Ahn 1 , Xing-Jiu Huang 1 , Sang Yup Lee 2 * , Yang-Kyu Choi , SMALL, vol 5 Issue 21, Pages 2407-2412, 10 aug 2009. file:///C:/Users/Owner/Documents/Assignments/INFLUENZA%20BIOSENSORS/May%20«%202009%20«%20All%20About%20Biosensors.htm Survival of Influenza Virus on Banknotes, Yves Thomas, 1,2* Guido Vogel, 3 Werner Wunderli, 1,2 Patricia Suter, 2 Mark Witschi, 4 Daniel Koch, 4 Caroline Tapparel, 1 and Laurent Kaiser 1,2, , Applied and Environmental Microbiology, May 2008, p. 3002-3007, Vol. 74, No. 10 .

Breathsafe- a biosensor designed for detecting H1N1

by Gayathri Vijayakumar

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