1. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. * ABTECH Scientific, Inc. Virginia Biotechnology Research Park 800 East Leigh Street, Suite 52 Richmond, Virginia 23219 USA Clinical Diagnostics Using Electroconductive Bio-Smart Thin Film Biosensors guiseppi@abtechsci.comhttp://www.abtechsci.com Anthony Guiseppi-Elie, Sc.D. A "Hands On" Workshop Theory and Practice of Point of Care Tests from Development Through Manufacturing Mar 31 - 2 Apr 2009.......... San Diego, CA

2. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. ABTECH Scientific, Inc. Founded in 1995 and located in the Biotechnology Research Park in Richmond, Virginia, ABTECH uses its platform electroactive polymer sensor technology (EPST™) to develop and deliver non- invasive, near-patient molecular diagnostic products of clinical significance.

3. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. ABTECH Scientific, Inc. Laboratory Products Group Advanced Products Group Microlithographically fabricated devices, related microelectrodes (pH and Ref), flow cell products (gas and liquid) and instruments that are used in research and development of electroconductive polymer sensor technology. Non-invasive, near- patient molecular diagnostic products of clinical significance and based on electroconductive polymer sensor technology.

4. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Scope The presentation introduces several of the device products of the Laboratory Products Group and illustrates how these devices and substrates may be used in chemical and biological sensor research and development around the use of inherently electroconductive polymers.

5. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 5  Biosensors Defined  Classification of Biosensor Systems  Based on molecular recognition, transduction principles or physical placement  Biosensor System – System’s Requirements  Challenges in Biosensor Systems Development and Application: Technical and Commercial  Examples of Biosensor Systems:  Voltammetric Biosensor …for lateral flow assay  Implantable Biochip …for intramuscular monitoring during trauma  Future of Biosensor Systems Outline

6. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Biosensors are test and measurement systems that include a biotransducer, which involves biological recognition and signal transduction, and instrumentation, which allows data capture and information presentation, and is a format that allows the performance of rapid, point-of- concern bioassays. Biosensors Defined The biosensor has emerged as a suitable platform for the point-of-concern screening and diagnostics in human and animal health, the environment and national security.

7. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Biosensors Defined  Biosensor -- Definition A biosensor is a measurement and/or monitoring system that uses biologically active molecules, cell fragments, whole cells or tissue as part of a biotransducer, which transforms chemical information ranging from concentration of a specific sample component to total composition analysis, into an analytically useful signal. A biosensor is therefore a class of chemical sensor  Chemical Biosensor -- IUPAC Definition A chemical biosensor is a device that transforms biochemical information ranging from concentration of a specific sample component to total composition analysis, into and analytically useful signal.

8. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. A Prototypical Biotransducer

9. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. A Prototypical Biosensor System

10. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA.  The goal of biosensor systems is to produce analytical data in context, i.e. useful information, that allows the end-user to make decisions and take actions.  Competitive advantage  “Foot print” (size of the device)  Convenience  Large enough market to justify the cost of development  Speed of the test to be performed Why and Where Biosensor Systems are Used

11. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Sensor Semantics Detector vs. Sensor A detector is generally not repeatable, is not updateable, is not quantitative and not regenerable. A sensor is repeatable, updateable, is quantitative and/or regeneragble. Advance®, Confirm®, Clear Blue Easy®, Answer®, and ept® Pregnancy Test Strips are Detectors of Human chorionic gonadotropin (HCG) produced by the placenta. Glucose biosensor for monitoring in critical care of diabetes or monitoring of a bioprocess.

12. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 12 Broad Applications of Biosensors Molecular diagnostics in patient health care measurement and monitoring – Blood metabolites (glucose, cholesterol), therapeutic drugs (theophyline), drugs-of-abuse (cocaine). Food and beverage industry – Microbial pathogens in food (Salmonella ssp.) Environmental and ecological monitoring – Assaying of polychlorinated biphenyls (PCBs) in soil samples Bioprocess monitoring – Monitoring of amino acids in mammalian cell culture

13. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Various Microarrays Formats Affymetrix/Roche CYP450 Nanogen Osmetech CF GE Healthcare Metrigenix’s Flow-Thru 4-D Chip BCI – SNPstream & Universal Capture plate Combimatrix BioDot’s Lateral Flow & Dispensing Amic Micronics’ lab card Brain Tumor beChip™

14. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. ABTECH’s Business and Technology Space  Enabling biochemical signal transduction with amplification  Uses electrochemical transducers (amperometric, voltametric, impedimetric)  Fashioned by microlithography with critical dimensions less than 25 microns (defines microelectrode electrochemistry)  Focused on point-of-concern clinical molecular diagnostics

15. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. microbore Microfabricated arrays for electrochemical transduction Borosilicate Glass 1. Surface Preparation 2. Photo Resist Application and soft bake Silicon Nitride (Si 3 N 4 ) Sputtered Au (100 nm) on Ti/W (10 nm) 3. Alignment and Exposure 4. Develop positive photoresist and post bake 5. Fluro-plasma etch (Si 3 N 4 ) layer 6. Strip photoresist layer

16. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Microfabricated Interdigitated Microsensor Electrodes (IMEs) IME XX50.5 M, CD, FD; Au, Pt, ITO XX = 05, 10, 15, 20  m IME XX25.3 M, CD, FD; Au, Pt, ITO M = Monolithic CD = Combined Differential FD = Full Differential AFM Profile of 15  m IME

17. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Sensor Test Clip (STC-4) Microfabricated Interdigitated Microsensor Electrodes (IMEs) Sensor Test Clip (STC-7)

18. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Microfabricated Interdigitated Microsensor Electrodes (IMEs) IME XX50.5 M, CD, FD; Au, Pt, ITO XX = 5, 10, 15, 20  m

19. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Microelectrodes Oxygen Microelectrodes pH and REF Microelectrodes

20. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. IME 1010.3 FD Au, Pt, ITO Liquid Flow Cell Detector

21. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Schematic illustration of the IME XX25.3 chips Collector Electrode Generator Electrode Digit width = spacing = 5, 10, 15 & 20 microns)

22. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Photomicrograph and AFM Images IME XX25.3 chips

23. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Ref: Analyst, 2007, 132, 365–370 m = number of the micro electrodes I = length of electrode, w = width, and g = gap distance Signal amplification via redox cycling IME XX25.3 chips

24. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Bioimmobilization of hydrogel membranes onto IME chips

25. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Hydrogel Layer Glass substrate SEM Photomicrograph of a Hydrogel Membrane of IME XX25.3 chips

26. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. MSRCVs obtained experimentally with and without hydrogel coating on the IMEs in the presence of 1 mM Fc-COOH prepared in 0.1 M TRIS + 0.1 M KCl (pH = 7.2).

27. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Simulated (modeled) MSRCV for the four IMEs with and without hydrogel in the presence of 1 mM Fc-COOH following Butler-Volmer kinetics.

28. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Comparison of voltammograms of IMEs at scan rate of 50 mV s -1

29. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. IME 1010.3 FD Au, Pt, ITO Gas / Vapor Flow Cell Detector

30. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Chips of 5μm, 10μm, 15μm and 20μm with electropolymerized PPy, PTh or PAn Sensor Arrays of VOC Responsive Polymers for the Electronic NOSE

31. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. IAME-co-IME 2-1 Au, Pt Independently Addressable Microband Electrodes and Microfrabricated Interdigitated Microsensor Electrodes)

32. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 4 bands 3 mm long 2 m lines, 1 m space 5 + 5 fingers 3 mm long 2 m lines, 1 m space IAME-co-IME 2-1 Au, Pt Independently Addressable Microband Electrodes and Microfrabricated Interdigitated Microsensor Electrodes)

33. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. IAME-co-IME 2-1 Au, Pt

34. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. IAME-co-IME 2-1 Au, Pt Independently Addressable Microband Electrodes and Microfrabricated Interdigitated Microsensor Electrodes) 2  line 1  space

35. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. MDEA XXX Au, Pt Microdisc Electrode Arrays 1

36. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. MDEA DEVICES Disc dimensions / Number of Discs Active Area Conductor MDEA 3600 D = 3,600  m 1 disc 0.10 cm 2 Au, Pt, ITO MDEA 250 D = 250  m 207 microdiscs 0.10 cm 2 Au, Pt, ITO MDEA 100 D = 100  m 1,296 microdiscs 0.10 cm 2 Au, Pt, ITO MDEA 050 D = 50  m 5,184 microdiscs 0.10 cm 2 Au, Pt, ITO 50  m100  m250  m x 50 3600  m x 50 Microdisc Electrode Arrays 2

37. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 50  m 100  m 250  m 3600  m Microdisc Electrode Arrays 3 5,184 microdiscs 1,296 microdiscs 207 microdiscs 1 macro disc

38. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. PAnCl on MDEA 50 Au x50 PAnCl on MDEA 100 Au x50 PAnPSS on MDEA 100 Au x50 PAnPSS on MDEA 50 Au x50  Total current density applied = 100 mA/cm 2  Total time = 20 s  Total charge density = 2 C/cm 2 Microdisc Electrode Arrays 4

39. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Independently Addressable Microband Electrodes (IAMEs) XX = 5, 10, 15, 20  m Microband Electrodes IAME XX04, Au, Pt or ITO

40. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Independently Addressable Interdigitated Microsensor Electrodes (IAIMEs) XX = 5, 10, 15, 20  m Microband Electrodes IAIME XX10, Au, Pt or ITO

41. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Design and Development of an Electrochemical Lateral Flow Assay

42. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Introduction to Lateral Flow Assays Definition: LFA’s are devices designed to detect the presence (or absence) of an analyte in a sample. Operating Principles: Antibody-Antigen Capture Interaction; Capillarity

43. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Applications, advantages and disadvantages of LFA LFA’s are used in  Home testing - Pregnancy tests  Rapid point of care testing – HIV, Troponin T, Malaria, Drugs of abuse, Fertility, Respiratory diseases.  Human and animal diagnostics  Field testing of various environmental and agricultural analytes  Laboratory use Advantages  User friendly format  Very short time to get test results  Long term stability over wide climatic ranges  Relatively inexpensive to make Disadvantages  Subjective interpretation of results  Non-quantitative… at best  Poor coefficient of variation  Lack of electronic documentation  Sensitivity limits

44. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Challenges with existing LFDs: Opportunities for New Technologies  Improved performance for Lateral flow  Improved sensitivity and specificity  Improved manufacturability  Quantitative and semi-quantitative rapid tests  Patent pressures leading to innovation As new applications for Lateral Flow Devices expand, there is demand for:

45. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Responding to the opportunity with novel technology  Improved quality of the flow field realized through the use of proprietary fiber bed technology.  Quantitative detection realized through the use of proprietary microfabricated array electroanalytical detection technology.

46. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Electrochemical Flow Through Assay

47. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. A B C D E F G Electrochemical Cell-on-a-Chip (ECC)

48. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Electrochemical Cell-on-a-Chip (ECC) XX = 05, 10, 15, 20  m ECC IME XX04, Au, Pt or ITO 2 cm 1 cm C1 C2 C2WE2 C2WE1 C2CE C1WE1 C1WE2 C1CE REF Schematic illustration of a microfabricaed, dual-channel, electrochemical cell-on-a-chip interdigitated microsensor electrode (ECC IME) device showing the fluid flow over the microelectrode arrays. A=C2W2, B= C2CE, C=C2W1, D=C1W1, E=C1CE, F=C1W2, G=REF. C2 is Cell 2, C1 is Cell 1, W is working electrode, CE is counter electrode and REF is reference electrode. A B C D E F G

49. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Counter Electrode Interdigitated WE1 and WE2 Reference Electrode, Ag/AgCl Si 3 N 4 Passivation Layer Unmodified Polypyrrole modified Unsilverized Silverized Electrochemical Cell-on-a-Chip (ECC)

50. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. An integrated biochip showing the microfabricated pattern of electrodes bonded and sealed into the Microncs’ microfluidic T-cassette. Electrochemical Cell-on-a-Chip (ECC) Integrated in a Microfluidic Cassette Polypyrrole modified

51. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 1mM fcCOOH for 30 s at 200 s 1mM fcCOOH for 10 s at 1350 s DI for 60 s at 1150 s Test Platform

52. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Design and Development of an Implantable Biochip for Physiologic Status Monitoring during Hemorrhage

53. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. PSMBioChip System (Physiologic Status Monitoring) An Implantable Biochip for Physiologic Status Monitoring 4 x 6 mm 4 x 1.5 x 0.5 cm Discrete Prototype Device ASIC Device Glucose, Lactate, pH and Temperature

54. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. PSMBioChip Biotransducer PSM Dual Microdisc Array Biotransducer Electrocond uctive “Bio- smart” Hydrogel On Microdisc Array 2 mm 4 mm ECC MDEA 5037 A. Guiseppi-Elie, S. Brahim, G. Slaughter and K. R. Ward, “Design of a Subcutaneous Implantable Biochip for Monitoring of Glucose and Lactate”, (2005) IEEE Sensor Journal, 5( 3), pp. 345-355.

55. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Si 3 N 4 Au Diffusion domain Microdisc 2r Front-end biotransducer for discrete component prototyping of the PSMBioChip 50x Counter electrode Reference electrode Working electrode Electrochemical-Cell-on-a-Chip Microdisc Electrode Array (MDEA) 50  m

56. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA.

57. ECC MDEA 5037 qualified by CV at 100 mV/s in 1.0 mM FcCOOH in TRIS Buffer Two counter electrodes Two working electrodes Single Reference electrode

58. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Performance enhancement of the microdisc electrode array format of the PSMBioChip Hydrogel CoatedUn-Coated G. Justin, A. R. Abdur Rahman, and A. Guiseppi-Elie, "Bioactive Hydrogel Layers on Microdisc Electrode Arrays: Cyclic Voltammetry Experiments and Simulations," Electroanalysis (2009) (accepted) Enhanced effective area with reduction of disc diameter Enhancement maintained for 50 µm device beneath hydrogel

59. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. HydrogelTissue/Solution GOx Fe 2+ COOH Glucose Gluconolactone Lactate Pyruvate e- LOx Fe 2+ COOH e- Au MDEA5037 Fabrication of the electrochemical p(HEMA)/p(pyrrole)/Ox biotransducer AuSi 3 N 4 Glass Walter Torres and Anthony Guiseppi-Elie, “Simulations of Redox Mediation within Bioactive Hydrogels of Amperometric Biosensors” Journal of Macromolecular Science, Part A: Pure and Applied Chemistry (2006) (12) 1923 - 1928

60. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. Custom Spot Production Microarray Array Fabrication BioDot AD 3400 Hydrogel Enzyme Cocktails

61. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA.

63. LOx or GOx Acr-PEG-NHS (Acr-PEG) n -GOx + ++ N-hydroxysuccinimide n Derivatization and functionalization of (PEGylation) of oxidoreductase enzymes 0:1, 2:1, 4:1, 6:1, 8:1, 10:1, 12:1 Substrate (glucose / Lactate) RT, 2.5 h HEPES buffer n = 79; 113; 170 for 3,500; 5,000; 7,500 Zhang M, Li X. H, Gong Y. D, Zhao N. M, Zhang X. F. “Properties and biocompatibility of chitosan films modified by blending with PEG”. Biomaterials (2002) 2 3:2641-2648 Vamsi K. Yadavalli, Won-Gun Koh, George J. Lazur, Michael V. Pishko” Microfabricated protein-containing poly(ethylene glycol) hydrogel arrays for biosensing” Sensors and Actuators B 97 (2004) 290–297.

64. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. In vitro Amperometric Characterization of MDEA 5037 Biotransducer Sense Region 1 (Lactate) Sense Region 2 (Glucose) 4 mm 2 mm BioSTAT Dual Potentiometer MDEA 5037

65. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. In vitro response of planar glucose biosensor incorporating bio-smart hydrogel membrane of composition 80:10:2.5:2.5:5.0 mol% (HEMA:TEGDA:PEGMA:MPC:Py) in 0.1 M PBKCl, pH 7.0 In vitro response of MDEA lactate biosensor incorporating bio-smart hydrogel membrane of composition 80:10:2.5:2.5:5.0 mol% (HEMA:TEGDA:PEGMA:MPC:Py) in 0.1 M PBKCl, pH 7.0 In vitro response curves for glucose and lactate 50  m 100  m 250  m

66. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. PSM Biochip Monitoring of Glucose and Lactate in Rats Real-Time In-Vivo Interstitial Fluid  Blood lactate and glucose concentration taken from drawn blood  BioChip capsule reading in the interstitial fluid of one rat

67. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. In vivo Amperometric performance of the PSMBioChip IMTRAMUSCULAR BLOO D

68. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA.

69. 69 Hemorrhage Remote Receiver Data Logger Ultimate Demonstration : 1. Conscious Hemorrhaging Animal 2. Comparison of VO 2 and Remote Glucose and Lactate Metabolic Chamber Signal surgically implanted in rat model Glucose and Lactate Monitoring and Data Transformation to oxygen debt Sensor- Transmitter PSM BioChip Implant Capsule

70. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 70 Biosensor System Requirements High degree of selectivity and sensitivity Rapid response Repeatability/reproducibility Rapid recovery from insult Stability Low drift Long lifetime Internal calibration and internal referencing Reagentless - no auxiliary reagents required No sample preparation Minimum cost per test+

71. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 71 Challenges in Biosensors Systems Development and Application: Technical Stable, long-term attachment of the bioactive layer to the solid state transducer Stability of the bioactive layer in the biotransducer Reproducible placement of small volumes of bioactive materials onto devices – Microarraying and microfluidic techniques. – Photolithographic patterning of bioactive layers Large scale manufacture - (The I-Stat story) Biocompatibility of in vivo biosensors

72. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 72 Challenges in Biosensor Systems Development and Application: Commercial Perceived marginal cost benefit over established tests Cost of reagents - bioactive reagents are expensive relative to typical reagents used in the the chemical industry. Measurement culture - entrenched, protected – Pathologists, Environmental Scientists, Food Processors Integrated biosensor systems that produce “analytically useful data”. – Understanding the end-user environment Distribution Channels

73. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. 73 Future of Biosensor Systems Developments in manufacturability – “Pick and play”; bio-inspired approaches Microfabrication techniques applied to biosensor production – Photolithographic patterning of bioactive layers Integration of biotransducers with microfluidic systems – Sample handling, preparation and delivery Improvement in sensitivity, stability and selectivity – Recombinant biomolecules – Stabilization of biomolecules in membranes and gels Integration of biosensors with effector/actuator devices – Controlled release

74. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. ABTECH’s Patents A. Guiseppi-Elie; US Patent No. 5, 766, 934; Issued on June 16, 1998. "Chemical and Biological Sensor Devices Having Electroactive Polymer Thin Films Attached to Microfabricated Devices and Possessing Immobilized Indicator Moieties". A. Guiseppi-Elie; US Patent No.: 5, 352, 574; Issued on: October 4th, 1994. "Electroactive Polymers with Immobilized Active Moieties". A. Guiseppi-Elie; US Patent No.: 5, 312, 762; Issued on: May 17th, 1994. "Method of Measuring an Analyte by Measuring Electrical Resistance of a Polymer Film Reacting with the Analyte".

75. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA. People Scientific Advisory Board Anthony Guiseppi-Elie, Sc.D. David Allara, Ph.D. Norman Sheppard, Jr. Ph.D. Management Team James W. Sharpe, MBA Chairman and CEO Anthony Guiseppi-Elie, Sc.D. President and Scientific Director James R. Funkey, MA, CPA VP and Controller Ann M. Wilson, Ph.D. Director, Applications Development Board of Directors James W. Sharpe, MBA Chairman Anthony Guiseppi-Elie, Sc.D. President and Scientific Director Annette Guiseppi-Elie, Ph.D. Secretary Cynthia Garwon-Burke, Ph.D. Patent and IP Council Leigh P. Gregory Patent Attorney Clemson, SC, USA

76. ABTECH Scientific, Inc. 800 East Leigh Street VA BioTech Center Richmond Virginia 23219-1535 USA.