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Enzyme Engineering 8. Applications(2): Sensor & Fuel Cell 8.1 Biosensor 8.2 Enzyme Fuel Cells.

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Presentation on theme: "Enzyme Engineering 8. Applications(2): Sensor & Fuel Cell 8.1 Biosensor 8.2 Enzyme Fuel Cells."— Presentation transcript:

1 Enzyme Engineering 8. Applications(2): Sensor & Fuel Cell 8.1 Biosensor 8.2 Enzyme Fuel Cells

2 8.1 Biosensor

3 Definition of biosensor Device for the detection of an analyte that combines a biological component with a physicochemical detector component (IUPAC)

4 Definition of biosensor  the sensitive biological element (biological material (eg. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc), a biologically derived material or biomimic) The sensitive elements can be created by biological engineering.  the transducer or the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified; associated electronics or signal processors that is primarily responsible for the display of the results in a user-friendly way Sensors 2008, 8,

5 Application  Clinical: glucose, cholesterol, pregnancy  Environmental monitoring: pesticides, heavy metal  Food analysis: Salmonella, E.coli O157  Defense: airborne bacteria, organophosphate  Industry: fermentation

6 Required characteristics  Selectivity  Limit of detection (LOD)  Response time  Reproducibility  Stability

7 Amperometric biosensor I. Willner and E. Katz, Bioelectronics 2005  First generation : The transduction of the biological reaction is by the oxidation or reduction (redox chemistry), at the electrode surface, or an electro-active product or reactant of the biological-recognition reaction.  Second generation: Use mediator molecules to transfer the electrons from the enzyme, after it reduces or oxidizes the substrate, to the electrode.  Third generation: Modified-electrode biosensor. In this generation, the surface of the electrode is modified by the addition of molecules which allow the direct oxidation or reduction of the enzyme at the electrode.

8 Amperometric biosensor  When a species is oxidized or reduced at an electrode, the current produced is directly related to the concentration of the species.

9 Immobilization of enzyme  Better contact and response  Improve enzyme stability  Electrode reusability  Less interferences  Loss of enzyme activity  Sensitivity of the electrochemical signal /BiosensorsBioelectronics_lecture5.pdf

10 Clark oxygen electrode Leland C. Clark (1918–2005) “Father of biosensor” Clark-type electrode: (A) Pt- (B) Ag/AgCl-electrode (C) KCl electrolyte (D) Teflon membrane (E) rubber ring (F) voltage supply (G) galvanometer O e − + 2 H 2 O → 4 OH −  A voltage of around 0.7 V is used to allow linearity between the measurements of the current and oxygen concentration.  Lifespan is limited to around 3 years due to the Teflon membrane, which becomes encoated with protein  Requires power source  Requires constant temperature

11 Glucose biosensor  In 1962, Clark described "how to make electrochemical sensors more intelligent" by adding "enzyme transducers as membrane enclosed sandwiches”.  Clark’s ideas became commercial reality in 1975 with the successful re-launch (first launch 1973) of the Yellow Springs Instrument Company (Ohio, USA) glucose analyzer based on the amperometric detection of hydrogen peroxide. YSI 23A

12 Glucose biosensor

13 Glucose + O 2 → Gluconolactone + H 2 O 2 The oxidase enzyme is inexpensive but requires oxygen as a cosubstrate. Consequently, as oxygen is depleted in the sample, performance decreases, whether one is monitoring oxygen depletion, or hydrogen peroxide production. Glucose + NAD + → Gluconolactone + NADH NAD + dependent GDH, on the other hand, is oxygen independent, and has the added attraction of being a well-established probe for monitoring biochemical reactions. The drawback is that the cofactors are relatively expensive. Glucose + PQQ(ox) → Gluconolactone + PQQ(red) PQQ-GDH is a particularly efficient enzyme system, with a rapid electron transfer rate, but it, too, is comparatively costly.

14 Glucose biosensor Figure. Calibration curves of the glucose biosensor based on glucose oxidase immobilized tin oxide electrode: (a) at various glucose concentrations (from 0 mM to 10 mM); Inset shows typical amperometric response of the glucose biosensor. (b) at low glucose concentrations (from 0 mM to 3 mM) which shows linear response in pH 7.0 potassium phosphate buffer (100 mM). Biotechnology and Bioprocess Engineering, 2008, 13,

15

16 NADH biosensor  Measuring NADH is very important because NAD(P) + is used as a cofactor for about 250 NAD + -dependent and 150 NADP + - dependent dehydrogenases.  It can be applied to analytical detection, fermentation, clinical practices, food industry, and dairy industry.  Direct electrochemical oxidation of NAD(P)H often occurs with high overpotential suffers from low sensitivity arises electrode fouling by its oxidation products

17 NADH biosensor NAD(P)HNAD(P) + electrode analyte red analyte ox amperometric detection

18 Electrochemical analysis Figure. (a) Linear sweep voltammograms of the Fe 2 O 3 /CB electrode with different NADH concentrations (0 mM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM; arranged from bottom to top, respectively) (insert: cyclic voltammograms in the absence and presence of 1 mM NADH) at a scan rate of 50 mV s −1 ; (b) linear sweep voltammograms of the Fe 2 O 3 /CB electrode at a scan rate of 10 mV s −1, 20 mV s −1, 30 mV s −1, 40 mV s −1, 50 mV s −1, 75 mV s −1, 100 mV s −1, 150 mV s −1, and 200 mV s −1 ; arranged from bottom to top, respectively (insert: dependence of the peak current on scan rate) in pH 7.5 potassium phosphate buffer (100 mM). Biosensors and Bioelectronics, in press

19 Electrochemical analysis Figure. (a) Nyquist plots for the Fe 2 O 3 /CB ( ● ), GC ( ○ ), and CB (  ) electrodes in the presence of 1 mM NADH in pH 7.5 potassium phosphate buffer (100 mM). This experiment was performed using a frequency range of 0.5–5 kHz, V, and 5 mV amplitude. (b) Calibration curve from the amperometric response of NADH at Fe 2 O 3 /CB ( ● ), GC ( ○ ), and CB (  ) electrodes.

20 Amperometric detection Figure. Calibration curve of NADH with different composition of iron oxide and carbon black 5:1 (  ), 10:1 (  ), and 15:1 (  ).

21 Linear range 10μM-1000μM (R 2 =0.993) Limit of detection (LOD) 10μM (S/N=3) Sensitivity 2.54 μA mM -1 Km=3.04mM Amperometric detection Figure. (a) Amperometric response for NADH oxidation at the Fe 2 O 3 /CB electrode after addition of different NADH concentrations into a stirred solution of 100 mM potassium phosphate buffer (pH 7.5) at V. (b) Calibration curve from the amperometric response of NADH.

22 Stability of electrode Figure. Amperometric response for NADH oxidation at the Fe 2 O 3 /CB electrode after addition of 1 mM NADH.

23 Response to NADPH Figure. Calibration curve from the amperometric response of NADH (  ) and NADPH (  ).

24 Performance of biosensor

25 Ethanol biosensor Figure. (a) Effect of pH on the amperometric response to NADH oxidation ( ■ ), activity of alcohol dehydrogenase ( ● ), and the amperometric response of the ethanol biosensor system (gray bar). (b) Calibration curve of the Fe 2 O 3 /CB electrode after addition of ethanol into a stirred solution that contained 2 mg alcohol dehydrogenase and 10 mM NADH in pH 7.5 potassium phosphate buffer at V.

26 8.2 Enzyme Fuel Cell

27 Enzymatic Biofuel Cell Microbial Biofuel Cell Fuel Cell A device to convert chemical energy to eletrical power Chemical Fuel Cell BioFuel Cell PMFC DMFC

28 Enzyme Biofuel Cell Enzymes are selected as catalysts. a power source for portable or implantable electronics, especially biomedical devices. the poor power density and the short life time. Fuel : glucose, alcohol, glycerol Anodic catalyst : glucose oxidase, glucose dehydrogenase alcohol dehydrogenase aldehyde dehydrogenase Cathodic catalyst : laccase bilirubin oxidase microperoxidase

29 Efforts for Efficient Electron Transfer Employment of the redox-mediator

30 Efforts for Efficient Electron Transfer Reconstitution of the enzyme using molecular or polymer relay unit

31 Possible Solutions for Practical Use Enzyme cascade (multienzyme system) Three-dimensional electrode architecture Oriented immobilization of enzyme on the electrode

32 Enzyme Cascade

33 Enzyme Cascade – case study Ref.) Electrochimica Acta 50 (2005) Alcohol dehydrogenase/Aldehyde dehydrogenase system

34 Three-dimensional electrode architecture Multidimensional and multidirectional pore structure Small pores : high loading density, enzyme stabilization Large pores : mass transport of liquid phase fuel

35 Three-dimensional electrode architecture – case study AnodeCathode Power (μW/cm 2 ) Ref. CatalystMediatorCatalystMediator Glucose oxidaseHQSBilirubin oxidaseABTS42 (Habrioux, Merle et al. 2008) Glucose oxidase1,1’-dicarboxylferroceneLaccase 1,1’- dicarboxylferrocene 28.4 (Liu and Dong 2007) Glucose oxidaseLaccase1.38 (Wang, Yang et al. 2009) Glucose oxidaseHQSLaccaseABTS27 (Brunel, Denele et al. 2007) Glucose oxidaseOsimium redox polymerLaccase Osimium redox polymer 10.0 (Barriere, Kavanagh et al. 2006) Glucose oxidaseFe(CN) 6 -3 LaccaseABTS110 (Zebda, Renaud et al. 2009) Laccase Pt wire10 (Zheng, Zhou et al. 2008) Glucose oxidaseTyrosinase (μW/cm 3 ) This study 3-dimensional glucose oxidase/SWNT/polypyrrole composite electrode

36 Three-dimensional electrode architecture – case study 3-dimensional glucose oxidase/SWNT/polypyrrole composite electrode ; Stability at working condition with continuous feeding of fuel AnodeCathodeFuel Power density (μW/cm 2 ) Stability (hr) Ref. GOx-epHOPGMP-11-epHOPG Glucose (2mM) 3.7<6 (Choi, Wang et al. 2009) Modified GC with GDH-polyBCB- SWNT Modified GC with BOD-BSA Glucose(40m M) 53.9<14(Gao, Yan et al. 2007) GOx/SWNT/Ppy composite Tyrosinase/CNPs/Ppy composite Glucose(1mM)157.4 μW/cm 3 29This study

37 Orient Immobilization Ref.) Journal of Molecular Catalysis B: Enzymatic 59 (2009)

38 Case study - glycerol/O 2 Biofuel Cell Ref.) Journal of Power Source 173 (2007)

39 Case study – Organell-based Biofuel Cell Ref.) Electrochimica Acta 53 (2008) Electrochimica Acta 54 (2009)

40 Case study – Organell-based Biofuel Cell Ref.) Electrochimica Acta 53 (2008) Electrochimica Acta 54 (2009)

41 Case study – Microfluidic Biofuel Cell Ref.) Electrochemistry Communications 11 (2009) Ref.) Journal of Power Source 178 (2008) 53-58

42 Case study – Hybrid Biofuel Cell Ref.) Journal of Power Source 188 (2009)

43 Case study – Hybrid Biofuel Cell Ref.) Biosensors and Bioelectronics 24 (2009)

44 Case study – Hybrid Biofuel Cell Ref.) Biosensors and Bioelectronics 24 (2009)


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