Introduction to Biosensor. Wei Shi DianHong Shi

Transcription

1 Introduction to Biosensor Wei Shi DianHong Shi

2 Outline The definition of biosensor The history of biosensor The development of biosensor The working principle of biosensor The application of the biosensors The future of biosensor Reference 2

3 Definition of Biosensor A biosensor is precisely defined, according to IUPAC definition, as a self-contained integrated device, capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element, which is retained in direct spatial contact with a transduction element 3

4 History of Biosensor 1956 Invention of the oxygen electrode by Leland C Clark 1969 First potentiometric biosensor: urease immobilised on an ammonia electrode to detect urea 1970 Invention of the Ion-Selective Field-Effect Transistor (ISFET) 1972/5 First commercial biosensor: Yellow Springs Instruments glucose biosensor 1976 First bedside artificial pancreas (Miles) 1980 First fibre optic ph sensor for in vivo blood gases 1982 First fibre optic-based biosensor for glucose 1983 First surface plasmon resonance (SPR) immunosensor 1984 First mediated amperometric biosensor: ferrocene used with glucose oxidase for the detection of glucose 1987 Launch of the MediSense ExacTech blood glucose biosensor 4

5 History of Biosensor (cont) 1990 Launch of the Pharmacia BIACore SPR-based biosensor system 1992 i-stat i STAT launches hand-held held blood analyser 1996 Glucocard launched 1998 Launch of LifeScan FastTake blood glucose biosensor 5

6 The development of biosensor There are three generations of biosensor First generation: The normal product of the reaction diffuses to the transducer and causes the electrical response Second generation: There is a specific 'mediators' between the reaction and the transducer in order to generate improved response Third generation: The reaction itself causes the response and no product or mediator diffusion is directly involved 6

7 The development of biosensor (cont) We use Amperometric biosensors as a example First generation: It was proposed by Clark and Lyons and implemented by Updike and Hicks Formula: Glucose +GOX-FAD(1) Gluconolactone+GOX-FADH2 GOX-FADH2 FADH2 + O2 GOX GOX-FAD +H2O2 GOX ( glucose oxidase ): Its function is to selectively oxidize analyte by the reduction of O2 to H2O2 GOX-FAD FAD and GOX-FADH2: represent the oxidized and reduced states of the glucose oxidase enzyme's flavin active site 7

8 The development of biosensor (cont) Second generation: It use an artificial electron mediator ( ferrocene, quinones, quinoidlike dyes, organic conducting salts, and viologens viologens ) which replaces O2 as the electron shuttle. Formula: GOX-FADH2 + Mediatorox GOX GOX-FAD + Mediatorred 8

9 The development of biosensor (cont) Third generation use of a freely diffusing mediator to a system where enzyme and mediator are coimmobilized at an electrode surface, making the biorecognition component an integral part of the electrode transducer Coimmobilization of enzyme and mediator can be accomplished by redox mediator labeling of the enzyme followed by enzyme immobilization, enzyme immobilization in a redox polymer, or enzyme and mediator immobilization in a conducting polymer 9

10 The development of biosensor (cont) Comparison of generations of biosensor Affected by low solubility of O2O in aqueous solutions 1st generation Yes ( sensor based on O2 O / H2O2H reaction ) 2nd generation No ( use high soluble artificial mediator ) 3rd generation No ( Integrate enzyme and mediator ) Use other oxidoreductase enzymes No ( need O2O as as an electron- accepting cosubstrate ) Yes ( O2O isn t t need by using mediator ) Yes ( Integrate enzyme and mediator ) 10

11 The development of biosensor (cont) Comparison of generations of biosensor ( cont ) Repeated measurements 1st generation No ( enzyme need to be added) 2nd generation No ( enzyme and mediator need to be added) 3rd generation Yes ( self-contained ) 11

12 The working principle of biosensor Biosensors are made up of two different but strictly connected components Bioreceptor: It is the part that selectively binds and recognizes the analyte Transduction: Transfer the signal from output domain of recognition system ( bioreceptor ) to physical measurable signal 12

13 The working principle of biosensor (cont ) 13

14 The working principle of biosensor (cont ) Anaylte: : generally any substance which is involved in a biochemical process Inorganic Gas Ions (PH) Heavy metals Organic acid Carbohydrates Urea Organic 14

15 The working principle of biosensor (cont ) Bioreceptor: Any biological substance that can attach itself to a particular analyte. Enzyme Microorganism Antigens & Antibodies Nucleic acids Catalytists 15

16 The working principle of biosensor (cont ) Bioreceptor: Any biological substance that can attach itself to a particular analyte. Catalytists Enzyme Microorganism Antigens & Antibodies Nucleic acids Use living microorganisms metabolic functions to detect anaylte 16

17 The working principle of biosensor (cont ) Bioreceptor: Any biological substance that can attach itself to a particular analyte. Catalytists Enzyme Microorganism Antigens & Antibodies Nucleic acids Use living microorganisms metabolic functions to detect anaylte Based on bio-affinity of binding & labeling 17

18 The working principle of biosensor (cont ) Bioreceptor: Any biological substance that can attach itself to a particular analyte. Catalysts Enzyme Microorganism Antigens & Antibodies Nucleic acids Use living microorganisms metabolic functions to detect anaylte Based on bio-affinity of binding & labeling Based on hybridization binding of a unique DNA/RNA sequence 18

19 The working principle of biosensor (cont ) Receptors: specificity vs. stability 19

20 The working principle of biosensor (cont ) Immobilization The technique used for the physical or chemical fixation of cells, organelles, enzymes, or other proteins (e.g. antibodies) onto a solid support, into a solid matrix or retained by a membrane, in order to increase their stability and make possible their repeated or continued use. 20

21 The working principle of biosensor (cont ) Immobilization techniques Covalent bond Entrapment Micro-encapsulation 21

22 Covalent bond The most intensely studied of the immobilization techniques. Based on the binding of enzymes and water- insoluble carriers by covalent bonds Two limit characteristics The binding reaction must be performed under conditions that do not cause loss of enzymatic activity The active site of the enzyme must be unaffected by the reagents used. 22

23 Covalent bond The functional groups that may take part: Amino group Carboxyl group Sulfhydryl group Hydroxyl group Imidazole group Phenolic group Thiol group Threonine group Indole group 23

24 Techniques for Entrapment Enzymes are well mixed with monomers/polymers and cross-linking agents in a solution. The solution is then exposed to polymerization promoters to start the process of gel formation. The solution is poured into a mold to achieve the desired shapes. 24

25 Entrapment Three commonly used entrapment media polyacrylamide The most widely used matrix for entrapping enzymes Non-ionic: properties of the enzymes are only minimally modified calcium alginate Polymer molecules are cross-linked by calcium ions. Because of this calcium alginate beads can be formed in extremely mild conditions, which ensure that enzyme activity yields of over 80% can be routinely achieved gelatin Requires only simple equipment Reagents are relatively inexpensive and nontoxic Enzymatic activities: typically 25-50% 50% of the original free enzyme 25

26 Micro-encapsulation Tiny particles are surrounded by a coating to give small capsules with many useful properties (e. g Isolated core from its surroundings to avoid chemical attack ) 26

27 The working principle of biosensor (cont ) Types of transduction: Electrochemical Optical Piezoelectric 27

28 The working principle of biosensor (cont ) Electrochemical: Potentiometry Amperometry Conductimetry Field Effect Transistors Difference between indicator and reference electrode potential at equilibrium (i.e. zero current) 28

29 Ion Ion-selective Electrode (ISE) 29

30 The working principle of biosensor Electrochemical: Potentiometry Amperometry Conductimetry (cont ) Field Effect Transistors Difference between indicator and reference electrode potential at equilibrium (i.e. zero current) Current flowing between working and reference electrode 30

31 Amperometry At t0 a voltage is applied over the electrodes, causing the cell to recharge. The diffusion rate of fresh Ox decreases. Fick s law: dc/dt = D * d2c/dc2 31

32 The working principle of biosensor Electrochemical: Potentiometry Amperometry Conductimetry (cont ) Field Effect Transistors Difference between indicator and reference electrode potential at equilibrium (i.e. zero current) Current flowing between working and reference electrode Transistors where the gate metal has been replaced by a chemically sensing surface 32

33 Field Effect Transistor 1. Silicon substrate 2. Insulator SiO2/Si3N4 3. Furrow Metals 4. Lacune (gap) 5. Selective coating 33

34 Optical Ultraviolet-visible absorption Luminescence Internal Reflection Spectroscopy Surface Plasmon Resonance (SPR) Laser light scattering 34

35 Piezoelectric Mechanical-piezoelectric Acoustical-piezoelectric Surface Acoustic Wave Transducer 35

36 The application of the biosensors Health industry Blood glucose sensors for diabetes patients ( 85% of the world market for biosensors) Food industry Determination of the food composition Detect of contamination Pathogens pesticides microorganisms toxins Natural environment Water quality control 36

37 The future of biosensor highly efficient electrocatalysts with nonprotein structure High sensitivity, selectivity and stability Employ more enzymes, proteins, biomimetic molecules New kinds of electronic communications, bimolecular immobilization, molecular architecture and sensor design More powerful of microprocessor. 37

38 Any Question? 38

39 Reference &ISSUE=0409&RELTYPE=PR&ORIGRELTYPE=HTS&P RODCODE= &PRODLETT=M muenchen.de/e25/members/garrido/mb- JASS/Munnikes.pdf 39