Enzyme based biosensors

Transcription

1 Enzyme based biosensors

2 Brief history; how it all started? 1916 First report on immobilization of proteins : adsorption of invertase on activated charcoal 1922 First glass ph electrode 1956 Clark published his definitive paper on the oxygen electrode First description of a biosensor: an amperometric enzyme electrodre for glucose (Clark) 1969 Guilbault and Montalvo First potentiometric biosensor:urease immobilized on an ammonia electrode to detect urea 1970 Bergveld ion selective Field Effect Transistor (ISFET) 1975 Lubbers and Opitz described a fibre-optic sensor with immobilised indicator to measure carbon dioxide or oxygen.

3 Introduction to Biosensors A biosensor is an analytical device which is used to determine the presence and concentration of a specific substance in a biological analyte Desired molecule Bioreceptor Transducer Signal Processing Display Biosample Biosensor

4 Introduction to Biosensors Bioreceptor Transducer Antibody Enzyme Optical Electrochemical Absorption Fluorescence Interference potentiometric amperometric conductimetric Nucleic Acid (DNA) Mass based Cell MIP Temperature based Electric & Magnetic Dielectric properties Permeability properties Voltage or Current

5 Bioreceptors Antibody Antibodies are biological molecules that exhibit very specific binding capabilities for specific structure (antigens). Antigen membrane It can be recognized by antibody.

6 Bioreceptors Bioreceptor Display

7 Bioreceptors Enzyme Enzyme is a large protein molecule that acts as a catalyst in chemical reactions. Enzymes are often chosen as bioreceptors based on their specific binding capabilities as well as their catalytic activity

8 Bioreceptors: Whole cell biosensor Bioreceptor Display

9 Immobilizatiom The immobilization is done either by physical entrapment or chemical attachment. Physical Entrapment Bioreceptor (Antibody, Enzyme, Cell, ) + polymer solution polymerization Adsorption adsorptive interactions such as ionic, polar or hydrogen bonding and hydrophobic interaction.

10 Immobilizatiom Covalent bounding formation of a stable covalent bond between functional groups of the bioreceptor components and the transducer Cross-linking bridging between functional groups on the outer membrane of the receptor by multifunctional reagents to transducer. The cells can be bounded directly onto the electrode surface or on a removable support membrane, which can be placed on the transducer surface

11 ELECTROCHEMICAL BIOSENSORS AI. Glucose Sensors - For determination of glucose in blood - For diagnosis and therapy of diabetes - Glucose oxidase is trapped between polyurathene and permselective membrane on a Pt WE AI. Glucose Sensors - The libration of H 2 O 2 in the enzymatic reaction is monitored at the electrode surface H 2 O 2 O 2 + 2H + + 2e - electrode - Enzymatic reaction can also be followed by consumption of O 2 Glucose + O 2 Gluconic acid + H 2 O 2 Glucose oxidase Hand-held glucose monitoring devices - Makes use of mediators - Disposable strips are PVC and screen-printed carbon electrodes - Contains a mixture of glucose oxidaze and a mediator - Applies potential-step (chronoamperometric) operation

12 AII. Ethanol Sensors (Ethanol Electrodes) - For amperometric sensing of ethanol - Employs immobilization of ADH and NAD + to C or Pt anode - Based on enzymatic reaction of ethanol with NAD + in the presence of ADH C 2 H 5 OH + NAD + C 2 H 5 O + NADH ADH AII. Ethanol Sensors (Ethanol Electrodes) - NADH is produced upon reduction of NAD + by alcohol - NAD + is regenerated by electrochemical oxidation of NADH NADH NAD + + 2e - + H + - The resulting anodic current is measured NAD + : nicotinamide adenine dinucleotide ADH: alcohol dehydrogenase

13 AIII. Urea Electrodes - For sensing urea in the presence of urease enzyme - Makes use of ammonium ion-selective electrode - Electrode is modified with a gel containing the urease enzyme NH 2 CONH 2 + 2H 2 O + H + 2NH 4+ + HCO 3 - urease AIV. Other Enzyme Electrodes - Cholesterol electrodes - Lactate electrodes - Penicillin electrodes - Uric acid electrodes

14 conductimetric

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16 Absorption I 1 /I 0 = e αlc Color reaction l is the pass length C is the concentration of absorbing material α is the absorption coefficient

17 Transducers-Optical methods- Florescence Fluorescence is a molecular absorption of light at one wavelength and its instantaneous emission of at longer wavelengths. Some molecules fluoresce naturally and others such as DNA can be modified for fluorescence detection by attachment of special f l u o r e s c e n t d y e s.. An optical system for Florescence measurement

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19 Transducers-Optical methods- Florescence Grating Antigens modified by florophor dye Planer waveguide Evanescent-field. Excitation light Florescence detector A device for florescence measurement

20 Transducers-Optical methods-refractive index Mach-Zehnder interferometer

21 Transducers-Electrochemical methods The underlying principle for this class of biosensors is that many chemical reactions produce or consume ions or electrons which in turn cause some change in the electrical properties of the solution which can be sensed out and used as measuring parameter

22 Transducers-Electrochemical methods-amperometric amperometric glucose biosensors

23 Transducers-Electrochemical methods-amperometric amperometric glucose biosensors

24 Transducers-Electrochemical methods-potentiometric Schematic diagram of an integrated enzyme-based flow-through glucose sensor.

25 Transducers-Electrochemical methods-potentiometric

26 Transducers-Electric methods Concept: Capture analyte and detect changes in electrical parameters of sample Immunosensing at micro-sized Au electrodes based on the change of conductivity between the Au strips upon binding of Au nanoparticles

27 Transducers-Electric methods F= 20KHz, AC amplitude of 40 mv peak to peak

28 Detection by cantilevers Transducers-Mass based methods

29 Detection by cantilevers Transducers-Mass based methods Microcantilever technology: a) with immobilised protein for a specific bacterium b) bending after adsorption of bacteria to the protein