Introduction of Biosensors Lecture April 17 Jeff T.H.Wang website: http://pegasus.me.jhu.edu/~thwang/ New course : BioMEMS and BioSensing (Spring 04 )
What s is a biosensor? Target 4.22 Signal Signal Analtye Receptor Transducer Signal processors Analyte: the substances to be measured Small molecules: Sugars, urea, cholesterol, glutamic acid, phosphate,.. Macro molecules : amino acids (DNA, RNA), peptide( protein, antibody, enzyme) Receptor: a sensing element that responds to the substances being measured, the interaction must be highly selective Enzyme, Antibody, Nucleic acids, Cells Transducer: a device that converts the physical or chemical changes due to analytereceptor reactions to another form of physical signal (in general, electronic signals) whose magnitude is proportional to the amount of the analyte Electrochemical (Potentiometric, Voltammetric, Conductimetric) Optical (Fluorescence, Absorbance, Light scattering, Refractive index) Field effect transistor (FET) Mechanical, Thermal, Piezoelectric, Surface acoustic waves, magnetic Signal processors: Amplification, filtration, correlation..
Performance Factors Sensitivity: Minimum amount of analyte that are able to be detected above the background Units: Concentration, number of analyte, density, weight Specificity/Selectivity: The ability to discriminate between substrates. This is function of biological component, principle, altough sometimes the operation of the transducer contributes to selectivity Molecular recognition, Separation scheme, Signal overlap, Example: SNPs detection Speed/Response Time: Sample preparation + Biological/Chemical reaction + Signal Processing Bench process : hours to weeks Chip process: minutes to hours Ultra-high temporal resolution, 10 ns, for real-time measurement of molecular kinetics Accuracy : False positive, False negative PCR amplification Simplicity, Cost, Lift time,..
Enzymatic Reaction & Biosensing for Small Molecules e - S + E(Red) k 1 k 2 ES E(ox) +P k -1 Enz (ox) Enz (red) S: substrate E: Enzyme ES : enzymesubstrate complex P: product (Substrate) e - (Product) rate of formation of complex = k 1 [S][E]-k -1 [ES] rate of breakdown of complex = k 2 [ES] (Steady-state equilibrium) k 1 [S][E]-k -1 [ES]-k 2 [ES]=0 As [E 0 ]=[E]+[ES], k 1 [S][E 0 ]-k 1 [S][ES]-k -1 [ES]-k 2 [ES]=0 [ES] = k 1 [E 0 ][S] k -1 +k 2 +k 1 [S] = [E 0 ][S] K M +[S] V max = k 2 [E 0 ] Reaction rate ~ Electric current /potential V max /2 where K M =(k -1 +k 2 )/k 1 v= d[p] dt d[s] = - = k 2 [ES] = k [E ][S] 2 0 dt K M +[S] K M Substrate concentration
Examples: glucose + O 2 + GOD ES gluconic acid + H 2 O 2 + GOD (NH 2 ) 2 CO + H 2 O + urease 2NH 3 + CO 2 + urease Reactant Cholesterol Esters Glucose Hydrogen peroxide Penicillin G Peptides Starch Sucrose Urea Uric acid Enzyme Cholesterol oxidase Chymotrypsin Glucose oxidase Catalase Penicillinase Trypsin Amylase Invertase Urease Uricase
Specific target Primary probe marker/report molecule Biosensing for Macromolecules immobilization secondary probe Non-specific molecule washing Enzyme-reaction Fluorescence ex./em. Steps: (1) Immobilization of primary probes (2) Mixing/incubation of the mixtures (3) Washing the non-specific bindings (4) Signal transduction Markers/Report molecules: (1) Enzymes (2) Fluorescence tags (3) Radioactive tags Molecular Recognition: Waston-Crick Base pairing : ATTGGCG (target) TAACCGC (probe) Antibody-Antigen binding : Ab + Ag Ab-Ag
Example 1: Generalized ELISA for protein detection
Transduction Methods: Electrochemical -Potentiometry : the measurement of a cell potential at zero currents -Amperometry in which an oxidizing (or reducing) potential is applied between the cell electrodes, and the cell current is measured Electrode (reference) Glass membrane (ion-sensitive) Electrode Working Electrode Reference electrode Auxiliary electrode -Conductometry where the conductance of the cell is measured sensor V V ref out Bridge circuit 5 cm 5 mm 10mm
Transduction Methods: Electrochemical -Field Effect Transistors Typical layout of a FET Ion-Sensitive Field Effect Transistor (ISFET) -Current from source to drain related the gate potential -Application of membrane to gate allows selective measurements -AD: Very small, array possible, high spatial resolution, short response tim -DA: Membrane needed, ph sensitivity, Drift, nonlinear
Transduction Methods : Optical Types of measurements -Absorbance(Oligo 260nm, Peptide280nm) -Fluorescence -Refractive index -Light scattering Changes measured -Intensity -Frequency -Phase shift -Polarization Types of components -Fiber optics -Wave guides -Photodiode -Spectroscopy -Charge coupled device (CCD) -Single photon APD -Interferometers
Optical Biosensor Basing on Evanescent Wave Evanescent Wave Separation between surface and bulk Noise reduction due to small observation volume Surface Plasmon Resonance (SPR) Light Source Prism Detector Evansescent wave High index (glass) Low index (Au or Ag) Sensing layer Flow cell α1 α2 Evanescent Wave Fiber Optic Sensor Biotinylated capture probe Biotinylated Fiber Streptavidin Biotin
Laser Induce Fluorescence (LIF) Based Detection Detector Pinhole Extremely High Sensitivity Dichoric beamsplitter Objective Band pass filter Laser Very small probe volume (< 10-15 L) -Less Raman scattering noise from solution -Less background luminescence Monochromatic excitation -Noise from light source can be efficiently filtrated Enhance Signal to Noise Ratio
Acoustic Wave & Nanomechanical Genosensor Acoustic Wave Genosensor Amplifier Quartz plate Target strand Probe strand Au An applied radiofrequency produces mechanical stress in the crystal Surface acoustic wave (SAW) induced SAW is received by the electrodes and is translated to voltage Nanomechanical Genosensor (Fritz,2000) Molecular binding surface tension Surface tension bend the cantilever beam Deflection of beam is optically measured
Advantages of Micro Biosensors Bench process Miniaturization µ-tas Parallel Processing Integration Automation
Multidisciplinary Expertise in Biosensor Development Chemistry Synthetic recognition Electrode Materials Polymers Disposable Membrane Immobilization Physics Optics Semiconductors -FET -Quantum dots Biology Biorecognition Protein engineering Receptor technology DNA ampl. technology Biosensor Electronics Optoelectronics Silicon technology Data processing Control Instrumentation Portable Miniaturization Discrete Data presentation Market On-line In-vivo in-vitro Disposable Re-usable Molecular electronics Bioelectronics Molecular electronics Neural sensing