Introduction of BiosensorsLecture April 17

Jeff T.H.Wangwebsite: http://pegasus.me.jhu.edu/~thwang/

New course : BioMEMS and BioSensing (Spring 04’)

What’s is a biosensor ?

Signal processors

TransducerReceptor

Signal

4.22Target

Signal

Analtye

Analyte: the substances to be measuredSmall 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, theinteraction must be highly selectiveEnzyme, Antibody, Nucleic acids, Cells

Transducer: a device that converts the physical or chemical changes due to analyte-receptor reactions to another form of physical signal (in general,electronic signals) whose magnitude is proportional to the amount of the analyteElectrochemical (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 contributesto 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

S + E(Red)

k1 k2ES E(ox) +Pk-1S: substrateE: Enzyme

ES : enzyme-substrate complex

P: product

Enz(ox) Enz(red)

(Substrate) (Product)

e-

e-

rate of formation of complex = k1[S][E]-k-1[ES]

rate of breakdown of complex = k2[ES]

(Steady-state equilibrium)

k1[S][E]-k-1[ES]-k2[ES]=0

As [E0]=[E]+[ES],

k1[S][E0]-k1[S][ES]-k-1[ES]-k2[ES]=0

[ES] =k1[E0][S]

k-1+k2+k1[S]

[E0][S]

KM+[S]=

where KM=(k-1+k2)/k1

v= d[P]

dt

d[S]

dt= - = k2[ES] =

k2[E0][S]

KM+[S]

Substrate concentration

Reaction rate ~ Electric current /potential

Vmax = k2[E0]

KM

Vmax/2

Examples: glucose + O2 + GOD ES gluconic acid + H2O2 + GOD

(NH2)2CO + H2O + urease 2NH3 + CO2 + urease

Reactant Enzyme

Cholesterol Cholesterol oxidase

Esters Chymotrypsin

Glucose Glucose oxidaseHydrogen peroxide Catalase

Penicillin G Penicillinase

Peptides Trypsin

Starch Amylase

Sucrose Invertase

Urea Urease

Uric acid Uricase

Biosensing for Macromolecules

Enzyme-reactionFluorescence ex./em.

washing

Primary probe

secondary probe

marker/report molecule

immobilization

Specific target

Non-specific molecule

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 :

•Antibody-Antigen binding :

ATTGGCG (target)TAACCGC (probe)

Ab + Ag Ab-Ag

Example 1: Generalized ELISA for protein detection

Transduction Methods: Electrochemical

-Potentiometry :the measurement of a cell potential at zero currents Glass membrane (ion-sensitive)

Electrode (reference)

Electrode

-Amperometryin which an oxidizing (or reducing)potential is applied between the cellelectrodes, and the cell current is measured

Working Electrode

Reference electrode Auxiliary electrode

VrefVout

sensor

Bridge circuit

-Conductometrywhere the conductance of the cell is measured

5 cm 5 mm 10mm

Transduction Methods: Electrochemical

-Field Effect Transistors

Ion-Sensitive Field Effect Transistor (ISFET)Typical layout of a FET

-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

•Changes measured-Intensity-Frequency-Phase shift-Polarization

•Types of measurements-Absorbance(Oligo 260nm, Peptide280nm)-Fluorescence-Refractive index-Light scattering

•Types of components-Fiber optics-Wave guides-Photodiode-Spectroscopy-Charge coupled device (CCD)-Single photon APD-Interferometers

Optical Biosensor Basing on Evanescent Wave

•Separation between surfaceand bulk

•Noise reduction due to smallobservation volume

Evanescent Wave

Light SourcePrism

Detector

High index (glass)

Flow cell

Low index (Au or Ag)Sensing layer

Evansescent wave

Surface PlasmonResonance (SPR)

α1 α2

StreptavidinBiotin

Biotinylated capture probe

Biotinylated Fiber

Evanescent Wave Fiber Optic Sensor

Laser Induce Fluorescence (LIF) Based Detection

LaserDichoricbeamsplitter

Objective

•Very small probe volume (< 10-15L)-Less Raman scattering noise from solution-Less background luminescence

•Monochromatic excitation-Noise from light source can be efficientlyfiltrated

Extremely High Sensitivity

Enhance Signal to Noise Ratio

Detector

Pinhole

Band pass filter

Acoustic Wave & Nanomechanical Genosensor

Acoustic Wave Genosensor

Au

Quartz plateAmplifier

Target strand

Probe strand

• An applied radiofrequency producesmechanical 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

Miniaturization

Parallel Processing

Integration

Automation

Bench process µ-TAS

Multidisciplinary Expertise in Biosensor Development

ChemistrySynthetic recognitionElectrode MaterialsPolymersDisposableMembraneImmobilization

BiologyBiorecognitionProtein engineeringReceptor technologyDNA ampl. technology

PhysicsOpticsSemiconductors

-FET-Quantum dots

ElectronicsOptoelectronicsSilicon technologyData processingControl

MolecularelectronicsBioelectronicsMolecular electronicsNeural sensing

InstrumentationPortableMiniaturizationDiscreteData presentation

MarketOn-lineIn-vivoin-vitroDisposableRe-usable

Biosensor