TOPICS IN (NANO) BIOTECHNOLOGY

Immunosensors

30th June

PhD Course

Overview • Last week we looked at what is DNA and

what is a gene.• We also looked at DNA replication and

protein synthesis, and the path from the gene to protein

• This week we will look at Recombinant DNA technology

• We will also look at the amplification of DNA and finally at its sequencing

Immunosensors

What is an antibody?

How do we produce polyclonal and monoclonal antibodies?

Polyclonal antibodies

- larger quantities may be produced at a time

- sometimes better selectivity and sensitivity due to recogintion of multiple epitopes

- no guarantee of batch to batch reproducibility

Monoclonal antibodies

- long and expensive process

- sometimes lower selectivity and sensitivity in comparison to Pabs observed

- once cell line established constant reproducible supply of antibodies …. forever

Sandwich assay

substrate

product

substrate

product

substrate

product

Concentration

Res

pon

se Useful for large molecules

Robust assay - all reagents in excess

Use with Pabs or different MAbs

Competition assay

substrate

product

substrate

product

Concentration

Res

pon

se Useful for small molecules

Reportedly less sensitive

Concentrations of reagents critical

Displacement assay

substrate

product

substrate

product

Concentration

Res

pon

se

One step assay

In practise difficulties to achieve - effect of non specific displacement

Sub-optimum haptens met with some success

History of immunosensors• 1964 - Fluorescence polarisation labeled Ab and Ag• 1967 - First voltammetric immunosensor (Purdy et

al)• 1976 - Use of FITC• 1972 - First PZ immunosensor (Shons et al)• 1975 - First potentiometric immunosensor (Janata)• 1976 - First report of EIA (Rubenstein et al)• 1976 - First amperometric immunosensor (Aizawa)• 1980 - First fluorescence immunoassay

Electrochemical transduction

Duan & Meyerhoff, 1994

• Gold coated microporous nylon membranes, serving as solid phase and working electrode

• Ab immobilised via SAM of thioctic acid on gold side of membrane

• Separation free sandwich assay - surface bound spatially resolved from excess conjugate in bulk

• Substrate introduced through back side of porous membrane

• Substrate diffuses rapidly through membrane first encountering ALP-Ab

• Enzymatically generated product detected immediately via oxidation at gold electrode

• Assay time of 30 minutes, measurement of 1 minute

Skladal et al, 1995

Reference electrode: silver paste

Working electrode

Isolating layer

Electrical contacts

Portable potentiostat

Lateral view micro-well

Electrochemical plate Disposable sensors

From Kaláb and Skládal, Anal. Chim. Acta, 304 (1995) 361-368

Nylon membranes

GasketCeramic support

Bauer et al, 1996

• FIA system using bienzyme recycling for detection of 2,4-D

• Clark-type electrode covered by membrane with PPO and PQQ-GDH

• 350-fold amplification observed

• 60 minute incubation with PP and zeptomole detection

Lu et al, 1997

• Electrically wired amperometric immunosensor

• Demonstrated for detection of biotin

• Redox polymer and antibody co-immobilised and competitive assay for biotin

• Only surface bound biotin-HRP ‘wired’

• L.O.D. One order of magnitude better than ELISA

Rishpon & Ivnitski, 1997

• Separation free enzyme channelling immunosensor

• Graphite pencil, Eapp = 0.0V

• Poly(ethylene)imine film to discriminate surface bound and bulk HRP

• Formats with I2, aminosalicylic acid

• 10-30 minute assay

Keay & McNeil, 1998

• Separation free immunosensor based on enzyme channelling

• Ab immobilised on Biodyne C membrane on SPEs

• 15 minute assay time, L.O.D. 0.012mg/L (12 p.p.t)

Wang, Tian & Rogers, 1998

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

Bi3+

Bi3

+PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGEPVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

Bi3+

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGEPVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

Bi3+

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

Bi3+

BiBiBi3+

3é3é

Stripping Deposition

Potentiometric stripping analysis

HSA used as model analyte

Bismuth metal ion label

30 minute incubation

HCl and Hg+ added to release metal label

10 minute deposition

Bäumner & Schmid, 1998

• Pioneering work patented by Durst (1996)

• Hapten tagged liposomes containing ascorbic acid

• Competition, - unbound labeled hapten passes detergent loaded membrane - releases ascorbic acid for electrodetection

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

Graphite electrode

Graphite electrode

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

Graphite electrode

Graphite electrode

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGEPVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGEPVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGEPVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGEPVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGEPVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

Ascorbic acid released

SIGNAL

SmartSenseTM

Ohmicron Co

SmartSense

Atrazine at p.p.b. Levels

15 minute assay time

H2O2

2 I -+ Polymer ox GOX labelled antigen

Antigen

Antibody

Reduced polymer

Oxidised polymer

I- / I2

Glucose

H2O2 + 2 I -+ 2 H + I2 + 2 H2O

I2 + Polymer red

Mo (IV)

Sample

Standard Standard

General features OHMICROM Co:

SmartSense™ Portable instrument Atrazine at ppb level

15 minutes total analysisThree-well cartridge

H2O2

2 I -+ Polymer ox GOX labelled antigen

Antigen

Antibody

Reduced polymer

Oxidised polymer

I- / I2

Glucose

H2O2 + 2 I -+ 2 H + I2 + 2 H2O

I2 + Polymer red

Mo (IV)

Sample

Standard Standard

General features OHMICROM Co:

SmartSense™ Portable instrument Atrazine at ppb level

15 minutes total analysisThree-well cartridge

Dequaire et al, 1999

•Sample and 2.4-D-ALP added to microwell-electrode format - 40 minute incubation

• Microwell-electrode format supported on magnet holding block

• Beads magnetically separated for 3 minutes and excess liquid removed

• Phosphoric acid ester of [[(4-hydroxyphenyl)amino]-carbonyl]cobaltecium hexafluorophosphate used as substrate

• Cationic phenol accumulated in Nafion film for 30 minutes

• L.O.D. of 10ng/L (p.p.t) of 2.4-D

Campbell et al, 1999

PVP{Os(bpy)2Cl}

HRP-LABELLED ANTIGEN

ANTIBODY

ANTIGEN (ANALYTE)

SUBSTRATE

PRODUCT

PEGDGE

2 H2O

HRP red 2 e - + 2 H+

H2O2

HRP oxWIRE Os II

WIRE Os III

• Ingenious assay - separation and reagentless immunosensor

• Choline oxidase does not interact with wire - produces H2O2 to act as substrate for HRP

• Washing not required as only surface bound HRP will be wired to electrode surface

•ChOX and avidin immobilised on redox hydrogel followed by biotinylated specific antibody

• An 18 minute assay - demonstrated with IgG

Kim et al, 2000

Attempted electrochemical detection of traditional immunochromatographic strips by measuring change

in conductance upon aggregation of colloidal gold labels

Direct detection - low sensitivity

Used gold colloids coated with polyaniline

Large improvement in sensitivity

Demonstrated with HSA

6 minute assay time

Benkert et al, 2000

Anti-analyte antibody

Redox labeled analyte

Analyte

Size exclusion layer MWCO 20,000

• SERI - size exclusion redox-labeled immunoassay

• Analyte competes with redox-labeled analyte for antibody binding

• Unbound redox-labeled passes therough the size exclusion layer and is indicated electrochemically

• Demonstrated with creatinine - low L.O.D.

Yang et al, 2001

Layer by layer (LbL) approach

Applied to enyme and immunosensors

Platform for fluorsecent immunosensors

Deposition of IgG

PS Microparticle Dye-labeled PS microparticle IgG conjugated dye-labeled PS microparticle

Consecutive assembly of PAH-FITC and PSS

Katz et al, 2001

• Sensing antibody using antigen monolayer electrode and anti idiotypic-HRP

• Biocatalytic precipitation of insoluble product - forms an insulating layer on electrode surface, decreases interfacial electron transfer rate constant

•Chronopotentiometry - measurement time of seconds, Faradaic impedance spectroscopy - 15 to 20 minutes

•

O’ Sullivan & Katakis, 2001

Quasi counter – reference Ag/AgCl electrode

Carbon working

electrode

Insulation Layer

Area for sample

application

• Competitive assay - immobilised antigen, labeled antibody

• Originally used ALP label and p-APP substrate - 22 minute assay time, mainly due to substrate

development

• Using Os-amine mediator and HRP label assay time of 10 minutes

Market drivers• The market drivers for the biosensors market, in order

of impact, are: high demand, expanding application areas high levels of research & development advancing technologies reducing production costs increased customer awareness legislation integrating partnerships between academia and industry innovative new product developments strong economy

Market predictions - 2001 to 2004

Biosensor Market Segments (Frost & Sullivan, 1998)

• Medical applications will continue to dominate

• Overall best growth rate of 6.8% predicted for environmental biosensors as applications will be realised for site characterisation and clean-up

• Growth attributed to development of immunosensors