Biosensors - ISOCS

Krishna Persaud, SCEAS, The University of Manchester, UK

Biosensors Krishna Persaud

• Biomolecules and their uses

• An Overview

Inspiration from Nature

Krishna Persaud, SCEAS, The University of Manchester, UK 2

Pit Viper


Thermoreceptors

From Runaway, by Michael Crichton.

Published by Tri-Star Pictures in 1985

Sniffing Robot

4

http://www.technovelgy.com/ct/AuthorSpecAlphaList.asp?BkNum=292

http://www.technovelgy.com/ct/AuthorTotalAlphaList.asp?AuNum=127

Father of the Biosensor

Professor Leland C Clark Jnr

1918–2005 5 Krishna Persaud, SCEAS, The University of Manchester, UK

Components of a Biosensor

Detector

6

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.

1962 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.

History of Biosensors

7 Krishna Persaud, SCEAS, The University of Manchester, UK

1975 First commercial biosensor ( Yellow springs

Instruments glucose biosensor)

1975 First microbe based biosensor, First immunosensor

1976 First bedside artificial pancreas (Miles)

1980 First fibre optic pH sensor for in vivo blood gases (Peterson)

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 glucose detection



1987 Blood-glucose biosensor launched by MediSense ExacTech

1990 SPR based biosensor by Pharmacia BIACore

1992 Hand held blood biosensor by i-STAT

1996 Launching of Glucocard

1998 Blood glucose biosensor launch by LifeScan FastTake

1998 Roche Diagnostics by Merger of Roche and Boehringer mannheim

2000-Current Quantum dots, nanoparticles, nanowires, nanotubes, etc



1. LINEARITY Linearity of the sensor should be high

forthe detection of high substrate

concentration.

2. SENSITIVITY Value of the electrode response per

substrate concentration.

3. SELECTIVITY Chemicals Interference must be

minimised for obtaining the correct

result.

4.RESPONSE TIME Time necessary for having 95%

of the response.

Basic Characteristics of a

Biosensor


Biomolecules

11

Peptides, polypeptides (chain of

amino acids)

Proteins

E.g. enzymes, antibodies

Nucleic acids

E.g. ribonucleic acid (uracil

not thymine),

deoxyribonucleic acid

Polysaccharides

E.g. cellulose

Lignin (plant cells)

…

http://upload.wikimedia.org/wikipedia/commons/c/c1/Protein-primary-structure.png

http://upload.wikimedia.org/wikipedia/en/3/3a/Piwi-siRNA-basepairing.png

http://upload.wikimedia.org/wikipedia/commons/6/68/Cellulose-2D-skeletal.svg

Key-Lock Mechanism – Selectivity

12

Enzyme to catalyse reaction

Antibody to neutralise bacteria and virus

http://en.wikipedia.org/wiki/Image:Induced_fit_diagram.svg

http://upload.wikimedia.org/wikipedia/commons/2/2d/Antibody.svg

Electrochemical biosensors

13

Blood glucose sensor

Diabetes common in

elderly people/obese

Millions sold since

1993

Disposable strips

Sold by Abbott et al.

http://upload.wikimedia.org/wikipedia/commons/e/e9/Glucose-2D-skeletal.png

http://en.wikipedia.org/wiki/Image:Suckale08_fig3_glucose_insulin_day.jpg

Electrochemical biosensors

14

Based on electrochemistry

Use enzymatic redox reaction

Plastic strips with polymers

and enzyme

Millions sold since 1993

2 electrode cell

http://upload.wikimedia.org/wikipedia/commons/3/38/Glucose_meters.jpg

http://upload.wikimedia.org/wikipedia/commons/e/e9/Glucose-2D-skeletal.png

Optical biosensors – I

15

Fluorescent dyes

Optical biosensors – Labelled antibodies

16

Inject antigen into host

(chicken, duck, mouse, rat,

goat, horse, sheep ..) and

obtain anti-body

Large molecule 150kDa

Label anti-body (e.g. anti-

goat) with fluorescent dye

Used for biochemical assays

Like ELISA

http://upload.wikimedia.org/wikipedia/commons/2/2d/Antibody.svg

http://upload.wikimedia.org/wikipedia/commons/a/a9/Antibody_IgG2.png

Competitive ELISA

17

Enzyme-linked immuno

sorbent array

Many welled micro-titer

plate

http://en.wikipedia.org/wiki/Image:Microtiter_plate.JPG

http://en.wikipedia.org/wiki/Image:Primary-Secondaryantibody.svg

Optical fibre biosensors - II

18

Surface plasmon resonance

sensor

Receptors as biosensors

19

7TM receptor/GPCR

Target protein

Measure conformal

change or cell pathway

http://en.wikipedia.org/wiki/Image:GPCR_mechanism.png

(Olfactory receptor (OR) membrane

proteins coupled with carbon

nanotubes)

Olfactory receptor biosensor

20

http://medgadget.com/2011/08/the-smell-of-success-researchers-embed-olfactory-receptors-onto-nanotubes.html



Cell based biosensors

21

Cytometers for

Red blood cells

Platelets

White blood cells

Bacteria

SAW resonators

Cantilever beams

…

1 ul blood = 5M erythrocytes, 500k thrombocytes, 10k leukocytes

http://upload.wikimedia.org/wikipedia/commons/2/24/Red_White_Blood_cells.jpg

Biosensor

Analyte

Sample handling/ preparation

Detection

Signal

Analysis

Response

Biosensor Components

1. The Analyte (What do you want to detect)

Molecule - Protein, toxin, peptide, vitamin, sugar,

metal ion

2. Sample handling (How to deliver the analyte to the sensitive region?)

(Micro) fluidics - Concentration increase/decrease),

Filtration/selection

Biosensor

4. Signal

(How do you know there was a detection)

3. Detection/Recognition

(How do you specifically recognize the analyte?)

Biosensor


Example of biosensors

Pregnancy test

Detects the hCG protein in urine.

Glucose monitoring device (for diabetes patients)

Monitors the glucose level in the blood.

Example of biosensors

Infectous disease biosensor

from RBS

Old time coal miners’ biosensor

electrode

substrate product

Enzyme

Apply voltage Measure current proportional

to concentration of substrate

Principle of Electrochemical Biosensors

E, V

time

E-t waveform

potentiostat

Electrochemical cell

counter

working electrode

N2

inlet

Protein film

reference

insulator electrode

material

Equipment for developing electrochemical biosensors

Cyclic

voltammetry


-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

-0.8-0.6-0.4-0.200.20.40.60.8

I ,

A

E, V vs SCE

Cyclic voltammogram (CV) at 100 mV s-1

and 25 oC of Mycobacterium

Tuberculosis KatG catalase-peroxidase in a thin film of

dimyristoylphosphatidylcholine on basal plane PG electrode, in anaerobic pH 6.0

buffer.

Oxidation

Of FeII

Reduction

Of FeIII Reversible

Peaks for

Direct electron

transfer

Cyclic Voltammetry

Electrode

enzyme

A lipid-enzyme film

Catalytic enzyme electrochemistry

a basis for biosensor - glucose oxidase

oxidation

Fc mediator

Fc + glucose

+ enzyme

I = f [glucose]

A. Cass, G. Davis, G. D. Francis, H. O. A. Hill, W. J. Aston, I. J. Higgins, E. V. Plotkin, L. D. L.

Scott, A. P. F. Turner, Anal. Chem. 56, 667-671 (1984).

Mediator shuttles

Electrons between

Enzyme and electrode

Scheme 2

Glucose + GO(FAD) + 2 H+ gluconolactone + GO(FADH2) (1)

GO(FADH2) + 2 Fc+ GO(FAD) + 2 Fc + 2 H

+ (4)

Fc Fc+ + 2 e

- (at electrode) (5)

Mechanism for catalytic oxidation of glucose

With Glucose oxidase (GO) and Fc mediator

Signal can also be measured by amperometry:

Hold const. E where oxidation occurs, measure I vs time

Fc = ferrocenecarboxylate

Commercial Glucose Sensors

• Biggest biosensor success story!

• Diabetic patients monitor blood glucose

at home

• First made by Medisense (early 1990s),

now 5 or more commercial test systems

• Rapid analysis from single drop of blood

• Enzyme-electrochemical device on a

slide

Patient Diabetes Management

Insulin secretion by pancreas regulated

by blood glucose, 4.4 to 6.6 mM normal

In diabetes, regulation breaks down

Wide swings of glucose levels

Glucose tests tell patient how much

insulin to administer


• Most sensors use enzyme called glucose oxidase (GO)

• Most sensors are constructed on electrodes, and use a

mediator to carry electrons from enzyme to GO

Fc = mediator, ferrocene, an iron complex

These reactions occur in the sensor:

Fc

Fc+ + e- (measured)

GOR + 2 Fc + --> GOox + 2 Fc

GOox + glucose --> GOR + gluconolactone

Reach and Wilson, Anal. Chem. 64, 381A (1992)

G. Ramsay, Commercial Biosensors, J. Wiley, 1998.

Glucose biosensor test strips (~$0.40-0.80 ea.)

Meter

Read glucose

Dry coating of GO + Fc

Patient adds drop of blood,

then inserts slide into meter

Output:

amperometry I

t

Patient reads glucose level on meter

e’s

electrodes

Research on glucose sensors

Non-invasive biosensors - skin, saliva

Implantable glucose sensors to

accompany artificial pancreas -

feedback control of insulin supply

Record is 3-4 weeks for implantable

sensor in humans


Other biosensors

Cholesterol - based on cholesterol oxidase

Antigen-antibody sensors - toxic substances, pathogenic bacteria

Small molecules and ions in living things: H+, K+, Na+, CO2, H2O2

DNA hybridization and damage

Micro or nanoarrays, optical abs or fluor.


Negative

surfacePolycation soln.,

then wash

+ + + + + + + + + + + +

soln. of negative protein

then wash

+ + + + + + + + + + + +

+ + + + + + + + + + + +

+ + + + + + + + + + + +

Repeat steps for desired

number of layers

Protein

layer

Polycation layersProtein

layer

Polycation soln.,

then wash

Figure 19

Layer by layer

Film construction:

PSS layer

SPAN layer

Enzyme

layer

Detection of hydrogen peroxide Conductive polymers efficiently wire

peroxidase enzymes to graphite

Xin Yu, G. A. Sotzing, F. Papadimitrakopoulos, J. F. Rusling,

Highly Efficient Wiring of Enzymes to Electrodes by Ultrathin

Conductive Polyion Underlayers: Enhanced Catalytic

Response to Hydrogen Peroxide, Anal. Chem., 2003, 75,

4565-4571.

e’s

(sulfonated polyaniline)

Horseradish Peroxidase (HRP)

100nm

50nm

Tapping mode atomic force microscopy (AFM) image

of HRP film

O2

H2O2

PFeIII PFeII PFeII-O2

2e-, 2H+

H2O2 + PFeII•PFeIV=O

active oxidant

+e-

-e-

PFeIII + H2O + O2

H2O2

Possible reduced species in red

Electrochemical Response of Peroxidases

-10

0

10

20

30

40

50

60

-0.8-0.6-0.4-0.200.2

I,A

E, V vs SCE

with SPAN

a

0

0.5

2

4

6

7.5

M H20

2

Catalytic reduction of H2O2 by peroxidase films

Catalytic cycles increase current

FeIII/FeII

reduction

0

0.5

1

0 100 200 300 400

I,

A

t, s

with PAPSA

without PAPSA

Rotating electrode amperometry at 0 V

HRP, 50 nmol H2O2 additions

span

No span

reduction

0

0.2

0.4

0.6

0.8

1

1.2

0 0.1 0.2 0.3 0.4 0.5 0.6

I,

A

[H2O

2], M

PAPSA/HRPPAPSA/Mb

Mb

HRP

Rotating electrode amperometry at 0 V

Sensitivity much higher with conductive polymer (SPAN);

Electrically wires all the protein to electrode

Span/HRP

Span/Mb

Part 2

Immobilisation Techniques


• Enzymes

• Antibodies

• DNA

• Receptors

• Organelles

• Microorganisms

• Animal and plant cells or tissues

Biological Sensing Elements


• Specificity

• Storage

• Operational and environmental stability

• Analyte to be detected, - chemical

compounds, antigens, microbes,

hormones, nucleic acids, or subjective

parameters such as smell and taste.

Choice of Biomaterial


• Thermal stability

• Ability to act in highly acidic, alkaline,

hydrophobic (organic solvent), or oxidizing

environments

• Extremophiles such as the thermophiles,

alkalophiles, and halophiles have gained

importance in the production of enzymes

for use in biosensors and other

applications

Environmental Conditions


Purified enzymes have been most

commonly used in the construction of

biosensors.

The major advantage of using a pure

enzyme is its high analytical specificity.

Disadvantages: High cost, poor stability

Enzymes


• Immobilization of whole cells has been

shown to be a better alternative to

immobilization of purified enzymes

• Avoids the lengthy and expensive

operations of enzyme purification

• Preserves the enzyme in its natural

environment, thus protecting it from

inactivation either during immobilization or

reuse

Whole Cells


• Permeabilized using physical, chemical,

and enzymatic approaches.

• The most common technique is the use of

organic solvent or detergents.

• Removes some of the lipids from the cell

membranes, thus creating minute pores,

allowing the free diffusion of small

molecular weight substrates/products

across the cell membrane

Cell Permeabilisation


• Permeabilization renders the cell

nonviable and also empties it of most of

the small molecular weight cofactors

• Minimises the unwanted side reactions

• Economic source of intracellular enzymes

for simple biosensor applications that do

not require cofactor regeneration or

metabolic respiration, such as glucose

oxidase, amino acid oxidase, and urease


• Immobilized viable cells in the fabrication

of biosensors

• Substrate assimilation capacity

• Respiratory metabolic activity - BOD

• Inhibition of microbial respiration -

Pollutants

• Genetic modification - organic and

pesticide contamination – luciferase

reaction

Immobilised viable cells


Source: http://www.whatsnextnetwork.com/technology/media/cell_adhesion.jpg

Whole Cell Sensors


Whole Cell Sensors

Harness normal genetic processes

May detect dozens of pathogens

Modifiable/customizable

Reports bioavailability

Temperature/pH sensitive

Short shelf-life


• Adsorption

• Entrapment

• Covalent binding

• Crosslinking

• Combination of all these techniques

Immobilisation techniques


• Passive trapping of cells into the pores of

membranes made up of cellulose or other

synthetic materials

• Retain the cells or enzymes in the close

proximity of the transducer surface using

dialysis membrane

• Entrapment in a variety of synthetic or

natural polymeric gels


Polyvinyl alcohol (PVA) is one of the most

widely studied polymers, because it can

form membranes, fibres, and so on.

Enzymes have been immobilized in these

membranes either by entrapment,

covalent binding, crosslinking, freezing

and thawing, γ-irradiation,

photocrosslinking, or entrapment followed

by crosslinking

Poly Vinyl Alcohol


• Membrane discs of PVA-glutaraldehyde

containing free carbonyl groups have been

prepared for the binding of biomaterials

through their free amine groups

• Modified polyvinyl chloride membranes

polyacrylonitrile membranes and albumin-

poly(ethylene glycol) hydrogel

• Albumin-poly(ethylene glycol) hydrogels –

highly biocompatible


• Biospecific reversible immobilization using

lectins or hydrophobic surfaces can also

be used for the introduction of biologic

catalysts into analytical systems

• Recently, novel sol-gel synthetic

techniques have been developed to

immobilize biologically active molecules in

stable, optically transparent, porous silica

glass matrix


• Nonviable cell preparations have been

immobilized in radiation polymerized

acrylamide.

• The major advantage of γ-ray

polymerization against chemical

polymerization is that the polymerization

can be carried out even under frozen

conditions.


Biosensor Based on Immobilized Indicator Cells

A B-lymphocyte cell line was encapsulated in a collagen gel matrix (Banerjee et al., 2007).

This assay measures alkaline phosphatase or lactate dehydrogenase released by cells infected with pathogens or exposed to different toxins.

The system was tested using different strains of Listeria, listeriolysin O, and enterotoxins from Bacillus species.

Banerjee et al., Laboratory Investigation

(2007) 1-11


The cryo-SEM images of pathogen- or toxin-induced damage of

Ped-2E9 cells in collagen gel matrix.

FROM:

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a

biosensor for rapid detection of pathogens and toxins (2007)

Pratik Banerjee, Dominik Lenz, Joseph Paul Robinson, Jenna L Rickus and Arun K Bhunia

Laboratory Investigation (2007) 1-11


Cell Viability – acridine orange


73

Neuron Based Biosensors

Definition: a biosensor that uses living neural cells to detect substance of interest

Why neuron based biosensors?

Key advantage: a single neuron-based sensor can potentially detect a vast number of chemical and biological agents

A healthy neuron generates voltage pulses (“action potentals”) spontaneously on the membrane of the axon.

Changes in environment (presence of chemicals or biological agents) modulate the neuron’s electrical activity.

Neuron exhibits a unique electrical response to particular agents

Image source: http://www.cic-caracas.org/departments/science/Topic11.php

Neuron Based Biosensor


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Review: Detection

Favored method of detection is Microelectrode Arrays (MEAs).

Electrodes fabricated on surface of device

Monitor signal externally; doesn’t damage cells

Neural signals typically in the range of 100s of uVpp

Many working neuron-based sensors utilize MEAs

Much research focused on improving control of neural growth on MEAs. Photo Source: Nam, et al., “Gold-coated microelectrode

array with thiol linked self-assembled monolayers for

engineering neuronal cultures.” IEEE Transactions on

Biomedical Engineering, 51:1 (2004) 158-165.


75

Analysis of Neural Response

MEA recordings of neural

activity

a) Spontaneous activity

b) Cyclothiazide

c) MK-801

d) NBQX

Source: Chiappalone et a. “Networks of neurons coupled to microelectrode

arrays: a neuronal sensory system for pharmacological applications”

Biosensor and Bioelectronics, 18:5-6 (2003), 627-634

Time domain analysis Characterize response for various

substances • Amplitude

• Duration of burst

• Time interval between bursts


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Analysis of Neural Response

Neural response to ethanol

Neural response to hydrogen peroxide

Source: Prasad et al. “Neurons as sensors: individual and cascaded

chemical sensing” Biosensor and Bioelectronics, 19:12 (2004), 1599-1610

Frequency domain analysis

Example: Prasad et al. 2004: Examine and

characterize frequency components of neural

response for particular substances


77

Challenges

Controlling interaction of living neuron to device.

Ideal of 1:1 association of neurons to electrodes is

difficult to achieve

Affects signal-to-noise ratio

Affects reproducibility and repeatability of response

Long term maintenance of cells in vitro

Stability of device (corrosion, biofouling, etc)


78

Review: Cell Patterning Techniques

Physical Immobilization

Topographical Patterning

Chemical Patterning

Dielectrophoresis

Source: James, et al., 2004.

Source: Craighead, et al., 1998.

Source: Tooker, et al., 2004.

Source: Prasad, et al., 2003.

Goal is to enhance detectibility of action potentials by patterning neurons over electrodes


79

Cell Patterning Using SAMs SAMs form a single layer of molecules on a

substrate.

Advantages: Creates a biocompatible membrane like

microenvironment

• Supporting structure for growth

• Directs growth

Relatively easy to create

Long term stability

Customizable

Many Types of SAMs

Recent research has focused on using thiols on gold substrates

Self Assembled Monolayers


80

Thiol-based SAMs

Structure:

Alkane chain, typically with

10-20 methylene units

Head group with a strong

preferential adsorption to

the substrate used. Eg:

Thiol (-SH) head groups

and Au(111) substrates

Tail group gives the SAM

its functionality

Source: “Self Assembled Monolayers”

http://www.ifm.liu.se/applphys/ftir/sams.html


81

Thiols on Au(111)

Thiol head group bonds

to the threefold hollow

site on gold surface.

Van der Waals forces

between alkane chain

causes them to lie at

30 degree angle Source: “Self Assembled Monolayers”

http://www.ifm.liu.se/applphys/ftir/sams.html

Commonly used SAMs:

MUA: 11-mercaptoundecanoic acid

11-AUT: 11-amino-1-undecanethiol


82

Nam et al. 2004

Contribution: Coated microelectrode arrays with gold in order to use alkanethiol-based SAM techniques

Techninque:

Coat MEAs with 50-80A of gold

Immerse in MUA solution for 2 hours to create SAM

Expose SAM to other compounds to produce layer of NHS esters

Use uCP to apply poly-D-lysine. Stable PDL layer created by covalent linking to SAM layer

Unstamped areas covered with chemical that inhibits cell growth

Source: Nam, et al., “Gold-coated microelectrode array

with thiol linked self-assembled monolayers for engineering

neuronal cultures.” IEEE Transactions on Biomedical

Engineering, 51:1 (2004) 158-165.


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Nam et al. 2004

Results: Demonstrated cell viability on PDL

linked gold surface

Good resolution stamped 100 x 100um grid pattern of 10um line width

Cells complied to pattern for > 2 weeks

Recording of spontaneous neural activity to verify cell activity.

Enhanced amplitudes up to 500uVpp (100-200uVpp typical)

Gold MEAs were not reusable Source: Nam, et al., “Gold-coated microelectrode array

with thiol linked self-assembled monolayers for engineering

neuronal cultures.” IEEE Transactions on Biomedical

Engineering, 51:1 (2004) 158-165.


84

Nam et al. 2006

Updated process different SAM 3-glycidoxypropyl

trimethoxysilane (3-GPS)

uCP for protein pattern stamping

Results Neurons complied to patterns for 2-3

weeks

Spontaneous neural activity recorded:

• Note SAM increased impedance by factor of 2-3

• Mean SNR of 6.5 at 2 weeks

• mean amplitude of extracellular spikes was 25uVpp at 7 DIV and 50uVpp at 20-24 DIV.

• Background noise 2.9uVpp

400ms

Source: Nam et al. “Epoxy-silane linking of biomolecules is simple

and effective for patterning neuronal cultures.” Biosensors and

Bioelectronics 22 (2006) 589–597


85

Palyvoda et al. 2007

Technique:

Create gold electrodes

Immerse in 11-AUT solution to create SAM

Studied effect of pad size on neural guidance

Contribution: used SAM to support and guide neural growth directly

No intermediate protien layer, e.g. polylysine (which is difficult to pattern, nonphysiological, toxic under some conditions)

Image of neurons on 50x50um SAM coated gold electrode

Source: Palyvoda, et. al., “Culturing neuron cells on

electrode with self-assembly monolayer”

Biosensor and Bioelectronics, 22 (2007) 2346-2350.


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Palyvoda et al. 2007

Results 50x50um pad size comes

close to single neuron immobilization, with error.

Source: Palyvoda, et. al., “Culturing neuron cells on

electrode with self-assembly monolayer”

Biosensor and Bioelectronics, 22 (2007) 2346-2350.


• Example Applications

• Cell Based Biosensors

Applications


Potential Applications

• Clinical diagnostics

• Food and agricultural processes

• Environmental (air, soil, and water) monitoring

• Detection of warfare agents.

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Food Analysis Study of biomolecules and their interaction Drug Development Crime detection Medical diagnosis (both clinical and laboratory use)

Environmental field monitoring Quality control Industrial Process Control Detection systems for biological warfare agents Manufacturing of pharmaceuticals and replacement organs

Application of Biosensors


Cell based biosensors

90

Cytometers for

Red blood cells

Platelets

White blood cells

Bacteria

SAW resonators

Cantilever beams

…

http://upload.wikimedia.org/wikipedia/commons/2/24/Red_White_Blood_cells.jpg

Quorum Sensing

Secretion of Signalling molecules that are

autoinducers

Other cells have receptors that bind to the

molecule

The result is gene transcription is mediated

The more cells – the more autoinducer –

more gene transcription – up regulation of

genes


Quorum sensing

Credit: Bonnie Bassler, et al., Princeton U. 92

Quorum Sensing Circuit

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Microprocessor

Chemical and

Biochemical

Processes

Cell-based Biosensor

Electro-

mechanical

Processes

Synthetic biology?

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Band dector (of chemical concentrations)

Credit: S. Basu, et al., Princeton U.

95

Credit: M.L. Simpson, M. L., et al. , Oak Ridge National Lab

Compounds Detected

• Napthalene

• TCE

• Tolulene

• Benzene

• Xylene

Sensitivity: <50 ppb

Cells glow when detect toxins. Luminescence is detected by an integrated photosensor circuit.

Cells

Photodetector Chip

Biochip for toxin detection

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97

Ca++

Analytes

Surface

Immunoglobulin

Signaling cascade

Aequorin

Data analysis

Cell-Based

Biosensor

Krishna Persaud, SCEAS, The University of Manchester, UK

Current Biosensor Technologies

• Bioreporters

o using cells or other organisms to detect relevant stimuli utilizes inherently rapid response times of cellular signaling easy to use reporting system needs to be established in advance organisms need to be maintained in their testing environment

Bomb-Sniffing Plants Colorado State University

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Phenol Detection

DmpR (Dimethyl phenol regulatory protein),

an NtrC-like regulatory protein for the

phenol degradation of Pseudomonas sp.

strain CF600

Strategy - modifying the phenol detection

capacity of DmpR by using mutagenic

PCR


Schematic illustration of sensing principle of whole cell biosensor (pRLuc42R) to detect phenol at

glance.

Gupta S, Saxena M, Saini N, Mahmooduzzafar , et al. (2012) An Effective Strategy for a Whole-Cell Biosensor Based on Putative

Effector Interaction Site of the Regulatory DmpR Protein. PLoS ONE 7(8): e43527. doi:10.1371/journal.pone.0043527

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043527


Linearity curve is presented as phenol concentration v/s NL value of luminescence.

Gupta S, Saxena M, Saini N, Mahmooduzzafar , et al. (2012) An Effective Strategy for a Whole-Cell Biosensor Based on Putative

Effector Interaction Site of the Regulatory DmpR Protein. PLoS ONE 7(8): e43527. doi:10.1371/journal.pone.0043527



• Many types of biosensors

• Many different transduction principles

• Future is bright

• Cell Based Biosensors – Can be widely used in environmental testing

• Genetically modified organisms can be tailored to detect specific analytes

• Both DEAD Cells as well as LIVE cells can be used as viable biosensors – dependent on the application

Conclusions


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