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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
Krishna Persaud, SCEAS, The University of Manchester, UK 3
Thermoreceptors
From Runaway, by Michael Crichton.
Published by Tri-Star Pictures in 1985
Sniffing Robot
4
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
History of Biosensors
8 Krishna Persaud, SCEAS, The University of Manchester, UK
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
History of Biosensors
9 Krishna Persaud, SCEAS, The University of Manchester, UK
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
10 Krishna Persaud, SCEAS, The University of Manchester, UK
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)
…
Key-Lock Mechanism – Selectivity
12
Enzyme to catalyse reaction
Antibody to neutralise bacteria and virus
Electrochemical biosensors
13
Blood glucose sensor
Diabetes common in
elderly people/obese
Millions sold since
1993
Disposable strips
Sold by Abbott et al.
Electrochemical biosensors
14
Based on electrochemistry
Use enzymatic redox reaction
Plastic strips with polymers
and enzyme
Millions sold since 1993
2 electrode cell
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
Competitive ELISA
17
Enzyme-linked immuno
sorbent array
Many welled micro-titer
plate
Optical fibre biosensors - II
18
Surface plasmon resonance
sensor
Receptors as biosensors
19
7TM receptor/GPCR
Target protein
Measure conformal
change or cell pathway
(Olfactory receptor (OR) membrane
proteins coupled with carbon
nanotubes)
Olfactory receptor biosensor
20
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
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
24 Krishna Persaud, SCEAS, The University of Manchester, UK
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
28 Krishna Persaud, SCEAS, The University of Manchester, UK
-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
34 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
37 Krishna Persaud, SCEAS, The University of Manchester, UK
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.
38 Krishna Persaud, SCEAS, The University of Manchester, UK
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
Krishna Persaud, SCEAS, The University of Manchester, UK 46
47 Krishna Persaud, SCEAS, The University of Manchester, UK
• Enzymes
• Antibodies
• DNA
• Receptors
• Organelles
• Microorganisms
• Animal and plant cells or tissues
Biological Sensing Elements
48 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
49 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
50 Krishna Persaud, SCEAS, The University of Manchester, UK
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
51 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
52 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
53 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
54 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
55 Krishna Persaud, SCEAS, The University of Manchester, UK
Source: http://www.whatsnextnetwork.com/technology/media/cell_adhesion.jpg
Whole Cell Sensors
56 Krishna Persaud, SCEAS, The University of Manchester, UK
Whole Cell Sensors
Harness normal genetic processes
May detect dozens of pathogens
Modifiable/customizable
Reports bioavailability
Temperature/pH sensitive
Short shelf-life
57 Krishna Persaud, SCEAS, The University of Manchester, UK
• Adsorption
• Entrapment
• Covalent binding
• Crosslinking
• Combination of all these techniques
Immobilisation techniques
58 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
59 Krishna Persaud, SCEAS, The University of Manchester, UK
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
60 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
61 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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
62 Krishna Persaud, SCEAS, The University of Manchester, UK
• 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.
63 Krishna Persaud, SCEAS, The University of Manchester, UK
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
64 Krishna Persaud, SCEAS, The University of Manchester, UK
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
65 Krishna Persaud, SCEAS, The University of Manchester, UK
Cell Viability – acridine orange
66 Krishna Persaud, SCEAS, The University of Manchester, UK
67 Krishna Persaud, SCEAS, The University of Manchester, UK
68 Krishna Persaud, SCEAS, The University of Manchester, UK
69 Krishna Persaud, SCEAS, The University of Manchester, UK
70 Krishna Persaud, SCEAS, The University of Manchester, UK
71 Krishna Persaud, SCEAS, The University of Manchester, UK
72 Krishna Persaud, SCEAS, The University of Manchester, UK
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
73 Krishna Persaud, SCEAS, The University of Manchester, UK
74
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.
74 Krishna Persaud, SCEAS, The University of Manchester, UK
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
75 Krishna Persaud, SCEAS, The University of Manchester, UK
76
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
76 Krishna Persaud, SCEAS, The University of Manchester, UK
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)
77 Krishna Persaud, SCEAS, The University of Manchester, UK
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
78 Krishna Persaud, SCEAS, The University of Manchester, UK
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
79 Krishna Persaud, SCEAS, The University of Manchester, UK
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
80 Krishna Persaud, SCEAS, The University of Manchester, UK
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
81 Krishna Persaud, SCEAS, The University of Manchester, UK
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.
82 Krishna Persaud, SCEAS, The University of Manchester, UK
83
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.
83 Krishna Persaud, SCEAS, The University of Manchester, UK
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
84 Krishna Persaud, SCEAS, The University of Manchester, UK
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.
85 Krishna Persaud, SCEAS, The University of Manchester, UK
86
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.
86 Krishna Persaud, SCEAS, The University of Manchester, UK
• Example Applications
• Cell Based Biosensors
Applications
87 Krishna Persaud, SCEAS, The University of Manchester, UK
Potential Applications
• Clinical diagnostics
• Food and agricultural processes
• Environmental (air, soil, and water) monitoring
• Detection of warfare agents.
88
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
89 Krishna Persaud, SCEAS, The University of Manchester, UK
Cell based biosensors
90
Cytometers for
Red blood cells
Platelets
White blood cells
Bacteria
SAW resonators
Cantilever beams
…
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
91 Krishna Persaud, SCEAS, The University of Manchester, UK
Quorum sensing
Credit: Bonnie Bassler, et al., Princeton U. 92
Quorum Sensing Circuit
93
Microprocessor
Chemical and
Biochemical
Processes
Cell-based Biosensor
Electro-
mechanical
Processes
Synthetic biology?
94
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
96
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
98
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
99 Krishna Persaud, SCEAS, The University of Manchester, UK
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
http://www.plosone.org/article/info: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
102 Krishna Persaud, SCEAS, The University of Manchester, UK
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