Assoc. Prof. Dr. smail H.

Boyacı
Hacettepe University
Departmet of Food Engineering
Ankara, Turkey
Analytical chemistry is the analysis of
material samples to gain an
understanding of their chemical
composition, structure and function.
 Spectroscopic Analysis

Electrochemical Analysis

Mass Analysis

Thermal Analysis

Separation Science

Data Acquisition and Analysis

Hybrid Techniques
 A biosensor is an analytical device which converts a
biological response into an electrical signal.

The biocatalyst (a) converts the substrate to product. This reaction is determined by the transducer (b) which converts it
to an electrical signal. The output from the transducer is amplified (c), processed (d) and displayed (e).
(http://www.lsbu.ac.uk/biology/enztech/biosensors.html)

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 ME ASURE M EN T
elect ric o u tpu t sig n al

e le c tr o de s: de te c tio ns:
tra n sisto rs (FE T) TR A N S D U C E R e le c tro c h e m ic a l
o p tic a l fib e rs
p h o to d io d e s o p tic a l
th e rm isto rs ph ysi co c he m ic al th e rm a l
sig nal
p ie zo d e v ic e s m a ss se n s itiv e
sen sin g l a yer

b io m o le c u la r re c o gn itio n

TA R G E T A N A L Y TE
c o m p le x m e d iu m

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Biosensors are;
important tools in food safety, diagnostics,
medical monitors, and detection systems for
biological warfare agents.
important devices offering analytical simplicity
both in and outside the analytical laboratory.
selective, rapid and sensitive instruments for
determination of chemical and biochemical
targets
7.3 Billion $ in 2003
10.2 Billion $ in
2007 with a growth
rate of about 10.4%

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 !
Types of Biological Recognition Name of the BIOSENSOR
Elements
Enzymes Enzyme electrode

Proteins

Antibodies Immunosensor

DNA DNA sensor

Organelles

Microbial cells Microbial sensor

Plant and animal tissues

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 !
Types of Transducers Measured Property
Electrochemical Potentiometric
Amperometric
Voltametric

Electrical Surface conductivity

Electrolyte conductivity
Optical Fluoresence
Adsorption
Reflection

Mass sensitive Rezonans frequency of piezocrytals

Thermal Heat of reaction
Heat of adsorpsion

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LINEARITY: Maximum linear value of the sensor
calibration curve. Linearity of the sensor must be high for
the detection of high substrate concentration.
SENSITIVITY: The value of the electrode response per
substrate concentration.
SELECTIVITY: Interference of chemicals must be
minimised for obtaining the correct result.
RESPONSE TIME: The necessary time for having 95% of
the response.
&
Professor Leland C Clark Jnr = the father of the biosensor

In 1956, Clark published his definitive paper on

the oxygen electrode.
In 1962, he described "how to make
electrochemical sensors more intelligent" by
adding "enzyme transducers as membrane
enclosed sandwiches”.

In 1975, YSI (Yellow Spring

Instruments Co., Ohio, USA )
produced the first of many
biosensor-based laboratory
analysers to be built by
1918–2005 companies around the world.
The picture was received from the artical of Prof. W. R. Heineman and W. B. Jensen
in Biosensor and Bioelectronics (2006, 21, 1403-1404)
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Glucose Measurement
Sucrose Measurement
Lactose Measurement
L-Lactate Measurement
Galactose Measurement
L-Glutamate Measurement
Choline Measurement
Ethanol Measurement
Methanol Measurement
Hydrogen Peroxide Measurement
Starch Measurement
&
Guilbault and Montalvo were the first to detail a potentiometric enzyme
electrode, a urea sensor based on urease immobilised at an ammonium-
selective liquid membrane electrode.
Lubbers and Opitz in 1975 to describe a fibre-optic sensor with
immobilised indicator to measure carbon dioxide or oxygen.
They extended the concept to make an optical biosensor for alcohol by
immobilising alcohol oxidase on the end of a fibre-optic oxygen sensor.
Route in 1975, bacteria could be harnessed as the biological element in
microbial electrodes for the measurement of alcohol.
In 1976, Clemens et al. incorporated an electrochemical glucose
biosensor in a bedside artificial pancreas and this was later marketed by
Miles (Elkhart) as the Biostator.
In the same year, La Roche (Switzerland) introduced the Lactate
Analyser LA 640 in which the soluble mediator, hexacyanoferrate, was
used to shuttle electrons from lactate dehydrogenase to an electrode.
In 1982, in vivo application of glucose biosensors was reported by
Shichiri et al. who described the first needle-type enzyme electrode for
subcutaneous implantation.
Ref: http://www.cranfield.ac.uk/biotech/sensors/history.htm
 &
In 1987, a pen-sized meter for home blood-glucose
monitoring formed the basis for the screen-printed
enzyme electrodes launched by MediSense (Cambridge,
USA).
The electronics have since been redesigned into popular
credit-card and computer-mouse style formats, and
MediSense's sales showed exponential growth, reaching
$175 million per annum by 1996, when they were
purchased by Abbott

The idea of building direct

immunosensors by fixing antibodies to a
piezoelectric or potentiometric transducer
had been explored since the early
seventies, but it was a paper by Liedberg
et al. The BIAcore (Pharmacia, Sweden)
produced commercial model of this
technology in 1990.

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&
Boehringer Mannheim,
Bayer and LifeScan now
have competing mediated
biosensors and the
combined sales of the
four companies dominate
the world market for
biosensors and are
rapidly displacing
conventional reflectance
photometry technology for
home diagnostics.

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Preparation of amperometric glucose electrode

Glucose oxidase
-D-Glucose + O2 Gluconic acid + H2O2

Pt-Ag/AgCl electrode
H2O2 O2 + 2H+ + 2e-
700 mV
 ' ' (

Enzeyme

Enzeyme

Enzeyme

Mutlu M., S. Mutlu, B. Alp, . H. Boyacı and E. Pi kin, Preparation of a Single Layer Enzyme Electrode by Plasma Polymerization
Technique, in "Plasma Processing of Polymers", Ricardo D'Agustino (ed.), NATO ASI Series, Kluwer Academic Publishers, Dordrecht,
477-485, (1997).

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' ' (

ELECTRODE DATA ACQUISITION

AND IMAGING

REACTOR nA

mM

10.0

VOLTAGE SOURCE
AND WATTMETER

A/D & D/A

CONVERTER PRINTER

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Lactate oxidase
Lactate + O2 Gluconic acid + H2O2

Invertase
Sucrose +H2O -D-Glucose + -D-Fructose
Mutarotase
-D-Glucose -D-Glucose
Glucose oxidase
-D-Glucose + O2 Gluconic acid + H2O2

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A characteristic current vs. time curves of amperometric measurement

) ' *
Infectious diseases account for nearly 40% of the total 50 million
annual estimated deaths worldwide.
Microbial diseases are the major cause of death in many developing
countries.
Drinking water is an essential part of the human diet, and
contamination of municipal and other supplied water with pathogenic
microorganisms constitutes a serious threat to public health.
Escherichia coli, a typical inhabitant of the human intestinal tract, is one
of the most dangerous food-borne and water-borne pathogens that can
also be a causative agent of intestinal and extra-intestinal infections.
Total coliforms, fecal coliforms, and E. coli are used as indicators of
fecal contamination of water supplies and recreational waters. Among
these, E. coli is usually regarded as the most reliable, because its
presence directly relates to fecal contamination.
Sensitive and rapid methods with low detection limits and high
accuracy to prevent false negatives and positives are needed for
detecting and providing early warning of fecal water contamination, for
process control, and for monitoring water quality in distribution
networks.
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*

Amperometric immunosensor (direct or

sandwich)
Optic immunosensor (direct or sandwich)
Piezoelectric biosensor immunosensor
Surface plasmon resonance immunosensor
DNA sensor
 ' , -

ELECTRODE DATA ACQUISITION

AND IMAGING

REACTOR nA

mM

10.0

VOLTAGE SOURCE
AND WATTMETER

A/D & D/A

CONVERTER PRINTER

ß-galactosidase
PAPG D-Galactose + PAP

PAPG
ß-glucorinadase
PAP
' , -

ELECTRODE DATA ACQUISITION

AND IMAGING

REACTOR nA

mM

10.0

VOLTAGE SOURCE
AND WATTMETER
E E
E E E E A/D & D/A
CONVERTER PRINTER

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Fluorescence
spectrophotometer

ß-galactosidase
MUG D-Galactose + MU
Emission
MUG at 440 nm
ß-glucorinadase

Excitation MU
at 360 nm

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Flow channel
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The response (RU) to binding of E. coli and regeneration. Injection of

A) E. coli solution (9.0×101 cfu ml-1); B) PBS; C) %1 SDS solution;
D) E. coli solution(1.8×103 cfu ml-1);
E) E. coli solution(1.8×105 cfu ml-1).
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Immunosensors using piezoelectric crystal
detectors ($ antigen determination)

The piezoelectric immunosensor is thought to

be one of the most sensitive analytical
instruments developed to date, being capable
of detecting antigens in the picogram range.
Moreover, this type of device is believed to
have the potential to detect antigens in the
gas phase as well as in the liquid phase.

∆F = frequency change in oscillating crystal in Hz, F = frequency of

piezoelectric quartz crystal in MHz, ∆M = mass of deposited film in g,
and A = area of electrode surface in cm2.

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Frequency,
MHz 10 -
Sample

9-
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Eschrichia coli Time, s

Antibody
Protein
Gold Electrode
Crystal substrate
Gold Electrode

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http://www.ornl.gov/info/ornlreview/rev29_3/text/biosens.htm
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Optic Fiber Probe

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Key words:(Biosensor and Escherichia coli)

All document types; Language=All languages;
Databases=SCI-EXPANDED, Timespan=1945-
2006
http://portal.isiknowledge.com/

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/ ' '
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Enumeration of bacteria where the sample is
found not in laboratory.
Determination of 1 bacteria in 100 mL sample
volume (it is official requirement for drinking
water).
Real time determination/enumeration of E. coli.
Low analyze cost.
Usability with common people (not requirement
of over qualified person).
/ ' '
, *

NO

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Development of a method carried out in
volume not on surface.
Using nanotechnology such as quantum dot
nanoparticles, nanowires or nanotubes.
Development of artificial antibodies such as
aptamer or peptides selected by phage
display method.
Development of Lab-on-a-chip for detection
of antigen.

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 - - - '
Aim of the development of Lab-on-a-chip
system is performing all stages on a chip.
Lab-on-a system consists of;
Microchanells and micropumps
Sample pretreatment units
Reservour for probes, substrates, buffers and
others,
Detectors,
Sampling more then one analyte or sample at the
same time

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http://www.nsbri.org/NewsPublicOut/Photos/lab-on-a-chip.jpg
http://www.chem.agilent.com/cag/feature/8-98/feature_graphics/chip.jpg
http://www.nyu.edu/classes/ytchang/book/n010/ChipCartRitn.jpg
http://www.umich.edu/news/MT/NewsE/archive_pics/AUG05Flugeno.jpg

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Agilent Technologies
This technology utilizes a network of channels
and wells that are etched onto glass or
polymer chips to build mini-labs.
Pressure or electrokinetic forces move pico liter
volumes in finely controlled manner through
the channels.
Lab-on-a-Chip enables sample handling,
mixing, dilution, electrophoresis and
chromatographic separation, staining and
detection on single integrated systems.
The main advantages of Lab-on-a-Chip are
ease-of-use, speed of analysis, low sample
and reagent consumption and high
reproducibility due to standardization and
automation.

The Agilent 2100 bioanalyzer is offering solutions for the analysis of DNA, RNA,
proteins and cells.

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NanoChip® Molecular
Biology Workstation
Nanogen is a technology company
specializing in integrating
microelectronics with molecular
biology - DNA arrays.
DNA arrays make it possible to
quickly determine characteristics
of an unknown DNA sample.
Applications range from studying
regulation of gene expression to
detection of genetic diseases.

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Dr. Marc Madou

An automated microfluidic compact disc

(CD) based system

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) 8 , 8*
Dudak F.C., .H. Boyacı, Development of an Immunosensor Based On Surface Plasmon
Resonance for Enumeration of Escherichia coli in Water Samples, Submitted To Food
Research International, (2006).
F.C. Dudak, .H. Boyacı, A. Jurkevica, M. Hossain, Z.P. Aguilar, H.B. Halsall, C.J. Seliskar,
W.R. Heineman. Determination of Escherichia coli Using Antibody-coated Paramagnetic
Beads with Fluorescence Detection, SUBMITTED TO Anal. Bioanal. Chem., (2006).
Boyacı .H., Z. P. Aguilar, M. Hossain, H. B. Halsall, C. J. Seliskar and W. R. Heineman,
Amperometric Enumeration of Escherichia coli using Antibody-coated Paramagnetic Beads,
Analytical and Bioanalytical Chemistry 382: 1234–1241 (2005).
Boyacı . H., U.Ö. . eker and M. Mutlu, “Determination of -glucan content of cereals with
amperometric glucose electrode”, European Food Research and Technology, Vol 215, 538-
541, (2002).
Boyacı . H. and M. Mutlu, “Development of A Biosensor for Simultaneous Determination of
Glucose, Sucrose and Lactose in A Mixture”, Nahrung, Vol 46, No 3, 174-178, (2002).
Biederman, H., . H. Boyacı, P. Bilkova, D. Slavinska, S. Mutlu, J. Zemek, M. Trchova, J.
Klimovic, M. Mutlu, “Characterization of glow-discharge-treated cellulose acetate membrane
surfaces for single-layer enzyme electrode studies”, Journal of Applied Polymer Science, Vol.
81, 1341-1352, (2001).
Mutlu M., S. Mutlu, B. Alp, . H. Boyacı and E. Pi kin, Preparation of a Single Layer Enzyme
Electrode by Plasma Polymerization Technique, in "Plasma Processing of Polymers", Ricardo
D'Agustino (ed.), NATO ASI Series, Kluwer Academic Publishers, Dordrecht, 477-485, (1997).
Mutlu M., S. Mutlu, . H. Boyacı, B. Alp and E. Pi kin, “High Linearity Glucose Enzyme
Electrodes for Food Industries by Plasma Polymerization Technique”, in Polymers in Sensors:
Theory and Practice, N. Akmal and A.M. Usmani, eds., American Chemical Society (ACS)
Symposium Series, 690, 57-65, (1998).

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) 8 0*
“Selection of Peptide Ligand That Bind To Staphylococcal
Enterotoxin B (Seb) and biosensor application of selected
peptide”, Supported by State Planning Organization Project,
No: 2006 K 120 640, Ankara, 2006 (continue).
“Detection of Escherichia coli By Using Antibodies
Conjugated with Semiconductor Nanoparticles”, Supported
by Turkish Scientific and Technical Research Council,
TUBITAK, Project No: 104 T 409, Ankara, 2004 (continue).
“Preparation of aflatoxin sensor using paramagnetic micro
particles”, Supported by Hacettepe University, Scientific
Research Center Project, Ankara, 2004, (continue).

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: 0 ,'

Dr. Ozcan Ceylan,

Dr. Esra Acar
Deniz Ba
Ceyda Dudak
Esen Sokulu
Hatice Akyurt

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http://yunus.hacettepe.edu.tr/~ihb

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