types and principle of various commercial biosensors and their application.

1. Presented By: PAVEL ROUT
M.Tech Student of
Dairy
Chemistry Department

2. DEFINITI
ON OF
BIOSENS
ORS.
 A biosensor is a device which reponds
to the presence of a specific analyte (a
chemical substance that needs to be
measured) by producing an electrical
signal proportional to the concentration
of the analyte.
 It is usually constructed from three
components: receptor, transducer and
electronics (amplification and display).

3.  Lenand C. Clarck invented (1962) the Clarck
Oxygen Electrode.
 A pivotal device that allows real time
monitoring of patients blood oxygen level and
made surgery safer and more successful for
millions throughout world
Generations
Lenand C. Clarck
(1918-2005).

4.  Specificity: Via biosensor, it’s possible to measure
specific analytes with great accuracy.
 Speed: Analyte tracers or catalytic products can be
directly and instantaneously measured.
 Simpltcity: Receptor and transducer are integrated
into one single sensor & the measurement of target
analytes without using reagents is possible.
 Continous Monitoring Capability: Biosensor
regenerate and reuse the immobilized biological
recognition component.

6.  A bioreceptor or a biorecognition element, which recognizes the target analyte
and a transducer, which converts the recognition event into a
measurable electrical signal. ( Velusamy et al.
2010)
receptors
Enzymatic
Immunological
Cellular
transducers
Potentiometric
Amperometric
Electrochemical
Optoelectric

7. CLASSIFICATION BY USE OF
PHYSIOLOGICAL PROPERTIES AND
METHODS

8. ELECTROC
HEMICAL
TRANSDUC
ERS
 Amperometry, Potentiometry, Impedance
and Conductivity methods are used in
this type.
 The most commonly used class of
biosensors are electrochemical-based
ones (Chaubey and Malhotra 2002).

9. AMPEROMETRIC TRANSDUCER
 Based on the measurement of a steady state current produced when
a constant potential is applied by a potentiostat.
 This current can be related to an electrochemical species that is
consumed or produced by the biological element…consists of a
working electrode (gold, platinum, glassy carbon, graphite or
carbon), a reference electrode (Ag/AgCl) and an auxiliary electrode,
(carbon or platinum)..
 The biological element can be directly
immobilized on the electrode and is
an enzyme.
 Most common.
 E.g. glucose biosensor, H2O2 biosensor…

10.  Potentiometric biosensors measure potential differences under zero current conditions.
 Antibody-antigen binding induces a small change in the charge of the proteins that can
be potentiometrically detected.
 Ion selective electrodes (ISEs) are based on potentiometric measurements and can be
used for mainly enzyme based biosensors.
The change in pH due to enzyme activity can be monitored
with a pH sensitive ISE. The potential that develops across
an ion selective membrane is measured.
 The first use by Guilbault and Montalvo for urea in 1969.
 More recent potentiometric devices are based on field-effect transistor (FET) devices.
(Mello and Kubota, 2007)

11. CONDUCTOMETRIC BIOSENSOR :
 Measure the change in conductance between
analate and refferance.
 Its sensitivity is lower than other electrochemical
biosensor.

12. OPTICAL TRANSDUCERS
 Optodes (or optrodes) can employ 0n absorbance, fluorescence, phosphorescence,
polarization, and surface plasmon resonance (SPR).
 The development of light emitting diodes (LEDs) allows the integration of these
small and often cheap devices into sensors.
SURFACE PLASMON RESONANCE (SPR):
 SPR occurs when light is internally reflected at the interface of a material with
high
refractive index and a material with low refractive index.
 An oscillating wave developed at this
interface can interact with electron packages
in the conductive layer.
 The plasmon excitation energy is lost from
the reflected light and can be measured with monochromatic light that is reflected

13.  Two waveforms are used for
piezoelectric biosensors.
 One is a Surface Acoustic Wave (SAW) device. The two electrodes are
placed on one side of the crystal and a standing surface wave is created.
 High frequencies of 30-200 MHz give the crystal a very good theoretical
sensitivity, but due to practical difficulties biosensors are mainly based on
Bulk Acoustic Wave (BAW) devices. (Leonard et
al., 2003).

14. THERMO
METRIC
TRANSDU
CERS
 Thermometric biosensors measuring
the change in temperature.
(Cock et al. 2009)
 Thermometric biosensors exploit the
change of heat during absorption or
evolution that occurs during
biochemical reactions.
 Sensitive thermistors are used to
monitor... Enzymatic reactions are
associated with enthalpy changes.
 Measurements can be improved by
co-immobilizing enzymes for signal
amplification or by using high-
protonation enthalpy buffers such as
TRIS. (Giese,
2002)

15. APPLICA
TION

16. COMMERCIAL USE OF BIOSENSOR IN
FOOD INDUSTRY…
 Suitable analalytical method for safety and quality purpose.
 Determine chemical and biological contaminants.
 Also in Analysis of amino acids , glucose, alcohol, flavors ,
sweetener .
 Detection of allergens, toxins, additives and pathogens.
 In food & fermentation process quick, cheap and safe analytical process is
needed.

17.  The presence of D-amino acids in food is associated with a decrease in
protein digestibility, thus affecting the bio-availability of essential amino
acids and seriously impairing the nutritional value of the food.
(Friedman 1999)
 An amperometric and a colorimetric biosensor to detect and quantify
D-amino acids selectively was constructed using DAAO from Rhodotorula
gracilis. For D-alanine concentration the range varies within of 0.2–3 mM
and 0.1–1 mM for the amperometric and colorimetric system, respectively.
(Sacchi et al. 1998)
 A graphite working electrode of a screen-printed strip modified with
Prussian Blue and Nafion layers. The electrode was modified with carbon
nanotubes to enhance the signal magnitude. A fast linear response was
observed for D-alanine in the concentration range from 5 to 200 mM .
. D-Amino acids were detected in fruit juices and milk samples. The
results were in a good agreement with those obtained by capillary
electrophoresis measurements. (Wcislo et al. 2007).

18.  Glucose biosensors are widely applied in the monitoring of fermentation products and in
dairy, wine, beer, and sugar industry. (Mao 2008)
 Measuring of O2 consumption or H2O2 production is performed during the catalytic
reaction using the substrate of interest, e.g., analyte
Glucose could be quantified in the concentration range between 50 μM and 10 mM.
(Zhu et al. 2002)
 An amperometric probe-type glucose sensor with Pt working electrode and an Ag/AgCl
reference one polarized at +650 mV. The electrode was used to determine the glucose
content in real samples. (Alp et al.2000)
 A biosensor electrode consisted of a thin film of ferric hexacyanoferrate (Prussian Blue)
electrodeposited on the glassy carbon electrode with immobilized glucose oxidase on a
Nafion polymer layer.Used for glucose in soluble coffee. (Mattos and Areias
2005)

19.  Using different transducers based on five various Solid Binding Matrices (SBMs). The
glucose biosensors based on the transducer with cholesteryl myristate SBM and ferrocene
mediator were used for determination of glucose in wine sample. (Svorc et al.
1997)
 Screen-printed electrodes to simultaneously detect D-glucose and L-lactate. They
immobilized glucose and lactate oxidase on a carbon working electrode. Ferricyanide ions
(electrochemically oxidized at a low voltage) were chosen as a mediator.
A linear range was found over a range of 1–100 mM (D-glucose) and 1–50 mM (Llactate).
Biosensor was applied after fermentation with lactic acid bacteria and with high-
performance liquid chromatography (HPLC). Method were completed within 5 min (Sato
and Okuma 2006).
 Immobilized enzyme reactor (IMER) and integrated it to a capillary electrophoresis (CE)
microchip.
Glucose was detected above 100 μM range using particles modified with glucose oxidase
packed at the end of the separation channel.
The applied procedure involved the separation of the target analyte by a CE, which is then
coupled to a post-column IMER that produced H2O2, which was finally detected at the surface
of a working electrode. The microchip-CE-IMER was used to quantify glucose in carbonated
beverages. (Blanes et al. 2007)

20. ANALYSIS OF TEA BY USING
BIOSENSOR
 Tea polyphenols are gaining importance due to their strong
antioxidant properties for nutrition and health.
 In this context cftri, mysore successfully developed an enzyme
based amperometric biosensor for the determination of total
polyphenol content in tea infusions.
 Both lab and industry trials were satisfactory
for tea polyphenols detection and tea biosensor
technology.
(Sujith Kumar et al., 2011)

21.  Conventional method- severel steps…time
consuming & laborious…
 Specific antibodies can be produced against
surface antigents of microbs.
 Mostly for confirming the absence of
pathogen.. salmonella & e. coli… causes
bloody diarrhea, renal failure, meningitis.
 Detects within minutes of sampling…

22.  The conductivity of the medium changes if micro organism
metabolize the substrate (e.g. carbohydrate) to intermediate
(e.g. lactic acid). By measuring the conductivity the micro
organism growth can be determined.
(Mello and Kubota, 2002)
 If pathogens are found with on- or near-line biosensors, then
food processors can make decisions more quickly about
applying treatments, minimizing the chance of a contaminated
final product. (Velasco and
Mottram, 2003)
 Very specific antibodies can be produced against surface
antigens of various microorganisms.
In this way, an immunosensor can discriminate between
different organisms.
In combination with different transducers (e.g. piezoelectric
materials or optical fibers) antibodies have been successfully
employed for the detection of microorganisms.
Generally salmonella and Escherichia are detected by this
operation. (Kuhnert et al.,
2000)

23. Flow Injection Potentiometric system has been
developed for simultaneous determination of
ascorbic acid with other parameters.
 This system is based on the reaction of the species with ascorbate oxidase
which is immobilized on alkaline glass beads
using glutaraldehyde. Fall in oxygen
consumption is detected by the electrode.
 Oxygen consumed is proportional to the
ascorbic acid content of the sample.
(Eshkenazi et al., 2000)

24. QUALIT
Y
CONTRO
L OF
MODIFI
ED
ATMOSP
HERE
PACKAG
ES
 Low package O2 also may promote growth of
dangerous pathogens (e.g. Clostridium
botulinum)… quality loss and eventually
product breakdown
 Detection of ethanol provide a sensitive
technique for low-O2 injury
 Commercial ethanol biosensor (with
chromagen) : Alcohol oxidase causes
oxidation of ethanol into acetaldehyde and
H2O2…peroxidase causes oxidation of the
chromagen …causing a colour change
 The biosensor detects ethanol to levels below
the human olfactory threshold (l0µl/l)
ethanol in gas phase at 50°C with a 15 s
exposure. The onset of low O2 injury was
detected in lightly processed lettuce,
cauliflower, broccoli und cabbage modified
atmosphere packages as measured by
accumulation of headspace ethanol.
(Smyth et al., 1996).

25.  Electronic toungue - A nanoparticle films with
embedded sensors in packaging in order to detect
pathogens. The technology will be able to detect and
alert the consumers if the food is contaminated by
triggering a colour change in the packaging.
(Selke, 2008)
 Machine vision technology has a hyperspectral
imaging ability which offer the additional benefits of
sensing the chemical composition of foods. By
employing high throughput chemometrics.
(Headwall photonics,2009)

26. FISH
FRESHNE
SS
AND
MEAT
QUALITY
 Fish freshness has been evaluated chemically and
expressed as K-value
 K-value is calculated from the concentrations of
inosine 5-monophosphate(IMP), inosine and
hypoxanthine (Hx)…with several kinds of
biochemical reagents & using human smell sense.
(Mitsubayashi et al., 2004).
 A four electrode array attached to a knife which can
be inserted into meat to measure the glucose
gradient immediately below the surface.

27. APPLICATI
ON OF
BIOSENSO
RS IN
DAIRY
INDUSTRI
ES
 On line lactose measurement… a multi-enzymatic
amperometric biosensor for lactose in fresh raw milk
 Microbial biosensor based on thick film technology for free
fatty acids…oxygen uptake by respiratory activity of the
immobilized microorganisms.
 having a short response high sensitivity and easy handling.
ONLINE
MEASUREMENT…

28. FOR
QUALIT
Y
CONTRO
L IN
MILK &
MILK
PRODUC
TS
 Urea biosensor is immobolized urease
yielding bacterial cell biomass Bacillus
sphaericus isolated from soil and coupled to
the ammonium ion selectively electrode of a
potentiometric transducer.
 Response time as low as two minutes
 To control the acidity of mozzarella cheese,
biosensor has been used to measure the
lactic acid.
 An electrochemical (flow-through flow-jet)
cell assembled with platinum sensor covered
with the immobilized lactate oxidase
enzyme connected to an amperometer.
 The amount of lactate in the curd is
detected by H2O2 probe. (Rajasekhar et al.,
2005)

29.  Lactose is hydrolyzed by the enzyme β-galactosidase to galactose and glucose.
Galactose can be further oxidized to galacto-hexodialdose:
 Amperometric lactose biosensor by immobilizing galactosidase and galactose oxidase
in Langmuir–Blodgett films of poly 3-hexyl thiophene/ stearic acid. The enzyme
electrodes showed linearity in the range 1–6 g dl ˉ ¹ for lactose and had a shelf life
more than 120 days...used in food and biological fluids.
(Sharma et al. 2004)
 β-galactosidase immobilized into polyelectrolyte membranes with the use of
organic solvents and perfluorosulfonated polymer.
The results of the analysis of milk whey in a flow-injection system that
included lactose biosensor, based on Berlin blue (as a signal
transducer) and polyelectrolyte membranes correlated well with
measurement data obtained by a standard chromatographic
technique. (Lukacheva et al. 2007)
ANALYSIS OF LACTOSE
CONCENTRATION

30.  The CDH(cellobiose dehydrogenase )-lactose sensor was successfully
used to quantify the content of lactose in pasteurized milk, buttermilk,
and low-lactose milk, using the standard addition method.
(Stoica et al.
2006)
 The bioelectrode based on the use of a 3-mercaptopropionic acid self-
assembled monolayer modified gold electrode.
Beta-galactosidase catalyzed the hydrolysis of lactose, and the
produced glucose was catalytically oxidized to gluconic acid and H2O2,
which was reduced in the presence of peroxidase.
The biosensor showed a useful lifetime of 28 days and was applied to
the determination of lactose in milk and other foodstuffs (chocolate,
butter, margarine, yogurt, cheese, and mayonnaise), and the results
obtained were validated using a commercial enzyme test kit.
(Conzuelo et
al. 2010)

31.  The lactate level is an indicator of the fermentative
processes and is related to the freshness, stability, and
storage quality.
Lactate determination by biosensors is typically based on
those reactions.
 An amperometric lactate biosensor based on a conducting
polymer, poly-5,20-50,200terthiophene-30-carboxylic acid
(pTTCA), and multiwalled carbon nanotube (MWNT)
composite present on a gold electrode. LDH and the
oxidized form of nicotinamide adenine dinucleotide (NAD+)
were subsequently immobilized onto the pTTCA/MWNT
composite film. The detection signal was amplified by the
pTTCA/ MWNT assembly with immobilized enzyme.
(Rahman et al. 2009).
 Biosensors were applied in the determination of lactate in
wine and beer, and results were in an agreement with those
obtained by an enzymatic-spectrophotometric assay kit
(Parra et al. 2006).

32.  A glassy carbon electrode modified with laponite/chitosan
hydrogels for LOx immobilization. Ferrocenemethanol was
utilized as an artificial mediator. The biosensor showed a
very short response time lower than 5 s and a detection limit
of 3.8 μM. (Zanini et
al.2011)
 Ballesta-Claver et al. prepared a chemiluminescencebased
one-shot biosensor tested for the analysis of lactate in
yoghurt.
The lactate recognition system was based on LOx and
the transduction system consisted of luminol, peroxidase from
Arthromyces ramosus, all immobilized in a poly-ion complex
membrane on a metallic aluminum electrode.
The performance of biosensor was validated by the
results obtained using an enzymatic reference procedure.
(Ballesta-Claver et al. 2008)

33. COMPANY ANALYTE RANGE STABILITY
Yellow Springs Instruments Glucose, lactate, ethanol 1-45, 0-15, 0-60 300 times
ZFWG, Berlin, Germany Glucose, Uric Acid 0.5-50, 0.1-1.2 >1000 times
Abbott,USA Glucose, Insulin 20-500
Roche, Switzeland Lactate, glucose, cholesterol 0.5-12, 20-600
Elite glucometer lactate 0.8-23
Nova diabetes glucose
Germaine laboratories, USA hemoglobin
Arkay, japan glucose 10-600
Pts, diagonostic, china cholesterol
Nova biomedical, USA glucose 20-600

34. Highly specific
Independent of factors like PH, stirring
Lenear response, tiny and biocompitable
Easy to use, durable
Require only small sample volume
Rapid, accurate, stable and sterilizable.
ADVANTA
GES…

35. Expensive
High maintainance
cost
pH temp parameter
Need supervision
Receptors must be
specific

36. On-site and on-line
monitoring is make
possible for biosensor
within few minutes.
Although Biosensor
concepts are a vast area of
research that continues to
develop rapidly.
It has a promising bright
future in food and
medicinal industry.

37.  Bergann T., Gifley K. and Abel P., 1999.Concentration of lactic acid in carcasses
and fresh meat estimation with an enzymatic-biosensors or measuring system.
Fleischwirtschaff, 79: 84- 487.
 Eshkenazi, Maltz E., Zio B. and J. Rishpon., 2000. Three cascaded enzymes
biosensor to determine lactose concentration in raw milk. J. Dairy Sci. 83(9):
1939–1945.
 Girta F., 1997. Use of on-line biosensors in food industry. J. of Qafqaz University,
1(1):138-150.
 Glaser R. W., 2000. Surface Plasmon Resonance Biosensors and their applications,
Kiuwer Academic/Plenum Publishers, New York.pp.195-212.
 Kumar Sujith., 2011. enzyme based amperometric biosensor for the determination
of total polyphenol content in tea infusions. Journal of food science and technology.
144-152

38. 