This document discusses enzyme electrode biosensors for analyzing carbohydrates like glucose, fructose, sucrose, and lactulose. It describes the basic principles of how these biosensors work using specific enzymes, mentions the historical development from first to third generation biosensors, and provides examples of how these biosensors have been applied to analyze foods like juices, jams, and milk. The biosensors allow for quick, reliable, and selective analysis of carbohydrates through electrochemical detection of products from enzyme-catalyzed reactions.

1. Enzyme Electrode Sensor for
Carbohydrate Analysis
Presented By
Ashok Suraj M.Tech (Agricultural
Processing Engineering)
2015604102
Shyamala.C M.Tech (Storage Engineering)
2015604605

2. Introduction
• Safety of food and environment has been a major
concern of food technologists and health scientists
in recent years.
• Biosensors present attractive, efficient alternative
techniques by providing quick and reliable
performances.

3. Prerequisites for a biosensor
• Selectivity
• Sensitivity
• Linearity of response
• Reproducibility of signal response
• Quick response time and recovery time
• Stability and operating life

4. Basic Principle of Carbohydrate Biosensor
Most electrochemical glucose biosensors are based on
the glucose oxidase (GOx) enzyme, which catalyzes the
oxidation of glucose to gluconolactone which is hydrolyzed to
gluconic acid and hydrogen peroxide.
The quantification of glucose can be achieved via
electrochemical detection of the enzymatic release of H2O2
Glucose+O2→H2O2+gluconicacid; H2O2→O2+2H+
+2e−

6. Historical Perspectives of Glucose Biosensors
First-generation of Glucose Biosensors

8. Second Generation

9. Third Generation

10. Enzyme Used In Bio-Sensor:

11. Electrochemical biosensors
• Potentiometric,
• Amperometric
• Conductometric
• Calorimetric

12. Electrochemical Biosensor
• An electrochemical biosensor uses an electrochemical
transducer where electrochemical signals are generated
during biochemical reactions and are monitored using
suitable potentiometric, amperometric or conductometric
systems of analyses.
• It is considered as a chemically modified electrode since
electronic conducting, semiconducting or ionic conducting
material is coated with a biochemical film.

13. Conductometric biosensors
• Many enzyme reactions, such as those of urease and many biological
membrane receptors may be monitored by ion conductometric or
impedimetric devices, using interdigitated microelectrodes.
• Because the sensitivity of the measurement is hindered by the parallel
conductance of the sample solution, usually a differential measurement
is performed between a sensor with enzyme and an identical one
without enzyme.
• Analytes like urea, charged species and oligonucleotides are detected
using this principle.

14. Calorimetric biosensors
• Calorimetric biosensors measure the change in temperature of the
solution containing the analyte following enzyme action and
interpret it in terms of the analyte concentration in the solution.
• Since most of the enzyme catalyzed reactions are exothermic, the
heat generated by the reaction is used to determine the analyte.
• Calorimetric biosensors are extensively used for the detection of
pesticides and other enzymatic reactions.

15. Potentiometric biosensors
• Potentiometric measurements are determined
based on potential difference between an indicator
and a reference electrode.
• The transducer may be an ion-selective electrode,
which is an electrochemical sensor based on thin
films or selective membranes as recognition
elements.
• The most common potentiometric devices are pH
electrodes; several other ion (F-, I-, CN-, Na+, K+,
Ca2+, NH4
+) or gas (CO2, NH3) selective electrodes
are available.

16. Amperometric biosensors
• Amperometry is based on the measurement of the
current resulting from the electrochemical oxidation or
reduction of an electroactive species.
• It is usually performed by maintaining a constant
potential at Pt, Au or C based working electrode or on
array of electrodes with respect to the reference
electrode, which may also function as the auxiliary
electrode, if currents are low (10−9 to 10−6 A).
• The resulting current is directly interrelated to the bulk
concentration of the electro active species or its
production or consumption rate within the adjacent
biocatalytic layer.

17. Enzyme Electrodes for Food Analysis
Glucose:
The method was applied to orange juice, several beverages and tonic
with good agreement to a conventional spectrophotometric method.
Glucose and sucrose were simultaneously determined by the use of an
enzyme sensor system consisting of a glucose sensing electrode based
on a lipid-modified glucose oxidase and a measuring cell which contains
an invertase/mutarotase-coimmobilized layer.

18. Cont…..
• After a period of 2 ± 6 s and 8 ±20 s from the injection of the
analytes, steady state currents were recorded and linear
calibration plots over the concentration ranges of 0.2µM - 3
mM glucose and of 10µM-6 mM sucrose, were, respectively,
constructed.
• The method was applied to jams and juices for the rapid
determination of glucose and sucrose and correlated well with
a reference spectrophotometric method.

19. Fructose:
Fructose is widely distributed in many fruits and vegetables, has greater
sweetness than glucose or sucrose and is frequently used in diabetic
sweeteners.
Enzyme electrodes for fructose determination are based on-fructose-5-
dehydrogenase (FDH) in the presence of a mediator. The enzyme
requires no additional cofactor as it contains PQQ and heme c as redox
active sites.
An amperometric fructose biosensor, based on FDH and the coenzyme
ubiquinone-6 immobilized in a membrane mimetic layer on gold..

20. The sensor exhibits a response time of less than 20 s, a sensitivity
of 15A/cm2 mM and a detection limit of less than 10M.
The membrane mimetic layer blocked effectively access of
ascorbates to electrode surface; a 4% positive error was recorded in
its presence.
Biosensor measurements of fructose in apple and orange juices
agreed to within a few percent with those made with an enzymic
spectrophotometric assay.

21. Sucrose
Sucrose determination requires a multi-enzyme system. Sucrose is
hydrolyzed enzymatically by the enzyme invertase,
Ivertase in combination with glucose oxidase may thus be used to
produce a sucrose enzyme electrode, however, a third enzyme,
mutarotase, is usually utilized in order to convert glucose to its -
isomer on which glucose oxidase is specific.
A multi-enzyme electrode obtained by a two-step immobilization of the
enzymes glucose oxidase, mutarotase and invertase was developed
for the determination of sucrose.

22. Cont…,
Glucose oxidase was entrapped in a poly-1,3-diaminobenzene film on a
platinum electrode by electrochemical polymerization and a combination of
mutarotase and invertase was cross-linked over the electrode via bovine
serum albumin and glutaraldehyde.
A second electrode, for glucose only, was constructed containing inactive
invertase, thus being used for signal subtraction.
 Application to a number of different soft drinks gave a good agreement
with a standard liquid chromatography (LC) method.

23. Lactulose
Lactulose, the epimerized lactose, is a synthetic disaccharide consisting of
galactose and fructose and is absent in raw milk.
It is formed in alkaline lactose solutions or by heating of milk due to the
epimerization of lactose. Therefore, it can be used as an indicator for the
severity of heat treatment of milk and to distinguish between pasteurized,
ultra-heat treated and sterilized milk.
Lactulose is enzymatically hydrolyzed to-galactose and fructose according
to the following reaction scheme-galactosidase in combination with
fructose dehydrogenase may thus be used to produce a lactulose enzyme
electrode.

24. Cont…
• An enzymatic procedure for the determination of lactulose was developed
by Bilitewski and co-workers.
• Lactulose was hydrolysed to fructose and galactose by using soluble -
galactosidase, and the amount of fructose was determined by using
immobilized FDH and K3 [Fe (CN)6]as mediator.
• The reduced mediator was re-oxidized at a screen-printed Pt-electrode at
385 mVAn automated flow method was applied to milk samples utilizing a
dialysis unit for separating the analyte from the sample matrix.
• Data correlates well to the official spectrophotometric method, except in
the case of condensed milk where a deviation of up to 20% was observed.
The effect of interferences was manipulated by subtracting the signal taken
after the addition of-galactosidase with that recorded before its addition.