Biosensor

Sensor systems

Biological materials

Biosensor

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BIOSENSOR

ENZYME

ENZYMATIC BIOSENSOR

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Enzymes

No change occur at the end of the reaction

Lowering activation energy

Acceleration of catalysis rate

Catalyst

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Fig . Enzyme working principle.

Enzymes

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Fig. Change in concentrations over time for enzyme E, substrate S, complex ES and product P

Turn over number indicates the

change of substrate molecules per

unit of time for each enzyme.

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Enzyme category Functions

Oxidoreductases Oxidation-reduction reactions

Transferazes Group transfer

Hydrolase

Hydrolysis reaction (transfer of functional group to water)

Lyase Addition or removal of groups to form double bonds

Isomerase Izomerization (intramolecular group transfer)

Ligase Joining of two molecules

Table. Six classes of enzymes and their functions used in the detection of analytes.

Fig . Pictorial representation of different immobilization techniques.

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Enzymes Based Biosensor

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Enzyme-based biosensors are made from the enzyme as a bioreceptor that is specific for

detecting the targeted analyte in the sample matrix. The lock and key stimulated fit

hypothesis can be applied to explain the mechanism of enzyme action which is highly

specific for such a biosensor. This specific catalytic reaction of the enzyme provides such

biosensors with the ability to find much lower limits than normal binding techniques. The

high specificity of enzyme-substrate interactions and the generally high turnover rate of

biocatalysts are the origin of sensitive and specific enzyme-based biosensor devices.

Enzymes are very sensitive molecules effected by external factors easily such as substrate

concentration, pH, temperature and inhibitors.

9 Fig.Factors affecting enzyme activity

Michaelis-Menten equation can be

used to further elucidate the

analytical performance of enzyme-

based biosensor.

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Enzyme inhibitors can be reversible or irreversible. Reversible inhibitors form an EI(enzyme-

inhibitor) complex that can be separated back to enzyme and free inhibitor. These inhibitors can

occur in 3 ways: competitive, non-competitive and uncompetitive. Irreversible inhibitors establish

covalent or very tight persistent bonds with amino acid at the active site of the enzyme and render

it inactive

CLARK and HISTORY

Clark and Lyons -oxygen electrode for glucose measurement in blood 1962.

Yellow Springs Instruments (YSI) developed the Clark and Lyon’s enzyme electrode system and

intoduced the amperometric device which measure glucose level by following hydrogen peroxide

concentration into the market in 1975.

(1918-2005)

Pioneer of enzyme-based biosensor

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Glucose Biosensor

For diabetes patients!

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Hexokinase

Glucose oxidase

Glucose-1-dehydrogenase

Measurements occur based on enzyme interactions

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Measurements occur based on enzyme interactions which can be hexokinase, glucose oxidase (GOx)

or glucose-1-dehydrogenase (GDH). In current glucometers, glucose oxidase which has higher

selectivity for glucose used for test strips. It has also additional advantages such as being cheap, easy

obtaining and withstanding to extreme conditions.

Mechanism of Glucose Biosensor

Glucose + GOx − FAD+ → Glucolactone + GOx − FADH2

GOx − FADH2 + O2 → GOx − FAD + H2 O2

H2O2 → 2H+ + O2 + 2e

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The mechanism of glucose biosensor works

according to oxidation of β-D-glucose

catalyzed by GOx through oxygen resulting in

gluconic acid and hydrogen peroxide

formation. A redox cofactor (Flavin Adenine

Dinucleotide) needed for GOx to work as

catalyst. FAD which acts as initial electron

acceptor reduced to FADH2. Then, oxygen

reaction leads to regeneration of FAD and

hyrogen peroxide production. Oxidation of

hydrogen peroxide occur platinum (Pt) anode

and electrode identify the amount of electron

transfer which is proportional to the amount of

glucose in blood.

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In literature, there are various

biosensor studies for glucose

monitoring which can be

invasive due to blood sample

needed or noninvasive

alternative to blood such as

saliva, sweat or tear samples.

(Du et al, 2016)

SWCNT / Chitosan / AuNP / GOx

Screen-printed platinum electrode

Electrochemical measurements

Detection of glucose

from saliva

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Fig. Cyclic voltammetry tests determined

steady-state calibration curve of biosensor

(Liu et al, 2018)

GOx,-

SWCNT-

Chitosan

Indium oxide (In2O3) field-

effect transistors

(FETs)

Inkjet printing

method

A wearable nanobiosensor

integrated on-chip

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Fig . Nanoribbon- and nanowire-based field-effect

transistor (FET) biosensor.

Working principle is provided by near-infrared light which change the color related to

amount of blood glucose as a result of interaction with the ink. This development

used for real-time monitoring for blood glucose level.

(MIT, 2011)

Sugar-sensitive

tattoo

Carbon nanotubes

Glucose test based on ink

Injectable

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Portable and wireless device is capable of controlled drug delivery through

temperature-responsive microneedles.

Working principle occur based on sweat generation with skin contacted

patch and glucose in collected sweat is analysed when the relative humidity

(RH) reach to 80% through humidity sensor.

(Lee et al, 2017)

Portable and wireless

pH and glucose monitoring from

sweat

Capable of controlled drug

delivery

Injectable

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Lactate Biosensor

Lactate is the key metabolite of the anaerobic metabolism pathway.

Lactate is a byproduct of anerobic glycolysis, a chemical process in which

anaerobic respiration breaks down into smaller molecular components of

sugar.

- food industry

- clinical diagnosis

- sports medicine

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Lactate control

Food industry

Lactate Control

Lactate in determining

the freshness and quality

of fermented products

such as yoghurt, cheese

and wine.

Clinical diagnosis

The increase in lactate

may be cause respiratory

disease, sepsis, heart

attacks, liver disease and

cancer.

Sport Medicine

Lactate accumulates in blood

and tissues when a person does

physical activities. It becomes

impotant when personalized

training programs is prepared

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Lactate detection

Electrochemical biosensors

Excellent

sensitivity

Good

selectivity

Low cost

Easy of

use

Lactate detection device

Fig. Sample mechanism of the test kit for lactate assays. (A) Electrode

system; (B) Hydrophobic layer

Reference electrode

Counter electrode

Working electrode

A lactate oxidase enzyme that attached to the working support material (polymer) is used.

When the sample (blood) is dropped here, a chemical reaction starts with oxygen and enzyme.

L-lactate + O2 Pyruvate + H2O2

H2O2 O2+2H+ + 2e-

L-lactate

oxidase

Electrically active substances Oxidation-reduction Electron production

Lactate Application

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Flexible biosensor

Monitoring of lactate in human sweat to early detection of pressure ischemia.

(Eva L. et al.,2017)

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Fig. Application of enzyme laminate

(Eva L. et al.,2017)

The core of the recognition system is a

highly flexible laminate containing two

high-grade porous polycarbonate

membranes, which are supported by lactate

oxidase enzyme (LOD), which is

immobilized by covalent cross-linking.

Fig. Final flexible sensor layout

Wang and colleagues , during real-time

noninvasive lactate detection on human skin using

a flexible printed transient tattoo electrochemical

biosensor fitted to the user's skin during prolonged

cycling exercise.

Fig. Shows that lactate measurement of

electrocehmical tattoo biosensor.

The device composes of a screen-printed

electrode on a flexible substrate, with

lactate oxidase immobilized onto the

working electrode with MWCNT acting as

the transducer surface.

Fig. Shows that wearable electrochemical

biosensor mechanism for lactate

determination. (S. R. Corrie et al.,2015)

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Pesticide Detection

Pesticides

High risk for environment

High insecticidal activity

Used in agriculture

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Acetylcholinesterase (AChE)

Active site of AChE found in hydrolase category consists of 3 amino acids which

are histidine, serine and aspartic acid.

29 (Pundir and Nidhi, 2012)

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AChE Catalysis

Attraction between positive charged

amonium group of ACh and anionic

binding site of triple aminoacid of

AChE active site.

AChE Inhibition

Covalent binding of nucleophilic

serin hydroxyl group of enzyme

active site to phosphorus atom of

pesticide.

(Gürsoy,2017)

(Pundir and Nidhi, 2012) 31

When the absence of pesticide, AChE provide convertion of

asetylthiocholine (ATCI) to thiocholine and acetic acid.

Convertion of asetylthiocholine (ATCI) to thiocholine which

has electrical activity.

(Pundir and Nidhi, 2012) 32

Fig . AChE inhibition with the presence of pesticide.

Prussian blue-modified electrode for

detecting organophosphorous pesticides.

Chitosan was chosen for AChE

immobilization.

Glutaraldehyde was used as crosslinker.

(Sun and Wang, 2010) 33

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The more pesticide concentration

increase, the less current obtained due

to enzyme inhibition.

(Pundir and Nidhi, 2012).

Fig . DPV of the NF/AChE–CS/Ag NPs–CGR–

NF/GCE in 0.1 M PBS containing 0.5 mM ATCl

after incubation with 0 (a), 10−13 M (b), 10−12 M

(c), 10−11 M (d), 10−10 M (e), 10−9 M (f) and

10−8 M (g) chlorpyrifos for 6 min.

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Table. Enzyme types and application areas