week 2 bioisensors 21sep2009 bio catalysis

8/4/2019 Week 2 Bioisensors 21Sep2009 Bio Catalysis

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Biocatalysis Based Biosensors, Bioaffinity BasedBiosensors & Microorganisms Based Biosensors,

Biologically Active Material and AnalytesTRANS

DUCER

AMPLIFIER DISPLAY

CH3 S

CH3 S

CH

2 S

CH3 S

CH3 S

CH2 S

CH3 S

MATRIXBIOMOLECULEANALYTE

Centre for NanoBioengineering & Spintronics,

Chungnam National University,Daejeon,Korea

10/5/2009 1


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10/5/2009 WCU Project, CNU,[email protected] 2

Biosensor

Biocatalysis based BiosensorsBiaffinity based Biosensors

Micoorganisms Based BiosensorsConclusions

Literature


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BIOSENSOR

IUPAC Nomenclature: A biosensor is a self-contained integrated device which is

capable of providing specific quantitative or semi-quantitative analytical

information using a biological recognition element (biochemical receptor) whichis in direct spatial contact with a transducer element.

What is a BIOSENSOR ?

Thebioreceptor.

Thetransducer or thedetector element

Associated electronics orsignal processors that isprimarily responsible for the display of the results

in a user-friendly way.

It consists of 3 parts



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Calorimetric (detect on the basis of heat evolved in biological reaction)

Piezoelectric (detect on the basis of electric dipoles generated due to mechanical

stress)

Optical (detect on the basis of change in light received ) Electrochemical such as Potentiometric, Conductometric and Amperometric

Classification of Biosensors

Characteristics of a Biosensor

Classification based on transducer system

Classification based on bio-recognition element

Antigen-antibody

(i) Selectivity, (ii) Recovery time (iii) Shelf-life (iv)Stability,

(v) Response time, (vi) Accuracy, (vii)Reusability

Enzyme

DNA

Cell



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Biocatalysis based sensor

Biocatalysis-based biosensors depend universally on the use of enzymes.

The field of biocatalysis is open. This frontier of research is racing

ahead, propelled by advances in the database-supported analysis of

sequences and structures as well as the designability of genes &proteins.

Biocatalytic processes differ from conventional chemical processes, owing

mainly to enzyme kinetics, protein stability under technical conditions andcatalyst features that derive from their role in the cells physiology, such as

growth, induction of enzyme activity or the use of metabolic pathways for

multistep reactions.



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Catalysts are substances that speed up chemical

reactions. Organic/bio-catalysts are called enzymes.

Reactions with enzymes are up to 10 billion times fasterthan those without enzymes.

Enzymes are specific for one particular reaction or group of

related reactions.

An enzyme-substrate complex forms when the enzymes

active site binds with the substrate like a key fitting a

lock. The shape of the enzyme must match the shape of the

substrate. Enzymes are ,therefore, very specific; they will only

function correctly if the shape of the substrate matches theactive site

The enzyme does not form a chemical bond with the

substrate. After the reaction, the products are released and theenzyme returns to its normal shape.

The enzyme molecule can be reused. Only a small amount of

enzyme is needed because they can be used repeatedly.



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Effect of Temperature

Increase in the temperature of a system results from increases in the kinetic energyof the system. This has several effects on the rates of reactions.

1. More energetic collisions

The greater the kinetic energy of the molecules in a system, the greater is the resulting chemical

potential energy when two molecules collide .

2 The number of collisions per unit time will increase.

In order to convert substrate into product, enzymes must collide with and bind to the substrate at

the active site. Increasing the temperature of a system will increase the number of collisions ofenzyme and substrate per unit time. Thus, within limits, the rate of the reaction will increase.

3 The heat of the molecules in the system will increase

As the temperature of the system is increased, internal energy of the molecules in the system will increase.

The internal energy of the molecules may include the translational energy, vibrational energy and rotational

energy of the molecules. Some of this may be converted into chemical potential energy. If this chemical

potential energy increase is great enough , some of the weak bonds that determine the three dimensional shape

of the active proteins may be broken. This could lead to a thermal denaturation of the protein and thus

inactivate the protein. Thus too much heat can cause the rate of an enzyme catalyzed reaction to decrease

because the enzyme or substrate becomes denatured and inactive.



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pH can affect the ionization of the amino acid side chain, which in turn change the

secondary, tertiary and quaternary structures of the protein molecule. This will

change the enzyme's active site and consequently its activity.

Effect of pH

Like most chemical reactions, the rate of an

enzyme-catalyzed reaction increases as the

temperature is raised. A ten degree Centigraderise in temperature will increase the activity of

most enzymes by 50 to 100%.

This figure shows that the reaction rate

increases with temperature to a maximum level,then abruptly declines with further increase of

temperature. Because most animal enzymes

rapidly become denatured at temperatures

above 40C, most enzyme determinations are

carried out somewhat below that temperature.



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Enzyme ClassificationThere are approximately 3000 known enzymes. These enzymes are classified into six categories

based on the type of reaction they catalyze.

1. Oxido- reductase: Oxidizes or reduces by transfer of hydrogen or electrons.(a) Dehydrogenases:

SH2 + A S + AH2 (S: Substrate, A: acceptor)

Example:Lactate dehydrogenase: L-lactate + NAD Pyruvate + NADH + H+

(b) Oxidases:SH2 + 1/2 O2 S + H2O or

SH2 + O2 S + H2O2Example:Glucose oxidase: -D-glucose + O2 Gluconolactone + H2O2

(c) Peroxidases:

2SH + H2O2 2S + 2H2O or

2S + 2H+ + H2O2 2S+ + 2H2O

Example:Horse radish peroxidase:

2[Fe(CN)6]4- + 2H+ 2[Fe(CN)6]

3- + 2H2O

(d) Oxygenases:

SH + DH + O2 S-OH + D + H2O

Example:Lactate 2-monooxygenase:L-lactate + O2 acetate + CO2 + H2O



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2. Transferase: Transfers C-, N-, P-, or S-containing functional groups such as aldehydes

and ketones, glycosils, acyls, phosphates, and sulfur containing groups.

AX + BA + BX

Example: Hexokinase:D-hexose + ATP D-hexose-6-phosphate + ADP

3. Hydrolase: Hydrolyses esters, anhydrides, peptide bonds, other C-N

bonds, glycosidesExample: Cholesterol esterase:

Cholesterol ester + H2O cholesterol + fatty acid

Glucoamylase:

Amylose + n H2O n -D-glucose

4. Lyase: Adds to double bonds:

> C = C C = O

> C = N

5. Isomerase: Isomerizes optical iomers

Example

Glucose isomerase:D-glucose D-fructose



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6. Ligase: Splits C-C, C-O, C-N, C-S and C-halogen bonds without hydrolysis or oxidation,

mostly with ATP

Example : Pyruvate Carboxylase:

Pyruvate + HCO3- + ATP oxaloacetate + ADP + Pi

Coenzymes, Prosthetic group, Effectors

Sometimes the surface cavity does not act as a catalytic site until it is modified by a second

incoming molecule. These participants known as the coenzymes are non-peptide moleculescapable of completing the binding site for the transition state. Other molecules that do the similar

function are prothetic group, and effectors.

Coenzyme: Coenzyme is a non-peptide molecule capable of completing the binding site for

the transition state. Examples include many vitamin derivative such as coenzyme A, thiamine,

pyrophosphate, vitamin B12

Prosthetic Group: Prosthetic group is the same as the coenzyme but are tightly bound tothe enzyme. When they are split off, the enzyme is mostly denatured. Examples include flavin

nucleotides and heme.

Effectors: Effectors accelerate (activators) or block (inhibitors) enzyme reactionExamples of activators include Mg++, Ca++, Zn++, K+, and Na+,

Examples for the inhibitors include Hg, and substrate analogs. Table 1 lists functions of someof the important coenzymes and prostshetic groups.



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Table1:Function of some important coenzymes and prosthetic groups



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Let us consider a catalytic reaction,

E + S ES E + Pk1 k2

k-1

where E, S, P and ES represent the enzyme,

substrate, product and transient complexesof the enzyme andk1 ,k-1 k2 are rate

constants (formation) and (breakdown),

So, Rate of ES formation d[ES]/dt= k1([E] - [ES])[S]

Rate of ES breakdown = k-1[ES] +k2[ES]

Now, the initial rate of reaction reflects a steady state in which [ES] is

constant, e.g. the rate of formation of ES is equal to the rate of its breakdown.

This is called the steady-state assumption.

k1([E] - [ES])[S] =k-1[ES] +k2[ES]

Or, k1[E] [S] -k1[ES][S] = (k-1 +k2 )[ES]

Or, k1[E] [S] = (k1[S] +k-1 +k2) [ES], {by addingk1[ES][S]}

Or, [ES] =(k1[S] +k-1 +k2)

k1[E] [S]

Or, [ES] =

[S] + (k-1 +k2)/k1

[E] [S]

The term (k2 +k-1) /k1 is defined as the Michaelisconstant,Km

ENZYME KINETICS



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Assumptions


The first key assumption in this derivation is the quasi-steady-state assumption (orpseudo-steady-state hypothesis), namely that the concentration of the substrate-bound

enzyme ([ES]) changes much more slowly than those of the product ([P]) and substrate

([S]). This allows us to set the rate of change of [ES] to zero and also write down the

rate of product formation:

The second key assumption is that the total enzyme concentration ([E]0) does not change

over time, thus we can write the total concentration of enzyme [E]0 as the sum of the free

enzyme in solution [E] and that which is bound to the substrate [ES]:
http://en.wikipedia.org/wiki/Steady_state_(chemistry)http://en.wikipedia.org/wiki/Steady_state_(chemistry)


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The validity of the following derivation rests on the reaction Scheme givenbelow and two key assumptions: that the total enzyme concentration andthe concentration of the intermediate complex do not change over time.

The most convenient derivation of the MichaelisMenten equation,

described by Briggs andHaldane, is obtained as follows (Note that often the experimentalparameter kcat is used but in this simple case it is equal tothe kinetic parameter k2):

The enzymatic reaction is assumed to be irreversible, and the product does

not bind to the enzyme.

WCU Project, CNU,[email protected]
http://en.wikipedia.org/wiki/J._B._S._Haldanehttp://en.wikipedia.org/wiki/J._B._S._Haldane


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GLUCOSE

CHOLESTEROL

UREA

BiocatalysisbasedBiosensoratNPL

10/5/2009WCU Project, CNU,[email protected]

16


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GLUCOSE SENSOR

GLUCOSE + GLUCOSE OXIDASEOXIDIZED

PRODUCT +GLUCOSE OXIDASE REDUCED

GLUCOSE OXIDASEREDUCED+MEDIATOROXIDIZED

MEDIATORREDUCED + GLUCOSEOXIDASEOXIDIZED

+ MEDIATORREDUCED

MEDIATOROXIDIZED+2e-


17

Gl O id d th bi h i l ti i l d


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Glucose Oxidase and the biochemical reactions involved

during the Glucose sensing

The enzymatic reaction catalysed by glucose

oxidase (GOx)

Structure of glucose oxidase

Active site structure of GOx enzyme


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Polythiophene Gold

Nanoparticles

Composite

Iron Oxide

Nanoparticles-

Chitosan

Composite

Au nanoparticle/

Polyaniline

Composite

Au-nanoparticles/

Polypyrrole

Composite

Matrices for

Glucose

biosensor


A N ti l / P l l C it


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Gold nanoparticles-polypyrrole thin film Covalently immobilized glucose oxidase (GOx) on

Gold nano particles-polypyrrole thin film

20 ml of 1mm chloro auricacid solution

Heat up to boiling

Tri sodiumcitrate

solution

Faint blue to

wine red color

Size ~ 10-20 nm

UV-vis spectra:

peak at 520nm

-1 000 -5 00 0 5 00 10 00 15 00

-200

0

2 00

4 00

6 00

8 00

(iii)

(ii)

(i )

I(A)

E(m V)

(i) PPy/ITO

(ii) GOx/AuNPs-PPy/ITO bioelectrode(iii) AuNPs-PPy/ITO electrode in phosphate

buffer .05M, pH7.0, at scan rate 20mV/s

Au Nanoparticles / Polypyrrole Composite

10/5/2009


20


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Cyclic voltammograms of

GOx/AuNPs-PPy/ITO bioelectrode

as a function of glucose

concentration (25mg/dL-300mg/dL).Gold nanoparticles

enhance the sensitivity of the

bioelectrode.

(i-vii) - current

Cyclic voltammograms of GOx-PPy/ITO

electrode as a function of different conc of

glucose (25mg/dL-200mg/dL) in phosphate

buffer .05M,pH 7.0, scan rate of 20mV/s



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Aniline

ITO

(AuNPs)

PANI/ITOAuNPs/PANI/ITO

N N N N NH-CO-EnzH H

N N N N NH H

H

+ H

N N N N NH H

H H

+ + H

HH

H

+NNNN

N N N N NH2H H

H H++

N N N N NH H H

N N N N NH H

H

+ + H

HH

H

+NNNN

EDC/NHS

HOOC-Enz

Au-nanoparticles / Polyaniline Composite

4000 3500 3000 2500 2000 1500 1000 500

b

c

a

2927

1290

1259

1247

1615

1627

1640

800

800

80 0

3186

3434

3434

3434

46 3

463

Trans

mittance(%)

Wavenumber (Cm-1

)

FTIR spectra of (a) PANI film (b) AuNPs-PANI (c) GOx/AuNPs-PANI

film on ITO

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

-1.5x10-3

-1.0x10-3

-5.0x10-4

0.0

5.0x10-4

1.0x10-3

1.5x10-3

2.0x10-3

2.5x10-3

c

b

a

Current(A)

Voltage (V)

Cyclic voltammogrammes (a) PANI/ITO film(b)AuNPS-PANI/ITO electrode

(c) GOx/AuNPS-PANI/ITO bioelectrode

Journal of Nanoscience and Nanotechnology, 2008, 8, 3158.



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Glucose Oxidase Activity Immobilized on AuNPs-PANI/ITO

Current response of GOx/AuNPs-PANI/ITO

bioelectrode as a function of glucose concentration.

Hanes plot of GOx/AuNPs-PANI/ITO bioelectrode as a

function of glucose concentration

GOx/PANI/AuNPs/ITO

Glucose + O2 Gluconic acid + H2O2.....Eq.1

Electrochemical oxidation2H+ + O2 + 2e

--

.........Eq.2H2O2



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Summary

Km value of immobilized enzyme on gold nanoparticles polyaniline composite films 2.2 mM(39.64 mg/dl)

Response time of GOx/AuNPsPANI/ITO bioelectrode ~ 10 s clearly indicate that self assembled goldnanoparticles in PANI matrix provide biocompatible environment to enzyme

Sensing property to glucose concentration 50300 mg/dl

GOx/AuNPsPANI/ITO bioelectrode retains more than 85% of the GOx activity even after 11 weeks.

0 2 4 6 8 10 12-2.0x10

-3

-1.0x10-3

0.0

1.0x10-3

2.0x10-3

Current(A)

Weeks

Shelf Life

Shelf life of GOx/AuNPs-

PANI/ITO bioelectrode with

time.



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Polythiophene Gold Nanoparticles

Composite

DTSP : dithiobissuccinnimidyl propionate

Schematic of Covalent immobilization of glucose

oxidase on bifunctionalized gold nanoparticles

FT-IR spectra of (a) regP3HT-AuNPs/Au film and (b)

bifunctionalized gold nanoparticles (regP3HT-DTSP-AuNPs-Au) film.

UV-Vis absorption of (a) citrate capped gold nanoparticles

(b) P3HT in toluene (c) P3HT - AuNPs in toluene.

Peak at 450 nm ~ P3HT moieties

Peak at 557 nm~ P3HT capped AuNPs

J. Applied Poly. Sci., 2008, DOI 10.1002/app.

10/5/2009


25

Enzyme activity studies using UV visible spectrophotometer


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0 50 100 150 200 250 300 350 400 450

0.002

0.004

0.006

0.008

0.010

0.012

0.014

Absorbance(500nm)

Conc (mg/dL)

Photometeric response of GOx-regP3HT-AuNPsDTSP/Au) bioelectrode as a function of analyte(glucose) concentration.

Hanes plots of GOx-regP3HT-AuNPsDTSP/Aubioelectrode as a function of analyte (glucose)concentration.

Enzyme activity studies using UV-visible spectrophotometer

Absorbance response of GOx-regP3HT-AuNPsDTSP/Au bioelectrode in PBS buffer (50mM, 0.9NaCl) of pH (i) 6.0 (ii) 6.5 (iii) 7.0 (iv) 7.4 (v) 8

Effect of temperature on response of GOx-regP3HT-AuNPsDTSP/Au bioelectrode10/5/2009 WCU Project, CNU,[email protected] 26


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Iron Oxide NanoparticlesChitosan Composite

Iron oxide nanoparticles (Fe3O4) has been prepared using co-precipitation method.

Nanocomposite of chitosan and Fe3O4 has been prepared using electrostatic

interaction between positively charged CH and negatively charged Fe3O4nanoparticles.

Schematic of formation of CH-Fe3O4 Nanocomposite and immobilization of glucose oxidase on nanocomposite matrix

Biosens. Bioelectron., DOI. 10.1016/j.bios.2008.06.032

10/5/2009WCU Project,

CNU,[email protected]


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a b c

SEM, CH/ITO SEM, CH-Fe3O4/ITO SEM, GOx/CH-Fe3O4/ITO

50

nm

b

Transmission Electron Micrograph of Fe3O4 nanoparticles

Km Value = 0.141 mM

Stability curve of GOx/CH-Fe3O4/ITO

bioelectrode as a function of absorbance with

respect to time (weeks)

The activity of the GOx/CH-Fe3O4/ITO bioelectrode stored at 4

o C has been measured at different time interval.

It has been observed that the activity of glucosel oxidase

immobilized onto the ITO surface shows stability upto 8 weeks10/5/2009 WCU Project, CNU,[email protected] 28


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The bioelectrode shows linearity within the

range of 50 to 400mg/dl of glucose with co-

relation factor of 0.99 and sensitivity of 0.1 x 10-3

mA/ (mg/dl).

Electrochemical Response Studies of GOx/PANI/ITO Bio-electrodes

DPV for GOx/NS-PANI/ITO bio-electrode asfunction of Glucose concentration


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Most urea biosensors are based on

urease Urs and n catalyticconversion of urea to hydrogen

bicarbonate and ammonium. It has

been observed that ammonium ions

easily diffuse in solution. Thus,

glutamate dehydrogenase, GLDHhas been used as an alternate since

it catalyzes the reaction between

ammonium ions, -ketoglutarate -KG

and nicotinamide adenine

dinucleotide NADH to produce L-glutamate and NAD+.

Estimation of urea in serum/blood/urine is important for diagnosis of renal and liver

diseases. An increase in urea level normal range is 820 mg/ dl in blood and urine causes

renal failure, urinary tract obstruction, dehydration, shock, burns, and gastrointestinal

bleeding. Moreover, reduced urea level may cause hepatic failure, nephritic syndrome,

and cachexia low-protein and high-carbohydrate diets.

Urea Biosensor



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Iron oxide-chitosan

nanobiocomposite

for urea sensor

P3HT - SAM

Zinc oxide-chitosan

nanobiocomposite

for urea sensor

Matrices for

Urea

biosensor


APPLIED PHYSICS LETTERS 93 163903 2008


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Zinc oxide-chitosan nanobiocomposite

XRD pattern of ZnO-CH

nanobiocomposite film. b

Scanning electron micrograph

of Urs-GLDH/ZnO-CH/ITO

bioelectrode

EIS of i) CH/ITO, ii) ZnO-CH/ITO, iii) Urs-

GLDH/ZnO-CH/ITO electrode,Electrochemical response of Urs-GLDH/ZnOCH/ ITO bioelectrode with respect to urea

concentration 5100 mg dl1 at scan rate of 10 mV s1

Km = 4.92 mg/ dl

Linearity =5100

mg/ dl,

Detection limit = 3

mg/ dl

APPLIED PHYSICS LETTERS 93, 163903 2008


Sensors and Actuators B 138 (2009) 572 580


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Iron oxide-chitosan nanobiocomposite

X-ray diffraction

pattern of Fe3O4nanoparticles.

Ur-GLDH/CH-Fe3O4nanobiocomposite/ITO electrode.

SEM images of CH-Fe3O4nanobiocomposite/ITO electrode

Sensors and Actuators B 138 (2009) 572580



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DPV of (a) CH/ITO, (b) CH-FeO4 nanobiocomposite/ITO and

(c) Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO bioelectrode

Cyclic voltammograms of (a) CH/ITO, (b) CH-Fe3O4nanobiocomposite/ITO and (c) Ur-GLDH/CH-Fe3O4nanobiocomposite/ITO bioelectrode

Electrochemical response of Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO

bioelectrode as a function of urea concentration (5100 mg/dL). The effect of interferents on electrctrochemical response of

Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO bioelectrode

10/5/2009 34


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Triglyceride

Cholesterol & Triglyceride Biosensor

http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/Triglyceride-2D-skeletal.png/800px-Triglyceride-2D-skeletal.pnghttp://upload.wikimedia.org/wikipedia/commons/thumb/3/32/Triglyceride-2D-skeletal.png/800px-Triglyceride-2D-skeletal.png


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Polythiophene Gold

Nanoparticles

Composite

P3HT - SAM

Polyaniline

Langmuir -Blodgett Films

Electrophoretically

deposited

MWCNTc/polyaniline

Electrophoretically

deposited nano-

structuredpolyaniline film

Matrices for

Cholesterol &

Triglyceridebiosensor

Cholesterol



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400 600 800 1000 12000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

(ii)

(i)

Absorbance(

Abs)

Wavelengh ()

Formation of polyaniline colloidal suspension

Electrophoretically deposited nano-structured

polyaniline film

Analytica Chimica Acta 6 0 2 ( 2 0 0 7 ) 244251

Electrophoretic deposition of polyaniline

from its colloidal suspension at 80V

Polyaniline chain

ITO

-NH+

Colloidal suspension

Film

Conformational analysis of polyaniline

analytica chimica acta 6 0 2 ( 2 0 0 7 ) 244251

10/5/2009 37


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Adduct-II

Adduct-I

HS

Pol aniline

EDCEnzyme

O

OHEnz CH

3CH

2-N=C=N-(CH

2)3N(CH

3)2

O

O

O

O

P NH2P NH

O

Amide bond formation

NEnzEnz

O

O N

NH

N-(CH2)3N(CH3)2

-CH2CH3

O O

OH

N

Enz

Covalent immobilization of cholesterol oxidase on electrophoretically deposited polyaniline films

50nm

SEM micrograph of

PANI/ChOx/ITO bio-electrode

Transmission electron microscopeimage of polyaniline fibre with the

associated protonating acid

100 nm

SEM micrograph of

electrophoretically deposited nano-

structured polyaniline film10/5/2009 WCU Project, CNU,[email protected] 38


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Photometric Response Studies of ChOx/PANI/ITO Bio-electrode

HRP

H2O2 + O- dianisidine (reduced) 2H2O + O- dianisidine (oxidized)

orange color

Photometric response of ChOx/NS-PANI/ITO bio-

electrode as a function of cholesterol concentration

ChOxoxiNS-PANIITO + Cholesterol + O2 ChOxredNS-PANIITO + 4-cholesten-3-one + H2O2

Optimum pH 6.5



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Electrophoretically deposited MWCNT-c/ Polyaniline Composite

(i)

(ii)

Revision submitted to Carbon

Carbon 46 (2008) 1727-1735

10/5/2009 40

Application of electrophoretically deposited MWCNT-c/polyaniline composite to


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free cholesterol sensing

UV-Vis spectra of electrophoretically deposited ES

and ES/MWNT-c films

AFM of Electrophoretically deposited Nanostructuredpolyaniline and ES /MWNT-c/Composite

CV comparing the electrochemical hysteresis of a pure

Polyaniline (ES) film to that of ES/MWNT-c composite

film



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Amperometric Response Studies of ChOx/PANI-CNT/ITO Bio-electrodes

CV for ChOx/PANI-MWCNT/ITO bioelectrode asfunction of cholesterol concentration

Amperometric response of ChOx/PANI-MWCNT/ITObioelectrode as a function of cholesterol concentration

Linearity 1.3 to 13mMSensitivity: 6700nA/mM

Schematic of reaction taking place at ChOx/PANI-MWCNT Bioelectrode


Polyaniline Langmuir Blodgett Films


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Polyaniline Langmuir-Blodgett Films

Schematic for electrode preparation10/5/2009 43


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FT-IR spectra of PANI/SA and PANI/Glut/ChOx LB film

bioelectrode

SEM of PANI/SA LB film

bioelectrode

SEM of ChOx/Glut/PANI-SA

LB film bioelectrode

-0.4 -0.2 0.0 0.2 0.4 0.6 0.8

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

(vii)

(vi)

(iv)

(v)

(iii)

(ii)

(i)Curre

nt(mA)

Potential(V)

sensitivity of 88.9 nA mg-1 dL

0 50 100 150 200 250 300 350 400 450

5

10

15

20

25

30

35

40

45

50

55

ChangeinCurrent[A]

Colesterol Concen tration [mg/dl]

Linearity 25-400mg/dl

1) LSV for ChOx/Glu/PANI-SA LB film as a function ofcholesterol concentration

2) Linear regression curve of ChOx /Glu/PANI-SA LB

film bioelectrode.

10/5/2009 44


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3 mMag: 1.0 K X Mag: 1.0 K X 3 m

300 nm

(i) (ii)

SEM images of PANI-NT/ITO(i) and LIP/Glu/PANI-

NT/ITO

(B) Effects of different interferents on the response

of LIP/Glu/PANI-NT/ITO bioelectrode.

Impedimetric response of LIP/Glu/PANI-NT/ITO bioelectrode for

tributyrin detection; inset shows the calibration plots derived fromthe impedimetric measurements as a function of tributyrin

concentration

Linearity : 25300 mg dL1,

Low Km :0.62 mM



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Bioaffinity Based Sensor

N

NH

NH2

O

N

N

NH

N

NH2

NH

NH

O

O

CH3

N NH

NH

N

NH2

O

N

NH

NH

N

NH2

O

OOOH

OH

O+

P

OOOH

OH

O+

P

OOOH

OH

O

P

O

O

OH

OH

OH

O+

P

OOOH

OH

O+

P

OOOH

OH

O+

P

CH3

CH3

CH3

CH3

NN

NH2

O

CH3


47

Deoxyribose Sugar

Phosphoric Acid

Nitrogenous Bases

Adenine

Guanine

Cytosine andThymine


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Among various affinity biorecognition elements DNA is known to have interesting

chemical and physical properties.DNA is a double stranded helix structure made up to sugar phosphate backbone with specific

sequences made up of nitrogen bases. since phosphate group of the backbone is negatively

charged, DNA is usually surrounded by positive counter-ion like hydrogen , sodium or

potassium in the solid state. In water, these so called counter ions can freely diffuse away leaving

behind a negatively charged DNA strand. This property of DNA makes it ideal for electrontransfer.

Physical properties of DNA is

also very important as with the

change in temperature and pH,two strands of DNA double helix

can be separated. The two

complementary strands of DNA

anneal when the conditions are

slowly brought to normal andthis process is called DNA

hybridization or annealing. This

process of annealing occurs due

to formation of hydrogen bonds

between the nitrogen bases of the complementary strands

DNA Biosensor



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Polythiophene Gold

Nanoparticles

Composite

P3HT - SAM

Nanostructured

Cerium Oxide Film

Based Immunosensorfor Ochratoxin-A

Detection

Electrochemically

deposited Polyaniline

film for N.Gonorrhoea

Polyaniline based

DNA biosensor for

Escherichia coli

Matrices for

DNA

biosensor


A t l A l ti l Ch i t 2007 79 6152 6158

Polyaniline based DNA biosensor for


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C=O C=O C=OC=O

N N N N

5biotin end

labeled BdEprobe

Avidin

Covalent bond between COOH of avidin and -NH ofPANI

PANI film ontoPt disc electrode

C=O

Hybridization withcomplementaryDNA

C=O C=OC=O

N N N N

Complementary target DNA

C=O

Immobilization of avidin onto PANI films coated onto Pt disc electrode using

EDC-NHS couplingImmobilization ofE. coli specific 5-biotin labelled BdE probe indirectly ontoavidin-PANICharacterization of prepared BdE-avidin-PANI bioelectrodes using DPV, SEM,Impedance spectroscopy, FT-IR etc.Hybridization detection of complementary, one base mismatched and non

complementary sequences via monitoring guanine and methylene blue oxidation.Detection of complementary sequences inE. coli genomic DNA and lysedE.colicells.

Arora et.al., Analytical Chemistry 2007, 79, 6152-6158Escherichia coli

E.coli is responsible for three types of infections in humans: urinary tract infections (UTI), Neonatal

meningitis, and intestinal diseases(gastroenteritis). These diseases depend on a specific array of

pathogenic(virulence) determinants.10/5/2009 WCU Project, CNU,[email protected] 51

Preparation of Electrochemically


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Preparation of Electrochemically

deposited Polyaniline filmFilm Wavenumber

(cm-1)

Assignment

PANI 1602 C=C double bond of quinoid

rings.

1482 C=C double bond associated

with the benzoid ring.

1305 Not as yet completely

understood. Perhaps linkedwith various stretching and

bending vibrations

associated with C-C single

bond.

1171 C-N double bond -

indicative of protonation.

Avidin-PANI 1565 & 1658 N-H amide bond.

BdNG-

Avidin-PANI

1067 Assymmetric stretching of

P-O-C vibration.

1243 Stretching vibration of P=O

of the phosphoric acidgroup.

1492 & 1606 Carbonyl Stretching

vibration band of C double

bonds in the purine &

pyrimidine rings.

dNG-Avidin-

PANI

Peak becomes more intense

due to complementry DNA

association.10/5/2009 52


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Polyaniline (PANI) BdNG-Avidin-PANI bioelectrode

DPV shows oxidation peak of

methylene blue at - 0.25V in

Phosphate buffer (0.05M, pH 7.0,0.9% NaCl).

There is increase in the

oxidation peak of methylene blue

observed with the decrease in

complementary DNA

concentration.

This bioelectrode can detect the

DNA upto 2 x 10-15g/l

concentration



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0 .8 0 .9 1 .0 1 .1 1 .2 1 .3 1 .4 1 .5 1 .6

2 .0x10-6

4 .0x10-6

6 .0x10-6

8 .0x10-6

1 .0x10-5

1 .2x10 -5

1 .4x10-5

1 .6x10-5

1 .8x10-5

2 .0x10-5 BdE-avid in-PANI

Hybridiza t ion wi th dE '

Hybridiza t ion wiht dE '1

Hybridiza t ion wi th dE 'nc

I(A

)

V (vo l ts )

DPV curves of BdE-avidin-PANI bioelectrodes in 0.05 M phosphate buffer pH 7.0 at at pulse height of 50 mV

and pulse width of 70 ms after hybridization with complementary probe (dE), one base mismatch probe

(dE1) and non-complementary probe (dEnc); (a) monitoring guanine oxidation, (b) monitoring methylene

blue oxidation.

-600 -500 -400 -300 -200 -100 0 100 200

-35

-30

-25

-20

-15

-10

-5

0

5

-35 -30 -25 -20 -15 -10 -5

2

4

6

8

10

12

14

16

I(A)

1/ln(II-dE' concentration in fmol)

I(A

)

V (mV)

I-dE' = 0.0007 fmol

I-dE' = 0.001 fmol

I-dE' = 0.005 fmol

I-dE' = 0.007 fmol

I-dE' = 0.01 fmol

I-dE' = 0.05 fmolI-dE' = 0.125 fmol

I-dE' = 0.25 fmol

DPV curves of BdE-avidin-PANI electrodes

after hybridization with dE probes (0.0005-

0.25 fmol) after 20 M MB pretreatment in

0.05 M phosphate buffer pH 7.0 at pulseheight of 50 mV and pulse width of 70 ms.

(Inset shows the linear plot of 1/ln(dE fmol)

as a function of peak height of MB in A.

Detection limit of BdE-avidin-PANI = 0.001 fmolIdE = - 0.49622 [1/ln (dE concentration)] + 0.0901



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Surface Plasmon Resonance based Nucleic Acid Biosensor for detection of M.

Tuberculosis

BK7 gold film

Nucleic acid immobilized onto

gold electrode

Hybridization signal due tochange in refractive index

upon the binding

Washing with acetone,

Ethanol and Piranha solution

Immobilization of 20 mer thiolated DNA

and 24 mer PNA probes specific to

M.Tuberculosis for

8500 sec. by SPR technique

Characterization of electrode

by contact angle measurement,

Impedance, Cyclic Voltametry,

Atomic force microscopy

techniques.

Study with the complementary,

one base mismatch and

non complementary targets

10/5/2009 55


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Total immobilized

thiolated DNA is 1200 of

refractive angle change is

corresponds to 1nanogram of

immobilized DNA ( 2380

= 1.98 ng/mm2 i.e.,

16.83ng/ spot or

PNA(204

0

= 1.7 ng/mm

2

i.e., 14.45 ng/spot)

Contact angle measurement with sesile drop method: bare gold 760, Thiolated DNA self assambled monolayer(600), Thiolated PNA self assambled monolayer 54.570


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ImmunosensorANTIBODY (immunoglobulin):A biological molecule

(protein) that specifically recognizes a foreign

substance (antigen) as a means of natural defence

Immunosensors transduce antigen-antibody

interactions directly into physical signals.

The design and preparation of an optimum interfacebetween the biocomponents and the detector material

is the key part of immunosensor development.



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Antibodies


Polyclonal Monoclonal

Antibodies that are collected

from sera of exposed animal

Recognize multiple antigenic

sites of injected biochemical.

Individual B lymphocyte hybridoma is

cloned and cultured.

Secreted antibodies are collected from

culture media

Recognize ONEantigenic site

of injected biochemical


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Fast , accurate and sensitive measurement are required,

Highest possible detection strength is required ,

Large numbers of samples are to be expected ,

Alternate of available expensive analytical methods.

Immunosensor becomes important when


Nanostructured Iron Oxide Film Based Immunosensor for ochratoxin-A Detection


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Ochratoxin-A (OTA) is one of the most abundant food contaminating mycotoxins. OTA is

found in tissues and organs of animals including human blood and breast milk and isknown to produce nephrotoxic, teratogenic, carcinogenic and immune toxic activity in

several animal species.

It affects humans mainly through

consumption of improperly stored food

products and causes carcinogenicity (Group

2B, possibly by induction of oxidative DNA

damage). OTA can also cause immuno-

supression and immuno toxicity.

Why Cerium oxide (FeO2) ?Superparamagnetics, Surface charged, High

adsorption capability, High electron transfer

capability, High affinity with the oxygen atom

of enzymes , Biocompatability

http://en.wikipedia.org/wiki/File:Ochratoxin_A.svg


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FTIR spectra of

(a) CH/ITO electrode

(b) CH-Fe3O4 nanobiocomposite

(c) IgGs/CH-Fe3O4 nanobiocomposite/ITO bioelectrode

(d) BSA/IgGs/CH-Fe3O4 nanobiocomposite/ITO bioelectrode



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X-ray diffraction pattern and transmission

electron microscopic studies of Fe3O4nanoparticles

CH-Fe3O4/ITO ; IgGs/ CH-Fe3O4/ITO and BSA/IgGs/ CH-Fe3O4/ITO10/5/2009 WCU Project, CNU,[email protected] 63

3 5x10-4 c


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-0.2 0.0 0.2 0.4 0.6 0.80.0

5.0x10-5

1.0x10-4

1.5x10-4

2.0x10-4

2.5x10

-4

3.0x10-4

3.5x10

d

b a

Current(A

)

Potential (V)

a) CH/ITO electrode,

b) CH-Fe3O4/ITO

c) r-IgGs /CH-Fe3O

4/ITO immunoelectrode

d) BSA/IgGs/CH-Fe3O4/ITO immunoelectrode

-0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.0

5.0x10-5

1.0x10

-4

1.5x10-4

2.0x10-4

2.5x10-4

3.0x10

-4

3.5x10-4

4.0x10-4

4.5x10-4

CLinear range: 0.5-6 ng dL-1

Detection limit: 0.5 ng dL-1

Sensitivity: 36 A/ng dL-1 cm-2Response time: 18 s

g

a

b

a

20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0

1.2

Potential(V)

Time (Second)

0 1 2 3 4 5 6

2.0x10-4

2.2x10-4

2.4x10-4

2.6x10-4

2.8x10-4

3.0x10-4

3.2x10-4

3.4x10-4

3.6x10-4

3.8x10-4

Current(A)

Concentration (ng dL-1)

Current(A)

Potential (V)

-0.2 0.0 0.2 0.4 0.6 0.8

2.0x10-7

4.0x10-7

6.0x10-7

8.0x10-7

1.0x10-6

1.2x10-6

1.4x10-6

1.6x10-6

1.8x10-6

2.0x10-6

2.2x10-6

2.4x10-6

0 20 40 60 80

0.150

0.155

0.160

0.165

0.170

0.175

Potential(V)

Time(s)

b

a

BLinear range: 1-6 ng dL-1

Detection limit: 1 ng dL-1

Sensitivity: 4.68 x 10-8

A/ng dL-1

cm-2

Response time: 35 s

0 1 2 3 4 5 61.5x10

-6

1.6x10-6

1.6x10-6

1.6x10-6

1.7x10-6

1.7x10-6

1.8x10-6

1.8x10-6

1.9x10-6

Current(A)

Concentration (ng)

g

aCurrent

(A)

Potential (A)10/5/2009 WCU Project, CNU,[email protected] 64


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Micro-organism based sensor



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Smallest to largest micro-organisms..

Prions

Viruses

Bacteria

Fungi



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Many types of microbial sensors have been developed as

analytical tools since the first microbial sensor was

studied by Karube et al. in 1977.

The microbial sensor consists of a transducer and

microbe as a sensing element. The characteristics of the

microbial sensors are a complete contrast to those of

enzyme sensors or immunosensors, which are highly

specific for the substrates of interest, although thespecificity of the microbial sensor has been improved

by genetic modification of the microbe used as the

sensing element.10/5/2009 WCU Project, CNU,[email protected] 67


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Microbial sensors have the advantages of tolerance to

measuring conditions, a long lifetime, and cost effectiveperformance, and have the disadvantage of a long

response time.

Microbial sensors result from the combination of a

microorganism with a transducer capable of detecting themetabolite involved.

Microorganisms possess enzymatic systems that effectbiological transformations. The immobilization of micro-organism on a transducer is first step in the construction

of a biosensor.10/5/2009 WCU Project, CNU,[email protected] 68

A self assembled monolayer based conductometric


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A self-assembled monolayer based conductometric

algal whole cell biosensor for water monitoring

Schematic representation of the immobilization of algal cells on the platinum

electrode modified by SAMs.

This unicellular green algae

has been chosen due to itsconsiderable ecological

advantages (it is ubiquist in all

dulcicol environments and is able

to accumulate large quantities of

pollutants).

Microchim Acta (2008) 163:179184

Bacterial whole-cell biosensors are very useful for toxicity measurements of various samples.

Semi-specific biosensors, containing fusions of stress-regulated promoters and reporter genes,

have several advantages over the traditional, general biosensors that are based on constitutively

expressed reporter genes.

Semi-specific biosensors are constantly being refined to lower their sensitivity and, incombination, are able to detect a wide range of toxic agents.



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The platinum electrodes modified by self-assembled monolayer

without (a) and with (b) immobilized algal cells

APA res (residual) for 30 min exposure to Cd2+ (10

mmol l1 TrisHCl, 50 mol l1 pNPP, pH 8.5)

Biosensor is sensitive to the presence of cadmium with a detection limit of

1 ppb.

It has been demonstrated that immobilization on a monolayer improves the

repeatability (RSD


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Conclusions:

Biocatalysis & Bioaffinity Sensors

Glucose,Urea,CholesterolDNAImmunosensorWhole Cell

Immunosensor

Some literature for Studies ( Week 2):


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Prospects of conducting polymers in biosensors, B.D Malhotra, A. Chaubey and S. P. Singh,

Analytica Chmica Acta , 578 (2006) 5974.Electrophoretically deposited conducting polymers for applications in organic electronics,Chetna

Dhand and B.D.Malhotra,Organic Electronics in Sensors & Biotechology, J.Shinar & Ruth Shinar(

Editors),McGraw-Hill),2008

Recent developments in urea biosensor, Gunjan Dhawan, G.Sumana and B.D.Malhotra, Biochemical

Engineering Journal ,2009 ,44 , pp. 42-52.

Electrocatalytic oxidation of hydrazine and hydroxylamine at gold nanoparticlepolypyrrole nanowire

modified glassy carbon electrode Jing Li, Xiangqin Lin, Sensors and Actuators B 126 (2007) 527535

Application of Polyaniline as glucose biosensor, K. Ramanathan, S. Annapoorni and B. D. Malhotra,

Sensors & Acturators B, 21, 1994, 165 69.Polythiophene gold nanoparticles composite film for application to glucose sensor, Pratibha Pandey,

Sunil K. Arya , Zimple Matharu, S. P. Singh, Monika Datta and B. D. Malhotra, Journal of Applied

Polymer Science , Vol. 110, 988994 (2008),

Cholesterol biosensor based on cholesterol esterase, cholesterol oxidase and peroxidase Immobilized on

conducting polyaniline films, Suman Singh, P. R. Solanki, M. K. Pandey and B. D. Malhotra, Sensors &Actuators B, 115,2006,pp534-541.

Microchim Acta (2008) 163:179184.

Fully integrated biocatalytic electrodes based on bioaffinity interactions, E Katz, V Heleg-Shabtai, A

Bardea, I Willner, Biosensors and Bioelectronics, 1998
http://linkinghub.elsevier.com/retrieve/pii/S0956566398000384http://linkinghub.elsevier.com/retrieve/pii/S0956566398000384


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Now,V0 is determined by the breakdown of ES to form product, which is determined by [ES]


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V0 =k2[ES] Substituting the value of V0, we haveOr, V0 = Km+ [S]

k2[E] [S]

This is the Michaelis-Menten equation, the rate

equation for a one-substrate enzyme-catalyzed reaction.

Km value determine s the affinity of biomolecule with the analyte . Lower is the value, higher isthe affinity

Or, V0 = Km +[S]

Vmax [S]

Now, Km +[S]

Vmax [S]V0

1=

[S]

Vmax [S]

Km

Vmax [S]V0

1= +

Km

Vmax [S]V0

1= +

1

Vmax

This form of the Michaelis-Menten equation is called

the Lineweaver-Burk equation



MichaelisMentenKinetics


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MichaelisMentenkinetics (alsoreferredtoasMichaelisMentenHenrikinetics)approximatelydescribesthekineticsofmany enzymes.

ItisnamedafterLeonorMichaelis andMaudMenten.Thiskineticmodelisrelevanttosituationswhereverysimplekineticscanbeassumed,(i.e.thereisnointermediateorproductinhibition,andthereisnoallostericity orcooperativity).

MorecomplexmodelsexistforthecaseswheretheassumptionsofMichaelisMentenkineticsarenolongerappropriate.

TheMichaelisMentenequationrelatestheinitialreactionratev0 tothesubstrateconcentration[S].Thecorrespondinggraphisahyperbolicfunction;themaximumrateisdescribedasvmax.

TheMichaelisMentenequationdescribestheratesofirreversiblereactions.AsteadystatesolutionforachemicalequilibriummodeledwithMichaelisMentenkineticscanbeobtainedwiththeGoldbeterKoshland equation.
http://en.wikipedia.org/wiki/Enzyme_kineticshttp://en.wikipedia.org/wiki/Goldbeter-Koshland_kineticshttp://en.wikipedia.org/wiki/File:Michaelis-Menten_saturation_curve_of_an_enzyme_reaction.svghttp://en.wikipedia.org/wiki/Goldbeter-Koshland_kineticshttp://en.wikipedia.org/wiki/Enzyme_kinetics

week 2 bioisensors 21sep2009 bio catalysis

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