Murthal Presentation

8/13/2019 Murthal Presentation

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An acrylamide biosensor based onimmobilization of hemoglobin ontomultiwalled carbon nanotube coppernanoparticles polyaniline hybrid film

Bhawna Batra, Suman Lata, Madhu Sharma, C.S. Pundir*

Department of Biochemistry

M.D.University, Rohtak-124001(Haryana)

*e-mail: [email protected]


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INTRODUCTIONAcrylamide is well known

neurotoxin and potential carcinogen.Acrylamide is formed in reaction between

reducing sugar such as glucose and aminoacid asparagine.

Maillard reaction mechanism has been

proposed to account for its formation in high-

starch foods during cooking at high temperatures.


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High level of this compound has been

found in potato crisps, french fries and

several other foods.


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Earlier methods for Acrylamide

determinationChromatography techniques

GC-MS

GC-MS-MS

HPLC-MS

LC-MS-MS


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Non specificity

Time consuming

Lack of sensitivity

Sample preparation

Cumbersome

Needed costly equipments, expertise handling & have

complicated assay system in case of chromatographic

methods

Radiolabel materials used in case of mass spectrometric

methods

Low reproducibility


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Microelectronics

Amplifier

Bio-compatible layer

Sample/ analyte

Enzymes, Microorganisms,DNA, Whole cells, Anti-

bodies,Synthetic/ Semi-syntheticbiomolecules etc

Electrochemical, Optical,

Mass, Temperature

Signal

Data processing

A bio senso r is an analyt ical

device which conver ts a

biological response into an

measu rable sig nal.

Biosensor is used to

determine the concentrat ion

of substances and other

parameters of biological

interest without using the

biolog ical system direct ly.

Bio senso r is a reagent less

system in wh ich reagents are

already immobi l ized in i t

therefore need not to be


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Earlier crylamide biosensors based ondifferent supportsSr.No References Type of

Biosensor

Support for

Immobilization

Detection limit

1

2

Stoviecka et al.,

(2007)

Silva et al.,(2008)

Voltammetric

Amperometric

A carbon-paste electrode

modified with hemoglobin

(Hb).

Membrane in the presence

of glutaraldehyde and an

ammonium ion-selective

electrode.

1.2 X 10-10M

4.48 X 10-3 M

3 Krajewska et al.,

(2008)

Amperometric Glassy carbon electrode

coated with single-walled

carbon nanotubes

(SWCNTs).

1.0 X 10-9M

4 Silva et al., (2011) Electrochemical PUR hydrogel matrix. & p-

HEMA matrix.

6.31 X10-4 M


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Sensitivity

Stability

Membrane supports used forimmobilization

Limited electron communicationSurface area

Response time

Major problems of earlier

biosensors


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Hypothesis

Use of MWCNT, Aniline,CuNPs is

expected to improve analytic

performance of amperometric

acrylamide biosensor


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Properties of multiwalled carbon nanotubes(MWCNTs)

High aspect ratio

High conductivity

High stability of immobilized

enzyme

Large surface area

Good biocompatibility

Chemical stability

Fast electron communication


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Properties of Polyanil ine (PANI)

Facile Synthesis

High conducting

Non-toxic

Adhesive ability

ANILINE
http://localhost/var/www/apps/conversion/tmp/scratch_4//upload.wikimedia.org/wikipedia/commons/f/fe/Aniline.svghttp://en.wikipedia.org/wiki/File:Aniline-3D-balls.pnghttp://en.wikipedia.org/wiki/File:Aniline-3D-balls.pnghttp://localhost/var/www/apps/conversion/tmp/scratch_4//upload.wikimedia.org/wikipedia/commons/f/fe/Aniline.svg


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Properties of CuNPs

High surface to volume ratio

Fast and direct electron transfer

High surface energy

Biocompatibility


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AIMS AND OBJECTIVES1. Preparation of CuNPs2. Preparation of Hb/CuNP/cMWCNT/PANI/PG

electrode

4. Optimization of acrylamide biosensor based

onto Hb/CuNP/cMWCNT/PANI/PG

5. Application of acrylamide biosensor indetermination of acrylamide in different varieties

of potato chips

3. Characterisation of Hb/CuNP/cMWCNT/PANI/PGelectrode by SEM, FTIR, EIS and CV.


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Preparation of CuNPs100 ml 0.4 M CuSO

100 ml 0.3M NaBH4 + 10ml 0.1M NaOH

stirring at 60 C

(

CuNPs colloid

Characterisation of CuNPs by XRD, TEM and UV

spectra

4


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Results

UV-visible spectra of (A) CuNPs (B) X-ray diffraction (XRD)

pattern of CuNPs (C) Transmission electron microscopic

(TEM) image of CuNPs


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Dispersion of cMWCNT

cMWCNT (1mg)Add 4ml H2SO4 and HNO3(3:1) Ultrasonicate for 4 h

cMWCNT SuspensionDilute with 4ml DW

Ultrasonicate for 24 h

Uniformly dispersed black coloured

cMWCNT solution


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EDC and NHS treatment of cMWCNT

0.1 ml dispersed cMWCNT

Mixture of 0.5ml 0.2 M EDC + 0.5ml 0.2 M NHS

Adjust pH to 6.0 and keep at RT for 1 hr

EDC=N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride

NHS= N-Hydroxysuccinimide


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Electropolymerisation of Aniline onto PG electrode

cleaned with 0.05m alumina

slurry

Dipped in mixture of 400 l of Aniline solution

+ 25ml of 1N KCl

Twenty polymerisation cycles at -0.1V to

0.2V at scan rate of 20mV/s

PG electrode

PANI/PG eletrode


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Electrodeposition of cMWCNT CuNPs onto PG ElectrodePANI/PG eletrode

Dipped in mixture of 1 ml of EDC and NHS treated

cMWCNT + 400 l CuNPs + 25 ml of 1N KCl

20 polymerisation cycles at -0.1V

to 0.6 V at scan rate 50mV/s

CuNP/cMWCNT/PANI/PG electrode


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Cyclic voltamogram for electrodeposition of cMWCNT/CuNPs

composite film. Supporting electrolyte: 1M KCl solution; Scan

rate: 20 mV/s


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Immobilization of Hb onto CuNP/cMWCNT/PANI/PG

electrode

CuNP/cMWCNT/PANI/PG electrode

Dipped in 2ml of sodium acetate

buffer(pH 5, 0.2 M) containing Hb

(1mg/ml)

Hb/CuNP/cMWCNT/PANI/PG electrode (workingelectrode)


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Scheme of fabrication of enzyme electrode

PG electrode


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SEM images of (a) bare PG electrode (b)

cMWCNT/CuNPs/PANI/PG (c) Hb/cMWCNT/CuNPs/PANI/PG

electrode

(a)

(b) (c)


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FTIR spectra of PANI/PG (i) cMWCNT/CuNP/PANI/PG (ii)

Hb/cMWCNT/CuNP/PANI/PG (iii)

(i) (ii)

(iii)


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Impedance spectra of (a) bare PG electrode (b) Hb/cMWCNT/CuNP/PANI/PG

electrode (c) cMWCNT/CuNP/PANI/PG electrode

0.01-105Hz

(a) RCT= 630

(b) RCT= 580 (c) RCT= 400


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Construction of Acrylamide biosensor


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Differential pulse voltametry response of Hb/cMWCNT/CuNP/PANI/PG on addition

of 100 l (3.5 M) acrylamide in 30 ml of 0.2 M sodium acetate buffer (pH 5.5) at the

different potential at a scan rate of 20 mV/s


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Optimization of Acrylamide biosensor

Optimum pH

Incubation temperature

Effect of substrate concentration


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Effect of pH on Hb/cMWCNT/CuNP/PANI/PG electrode.


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Effect of temperature on Hb/cMWCNT/CuNP/PANI/PG

electrode


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Effect of acrylamide concentration on response of acrylamide biosensor based

on Hb/cMWCNT/CuNP/PANI/PG electrode


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Linearity5 nM to 75 mM

Detection Limit0.2 nM

Sensitivity72.5 A/nM/cm

2

Analytical performance of

Hb/cMWCNT/CuNPs/PANI/PG electrode

A l i l f dd d l id i h i


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Acrylamide

added (nM)

Acrylamide

found (nM)

% Recovery

- 12.5 -

20 31.90 95.402.7

40 52.19 97.56 3.1

Analytical recovery of added acrylamide in the potato crisps

as measured by Hb/cMWCNT/CuNP/PANI/PG electrode


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N Acrylamide(nM) CV (%)Within assay (6)

70 72.16667 2.35

72

75

71

73

72

Between assay (6)

78 78 4.5

80

80

77

80

73

Within and between assay coefficients of variation for

determination of acrylamide in potato crisps as measured by

Hb/cMWCNT/CuNP/PANI/PG electrode


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Application of biosensorPotato crisp (4g)

Shaking for 1hour at 60 c &Centrifuge for 20 min at 4500 rpm

Use for response measurement by biosensor (current, mA)

Acrylamide conc. extrapolated from standard curve b/w

acrylamide conc. v/s current

Homogenise in 100 ml deionised

water for 20 min for swelling

Homogenate (Acrylamide + DW etc.)

Homogenate (Acrylamide etc.)Added 2.5ml of carez I and

carrez II

A l id l l i diff t b d f t t i


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S.No. Brand nameAcrylamide

Concentration

(nM) MeanS.D.

(n=4)

1. A 85.70.3

2. B 73.090.4

3. C 68.340.2

4. D 55.450.1

Acrylamide levels in different brands of potato crisps as

measured by Hb/cMWCNT/CuNP/PANI/PG electrode


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Conclusion

The present electrode resulted in an improved analyticalperformance of acrylamide biosensor in terms of: High sensitivity 72.5 A/nM/cm2) Low applied potential 0.194V) Low detection limit 0.2nM) Wider working range 5nM-75mM )


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My Sincere thanks toMy advisor Prof. C.S. Pundir for his guidanceand encouragement during entire period of

research workHOD, Biochemistry for providinginfrastructure.CSIR, New Delhi for research fellowship.

Acknowledgement


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