An acrylamide biosensor based on immobilization of hemoglobin onto multiwalled carbon nanotube/copper nanoparticles/polyaniline hybrid film

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

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

*e-mail: chandraspundir@gmail.com

INTRODUCTION
Acrylamide is well known

neurotoxin and potential carcinogen.

reducing sugar such as glucose and amino acid asparagine.

Acrylamide is formed in reaction between

Maillard reaction mechanism has been

proposed to account for its formation in high-

starch foods during cooking at high temperatures.

High level of this compound has been found in potato crisps, french fries and several other foods.

Earlier methods for Acrylamide determination

Chromatography techniques
GC-MS GC-MS-MS

HPLC-MS
LC-MS-MS

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

Sample/ analyte

Enzymes, Microorganisms, DNA, Whole cells, Antibodies, Synthetic/ Semi-synthetic biomolecules etc Bio-compatible layer Electrochemical, Optical, Mass, Temperature

A biosensor is an analytical device which converts a biological response into an measurable signal. Biosensor is used to determine the concentration of substances and other parameters of biological interest without using the biological system directly. Biosensor is a reagent less system in which reagents are already immobilized in it therefore need not to be

Amplifier

Signal

Microelectronics

Data processing

Earlier Acrylamide biosensors based on different supports

Sr.No References
1 Stoviecka et al., (2007)

Type of Biosensor
Voltammetric

Support for Immobilization

Detection limit

A carbon-paste electrode 1.2 X 10-10 M modified with hemoglobin (Hb). Membrane in the presence 4.48 X 10-3 M of glutaraldehyde and an ammonium ion-selective electrode. Glassy carbon electrode coated with single-walled carbon nanotubes (SWCNTs). 1.0 X 10-9 M

Silva et al., (2008)

Amperometric

Krajewska et al., (2008)

Amperometric

Silva et al., (2011)

Electrochemical

PUR hydrogel matrix. & p- 6.31 X10-4 M HEMA matrix.

Major problems of earlier biosensors

Sensitivity Stability Membrane supports used for

immobilization Limited electron communication Surface area Response time

Hypothesis
Use of MWCNT, Aniline,CuNPs is expected to improve analytic performance of amperometric acrylamide biosensor

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

Properties of Polyaniline (PANI)

Facile Synthesis

High conducting
ANILINE

Non-toxic Adhesive ability

Properties of CuNPs
High surface to volume ratio
Fast and direct electron transfer High surface energy Biocompatibility

AIMS AND OBJECTIVES

1. Preparation of CuNPs 2. Preparation of Hb/CuNP/cMWCNT/PANI/PG electrode 3. Characterisation of Hb/CuNP/cMWCNT/PANI/PG electrode by SEM, FTIR, EIS and CV. 4. Optimization of acrylamide biosensor based onto Hb/CuNP/cMWCNT/PANI/PG 5. Application of acrylamide biosensor in determination of acrylamide in different varieties of potato chips

Preparation of( CuNPs

100 ml 0.4 M CuSO 4
100 ml 0.3M NaBH4 + 10ml 0.1M NaOH stirring at 60 C

CuNPs colloid

Characterisation of CuNPs by XRD, TEM and UV spectra

Results

UV-visible spectra of (A) CuNPs (B) X-ray diffraction (XRD) pattern of CuNPs (C) Transmission electron microscopic (TEM) image of CuNPs

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

cMWCNT Suspension
Dilute with 4ml DW Ultrasonicate for 24 h

Uniformly dispersed black coloured cMWCNT solution

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

Electropolymerisation of Aniline onto PG electrode

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

PANI/PG eletrode

Electrodeposition of cMWCNT &CuNPs onto PG Electrode

PANI/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

Cyclic voltamogram for electrodeposition of cMWCNT/CuNPs composite film. Supporting electrolyte: 1M KCl solution; Scan rate: 20 mV/s

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 (working electrode)

Scheme of fabrication of enzyme electrode

PG electrode

(a)

(b)

(c)

SEM images of (a) bare PG electrode (b) cMWCNT/CuNPs/PANI/PG (c) Hb/cMWCNT/CuNPs/PANI/PG electrode

(i)

(ii)

(iii)

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

0.01-105 Hz (a) RCT = 630 (b) RCT = 580 (c) RCT = 400

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

Construction of Acrylamide biosensor

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

Optimization of Acrylamide biosensor

Optimum pH Incubation temperature
Effect of substrate concentration

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

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

Effect of acrylamide concentration on response of acrylamide biosensor based on Hb/cMWCNT/CuNP/PANI/PG electrode

Analytical performance of Hb/cMWCNT/CuNPs/PANI/PG electrode

Linearity
5 nM to 75 mM

Detection Limit
0.2 nM

Sensitivity
72.5 A/nM/cm2

Analytical recovery of added acrylamide in the potato crisps as measured by Hb/cMWCNT/CuNP/PANI/PG electrode Acrylamide added (nM) Acrylamide found (nM) 12.5 % Recovery

20

31.90

95.40 2.7

40

52.19

97.56 3.1

Within and between assay coefficients of variation for determination of acrylamide in potato crisps as measured by Hb/cMWCNT/CuNP/PANI/PG electrode
N Within assay (6) 70 72 75 71 73 72 Between assay (6) 78 80 80 77 80 73 Acrylamide(nM) 72.16667 CV (%)

2.35

78

4.5

Application of biosensor
Potato crisp (4g)
Homogenise in 100 ml deionised water for 20 min for swelling

Homogenate (Acrylamide + DW etc.)

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

Homogenate (Acrylamide etc.)

Added 2.5ml of carez I and carrez II

Use for response measurement by biosensor (current, mA) Acrylamide conc. extrapolated from standard curve b/w acrylamide conc. v/s current

Acrylamide levels in different brands of potato crisps as measured by Hb/cMWCNT/CuNP/PANI/PG electrode

S.No.

Brand name

Acrylamide Concentration (nM) MeanS.D. (n=4)

1.
2. 3. 4.

A
B C D

85.70.3
73.090.4 68.340.2 55.450.1

Conclusion

The present electrode resulted in an improved analytical performance 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 )

Acknowledgement
My Sincere thanks to My advisor Prof. C.S. Pundir for his guidance and encouragement during entire period of research work HOD, Biochemistry for providing infrastructure. CSIR, New Delhi for research fellowship.