murthal presentation
TRANSCRIPT
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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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