research article fabrication and optimization of che/cho...

Research ArticleFabrication and Optimization ofChEChOHRP-AuNPsc-MWCNTs Based Silver Electrode forDetermining Total Cholesterol in Serum

Kusum Lata1 Vikas Dhull2 and Vikas Hooda1

1Centre for Biotechnology Maharshi Dayanand University Rohtak Haryana 124001 India2Department of Bio amp Nano Technology Guru Jambheshwar University of Science amp Technology HisarHaryana 125001 India

Correspondence should be addressed to Vikas Hooda vikascbtmdugmailcom

Received 12 October 2015 Accepted 20 December 2015

Academic Editor Hans-Jurgen Apell

Copyright copy 2016 Kusum Lata et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The developed method used three enzymes comprised of cholesterol esterase cholesterol oxidase and peroxidase for fabrication ofamperometric biosensor in order to determine total cholesterol in serum samples Gold nanoparticles (AuNPs) and carboxylatedmultiwall carbon nanotubes (cMWCNTs) were used to design core of working electrode having covalently immobilized ChOChE and HRP Polyacrylamide layer was finally coated on working electrode in order to prevent enzyme leaching Chemicallysynthesised Au nanoparticles were subjected to transmission electron microscopy (TEM) for analysing the shape and size of theparticlesWorking electrode was subjected to FTIR and XRDThe combined action of AuNP and c-MWCNT showed enhancementin electrocatalytic activity at a very lowpotential of 027 VThepH7 temperature 40∘C and response time of 20 seconds respectivelywere observed The biosensor shows a broad linear range from 05mgdL to 250mgdL (001mMndash583mM) with minimumdetection limit being 05mgdL (001mM) The biosensor showed reusability of more than 45 times and was stable for 60 daysThe biosensor was successfully tested for determining total cholesterol in serum samples amperometrically with no significantinterference by serum components

1 Introduction

With increasing mortality rate due to cardiovascular diseases(CVDs) in present scenario it is necessary to develop moreadvanced methods for diagnosis These advanced diagnosticmethods should sense the disease at an early stage andprevent it from being fatal Cholesterol is considered asrisk factor when the blood cholesterol is above the normallevel and so causes the risk of cardiovascular disease (CVD)[1] In addition high blood pressure or diabetes may alsoincrease the risk even more Elevated level of cholesterolin blood is one of the factors responsible for coronaryartery disease hypertension nephritic syndrome or cirrhosisatherosclerosis heart attack and stroke [2] When too muchof cholesterol circulates in the blood it slowly builds uplayer inside the inner walls of the arteries which makes themnarrow and less flexible [2]

There are several methods such as calorimetric method[3] liquidgas chromatographic method HPLC spectropho-tometric methods and thermistor based assays used to deter-mine cholesterol concentration [4ndash8]The abovemethods arefast accurate and sensitive but suffer from limitations theyrequire trainedmanpower they require sample pretreatmenta lot of time is required and they are only confined tolaboratory lacking on-site monitoring A promising alternateto these methods is a biosensor which is able to determinethe cholesterol more rapidly and to a more sensitive levelwith a plus point of portability [9] The biosensor assemblycomprised three components biorecognition layer (enzymein this case) which specifically interacts with analyte andgenerates the biological response a transducer which sensesthese responses and processes the biological response to ameasurable signal and a display unit which is the thirdcomponent Transducers are an important part of assembly

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2016 Article ID 1545206 11 pageshttpdxdoiorg10115520161545206

2 Biochemistry Research International

of biosensor in determining accuracy and sensitivity [10]Immobilizing ChO on various supports helps in directlydetermining concentration of cholesterol in different sam-ples As serum cholesterol is present in the form of ester inorder to determine total cholesterol ChE is required alongwith ChO Cholesterol esterase first hydrolyses the esterifiedcholesterol and is further oxidized by cholesterol oxidasefor producing cholest-4-en-3-one and hydrogen peroxide(H2O2) In amperometric biosensors electric potential is

applied onH2O2which get oxidized to produce electrons and

the flow of electrons produces current The current gener-ated is directly proportional to cholesterol concentration inserum

Cholesterol ester +H2O

Cholesterol Esterase997888997888997888997888997888997888997888997888997888997888997888997888997888997888rarr Cholesterol + fatty acids

Cholesterol +O2

Cholesterol Oxidase997888997888997888997888997888997888997888997888997888997888997888997888997888997888rarr Cholest-4-en-3-one +H

2O2

H2O2

Electric Potential997888997888997888997888997888997888997888997888997888997888997888rarr 2H+ +O

2+ eminus

(1)

A new approach to reduce interference is the use ofhorseradish peroxidase (HRP) HRP has redox centre associ-ated with ferrohemeferriheme pair Conversion of reducedstate to oxidized state is done through direct transfer ofelectrons In its 3D configuration HRP has heme grouppresent in outer region which enhances the direct transfer ofelectrons between redox centre and conducting sites presenton transducer [11] Regeneration of HRP is done via directelectron transfer

H2O2+ 2H+ +HRPRed 997888rarr 2H2O +HRPOxd

HRPOxd + 2eminus

larrrarr HRPRed

(2)

Immobilizing enzyme on suitable support may lower theMichaelis constant 119870

119898or it may enhance bimolecular rate

constant which improves the biosensor performance [12ndash15]Cholesterol hydrolysing enzymes ChO and ChE have beenimmobilized on conducting polymers [16] like polypyrrolefilms [17] PVC Strip [18] PBpolypyrrole (PPy) compositefilm [19] cellulose acetatecarbon electrode [20] celluloseacetatePt electrode [21] electropolymerised PPy films [22]poly 3-thiopheneacetic acid filmPt electrode [23] and PANIndashpTSA-AgITO [11] and sol-gel films [24] Evolution ofnanoscale materials has revolutionised the field of biosensorsalsoThenanoscale dimensions their graphitic surface chem-istry and electrocatalytic properties of carbon nanotubesmake them an interesting material for sensing purposeSingle andmultiwalled carbon nanotubes [25 26] MWCNTs[27] CNT-chitosanGCE [28] Fe

3O4-SiO2MWCNT [29]

graphene modified graphite electrode [30] graphenePt NPs[31] PtAu ZnO nanorods [32] NSPANI-AuNP-GRITO[33] AuNPsAu electrode [34] graphite-teflon matrix [35]and Pt-Pd bimetallic nanoparticle decorated grapheme [36]

are some of the supports which have been used for thetransduction of generated biological signals to electricalsignals for cholesterol determination PVC has also beenused to immobilize cholesterol hydrolysing enzymes ChOand ChE were covalently immobilised on surface of PVCbeaker which act as reaction cell and HRP was incorporatedin carbon electrode [37]

In the present method the combination of three enzymeswas used for the development of electrochemical biosensorby immobilization of three enzymes (ChO ChE and HRP)on AuNPs and c-MWCNT based electrode for determiningcholesterol in serum cholesterol

2 Materials and Methods

21 Chemicals and Reagents Cholesterol esterase was puri-fied from Pseudomonas species (1658Unitsg) 4-amino-phenazone Triton X-100 cholesteryl acetate and carboxy-lated multiwalled carbon nanotubes (c-MWCNTs) were pur-chased from Sigma Chemical Co USA Cholesterol oxidasefrom Streptomyces sp (500 units10mg) and horseradishperoxidase (HRP) (80Umg) were obtained from SISCOResearch Laboratory Pvt Ld Mumbai Silver wire was pur-chased from local market All other chemicals used duringthe experimentation were of analytical grade Gold nanopar-ticles (AuNPs) were synthesised at Centre for BiotechnologyMaharshi Dayanand University Rohtak

22 Instrumentation Potentiostat (PSTAT mini 910 Met-rohm Switzerland) was used for all electrochemical stud-ies Ultrasonication was done with Chrom Tech UltrasonicLiquid Processor For TEM JEM-2100F microscope (AIRFJNU New Delhi) was used Varian 7000 FTIR spectrometer(AIRF JNU New Delhi) was used for performing Fouriertransform infrared (FTIR) spectroscopy X-ray diffraction(XRD) facility for gold nanoparticles and c-MWCNTs wasprovided byDepartment of PhysicsMD University RohtakShimadzu corporationUV 2450 spectrophotometer was usedfor spectrophotometric measurement at Centre for Biotech-nology

23 Synthesis of Gold Nanoparticles Chemical synthesis ofgold nanoparticles was done using citrate reduction method[38] 100mL of 0001 gold chlorous acid (HAuCl

4) was

boiled at 97∘C with continuous stirring using magnetic stir-rer Then the 1 sodium citrate solution was added within 4to 5min solution turns wine red and was further heated for 4to 5min and then cooled down at room temperature Finallycentrifuged pellets were dried for further use Transmissionelectron microscopy (TEM) of the synthesised AuNPs wasdone on commercial basis at AIRF JNU New Delhi for theconfirmation of shape of newly synthesised nanoparticles andsize range of particles

24 Fabrication of ChEChOHRP-AuNPsc-MWCNT Modi-fied Silver Working Electrode Fabrication strategy of work-ing electrode comprised mixing c-MWCNTs and goldnanoparticles (AuNPs) in paraffin oil in fixed proportion

Biochemistry Research International 3

until a consistent paste is obtained A plastic hollow tube(3 cm times 4mm) was filled with the nanomaterial passedobtained aboveThe silver (Ag) wire was cleanedwith ethanoland ddH

2O by sonication and then inserted in the paste

filled tube for achieving the electrical contactThen the aboveelectrode was allowed to dry and then immersed in amixtureof ChO ChE and HRP solution for 2 hrs so that the enzymecan bind on electrode surface The carboxyl (COOH) grouppresent on MWCNTs forms amide bond with the aminogroup present on the enzyme leading to the formation ofcovalent bondThen the electrode surface was covered with athin film of polyacrylamide (PAA) which helps in preventingenzyme from leaching

25 Characterisation of Carbon BasedWorking Electrode Thenanomaterial based core of working electrode (AuNPsc-MWCNTsAg electrode) and enzyme bound electrode(ChEChOHRP-AuNPc-MWCNTsAg electrode) werecharacterised using Varian 7000 FTIR spectrometer (atAIRF JNU New Delhi) before coating with PAA Fouriertransform infrared (FTIR) spectroscopy sample preparationwas done in KBr X-ray diffraction (XRD) studies were alsoperformed to analyse the stability of c-MWCNTs on mixingwith AuNPs Scanning ElectronMicroscopy (SEM) was donefor analysing the modifications in the surface morphologyof the working electrode at different stages of fabrica-tion

26 Assembly of Cholesterol Biosensor An amperometriccholesterol biosensor was assembled using ChEChOHRP-AuNPsc-MWCNTAg as enzyme immobilized working elec-trode AgAgCl pure as reference and Pt wire as auxiliaryelectrode connected via Potentiostat

27 Electrochemical Study of ChOChEHRP-AuNPsc-MWCNT Ag Electrode Cyclic voltammetry (CV) studiesElectrochemical Impedance Spectroscopy (EIS) and all otheramperometric detections throughout the experiment wereperformed on a Potentiostat (PSTAT mini 910 Metrohm)using three-electrode system in electrochemical cell Allof the cyclic voltammetric measurements were performedat room temperature and were continuously recordedfrom minus04 to +04V with different scan rates (25 50 and100mVs)

28 Kinetic Study of Present Method Kinetic propertiesof newly developed method were studied which includeoptimum pH temperature response time effect of substrate(cholesteryl acetate) concentration119870

119898 and 119868max

29 Evaluation of the Present Method Evaluation of presentmethod was carried out with respect to linearity minimumdetection limit and percent analytical recovery along withprecision and accuracy The effect of interfering species onperformance of biosensor was studied The storage stabilitywith time and repeatability of present method were alsoanalyzed

291 Linear Working Range and Minimum Detection LimitLinearity range and minimum detection range were calcu-lated by plotting values against the values of standard graph

292 Analytical Recovery Reliability of the method wastested using different concentrations of cholesteryl acetate(100mgdL and 200mgdL) (233mM and 466mM) byspiking the serum samples and mean analytical recoveries ofcholesteryl acetate were determined

293 Precision The reproducibility of the present methodand the total cholesterol level was determined in the sampleon the same day (within batch) and in the same sampleafter storage at 4∘C for one week (between batches) coef-ficients of variation (CVs) were calculated for the presentmethod

294 Accuracy For determining accuracy of newly devel-oped method the 10 serum samples were spiked withcholesteryl acetate and then tested by standard Bayerrsquos enzokit (119909) and also by present method (119910) then the valuesobtained by both methods were co-related and regressionequation was obtained

295 Effect of Interfering Substances The response of thepresent method was analyzed in the presence of interferingsubstances found in serum such as pyruvate glucose citrateCa2+ uric acid ascorbic acid acetone urea and bilirubinThe effect of interference by these substances was determinedby adding the interfering species in the reaction mixture oneby one at their physiological concentration

210 Storage Stability and Reusability of the Present MethodBefore every use the enzyme electrode was cleaned usingwashing buffer (001M phosphate buffer saline pH 72 with01 tween 20) The stability of the working electrode wasinvestigated over a period of 60 days when stored at 4∘CTheresponse of working electrode was measured once in every 5days

211 Application of the Newly Developed Method Bloodsamples (1mL each) with different age and sex group werecollected from healthy persons and persons suffering fromdisease due to elevated cholesterol level after 12 hours offasting at Pt BDS PGIMS Rohtak The blood samples werecentrifuged at 1500timesg for 5min and resulting supernatant(serum) was collected for determination of cholesterol levelThe test for the serum total cholesterol was carried outby the present method ChO ChE and HRP immobilizedonto the working electrode surface catalyze the hydrolysisof cholesteryl acetate and produce H

2O2which is then

oxidized by HRP HRP itself is regenerated as it passes theelectron to the electrode directly Current produced in theprocess is directly proportional to the concentration of theH2O2produced which itself is directly proportional to total

cholesterol


(a) (b)

116nm266nm 182nm

126nm

(c)

Figure 1 Transmission electron microscopy (TEM) of gold nanoparticles (a) Aggregates of gold nanoparticles (b) dimensions of a tuft ofnanoparticles and (c) enlarged image for size analysis

3 Results and Discussion

31 Characterization of Gold Nanoparticles (AuNPs) Thelab synthesised AuNPs were characterized by transmissionelectron microscopic (TEM) study The data showed thatnanoparticles were of spherical shape ranging from 10 to30 nm in size (Figures 1(a) 1(b) and 1(c))

32 Analysis of Gold Nanoparticles Decorated c-MWCNTsby XRD Structural stabilization of MWCNTs and workingelectrode (AuNPsc-MWCNT) was examined by XRD char-acterization as analysed characteristic peaks of c-MWCNTs(Figure 2 Curve (a)) persisted even in the diffraction peaksof AuNPs and c-MWCNT paste (Figure 2 Curve (b)) Peaksappeared at 196 and 215 which are the unique characteristicpeaks of c-MWCNTs which also are present in Curve (b)ensuring that the typical graphitic signature structure of c-MWCNTs is stable even when AuNPs were mixed with c-MWCNT

33 Confirmation of Covalent Immobilization by FTIR FTIRspectra of AuNPsc-MWCNTs showed absorption peaks near

Relat

ive i

nten

sity

(au

)

(a)

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

100

002 10

1

102

110

103

112

10 20 30 40 50 60 70 8002120579 (deg)

Figure 2 X-ray diffraction patterns of c-MWCNTs (a) andAuNPc-MWCNTs (b)

1011 cmminus1 1545 cmminus1 and 2250 cmminus1 which are signaturepeaks of CNTs After immobilization of enzyme that isChEChOHRP-AuNPsc-MWCNTs a new signature peak


(a) (b)

Figure 3 SEM images of (a) electrode without enzyme and (b) with enzyme

appeared at 1537 cmminus1 and 1630 cmminus1 and a broad peak rose at3287 cmminus1 which is due to carbonyl stretch and amide bond

34 Surface Morphology of the Newly Fabricated ElectrodeSurface morphology of the working electrode was analysedusing Scanning Electron Microscopy at the different stagesof fabrication A visual change had been noticed in themorphological characteristic of the electrode surface withthe deposition of material The SEM images of workingelectrode without and with enzyme have been shown inFigure 3

35 Electrochemical Analysis of ChEChOHRP-AuNPsc-MWCNT Ag Electrode The electrochemical study of work-ing electrodewas carried out by using cyclic voltammetry Fordetermination of reduction potential of HRP immobilized onelectrode core it was immersed in 001M H

2O2for 30min

and then washed with distilled water before performingcyclic voltammetry Cyclic voltammetry of ChEChOHRP-AuNPsc-MWCNT Ag electrode was reported from minus04and +08V at particular scanned rates of 25mVs 50mVsand 100mVs (Figure 4) An optimum scan rate of 50mVswas perceived for the above fabricated biosensor The repro-ducibility of the fabricated biosensor was examined by run-ning four CV cycles at scan rate of 50mVs (Figure 5) Duringthe entire examination a sharp peak at 027V was observedthis arises as a result of oxidation ofHRPpresent on electrodeSo it was estimated that ChEChOHRP-AuNPsMWCNTAg electrode offered ultimate signal and least noise at 027Vand was further employed to analytical determinationsThe biosensor works at lower potential in comparison toother available biosensors which is due to the large sur-face area of gold nanoparticles and their highly conductivenature

36 Kinetic Study of the Present Method To improve theworking performance of the biosensor various parameterssuch as working potential temperature time and concen-tration of substrate and pH value on the fabricated biosensorwere analyzed

361 Response for Working Potential The response of cur-rent with varying applied potential on the biosensor had

350

300

250

200

150

100

50

0

minus04 minus02 0 02 04 06 08

E (V)

I(m

A)

Figure 4 Cyclic voltammogram of ChEChOHRP-AuNPsc-MWCNTs Ag electrode at various scan rates

350

300

250

200

150

100

50

00 minus02 0 02 04 06 08

E (V)

I(m

A)

A

B

Figure 5 Cyclic voltammogram (A) ChEChOHRP-AuNPsc-MWCNTs (B) bare silver electrode

been shown in Figure 6 The working potential was skippedfrom 00V to 07 V With the increase in working poten-tial increase in steady-state current response was observedFirstly it showed a significant increase in current value from+01 V to +027V and then reached a level from +027V to+05 V and declined slightly after +05 V Therefore +027V


0

005

01

015

02

025

03

035

Curr

ent (

mA

)

01 02 03 04 05 06 070Applied potential (V)

Figure 6 Response of current with applied potential

00002000400060008

001001200140016

Curr

ent (

mA

)

64 66 68 7 72 74 76 7862pH

Figure 7 Effect of pH on current response of the present method

was selected as the working potential for detection of choles-terol by the biosensor

362 Response for pH The pH of reaction buffer was variedin the pH range pH 45 to 80 to find the optimum pHusing sodium succinate buffer (pH 45ndash55) and sodiumphosphate buffer (pH 60ndash80) at a final concentration of002M All other variants were of standard assay conditionexcept pH Optimum pH was found to be 7 for the biosensor(Figure 7)

363 Response for Temperature The response of biosensorfor continuous raise of temperature by 5∘C from 20∘Cto 60∘C was interrogated It was found that the biosen-sor showed maximum response at 40∘C (Figure 8) Themicroenvironment provided by support used for immobiliza-tion makes it thermally stable and maintains its biologicalactivity

364 Response with Time The amperometric response wasmeasured from 5 s to 60 s at interval of 10 sThe response timeincreases from 5 to 20 s and later attains stability (Figure 9)

365 Response for Substrate Concentration A linear relation-ship among the substrate concentration from 05mgdL to250mgdL and current was observed The current approach

25 30 35 40 45 50 55 6020Temperature (∘C)

05

1015202530354045

Curr

ent (

mA

)

Figure 8 Effect of incubation temperature on the response ofpresent method

minus0050

00501

01502

02503

03504

Resp

onse

(mA

)

0 10Response time (s)

20 30 40 50

Figure 9 Response time of the current method

100 200 300 400 500 600 7000Concentration (mgdL)

0

005

01

015

02

025

03

035

04

Curr

ent (

mA

)

Figure 10 Effect of cholesteryl acetate concentration on the presentmethod

gave a hyperbolic curve between current response andcholesteryl acetate concentration A significant response wasobserved up to a concentration of 500mgdL (Figure 10)

366 Resolving 119870119898

and 119868max Values The 119870119898

(app) and119868max (app) values were resolved by estimating the slope andintercept for the reciprocal plot of current versus cholesterylacetate concentrations that is double reciprocal plot or


y = 65231x + 11096

minus10123456789

0 002 004 006 008 01 012minus002(sminus1)

(Vminus1)

Figure 11 Lineweaver-Burk plot of the present method

Table 1 Analytical recovery calculated by using added cholesterylacetate in serum sample

Cholesteryl acetateadded (mgdL)

Cholesteryl acetatefound (mgdL)(mean (119899 = 5))

recovery SD

Nil 17374 mdash mdash

100 27133 9911 088

200 36879 9867 093

Lineweaver-Burk plotThe119870119898and 119868max values obtained were

587mgdLminus1 and 09mAsminus1 respectively (Figure 11)

37 Assessment of the Current Method

371 Linear Range for Working and Minimum Limit of Detec-tion Both the linear range for working and minimum limitof detection of a biosensor are considered while interrogatingthe performance of a biosensor In the current methodthe standard graph between substrate concentration andcurrent response was used for estimation of linear rangefor working and minimum limit of detection The linearrange for working offered by current biosensor is 05mgdLndash250mgdL andminimum limit of detection limit is 05mgdLwhich is far better than previous announced biosensors(Table 1)

372 Analytical Recovery By analytical recovery of addedenzyme cholesteryl acetate reliability of the current biosensorwas calculated (Table 1) The mean analytical recoveries for100mgdL and 200mgdL of added cholesteryl acetate were991 and 986 respectively

373 Precision The concentration of total cholesterol wascalculated on the same day (within batch) and in thesame sample after storage at 4∘C for one week (betweenbatches) in the serum sample repeatedly to examine thereproducible nature of the current biosensor The values forcoefficients of variation (CVs) were lt 061 and lt 098 forwithin batch and between batches respectively (Table 2)The

Table 2 Within batch and between batches coefficients of variationfor determination of total cholesterol in serum samples

119899

Total cholesterol(mgdL)

(mean plusmn SD)CV ()

Within batch (6) 17002 plusmn 104 061Between batches (6) 16996 plusmn 16 098

Chol

este

ryl a

ceta

te co

ncen

trat

ion

(gd

L)

R2 = 09885

y = 09424x + 00151

02 04 06 08 1 120Cholesteryl acetate concentration (gdL)

in serum by enzo kit method

0

02

04

06

08

1

12

in se

rum

by

pres

ent m

etho

d

Figure 12 Correlation between cholesteryl acetate concentrationsin serum determined by standard enzo kit method (119909-axis) and bythe present method (119910-axis)

results were far better than various earlier reported methods(Table 6)

374 Accuracy The level of cholesteryl acetate added toserum samples was computed by using the standard methodthat is enzo kit (119909) and current method (119910) and accu-racy of the current biosensor was analysed (Figure 12) Theregression equation 119910 = 09424119909 + 00151 was used toattain cholesteryl acetate results for newly fabricated workingelectrode with a good correlation (1198772 = 0988) as comparedto standard method All these results showed that the currentbiosensor offers excellent accuracy

375 Interference Study Among the various serum sub-stances investigated for possible interference on the responseof the present method none caused any significant interfer-ence on the performance of cholesterol biosensor Effect ofdifferent substances on the working of biosensor has beenshown in Table 3

38 Reliability and Stability The biosensor was reliable andstable When stored at 4∘C the sensor was stable up to onemonth and after 35 uses in second month its performancewas reduced (Figure 13) Only 45 activity of the biosensorremained at the end of the 2nd month (Figure 14) In com-parison to the reported biosensors it showed better stability(Table 6) The immobilization of ChO ChE and HRP onAuNPsc-MWCNTs Ag based working electrode was cred-ited by good stability reliability and reproducibility Furtherenzyme degrading and leaching was prevented by coating


Table 3 Effect of different serum substances on the working of CHbiosensor

Compoundsadded

Final conc(physiologicalconc) (gL)

relative response

None mdash 100Glucose 090 99Uric acid 003 100Ascorbic acid lt17 101Urea 010 98Ca2+ 115 99Acetone 002 98Bilirubin 22 100

0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)

10 20 30 400Number of uses

Figure 13 Reusability of the present method

0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)

10 20 30 40 50 60 700Storage time (days)

Figure 14 Storage stability of the present method

the biosensor with polyacrylamide which also increases thestability of biosensor

39 Application of the Newly Developed Method Concen-tration of cholesterol was determined in different samplesby the newly developed biosensor Table 4 represents thetotal cholesterol in serum of probably healthy individualsincluding males and females of different age group computedby the current biosensor The total cholesterol level wasfound between 15417 and 22589mgdL for males and 14456and 22558mgdL for females which is in normal range

Table 4 Total cholesterol level in serum of probably healthyindividuals calculated by current biosensor

Age group(119899 = 08) Sex

Total cholesterol inserum mgdL(mean plusmn SD)

lt10 M 15417 plusmn 204F 14456 plusmn 305

11ndash20 M 17014 plusmn 657F 16395 plusmn 431





61 amp above M 22589 plusmn 605F 22558 plusmn 903

Table 5 Working parameters of the newly developed method

Parameters Present methodpH 7Temperature (∘C) 40Working potential (V) +027119870119898

(app) (mgdL) 587 (136mM)119868max (app) (mA) 09Detection limit (mgdL) 05 (001mM)Linearity (mgdL) 05ndash250 (001mMndash58mM)Response time (sec) 20Storage stability (days) 60

Table 5 outlines the different working parameters of freshlyfabricated biosensor

4 Conclusion

A fresh biosensor was fabricated exploiting the con-ductive properties of Au nanoparticles and c-MWCNTpaste Covalent immobilisation of ChO ChE and HRPon the working electrode was insured by FTIR ThisChEChOHRP-AuNPsc-MWCNTs modified Ag electrodeexhibits enhanced sensitivity in a linear range of 05mgdLndash250mgdL (001mMndash583mM) quick response time (lt20 s)low limit of detection (05mgdL) (001mM) reproducibilityof more than 55 times and stability of 2 months A goodcorrelation (1198772 = 0988) was obtained with that of standardmethod Further the working electrode was coated withpolyacrylamide polymer which provides long time stabilityand high reusability to the biosensor The work contributeda competent amperometric approach for detection of totalcholesterol in serum


Table 6 Comparison of the present method with previously reported biosensor for total cholesterol determination

TransducerMethod ofenzyme

immobilization

Workingpotential

Responsetime

Detectionlimit Linearity Storage

stabilityReference

Laponite claynanoparticles-pol((12-pyrrol-1-dodecyl)triethylammoniumtetrafluoroborate)Pt disk electrode

ChO ChEenzyme

Entrapment

053V versusAgAgCl 50 sec 20 120583M mdash 20 days [39]

Screen printed graphite electrode

ChO ChE HRPK4Fe(CN)6Physical

adsorption

minus02V versusAgAgCl mdash 281mM 281ndash13mM mdash [40]

PolydiaminonaphthalenePt disk ChO ChEEntrapment

07 V versusAgAgCl 15 sec 97 120583M Up to 08mM mdash [41]

MWCNscreen printed carbonelectrode


adsorption

03 V versusAgAgCl 180 sec 100mgdL 100ndash

400mgdL 2 months [27]

3-Aminopropyl-modifiedcontrolled-poreglass(APCEG)rotating disk

ChO ChE HRPCovalent

cross-linking viaGlutaraldehyde

minus015 Vversus

AgAgCl withTBC asmediator

mdash 119 nM 12120583Mndash1mM 25 days [42]

PANIITO

ChO ChECovalent



500mgdL 6 weeks [43]

HRP incorporated carbon paste

ChO ChECovalent

cross-linking onPVC beaker

minus05 V versusAgAgCl 20 sec 25mgdL 50ndash

550mgdL 100 days [38]

Nanoporous Au networks directlygrown on a titanium substrate

ChO ChE HRPPhysical

adsorptionChitosan used

as glue

Cyclicvoltammetry mdash 05mgdL 097ndash78mM 60 days [44]

ZnOndashCuO composite matrix grownonto ITO coated corning glass

ChO ChEPhysical

adsorption

Cyclicvoltammetry 5 sec 05mM 05ndash12mM mdash [45]

c-MWCNTAuNP

ChO ChE HRPCovalent

cross-linking viac-MWCNT

027V versusAgAgCl 20 sec 05mgdL 05ndash

300mgdL 60 days This work

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to Department of Physics Mahar-ishi Dayanand University Rohtak for providing the XRDfacility and Jawaharlal Nehru University New Delhi forproviding facilities for SEM TEM and FTIR analysis Specialthanks are due to PGIMS Rohtak for providing serumsamples

References

[1] M Kratz ldquoDietary cholesterol atherosclerosis and coronaryheart diseaserdquo in Atherosclerosis Diet and Drugs vol 170 ofHandbook of Experimental Pharmacology pp 195ndash213 SpringerBerlin Germany 2005

[2] G J Miller ldquoHigh density lipoproteins and atherosclerosisrdquoAnnual Review of Medicine vol 31 pp 97ndash108 1980

[3] S Kroger and B Danielsson ldquoCalorimetric biosensorsrdquo inHandbook of Biosensors and Electronic Noses Medicine Foodand the Environment pp 279ndash298 CRC Press Oxford SciencePublications New York NY USA 1997

[4] Y Kayamori H Hatsuyama T Tsujioka M Nasu and YKatayama ldquoEndpoint colorimetric method for assaying total


cholesterol in serum with cholesterol dehydrogenaserdquo ClinicalChemistry vol 45 no 12 pp 2158ndash2163 1999

[5] D M Amundson and M Zhou ldquoFluorometric method for theenzymatic determination of cholesterolrdquo Journal of Biochemicaland Biophysical Methods vol 38 no 1 pp 43ndash52 1999

[6] T C Huang V Wefler and A Raftery ldquoA simplified spec-trophotometricmethod for determination of total and esterifiedcholesterol with tomatinerdquo Analytical Chemistry vol 35 no 11pp 1757ndash1758 1963

[7] C C Allain L S Poon C S Chan W Richmond and P C FuldquoEnzymatic determination of total serum cholesterolrdquo ClinicalChemistry vol 20 no 4 pp 470ndash475 1974

[8] V Raghavan K Ramanathan P V Sundaram and B Daniels-son ldquoAn enzyme thermistor based assay for total and freecholesterolrdquo Clinica Chimica Acta vol 289 no 1-2 pp 145ndash1581999

[9] A Vikas and C S Pundir ldquoBiosensors future analytical toolssensors amp transducersrdquo FJournal vol 76 pp 935ndash936 2007

[10] S V Dzyadevych V N Arkhypova A P Soldatkin A VElrsquoskaya C Martelet and N Jaffrezic-Renault ldquoAmperometricenzyme biosensors past present and futurerdquo IRBM vol 29 no2-3 pp 171ndash180 2008

[11] R K Basniwal R P S Chauhan S Parvez and V K JainldquoDevelopment of a cholesterol biosensor by chronoamperomet-ric deposition of polyaniline-Ag nanocompositesrdquo InternationalJournal of Polymeric Materials and Polymeric Biomaterials vol62 no 9 pp 493ndash498 2013

[12] U Hanefeld L Gardossi and E Magner ldquoUnderstandingenzyme immobilisationrdquo Chemical Society Reviews vol 38 no2 pp 453ndash468 2009

[13] A Sassolas L J Blum and B D Leca-Bouvier ldquoImmobiliza-tion strategies to develop enzymatic biosensorsrdquo BiotechnologyAdvances vol 30 no 3 pp 489ndash511 2012

[14] C Spahn and S D Minteer ldquoEnzyme immobilization in bio-technologyrdquoRecent Patents on Engineering vol 2 no 3 pp 195ndash200 2008

[15] S Datta L R Christena Y Rani and S Rajaram ldquoEnzymeimmobilization an overview on techniques and support mate-rialsrdquo 3 Biotech vol 3 no 1 pp 1ndash9 2013

[16] X Wang and S Uchiyama ldquoPolymers for biosensors construc-tionrdquo in State of the Art in BiosensorsmdashGeneral Aspects TRinken Ed chapter 3 pp 67ndash84 InTech Rijeka Croatia 2013

[17] S Singh A Chaubey and B D Malhotra ldquoAmperometriccholesterol biosensor based on immobilized cholesterol esteraseand cholesterol oxidase on conducting polypyrrole filmsrdquo Ana-lytica Chimica Acta vol 502 no 2 pp 229ndash234 2004

[18] A Gahlaut A K Chhillar Ashish and V Hooda ldquoDevelop-ment of analytical method based on enzymatic PVC strip formeasurement of serum total cholesterolrdquo International Journalof Biotechnology amp Biochemistry vol 2 pp 185ndash195 2012

[19] J-C Vidal E Garcia and J-R Castillo ldquoDevelopment of aplatinized and ferrocene-mediated cholesterol amperometricbiosensor based on electropolymerization of polypyrrole in aflow systemrdquo Analytical Sciences vol 18 no 5 pp 537ndash5422002

[20] V Hooda and C S Pundir ldquoCholesterol biosensor based onHRP incorporated carbon paste electrode wrapped with CAmembrane enzyme laminaterdquo International Journal of Biotech-nology amp Biochemistry vol 7 pp 617ndash635 2011

[21] Vikas and C S Pundir ldquoFabrication of Pt based amperometriccholesterol biosensor using cellulose acetate membranerdquo Jour-nal of Scientific and Industrial Research vol 67 no 4 pp 299ndash306 2008

[22] A Kumar P Rajesh A Chaubey S K Grover and B D Mal-hotra ldquoImmobilization of cholesterol oxidase and potassiumferricyanide on dodecylbenzene sulfonate ion-doped polypyr-role filmrdquo Journal of Applied Polymer Science vol 82 no 14 pp3486ndash3491 2001

[23] P-C Nien P-Y Chen and K-C Ho ldquoFabricating an amper-ometric cholesterol biosensor by a covalent linkage betweenpoly(3-thiopheneacetic acid) and cholesterol oxidaserdquo Sensorsvol 9 no 3 pp 1794ndash1806 2009

[24] S Singh R Singhal and B D Malhotra ldquoImmobilization ofcholesterol esterase and cholesterol oxidase onto sol-gel filmsfor application to cholesterol biosensorrdquo Analytica ChimicaActa vol 582 no 2 pp 335ndash343 2007

[25] P Norouzi F Faridbod E Nasli-Esfahani B Larijani and MR Ganjali ldquoCholesterol biosensor based on MWCNTs-MnO

2

nanoparticles using FFT continuous cyclic voltammetryrdquo Inter-national Journal of Electrochemical Science vol 5 no 7 pp1008ndash1017 2010

[26] J-Y Yang Y Li S-M Chen and K-C Lin ldquoFabrication of acholesterol biosensor based on cholesterol oxidase and multi-wall carbon nanotube hybrid compositesrdquo International Journalof Electrochemical Science vol 6 no 6 pp 2223ndash2234 2011

[27] G Li J M Liao G Q Hu N Z Ma and P J Wu ldquoStudy ofcarbon nanotube modified biosensor for monitoring totalcholesterol in bloodrdquo Biosensors and Bioelectronics vol 20 no10 pp 2140ndash2144 2005

[28] H Zhang R Liu and J Zheng ldquoSelective determination ofcholesterol based on cholesterol oxidase-alkaline phosphatasebienzyme electroderdquo Analyst vol 137 no 22 pp 5363ndash53672012

[29] T T Baby and S Ramaprabhu ldquoNon-enzymatic glucose andcholesterol biosensors based on silica coated nano iron oxidedispersed multiwalled carbon nanotubesrdquo in Proceedings ofthe International Conference on Nanoscience Technology andSocietal Implications (NSTSI rsquo11) pp 1ndash6 IEEE BhubaneswarIndia December 2011

[30] RManjunatha G Shivappa Suresh J SavioMelo S F DrsquoSouzaand T Venkatarangaiah Venkatesha ldquoAn amperometric bienzy-matic cholesterol biosensor based on functionalized graphenemodified electrode and its electrocatalytic activity towards totalcholesterol determinationrdquo Talanta vol 99 pp 302ndash309 2012

[31] V N Psychoyios G-P Nikoleli N Tzamtzis et al ldquoPoten-tiometric cholesterol biosensor based on ZnO nanowalls andstabilized polymerized lipid filmrdquo Electroanalysis vol 25 no 2pp 367ndash372 2013

[32] R Khan A Kaushik P R Solanki A A Ansari M K Pandeyand B D Malhotra ldquoZinc oxide nanoparticles-chitosan com-posite film for cholesterol biosensorrdquo Analytica Chimica Actavol 616 no 2 pp 207ndash213 2008

[33] D Saini R Chauhan P R Solanki and T Basu ldquoGold-nano-particle decorated graphene-nanostructured polyaniline nano-composite-basedbienzymatic platformform cholesterol sens-ingrdquo ISRN Nanotechnology vol 2012 Article ID 102543 12pages 2012

[34] M Zhang R Yuan Y Chai et al ldquoA biosensor for cholesterolbased on gold nanoparticles-catalyzed luminol electrogener-ated chemiluminescencerdquo Biosensors and Bioelectronics vol 32no 1 pp 288ndash292 2012


[35] N Pena G Ruiz A J Reviejo and J M Pingarron ldquoGraphite-teflon composite bienzyme electrodes for the determination ofcholesterol in reversed micelles Application to food samplesrdquoAnalytical Chemistry vol 73 no 6 pp 1190ndash1195 2001

[36] S Cao L Zhang Y Chai and R Yuan ldquoElectrochemistryof cholesterol biosensor based on a novel Pt-Pd bimetallicnanoparticle decorated graphene catalystrdquo Talanta vol 109 pp167ndash172 2013

[37] V Hooda A Gahlaut H Kumar and C S Pundir ldquoBiosensorbased on enzyme coupled PVC reaction cell for electrochemicalmeasurement of serum total cholesterolrdquo Sensors and ActuatorsB Chemical vol 136 no 1 pp 235ndash241 2009

[38] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951

[39] J-L Besombes S Cosnier P Labbe and G Reverdy ldquoImprove-ment of the analytical characteristics of an enzyme electrode forfree and total cholesterol via laponite clay additivesrdquo AnalyticaChimica Acta vol 317 no 1ndash3 pp 275ndash280 1995

[40] R Foster J Cassidy and E OrsquoDonoghue ldquoElectrochemicaldiagnostic strip device for total cholesterol and its subfractionsrdquoElectroanalysis vol 12 no 9 pp 716ndash721 2000

[41] E Garcıa-Ruiz J C Vidal M T Aramendıa and J RCastillo ldquoDesign of an interference-free cholesterol ampero-metric biosensor based on the electrosynthesis of polymericfilms of diaminonaphthalene isomersrdquo Electroanalysis vol 16no 6 pp 497ndash504 2004

[42] E Salinas V Rivero AA J TorrieroD BenuzziM I Sanz andJ Raba ldquoMultienzymatic-rotating biosensor for total choles-terol determination in a FIA systemrdquo Talanta vol 70 no 2 pp244ndash250 2006

[43] S Singh P R Solanki M K Pandey and B D MalhotraldquoCovalent immobilization of cholesterol esterase and choles-terol oxidase on polyaniline films for application to cholesterolbiosensorrdquoAnalyticaChimicaActa vol 568 no 1-2 pp 126ndash1322006

[44] A Ahmadalinezhad and A Chen ldquoHigh-performance elec-trochemical biosensor for the detection of total CholesterolrdquoBiosensors and Bioelectronics vol 26 no 11 pp 4508ndash4513 2011

[45] N Batra M Tomar and V Gupta ldquoZnOndashCuO compositematrix based reagentless biosensor for detection of total choles-terolrdquo Biosensors and Bioelectronics vol 67 pp 263ndash271 2015

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of biosensor in determining accuracy and sensitivity [10]Immobilizing ChO on various supports helps in directlydetermining concentration of cholesterol in different sam-ples As serum cholesterol is present in the form of ester inorder to determine total cholesterol ChE is required alongwith ChO Cholesterol esterase first hydrolyses the esterifiedcholesterol and is further oxidized by cholesterol oxidasefor producing cholest-4-en-3-one and hydrogen peroxide(H2O2) In amperometric biosensors electric potential is

applied onH2O2which get oxidized to produce electrons and

the flow of electrons produces current The current gener-ated is directly proportional to cholesterol concentration inserum

Cholesterol ester +H2O

Cholesterol Esterase997888997888997888997888997888997888997888997888997888997888997888997888997888997888rarr Cholesterol + fatty acids

Cholesterol +O2

Cholesterol Oxidase997888997888997888997888997888997888997888997888997888997888997888997888997888997888rarr Cholest-4-en-3-one +H

2O2

H2O2

Electric Potential997888997888997888997888997888997888997888997888997888997888997888rarr 2H+ +O

2+ eminus

(1)

A new approach to reduce interference is the use ofhorseradish peroxidase (HRP) HRP has redox centre associ-ated with ferrohemeferriheme pair Conversion of reducedstate to oxidized state is done through direct transfer ofelectrons In its 3D configuration HRP has heme grouppresent in outer region which enhances the direct transfer ofelectrons between redox centre and conducting sites presenton transducer [11] Regeneration of HRP is done via directelectron transfer

H2O2+ 2H+ +HRPRed 997888rarr 2H2O +HRPOxd

HRPOxd + 2eminus

larrrarr HRPRed

(2)

Immobilizing enzyme on suitable support may lower theMichaelis constant 119870

119898or it may enhance bimolecular rate

constant which improves the biosensor performance [12ndash15]Cholesterol hydrolysing enzymes ChO and ChE have beenimmobilized on conducting polymers [16] like polypyrrolefilms [17] PVC Strip [18] PBpolypyrrole (PPy) compositefilm [19] cellulose acetatecarbon electrode [20] celluloseacetatePt electrode [21] electropolymerised PPy films [22]poly 3-thiopheneacetic acid filmPt electrode [23] and PANIndashpTSA-AgITO [11] and sol-gel films [24] Evolution ofnanoscale materials has revolutionised the field of biosensorsalsoThenanoscale dimensions their graphitic surface chem-istry and electrocatalytic properties of carbon nanotubesmake them an interesting material for sensing purposeSingle andmultiwalled carbon nanotubes [25 26] MWCNTs[27] CNT-chitosanGCE [28] Fe

3O4-SiO2MWCNT [29]

graphene modified graphite electrode [30] graphenePt NPs[31] PtAu ZnO nanorods [32] NSPANI-AuNP-GRITO[33] AuNPsAu electrode [34] graphite-teflon matrix [35]and Pt-Pd bimetallic nanoparticle decorated grapheme [36]

are some of the supports which have been used for thetransduction of generated biological signals to electricalsignals for cholesterol determination PVC has also beenused to immobilize cholesterol hydrolysing enzymes ChOand ChE were covalently immobilised on surface of PVCbeaker which act as reaction cell and HRP was incorporatedin carbon electrode [37]

In the present method the combination of three enzymeswas used for the development of electrochemical biosensorby immobilization of three enzymes (ChO ChE and HRP)on AuNPs and c-MWCNT based electrode for determiningcholesterol in serum cholesterol

2 Materials and Methods

21 Chemicals and Reagents Cholesterol esterase was puri-fied from Pseudomonas species (1658Unitsg) 4-amino-phenazone Triton X-100 cholesteryl acetate and carboxy-lated multiwalled carbon nanotubes (c-MWCNTs) were pur-chased from Sigma Chemical Co USA Cholesterol oxidasefrom Streptomyces sp (500 units10mg) and horseradishperoxidase (HRP) (80Umg) were obtained from SISCOResearch Laboratory Pvt Ld Mumbai Silver wire was pur-chased from local market All other chemicals used duringthe experimentation were of analytical grade Gold nanopar-ticles (AuNPs) were synthesised at Centre for BiotechnologyMaharshi Dayanand University Rohtak

22 Instrumentation Potentiostat (PSTAT mini 910 Met-rohm Switzerland) was used for all electrochemical stud-ies Ultrasonication was done with Chrom Tech UltrasonicLiquid Processor For TEM JEM-2100F microscope (AIRFJNU New Delhi) was used Varian 7000 FTIR spectrometer(AIRF JNU New Delhi) was used for performing Fouriertransform infrared (FTIR) spectroscopy X-ray diffraction(XRD) facility for gold nanoparticles and c-MWCNTs wasprovided byDepartment of PhysicsMD University RohtakShimadzu corporationUV 2450 spectrophotometer was usedfor spectrophotometric measurement at Centre for Biotech-nology

23 Synthesis of Gold Nanoparticles Chemical synthesis ofgold nanoparticles was done using citrate reduction method[38] 100mL of 0001 gold chlorous acid (HAuCl

4) was

boiled at 97∘C with continuous stirring using magnetic stir-rer Then the 1 sodium citrate solution was added within 4to 5min solution turns wine red and was further heated for 4to 5min and then cooled down at room temperature Finallycentrifuged pellets were dried for further use Transmissionelectron microscopy (TEM) of the synthesised AuNPs wasdone on commercial basis at AIRF JNU New Delhi for theconfirmation of shape of newly synthesised nanoparticles andsize range of particles

24 Fabrication of ChEChOHRP-AuNPsc-MWCNT Modi-fied Silver Working Electrode Fabrication strategy of work-ing electrode comprised mixing c-MWCNTs and goldnanoparticles (AuNPs) in paraffin oil in fixed proportion









119898 and 119868max









2O2which is then


cholesterol


(a) (b)

116nm266nm 182nm

126nm

(c)






Relat

ive i

nten

sity

(au

)

(a)

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

100

002 10

1

102

110

103

112

10 20 30 40 50 60 70 8002120579 (deg)




(a) (b)





2O2for 30min




350

300

250

200

150

100

50

0

minus04 minus02 0 02 04 06 08

E (V)

I(m

A)


350

300

250

200

150

100

50

00 minus02 0 02 04 06 08

E (V)

I(m

A)

A

B




0

005

01

015

02

025

03

035

Curr

ent (

mA

)



00002000400060008

001001200140016

Curr

ent (

mA

)

64 66 68 7 72 74 76 7862pH







25 30 35 40 45 50 55 6020Temperature (∘C)

05

1015202530354045

Curr

ent (

mA

)


minus0050

00501

01502

02503

03504

Resp

onse

(mA

)


20 30 40 50



0

005

01

015

02

025

03

035

04

Curr

ent (

mA

)



366 Resolving 119870119898




y = 65231x + 11096

minus10123456789

0 002 004 006 008 01 012minus002(sminus1)

(Vminus1)





recovery SD


100 27133 9911 088

200 36879 9867 093








119899




Chol

este

ryl a

ceta

te co

ncen

trat

ion

(gd

L)

R2 = 09885

y = 09424x + 00151



0

02

04

06

08

1

12

in se

rum

by

pres

ent m

etho

d








Compoundsadded


relative response


0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



















4 Conclusion





immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









119898 and 119868max









2O2which is then


cholesterol


(a) (b)

116nm266nm 182nm

126nm

(c)






Relat

ive i

nten

sity

(au

)

(a)

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

100

002 10

1

102

110

103

112

10 20 30 40 50 60 70 8002120579 (deg)




(a) (b)





2O2for 30min




350

300

250

200

150

100

50

0

minus04 minus02 0 02 04 06 08

E (V)

I(m

A)


350

300

250

200

150

100

50

00 minus02 0 02 04 06 08

E (V)

I(m

A)

A

B




0

005

01

015

02

025

03

035

Curr

ent (

mA

)



00002000400060008

001001200140016

Curr

ent (

mA

)

64 66 68 7 72 74 76 7862pH







25 30 35 40 45 50 55 6020Temperature (∘C)

05

1015202530354045

Curr

ent (

mA

)


minus0050

00501

01502

02503

03504

Resp

onse

(mA

)


20 30 40 50



0

005

01

015

02

025

03

035

04

Curr

ent (

mA

)



366 Resolving 119870119898




y = 65231x + 11096

minus10123456789

0 002 004 006 008 01 012minus002(sminus1)

(Vminus1)





recovery SD


100 27133 9911 088

200 36879 9867 093








119899




Chol

este

ryl a

ceta

te co

ncen

trat

ion

(gd

L)

R2 = 09885

y = 09424x + 00151



0

02

04

06

08

1

12

in se

rum

by

pres

ent m

etho

d








Compoundsadded


relative response


0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



















4 Conclusion





immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

116nm266nm 182nm

126nm

(c)






Relat

ive i

nten

sity

(au

)

(a)

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

100

002 10

1

102

110

103

112

10 20 30 40 50 60 70 8002120579 (deg)




(a) (b)





2O2for 30min




350

300

250

200

150

100

50

0

minus04 minus02 0 02 04 06 08

E (V)

I(m

A)


350

300

250

200

150

100

50

00 minus02 0 02 04 06 08

E (V)

I(m

A)

A

B




0

005

01

015

02

025

03

035

Curr

ent (

mA

)



00002000400060008

001001200140016

Curr

ent (

mA

)

64 66 68 7 72 74 76 7862pH







25 30 35 40 45 50 55 6020Temperature (∘C)

05

1015202530354045

Curr

ent (

mA

)


minus0050

00501

01502

02503

03504

Resp

onse

(mA

)


20 30 40 50



0

005

01

015

02

025

03

035

04

Curr

ent (

mA

)



366 Resolving 119870119898




y = 65231x + 11096

minus10123456789

0 002 004 006 008 01 012minus002(sminus1)

(Vminus1)





recovery SD


100 27133 9911 088

200 36879 9867 093








119899




Chol

este

ryl a

ceta

te co

ncen

trat

ion

(gd

L)

R2 = 09885

y = 09424x + 00151



0

02

04

06

08

1

12

in se

rum

by

pres

ent m

etho

d








Compoundsadded


relative response


0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



















4 Conclusion





immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)





2O2for 30min




350

300

250

200

150

100

50

0

minus04 minus02 0 02 04 06 08

E (V)

I(m

A)


350

300

250

200

150

100

50

00 minus02 0 02 04 06 08

E (V)

I(m

A)

A

B




0

005

01

015

02

025

03

035

Curr

ent (

mA

)



00002000400060008

001001200140016

Curr

ent (

mA

)

64 66 68 7 72 74 76 7862pH







25 30 35 40 45 50 55 6020Temperature (∘C)

05

1015202530354045

Curr

ent (

mA

)


minus0050

00501

01502

02503

03504

Resp

onse

(mA

)


20 30 40 50



0

005

01

015

02

025

03

035

04

Curr

ent (

mA

)



366 Resolving 119870119898




y = 65231x + 11096

minus10123456789

0 002 004 006 008 01 012minus002(sminus1)

(Vminus1)





recovery SD


100 27133 9911 088

200 36879 9867 093








119899




Chol

este

ryl a

ceta

te co

ncen

trat

ion

(gd

L)

R2 = 09885

y = 09424x + 00151



0

02

04

06

08

1

12

in se

rum

by

pres

ent m

etho

d








Compoundsadded


relative response


0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



















4 Conclusion





immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

005

01

015

02

025

03

035

Curr

ent (

mA

)



00002000400060008

001001200140016

Curr

ent (

mA

)

64 66 68 7 72 74 76 7862pH







25 30 35 40 45 50 55 6020Temperature (∘C)

05

1015202530354045

Curr

ent (

mA

)


minus0050

00501

01502

02503

03504

Resp

onse

(mA

)


20 30 40 50



0

005

01

015

02

025

03

035

04

Curr

ent (

mA

)



366 Resolving 119870119898




y = 65231x + 11096

minus10123456789

0 002 004 006 008 01 012minus002(sminus1)

(Vminus1)





recovery SD


100 27133 9911 088

200 36879 9867 093








119899




Chol

este

ryl a

ceta

te co

ncen

trat

ion

(gd

L)

R2 = 09885

y = 09424x + 00151



0

02

04

06

08

1

12

in se

rum

by

pres

ent m

etho

d








Compoundsadded


relative response


0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



















4 Conclusion





immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


y = 65231x + 11096

minus10123456789

0 002 004 006 008 01 012minus002(sminus1)

(Vminus1)





recovery SD


100 27133 9911 088

200 36879 9867 093








119899




Chol

este

ryl a

ceta

te co

ncen

trat

ion

(gd

L)

R2 = 09885

y = 09424x + 00151



0

02

04

06

08

1

12

in se

rum

by

pres

ent m

etho

d








Compoundsadded


relative response


0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



















4 Conclusion





immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Compoundsadded


relative response


0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



0005

01015

02025

03035

04045

Relat

ive r

espo

nse (

mA

)



















4 Conclusion





immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




immobilization

Workingpotential

Responsetime


stabilityReference


ChO ChEenzyme

Entrapment




adsorption






adsorption




ChO ChE HRPCovalent


minus015 Vversus



PANIITO

ChO ChECovalent





ChO ChECovalent



550mgdL 100 days [38]


ChO ChE HRPPhysical


as glue



ChO ChEPhysical

adsorption


c-MWCNTAuNP

ChO ChE HRPCovalent






Acknowledgments


References




























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























2






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article fabrication and optimization of che/cho...

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