Research Article Amino-Functionalization of Carbon...

Research ArticleAmino-Functionalization of Carbon Nanotubes byUsing a Factorial Design Human Cardiac Troponin TImmunosensing Application

Tatianny A Freitas Alessandra B Mattos Baacuterbara V M Silva and Rosa F Dutra

Biomedical Engineering Laboratory Federal University of Pernambuco Avenida Professor Moraes Rego 123550670-901 Recife PE Brazil

Correspondence should be addressed to Rosa F Dutra rosadutraufpebr

Received 25 February 2014 Revised 4 May 2014 Accepted 17 June 2014 Published 13 July 2014

Academic Editor Pablo Avanzas

Copyright copy 2014 Tatianny A Freitas et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A simple amino-functionalization method for carbon nanotubes and its application in an electrochemical immunosensor fordetection of the human cardiac troponin T are described Amino-functionalized carbon nanotubes allow oriented antibodiesimmobilization via their Fc regions improving the performance of an immunosensor Herein multiwalled carbon nanotubes wereamino-functionalized by using the ethylenediamine reagent and assays were designed by fractional factorial study associated withDoehlert matrix Structural modifications in the carbon nanotubes were confirmed by Fourier transform infrared spectroscopyAfter amino-functionalization the carbon nanotubes were attached to screen-printed carbon electrode and a sandwich-typeimmunoassay was performed for measuring the cardiac troponin T The electrochemical measurements were obtained throughhydrogen peroxide reaction with peroxidase conjugated to the secondary antibody Under optimal conditions troponin Timmunosensor was evaluated in serum samples which showed a broad linear range (002 to 032 ngmLminus1) and a low limit ofdetection 0016 ngmLminus1 This amino platform can be properly used as clinical tool for cardiac troponin T detection in the acutemyocardial infarction diagnosis

1 Introduction

The screen-printing technology is a well-established andpractical approach ideal for development of electrochemicalpoint-of-care testing [1] Screen-printed electrode (SPE) pro-vides advantages such as easy miniaturization and portableinstrumentation making possible the on-site detection ofdifferent target analytes [2] When compared with conven-tional electrodes SPEs are inexpensive being used as dis-posable due to their large-scale production capability Severalmethods have been devoted to increase the surface area ofthe SPEs and enhance their sensitivity for electrochemicaldetection including the application of nanomaterials [3ndash5]Carbon nanotubes (CNTs) have attracted the interest ofresearchers in the field of electrochemical SPE immunosen-sors [6ndash8] These nanomaterials specially combine severalproperties that improve the electrochemical performance

such as easy surface functionalization and increase in theamount of immobilized biomolecules and in the electron-transfer charge on the electrode surface [9]

The surface functionalization of the CNTs by linkingspecific functional groups has been a fundamental point forantibodies attaching as recognition element for immunosens-ing application Carboxyl-terminated nanotubes have beenalso explored as essential functionalization strategy for cova-lent linkage of antibodies However this approach is limitedbecause most antibodies contain amino groups randomlydistributed leading tomultiple attachment sitesThe randomnatures of this attachment sites can cause some loss of theantigen-binding activity due to the steric hindrance [10 11]The carboxylic groups present in Fc regions of the antibodiescan be conveniently explored for oriented immobilization byexposing the Fab regions that exhibit a high affinity towardepitopes of the antigens These groups can form stable amide

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 929786 9 pageshttpdxdoiorg1011552014929786

2 BioMed Research International

bonds with the amino groups of the CNTs [12] Thus amino-functionalized nanotubes improve the reactivity of antigen-antibody recognition and the efficiency in immobilizationprocess

Techniques that are specifically intended for CNTs func-tionalization with amino groups comprise chemical treat-ments using acids sheathing or wrapping of the CNTs withpolymer chains [13 14] grafting of CNTs with a thin layerof polymer chains based on plasma [15] or a combinationof these [16] However these methods are generally a com-plicated process involving a long reaction time and severalcoupling reagents and require strictly controlled reactionconditions [17 18] In this work a simple method basedon fractional factorial design has been proposed for amino-functionalization of the CNTs using the ethylenediamine(EDA) as crosslink amino reagent Under optimization pro-cess of functionalization amino-CNTs were employed todevelop an immunosensor for cardiac troponin T (cTnT) animportant marker for acute myocardial infarction

Cardiovascular diseases are the leading cause of deathglobally according to the World Health Organization statis-tics Among the cardiovascular diseases acute myocardialinfarction (AMI) is one of the most serious diseases thatextremely affect peoplersquos health [19] In the past decadescardiac troponins (cTnT and cTnI) have been recommendedas the biomarkers of choice for the serological diagnosisand prognosis of AMI because of their high sensitivity andspecificity [20ndash23] In particular the cTnT levels increase2ndash4 h after the AMI symptoms and could be elevated upto 14 days after the acute episode of myocardial damage[24 25] Thus the development of a rapid and practicalimmunosensor for the detection of the cTnT in serumsamples from patients with myocardial infarction is desirabledue to its roles in cardiospecific diagnosis risk stratificationprognostic risk assessment and therapeutic choices

2 Materials and Methods

21 Reagents and Materials Mouse monoclonal antibodyagainst cTnT (mAb-cTnT) cTnT and peroxidase conju-gated mouse monoclonal antibody against cTnT (mAb-cTnT-HRP) were purchased from Calbiochem (DarmstadtDEU) COOH-functionalizedmultiwalled carbon nanotubes(MWCNTs) were obtained from Dropsens (Oviedo ESP)EDA N-ethyl-N1015840-(3-dimethylaminopropyl) carbodiimide(EDC) N-hydroxysuccinimide (NHS) dimethylformamide(DMF) and glycine were acquired from Sigma-Aldrich (StLouis USA) Ethanol sulfuric acid (H

2

SO4

) (98 wv) andhydrogen peroxide (H

2

O2

) (30 wv) were obtained from FMaia (Cotia BRA) All reagents were of analytical gradeThewater used to prepare all solutions was obtained from aMilli-Qwater purification system (Billerica USA) All the solutionswere freshly prepared prior to each experiment

22 Serum Samples The serum samples of cTnT wereobtained from venous blood and immediately centrifugedfor 120 s at 1150 rads Aliquots of serum were quantifiedin an automatic system Roche Elecsys 2010 immunoassay

analyzer based on electrochemiluminescence immunoassay(ECLIA) and the remaining serum stored at minus20∘C was usedfor the electrochemical measurements The venous bloodsamples were collected from donorrsquos patient from the CardiacEmergency of Pernambuco (PROCAPE) the Hospital ofPernambuco State University according to ethics committeersquosrecommendations

23 Apparatus and Measurements Electrochemical studieswere performed by using a 120583Autolab III analysis system withGPES 49 software Eco Chemie (Utrecht NLD) Screen-printed carbon electrodes (SPCEs) were purchased fromDropSens (Oviedo ESP) These electrodes (reference 110)incorporate a conventional three-electrode configurationprinted on ceramic substrates (34 cm times 10 cm) Both work-ing (disk-shaped 40mm diameter) and counter electrodeswere made of the carbon inks whereas the pseudoreferenceelectrode and electric contacts were made of silver

Cyclic voltammetry (CV) was performed at 100mV sminus1scan rate in presence of PBS (001mol Lminus1 pH 70) used in allexperiments as electrolyte support The chronoamperometrywas carried out in the same electrolyte at the potentialof +03 V at 120 s All experiments were executed at roomtemperature (24∘C)

24 Characterization of the MWCNTs The structural char-acterization of the MWCNTs was evaluated by Fouriertransform infrared (FTIR) spectroscopy The spectra wereobtained by using a Bruker IFS 66model FT-IR spectrometerin the region 4000 to 400 cmminus1 by the standard KBr pellettechniqueThemodification of the SPE surface by depositionof the MWCNTs was characterized by scanning electronmicroscopy (SEM) using Philips XL30 FEG FE-SEM at 10 kV

25 Amino-Functionalization of the MWCNTs The car-boxylic groups of the MWCNTs were activated by using asolution of 01mol Lminus1 acetate buffer (pH 48) containing01mol Lminus1 EDC and 02mol Lminus1 NHS [1 1] At the sametime amino groups of the EDA were submitted to acidtreatment in H

2

SO4

solution (01mol Lminus1) for 2 h understirring conditions at 60∘C changing their protonation stateAfter that 10mg activatedMWCNTs was dispersed in 10 120583LEDA treated and it was stirred for 25 h at room temperatureThe sample was centrifuged at 2000timesg and washed five timeswith deionized water to remove acid residues The resultingamino-MWCNTs were dried at 150∘C and then dispersed inDMF under sonication during 2 h

26 Electrode Preparation Prior to use the SPCE surface(0125 cm2) was sonicated with ethanol and deionized waterrespectively for 2min to remove any organic contaminantSPCE was coated with 30 120583L of the amino-MWCNTs on theworking electrode surface and dried at 40∘C for 5min forabsolute evaporation

27 mAb-cTnT Immobilization After being coated withamino-MWCNTs the SPCE surface was incubated with

BioMed Research International 3

an aliquot (30 120583L) of mAb-cTnT (10 120583gmLminus1) for 1 h Inthis concentration the maximal current was measured asa result of antigen-antibody equilibrium demanded [26]Subsequently the electrode was rinsed by exhaustive PBS(pH 70) washing with the purpose of removing unboundmAb-cTnT To avoid nonspecific binding the modified elec-trode surface was blocked with 50mmol Lminus1 glycine solutionfor 2 h

28 Analytical Measurements For evaluating the analyticalresponse of the immunosensor the modified electrodes wereincubated with human serum samples spiked with differentcTnT concentrations (002 004 008 016 and 032 ngmLminus1)for 30min After that the electrode was incubated withmAb-cTnT-HRP for 1 h The immunosensor response wasevaluated by chronoamperometric assays at +03 V during120 sThe amperometric detection of the cTnTwasmonitoredthrough the electrocatalytic reduction reaction of the H

2

O2

by the HRP-labeled secondary mAb-cTnT The measure-mentswere performed in 01mol Lminus1 PBS (pH70) containing15mmol Lminus1 H

2

O2

at room temperature

29 Multivariate Optimization The screening of variableswas accomplished using a 25ndash1 fractional factorial design Fivevariables were examined in two levels lower (minus) and upper(+) The subsequent factors and their levels were as followsEDA concentration (50ndash100) H

2

SO4

concentration (01ndash10mol Lminus1) time of acid treatment of the EDA (1-2 h) ami-nation time (2ndash4 h) and amino-MWCNT dispersion time(2ndash4 h) (Table 1) After establishing the variables a Doehlertdesign was used for final optimization and then the responsesurface was obtainedThese experiments were performed in arandom order and themonitored parameter was the cathodiccurrent established by CV Data was processed using theSTATISTICAL package program (version 60 Stat Soft IncTulsa OK USA)

3 Results and Discussion

31 Multivariate Optimization of the Amino-MWCNTsMethods for chemical functionalization of MWCNTs havebeen reported by using different reactive groups such as ndashNH2

ndashCOOH ndashOH and ndashSH Among these the use ofthe ndashNH

2

groups has been a viable alternative to attachantibodies to sensing surfaces in an oriented manner viaamide bonds [27] In this study the amino-functionalizationof the MWCNTs with EDA was investigated by using afractional factorial design which shows the influence ofvarious factors on the experimental results and the optimumsetting for each factor

To optimize the different variables involving the amino-functionalization process of MWCNTs a fractional fac-torial design associated with Doehlert matrix was usedEDA concentration (50ndash100) H

2

SO4

concentration (01ndash10mol Lminus1) time of acid treatment of the EDA (1-2 h)amination time (2ndash4 h) and amino-MWCNT dispersiontime (2ndash4 h)were established according to the results attained

Fitted surface variable resposta2 factors 1 blocks and 7 runs MS residual = 0005486

DV resposta

gt06

lt06

lt04

lt02

lt 0

ltminus02

minus04

minus02

00

02

04

06

08

020

4060

80100

120

EDA concentration 45

40

35

30

25

20

15

10

05

Tim

e of a

cid tr

eatm

ent o

f EDA

ΔI120583

A

Figure 1 Surface response obtained from the Doehlert designemployed for the optimization of the EDA concentration and time ofacid treatment of the EDA in the amino-functionalizationMWCNTprocedure The optimum conditions correspond to 70 and 25 hrespectively

from the 25ndash1 fractional factorial design and carried out induplicate

The Pareto diagram demonstrated that the most signifi-cant effects in the cathodic current were EDA concentrationand time of acid treatment of the EDA The positive valuesobtained in this study indicate that by increasing thesefactors the analytical signal will increase tooThe factorswereselected and simultaneously optimized by Doehlert design

The EDA concentration was evaluated at five levels (1025 50 75 and 100) and time of acid treatment of the EDAat three levels (1 2 and 4 h) Figure 1 shows the responsesurfaces obtained for the experiments considering the previ-ously obtained effects By analyzing the response surfaces theoptimum conditions of the experimental design generatedthe highest current peak These conditions correspond to70 and 25 h of the EDA concentration and time of acidtreatment of the EDA respectively

32 Characterization of the MWCNTs FTIR spectroscopyhas proved to be a powerful tool to comply with the purposeof comprehensive characterization The spectrum of theCOOH-MWCNT in Figure 2(a) shows typical bands of thecarboxylic groups at 3445 cmminus1 corresponding to molecular


Table 1 Experimental factors and their levels employed in 25minus1 fractional factorial design for amino-functionalization of the MWCNTprocedure

ExperimentEDA

concentrationminus (50)+ (100)

H2SO4concentrationminus (01mol Lminus1)+ (10mol Lminus1)

Time of acidtreatment of the EDAminus (1 h)+ (2 h)

Amination timeminus (2 h)+ (4 h)

Amino-MWCNTdispersion timeminus (2 h)+ (4 h)

Δ119868 (120583A)mean values

1minus minus minus minus + 05757

2 + minus minus minus minus 043283

minus + minus minus minus 028034 + + minus minus + 028035

minus minus + minus minus 041316 + minus + minus + 042787

minus + + minus + 063838 + + + minus minus 030499

minus minus minus + minus 0406510 + minus minus + + 0344311

minus + minus + + 0431912 + + minus + minus 0298313

minus minus + + + 0249214 + minus + + minus 0213115

minus + + + minus 0482016 + + + + + 04475

4000 3500 3000 2500 2000 1500 1000

055

060

065

070

075

1200

1335

1520

3170

2989

29953420

Abso

rban

ce (

)

Wavenumber (cmminus1)

(a)

(b)

3445

3170

Figure 2 FTIR spectra of theMWCNTs (a) before and (b) after EDAtreatment

stretching of ndashOH groups The presence of the small peakat 1710 cmminus1 is associated with the C=O stretching Theseresults were used as control of the amino-functionalizationprocedure Figure 2(b) exhibits the spectra of the MWC-NTs after EDA treatment The peaks in the regions 3420and 3170 cmminus1 can be attributed to NndashH stretch of theamino group In addition two peaks around 2995 cmminus1 and2989 cmminus1 evidence the CminusH stretching mode of the EDAmolecule [28] The appearance of new absorption bands at1520 and 1340 cmminus1 corresponds to NndashH stretch of the amino

groupThe peak at 1120 cmminus1 is ascribed to CndashN stretching ofamide groups [29]

The presence and location of the ndashNH2

and CndashN bandsin this spectrum provide strong evidence of the introduc-tion of EDA moieties onto the MWCNT walls Then theobtained results showed a successful method for amino-functionalization of the MWCNTs

The SEM images were employed to morphologicallycharacterize the modification of the electrode surface Thebare surface of the SPCE is showed in Figure 3(a) It wastypically characterized by a great content of graphite particlescovered with polymeric binder from the carbon ink [30]The distribution of the graphite particles in the bare SPCEsurface demonstrated a porous structure The morpholog-ical changes in the sensing interface after treated amino-MWCNTs modification can been seen in Figure 3(b) Thisimage reveals a homogeneous mesh of the amino-MWCNTsin the form of small bundles of tubes The adsorbed amino-MWCNTshave resulted in a higher roughness as compared tounmodified SPCE by favoring an increased active surface tofurther antibody immobilization SEM analysis also showedthat chemical modification of the MWCNTs in the amino-functionalization procedure did not affect the adsorption andformation of nanostructured film

33 Immunosensor Preparation The scheme of stepwiseimmunosensor fabrication is illustrated in Figure 4The acti-vation of carboxylic acids of the MWCNTs with EDCNHSchemical provides a reactive group for interaction withEDA (Figure 4(a)) The EDCNHS enables the conversion ofcarboxylic groups to amino-reactive esters of the NHS [31]


(a) (b)

Figure 3 SEM image of working area of the SPCE (a) bare and (b) modified with amino-MWCNTs

(SPCE)

(mAb-cTnT)

(amino-MWCNTs)

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

C

C=O

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

C=O C=O C=O C=O C=O

(COOH-MWCNTs)

CO OH

ClNH

N

NC

CO O

CO O

O

O

(EDC)

(activated MWCNTs)

(EDA acid treated)

+ C=ON-H

CO O

O

O

(amino-MWCNTs)

(NHS)

minus

+

OHN OO

NN HH

NH2+

NH2+

C=OC=O

NH2+

NH2+

NH2+

Cl NH

N

NC

minus

+

N

N

(a)

(b) (c)

Figure 4 Schematic illustration of stepwise of the immunosensor (a) activation of the MWCNTs (b) amino-functionalization procedureand (c) mAb-cTnT immobilization

These groups are susceptible to nucleophilic attack fromamino group with charge excess of the EDA (Figure 4(b))The reaction resulted in the formation of a stable covalentbond between the materials

The use of the EDA to functionalize the MWCNTs with ndashNH2

groups showed a viable alternative to attach the ndashCOOH

groups present in Fc regions of the mAb-cTnT via amidebondsThe oriented immobilization via amide covalent bind-ing influences the conformational stability and the activityof immobilized antibodies Although more complex thanother immobilization methods (ie physical adsorption)the attachment covalent provides a most-stable strategy for


45 50 55 60 65 70 75 80

020

025

030

035

040

045

ΔI c(120583A)

pH

(a)

0 5 10 15 20

000

005

010

015

020

025

030

035

040

045

Ionic strength (mmol Lminus1)

ΔI c(120583A)

(b)

0 1 2 3 4 5

030

035

040

045

050

CmAb-cTnT -HRP (120583g mLminus1)

ΔI c(120583A)

(c)

Figure 5 Influence of (a) pH values (b) ionic strength of the PBS and (c) the amount of the mAb-cTnT-HRP on the cathodic peak currentof the SPCE Measurements obtained by CVs experiments in presence of the 15mmol Lminus1 H

2

O2

irreversible immobilization of the biomolecules [32 33]The advantage of low protein loss by desorption improvesthe stability and reproducibility of the device indicatingthat covalent immobilization is most desirable for severalbiosensor technologies [33] The reactions of the mAb-cTnTimmobilization procedure are demonstrated in Figure 4(c)

34 Optimization of the Experimental Conditions Influenceof the buffer pH is essential to analytical performance of theimmunosensor because the pH affects not only the electro-chemical behavior of the sensor but also the bioactivity of thebiomolecules [34] The pH value of the 01mol Lminus1 PBS wasinvestigated in the range of 50 to 80Themeasurements wereobtained through CV assay in presence of the 15mmol Lminus1H2

O2

The results in Figure 5(a) showed the effect of the pHon the reduction current of the SPCE The cathodic current

reached a maximum value at pH 70 Thus the optimum pHwas chosen for subsequent studies

The ionic strength studies of the PBS were investi-gated These experimental parameters can influence thecharge transport of the electrolyte support in the electro-chemical measurements of the SPCE [29] For this assaythe SPCE was submitted to amperometric measurementsin 15mmol Lminus1 H

2

O2

solution diluted in different ionicstrengths of PBS (001 01 10 25 50 75 100 125 150175 and 200mmol Lminus1) The results exhibited a maximumreduction current at 001mol Lminus1 of the PBS (Figure 5(b))

In order to obtain an optimal response with the minimalamount of HRP-labeled antibody the concentrations ofthe mAb-cTnT-HRP were varied Herein after the mAb-cTnT SPCE incubation with cTnT (25 ngmLminus1) for 1 h theimmunosensor was incubated at different concentrations ofthe mAb-cTnT-HRP (001 to 50 120583gmLminus1) As can be seen in


0 1 2 3 4 5 6 7 8 9 10 11

004

006

008

010

012

014

Electrodes (n)

ΔI c(120583A)

(a)

0 5 10 15 20

020

022

024

026

028

030

032

034

Cycles (n)

ΔI c(120583A)

(b)

Figure 6 (a) Reproducibility and (b) repeatability of the SPCE to 25 ngmLminus1 cTnT

000 005 010 015 020 025 030 035

minus045

minus040

minus035

minus030

minus025

CcTnT (ng mLminus1)

ΔI c(120583A)

Figure 7 Calibration curve of the resulting immunosensor for cTnTdetection in serum samples

Figure 5(c) the reduction current increased with the increaseof the anti-cTnT-HRP concentration up to 10120583gmLminus1

35 Reproducibility and Stability A key problem ofimmunosensors is their reproducibility and how longtheir devices can operate with a reproducible response [35]In the reproducibility study 10 different electrodes wereprepared under the same conditions and evaluated withcTnT samples (25 ngmLminus1) The immunosensor reached anacceptable reproducibility with a relative standard deviation(RSD) of 25 (Figure 6(a)) Regarding the repeatabilityimmunosensor has been shown to be significantly stablefor 20 measurements in the same electrode registered ineach 1min interval This statement was confirmed by lowRSD (134) (Figure 6(b)) These results can be attributed

to strong interaction between the amino-MWCNT film andimmobilized mAb-cTnT

36 Determination of cTnT in Human Serum Human serumsamples spiked with different cTnT concentrations wereanalyzed by chronoamperometric assays fixing the work-ing potential at minus03 V at 120 s (Figure 7) The amount ofcTnT of serum samples was previously measured by RocheElecsys 2010 immunoassay analyzer based on ECLIA Theimmunosensor measurements showed a good agreementwith the ECLIA method at 95 confident level when pairedt-test was applied As shown in Figure 7 the calibrationplot exhibited a good linear correlation between 002 and032 ngmLminus1 cTnT (119903 = 0985 119899 = 5 and 119875 lt 0001)The limit of detection (LOD) of the SPCE was calculatedaccording to the following equation

LOD = 3SD119898

(1)

where SD is standard deviation of the blankmeasurementand119898 is the slope of the linear part of the calibration curve [36]The LOD was estimated in 0016 ngmLminus1 showing a highsensitivity thereby this immunosensor can be useful for cTnTdetermination in clinical routine since the cutoff is found atapproximately 002 ngmLminus1 in the AMI diagnosis Since nomediators have been added in the electrochemical detectionof the cTnT based on HRP reaction the SPCE exhibiteda LOD comparable with other cTnT immunosensors thatmake use of the mediators such as hydroquinone [26]The properties of the MWCNTs at the sensor interface candispense the employ of the mediators due to the ability ofthe nanomaterials to improve the electron-transfer reactionsThe use of mediators can cause damage or passivation on theelectrode surface reducing its lifetime [37]


4 Conclusions

A simpler method for amino-functionalization of the MWC-NTs than the previously described was proposed It doesnot require time-consuming and costly multistep reactionsseveral coupling reagents or strictly controlled processesTheexperimental conditions were optimized using a fractionalfactorial design showing that protonation time and EDA con-centration are dependent parameters for successful MWC-NTs functionalization Oriented immobilization of anti-cTnTand development of an immunosensor for cTnT with highsensitivity and reproducibility were possible

Conflict of Interests

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

Acknowledgments

This work was supported by the National Council of Techno-logical and ScientificDevelopment (CNPq) Brazilian agencyT A Freitas PhD student is grateful to FACEPE (Brazil) forscholarship during thisworkThe assistance of the PROCAPE(Cardiac Emergency of Pernambuco Recife PE Brazil) isalso acknowledged

References

[1] M Li Y T Li D W Li and Y T Long ldquoRecent developmentsand applications of screen-printed electrodes in environmentalassaysmdasha reviewrdquo Analytica Chimica Acta vol 734 pp 31ndash442012

[2] N Serrano J M Dıaz-Cruz C Arino andM Esteban ldquoEx situdeposited bismuth bilm on screen-printed carbon electrodea disposable device for stripping voltammetry of heavy metalionsrdquo Electroanalysis vol 22 no 13 pp 1460ndash1467 2010

[3] M Dıaz -Gonzalez D Hernandez-Santos M B Gonzalez-Garcıa and A Costa-Garcıa ldquoDevelopment an immunosen-sor the determination rabbit IgG using streptavidin modifiedscreen-printed carbon electrodesrdquo Talanta vol 65 no 2 pp565ndash573 2005

[4] P Ekabutr O Chailapakul and P Supaphol ldquoModificationof disposable screen-printed carbon electrode surfaces withconductive electrospun nanofibers for biosensor applicationsrdquoJournal of Applied Polymer Science vol 130 no 6 pp 3885ndash3893 2013

[5] C I L Justino T A P Rocha-Santos and A C DuarteldquoAdvances in point-of-care technologies with biosensors basedon carbonnanotubesrdquoTrACTrends inAnalytical Chemistry vol45 pp 24ndash36 2013

[6] I Willner and B Willner ldquoBiomolecule-based nanomaterialsand nanostructuresrdquoNano Letters vol 10 no 10 pp 3805ndash38152010

[7] C B Jacobs M J Peairs and B J Venton ldquoReview carbonnanotube based electrochemical sensors for biomoleculesrdquoAnalytica Chimica Acta vol 662 no 2 pp 105ndash127 2010

[8] S K Vashist D Zheng K Al-Rubeaan J H T Luong and F-S Sheu ldquoAdvances in carbon nanotube based electrochemical

sensors for bioanalytical applicationsrdquo Biotechnology Advancesvol 29 no 2 pp 169ndash188 2011

[9] C Singh S Srivastava M A Ali et al ldquoCarboxylated multi-walled carbon nanotubes based biosensor for aflatoxin detec-tionrdquo Sensors and Actuators B Chemical vol 185 pp 258ndash2642013

[10] P Peluso D S Wilson D Do et al ldquoOptimizing anti-body immobilization strategies for the construction of proteinmicroarraysrdquo Analytical Biochemistry vol 312 no 2 pp 113ndash124 2003

[11] C Chen A Baeumner and R Durst ldquoProtein G-liposomalnanovesicles as universal reagents for immunoassaysrdquo Talantavol 67 no 1 pp 205ndash211 2005

[12] YWan Y Su X Zhu G Liu and C Fan ldquoDevelopment of elec-trochemical immunosensors towards point of care diagnosticsrdquoBiosensors and Bioelectronics vol 47 pp 1ndash11 2013

[13] P W Chiu G S Duesberg U Dettlaff-Weglikowska andS Roth ldquoInterconnection of carbon nanotubes by chemicalfunctionalizationrdquo Applied Physics Letters vol 80 no 20 pp3811ndash3813 2002

[14] D E Hill Y Lin A M Rao L F Allard and Y-P SunldquoFunctionalization of carbon nanotubes with polystyrenerdquoMacromolecules vol 35 no 25 pp 9466ndash9471 2002

[15] D Shi J Lian P He et al ldquoPlasma coating of carbon nanofibersfor enhanced dispersion and interfacial bonding in polymercompositesrdquo Applied Physics Letters vol 83 no 25 pp 5301ndash5303 2003

[16] S W Kim T Kim Y S Kim et al ldquoSurface modifications forthe effective dispersion of carbon nanotubes in solvents andpolymersrdquo Carbon vol 50 no 1 pp 3ndash33 2012

[17] M R S Castro and H K Schmidt ldquoPreparation and charac-terization of low- and high-adherent transparent multi-walledcarbon nanotube thin filmsrdquo Materials Chemistry and Physicsvol 111 no 2-3 pp 317ndash321 2008

[18] W-M Chiu and Y-A Chang ldquoChemical modification ofmultiwalled carbon nanotube with the liquid phase methodrdquoJournal of Applied Polymer Science vol 107 no 3 pp 1655ndash16602008

[19] H D White and D P Chew ldquoAcute myocardial infarctionrdquoTheLancet vol 372 no 9638 pp 570ndash578 2008

[20] A A Mohammed and J L Januzzi ldquoClinical applications ofhighly sensitive troponin assaysrdquo Cardiology in Review vol 18no 1 pp 12ndash19 2010

[21] F S Apple A H B Wu and A S Jaffe ldquoEuropean Society ofCardiology and American College of Cardiology guidelines forredefinition ofmyocardial infarctionHow to use existing assaysclinically and for clinical trialsrdquo The American Heart Journalvol 144 no 6 pp 981ndash986 2002

[22] B Zhang A Morales R Peterson L Tang and J Y Ye ldquoLabel-free detection of cardiac Troponin I with a photonic crystalbiosensorrdquo Biosensors and Bioelectronics vol 58 pp 107ndash1132014

[23] M J Guy Y C Chen L Clinton et al ldquoThe impact ofantibody selection on the detection of cardiac troponin IrdquoClinica Chimica Acta vol 420 pp 82ndash88 2013

[24] J Shen W Huang L Wu Y Hu and M Ye ldquoThermo-physicalproperties of epoxy nanocomposites reinforced with amino-functionalized multi-walled carbon nanotubesrdquo Composites AApplied Science andManufacturing vol 38 no 5 pp 1331ndash13362007


[25] Z Yang and D Min Zhou ldquoCardiac markers and their point-of-care testing for diagnosis of acute myocardial infarctionrdquoClinical Biochemistry vol 39 no 8 pp 771ndash780 2006

[26] A B Mattos T A Freitas L T Kubota and R F Dutra ldquoAn o-aminobenzoic acid film-based immunoelectrode for detectionof the cardiac troponin T in human serumrdquo BiochemicalEngineering Journal vol 71 pp 97ndash104 2013

[27] M-L Sham and J-K Kim ldquoSurface functionalities of multi-wall carbon nanotubes after UVOzone and TETA treatmentsrdquoCarbon vol 44 no 4 pp 768ndash777 2006

[28] J Xiong Z Zheng X Qin M Li H Li and X Wang ldquoThethermal and mechanical properties of a polyurethanemulti-walled carbon nanotube compositerdquo Carbon vol 44 no 13 pp2701ndash2707 2006

[29] J Shen W Huang L Wu Y Hu and M Ye ldquoThermo-physicalproperties of epoxy nanocomposites reinforced with amino-functionalized multi-walled carbon nanotubesrdquo Composites AApllied Science and Manufacturing vol 38 no 5 pp 1331ndash13362007

[30] R O Kadara N Jenkinson and C E Banks ldquoCharacterisationof commercially available electrochemical sensing platformsrdquoSensors and Actuators B Chemical vol 138 no 2 pp 556ndash5622009

[31] Z Pei H Anderson A Myrskog G Duner B Ingemars-son and T Aastrup ldquoOptimizing immobilization on two-dimensional carboxyl surface pH dependence of antibody ori-entation and antigen binding capacityrdquoAnalytical Biochemistryvol 398 no 2 pp 161ndash168 2010

[32] R A Williams and H W Blanch ldquoCovalent immobilization ofprotein monolayers for biosensor applicationsrdquo Biosensors andBioelectronics vol 9 pp 159ndash167 1994

[33] A Makaraviciute and A Ramanaviciene ldquoSite-directed anti-body immobilization techniques for immunosensorsrdquo Biosen-sors and Bioelectronics vol 50 pp 460ndash471 2013

[34] L Ruiyi X Qianfang L Zaijun S Xiulan andL Junkang ldquoElectrochemical immunosensor forultrasensitive detection of microcystin-LR based ongraphenegold nanocompositefunctional conductingpolymergold nanoparticleionic liquid composite filmwith electrodepositionrdquo Biosensors and Bioelectronics vol 44no particle pp 235ndash240 2013

[35] N B Ramırez A M Salgado and B Valdman ldquoThe evolutionand developments of immunosensors for health and envi-ronmental monitoring problems and perspectivesrdquo BrazilianJournal of Chemical Engineering vol 26 no 2 pp 227ndash2492009

[36] G L Long and J D Winefordner ldquoLimit of detection a closerlook at the IUPAC definitionrdquo Analytical Chemistry vol 55 no7 pp 713Andash724A 1983

[37] S L R Gomes-Filho A C M S Dias M M S Silva B VM Silva and R F Dutra ldquoA carbon nanotube-based electro-chemical immunosensor for cardiac troponin TrdquoMicrochemicalJounal vol 109 pp 10ndash15 2013

Submit your manuscripts athttpwwwhindawicom

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International Journal of

Volume 2014

Zoology


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BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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Virolog y

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


bonds with the amino groups of the CNTs [12] Thus amino-functionalized nanotubes improve the reactivity of antigen-antibody recognition and the efficiency in immobilizationprocess

Techniques that are specifically intended for CNTs func-tionalization with amino groups comprise chemical treat-ments using acids sheathing or wrapping of the CNTs withpolymer chains [13 14] grafting of CNTs with a thin layerof polymer chains based on plasma [15] or a combinationof these [16] However these methods are generally a com-plicated process involving a long reaction time and severalcoupling reagents and require strictly controlled reactionconditions [17 18] In this work a simple method basedon fractional factorial design has been proposed for amino-functionalization of the CNTs using the ethylenediamine(EDA) as crosslink amino reagent Under optimization pro-cess of functionalization amino-CNTs were employed todevelop an immunosensor for cardiac troponin T (cTnT) animportant marker for acute myocardial infarction

Cardiovascular diseases are the leading cause of deathglobally according to the World Health Organization statis-tics Among the cardiovascular diseases acute myocardialinfarction (AMI) is one of the most serious diseases thatextremely affect peoplersquos health [19] In the past decadescardiac troponins (cTnT and cTnI) have been recommendedas the biomarkers of choice for the serological diagnosisand prognosis of AMI because of their high sensitivity andspecificity [20ndash23] In particular the cTnT levels increase2ndash4 h after the AMI symptoms and could be elevated upto 14 days after the acute episode of myocardial damage[24 25] Thus the development of a rapid and practicalimmunosensor for the detection of the cTnT in serumsamples from patients with myocardial infarction is desirabledue to its roles in cardiospecific diagnosis risk stratificationprognostic risk assessment and therapeutic choices

2 Materials and Methods

21 Reagents and Materials Mouse monoclonal antibodyagainst cTnT (mAb-cTnT) cTnT and peroxidase conju-gated mouse monoclonal antibody against cTnT (mAb-cTnT-HRP) were purchased from Calbiochem (DarmstadtDEU) COOH-functionalizedmultiwalled carbon nanotubes(MWCNTs) were obtained from Dropsens (Oviedo ESP)EDA N-ethyl-N1015840-(3-dimethylaminopropyl) carbodiimide(EDC) N-hydroxysuccinimide (NHS) dimethylformamide(DMF) and glycine were acquired from Sigma-Aldrich (StLouis USA) Ethanol sulfuric acid (H

2

SO4

) (98 wv) andhydrogen peroxide (H

2

O2

) (30 wv) were obtained from FMaia (Cotia BRA) All reagents were of analytical gradeThewater used to prepare all solutions was obtained from aMilli-Qwater purification system (Billerica USA) All the solutionswere freshly prepared prior to each experiment

22 Serum Samples The serum samples of cTnT wereobtained from venous blood and immediately centrifugedfor 120 s at 1150 rads Aliquots of serum were quantifiedin an automatic system Roche Elecsys 2010 immunoassay

analyzer based on electrochemiluminescence immunoassay(ECLIA) and the remaining serum stored at minus20∘C was usedfor the electrochemical measurements The venous bloodsamples were collected from donorrsquos patient from the CardiacEmergency of Pernambuco (PROCAPE) the Hospital ofPernambuco State University according to ethics committeersquosrecommendations

23 Apparatus and Measurements Electrochemical studieswere performed by using a 120583Autolab III analysis system withGPES 49 software Eco Chemie (Utrecht NLD) Screen-printed carbon electrodes (SPCEs) were purchased fromDropSens (Oviedo ESP) These electrodes (reference 110)incorporate a conventional three-electrode configurationprinted on ceramic substrates (34 cm times 10 cm) Both work-ing (disk-shaped 40mm diameter) and counter electrodeswere made of the carbon inks whereas the pseudoreferenceelectrode and electric contacts were made of silver

Cyclic voltammetry (CV) was performed at 100mV sminus1scan rate in presence of PBS (001mol Lminus1 pH 70) used in allexperiments as electrolyte support The chronoamperometrywas carried out in the same electrolyte at the potentialof +03 V at 120 s All experiments were executed at roomtemperature (24∘C)

24 Characterization of the MWCNTs The structural char-acterization of the MWCNTs was evaluated by Fouriertransform infrared (FTIR) spectroscopy The spectra wereobtained by using a Bruker IFS 66model FT-IR spectrometerin the region 4000 to 400 cmminus1 by the standard KBr pellettechniqueThemodification of the SPE surface by depositionof the MWCNTs was characterized by scanning electronmicroscopy (SEM) using Philips XL30 FEG FE-SEM at 10 kV

25 Amino-Functionalization of the MWCNTs The car-boxylic groups of the MWCNTs were activated by using asolution of 01mol Lminus1 acetate buffer (pH 48) containing01mol Lminus1 EDC and 02mol Lminus1 NHS [1 1] At the sametime amino groups of the EDA were submitted to acidtreatment in H

2

SO4

solution (01mol Lminus1) for 2 h understirring conditions at 60∘C changing their protonation stateAfter that 10mg activatedMWCNTs was dispersed in 10 120583LEDA treated and it was stirred for 25 h at room temperatureThe sample was centrifuged at 2000timesg and washed five timeswith deionized water to remove acid residues The resultingamino-MWCNTs were dried at 150∘C and then dispersed inDMF under sonication during 2 h

26 Electrode Preparation Prior to use the SPCE surface(0125 cm2) was sonicated with ethanol and deionized waterrespectively for 2min to remove any organic contaminantSPCE was coated with 30 120583L of the amino-MWCNTs on theworking electrode surface and dried at 40∘C for 5min forabsolute evaporation

27 mAb-cTnT Immobilization After being coated withamino-MWCNTs the SPCE surface was incubated with




2

O2


2

O2

at room temperature


2

SO4





2



2

SO4



DV resposta

gt06

lt06

lt04

lt02

lt 0

ltminus02

minus04

minus02

00

02

04

06

08

020

4060

80100

120


40

35

30

25

20

15

10

05

Tim

e of a

cid tr

eatm

ent o

f EDA

ΔI120583

A








ExperimentEDA















minus + + + minus 0482016 + + + + + 04475

4000 3500 3000 2500 2000 1500 1000

055

060

065

070

075

1200

1335

1520

3170

2989

29953420

Abso

rban

ce (

)


(a)

(b)

3445

3170









(a) (b)


(SPCE)

(mAb-cTnT)

(amino-MWCNTs)

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

C

C=O

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

C=O C=O C=O C=O C=O

(COOH-MWCNTs)

CO OH

ClNH

N

NC

CO O

CO O

O

O

(EDC)

(activated MWCNTs)

(EDA acid treated)

+ C=ON-H

CO O

O

O

(amino-MWCNTs)

(NHS)

minus

+

OHN OO

NN HH

NH2+

NH2+

C=OC=O

NH2+

NH2+

NH2+

Cl NH

N

NC

minus

+

N

N

(a)

(b) (c)







45 50 55 60 65 70 75 80

020

025

030

035

040

045

ΔI c(120583A)

pH

(a)

0 5 10 15 20

000

005

010

015

020

025

030

035

040

045


ΔI c(120583A)

(b)

0 1 2 3 4 5

030

035

040

045

050


ΔI c(120583A)

(c)


2

O2



O2




2

O2




0 1 2 3 4 5 6 7 8 9 10 11

004

006

008

010

012

014

Electrodes (n)

ΔI c(120583A)

(a)

0 5 10 15 20

020

022

024

026

028

030

032

034

Cycles (n)

ΔI c(120583A)

(b)


000 005 010 015 020 025 030 035

minus045

minus040

minus035

minus030

minus025

CcTnT (ng mLminus1)

ΔI c(120583A)






LOD = 3SD119898

(1)



4 Conclusions




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2

O2


2

O2

at room temperature


2

SO4





2



2

SO4



DV resposta

gt06

lt06

lt04

lt02

lt 0

ltminus02

minus04

minus02

00

02

04

06

08

020

4060

80100

120


40

35

30

25

20

15

10

05

Tim

e of a

cid tr

eatm

ent o

f EDA

ΔI120583

A








ExperimentEDA















minus + + + minus 0482016 + + + + + 04475

4000 3500 3000 2500 2000 1500 1000

055

060

065

070

075

1200

1335

1520

3170

2989

29953420

Abso

rban

ce (

)


(a)

(b)

3445

3170









(a) (b)


(SPCE)

(mAb-cTnT)

(amino-MWCNTs)

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

C

C=O

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

C=O C=O C=O C=O C=O

(COOH-MWCNTs)

CO OH

ClNH

N

NC

CO O

CO O

O

O

(EDC)

(activated MWCNTs)

(EDA acid treated)

+ C=ON-H

CO O

O

O

(amino-MWCNTs)

(NHS)

minus

+

OHN OO

NN HH

NH2+

NH2+

C=OC=O

NH2+

NH2+

NH2+

Cl NH

N

NC

minus

+

N

N

(a)

(b) (c)







45 50 55 60 65 70 75 80

020

025

030

035

040

045

ΔI c(120583A)

pH

(a)

0 5 10 15 20

000

005

010

015

020

025

030

035

040

045


ΔI c(120583A)

(b)

0 1 2 3 4 5

030

035

040

045

050


ΔI c(120583A)

(c)


2

O2



O2




2

O2




0 1 2 3 4 5 6 7 8 9 10 11

004

006

008

010

012

014

Electrodes (n)

ΔI c(120583A)

(a)

0 5 10 15 20

020

022

024

026

028

030

032

034

Cycles (n)

ΔI c(120583A)

(b)


000 005 010 015 020 025 030 035

minus045

minus040

minus035

minus030

minus025

CcTnT (ng mLminus1)

ΔI c(120583A)






LOD = 3SD119898

(1)



4 Conclusions




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



ExperimentEDA















minus + + + minus 0482016 + + + + + 04475

4000 3500 3000 2500 2000 1500 1000

055

060

065

070

075

1200

1335

1520

3170

2989

29953420

Abso

rban

ce (

)


(a)

(b)

3445

3170









(a) (b)


(SPCE)

(mAb-cTnT)

(amino-MWCNTs)

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

C

C=O

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

C=O C=O C=O C=O C=O

(COOH-MWCNTs)

CO OH

ClNH

N

NC

CO O

CO O

O

O

(EDC)

(activated MWCNTs)

(EDA acid treated)

+ C=ON-H

CO O

O

O

(amino-MWCNTs)

(NHS)

minus

+

OHN OO

NN HH

NH2+

NH2+

C=OC=O

NH2+

NH2+

NH2+

Cl NH

N

NC

minus

+

N

N

(a)

(b) (c)







45 50 55 60 65 70 75 80

020

025

030

035

040

045

ΔI c(120583A)

pH

(a)

0 5 10 15 20

000

005

010

015

020

025

030

035

040

045


ΔI c(120583A)

(b)

0 1 2 3 4 5

030

035

040

045

050


ΔI c(120583A)

(c)


2

O2



O2




2

O2




0 1 2 3 4 5 6 7 8 9 10 11

004

006

008

010

012

014

Electrodes (n)

ΔI c(120583A)

(a)

0 5 10 15 20

020

022

024

026

028

030

032

034

Cycles (n)

ΔI c(120583A)

(b)


000 005 010 015 020 025 030 035

minus045

minus040

minus035

minus030

minus025

CcTnT (ng mLminus1)

ΔI c(120583A)






LOD = 3SD119898

(1)



4 Conclusions




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)


(SPCE)

(mAb-cTnT)

(amino-MWCNTs)

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

NH2+

C=O

N-H

C

C=O

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

N-H

C=O

N-H

C=O C=O C=O C=O C=O

(COOH-MWCNTs)

CO OH

ClNH

N

NC

CO O

CO O

O

O

(EDC)

(activated MWCNTs)

(EDA acid treated)

+ C=ON-H

CO O

O

O

(amino-MWCNTs)

(NHS)

minus

+

OHN OO

NN HH

NH2+

NH2+

C=OC=O

NH2+

NH2+

NH2+

Cl NH

N

NC

minus

+

N

N

(a)

(b) (c)







45 50 55 60 65 70 75 80

020

025

030

035

040

045

ΔI c(120583A)

pH

(a)

0 5 10 15 20

000

005

010

015

020

025

030

035

040

045


ΔI c(120583A)

(b)

0 1 2 3 4 5

030

035

040

045

050


ΔI c(120583A)

(c)


2

O2



O2




2

O2




0 1 2 3 4 5 6 7 8 9 10 11

004

006

008

010

012

014

Electrodes (n)

ΔI c(120583A)

(a)

0 5 10 15 20

020

022

024

026

028

030

032

034

Cycles (n)

ΔI c(120583A)

(b)


000 005 010 015 020 025 030 035

minus045

minus040

minus035

minus030

minus025

CcTnT (ng mLminus1)

ΔI c(120583A)






LOD = 3SD119898

(1)



4 Conclusions




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


45 50 55 60 65 70 75 80

020

025

030

035

040

045

ΔI c(120583A)

pH

(a)

0 5 10 15 20

000

005

010

015

020

025

030

035

040

045


ΔI c(120583A)

(b)

0 1 2 3 4 5

030

035

040

045

050


ΔI c(120583A)

(c)


2

O2



O2




2

O2




0 1 2 3 4 5 6 7 8 9 10 11

004

006

008

010

012

014

Electrodes (n)

ΔI c(120583A)

(a)

0 5 10 15 20

020

022

024

026

028

030

032

034

Cycles (n)

ΔI c(120583A)

(b)


000 005 010 015 020 025 030 035

minus045

minus040

minus035

minus030

minus025

CcTnT (ng mLminus1)

ΔI c(120583A)






LOD = 3SD119898

(1)



4 Conclusions




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1 2 3 4 5 6 7 8 9 10 11

004

006

008

010

012

014

Electrodes (n)

ΔI c(120583A)

(a)

0 5 10 15 20

020

022

024

026

028

030

032

034

Cycles (n)

ΔI c(120583A)

(b)


000 005 010 015 020 025 030 035

minus045

minus040

minus035

minus030

minus025

CcTnT (ng mLminus1)

ΔI c(120583A)






LOD = 3SD119898

(1)



4 Conclusions




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


4 Conclusions




Acknowledgments


References















































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 Amino-Functionalization of Carbon...

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Transcript of Research Article Amino-Functionalization of Carbon...