synthesis of carbon nanofibers for mediatorless sensitive detection of nadh

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Synthesis of Carbon Nanofibers for Mediatorless SensitiveDetection of NADH

Yang Liu,a Haoqing Hou,b Tianyan Youa*a State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Graduate School of the ChineseAcademy of Sciences, Jilin 130022, P. R. China

b College of Chemistry and Chemical Engineering, Jiangxi Normal University, Jiangxi 330027, P. R. China*e-mail: [email protected]

Received: February 27, 2008Accepted: April 29, 2008

AbstractHighly sensitive amperometric detection of dihydronicotinamide adenine dinucleotide (NADH) by using novelsynthesized carbon nanofibers (CNFs) without addition of any mediator has been proposed. The CNFs were preparedby combination of electrospinning technique with thermal treatment method and were applied without any oxidationpretreatment to construct the electrochemical sensor. In amperometric detection of NADH, a linear range up to11.45 mM with a low detection limit of 20 nM was obtained with the CNF-modified carbon paste electrode (CNF-CPE). Good selectivity was exhibited for the simultaneous detection of NADH and its common interferent ofascorbic acid (AA) by differential pulse voltammogram. The attractive electrochemical performance and the versatilepreparation process of the CNF-CPE made it a promising candidate for designing effective NADH sensor.

Keywords: Carbon nanofibers, Amperometric sensor, NADH, Electrocatalysis, Nonmediator

DOI: 10.1002/elan.200804242

1. Introduction

In recent decades, dihydronicotinamide adenine dinucleo-tide (NADH) has attracted considerable interest due to itssignificant role as cofactor formore than 300 dehydrogenaseenzymes and in the electron-transfer chain in biologicalsystem [1, 2]. Since the sensing ofmany important biologicalanalytes is based on the electrochemical detection ofenzymatically generated NADH, a highly sensitive andselective NADH sensor is increasingly desirable [3]. How-ever, the direct oxidation of NADH at conventionalelectrodes such as platinum and carbon requires largeoverpotential (>1.0 V), which causes notably increasedbackground current, making it difficult to obtain a well-defined limiting current, especially at low NADH concen-trations [2, 4]. Therefore, various mediators, such as theadenine derivatives [5, 6], aromatic compounds containingcatechol or its derivative group [7, 8], phenoxazine dyes [9],organic salt [10], metal complexes [11], and conductingpolymers [12, 13], were proposed to improve the electro-chemical response by lowering the overpotential. Althoughthe use of mediators is promising, such biosensors havecertain drawbacks including mediator leaching from theelectrode surface especially in a continuous flow system,lacking of long-term stability and requiring consideration ofmediator toxicity, which may limit their analytical applica-tion [14, 15].Recently, with the great development of nanotechnology

in material science, carbon nanotubes (CNTs) and carbon

nanofibers (CNFs) have been widely used in electrochem-istry due to their unique nanostructure and electronicproperties [16 – 19]. In fabrication of enzyme biosensors,CNFs are more favorable compared with CNTs becausethe whole surface could be activated and then the oxygen-containing activated sites are ideal for immobilization ofenzymes [20]. Additionally, CNFs, which have cylindricalnanostructureswith graphite layers arranged as cones, cupsor plates, possess large amount of edge plane sites that mayplay an important role in electrocatalysis toward manybiological molecules [21]. In amperometric detection ofNADH, Arvinite et al. reported a detection limit of 11 mMby using CNF-modified glassy carbon electrode [17]. Wuet al. proposed soluble CNFs with a low overpotential tocatalyze the NADH oxidation, while the high backgroundcurrent due to the presence of oxygen-rich groups on thesurface of CNFs made it difficult to detect NADH down tonmol L�1-level [18]. Additionally, long time oxidationtreatment of either CNFs or CNTs may degrade thestructural backbone and even have negative effect on theirelectronic properties [22].In this work, CNFs were produced by combination of

electrospinning technique with subsequent thermal treat-ment, andwere usedwithout any oxidation pretreatment fordesigning mediatorless NADH sensor with a low detectionlimit. In addition, good selectivity could be obtained for thedetection of NADH in presence of ascorbic acid (AA) withthe CNF-modified carbon paste electrode (CNF-CPE).

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Electroanalysis 20, 2008, No. 15, 1708 – 1713 F 2008 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

2. Experimental

2.1. Chemicals and Reagents

Polyacrylonitrile (PAN), dimethylformamide (DMF),NADH and graphite powder (2 mm) were purchased fromAldrich. Mineral oil and AA were obtained from BeijingChemical Co. (China). All other reagents were of analyticalgrade andwereusedwithout further purification. Phosphatebuffer solution (PBS, 0.1 M, pH 7.0)was prepared bymixingsolution of Na2HPO4 and NaH2PO4.

2.2. Apparatus

The X-ray photoelectron spectroscopy (XPS) was recordedon an ESCALAB-MKII spectrometer (VGCo., UK) with aMo Ka X-rays radiation as the X-ray source for excitation.The scanning electronmicroscopy (SEM) experiments weremadeonaPHILIPSXL-30ESEMat anaccelerating voltageof 20 kV. Electrochemical impedance spectroscopy (EIS)was performed with a CHI 660 electrochemical analyzer in5 mM K3Fe(CN)6/K4Fe(CN)6 (1 : 1) with 0.5 M KCl assupporting electrolyte, using an alternating current voltageof 5 mV within a frequency range of 0.01 – 105 Hz. Allelectrochemical measurements were performed on a CHI832 electrochemical workstation (Shanghai, China) with aconventional three-electrode system composed of a plati-num auxiliary, a Ag/AgCl (saturated KCl) reference, and abare or modified CPE working electrode. Amperometricmeasurements were carried out in a continuous stirringsolution of 2 mL PBS (pH 7.0) using a magnetic stirrer.Differential pulse voltammogram (DPV) experiments wereperformed with pulse potential 50 mV, pulse width 50 msand pulse period 0.2 s.

2.3. Synthesis of CNFs

The CNFs were prepared by carbonizing the PAN fibers,which were made via an electrospinning technique by using8% wt. PAN solution in DMF. The electrospinning processwas performed in the electric fields of the order of 100 kVm�1, from a 30 kV voltage applied to a 30 cm gap betweenthe spinneret and the collector. The stabilization andcarbonization of PAN fibers were completed in a hightemperature furnace by the following steps: 1) 230 8Cannealing in air for 3 h to oxidize PAN partially; 2) heatingup to 300 8C at a rate of 5 8C min�1 and stayed at thistemperature in H2 and Ar mixture (H2/Ar¼ 1/3) for 2 h forthe shape stabilization of the fibers; 3) heating up to 1100 8CinAr at a rate of 5 8Cmin�1 to carbonize the fibers, staying atthe highest temperature for half an hour, and then coolingdown to room temperature in Ar.

2.4. Preparation of Electrodes

CPE was made by packing 70/30 (w/w) graphite powder/mineral oil paste into a pipette tube (1.2-mm CID; 1-cmdepth) and inserting copper wire for electrical contact. Thesurface of the resulting CPE was smoothed and rinsed withdouble-distilled water. OnemilligramCNFs were dispersedin 1 mL water with vigorous agitation to form uniformsuspension. The CNF-CPE was obtained by applying theCNF suspension (8 mL) to the surface of the CPE, followedby drying at room temperature in a desiccator. (Note: inpreliminary NADH electrooxidation experiments withCNF-CPEs prepared using different volumes of CNFsuspension, 8 mL was identified as the optimum for max-imum catalytic current without significant peak broadeningor background current increasing.)

3. Results and Discussion

3.1. Synthesis and Characteristics of CNFs

Electrospinning has been extensively investigated as anefficient and versatile approach to produce long polymerfibers with the diameter ranging from tens of nanometers toseveral micrometers, and subsequent carbonization of theelectrospun polymer fibers at high temperature could beused to prepare CNFs [23 – 25]. Herein, PAN fibers, whichwere commonly used as precursor of CNFs, were extrudedfrom the polymer solution via electrostatic forces in theprocess of electrospinning.And then, large-scaleCNFswereproduced by the carbonization of the electrospun PANfibers at high temperature as expected. The XPS character-ization was used to investigate each element existed in theCNFs (Fig. 1). The wide range spectra of the CNFs indicatethat in addition to the characteristic band of carbon, itexhibits a small N1s peak at 401.5 eVand a weak O1s signalat 532.6 eV. Since the CNFs were produced by usingpolyacrylonitrile (PAN) as precursor, the low content ofN1s may be attributed to a few nitrogen subsisted in theprocess of carbonization. In addition, the oxygen to carbon

Fig. 1. Wide range XPS spectra of the CNFs.

1709Mediatorless Sensitive Detection of NADH

Electroanalysis 20, 2008, No. 15, 1708 – 1713 www.electroanalysis.wiley-vch.de F 2008 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

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atomic ratio (O1s/C1s) is calculated to be 5.6%, which iscomparable to that obtained with other carbon-basedelectrodes before the oxidation treatment, such as 5.3%with the electron-cyclotron-resonance plasma sputteredcarbon (ECR-SC) film [26] and 7.0% with the screen-printed carbon electrode (SPCE) [21]. Therefore, the lowcontent of oxygen may be attributed to the existence ofphysisorbed oxygen. In addition, the morphology of theCNFs were investigated by SEM and TEM (Fig. 2). Theresults show that the as-prepared CNFs have a diameterfrom 200 to 500 nm with tens of micrometers in length, andthe rough and porous surface of the CNFs, which may havesignificant effect on their electrochemical properties, couldbe observed in the TEM image.Since CNFs are highly conductive and are often used as

catalysts or catalyst supports due to the unique nano-structure, electronic and mechanic properties [27], wesimply prepared CNF-CPE to investigate the electrochem-ical performance of the CNFs by using 5 mM [Fe(CN)6]

3�/4�

redox probe. As shown in Figure 3a, a pair of redox peakswith peak-to-peak separation (DEp) of 151.8 mV wasobserved at CPE, while at CNF-CPE, the DEp decreasedto 77.4 mVand the anodic peak currentwas two times higherthan that at CPE (Fig. 3b), indicating the improved rever-sibility of the redox reaction and the enlarged electroactivesurface area of the modified electrode. Additionally, EISillustrates a notable decrease in resistance (Rct) atCNF-CPEas compared with CPE (Fig. 4), further confirming that theCNFs were highly conductive and facilitated electron-transfer kinetics.

3.2. Amperometric Detection of NADH

Since Wang et al. proposed low-potential NADH detectionat CNT-modified glassy carbon electrodes [16], much workconcerning CNT-based electrodes for determination ofNADH have been reported [4, 28 – 30]. Although theoverpotential was notably decreased and the sensitivityincreased to some extent because of the electrocatalyticeffect of the mediators, effective NADH sensing was still a

challenge because the leaching of the mediators and theinterference of other electroactive compounds were inevi-table [3, 14]. Instead of CNT-composites, pristine CNTs orCNFs avoided the problem of mediator leaching, however,the high background current due to the oxygen-containinggroups that were produced by oxidation pretreatmentmadeit difficult to lower the detection limit down to nmol L�1-level [16 – 18]. Herein, our interest is focused on thedevelopment of mediatorless sensor for detecting NADHat low concentrations by using the novel synthesized CNFs.As shown inFigure 5, theCNF-CPEdisplayed a remarkableincrease of the anodic peak current by two times and anegative shift of the oxidation potential by 144 mV ascompared with the CPE. Since the sensitivity estimatedaccording toCVwasmuchhigher than that of the previouslyreported CNF-modified electrodes [17, 18], the novel CNFswere expected to exhibit excellent electrocatalytic activitytoward NADH oxidation.In order to obtain the maximum catalytic current, the

effect of applied potential on the steady-state current ofNADH at the CNF-CPE was investigated.With the applied

Fig. 2. SEM image of CNFs. Inset shows TEM image of CNFs. Fig. 3. CVs of 0.5 M KCl solution containing 5 mM K3Fe(CN)6/K4Fe(CN)6 at (a) the CPE and (b) the CNF-CPE. Scan rate: 0.1 Vs�1.

Fig. 4. EIS of the CNF-CPE in 0.5 M KCl solution containing5 mM K3Fe(CN)6/K4Fe(CN)6. Inset shows EIS of the CPE in thesame solution.

1710 Y. Liu et al.



potential shifting from 0.2 to 0.6 V, the steady-state currentincreased until reach a plateau at 0.45 V (not shown).Therefore, 0.45 V was identified as the optimum in thefollowing experiments. Figure 6a indicates the amperomet-ric response of CNF-CPE to successive addition of certainconcentrations of NADH into the stirring buffer solution.After each injection ofNADH, the anodic current increasedimmediately and reached a steady state within 5 s. Itexhibited a linear range from 0.02 to 11.47 mM with acorrelation coefficient of 0.9981 and a sensitivity of 10 nAmM�1 (Fig. 6a). The linear range is much wider than that of0.01 – 0.5 mM reported previously with boron-doped dia-mond electrode at operating potential of 0.58 V [14]. Thedetection limit of 20 nM (S/N¼ 3) (Fig. 6b), which isattributed to the extremely low background current, isamong the lowest ones reported for amperometric detectionofNADH.Tunon et al. reported an extremely low detectionlimit of 2.5 nMbyusing adenosine-5M-diphosphate (ADP) asprecursor for NADH electrocatalysis [5]. Rao et al. pro-posed an amperometric detection limit of 10 nM by usingboron-doped diamond electrodes [14]. Jena et al. reported abiosensor for low-potential oxidation of NADH based onsilicate network supported Au nanoparticles with an am-perometric detection limit of 5 nM [31]. Recently, Tu et al.synthesized novel quinone-amine polymer/carbon nano-tubes composite for electrocatalytic oxidation of NADH

with a detection limit of 6.4 nM [28]. While the electrodematerials mentioned above require either delicate fabrica-tionmethod or multi-step synthetic procedure. In this work,without pretreatment or combination of any mediator, weobtained a low detection limit at the electrospun CNFs,which was much lower than that at commercial CNFs [17],soluble CNFs [18], edge plane pyrolytic graphite [32],ordered CNTs [33], and comparable to that at the boron-doped diamond electrode [14] (Table 1). Therefore, theproposed CNF is a promising candidate for designingconvenient and sensitive NADH sensor.

Fig. 5. CVs of 0.1 M PBS (pH 7.0) a) plain and b) containing1 mM NADH at the CNF-CPE. c) Corresponding CV of (b) withthe CPE. Scan rate: 50 mV s�1.

Table 1. Analytical parameters for the detection of NADH at different carbon-based electrodes.

Carbon material Detection limit (mM) Peak separation betweenNADH and AA (mV)

Reference

Electrospun CNFs 0.02 330 Our workCommercial CNFs 11 – [17]Soluble CNFs 0.11 – [18]CNT-nanobiocomposite 0.5 100 [4]Edge plane pyrolytic graphite 0.33 260 [32]Ordered CNTs 0.5 – [33]Boron-doped diamond 0.01 130 [14]

Fig. 6. a) Current – time responses of the CNF-CPE uponsuccessive injection of certain concentration of NADH into0.1 M PBS (pH 7.0). Inset shows the calibration curve forNADH detection. b) Amplified response curve of (a) at low-concentration injection. Applied potential: 0.45 V.




3.3. Interference Study

Another attractive performance of the CNF-CPE is thegood selectivity for simultaneous detection of NADH andAA. AA is the common electroactive interferent foramperometric detection of NADH because of their similaroxidation potentials [14, 32]. One way to alleviate thisproblem was by using a membrane like Nafion, which couldelectrostatically exclude anions, while the drawback was areduction in sensitivity since NADH was also negativelycharged at neutral pH [34]. Another effective approach wasto apply ascorbate oxidase, which was an enzyme capable ofselectively oxidizingAA in presence of oxygen [35]. Herein,we investigated the current response to the mixture ofNADH and AA with the CNF-CPE by DPV, which wascommonly used as a well-resolved electrochemical tech-nique. As shown in Figure 7a, the peak separation foroxidation of NADH and AA was not large enough forselective detection ofNADHat theCPE.While at theCNF-CPE, two well-defined peaks at 417 mV and 87 mV,corresponding to NADH and AA oxidation respectively,were observed (Fig. 7b). The peak separation of 330 mVbetween NADH and AA was larger than the previousreports (Table 1), which suggested the feasibility for simul-taneous sensing of both NADH andAAwith this electrode.Interestingly, the sensitivity of the CNF-CPE towardsNADH remained constant with the increasing concentra-tion of AA (Fig. 7b, 1 – 3), indicating the oxidation processof NADH and AAwere independent from each other andthus NADH determination in presence of AA could berealized at the CNF-CPE.

4. Conclusions

Novel CNFs were prepared by electrospinning techniqueand thermal treatment method. In electrochemical applica-tion to detect NADH, the pristine CNF-CPE exhibited low

detection limit down to nmol L�1-level with wider linearrange and good selectivity for determination of NADH inpresence of AA. As a novel CNF material, the excellentelectrochemical properties and the versatile preparationmethod make it a promising candidate for practical design-ing of effective biosensors.

5. Acknowledgements

We are grateful for the financial support from the NationalNatural Science Foundation of China (20605020), ChineseAcademy of Sciences (KJCX2-YW-H11) and the Founda-tion of Distinguished Young Scholars of Jilin Province(20060112).

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Fig. 7. DPVs of a) CPE in 0.1 M PBS (pH 7.0) containing 1 mMNADH and 0.5 mM AA, and b) CNF-CPE in 0.1 M PBS (pH 7.0)containing 1 mM NADH and different concentrations of AA. Thenumber of 1 – 3 corresponds to 0.2 mM, 0.3 mM, 0.4 mM AA,respectively.

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synthesis of carbon nanofibers for mediatorless sensitive detection of nadh

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