Sensors and Actuators B 117 (2006) 308–313

Letter to the Editor

Novel lactate and pH biosensor for skin and sweat analysisbased on single walled carbon nanotubes

Abstract

Carbon nanotubes (CNTs) were functionalized and employed in an electrochemical cell to serve as a biosensor to specifically detect either lactateor pH in an electrolyte solution of artificial sweat. They were functionalized with the carboxyl group ( COOH) to detect pH and the enzyme lactateoxidase (LOX) to detect lactate. All CNT samples were characterized to compare the electrodes before and after functionalization. Fourier transforminfrared spectroscopy (FTIR) was used to verify the attachment of both COOH and LOX to the respective carbon nanotubes samples. Scanningelectron microscopy (SEM) was used to examine the structure of the CNT–lactate electrode. Square wave voltammetry proved to be the besttemplate to use to sense these target analytes. The functionalized CNT–COOH electrode displayed a linear response to pH 1–10, with a negativevoltage shift corresponding to an increase in pH. Two types of lactate sensors were fabricated, both of which exhibited an increase in currentcorresponding to an increase in lactate concentration. The functionalized CNT–LOX on a glassy carbon electrode displayed an amperometricresponse in the range of 1–4 mM lactate. The CNT–LOX on a silicon/indium tin oxide (Si/ITO) substrate displayed an amperometric response inthe range of 0.01–0.05 M lactate.© 2006 Elsevier B.V. All rights reserved.

Keywords: Lactate oxidase; pH; Square wave voltammetry; Carbon nanotube functionalization; Sweat biosensor

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. Introduction

There are several molecules and ions in human sweat that,hen excreted in abnormal amounts, may be an indication ofseparate pathological disorder. Likewise, pH and lactate are

nalytes present on the skin and excreted in sweat that may beetected and evaluated for diagnostic purposes [1]. The mea-urement of pH is one of the most common tasks in clinicalnalysis. A slightly acidic pH of the skin (4–6) helps deter colo-ization by non-resident bacteria and pathogens because manyacteria can survive only in a narrow pH range near neutral. Ifhe acid mantle is lost or disrupted, the skin becomes more proneo damage and infection. While pH tests most likely utilize thetandard glass membrane electrode, the need to miniaturize thelectrode for fast, local measurements is emerging. Sweat lac-ate is a function of sweat gland energy metabolism, therefore aecrease in oxygen supply induces a rise in sweat lactate concen-ration and a decrease in sweat gland activity. The developmentf pressure sores, or bedsores, has implications on the health ofbedridden individual as well as requirements for health care

rofessionals [2]. Previous studies have evaluated the reduction

As an electrode material, CNTs are not reduced or oxidized overa large range of potentials and demonstrate catalytic activitiestowards many electrochemical reactions [5,6]. Potentiometricstudies of materials such as activated carbon and carbon blackhave shown that the potential of such material decreases withincreasing pH [7]. For biological sensing, CNT endcaps andsidewalls can be modified with a functional group which mayreact with other reagents [8,9]. Single walled carbon nanotubes(SWNTs) were further explored for pH sensing with an enzymelayer immobilized by a sol–gel hybrid material [10]. To date,there has been no published literature on the functionalizationof carbon nanotubes as lactate sensors. However, several suc-cessful attempts of the fabrication of a lactate sensor utilizingthin films and micro electromechanical systems (MEMS) tech-nology has been achieved. Immobilization of lactate oxidaseand lactate dehydrogenase has been prepared on electrochemi-cally conducting polymer films [11]. A microsystem biosensormicroarray has been formed for the detection of lactate and threeother molecules [12]. A flow-injection biosensor for simultane-ous sampling of glucose, l-lactate and pyruvate has recentlybeen developed for rat brain monitoring [13]. An enzyme field-

n oxygen delivery in the tissue of patients with peripheral arte-ial occlusive disease [3]. This research concluded that lactaten calf sweat is a good indicator in the evaluation of the sever-ty of peripheral occlusive arterial disease and in assessing thefficiency of vasoactive drug treatment.

effect transistor (ENFET) using MnO2 nanoparticles and LOXhas also been assembled. The use of the MnO2 nanoparticlesincreased the sensitivity of the sensor by a factor of 50 [14]. Thispaper reports the investigation on SWNTs with respect to pHand lactate sensitivity. SWNTs functionalized with the COOHgv

First discovered in 1991 [4], carbon nanotubes (CNTs) dis-lay excellent qualities to be used as electrochemical sensors.

925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2005.12.025

roup were prepared and utilized as pH biosensors. Square waveoltammetry (SWV) was used to investigate the potentiometric

Letter to the Editor / Sensors and Actuators B 117 (2006) 308–313 309

response of the modified SWNTs with respect to shifts in pH.A linear response was observed in buffer solutions of pH 1–10.In this study, we also investigated the amperometric responseof SWNTs to lactate. The carbon nanotubes displayed a nearlylinear response to lactate in the range of 1–4 mM when immo-bilized on a glassy carbon electrode via physical adsorption.

2. Experimental

2.1. Materials

SWNTs were obtained from Aldrich, with 50–70 vol% sam-ple purity. The tubes occur in bundles with bundle length ofabout 20 �m. All pH measurements were carried out in a 0.1 Mphosphate buffer solution (PBS). The electrolyte was made indeionized water with K2HPO4 (purity 98%, Sigma–Aldrich) andKH2PO4 (purity 99%, Sigma–Aldrich). A solution of 50% HClor a solution of 0.1 M NaCl was used to adjust the pH of the phos-phate buffer. Lactate oxidase (LOX, E.C. 1.13.12.4 from Pedio-coccus species, 49 U/mg) was purchased from Sigma–Aldrich.Lactate measurements were carried out in a 0.1 M phosphatebuffer solution with a pH of 7.23 at room temperature. Lactate(Fluka) was systematically added to the PBS with a pipette.

2.2. Apparatus

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SWNTs were collected on a membrane, then placed in 100 mLof ethanol, sonicated for 20 min, and then filtered again. Duringthe filtration, a large amount of ethanol was repeatedly used tocompletely wash off the unreacted peroxides and the reactionbyproducts. For the construction of the lactate sensor, the pre-viously functionalized carbon nanotubes were further modifiedto be sensitive only to lactate. One milliliter of SWNT–COOHwas placed in a 10-mL beaker and dried in an oven overnightat 30 ◦C. The carbon nanotubes were then rinsed with 1 mL ofdeionized water and again dried overnight at 30 ◦C. From thedried batch of nanotubes, 6.5 mg of CNTs were weighed andplaced in a separate test tube with 75 �L deionized water andshaken for 5 min. Ten IU of LOX were added to the CNTs sus-pended in 75 �L of deionized water. This tube was then shakenfor 3 h to ensure adequate functionalization. FTIR verified thefunctionalization of COOH and LOX on the nanotubes. LOXcan be seen present through the amine group (Fig. 1).

2.4. Construction of the modified electrode

2.4.1. CNT–COOH electrodeThe 3-mm diameter glassy carbon electrode was polished

with 1.0 �m alpha alumina powder and rinsed with deionizedwater. It was then polished again with 0.3 �m alpha aluminapowder. Previously prepared SWNTs modified with the car-boxyl group ( COOH) were suspended in an ethanol solution.Itsoa

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The pH of the buffer solutions was measured with a Corn-ng Scholar 425 pH meter to verify the results obtained fromhe carbon nanotube pH sensor. Square wave voltammetry mea-urements were performed using a Princeton Applied ResearchARSTAT 2263 advanced electrochemical analyzer. All elec-rochemical measurements were performed in a standard three-lectrode system. The modified SWNT/glassy carbon electrodected as the working electrode, an Ag/AgCl (3 M KCl) acteds the reference electrode, and a platinum wire (CH Instru-ents) acted as the counter electrode. All measurements were

erformed at room temperature. The potential scan rate was.1000 mV/s and the step height was 2.0000 mV. Fourier trans-orm infrared spectroscopy (FTIR) analysis was performed onPerkin-Elmer Spectrum One Spectrometer. Scanning electronicroscopy (SEM) images were obtained from a Hitachi 800canning Electron Microscope.

.3. Electrode functionalization

All carbon nanotubes used in the electrode fabrications wereingle walled and functionalized in the laboratory. SWNTs wererst functionalized with the carboxylic acid hydrogen bondedimer. The presence of this type of functional group allows forhe attachment of several other biomolecules, including LOX.he COOH group enables amine bonding through covalentonding at the sidewalls. The SWNTs were chemically function-lized by a previously described technique [15]. Fifty milligramf SWNTs were placed in a flask filled with 50 mL of dry o-ichlorobenzene and sonicated for 30 min to obtain a SWNTuspension solution. The solution was heated at 80–90 ◦C for 10ays while each day 0.5 g of peroxide was added. Functionalized

n 200 �L of deionized water, 5 �L of the modified SWNT solu-ion was added. From this new solution, 5 �L was applied to theurface of the polished glassy carbon electrode via the methodf physisorption. The electrode was dried in an oven at 30 ◦C tollow the solvent and water to evaporate.

.4.2. CNT–LOX electrodeIndium tin oxide (ITO) was sputtered onto a 2-in diameter

ilicon substrate to a thickness of 400 A. From this wafer, sev-ral 1.5 cm × 0.5 cm pieces were cut. Silver paint (Ted Pella)as used to bond the carbon nanotubes to the Si/ITO sub-

trate. A thin layer of paint was brushed on top of the ITO andhen 10 �L of CNT–COOH–LOX was dropped onto the paint

Fig. 1. FTIR spectra of CNT–COOH (top) and CNT–LOX (bottom).

310 Letter to the Editor / Sensors and Actuators B 117 (2006) 308–313

Fig. 2. Schematic drawing of CNT–LOX on Si/ITO electrode.

(Fig. 2). This modified electrode was allowed to dry at roomtemperature overnight. The fabrication of the lactate sensor onglassy carbon is similar to that of the pH sensor. Five microliter ofCNT–COOH–LOX were dropped onto a 3-mm diameter glassycarbon electrode. The modified electrode was then allowed todry at room temperature overnight.

3. Results

3.1. Electrochemical behavior of CNT–COOH electrode

The CNT–COOH sensor exhibited extremely high accuracywith only about 1.22% error. A linear response was observedfor pH 1–10 (Fig. 3). For each increase in pH, the electrodeexperiences a negative shift in potential. The calibration linewas created by taking the voltage values at the peaks of eachcurve in Fig. 3(a and b) and plotting this voltage value by its

respective pH value. Note that the inconsistent vertical spacingof the current between the peak values does not affect the results.Only the horizontal shifts relating to voltage are considered. Thepotential range is from −0.70 to +0.70 V because this is therange of detection. This range was chosen experimentally byperforming the potentiometry and determining the active voltagefor each value of pH. For pH 1–10 the active potential rangeis about −0.3 to 0.5 V; therefore, a voltage range larger than−0.70 to +0.70 V is not necessary. There is a small offset at pH7 where the potential is not quite equal to zero. In order to verifythe stability of the sensor, 20 potentiometric experiments wereperformed at pH 7 with the same electrode. The voltage rangefrom these experiments is −0.008 to 0.11 V with an averagevoltage reading of 0.007 V. From this data the electrode wasfound to be stable.

3.2. Electrochemical behavior of CNT–LOX electrodes

The lactate sensor worked in the range of 0.01–0.05 M lacticacid (Fig. 4). The following electrochemical reaction occurs atthe CNT–LOX electrodes:

Lactate + O2LOX−→Pyruvate + H2O2

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ig. 3. (a) Voltammetric response of CNT–COOH pH electrode for pH 1–6; (b) voltaalibration line.

mmetric response of CNT–COOH pH electrode for pH 7–10; (c) pH electrode

Letter to the Editor / Sensors and Actuators B 117 (2006) 308–313 311

Fig. 4. (a) Amperometric response of CNT–LOX on Si/ITO substrate in the range of 0.01–0.05 M concentrations of lactic acid; (b) CNT–LOX on Si/ITO calibrationline.

Furthermore, hydrogen peroxide is oxidized as follows:

H2O2 → O2 + 2H+ + 2e−

The sensor works by measuring how many electrons are trans-ferred to the working electrode. In this case, for every twotransferred electrons to the working electrode, one moleculeof lactate is detected. However, by continually increasing theamount of lactic acid in the electrochemical cell, the silver paintlost adhesion to the ITO thin film and the silver paint with theimmobilized carbon nanotubes peeled off of the Si/ITO sub-strate. Upon examination by SEM, the silver paint was observedto be extremely porous (Fig. 5). It is important to note that failureoccurred at higher lactate concentrations due to loss of contactbetween the LOX functionalized carbon nanotubes and the ITOthin film via the silver paint. The peaks are not as well definedas they could be due to the silver paint interfering and reactingin the electrochemical cell. This interference was caused by a

redox reaction that occurred with the silver paint. The oxidationand reduction potentials of Ag are −0.080 and +0.80 V, respec-tively. The potential range for this experiment was from −0.90to +0.90 V, which was just large enough to cause the Ag paintredox reaction as well as the LOX reaction with lactate. Thisphenomenon also caused the peaks to not be vertically linearlyspaced with respect to current.

When dried on a glassy carbon electrode, the LOX function-alized carbon nanotubes gave a much better response to changesin lactate at lower concentrations. The response curves forthis experiment provided better overall resolution (Fig. 6). Theresponse current increases linearly with increasing concentra-tion of lactate up to 4 mM, after which steady state was reached.The calibration lines were calculated by taking the maximumcurrent reading for each concentration in Figs. 4(a) and 6(a) andsubsequently plotting that current value versus lactate concen-tration in Figs. 4(b) and 6(b), respectively. The potential rangeof −0.90 to +0.90 V was used for the Si/ITO electrode and a

of C

Fig. 5. (a) SEM image of porous lactate electrode surface; (b) SEM image NTs suspended in silver paint [magnification of selected area in part (a)].

312 Letter to the Editor / Sensors and Actuators B 117 (2006) 308–313

Fig. 6. (a) Amperometric response of CNT–LOX on glassy carbon in the range of 1–4 mM concentrations of lactic acid; (b) CNT–LOX on glassy carbon calibrationline.

potential range of −0.75 to −0.40 V was used for the CNT–LOXglassy carbon electrode. A smaller potential range was chosenand depicted in Fig. 6(a) for the CNT–LOX glassy carbon elec-trode because it was seen that LOX reacts at about −0.53 Vfrom the previous experiment and a larger potential range is notdesirable.

4. Conclusion

In this work, electrochemical sensors designed to target twotypes of analytes in human sweat were fabricated. Due to theirelectrical properties, carbon nanotubes were selected as the elec-trode material for the detection of pH and lactate. They are ableto detect low level concentrations and respond well to electro-chemical characterization. Single walled carbon nanotubes werechemically functionalized to introduce carboxylic acid groupsto the sidewalls of the tubes. The ability to functionalize thesidewalls introduced more sites for the adsorption of lactateoxidase onto the tube and consequently allowed more currentto be detected. The large amount of LOX immobilized on thenanotube greatly increased the stability of the sensor. In orderto verify that the carbon nanotubes were properly functional-ized with carboxylic acid and lactate oxidase FTIR spectra weretaken from several samples. The carboxylic acid functionalizednanotubes displayed a broad peak at 2879 cm−1 correspondingt −1

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paint lost its adhesion in the solution at about 0.6 M lactate. Thiselectrode did not display as good of resolution as the CNT–LOXon glassy carbon due to the use of the silver paint. SEM imagesverified the porosity of the silver paint. Much better resolu-tion was shown on the CNT–LOX glassy carbon electrode. Thiselectrode detected up to 4 mM lactate concentration with a lin-ear amperometric response. Future work in this area involvespatterning an array of carbon nanotubes on a substrate by chem-ical vapor deposition (CVD). Two separate arrays could thenbe functionalized to detect two separate analytes, such as pHand lactate. The sensor array would then be tested to simultane-ously detect several analytes in human sweat. The developmentof a lab-on-a-chip device such as this has several clinical-basedapplications.

Acknowledgement

This research was partially funded by the National ScienceFoundation IGERT Grant DGE 0221681.

References

[1] N. Nakagawa, S. Sakai, M. Matsumoto, K. Yamada, M. Nagano,T. Yuki, Y. Sumida, H. Uchiwa, Relationship between NMF (lactateand potassium) content and the physical properties of the stratum

o the O–H stretch bands and a sharp peak at 1610 cm corre-ponding to the C O band. The LOX functionalized nanotubesisplayed peaks corresponding to the presence of amine groups.–H bend was shown at 1558 cm−1. N–H out-of-plane bending

bsorption was also observed at 794 cm−1. C–N stretch occurredt 1190 cm−1. Carbon nanotubes functionalized with COOHere extremely sensitive to changes in pH of a phosphate buffer

olution. This sensor exhibited a linear response to pH shifts.s expected, a negative shift in voltage was displayed for each

ncrease in pH. Carbon nanotubes further functionalized withOX also displayed sensitivity to changes of lactate in a phos-hate buffer solution. The CNT–LOX on Si/ITO appeared tohow a nearly linear response up to 0.5 M lactate. The silver

corneum in healthy subjects, J. Invest. Dermatol. 122 (2004) 755–763.

[2] A. Polliack, R. Taylor, D. Bader, Sweat analysis following pressureischaemia in a group of debilitated subjects, J. Rehabil. Res. Dev. 34(1997) 303–308.

[3] N. Fellmann, R. Fabry, J. Coudert, Calf sweat lactate in peripheral arte-rial occlusive disease, Am. J. Physiol. Heart Circ. Physiol. 257 (1989)395–398.

[4] S. Iijima, Helical microtubules of graphite carbon, Nature 354 (1991)56–58.

[5] J. Robertson, Realistic applications of CNTs, Mater. Today 7 (2004)46–52.

[6] B.S. Sherigara, W. Kutner, F. D’Souza, Electrocatalytic properties andsensor applications of fullerenes and carbon nanotubes, Electroanal 15(2003) 753–772.

Letter to the Editor / Sensors and Actuators B 117 (2006) 308–313 313

[7] H. Jankowska, S. Neffe, A. Swiatkowski, Investigations of the electro-chemical properties of activated carbon and carbon black, Electrochim.Acta 26 (1981) 1861–1866.

[8] C.G. Hu, W.L. Wang, S.X. Wang, W. Zhu, Y. Li, Investigation on elec-trochemical properties of carbon nanotubes, Diamond Relat. Mater. 12(2003) 1295–1299.

[9] K. Besteman, J. Lee, F.G.M. Wiertz, H.A. Heering, C. Dekker, Enzyme-coated carbon nanotubes as single-molecule biosensors, Nano Lett. 3(2003) 727–730.

[10] Z. Xu, X. Chen, X. Qu, J. Jia, S. Dong, Single-walled carbon nanotube-based voltammetric sensor and biosensor, Biosens. Bioelectron. 20(2004) 579–584.

[11] A. Chaubey, K.K. Pande, V.S. Singh, B.S. Malhotra, Co-immobilizationof lactate oxidase and lactate dehydrogenase on conducting polyanilinefilms, Anal. Chim. Acta 407 (2000) 97–103.

[12] I. Moser, G. Jobst, G.A. Urban, Biosensor arrays for simultaneousmeasurement of glucose, lactate, glutamate, and glutamine, Biosens.Bioelectron. 17 (2001) 297–302.

[13] T. Yao, T. Yano, H. Nishino, Simultaneous in-vivo monitoring of glu-cose, l-lactate, and pyruvate concentrations in rat brain by a flow-injection biosensor system with an on-line microdialysis sampling, Anal.Chim. Acta 510 (2004) 53–59.

[14] J.J. Xu, W. Zhao, X.L. Luo, H.Y. Chen, A sensitive biosensor for lactatebased on layer-by-layer assembling MnO2 nanoparticles and lactate oxi-dase on ion-selective field-effective transistors, Chem. Commun. (2005)792–794.

[15] H. Peng, L.B. Alemany, J.L. Margrave, V.N. Khabashesku, Sidewallcarboxylic acid functionalization of single-walled carbon nanotubes, J.Am. Chem. Soc. 125 (2003) 15174–15182.

Jessica Weber a,b

Arun Kumar c

Ashok Kumar a,b,∗Shekhar Bhansali b,d

a Department of Mechanical Engineering, Universityof South Florida, Tampa, Florida, USA

b Nanomaterials and Nanomanufacturing Research Center,University of South Florida, Tampa, Florida, USA

c Department of Internal Medicine, University of SouthFlorida, Tampa, Florida, USA

d Department of Electrical Engineering, University of SouthFlorida, Tampa, Florida, USA

∗ Corresponding author. Tel.: +1 813 974 3942;fax: +1 813 974 3610.

E-mail address: akumar@eng.usf.edu (A. Kumar)

12 August 2005Available online 23 January 2006