Molecular Sensors and Sensor Arrays Based onPolyaniline Microtubules

S. Sukeerthi† and A. Q. Contractor*

Department of Chemistry, Indian Institute of Technology, Bombay, Mumbai 400 076, India

This paper describes the fabrication of microtubularbiosensors and sensor arrays based on polyaniline withsuperior transducing ability. These sensors have beentested for the estimation of glucose, urea, and triglycer-ides. As compared to that of a macro sensor, the responseof the microtubular sensor for glucose is higher by a factorof more than 103. Isoporous polycarbonate membraneshave been used to fabricate inexpensive devices by simplethermal evaporation of gold using appropriate machinedmasks. Polyaniline deposition and enzyme immobilizationhave been done electrochemically. Electrochemical po-tential control has been used to direct enzyme im-mobilization to the chosen membrane device and avoidcross talk with adjacent devices. This has enabled theimmobilization of a set of three different enzymes on threeclosely spaced devices, resulting in a microtubule arraythat can analyze a sample containing a mixture of glucose,urea, and triglycerides in a single measurement. This, inessence, is an “electronic tongue”.

Conducting polymer-based biosensors for various biologicallysignificant molecules have evoked considerable attention recently.1-5

Early reports were concerned with the use of polypyrrole for theamperometric detection of glucose.6 The idea essentially was toprovide an electronically conducting polymer matrix for theimmobilization of enzyme glucose oxidase. We have7-9 describeda new generic concept which is based on the fact that theconductivity of this class of polymers is very sensitive to thechemical potential and pH10-13 of the microenvironment within

the polymer matrix. In this concept, the conducting polymer actsas the immobilization matrix as well the physicochemical trans-ducer to convert a chemical signal (change of chemical potentialof the microenvironment) into an electrical signal. Devices basedon conductivity changes arising from conformational changes inthe polymer have also been described.14 These changes inconformation could be brought about by binding events occurringalong the backbone and can also be influenced by the morphologyof the polymer growth. It has been found that changes inconductivity are larger when the polymer is disordered ascompared to when it is ordered.15

Martin et al. have described applications of conductingpolymers synthesized within the pores of various microporousmedia using the concept of template synthesis.16-19 This approachhas been adapted by us to fabricate microelectrochemical de-vices.15

We previously reported the functioning of a biosensor array9

for the detection of a specific substrate from a mixture ofsubstrates using a few microliters of sample. These sensors weremade using interdigitated microelectrodes. In this report, wedescribe the extension of the same general idea to isoporouspolycarbonate membrane based devices which involve very simpleand inexpensive fabrication techniques. These biosensors arebased on polyaniline with superior transducing abilities.15

EXPERIMENTAL SECTIONFabrication of Electrodes. The sensors were constructed

with an isoporous membrane as support. Gold films deposited onthe opposite faces of the membrane acted as electrodes for growthof the polymer. The polymerization was continued till the two faceswere bridged by tubules of the polymer growing through thepores. The process is described schematically in Figure 1.Isoporous membranes with two different pore diameters wereused, 1.2 and 0.2 µm. Thermal evaporation of gold was carriedout in a 15F6 HINDIVAC vacuum evaporation system using amask. The mask was made by cutting equidistant lines of 0.85mm width separated by 1.0 mm on an aluminum sheet. Gold films

* Author for correspondence. E-mail: aliasgar@ether.chem.iitb.ernet.in.† Current address: W. M. Kneck Center for Molecular Electronics, Syracuse

University, Syracuse, NY.(1) Nishizawa, M.; Miwa, Y.; Matsue, T.; Uchida, I. J. Electrochem. Soc. 1993,

140, 1650.(2) Nishizawa, M.; Matuse, T.; Uchida, I. Anal. Chem. 1992, 64, 2642.(3) Bartlett, P. N.; Birkin, P. R. Anal. Chem. 1993, 65, 1118.(4) Bartlett, P. N.; Birkin, P. R. Anal. Chem. 1994, 66, 1552.(5) Matsue, T.; Nishizawa, M.; Sawaguchi, T.; Uchida, I. J. Chem. Soc., Chem.

Commun. 1991, 1029, 132.(6) Umana, M.; Waller, J. Anal. Chem. 1986, 58, 2980.(7) Hoa, D. T.; Suresh Kumar, T. N.; Srinivasa, R. S.; Lal, R.; Punekar, N. S.;

Contractor, A. Q. Anal. Chem. 1992, 64, 2645.(8) Contractor, A. Q.; Srinivasa, R. S.; Suresh Kumar, T. N.; Narayanan, R.;

Sukeerthi, S.; Lal, R. Electrochim. Acta 1994, 39, 1321.(9) Sangodkar, H.; Sukeerthi, S.; Lal, R.; Srinivasa, R. S.; Contractor, A. Q. Anal.

Chem. 1996, 68, 779.(10) Ochmanska, J.; Pickup, P. G. J. Electroanal. Chem. Interfacial Electrochem.

1991, 297, 211.(11) Travers, J. P.; Nechtschein, M. Mol. Cryst. Liq. Cryst. 1987, 21, 135.

(12) MacDiarmid, A. G.; Chiang, J. C.; Huang, W.; Humphrey, B. D.; Somasiri,N. L. D. Mol. Cryst. Liq. Cryst. 1985, 125, 30.

(13) Gholamian, M.; Suresh Kumar, T. N.; Contractor, A. Q. Proc. Indian Acad.Sci. 1986, 457.

(14) Dabke, R. B.; Singh, G. D.; Dhanabalan, A.; Lal, R.; Contractor, A. Q. Anal.Chem. 1997, 69, 724.

(15) Sukeerthi, S.; Contractor, A. Q. Chem. Mater. 1998, 10, 1412.(16) Martin, C. R. Acc. Chem. Res. 1995, 28, 61.(17) Parthasarathy, R.; Martin, C. R. Chem. Mater. 1994, 6, 1027.(18) Martin, C. R. Chem. Mater. 1996, 8, 1739.(19) Parthasarathy, R.; Martin, C. R. Nature 1994, 369, 298.

Anal. Chem. 1999, 71, 2231-2236

10.1021/ac9810213 CCC: $18.00 © 1999 American Chemical Society Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2231Published on Web 04/30/1999

of 1000 Å thickness were evaporated on both sides of themembrane. The masks were aligned manually such that the goldlines on opposite faces superimposed on each other. Each goldline was used as an electrode. The electrode holder comprised asimple plastic clip with Pt contacts from which connections to theinstrumentation were made.

Deposition of the Polymer and Immobilization of En-zymes. Prior to polymerization, electrochemical cleaning of theAu-coated membrane was carried out in 0.5 M H2SO4 betweenthe potentials -0.2 and +1.6V vs SCE. Two different procedureswere adopted to synthesize polyaniline electrochemically. Inprocedure A, polyaniline was deposited on the membrane deviceby electropolymerization from a solution containing 0.1 M anilinein 0.5 M H2SO4. The electrode potential was cycled between -0.2and +0.8 V vs SCE. In procedure B, the membrane was treated

with 0.01% Triton-X 100 for 20 min prior to polymerization.Polyaniline was then grown electrochemically as mentioned above.

Glucose oxidase, EC 1.1.3.4 (Asperigillus niger), was im-mobilized in the conducting polymer matrix by electropolymer-ization from pH 4.0 phthalate buffer containing 0.1 M aniline and250 units/mL Gox as described earlier.7 Lipase (triglycerolacylhydrolase), EC 3.1.1.3 (Pseudomonas sp), and urease, EC3.5.1.5 (Jackbean meal), were immobilized from pH 5.2 acetatebuffer by physical entrapment in a polyaniline-bridged device asdescribed earlier.8 In the array configuration, multiple lines wereused, so during immobilization of an enzyme onto a particulardevice, the adjacent devices where immobilization was not desiredwere maintained at -0.2 V vs SCE.

Chemicals and Instrumentation. The monomer aniline wasAR grade, which was distilled and stored under dry nitrogen. The

Figure 1. Sequence of steps leading to the membrane-based biosensor: (a) view of the cross-section through a polycarbonate membraneshowing a pore and gold films deposited on the faces of the membrane; (b) electropolymerization of aniline at one of the gold-coated faces; (c)growth of PANI along the pore walls; (d) bridging of the two faces by PANI growing through the pore; (e) enzyme immobilization underelectrochemical potential control; (f) enzyme-loaded PANI tubule; (g) enzyme-catalyzed reaction of the substrate molecules.

2232 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

sulfuric acid was Electronic grade. Salts for the preparation ofbuffers were AR grade and were used without further purification.Glucose oxidase and urease were obtained from Sisco ResearchLabs, and lipase was purchased from Amano Chemicals. Glucose,urea, and triolein were obtained from SRL. All solutions wereprepared in water purified from a Milli-Q water purification system.

Polycarbonate membranes having pores of 1.2 and 0.2 µmdiameters were procured from Millipore Inc.

Electrochemical potential control was provided using an EG& G PARC model 273 potentiostat, and the polymerization processwas monitored by recording voltammetric traces on a Linseis LY16100 XYt recorder. In situ resistance measurement of the polymerfilm was made using a PINE AFRDE4 bipotentiostat. Theconductance was obtained by taking the inverse of the measuredresistance, assuming that it is a reasonable estimate of theconductance of the polymer film. The sensor “response” isrepresented by ∆g/g0 where g0 is the conductance of the sensorin the buffer without the substrate and ∆g ) g - g0, where g isthe conductance of the sensor in the presence of the substrate.Representing the response as the ratio ∆g/g0 normalizes thesensor response and minimizes variations from sensor to sensor.In case of the urea and triglyceride sensors, the sensor responseis represented as ∆r/r0, where r represents the resistance of thepolymer film.

Types of Sensors. In this paper, we compare the response ofthree different sensor structures. Our early studies were carriedout with “twin-wire” electrode configurations;7,8 these are called“macro sensors”. The next phase of our work9 used interdigitatedmicroelectrodes and these are called “micro sensors”. The presentwork uses a structure based on isoporous membranes,15 and theseare called “microtubular sensors”.

RESULTS AND DISCUSSIONThe response of these membrane devices was measured by

the dc conductivity technique20-22 as shown schematically inFigure 2. Here the electrochemical state of the polymer could becontrolled by performing the measurements under potentialcontrol. VG was varied from -0.2 to +0.5 V vs SCE with a constantVD of +20 mV. The sensor responses were found to be maximumat -0.2 V; hence, results for this potential are reported here. (SeeTable 1.) The potential dependence of sensor response will bediscussed separately.

In the case of the glucose sensor, the products of the enzyme-catalyzed reaction are gluconic acid and H2O2. The production ofgluconic acid brings about a decrease in pH in the polyanilinemicroenvironment and hence an increase in its conductance.Figures 3 and 4 show the response of the glucose sensors basedon 1.2 and 0.2 µm polyaniline tubules. The sensitivities (definedas the change in response of the sensor per unit change inconcentration of the substrate) of the microtubular sensors were6.0/mM for 1.2 µm pores and 7.5/mM for a similar sensor basedon 0.2 µm pores as compared to 0.06/mM for macro sensors and0.6/mM for micro sensors. Sensors based on polyaniline micro-tubules in which the polymer was largely disordered showed even

higher sensitivities. The sensitivity for the 1.2 µm sensor was 53.3/mM, and that for the 0.2 µm was 200/mM. The sensor responsesare shown in Figures 5 and 6. This tremendous increase in thesensitivity of the sensor is due to a combination of factors. It hasbeen found that improved transduction ability results from ashorter source-to-drain distance in the device.25 The source-to-drain distance in the microtubular device is determined by themembrane thickness which is 10 µm as compared to the microsensors where the source-to-drain distance was 60 µm.9 It has alsobeen shown15 that the polymer synthesized by procedure B ismore disordered and exhibits a larger change in conductivity onthe transition from the insulating to the conducting state. Ad-ditionally, one may have higher effective enzyme loading in thedisordered polymer tubules.

(20) Paul, E. W.; Ricco, A. J.; Wrighton, M. S. J. Phys. Chem. 1985, 89, 1441.(21) Kittelson, G. P.; White, H. S.; Wrighton, M. S. J. Am. Chem. Soc. 1984,

106, 7389.(22) Ofer, D.; Crooks, R. M.; Wrighton, M. S. J. Am. Chem. Soc. 1990, 112,

7869.

(23) MacDiarmid. A. G.; Epstein, A. J. Synth. Met. 1994, 65, 103.(24) Avlyanov, J. K.; Min, Y.; MacDiarmid, A. G.; Epstein, A. J. Synth. Met. 1995,

72, 65.(25) S. Sukeerthi, Ph.D. Thesis, IIT, Bombay, 1998.

Figure 2. Measurement of sensor response. The sensor responsewas determined by measuring the conductance of the polymer tubulesin the transistor mode. Device conductance ) ID/VD. Sensor responseis ∆g/g0, where ∆g ) g - g0, and g is the conductance of the devicein the presence of the substrate, and g0 is the device conductance inthe absence of the substrate. For substrates which caused a decreasein the device conductance, the sensor response is represented by∆r/r0 where r ) 1/g is the device resistance.

Table 1. Comparison of Sensitivities (mM-1) of Macro,Micro, and Microtubular Sensors with Ordered (A) andDisordered (B) PANI at -0.2 V vs SCE (∆g/g0 forGlucose Sensors and ∆r/r0 for Urea and TriglycerideSensors)

sensor glucose urea triglyceride

macro 0.06 0.015 0.009micro 0.625 0.018 0.017microtubular, 1.2 µm (A) 6.0 0.017 0.019microtubular, 0.2 µm (A) 7.5 0.019 0.025microtubular, 1.2 µm (B) 53.3 0.66 0.087microtubular, 0.2 µm (B) 200 0.8 0.12

Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2233

The urease-catalyzed reaction results in hydrolysis of urea toNH3 and CO2. This causes a net increase in pH and hence adecrease in polymer conductivity. The sensitivity of the ureamicrotubular sensor was 0.018/mM for the 1.2 µm pore diameterand 0.025/mM for the 0.2 µm pore diameter. In the case of thesurfactant-treated membranes, the sensitivity rose to 0.087/mMfor the 1.2 µm pores and 0.38/mM for the 0.2 µm pores (Figure7). The urea microtubule sensor showed linearity up to aconcentration of 80 mM, above which it saturates. Since theenzyme-catalyzed reaction brings about an increase in pH of themicroenvironment in the urea and triglyceride sensors, there isan increase in the resistance of the polymer film; therefore, thesensor response is denoted as ∆r/r0.

In the triglyceride sensor, lipase catalyzes the hydrolysis toglycerol. The production of glycerol in the presence of TritonX100 in the polymer microenvironment causes an increase in pHand hence a decrease in polymer conductance.9 The triglyceridemicrotubular sensor showed linearity up to 80 mM. The sensitivity

for the untreated membrane sensor was about 0.017/mM for the1.2 µm pores and 0.019/mM for the 0.2 µm pores. But thesurfactant-treated membrane sensors showed an order of mag-nitude increase in sensitivity. The sensitivity of the 1.2 µm sensorwas now 0.66/mM, and that of the 0.2 µm was 0.80/mM (Figure8).

The sensor response as a function of repeated use wasexamined for both the untreated and surfactant-treated membranesensors. The sensor response was observed to be fairly constant.The response was stable even after 10 independent measurements,which included washing with buffer solution between every twomeasurements. These observations are consistent with our earlierconclusion9 that the enzyme is entrapped in the polymer matrixand not just adsorbed on the surface.

On the basis of the results from the microelectrodes andsubsequently with the membrane-based devices, we fabricated amicrotubular sensor array consisting of glucose, urea, and tri-

Figure 3. Response of the glucose microtubular sensor based on1.2 µm pores (polymer grown by procedure A; glucose in a phosphatebuffer, pH 7.0).

Figure 4. Response of the glucose microtubular sensor based on0.2 µm pores (polymer grown by procedure A; glucose in a phosphatebuffer, pH 7.0).

Figure 5. Response of the glucose microtubular sensor based on1.2 µm pores (polymer grown by procedure B; glucose in a phosphatebuffer, pH 7.0).

Figure 6. Response of the glucose microtubular sensor based on0.2 µm pores (polymer grown by procedure B; glucose in a phosphatebuffer, pH 7.0).

2234 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

glyceride sensors as well as a reference sensor. At first, all fourgold lines on the membrane were coated with polyaniline. Glucoseoxidase, urease, and lipase were immobilized from their respectivesolutions on one PANI gold line each, while the fourth line wasused a reference sensor. Chemical cross talk between PANI-coatedneighboring lines was prevented by maintaining the lines at whichimmobilization was not desired at -0.2 V vs SCE. At this potential,the polymer is insulating and is in a compact coil state.23,24

The sensor response to each substrate was determinedindependently, and the resulting calibration plots were used toobtain the concentrations of these substrates when present in amixture. Each line was independently addressed, and the con-ductance of each polymer-coated line could be determined whileall four lines were exposed to a common pool of analyte solution.Figures 9 and 10 show the results of the analyses of three mixturesof glucose, urea, and triglyceride. The concentrations calculated

from the sensor response are plotted against the concentrationsas prepared. The experimental data points are in excellentagreement with a line of unity slope, which is to be expected ifthe microtubule array behaved ideally.

CONCLUSIONSMicrotubular sensors and microtubular sensor arrays with

superior performance capabilities have been described. A com-bination of factors determines the sensitivity of these sensors.These are (i) a smaller source-to-drain separation, (ii) a disordered

Figure 7. Response of the urea microtubular sensor based on 0.2µm pores (polymer grown by procedure B; urea in an acetate buffer,pH 5.2).

Figure 8. Response of the triglyceride microtubular sensor basedon 0.2 µm pores (polymer grown by procedure B; triglyceride in anacetate buffer, pH 5.2).

Figure 9. Response of the microtubular sensor array (1.2 µm).Compositions of the three solutions analyzed, glucose:urea:triglyc-eride: (i) 1:5:9; (ii) 5:2:6; (iii) 7:3:4. The substrates were dissolved inphosphate buffer, pH 6.0.

Figure 10. Response of the microtubule sensor array (0.2 µm).Compositions of the three solutions analyzed, glucose:urea:triglyc-eride: (i) 1:5:9; (ii) 5:2:6; (iii) 7:3:4. The substrates were dissolved inphosphate buffer, pH 6.0.

Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2235

polymer microstructure resulting in larger changes in conductivityon switching of polyaniline, and (iii) the immobilization of enzymesinto a disordered polyaniline matrix which may result in higherenzyme loading and faster diffusion of the substrates into thematrix and hence a greater extent of enzymatic conversion.

There is great potential for developing such simple devicesthat are inexpensive, easy to fabricate, disposable, and highlysensitive. These can prove to be simple miniaturized diagnostictools for various important state-of-health indicators.

ACKNOWLEDGMENTIt is a pleasure to acknowledge the numerous discussions with

Drs. S. S. Talwar, S. Major, and R. Lal which contributed to thiswork. S.S. acknowledges an SRF award from the CSIR.

Received for review September 14, 1998. AcceptedFebruary 10, 1999.

AC9810213

2236 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999