microtubule sensors and sensor array based on polyaniline synthesized in the presence of...

Microtubule Sensors and Sensor Array Based onPolyaniline Synthesized in the Presence ofPoly(styrene sulfonate)

Mandakini Kanungo, Anil Kumar, and A. Q. Contractor*

Department of Chemistry, Indian Institute of Technology, Bombay, Mumbai-76, India

Microtubule sensors for glucose, urea, and triglyceridewere fabricated based on poly(styrene sulfonate)-poly-aniline (PSS-PANI) composites synthesized within thepores of track-etched polycarbonate membranes. Thesynthesis of a sufficiently thick and conducting PSS-PANIfilm at pH 5 provided the advantage of immobilizingenzymes during polymerization. This resulted in theimprovement of sensor response for urea and triglycerideby a factor of ∼102 with a significant increase in the linearregion of response compared to polyaniline-based sen-sors, where the enzymes were immobilized by physicaladsorption after the polymerization. The sensors basedon urea and triglyceride were found to have a higher linearrange of response, better sensitivity, improved multipleuse capability, and faster response time compared to thepotentiometric and amperometric sensors based on poly-aniline. A microtubule sensor array for glucose, urea, andtriglyceride based on PSS-PANI was fabricated by im-mobilization of three different sets of enzymes on threeclosely spaced devices and its response was found to befree from cross-interference when a sample containing amixture of the above analytes was analyzed in a singlemeasurement.

Organic conducting polymers have emerged as promisingmaterials in the development of compact and portable probes forthe detection of biologically significant molecules.1,2 Early reportsof using conducting polymer matrix for the immobilization ofbiological substrates were concerned with the use of polypyrrolefor the amperometric detection of glucose.3 Since then, variousresearch pursuits have resulted in a variety of sensors based onconducting polymers. Among various conducting polymers, poly-aniline has a unique position due to its easy synthesis, environ-mental stability, and reversible acid-base chemistry in aqueoussolution. The application of polyaniline in biosensors is verypromising. Polyaniline has been used both as an immobilizationmatrix and as a physicochemical transducer to convert a chemicalsignal into an electrical signal. A change in the pH of themicroenvironment or a change in the conformation of the polymer

caused by a binding event in the polymer matrix results in achange in the electronic conductivity of the polymer. This propertyof polyaniline has been explored in fabrication of conductometricsensors for the determination of various biomolecules/ions in ourlaboratory.4-7

Polyaniline loses its electrochemical activity in solutions of pHgreater than 4.8,9 Therefore, adaptation of polyaniline to neutralpH is an important problem. Attempts have been made to modifythe properties of polyaniline in order to extend its conductivity toneutral pH. The first attempt in this direction was by Epstein andco-workers wherein sulfonic acid groups were introduced on thepolyaniline backbone by sulfonation of the emeraldine andleucoemeraldine states of polyaniline to get self-doped polyani-line.10-12 Self-doped polyanilines were also synthesized electro-chemically by copolymerization of aniline and metanilic acid wherethe redox activities of the polymers was maintained up to pH 9.13

Recently Kumar and co-workers have reported the successfulhomopolymerization of metanilic acid to get 100% sulfonatedpolyaniline.14 Another strategy in which the conductivity ofpolyaniline can be extended to neutral pH is to synthesize thepolymer in the presence of anionic polyelectrolyte with a sulfonategroup.15 Several potentiometric and amperometric sensor deviceshave been developed using the modified polyanilines, which areresponsive to glucose, urea, and NADH and can be operated inpH 7 buffer solution.16-20

* Corresponding author. E-mail: [email protected].(1) McQuade, D.; Tyler, Pullen. A. E.; Swager, T. M. Chem. Rev. 2000, 100,

2537.(2) Bartlett, P. N.; Astier, Y. Chem. Commun. 2000, 105.(3) Umana, M.; Waller, J. Anal. Chem. 1984, 106, 7389.

(4) Hoa, D. T.; Suresh Kumar, T. N.; Srinivasa, R.S.; Lal, R.; Punekar, N. S.Contractor, A. Q. Anal. Chem. 1992, 64, 2645.

(5) Sangodhkar, H.; Sukeerthi, S.; Lal, R.; Srinivasa, R. S.; Contractor, A. Q.Anal. Chem. 1996, 68, 779.

(6) Sukeerthi, S.; Contractor, A. Q. Anal. Chem. 1999, 71, 2231.(7) Dabke, R. B.; Singh, G. D.; Dhanabalan, A.; Lal, R.; Contractor, A. Q. Anal.

Chem. 1997, 69, 724.(8) Gospodinova, N.; Terlemyzyan, L.; Mokreva, P.; Kossev, K. Polymer 1993,

34, 2434.(9) Gospodinova, N.; Mokreva, P.; Terlemezyan, L. Polymer 1994, 35, 3102.

(10) Yue, J.; Epstein, A. J.J. Am. Chem. Soc. 1990, 112, 2800.(11) Yue, J. W.; Jhao, H.; Cromack, K. R.; Epstein, A. J.; MacDiarmid, A. G. J.

Am. Chem. Soc. 1991, 113, 2265. 12.(12) Wei, X.; Wang, Y. Z.; Lang, S. M.; Bobeczcko, C.; Epstein, A. J. J. Am. Chem.

Soc. 1996, 118, 2545.(13) Karyakin, A. A.; Strakhova, A. K.; Yatimirsky, A. K. J. Electroanal. Chem.

1994, 371, 259.(14) Krishnamoorthy, K.; Contractor, A. Q.; Kumar, A. Chem. Commun. 2002,

240.(15) Austria, G.; Jang, G. W.; MacDiarmid, A. G.; Doblhofer, K.; Zhang, C. Ber.

Bunsen-Ges. Phys. Chem. 1991, 95, 1381.(16) Castillo-Ortega, M. M.; Rodriguez, D. E.; Encinas, J. C.; Plascencia, M.;

Mendez-Velarde, F. A.; Olayo, R. Sens. Actuators, B 2002, B85(1-2), 19.

Anal. Chem. 2003, 75, 5673-5679

10.1021/ac034537h CCC: $25.00 © 2003 American Chemical Society Analytical Chemistry, Vol. 75, No. 21, November 1, 2003 5673Published on Web 09/12/2003

Since conducting polyaniline could only be synthesized fromacidic solutions, most of the sensor devices are fabricated by thephysical adsorption of the enzyme/host species after the polym-erization. This restricts the amount of loading, which then resultsin poor sensitivities. Furthermore, the multiple-use capability ofthese devices is also very poor due to the easy leaching out ofthe enzyme/host species because of the poor interaction due tothe physical adsorption. These problems can be circumvented ifthe enzyme/host species could be loaded during the polymeri-zation, which will then result in higher loading levels and betterentrapment. This cannot be achieved because most of the enzymesare unstable in acidic conditions required for the polymerizationof aniline. Recently, Tripathy and co-workers have come up witha novel strategy for enzymatic synthesis of conducting polyanilinein the presence of a strong acidic polyelectrolyte such as poly-(styrene sulfonate) at pH 4.3-5.5 phosphate buffer in the presenceof the enzyme horseradish peroxidase.21-23 Since acidic polyelec-trolytes attract hydrogen ions electrostatically, the pH at the acidicpolyelectrolyte surfaces is much lower than that of bulk aqueousmedium.24 This provides a suitable microenvironment for theformation of conducting polyaniline. On the basis of these reports,we envisaged that if the polyaniline could be synthesized at higherpH then this would enable us to immobilize the enzymes duringpolymerization. This should then result in significant improvementin sensitivity and recycling ability. In this paper, we report on thesynthesis and characterization of a sufficiently thick conductingfilm at a biocompatible pH 5. Since most of the enzymes are fairlystable at this pH, we could immobilize the enzymes duringpolymerization. This goal was achieved by synthesizing thepolyaniline in the presence of an anionic polyelectrolyte, poly-(styrene sulfonate) (PSS). A strong acidic polyelectrolyte suchas PSS is the most favored because it provides a sufficiently lowlocal pH microenvironment for the formation of a conductingpolymer film at high bulk pH. The resulting devices showed asignificant increase in the sensitivity and linear range of responseespecially in the case of urea and triglyceride compared to thatof polyaniline. A sensor array was fabricated, which was used todetermine the concentration of glucose, urea, and triglyceride ina single measurement. The sensors based on urea and triglyceridewere found to have a higher linear range of response, bettersensitivity, improved recycling ability, and faster response timecompared to the potentiometric and amperometric sensors basedon polyaniline.

EXPERIMENTAL SECTIONChemicals and Materials. Freshly distilled aniline (Merck)

was used for preparing monomer solution. The sulfuric acid usedwas MOS grade with 99.9% purity. The sodium salt of poly(styrenesulfonate) (Mw ) 70,000) was obtained from Aldrich. The enzymesglucose oxidase from Aspergillus niger (EC 1.1.3.4), urease fromJack bean meal (EC 3.5.1.5), and peroxidase (EC 1.11.1.7) wereobtained from Sigma. Lipase from triglycerol acylhydrolase andglucose, urea, and triolein was obtained from SRL (Sisco ResearchLaboratories). Salts used for the preparation of buffers were ofanalytical grade and were used without further purification. Track-etched polycarbonate membranes having a pore diameter of 1.2µm and thickness of 10 µm were obtained from the Millipore Inc.The pore densities of the membrane were calculated with the helpof the scanning electron micrographs of the polycarbonatemembranes. Around six pictures of various regions were taken,and the numbers of pores per cubic centimeter were calculated.The average pore densities were found to be 1.2 × 107 pores/cm2.

Fabrication of Sensor Devices. Track-etched polycarbonatemembranes were used for the fabrication of sensor devices. Goldfilms were deposited on the two sides of the membrane by vacuumevaporation using a mask in a homemade vacuum evaporationsystem.25 The mask was made by cutting equidistant lines of 1-mmwidth separated by 1 mm on an aluminum sheet. The two goldlines at the opposite faces of the membrane exactly overlappedwith each other and were used as two electrodes for the growthof the polyaniline. Each gold line was used as an electrode. Theelectrodes were held by a plastic clip holder with platinum contactsfrom which connections to the instruments were made. PSS-polyaniline (PANI) was synthesized from pH 0.6 solution (0.1 Maniline in 0.5 M H2SO4 + 50 mM PSS) and from pH 5.0 solution(0.1 M aniline in pH 5 buffer + 50 mM PSS) within the pores(1.2 µm) of gold-coated polycarbonate membranes. PSS-PANIwas deposited electrochemically from pH 0.6 by scanning thepotential between -0.2 and 0.8 V versus SCE at a scan rate of50 mV/s. For the polymerization from pH 5 solution, thepotentiodynamic growth of the polymer was carried out byscanning the potential between -0.2 and 1.0 V versus SCE.However, the potentiodynamic growth of the polymer at pH 5phthalate buffer was very slow and therefore a potentiostaticgrowth of the polymer at 1.0 V was preferred. It took ∼25 min tobridge the two sides of the membrane. Polyaniline films in theabsence of PSS were synthesized by scanning the potentialbetween -0.2 and 0.8 V versus SCE at a scan rate of 50 mV/s for10 min. The synthesis of polyaniline in the absence of PSS atpH g4 resulted in a very slow kinetics of polymerization with theformation of a very thin and poorly conducting film.

The sensor devices were fabricated in two ways. For PSS-PANI-A and PANI devices, the electropolymerization was doneat pH 0.6. The immobilization of glucose oxidase (GOx) wascarried out by formation of a two-layer film, polymer, and polymer+ GOx.4,5 The immobilization of urease and lipase was carriedout by physical adsorption after polymerization in the samemanner as described earlier.4,5 For PSS-PANI-B devices, theelectropolymerization was done at pH 5 in the presence of theenzyme by potentiostatic polymerization at 1.2 V versus SCE for

(17) Bartlett, P. N.; Birkin. P. R.; Wallace, E.N. K. J. Chem. Soc., Faraday Trans.1997, 93, 1951.

(18) Tatsuma, T.; Ogawa, T.; Sato, R.; Oyama, N. J. Electroanal. Chem. 2001,501 (1-2), 180.

(19) Karyakin, A. A.; Lukachova, L. V.; Karyakina, E. E.; Orlov, A. V.; Karpachova,G.; Wang, J. Anal. Chem. 1999, 71, 2534.

(20) Raitman, O. A.; Katz, E.; Bueckmann, A. F.; Willner, I. J. Am. Chem. Soc.2002, 124 (22), 6487.

(21) Liu, W.; Kumar, J.; Tripathy, S. K.; Senecal, K. J.; Samuelson, L. J. Am. Chem.Soc. 1999, 121, 71.

(22) Liu, W.; Cholli, A. L.; Nagarajan, R.; Kumar, J.; Tripathy, S.; Bruno, F. F.;Samuelson, L. J. Am. Chem. Soc. 1999, 121, 11345.

(23) Nagarajan, R.; Tripathy, S.; Kumar, J.; Bruno, F. F.; Samuelson, L.Macromolecules 2000, 33, 9542.

(24) (a) Manning, G. S. Acc. Chem. Res. 1979, 12, 443. (b) Manning, G. S. J.Chem. Phys. 1988, 89, 3722. (25) Sukeerthi, S.; Contractor, A.Q. Chem. Mater. 1998, 10, 1412.

5674 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

∼35 min. The potential was kept at 1.2 V because the presenceof enzymes in the monomer solution slows the rate of polymer-ization, probably due to inhibition by adsorption of enzyme. Itshould be noted that phthalate buffer was used for the chracter-ization of the polymer but the buffer was changed to phosphatefor biosensor studies because our earlier work on the biosensorused this buffer. Changing the buffer from phthalate to phosphatedid not have any effect in the device studies.

Fabrication of Sensor Array for Glucose, Urea, andTriglyceride. Microtubular sensor arrays were fabricated on onesingle device consisting of glucose, urea, and triglyceride im-mobilized on polyaniline synthesized in the presence of PSS atpH 5. Four gold lines were taken and were held by a plastic clipholder with platinum contacts. Among the four gold lines, threelines were used as sensor devices and the fourth line was usedas a reference sensor. The reference sensor was coated with PSS-PANI in the absence of enzyme. Glucose oxidase, urease, andlipase were immobilized on individual lines one after another bysynthesizing PSS-PANI from pH 5 buffer in the presence of therespective enzymes. All the other neighboring lines coated withPSS-PANI (in case of the reference device) or PSS-PANI-enzyme films were maintained at -0.2 V. At -0.2 V, the polymerremains in a compact form and hence it minimizes the possibilityof cross-immobilization of enzymes on the polymer matrix.

Characterization of the Polymer. PSS-PANI films werecharacterized by cyclic voltammetry, in situ conductance measure-ments, spectroelectrochemistry, and scanning electron micros-copy. Cyclic voltammograms of the polymer were recorded withthe help of an EG & G PARC 362 potentiostat/galvanostat coupledto a Linseis XY-t recorder. In situ resistance measurements werecarried out on polymer formed on gold-coated 1.2-µm-porediameter polycarbonate membranes in the transistor mode. Thetwo sides of the membrane electrode act as “source” and “drain”of the electrochemical transistor. The conductance of the polymerwas taken as the reciprocal of resistance. An AFRDE4 Pinebipotentiostat coupled with a Philips multimeter was used forcarrying out the resistance/conductance measurements. The insitu UV-visible spectroscopy of the PSS-PANI film was carriedout with the help of a Shimazdu 2100 UV-visible spectrophotom-eter. The potential was controlled with the help of an EG &GPARC 362 potentiostat. The scanning electron microscopy of thepolymer tubules was recorded with the help of a JEOL JSM6400microscope after dissolving the template membrane in dichlo-romethane.

Activity of the Immobilized Enzymes. The immobilizedenzymes on PANI and PSS-PANI films were checked for activity.To check the activity of the polymer-GOx film (0.5 cm2), the filmswere immersed in 2.6 mL of 0.1 M sodium phosphate buffer(pH 7) containing 2 units of peroxidase (POD), 50 µmol ofD-glucose and o-anisidine.26 The film was maintained for 30 minat 298 K with vigorous stirring and was further incubated for30 min at 310 K. The change in absorbance was recorded at 520nm. The polymer-urease was immersed in pH 5.2 acetate buffercontaining 50 µmol of urea. The film was maintained for 30 minat 298 K with stirring followed by 30-min incubation at 310 K.

Then the assay for urea was done by the conventional procedure.27

A titrimetric assay was carried out to check the activity ofimmobilized lipase on the polymer film.28 The polymer-lipase filmwas immersed in 5 mL of 20-µmol triolein solution in 0.01% TritonX for 30 min, and 5 mL of methanol was added to stop the reaction.The liberated fatty acid was titrated with 0.1 M KOH. Controlexperiments were done in all the above cases for polymer film inthe absence of enzyme, and the values obtained were subtractedfrom the experiments conducted in the presence of enzyme tocalculate the activity of immobilized enzyme. Enzyme activity wascalculated in terms of the moles of substrate oxidized in the aboveassay. The immobilized enzyme activity for the various enzymesinto different polymer devices was determined, and the valuesare reported in Table 1. In the case of PANI and PSS-PANI-A,enzyme immobilization was carried out for 2 h by a physicalabsorption method. In the case of PSS-PANI-B, the enzyme wasloaded during the polymerization (pH 5) that was carried out for40 min, i.e., the time taken to form a sufficiently thick film on theelectrode surface. As can be seen in the Table 1, the enzymeactivities are significantly higher in PSS-PANI-B devices thoughthe loading time was smaller. This further confirms the efficientloading of the enzymes during polymerization. Enzyme activitiesin PSS-PANI-A devices, where the enzymes were loaded afterthe polymerization by physical adsorption, were comparable tothat of PANI.

Sensor Measurements. Sensor response for glucose isrepresented by ∆g/go, where go is the conductance of the sensorin the absence of the substrate and ∆g ) g - go, where g is theconductance in the presence of the substrate. Representing theresponse in this manner normalizes it for variations in conductancefrom sensor to sensor. The response of urea and triglyceridesensors is represented by ∆r/ro, where ro is the resistance of thesensor in the absence of the substrate and ∆r ) r - ro, where ris the resistance in the presence of the substrate. The variable gwas chosen in the case of glucose and r in the case of urea andtriglyceride so that the sensor response had a positive slope whenplotted versus concentration of the substrate in all the three cases.

RESULTS AND DISCUSSIONSynthesis and Characterization of PSS-PANI Film. PSS-

PANI film was synthesized both at pH 0.6 (PSS-PANI-A) and atpH 5 (PSS-PANI-B). Here, PSS acts as a charge compensatorand the rate of polymerization of aniline is enhanced in thepresence of PSS.29 PSS-PANI film was synthesized from pH 5

(26) Kunst, A.; Drager, B.; Ziegenhorn, J. In Methods of Enzymatic Analysis;Bergmeyer, H. U., Bergmeyer, J., Grassi, M., Eds.; Verlag Chemie:Weinheim, Germany, 1984; Vol. 6, p 178.

(27) Kerscher, L.; Ziegenhorn, J. In Methods of Enzymatic Analysis, 3rd ed.;Bergmeyer, H. U., Editor-in-Chief, Bergmeyer, J., Grabi, M., Eds.; VCHPublishers: Weinheim, Germany, 1985; Vol. 8, p 449.

(28) Borgstrom, B.; Brockman, L. H. Lipases; Elsevier Science Publishers:Amsterdam, The Netherlands, 1984.

(29) Michaelson, J. C.; McEnvoy, A. J.; Kuramoto, N. Reactive Polym. 1992, 17,197.

Table 1. Activities of the Immobilized Enzymes

enzyme activity (µmol)

polymer urea lipase glucose

PANI 0.8 0.6 2PSS-PANI-B 2.6 1.7 3.2

Analytical Chemistry, Vol. 75, No. 21, November 1, 2003 5675

buffer solution by potentiostatic polymerization at 1.0 V versusSCE. The formation of conducting polyaniline at pH 5 wasattributed to the presence of aniline in the regions, which arelocally more acidic than the bulk solution.30 In other words, PSSprovides the necessary alignment of the aniline monomer and asufficiently low local pH for the formation of a conductingpolyaniline.21-23 Since the bulk pH is high enough to prevent thedenaturation of enzymes, they can be added to the monomersolution and therefore can be immobilized into the polymer filmsduring polymerization. However, the synthesis of a conductingpolyaniline in the presence of PSS was also found to depend onthe pH of the monomer solution. In our case, conductingpolyaniline film could not be formed at pH greater than 5.3. Thiswas further supported by the observation of Liu et al., who havereported the formation of a highly branched and insulatingpolyaniline at pH g6.21,22 Figure 1 shows the micrographs of PSS-PANI-B tubules formed inside the pores of the membrane. Thetubules are 8-9 µm in length. The formation of the polymerictubules inside the pores of the membrane indicates that PSS-PANI synthesized at pH 5 phthalate buffer solution is sufficientlyconducting in nature to allow the growth of a bridge across themembrane. The in situ conductance measurements of the PSS-PANI-B synthesized at pH 5 were carried out at different potentialsand pHs. Figure 2 shows the change in conductance as a functionof pH for 1.2-µm PSS-PANI-B tubules at different gate potentials.Similar trends were observed with PSS-PANI-A film synthesizedat pH 0.6. The conductance of the PSS-PANI decreases graduallywith increase in pH. This is in contrast to PANI, where theconductance of the polymer film decreases sharply with increasein pH from pH 1 to 6 after which there is no significant change inthe conductance of the polymer with pH. However, unlike thesulfonated PANI films, the conductance of the PSS-PANI is notentirely independent of pH.12 This may be because in sulfonatedpolyaniline sulfonate groups are bound covalently to the polymer.Therefore, the polymer will not be in a completely doped state atpH 5. This results in an increase in conductance of around 1-1.5

order of magnitude while going from pH 5 to pH 1. The in situUV-visible spectrum of PSS-PANI-B also shows the formationof strong absorption bands at 430-440 and 800 nm, which areindicative of the formation of a conducting film. The 800-nm bandincreases with increase in potential until 0.3 V, after which itdecreases because of the presence of a fully oxidized PANI athigher potential.

Sensor Response. The responses of the membrane deviceswere measured by dc conductance measurements as describedearlier.25 The sensor response was measured at three differentgate potentials -0.2, 0, and 0.2 V versus SCE with a 20-mV drainpotential. The sensitivity (defined as change in response of thesensor per millimolar change in the concentration of the substratein the linear region of response) was found to be highest at 0 Vfor PSS-PANI-based sensors and at 0.2 V for PANI-based sensors.All the experiments were repeated three to four times with filmsprepared under nominally identical conditions. The extents ofvariation between the experiments are mentioned in the graphsas the +ve and -ve variation from the mean values of measure-ment obtained from three different experiments. In the case ofthe glucose sensor, the enzyme-catalyzed reaction results in theformation of gluconic acid and H2O2. The production of gluconicacid results in a decrease in the pH of the microenvironment andin turn an increase in the conductance of the polymer. Figure 3shows the response based on PSS-PANI-B, PSS-PANI-A, andPANI devices.

(30) Kuramoto, N.; Michaelson, J. C.; McEnvoy, A. J. Gratzel, M. J. Chem.Commun. 1990, 1478.

Figure 1. Scanning electron micrographs of 1.2-µm PSS-PANI-Btubules formed on the pores of polycarbonate membranes. Thetubules are 8-9 µm in length. The thickness of the polycarbonatemembrane used was 10 µm.

Figure 2. Change in conductance of 1.2-µm PSS-PANI and PANItubules as a function of pH at various gate potentials. [, 9, and 2

are for PSS-PANI at a gate potential of -0.2, 0.0, and 0.2 V,respectively, and b for PANI at a gate potential of 0.2 V. PSS-PANIwas polymerized from pH 5 buffer solutions.

Figure 3. Response of glucose microtubules based on PANI (×),PSS-PANI-A (2), and PSS-PANI-B (b) (PSS-PANI-A and PANIshown in the inset) along with the error bars (glucose in phosphatebuffer of pH 7). Data are given as mean ( (variation from the mean)for three experiments.


The enzyme-catalyzed reaction for the urease-loaded film inthe presence of urea results in the formation of NH3. Theproduction of NH3 (pKa ) 9.25) would raise the pH of themicroenvironment of the polymer matrix and would consequentlylower the conductance of the film. The sensor response of ureabased on PSS-PANI-B, PSS-PANI-A, and PANI devices is shownin Figure 4.

The enzyme-catalyzed reaction for lipase-loaded film resultsin the formation of oleic acid and glycerol. Oleic acid, beinginsoluble in water, either forms micelles or remains solubilizedby 0.01% Triton X 100 while the glycerol goes into the solution.An independent control experiment shows that addition of glyceroland Triton X into the buffer in the concentration similar to thatused here results in an increase in the pH of the solution andhence a lowering of the conductance of the film. This is inagreement with our present observation. Figure 5 shows thesensor response of triolein for PSS-PANI-B, PSS-PANI-A, andPANI films. Control experiments for PANI and PSS-PANI werecarried out in all the above cases. The polymer film was exposedto different concentrations of the analyte in the absence of enzyme.The small and constant response of the reference film shows thatthe sensor response is due to the presence of enzyme-catalyzedreaction on the polymer matrix.

The sensitivity and linear range of response of glucose, urea,and triglyceride sensors based on PSS-PANI-B, PSS-PANI-A,and PANI films are given in Table 2. It is clear from theobservations that the sensitivity for urea and triglyceride sensorsis highest for PSS-PANI-B followed by PSS-PANI-A and PANIfilm. On the other hand, for the glucose sensor, the sensitivity

was found higher in PANI followed by PSS-PANI-B and PSS-PANI-A. The linear range of response for urea and triglyceridesensors were found more for PSS-PANI-based sensors comparedto that of PANI. The sensor response depends on multiplefactors: amount of active enzyme immobilized on the polymermatrix, pH-dependent conductance of the polymer, and diffusionof substrates into the polymer film. In the present case, ureaseand lipase were immobilized into PANI and PSS-PANI-A filmsby physical adsorption after polymerization and into PSS-PANI-Bfilm during polymerization. For PSS-PANI, the conductancechanges with pH are sharper on going to higher pH from pH 5 incomparison to that of PANI (Figure 2). Thus, there is an increasein the sensitivity of a sensor based on PSS-PANI-A film comparedto PANI film both for urea and triglyceride sensors. It should benoted here that, in both the cases, the enzyme immobilizationwas carried out by physical adsorption. The sensitivity of thesensor further increases in the case of PSS-PANI-B, and this wasattributed to the higher enzyme loading on the polymer matrixby immobilizing the enzymes during polymerization. On the otherhand, for the glucose sensor, the sensitivity was found highestfor the PANI-based sensor followed by PSS-PANI-B and PSS-PANI-A. This is because in the glucose sensor, the pH of themicroenvironment decreases due to the production of gluconicacid and the conductance of PANI shows a sharp increase withpH in the region of pH 1-6 (Figure 2). Though the enzymeloading was found more in PSS-PANI-B compared to PANI film,the sharp change of conductance of PANI in the range of pH 1-6explains the increase in sensitivity of the sensor in the latter. ForPSS-PANI-A film, the sensitivity was found still lower. Therefore,

Table 2. Comparison of Sensitivity and Linear Range of Glucose, Urea, and Triglyceride Sensors Based on PANIand PSS-PANI Microtubules

substrate

urea triglyceride glucose

polymersensitivity(mM-1)

linear range(mM)

sensitivity(mM-1)

linear range(mM)

sensitivity(mM-1)

linear range(mM)

PANI 0.02 ( 0.001 0-30 0.03 ( 0.004 0-30 2.4 ( 0.081 0-50PANI-A 0.09 ( 0.003 0-40 0.15 ( 0.005 0-40 0.19 ( 0.011 0-50PANI-B 1.72 ( 0.056 0-60 0.98 ( 0.037 0-60 0.72 ( 0.033 0-60

Figure 4. Response of the urea microtubule sensor based on PSS-PANI-B (b), PSS-PANI-A (2), and PANI (×) (PSS-PANI-A andPANI shown in the inset), urea in pH 5.2 buffer. Data are given asmean ( (variation from the mean) for three experiments.

Figure 5. Response of triolein microtubule sensor based on PSS-PANI-B (b), PSS-PANI-A (2), and PANI (×) (PSS-PANI-A andPANI shown in the inset), triolein in pH 5.2 buffer solubilized by 0.01%Triton X 100. Data are given as mean ( (variation from the mean)for three experiments.


the sensitivity and linear range of urea and triglyceride sensorscan be improved by modifying the pH-dependent conductancebehavior of the polymer and further by increasing the effectiveenzyme loading in the polymer matrix by immobilizing theenzymes during polymerization. The current increases (in the caseof a glucose sensor) or decreases (in the case of urea andtriglyceride sensors), when the enzyme-modified electrode wasexposed to the appropriate analyte solution, then it reaches a stable

value (less than 5% variation over a period of 3 min) in 6-8 s forglucose and urea and 10 s for triglyceride. The time taken for thesensor to reach this stable value is called here the response timeof the sensor. The slower response time found in the triglyceridesensor may be attributed to the slow diffusion of the triglyceridemolecule to the polymer electrode because of the solubilizationof triglyceride by Triton X 100. To study the reproducibility andthe stability of the sensor devices, the PSS-PANI-B sensors wereused for repeated measurements for a 30 mM urea and 20 mMtriglyceride solution. The results are shown in Figure 6. Eachmeasurement was preceded by rinsing the sensor in buffersolution. The sensor response was fairly reproducible for boththe urea and triglyceride sensors even after 20 runs with a lowstandard deviation of 4%, which indicates that the enzyme istrapped in the polymer film and does not leach out. The ureasensors based on PSS-PANI-B and PANI were tested for shelflife by keeping the polymer-enzyme film at 4 °C in buffersolutions. PSS-PANI-based urea sensors were found to maintain75-80% activity even after 7 days. On the other hand, the PANI-based urea sensors retained only 15-20% activity after 7 days.The PSS-PANI-B sensor devices were found to retain 50% of theiractivity after storing the film in the buffer solution for 2 months.

Figure 7 shows the sensor response of urea for PSS-PANI-Band PANI as a function of number of days. Thus, the stabilityand the shelf life of the sensors were found to be improvedsignificantly by immobilizing the enzyme during polymerization.

Response of Sensor Array for Glucose, Urea, and Tri-glyceride. The detailed explanation about the fabrication of thesensor array is given in the Experimental Section. The sensorarray was exposed to a mixture containing all three substrates.Data for three such mixtures are presented here, and thecompositions of the three mixtures are given in Table 3. Thesensors were addressed sequentially while maintaining the sen-sors that were not being addressed at -0.2 V to minimize leachingout of the respective enzyme. The concentration of each compo-nent was estimated by comparison with the respective calibrationplot. Concentrations thus obtained are plotted versus the knownconcentrations of that component and shown in Figure 8. The

Figure 6. Repeatability of the urea and lipid microtubule sensorsto 20 independent measurements (a) Response of the urea sensorsto 30 mM urea solution ([). ((b) Response of the lipid sensor to 20mM lipid solution (9).

Figure 7. Sensor response for urea for PSS-PANI-B (9) and PANI([) as a function of number of days. The sensors were stored in bufferat 4 °C.

Figure 8. Response of the microtubule sensor array for PSS-PANI-B.


data points are in good agreement with a line of unity slope thatwould be expected ideally.

CONCLUSIONSIn this paper, we have shown that conducting polyaniline can

be synthesized at a more biocompatible pH 5 in the presence ofpoly(styrene sulfonate). The PSS-PANI films showed a differentpH-dependent conductivity compared to polyaniline. Here theconductivity change with pH was more gradual compared topolyaniline. The immobilization of enzymes during polymerizationincreased the effective enzyme loading, sensitivity, and linearresponse of the sensor devices. The linear range (0-60 mM) andresponse time (6-8 s) of the present urea sensors based on PSS-PANI were found to be significantly higher compared to thepotentiometric urea sensor based on processible polyaniline (linearrange of 1-10 mM with response time of 50 s).19 On the other

hand, the linear range (0-60 mM) and response time (6-8 s) ofthe glucose sensors were also found to be better than the reportedlinear range of 0.1-30 mM with a response time of 1-2 min.19

Furthermore, the linear range of our glucose sensor is better thanthe linear range of some commercial instruments that have anassay range of 20-600 mg/dL (1.1-33.3 mM). Bartlett and Wangreported on glucose sensors based on PSS-PANI composite filmsoperated at neutral pH 7.31 The response time of the sensors was100 s, and the sensors showed good stability for 58 consecutivemeasurements. On the basis of these facts, we can conclude thatthe loading of the enzymes during polymerization improves theloading of the enzymes, which in turn enhances the performanceof the devices. It is interesting to note that the sensor responseis the result of the change in conductivity resulting from a changein pH though the solutions are buffered. This is because thechange in conductance is due to the polymer deposited on thepore wall, and since the pores are extremely small (diameter 1.2µm and length 10 µm), movement and equilibration of H+ ionswith the bulk solution is very slow. Therefore, though the pH inthe bulk is buffered, that in the pores is not.

ACKNOWLEDGMENTWe thank MHRD, India for the financial help and Prof. Rakesh

Lal for numerous discussions.

Received for review May 21, 2003. Accepted August 14,2003.

AC034537H(31) Bartlett, P. N.; Wang, J. H. J. Chem. Soc., Faraday. Trans. 1996, 92, 4137.b

Table 3. Response of Sensor Array for Glucose, Urea,and Triglyceride

concentrationsas prepared (mM)

concentrationsas measured (mM)

solution glucose urea triglyceride glucose urea triglyceride

A 10 20 30 8 22 28B 30 40 10 27 38 11C 40 30 20 37 27 18


microtubule sensors and sensor array based on polyaniline synthesized in the presence of...

Documents