Enhanced Sensitivity of a Galactose Biosensor Fabricated with aBundle of Conducting Polymer Microtubules

Kyoung Neung Lee,a Youngkwan Lee,b Yongkeun Son*a

a Department of Chemistry and BK21 School of Chemical Materials Science, Sungkyunkwan University, Suwon 440-746, SouthKorea82-31-290-7068 (T)

b Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea*e-mail: ykson@skku.edu

Received: March 30, 2011;&Accepted: June 18, 2011

AbstractA bundle of conducting polymer tubules was employed to build a biosensor of improved sensitivity. The tubulestructure was able to provide larger enzyme encapsulating space per unit area of the sensor electrode compared toother immobilizing methods. The bundle structure was prepared by using electrochemical polymerization of EDOTinto the template on ITO. After dosing of enzyme into the tubule, it was covered with PPDA channels. The ampero-metric response of the sensor to galactose showed linear behavior in the range of 0.1–1 mM with an average sensi-tivity of 6.37 mA/mM cm2. The response time was 30–40 s. After keeping the sensor in phosphate buffer solution at4 8C for a week, it showed about 82% of its original sensitivity. The detection limit was 0.01 mM based on S/N=3.

Keywords: Galactose biosensors, Conducting polymers, Microtubules, Amperometric detection, PEDOT,Electrochemical polymerization

DOI: 10.1002/elan.201100183

1 Introduction

Prevention of disease has been known to be more impor-tant than treatment of the ailment. The prevention issometimes possible only through diagnosis. Point-of-caretesting is very important because it is a diagnostic testingat or near the site of patient care, therefore the testingshould be simple, reasonable in price, and done in shortperiod. The fast and accurate determination of the galac-tose level in human urine or blood is a very importantissue in the field of food science, human nutrition, andpharmacy because it could be used as a criterion of fatalgalactosemia. When the blood level is more than 1.1 mMin neonatal infant it becomes fatal galactosemia and leadsto vomiting, diarrhea, jaundice, cataract, intellectual disa-bility etc. [1]. A little percentage of neonatal infants isborn with this disease and more than 90% of them couldbe cured if the disease is diagnosed at an early stage. Ifnot 75 % of the neonatal infant patients end up withdeath [2]. So development of a simple and less expensivegalactose sensor is necessary for point-of-care tests.

A lot of research firms in industry and academia aretrying to produce sensors of good sensitivity for accuratediagnosis and are reporting many electrochemical galac-tose biosensors using galactose oxidase to help diagnosinghuman galactosemia [3–5]. Some of them adopted elec-trochemical quantification methods of hydrogen peroxide,which had been produced by the enzyme reaction of gal-actose [6–8]. Most of them were using encapsulation of

enzyme simply using a polymer composite and membranefor dialysis. Sensors of this type are relatively large in sizeand low in sensitivity, so they are hardly used in a point-of care system.

In this study we like to show a slightly different way ofenzyme encapsulation to produce a simple galactose bio-sensor of high sensitivity. During this process a wellknown conducting polymer microstructure which wasformed on top of a base electrode via template electro-chemical fabrication was used for encapsulation of galac-tose oxidase. The tubule structure was able to immobilizea higher amount of enzymes into the tubule compared toother immobilization methods [9]. In other words, theloading amount of the enzyme per unit area could be in-creased freely. After loading the enzyme, the open mouthof the tubule was sealed with channels of functional mate-rial which can allow the substrate to pass through it. Theschematic feature was illustrated and found elsewhere[10]. The tubule structure here was providing two impor-tant rolls simultaneously. These are: providing plenty ofspace for loading enzyme species as a bundle of tubules,and supplying a large area of current collector for gooddetection of electrochemical oxidation of hydrogen per-oxide.

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2 Experimental

2.1 Reagents and Apparatus

3,4-Ethylenedioxythiophene (EDOT) was purchasedfrom Sigma-Aldrich and Baytron P 4083(PEDOT/PSSdispersion) was obtained from Bayer. Polyvinyl alcohol(PVA) was gratefully donated by OCI (Korea) and galac-tose oxidase (GAO, 1KU/G) was the product of Sigma.Other reagents, d-galactose (98 %), l-ascorbic acid(99 %), uric acid (99%) and phenylenediamine (99%)were obtained from Sigma-Aldrich. The rest of the chem-icals were ACS grade and used as obtained with furtherpurification. Phosphate buffer saline (PBS) solution wasprepared with 0.1 M Na2HPO4, 0.1 M NaH2PO4 and0.15 M NaCl and the pH was kept at 7.4 with 3.0 MNaOH solution.

The indium tin oxide (ITO) glass electrode was donat-ed by Samsung-Corning and used as a base electrodeafter several cleaning procedures. A platinum disk elec-trode was used as counter electrode and an Ag/AgCl (sa-turated with KCl) electrode was used as reference elec-trode. All potential values in this paper are based on thisreference electrode. The track etched porous poly-carbonate (PC) membrane filters (Millipore) used in thisstudy possess 1.2 and 0.05 mm diameter pores with athickness of 10 mm. Each surface of the membrane wasdifferent from the other, one was shiny and the other wasflat. The flat side was glued on top of the ITO base elec-trode with a house made conducting glue. Spin coatingsof the conducting glue were done with a high speed spincoater (Headway) equipped with a vacuum pump. Elec-trochemical synthesis and measurements were performedwith a potentiostat BAS 100B (Bioanalytical System).SEM images were taken with an instrument JSM6700FFE-SEM (JEOL).

2.2 Preparation of Microtubule Sensor Electrode

The flat side of the PC membrane was sputtered withplatinum to provide a good electrical connection and itwas cut into pieces of 1 �2 cm size. The ITO base elec-trode of 1 �3 cm was fixed on the spin coater by vacuum,later conducting glue was spin coated on the surface ofthe ITO at a rate of 6000 rpm. A piece of the PC mem-brane was placed on the glue directly. The Pt-side of themembrane met the glue. The glue was allowed to dry fora day. The PC membrane attached on the ITO was usedas a template for the fabrication of microtubules by elec-trochemical polymerization. After drying, the templateelectrode was immersed into a polymerization solutionconsisting of a 0.1 M EDOT and 0.1 M LiClO4 solution inacetonitrile and water mixture (water contents 30 %). Thesweeping voltage was applied to the template electrodefrom 0.3 to 1.3 V with a sweep rate of 100 mV/s. Thethickness and the length of the tubule inside the porewere directly depending on the number of the cycles.Polymerization was finished after the 10th cycle. In this

way the poly-EDOT (PEDOT) starts to form at the deepinside of the pore of 10 mm long and grows towards outerdirection. When the polymerization occurs, the polymerused to deposit onto the pore wall of the PC membraneand finally PEDOT would be in the form of a tubule [11].The shape of the tubule produced was investigated with aSEM image taken after the PC template was removedwith methylene chloride.

A 10 mL dose of enzyme solution (2000 U/mL, galac-tose in phosphate buffer) was placed on top of the micro-tubules to stay inside the template membrane by using amicrosyringe. After adding enzyme solution, the electrodewas kept in a dish filled with water vapor for 30 min toallow the solution to be sucked down into the tubules.After drying, the surface of the template was cleanedwith a sheet of wet Kimwipes three times. To prevent theenzyme dissolute out of the tubule, the open mouth ofthe tubule was covered with a functional cap. To cover upthe mouth, a PC membrane having 0.05 mm pores wasused as a matrix of the functional cap. The matrix was en-crusted with the conductive glue with the spin coater andput on top of the open mouth of the template membrane.The mouth-covered electrode was then dipped into apolymerization solution of 0.01 M 1,3-phenylenediaminein 0.1 M LiClO4/acetonitrile. Electrochemical polymeri-zation was performed with potential cycling between 0.3and 1.3 V at a rate of 100 mV/sec. After three times of cy-cling, the polymerization was finished. This preparationprocedure could be found in detail elsewhere [10].

2.3 Sensor Test

The sensor system was assembled as in Figure 1. The elec-trochemical cell was composed of two different Teflonparts and a sensor electrode residing between them. Thetop part has a hole at the bottom. An O-ring of 4 mminner diameter just fit around the hole. The bottom partwas a Teflon plate holding another O-ring identical to thetop one. The final sensor electrode was place between thetwo Teflon parts as shown in the figure. Four pairs of boltand nut were used to assemble the cell. The cell wasfilled with 10.0 mL of phosphate buffer solution and latergalactose test solution was added to the buffer. A hydro-gen peroxide oxidation potential of 0.6 V was applied tothe sensor electrode (WE) after testing several potentialsto measure the responding current. The galactose concen-tration was controlled by adding a certain volume ofhighly concentrated galactose stock solution consecutivelyto the testing buffer solution. After investigating the sen-sitivity of the sensor, interference effects from other inter-fering species such as ascorbic acid and uric acid weretested.

3 Results and Discussion

After the template was fixed on the base ITO, the elec-trode was dipped into a polymerization solution. A cy-

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cling potential was applied to the electrode to preparethe PEDOT tubule into the template. The applied poten-tial range was 0.3–1.2 V with a scan rate of 100 mV/s. Thecyclic voltammogram is shown in Figure 2. The electro-chemical current response was plotted as function of theapplied potential. The response current shows only asmall charging response at the beginning but it increasesrapidly towards the higher potential region. The rapidcurrent response at the right was due to the oxidation ofthe monomer, the only oxidizing species in the cell in thatpotential range. Potential switching allows the current todecrease but it shows a flat capacitive response later. Asthe number of cycles increased, the current responsesalso increased. This indicates that the polymer is growinggradually by the electrochemical process. The capacitiveresponse is also rowing and indicates that the polymerformed inside the template possesses good electrical con-ductivity [14]. The cycling window was set a little bitnarrow. In other words, the reduction switch potentialwas set to the value before the polymer was turned intothe full reduction state to keep the produced polymer inthe conductive state during the polymerization state. Theelectrochemical polymerization produced nice PEDOTtubule into the template as shown in Figure 3. The imagewas obtained after removing the PC template with meth-ylene chloride and washing with water for 30 minutes toobserve the tubule shape. The PEDOT product was inreal tubular structure and leaned over each other. Theyare insoluble in water even after 30 minute water treat-ment.

The whole bundle of tubules imbedded into the PCtemplate was filled with enzyme solution as describedearlier. The top was capped with a PC membrane withmuch smaller pores of 0.05 mm by use of conductive glue.After gluing the membrane, electrochemical polymeri-

zation was performed similar to the previously describedprocedure with a different polymerization solution. Thepolymerization was done in a solution of 0.01 M 1,3-phe-nylenediamine (PDA) and 0.1 M LiClO4 in acetonitrile.Polymerization was achieved with a potential cyclingrange of 0.3–1.3 V at a scan rate of 100 mV/s. The currentresponse of the polymerization is shown in Figure 4a. Theshape of the CV is a little different from the one inFigure 2. Polymerization starts at 0.7 V and the reactionrate increases exponentially at higher potentials. Whenthe scanning direction was switched, a sharp decrease ap-peared and finally a very small charging current responsefollowed. Current responses for the second and thirdscans decreased compared to the first one, because thePPDA formed in this way was a nonconductive polymer[10]. This is a representative irreversible polymerizationCV for a nonconductive polymer formation. The PPDAformed in 0.05 mm pores produced the SEM image depict-ed in Figure 4b. This image was obtained after removingthe PC template of 0.05 mm pore. The whole bunch of0.05 mm size threads was tangled together and a smallportion of them are covered with small flat PPDA piecesformed by the meeting of the thread ends together. Thetangled threads were considered to be formed during theearly stage inside the pore, and the flat pieces are thepolymer product formed on top of the tubule during laterstage. When the growing threads overflowed the tubulethey can meet each other and fuse to build flat areas.This implies that the tubules were capped completely bythe thin PPDA threads.

The amperometric response of this biosensor to galac-tose is depending on the applied potential. Potentials of0.4, 0.5, 0.6, and 0.7 V were applied to the sensor, respec-tively, and the amperometric responses to a successive ad-dition of galactose stock solution were measured. As theoxidation potential increased, the amperometric responseincreased except at 0.7 V as shown in Figure 5. Thereason for this is under further exploration in this lab. Apotential of 0.6 V showed maximum response in the

Fig. 1. Galactose biosensor cell system. A Pt counter electrodeand a Ag/AgCl (Sat�d KCl) reference electrode are positioned atthe cell top. Two different Teflon pieces are serving as a cell con-tainer and a base plate. The sensor electrode is attached to thecell container with two O-rings.

Fig. 2. Cyclic voltammograms for electrochemical polymeri-zation of EDOT into the pores of the PC membrane template.

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Galactose Biosensor Fabricated with a Bundle of Conducting Polymer Microtubules

given circumstance. This potential value was adopted totest the biosensor. The calibration curve for galactose wasdetermined with a test cell described above. The test solu-tion was a 10 mL of phosphate buffer and a small amountof galactose solution was added successively every 60 s byusing a microsyringe. Figure 6 shows the result of the cali-bration. In this measurement each increment of galactoseconcentration was 0.1 mM. The curve shows linear re-sponse up to a concentration of 1.0 mM. Every single ad-dition step shows so fast response that the current in-crease looks like a step of right angle. This is very impor-tant if we consider about point-of-care test, because inthis concentration range, the sensor reaction occurswithin 30 sec. As indicated in the figure this biosensorshows a slope of 0.80 mA/mM (6.37 mA/mM cm2) with

good linearity in the range of 0–1.0 mM. This value islarger than results any other enzyme immobilizationmethod could provide as presented in Table 1. The detec-tion limit appeared to be 0.01 mM based on a signal tonoise ratio (S/N) of 3. We think that there are two majorreasons for the sensitivity improvement. Firstly the micro-tubule structure can provide a large space for holdingenzyme in even small area. Secondly the tubules can holdfree enzyme in them without any enzyme modification.This sensor showed 82% of the original sensitivity afterbeing stored in PBS at 4 8C for a week.

This biosensor was also tested in a solution containingseveral interfering species beside galactose. Ascorbic anduric acid were chosen because they are easily found inhuman blood. When each teaser was added no current re-sponse was detected as shown in Figure 7. This has beenknown as screen effect of the PPDA capping film [15].

Fig. 3. SEM image of the PEDOT tubules formed into the PCtemplate. The image was taken after removing the PC membranetemplate.

Fig. 4. Formation and shape of mouth covering PPDA. (a) Cyclic voltammogram for polymerization; (b) SEM image of the cappingPPDA produced by electrochemical method after removing PC with methylene chloride. Rods and tubules formed electrochemicallywere tangled together.

Fig. 5. Amperometric responses of the sensor with differentsensing potentials of 0.4, 0.5, 0.6, and 0.7 V. 0.6 V provides thehighest response.

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4 Conclusions

Electrochemical template synthesis of a conducting poly-mer microtubule structure was applied to a biosensorsystem to improve its sensitivity. The tubule plays two im-

portant rolls simultaneously: as a container which cankeep free enzymes without modifications and as a currentcollector for the electrochemical oxidation of hydrogenperoxide, the enzyme reaction product. The amperomet-ric response of this sensor to varying galactose concentra-tion showed linear response in the range of �1.0 mMwith an average sensitivity of 6.37 mA/mM cm2. The re-sponse time was 30 – 40 s. Several interfering materialssuch as uric acid or ascorbic acid did not produce anynoise response because of the presence of the PPDA cap-ping which has good selectivity. 82 % of the original sensi-tivity was reproduced even after keeping the sensor inPBS at 4 8C for a week. Sensors of this type did not showany cross talk with glucose (not shown).

Acknowledgements

This work was supported by the Energy & Resource Re-cycling of the Korea Institute of Energy Technology Eval-uation and Planning (KETEP) grant funded by the Minis-try of Knowledge Economy, Republic of Korea (No.2010501010002B).

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Fig. 6. Responses to successive addition of 0.1 mM galactose.

Fig. 7. Sensor responses to interfering materials compared tothe galactose substrate. 0.1 M ascorbic acid and 0.1 M uric acidwere used.

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Immobilization with microtubule structure Thiswork

6.37 mA/mMcm2

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[1] 3.748 mA/mMcm2

Immobilizing galactose oxidase in polyvi-nylferrocenium matrix coated on a Pt elec-trode surface

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