P SREE DIVY A, D SA VITRI and CHANCHAL K MITRA *

Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

*Corresponding author (Fax, 91 40 3010 120/3010 145,' Eniail, ckmsl@uohyd.ernet.in).

The aim of the present work is to design an electrode for biosensors by covalent immobilization of theredox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surfaceand this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methodsof covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horseradish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on theintended use. A three step procedure that includes (i) heat treatment of matrix at lOO-l10°C to removevolatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO2, -Br etc.followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated mati x after subsequent reduction)or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylatedmatrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated byspectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simplenitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxidepretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrodewas tested in a rotating disk experiment for its response with the substrate.

Introduction

The optimum immobilization technique for a particularpurpose is arrived at by a number of technical, commercialand practical considerations. However, covalent immo-bilization best suits the purpose where long term stabilityof the immobilized enzyme is particularly important. Theformation of a covalent bond between the biocatalystand a support matrix creates a stable conjugate whichis unlikely to dissociate during normal use and hencethe problem of enzyme leaching out often encounteredin other immobilization methods (like adsorption) iscompletely avoided. Moreover, since long term stabilityand efficient operation are major requirements of many

immobilized enzyme systems, there has been a prolifera-tion of covalent binding techniques each seeking toprovide a simple, efficient and environmentally safemethod of immobilization. Covalent immobilization hasbeen worked out with various kinds of supporting matrices(mostly polysaccharides and other organic polymers)following several different activation and coupling pro-cedures.

Immobilization on a conducting matrix (e.g., a metalelectrode) is generally difficult but is desirable in biosen-sor application. Several enzyme biosensors have beendesigned and constructed using various kinds of immo-bilization techniques. Recent works dealing with chemi-cally modified electrodes have made possible the

Keywords. Covalent coupling; horse radish peroxidase; carbodiimide coupling; enzyme biosensor

Abbreviations used: -Br: Bromo; CM: a-carboxy methyl; DCC: diocyclohexyl carbodiimide; EDA 2HCl: ethylenediaminedi-hydrochloride; EDC: l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; HRP: horse radish perxodiase; ~O2: hydrogenperoxide; KOH: potassium hydroxide; NaCl: sodium chloride; NaOH: sodium hydroxide; -NH2: amino; -NO2: nitro; -OH:hydroxyl.

Biosci., 23, No.2, June 1998, pp 131-136. @ Indian Academy of Sciences 131

P Sree Divya, D Savitri and Chanchal K Mitra132

temperatur Werke, Germany. l-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) was obtainedfrom Aldrich Chemical Co. Di-o-anisidine and HRP wereprocured from Sigma Chemical Co. (USA). DCC andsodium borohydride were obtained from Spectrochem.All other chemicals were AR grade from Qualigens

(Mumbai).

2.2 Methods

We have followed a three step procedure to covalentlyimmobilize the enzyme HRP that includes (i) heat treat-ment of carbon matrix, (ii) chemical pretreatment tointroduce functional groups .like -NO2, -OR and -Br

groups onto the matrix followed by (iii) enzyme coupling.

Step 1 (heat treatment): The gJassy carbon matrix washeated in a hot air oven at 100-110°C for 1-2 h. Thishelps in removing the volatiles and other absorbatesfrom the surface of the glassy carbon particles so thatthe full surface is available for the next chemical pre-treatment step. This heat treated matrix has been usedin all further experiments. Washing the matrix withorganic solvents prior to this procedure also helps inremoving the tar and other waxy (non-volatile) materialpresent on .the carbon surface.

Step 2 (chemical pretreatment): Three different andindependent chemical treatments procedures have been

attempted.

permanent chemical modification of various .electrOdematerials (Watkins et at 1975; Dautartas et at 1979).For example, gold electrodes have been covalently modi-fied using thiol compounds (Katz et at 1997). Glassycarbon is not an attractive material for enzyme immo-bilization as its chemical inertness towards couplingreagents is very high. Enzyme adsorptions on graphiteor activated carbon followed by glutaraldehyde (Liu etat 1975) or soluble carbodiimide (Cho and Bailey 1977,1979) cross-linking have been reported. Recently covalentimmobilization of the glucose oxidase enzyme onto gra-phite powder by carbodiimide treatment was reported(Gorton 1995). The direct covalent binding of the enzymeby carbodiimide coupling onto glassy carbon after initialactivation by electrochemical oxidation was reported byBourdillion et at (1980). Relatively fewer reports areavailable on carbon paste electrodes used as biosensors.

We have attempted to covalently immobilize enzymesonto a glassy carbon supporting matrix to design a pasteelectrode system. The initial derivatization of the matrixin our case was done by a chemical oxidation method.We have used the enzyme horse radish peroxidase (HRP)as a model in our present studies. The matrix wassubjected to several chemical and heat pretreatments toremove moisture and other volatiles. This would alsohelp to introduce functional groups that can be used inenzyme coupling. The chemical pretreatments like nitra-tion, bromination and peroxidation introduce functionalgroups nitro (-NOJ, bromo (-Br) or' hydroxyl (-OH)groups respectively. The NO2 matrix was subsequentlyreduced to -NH2 matrix and the enzyme was coupledusing glutaraldehyde. The Br matrix was converted tohydroxy derivative by subsequent hydrolysis using con-centrated potassium hydroxide (KOH). The -OH matrixwas modified by carboxymethylation (using chloroaceticacid) to a-carboxymethyl (CM)-matrix. Enzyme wascoupled to this matrix by l-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC)/dicyclohexylcarbodiimide (DCC) coupling with and without spacerarm.

An assay was performed to estimate the amount ofenzyme loaded onto the surface. We report here thedifferent sets of conditions we followed for enzymeimmobilization and compare the efficiencies of the tech-niques employed. The protocol for the immobilizationprocess can be used for any other suitable enzyme e.g.glucose oxidase. The covalent electrode offers betterperformance in an electrochemical flow-system than anadsorbed (enzyme) electrode.

(i) Nitration followed by reduction of nitrated matrix:The heat treated matrix was subjected to nitration bytreatment with nitration mixture (1 : 3 HNO3: ~SO4) at100°C for 3-4 h. Afterwards, the matrix was suspendedin sodium hydroxide (NaOH) to neutralize the acid priorto centrifugation. Further washing using dilute sulfuricacid was done to remove the excess alkali. The nitrogroups on the matrix were reduced with sodium boro-hydride. 300-500 mg of sodium borohydride (for 250mg matrix) dissolved in 1 inl of water was added to thenitrated matrix and incubated overnight at room tem-perature. The matrix was thoroughly washed by repeatedcentrifugation to remove excess reagent.

Treatment with nitration mixture is expected to intro-duce -NO2 groups on the matrix. Sodium borohydridereduces the nitro group to amino (-NHJ group. Thematrix after the reduction step was used for enzymecoupling using a bifunctional reagent (glutaraldehyde).

(ii) Bromination followed by hydrolysis with alcoholicpotassium hydroxide: The heat treated matrix (250 mgof matrix) was suspended in 5 ml of bromine solutionin carbon tetrachloride (20% v/v) and incubated at roomtemperature till all the bromine evaporated from the

2. Materials and methods

Materials2.

The glassy carbon powder was procured from Hoch-

133Enzyme immobilization on conducting matrix

chemical treatment procedures i.e., the -OR matrix wasmodified by carboxymethylation (Inman 1985). Thematrix was suspended in an equal volume of 0.1 Msodium chloride (NaCI) solution and was put in anicebath. NaOH (6 M) solution was added slowly andstirred until the temperature reached 15°C. The mixturewas removed from icebath and 5 ml of monochloroaceticacid solution (2% solution) was added and incubated for70 min at 25°C with constant stirring. The matrix waswashed with 0.1 M NaCI 3 to 4 times to remove excessreagent. The resulting CM-matrix was stored in 0.1 MNaCI containing 0.04% (w/v) sodium azide (to preventbacterial growth on prolonged storage).

In the presence of chloroacetic acid and alkali (NaOH),hydroxyl groups on the matrix are converted to carboxy-methyl functions. Large excess of chloroacetic acid wasused in order to reduce the time of exposure of thematrix to alkali.

The HRP enzyme was coupled to the CM-matrix inthe next step. We have used two coupling reagentsnamely EDC and DCC.

2.2c DCC coupling: To the CM-matrix suspension(roughly 250 fig), 40-60 mg of DCC dissolved in di-methyl sulfoxide was added with constant stirring. Oneml of HRP at a concentration of 3 mgiml was addedand left for incubation at 25°C for 4 h. The matrix waswashed with 0.1 M NaCl solution and evaporated todryness under yacuum. The pH was maintained at 5 :t: 0.2throughout the course of the reaction (Dean et al 1985).

solution (approximately 3 days). It was then washedthoroughly with water to remove any traces of bromineand subjected to hydrolysis. The matrix was heated withalcoholic KOH (20% solution in 90% ethanol) for 2 hon a boiling water bath. The matrix was then washedwith water and treated for further chemical modification(carboxymethylation).

Reaction with bromine introduces bromine atoms ontothe matrix surface. Treatment with alcoholic KOH hy-drolyses the halogen moieties to -OH groups.

(iii) Peroxidation: The heat treated matrix was sus-pended in 30% H202 solution for 48 h at room tempera-ture. Treatment with hydrogen peroxide introduceshydroxyl groups onto the matrix. The matrix was washedwith distilled water 3-4 times before proceeding for thenext step.

In case 1, we obtained amino derivatives of the matrix.In the other two cases, hydroxylated derivatives wereobtained. In all the three chemical pretreatments, carbonmatrix samples were collected at the end of each of thechemical reactions. All these samples were used ascontrols and were processed along with the enzymecoupled matrix for the activity assay. All washings werecarried out by addition of 10 to 15 ml of double distilledwater and centrifugation at 12,000 g for 20 min at leasttwice. The supernatant was discarded and the pellet wasprocessed for the next step.

Step 3 (enzyme coupling): The pretreated matrix wasprocessed for the final enzyme coupling step. Two dif-ferent techniques were used for this purpose: (i) glu-taraldehyde (for the amino derivatives) or (ii) carboxy-methylation (for the hydroxylated matrix) followed bycarbodiimide (either DCC or EDC) treatment.

2.2a Coupling using a bifunctional reagent: The aminoderivative of the matrix was incubated with 2% glutaral-dehyde solution for 12 h at 4°C. The matrix was washedwith distilled water 3-4 times to remove excess glu-taraldehyde. The matrix was suspended in 1 to 2 m1water and 3 mg of HRP enzyme (for 250 mg matrix) in1 ml of phosphate buffer was added and incubated for12 h at 4°C. The matrix after enzyme coupling waswashed with distilled water and dried under vacuum.

Glutaraldehyde attaches to the matrix via the -NH2group of the latter and the other end remains free tocouple to enzyme. Enzyme is coupled to the free endof the glutaraldehyde (to -CHD group). The mechanismof reaction of glutaraldehyde with arnines is the subje;ctof much speculation (Branner-Jorgenson 1978) and mayinvolve condensation and subsequent reaction betweenthis polymer and the amino group rather than reactionwith a simple aldehyde.

2.2d EDC coupling: In this step a support bearingprimary amino groups was prepared by the reaction ofthe CM-matrix with ethylenediamine in the presence ofEDC and the enzyme was coupled to these amino groups(Inmann 1985). The CM-matrix was washed 3 timeswith an aqueous solution of 1.5 M ethylenediaminedi-hydrochloride (EDA. 2HCl). A water soluble carbodi-imide, EDC, was employed as condensing agent. Thematrix was suspended in 5 ml of 1.5 M solution of EDA2HCl and the pH was adjusted to 4.6. EDC (40 mg)was added in portions, with constant stirring and thiswas incubated at 30°C for 1 h. Another 40 mg of EDCwas added as in the above step and stirring was maintainedfor 4 h after the addition. The matrix was washed with0.1 M NaCI solution several times and was used for theenzyme coupling as described in the DCC coupling step.

To the CM-matrix (250 mg) suspended in I rnl of0.1 M NaCI solution, 40-60 mg of EDC was added inportions yiith constant stirring. HRP was coupled in anidentical fashion as described in § 2.2c.

A large excess of ethylenediamine was employed inthe previous step in order to promote a one endedreaction: a single amide link was formed leaving anuncombined amino group. The pH is a critical factor2.2b Carboxymethylation. The matrix from other two

134 P Sree Divya, D Savitri and Chanchal K Mitra

for this step. At pH 4.6, the reactivity of the proximal(reactive) amino group is considerably enhanced withrespect to the amino group remaining after attachment.Use of ethylene diamine provides a longer spacer armfor the enzyme.

The EDC/DCC coupling is a standard protocol tocouple a carboxyl and an amino group. The carboxylgroup can be either on the matrix (then coupling to theamino group of the enzyme takes place) or on theenzyme (then coupling with the amino group of thematrix takes place). In our case the DCC coupling ofthe enzyme was done with CM-derivative of the matrix;while the enzyme coupling onto the matrix using EDCas condensing agent was done with the aminoethylcarboxymethyl (AECM) matrix.

2.3 Enzyme activity assay

The enzyme activity that is immobilized onto the carbonmatrix was assayed colorimetrically following a modifi-cation of the procedure provided by Sigma Chern. Co.The principle of the reaction is as follows:

HRP

dures followed for covalent immobilization of enzyme.The glassy carbon matrix was initially hydrophobic (notreadily wetted by water). This was clearly observed whenthe glassy carbon powder was suspended in water, asit was not wetted and remained at the surface. Thematrix samples after the chemical pretreatments easilyformed a suspension. This suggests that the chemicaltreatments have introduced some hydrophilic functionalgroups onto the matrix surface. A sensitive chemicalanalysis would in principle give an idea about the degreeof surface modification of the matrix. Standard CHNanalysis is however not sufficiently sensitive as thematrix is -100% pure carbon and the contribution fromthe surface modifications is negligible.

Qualitative assay of the coupled protein has beencarried out using micro BSA reagent. Standard techniquesfor protein estimation (e.g., A280 or other colorimetricmethods) does give quantitative results as the protein isimmqbilized and the matrix is difficult to separate com-pletely (the protein is covalently coupled to the matrix).This gave us a relative idea of the degree of couplingof the protein in various systems. .

The results of the activity assay are summarized intable 1. The activity of the enzyme estimated from theassay shows that the activity is highest for the peroxidatedmatrix. For the nitration under drastic conditions theenzyme loading was the lowest. The matrix subjectedto bromination showed higher amounts of enzyme load-ing-the activity was approximately thrice the amountof activity' observed for the nitrated sample. Accordingto the literature provided by the manufacturer, the glassycarbon is resistant to hot concentrated nitric acid forextended periods, and the electron microscopic pictureshows only slight modification of the surface structure(Hochtemperatur-Werkostoffe GmbH, Catalogue forSigradur glassy carbon, Gemeindewald 41, D-86672Thierhaupten, Germany).

Different coupling methods provide different spacerarm length for the enzyme. A large spacer is desirablefor proper enzyme function and activity but a largespacer may also decrease the electron transfer rate withthe base matrix. However, a proper comparison requiresthat identical amounts of enzyme must be immobilizedso that the observed effect is solely due to the effectof the spacer arm and not due to different enzymeconcentration. This is however, difficult to realize in the

Table 1. Summary of enzyme activity assay.

Activity of HRP enzyme(units/mg of carbon matrix)Procedure

Nitration under drasticconditions

BrominationPeroxidation

0.005

0.0170.050

H202 + o-dianisidine (reduced) ~

o-dianisidine (oxidized) + H2O.

The dye, di-o-anisidine, is oxidized to a coloured complexin the presence of hydrogen peroxide in a reactioncatalyzed by HRP. The colour is measured at 500 nmagainst a reagent blank. As the enzyme activity on thematrix is rather low, a relatively long reaction time isrequired. However, it is convenient as we have to cen-trifuge the matrix suspension and take the supernatantfor absorbance measurements. The reaction virtually stopsafter centrifugation as all the enzyme is removed fromthe reaction mixture.

For the assay of enzyme activity immobilized ontothe matrix, 3 mg of the matrix was suspended in 1 mlof water and 1 ml of 1 mM H202 and 2.5 ml of dyewere added. The contents were mixed well and centrifugedat 10,000 g for 20 min. The supernatant was taken tomeasure the absorbance.

Soluble enzyme at different units of activity was takento make the standard for comparison. The activity ofthe immobilized enzyme was calculated from standardvalues and was expressed as units per mg of carbonmatrix. Ideally, some function independent method (suchas 1125 labelling) could have been done for the deter-mination of the actual amount of enzyme coupled to thematrix. However, such methods do not take into accountthe fact that a part of the enzyme may be deactivatedduring the coupling process.

3. Results and discussion

The experiments performed were aimed at comparingthe efficiency of enzyme loading in the different proce-

135Enzyme immobilization on conducting matrix

1.-+"'c::~L-::J()

0 5 10 15 20 25 30 35 40

Figure 1. Plot of 10 as a function of square root of rpm ata bias potential of -300 mv (vs. saturated calomel electrode).All the experiments have been carried out in 0.] M KCl solutionwith substrate (H2O2) concentration indicated in the figure. Allthe graphs show clear curvature at high rotational speedssuggesting that bulk diffusion is not rate limiting in all cases.The solid lines are calculated from the experimental data pointsusing the non-linear equation I D = a .(J)1/2/(b + (J)1/1. where aand b are empirical parameters. The agreement between theempirical equation and the experimental points are good.

electrode., The response of the prepared electrode waschecked in a rotating disc experiment with differentconcentrations of substrate ~02' Different concentrationsof "2°2 from 0.1 rnM to 1 mM were used in the ex-periment.

Figure 1 shows the plot of the rotations per minute(rpm) against current for the peroxidase paste electrodewith three different substrate (~OJ concentrations. Thesolid lines are fitted from the experimental valuesusing the modified Michaelis-Menten equationID = a .WI/2/(b + WI/2), where ID is diffusional current andW is angular velocity. It is clearly seen that the enzymekinetics is the rate limiting factor and an approximatehyperbolic graph is seen. In a rotating disk electrodeset-up, the effective concentration of the substrate closeto the electrode surface is proportional to the squareroot of angular velocity. Also the current flowing at theelectrode surface is proportional to the rate. of enzymesubstrate reaction at the surface. The electrode showsan increase in current with increase in substrate concen-tration, as predicted by the Michaelis-Menten equation.This graph confirms that the immobilized enzyme isshowing significant amount of activity. The graph wasplotted and the curve was fitted using a commercialsoftware (Sigmaplot, Jandel Scientific).

The background current was observed to be lower (bya factor of 10) with the modified electrode comparedto the electrode prepared from chemically untreated ma-trix. The pla~sible reason is the chemical treatmentsmight have removed some of the impurities or haveblocked some of the surface functional groups leadingto a lower background current.

We have prepared a covalently coupled glucose oxidaseelectrode following the sam~ protocol. The electroderesponse is found to be stable at least over a period of60 days. The response of the designed glucose oxidasebiosensor is found to be satisfactory in terms of linearitywith the substrate (data not presented).

Acknowledgements

One of the authors (DS) wishes to thank the UniversityGrants Commission, New Delhi for a research fellowship.This paper is dedicated to the memory of Prof. B KBachhawat.

References

present experiment. The overall performance shows thatthe enzyme has been successfully immobilized onto thematrix by covalent methods. Although a strict comparisoncannot be made due to heterogeneous nature of theimmobilized enzyme, the trends are clear and significant.

Taking the molecular weight of HRP to be 40 kDa,(assuming it to be a sphere with specific gravity of 1.2)its footprint size is estimated to be 20 nm2 (i.e., 20 x 10-18m2). Assuming that all glassy carbon particles are ofequal size of 1nm (from the data provided by themanufacturer), we estimate that each mg of carbon hasa surface area of 22 cm2 and the enzyme loading isequivalent to 12.9 ngicm2 or 4% of the surface coverage(the enzyme loading in the peroxidation case -0.05units/mg of matrix was taken for calculation).

The reasons for the low enzyme loading on the matrixcannot be exactly explained at this juncture. We presumethat some of the chemical treatments are not effectiveenough to introduce sufficient number of hydroxyl groupsthat can be used for enzyme coupling. Efforts are beingmade to increase the enzyme loading by modifying thevarious steps in the i~obilization procedure.

Another aspect that is to be checked to establish theefficacy of the technique is sustained activity of theenzyme, that too over a period of time. As mentionedearlier, the modified carbon matrix has wide applicationsin the field of biosensors. A paste electrode has beenconstructed with the modified matrix. The matrix wasmade into smooth paste using silicone oil and siliconegrease as pasting liquids and packed into a small disc

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MS received 20 October 1997,' accepted 24 April 1998

ColTesponding editor: M S SHAlLA