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A reusable amperometric biosensor based on a novel silver-epoxy electrode for immunoglobulin detection

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A renewable immunosensor consisting of an `epoxygraphite' biocomposite containing silver and tetracyanoquinodimethane (TCNQ) is described. These compounds enhance conductivity allowing the use of a smaller potential (0.28 v) which, in turn,
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   Biotechnology Letters  22:  579–583, 2000.© 2000  Kluwer Academic Publishers. Printed in the Netherlands.  579 A reusable amperometric biosensor based on a novel silver-epoxyelectrode for immunoglobulin detection R.F. Dutra 1 , 2 , G.D. Coelho 2 , V.L. Silva 3 , W.M. Ledingham 5 & J.L. Lima Filho 2 , 4 , ∗ 1  Departamento de Patologia, ICB – ESEF/UFPE, Recife – PE, Brazil 2  Laborat´ orio de ImunopatologiaKeizo Asami (LIKA)/UFPE, Recife – PE, Brazil 3  Departamento de Engenharia Qu´ımica/UFPE, Recife – PE, Brazil 4  Departamento de Bioqu´ımica, CCB/UFPE, Recife – PE, Brazil 5  Department of Life Science, University of St. Andrews, Scotland  ∗  Author for correspondence (E-mail: Jose − luiz60@hotmail.com) Received 11 November 1999; Revisions requested 7 December 1999; Revisions received 10 February 2000; Accepted 11 February 2000 Key words:  biocomposite, epoxy graphite, immunoglobulin,immunosensor, TCNQ Abstract A renewable immunosensor consisting of an ‘epoxygraphite’ biocomposite containing silver and tetracyanoquin-odimethane (TCNQ) is described. These compounds enhance conductivity allowing the use of a smaller potential(0.28v) which, in turn, enhances selectivity. This sensor, which may be renewed by simple polishing of its surface,was employed to detect human IgG using peroxidase-coupledanti-human IgG. Introduction Animmunosensorisa devicecomprisinganantigenorantibody species coupled to a signal transducer whichdetects the binding of the complementary species(Morgan  et al . 1996). Immunosensors are categorisedaccording to the detection principles employed, e.g.,immunoelectrodes (electrochemical immunosensors);piezoelectric immunosensors, or as sensors based onoptical detection of the immunoreaction (Ghindilis et al.  1997). There is a preference for optical sensorssuch as those using surface plasmon ressonance (SPR)technology (Morgan  et al.  1996, Fontana  et al.  1990).However, no commercial applications have been re-ported due to the limited sensitivity (Hock 1997).In contrast, the electrochemical detection of an im-munoanalytical reaction allows easier discriminationof specific and non-specific binding (Tiefenauer  et al. 1997). The use of antibodies, labelled with enzyme,is capable of generatingelectroactive species (Monroe1984), providing more robust and selective analyticalmethodologies(Santandreu  et al.  1997). Electrochem-ical immunosensors have been reported employingdifferent principles, e.g., silk screening graphite painton cardboard (Weetall & Hotaling 1987), highly dis-persed carbon material (Krishnan  et al.  1996a, 1996b,Ghindilis  et al.  1997), glassy carbon electrodes (Lu et al.  1997), etc. Recently, a new generation of elec-trochemical immunosensors was described employ-ing conductive biocomposites based on different rigidpolymer matrices (Santandreu  et al.  1998). The bio-composites act not only as a support for biomaterialimmobilization but also as transducers. Moreover, therigid matrices allows regeneration of the biocompos-ites by simple polishing of the surface, without the ne-cessity of complicated procedures for dissociation of antigen-antibody complexes established (Uttenthaler et al.  1998). This approach has also been describedfor enzymatic biosensors for glucose determination(Galan-Vidal  et al . 1997). Therefore, these aforemen-tioned graphite-epoxy biocomposites require a highworking potential, close to 1 V, which implies poorselectivity due to interfering ions present.This work describes a reusable modified epoxy-graphite biocomposite employing silver as metal dis-persed in association with tetracyanoquinodimethane(TCNQ), as an electron transfer mediator. The work-ing potential is reduced (260mV) achieving responses  580 Fig. 1.  The flow injection analysis system: (1) buffer reservoir;(2) peristaltic pump; (3) injector; (4) sample input; (5) immunoeletrode and reference (Ag/AgCl); (6) poloragraph; (7) chart record;(8) electrode surface. that are more selective. Analytical properties of thissensor have been evaluatedby detectionof humanIgG(hIgG)in a‘sandwich’immunoassayusingperoxidaseas label. Protein A was immobilized into modifiedepoxy-graphite biocomposite and used as a model tobindhIgG(Santandreu et al.  1997).The amperometricresponses are measured by a potentiostat coupled to aFlow Injection Analysis system (FIA). Experimental  Materials Protein A, hIgG and peroxidase conjugated anti-human IgG, casein and tetracyanoquinodimethane(TCNQ) were from Sigma (USA). Graphite pow-der (particle size 1–2  µ m) was obtained from Fluka.Silver-epoxy hardener and silver-epoxy resin werepurchased from World Precision Instruments (USA).All other reagents were of analytical grade. Preparation of the working electrode The compounds were mixed in a small reservoir inthe following proportion (w/w%): silver epoxy hard-ener(40%), silver epoxyresin (40%), graphitepowder(10%), TCNQ (5%) and protein A (5%). The pastewas firmly packed in a slot at the end of an electrodebody(2 mmdiameter; 3 mm deep)which hada copperwire lead to make the electrode connection. The elec-trodewas subsequentlycuredfor72hat 28 ◦ C. Duringmeasurements the top of the electrode was coveredwith a dialysis membrane (molecular weight cut off:12000–14000) to decrease the baseline instability. Adiagram of the electrode system is shown in Figure 1.The electrode was stored at  − 20  ◦ C.  Apparatus and procedures Amperometric measurements were performed with apotentiostat (Radelkis, model OH 107) coupled toa three electrode system. A platinum wire and anAg/AgCl electrode were used as counter and refer-ence electrode, respectively.The three electrodeswereimmersed in a chamber reaction (700  µ l). The FIAsystem apparatus consisted of a peristaltic pump, aheating system for the samples, carrier buffer, and aninjection valve (Figure 1).A typical procedureto determineamperometricre-sponse for hIgG is as follows: the working electrodewas polished with emery paper until a smooth sur-face was obtained. The electrode was incubated with2% casein in 0.01 M phosphate buffer saline (PBS)pH 7.2 for 2 h at 28  ◦ C to reduce non-specific binding.The electrode was immersed for 1 h at 28  ◦ C withIgG. Non-specific bound molecules were removed bywashing with PBS-buffer. The sandwich reaction wascarried out with human anti-IgG conjugate to peroxi-dase after immersing the electrode in hIgG for 1.5 hat 28  ◦ C. The FIA system was maintained at 32  ◦ Cwith 0.1 M citrate phosphate buffer (CPB) pH 6.0 ascarrier buffer at a flow rate of 330  µ l min − 1 with aloop injection of approximately 150  µ l and an appliedpotential between the electrodes of 260 mV. Fifty mMhydrogen peroxide, used as substrate, was prepared inthe carrier buffer. Results and discussion  Electrochemical behavior of the electrode A cyclicvoltammetricstudywas performedin ordertodetermine a new operating potential for the modifiedepoxy-graphite composites. The electrode was testedover the potential range from  − 100 mV to 860 mVat a sweep rate 2 m V s − 1 (Figure 2). A potentialof 260 mV was selected for subsequent studies. Alarge anodic potential window has been observed forrigid graphitecomposites (Kauffman et al.  1998).Fur-thermore, this behavior may be due to irregularity onthe electrode surface, therefore polishing will be nec-essary. An alternative may be the use of thick-filmtechnology such as the screen-printable techniqueswhich promote more regular, reproducible electrodes(Galan-Vidal  et al.  1997).  581 Fig. 2.  Cyclic voltammograms for hydrogen peroxide 2 mM (A),50 mM (B) and 100 mM (C), using a modified epoxy-graphiteelectrode vs. Ag/AgCl reference electrode. Sweep rate at 2 m Vs − 1 . The conductivity of an epoxy-graphite compositeis determined by its graphite content. If the graphitecontent is lowered, a decrease in conductivity andsensitivity will result in poor amperometric response.Previous studies using graphite compositions of 5,10, 15 and 20% showed that the optimal proportionwas 20% (Galan-Vidal 1997, Magalhães  et al.  1997).The applied potential determined for a 20% graphiteelectrode was between 800 and 1200 mV (Santan-dreu  et al.  1997, Céspedes  et al.  1994), relativelyhigh for use in complex samples such as biologi-cal fluids. Here, the potential used for this modified Fig. 3.  Plot of current response of the electrode with the injectionof hydrogen peroxide at different concentrations from 2 mM to500 mM. graphite-epoxy composite using silver in associationwith TCNQ is very low, increasing the electrode con-ductivity. A high applied potential presents problemssuch as interference arising from the electrochemicalactive species present in the liquid medium, and theimpossibility of a clear discrimination from the ox-idation potential of water (approximately 1000 mV)(Céspedes  et al.  1993). Another alternative to improvethe conductivity of graphite epoxy rigid composite in-cludes adding platinum to the graphite-epoxy mixture(Morales  et al.  1996). This biosensor is cheaper andthe nature of the composite material eases the mixtureand allows the absorption of other materials, such aschemical mediators, reducing the necessary appliedpotential. Composites modified by tetrathiafulvalene(TTF),aredoxmediator,havebeenreportedwithapo-tential of 150 mV (Céspedes  et al.  1994). In this work,we tested a modified silver-graphite-epoxy electrodeusingTTF, instead ofTCNQ. However,TTF-electrodeshowed more instability on baseline response andhigher applied potential comparing to TCNQ, when acyclic voltammetric study was performed with hydro-gen peroxide as describe above. Kinetic studies In order to establish the hydrogenperoxide concentra-tion for an incubated peroxidase conjugate electrode,solutions of2 mM, 10mM, 50 mM,100mM, 250mMand 500 mM H 2 O 2  (Figure 3) were evaluated. A con-centration of 50 mM was selected, because the use of more concentrated solutions affected the performanceof the electrode by changing the electrode porosity  582(Galan-Vidal  et al.  1997). It was observed that thebaseline after the response is shifted, probably due tothe charge distribution on the surface of the electrode.Studies using different pHs on the peroxidase-coupled electrode have demonstrated an optimumworking pH of 7.0 (Martorell  et al.  1994, Galan-Vidal et al.  1997). Although the current response increasesconsiderablyathighpHduetoanincreaseofoxidationpotential caused by protondeficit, the optimumpH forthesystemalso dependsonimmobilizedenzymeprop-erties (Céspedes  et al.  1993).In this work, if a highpHis used, not only the enzyme peroxidase is denaturedbutit will be difficultto maintainthe immunocomplex.On the otherhand, the choice of peroxidaseas enzymetag (optimum pH between 5.0 and 6.0), instead of al-kaline phosphatase (optimum pH  >  8.0), allows theuse of the pH 6.0, which is close to optimum pH forthe system.It has been reported that epoxy in carbon fiber in-creases its electrical resistivity during the curing of the epoxy due to residual compressive stress that in-creases with the increasing curing temperature (Wang& Chung 1997). For this modified graphite-epoxyelectrode,thebestamperometricresultwasobtainedat28  ◦ C. This temperature too helps in the constructionof the immunoelectrode, because the protein A can beadsorbedintothe compositeduringthe curingprocess,as it is not denatured at this temperature.The thermal behavior in relation to the electricalresistivity of the graphite-epoxy composite is not wellunderstood. All solid states electrodes are complexdevices that include materials with different electricaland thermal expansion coefficients, as well as severalinner electric contacts (Magalhães  et al.  1997). A rea-sonable explanation relates to the degree to which theconductingparticles inside the polymericmatrix makecontact with each other (Alegret 1996). In order todetermine the optimal working temperatures, experi-ments were conducted at a flow rate of 330  µ l min − 1 ,at different temperatures. The results showed that theslope of the residual current decreases with temper-ature (Figure 4). This non-Nerstian behavior, welldescribed by other authors (Magalhães  et al.  1997),may be explained due to the complex variation of theconducting properties of the composites employed. Calibration curve A calibration curve for IgG is plotted in Figure 5for the immunosensor. Zero values of human IgGwere obtainedincubatingthe electrodewith phosphate Fig. 4.  Variation of the slope of residual current and temperature upto 28  ◦ C. Fig. 5.  Representative of human IgG calibration curve for epoxymodified immunosensor using sandwich immunoassay as model.Slope  − 0.05 mg ml − 1 µ  /A; y intercept 0.65,  r  = − 0 . 99 ( n  =  5; p <  0 . 05). salinebufferinsteadofhumanIgG.Themeasurementswere performed in replicate with the same electrodeand with differentelectrodes. The range is up to 80  µ gml − 1 and the limit of detection is in the order of a 100 ng ml − 1 , a greater sensitivity than previouslyachieved in epoxy-graphite biocomposites (Santan-dreu  et al.  1998). Complementarymeasurements weremade using a series of human serum concentrationsshowing minimal non-specific binding. The sensitiv-ity of this immunosensor is with most conventional  583ELISA methods, capable of detection down to a fewmicrograms of immunoglobulin in human serum. Thecombination of inert and conductive materials in theconstruction of electrochemical immunosensors rep-resents an advance in amperometric developments.The epoxy-modified electrode described in this paperrepresents an immunosensor with excellent featuresof simplicity and reusability. This work also pointsthe way to the development of new immunosensorsusing known thick-film technology based on screen-printable biocomposite techniques for electrode con-struction. These electrodes are more reproduciblethanthe others described in this area, similar approacheshave been described in graphite-epoxy biocompos-ite electrodes for glucose determination (Galan-Vidal et al.  1997). Acknowledgements The authors acknowledge the financial support of the LIKA, CAPES, BNB and CNPq and FACEPE.We also gratefully acknowledge Prof Dr ElizabethMalagueño for important suggestions during thiswork. References Alegret S (1996) Rigid carbon-polymer biocomposites for electro-chemical sensing – a review.  Analyst   121 : 1751–1756.Céspedes F, Martínez-Fàbregas E, Bartrolí J, Alegret S (1993) Am-perometric glucose biosensor based on an electrocatalyticallybulk-modified epoxy-graphite biocomposite.  Anal. Chim. Acta 273 : 409–417.Céspedes F, Martínez-Fàbregas E, Bartrolí J, Alegret S (1994)Amperometric glucose biosensor based on a tetrathiafulvalene-mediated epoxy-graphite biocomposite.  J Electroanal.  6 : 759–763.Fontana E, Pantell RH, Strober S (1990) Surface plasmon im-munoassay.  Appl. 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