electrochemical enzyme immunoassay using immobilized antibody on gold film with monitoring of...

Analytica Chimica Acta 463 (2002) 219–227

Electrochemical enzyme immunoassay using immobilizedantibody on gold film with monitoring of surface

plasmon resonance signal

Kei Toda∗, Masato Tsuboi, Noriko Sekiya, Misuzu Ikeda, Ken-Ichi YoshiokaDepartment of Environmental Science, Faculty of Science, Kumamoto University, 2-39-1,

Kurokami, Kumamoto 860-8555, Japan

Received 8 January 2002; received in revised form 2 May 2002; accepted 14 May 2002

Abstract

Sandwich immunoassay was conducted on a thin gold film set in a surface plasmon resonance (SPR) cell. Monochronalantibody (anti-IgG) was immobilized onto the gold film via 4,4′-dithiodibutyric acid (DDA) and avidin–biotin bonding. Next,IgG sample and alkaline phosphatase-conjugated anti-IgG (ALP anti-IgG) were introduced into the cell successively. Finally,p-aminophenyl phosphate (PAPP) was injected as an enzyme substrate, and the producedp-aminophenol (PAP) was elec-trochemically measured. Flow did not need to be stopped for incubation for the enzyme reaction, because of the thinnessof the cell. In these processes, all the antigen–antibody reactions took place on the gold film. Therefore, the immobilizationwas performed quickly, and each process could be confirmed by SPR signal. This system had the advantage that the middleof the complicated process could be monitored. For example, the amount of antibody immobilized, which affected on thefinal electrochemical signal, could be confirmed in the course of immobilization. It was also convenient to investigate processconditions, such as removal of used antigens and labeled antibodies. Good correlation was obtained between the electrochem-ical current and the SPR signals due to the adsorption of IgG and ALP anti-IgG, and the sensitivity of the electrochemicalmeasurement was much higher than the SPR’s. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Electrochemical enzyme immunoassay; Surface plasmon resonance (SPR); IgG; Gold film

1. Introduction

Measurement methods with antigen–antibody bind-ings are widely used for determining virus, bacteriaand environmental pollutants such as DDT and poly-chlorinated biphenyls (PCBs). Most enzyme-linkedimmunosorbent assay (ELISA) kits available on themarket are based on absorbance or fluorescence mea-surement and need a special instrument designedfor this purpose. On the other hand, electrochemical

∗ Corresponding author. Tel.:+81-96-342-3389;fax: +81-96-342-3389.E-mail address: [email protected] (K. Toda).

detection does not need a specific optical system, andmeasurement can be carried out with only a smalldetection cell and a simple current meter. Such elec-trochemical enzyme immunoassay (EEIA) have beenapplied to measurement of digoxin[1], progesteronein cow’s milk [2], mouse IgG with an interdigitatedarray electrode (IDAE)[3–6], ferritin [7] and bacteriasuch asE. Coli andSalmonella [8].

Recently, attempts have been made to measurephysiological species and environmental pollutantsusing surface plasmon resonance (SPR)[9,10]. In theSPR method, a specific antibody is immobilized ontoa gold film. An antigen is then introduced into the celland bound onto the antibody. These processes alter

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0003-2670(02)00419-1

220 K. Toda et al. / Analytica Chimica Acta 463 (2002) 219–227

the refractive index near the gold film. This changesthe incidence angle where the horizontal componentof the light alters to the evanescent light. By mon-itoring the resonance angle, the antigen–antibodyreaction can be observed continuously.

Suzuki et al. combined electrochemical sensorsand SPR together[11]. They measured IgG with SPRand determined glucose and lactate electrochemically.Two kinds of detectors played roles for different an-alytes. In our work, immunoassay was conducted ona gold film and was analyzed with both of SPR andelectrochemical approach. Immobilization of anti-body, antigen–antibody reactions and refreshment ofthe immobilized antibody were monitored using SPR,and the final products generated on the gold film inan SPR cell were introduced into an electrochemi-cal cell connected just after the SPR cell, and thusdetermined. With the SPR instrument used, sampleand reagent injections are conducted easily throughcomputer operation. In usual enzyme immunoassay(EIA), antibody is immobilized onto a glass plate,small beads or polystyrene material. In this work,antigen–antibody immobilized onto a gold film wasapplied to the electrochemical analysis. As the goldfilm was used as a support for immobilization, wecould analyze from both of SPR and electrochemi-cal analysis. Furthermore, the immobilization ontothe gold film did not require long incubation, whileit takes several hours in conventional methods[12].And since the immobilization was conducted in aflow system, very small amount of antibody could beused repeatedly with the removal of the used samples.Here, the combined method has been evaluated usingan IgG determination system.

2. Experimental

2.1. Reagents

Antigen and antibodies were obtained from Jack-son Immuno Research Laboratory. They comprisedaffinipure sheep anti-mouse IgG, F(ab′)2 (2.4 mg/ml), biotin–SP-conjugated affinipure sheep anti-mouseIgG, F(ab′)2 (2.0 mg/ml), chrompure mouse IgG(whole molecule, 11.8 mg/ml), alkaline phosphatase-conjugated affinipure rat anti-mouse IgG (H+ L)(0.8 mg/ml) and streptavidin (unconjugated, 1 mg).

Bovine serum albumin (BSA) was obtained fromBoehringer Mannheim. The running buffer comprisedphosphate buffer salts pH 7.3 prepared from PBStablets purchased from Roman Industry Co. A 4,4′-dithiodibutyric acid (DDA) was obtained fromAldrich, and 1-ethyl-3-(3-dimethylaminopropyl)car-bodiimide hydrochloride (EDC),N-hydroxysuccini-mide (NHS) and 1,4-dioxan were purchased fromWako. As a standard of the enzyme reaction product,p-aminophenol (PAP) was obtained from KatayamaChemical. Substratep-aminophenyl phosphate (PAPP)was obtained from Biomedical Inc. or preparedfrom p-nitrophenyl phosphate (PNPP) obtained fromNacalai Tesque by catalytic hydrogen reduction[13]or reduction with Na2S [14]. In the former method,hydrogen gas was bubbled for 1 h through the PNPPsolution to which palladium carbon catalyst wasadded. This procedure, involving an explosive gas,had to be conducted with care. Furthermore, the finalconcentration could not be obtained accurately be-cause the product was not crystallized in the formermethod. On the contrary, the latter needed a longertime but was capable of relatively large-scale syn-thesis, and the obtained product could be used withweighing out in each examination. Accordingly, thelatter method was finally adopted.

2.2. Apparatus

A series of processes was conducted using an SPRinstrument SPR670-Y (Nippon Laser & ElectronicsLab.) with some modifications as shown inFig. 1. Therunning buffer was caused to flow through the SPRcell by a double plunger pump. Reagents and sampleswere introduced into the cell by a six-port sample in-jector via a 60-�l sample loop. The flow channel ofthe SPR cell was designed for the reaction field ofthe EEIA to greatly enlarge the immobilization area.Just downstream of the SPR cell, an electrochemicalflow cell (BAS) was connected with a 15-cm PEEKtube. A working electrode made of glassy carbon wasplaced in a 25-�m-thick flow channel. Downstream ofthe working electrode, an Ag/AgCl reference electrodeand a stainless steel pipe were put into the cell-block.The stainless steel pipe acted as both a drain tube anda counter electrode. These electrodes were connectedto an electrochemical analyzer, model 701A (ALS).

K. Toda et al. / Analytica Chimica Acta 463 (2002) 219–227 221

Fig. 1. Schematic diagram of the flow system. RB: running bufferbottle; DG: degasser; P: plunger pump; SI: six-port sample injec-tor; SL: sample loop (60�l); SP: syringe pump; SPR: SPR cell;WE: working electrode made of glassy carbon or gold; RE: ref-erence electrode Ag/AgCl; CE: counter electrode (stainless steeltube); EA: electrochemical analyzer; WB: waste bottle; AB: an-tibody solution; B: blocking buffer; S: antigen samples; ALP:ALP-conjugated antibody solution; PAPP: PAPP solution; RA: re-moving agent.

2.3. Procedure

A glass chip with a 50-nm gold film deposited onwas first washed in acetone, next dipped into 10-mMDDA ethanol solution for 30 min, then rinsed inethanol twice and finally dried. The DDA was acti-vated for 1 h in a mixture comprising 1 ml of aqueousEDC solution (25 mg/ml) and 9 ml of 1,4-dioxan con-taining 15 mg NHS. After that, 10 ml of water wasadded and the chip was left for 5 min, then washedwith water, dried and mounted on a prism cartridge.

The electrochemical cell was disconnected fromthe SPR cell beforehand. The running buffer wascaused to flow through the SPR cell at the rate of15�l/min (1.3 mm/s). After baseline stabilization,100-mg/ml streptavidin was injected twice, givinga total volume of 120 ml. The biotinylated anti-IgGwas immobilized by avidin–biotin bonding, and the

remaining DDA was then blocked with 1% BSA.Next, a series of processes was started at a flow rateof 5�l/min (0.42 mm/s). An IgG sample and ALPanti-IgG solutions were injected successively. Thenthe electrochemical cell was connected downstreamof the SPR cell, and 1 mM PAPP was injected at a rateof 100�l/min while the oxidation current was moni-tored. The electrochemical measurement was repeatedby re-injections of the PAPP. After obtaining an ox-idation current peak due to the PAP produced, theelectrochemical cell was disconnected, and the IgGand ALP anti-IgG were removed by 0.2 M glycineadjusted to pH 2.2 with HCl. If further sample mea-surement was required, the series was started againfrom the injection of the IgG sample.

3. Results and discussion

3.1. Electrochemical investigation

In optical EIA with ALP, PNPP is widely usedas the substrate. On the other hand, PAPP is mostlyutilized for electrochemical detection. New substratesproposed by Kreuzer et al.[15] were attractive, butPAPP was relatively easy to obtain, and this con-ventional substrate was used in this experiment. ThePAPP was commercially available, but expensiveand unsuitable for long storage at room tempera-ture because of its instability. Therefore, PAPP wassynthesized from PNPP.

Cyclic voltammograms (CVs) of PAPP and its en-zyme reaction product PAP are shown inFig. 2. Thesolutions of PAPP and PAP were prepared as phos-phate buffer salts, as for the enzyme reaction. WhilePAPP was oxidized at >0.4 V versus Ag/AgCl, theredox potential of PAP was 0.12 V versus Ag/AgCl.For PAPP, the shape of the CV curve would changewith repeated scanning. This was because the elec-trode surface was covered with the PAPP products,and we had to attend to this matter. The electrode ac-tivity was often checked with Fe(CN)6

4− solution. Ifthe electrode was damaged, its surface was polishedand cleaned. Results obtained in chronoamperometry(CA) using glassy carbon and gold electrodes areshown inFig. 2 (b) and (c), respectively. The resultsobtained with the two electrodes were similar, andPAP/PAPP current ratio with gold electrode was larger


Fig. 2. Electrochemical investigation of PAP detection: (a) CVs of PAP and PAPP obtained with a glassy carbon electrode, both concentrationswere 1 mM in PBS; (b) and (c) show CA results using a glassy carbon and a gold electrodes, respectively. A 20-�l of 0.1 mM PAP (�) orPAPP (�) solution was injected into PBS buffer carrier flowing at 100�l/min. The symbol (�) indicates the ratio of PAP current to PAPP’s.


than the ratio with glassy carbon electrode. But theglassy carbon was better than the gold from the view-point of electrode contamination[15]. Also in otherreports, glassy carbon electrode has been used withantibody directly immobilized onto the electrode forthe same reason[2,4,14,16]. Thus, the glassy carbonwas adopted in this experiment. FromFig. 2 (b), PAPcould be determined by selecting a suitable potentialof 0.2 V versus Ag/AgCl. In this condition, a goodcalibration curve of PAP from 50 nM to 100�M wasobtained with a correlation coefficient 0.9985 and adetection limit of 50 nM (S/N= 3).

3.2. Flow rate

Figure 3 shows the relationship between the car-rier flow rate and the response obtained when 20�lof 100-�M Fe(CN)64− or PAP was injected. Theordinate is current change at the peak obtained foreach injection. As PAP easily decomposed at roomtemperature[17], the PAP sample had to be kept inice water during the experiment. For PAP at 0.4 V

Fig. 3. Effect of flow rate on electrochemical detection: (�)100�M Fe(CN)64− at 0.6 V; (�) 100�M PAP at 0.2 V; (�)100�M PAP at 0.4 V vs. Ag/AgCl. The PAP solution was preparedwith a degassed buffer and stored in cold water with ice duringthe measurement.

versus Ag/AgCl, the current increased with flow ratesame as for the Fe(CN)6

4− detection. The smallerthe flow rate, the smaller the supply of the speciesto the electrode surface. However, when the potentialwas lower than the former for PAP, i.e. 0.2 V versusAg/AgCl, which was the optimum potential for theselective detection of PAP, the response was indepen-dent of the flow rate >100�l/min. In order to obtaina larger response to PAP and to avoid reducing thecell-passing time too much which was correspondingto the enzyme reaction time, 100�l/min was adoptedfor the electrochemical measurement, while 5�l/minor 15�l/min was chosen to obtain a longer reactiontime for antigen–antibody reactions.

3.3. Monitoring of immobilization andimmunoreactions

The immobilization methods were described inSection 2. The DDA monolayer film was formed onthe thin gold film and the aldehyde group at the endof the DDA was activated with NHS and EDC. Next,anti-IgG was immobilized by amino coupling directlyor through avidin–biotin bonding.

In this work, as SPR cell was used as a reactionfield, immobilization and its progress could be ob-served from the SPR signal. An example is shown inFig. 4(a). In the SPR instrument, there were two cellsin parallel; they were called as sensor (S) and refer-ence (R). The samples and reagents are flowed throughboth of the cell, except anti-IgG which was flowedthrough only S cell. The response shown inFig. 5(a)was S signal, and the height difference between be-fore and after the each injection was correspondingto the amount of the immobilization. Streptavidin wasinjected twice and the each amount of the bindingwas almost same. Then, biotinylated anti-IgG was in-troduced twice. It can be seen that the immobilizedstreptavidin was almost saturated with the anti-IgG inthe first injection. After the BSA injection, the signaljumped up and then became steady. After the immobi-lization of the antibody, the difference between S andR signals represented the adsoption and desorption ofIgG and ALP anti-IgG. This difference cancelled thesolvent effect on the SPR signal, and the transitionalprogress could be obtained asFig. 4 (b). The reac-tion of IgG with anti-IgG was relatively fast, and thesignal increased suddenly at the injection point and


Fig. 4. SPR response curves show (a) antibody immobilization pro-cess obtained by S signal. Streptavidin (SA, 100�g/ml), biotiny-lated anti-IgG (BA, 40�g/ml) were injected twice at 15�l/minand then it was treated with 1% BSA at 5�l/min; (b) a differentialsignal (S–R) of antigen–antibody reaction. IgG (10�g/ml), ALPlabeled antigen (10�g/ml) were injected into a PBS buffer streamat the flow rate of 5�l/min. Remover (0.2 M glycine adjusted pH2.2 with HCl) was introduced at 15�l/min. Characters I, A andR indicate injection points of IgG, labeled antigen and remover.Time scale of (b) is same as (a).

moved toward saturation. However, adsorption of theALP anti-IgG was relatively slow and the SPR signalincreased almost linearly with the reaction time. Afterexposure to IgG or ALP anti-IgG for 12 min each, thesignal became constant by washing with PBS buffer.When the removing reagent was injected, the signalreturned to the original baseline. Series of these in-jections were conducted twice and good repeatabilitywas obtained.

3.4. Calibration curve

Calibration curves based on the PAP current areshown inFig. 5. The symbols (�) and (�) indicatepeak current in the case of direct and avidin–biotin im-mobilizations, respectively. The symbols (�) and (�)

Fig. 5. Calibration curves of IgG using immobilized antigen ontoDDA/Au (�) and through avidin–biotin bonding (�). The symbols(�) and (�) indicate the each blank level.

indicate the blank levels. Even though there was noflow-stop for the incubation of enzyme reaction, goodresponses were obtained up to 3�g/ml in the case ofthe avidin–biotin bonding. The linearity over 3�g/mlwith the anti-IgG bonded by avidin–biotin could beimproved by increasing the labelled antigen concentra-tion, but the blank level became larger. For example, 10times higher concentration of ALP anti-IgG made boththe measurable range and blank current three timeslarger. Experiments with incubation for 2, 5 and 10 minwere also examined, but no conspicuous response im-provement was obtained. Moreover, the series of pro-cedures became complicated, and reproducibility andlinearity deteriorated with the incubation. The reactioncell volume was small (about 6�l). Thus, the solutionkept in the cell might be pushed out by the remainingpressure, if the solution was not completely stopped.Therefore, better results could be obtained by the sim-pler procedure without the liquid stop. The SPR cellwas 0.1 mm in thickness, so the surface-to-volume ra-tio was large and the enzyme reaction could be carriedout without the liquid stop for the incubation.

Originally, anti-IgG was immobilized onto DDAdirectly with amino coupling. In this case, as can


be seen inFig. 5, the current change was small andthe blank current was large. It was thought that themain reasons for the large blank were non-specificadsorption (NSA) of the ALP anti-IgG and defect ofthe blocking. The ALP anti-IgG adsorbed not onlyonto the IgG but also onto the remaining DDA, goldfilm directly and the tubing material. If the contribu-tion onto the remaining DDA was large, it would besolved by improving the blocking of the remainingDDA. In order to improve the blocking, the DDAwas treated with BSA, and then a small moleculeagent ethanolamine–HCl was injected to block thefree DDA presented in the aperture between BSAs.This two-step blocking was the best, but the improve-ment was not remarkable. Also tested blocking agentswere γ -globulin, gelatin, ethanolamine, ethylene di-amine, glycylglycine, glycine, Tween-20[18], butthey did not show a better result. Accordingly, it wasthought that the contribution of adsorbing onto thegold and tubing material was not negligible. Whenthe anti-IgG was immobilized onto a glass substrateand not onto the gold film, the background currentwas smaller. Gold film must be used in the SPR ex-amination. However, the gold interacts with manycompounds, so it is not always a good material forantigen–antibody examinations if optimal utilizationis not adopted. When the adsorption to the tubing wasnot negligible, it was solved by the washing of thetubing from the injector to the just before the SPR cellwith the removing agent before the injection of PAPPif necessary.

After the investigation of the blocking, the immo-bilization was changed to avidin–biotin method andwas compared to the original. Biotinylated IgG wasimmobilized through avidin–biotin bond after strepta-vidin was attached to DDA. In this case, the currentchange became 2.5 times greater and the blank currentbecame one-third that of the direct immobilization, ascan be seen inFig. 5. The advantage of the immobi-lization with the avidin–biotin bond was reconfirmedby this experiment. Schematics of the two kinds ofbindings are shown inFig. 6. The antibody immobi-lized directly to the DDA is close to the gold film,and the interaction of the antigen and the labelled an-tibody to the gold film is not negligible. Furthermore,the antibody arrangement became random and did notalways act well as the antigen receptor. On the otherhand, the antibody immobilized via the avidin–biotin

Fig. 6. Schematic of the binding reactions: (a) direct immobiliza-tion of anti-IgG; (b) with avidin–biotin bonding. S: glass substrate;G: gold film with chromium as an adhesive layer; D: DDA; A:anti-IgG; I: IgG; AV: streptavidin; B: biotin; and LS: long spacer.

coupling was placed far from the gold film and anti-body arrangement was good[19]. For these reasons,the antibody, through avidin–biotin bonding, had ad-vantages of high sensitivity and low background levelin the collecting antigen.

The anti-IgG could be used repeatedly by removingthe IgG and ALP-labeled anti-IgG adsorbed onto theimmobilized antibodies. Some removing agents weretested, for example: 0.2 M glycine–HCl, pH 2.2; 0.2 Mglycine–HCl+ 10% 1,4-dioxane, pH 2.2; 4 M MgCl2,pH 3.2; and 8 M urea, pH 7.7. The normal agent 0.2 Mglycine–HCl was enough for the purpose. Removalcharacteristics were confirmed by the SPR signal asin Fig. 4 (b). The temperature of the SPR cell wascontrolled with a Peltier device, and the immobilizedanti-IgG with avidin–biotin bond could be used for>48 h at 25◦C.

3.5. SPR signal and electrochemical measurement

Fig. 7 shows the correlation between the PAP de-tection current and the SPR responses to the antigenand ALP-conjugated antibody. These show good cor-relation between the SPR and current signals. TheSPR is convenient for observing the reaction process,but it lacks reliability in the low concentration range.In this work, the PAP signal could be obtained evenat low antigen concentration that could not be deter-mined with SPR. For the electrochemical immunoas-say, however, the results would be obtained at the end


Fig. 7. SPR signal and current change. Symbols (�) and (�) are SPR signal changes corresponding to IgG and ALP-conjugated antigen,respectively, and (�) is the current response in the electrochemical measurement.

of a complicated process, and it cannot be determinedwhether the middle of the process is going well. There-fore, it is useful to combine SPR with EEIA. Reagentsand samples of immunoassays are very expensive, andit is desirable to do the experiments while observingthe whole process from the start of antibody immo-bilization as shown inFig. 4. The amount of antigenimmobilized first affects the sensitivity.

The EEIA is usually conducted with antigen or anti-body immobilization onto a glass or polystyrene sub-strate. Here, it can be conducted on a gold film. Theimmobilization onto the glass needs a long incubationtime, e.g. 1 day, because the surface should be treatedwith alkaline, silane coupler and glutalaldehyde, suc-cessively[20]. On the polystyrene, passive adsorptionwas adopted[21]. On the contrary, DDA binding re-action onto gold is fast, and it needs only tens of min-utes. Binding of anti-IgG to DDA or avidin also couldbe conducted within several minutes in the flow cell.It enables rapid determination of the EEIA. Immobi-lization onto a gold film has been used in some ap-plications using a microporous membrane[22], flow

injection analysis[23] and capacitance measurement[24], and here it was demonstrated for the EEIA.

4. Conclusion

EEIA has been carried out with monitoringantigen–antibody reactions in an SPR cell. As a resultof the investigation with the EEIA/SPR instrument,it was found that immobilization with avidin–biotinbond was more effective in preventing NSA than theimprovement of blocking material and buffer solu-tion. EEIA with SPR monitoring is convenient anduseful. Recently, immunoassay with a micro-devicefabricated on a glass chip with several tens to a fewhundreds micrometer flow channel has been widelyinvestigated[25–27]. The results obtained using thecombination of EEIA and SPR can be applied to amicro-device system. We are also examining a needletype device, which is capable to sample with directinsertion into living body, based on the investigationusing EEIA/SPR.


Acknowledgements

This work was funded by the Japanese Ministryof Education, Science and Culture (Grant-in-aid forUniversity and Society Collaboration (2) 11793003.

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electrochemical enzyme immunoassay using immobilized antibody on gold film with monitoring of...

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