flow injection electrochemical enzyme immunoassay based on the use of an immunoelectrode strip...

Analytica Chimica Acta 488 (2003) 61–70

Flow injection electrochemical enzyme immunoassaybased on the use of an immunoelectrode strip

integrate immunosorbent layer and ascreen-printed carbon electrode

Qiang Gao, Ying Ma, Zhiliang Cheng, Weidong Wang, Xiurong Yang∗State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,

Remin Street 159, Changchun, Jilin 130022, China

Received 15 November 2002; received in revised form 5 May 2003; accepted 15 May 2003

Abstract

A flow injection amperometric immunoassay system based on the use of screen-printed carbon electrode for the detection ofmouse IgG was developed. An immunoelectrode strip, on which an immunosorbent layer and screen-printed carbon electrodewere integrated, and a proposed flow cell have been fabricated. The characterization of the flow immunoassay system andparameters affecting the performance of the immunoassay system were studied and optimized. Amperometric detection at0.0 V (versus Ag/AgCl) resulted in a linear detection range of 30–700 ng ml−1, with a detection limit of 3 ng ml−1. The signalvariation among electrode strips prepared from variant batch did not exceed 8.5% (n = 7) by measuring 0.5�g ml−1 antigenstandard solution.© 2003 Elsevier Science B.V. All rights reserved.

Keywords:Enzyme immunoassay; Screen-printed carbon electrode; Flow injection

1. Introduction

Enzyme immunoassay (EIA) has been widely ap-plied in bioanalytical chemistry owing to its high sen-sitivity and specificity, availability of many enzymemarkers, long-term stability of the labeled reagentsand operational safety[1,2]. With the extension ofthe applications of EIA, including electrochemicalenzyme immunoassay, there has been growing inter-ests in the development of flow injection EIA systemdue to a few important advantages of flow injection

∗ Corresponding author. Tel.:+86-431-568-9278;fax: +86-431-568-9711.E-mail address:[email protected] (X. Yang).

analysis (FIA)[3–5]. Enzyme immunoassay couplingwith a flow injection system and an amperometricdetection has become a powerful analytical tool forthe determination of low levels of analytes such asatrazine [6], bacteria [7], cortisol [8], cephalexin[9], gentamicin[10], human chorionic gonadotrophin[11] and progesterone[12] in different biologicalfluids.

As some special designs, Ghindilid et al. presenteda kind of flow-through immunosensor based on ahigh-surface-area carbon immunoelectrode. Dispersedcarbon material serves as a carrier for immobilizedantibodies and at same time as an electrode material[13]. The same group have developed a flow injectionamperometric immunofiltration assay system. The

0003-2670/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0003-2670(03)00578-6

62 Q. Gao et al. / Analytica Chimica Acta 488 (2003) 61–70

system was based on the use of disposable porousnylon membranes, which acted as a support for theimmobilization of antibody, and an amperometric de-tector [7]. Liu et al. [14] proposed a flow injectionsolid-phase chemiluminescent immunoassay using amembrane-based reactor. The membrane to whichantigen was attached was mounted in a flow cell andcould be replaced after each measurement. In theirsystem, a disposable immunosensor containing animmobilized antibody was used on-line with an FIAsystem. The system was capable of continuously car-rying out each of the steps involved in solid-phasesandwich immunoassays, including the immune re-action, the washing, the sandwich reaction, and theenzymatic reaction.

One of the advantages of screen-printing as ameans of sensor production is its ability to producesensor sufficiently cheaply and reproducibly to allowthem to be used for a few or single determinationin batch analysis[15]. There have been a lot ofreports about enzyme biosensor or immunosensor us-ing screen-printed electrode[16–19], some of theseworks involved a flow injection system. A study in-volving flow injection analysis of organophosphatepesticides incorporated a cobalt phthalocyanine mod-ified screen-printed carbon electrode into a thin-layercell, demonstrated that an on-line approach usingscreen-printed electrodes would be feasible[20].Collier et al. [21] presented a special design ofscreen-printed biosensor and corresponding wall-jetflow cell. The work reported by Killard et al.[22]has indicated the possibility of using a screen-printedcarbon electrode-based immunosensor in an amper-ometric flow cell. And then, the reports about planarworking electrodes employed in flow immunoassaysystems have been published, including gold filmelectrodes [23] and the screen-printed electrodes[12,24].

In the present work, an electrochemical flow im-munoassay system based on the screen-printed elec-trode-based immunoelectrode strip was presented. Aproposed immunoelectrode strip and flow cell with athree-electrode configuration were used for the im-munoreaction steps and the electrochemical detection.The optimization of determination condition usingmouse IgG as a model analyte was presented andanalytical characteristics of the immunoassay systemwere evaluated.

2. Experimental

2.1. Chemicals and reagents

Goat anti-mouse IgG (whole molecular, M8642),mouse IgG (MIgG, I5381), goat anti-mouse IgG-HRPconjugates (anti-MIgG-HRP, A4416) were purchasedfrom Sigma. NaI, bovine serum albumin (BSA),poly-oxyethylene-sorbitan monolaurate (Tween 20)and glutaraldehyde (25% solution) were obtained fromSigma. Carbon ink (Electrodag 423SS) was obtainedfrom Acheson Colloids (Japan). Hydrogen peroxide(30% (v/v) aqueous solution) was purchased fromBeijing Chemical Reagent Factory (Beijing, China).

Unless otherwise stated all chemicals and reagentsused are of analytical grade. Buffers were preparedusing water from a Milli-Q ultra-pure water systemand deaerated by bubbling nitrogen before use. Phos-phate buffer (0.1 M) was prepared by mixing the stocksolution of potassium dihydrophosphate and sodiumhydroxide and then adjusted to required pH with hy-drochloric acid.

2.2. Apparatus

The flow injection system comprised an ISIF-Cbrainpower flow injection analyzer (Ruimai Company,Xi’an, China) equipped with two three-channel peri-staltic pump and an eight-port rotary injection valvewith two 50�l loops and a 200�l loop, two three-waystopcocks and a home-made electrochemical flow cell.All the tubes and connectors were of PVC or PTFE.The manifold of the complete flow injection systemand the valve configuration were shown inFig. 1Aand B, respectively. Inflow to the flow cell could beswitched using the three-way stopcock between reser-voirs of either carrier or washing solution. Anotherthree-way stopcock was used to switch solution ofHRP-conjugated antibody or substrate solution into50�l loop in turn. Solution of antigen was injectedinto 200�l sample loop manually with micropipette.

A purposed three-electrode flow cell (20�l volume)was used for the immunoreaction steps and the elec-trochemical detection. First, the Ag/AgCl referenceelectrode was inserted into cell for reference elec-trode, which filled with saturated KCl solution. Andthen, the counter electrode and immunoelectrode stripwere inserted into the flow-through channel. After

Q. Gao et al. / Analytica Chimica Acta 488 (2003) 61–70 63

Fig. 1. Manifold of flow injection immunoassay system (A) and the valve configuration (B). C, carrier; W, washing solution; V, three-waystopcock; P, pump; EFC, electrochemical flow cell.

three electrodes were connected with a CHI 832 po-tentiostat (Shanghai, China), the FIA programmableautomate was used to control the incubation steps, thewashing step and the current measurement step. Thepeak height of reduction current was used as a signal.A thermostated bath was used to control the reactiontemperature at 25±1◦C, and the flow cell was soakedin the water beside the top.

2.3. Preparation of base electrodes and antibodymodified electrodes

The screen-printed carbon electrodes used asbase electrode were prepared by the method de-scribed in the literature[25]. The immunoelectrodestrips were prepared by immobilizing the antibodyonto the screen-printed carbon electrode strips inone-step cross-linking reaction. Seventeen micro-liters of the antibody solution containing antibody(5 mg ml−1), BSA (5 mg ml−1) and glutaraldehyde(0.1%) was coated onto the surface of the epoxysubstrate (1 mm× 17 mm) of the screen-printed car-

bon electrode strip, as shown inFig. 2A. The stripswere placed in air at room temperature for dry andthen washed with phosphate buffer (0.1 M, pH 7.4).Prepared immunoelectrode strips were stored at 4◦Cbefore use.

2.4. Amperometric enzyme-linking immunoassay

A sandwich scheme of immunoassay was em-ployed. First, the immunoelectrode strip was insertedinto the flow channel, and then a washing step whichaccomplished by flowing a washing buffer (0.1 M PB,pH 7.4) containing 1% BSA and 5 mM NaI for a dura-tion of 3 min with flow rate of 200�l min−1. At sametime, 200�l antigen solution in 0.1 M PB (pH 7.4)containing 0.15 M NaCl was load in 200�l loop. Af-ter that, antigen solution was injected into flow celland flow over the surface of the immunoelectrodestrip for a duration of 10 min. This represented thefirst stage of immuno-interaction and resulted in thecapture of the antigen by the immobilized antibodies.And then, second washing step was performed for a


Fig. 2. Schematic diagrams of the immunoelectrode strip (A) and electrochemical flow-through cell (B). (a) Insert point of workingelectrode and counter electrode; (b) insert point of reference electrode; (c) outlet; (d) inlet; (e) Ag/AgCl reference electrode; (f) glass frit,(g) counter electrode; (h) working electrode; (i) immobilized antibody layer; (j) cell for reference electrode.


duration of 3 min to remove any unbound antigen.At same time, 50�l horseradish peroxidase labeledconjugate solution (1:100 diluted with 0.1 M PB, pH7.4, containing 0.15 M NaCl, 1% BSA and 0.05%Tween 20) was loaded into 50�l of loop. And then,conjugate solution was injected into flow cell andflow over the immunoelectrode strip for a durationof 15 min. This represented the second stage of theimmuno-interaction and resulted in the formation ofthe sandwich complex. This was followed by a wash-ing procedure for a duration of 4 min. Carrier was0.1 M phosphate buffer (pH 7.4).

The last step was the electrochemical measurement.A polarization potential of 0.0 V was applied to obtaina stable baseline, and then 50�l of substrate solutioncontaining H2O2 and NaI in deaerated PB (0.1 M, pH6.0) was injected. Amperometric signal was monitoredwith the reduction of enzymatic iodine on the electrodeat the downstream.

3. Results and discussion

3.1. Construction of the immunoelectrode stripsand electrochemical flow-through cell

The immunosorbent reactor with separated elec-trochemical detector (downstream of immunoreactor)coupled to flow injection system was powerfullypromoted the development of FIA-EIA[26–28]. Ac-cording to the similar principle, a deviation from thetraditional “stacked layers” of the biosensor geometrywas adopted in this study. The construction of theimmunoelectrode strip was shown inFig. 2A. Theimmunoelectrode strip included immunosorbent layer(1 mm × 17 mm) with immobilized antibody andthe screen-printed carbon electrode which acted as adetector (1 mm× 3 mm). The immunosorbent layerwas located at the upstream of the electrode whenimmunoelectrode stripe was inserted in the flow cell.This design was similar to that of Gooding and Hall[29]. The immunoreaction and enzymatic reactiontook place on the immunosorbent layer and the iodinegenerated from the enzyme reaction was carried tothe electrode at downstream, and then was detectedelectrochemically.

The main principle of the design of the flow-throughcell has been described previously[25]. The schematic

diagram of the flow cell was shown inFig. 2B. Theflow cell comprised a channel unit and cell for areference electrode, both separated by a glass frit.The channel had dimensions of 1 mm× 1 mm afterthe counter electrode and working electrode were in-serted into the channel. The depth of the channel was20 mm. The improvement of flow cell compared toprevious one was an accession of cell for the refer-ence electrode, so a three-electrode configuration wasused instead of previous two-electrode configuration.The cell was operated using a three-electrode configu-ration comprising the screen-printed carbon electrodeas working electrode, a stainless steel counter elec-trode and an Ag/AgCl reference electrode. Because itis an open flow cell, so disassembly of flow cell wasnot necessary when the immunoelectrode strip needsto be replaced. After the measurement, the immuno-electrode strip may be replaced with a fresh one if de-sired, the replacement procedure is simple and rapid,usually taking only about 1 min.

3.2. Characterization of the flow injectionimmunoassay system

In order to evaluate the present system, it was nec-essary to prove reliability of the electrochemical flowimmunoassay system.Fig. 3 compared the ampero-metric response to the injection of substrate solutionwith the non-incubated immunoelectrode strip (a) andthe incubated imunoelectrode strip with 1�g ml−1 ofantigen solution (b). Small currents resulting from io-dide auto-oxidation were found when a non-incubatedimmunoelectrode strip was used, as shown inFig. 3,(a), which was decreased with the decrease of NaI con-centration. The much larger responses to the injectionof substrate solution, as shown inFig. 3, (b), camefrom the enzymatic iodine that proceeded on the in-cubated immunoelectrode strip. The experimental re-sults confirmed the feasibility of the immunoelectrodestrip to antigen analysis in the flow injection system.The background current due to the presence of iodinesin the substrate solution was corrected by subtractingthe background current from the current responses ob-tained in the followed immunoassay.

Good reproducibility (R.S.D. = 1.7%,N = 5) wasobtained in the successive injection of substrate so-lution with incubated immunoelectrode strip, whichmeant the sandwich complex formed on the sensing


Fig. 3. Current response of the immunoelectrode strip without incubated (a) and incubated (b). Concentration of NaI: 10 mM (1); 7 mM(2); 5 mM (3) and 3 mM (4). Concentration of H2O2: 0.6 mM. Potential: 0.0 V (vs. Ag/AgCl). Flow rate: 200�l min−1. Carrier: 0.1 Mphosphate buffer (pH 7.4). Electrolyte: 0.1 M phosphate buffer (pH 6.0).

surface of the electrode did not leak in the flowing so-lution after continuous operation and implied stabilityof the cross-linking antibody film. It demonstrated thatthe immunoelectrode strip could be used in on-linedetermination of antigen.

3.3. Flow injection enzyme-linking immunoassay

3.3.1. Optimization of the assay procedureThe optimization of the assay procedure was con-

ducted using a constant concentration of mouse IgG(1�g ml−1). The pH of substrate solution effect on thesensitivity of system lies in the activity of HRP lab.Therefore, an optimum pH between 5.0 and 8.0 wasexplored, and the pH effect on the sensor responsewas illustrated inFig. 4. The sensor shows a large re-sponse on pH between 5.0 and 8.0, and the current re-sponse reaches a maximum value at pH 6.0. To obtainmaximum sensitivity, we used the phosphate buffer(0.1 M, pH 6.0) containing H2O2 and NaI as substratesolution.

The dependence of the immunoelectrode strip re-sponse on the concentration of substrates was inves-tigated. The results indicated that the amperometric

signal started to increase gradually with the increaseof the hydrogen peroxide concentration from 0.1 to0.4 mM, and then reached a plateau over the range ofhydrogen peroxide concentration from 0.4 to 1.0 mM.Therefore, 0.6 mM of hydrogen peroxide was chosenin the substrate solution.Fig. 3showed the effect of io-dide solution concentration (varied from 3 to 10 mM)on the amperometric signal. It can be seen that theincrease of the iodide concentration from 3 to 10 mMresulted in an overall increase in the amperometricresponse. However, the background current increasedalso due to the presence of iodine in the preparationof substrate solution. Consideration of sensitivity andbackground correction, 10 mM of NaI concentrationwas chosen as optimal concentration. As a result ofthe study, 10 mM iodide and 0.6 mM hydrogen per-oxide were selected as the optimal working rangeof substrate concentrations for the electrochemicalstage.

The effect of substrate solution flow rate on theamperometric signal was investigated over the rangefrom 50 to 400�l min−1. The experiment results wereshown in Fig. 5. The current response increased atthe beginning and then decreased gradually with the


Fig. 4. Dependence of the reduction current on pH of substrate solution. Carrier: 0.1 M phosphate buffer (pH 7.4).

Fig. 5. Dependence of the reduction current on flow rate of substrate solution. Carrier: 0.1 M phosphate buffer (pH 7.4). Electrolyte: 0.1 Mphosphate buffer (pH 6.0).


increase of flow rate. So 200�l min−1 of flow rate waschosen to be optimal flow rate.

3.4. Non-specific binding of conjugate to antibodymodified electrodes

Non-specific binding of the conjugate molecules tothe solid surface of the immunosorbent was a crucialfactor affecting the development of the immuoassaysince it presented the major obstacle in decreasingthe lower detection limit. Blocking reagents such asTween 20, BSA, Casein or fat-free milk were usuallyadded to the rinsing and/or incubation buffer solutionto decrease non-specific binding[30]. In the presentstudy, the solution containing BSA was chosen to beblocking solution. Prior to the evaluation of the im-munoassay system performance, the immunoelectrodestrip was tested for non-specific binding. The elec-trode response towards BSA instead of target analytewas obtained and assigned as background signal. Thisbackground signal coincided in value with the amper-ometric signal obtained in a control experiment whereno immunoconjugate was introduced into the system.It indicates that the nature of the background was dueto iodide auto-oxidation. These experiments demon-

Fig. 6. Calibration curve for the immunoelectrode strips with antigen concentration expressed in logarithmic scale. Concentration of antigenstudied in a range of 0–3�g ml−1. Flow rate of substrate solution: 200�l min−1. Potential: 0.0 V (vs. Ag/AgCl).

strated that the effect of the non-specific adsorptionon the electrode response was negligible.

3.5. Measurements with the immunoelectrodestrips

Fig. 6 showed the calibration curve obtained usingantigen standards solution under optimal experimentalconditions. A linear relationship between the currentresponses and concentration of antigen was obtainedand with a dynamic range of 30–700 ng ml−1 IgG(I = 136 logc − 142; R2 = 0.992). The detec-tion limit using this method was 3 ng ml−1, whichis based on the minimum concentration of antigenthat gave a signal at least one time larger than thesignal from the control experiment. The error barsin Fig. 6 represented the standard deviation values,which was calculated from the amperometric responseusing three independent measurements with new im-munoelectrode strip for each antigen concentration.The immunoelectrode strips demonstrate good repro-ducibility of the amperometric response for the set ofimmunoelectrode strips from same or variant batch.The average deviation for six measurements (eachperformed with a separate immunoelectrode strip) of


Fig. 7. Regeneration of immunoelectrode strip by washing with 100 mM glycine/HCl at pH 2. Each regeneration was followed by a newincubation step and the electrochemical measurement procedure.

0.5�g ml−1 IgG concentration is equal to 6.3% forwithin batch and 8.5% for out batch.

The duration of the amperometric measurementstage is usually 3–4 min. So, in general, the measur-ing procedure is limited by the sum of the durationof sample flowing, conjugate flow and amperomet-ric measurement. Thus, the overall assay time is notlonger than 40 min.

In addition, the immunoelectrode strips were suc-cessfully regenerated by occasional elution with re-generation solution containing 100 mM glycine/HClat pH 2. Results by measuring 0.5�g ml−1 antigenstandard solution indicated that current response wasapproximately equal after using the immunoelectrodestrip six times, results were shown inFig. 7. Differ-ent pieces of immunoelectrode strips from the samebatch gave a series of calibration curve between 30 and700 ng ml−1 and standard deviations of curve slopesless than 9.2% (n = 7).

4. Conclusion

In this work, a flow injection enzyme-linking im-munoassay system with electrochemical detection was

developed using the immunoelectrode strips basedon the screen-printed carbon electrode. The proposedelectrochemical flow cell with the three-electrodeconfiguration showed the preferable characterizationand practicability. The feasibility of the idea aboutintegrating biorecognition layer and electrochemi-cal detector on the same electrode stripe but placingrespectively at upstream and downstream in a flowsystem has been demonstrated.

Acknowledgements

This work was supported by the National Key BasicResearch Development Project “Research on HumanMajor Disease Proteomics” (2001CB5102) and theNational Natural Science Foundation of China (No.20299030).

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flow injection electrochemical enzyme immunoassay based on the use of an immunoelectrode strip...

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