Analytica Chimica Acta 558 (2006) 110–114

Scanning electrochemical microscopy with enzyme immunoassayof the cancer-related antigen CA15-3

Xiaoli Zhang, Xuewei Peng, Wenrui Jin∗School of Chemistry and Chemical Engineering, State Key Laboratory of Crystal Materials,

Shandong University, Jinan 250100, China

Received 10 September 2005; received in revised form 5 November 2005; accepted 8 November 2005Available online 19 December 2005

Abstract

Scanning electrochemical microscopy (SECM) with enzyme immunoassay was applied to detect the cancer related antigen CA15-3 (Ag). Inthis method, CA15-3 was concentrated and immobilized on a plane substrate via a sandwich method employing two corresponding antibody (Abcaptured on a streptavidin-coated substrate and Ab* labeled with horseradish peroxidase (HRP)). In the presence of hydroquinone (H2Q) andH2O2, HRP on the complex of CA15-3 with Ab and Ab* , Ab–Ag–Ab* , converted H2Q to the electroactive product benzoquinone (BQ) throught A15-3 wasm –Ag–Abw©

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he HRP-catalyzed reaction. The reduction current of BQ generated by the HRP-catalyzed reaction corresponding to the amount of Conitored and imaged by SECM. With a view of improving the sensitivity of SECM-enzyme immunoassay to meet our objective, Ab*

as concentrated on the substrate via a microcell. The detection limit of this method was 2.5 U/mL for CA15-3.2005 Elsevier B.V. All rights reserved.

eywords: Enzyme immunoassay; Scanning electrochemical microscopy; Cancer related antigen; CA15-3

. Introduction

Breast cancer is the most common life-threatening malignantesion in women of many countries. CA15-3 is a circulating anti-en, which is relatively specific for human breast tissue, and isefined by two monoclonal antibodies: DF3 and 115D8[1]. It isne of tumor markers which can help clinician identify and diag-ose if breast cancer patients will have aggressive disease andave an indolent course. Compared with other markers, CA15-is most useful for monitoring patients post-operatively for

ecurrence, and more sensitive than carcinoembryonic antigenn the evaluation of patients with both primary and metastaticreast cancer. For healthy human, the concentration levels ofA15-3 are lower than 30 U/mL. CA15-3 levels are often mea-ured by radioimmunometric assay[2,3], enzyme immunoassay4], microparticle enzyme immunoassay[4,5], chemilumines-ence immunoassay[4] and immunofluorometric assay[6,7].hese conventional immunoassays have some shortcomingsuch as being time consuming, having high reagent consumption

and complicated operation. Recently, capillary electrophoenzyme immunoassay with electrochemical detection hasalso used to detect CA15-3 in our laboratory[8].

Scanning electrochemical microscopy (SECM)[9,10] is akind of scanning probe microscopy, equipping a microetrode to afford information of local electrochemical and belectrochemical properties of functional surfaces[11–17]. Mat-sue’s group combined SECM with immunoassay for detecmicrosport of trace antigen molecules[18], leukocidin[19] andmultiple analytes[20]. Heineman and co-workers employSECM to image the immobilized antibody layers[21] and themicrospot formed using Ab-coated magnet heads[22].

In the present work, we attempt to assay CA15-3 by mof the SECM-enzyme immunoassay. CA15-3 is immobilon a substrate via a sandwich method, in which first antib(Ab) on the modified solid substrate selectively capturescorresponding antigen CA15-3 (Ag), further binding an enzylabeled second antibody (Ab* ) results in the configurationAb–Ag–Ab* on the substrate. The labeled enzyme, horserperoxidase (HRP), can convert enzyme substrate hydroqu(H2Q) into benzoquinone (BQ) at a relatively high reaction r

∗ Corresponding author. Fax: +86 531 88565167.E-mail address: jwr@sdu.edu.cn (W. Jin).

which can be detected by SECM. With a view of improvingthe sensitivity of SECM-enzyme immunoassay, Ab–Ag–Ab* is

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.11.032

X. Zhang et al. / Analytica Chimica Acta 558 (2006) 110–114 111

formed in a microcell and concentrated on the substrate. Com-pared to the SECM-immunoassay directly on a plane substratewith a detection limit of 15 U/mL, the sensitivity of the methoddescribed here is enhanced to 2.5 U/mL, which is suitable fordetecting CA15-3 samples.

2. Experimental

2.1. Reagents and solutions

The CA15-3 EIA Kit (No. 200-10) was purchased fromCanAg Diagnostics AB (Gothenburg, Sweden), which con-sisted of a series of CA15-3 standard solutions with differ-ent concentrations from 0 to 250 U/mL, two CA15-3 con-trols, tracer (anti-CA15-3 monoclonal antibody from mouselabeled with horseradish peroxidase (HRP)), biotinylated anti-CA15-3 monoclonal antibody from mouse, streptavidin-coatedmicrotiter plates (12× 8 wells) and concentrated wash solution.The standard solution of 185 U/mL CA15-3 was prepared bymixing 37�L of 250 U/mL standard solution with 13�L of0 U/mL standard solution. The kit was stored at 4◦C. H2Q, BQand H2O2 (content≥ 30%) were from Shanghai Chemical Co.(China) and used without further purification. The phosphatebuffer solution (PBS) (pH 7.0) consisted of 0.10 mol/L KCl,0.030 mol/L Na2HPO4, and 0.020 mol/L NaH2PO4. Wash solu-t tioni re oa pplieA atera

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2.3. Immunoassay procedure

Biotinylated anti-CA15-3 (Ab) of 3.0�L was added to themicrocell under an optical inverted microscope, and incubatedfor 60 min at 25± 2◦C in a constant-humidity chamber to pre-vent evaporation. Then, the microcell was rinsed with washsolution to remove any nonspecifically adsorbed Ab. Unre-acted streptavidin groups were deactivated by treating with1.0× 10−9 mol/L of biotin for 30 min. Then, 1.5�L of the sam-ple containing CA15-3 (Ag) was dropped into the microcelland shaken for 60 min by an ultrasonicator. After washing themicrocell with wash solution, an excess (2.0�L) of HRP-labeledanti-CA15-3 (Ab* ) was allowed to react with the immobi-lized CA15-3 for 60 min. Between each step, the microcell waswashed thoroughly with wash solution, and all immunoreactionwas carried out in a constant-humidity chamber to minimizeevaporation. After the immunoreaction was completed, the com-plex with HRP-labeled sandwich structure (Ab–Ag–Ab* ) wasconstructed on the substrate of the microcell. The resulting sub-strate had a surface architecture shown inFig. 2. The waterprooftapes were, then, removed, i.e. the microcell was dismantled. Thesubstrate with Ab–Ag–Ab* was placed in the electrochemicalcell for SECM measurement.

2.4. SECM experiments

medw (CHi de( hea gCl( erec ith

F stratea

ion was obtained by diluting the concentrated wash solun water by 1:25. Unless stated otherwise, all reagents wenalytical grade and purchased from standard reagent sull aqueous solutions were prepared with doubly distilled wnd were stored at 4◦C until use.

.2. Microcell fabrication

The streptavidin-coated microtiter plates consisting oells from the CA15-3 EIA Kit were separated. The wall of was cut away, to obtain the well substrate coated with stridin. The substrate was covered by a piece of waterproof30�m thick) with a hole (1 mm diameter) punched previouhen, another piece of thick waterproof tape (1.5 mm thick)bigger hole (1.5 mm diameter) was attached onto the first

esulting in a microcell shown inFig. 1. Thus, the microcell wit.5 mm diameter at the top and 1 mm diameter at the bottom

abricated. Subsequently, the immunoreaction was performhis microcell.

Fig. 1. Schematic representation of fabricating the microcell.

fr.

-e

,

sn

Approach curves and SECM images were perforith a CHI900 scanning electrochemical microscope

nstruments, Austin, TX, USA). An Au ultramicroelectroUME) of 10�m radius was employed as the SECM tip. Tuxiliary and reference electrode were a Pt wire and an Ag/A1 mol/L KCl) electrode, respectively. All measurements warried out at 25± 2◦C. The SECM image was obtained w

ig. 2. Assembly of the sandwich structure on the streptavidin-coated subnd the schematic of SECM detection.

112 X. Zhang et al. / Analytica Chimica Acta 558 (2006) 110–114

a generation-collection mode. To obtain the reliable results,the SECM tip should be placed the same position above thesubstrate before scanning. During adjusting the tip position, thenegative feedback mode was used. The experiment procedurewas as follows: The substrate with Ab–Ag–Ab* was positionedon the bottom of a Petri dish as the SECM cell with thereference electrode and the auxiliary electrode, PBS containing1.0× 10−3 mol/L H2Q was added into the cell. The Au tip of10�m radius was positioned above the substrate and immersedin the solution. A potential of 0.8 V was applied to the tip tooxidize H2Q. Then, the tip was moved slowly and verticallydown to the bottom, and the curve of the tip current (i) versus thedisplacement was recorded. Wheni was 80% of the steady-statecurrent of the tip, the tip was stopped. In this case, the distancebetween the tip and the substrate surface could be estimatedto be ∼20�m using the fit of the normalized experimentalapproach curve to theory. Subsequently, H2O2 was added tothe solution with a final concentration of 1.0× 10−3 mol/L andallowed to react with H2Q for 6–8 min via the HRP-catalyzedreaction at Ab–Ag–Ab* . The tip potential was switched to−0.4 V to reduce the reaction product BQ. The tip was scannedabove the substrate and SECM image was recorded.

3. Results and discussion

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n, thee rod-u lowerr c-t goalw er tion.T scanc ehav-i fecto bsB d toH y-s ed ato andt wao foreiB

H cen-t ft thecwt1

Fig. 3. The process of finding the center of an Ab–Ag–Ab* spot.

3.2. Finding the center of CA15-3 complex spot

Before SECM scan curve was recorded, the center of immobi-lized Ab–Ag–Ab* spot on the substrate should be found accord-ing to the following procedure (seeFig. 3): When the tip held at−0.4 V was moved along thex-axis (solid line 1 inFig. 3) abovethe Ab–Ag–Ab* spot in PBS containing H2Q and H2O2, the tipcurrent,i, increased greatly and there was a peak on thei–x curve.Wheni decreased, implying that the tip was across the spot, thetip was moved back (dashed line 2 inFig. 3) and stopped at theposition of the maximum current. Then, the tip moved alongy-axis (dashed line 3 inFig. 3) and across the whole spot. Finally,the tip was moved back along the same trace (solid line 4 inFig. 3), andi–y curve was recorded. Thus, the one-dimensionalcurrent profile above the spot was obtained.

3.3. SECM imaging of CA15-3 spot with collection mode

Fig. 4shows a SECM image of Ab–Ag–Ab* spot fabricatedwith 125 U/mL CA15-3. The appearance of the circular spot withlarge reduction currents was attributed by the HRP-catalyzedreaction of the spot, which indicated the localized presence of

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.1. Electrochemical behavior of BQ and H2Q

In the method based on the enzyme-catalyzed reactionzyme substrate H2Q was chosen because its reaction pct BQ has excellent electrochemical properties such aseduction potential (−0.4 V versus Ag/AgCl), negligible elerode fouling, and reversible electrochemical behavior. Ouras to image the spot of Ab–Ag–Ab* through detecting th

eduction current of BQ generated by HRP-catalyzed reacherefore, before mapping the SECM images and recordingurves of the spots, we investigated the electrochemical bor of both H2Q and BQ in PBS and the concentration eff H2Q and H2O2 on the SECM measurements of Ab–Ag–A*

pots. The cyclic voltammogram of PBS containing H2Q andQ at an Au tip (not shown) indicated that BQ was reduce2Q at the potential more negative than∼0.0 V and the steadtate reduction current on the voltammogram was observhe potentials more negative than−0.3 V, and that H2Q wasxidized to BQ at the potential more positive than 0.6 V

he steady-state oxidization current on the voltammogrambserved at the potentials more positive than +0.7 V. There

n SECM experiments, the tip was held at−0.4 V for detectingQ and at +0.8 V for measuring H2Q.In the SECM measurements, the concentration of H2Q and

2O2 affected the image. It was found that when the conration of H2Q was larger than 1.0× 10−3 mol/L, the profile ohe images of Ab–Ag–Ab* spot was almost independent ononcentration. A similar tendency was also observed for H2O2,hen the concentration was higher than 1.0× 10−3 mol/L. In

he subsequence experiments, the 1.0× 10−3 mol/L H2Q and.0× 10−3 mol/L H2O2 were used for detecting CA15-3.

t

s,

ig. 4. SECM image of an immobilized Ab–Ag–Ab* spot of 1.0 mm diameteabricated using 125 U/mL CA15-3. PBS containing 1.0× 10−3 mol/L H2Q and.0× 10−3 mol/L H2O2; Au microelectrode tip of 10�m-radius; tip potentia0.4 V (vs. Ag/AgCl); scan rate: 50�m/s. Distance between the tip and

ubstrate surface: 20�m.

X. Zhang et al. / Analytica Chimica Acta 558 (2006) 110–114 113

Fig. 5. Comparison between SECM scan curves for: (1) Ab–Ag–Ab* spotsfabricated directly on the substrate and (2) using the microcell. Experimentalconditions as inFig. 4except for 50 U/mL CA15-3 and 3�m/s scan rate.

CA15-3. The peak current on the SECM image could sensi-tively afford the information on the mount of CA15-3. Becauseof diffusion of BQ toward the bulk solution, the image wassomewhat larger than the lateral width of Ab–Ag–Ab* spotdiameter (∼1.0 mm). We compared SECM measurements fortwo spots fabricated at different immunoreaction circumstances.The two Ab–Ag–Ab* spots were fabricated directly and usingthe microcell on the substrate, respectively. The correspondingSECM scan curves at the center across both spots are shown inFig. 5. The signal of the spot fabricated using the microcell wasapproximately six times larger than that fabricated directly onthe substrate for 50 U/mL CA15-3. This was because that whenthe microcell was used, a dense Ab–Ag–Ab* layer was formedon the substrate due to accumulation.

3.4. Linear range, limit of detection and determination ofcontrol

The dependence of the reduction current on the CA15-3 con-centration was investigated in detail.Fig. 6shows the reductioncurrent profiles for the spots fabricated at different concentra-tions of CA15-3. Curve 1 inFig. 6shows the control experiment,in which no Ag was added, implying no Ab–Ag–Ab* on the spot.Clearly, no signal was detected at this spot. In curves 2–6, thepeak current rose and was proportional to the concentration ofC ithC cor-r L.T 5.7%( tov tedao on-t /mLf byt

Fig. 6. SECM scan curves across the Ab–Ag–Ab* spots fabricated by differentCA15-3 concentrations. CA15-3 concentration (U/mL): (1) 0, (2) 15, (3) 50, (4)125, (5) 185, (6) 250. Spot size: 1.0 mm diameter. Other conditions as inFig. 5(2).(Insert) Relationship between peak current and CA15-3 concentration.

4. Conclusion

CA15-3 can be determined by the SECM-enzyme immunoas-say. This method can also be used to quantitatively determineantigens by recording SECM scan curve on the spot of the anti-gen complex with enzyme-labeled antibody in the presence ofenzyme substrates. When the complex is formed in a micro-cell, the wall of which is made by waterproof tapes and can beremoved, the sensitivity of detection was significantly enhanced.The method for fabricating a microcell on a plane substrate israther simple and can reduce reagent consumption.

Acknowledgements

This project was supported by the National Natural ScienceFoundation of China (No. 20235010, 20475033), the NaturalScience Foundation of Shandong Province (No. Y2003B04)and the State Key Laboratory of Electroanalytical Chemistry,Changchun Institute of Applied Chemistry, Chinese Academyof Sciences.

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