Analytica Chimica Acta 436 (2001) 103–108

Determination of human serum albumin by chemiluminescenceimmunoassay with luminol using a

platinum-immobilized flow-cell

Satoka Aoyagi a, Takeshi Iwata a, Takehiro Miyasaka b, Kiyotaka Sakai a,∗a Department of Chemical Engineering, Waseda University, 3-4-1 Okubo Shinuku-ku, Tokyo 169-8555, Japan

b Department of Physiology, Kawasaki Medical School, 577 Matsushima, Kurashiki City, Okayama 701-0192, Japan

Received 25 May 2000; accepted 2 February 2001

Abstract

A new technique for analysis by means of chemiluminescence (CL) is reported. Conventional CL methods for analysisusing a homogeneous catalyst can be unsuitable for stable analysis, because the conditions of mixing of the reactant solutionsgreatly affects the results. Though, electrochemiluminescence (ECL) is capable of solving this problem, it requires morecomplicated equipment than CL. A platinum plate-immobilized flow-cell is capable of stably controlling the CL from luminolas in ECL and has the advantage of requiring no electrical equipment. Human serum albumin (HSA) concentrations rangingfrom 0 to 100 �g ml−1 in water and in dialysate are measured by means of the CL intensity enhancement of luminol-labeledanti-HSA antibody caused by an antigen–antibody reaction in the presence of hydrogen peroxide using the Pt-immobilizedflow-cell. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Chemiluminescence immunoassay; Platinum plate; Flow-cell; Luminol; Hydrogen peroxide; Albumin

1. Introduction

Radical anions cause luminescence of luminol[1–3]. Radicals generated by chemical reactionsor electrode reactions attack luminol to emit light.Chemiluminescence (CL) of luminol requires cata-lysts such as peroxidase on iron ions for generatingradicals [4]. Since the CL of luminol occurs at the mo-ment of mixing reagents and catalyst, it is influencedby the conditions of mixing [5]. On the other hand,electrochemiluminescence (ECL) occurs only at anelectrode when the potential is applied. Homogeneousimmunoassay by means of the ECL of luminol wasreported by Aizawa et al. [6] in 1989, and it became a

∗ Corresponding author. Fax: +81-3-3209-7957.E-mail address: kisakai@mn.waseda.ac.jp (K. Sakai).

widely used method. Authors also reported [7,8] thatthe change of the ECL intensity from luminol-labeledantibody responded to changes in the concentrationsof the antigen. CL, however, has the advantage that itrequires simpler equipment than ECL.

We found that luminol emitted light withoutapplying a potential when using a flow-cell in-cluding a platinum electrode (Pt flow-cell). Inaddition, the CL intensity of luminol dependedon the luminol concentration. Therefore, we ap-plied the Pt flow-cell to CL immunoassay with aluminol-labeled antibody. Human serum albumin(HSA) was chosen as a test solute, because leak-age of HSA into dialysate was becoming a bigproblem. This method will be appropriate for mon-itoring dialysate, because of its simple operationwhere HSA, luminol-labeled anti-HSA antibody, and

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0 0 0 3 -2 6 70 (01 )00886 -8

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hydrogen peroxide were mixed and reacted in the Ptflow-cell.

2. Experimental

2.1. Luminol labeling

Luminol powder (Wako, Osaka, Japan) was dis-solved into 0.2 M NaOH and diluted with reverseosmosis (RO)-treated water. Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC: Pierce, Rockford, IL) was dissolved in theluminol solution and reacted for 2 h with stirring.The reacted luminol solution and the treated waterwere added to a anti-HSA antibody solution (EY Lab,CA) and reacted for 20 h at 277 K. N-ethylmaleimide(Wako) was added to the solution to stop the reaction,and the solution diluted with the RO water.

2.2. Measurement of chemiluminescence fromluminol with the Pt flow-cell

Fig. 1 shows the apparatus for measuring CL witha Pt flow-cell. Sample solutions including luminol

Fig. 1. Schematic diagram of the apparatus for measurement of chemiluminescence with a Pt flow-cell.

or luminol-labeled anti-HSA antibody and HSA, and30 mM hydrogen peroxide were pumped to the Ptflow-cell at flow rates of 0–20 m h−1 with a syringepump TE-311 (Terumo, Tokyo). Fig. 1 also shows adetailed schematic diagram of the Pt flow-cell. Themeasurement was carried out in a dark box at 295 K.Emission from luminol was detected by a photomulti-plier tube R269 (Hamamatsu Photonics, Hamamatsu,Japan) and changed to an electronic signal via a pho-ton counting unit U3997 (Hamamatsu Photonics) anda computer 386GS (Epson, Tokyo). Photons werecounted and integrated for 20 s after emission attaineda steady state.

2.3. Determination of HSA in dialysate

HSA solution was diluted with Kindary AF-2Pdialysate (Fuso Chemicals, Osaka) to give a rangeof concentrations from 0 to 100 �g ml−1. Luminol(33.9 �M), anti-HSA antibody (120 �g ml−1) and hy-drogen peroxide (30 mM) were added to the HSA so-lution. The CL intensity was measured by the methoddescribed above. The components of the KindaryAf-2P dialysate are shown in Table 1.

S. Aoyagi et al. / Analytica Chimica Acta 436 (2001) 103–108 105

Table 1Glucose and electrolyte ion concentration in Kindaly AF-2P dialysate

Electrolytic ion (meq. l−1) Glucose (C6H12O6) (mg dl−1)

Na+ K+ Ca2+ Mg2+ Cl− CH3COO− HCO3−

140 2 3 1 110 8a 30 100

a Containing 2 meq. l−1 CH3COO− of glacial acetic acid for pH regulation.

3. Results and discussion

3.1. Luminol determination

Fig. 2 shows the time courses of CL emission fromluminol at various luminol concentrations with the Ptflow-cell. According to the generally accepted reac-tion scheme, a luminol anion is initially oxidized, mostprobably to a radical which, depending on the exper-imental conditions, reacts with oxygen or hydrogenperoxide to yield a peroxide intermediate [1]. Hydro-gen peroxide is decomposed by a platinum catalyst andelectrochemically oxidized at a Pt electrode [9,10]. Areaction between hydrogen peroxide and Pt generatesmainly oxygen and partly free radicals. Therefore, lu-minol in hydrogen peroxide solution emits light in thePt flow-cell.

The CL intensity from luminol was very high ini-tially and decreased gradually until a steady statewas attained, because adsorption of oxygen gas,

Fig. 2. Time-course of chemluminescence intensity dependence onluminol concentration (H2O2 concentration: 30 mM).

generated by oxidation of hydrogen peroxide, on thePt plate caused a decrease in its available area forgenerating radicals to excite luminol. The CL inten-sity increased with luminol concentration in terms ofeither peak height or integrated number of photonsfor 20 s after CL attained the steady state. The peakheight, however, was not always stable, therefore CLintensity was calculated by integrating the number ofphotons.

Fig. 3 shows a relationship between the number ofintegrated photons and luminol concentration. The CLfrom luminol with the Pt flow-cell and the luminolconcentration had a linear correlation at luminol con-centrations ranging from 0 to 50 �M. The amount ofhydroxyl radicals generated by the oxidation of hy-drogen peroxide on the Pt plate is enough to stimulatelight emission from luminol. In addition, the emissionis stable. Therefore, the Pt flow-cell is capable of mea-suring luminol concentration by means of CL fromluminol with the same advantage of ECL of indepen-dence of mixing conditions, and it enables simpler op-eration than an instrument for ECL.

Fig. 3. Relationship between chemluminescence intensity and lu-minol concentration (H2O2 concentration: 30 mM; n = 3).

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Fig. 4. Comparison of the chemiluminescence enhancement due to the presence of HSA at various luminol concentrations (H2O2

concentration: 30 mM; n = 3).

3.2. HSA determination

In order to obtain reproducible and sensitivedata, optimum concentrations of luminol andluminol-labeled anti-HSA antibody were evaluated.Fig. 4 shows the effect of luminol concentration on CLintensity at HSA concentrations of 0 and 100 �g ml−1

of the luminol concentrations investigated. Therefore,the optimum luminol concentration is 33.6 �M, be-

Fig. 5. Comparison of the chemiluminescence enhancement due to the presence of HSA at different anti-HSA antibody concentrations(H2O2 concentration: 30 mM; n = 3).

cause HSA response is as great as at 678 �m and thereproducibility is better.

Fig. 5 shows the effect of luminol-labeledanti-HSA antibody concentration on CL inten-sity at HSA concentrations of 0 and 100 �g ml−1.When luminol-labeled anti-HSA antibody con-centration was 120 �g ml−1, the CL intensity in-creased more due to the presence of HSA than at60 �g ml−1 antibody. The CL intensity without HSA

S. Aoyagi et al. / Analytica Chimica Acta 436 (2001) 103–108 107

Fig. 6. Relationship between chemluminescence intensity andHSA concentration ranging from 0 to 100 �g ml−1 in treatedwater (H2O2 concentration: 30 mM; luminol concentration:33.9 �g ml−1; anti-HSA antibody concentration: 120 �g ml−1;n = 3).

was slightly greater at the higher anti-HSA anti-body concentration. The higher concentration ofluminol-labeled anti-HSA antibody is suitable forobtaining highly sensitive response to HSA. Sinceantibody is expensive, a large amount of the anti-body for each measurement is undesirable in termsof the cost. Therefore, 120 �g ml−1 is a suitableconcentration of anti-HSA antibody for this measure-ment.

3.3. HSA calibration in treated water and in dialysate

Fig. 6 shows a relationship between CL intensityfrom luminol and HSA concentration and also a lineardependence of the CL intensity on HSA concentrationsranging from 0 to 100 �g ml−1. Fig. 7 also shows therelationship between CL intensity from luminol andHSA concentration ranging over 4 orders of magnitudeup to 100,000 �g ml−1.

Fig. 8 shows the relationship between CL intensityfrom luminol and HSA concentration in the KindaryAF-2P dialysate. A linear dependence of CL inten-sity on HSA concentration from 0 to 100 �g ml−1

was obtained. Though, the calibration slope for thedialysate is lower than that for the treated water shownin Fig. 7, it is satisfactory for HSA determination. Themain reason why the slope is lower is inhibition of theCL reaction by components in the dialysate. Furtherstudy to determine what inhibits the CL reaction is

Fig. 7. Relationship between chemluminescence intensity andHSA concentration ranging from 0 to 105 �g ml−1 in treatedwater (H2O2 concentration: 30 mM; luminol concentration:33.9 �g ml−1; anti-HSA antibody concentration: 120 �g ml−1;n = 3).

Fig. 8. Relationship between chemluminescence intensity and HSAconcentration in dialysate (H2O2 concentration: 30 mM; lumi-nol concentration: 33.9 �g ml−1; anti-HSA antibody concentration:120 �g ml−1; n = 3).

necessary in order to obtain a calibration for a lowHSA concentrations (ca. 10 �g ml−1).

4. Conclusions

The CL immunoassay with the Pt flow-cell enablesstable measurements to be made without electrolysis

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equipment. HSA concentrations ranging from 0 to100 �g ml−1 in treated water and dialysate are mea-surable from data on CL intensity enhancement of120 �M luminol-labeled anti-HSA antibody (luminolconcentration: 33.6 �M) mixed with 30 mM hydro-gen peroxide using the Pt flow-cell. This is a promis-ing method of continuously monitoring the concen-tration of biochemical substances such as HSA in adialysate.

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