2404 Anal. Chem. 1990, 62, 2404-2405

CORRESPOCYDHWE

Enzyme Immunoassay of Insulin by Semiconductor Laser Fluorometry

Sir: A laser has a large photon flux and is useful as a light source in fluorescence spectrometry. There are many reports to show analytical advantages of laser fluorometry. For ex- ample, a biological molecule such as insulin has been deter- mined a t ultratrace levels by laser-induced fluorescence im- munoassay ( I ) and enzyme immunoassay (2, 3) . However, laser fluorometry has seldom been used in practical trace analysis. This is due to expensiveness, complexity, poor re- liability, etc. of the laser. However, a recently developed semiconductor laser has no such limitations and can possibly be used in practical applications ( 4 ) .

In biological assay, the semiconductor laser has been used in fluorometric determination of protein labeled with fluor- escent dyes in the near-infrared (5) and deep-red regions (6). An enzyme reaction has been monitored by the fluorescence quenching effect: the fluorescence intensity of indocyanine green (ICG) decreases with an increase in the concentration of the OH radical produced from HzOz by a catalytic reaction. Thus an enzyme reaction producing HzOz can be monitored (7).

In this study we report enzyme immunoassay of insulin based on sandwich assay. The enzyme activity is determined by measuring the fluorescence quenching effect of ICG by an OH radical produced by peroxidase from Hz02 The analytical advantage of semiconductor laser fluorometry in immuno- logical assay is further discussed.

EXPERIMENTAL SECTION Apparatus. The apparatus used is already reported in detail

elsewhere (7) and is briefly described here. The GaAlAs semi- conductor laser oscillating at 780 nm (Sharp, LT 024MD, 20 mW) was used as an exciting source in fluorometry. The output power was regulated by an integrated circuit (Sharp, IR3C02N) for feedback control. Fluorescence was collected by a glass lens, transmitted through an interference filter (Ditric, 15-20785, transmission maximum 830 nm, transmittance 45%) and mea- sured by a monochromator (Jasco, CT-10) equipped with a red- sensitive photomultiplier (Hamamatsu, R928).

Procedure. The pH dependence of ICG fluorescence was investigated by diluting 20 pL of 2.8 x 10" M ICG to 5 mL with various buffer solutions, which were prepared by following the protocol given in ref 8: pH 1-5, 1 M CH,COONa + HCl; pH 6-8, 0.2 M KH2P04 + NaOH; pH 9 and 10,0.2 M H3B03 + KCl + NaOH; pH 12.5, NaOH. The fluorescence intensity was measured by using an 1-cm quartz cuvette.

For determination of enzyme activity, a specified amount of peroxidase, ICG (1 x 10" M, 100 pL), and H202 (2 x lo5 M, 200 pL) were mixed and the solution was diluted to 5 mL with a buffer (pH 7). The buffer contained albumin (500 mg), a preservative of sodium salicylate (100 mg), KHzPOl (1.4 g), NaHC03 (0.58 g), and NaCl(O.9 g) in water (100 mL). The fluorescence intensity was measured at 15 min after initiation of the enzyme reaction.

The experimental procedure for enzyme immunoassay was mostly followed by the protocol attached to the commercial kit for enzyme immunoassay of insulin based on colorimetry (Insulin B test, Wako Pure Chemical). Insulin (0-160 punits/mL, 0.5 mL), peroxidase-labeled antibody (kit reagent, 0.5 mL), and an im- munobead were reacted for 1 h at 37 "C. The solution was rinsed three times with a copious amount of 0.9% NaCl solution to remove an excess amount of enzyme-labeled antibody. The im- munobead was transferred to a new test tube and was reacted with a solution consisting of ICG (4 X 10" M) and H,OP (1 X

M) for 15 min at 37 "C. The immunobead was taken out of the test tube, and the solution was mixed with 2 mL of buffer solution (pH 7). The fluorescence intensity of ICG was measured at 810 nm by the semiconductor laser fluorometer.

Reagents. A fluorescent dye of ICG was purchased from Daiichi Seiyaku. Insulin, peroxidase, and other chemical reagents for immunoassay (e.g. enzyme-labeled antibody, immunobead, etc.) were obtained from Wako Pure Chemical.

RESULTS AND DISCUSSION Effect of pH. The optimum pH for measurement of ICG

fluorescence was investigated from pH 1 to pH 12.5. The fluorescence intensity rapidly increased from pH 2 and reached an almost constant value at pH 5. The signal intensity slightly increased above pH 5, but the fluorescence intensity was less stable above pH 8. Then, the fluorescence mea- surement was carried out between pH 5 and pH 7 throughout this experiment.

Determination of Peroxidase. In the previous study, ICG was found to be quenched by an OH radical produced from HzOz in the presence of a catalyst, an Fe(I1) ion (7). A me- tabolite of xanthine producing H202 was indirectly measured by using this reaction scheme. In this study, we more directly measured peroxidase by producing an OH radical from HzOz: i.e. peroxidase acts as a catalyst. The fluorescence quenching effect was evaluated by calculating (Io - O / I , where Io and I are initial and final fluorescence intensities (7). A straight analytical curve (correction coefficient 0.990) was observed from 0 to 400 punits/mL, and the signal changed from 0 to 8.3. The detection limit was -20 punits.

Determination of Insulin. According to the procedure described in the experimental section, insulin was determined at trace levels. The constructed analytical curve is shown in Figure 1. The insulin concentration is measured from 0 to 160 punits/mL, the detection limit being -10 Munits/mL. The detection sensitivity was limited by a base line drift of the fluorescence intensity for ICG. The analytical range and the detection sensitivity are similar to those for the conven- tional method using standard absorption spectrometry. No optimization in the analytical procedure was carried out in this study, and further investigation may be necessary for substantial improvement of the sensitivity, e.g. a search of a stabilizing reagent giving stable ICG fluorescence, an opti- mization of the ICG concentration, etc.

Analytical Advantage. In clinical assay, radioimmu- noassay is most frequently used due to its high sensitivity. Since radioactive substances are quite few in the environment, the background signal is almost completely negligible. Re- cently, enzyme immunoassay has become more popular, which might be due to safety in the use of a nonradioactive substance and a signal enhancement by an enzyme catalytic reaction. Needless to say, the background signal should be reduced as much as possible in enzyme immunoassay for the best use of its high sensitivity, though impurity fluorescence can be substantially reduced through a washout process. To our best knowledge, only a polymethine dye is fluorescent in the near-infrared region (9), so that near-infrared semiconductor laser fluorometry is potentially useful due to its high sensitivity and low background signal, as in the case of radioimmu- noassay.

0003-2700/90/0362-2404$02.50/0 0 1990 American Chemical Society

Anal. Chem. 1990, 62, 2405-2408 2405

furthermore, the B/F separation in immunoassay will be achieved by polarization or time-resolved fluorometry. For this purpose, a new fluorometric reagent is necessary for la- beling protein in the near-infrared region.

Registry No. Insulin, 9004-10-8.

LITERATURE CITED (1) Lidofsky, S. D.; Imasaka, T.; &re, R. N. Anal. Chem. 1979, 57,

1602. (2) Lidofsky, S. D.; Hinsberg, W. D., 111.; Zare, R. N. Roc. Natl. Acad.

Sci. U.S .A . 1981, 78, 1901. (3 ) Hinsberg, W. D., 111.; Milby, K. H.; Zare, R. N. Anal. Chem. 1981, 53,

1509. (4) Imasaka, T.; Ishibashi, N. Anal. Chem. 1990, 62, 363A. (5) Sauda, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1986, 58, 2649. (6) Imasaka, T.; Tsukamoto, A,; Ishibashi, N. Anal. Chem. 1989, 67,

(7) Imasaka, T.; Okazaki, T.; Ishibashi, N. Anal. Chim. Acta 1988, 208, 325.

2285.

1 .o

- . - - '0

0.5

I

0 50 100 150 Concentration/(pU/mi)

Figure 1. Analytical curve for insulin. I, and I are fluorescence intensities at 0 and 15 min after initiation of the enzyme reaction, respectively.

In heterogeneous enzyme immunoassay, an additional in- cubation time is required for the enzyme reaction. However, more rapid competitive binding assay is used in radioimmu- noassay: the sample is readily measured after the immuno- logical reaction and the succeeding phase separation of bound and free (B/F) antigens. Competitive binding fluorescence immunoassay based on semiconductor laser spectrometry may provide us with a more practical means for fluorometric de- termination of protein: low background fluorescence in the near-infrared region is essential in ultratrace analysis, and

(8) Handbook of Chemical Substances (Kagaku Binran), Fundamental I I ; The Chemical Society of Japan, Ed.; Maruzen: Tokyo, 1975; p. 1490.

(9) Imasaka, T.; Yoshitake, A.; Ishibashi, N. Anal. Chem. 1984, 56, 1077.

To whom correspondence should be addressed

Totaro Imasaka Hiroyuki Nakagawa

Takashi Okazaki Nobuhiko Ishibashi*

Faculty of Engineering Kyushu University Hakozaki, Fukuoka 812, Japan

RECEIVED for review May 7, 1990. Accepted July 19, 1990. This research is supported by Grants-in-Aid for Scientific Research from the Ministry of Education of Japan and Naito Foundation.

TECHNICAL NOTES

Reversed Injector Loading Technique for Simultaneous Determinations by Flow Injection Analysis

Jose Luis PBrez Pavbn,* Carmelo Garcia Pinto, Bernard0 Moreno Cordero, and Jesiis Hernandez MBndez

Department of Analytical Chemistry, Bromatology and Food Sciences, University of Salamanca, Salamanca, Spain

Usually flow injection methods are based on the measure- ment of a single signal depending on the analyte concentration. However, this methodology also permits multidetection and multidetermination, the difference between these two terms having been established by Luque de Castro et al. (I). The same authors have reviewed the proposed configurations al- lowing multidetection and multidetermination by flow in- jection analysis (FIA) (2).

The more usual ways to carry out multidetermination are sequential injection and sample splitting ( 3 , 4 ) . The use of a two-valve injector (5, 6) or an eight-port valve (7) allow simultaneous determinations in FIA. In this paper a six-port valve is used for the first time to carry out multidetermination by a single injection.

PRINCIPLE The term "reversed injector loading technique" is used to

indicate that in the inject mode the flow through the sample loop is opposite to the flow in the loading mode (Figure 1). If a chemical reactor (i.e. a reducing column) is included in

the loop, when the valve in turned to the inject mode, two zones of sample are inserted into the carrier stream, one of them having undergone a differentiating chemical process, thus originating two signals in the detector.

EXPERIMENTAL SECTION

M) prepared by dissolving appropriate amounts of uranyl nitrate hexahydrate (Merck) and thorium nitrate pentahydrate (Merck) in water. Stock solutions (lo-* M) of Fe(II1) and Fe(I1) were prepared by dissolving appropriate amounts of their chlorides in 0.1 M HC1. Stock solutions of nitrate and nitrite (10" M) were also prepared from sodium nitrate (Panreac) and sodium nitrite (Panreac) in aqueous 1% NH4C1 (Panreac).

Carrier solutions: 3.6 M HCl for the spectrophotometric de- termination of Th and U; 0.1 M HC1 in 0.3 M NaCl for the spectrophotometric simultaneous determination of Fe(I1) and Fe(II1); aqueous 1 % NHICl for the amperometric determination of nitrate and nitrite.

Reagent solutions: 2.0 X 10"' M Arsenazo I11 in 3.6 M HC1 (in the presence of 1% Triton X-100) for the spectrophotometric

Reagents. Stock solutions of uranium and thorium (2.0 X

0003-2700/90/0362-2405$02.50/0 0 1990 American Chemical Society