Anal. Chem. 1990, 62, 2404-2405
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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 example, a biological molecule such as insulin has been determined a t ultratrace levels by laser-induced fluorescence immunoassay ( I ) and enzyme immunoassay (2, 3 ) . However, laser fluorometry has seldom been used in practical trace analysis. This is due to expensiveness, complexity, poor reliability, 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 fluorescent 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 HzOzby a catalytic reaction. Thus an enzyme reaction producing HzOzcan 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 immunological assay is further discussed. EXPERIMENTAL SECTION Apparatus. The apparatus used is already reported in detail elsewhere (7) and is briefly described here. The GaAlAs semiconductor 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 measured by a monochromator (Jasco, CT-10) equipped with a redsensitive 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 (1x 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 (100mg), 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 immunobead were reacted for 1h 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 immunobead 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 0003-2700/90/0362-2404$02.50/0
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. R E S U L T S 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 measurement 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 HzOzin the presence of a catalyst, an Fe(I1) ion (7). A metabolite of xanthine producing H202was 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 conventional 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 optimization of the ICG concentration, etc. Analytical Advantage. In clinical assay, radioimmunoassay 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. Recently, 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 radioimmunoassay. 0 1990 American Chemical Society
Anal. Chem. 1990, 62, 2405-2408
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 labeling protein in the near-infrared region. Registry No. Insulin, 9004-10-8.
1 .o
LITERATURE CITED
. -'0
0.5
I
0
2405
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 incubation time is required for the enzyme reaction. However, more rapid competitive binding assay is used in radioimmunoassay: the sample is readily measured after the immunological 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 determination of protein: low background fluorescence in the near-infrared region is essential in ultratrace analysis, and
(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. R o c . 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, 2285. (7) Imasaka, T.; Okazaki, T.; Ishibashi, N. Anal. Chim. Acta 1988, 208, 325. (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 a n d Food Sciences, University of Salamanca, Salamanca, Spain Usually flow injection methods are based on the measurement 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 allowing multidetection and multidetermination by flow injection 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 Reagents. Stock solutions of uranium and thorium (2.0 X 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 determination 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
0003-2700/90/0362-2405$02.50/00 1990 American Chemical Society
