Anal. Chem. 1989, 6 1 , 2285-2288
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Visible Semiconductor Laser Fluorometry Totaro Imasaka, Atsushi Tsukamoto, and Nobuhiko Ishibashi* Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
A vlslMe semkonductor laser oscliiatlng at 670 nm Is used as a light source for fluorescence spectrometry. The detectlon llmit of rhudamlne 800 Is 4 X IO-'* M, which is slightly better than that obtained by near-Infrared semlconductor laser fluorometry. Several dyes with reactive sltes, available in the deep-red reglon, are used as labellng reagents. Nile blue with a primary amino group Is most efficlently bound to albumin with water-soluble carbodlimlde, but the absorptlon maxlmum Is located at 640 nm and Is less efficiently detected. Oxarlne 750 Is less reactive to protein due to As attached ethyl group but glves the largest slgnai. This is ascribed to good coincidence between the absorption maximum (670 nm) and the laser wavelength. Methylene blue with a tertiary amino group Is also used for labeling proteln by electrostatic adsorption.
Laser fluorometry provides an ultrasensitive means for chemical analysis. Recently, efforts have been much more concentrated on the determination of trace biological molecules. The detection limits achieved for the fluorescein isothiocyanate derivatives of amino acids are reported to be 0.009 amol in capillary zone electrophoresis (1). Even in indirect fluorometry 70 amol of nucleotides is detected by using sodium salicylate as a fluorescent reagent in the carrier (2). A laser has great performance, but it has not yet been used as a light source in a commercial spectrometer. This is probably due to the large dimension and expensiveness of the laser. Furthermore, maintenance and operation costs may be additional problems in practical applications. On the other hand, a recently developed semiconductor laser is small and less expensive, and it is simply driven by an integrated circuit or a small battery. The output power of the semiconductor laser was recently reported to 38 W, which exceeds that given by a commercial high-power argon or krypton ion laser ( 3 ) . Based on near-infrared semiconductor laser fluorometry, ultratrace analysis has already been demonstrated (4). The detection limit reported for a polymethine dye is 5 x 10-l2 M or 12 fg (5,6). High-performance liquid chromatography based on near-infrared semicondudor laser fluorometry has also been demonstrated, and protein in human serum was determined after labeling it with a near-infrared dye by an electrostatic force (adsorption) (7,8). The polymethine dye of indocyanine so that an green forms a nonfluorescent species with H202, is monitored (9). enzyme reaction producing H202 T o our best knowledge, all the dyes fluorescent in the near-infrared region belong to a group of polymethine dyes. Three thousand of these molecules are commercially available, but they have no active sites to covalently bind to biological molecules such as protein. Furthermore, the fluorescence intensity of the polymethine dye decreases rapidly in an aqueous solution, which is considered to be due to formation of nonfluorescent dimers (10). The oscillating wavelength of the semiconductor laser used in fluorometry was limited to 750-1500 nm, and it was difficult to find a suitable organic dye for labeling by a covalent bond. It prevents wide use of
* Author to whom correspondence should be addressed. 0003-2700/89/036 1-2285$01.50/0
semiconductor laser fluorometry in practical work such as the fluorescence immunoassay. In this study we report the first application of a visible semiconductor laser oscillating at 670 nm to fluorescence spectrometry to overcome the above problems. There are several stable and strongly fluorescent dyes with reactive sites useful for labeling in the deep-red region, e.g. thionine, oxazine, and similar analogues (11, 12). We bind these organic dyes to protein by a covalent or noncovalent bond and discuss their labeling and detection efficiencies.
EXPERIMENTAL SECTION Apparatus. A block diagram of the experimental apparatus is shown in Figure 1. A sample is injected at the top of a column (Pharmacia Fine Chemicals, K 9/30,9-mm id., 30 cm long) and is separated by a gel (Pharmacia Fine Chemicals, Shephadex G25) by flowing a phosphate buffer (pH 6.4) using a pump (Shimadzu, LC-5A). The eluent is detected by a commercial absorbance spectrometer (Jasco, Uvidec-lOO-LV), a commercial fluorescence spectrometer (Kyowa, KLF-3080), and a homemade fluorescence spectrometer using a visible semiconductor laser. Details of the laser fluorometric system constructed are shown in Figure 2. The semiconductor laser (NEC, NDL 3200) has an output power of 3 mW and an oscillating wavelength of 670 nm. The laser has a high slope efficiency (-1 W/A), and the diode current should be carefully controlled by not allowing the output power to exceed the maximum rating (4 mW). The laser power is regulated by an automatic power control circuit specified by the manufacturer (13). The laser beam is collimated by an objective lens for a microscope (Olympus, LWD MS Plan 50) mounted on a micrometer-controlled stage (Sigma, 2-2078). It is focused into a homemade quartz flow cell (1-mm i.d., 10 mm long) or a conventional quartz cuvette for fluorescence spectrometry (1 X 1 X 5 cm3). Fluorescence from the sample is collected by a pair of glass lenses onto the slit of a monochromator (Jasco, CT-10). A R928 photomultiplier (Hamamatsu) is used for construction of an analytical curve for rhodamine 800 and is replaced by Model R636 (Hamamatsu) with a flatter spectral response for comparison of the fluorescence intensities in chromatography. The signal is amplified 500 times by a homemade amplifier and is directly displayed by a three-pen chart recorder (Rikadenki, R304). For recording the emission spectrum of the semiconductor laser, a double monochromator (Jasco, CT-40D) was used to reduce stray light. The double monochromator was necessary to measure weak photoemission in the vicinity of the strong oscillating line. The absorption and fluorescence spectra were measured by a double-beam spectrophotometer (Shimadzu, UV-140-02) and a fluorescence spectrophotometer (Hitachi, MPF-4), respectively. Reagents. Chemical structures of the organic dyes used in this study are shown in Figure 3. Rhodamine 800 (LC 8000) and oxazine 750 (LC 7271) were purchased from Lambda Physik. The other dyes, thionine (Lauth's Violet), nile blue, and methylene blue, were supplied from Tokyo Kasei, Wako Pure Chemical Industries, and Kanto Chemical, respectively. Albumin (from egg, 018-09882) was obtained from Wako. A bifunctional reagent of water-soluble carbodiimide (l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide hydrochloride) purchased from Dojindo Laboratories was used for the condensation reaction of COOH and NH2 (or NHR). This reaction scheme is shown in Figure 4. Procedure. For construction of the analytical curve, rhodamine 800 was diluted stepwise with ethanol. The fluorescence measurement was carried out by using a cuvette. Water-soluble carbodiimide, which has currently been used as a binding reagent for peptide (14,15),was used to label protein with nile blue, 27 0 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 20, OCTOBER 15, 1989 HPLC Pump
Buffer
3
Filtration Column
the 9 mL of phosphate buffer (pH 6.4). This solution was mixed with methylene blue dissolved in the phoaphate buffer prepared at a specified concentration. The 0.1-mL sample solution was injected into the gel filtration column, and absorption and fluorescence signals were simultaneously recorded. The column was rinsed with concentrated NaOH and a copious amount of buffer to remove dyes that remained at the top of the column.
Monochro-
I-
Monitor
I
RESULTS AND DISCUSSION Analytical Curve of Rhodamine 800. In order to investigate the sensitivity of visible semiconductor laser fluorometry, an analytical curve was constructed for rhodamine 800. In this measurement the fluorescence wavelength of the monochromator was adjusted to 700 nm. The absorption maximum was located a t 682 nm, which was close to the oscillating wavelength of the semiconductor laser (670 nm). The molar absorptivity is reported to be 8.95 x 104 for ethanol solution (11)and the fluorescence quantum yield to be 39% for dichloroethane solution; the value for ethanol solution is unknown but is estimated to be -10% (16). The constructed analytical curve was straight from lo-" M to lo* M. The signal was saturated above this range owing to concentration M, which was quenching. The detection limit was 4 X limited by scattering of the exciting light. To investigate the source of noise, the emission spectrum of the semiconductor laser was measured by a monochromator. It was found that a broad background emission was appreciable a t the wavelength of fluorescence detection. This is probably due to nonlasing photoemission from the semiconductor laser. The present detection limit is slightly better than the values achieved by near-infrared semiconductor laser fluorometry: 5X M by a digital photon counting system and 5 X by an analog lock-in amplifier (5). It is worth mentioning that the present detection limit is achieved by a simple direct current amplifier with a time constant of
