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Anal. Chem. 1984, 56,1077-1079
Semiconductor Laser Fluorimetry in the Near-Infrared Region Totaro Imasaka, Akinori Yoshitake, and Nobuhiko Ishibashi* Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
Ultrasensltive detection of 3,3’-dlethyl-2,2‘-(4,5;4’,5’-dlbenz0)thlatrlcarbocyanlne iodide (DDTC) was demonstrated In the near-Infrared region by using a semiconductor laser fluorimeter comblned with a photon countlng system. The detection ilmlt was 5 X lo-’* M, which was 2 orders of magnltude better than the value obtalned by a conventional fluorlmeter equlpped with a xenon lamp and an analog detector system. The near-infrared dye of DDTC was found to be extracted Into benzene with surfactants of sodlum lauryl sulfate (SDS) or sodlum dodecyl benrenesulfonate (DBS). The achieved detection limits for the surfactants were lo-’ M. The analytical advantages of the semiconductor laser fluorlmetry were dlscussed.
Laser fluorimetry has great performances for sensitive and selective determinations of trace species (1). However, no commercial instrument has been developed for practical applications, perhaps because of the laser’s large dimensions, expense, and poor reliability. Our strategy to overcome these problems is the use of a semiconductor laser as a light source for fluorimetry. For data communication a semiconductor laser has been rapidly developed and is now commercially available at a low price. It is as small as a photodiode, and it can be reliably operated for more than lo00 h. Then, the semiconductor laser may have the ideal characteristics required of a light source in practical instruments. A very few analytical applications of the near-infrared semiconductor laser have been reported. The first study is the use as a light source in photoacoustic spectrometry for the determination of phosphorus based on the molybdenum blue method (2). A new type of high-power solid-state emitter has been also developed by the technology related to that in a semiconductor laser. This emitter is not a laser but is very useful as a light source for spectrophotometry (3). Recently, absorption spectral lines were measured for NH3 and H 2 0 in the vapor phase with a 1.5 pm semiconductor laser for the purpose of pollutant gas monitoring (4). The wavelength of the semiconductor laser commercially available is limited to the near-infrared region (780-1300 nm). However, it is emphasized that the blank fluorescence from a solvent, which sometimes determined the detection limit in laser fluorimetry, can be greatly reduced in this near-infrared region. A biomedical assay based on a fluorescence labeling technique should be carried out in the long wavelength region to eliminate blank fluorescence from the various fluorescent impurities in serum or other matrices (5). Some of polymethine dyes are known to have an absorption band around 600-900 nm. They have very large molar absorptivities and are strongly fluorescent. Therefore, they might be detected at ultratrace levels by semiconductor laser fluorimetry. A polymethine dye seems to be used as a fluorescent probe based on ion-pair solvent extraction in practical analysis. Since a polymethine molecule has a positive charge, it might form an ion pair with a large molecule with a negative charge and be extracted into the organic solvent. If so, the sample with a negative charge can be sensitively
detected by measuring fluorescence from the polymethine dye. In this study we constructed a very sensitive semiconductor laser fluorimeter and compared its performances with a conventional one. We demonstrated trace analysis of surfactants based on ion-pair extraction with a polymethine dye. The analytical advantage of semiconductor laser fluorimetry is also discussed.
EXPERIMENTAL SECTION Apparatus. A block diagram of the experimental apparatus is shown in Figure 1. The light source is a double hetero structure AlGaAs laser (NEC, NDL 3000,5 mW, 786 nm, fwhm = 1nm). The illumination area of the laser diode is so small that the beam divergence exceeds 12 X 40 degree by diffraction. A lens with a short focal length (7 mm) is placed 8 mm from the semiconductor laser. The focal point is adjusted to the center of the sample cell by changing the position of the lens and the angle of the mirror. Fluorescence is collected by a lens (focal length = 20 mm) and imaged onto the entrace slit of a double monochromator (JASCO, CTdOD). Since the photomultiplier used in a conventional fluorimeter has low sensitivity around 700-900 nm, a photomultiplier with a GaAs photocathode (Hamamatsu Photonics, R636, 186-930 nm) is used in this study. The typical applied voltage was 1400 V. The output analog signal was measured by a lock-in amplifier (NF Electronic Instrument, LI-570) equipped with a strip chart recorder. For sensitive determination of the sample species, the photoelectron pulses were amplified 200 times by a preamplifier (NF, IA-551) and recorded by a photon counter (NF, PC-545A) as shown in Figure 1. The signals were accumulated in 50 s and recorded by a digital printer (Nada Electronic Lab, LTD., DP-102). A He-Ne laser (NEC, GLG 2026,l mW) was used for comparison with a semiconductor laser. The commercial fluorimeter was Hitachi MPF-4 fluorescence spectrophotometer. Reagents. A polymethine dye of 3,3’-diethyl-2,2’-(4,5;4’,5’dibenz0)thiatricarbocyanine iodide (DDTC) was obtained from the Japanese Research Institute for Photosensitizing Dyes Co., Ltd. Sodium lauryl sulfate (SDS) and sodium dodecyl benzenesulfonate (DBS) were used as typical surfactants. Both were obtained from Wako Pure Chemical Industries, Ltd. The methanol was an extra pure grade (not spectral grade) from Kishida Chemical Co., Ltd., and used without further purification. The water was doubly distilled and deionized. The glassware was washed with soap and a chromic acid mixture and then rinsed with copious amounts of water. Procedure. In order to evaluate the sensitivity of the fluorimetric system DDTC was diluted stepwise with methanol, and the fluorescence intensity was measured. For the determination of the surfactants, the samples of SDS and DBS were prepared by adding the aqueous solution of the surfactant and the saturated solution of DDTC (absorbance = 0.08) to a test tube with ground stopper. After the samples were mixed, the solvent benzene (7 mL) was added to the aqueous solution (13 mL). The mixture solution was shaken during 5 min, and the ion pair of the surfactant and DDTC was extracted into the organic phase. The upper part (organic phase) of the separated solution was taken into a quartz cuvette, and the fluorescence intensity was measured by a conventional or laser fluorimeter.
RESULTS AND DISCUSSION Spectrum of DDTC. As shown in Figure 2 the molecule of DDTC has a large conjugated structure, and it has a strong absorption band in the near-infrared region. The DDTC molecule in methanol was found to have a molar absorptivity of 150 000. It is noted that this value is comparable to that
0003-2700/84/0358-1077$01.50/00 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 7, JUNE 1984
: I T ~ A eh m =~5nm & ~
=
l
= 6 a,
E 4 g 2
-3
Lens
Preamplifier
u O
Current Modulator
Photon Counter I
[
Digital Printer
a5
800
I
1
Figure 1. Block diagram of semiconductor laser fluorimeter.
Wavelengthlnm
Wavelengthlnm
(A)
(5)
Flgure 4. Fluorescence spectrum of DDTC measured by (A) conventional fluorimeter (Hltachi, MPF-4) and (B) semiconductor laser fluorimeter equipped with lock-In amplifier. Table I. Detection Limit of DDTC in Methanol
Flgure 2. Chemical structure of 3,3'-diethyl-2,2'-(4,5:4',5'-dibenzo)thlatricarbocyanine iodide (DDTC). I
I
method
detection limit, M
conventional fluorimeter (Hitachi MPF-4) semiconductor laser fluorimeter lock-in amplifier photon counter
3 x 10-'0
5 x 10-11 5 x 10-*2
Table 11. Fluorescence Intensity of SDS-DDTC Ion Pair Extracted into Various Organic Solvents
Waveiengthlnm Figure 3. Excitation and emission spectra of DDTC in methanol: excitation wavelength, 700 nm; emission wavelength, 860 nm. of porphyrin molecules (200000-500 000), which are known to have a very large molar absorptivity and are used as a sensitive colorimetric reagent. Figure 3 shows excitation and emission spectra of DDTC in methanol. It has several bands in the excitation spectrum, and absorbs 786-nm emission of the semiconductor laser. The fluorescence maximum appears a t around 820 nm when measured by a conventional fluorimeter but the true emission maximum is located around 840 nm because the photomultiplier spectral response decreases rapidly with increasing wavelength. The DDTC dye molecule was less soluble in water and was completely nonfluorescent. No appreciable fluorescence could be detected by a conventional fluorimeter even for the concentrated sample. On the other hand it was strongly fluorescent in methanol or benzene and was used as a laser material (6). It is noted that more than 2000 dyes in this group have been commercially available and may be conveniently used for various applications (7). Ultratrace Analysis of DDTC. Figure 4A shows a fluorescence spectrum measured by a conventional fluorimeter near the detection limit. The resolution of the excitation and fluorescence monochromator is decreased to 20 nm to increase the sensitivity of the measurement. When a semiconductor laser is used as a light source, an order of magnitude lower sample concentration could be measured as shown in Figure 4B. In this case the measurement was carried out with a fluorescence spectral resolution of 5 nm, since it was the poorest limit of the monochromator in this study. The detection limit was determined by the radiant power of the light source and by the dark current noise of the photomultiplier. An analytical curve was constructed by using a photon counting detection system. I t could detect 5 X M of
organic solvent
fluorescence intensity
benzene toluene o-xylene m-xylene p-xylene cyclohexane
100
cct,
52 29 22
19 0 0
DDTC. It is emphasized that blank fluorescence was almost negligible in the measurement, and minimum detectability was determined by the dark current noise of the photomultiplier. Further improvement of the detection limit might be readily achieved by using a semiconductor laser with a larger output power or a cooled photomultiplier. In this study no special grade solvent was required. This result arises from the fact that a very few substances are fluorescent in the near-infrared region. This is an apparent analytical advantage of near-infrared fluorimetry. Comparison of Sensitivity. The detection limits of DDTC in methanol measured by various fluorimetric methods are listed in Table 1. The time constant of the conventional fluorimeter and the semiconductor laser fluorimeter with a lock-in amplifier is adjusted to 1 s. The detection limit is an order of magnitude better for semiconductor laser fluorimetry. The time constant of the photon counting system was set to 50 s, so the detection limit was an order of magnitude lower than a lock-in amplifier system. If the spectral resolution is taken into account, the last two methods would be 2 and 3 orders of magnitude more sensitive than the conventional method. The detection limit was also measured by using a He-Ne laser as an exciting source. However, it was 2 orders of magnitude poorer than that obtained by using a semiconductor laser. This result may come pmtly from the low output power of the He-Ne laser (1/8 times) and partly from mismatch of the absorption maximum and the lasing wavelength (1/8 times). Trace Analysis of Surfactant. The ion pair of SDS-DDTC was extracted into various organic solvents, and the fluorescence intensity was measured. The result is shown in Table 11. The ratio of the SDS-DDTC fluorescence signal to the blank was almost identical for benzene, toluene, and
ANALYTICAL CHEMISTRY, VOL. 56, NO. 7, JUNE 1984
xylenes, and therefore benzene which gave a largest signal intensity was used for the determinations of SDS and DBS. The analytical curve was constructed for SDS, and it was found to be a straight line from 0 to 7 X M. Observed blank fluorescence corresponded to 3 X lo-’ M of SDS. The DDTC dye dissolved in the aqueous phase was located at the boundary of the aqueous and organic phases. After the organic phase was taken to a different test tube, water was added to the sample solution and an attempt was made to extract the blank species back to the aqueous phase. This attempt was unsuccessful since the fluorescence signal substantially decreased though the blank signal also decreased. The ratio of the fluorescence background intensities could not be improved. The blank seems to arise from impurity surfactant that remained at the surface of the glassware. The minimum detectability of SDS was l X lo-‘ M. A similar result was obtained for DBS. The straight analytical curve was obtained from 0 to 6 X M, and the background signal corresponded to 2 X lo-’ M of DBS. The detection limit of DBS was 1 X M. The practical analytical range of the currently used methylene blue and its related methods based on spectrophotometry is (0.2-5) X lo4 M (8). The present detection limit is similar to or slightly better than the conventional method. Advantage of Semiconductor Laser Fluorimetry. The semiconductor laser has various advantages as a light source in fluorimetry. It has an output power (5-40 mW) comparable or larger than a He-Ne laser. It is noted that a semiconductor laser with an output power exceeding 1.5 W has already been constructed (9). The semiconductor laser is monochromatic and its narrow line width allows the efficient discrimination of unwanted scattered emission by a simple optics. The laser is compact and has a high conversion efficiency (4%), which is much larger than that of an argon ion laser (0.1%). It is noted that the energy conversion efficiency of 39% for one direction of the laser (78% for both laser facets) has been achieved for a high-power semiconductor laser (9). The power of the semiconductor laser can be readily modulated from very low to very high frequencies by controlling the electric current, and it can be stabilized by monitoring its output power with a photodiode in the package of the laser diode and by the feedback of the driving current. These characteristics of the semiconductor laser are very important for its use in practical applications. The emission wavelength of the laser is located at 780-820 nm; the shortest limit of the oscillating wavelength for a continuous laser is 683 nm (10) and that for a pulsed laser is 621 nm (11).The long wavelength of the laser may restrict
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the analytical samples to be measured, since most of the samples may have an absorption band in visible and ultraviolet regions but have no absorption band in the near-infrared region, and so very few molecular species can be directly determined by semiconductor laser fluorimetry. In most applications some analytical technique such as fluorescence tag labeling with a near-infrared dye may be essential for its practical use. Though many fluorescent tags such as fluorescein and rhodamine analogues are used in fluorescence immunoassay and other applications (12),the near-infrared labeling reagent has, to the best of our knowledge, not been developed. If hormones or metabolites can be labeled with a near-infrared dye such as a polymethine derivative, the present method might be very widely used in biochemical and clinical studies. A harmonic generation technique is now being developed by using a thin organic film for the use to the semiconductor laser (13). If this attempt is made in a high conversion efficiency, the emission wavelengths at 420 nm (second harmonic) and 280 nm (third harmonic) will be available for analysis. It may greatly extend the analytical usefulness of semiconductor laser fluorimetry. Registry No. DDTC, 20682-18-2; SDS, 151-21-3; DBS, 25155-30-0; benzene, 71-43-2.
LITERATURE CITED (1) Kllger, D. S. “Ultrasensitive Laser Spectroscopy”; Academic Press: New 1983. . York. . (2) Kawabata; Y:;-Kamikubo, T.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1983, 5 5 , 1419. (3) Imasaka, T.; Kamikubo, T.; Kawabata, Y.; Ishibashi, N. Anal. Chlm. Acta 1983, 753, 261. (4) Ohtsu, M.; Kotani, H.; Tagawa, H. Jpn. J . Appl. Phys. 1983, 2 2 , 1553. (5) Soini, E.; Hemmiia, I.Clin. Chem. (Winston-Salem, N . C . ) 1979, 2 5 , 353. (6) Maeda. M.; Banno, M.; Ogo, Y. Bull. Nippon Kanko Shiklso Kenkyusho 1973. (7) “Organic Chemicals List”; Nlppon Kanko-Shikiso Kenkyusho: Okayama, 1969 (supplement 1974). (8) Kimura, K., Ed. ”Handbook of Analytical Chemistry”; Maruzen: Tokyo, 1971. (9) Scifres, D. R.; Burnham, R. D.; Lindstrom, C.; Streifer, W.; Paoli, T. L. Appl. Phys. Lett. 1983, 42, 645. (IO) Yamamoto, S.; Hayaphi, H.; Hayakawa, T.; Miyauchi, N.; Yano, S.; Hijikata, T. I€€€ J. Quantum. Elecfron. 1983, Q€-79. 1009. (11) Yasuda et ai. ‘Nikkei Electronics, Sept 27, 1982, p 127. (12) Goidman, M. “Fluorescent Antibody Methods”; Academic Press: New York and London, 1968. (13) Hewig, G. H.; Jain, K. Opt. Commun. 1083, 4 7 , 347.
RECEIVED for review November 14,1983. Accepted February 3, 1984. This research is supported by a Grant-in Aid for Scientific Research from the Ministry of Education of Japan and by a Kajima Foundation Research Grant.
