Anal. Chem. 1994'66, 3102-3107
Instrument To Detect Near- Infrared Fluorescence in Solid-Phase Immunoassay Richard J. Wllllams, Naraslmhacharl Narayanan, Gulllermo A. Casay, Malgorzata Llpowska, Lucjan Strekowskl, and Gabor Patonay' Department of Chemistry, University Plaza, Georgia State University, Atlanta, Georgia 30303
Jose Mauro Peralta Departmento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil Victor C. W. Tsang Division of Parasitic Diseases, Centers for Disease Control, Atlanta, Georgia 3034 1
The construction of a near-infrared (near-IR) fluorescence detector for measuring picomolar levels of near-IR laser dyes is described. The detectoris designed for use in an immunoassay technique that employs antibodies labeled with near-IR polymethine cyanine dyes. These dyes possess spectral properties that are exclusive to the near-IR region (650-1100 nm). The instrumentation is characterized, including its hardware and data acquisitionsoftware components. The detector is capable of measuring fluorescence in both solution and solid-phase environments. Data on the detector's performance is presented. The use of laser-induced fluorescence in the development of immunoassays has been widely reported.' The limitations of laser-induced fluorescence depend mainly on the excitation source. These limitations include: the high price and maintenance cost of lasers, their large size, and their limited ability for wavelength selection.lI2 Recent advances in semiconductor laser technology have made the use of lasers as an excitation source more p r a c t i ~ a l . ~Near-IR-emitting -~ semiconductor laser diodes have become more readily available because of their increased use in telecommunications and data handlinga2This type of laser is inexpensive, small and compact in size, and reliable. The GaAlAs laser diode has drawn interest because its emission wavelength of 800 nm is compatible with the excitation wavelength of several classes of polymethine cyanine dyes that fluoresce in the near-infrared region. The spectral properties of these classes of polymethine cyanine dyes offer advantages for detection in the near-IR regiorg-12 These near-IR dyes typically possess large molar ( I ) HemmilB.1. A. Applicarions of Fluorescence in Immunoassays; John Wiley and Sons, Inc.: New York, NY, 1991. (2) Svelto.0. Principles of Luasers, 3rd ed.; Plenum Press: New York, 1989. (3) Imasaka, T.; Yoshitake, A.; Ishibashi, N. Anal. Chem. 1984.56.1077-1079. (4) Sauda, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1986.58, 2649-2653. (5) Imasaka, T.; Yoshitake, A,; Hirata, K.; Kawabata, Y.; Ishibashi, N. Anal. Chem. 1985,57,947-949. (6) Imasaka, T.; Ishibashi, N. Anal. Chem. 1990, 62, 363A-371A. (7) Imasaka, T.; Tsukamoto, A.; Ishibashi, N. Anal. Chem. 1989,61,2285-2288. (8) Imasaka, T.; Nakagawa, H.; Okazaki, T.; Ishibashi, N. Anal. Chem. 1990, 62, 2404-2405. (9) Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 63, 321A-326A. (10) Boyer, A. E.; Lipowska, M.; Zen, J. M.; Patonay, G.; Tsang, V. C. W. Anal. Lett. 1992, 25, 415-428.
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Table 1. Some Spectral Propertlor of Dyes Uwd In Thls Study
molar absorptivity (L mol-' cm-l) Xabs max
Xflr max
quantum yield (%)
.
MeOH
H20
179 260 168 785 34.8
186 866 774 791 24.9
absorptivities and above average quantum yields. In the nearIR region, there is very little background interference from molecules other than the molecules of interest. The detection limits of such molecules are more dependent on the performanceof thedetection instrumentation and less on background interference. The near-IR heptamethine cyanine dyes are a particularly interesting class of near-IR polymethine cyanine dyes because they can be tailored for different a p p l i c a t i ~ n s . ~ J By ~**~ changing the functional moieties added, derivatives of these dyes have been used for several different tasks including pH determination, metal ion analysis, DNA labeling, etcag-l1~14,15 Their synthesis has been reported previously in the literature.13J6 Some spectral properties of the heptamethine dye called "dye 1" are shown in Table 1. This near-IR dye has a maximum absorbance at 778 nm and a fluorescence spectrum that extends beyond 840 nm. The dye possesses a high molar absorptivity and a high quantum yield in an aqueous environment. The dye also possesses a relatively short fluorescence lifetime (400-700 ps) compared to that of conventional visible dyes used as labels (40-60 ~ s ) . ~ This J' short lifetime suggests that cyanine dyes similar to dye 1 are less susceptible to photodestruction and photobleaching. These (1 1) Williams, R. J.; Lipowska, M.; Patonay, G.; Strekowski, L. Anal. Chem. 1993,
65, 601-605. (12) Fabian, J.; Nakazumi, H.; Matsuoka, M. Chem. Reu. 1992, 92, 1197-1226. ( 1 3) Strekowski, L.; Lipowska, M.; Patonay, G. Synrh. Commun. 1992.22.25932598. (14) Zen, J.; Patonay, G. Anal. Chem. 1991, 63, 2934-2938. (1 5) Patonay, G.; Antoine, M. D.; Devanathan, S.;Strekowski, L. Appl. Specrrosc. 1991, 45, 457461. (16) Strekowski, L.; Lipowska, M.; Patonay, G. J. Org.Chem. 1992, 57, 45784580. (17) Soper, S. A.; Mattingly, Q.L.; Vegunta, P. Anal. Chem. 1993.65, 740-747. 0003-2700/04/0366-3 102$04.50/0
0 1994 American Chemical Society
Laser housing
'
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Figure 1. The instrument used for measuring the near-infrared fluorescence in this study consists of four main components: the excitationsource, the detector, the sample-holdingapparatus, and the data acquisition interface.
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1UG2A dyes are also very soluble in water. This solubility can be P enhanced by the addition of up to four sulfonate groups per Casw molecule. Diodes *.-... 5 A photoinduced quenching effect was observed for dye 1 LlO24MFO 1 f lclF when it was excited by a low-powered semiconductor laser. That is, the maximum fluorescence signal rapidly decreased .sv after the laser light was held fixed on the sample that contained Figure 2. The optical arrangement for the excitation source of the dye. This implied photodestructionof the dye had occurred. detector. Laser light is collimated through an interference filter and However, when the light was removed and returned an instant then focused onto sample. A diagram of the feedback control circuit used to power the semiconductor laser is also shown. later, the fluorescence signal returned to its maximum signal. This phenomena will be discussed later in this paper. Since silicon photodiodes generally have good responsivity between 780 and 790 nm. The technical specifications of the in the near-IR region, a simple optical system using a silicon LT024MFO are described in the current literature.2120 photodiode as the detector should give reasonable r e ~ u l t s . ~ * ~ J ~ J ~ Laser oscillations inside of the optical cavity of the FabryA detection system consisting of a near-IR semiconductor Perot resonator create standing waves due to constructive laser diode, simple optics, and a silicon photodiode was interference. Also, laser oscillation occurs at the wavelength proposed for an immunoassay that uses near-IR-labeled that corresponds with the maximum gain. The maximum antibody. Of course, this immunoassay should have a detection gain depends on the band-gap energy,and the band-gap energy sensitivity comparable to what is currently available with the varies with temperature. Variations in temperature during enzyme-linked immunosorbent assay (ELISA) technique. operationcan result in wavelength shifts of the emerging beam. Optical filters at the desired wavelength of operation must be INSTRUMENTATION used to compensate for these effects and produce pure laser The instrument used for measuring the near-infrared light at the required wavelength of excitation. fluorescence in this study consisted of four main components: The focusing optics for the excitation source is shown in the excitation source, the detector, the sample-holding apFigure 2. The laser beam was collimated with a plano-convex paratus, and the data acquisition interface (Figure 1). The lens (19.0-cm focal length, 12.7-cm diameter) from the excitation source was a semiconductor laser that emitted in Newport Corporation (Fountain Valley, CA) through a 12.5the 800-nm range. A fiber-optic cable was added to control mm diameter, 790-nm interference filter from the Andover the geometry of the excitation source. The detector was a Corporation (Salem, NH) and then focused onto the sample single-source silicon photodiode. The sample-holding ap(located in either a stationary cuvette or a z-direction paratus was designed to support a fluorometer cuvette for translating sample holder) with a duplicate plano-convex lens. single stationary measurementsin solution or to support solid Variations in the ambient temperature cause fluctuations matrix plates that can be directionally scanned for several in laser power accompanied by changes in the laser energy solid-phase measurements of different samples on the same output. A feedback control circuit was used to maintain a plate. constant power output in an environment where the temThe excitation source was a semiconductor laser diode, perature varies. This was done by comparing the optical output type LT024MFO from Sharp Electronics (Sharp Corporation, of the laser with a monitor photodiode built into the laser Osaka, Japan). This type of laser diode is a direct gap gallium apparatus. A diagram of the feedback control circuit used aluminum arsenide (GaAlAs) semiconductor that emits to power the semiconductor laser is shown in Figure 2. The 1C3C02A current regulator from Sharp Electronics (Sharp (1 8) Lakowicz, J. R. Principles of FluorescenceSpectroscopy;Plenum Press: New ~
York, 1983. ( 19) Schulman,S. G. Fluorescence and Phosphorescence Spectroscopy: Physi-
cochemical Principles and Practice; Pergamon Press: Elmsford, NY, 1977.
(20) Sharp Laser Diode User's Manual; Sharp Electronics Corporation: Osaka, Japan, 1992.
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Corporation, Osaka, Japan) was used to insure an uninterrupted delivery of laser energy. In addition, the laser diode was attached to a heat sink to minimize variations in ambient temperature. A Peltier cooling element may be added if additional temperature control is desired. The silicon photodiode is an ideal detector for near-infrared fluorescence radiation because it offers good sensitivity well into the near-IR region, it can operate efficiently at room temperature, and it does not require a restrictive power source or an elaborate control setup to operate The silicon photodiode was chosen because it offers the simplest and most practical solution in detecting faint amounts of nearIR radiation. The responsivity of silicon photodiodes is typically above 0.5 A/W for the near-IR region up to 1000 nm.1J7921722 The responsivity performance is generally better than that of conventional photomultiplier tubes (PMT’s). In addition, the silicon photodiode does not require special high voltage power sources in order to operate. Typically, PMT’s require power sources in the 750-1000-V range. In the nearIR region, the sensitivity of avalanche silicon photodiodes is better than that of silicon photodiodes. However, at low light levels, the temperature control required to reduce dark current noise can add considerable cost to a detector setup using an avalanche photodiode. The focusing optics for the fluorescence detector is shown in Figure 3. To minimize scattered light effects, fluorescence was detected above 820 nm, more than 30 nm away from the excitation wavelength of the laser. A UV-100BG photodiode from EG&G (Vaudreuil, Quebec, Canada) was the main component of the detector. Fluorescence radiation was collimated with a plano-convex lens (25.4-mm focal length, 25.4-mm diameter) from Rolyn Optics (Covina, CA) through two 25-mm, 820-nm and one 25-mm, 830-nm interference filters from Omega Optical (Brattleboro, VT) and focused onto the photodiode with a plano-convex lens (3 1.7-mm focal length, 25.4-mm diameter) from JML Direct Optics (Rochester, NY). A diagram of the detector-amplifier circuit is shown in Figure 3. The signal from the photodiode was measured as voltage using a current-to-voltage circuit with a built in amplifier. Nine operational amplifiers (op amps) from Analog Devices (Norwood, MA) were used as both first- and secondstage amplifiers. An AD549 op amp was used for the first stage and an AD707 op amp was used for the second stage. Signal sensitivity was regulated by the resistance across the feedback loop of the AD549 operational amplifier. Resistors with resistances greater than 1 Gohm gave optimal results. Resistors from Morganite Resistors, Ltd. (Durham, England) with resistances of 1and 9 Gohms were chosen for the detector used in this study. Resistor “Johnson” noise associated with random thermal motion from the flow of charge carriers was negligible compared to noise associated with the circuit. To minimize the observed photoinduced quenching effect, the samplecontaining dye 1was moved in they direction past the laser and detector. This was accomplished by coupling an x-y slide platform from a light microscope with a motorized y translator. The sample movement was motorized in the y (21) Li, L. Q.;Davis, L. M. Rev. Sci. Instrum. 1993, 64, 1524-1529. (22) Mielenz,K. D., ed. Optical Radiation Measurements: Volume3: Measurement of Photoluminescence, Academic Press, Inc.: New York, NY, 1982.
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detector
sample
NIR Detector Circuit to offset circutt
Signal out
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Flgure 3. The optical arrangement for the fluorescence detector. Interference filters at 820 and 830 nm are used to reduce scattered light. The measured fluorescence is confined to the region where the filters overlap. A diagram of the circuit for the detector is also shown. A 9 Gohm resistor is used to increase the sensitivity of the currentto-voltage signal. A secondamplifier is also included.(The circuit diagram appears courtesy of Analog Devices, Inc., Norwood,MA.)
direction only. The y-axis speed was controlled by a variable power source and reversible switches that allowed translation of the entire platform length to take place in both directions. Translation in the x direction covered the entire width of the platform. It was manually controlled and used to optimize the fluorescence signal. In moving the sample past the excitation source and detector, a voltage peak was created that was proportional to the concentration of dye contained in the area scanned (Figure 4). Either the peak height or the area under the peak could have been used as a measurement of the fluorescenceintensity associated with dye concentration. The analog voltage generated by the photodiode circuit was converted to a digital signal and then interfaced with a personal computer for data acquisition. The analog-to-digital converter board used in this instrument setup was the DAS-8, a 12 bit, ADC converter board from Keithley Metrabyte (Tauton, MA). The data acquisition software used was the Easyest LX software, also from Keithley Metrabyte. The personal computer used in this instrumentation setup was a 386sx, 33-MHz, IBM-compatible personal computer from Gain Systems (Norcross, GA).
EXPERIMENTAL SECTION: The near-IR fluorescence detector described above was characterized by measuringthe fluorescence intensity of nearIR dye adsorbed on solid surface^.^^-^^ The relationship between the fluorescenceintensity signal to the blank signal
I NIR dye spots on nitrocellulose 1)
I signal I scan
~
'I ~
Flgure 4. A voltage peak is created that corresponds to the concentration of dye contained in the area scanned as the sample is moved past the excitationsource and detector. The peak height or the area under the peak can be used as a measurement of the fluorescence intensity that is directly related to the concentration of dye.
ratio ( F I B ) and laser power was determined to find the optimum operating range of the instrument. The relationship between the angle of detection was compared to the FIB in order to find the angle between the detector and the excitation sourcewhere undesired reflected and scattered light from the laser was minimized. To minimize the photoinduced quenching effect, FIB was compared to the instrumentscanning speed. Finally, the detection limits of dye 1 were determined in an aqueous solution and on a solid-phase matrix of nitrocellulose. Most materials generate some background fluorescence.17-19 Fluorescence lifetimes are usually shorter for near-IR molecules.17J8 This is mainly because of the smaller differences between vibrational energy levels in the near-IR region. For the same reason, ultraviolet-visible (UV-vis) fluorophores generally have longer lifetimes than near-IR fluorophores. Because the fluorescence lifetimes of molecules from these respective regions are different, the rate of photobleachingis much less for some near-IR fluorophores, such as the polymethine cyanine dyes. Laser power and scan speed were optimized to determine the instrument conditions that take advantage of the fluorescence lifetime differences between the near-IR fluorophores and those of the materials exhibiting background fluorescence so that background fluorescence could be minimized. F/B vs Laser Power. Nitrocellulose was chosen as the solid matrix because of its high protein affinity and its low reflective surface.26 Strips of nitrocellulose containing dye 1 were prepared by incubating 1-cm2strips of nitrocellulose in M solutions of dye in the wells of a 24-well 500-pL of polystyrene reaction plate for 1 h. Blanks were prepared by incubating 1-cm2nitrocellulose strips in wells containing 500pL of water. The strips were then washed three times at 5 min per wash in phosphate buffered saline (PBS) solution (pH 7.2). The fluorescence intensity was determined for the (23) Larson, A. P.;Ahlberg, H.; Folestad, S. Appl. Opt. 1993, 32, 794-805. (24) Mathies, R. A.; Peck, K.; Stryer, L. Anal. Chem. 1990, 62, 1786-1791. (25) White, J. C.; Stryer, L. Anal. Biochem. 1987, 161, 442452. (26) Brown, W. R.; Dierks, S. E.; Butler, J. E.; Gershoni, J. M. Immunoblotting:
Membrane Filters as the Solid Phase for Immunoassays. In Immunochemistry ofSolid-phaseImmunoassay; Butler, J. E., Ed.; CRC PressJnc.: Boca Raton, FL, 1991; pp 151-171.
nitrocellulose strips at various laser powers that ranged from mW. FIB was then plotted vs laser power. 18 to 18 X F / B vs Detection Angle. Nitrocellulose strips were prepared as described above. The operating power of the semiconductor laser was set at 20 mW. Fluorescence intensity was determined for both blank strips and strips that contained dye at various angles of detection that ranged from 60' to 160'. FIB was then plotted vs the angle of detection. F / B vs Scanning Speed. Nitrocellulose strips were prepared as described above. Fluorescence intensity was determined for the nitrocellulose strips at various scanning speeds that ranged from 0.33 to 0.10cm/s, while keeping the operating power of the laser at 20 mW. FIB was then plotted vs scanning speed. Detection Limits. The detection limits were determined by measuring the fluorescenceintensity of different concentrations of dye 1 in aqueous solutions and on a nitrocellulose solid-phase Only the solid-phase matrix was scanned. Solutions of sample were housed in a stationary quartz fluorometer cell (12.5 X 12.5 X 10 mm, volume = 3 mL) for the liquid-phase measurement of fluorescence. A stock solution of dye 1 was prepared with a concentration of 1 mg/mL in water. Dilutions were made for concentrations extendingto 10-loM. The solutions were placed in the sample fluorometer cell, and the fluorescence was measured for each concentration. The detection limits were determined from the results. The width of the laser excitation beam was calculated from the optical arrangement as outlined by Larson et al.23and used to determine the working volume of the spot illuminated by the laser. This volume was used to determine the number of excited dye molecules that corresponded to the minimum fluorescence signal. The effective width of the spot size of the excitation laser beam was determined to be 1 f 0.03 pm. This corresponded to working volumeof 3.9 X 10-loL for a solution in the cuvette. The given spot size allowed the complete saturation of the photodiode surface after magnificationfrom the detector optics. Any spot size larger than 1 pm resulted in truncation at the photodiode surface. For the solid-phase detection limit determination, 1-cm2 strips of nitrocellulose were prepared with dye 1 as outlined above with different concentrations of dye. A stock solution containing 1 mg/mL of dye 1 in water was diluted in water to create various concentrations of dye 1 to 10-lo M. The volume of nitrocellulose illuminated by the laser was determined to be 5.7 X L.
RESULTS AND DISCUSSION The results of the comparison of FIB and laser power is shown in Figure 5. A sharp increase in the ratio occurred at 18 mW near the peak operating power of the laser. This was a result of the relative fluorescence lifetimes and photostabilities of dye 1and the nitrocellulose from the solid matrix. The lifetime of nitrocellulose is much shorter than that of the near-IR laser dye. This implied that photobleaching of nitrocellulose occurred at a lower laser power than the nearIR laser dye. The sharp FIB increase observed at 18 mW was (27) Johnson, P. A.; Barber, T. E.; Smith, B. W.; Winefordner, J. D. Anal. Chem. 1989,61, 861-863. (28) Long, G. L.; Winefordner, J. D. Anal. Chem. 1983,55,712A-724A.
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I
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Scan Speed (mmlsec)
Power (watts) Figure 5. Comparison of the signal-to-blank ratio and laser power. The sharp ratio increase observed at 18 mW is an indication that the background fluorescence from nitrocellulose molecules on the solid matrix has undergonesignificant photobleachingwhile dye 1molecules remain photostable.
'c 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Angle of Detection (degrees) Figure 6. Relationship between the angle of detection and the signalto-blank ratio. The largest signal-to-blank ratio occurred at 64'. The minimum ratio occurred at 90'. Scattered light is minimized near 60° and a maximum at 90'.
an indication that the background fluorescence from nitrocellulose molecules on the solid matrix had undergone significant photobleaching while dye 1 molecules remained photostable. The optimum operating power of the semiconductor laser was determined to be 18 mW. By operating the laser above 18 mW, background fluorescence from nitrocellulose was significantly reduced and the detection of the fluorescence intensity of dye 1 became more instrument dependent. The relationship between the angle of detection and F / B can be determined from the results shown in Figure 6. The largest FIB occurred at 64'. The minimum ratio occurred at 90'. This implied undesired reflected and scattered light from the laser source was minimized near 60' and a maximum at 90'. By the optical laws of reflection,the minimum observed at 90" was due to the mirror effect of incident radiation. The maximum intensity of scattered light was being reflected at this angle. By the same optical effect, the scattered light intensity was minimized near 60'. Here, the amount of scattered light reaching the surface of the photodiode detector had been minimized while the fluorescence radiation reaching 3106
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Figure 7. Relationship between scan speed and the signal-to-blank ratio. As the scan speed increases, the photostability effects are minimized and the limitations of the instrument become the more dominant factor. The ratio reaches a maximum at 3.2 mm/s and decreases at higher speeds.
the photodiode surface was at its peak. The constant ratio observed past the 90' angle of detection was an indication that less light was reaching the surface of the photodiode detector at these angles. The optimum scan speed was determined to be 3.2 mm/s. The relationship between scan speed and FIB is shown in Figure 7. This relationship depended mainly on the relative photostabilities of the dye 1 and nitrocellulose molecules, the photoinduced quenching observed for dye 1, and the limits of the time constant associated with the photodiode detector. In the region of slower scan speeds, the large ratio observed at the slowest speed was due to the relative differences in the fluorescence lifetimes of dye 1 and nitrocellulose molecules. At this speed, both photoinduced quenching and photobleaching effects were at work respectively for dye 1 and nitrocellulose. The high ratio indicated the photobleaching of the background fluorescence of nitrocellulose was occurring at a much faster rate than the photoinduced quenching of the fluorescence of dye 1. As the scan speed increased, the photostability effects were minimized and the performance of the instrument became the more dominant factor. The ratio reached a maximum at 3.2 mm/s and decreased at higher speeds. In this region, the time constant limits of the detector imposed a slow response time for the detector to reach saturation. This high time constant helped to smooth signal noise and increase detection sensitivity. The photoinduced quenching effect observed appeared to be the result of some sort of dimerization process.29 At first, this effect was thought to be due to photobleaching of dye 1 molecules. However, the thermodynamic requirements of the photobleaching process suggested it would be irreversible. The results observed for excited dye 1 molecules clearly showed that the fluorescence intensity signal returned to its maximum after the laser was removed and returned sometime later. The energetic requirements of dimerization of these near-IR dyes were well within the range of the energy produced by laser excitation and also low enough to be a reversible process. Since dimerization produces a known quenching effect, it was (29) West, W.; Pearce, S. J. Phys. Chem. 1965,69, 1894-1903.
thought to be the mechanism chiefly responsible for the reversible process that was observed here. The cause of this photoinduced quenching effect will be further studied in future research. A detection limit of 175 000 molecules was observed for an aqueous solution of dye 1, while a detection limit of 65 000 molecules was observed for dye 1 on the nitrocellulose matrix for volumes corresponding to a spot size of 1 f 0.03 pm. Previous literature has presented results with detection limits of less than 45 000 molecules in solution for other polymethine cyanine This implies further optimization of the detector is necessary for optimum results. The lower detection limit for the solid phase was expected because, in solid-phase fluorescence detection, more fluorescent molecules are concentrated in an area without the quenching effects of solvent molecules.
CONCLUSIONS An instrument designed to detect the near infrared fluorescence signal generated by near-IR polymethine dyes with a simple optical system was constructed. It was constructed of readily available components and was optimized to take advantage of the unique spectral properties of the near-IR laser dyes. The excitation source was a semiconductor laser diode that operates at room temperature and generates a stable output and requires little maintenance. The detector was a semiconductor photodiode. The reponsivity of the photodiode in the near-IR region was superior to commercially available photomultiplier tubes. The detector was operated at room temperature without any special power requirements. The instrument, as constructed, detected ultra-trace levels of near-IR polymethine cyanine dyes through the use of an analog-to-digital converter and a personal computer. Voltage signals corresponding to the fluorescence intensity of the nearIR laser dyes were obtained by scanning the area of a solidphase nitrocellulose surface that contained the dye. These (30) Middendorf, L. R.;Bruce, J. C.; Bruce, R.C.; Eckles, R. P.; Grone, D. L.; R o w " , S. C.; Sloniker, G.D.; Steffens, D. L.; Brumbaugh, J. A,; Patonay, G. Electrophor. J . 1992, 13, 481492.
signals were converted to peaks that corresponded to the concentration of near-IR dye with the ADC converter and a simple data acquisition software package. The fluorescence intensity was recorded as peak height and related directly to the concentration of near-IR dye present on the nitrocellulose strips. The detection limits of this instrument allowed the detection of low levels of dye 1. Concentration levels approximating 100 pmol/L were detected for a spot size of 1 f 0.03 pm. Detection limits well past the picomole per liter range can be obtained by further optimizing the instrument optics and by adding a more sensitive detector. The instrument described was designed to be the detector in a near-IR solid-phase fluorescence immunoassay. The immunoassay will make use of near-IR fluorophores, similar to dye 1, as dye labels. The minimal interference incurred in the near-IR region and the low detection levels of the near-IR fluorophores should potentially allow detection limits of antigen that are comparable to those of the conventional enzymelabeled immunoassay techniques (i.e. ELISA). A solid-phase matrix such as nitrocellulose is preferred because it can generate a strong and consistent fluorescence signal with a minimum of interference. When using this near-IR fluorescence immunoassay in place of conventional techniques such as ELISA, total assay time can be reduced by removing two time-consuming steps because no substrate is involved and the measurement of the quantifying signal does not have to take place in a wet solution.
ACKNOWLEDGMENT This work was supported in part by a grant from the National Institutes of Health (Grant 1 R01 AI 28903-01A2). Received for review February 17, 1994. Accepted June 15, 1994." ~
*Abstract published in Advance ACS Absrrucrs, August, 1, 1994.
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