Development and application of a compact long wavelength fluorescence detection system

Long wavelength (>600nm) fluorescence has many advantages in analysis, including the presenting possibility of building compact, robust, yet sensitive instrumentation, where measurements can be made with minimal autofluorescence and scattered light from biological samples. With the requirement for high sensitivity in immunoassays, e.g., for environmental monitoring, clinical analysis and therapeutic drug monitoring, a novel, compact, fluorescence detection system (NFDS) was successfully constructed using several pulsed diode lasers as excitation sources, and a photodiode as optoelectronic sensor. It has the following characteristics: 1. Excitation source range: 600 nm- 900 run, and emission wavelength range: 650 run -1000 run. 2. Utilisation of various cut-off filters to eliminate undesirable background radiation and to define the fluorescence wavelength band. 3. Emission beam detection by means of an efficient, high speed and large area silicon photodiode. 4. An adjustable laser pulse frequency and linear optoelectronic amplification. 5. Digital display, output for a digital multimeter or a chart recorder, and analogue to digital converter (ADC) for connecting to a PC with a suitable data handling package. A 635 run-laser diode with the output power of 2 m Wand a 650 nm cut-off filter were used to test the detection limit of the naphthofluorescein fluorescence dye (NF) in 0.50 M Tris buffer (PH 8.8), containing 2.5% (w/v) 3-[(3-cholamidopropy)dimethylammonio ]-l-propanesulfonate (CHAPS). A 645 nm laser diode with the output power of 2m Wand a 665 nm cut-off filter were used to test the detection limit of Cy5 monofunctional dye (Cy5) in 0.50 M Tris buffer (pH8.8). A 670 nm laser diode with the output power of 2mW and a 690 nm cut-off filter were used to test the detection limit of Cy5.5 bisfuncational dye (Cy5.5) in 0.50 M Tris buffer (PH 8.8). A comparative test was carried out to assess the detection limit of Cy5 in 0.50 M Tris buffer (PH 8.8), using the NFDS with the RACALL-DANA 4009 digital multimeter and the Hitachi F-4500 commercial research grade fluorescence spectrometer. A flow injection immunoassay was developed using the a-Interferon as the analyte and Cy5 as the label. A calibration curve was obtained using the NFDS with the FlowTEK data capture software. The potential of this novel fluorescence detector has been demonstrated through hardware experimentation and practical investigation of detection limits and a flow injection immunoassay (FIlA). Its application could be extended by the use of superluminescent light emitting diodes (SLEDs) at shorter wavelengths (450 nm - 600 nm); a microprocessor based electronic system, and the LabVIEW 5.0 software for data capture