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|Title: ||Development and application of a compact long wavelength fluorescence detection system|
|Authors: ||Hu, Sijung|
|Issue Date: ||2000|
|Publisher: ||© Si Jung Hu|
|Abstract: ||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
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
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
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.|
|Description: ||A Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.|
|Appears in Collections:||PhD Theses (Chemistry)|
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