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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.

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The use of digital microfluidics (DMF) for sample handling in a microarray immunoassay was investigated. A two plate DMF device was used, with the top plate being used for the immobilization of antibodies for a sandwich immunoassay. A patterning procedure was developed for the top plate to expose patches of glass that were chemically modified, using silane chemistry, to allow for the covalent immobilization of antibodies. For creating microarrays, a set of parallel microchannels were used for the high density patterning of proteins onto the functionalized surface of the top plate. This patterning procedure was optimized to ensure the reproducibility of the immobilization and the physical integrity of the top plate. Preliminary work for a multiplexed immunoassay such as verification of cross-reactivity and detection schemes was also conducted. This work represents the initial efforts towards a microarray immunoassay on DMF, which has the potential to improve high throughput analysis.

The use of digital microfluidics (DMF) for sample handling in a microarray immunoassay was investigated. A two plate DMF device was used, with the top plate being used for the immobilization of antibodies for a sandwich immunoassay. A patterning procedure was developed for the top plate to expose patches of glass that were chemically modified, using silane chemistry, to allow for the covalent immobilization of antibodies. For creating microarrays, a set of parallel microchannels were used for the high density patterning of proteins onto the functionalized surface of the top plate. This patterning procedure was optimized to ensure the reproducibility of the immobilization and the physical integrity of the top plate. Preliminary work for a multiplexed immunoassay such as verification of cross-reactivity and detection schemes was also conducted. This work represents the initial efforts towards a microarray immunoassay on DMF, which has the potential to improve high throughput analysis.

Immunoassayer är en väldigt vanlig metod för koncentrationsbestämning av biomolekyler. Denna metod nyttjar antikroppars affinitet för vissa analyter. I detta projekt har det undersökts om Gyros AB:s detektionsinstrument kan göras känsligare. Flera förslag har tagits fram genom efterforskning i artiklar och intervjuer med personer insatta inom området. De metoder som projektgruppen valt att presentera för företaget är enzymbaserad immunoassay med fluorescerande substrat samt multipel inmärkning av antikroppar, vilket vi tror kommer bidra till att försärka den fluorescerande signalen. Immobilisering av antikroppar med hjälp av traptavidin är ytterligare ett framtaget förbättringsförslag som syftar till att bidra med starkare och förlängd immobilisering av antikroppar till den fasta fasen i immunoassay. Flertalet andra metoder har undersökts såsom detektion med hjälp av radioaktivitet och kemiluminiscens. Inga fortsatta studier gjordes på grund av svårigheter att anpassa metoderna till de nuvarande instrumenten. Vi tror att de förslag på metodförbättringar som vi föreslår kan förbättra känsligheten på Gyros mätningar om de implementeras på den nuvarande plattformen.

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

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