Studies On Microfluidic Chip-based Sensitive Laser Induced Fluorescence Detection System And Its Applications To Integrated Biochemical Analysis Systems

In recent years, microfluidic chip based analysis has made great progress in realizing the lab-on-a-chip orμTAS concept of integrating all components in analytical system onto a single chip. However, highly sensitive detection techniques, such as laser induced fluorescence (LIF), have to be employed in such systems in order to detect the minute amounts of analyte in the microchannels in micrometer scale in the chips.In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration with a 1.5 mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. An optimized fluorescence collection angle of 45°in the chip plane was chosen in the LIF system with a scattered light intensity of 1/38 of that obtained at angle of 90°. Sodium fluorescein and fluorescein isothiocyanate (FITC) labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N=3) of 1.1×10^-12 M fluorescein was obtained, which is comparable to optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness and ease in building.In the subsequent work, a miniaturized on-chip flow cytometry and an integrated amino acid bioanalyzer were developed based on the orthogonal LIF detection systems. The fluorescent beads flowed through the microchannel in the chip confined in a gravity-driven sheath flow formed in the channel. The counting of the beads was performed by detecting the forward scattered light at the angle of 6°. The detection of the side scattered light and fluorescence was achieved by collecting the light emitted from the microchannel through the sidewall of the chip with a collecting angle of 90°for side scattered light and 45°for fluorescence light, respectively. A maximum analysis throughput of 80 particles per second was achieved with a signal sampling rate of 1000 Hz. The detection limit of the system for sodium fiuorescein was 100 pM (S/N=3).The orthogonal LIF detection system was also applied in the development of an microfluidic chip-based amino acid bioanalyzer, which integrated capillary electrophoresis chip, miniaturized fluorescence detector, high voltage power supply for CE separation and control systems. A mixture of 9 amino acid solutions was used as a model sample to demonstrate the performance of the amino acid bioanalyzer. The mixture of amino acid solutions can be well separated within 3 minutes. The size of the integrated bioanalyzer was only 300 mm X 200 mm X 200 mm.