| Summary: | 博士 === 國立成功大學 === 醫學工程研究所碩博士班 === 98 === In the conventional microfluidic system, detection of a single fluorescent is an established technique in capillary electrophoresis (CE) and cytometry applications. In order to increase the throughput in multi-sample detection, multi-lanes and multi-wavelengths systems have been developed. However, delicate optical components and various optical filters and photo-detectors such as photo-multiplier tubes (PMT) make these systems relatively bulky and expensive when increasing the number of detection samples. Therefore, this research presents a simple and novel configuration, composed of dark-field illumination and a spectral detector, to increase detection throughput for multiple bio-detection in a single microfluidic channel.
In the preliminary experiment, a commercial dark-field condenser is used to demonstrate the wavelength-resolved fluorescence detection for high-throughput analysis of bio-samples in a micro-CE chip. Instead of using a conventional laser-induced-fluorescence microscope equipped with delicate spatial filters and complex control systems, this experiment adopts a dark-field illumination combined with a continuous wavelength light source (400 nm to 900 nm) for exciting fluorescence in a microchannel and an ultraviolet-visible-near infrared (UV-Vis-NIR) spectrometer to detect the emission signals. The proposed system is simple and economical since no sophisticated optical filter sets and laser sources are required for excitation and detection. Experimental results show that the proposed system is feasible for simultaneously detecting a mixed sample composed of Atto610, Rhodamine B and FITC fluorescent dyes in a single test run. Furthermore, a mixed bio-sample composed of two single-strand DNA (ssDNA) samples labeled with Cy3 and FITC fluorescent dyes is also successfully separated and detected with the proposed system. The measured detection limit for detecting FITC fluorescein of the proposed system can be as low as 10^-5 M (SNR = 5.56).
In order to improve excitation performance and increase the detection limit, an objective-type dark-field technique is developed. The proposed system includes an objective-type dark-field condenser comprised of a high NA objective and light stop-film to excite fluorescence and a UV-Vis-NIR spectrometer via a low NA objective to detect emitted fluorescent signals. An optimized stop-film pattern for obtaining the best fluorescence excitation and reducing scattered light from the channel wall is determined by experimental results. This advanced configuration has a measured detection limit up to 5×10^-8 M (SNR = 3) while detecting a standard fluorescence of 2’,7’-dichlorofluoresein, a limit which is capable of detecting fluorescence samples in general applications.
In the CE application of this advanced configuration, experimental results show that the proposed system can effectively increase the fluorescent signals and simultaneously detect a mixed sample composed of 2’,7’-dichlorofluoresein, Rhodamine B, Atto610, and Atto647N. Furthermore, this advanced configuration also successfully separates and detects a mixed fluorescence-labeled bio-sample composed of three ssDNA samples in a single channel. In addition, a simple and fast calculation removes scattering noise and spectral cross-effect to obtain reliable electropherograms for analysis.
In a cytometric application, using the configuration mentioned above, continuous cell counting and spectra analysis are realized in a single channel without using any spatial filters or delicate optical components. Results show that the developed system is capable of identifying different labeled particles and cell samples by extracting the side-scatter, absorption, and fluorescence signals from the information-rich spectra. This proposed detection technique has successfully counted and classified a mixed dummy sample composed of three fluorescence-labeled particles and one non-labeled particle, with sizes from 2 μm to 15 μm. Furthermore, a mixed bio-sample composed of Tripan-blue, Erythrosin-blush, and non-labeled AGS cells (gastric epithelial cells) can be successfully discriminated using this proposed system. Unlike the traditional filter technique, the continuous wavelength measurement enables efficient multi-color detection, identification, and classification for micro-flow cytometer applications. The developed diascopic illumination system will substantially impact the development of high performance CE and micro-flow cytometers using this simple and straightforward technique of spectral detection and analysis.
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