在傳統的微流體系統偵測中，單一螢光偵測技術早已成熟發展並應用於毛細管電泳和流式細胞儀上，其有操作效能較為低落之缺點。為了提升可同時偵測的螢光樣本數量，研究上常以多重管道及多重螢光偵測的方式實現。然而，此類系統需使用較為複雜且精密的光學零組件、多組濾鏡系統和光感測器來提高可偵測的樣本數，而增加系統成本及體積。因此，本研究利用暗視野光源的特性，發展一種簡單且具創新性的光學架構，應用於微流體管道內的生物樣本偵測，藉由多波長激發及多波長量測技術，提高同時可偵測螢光和生物樣品之數目。
本研究初步驗證暗視野光源在多波長偵測上的可行性，以一商用暗視野聚焦鏡，創造一中空圓錐狀的聚焦光源取代傳統雷射誘導螢光技術。此暗視聚焦光源可有效激發微管道內的螢光分子並避免激發光源進入接收物鏡，配合一紫外-可見光-近紅外線光譜儀做為訊號接收元件，取代原有的濾鏡組件和複雜的控制系統，用以達到多波長多樣本的偵測。研究中採用一連續波長的可見光源做為激發光（400 nm到900 nm），以激發不同激發波長的螢光分子。實驗結果顯示，此暗視野架構成功在單一微管道下同時偵測一包含Atto610、Rhodamine B和FITC三種螢光染劑之混合螢光樣本。除此之外，本系統亦成功分離且同時偵測分別標定Cy3和FITC螢光之單股DNA混合樣本。雖然本研究架構中的偵測極限低於傳統的雷射誘導螢光偵測系統，約為10-5 M（SNR=5.56以FITC濃度為參考），但此系統在平行偵測及多波長解析的潛力卻是值得被注意以及深入探討的。
有鑑於利用商用暗視野聚焦鏡所聚焦之光源，由於其光點大小和數值孔徑（numerical aperture, N.A.）皆無法任意改變的情況下，用以激發微管道內螢光時，管壁內所產生的大量散射而造成螢光訊噪比低落的缺點。本研究利用一高數值孔徑物鏡和塑膠光罩光擋片，創造出一自製的高效能暗視野聚焦光系統鏡以改善商用暗視野聚光鏡之聚光效果，期能達到較佳的螢光偵測效率。研究中利用實驗找出光擋片設計之最佳參數，以降低管道內散射問題並達到良好的激發效能。螢光量測結果顯示，經最佳化的光擋片可量測到5×10-8 M之螢光染料（SNR=3，以2',7’-dichlorofluorsin，2',7’-DCF濃度做為參考），其效能與利用商用暗視野聚光鏡之架構高出約一百倍。
將此物鏡式暗視野架構應用於電泳偵測時，顯示出新架構對螢光分子的偵測更為靈敏且有效率。實驗利用一包含2',7’-DCF、Rhodamine B、Atto610和Atto647N之混合螢光樣本進行單一管道之電泳偵測，及使用螢光標定之單股DNA混合樣本，進行系統效能驗證。除此之外，本研究更提出一簡單且快速的計算和分析方式，其可有效對電泳圖譜訊號之雜訊與螢光光譜交互干擾進行濾除，以達到快速解析多樣本之目的。
最後，將此高效能之暗視野聚光架構應用在流式細胞偵測晶片上，以進行連續流體中的細胞計數，並將所偵測之光譜訊號進行波長解析，進而達到多變數偵測目的以利細胞分類及辨識。本研究利用此光學架構可偵測不同大小和帶有不同螢光的微米小球，依據微米小球在暗視野下的光譜訊號，可將其側向散射、吸收和螢光等參數值計算並量化，並做為細胞分類和辨識的基礎，實驗成功將一混合螢光小球樣本有效分類並計數。此外，本研究亦使用腸道上皮癌細胞做為實際細胞偵測的應用，並將細胞標定上Tripan-blue和Erythrosin-blush等染劑，並成功以所發展出之系統對該樣本進行快速分類與數量統計。
由以上研究結果中指出，此暗視野偵測架構應用於晶片電泳和流式細胞計數器時，皆表現出其多樣性的偵測潛力，對於微管道內的生物樣本偵測提供一快速且低成本的方法。未來亦可藉由光源系統的變更以及分析計算的精進，將此偵測架構實際應用於生物、醫藥和病理等偵測。

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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