Objective-type dark-field system applied to multi-wavelength capillary electrophoresis for fluorescent detection and analysis

This paper presents an advanced diascopic illumination technique for simultaneous multi-wavelength fluorescence excitation and detection without using any spatial filter sets. The proposed system includes a home-built dark-field condenser comprising a high N.A. objective and a light stop-film to excite fluorescence and an ultraviolet–visible–near infrared (UV–VIS–NIR) spectrometer to detect emitted signals. Since no direct light source enters the optical detection system, no complex optical filter is required for multi-wavelength fluorescence detection. This study also designs an optimized stop-film pattern to obtain the best performance in exciting fluorescent samples and reduce background light. Experimental results show that the proposed system can effectively increase the fluorescent signal and simultaneously detect a mixed sample composed of 2′,7′-dichlorofluorescein, Rhodamine B, Atto610, and Atto647N. Furthermore, this proposed system successfully separates and detects a mixed bio-sample composed of three single-stranded DNA samples labeled with Cy3, FITC, and Alexa647 fluorescence in a single channel. A simple and fast calculation removes noise and fluorescent cross-effect for conveniently observing the electropherograms. The proposed system has a measured detection limit up to 5 × 10−8 M (S/N = 3) while detecting a standard fluorescence of 2′,7′-dichlorofluoresein, which is capable of detecting fluorescence samples in general applications. The proposed method provides a simple and straightforward way to detect multi-wavelength fluorescence for CE analysis.