A simple and highly sensitive spectroscopic fluorescence-detection system for multi-channel plastic-microchip electrophoresis based on side-entry laser-beam zigzag irradiation

Abstract

A five-color fluorescence-detection system for eight-channel plastic-microchip electrophoresis was developed. In the eight channels (with effective electrophoretic lengths of 10 cm), single-stranded DNA fragments were separated (with single-base resolution up to 300 bases within 10 min), and seventeen-loci STR genotyping for forensic human identification was successfully demonstrated. In the system, a side-entry laser beam is passed through the eight channels (eight A channels), with alternately arrayed seven sacrificial channels (seven B channels), by a technique called "side-entry laser-beam zigzag irradiation." Laser-induced fluorescence from the eight A channels and Raman-scattered light from the seven B channels are then simultaneously, uniformly, and spectroscopically detected, in the direction perpendicular to the channel array plane, through a transmission grating and a CCD camera. The system is therefore simple and highly sensitive. Because the microchip is fabricated by plastic-injection molding, it is inexpensive and disposable and thus suitable for actual use in various fields.

An ultra-small, multi-point, and multi-color photo-detection system with high sensitivity and high dynamic range

“…As for the sixth, when multiple emission points are not in the capillary array or the microchip channel array, but on plates, it is necessary to combine the developed system with a fluorescence-excitation technique other than side-entry laser-beam irradiation. 14,18,27 As explained below, one promising technique is to use an array of epi-illumination systems. The long-pass filter in the system shown in Fig.…”

“…As for Sanger DNA sequencing, 14,15 plural DNA fragments labeled with different fluorophores for four kinds of nucleotide at their terminals are prepared from a sample and electrophoretically separated in each capillary of a capillary array, 14 or in each channel of a microchip-channel array, 15 and fluorescence from each capillary or channel is simultaneously (or sequentially) and spectroscopically detected. STR genotyping [16][17][18] is carried out by using similar analyzers to those used for Sanger DNA sequencing. As for next-generation DNA sequencing, 19 plural DNA fragments are separately colonized on a plate and incorporated step-by-step by any of four kinds of nucleotide labeled with different fluorophores, and at each step, fluorescence from each colony is simultaneously and spectroscopically detected.…”

“…The spectroscopic means can be categorized as time-division filters 9,12,19 dichroic mirrors, 4,5,15,16,[20][21][22] or wavelength dispersion. 8,10,17,18,23 With time-division filters, each of the different plural wavelength-band-pass filters is mechanically and sequentially inserted. With dichroic mirrors, a single dichroic mirror or a series of plural dichroic mirrors is used to split the fluorescence flux into plural fluxes with different wavelength bands.…”

“…As a representative conventional optical system for detecting multi-point and multi-color fluorescence with high fluorescence-detection sensitivity, the optical system of a Sanger DNA sequencing analyzer (e.g., Applied Biosystems™ 3500xL Genetic Analyzer) is explained below. 18,27 Fluorescence emitted from 24 points with intervals of 0.37 mm (forming an 8.5 mm-wide one-dimensional array in which 24 capillaries are irradiated by a laser beam) is collimated by a camera lens (f = 50 mm and F = 1.4, i.e., effective diameter of 35.7 mm) to form a fluorescence flux. The flux is passed through a long-pass filter and a transmission grating and then focused by another camera lens (f = 50 mm and F = 1.4) to form spectrum images of fluorescence (500-700 nm) from the 24 points on a two-dimensional CCD.…”

Study of the peak broadening due to detection in the electrophoretic separation of DNA by CE and microchip CE and the application of image sensor for ultra‐small detection cell length

“…For example, image sensors easily realized parallel monitoring of electrophoresis in capillary arrays [38] and microchannel array chips [10,[39][40][41][42][43][44][45]. In some other works, the second dimension of pixel array was adopted for the multi-wavelength or spectral detection [17,[46][47][48][49][50]. Recently, we pointed out that image sensor could dramatically decrease the detecion cell length of CE [51].…”