The capillary electrophoresis (CE) with laser induced fluorescence detection (LIFD) system was founded according to confocal theory. The 3-D adjustment of the exciting and collecting optical paths was realized. The photomultiplier tube (PMT) is used and the signals are processed by a software designed by ourselves. Under computer control, high voltage is applied to appropriate reservoirs and to inject and separate DNA samples respectively. Two fluorescent dyes Thiazole Orange (TO) and SYBR Green I were contrasted. With both of the dyes, high signals-to-noise images were obtained with the CE-LIFD system. The single-bases can be distinguished from the electrophoretogram and high resolution of DNA sample separation was obtained.
Laser-induced fluorescence (LIF) is widely used in biological detection system in characteristic of high sensitivity and selectivity, especially for microarray biochip readout and capillary electrophoresis detection. In these systems, fluorescence separation from background noise is necessary. In this paper, two methods of fluorescence separation were investigated. One adopts a total reflection mirror with a hole at the center; the other uses a dichroic mirror. For dichroic mirror system, fluorescence could transmit through the filter or be reflected by it. Signal to noise ratio depends on dichroic mirror transmitting spectra and reflecting spectra. For center hole mirror system, partial fluorescence loses during propagating through the center hole directly. Detected fluorescence is the part that reflected by the mirror outside the center hole. Size of the hole in the mirror must be changed in different systems. Performance of system with an f-theta lens as scanning lens for laser focus and fluorescence collecting was simulated. Collinear systems with above-mentioned two methods were set up and compared. Simulated results were verified by experiments.
High speed imaging technology has been applied on biomedical research for a long history. Suspension array technology
is a new generation of biochip, which was widely used in fields of life science and analytical chemistry, and was
developed quickly. This study present a detecting system based on framing camera for suspension array. In suspension
array microspheres were used as the carrier of bio-probes and microchannels were used as analyzing platform. By
pre-dyeing of fluorophores in microbeads, the addressing of microbeads was implemented by optical coden. Bio-probes
attached to microbeads were distinguished by intensity of fluorescence. Suspension array was usually detected with flow
cytometry serially, which was slow relatively. Then a 2D parallel measurement system based on framing camera for
suspension array was established in order to increase the measurement speed. Liquid sample containing microsphere was
injected into microchannel by a 100ul syringe connected by a capillary. Microspheres flowing in the microchannel form
a 2D layer, which was illuminated freezingly by a pulsed Xenon lamp and imaged by a microscopy objective in parallel.
The microfluidic channel was designed and fabricated, which was a rectangle microchannel of 1mm×50um in
cross-section. The image was captured by CCD and transmitted into computer by frame grabber. Image was processed to
distinguish microspheres extract information from the background. Thus area measurement of suspension array in
microchannel was realized. Compared with flow cytometry, this technology increased analyzing rate greatly, which could
be thousands of microspheres per second.
Movement in fluid field is a significant facet in suspension biochip detection and can be analyzed by the non-intrusive
technique-Particle image velocimetry (Ply). A special flat channel oftest sample was designed in the suspension biochip
detecting system to form a two-dimension fluid field. Serial images ofthe suspension biochip fluid field were acquired by
high-sensitivity CCD at ten frames per second and analyzed through Ply technique based on cross-correlation. First,
image was preprocessed to remove noise and became a binary image. Second, the preprocessed image was divided into a
proper number of area units, and then velocity vector was obtained by calculating the cross-correlation of corresponding
areas between two images. All velocity vectors were synthesized to reconstruct a whole velocity distribution map of fluid
field. Two simulated particle images have been made and the velocity distribution was reconstructed based on PIV
technique. The result shows that PIV technique can effectively obtain velocity distribution offluid field through which we
can improve the structure of fluid field channel and accomplish continuous analysis by processing different area in
two-dimension fluid field according to their relative velocity.
In this paper, a 2D parallel measurement technology for suspension array was presented. Suspension array technology was
a new type of biochip, in which microspheres were used as the carrier of bio-probes. It was usually detected by flow
cytometry serially. To measure it in parallel, microchannels were used as analyzing platform. Microspheres flowing in the
2D microchannel were freezingly imaged by pulsed Xenon lamp and a microscopy objective in parallel. The image was
captured with CCD. The microfluidic channel was designed and fabricated, which was a rectangle microchannel of 1mm
x5Oum in cross-section. System performance design was derived. After the selection of CCD, relationship between the
limitation of detection and the power of pulsed Xenon lamp was given. System parameter was provided. Some
photography of experimental result was presented. Area measurement of suspension array in microchannel was realized.
Compared with flow cytometry, this technology increased analyzing rate greatly, which could be thousands of
microspheres per second.
Optical transfer function is widely used to evaluate the imaging performance of an optical system. Combined with confocal scanning technology, f-theta lens can increase the reading speed for microarrays greatly in guarantee of sufficient resolution and fluorescence collection efficiency, compared with micro-array analyzers that adopting mechanical scanning. In this paper, the characteristics of a confocal scanning f-theta objective lens, which was used in micro-array analyzing instrument, were analyzed by means of optical transfer function. In the whole system, laser passed through the f-theta lens, and arrived at the microarray slide where fluorophores were excited. Fluorescence emitting from the micro-array slide was collected by the same f-theta lens, and was captured by a detector. As a laser illumination system, the objective lens had a smaller stop aperture. As a fluorescence collection system, it had a bigger stop aperture. In conclusion, optical transfer function for the whole system, from source to detector, is the combination of that of the laser illumination, a coherent system, and that of the fluorescence collection system, an incoherent system. Uniformity of laser illumination at the micro-array slide was analyzed using optical transfer function during the course of scanning. The influence of aberrations on optical transfer function is given. The simulating results for above characteristics are also presented.
This paper presents a novel method for establishing a two-dimensional laminar fluidic suspension array which is analyzed by using time delay integration (TDI) CCD imaging technology in parallel. The method will make suspension array technology (SAT) bear high throughput as well as its flexibility. Basically, bioassays are conducted on the surface of fluorescent-dyed beads. With each bead set (i.e., multiple beads with the same fluorescent signature) having a slightly different fluorescent signature, probes are first attached to a particular bead set and then hybridized with labeled samples or targets. Two different kinds of encoding dyes are excited by red laser (635 nm, 20mw), their emission wave length are 660nm, 720nm, respectively. Fluorescent dye of reporter molecules was excited by green laser (532nm, 20mw), emitted at 580 nm. The liquid sample was pumped into micro-reservoir by a linear motor. As the velocity of liquid sample is so slow (10mm/s) it is easy to form a laminar fluidic field in the middle of the micro-reservoir. In the direction of laser propagation the size of reservoir is 0.1mm so the laminar liquid can be treated as a two-dimensional fluidic plane. The size of detection area depends on size of micro-sphere and CCD imaging area. The three kinds of fluorescence signals were focused by a lens and then split by mirrors. Fluorescence pass through three band-pass filters (±20nm) before collected by three TDI-CCDs respectively. With these high-quality filters the cross-talk between signals was diminished significantly. The analysis speed is about 2x103 micro-spheres per second, which is much higher than that obtained from currently cytometry method (about 102 micro-spheres to the same size micro-spheres).
The most successful biochip technologies today are flat microarray and suspension microarray. Usually probes are fluorescence labeled. The fluorophores are excited by laser with a special wavelength. Because the fluorescence signal is very weak, it is hard to detect. The limitation of detection (LOD) is an important index of microarray analyzer. The dependence of LOD of flat and suspension microarray analyzer based on CCD and the fluorescent intensity on characters of excitation light optical system and fluorescence collection optical system as well as the parameters of elements system has been analyzed in detail based on the system configuration. A formula of LOD and fluorescence signal intensity depending on those parameters has been established. The study analyzed system limitation of detection (LOD). Also present a formula of minimal detectable fluorescent molecule numbers as the function of each parameter of microarray analyzer based on CCD. Estimated LOD of our suspension microarray detection system is about 7.9 fluorophores/μm2 at exposure time 1s.