Multiple-Point Fluorescent Detection

Multiple-point fluorescent deteclion has been proposed to enhance detection sensitivity. This method is based on the use of a detector function, such as the Shah function. The time-domain signals were first detected, and they were converted into a frequency-domain plot by Fourier transformation. Therefore, this technique was dubbed Shah convolution Fourier transform detection (SCOFT). As a comparison, the single-detection point time-domain response is commonly known as the electropherogram [698,699,701]. 

FIGURE 7.10 Micromachined glass electrophoresis chip for SCOFT detection (a) schematic showing the solution reservoirs, one electrophoresis channel (4.5 cm long, 15 pm deep, 50 im wide at the top, 20 pm wide at the bottom), and patterned Cr layer with micromachined slits (300 pm-wide, 700 pm-spaced center to center) (b) scanned image of the actual 75-mm x 75-mm chip. The six different channel systems can be seen [698]. Reprinted with permission from the American Chemical Society. 

The use of multiple sample injections (up to a maximum of three) was found to enhance S/N, i.e., the S/N is slightly higher than the square root of the number of injected sample plugs. In addition, multipoint detections of a two-component sample [700,701] or four-component sample [699] were also achieved, but the separation resolution was not as good as that obtained from the conventional single-point detection [701]. Besides Fourier transform, wavelet transform was also used in multipoint fluorescent detection to retain some time information in addition to the frequency information [702]. 

FIGURE 7.11 One-component injection detected by SCOFT. (a) Time-domain data produced when a fluorescein sample is injected down the separation channel 55 peaks, spaced at 0.58 s, superimposed on the Gaussian distribution of the expanded laser beam, (b) Fourier transformation of part (a). Peaks for 1.74 Hz (fundamental) and 3.43 Hz (second harmonic) can be seen. The frequency of 1.74 Hz was correlated to the migration time of 0.58 s [698]. Reprinted with permission from the American Chemical Society. 

Instead of realizing the Shah detector function using the multiple slits, multiple excitation points were created. This was achieved by monolithically integrating multiple planar waveguide beam splitters on a fluidic channel. In this way, 128 excitation points were created on the fluid channel for multipoint fluorescent detection [413]. 

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JBPDS The Joint Biological Point Detection System (JBPDS) is in development to replace and outperform the Army BIDS and Navy IBAD systems. It is planned to enter development of Block II in FY 2004. JBPDS comprises trigger, sampler, detection, and identification subsystems to meet Joint Operational Requirements Document (JORD) specifications. It is designed to be able to identify multiple BW agents in less than 15 minutes, at 1 ACPLA sensitivity, and have less than 2 percent error in identification." Generic UV laser-induced fluorescence detection capability (BAWS) improves system performance while reducing operations and support costs. 

Con focal fluorescence microscopy has also been connected with resolution improvement (Fig. 19.1a) [15-17]. Illuminating with a diffraction limited focused spot and detecting with a S3mimetrically arranged point detector, the effective focal spot, i.e., the effective PSF of this microscope is described by the product of the diffraction pattern for illumination and for detection I x,y, z) z) P x,y,z). The multiplication of intensities and the... 

Rohthmann, X Engel, H. Laue, X. Simultaneous detection of multiple nucleic acid sequences in amplification reaction by probes containing multiple fluorescent labels and by different melting points for probes containing same label. Eur. Pat. Appl. EP 2116614, 2009. 

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