This paper presents a novel way of designing a flow focusing channel for microchip flow cytometers. With this method we increased throughput and sensitivity of particle detection at the same time. Generally, to increase the detection throughput of a flow cytometer, the speed of the flow inside the focusing channel needs to be increased, hence reducing the time of exposure to laser beam. With the shorter exposure time, both the fluorescence and scatter signal from the target particles become dimmer. To increase the sensitivity of signal detection, however, the speed of the flow should be decreased so as to decrease throughput of detection. To overcome this dilemmatic problem, we integrated an expansion channel inside a focusing channel. Signals from particles in an expansion channel were about 10 times brighter than those in a normal channel. With this enhanced sensitivity, we could also speed up the inlet flow, which in turn increases the overall throughput of detection.
Flow cytometry and microfluidic system are versatile tools to study the functional genomics at the single-cell level in the postgenomic era. We have developed a microfluidics-based benchtop flow cytometry system incorporating a disposable plastic microchip and low-power diode lasers. The plastic microfluidic chip is designed and fabricated using soft lithography which enables the development of inexpensive and flexible miniaturized fluidic devices. The microchip contains the hydrodyamic focusing chamber where the sample and sheath fluids are driven by a pressure difference. The performance of the combined fluidics and optics is studied systematically to evaluate the detection accuracy and efficiency of our flow cytometry system. The interactions of the biological particles with surrounding squeezed flow and focused laser beam are investigated to optimize the design of the microchannel network as well as the optical characteristics of the instrument for efficient single-cell manipulation and detection.
Although CD4+ T-cells are an important target of HIV detection, there have been still major problems in making a diagnosis and monitoring in the third world and the region with few medical facilities. Then, it is necessary to use portable diagnosis devices at low cost when you put an enumeration of CD4+ T-cells. In general, the counting of CD4 below 200cells/uL makes it necessary to initiate antiretroviral treatment in adults (over 13 years old). However, lymphocyte subsets (including CD4 counts) of infants and young children are higher than those of adults. This fact shows the percentage of CD4+ T-cells of blood subsets, i.e., CD4/CD45%, CD4/CD8% or CD4/CD3% means a more reliable indicator of HIV infection than absolute counts in children. To know the percentage of CD4+ T-cell by using two fluorescent dyes of different emission wavelength, at least, one laser and two PMT detectors are in general needed. Then, it is so hard to develop a portable device like a 'toaster size' because this makes such a device more complex including many peripheral modules. In this study, we developed a novel technique to control the intensity of fluorescent dye-doped silica nanoparticles. I synthesized FITC-doped silica nanoparticles conjugated CD4 antibody 10 times brighter than FITC-conjugated CD45 antibody. With the difference of intensity of two fluorescent dyes, we measured two parameters by using only a single detector and laser. Most experiments were achieved with uFACS (microfabricated fluorescence-activated cell sorter) on an inverted microscope (IX71, Olympus). In conclusion, this method enables us to discriminate the difference between CD4 and CD45 in an intensity domain simultaneously. Furthermore, this technique would make it possible develop much cheaper and smaller devices which can count the number of CD4 T-cells.
Today's pharmaceutical industry is facing challenges resulting from the vast increases in sample numbers produced by high-throughput screening (HTS). In addition, the bottlenecks created by increased demand for cytotoxicity testing (required to assess compound safety) are becoming a serious problem. We have developed a polymer PDMS (polydimethylsiloxane) based microfluidic device that can perform a cytotoxicity test in a rapid and reproducible manner. The concept that the device includes is well adjustable to automated robots in huge HTS systems, so we can think of it as a potential dilution and delivery module. Cytotoxicity testing is all about the dilution and dispensing of a drug sample. Previously, we made a PDMS based microfluidic device which automatically and precisely diluted drugs with a buffer solution with serially increasing concentrations. This time, the serially diluted drug solution was directly delivered to 96 well plates for cytotoxicity testing. Cytotoxic paclitaxel solution with 2% RPMI 1640 has been used while carrying out cancerous cell based cytotoxicity tests. We believe that this rapid and robust use of the PDMS microchip will overcome the growing problem in cytotoxicity testing for HTS.
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