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This work develops a microflow cytometer, based on a microfluidic chip for three-dimensional (3D) hydrodynamic focusing and a binary optical element (BOE) for shaping and homogenizing a laser beam. The microfluidic chip utilizes sheath flows to confine the sample flow along the channel centerline with a narrow cross section. In addition to hydrodynamic focusing, secondary flows are generated to strengthen the focusing in the vertical direction. In experiments, the chip was able to focus the sample flow with cross sections of 15 μm high and 8–30 μm wide at 5 m/s, under the condition of the sample flow rates between 10 and 120 μL/min. Instead of using the conventional elliptical Gaussian spot for optical detection, we used a specially designed BOE and obtained a 50 μm × 10 μm rectangular quasi-flat-top spot. The microflow cytometer combining the chip and the BOE was tested to count 3, 5, and 7 μm fluorescence microbeads, and the experimental results were comparable to or better than those derived from two commercial instruments.

The miniaturization of flow focuser is a challenge in developing microflow cytometers. Most previously reported microfluidic cell focusers require complex structures or external force fields to achieve the 3D cell focusing. Herein, we propose a novel circular-channel particle focuser utilizing viscoelastic focusing. The circular-channel particle focuser is fabricated using a simple and low-cost microwire molding technique. Whole PDMS channels with perfect circular cross-sections can be fabricated using this protocol. We then characterize the particle focusing performances in our circular-channel particle focuser and discuss the effects of particle size, operating flow rate, cross-sectional dimension and fluid rheological property on particle focusing. The experimental results show that a perfect single-line focusing can be achieved exactly at the channel centerline. Finally, our circular-channel particle focuser is employed for the focusing of blood cells. As it offers special advantages of simple structure, easy fabrication, and sheathless operation, our circular-channel particle focuser may serve as a potential flow focuser for microflow cytometers.

The eutrophication of aquatic ecosystems caused by rapid human urbanization has led to an increased production of potentially hazardous bacterial populations, known as blooms. One of the most notorious forms of these aquatic blooms are cyanobacteria, which in sufficiently large quantities can pose a hazard to human health through ingestion or prolonged exposure. Currently, one of the greatest difficulties in regulating and monitoring these potential hazards is the early detection of cyanobacterial blooms, in real time. Therefore, this paper presents an integrated microflow cytometry platform for label-free phycocyanin fluorescence detection, which can be used for the rapid quantification of low-level cyanobacteria and provide early warning alerts for potential harmful cyanobacterial blooms. An automated cyanobacterial concentration and recovery system (ACCRS) was developed and optimized to reduce the assay volume, from 1000 mL to 1 mL, to act as a pre-concentrator and subsequently enhance the detection limit. The microflow cytometry platform utilizes an on-chip laser-facilitated detection to measure the in vivo fluorescence emitted from each individual cyanobacterial cell, as opposed to measuring overall fluorescence of the whole sample, potentially decreasing the detection limit. By applying transit time and amplitude thresholds, the proposed cyanobacteria detection method was verified by the traditional cell counting technique using a hemocytometer with an R2 value of 0.993. It was shown that the limit of quantification of this microflow cytometry platform can be as low as 5 cells/mL for Microcystis aeruginosa, 400-fold lower than the Alert Level 1 (2000 cells/mL) set by the World Health Organization (WHO). Furthermore, the decreased detection limit may facilitate the future characterization of cyanobacterial bloom formation to better provide authorities with ample time to take the appropriate actions to mitigate human risk from these potentially hazardous blooms.

NRC publication: Yes

A novel microflow cytometer is proposed in which the sample stream is focused initially in the horizontal (X–Y) plane by two sheath flows and is then focused in the vertical (Y–Z) plane by a sequential micro-weir structure before entering the detection region of the device. The proposed device is fabricated using a modified glass fabrication process, and is then characterized both numerically and experimentally using fluorescent polystyrene beads with diameters of 7 and 15 μm, respectively. The experimental and numerical results confirm the effectiveness of the hydrodynamic sheath flows in centralizing the particles in the horizontal plane prior to entering the sequential micro-weir structure. Furthermore, it is shown numerically that the micro-weir structure confines the particle stream to the center of the vertical plane such that the particles pass through the detection region in a one-by-one fashion can be counted with a high degree of reliability. The experimental results confirm that the proposed 3-D focusing scheme enables the fluorescent beads with different diameters to be reliably sorted and counted.

With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ∼35 μm × 40 μm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 μm × 125 μm) successfully minimized the risk of clogging. The device could withstand pressures greater than 100 psi without leaking. Assays using both coded microparticles and cells were demonstrated using the microflow cytometer. Multiplexed immunoassays detected nine different bacteria and toxins using a single mixture of coded microspheres. A549 cancer cells processed with locked nucleic acid probes were evaluated using fluorescence in situ hybridization. [graphic removed]

Micron-sized particles have primarily been used in microfabricated flow cytometers for calibration purposes and proof-of-concept experiments. With increasing frequency, microparticles are serving as a platform for assays measured in these small analytical devices. Light scattering has been used to measure the agglomeration of antibody-coated particles in the presence of an antigen. Impedance detection is another technology being integrated into microflow cytometers for microparticle-based assays. Fluorescence is the most popular detection method in flow cytometry, enabling highly sensitive multiplexed assays. Finally, magnetic particles have also been used to measure antigen levels using a magnetophoretic micro-device. We review the progress of microparticle-based assays in microflow cytometry in terms of the advantages and limitations of each approach. [graphic removed]

A novel on-chip optical flow cytometer concept is reported for fluorescence detection, enumeration, and sizing of microparticles in a poly-dimethylsiloxane (PDMS) microchip. The detection system integrates a pair of external optical fibers and other optical components for particle counting, sizing and fluorescence analysis in each measurement simultaneously. The scattered light signal indicates the total number and the size of the particles passing through the detection window, whereas the concurrent backward fluorescence signal shows only the number of fluorescence particles. In the experiments, microparticles of four different sizes with diameters ranging from 3.2 to 10.2 µm were discriminated and counted based on the fluorescence and scattered light intensity. The relative percentage of the fluorescence-labeled particles can be analyzed by the ratio of the events of fluorescence signals to forward scattered signals.

This study reports on an integrated microfluidic system capable of counting CD4 + /CD8 + T lymphocytes from a whole blood sample, which may be further applied for the rapid screening of the human immunodeficiency virus (HIV) infection. This system is composed of a sample incubation module for fluorescence-labeling of the target cells and a micro-fabricated flow cytometry module for cell counting. First, a pneumatically driven, vortex-type micro-mixer has been adopted for the fluorescence-labeling of CD4 + /CD8 + T lymphocytes from whole blood. After the labeling process, different laser-excited fluorescent signals are detected and are used for counting of CD4 + /CD8 + T lymphocytes as they pass through the detection region of the microflow cytometer. A concentration of 963 cells/μl is counted for cultured CD4 + T lymphocytes with a reference concentration of 1000 cells/μl. The ratio of CD4 + /CD8 + T lymphocytes is then calculated. Experimental results show that the results from the microsystem are in agreement with the ones from large-scale flow cytometers. In addition, the entire diagnostic procedure, including the sample incubation and the cell counting, can be automatically performed within 35 min. Therefore, this may become a powerful tool for further biomedical applications, especially for fast screening of HIV infection.

This paper describes a novel concept of integrated on-chip fiber free laser-induced fluorescence detection system. The poly-dimethylsiloxane (PDMS) chip was fabricated using soft lithography and was bonded with a glass substrate of 150 μm thickness that reduced the distance of channel-to-sidewall to less than 180 μm. The cells and particles detection was conducted by an external single fiber close to the glass substrate that transmitted laser light for simultaneous excitation and receipt of the emission light signals. The performance of the proposed device was demonstrated using fluorescence beads, stained white blood cells, and yeast cells. The experimental results showed the simplicity and flexibility of the proposed device configuration which can provide convenient on-chip integration interface for fast, high throughput, and low-cost laser-induced fluorescence detection micro flow cytometer.