Studies On Single Cell Analysis On A Microfluidic Chip

Single-cell analysis is of significant interest to the biological, medical, and pharmaceutical communities, because it is essential to a better understanding of basic cellular functions and intra- and intercellular communications. It can also provide information about the cell-to-cell variation in large populations of cells.However, detection of low concentrations of cell constituents needs a highly sensitive detection method such as laser-based fluorescence. A large number of species in cells are no fluorescent. Therefore, microderivatization of living cells became a challenge in single cell analysis.Recently the exploitation of microfluidic chip-based systems for biological cell studies is attracting broad interest. Such systems, more generally referred to as miniaturized total analysis systems (μTAS), had gone through rapid development in the past decade. The micrometer channel dimensions of microfluidic chips are ideally suited for the sample introduction, manipulation, reaction, separation and detection of single cells.In this paper, a microfluidic chip-based approach for the quantitative determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip, with integration of operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection. Reactive oxygen species (ROS) are known not only to mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential in understanding the mechanisms of cellular alterations. Nonfluorescent dihydrorhodamine 123 (DHR 123), which can penetrate into cell and be oxidized intracelluarly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as fluorogenic reagent. The effect of pH on the migration time of Rh 123 was discussed. The present method minimized dilution of intracellular ROS during reaction and determination, as a result, an extremely low detection limit of 0.74 amol has been achieved. A throughput of 2 min per single cell,a migration time precision of 2.1% RSD was obtained for 6 consecutively injected cells.Introducing cells into microchannels is a precondition for cell analysis in microchip. However, unless those cells are chosen specifically, most cells tend to adhere to the surface of plastic or glass material. Cell adhesion in chips had become a major difficulty in single cell and multiple cell analysis. A novel multi-depth microfluidic chip was fabricated on glass substrate by use of conventional lithography and 3-step etching technology. The sampling channel on the microchip was 37 urn deep, while the separation channel was 12 urn deep. A 1 mm long weir was constructed in the separation channel, 300 um down the channel crossing. The channel at the weir section was 6 |im deep. 0.4% HPMC was added to PBS to obtain a relatively stable suspension with cell population of 1.2 x 105 cells/mL. By using the multi-depth microfluidic chip, human carcinoma cells, which easily aggregate, settle and adhere to the surface of the channel, can be driven from the sample reservoir to the sample waste reservoir by hydrostatic pressure generated by the difference of liquid level between sample and sample waste reservoirs. Single cell loading into the separation channel was achieved by applying a set of pinching potentials at the four reservoirs. The loaded cell was stopped by the weir and precisely positioned within the separation channel. The trapped cell was lysed by SDS containing buffer solution in 20 s. This approach reduced the lysing time and improved the reproducibility of chip-based electrophoresis separations. Reduced glutathione (GSH) and ROS were used as model intracellular components in single human carcinoma cells, and the constituents were separated by chip-based electrophoresis and detected by LIF. A throughput of 15 samples/h, a migration time precision of 3.1% RSD for ROS and 4.9% RSD for GSH were obtained for 10 consecutively injected cells. The proposed microfluidic chip provides an effective and efficient platform for analysis of intracellular constituents in single carcinoma cells, which are easy to assemble, settle, and adhere to microchannels.Detection of low concentrations of cell constituents often needs derivatization. To avoid dilution of the trace level analytes of a single cell during derivatization,intracellular derivatization was widely used. However, most of derivatization reagents with charges, such as FITC, can not penetrate into cells. In the present work, a novel method was developed to deliver fluorescent dyes and other polar materials into cells by nanometer sized-liposomes. An average 100 nm diameter of liposomes was produced by an ultrasonic method, and the influence factor during preparation and the liposomes stability were discussed. Fluorescence images demonstrated that liposomes can transfer fluorescent dyes ( FITC, Rhodamine B), which are not cell membrane permeable, into cells. Fluorescence intensity in cells increased with increasing the incubation time and got a highest value in 2 h. Experiments showed that liposomes has no negative effect on cell viability. Derivatization of amino acids and proteins in cells by FITC was proved by chip based capillary electrophoresis with LIF.Superoxide dismutase (SOD), a kind of metal protein enzyme existed widely in biological bodies, can catalyze and eliminate superoxide radicals effectively in cells. However, the results of animal and clinical studies of the therapeutic usage of SOD showed only a modest protective effect against oxidative stress attribute to inadequate delivery of the enzyme to the sites of therapeutic action. SOD-liposomes were prepared by ultrasonic method and its ability to eliminate superoxide radicals was detected by chip based capillary electrophoresis with LIF detection. It was found that ROS in cells decreased and GSH increased after the cells incubation with SOD lipsomes for 2 h, and the results were consistent with flow cytometry's. It can be concluded that SOD still maintain a good viability after encapsulation in liposomes by ultrasonic method and had a good ability to eliminate superoxide radicals.