CN1588088A - Micro flow control chip detecting system for flowing cell detection - Google Patents

Abstract

The invention relates to a microfluidic chip system for flow cytometric detection, which is characterized in that it includes a gravity-driven microfluidic chip, a confocal laser-induced fluorescence detection system that can be connected to a computer, and a charge-coupled detector. and a high-voltage power supply; the main channel and the auxiliary channel of the microfluidic chip are opened on the upper surface of the microfluidic chip, and are respectively connected to the sample pool and the buffer solution pool located on the upper surface of the microfluidic chip, and the three cell collectors are connected to each other. At the end of the main channel, the microfluidic chip is vertically fixed on a base, the confocal laser-induced fluorescence detection system is located on one side of the microfluidic chip, and its detection area is located in the middle and lower part of the main channel, and the charge-coupled detector is located at the bottom of the main channel. On the other side of the microfluidic chip, the positive and negative voltages output by the high-voltage power supply are respectively connected to the cell collectors on both sides of the microfluidic chip through wires. The present invention can save auxiliary equipment such as an external pressure pump, simplify equipment structure, reduce equipment volume, and reduce the consumption of samples and reagents.

Description

A microfluidic chip detection system for flow cytometry

technical field

The invention relates to a microfluidic chip detection system, in particular to a gravity-driven microfluidic chip detection system for flow cytometry.

technical background

The microfluidic chip technology in the micro-total analysis system (μ-TAS) has received widespread attention in recent years, and it has developed rapidly in China, and has applied for many patents. Such as the cooperation between HP and Caliper, Hitachi, Nanogen, etc.

Flow Cytometry (FCM) is a detection method for quantitative analysis and sorting of single cells or other biological particles at the functional level. It has the advantages of high speed, high precision, and good accuracy. Its work The principle is to make a single cell suspension after the cells to be tested are stained. The sample to be tested is pressed into the flow chamber with a certain pressure, and the phosphate buffer solution without cells is sprayed from the sheath fluid tube under high pressure to form a stream of a certain shape. The cells to be tested are arranged in a single row under the coating of the sheath fluid, and then through the detection zone. Flow cytometers usually use laser light as the excitation light source, which is irradiated vertically on the sample flow. Under the irradiation of the laser beam, the fluorescently stained cells produce scattered light and excited fluorescence, and are multiplied by the forward photodiode and the 90° photoelectric multiplier at the same time. tube reception. Cell sorting in flow cytometry is accomplished by isolating droplets containing single cells. The nozzle of the flow chamber is equipped with an ultra-high-frequency transistor, which vibrates after being charged, so that the ejected liquid flow is broken into uniform droplets, and the cells to be measured are dispersed in these droplets. These droplets are charged with different positive and negative charges. When the droplets flow through the deflection plate with several thousand volts, they are deflected under the action of a high-voltage electric field and fall into their respective collection containers. The uncharged droplets fall into the waste container in the middle, so as to realize the separation of cells. However, the traditional flow cytometer has disadvantages such as expensive, bulky, cumbersome operation, large amount of samples and reagents, easy to pollute and difficult to clean.

Due to the above shortcomings of traditional flow cytometers, the use of microfluidic chip systems for cell analysis has become a hot spot in recent years and has attracted more and more attention. The microfluidic chip system has the characteristics of high miniaturization, integration and flexible design. Through ingenious design and precise processing, it can realize the functions of cell culture, apoptosis and detection. It combines microfluidic chip technology with flow cytometry technology. It can greatly reduce the volume of the instrument, reduce the probability of retention and cross-infection, and also reduce the cost. Recently, this technology is gradually receiving more widespread attention.

Different from the traditional flow cytometer, the microfluidic chip system uses a variety of cell manipulation and driving modes including negative pressure, electroosmosis (electrophoresis) and two-dimensional electrophoresis in addition to positive pressure drive. Each has advantages and disadvantages. Among them, the electric field has a certain degree of killing effect on the cells, which will produce false positive results when detecting the survival probability of the cells. Pressure driving is a method commonly used in traditional cell detection instruments, but when it is used in a microfluidic chip system with a small channel size, the flow rate provided by the pressure pump is required to be low enough and stable to meet the requirements of chip cell analysis. The cell-driven mode based on two-dimensional electrophoresis is a very suitable method for cell manipulation on a chip. It has the characteristics of small damage and accurate control. However, the cost of processing microelectrode arrays on a chip is relatively high. Gravity-driven microfluidic chips have been used in microfluidic chips. Such systems have the characteristics of simple equipment, but microfluidic chips driven by gravity have not been used in flow cytometry.

Contents of the invention

In view of the above problems, the object of the present invention is to provide a gravity-driven microfluidic chip detection system for flow cytometry that can reduce the volume of the device, reduce the cost of the device, and reduce the amount of samples and reagents.

To achieve the above object, the present invention adopts the following technical solutions: a microfluidic chip system for flow cytometric detection, characterized in that it includes a gravity-driven microfluidic chip, a confocal laser that can be connected to a computer An induced fluorescence detection system, a charge-coupled detector and a high-voltage power supply; the main channel and the auxiliary channel of the microfluidic chip are all opened on the upper end surface of the microfluidic chip, and are respectively connected to the upper end surface of the microfluidic chip. The sample pool and the buffer solution pool, the three cell collectors are respectively connected to the end of the main channel, the microfluidic chip is vertically fixed on a base, and the confocal laser-induced fluorescence detection system is located in the microfluidic One side of the control chip, and its detection area is located in the middle and lower part of the main channel, the charge-coupled detector is located on the other side of the microfluidic chip, and the positive and negative voltages output by the high-voltage power supply pass through the wires Connect the cell collectors on both sides of the microfluidic chip respectively.

The confocal laser-induced fluorescence detection system includes a laser light source, an excitation light filter, a dichroic mirror, a microscope objective lens, an emission light filter, a focusing lens, a pinhole and a photomultiplier tube installed in sequence in the light path.

The microfluidic chip is made of glass.

The microfluidic chip is made of high molecular polymer material which is transparent to visible light.

The high molecular polymer material is polydimethoxysilane.

The high molecular polymer material is polymethyl methacrylate.

Because the present invention adopts the above technical scheme, it has the following advantages: 1. The present invention is different from other microfluidic chip flow cytometric detection systems, because the main channel and the auxiliary channel are all opened on the upper end surface of the microfluidic chip, which is different from that of other microfluidic chip flow cytometric detection systems. The sample pool also arranged on the upper surface is connected to the buffer solution pool. Therefore, during operation, the chip is vertically placed and fixed on the base of the detection platform, and the movement in the channel of the chip can be carried out with the gravity of the cells as the driving force. Quantitative analysis and sorting of cells, eliminating the need for external pressure pumps and other auxiliary equipment, reducing the size of the equipment. 2. Since the density of cells is much higher than that of water, they will settle down quickly in aqueous solution. Therefore, when cells are used as analysis samples, gravity can be used as the driving method to obtain faster analysis speed. There can be problems with sample diffusion. 3. Since the present invention is provided with a confocal laser-induced fluorescence detection system that can be connected to a computer, a charge-coupled detector and a high-voltage power supply, the present invention, which is very small in size, can be carried around, and where there is a computer, install software to connect various devices After that, it can be operated, and the operation process can be monitored in real time through the cooperation of the charge-coupled detector and the computer. 4. The present invention can apply a certain electric field to the cell collector by controlling the high-voltage power supply through the computer. By controlling the magnitude and direction of the electric field, the cells with fluorescence and without fluorescence can be separated under the combined action of gravity and electrophoresis, which not only makes the cells The damage is small and can be accurately controlled. The invention can be widely used in the detection of quantitative analysis and sorting of various single cells or other biological particles.

Description of drawings

Figure 1 is a schematic diagram of the microfluidic chip structure

Figure 2 is a schematic diagram of the partial structure of a traditional flow cytometry device

Fig. 3 is to adopt the structure schematic diagram of the present invention

Fig. 4 is the semi-quantitative detection example that the present invention is applied to HeLa cell damage by ultraviolet light irradiation

Detailed ways

As shown in Figure 1, the microfluidic chip 1 of the present invention is processed by using glass or polydimethoxysilane (PDMS), polymethyl methacrylate (PMMA) and other high molecular polymer materials or plastic materials that can transmit visible light. become. The microchannel directly connected with the sample pool 2 is the main channel 3, and auxiliary channels 4 are arranged on both sides of the main channel 3, and the two auxiliary channels 4 are respectively connected with the buffer solution pool 5, and the cell collectors C1, C2, and C3 are arranged in the main channel. end of channel 3. The laser-induced fluorescence detection area 6 is located in the middle and lower part of the main channel 3 . Since the main channel 3 and the auxiliary channel 4 are all opened on the upper surface of the chip 1, the chip 1 is placed vertically during operation, and the cells and buffer solution in the sample pool 2 and the buffer solution pool 5 can enter the main channel under the action of their own gravity 3. Fluorescent quantitative analysis and sorting of cells.

As shown in Figure 2, in a traditional flow cytometer, the cells in the sample pool 21 and the buffer solution in the buffer solution pool 22 enter the flow cytometer under the push of the pressure system 23, and the cells are arranged in a single row by controlling the pressure and flow rate It is sprayed out from the nozzle 24 to detect the fluorescent signal, and the cells are sorted by applying positive and negative high-voltage electric fields. The present invention integrates the above-mentioned parts 21, 22 and 24 of the traditional flow cytometer into a chip with a square of several centimeters, which greatly reduces the volume of the instrument and reduces the cost. At the same time, because the present invention relies on gravity to drive the movement of cells in the microchannel of the chip, the pressure system 23 is omitted, and the device is further simplified.

As shown in FIG. 3 , the detection system of the present invention includes: a microfluidic chip 1 , a confocal laser-induced fluorescence detection system, a high-voltage power supply 15 , and a charge-coupled detector (CCD) 16 . The microfluidic chip 1 is vertically fixed on the base of the detection platform (not shown in the figure), and the confocal laser-induced fluorescence detection system includes a microscopic objective lens 10, a dichroic mirror and 9. An emission filter 11 , a focusing lens 12 , a pinhole 13 and a photomultiplier tube 14 , and a light source 7 and an excitation filter 8 arranged below the dichroic mirror 9 . The charge-coupled detector 16 is set at the corresponding position on the other side of the microfluidic chip 1, and the output end of the high-voltage power supply 15 is connected and fixed on the cell collectors C1 and C3 through wires, and is input to the cell collectors C1 and C3. 600V and 0V voltage, simultaneously, photomultiplier tube 14, high-voltage power supply 15 and charge-coupled detector 16 are also respectively connected to a computer 17 by circuit connection, can not comprise computer 17 in the equipment of the present invention, only have various connecting wires, when there is Where the computer is installed, it can be operated by installing the control software.

During operation, the laser 7 emits a beam of laser light, filters stray light through the excitation light filter 8, reflects it through the dichroic mirror 9, and then focuses it on the laser-induced fluorescence detection area 6 of the main channel 3 of the chip through the microscope objective lens 10. When cells marked with fluorescent dyes pass by, they are excited by laser light to emit fluorescence of corresponding wavelengths, and the fluorescent signals are collected by the microscope objective lens 10, transmitted through the dichroic mirror 9, and then passed through the emission filter 11 to remove the interference of the excitation light , through the focusing lens 12 and the pinhole 13, it is collected and amplified by the photomultiplier tube 14 and converted into an electrical signal, which is finally recorded by the software of the computer 17, and the signal is processed. The movement in the channel is monitored in real time.

In the present invention, a certain concentration of dispersion medium can be added to the cell sample solution and the buffer solution, so that the cells are well dispersed in the solution, reducing aggregation and sedimentation, so the cells can enter the channel one by one without blocking the entrance of the channel. Phenomenon. The cell sorting process is realized by computer 17 online control of the magnitude and direction of the output voltage of the high-voltage power supply 15. After the cells pass through the detector, they enter the sorting area, and the high-voltage power supply 15 outputs 600V and 0V to C1 and C3 respectively (as shown in Figure 1 As shown), the cells will enter into C1 and C3 respectively according to the positive and negative properties of the surface charge. When the detector gets a fluorescent response signal exceeding a certain intensity, the high voltage control program will be turned off, so the cells will only enter into C2 under the action of gravity , to achieve cell separation.

Example: After HeLa cells grow into the logarithmic growth phase, remove the lid of the culture dish, suck off the culture medium, irradiate with ultraviolet light (254nm) for 10, 20, and 40 minutes respectively, digest and collect the cells and label them with TO-PRO-3 for 1 hour . During detection, take 40 μl of labeled single cell suspension and add 200 μl of phosphate buffer solution containing 0.4% HPMC as the sample solution, add 20 μl of phosphate buffer solution to the buffer solution pool 5 of the microfluidic chip 1 respectively, and add 20 μl of phosphate buffer solution in the sample pool 2 Add an equal amount of labeled cell samples to the assay for fluorescence detection. The results are shown in Figure 4. From bottom to top in the figure are the detection results after 10min, 20min and 40min of ultraviolet irradiation. It can be seen from the figure that as the irradiation time increases, the number of apoptotic cells and the degree of apoptosis increase, and the number of detected cells and the fluorescence intensity also increase.

Claims (6)

1. A microfluidic chip system for flow cytometry, characterized in that it includes a gravity-driven microfluidic chip, a confocal laser-induced fluorescence detection system that can be connected to a computer, a charge-coupled detector and High-voltage power supply; the main channel and the auxiliary channel of the microfluidic chip are all opened on the upper end surface of the microfluidic chip, and are respectively connected to the sample pool and the buffer solution pool located on the upper end surface of the microfluidic chip, three The cell collectors are respectively connected to the ends of the main channels, the microfluidic chip is vertically fixed on a base, the confocal laser-induced fluorescence detection system is located on one side of the microfluidic chip, and its detection area Located in the middle and lower part of the main channel, the charge-coupled detector is located on the other side of the microfluidic chip, and the positive and negative voltages output by the high-voltage power supply are respectively connected to the microfluidic chip through wires. Cell collectors on both sides. 2. A microfluidic chip detection system for flow cytometry as claimed in claim 1, characterized in that: the confocal laser-induced fluorescence detection system includes a laser light source, an excitation filter Light sheets, dichroic mirrors, microscope objectives, emission filters, focusing lenses, pinholes, and photomultiplier tubes. 3. A microfluidic chip detection system for flow cytometric detection according to claim 1 or 2, characterized in that: said microfluidic chip is made of glass. 4. A microfluidic chip detection system for flow cytometric detection according to claim 1 or 2, characterized in that: said microfluidic chip is made of a polymer material that is transparent to visible light. 5. A microfluidic chip detection system for flow cytometry as claimed in claim 4, characterized in that: the polymer material is polydimethoxysilane. 6. A microfluidic chip detection system for flow cytometry as claimed in claim 4, characterized in that: said high molecular polymer material is polymethyl methacrylate.

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