CN105699671A - Small micro-fluidic chip system for biological particle parting analyzing - Google Patents

Abstract

The invention relates to a small-sized microfluidic chip system for type analysis of biological microparticles, which belongs to the field of biological sample detection. Including exciting optical fiber channel, exciting on-chip microlens, collecting on-chip microlens, and collecting optical fiber channel; the exciting on-chip microlens and collecting on-chip microlens are respectively placed on both sides of the waste liquid microchannel; The optical axis and the waste liquid microchannel are perpendicular to each other; the optical axis of the collecting on-chip microlens forms an included angle with the waste liquid microchannel; the exciting optical fiber channel and the collecting optical fiber channel are set on the chip. The microfluidic chip system used in the present invention has simple structure, small size, low cost, low power consumption, high sensitivity, accurate detection, convenient operation, no need for optical calibration, and the on-chip microlens on the microfluidic chip - The optical fiber detection module can process several parallel jobs, which helps to achieve high-throughput detection. Therefore, the system is suitable for the analysis and detection of various biological samples, especially biological particles.

Description

A small microfluidic chip system for the typing and analysis of biological particles

technical field

The invention relates to a small-sized microfluidic chip system for type analysis of biological particles, which belongs to the field of biological sample detection.

Background technique

Flow cytometry is a routine detection technique for typing analysis of biological particles, and is widely used in clinical diagnosis, biochemistry, biology and environmental monitoring and other fields. Although flow cytometry technology has been very advanced, traditional flow cytometers are bulky, have high power consumption, are expensive, and are cumbersome to operate. These devices cannot function normally in some extreme environments and conditions, such as emergency rescue, Space station, border post, remote village, etc. Miniaturized total analysis system (μ-TAS), also known as Laboratory-on-a-Chip (LOC for short), was first proposed by Manz and Widmer of CibaGeigy in Switzerland in 1990, and has developed rapidly since then. After decades of development, microfluidic chips have been widely used in the typing and analysis of biological particles due to their advantages such as fast speed, high throughput, low power consumption, portability, and easy integration. The miniaturization of particle detection instruments puts forward new ideas. David Holmes of the University of Southampton in the United Kingdom proposed a microfluidic flow cytometry system that integrates electrodes on a microfluidic chip and detects the fluorescence signal of biological particles off-chip, realizing the simultaneous measurement of optical and electrical information of biological particles (HolmesD ., PettigrewD., RecciusC.H.etal.LeukocyteAnalysisandDifferentiationUsingHighSpeedMicrofluidicSingleCellImpedanceCytometry[J].LabOnaChip,2009,9(20):2881-2889.). As shown in Figure 1, although the volume of the microfluidic chip is small, it has a bunch of complex auxiliary equipment, such as lasers, detectors, liquid drive devices and electronic parts, so that the final device is still quite bulky, and The integration and calibration of spatial optical components and microfluidic chips is difficult, resulting in high power consumption and instrument costs, which completely offset the advantages brought by microfluidic chips, and instead make sample pretreatment steps cumbersome However, the counting accuracy and detection sensitivity are not satisfactory, and the real low cost, miniaturization, rapidity and portability have not been realized.

Therefore, this patent mainly aims at the technical characteristics of microfluidic chips, and has invented a microfluidic chip that is miniaturized, low cost, low power consumption, easy to integrate with microfluidic chips, easy to operate, and does not require optical calibration. Detection of microfluidic chip devices.

Contents of the invention

The purpose of the present invention is to provide a small microfluidic chip system for biological microparticle typing analysis in order to solve the problems of large volume, complicated operation and difficult integration with microfluidic chips in the prior art.

The purpose of the present invention is achieved through the following technical solutions.

A small microfluidic chip system for biological microparticle typing analysis, including sheath liquid microchannel, sample microchannel, waste liquid microchannel; also includes excitation optical fiber channel, excitation on-chip microlens, collection on-chip microlens, Collect the optical fiber channel; the excitation on-chip microlens and the collection on-chip microlens are respectively placed on both sides of the waste liquid microchannel; the optical axis of the excitation on-chip microlens is perpendicular to the waste liquid microchannel; the light collected by the on-chip microlens The axis forms an angle with the waste liquid microchannel, and it is necessary to ensure that the included angle is not 90°; the excitation fiber channel and the collection fiber channel are set on the chip, wherein the excitation fiber channel is coaxial with the optical axis of the excitation on-chip microlens, The collection fiber channel is coaxial with the optical axis of the collection on-chip microlens.

Working process: It is to use MEMS technology to process a microfluidic chip device that can perform multi-parameter detection of biological particles, and use it for typing analysis of various biological particles. The microfluidic chip system adopts a two-dimensional entrainment channel for sample injection, and the biological particle samples are arranged in a row and pass through the detection area one by one under the sheath fluid wrapping. The laser light enters the excitation fiber through the fiber coupler to the microfluidic chip, and forms a light spot with a diameter similar to that of the detected biological particle in the center of the channel through the excited on-chip microlens. When a single detected biological particle passes through the detection area, forward scattered light is generated. , side-scattered light and fluorescence are collected to the photodetector by the collection microlens on the opposite side of the channel and the collection fiber respectively. Through the data acquisition card, the collected data is transmitted to the PC for analysis and display.

Beneficial effect

The microfluidic chip system used in the present invention has simple structure, small size, low cost, low power consumption, high sensitivity, accurate detection, convenient operation, no need for optical calibration, and the on-chip microlens on the microfluidic chip - The optical fiber detection module can process several parallel jobs, which helps to achieve high-throughput detection. Therefore, the system is suitable for the analysis and detection of various biological samples, especially biological particles.

Description of drawings

Figure 1 is a system structure diagram proposed in the document "LeukocyteAnalysisandDifferentiationUsingHighSpeedMicrofluidicSingleCellImpedanceCytometry";

Fig. 2 is the specific assembly structure schematic diagram of the present invention;

3 is a schematic diagram of the specific structure of the microfluidic chip;

Fig. 4 is the collection result of the forward scattered light signal of the 10 μm diameter microsphere.

Among them, 1—sheath liquid microchannel, 2—sample microchannel, 3—waste liquid microchannel, 4—excitation optical fiber channel, 5—excitation on-chip microlens, 6—collection on-chip microlens, 7—collection optical fiber channel.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example 1

A small microfluidic chip system for the typing analysis of biological particles, as shown in Figure 3, includes a sheath liquid microchannel 1, a sample microchannel 2, and a waste liquid microchannel 3; it also includes an excitation fiber channel 4, an excitation sheet Carrying microlens 5, collecting on-chip microlens 6, and collecting fiber optic channel 7; the excitation on-chip microlens 5 and collecting on-chip microlens 6 are respectively placed on both sides of the waste liquid microchannel 3; the excitation on-chip microlens 5 The optical axis and the waste liquid microchannel 3 are perpendicular to each other; the optical axis of the collecting on-chip microlens 6 forms an included angle with the waste liquid microchannel 3, and it is necessary to ensure that the angle of the included angle is not 90°; the exciting optical fiber channel 4 and the collecting optical fiber channel 7 is set on the chip, wherein the excitation optical fiber channel 4 is coaxial with the optical axis of the excitation on-chip microlens 5, and the collection optical fiber channel 7 is coaxial with the optical axis of the collection on-chip microlens 6.

1. Substrate design

In order to let the biological particles line up in the outflow channel and pass through the detection area one by one to realize single particle sampling, the microfluidic channel structure and inlet flow rate were simulated and designed by using fluid dynamics simulation software. In order to introduce the 633nm laser into the microfluidic chip device and form a detection spot similar to the size of the particle in the outflow channel, the optical simulation software was used to simulate the exiting beam from the end face of a single-mode fiber (4μm core diameter 0.13NA) in the microfluidic chip. The on-chip transmission path was designed and fabricated for exciting on-chip microlenses and collecting on-chip microlenses.

2. Substrate fabrication

【1】Mask Design

Use drawing software to design photolithographic mask, design microfluidic channel, optical fiber channel and on-chip microlens structure on the mask, and output mask film with laser imagesetter.

【2】Reject glue

The amount of SU-8 photoresist spin coating is 1ml/inch, and a 3-inch silicon wafer is used, so 3ml SU-8 photoresist is poured on the center of the silicon wafer, and the glue is sprayed with an all-in-one machine: 500r/min Spin coating for 10s, Spin coating at 1375r/min for 30s, and let stand for 10 minutes to eliminate the edge effect to obtain a 130μm thick glue surface.

【3】Pre-baking: The purpose of pre-baking is to volatilize the solvent in the SU-8 photoresist and to take away the fine bubbles in the photoresist, so rate control is the key. During the experiment, the temperature was raised from room temperature to 95°C at a rate of 5°C/min on the heating platform, during which the temperature was maintained at 65°C and 95°C for 5 min and 25 min, respectively, and then the temperature was slowly lowered to below 75°C.

【4】Lithography: The theoretical formula of exposure time is: exposure time = exposure dose/exposure machine power, the power of mercury lamp in the laboratory is 20mW/cm2, and the exposure dose of 130μm thick glue is between 350mJ/cm2～550mJ/cm2 During the experiment, 450mJ/cm2 was used in the experiment, so the exposure time was 32.5s.

[5] Post-exposure bake: Since SU-8 is a negative photoresist, it is necessary to use post-exposure bake to strengthen the bonding of the light-receiving part to prevent the pattern from being dissolved during development. During the experiment, the temperature was raised from room temperature to 95°C at a rate of 5°C/min on the heating platform, during which the temperature was kept at 65°C and 95°C for 5min and 12min, respectively, and then slowly lowered to room temperature.

【6】Development and fixation: Put the silicon substrate into the developer solution, and at the same time shake it gently to make the development more complete. After the development, rinse or soak with isopropanol for fixation, then clean it with plasma water, and dry it with nitrogen. If it is found that white stains remain on the silicon substrate, it means that the development is insufficient, and the development and fixing process need to be repeated.

【7】Hard baking: Place the silicon wafer on a hot plate and bake it at 120°C for 10 minutes, so that the photoresist can firmly adhere to the surface of the silicon wafer to ensure the reusability of the mold.

[8] Silanization: In order to make the PDMS easy to peel off from the mold, the mold was silanized for 1 hour to complete the entire mold making.

[9] Molding: Place the clean silicon substrate on a horizontal aluminum plate and wrap a circle of tape around it to prevent PDMS from leaking out. On the mold, let it stand for 5 minutes to make it self-levelling. Thereafter, it was transferred to a vacuum oven at 100°C and cured for 1 hour.

[10] Bonding: After cooling, release the PDMS structure from the SU-8 mold, and punch holes at the sample outlet and inlet, and then place it together with the cleaned PDMS/glass/PMMA cover slip 2 In the oxygen plasma surface treatment instrument, the surface was treated for 30s, and after taking it out, the two were quickly bonded to form a closed microchannel (note the extrusion bubbles), and the chip production was completed.

3. Assembly,

In order to introduce the biological microparticle sample and the sheath fluid into the microchannel, the biological microparticle sample and the sheath fluid are pumped into the microchannel by using a microflow pump. The invention can simultaneously detect three parameters of biological microparticles: forward scattered light, side scattered light and fluorescence. The light source required for the detection of scattered light and fluorescence is provided by the same 633nm laser. A single-mode optical fiber is installed in the excitation optical fiber channel 4, and the laser output is led to the microfluidic chip device; a multi-mode optical fiber is installed in the collection optical fiber channel 7 to collect the scattering and light generated by a single biological particle passing through the detection area, and transmit it to the photoelectric detector device. With the microfluidic chip as the center, photodetectors and related equipment are placed around the chip. The overall structure of the device is shown in Figure 2.

4. System performance

Use a microflow pump to pump the polystyrene microsphere sample with a diameter of 10 μm and the sheath liquid into the sample microchannel 1 and the sheath liquid microchannel 2 respectively, by controlling the injection speed at the sample inlet and the sheath liquid inlet. Ratio to realize the injection of a single biological particle, the excitation optical fiber in the excitation fiber channel 4 guides the laser to the microfluidic chip, and converges to the center of the waste liquid microchannel 3 through the excitation chip-mounted microlens 5, forming a particle size similar to the diameter of the biological particle The light spot is detected. When a single biological particle passes through the detection area, forward scattered light, side scattered light and side fluorescence are generated. The scattered light and fluorescence are collected by the on-chip microlens 6, coupled to the collection optical fiber and detected by the photodetector, thereby realizing Multiparameter detection and analysis of biological particle samples. Figure 4 shows the forward scattered light signal of microspheres with a diameter of 10 μm collected by this system. According to statistical calculations, the coefficient of variation CV of this system is about 18%, and the signal-to-noise ratio SNR is about 20dB. Compared with the biological particle typing analysis system (CV: 20% ~ 30%, SNR ~ 9dB) controlled by the chip, the present invention has good response consistency and stability to uniform microspheres of a certain size, and is more superior system performance.

Claims (2)

1. for a small-sized micro-fluidic chip system for biological particle phenotypic analysis, including sheath fluid microchannel (1), sample microchannel (2), waste liquid microchannel (3)；It is characterized in that: also include excitation fiber passage (4), excite sheet load lenticule (5), collecting tab load lenticule (6), collect optical-fibre channel (7)；Described excite sheet load lenticule (5) with collecting tab load lenticule (6) be respectively placed in waste liquid microchannel (3) both sides；The optical axis exciting sheet load lenticule (5) is mutually perpendicular to waste liquid microchannel (3)；The optical axis of collecting tab load lenticule (6) and waste liquid microchannel (3) have angle, and need to ensure that the angle of this angle is not 90 °；Excitation fiber passage (4) is opened on chip with collecting optical-fibre channel (7), wherein excitation fiber passage (4) and the light shaft coaxle exciting sheet load lenticule (5), collect the light shaft coaxle of optical-fibre channel (7) and collecting tab load lenticule (6)。

2. a kind of small-sized micro-fluidic chip system for biological particle phenotypic analysis as claimed in claim 1, it is characterized in that: the work process of described system: be in that to utilize MEMS technology to process the micro flow control chip device that biological particle can carry out multiparameter detection, use it for the phenotypic analysis of various biological particle；This micro-fluidic chip system adopts two dimension folder circulation road sample introduction, and biological particle sample, under the parcel of sheath fluid, forms a line and passes through detection zone one by one；Laser is through fiber coupler entrance excitation fiber to micro-fluidic chip, formed through exciting sheet load lenticule to entreat in the channel and detect the hot spot that biological particle diameter dimension is close, when single detection biological particle is by detection zone, produce forward scattering light, side scattered light and fluorescence, and carried lenticule by the collecting tab of passage offside respectively and collect optical fiber and collect to photodetector；Through data collecting card, the data collected are reached PC and be analyzed display。