Single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing and preparation method thereof

Abstract

The invention discloses a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing and a preparation method thereof, and belongs to the technical field of single-cell detection.The preparation method comprises the steps that 1, a mask is formed; step 2, corroding an alignment mark; step 3, spin coating of photoresist, pre-baking, extensive exposure, post-baking and hardening; step 4, spin-coating a photoresist, pre-baking, exposing, post-baking, not developing, cooling, then spin-coating the photoresist, pre-baking, exposing, post-baking, developing, and hardening to form a microfluidic mold; step 5, mixing the prepolymer of the polydimethylsiloxane polymer with a curing agent, and pouring the mixture on a mold after vacuum degassing; and step 6, aligning and bonding two pieces of PDMS with the same structural parameters, and then bonding the two pieces of PDMS with the quartz substrate. According to the invention, high-sensitivity electrical detection and high-resolution image detection can be carried out at the same time, the problem of imaging blurring caused by a three-dimensional focusing channel manufactured by 3D printing is solved, the manufacturing efficiency is improved, and the manufacturing cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of single cell detection, and in particular relates to a single cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing and a preparation method thereof. Background Art

[0002] Blood is an important component of the human body, and plays a role in nutrient transport and regulating organ activities. Peripheral blood cells can be mainly divided into red blood cells, white blood cells and platelets. As immune cells, white blood cells play an important role in the human immune system, helping the body resist infection and cancer. They are mainly divided into five types: neutrophils, basophils, eosinophils, monocytes and lymphocytes. The number and status of white blood cells are closely related to the health status of the human body and the progression of the disease. Therefore, the classification and counting of white blood cells can provide important references for clinical diagnosis and treatment.

[0003] Microscopic examination is the "gold standard" for leukocyte identification. After the prepared blood smear is stained, the cell morphology is observed under a microscope and classified and counted. However, the smear-based method has low detection throughput, slow speed (~10 samples / hour), and relies on expert experience and manually defined features, and the accuracy of leukocyte classification is limited.

[0004] In response to the clinical needs of rapid classification and typing of white blood cells, commercial blood cell analyzers classify and count cells based on the principles of electrical impedance and light scattering. When a single cell passes through the detection area quickly, it causes changes in scattered light and impedance waveforms, and has a high detection flux. However, since the volume of cells in the detection area is small, the electrical detection sensitivity is low; and the cell structure information obtained by light scattering is far less than that obtained by cell imaging, which has limited help in improving the accuracy of white blood cell identification and classification.

[0005] The microfluidic detection platform based on micromachining technology mainly includes two methods: compression and non-compression channels. Among them, the single-cell detection platform based on compression channels uses microchannels that are smaller than the geometric size of the cells. When the cells to be tested pass through the compression channels, the electric field lines can be effectively blocked, causing a large impedance change; and at the entrance of the compression channel, the cells are confined to a relatively small detection plane, which can be imaged with high definition. However, due to the limitation of channel size, the compression channel can only adapt to cells within a certain size range, and there are problems such as channel blockage and low detection flux in actual application. For non-compression microchannels with a cross-sectional area larger than the cell size, the risk of blockage can be reduced, cells of more sizes can be adapted, and the detection flux can be increased. At the same time, in terms of electrical detection, the electric field lines blocked by the cells to be tested are relatively small, and no obvious impedance changes can be obtained; in terms of image detection, cells are easily out of focus, resulting in blurred imaging.

[0006] In order to achieve single-cell high-sensitivity electrical detection and high-quality image detection while increasing detection throughput and reducing the risk of clogging, the patent CN116026897A was cited to produce an integrated sheath flow focusing device, which achieves high-sensitivity electrical detection by surrounding the conductive sample liquid with insulating sheath liquid, and image detection can be achieved by fixing the cells near the focal plane through three-dimensional focusing. However, the device is formed by printing insulating resin materials using 3D printing technology. Due to the resolution limitations of 3D printing technology, the influence of layer patterns and the interference caused by the color of the resin itself, the imaging resolution will be reduced and the imaging quality will be affected. In addition, when the same chip is used for a long time, the channel will be damaged and cracked, resulting in a decrease in detection performance, and the chip needs to be replaced when the channel is blocked. 3D printing technology can only print one chip at a time. Compared with the method of manufacturing microfluidic chips based on molds, it has the disadvantages of high cost and low manufacturing efficiency.

[0007] Therefore, it is very meaningful to propose a preparation method for a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing, and to make the detection chip based on soft lithography technology, which can reduce manufacturing costs and improve manufacturing efficiency. At the same time, it can achieve high-throughput single-cell high-sensitivity electrical detection and high-resolution image detection to improve cell classification accuracy. Summary of the invention

[0008] In combination with the technical problems raised in the background technology, the present invention uses microfluidic technology to invent a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing and its preparation method. Compared with the prior art, this invention has two improvement goals: (1) Improving the quality of single-cell optical imaging. The three-dimensional fluid focusing structure based on 3D printing technology will reduce the imaging resolution and affect the imaging quality due to the limitation of the resolution of 3D printing technology, the influence of layer patterns and the interference caused by the color of the resin itself. (2) Improving chip manufacturing efficiency and reducing manufacturing costs. When the same chip is used for a long time, the channel will be damaged and cracked, resulting in a decrease in detection performance, and the chip needs to be replaced when the channel is blocked. 3D printing technology can only print one chip at a time. Compared with the method of manufacturing microfluidic chips based on molds, it is expensive and has low manufacturing efficiency.

[0009] The technical solution adopted by the present invention is as follows:

[0010] The method for preparing a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing comprises the following steps:

[0011] Step 1: First, chromium metal is sputtered on the silicon wafer, and then photoresist is spin-coated, and a mask is formed after pre-baking, exposure, and development;

[0012] Step 2: Then, a chromium etching solution is used to etch out the alignment marks required for subsequent manufacturing;

[0013] Step 3, then spin-coat the photoresist, and perform pre-baking, flood exposure, post-baking, and hardening;

[0014] Step 4, spin-coating photoresist, and performing pre-baking, exposure, post-baking, without developing, and then spin-coating photoresist after cooling, and performing pre-baking, exposure, post-baking, and developing, and forming the required microfluidic mold after hardening the film;

[0015] Step 5, then mixing the prepolymer of the polydimethylsiloxane polymer and the curing agent, and pouring the mixture on the prepared mold after vacuum degassing, and then curing, demoulding, punching, and cleaning;

[0016] Step 6: First, align and bond two pieces of PDMS with the same structural parameters, and then bond them to a quartz substrate to obtain a single-cell photoelectric detection chip based on three-dimensional focusing of a rectangular sleeve.

[0017] In the above technical solution, in step 1, the photoresist is SPR220-3.0 photoresist.

[0018] In the above technical solution, in step 3, the photoresist is SU 8-5 photoresist with a thickness of 5 um.

[0019] In the above technical solution, in step 4, the spin-coated photoresist is SU 8-25 with a thickness of 25 um, and after cooling, the spin-coated photoresist is SU 8-75 photoresist with a thickness of 75 um.

[0020] In the above technical solution, in step 5, the mixing ratio of the prepolymer of the polydimethylsiloxane polymer and the curing agent is 10:1 by volume.

[0021] The single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing was prepared by the above method.

[0022] Beneficial effects:

[0023] (1) The present invention uses soft lithography to produce a three-dimensional focusing channel based on a rectangular sleeve, which can simultaneously perform high-sensitivity electrical detection and high-resolution image detection, solves the problem of imaging blur caused by the three-dimensional focusing channel produced by 3D printing, and improves manufacturing efficiency and reduces manufacturing costs.

[0024] (2) The present invention improves the quality of single-cell optical imaging. The microfluidic chip manufactured using soft lithography technology is composed of PDMS and quartz, has high optical transmittance and low fluorescence background noise, and can meet the needs of high-definition and high-resolution cell imaging. It solves the problem of resolution limitation of 3D printing technology, layer texture influence and the problem of the color of the resin itself reducing the quality of cell imaging.

[0025] (3) The present invention improves chip manufacturing efficiency and reduces manufacturing costs. The mold manufactured by micromachining is based on photolithography technology and has high dimensional accuracy. Multiple chips can be manufactured at one time, and the mold can be reused. Compared with the disposable microfluidic channel made by 3D printing, it effectively improves the chip manufacturing accuracy and efficiency and reduces the chip manufacturing cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Connect hardware devices and modules;

[0027] Figure 2 It is a rectangular sleeve three-dimensional focusing module;

[0028] Figure 3 For the chip manufacturing process.

[0029] Among them: rectangular sleeve three-dimensional focusing module 1, fluid control module 2, impedance detection module 3, image acquisition module 4;

[0030] Sample flow injection channel 1-1, left electrode inlet 1-2, right electrode inlet 1-3, mixed fluid outlet 1-4, sheath flow injection channel 1-5, rectangular sleeve three-dimensional focusing channel 1-6, photoelectric detection area 1-7; DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example

[0033] This embodiment is mainly divided into hardware devices and experimental procedures.

[0034] (1) The hardware devices required for this embodiment are as follows: Figure 1 As shown, it mainly includes four parts: a rectangular sleeve three-dimensional focusing module 1, a fluid control module 2, an impedance detection module 3 and an image acquisition module 4.

[0035] The rectangular sleeve three-dimensional focusing module 1 is used to achieve the focusing of the sample flow and the cells therein, and is made using soft lithography technology. The overall structure of the chip is as follows Figure 2-Ⅰ As shown, the rectangular sleeve three-dimensional focusing channel 1 is mainly composed of a sample flow injection channel 1-1, a sheath flow injection channel 1-5, a rectangular sleeve three-dimensional focusing channel 1-6, a left electrode inlet 1-2, a right electrode inlet 1-3 and a mixed fluid outlet 1-4. Figure 2-ⅡIt is a top view of the key area of ​​the microfluidic chip, in which the sample flow injection channel 1-1 is introduced into the high conductivity sample solution whose flow rate is controlled by the fluid control module 2. The cells to be tested are suspended in the sample flow solution, and its cross-section is a rectangle with a width of 600um and a height of 200um, and gradually changes to a rectangle with a width of 50um and a height of 50um when approaching the rectangular sleeve three-dimensional focusing area 1-6, which is much larger than the diameter of most peripheral blood cells (10-25um); the sheath flow injection channel 1-5 is two symmetrically distributed rectangular channels, and the two channels have the same inlet. The channel is used to introduce the low conductivity sheath flow solution whose flow rate is controlled by the fluid control module 2. When entering the rectangular sleeve three-dimensional focusing area 1-6, the cross-section is a rectangle with a width of 100um and a height of 200um; the rectangular sleeve three-dimensional focusing area 1-6 is used to focus the sample flow, and the cross-section is as shown in FIG. Figure 2-III As shown, the sample flow is focused on the central axis of the channel with an approximately circular cross-section. The length of the focusing channel is 100um, and the diameter of the sample flow after focusing can vary from 0 to 50um; thereafter, the sample flow enters the photoelectric detection area 1-7 with a width of 50um, a height of 50um, and a length of 50um to collect the impedance and image information of the cells; finally, the sample flow is expanded and discharged through the mixed fluid outlet 1-4 channel with a width of 600um and a height of 200um.

[0036] The microfluidic chip processing flow is as follows Figure 3 Specifically, 1000 nanometers of silicon are first sputtered on the silicon wafer. The chromium metal is then spin-coated with SPR220-3.0 photoresist. After pre-baking, exposure, and development, a mask is formed. Subsequently, a chromium etching solution is used to etch out the alignment marks required for subsequent manufacturing, such as Figure 3 (a)-(c) As shown; in order to increase the adhesion between the substrate and the photoresist, a 5um thick SU 8-5 photoresist is then spin-coated, and pre-baking, flood exposure, post-baking, and hardening are performed. Thereafter, a 25um thick SU 8-25 photoresist is spin-coated, and pre-baking, exposure, and post-baking are performed without developing. After cooling, a 75um thick SU 8-75 photoresist is then spin-coated, and pre-baking, exposure, post-baking, and development are performed. After hardening, the desired microfluidic mold is formed, as shown in FIG. Figure 3 (d)-(g) As shown. Then, the prepolymer of polydimethylsiloxane polymer (PDMS) and curing agent are mixed in a ratio of 10:1, and the mixture is poured on the prepared mold after vacuum degassing. After curing, demoulding, drilling, and cleaning, two pieces of PDMS with the same structural parameters are first aligned and bonded, and then bonded to the quartz substrate to obtain a completed microfluidic chip, as shown in FIG. Figure 3 As shown in (h)-(j).

[0037] The fluid control module 2 is used to control the flow rate of the sample flow and the sheath flow, and includes a fluid injection pump and a liquid guide tube. The fluid injection pump can output the required flow rate through manual control. For a syringe with a capacity of 1 ml, the fluid injection pump can provide an adjustable flow rate within the range of 0.0033ul / min-205.30ml / h, and is connected to the aforementioned rectangular sleeve three-dimensional focusing module 1 through the liquid guide tube.

[0038] The impedance detection module 3 is used to collect the impedance signal caused by the cell passing through the photoelectric detection area 1-7, including a phase-locked amplifier and a data acquisition card. According to the embodiment, the impedance signal at four frequencies can be detected, the output frequency is 100000 sampling points / s, and the electrode inlet on the left side 1-2 and the electrode inlet on the right side 1-3 of the aforementioned rectangular sleeve three-dimensional focusing module 1 are connected by inserting the electrode line.

[0039] Image acquisition module 4 is used to collect image information of cells passing through photoelectric detection areas 1-7, including an inverted microscope, a bright field light source and a high-speed camera. The bright field light source provides the incident light required for imaging, and the high-speed camera can record 128 8-1920 The video has a resolution of 1200 and a frame rate of 730-400000. The minimum exposure time is 1us. When combined with an inverted microscope, it can record image information of the cell migration process.

[0040] (2) The specific experimental process of this example is as follows:

[0041] First, connect the rectangular sleeve three-dimensional focusing module 1, the fluid control module 2, the impedance detection module 3 and the image acquisition module 4. The fluid control module 2 is connected to the sample flow inlet 1-1 and the sheath flow inlet 1-5 respectively through the liquid guide hose and the flat joint; the electrode wires of the impedance measurement module 3 are respectively connected to the left electrode inlet 1-2 and the right electrode inlet 1-3 of the rectangular sleeve three-dimensional focusing module 1; the optical path of the image acquisition module 4 is placed in the photoelectric detection area 1-7 in the rectangular sleeve three-dimensional focusing module 1, and imaging is performed through a 40X objective lens.

[0042] Before the sample is introduced, all channels in the microfluidic chip are filled with anhydrous ethanol. The purpose is to first use the organic solvent to infiltrate the hydrophobic PDMS inner wall to ensure that no bubbles are generated when the sample flow is introduced.

[0043] Then, a high-conductivity sample flow is added to the sample flow introduction channel 1-1 using the fluid control module 2, and cells of a certain concentration are suspended, and a low-conductivity solution is added to the sheath flow introduction channel 1-5, and sucrose is dissolved in the sheath flow to maintain an osmotic pressure equal to that of the cells, so as to avoid cell rupture or shrinkage caused by osmotic pressure mismatch. The flow rates of the sample flow and the sheath flow are controlled by the fluid control module 2, and the sample flow is three-dimensionally focused into a nearly circular shape by the sheath flow, and at the same time, most of the electric field lines generated by the alternating current are confined to the high-conductivity sample flow, and the size of the focused sample flow is controlled by adjusting the flow rate ratio of the sample flow to the sheath flow.

[0044] Next, driven by the fluid, the cells suspended in the sample flow pass through the rectangular sleeve focusing area 1-6, the photoelectric detection area 1-7 and the mixed fluid outlet 1-4 in sequence. The image acquisition module 4 is used to collect image video information of the cells when they pass through the photoelectric detection area, and the impedance detection module 3 is used to collect impedance information of the cells when they pass through the photoelectric detection area.

[0045] In this embodiment, the sheath flow is an insulating deionized water solution in which sucrose is dissolved, and a low-conductivity solution that is miscible with the water system does not affect the realization of basic functions; the sample flow is a phosphate buffered saline (PBS for short), and a conductive solution such as iodixanol prepared with physiological saline, cell culture medium, and PBS does not affect the realization of basic functions.

[0046] In this embodiment, the substrate is made of quartz glass material. Those skilled in the art should understand that, in addition to quartz glass, the substrate can be a transparent material such as ordinary glass, polymethyl methacrylate (PMMA, also known as acrylic, acrylic, or organic glass) or polydimethylsiloxane (PDMS) sheet.

[0047] In this embodiment, the rectangular sleeve three-dimensional focusing module is processed using soft lithography technology based on SU 8, but the three-dimensional fluid focusing module can also be formed using soft lithography based on silicon etching and femtosecond laser quartz processing technology.

[0048] In this embodiment, the shape and size of the interface between the sample flow channel inlet, sheath flow channel inlet, left (right) side electrode inlet, and mixed fluid outlet in the rectangular sleeve three-dimensional focusing module and the fluid control module do not affect the realization of basic functions.

[0049] In this embodiment, the rectangular sleeve three-dimensional focusing demonstrated is the basic unit of this method, which can be easily arranged in parallel and series in the direction of cell migration, and even the combination of certain structures will bring different effects.

[0050] In this embodiment, the channel cross-section in the rectangular sleeve three-dimensional focusing module is rectangular, and can also be replaced with a circular or semicircular shape without affecting the realization of basic functions.

[0051] In this embodiment, fluid is used to drive the cell solution through the channel, but other methods may also be used, such as applying negative pressure to the sheath flow outlet channel and the sample flow outlet channel.

[0052] The present invention does not elaborate on some of the common technologies of those skilled in the art. The above-described embodiments are only descriptions of preferred implementations of the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementations described. Without departing from the design spirit of the present invention, various modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for preparing a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing, characterized in that: The steps include: Step 1: First, chromium metal is sputtered on the silicon wafer, and then photoresist is spin-coated, and a mask is formed after pre-baking, exposure, and development; Step 2: Then, a chromium etching solution is used to etch out the alignment marks required for subsequent manufacturing; Step 3, then spin-coat the photoresist, and perform pre-baking, flood exposure, post-baking, and hardening; Step 4, spin-coating photoresist, and performing pre-baking, exposure, post-baking, without developing, and then spin-coating photoresist after cooling, and performing pre-baking, exposure, post-baking, and developing, and forming the required microfluidic mold after hardening the film; Step 5, then mixing the prepolymer of the polydimethylsiloxane polymer and the curing agent, and pouring the mixture on the prepared mold after vacuum degassing, and then curing, demoulding, punching, and cleaning; Step 6: First, align and bond two pieces of PDMS with the same structural parameters, and then bond them to a quartz substrate to obtain a single-cell photoelectric detection chip based on three-dimensional focusing of a rectangular sleeve.

2. The method for preparing a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing according to claim 1, characterized in that: In step 1, the photoresist is SPR220-3.0 photoresist.

3. The method for preparing a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing according to claim 1, characterized in that: In step 3, the photoresist is SU 8-5 photoresist with a thickness of 5 um.

4. The method for preparing a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing according to claim 1, characterized in that: In step 4, the spin-coated photoresist is SU 8-25 with a thickness of 25 um, and after cooling, the spin-coated photoresist is SU 8-75 photoresist with a thickness of 75 um.

5. The method for preparing a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing according to claim 1, characterized in that: In step 5, the mixing ratio of the polydimethylsiloxane polymer prepolymer and the curing agent is 10:1 by volume.

6. A chip obtained by the method for preparing a single-cell photoelectric detection chip based on rectangular sleeve three-dimensional focusing according to any one of claims 1 to 5.

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