Integrated preparation method of micro cell detection chip

Abstract

The application provides an integrated preparation method of a micro cell detection chip, and aims at solving the technical problems of the prior preparation method, such as being complicated, the micro optical element in the integrated chip being difficult to realize high-quality imaging, and poor high-temperature and high-pressure resistance, etc. The preparation method comprises the following steps: S1, preparing a sample according to the size requirement of the micro cell detection chip; S2, compiling the moving path of a workbench according to the structure and size requirement of the micro cabin and micro channel of the micro cell detection chip; S3, fixing the sample on the workbench; S4, starting a femtosecond laser and the workbench, moving the workbench along the precompiled moving path, and scanning the sample after focusing by the femtosecond laser through an optical microscope to obtain a modified area of the micro cabin and the micro channel; S5, sequentially etching and polishing the modified area; S6, placing microspheres in the micro cabin to form a micro optical element; and S7, packaging to obtain the micro cell detection chip.

Description

Technical Field

[0001] This invention relates to a method for fabricating a detection chip, and more particularly to an integrated method for fabricating a micro cell detection chip. Background Technology

[0002] Microfluidics technology integrates the basic operational units of sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes onto a single chip at the micrometer scale, automating the analysis process. Micro / Nano Optics components refer to optical elements with micrometer / nanometer-scale feature sizes, such as light sources, optical waveguides, microlenses, and detectors, which can be applied in numerous fields including sensing and imaging, display and lighting, and communication and interconnection.

[0003] Key components in traditional biological detection systems, such as optical systems that enable optical imaging and fluorescence detection, cannot be effectively integrated into biochips, resulting in high manufacturing costs for the entire biological detection system. Furthermore, the low integration level leads to a large size of the biological detection system.

[0004] With the continuous development of micro-nano fabrication technology, integrated chips formed by integrating micro-nano optical elements and microfluidic chips have attracted widespread attention, effectively solving the problems of large size and low integration in biological detection systems. These integrated chips can be fabricated using methods such as photoresist thermal reflow, photopolymer inkjet printing, and ultra-precision diamond milling. However, these methods are all relatively cumbersome, and the micro-optical elements in the resulting integrated chips are difficult to achieve high-quality imaging. Furthermore, these methods generally use polymers as substrates, which, although possessing good chemical inertness, result in integrated chips with poor high-temperature and high-pressure resistance. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated fabrication method for a micro cell detection chip, which solves the technical problems of existing fabrication methods being cumbersome, the micro-optical elements in the fabricated integrated chip being difficult to achieve high-quality imaging, and the poor performance of high temperature and high pressure resistance.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] An integrated fabrication method for a microcell detection chip, the microcell detection chip comprising a substrate, a microchamber disposed on the upper part of the substrate, microspheres disposed within the microchamber, and microchannels disposed on the lower part of the substrate, characterized by comprising the following steps:

[0008] S1. Prepare the substrate sample in advance according to the size requirements of the micro cell detection chip;

[0009] S2, based on the structural and dimensional requirements of the microcells and microchannels of the microcell detection chip, the movement path of the worktable is pre-compiled in the five-axis machining system; the worktable is the five-axis worktable of the five-axis machining system; the movement path includes the movement path of the worktable during the modification of the microcells and microchannels; the modification is performed by scanning modification using a femtosecond laser focused by an optical microscope;

[0010] S3, fix the substrate sample from step S1 on the worktable of step S2, and position the focus of the optical microscope at the initial position of the microchamber modification area on the substrate sample.

[0011] S4, start the femtosecond laser and stage. The stage moves along the pre-compiled movement path. The femtosecond laser is focused by an optical microscope and scans the substrate sample, and the modified areas of microchambers and microchannels are obtained sequentially on the substrate sample.

[0012] S5, the modified areas of the microchambers and microchannels from step S4 are sequentially etched and polished to obtain smooth microchambers and microchannels.

[0013] S6, place the pre-prepared microspheres into the microchamber obtained in step S5 to form a micro-optical element; the size of the microspheres matches the bottom size of the microchamber;

[0014] S7. The micro-optical elements obtained in step S6 are packaged to obtain a micro cell detection chip.

[0015] Furthermore, in S1, the substrate sample is a quartz glass sample.

[0016] Furthermore, in S2, the cross-section of the micro-chamber is U-shaped, and the moving path of the workbench when modifying the micro-chamber is: a moving radius of R from bottom to top. i The movement height is h i A circular path, in which,

[0017] R i =Ri*x

[0018]

[0019] In the above formula, R is the radius of the top of the micro-chamber U-shaped structure, i is the number of modified layers set according to the micro-chamber size requirements, i≥0, and x is the radius reduction of each layer during modification, with x ranging from 0.5 to 5 μm.

[0020] The microchannels are either straight or spiral channels.

[0021] Further, in S5, the etching involves immersing the substrate sample modified in step S4 in a hydrofluoric acid solution with a concentration of 5-8%, and then etching it by ultrasonication for 2-3 hours.

[0022] Furthermore, in S5, the polishing process employs an oxyhydrogen annealing hot polishing process, specifically:

[0023] a) Preheat the etched substrate sample at a temperature of 800℃~1100℃ for 12~17s;

[0024] b) The preheated substrate sample is polished using a flame gun of a thermal polishing system at a temperature of 1200–1300°C for 12–15 seconds.

[0025] c) After polishing, the substrate sample is cooled and cured in a natural environment. After the temperature reaches room temperature, it is tested using a confocal microscope to determine whether the surface roughness of the microchamber and microchannel meets the expected requirements. If it does, proceed to step S6; otherwise, return to step a).

[0026] Further, step S6 specifically involves placing the pre-prepared microspheres inside the microchamber and dripping deionized aqueous solution onto the inner surface of the microchamber drop by drop until the microspheres sink to the bottom of the microchamber along with the deionized aqueous solution. Then, the deionized aqueous solution on the inner surface of the microchamber and the surface of the microspheres is dried to obtain the micro-optical element.

[0027] Furthermore, in step S6, the microspheres are selected from barium titanate solid microspheres with a refractive index of 1.8 to 2.2.

[0028] Further, in step S7, the encapsulation process is as follows:

[0029] 7.1) Cover one side of the substrate sample modified microchamber with a transparent film, and leave a liquid inlet on the transparent film above the microchamber;

[0030] 7.2) Connect a catheter to the inlet and outlet of the microchannel for the input and output of the cell solution to be tested.

[0031] Furthermore, the transparent film is selected from one of thin quartz glass sheet, PMMA film, and PDMS film.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. This invention uses a five-axis machining system and a femtosecond laser, and by pre-compiling the movement path of the worktable, it achieves high-precision integrated machining of micro-chambers and micro-channels. The machining method is simple, and the resulting micro-cell detection chip has a high degree of integration.

[0034] 2. This invention selects quartz glass as the substrate sample. Quartz glass has high temperature and high pressure resistance as well as corrosion resistance, which not only realizes high-quality imaging of micro-optical elements, but also realizes the integrated fabrication of micro-chambers and microchannels on hard and brittle materials, thereby improving the integration of micro-optical elements and microfluidic chips.

[0035] 3. This invention modifies micro-chambers and micro-channels using femtosecond lasers. Femtosecond lasers have the characteristics of ultra-short pulse time, ultra-high peak power, strong controllability, and high processing precision, thereby improving processing efficiency and processing quality.

[0036] 4. By rationally controlling the process parameters of etching and polishing, this invention further improves the processing quality of micro cell detection chips, thereby improving the detection accuracy of the chips. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the radial path of the femtosecond laser scanning layer by layer in an embodiment of the present invention.

[0038] Figure 2 This is a schematic diagram of the axial path of the femtosecond laser scanning layer by layer in an embodiment of the present invention.

[0039] Figure 3 This is a schematic diagram of the micro-chamber and microchannel modification area in an embodiment of the present invention.

[0040] Figure 4 This is a schematic diagram of the polished microchamber and microchannel structure in an embodiment of the present invention.

[0041] Figure 5 This is a schematic diagram of the packaged micro cell detection chip structure in an embodiment of the present invention.

[0042] Figure 6 This is a reference diagram showing the usage status of the micro cell detection chip prepared according to an embodiment of the present invention.

[0043] Figure 7 This is a schematic diagram of observing biological cells using the microcell detection chip prepared according to the embodiments of the present invention.

[0044] The attached figures are labeled as follows:

[0045] 1-Substrate, 2-Microchamber, 3-Microsphere, 4-Microchannel, 5-Transparent film, 6-Infusion inlet. Detailed Implementation

[0046] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] Currently, integrated chips, which combine micro / nano optical elements and microfluidic chips, offer a core advantage for biological research, providing crucial support for cell counting, cell observation, and nucleic acid and protein detection. However, existing integrated chip fabrication generally uses polymers as substrates, and quartz glass has not yet been used as a substrate. This is because the anisotropy that occurs during the etching of quartz glass makes it difficult to fabricate micro-optical elements on it. This embodiment mainly studies an efficient and high-quality fabrication method based on femtosecond laser micro / nano fabrication technology to integrate quartz-based micro-optical elements and microfluidics into a biodetection chip.

[0048] This embodiment provides an integrated fabrication method for a microcell detection chip, wherein the microcell detection chip includes a substrate 1, a microchamber 2 disposed on the upper part of the substrate 1, microspheres 3 disposed within the microchamber 2, and microchannels 4 disposed on the lower part of the substrate 1. The fabrication method of the microcell detection chip includes the following steps:

[0049] S1. Prepare a substrate sample in advance according to the size requirements of the micro cell detection chip. In this embodiment, the substrate sample used is a quartz glass sample. Quartz glass has high temperature resistance and corrosion resistance, which can further improve the detection performance of the chip.

[0050] S2. Based on the structural and dimensional requirements of the microcell 2 and microchannel 4 of the microcell detection chip, a TCL program is pre-compiled in the five-axis machining system. This program includes the movement path of the worktable.

[0051] The worktable used in this embodiment is a five-axis worktable of a five-axis machining system; the movement path includes the movement path of the worktable when modifying the micro-chamber 2 and the micro-channel 4; the modification is performed by scanning modification using a femtosecond laser after focusing through an optical microscope; the optical microscope can be the microscope of a five-axis machining system.

[0052] like Figure 1 and Figure 2 As shown, the cross-section of the microchamber 2 of the microcell detection chip in this embodiment is U-shaped. When modifying the microchamber 2, the moving path of the stage is from bottom to top with a moving radius of R. i The movement height is h i A circular path, in which,

[0053] R i =Ri*x

[0054]

[0055] In the above formula, R is the radius of the top of the U-shaped structure of the micro-chamber. i is the number of modified layers set according to the size requirements of micro-chamber 2, i≥0; when i=0, the workbench is at the initial height, R i=R0=R, meaning the laser scanner scans a circular modification area with radius R on the substrate sample; when i=0, the stage moves up one layer, and so on. The more layers, the higher the processing accuracy. x is the step reduction amount (i.e., radius reduction amount) of each layer during modification, and the value of x ranges from 0.5 to 5 μm.

[0056] The microchannel 4 can be a straight channel or a spiral channel. When it is a straight channel, the movement path of the worktable is linear; when it is a spiral channel, the movement path of the worktable is spiral. The specific movement path of the worktable is set according to the actual processing requirements.

[0057] S3. Fix the substrate sample from step S1 onto the worktable of step S2, and position the focal point of the optical microscope at the initial position of the modified microchamber 2 region on the substrate sample. After fixing the substrate sample, the flatness of the substrate sample generally needs to be adjusted using the Rx and Ry rotation axes of the five-axis machining platform to ensure its machining accuracy.

[0058] S4, the femtosecond laser and stage are activated. The stage moves along a pre-programmed path. The femtosecond laser, focused by an optical microscope, scans the substrate sample, sequentially obtaining the modified regions of microchamber 2 and microchannel 4 on the substrate sample, such as... Figure 3 As shown.

[0059] S5, the modified areas of microchamber 2 and microchannel 4 in step S4 are etched sequentially. The etching is performed by immersing the substrate sample modified in step S4 in a hydrofluoric acid solution with a concentration of 8% and ultrasonically etching for 2 hours to remove the modified areas in a localized manner, thereby forming U-shaped microchamber 2 and through microchannel 4.

[0060] After etching, the surface structures of microchambers 2 and microchannels 4 become rough, affecting the imaging quality of the micro-optical elements. Therefore, polishing is required to reduce the surface roughness of microchambers 2 and microchannels 4. After polishing, smooth microchambers 2 and microchannels 4 are obtained, such as... Figure 4 As shown.

[0061] The polishing process in this embodiment adopts an oxyhydrogen annealing hot polishing process, specifically as follows:

[0062] a) Preheat the substrate sample after etching in step 3 at a temperature of 800℃~1100℃ for 15s.

[0063] b) The preheated substrate sample was polished for 15 seconds at a temperature of 1200-1300℃ using a flame torch of a thermal polishing system.

[0064] c) After polishing, the substrate sample is cooled and cured in a natural environment. After the temperature reaches room temperature, it is tested using a confocal microscope to determine whether the surface roughness of the inner surfaces of the microchamber 2 and the microchannel 4 meets the expected requirements. If it does, proceed to step S6; otherwise, return to step a).

[0065] S6. Place the pre-prepared microspheres into the microchamber 2 obtained in step S5 to form a micro-optical element.

[0066] The microsphere 3 is made of barium titanate solid microspheres with a refractive index of up to 2.2. The specific size of the microsphere 3 must be matched with the bottom size of the microchamber 2.

[0067] The specific method for placing the microspheres 3 is as follows: place the pre-prepared microspheres 3 inside the microchamber 2, and drip the deionized aqueous solution into the inner surface of the microchamber 2 drop by drop. Due to the fluidity of the deionized aqueous solution, the microspheres 3 sink into the bottom of the microchamber 2 along with the deionized aqueous solution. Then, dry the deionized aqueous solution on the inner surface of the microchamber 2 and the surface of the microspheres 3 to obtain the micro-optical element.

[0068] Currently, the microchamber 2 is sized to accommodate only one microsphere 3, but the fabricated dimensions of the microchamber 2 can be adjusted according to the size of the microsphere 3. This method allows microspheres 3 with diameters ranging from 50 μm to 1 mm to be combined with corresponding microchambers 2 to form micro-optical elements.

[0069] Simultaneously, using this method, multiple microchambers 2 can also be prepared on quartz glass samples (the specific method is the same as the steps above), and microspheres 3 of corresponding sizes can be matched to form multiple micro-optical structures, so as to simultaneously detect biological samples in microchannels 4, expand the detection range, and improve detection efficiency.

[0070] S7, the micro-optical elements obtained in step S6 are packaged to obtain a micro-cell detection chip (e.g., Figure 5 (As shown).

[0071] The specific encapsulation method is as follows:

[0072] 7.1) Cover one side of the modified microcompartment 2 of the substrate sample with a transparent film 5, and leave a liquid inlet 6 on the transparent film 5 above the microcompartment 2. The transparent film 5 can be selected from thin quartz glass, PMMA film, or PDMS film, or other materials as needed. The transparent film 5 can be tightly attached to the side of the quartz glass with the microcompartment 2 by means of adhesives, bonding, etc.

[0073] 7.2) Connect a catheter to the inlet and outlet of microchannel 4 for the input and output of the cell solution to be tested.

[0074] Combination Figure 6 and Figure 7As shown, the method for observing microbial cells using the microcell detection chip prepared in this embodiment is as follows:

[0075] Figure 6 This is a schematic diagram illustrating the use of the microcell detection chip to observe microbial cells. First, the biological sample to be tested is injected through the microchannel inlet. The micro-optical elements, consisting of microspheres 3 and microchambers 2, can magnify the biological sample within the microchannel 4. Then, by using a simple digital microscope or low-power microscope, the detection of microbial cells and even viral cells in the biological sample can be achieved using a low-power objective lens.

[0076] This embodiment utilizes the microcell detection chip (prepared by the method of this invention) in conjunction with a low-magnification (10x) objective lens to achieve the observation and analysis of red blood cells with a diameter of 5-7 μm. The experimentally measured micro-optical elements can magnify biological samples within the microchannel by 2-5 times. Figure 7 As shown in the image, biological samples can be magnified 20-50 times when used with a 10x microscope lens.

[0077] In this embodiment, liquids with different refractive indices (such as water, or sucrose solutions, ethanol solutions, etc. of different concentrations) can be injected into the microchamber 2 through the injection inlet 6, thereby changing the refractive index of the medium in contact with the microsphere 3, and thus realizing the function of adjustable focal length of the micro-optical element.

[0078] Furthermore, based on the results observed by the aforementioned micro-cell detection chip, the morphology of microbial cells at different locations can be photographed and two-dimensional images can be obtained using devices such as CCD cameras. By comprehensively utilizing image processing, visual computing, and other technologies, the three-dimensional information of the object can be reconstructed by computer, thereby realizing the 3D morphological observation of microbial cells.

Claims

1. A method for integrally fabricating a micro cell detection chip, the micro cell detection chip comprising a substrate, a microchamber disposed on an upper portion of the substrate, a microsphere disposed in the microchamber, and a microchannel disposed on a lower portion of the substrate, the method comprising: Includes the following steps: ​ S1. Prepare a substrate sample in advance according to the size requirements of the micro cell detection chip; the substrate sample is a quartz glass sample. S2, based on the structural and dimensional requirements of the micro-chambers and microchannels of the microcell detection chip, the movement path of the worktable is pre-compiled in the five-axis machining system; the worktable is the five-axis worktable of the five-axis machining system; the movement path includes the movement path of the worktable during the modification of the micro-chambers and microchannels; the modification is performed by scanning modification using a femtosecond laser focused through an optical microscope; the cross-section of the micro-chamber is U-shaped, and the movement path of the worktable during the modification of the micro-chamber is: a movement radius from bottom to top of [missing information]. The moving height is A circular path, in which, ； ； In the above formula, Let be the radius of the top of the U-shaped structure of the micro-cabin, and i be the number of modified layers set according to the size requirements of the micro-cabin, i≥0. x represents the reduction in radius of each layer during modification, with x ranging from 0.5 to 5 μm. S3, fix the substrate sample from step S1 on the worktable of step S2, and position the focus of the optical microscope at the initial position of the microchamber modification area on the substrate sample. S4, start the femtosecond laser and stage. The stage moves along the pre-compiled movement path. The femtosecond laser is focused by an optical microscope and scans the substrate sample, and the modified areas of microchambers and microchannels are obtained sequentially on the substrate sample. S5, the modified areas of the microchambers and microchannels in step S4 are sequentially etched and polished to obtain smooth microchambers and microchannels; the etching is performed by immersing the substrate sample modified in step S4 in a hydrofluoric acid solution with a concentration of 5-8% and etching by ultrasonic etching for 2-3 hours. The polishing process employs an oxyhydrogen annealing hot polishing process, specifically: a) Preheat the etched substrate sample at a temperature of 800℃~1100℃ for 12~17s; b) The preheated substrate sample was polished using a flame gun of a thermal polishing system at a temperature of 1200~1300℃ for 12~15s. c) After polishing, the substrate sample is cooled and cured in a natural environment. After the temperature drops to room temperature, it is tested using a confocal microscope to determine whether the inner surface roughness of the microchambers and microchannels meets the expected requirements. If it does, proceed to step S6; otherwise, return to step a). S6. Place the pre-prepared microspheres into the microchamber obtained in step S5, and drip deionized aqueous solution into the inner surface of the microchamber until the microspheres sink to the bottom of the microchamber with the deionized aqueous solution. Then dry the deionized aqueous solution on the inner surface of the microchamber and the surface of the microspheres to obtain the micro-optical element. The size of the microspheres matches the bottom size of the microchamber. The microspheres are barium titanate solid microspheres with a refractive index of 1.8~2.

2. S7. The micro-optical elements obtained in step S6 are packaged to obtain a micro cell detection chip. The encapsulation process is as follows: 7.1) Cover one side of the modified microchamber of the substrate sample with a transparent film, and leave a liquid inlet on the transparent film above the microchamber; inject liquids with different refractive indices into the microchamber through the liquid inlet to change the refractive index of the medium in contact with the microsphere, so as to realize the function of tunable focal length of the micro-optical element. 7.2) Connect a catheter to the inlet and outlet of the microchannel for the input and output of the cell solution to be tested.

2. The integrated fabrication method of the micro cell detection chip according to claim 1, characterized in that: In S2, the microchannel is a straight channel or a spiral channel.

3. The integrated fabrication method of the micro cell detection chip according to claim 2, characterized in that: The transparent film is selected from one of the following: thin quartz glass sheet, PMMA film, and PDMS film.

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