Liquid crystal optical flow control chip, optical imaging device and application

Abstract

The invention provides a liquid crystal optical flow control chip, an optical imaging device and application, the liquid crystal optical flow control chip comprises a substrate, the substrate is provided with a closed microstructure, the microstructure comprises a plurality of sample pools and a plurality of optical pools, and the sample pools and the optical pools are the same in number and are in one-to-one correspondence; each sample cell is communicated with one optical cell corresponding to the sample cell through a micro-mixing channel; each sample cell is provided with an inlet / outlet pipeline I, and each optical cell is provided with an inlet / outlet pipeline II; the micro-mixing channel comprises a first channel and a second channel which are communicated with each other, and the first channel is provided with a sample introduction pipeline. The liquid crystal optical flow control chip and the optical imaging device are developed on the basis of an adapter conformation change principle in combination with liquid crystal birefringence performance, different liquid crystal optical morphologies of a sample on the liquid crystal optical flow control chip are observed through a polarizing microscope, and then the purpose of detecting exosomes in the sample is achieved. The kit has the advantages of high throughput, rapid detection and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biochip and cell detection technology, and in particular to a liquid crystal optofluidic chip, an optical imaging device and applications. Background Art

[0002] The determination of exosome content has a wide range of applications for early diagnosis and treatment of cancer in patients: in the research of completed clinical trials, the application areas of exosomes include: early diagnosis of cancer and eclampsia, Parkinson's disease, platelet function and coagulation, and tumor vaccines; in the clinical trials currently being recruited, the application areas of exosomes include: early diagnosis of cancer, tumor prognosis monitoring, immunotherapy, diagnosis of polycystic ovary syndrome, tissue repair, etc. Therefore, the research and detection of exosomes have always received widespread attention.

[0003] The methods reported for detecting exosomes include transmission electron microscopy (TEM), flow cytometry (FCM), Western Blot detection, and nanoparticle tracing technology (NTA). After comprehensive analysis of the above methods, we found that these detection methods have certain defects when detecting exosomes in biological samples. For example, flow cytometry (FCM) has a poor detection limit for exosomes, and it is necessary to enrich exosomes and increase the concentration before detection, and it shows general sensitivity. In addition, there are some methods such as transmission electron microscopy (TEM) and nanoparticle tracing technology (NTA) that show ideal analytical performance, but usually require precision instruments and strict manual operation. In addition, the current methods have long test cycles, are labor-intensive, have complex instruments, and are not suitable for rapid, sensitive, and selective detection of exosomes.

[0004] Liquid crystal biosensors use liquid crystal molecules as signal conversion elements of biosensors to convert specific recognition and binding events between biomolecules into amplified optical signal outputs. The detection principle is that the arrangement and orientation of liquid crystal molecules on the functionalized sensing interface will change with the addition of specific target molecules, thereby causing the liquid crystal to change the direction of light refraction, which is further amplified into a change in the optical image morphology of the liquid crystal under a polarizing microscope. Due to its advantages such as simple operation and sensitive response, it has certain applications in small molecule drug control and protein detection. The present invention attempts to explore liquid crystal biosensors that can be used for the detection of organisms such as exosomes. Summary of the invention

[0005] The present invention provides a liquid crystal optofluidic chip, an optical imaging device and applications, with the aim of developing a new liquid crystal optofluidic chip and an optical imaging device, and exploring their possibilities in exosome detection.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a liquid crystal optofluidic chip, comprising a substrate having a closed microstructure thereon, wherein the microstructure comprises a plurality of sample pools and a plurality of optical pools of equal number and one-to-one correspondence, each of the sample pools being connected to an optical pool corresponding to the sample pool through a micro-mixing channel; each of the sample pools is provided with an inlet and outlet pipe one, and each of the optical pools is provided with an inlet and outlet pipe two; the micro-mixing channel comprises a first channel and a second channel connected to each other, wherein the first channel is provided with an injection pipe.

[0008] Preferably, the sample pool is provided with a plurality of small molecule filtering holes, and the small molecule filtering holes are located on the side of the sample pool connected to the micro-mixing channel; and / or, the optical pool is provided with a plurality of slots for loading optical components; preferably, the cross-section of the slots is a rectangular, regular hexagonal, regular triangle, or circular structure; more preferably, a plurality of the slots are arranged to form a square matrix.

[0009] Preferably, the first channel is a hierarchical tree-like channel; and / or the second channel includes a plurality of third channels, each of the third channels is respectively connected to one of the sample cells and an optical cell corresponding to the sample cell.

[0010] Preferably, the third channel is connected to the sample cell and the optical cell respectively through pipelines; more preferably, the third channel is a curved channel.

[0011] Preferably, the substrate is a PDMS flexible substrate; further preferably, the chip further comprises a shell, and more preferably, the shell is a transparent structure.

[0012] In a second aspect, the present invention provides a method for preparing the liquid crystal optofluidic chip according to the first aspect, comprising the following steps:

[0013] Step 1, designing a microstructure pattern through a mask;

[0014] Step 2, transferring the microstructure pattern on the mask to the silicon wafer;

[0015] Step 3: Transfer the microstructure pattern on the silicon wafer to the substrate.

[0016] Preferably, in step 2, transferring the microstructure pattern on the mask to the silicon wafer comprises the following steps:

[0017] Step 2-1, coating the silicon wafer once, preferably by spin coating, with a spin coating speed of 500 rpm, an acceleration of 500 rpm·s-1, and a time of 5 s; preferably using SU8-3000 series photoresist;

[0018] Step 2-2, secondary coating of the silicon wafer, preferably by spin coating, with a spin coating speed of 1800-2500 rpm, an acceleration of 1800-2500 rpm·s-1, and a time of 60 s;

[0019] Step 2-3, pre-baking the silicon wafer after the second coating, cooling it to room temperature, stacking the silicon wafer and the mask plate, and then UV-exposing it, and then post-baking the silicon wafer;

[0020] Step 2-4, developing the silicon wafer, preferably using PGMEA developer for 4-10 minutes; cleaning and drying after development;

[0021] Step 2-5, further heating the silicon wafer at 120-150° C. for 20-30 min, and cooling to room temperature to obtain a silicon wafer with a microstructure;

[0022] Further preferably, in step 2-3, the stepwise pre-baking comprises: the first step of heating from room temperature to 50±5°C and keeping for 10-15 min; the second step of heating to 65±5°C and keeping for 10-15 min; the third step of heating to 95±5°C and keeping for 10-15 min; the fourth step of cooling from 95°C to room temperature; and / or,

[0023] The step-by-step post-baking includes: the first step is heating from room temperature to 50±5℃, and keeping at 50±5℃ for 10 minutes; the second step is heating from 50±5℃ to 65±5℃, and keeping at 65±5℃ for 10 minutes; the third step is heating from 65±5℃ to 80±5℃, and keeping at 80±5℃ for 10 minutes; the fourth step is cooling from 80±5℃ to room temperature; and / or,

[0024] The control parameters of the UV exposure are: exposure time 25-30s, UV intensity 120-130μM·cm -2 .

[0025] Preferably, in step 3, the microstructure pattern on the silicon wafer is transferred to a substrate; the substrate is a PDMS flexible substrate, and two PDMS flexible substrates are used, one of which has a sample pool, an optical pool, and a first channel in the microstructure, and the other PDMS flexible substrate has a second channel in the microstructure, specifically comprising the following steps:

[0026] Step 3-1, PDMS and curing agent are mixed in a mass ratio of 10:1 and bubbles are eliminated to obtain liquid PDMS;

[0027] Step 3-2, covering the liquid PDMS on the silicon wafer and performing thermal curing, preferably, the curing parameters include: temperature 80-90° C., time 30-40 min, and cooling to room temperature; after the curing is completed, the PDMS is peeled off from the silicon wafer to obtain two PDMS flexible substrates with corresponding microstructures;

[0028] Step 3-3, bonding two PDMS flexible substrates, wherein the PDMS flexible substrate having the sample pool, the optical pool, and the first channel in the microstructure is located at the lower layer, and the PDMS flexible substrate having the second channel in the microstructure is located at the upper layer;

[0029] Further preferably, the preparation method further comprises loading the bonded PDMS flexible substrate into a shell.

[0030] In a third aspect, the present invention provides an optical imaging device, comprising the liquid crystal optofluidic chip described in the first aspect and a polarizing microscope, wherein the polarizing microscope is used to observe optical phenomena in an optical cell of the liquid crystal optofluidic chip.

[0031] In a fourth aspect, the present invention provides the application of the liquid crystal optofluidic chip described in the first aspect and / or the optical imaging device described in the third aspect in the detection of organisms. Organisms include cells, viruses, and small biological molecules such as proteins and nucleic acids.

[0032] In a fifth aspect, the present invention provides a method for detecting exosomes, which uses the optical imaging device described in the third aspect, and comprises the following steps:

[0033] Mixing liquid crystal with a cationic surfactant solution to obtain a liquid crystal mixed solution, wherein the concentration of the cationic surfactant solution is less than 0.025 mM;

[0034] Injecting the organism to be detected into the sample pool and introducing it into the micro-mixing channel, and injecting the aptamer solution into the micro-mixing channel to mix with the organism to be detected; preferably, the concentration of the aptamer solution is 45-50nmol / L;

[0035] further introducing the mixture into an optical cell, and injecting a liquid crystal mixed solution into the optical cell to further mix with the mixture;

[0036] The optical cell was placed under a polarizing microscope for detection, and the optical images obtained were used for exosome analysis.

[0037] Preferably, the cationic surfactant is selected from at least one of cetyltrimethylammonium bromide, dodecyldimethylbenzylammonium bromide, octadecyltrimethylammonium chloride, and methyl ditallowylethyl-2-hydroxyethylammonium methyl sulfate.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention combines traditional microfluidic technology with liquid crystal optical sensing technology. Based on the principle of aptamer conformational change and liquid crystal birefringence, a new liquid crystal optofluidic chip and optical imaging device that can achieve high-throughput detection are developed. The different liquid crystal optical morphologies of the sample on the liquid crystal optofluidic chip are observed by polarizing microscope, thereby achieving the purpose of detecting exosomes in the sample. The liquid crystal optofluidic chip and optical imaging device are used to detect exosomes with the advantages of high throughput, rapid detection, and high sensitivity. The entire operation process is simple, the reagent consumption is low, and the sample processing time is short. It solves the technical problems of the existing detection methods such as complexity and high cost, and broadens the application scope of liquid crystal optofluidic chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the structure of the liquid crystal optofluidic chip in Example 1 of the present invention.

[0041] Figure 2 Schematic diagram of the structure of the sample pool in Example 1 of the present invention.

[0042] Figure 3 Schematic diagram of the structure of the optical pool in Example 1 of the present invention.

[0043] Figure 4 This is a design structure diagram of the mask in Example 2 of the present invention.

[0044] Figures 1 to 4 In the figure, 1-substrate, 2-sample pool, 3-optical pool, 4-inlet and outlet pipe 1, 5-inlet and outlet pipe 2, 6-first channel, 7-second channel, 8-injection pipe, 9-third channel, 10-pipe connecting sample pool and micro-mixing channel, 11-pipe connecting micro-mixing channel and optical pool, 12-diversion port 1, 13-diversion port 2, 14-diversion port 3, 15-square matrix formed by pores and grooves, 16-macromolecule filtering hole, 17-square column.

[0045] Figure 5 These are different cross-sectional structural diagrams of the hole grooves in Example 2 of the present invention (all forming rectangular arrays), wherein, in Figure A, the hole grooves are regular hexagons, in Figure B, the hole grooves are squares, in Figure C, the hole grooves are regular triangles, and in Figure D, the hole grooves are circular.

[0046] Figure 6Polarized light microscope photographs and grayscale gradient diagrams of the optical cell of the liquid crystal optofluidic chip added with cetyltrimethylammonium bromide solutions of different concentrations in Example 3 of the present invention; the concentrations of the cetyltrimethylammonium bromide solution are: 0.02mM, 0.0225mM, 0.025mM, 0.03mM, and 0.04mM; wherein, Figure A: polarized light microscope photograph of the liquid crystal modified by 0.02mM cetyltrimethylammonium bromide solution; Figure B: polarized light microscope photograph of the liquid crystal modified by 0.0225mM cetyltrimethylammonium bromide solution; Figure C: polarized light microscope photograph of the liquid crystal modified by 0.025mM cetyltrimethylammonium bromide solution; Figure D: polarized light microscope photograph of the liquid crystal modified by 0.03mM cetyltrimethylammonium bromide solution; Figure E: polarized light microscope photograph of the liquid crystal modified by 0.04mM cetyltrimethylammonium bromide solution; and F: grayscale gradient diagram.

[0047] Figure 7 This is a diagram of the mechanism of detecting exosomes using an optical imaging device in Example 3 of the present invention, wherein Figure A: a liquid crystal optical morphology diagram in which there are no exosomes and aptamers in the optical cell; Figure B: a liquid crystal optical morphology diagram in which there are aptamers in the optical cell; Figure C: a liquid crystal optical morphology diagram in which there are exosomes and aptamers in the optical cell.

[0048] Figure 8 Polarized light microscope photos and grayscale gradient images of the optical pool of the liquid crystal optofluidic chip in which different concentrations of nucleic acid aptamer solutions and 0.02 mM cetyltrimethylammonium bromide solution are added in Example 4 of the present invention; the concentrations of the nucleic acid aptamer solution are: 50 nmol / L, 40 nmol / L, 20 nmol / L, and 10 nmol / L; Figure A: Liquid crystal polarized light display of the reaction solution of 10 nmol / L nucleic acid aptamer solution and 0.02 mM cetyltrimethylammonium bromide solution Microscope photo; Figure A: Liquid crystal polarizing microscope photo of the reaction solution of 20nmol / L nucleic acid aptamer solution and 0.02mM cetyltrimethylammonium bromide solution; Figure C: Liquid crystal polarizing microscope photo of the reaction solution of 40nmol / L nucleic acid aptamer solution and 0.02mM cetyltrimethylammonium bromide solution; Figure D: Liquid crystal polarizing microscope photo of the reaction solution of 50nmol / L nucleic acid aptamer solution and 0.02mM cetyltrimethylammonium bromide solution; Figure E: Grayscale gradient image.

[0049] Fig. 9Liquid crystal polarizing microscope photographs of the optical cell of the liquid crystal optofluidic chip added to the reaction solution of different concentrations of exosome standard solution, 0.02mM cetyltrimethylammonium bromide solution and 100nmol / L nucleic acid aptamer solution in Example 5 of the present invention, wherein Figure A: Liquid crystal polarizing microscope photograph of the exosome standard solution with a concentration of 9.8×10^6 / mL; Figure B: Liquid crystal polarizing microscope photograph of the exosome standard solution with a concentration of 1.95×10^7 / mL; Figure C: Liquid crystal polarizing microscope photograph of the exosome standard solution with a concentration of 3.9×10^7 / mL; Figure D: Linear curve of grayscale value of liquid crystal picture under polarizing microscope vs. exosome concentration.

[0050] Fig.10 These are experimental polarization images of the optical imaging device for detecting exosomes in Example 6 of the present invention, wherein: Figure A: liquid crystal is introduced; Figure B: aptamer is introduced; Figure C: a mixed solution of exosomes and aptamers is introduced. DETAILED DESCRIPTION

[0051] 4-Cyano-4'-pentylbiphenyl (5CB) in the examples of the present invention was purchased from Tokyo Jinsei Industry Co., Ltd. In addition, if the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0052] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0053] The nucleic acid aptamer sequence used below is CD63 aptamer (shown in SEQ ID NO. 1): 5′-CACCCCACCTCGCTCCCGTGACACTAATGCTATTTTTTTTTTTTTTT-3′-FAM.

[0054] Other nucleic acid aptamers that can be used include: CD63-2 aptamer (shown in SEQ ID NO. 2): 5′-TAACCACCCCACCTCGCTCCCGTGACACTAATGCTAATTCCAA-3′;

[0055] CD9-26 aptamer (shown in SEQ ID NO.3):

[0056] 5′-ATAGTCCCTTGGCGTGCTTCACAACCTTGAACTTGACGCAGGATCGTTCAGTGCGCACTAGAGCAGGTACGGTGTCA-3′.

[0057] The components of the PBS buffer in the embodiment are as follows: 137 mol / L NaCl, 2.7 mmol / L KCl, 4.3 mmmol / L Na2HPO4, 1.4 mmol / L KH2PO4, the solvent is water, and the pH is adjusted to 7.4 with dilute hydrochloric acid.

[0058] Example 1

[0059] This embodiment provides a liquid crystal optofluidic chip, whose structure is as follows Figure 1-5 As shown, it includes a PDMS flexible substrate 1, on which a closed microstructure is provided, and the microstructure includes a plurality of sample pools 2 and a plurality of optical pools 3 of the same number and one-to-one correspondence, and each of the sample pools 2 and an optical pool 3 corresponding to the sample pool 2 are communicated through a micro-mixing channel. Each of the sample pools 2 is provided with an inlet and outlet pipe 1 4, and each of the optical pools 3 is provided with an inlet and outlet pipe 2 5; the micro-mixing channel includes a first channel 6 and a second channel 7 that are communicated with each other, wherein the first channel 6 is provided with a sample injection pipe 8. There are no special requirements for the number of sample pools 2 and optical pools 3, which can be specified according to the flux requirements, such as 8, 9, 10, 20, 30, 50, 100, etc. Therefore, the liquid crystal optofluidic chip of the present invention can realize high-throughput and different concentrations of biological sample measurements. In all embodiments of the present invention, a microstructure consisting of 8 sample pools 2 and 8 optical pools 3 is used.

[0060] Furthermore, if Figure 2 As shown, the sample pool is provided with a plurality of small molecule filtration holes 16, and the small molecule filtration holes 16 are located on the side where the sample pool 2 is connected to the micro-mixing channel. In this embodiment, the small molecule filtration holes 6 are formed by setting a plurality of square columns 17 in the sample pool, and the small molecule filtration holes 16 are formed between the square columns 17. In this embodiment, the size of the small molecule filtration holes 16 is set below 1um, and the small molecule detection object ( Figure 2 The spherical particles in the middle represent small molecule detection objects, and the flake and central spherical particles represent cell cultures) are usually at the nanometer level, such as exosomes and other secretions at the 100-150nm level. The small molecule filter hole allows small molecules to pass through, while retaining macromolecular cells, which refer to cells with a size of more than 10um. The size of the small molecule filter hole 16 can be flexibly adjusted according to the size of the components to be detected in the sample. In addition, there is no special requirement for the size of the sample pool. In this embodiment, the sample pool 2 is 1.7cm long, 3mm wide, and 100um high.

[0061] like Figure 3 , 5As shown, the optical pool 3 is provided with a plurality of holes 15 for loading optical components; the hole cross section can be designed as a rectangular (cube), regular hexagon (regular hexagonal prism), regular triangle (regular triangular prism), or circular structure. Correspondingly, different hole structures are cube, regular hexagonal prism, regular triangular prism, and cylindrical structure, and the optical images formed under the polarizing microscope are rectangular, regular hexagonal, regular triangle, and circular, respectively. In this embodiment, a square hole structure is adopted. Figure 3 to Figure 6 The optical image formed by each hole groove is therefore square. In addition, the optical image formed by the holes grooves arranged in a regular shape is more uniform and convenient for observation. Therefore, the holes grooves of this embodiment are arranged to form a square matrix. In addition, there is no special requirement for the size of the optical pool. In this embodiment, the optical pool 3 is 1.6 cm long, 3 mm wide, and 100 um high. The square hole groove has a side length of 80 μm and a height of 25 μm.

[0062] Furthermore, if Figure 4 As shown, the first channel 6 is a hierarchical tree-like channel, the number of levels is ≥ 2, and the number of levels is preferably controlled between 2 and 5, which can control the uniformity and efficiency of the injection. In all embodiments of the present invention, a 3-level tree-like channel is adopted, the first level is provided with an injection pipeline 8 and 2 diversion ports 12, the second level is provided with 4 diversion ports 2 13, and the third level is provided with 8 diversion ports 3 14, and the diversion ports 3 14 are connected to the third channel 9 of the second channel 7. The components to be introduced enter from the injection pipeline 8 through the diversion port 1 12 into the second level, and then enter the third level through the diversion port 2 13, and then enter the second channel 7 through the diversion port 3 14.

[0063] The second channel 7 includes a plurality of third channels 9, each of which is connected to one of the sample pools 2 and an optical pool 3 corresponding to the sample pool 2, that is, the number of third channels 9 is the same as that of the sample pool 2 and the optical pool 3. In all embodiments of the present invention, 8 third channels 9 are used. The two ends of the third channel 9 are connected to the sample pool 2 and the optical pool 3 through pipes 10 and 11, respectively. In this embodiment, the structure of each of the third channels 9 is that a "U-shaped" channel is connected to a spiral channel, wherein the "U-shaped" channel corresponds to and is connected to the diversion port 14 one by one, the "U-shaped" channel is located on one side of the sample pool 2, and the spiral channel is located on one side of the optical pool 3.

[0064] In order to better protect the liquid crystal optofluidic chip, a housing may be provided. The housing may be an integral structure with the chip or a detachable structure. When the housing and the chip are an integral structure, the housing is preferably a transparent structure. The function of the housing is to prevent the liquid crystal optofluidic chip from being contaminated or damaged. Figure 1 The structure shown in the figure is without the housing.

[0065] The liquid crystal optofluidic chip of this embodiment can achieve uniform mixing of different components in the micro-mixing channel, split multi-step, complex experiments into a pipeline-like structure, accurately control the volume of the injected solution, quickly complete the mixing, and solve the volume error problem and cumbersome operation caused by manual mixing. The mixture is then collected in the optical area, and optical components such as liquid crystals can be added to the optical area at the same time, so as to use different optical phenomena to detect, identify and distinguish the substances in the components. The chip can conduct multiple groups of experiments at the same time and observe multiple groups of experimental phenomena at the same time, which is convenient, fast and efficient.

[0066] Example 2

[0067] The preparation method of the liquid crystal optofluidic chip and the assembly of the optical imaging device of Example 1 are as follows:

[0068] (1) Use AutoCAD software to draw geometric patterns of different sizes and shapes, and customize them into masks with a size of 5 inches × 5 inches × 2.3 mm. Figure 4 A mask design diagram of a microstructure is shown, where black represents the opaque part and white represents the transmissive part.

[0069] (2) Photolithography microstructure process: Glue coating: SU8-3000 series photoresist is used for spin coating on silicon wafers. The spin coating is divided into two steps. The first step is: speed 500 rpm, acceleration 500 rpm·s -1 , time 5s; the second step of spin coating is: speed 1800-2500rpm, acceleration 1800-2500rpm·s -1 , time 60s, spin coating thickness is about 20-60μm; then step-by-step pre-baking is performed and naturally cooled to room temperature; the mask is aligned with the silicon wafer coated with glue in the photolithography machine for UV exposure, and the UV exposure parameters are: exposure time 25s, UV intensity 126μM·cm -2 ; Then carry out post-baking in steps; Development: Use PGMEA developer for 4-10 minutes; After development, wash with isopropyl alcohol, blow dry the silicon wafer with nitrogen and observe the photolithography under a microscope; Harden the film: Put the photolithographic silicon wafer on a 120℃ hot plate for 30 minutes, and then cool it naturally to room temperature.

[0070] In the above process, the step-by-step pre-baking includes: the first step is heating from room temperature to 50±5°C and keeping it for 10 to 15 minutes; the second step is heating to 65±5°C and keeping it for 10 to 15 minutes; the third step is heating to 95±5°C and keeping it for 10 to 15 minutes; the fourth step is cooling from 95°C to room temperature.

[0071] The step-by-step post-baking includes: the first step is heating from room temperature to 50±5℃, and maintaining at 50±5℃ for 10 minutes; the second step is heating from 50±5℃ to 65±5℃, and maintaining at 65±5℃ for 10 minutes; the third step is heating from 65±5℃ to 80±5℃, and maintaining at 80±5℃ for 10 minutes; the fourth step is cooling from 80±5℃ to room temperature.

[0072] (3) PDMS mold pouring: PDMS and curing agent are fully stirred and mixed at a mass ratio of 10:1, and then the bubbles are eliminated by vacuum to obtain liquid PDMS; the liquid PDMS is then evenly poured on the silicon wafer for thermal curing. The curing parameters include: temperature 90°C, time 30 minutes, and natural cooling to room temperature; after the curing is completed, the PDMS is peeled off from the silicon wafer and cut into two pieces of PDMS. The two pieces of PDMS flexible substrates are bonded, specifically bonding, wherein the PDMS flexible substrate with the sample pool, optical pool, and first channel in the microstructure is located in the lower layer, and the PDMS flexible substrate with the second channel in the microstructure is located in the upper layer. At the same time, the non-microchannel part of the PDMS flexible substrate is cleaned. The instrument uses a plasma cleaning machine, in which the plasma gas is air, and the time is 6 minutes. The microstructure is masked with 3M tape during the plasma cleaning process. The masking materials of the upper and lower substrates need to be removed during the bonding process.

[0073] After bonding, a hole punch with an outer diameter of 1.25 mm was used to set the ports for connecting the inlet and outlet pipes 1, 2, and the sample injection pipe, as well as the ports for connecting the sample pool with the micro-mixing channel, and the micro-mixing channel with the optical pool. The liquid crystal optofluidic chip was obtained and used for standby. In this embodiment, the liquid crystal optofluidic chip was not loaded with a housing.

[0074] Assembly of optical imaging device: Use silicone hose with inner diameter of 0.5mm and outer diameter of 1.5mm for inlet and outlet pipe 1, inlet and outlet pipe 2, and sample injection pipe, and connect inlet and outlet pipe 1 and inlet and outlet pipe 2 to injection device, which is a fluid delivery device composed of injection pump and injection needle. Then add polarizing microscope, and place the optical area of ​​LC optofluidic chip under the polarizing microscope. The two linear polarizers of polarizing microscope are vertically crossed. One side of the optical pool of LC optofluidic chip receives the detection beam, and the detection is realized according to the change of the beam passing through the optical pool on the other side, and the optical image of LC optofluidic chip is received by CCD electronic components.

[0075] Example 3

[0076] Functional test of liquid crystal optofluidic chip:

[0077] Using the optical imaging device of Example 2, first inject liquid crystal into the optical pool of the liquid crystal optofluidic chip through the second inlet and outlet pipes, and the amount added is about 2 μL per optical pool;

[0078] Prepare cetyl trimethylammonium bromide solution, dissolve cetyl trimethylammonium bromide in PBS buffer, ultrasonically dissolve for 30 minutes, and prepare cetyl trimethylammonium bromide solutions with concentrations of 0.02mM, 0.0225mM, 0.025mM, 0.03mM, and 0.04mM, respectively. Add the cetyl trimethylammonium bromide solutions of different concentrations into the optical cell of the liquid crystal optofluidic chip through different inlet and outlet pipes, respectively. The added volume of each optical cell is 110μL. Observe using a polarizing microscope. The results are as follows Figure 6 As shown, when the concentration of the cetyltrimethylammonium bromide solution is less than 0.025mM, the liquid crystal presents a bright appearance within 5 minutes; when the concentration of the cetyltrimethylammonium bromide solution is ≥0.025mM, the liquid crystal presents a dark appearance within 5 minutes. Therefore, the concentration of the cetyltrimethylammonium bromide solution is controlled to be less than 0.025mM, preferably 0.02mM. Cetyltrimethylammonium bromide is a cationic surfactant. The present invention can also select dodecyldimethylbenzyl ammonium bromide, octadecyltrimethylammonium chloride, methyl ditallow ethyl-2-hydroxyethyl methyl ammonium sulfate.

[0079] In the above process, the amount of the liquid crystal added is not specifically limited, as long as the prepared liquid crystal biosensor is a uniform dark field under a polarizing microscope.

[0080] The present invention uses an optical imaging device to detect exosomes. Figure 7 As shown: When there are no exosomes and aptamers in the optical pool of the liquid crystal optofluidic chip, the monolayer of cetyltrimethylammonium bromide at the interface still maintains the vertical arrangement of liquid crystal molecules, and the liquid crystal image under the polarizing microscope shows a dark optical morphology ( Figure 7 When the aptamer is added, the concentration of cetyltrimethylammonium bromide at the interface is reduced, thereby destroying the previously formed cetyltrimethylammonium bromide monolayer, causing the liquid crystal molecules to change from vertical arrangement to parallel / tilted arrangement. The liquid crystal image under the polarizing microscope shows a bright optical morphology ( Figure 7 When the aptamer and exosome mixed solution is added, due to the specific binding of exosomes and aptamers, the monolayer of cetyltrimethylammonium bromide at the interface still maintains the vertical arrangement of liquid crystal molecules, and the liquid crystal image under the polarizing microscope shows a dark optical morphology ( Figure 7 Based on the above principles, combined with the grayscale-concentration standard curve of Example 5, the qualitative and quantitative detection process of exosomes can be obtained.

[0081] In addition, the sample pool can also be used as a cell culture unit as needed, and the holes and slots in the optical pool for loading optical components can also be adjusted according to different requirements, thereby changing the shape and size of the grid in the formed liquid crystal image.

[0082] Example 4

[0083] Determination of aptamer concentration:

[0084] Using the optical imaging device of Example 2, first inject liquid crystal into the optical pool of the liquid crystal optofluidic chip through the second inlet and outlet pipes, and the amount added is about 2 μL per optical pool;

[0085] The CD63 aptamer freeze-dried powder was dissolved in PBS buffer and vortexed to prepare aptamer solutions with concentrations of 50 nmol / L, 40 nmol / L, 20 nmol / L, and 10 nmol / L, respectively. The aptamer solutions with different concentrations were vortexed with 0.02 mM cetyltrimethylammonium bromide solution (preparation method is the same as in Example 3), respectively, and incubated at 37°C for 30 min before adding to the array optical cell of the liquid crystal biosensor. The drop volume of each optical cell was 110 μL. Polarized light microscope was used for observation. The results are as follows: Figure 8 As shown, when the concentration of the aptamer solution is ≥50nmol / L, the liquid crystal is in a completely bright morphology; when the aptamer concentration of the solution is gradually reduced from 50nmol / L to 10nmol / L, the morphology of the liquid crystal gradually changes from completely bright to completely dark, and the bright area gradually decreases. Therefore, it is appropriate to control the concentration of the aptamer solution to 45-50nmol / L, preferably 50nmol / L.

[0086] Example 5

[0087] Obtaining the grayscale-concentration standard curve of exosomes:

[0088] Using the optical imaging device of Example 2, first inject liquid crystal into the optical pool of the liquid crystal optofluidic chip through the second inlet and outlet pipes, and the amount added is about 2 μL per optical pool;

[0089] Extraction of exosomes: Select 3rd-4th generation human umbilical cord mesenchymal stem cells (MSC) in good condition. When the MSC is fused to about 75%, replace with fresh serum-free medium. Collect the supernatant after 48 hours of culture. Place the supernatant in a centrifuge tube and centrifuge at 4°C and 2000g for 30 minutes to remove cell debris. The retained supernatant is filtered through a sterile filter membrane (0.45μm) and placed in an ultracentrifuge tube. Centrifuge at 4°C and 1000g for 70 minutes to obtain an exosome concentrate. Move the concentrate to a new ultracentrifuge tube and centrifuge at 4°C and 100000g for 70 minutes. Dilute the precipitate with PBS and centrifuge again at 4°C and 100000g for 70 minutes. The exosome concentrate obtained is filtered through a sterile filter membrane (0.22μm), resuspended in a small volume of PBS, and refrigerated at -80°C for subsequent experiments to obtain a laboratory-made exosome standard.

[0090] The exosome standard was vortexed and dissolved in PBS buffer to prepare exosome standard solutions with concentrations of 3.9×10^7 / mL, 1.95×10^7 / mL, and 9.8×10^6 / mL; CD63 nucleic acid aptamer solution was added to the above exosome standard solutions of different concentrations and mixed evenly. The volume ratio of nucleic acid aptamer to exosome standard solution was 1:1, and the final concentration of nucleic acid aptamer in the mixed solution was 100nmol / L. The mixture was reacted at 4°C for 60min to obtain reaction solution ①. The above reaction solution ① was mixed evenly with cetyltrimethylammonium bromide solution. The volume ratio of cetyltrimethylammonium bromide solution to reaction solution ① was 1:1. The final concentration of cetyltrimethylammonium bromide in the mixed solution was 0.02mM. The mixture was reacted at 37°C for 30min to obtain reaction solution ②. The reaction solution ② was added to the optical cell. The drop volume of each optical cell was 110μL. The reaction was observed using a polarizing microscope. The results are as follows: Fig. 9 As shown, when the concentration of the exosome standard solution is ≥3.9×10^7 / mL, the liquid crystal presents a dark morphology; when the concentration of the exosome standard solution is ≤9.8×10^6 / mL, the liquid crystal presents a bright morphology; when the concentration of the exosome standard solution gradually decreases from 3.9×10^7 / mL to 9.8×10^6 / mL, the morphology of the liquid crystal gradually changes from all dark to bright. The collected optical images were analyzed by pixel analysis using MATLAB (version9.6.0, R2019a) software to obtain the average grayscale value of the liquid crystal image and plotted using OriginPro9.1 software. In the concentration range of 9.8×10^6 / mL to 3.9×10^7 / mL, the grayscale value of the liquid crystal image has a good linear relationship with the exosome concentration, with a correlation coefficient of 0.9996( Fig. 9 (D in the middle). The liquid crystal biosensor can detect exosomes with a detection limit of 1.86×10^6 / ml.

[0091] Example 6

[0092] The optical imaging device of Example 2 was used to detect exosome samples:

[0093] First, inject the liquid crystal into the optical pool of the liquid crystal optofluidic chip through the inlet and outlet pipes 2, and the amount added is about 2 μL per optical pool, forming a liquid crystal film of a certain thickness while anchoring the liquid crystal molecules on the interface of the liquid crystal film to be arranged vertically. Fig.10 Figure A in the middle shows the POM image of the vertically anchored microstructure of the liquid crystal.

[0094] Then, 0.01 mM hexadecyltrimethylammonium bromide solution was added to injection needle 1, fixed in injection pump 1, and the injection volume was 30 μL.

[0095] Exosomes were obtained by referring to the method for extracting exosomes in Example 5, and resuspended with PBS to make the exosome concentration 3.9×10^7 / mL, added to the injection needle 2, fixed in the injection pump 2, and the injection volume was 30 μL.

[0096] The injection needle of a solution prepared with PBS buffer solution containing CD63 aptamer at a concentration of 40 nmol / L was fixed in the injection pump, and the injection volume was 60 μL.

[0097] The three liquids were injected into the optical pool, sample pool, and micro-mixing channel at flow rates of 25 μL·min-1, 150 μL·min-1, and 80 μL·min-1, respectively, and finally mixed in the optical pool. The results observed using a polarizing microscope are shown in the figure. Fig.10 As shown in Figures B and C, the collected optical images were analyzed by MATLAB (version 9.6.0, R2019a) software, and the calculated exosome concentration was also 3.9×10^7 / mL, combined with the grayscale-concentration standard curve of exosomes. The exosomes were further prepared into solutions with the following concentrations: 4.5×10^7 / mL, 5.0×10^7 / mL, 5.5×10^7 / mL, and 6.0×10^7 / mL. The above experimental process was repeated to verify the results. The calculated results were consistent with the target concentration. The results of the embodiment prove that this liquid crystal sensor has high accuracy for exosome detection.

[0098] This result shows that the liquid crystal biosensor assembly of the present application can effectively detect exosomes in liquid samples. The method of the present invention can be used to quickly, sensitively, simply, and array-basedly detect trace amounts of exosomes, and the entire detection process takes about 10 minutes.

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Liquid crystal optofluidic chip, characterized in that: The invention comprises a substrate having a closed microstructure, wherein the microstructure comprises a plurality of sample pools and a plurality of optical pools of the same number and corresponding to each other, each of the sample pools and an optical pool corresponding to the sample pool are communicated with each other through a micro-mixing channel; each of the sample pools is provided with an inlet and outlet pipe 1, and each of the optical pools is provided with an inlet and outlet pipe 2; the micro-mixing channel comprises a first channel and a second channel communicated with each other, wherein the first channel is provided with a sampling pipe.

2. The liquid crystal optofluidic chip according to claim 1, characterized in that: The sample pool is provided with a plurality of small molecule filtering holes, and the small molecule filtering holes are located on the side of the sample pool connected to the micro-mixing channel; and / or, the optical pool is provided with a plurality of slots for loading optical components; preferably, the cross-section of the slots is a rectangular, regular hexagonal, regular triangle, or circular structure; more preferably, a plurality of the slots are arranged to form a square matrix.

3. The liquid crystal optofluidic chip according to claim 1, characterized in that: The first channel is a hierarchical tree-like channel; and / or the second channel includes a plurality of third channels, each of which is connected to one of the sample cells and an optical cell corresponding to the sample cell.

4. The liquid crystal optofluidic chip according to claim 3, characterized in that: The third channel is connected to the sample pool and the optical pool through pipelines respectively; preferably, the third channel is a curved channel.

5. The liquid crystal optofluidic chip according to any one of claims 1 to 4, characterized in that: The substrate is a PDMS flexible substrate; preferably, the chip further comprises a shell, and more preferably, the shell is a transparent structure.

6. The method for preparing a liquid crystal optofluidic chip according to any one of claims 1 to 5, characterized in that: The steps include: Step 1, designing a microstructure pattern through a mask; Step 2, transferring the microstructure pattern on the mask to the silicon wafer; Step 3: Transfer the microstructure pattern on the silicon wafer to the substrate.

7. The preparation method according to claim 6, characterized in that: In step 2, transferring the microstructure pattern on the mask to the silicon wafer comprises the following steps: Step 2-1: Spin coating is preferably used, with a spin coating speed of 500 rpm and an acceleration of 500 rpm·s -1 , time is 5s; preferably SU8-3000 series photoresist; Step 2-2: Secondary coating of silicon wafer, preferably by spin coating, with a spin coating speed of 1800-2500 rpm and an acceleration of 1800-2500 rpm·s -1 , time is 60s; Step 2-3, pre-baking the silicon wafer after the second coating, cooling it to room temperature, stacking the silicon wafer and the mask plate, and then UV-exposing it, and then post-baking the silicon wafer; Step 2-4, developing the silicon wafer, preferably using PGMEA developer for 4-10 minutes; cleaning and drying after development; Step 2-5, further heating the silicon wafer at 120-150° C. for 20-30 min, and cooling to room temperature to obtain a silicon wafer with a microstructure; Preferably, in step 2-3, the stepwise pre-baking comprises: the first step of heating from room temperature to 50±5°C and keeping for 10-15 minutes; the second step of heating to 65±5°C and keeping for 10-15 minutes; the third step of heating to 95±5°C and keeping for 10-15 minutes; the fourth step of cooling from 95°C to room temperature; and / or, The step-by-step post-baking includes: the first step is heating from room temperature to 50±5℃, and keeping at 50±5℃ for 10 minutes; the second step is heating from 50±5℃ to 65±5℃, and keeping at 65±5℃ for 10 minutes; the third step is heating from 65±5℃ to 80±5℃, and keeping at 80±5℃ for 10 minutes; the fourth step is cooling from 80±5℃ to room temperature; and / or, The control parameters of the UV exposure are: exposure time 25-30s, UV intensity 120-130μM·cm -2 .

8. The preparation method according to claim 6 or 7, characterized in that: In the step 3, the microstructure pattern on the silicon wafer is transferred to a substrate; the substrate is a PDMS flexible substrate, and two PDMS flexible substrates are used, one of which has a sample pool, an optical pool, and a first channel in the microstructure, and the other PDMS flexible substrate has a second channel in the microstructure, specifically comprising the following steps: Step 3-1, PDMS and curing agent are mixed in a mass ratio of 10:1 and bubbles are eliminated to obtain liquid PDMS; Step 3-2, covering the liquid PDMS on the silicon wafer and performing thermal curing, preferably, the curing parameters include: temperature 80-90° C., time 30-40 min, and cooling to room temperature; after the curing is completed, the PDMS is peeled off from the silicon wafer to obtain two PDMS flexible substrates with corresponding microstructures; Step 3-3, bonding two PDMS flexible substrates, wherein the PDMS flexible substrate having the sample pool, the optical pool, and the first channel in the microstructure is located at the lower layer, and the PDMS flexible substrate having the second channel in the microstructure is located at the upper layer; Preferably, the preparation method further comprises loading the bonded PDMS flexible substrate into a shell.

9. An optical imaging device, characterized in that: It comprises the liquid crystal optofluidic chip according to any one of claims 1 to 5 and a polarizing microscope, wherein the polarizing microscope is used to observe optical phenomena in an optical pool of the liquid crystal optofluidic chip.

10. Application of the liquid crystal optofluidic chip according to any one of claims 1 to 5 and / or the optical imaging device according to claim 9 in biological detection.

11. A method for detecting exosomes, characterized in that: The optical imaging device according to claim 9 comprises the following steps: Mixing liquid crystal with a cationic surfactant solution to obtain a liquid crystal mixed solution, wherein the concentration of the cationic surfactant solution is less than 0.025 mM; Injecting the organism to be detected into the sample pool and introducing it into the micro-mixing channel, and injecting the aptamer solution into the micro-mixing channel to mix with the organism to be detected; preferably, the concentration of the aptamer solution is 45-50nmol / L; further introducing the mixture into an optical cell, and injecting a liquid crystal mixed solution into the optical cell to further mix with the mixture; The optical cell was placed under a polarizing microscope for detection, and the optical images obtained were used for exosome analysis.

12. The method for detecting exosomes according to claim 11, characterized in that: The cationic surfactant is at least one selected from cetyltrimethylammonium bromide, dodecyldimethylbenzylammonium bromide, octadecyltrimethylammonium chloride, and methyl ditallowylethyl-2-hydroxyethylammonium methyl sulfate.

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