Large-area flow pool chip and application thereof

Abstract

The invention provides a large-area flow pool chip and application thereof, the large-area flow pool chip comprises: a prism, a gold film and a flow pool, the flow pool comprises an upper flow pool and a lower flow pool which are laminated, the lower flow pool is provided with a channel, one side of the lower flow pool provided with the channel is attached to the gold film, and the upper flow pool and the lower flow pool are laminated. The upper flow pool is provided with a liquid path inlet, a first upper flow pool liquid path channel, a second upper flow pool liquid path channel and a liquid path outlet, the lower flow pool is provided with a lower flow pool liquid path inlet and a lower flow pool liquid path outlet, and the two ends of the channels are connected with the lower flow pool liquid path inlet and the lower flow pool liquid path outlet respectively. The liquid path inlet, the first up-flow pool liquid path channel and the down-flow pool liquid path inlet are communicated, the liquid path outlet, the second up-flow pool liquid path channel and the down-flow pool liquid path outlet are communicated, and the area of the channels is not smaller than 60 mm < 2 >. According to the chip, on the basis of ensuring the mechanical strength of a flow pool, the design area of a channel can be increased, and molecular fishing is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and in particular, to a large-area flow cell chip and its application. Background Art

[0002] A surface plasmon resonance instrument (SPR) can detect the affinity, specificity, kinetics, and binding mechanism (competition / allosteric) between biomolecules, and is widely used in research fields such as biological structure and disease mechanism research, discovery of active ingredients in traditional Chinese medicine compounds, target fishing, high-throughput drug screening, drug structure-activity relationship, structure optimization of lead compounds, drug action mechanism, epitope analysis, and detection of active concentration of biomolecules.

[0003] Molecular fishing is an experimental technique for finding specific molecules that interact with known biomolecules from complex sample systems, and plays an important role in biological research, traditional Chinese medicine research, discovery of active drugs, etc. The traditional magnetic bead fishing method is an end-point technique, which has disadvantages such as high non-specificity, difficult to explore elution conditions, and cumbersome operation. Some commercial surface plasmon resonance instruments (such as Biacore T200 of Cytiva) can perform real-time dynamic and monitorable molecular fishing experiments. However, the flow cell volume of existing SPR chips is relatively small, the detection area is limited, and the same chip needs to be combined many times to collect enough samples for mass spectrometry detection. The experimental time is usually more than 12 hours, and during the repeated use of the chip, the binding efficiency will become lower and lower.

[0004] At the same time, the crimping method of the SPR instrument and the chip is four-point crimping corresponding to the instrument flow path. The pressure on the flow cell is concentrated on the central 4 points. The flow cell for molecular fishing has a larger contact area with the gold film compared to the ordinary flow cell, and at the same time increases the hollow area of the flow cell itself, reducing the strength and durability of the flow cell, making the flow cell more likely to collapse due to excessive pressure, or the patch edge falls off due to uneven pressure, resulting in sample leakage. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the prior art to some extent. For this reason, an object of the present invention is to provide a large-area flow cell chip and its application.

[0006] In the first aspect of the present invention, a large-area flow cell chip is proposed, which includes: a prism, a gold film, and a flow cell. The flow cell includes a stacked upper flow cell and a lower flow cell. A channel is provided on the lower flow cell. One side of the lower flow cell provided with the channel is attached to the gold film. A liquid path inlet, a first upper flow cell liquid path channel, a second upper flow cell liquid path channel, and a liquid path outlet are provided on the upper flow cell. A lower flow cell liquid path inlet and a lower flow cell liquid path outlet are provided on the lower flow cell. Two ends of the channel are respectively connected to the lower flow cell liquid path inlet and the lower flow cell liquid path outlet. The liquid path inlet, the first upper flow cell liquid path channel, and the lower flow cell liquid path inlet are connected. The liquid path outlet, the second upper flow cell liquid path channel, and the lower flow cell liquid path outlet are connected. The area of the channel is not less than 60 mm 2 .

[0007] According to the above large-area flow cell chip provided by the present invention, a double-layer structure including an upper flow cell and a lower flow cell is included, which can disperse the four-point pressure from the instrument to the entire surface of the flow cell, making the part where the lower flow cell is bonded to the chip gold film more stable, increasing the stability of the flow cell, and avoiding the collapse and blockage of the flow path or the detachment of the patch edge. On this basis, on the premise of ensuring the mechanical strength of the flow cell, the design area of the channel can be increased, specifically not less than 60 mm 2 , so as to realize a large-area flow cell chip dedicated to molecular fishing. The detection area is 5.5 times that of the flow cell of the existing two-channel SPR chip, greatly improving the use efficiency of the chip, increasing the fixation amount and capture amount of the sample, and shortening the molecular fishing experiment time to 1-2 hours, significantly improving the work efficiency.

[0008] In some embodiments of the present invention, the total area of the channels is 40-60 mm 2 .

[0009] In some embodiments of the present invention, the diameter of the channel is 0.5-0.7 mm.

[0010] In some embodiments of the present invention, the channels are evenly distributed on the lower flow cell.

[0011] Preferably, the channels are symmetrically distributed in a zigzag structure.

[0012] In some embodiments of the present invention, a reference liquid inlet and a reference liquid path channel are further provided on the upper flow cell. A reference path is provided on the lower flow cell. The reference liquid inlet, the reference liquid path channel, and the reference path are connected in sequence. The first upper flow cell liquid path channel extends and is connected to the reference path.

[0013] In the second aspect of the present invention, a surface plasmon resonance instrument is proposed, which includes the above large-area flow cell chip.

[0014] In the third aspect of the present invention, the present invention proposes the application of the above surface plasmon resonance instrument in molecular detection.

[0015] In some embodiments of the present invention, glucose is modified on the gold film surface of the large-area flow cell chip, and then carboxyl modification is carried out on the glucose to obtain a carboxyl-modified large-area flow cell chip.

[0016] In some embodiments of the present invention, the method for immobilizing biomolecules on the carboxyl-modified large-area flow cell chip includes: putting the carboxyl-modified large-area flow cell chip into an SPR instrument, and immobilizing proteins by the method of amino coupling or immobilizing nucleic acids or polypeptides by the method of streptavidin-biotin binding.

[0017] In some embodiments of the present invention, the method for immobilizing small molecule compounds on the carboxyl-modified large-area flow cell chip includes: modifying the carboxyl-modified large-area flow cell chip with the small molecule compounds by means of photocrosslinking. The small molecule compounds are coupled with the large-area flow cell chip through photocrosslinking active molecules. The photocrosslinking active molecules contain an amino terminus and a diazirine structure, and the ultraviolet light wavelength used for photocrosslinking is 365 nm; Preferably, the photocrosslinking modification uses a polytetrafluoroethylene box, which has 1-25 independent chip placement grooves, and the volume of the reaction solution that can be contained in the grooves is 80-120 μL.

[0018] In some embodiments of the present invention, the specific process of the large-area flow cell chip for molecular fishing is as follows: (1) Immobilize a biomolecule on the large-area flow cell chip; (2) In the SPR instrument, suck the analyte sample through the injection needle, flow through the surface of the large-area flow cell chip and bind to the biomolecules on the surface of the large-area flow cell chip, and the unbound sample flows through the surface of the large-area flow cell chip; (3) Use the eluent to elute the sample bound to the biomolecules immobilized on the large-area flow cell chip; (4) Collect the eluted sample through the recovery pipeline of the instrument; (5) Identify the sample by mass spectrometry; (6) For the identified molecules, prepare pure products and further verify them by SPR.

[0019] The present invention has at least the following technical effects: (1) Based on SPR technology, the present invention develops a large-area flow cell chip dedicated to molecular fishing. On the basis of ensuring the mechanical strength of the flow cell, the design area of the channel can be increased, specifically not less than 60 mm 2, thus realizing a large-area flow cell chip dedicated to molecular fishing. The detection area is 5.5 times that of the flow cell of the existing two-channel SPR chip, greatly improving the chip usage efficiency, increasing the sample fixation amount and capture amount. The molecular fishing experiment time can be shortened to 1 - 2 hours, significantly enhancing the work efficiency.

[0020] (2) On the basis of ensuring the molecular fishing area, the large-area flow cell chip of the present invention adds a reference channel. The image of molecular fishing will have analytical significance and the signal will be more accurate. By using the upper flow cell for transition, the reference channel can be designed at the edge of the chip, and most of the other space will be used for the fishing channel to couple and fish samples.

[0021] (3) The present invention provides a polytetrafluoroethylene box for the chemical reaction of the large-area flow cell chip. This box can hold 1 - 25 SPR chips, and the reaction solution volume is 80 - 120 μL, greatly saving the usage volume and cost of reaction reagents.

[0022] (4) The present invention provides an experimental method for surface chemical modification and fixation of various biomolecules on the large-area flow cell chip, and its application in the field of molecular fishing. This method has advantages such as non-labeling, real-time detection, fast speed, simple operation, and easy popularization. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a schematic diagram of the large-area flow cell structure of an embodiment provided by the present invention; Figure 2 It is a schematic diagram of the large-area flow cell structure of another embodiment provided by the present invention; Figure 3 It is a physical diagram of the polytetrafluoroethylene box provided by the present invention; Figure 4 It is a process flow chart of the preparation of the photoactive molecule provided by the present invention; Figure 5 It is a schematic diagram of the process of the molecular fishing experiment using the large-area flow cell chip of the present invention; Figure 6 It is a GO analysis diagram of the proteins identified by the biomass spectrometry in Example 3 of the present invention; Figure 7 It is two modes of the fishing experiment in Example 3 of the present invention. Detailed Embodiments

[0025] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0026] In a first aspect of the present invention, the present invention provides a large-area flow cell chip, which includes: a prism, a gold film, and a flow cell. Refer to Figure 1 , the flow cell includes a stacked upper flow cell 1 and a lower flow cell 2. A channel 3 is provided on the lower flow cell 2. One side of the lower flow cell 2 provided with the channel 3 is attached to the gold film. A liquid path inlet 4, a first upper flow cell liquid path channel 5, a second upper flow cell liquid path channel 6, and a liquid path outlet 7 are provided on the upper flow cell 1. A lower flow cell liquid path inlet 8 and a lower flow cell liquid path outlet 9 are provided on the lower flow cell 2. Two ends of the channel 3 are respectively connected to the lower flow cell liquid path inlet 8 and the lower flow cell liquid path outlet 9. The liquid path inlet 4, the first upper flow cell liquid path channel 5, and the lower flow cell liquid path inlet 8 are connected. The liquid path outlet 7, the second upper flow cell liquid path channel 6, and the lower flow cell liquid path outlet 9 are connected. The area of the channel 3 is not less than 60 mm 2 .

[0027] According to the above large-area flow cell chip provided by the present invention, which includes a double-layer structure of an upper flow cell and a lower flow cell, the four-point pressure from the instrument can be dispersed to the entire surface of the flow cell, making the part where the lower flow cell is bonded to the chip gold film more stable, increasing the stability of the flow cell, and avoiding the collapse and blockage of the flow path or the detachment of the patch edge. On this basis, on the premise of ensuring the mechanical strength of the flow cell, the design area of the channel can be increased, specifically not less than 60 mm 2 , so as to realize a large-area flow cell chip dedicated to molecular fishing. The detection area is 5.5 times that of the flow cell of the existing two-channel SPR chip, greatly improving the use efficiency of the chip, increasing the fixed amount and capture amount of the sample, and shortening the molecular fishing experiment time to 1-2 hours, significantly improving the work efficiency.

[0028] Specifically, the specific flow direction of the liquid in the above large-area flow cell is that the liquid enters the channel 3 from the liquid path inlet 4 through the first upper flow cell liquid path channel 5, then enters through the lower flow cell liquid path inlet 8, and after passing through the channel 3, it flows out through the second upper flow cell liquid path channel 6 from the lower flow cell liquid path outlet 9 and finally through the liquid path outlet 7.

[0029] In some embodiments of the present invention, the total area of the channel is 40-60 mm 2 .

[0030] In some embodiments of the present invention, the diameter of the channel is 0.5-0.7 mm.

[0031] In some embodiments of the present invention, the channels are evenly distributed on the downstream cell.

[0032] Preferably, the channels are symmetrically distributed in a "Ji" character structure.

[0033] In some embodiments of the present invention, referring to Figure 2 , a reference liquid inlet 10 and a reference liquid path channel 11 are further provided on the upper flow cell 1, a reference path 12 is provided on the downstream flow cell 2, the reference liquid inlet 10 and the reference liquid path channel 11 are connected, the reference liquid path channel 11 is connected to the reference path 12 through a first through port 13, and the first upper flow cell liquid path channel 5 extends and is connected to the reference path 12 through a second through port 14. The reference path 12 is attached to the gold film.

[0034] In the double-flow cell design, through the design of the upper flow cell, the starting and ending points of the lower flow cell can be freely designed without being restricted by the instrument flow path interface, increasing the scalability of flow cell customization. Usually, in a molecular fishing flow cell, each flow cell is connected in series, and all flow cells are coupled with samples for molecular fishing. If a reference channel is added on the basis of ensuring the molecular fishing area, the molecular fishing image will have analytical significance and the signal will be more accurate. However, due to the limitation of the instrument flow path interface, setting a reference channel will greatly affect the design and arrangement of the fishing channel. In the present invention, by using the upper flow cell for transition, the reference channel can be designed at the edge of the chip, and most of the other space will be used for the fishing channel to couple and fish for samples.

[0035] The specific process is as follows: First, a biomolecule is fixed on the gold film in the channel 3. The sample liquid flows from the liquid path inlet 4 through the first upper flow cell liquid path channel 5 into the channel 3 and then flows out from the liquid path outlet 7, realizing the fixation of the biomolecule in the channel 3. At this time, the reference liquid inlet 10 is closed, so the sample liquid will not flow into the reference path 12. Then, the sample to be measured enters from the reference liquid inlet 10, first passes through the reference liquid path channel 11, and then enters the channel 3 from the downstream flow cell liquid path inlet 8 through the second through port 14 and the first upper flow cell liquid path channel 5, and finally flows out from the liquid path outlet 7. At this time, the liquid path inlet 4 is closed. Through the reference path 12, the exclusion of impurity adsorption can be realized, improving the measurement accuracy.

[0036] In the second aspect of the present invention, the present invention provides a surface plasmon resonance instrument, which includes the above-mentioned large-area flow cell chip.

[0037] In the third aspect of the present invention, the present invention provides the application of the above-mentioned surface plasmon resonance instrument in molecular detection.

[0038] In some embodiments of the present invention, glucose is modified on the surface of the gold film of the large-area flow cell chip, and then carboxyl modification is performed on the glucose to obtain a carboxyl-modified large-area flow cell chip.

[0039] In some embodiments of the present invention, the process of modifying the surface of the gold film of the large-area flow cell chip with glucose and then carboxyl-modifying the glucose includes: The chip can be placed in a beaker or a polytetrafluoroethylene box and soaked in piranha solution (98% H2SO4 / 30% H2O2, volume ratio 7:3) at room temperature for 2 - 12 h. The chip is rinsed clean with ultrapure water and 80% ethanol, then placed in a beaker, 16-mercapto-1-hexadecanol solution is added, and it is placed on a shaker, shaken overnight at 40 °C and 100 rpm. The chip is rinsed clean with ultrapure water and 80% ethanol, then placed in a beaker, 2 - 10 mM epichlorohydrin solution is added, and it is placed on a shaker and incubated at 25 °C for 3 - 10 h. The chip is rinsed clean with ultrapure water and 80% ethanol, then placed in a beaker, dextran solution (0.1 - 0.5 g / mL, containing 0.1 M NaOH) is added, and it is placed on a shaker and incubated at 37 °C for 12 - 36 h. The chip is rinsed clean with ultrapure water and 80% ethanol, then placed in a beaker, bromoacetic acid / NaOH solution (10 M bromoacetic acid / 2 M NaOH, mixed solution with a volume ratio of 1:9) is added, and it is placed on a shaker and incubated at room temperature for 12 - 36 h. The chip is taken out, placed in a petri dish filled with ultrapure water, washed with water, dried with nitrogen, placed in a chip box, and stored at 4 °C.

[0040] In some embodiments of the present invention, the method for immobilizing biomolecules on the carboxyl-modified large-area flow cell chip includes: The carboxyl-modified large-area flow cell chip is placed in an SPR instrument, and proteins are immobilized by amino coupling or nucleic acids or polypeptides are immobilized by the streptavidin-biotin binding method.

[0041] In some embodiments of the present invention, proteins can be immobilized on the chip surface by the method of amino coupling. The chip can be placed in an SPR instrument to detect the protein coupling process in real time. The amino coupling method includes: Activating the above carboxyl-modified flow cell chip by mixing 0.1 M NHS and 0.4 M EDC in a 1:1 ratio, with a flow rate of 5 - 10 μL / min for 7 - 20 min. Then, a protein solution with a concentration of 10 - 100 μg / mL is prepared with 10 mM sodium acetate solution at pH 4 - 6, with a flow rate of 5 - 10 μL / min, and injected for 1 - 20 min. Then, it is blocked with 1 M ethanolamine at pH 8.5 at a flow rate of 5 - 10 μL / min for 7 - 20 min, so that as much protein as possible can be immobilized on the flow cell chip.

[0042] In some embodiments of the present invention, a biotin-labeled nucleic acid aptamer or polypeptide is immobilized on a flow cell chip by means of a streptavidin-biotin reaction. The flow cell chip can be placed in an SPR instrument to detect the nucleic acid and protein coupling process in real time. The coupling process includes first immobilizing streptavidin protein on the chip, and then diluting the biotin-modified nucleic acid aptamer or polypeptide to 5-10 μg / mL with running buffer PBS (pH = 7.4), injecting at 5-10 μL / min for 30-300 s, and the nucleic acid aptamer or polypeptide can be immobilized on the streptavidin-modified large-area flow cell chip.

[0043] In some embodiments of the present invention, the method for immobilizing small molecule compounds on the carboxyl-modified large-area flow cell chip includes: modifying the carboxyl-modified large-area flow cell chip with the small molecule compound by means of photocrosslinking. The small molecule compound contains an amino terminus and a diazirine structure, and the ultraviolet light wavelength used for photocrosslinking is 365 nm.

[0044] In some embodiments of the present invention, the photocrosslinking method requires the use of a photocrosslinking active molecule containing an amino terminus and a diazirine structure. The amino group of the photocrosslinking active molecule is used for covalent coupling with the carboxyl group on the surface of the large-area flow cell chip. The diazirine group can form a carbene carbon radical under 365 nm ultraviolet light irradiation, and can form a carbon-carbon bond with the small molecule in a random orientation to covalently immobilize the small molecule on the flow cell chip. The random orientation can maintain as much as possible the binding activity of the small molecule itself. The reaction process refers to placing the above-mentioned carboxyl-modified flow cell chip with the gold film facing down in a polytetrafluoroethylene box, adding a 1:1 mixture of NHS (0.1 M) and EDC (0.4 M), and reacting for 1 h. Then suck away the reaction solution, add 5-10 mM of the above-mentioned photocrosslinking active molecule (dissolved in DMF) and 50 mM N,N-diisopropylethylamine (DIPEA), and react in the dark for 6 h. Then block the flow cell chip with 1 M ethanolamine DMF solution for 1 h. Wash the surface of the chip with ethanol and water respectively to remove the unreacted substances. Then take 50 μL of 10 mM small molecule dissolved in DMSO and drop it on the photocrosslinked flow cell chip, place it in an oven at 40 °C overnight, irradiate it with a 365 nm ultraviolet lamp for 30 minutes, and wash it with ethanol and water respectively, and the small molecule can be immobilized on the large-area flow cell chip in a random orientation.

[0045] For small molecules, when immobilized on an SPR chip, it is usually necessary to label the small molecules with biotin. However, labeling small molecules with biotin has high technical difficulties and thresholds. Some biomolecules lack reactive groups and are difficult to label with biotin, and if the labeling site happens to be the active site of the biomolecule, the biomolecule will lose its activity. Therefore, a photocrosslinking flow cell chip method has been developed. Without chemical modification, small molecules can be immobilized on the chip through a photocrosslinking reaction under irradiation with a 365 nm ultraviolet lamp for target fishing experiments. As an example, the structure of the photocrosslinking active molecule is as follows: 。

[0046] In some embodiments of the present invention, as Figure 3 shown, the photocrosslinking modification uses a polytetrafluoroethylene box, and the polytetrafluoroethylene box has 1-25 independent chip placement grooves ( Figure 3 There are 25 grooves in ), and the volume of the reaction solution that the groove can hold is 80-120 μL. This polytetrafluoroethylene box can be used for the chemical reaction of one SPR chip or can place multiple SPR chips simultaneously for chemical reactions, and has the advantages of inertness, high throughput, sample saving, and easy operation.

[0047] In some embodiments of the present invention, the large-area flow cell chip is used for molecular fishing, and the specific process is as follows: (1) Immobilize a biomolecule on the large-area flow cell chip; (2) In an SPR instrument, suck the analyte sample through an injection needle, flow through the surface of the large-area flow cell chip and bind to the biomolecule on the surface of the large-area flow cell chip, and the unbound sample flows through the surface of the large-area flow cell chip; (3) Use an eluent to elute the sample bound to the biomolecule immobilized on the large-area flow cell chip; (4) Collect the eluted sample into a 1.5 mL EP tube through the recovery pipeline of the instrument; (5) Identify the sample by mass spectrometry; (6) For the identified molecules, prepare pure products and further verify them by SPR.

[0048] In some embodiments of the present invention, in step (1), when immobilizing a biomolecule on the large-area flow cell chip, if the biomolecule is a protein, nucleic acid or polypeptide, first attach a multi-channel flow cell to the chip, and then put it into an SPR instrument, and fix the protein by amino coupling or fix the nucleic acid or polypeptide by streptavidin-biotin binding; if the biomolecule is a small molecule, the chip is modified with the small molecule by a photocrosslinking method, then a multi-channel flow cell is attached, and then put into an SPR instrument for a molecular fishing experiment.

[0049] In some embodiments of the present invention, in step (2), the analyte sample includes at least one of traditional Chinese medicine extracts, plasma, cells, cell membrane lysates, tissue lysates, lymph fluid, and cerebrospinal fluid. The SPR experimental method can detect the dynamic process of the binding of the analyte sample to the molecules on the chip in real time. The flow rate of the SPR experimental method is set to 5 - 10 μL / min, the injection time is 60 - 600 s, and the instrument running buffer can be adjusted according to experimental needs, including but not limited to PBS, HEPES, Tris-HCl, PBS containing DMSO and / or tween 20, etc.

[0050] In some embodiments of the present invention, in step (3), the eluent includes but not limited to PBS, HEPES, PBS containing DMSO and / or tween 20, trifluoroacetic acid, formic acid, acetic acid, glycine, hydrochloric acid, sodium dodecyl sulfate (SDS), etc. The elution flow rate is set to 5 - 30 μL / min, and the injection time is 30 - 300 s. The elution includes two modes: (a) For analyte samples with fast dissociation, no regeneration is required, and the buffer can be used for elution. In this case, the buffer is directly used for flushing and recovery after the sample; (b) For analyte samples with slow dissociation, regeneration is generally required, and the regeneration solution can be used as the eluent. In this case, the regeneration solution is first introduced after the sample, then the regeneration solution is collected, and then the buffer is run.

[0051] In some embodiments of the present invention, in order to fish out a sufficient amount of sample for mass spectrometry detection, steps (2)-(4) can be repeated 1 - 10 times in a cycle.

[0052] In some embodiments of the present invention, in step (5), if the analyte sample is a small molecule sample such as a traditional Chinese medicine extract, it can be identified by high performance liquid chromatography - mass spectrometry (such as Thermo Q Exactive HF-X); if the analyte sample is a biological fluid, cell or tissue sample such as plasma, cells or cell membrane lysates, tissue lysates, lymph fluid, cerebrospinal fluid, etc., it can be identified by proteomics (such as Thermo Fusion Lumos mass spectrometer).

[0053] In some embodiments of the present invention, in step (6), for the identified molecules, verification by SPR means preparing pure products and detecting their affinity with the molecules on the chip for verification. If there is indeed binding, the affinity K D and kinetic parameters (such as k on and k off ) values can be given.

[0054] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0055] Example 1 This example provides an experimental method for modifying carboxyl dextran on a large-area flow cell chip.

[0056] (1) Modifying dextran on the surface of the large-area flow cell chip: Place the chip with the gold film facing down in a Teflon box, pour in the piranha solution (98% H2SO4 / 30% H2O2 = 7:3), and soak at room temperature for 10 h. Then rinse the chip thoroughly with ultrapure water and rinse 4 times with 80% ethanol. Place the chip with the gold film facing up in a beaker, add a 6 mM 16-mercapto-1-hexadecanol solution, cover it with a sealing film, place it in a shaker, and shake overnight at 40 °C and 100 rpm. Take out the chip, place it in a petri dish containing 80% ethanol, wash 4 times with 80% ethanol, rinse thoroughly with ultrapure water, and dry with nitrogen. Then place the chip with the gold film facing up in a beaker, add a 0.6 M epichlorohydrin solution, place it in a shaker, and incubate at 25 °C for 6 h. Take out the chip, place it in a petri dish containing ultrapure water, rinse once with water, wash 4 times with 80% ethanol, rinse thoroughly with ultrapure water, and dry with nitrogen. Place the chip with the gold film facing up in a beaker, add a dextran solution (0.4 g / mL, containing 0.1 M NaOH), cover it with a sealing film, place it in a shaker, and incubate at 37 °C for 24 h. Take out the chip, place it in a petri dish containing ultrapure water, wash it clean with water, and dry with nitrogen.

[0057] (2) Modifying carboxyl groups on the dextran of the large-area flow cell chip: Place the chip with the gold film facing up in a beaker, add a bromoacetic acid / NaOH solution (a mixed solution of 10 M bromoacetic acid / 2 M NaOH = 1:9), place it in a shaker, and incubate at room temperature for 24 h. Take out the chip, place it in a petri dish containing ultrapure water, wash it with water, dry with nitrogen, place it in a chip box, and store it at 4 °C.

[0058] Example 2 This example provides an experimental method for immobilizing biomolecules and small molecule compounds on a large-area flow cell chip.

[0059] (1) Immobilize the protein (carbonic anhydrase) on the large-area flow cell chip by amino coupling Taking the surface plasmon resonance instrument (MI-S200F) of Beijing Yingbo Biotechnology Co., Ltd. as an example, on the MI-S200F instrument, first, activate the above carboxyl-modified large-area flow cell chip with a 1:1 mixture of 0.1 M NHS and 0.4 M EDC (10 µL / min, 15 min). Then, prepare a protein solution with a concentration of 50 μg / mL using a 10 mM sodium acetate solution at pH 5.0, inject it at a rate of 10 µL / min for 10 min, and then block it with 1 M ethanolamine at pH 8.5 at a rate of 10 µL / min for 15 min, so that as much protein as possible can be immobilized on the large-area flow cell chip.

[0060] (2) Immobilize biotin-labeled nucleic acid aptamers or polypeptides on the large-area flow cell chip through the streptavidin-biotin reaction Taking the surface plasmon resonance instrument (MI-S200F) of Beijing Yingbo Biotechnology Co., Ltd. as an example, on the MI-S200F instrument, first, activate the above carboxyl-modified large-area flow cell chip with a 1:1 mixture of 0.1 M NHS and 0.4 M EDC (10 µL / min, 15 min). Then, prepare a streptavidin solution with a concentration of 50 μg / mL using a 10 mM sodium acetate solution at pH 4.5, inject it at a rate of 10 µL / min for 10 min, and then block it with 1 M ethanolamine at pH 8.5 at a rate of 10 µL / min for 15 min, so that streptavidin can be immobilized on the large-area flow cell chip. Dilute biotin-modified nucleic acid aptamers or polypeptides to 10 µg / mL with running buffer PBS (PH = 7.4), inject it at a rate of 10 µL / min for 180 s, so that the nucleic acid aptamers or polypeptides can be immobilized on the streptavidin-modified large-area flow cell chip.

[0061] (3) Immobilize small molecules on the large-area flow cell chip by photochemical cross-linking method (a) Prepare a photoactive molecule ( ) Dissolve 1,2-bis(2-aminoethoxy)ethane (1.96 g) in dichloromethane (CH2Cl2), then cool it to 0 °C in an ice bath, and dropwise add a CH2Cl2 solution of di-tert-butyl dicarbonate (0.30 g) to the flask under nitrogen protection. The dropping process lasts for 3 h, and then the reaction solution is stirred overnight at room temperature. Extract the reaction solution with saturated NaCl and CH2Cl2, dry the organic phase with Na2SO4, filter and concentrate it to obtain a colorless transparent liquid, which is compound 1.

[0062] To a solution of trifluoromethylphenyl bisaziridine (TAD, 0.25 g) in CH2Cl2 was added 1,1'-carbonyldiimidazole (CDI, 0.40 g), and the mixture was stirred at 0 °C for 1 h. The carboxyl activation process was monitored by TLC. After carboxyl activation, compound 1 dissolved in CH2Cl2 was added dropwise. The reaction solution was stirred overnight at room temperature. The mixture was extracted with saturated NaCl, NaHCO3, and CH2Cl2, and the organic phase was dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound 2 as a yellow oily solid.

[0063] To a solution of compound 2 (0.21 g) in MeOH was added 4 M HCl, and the mixture was stirred overnight at room temperature. After monitoring the completion of the reaction by TLC, it was extracted with saturated NaCl, NaHCO3, and CH2Cl2, and then the organic phase was dried over Na2SO4, filtered, and concentrated to obtain compound 3 as a pale yellow viscous liquid ( ). The specific reaction process can be referred to Figure 4 .

[0064] (b) Preparation of a photo-crosslinked large-area flow cell chip The above carboxyl-modified large-area flow cell chip was placed with the gold film facing down in a Teflon box, and a 1:1 mixture of NHS (0.1 M) and EDC (0.4 M) was added, and the reaction was carried out for 1 h. Then the reaction solution was aspirated, and 10 mM of the above compound 3 (dissolved in DMF) and 50 mM N,N-diisopropylethylamine (DIPEA) were added, and the reaction was carried out in the dark for 6 h. Then the large-area flow cell chip was blocked with a 1 M ethanolamine DMF solution for 1 h. The chip surface was washed with ethanol and water respectively to remove the unreacted substances.

[0065] (c) Fixing small molecules on the large-area flow cell chip by photo-crosslinking A 10 mM small molecule solution was prepared with DMSO. 50 μL was added dropwise onto the photo-crosslinked large-area flow cell chip, and then it was placed in an oven at 40 °C overnight, irradiated with a 365 nm ultraviolet lamp for 30 minutes, and washed with ethanol and water respectively. Then the small molecules can be fixed on the large-area flow cell chip in a random orientation.

[0066] Example 3 This example provides the process and method for performing a molecular fishing experiment using a large-area flow cell chip.

[0067] (1) The schematic diagram of the process for performing a molecular fishing experiment using a large-area flow cell chip is shown in Figure 5 .

[0068] Taking the experiment of fixing small molecules on the chip and fishing for protein targets as an example.

[0069] First, attach the flow cell to the photo-crosslinking large-area flow cell chip with the small molecules already fixed above, and then place it into a surface plasmon resonance instrument (MI-S200F). Aspirate the analyte sample (such as cell lysate) through the injection needle, and then let it flow through the surface of the large-area flow cell chip. The proteins bound to the small molecules will bind to the small molecules, and the unbound proteins will flow through the chip surface. Then, elute the proteins bound to the small molecules with the eluent, and collect the eluted proteins into a 1.5 mL EP tube through the recovery pipeline of the instrument, and identify them by biomass spectrometry. Figure 6 To perform GO analysis on the proteins identified by biomass spectrometry, potential protein targets can be analyzed in combination with the research direction. For the identified potential protein targets, their affinity with small molecules can be further verified by SPR detection.

[0070] (2)According to the dissociation speed of the analyte, the fishing experiment is divided into two modes: (a)For analyte samples with fast dissociation, no regeneration is required, and the buffer can be used for elution. In this case, the buffer is directly used for flushing and recovery after the sample, such as Figure 7 A in (b)For analyte samples with slow dissociation, regeneration is generally required, and the regeneration solution can be used as the eluent. In this case, the regeneration solution is first passed after the sample, then the regeneration solution is collected, and then the buffer is passed (such as Figure 7 B in

[0071] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A large-area flow cell chip, characterized in that, Including: A prism, a gold film, and a flow cell. The flow cell includes a stacked upper flow cell and a lower flow cell. A channel is provided on the lower flow cell, and one side of the lower flow cell provided with the channel is attached to the gold film. A liquid path inlet, a first upper flow cell liquid path channel, a second upper flow cell liquid path channel, and a liquid path outlet are provided on the upper flow cell. A lower flow cell liquid path inlet and a lower flow cell liquid path outlet are provided on the lower flow cell. Two ends of the channel are respectively connected to the lower flow cell liquid path inlet and the lower flow cell liquid path outlet. The liquid path inlet, the first upper flow cell liquid path channel, and the lower flow cell liquid path inlet are connected. The liquid path outlet, the second upper flow cell liquid path channel, and the lower flow cell liquid path outlet are connected. The area of the channel is not less than 60 mm 2 .

2. The large-area flow cell chip according to claim 1, wherein The total area of the said channel is 40 - 60 mm 2 ; And / or, the diameter of the channel is 0.5 - 0.7 mm.

3. The large-area flow cell chip according to claim 1, wherein The channels are evenly distributed on the downstream cell; Preferably, the channels are symmetrically distributed in a "Ji" character structure.

4. The large-area flow cell chip according to any one of claims 1-3, characterized in that A reference liquid inlet and a reference liquid path channel are further provided on the upstream cell, a reference path is provided on the downstream cell, the reference liquid inlet, the reference liquid path channel and the reference path are connected in sequence, and the first upstream cell liquid path channel extends and is connected to the reference path.

5. A surface plasmon resonance instrument, characterized in that, Including the large-area flow cell chip according to any one of claims 1 - 4.

6. Application of the surface plasmon resonance instrument according to claim 5 in molecular detection.

7. The application according to claim 6, wherein Modify glucose on the gold film surface of the large-area flow cell chip, and then perform carboxyl modification on the glucose to obtain a carboxyl-modified large-area flow cell chip.

8. The application according to claim 7, wherein The method for immobilizing biomolecules on the carboxyl-modified large-area flow cell chip includes: putting the carboxyl-modified large-area flow cell chip into an SPR instrument, and immobilizing proteins by an amino coupling method or immobilizing nucleic acids or polypeptides by a streptavidin-biotin binding method.

9. The application according to claim 7, wherein The method for immobilizing small molecule compounds on the carboxyl-modified large-area flow cell chip includes: modifying the carboxyl-modified large-area flow cell chip with the small molecule compounds by a photocrosslinking method, the small molecule compounds are coupled with the large-area flow cell chip through a photocrosslinking active molecule, the photocrosslinking active molecule contains an amino terminal and a diazirine structure, and the ultraviolet light wavelength used for photocrosslinking is 365 nm; Preferably, the photocrosslinking modification uses a polytetrafluoroethylene box, the polytetrafluoroethylene box has 1 - 25 independent chip placement grooves, and the volume of the reaction solution that can be contained in the grooves is 80 - 120 μL.

10. The application according to claim 6, wherein The specific process of using the large-area flow cell chip for molecular fishing is as follows: (1) Immobilize a biomolecule on the large-area flow cell chip; (2) In an SPR instrument, suck an analyte sample through a sampling needle, flow through the surface of the large-area flow cell chip and bind to the biomolecules on the surface of the large-area flow cell chip, and the unbound sample flows through the surface of the large-area flow cell chip; (3) Use an eluent to elute the sample bound to the biomolecules immobilized on the large-area flow cell chip; (4) Collect the eluted sample through the recovery pipeline of the instrument; (5) Identify the sample by mass spectrometry; (6) For the identified molecules, prepare pure products and further verify them by SPR.

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