A method for verifying cleaning effect of a photonic crystal fluorescence-enhanced printing synthetic DNA chip

Abstract

The application discloses a kind of photonic crystal fluorescence enhancement's printing synthetic DNA chip's cleaning effect verification method, comprising: S1, preparation photonic crystal microchip, form hydrophobic substrate and hydrophilic detection area;S2, build chip test platform, collect specific time's reaction cleaning liquid sample, and drop the sample to be measured on the surface of photonic crystal microchip;S3, heat photonic crystal microchip, after sample solution drying, the detection object is enriched in hydrophilic detection area;S4, the fluorescence spectrum signal that the detection object emits is detected by fluorescence spectroscopy, to realize the detection of detection object;S5, the cleaning effect of chip is judged using fluorescence intensity change.The verification method of the application can not only use fluorescence intensity change to judge the cleaning effect of chip, but also be suitable for trace detection under very low concentration condition, realize higher sensitivity than ordinary substrate such as glass, be favorable to reduce detection limit, realize more accurate analysis under low concentration.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chips, and in particular, relates to a photonic crystal fluorescence-enhanced printing synthetic DNA chip cleaning effect verification method. BACKGROUND

[0002] High-throughput DNA synthesis chip is an important research direction in the fields of biology and chip-based DNA synthesis technology. The chip provides suitable reaction sites for DNA synthesis and has an important influence on the synthesis effect as a DNA synthesis carrier.

[0003] In the process of printing synthetic DNA, the cleaning effect is one of the key factors for the successful operation of high-throughput DNA synthesis chip, which directly affects the accuracy, coupling efficiency and cost of synthesis. Since the entire DNA synthesis process involves multiple reaction steps in multiple cycles, different reaction steps will affect each other, so the cleaning process on the chip surface after each step is very important. If the cleaning is insufficient, the residual chemicals may interfere with the subsequent reaction, resulting in reduced reaction efficiency or even aborted reaction in the subsequent steps, affecting the effectiveness and stability of the synthesis, limiting the length and load of the synthesis, and also affecting the quality of the synthesis product due to error accumulation. If the cleaning is excessive, it may not only increase the interval time of the synthesis steps, thereby reducing the reaction efficiency, but also increase the time and cost of the synthesis process. Especially in a large-scale synthesis process, the use of a large amount of organic reagents and the generation of chemical waste liquid will greatly increase the cost and environmental burden.

[0004] Therefore, determining the optimal cleaning method and time is not only beneficial to improving the synthesis efficiency and quality, but also conducive to realizing the low cost and environmental protection of the synthesis process. However, due to the low content of chemicals in the cleaning solution, it is difficult to accurately quantify and analyze them, and how to effectively identify the cleaning effect is a difficult problem.

[0005] Currently commonly used detection methods such as colorimetric method, chemical titration method, spectrophotometric method, chromatographic method, mass spectrometry method, etc. have the disadvantages of detection limit, sensitivity, and being easily affected by the environment, etc. It is difficult to ensure the accuracy of detection for low-concentration solutions. Fluorescence detection technology has relatively high sensitivity and anti-interference compared to other methods, and is more suitable for detection under low-concentration conditions, but it is still difficult to apply to trace detection under extremely low-concentration conditions. SUMMARY

[0006] In view of the problem that the cleaning effect of the chip surface is difficult to determine, resulting in low synthesis yield, and the stability and parallelism of different batches are difficult to maintain in the process of chip DNA synthesis, the application provides a printing synthetic DNA chip cleaning effect verification method based on photonic crystal fluorescence enhancement, which detects the fluorescence spectrum signal emitted by the to-be-detected substance by means of the fluorescence enhancement characteristic of the photonic crystal, judges the cleaning effect of the chip by means of the fluorescence intensity change, and provides support for determining the optimal cleaning method in the process of chip DNA synthesis.

[0007] In order to achieve the above-mentioned purpose, the application provides the following technical scheme:

[0008] A printing synthetic DNA chip cleaning effect verification method based on photonic crystal fluorescence enhancement, comprising the following steps:

[0009] S1, preparing a photonic crystal microchip to form a hydrophobic substrate and a hydrophilic detection area;

[0010] S2, building a chip test platform, collecting a reaction cleaning liquid sample at a specific time, and dropping the to-be-detected sample solution on the surface of the photonic crystal microchip;

[0011] S3, heating the photonic crystal microchip, and after the sample solution is dried, the to-be-detected substance is enriched in the hydrophilic detection area;

[0012] S4, detecting the fluorescence spectrum signal emitted by the to-be-detected substance by means of the fluorescence enhancement characteristic of the photonic crystal, so as to realize the detection of the to-be-detected substance;

[0013] S5, judging the cleaning effect of the chip by means of the fluorescence intensity change.

[0014] In an embodiment of the application, in the step S5, the step of judging the cleaning effect of the chip by means of the fluorescence intensity change comprises:

[0015] When a weak fluorescence signal is detected by the photonic crystal microchip, it is determined that the chip is not cleaned completely, and the concentration of the liquid sample at this time point or the residual amount of the liquid sample at 0s after the cleaning is started is calculated;

[0016] When no obvious fluorescence signal can be detected by the photonic crystal microchip, it is determined that the cleaning is complete and no residue is left.

[0017] In an embodiment of the application, in the step S5, the step of judging the cleaning effect of the chip by means of the fluorescence intensity change comprises:

[0018] S51, preparing a series of gradient concentration acetonitrile or water solutions of fluorescent dyes, and the solution concentration is 1-10 -10mM, for example 0.1 mM, 0.01 mM, 10 -4 mM, 10 -6 mM, 10 -8 mM, 10 -10 mM; the fluorescence intensity of the solution with different concentrations of fluorescent dyes is measured by using the photonic crystal microchip, and a fluorescence intensity-solution concentration change curve is drawn;

[0019] S52, the fluorescence intensity of the sample received at different cleaning times is tested by using the photonic crystal microchip, a fluorescence intensity-cleaning time change curve is drawn, and the concentration or concentration range of the liquid sample received at different cleaning times is analyzed in combination with the fluorescence intensity-solution concentration change curve; or the sample received at 0s after the start of cleaning is taken as the initial concentration, and whether there is a residue or the corresponding residue amount after different cleaning times is calculated.

[0020] In an embodiment of the present application, the step of preparing the photonic crystal microchip in step S1 comprises:

[0021] S11, a mixed solution (such as a PDMS mixed solution) for forming a hydrophobic substrate is prepared, the mixed solution is spin-coated on the surface of a glass slide, the glass slide is heated for a certain time, and a hydrophobic surface (such as a PDMS surface), also referred to as a hydrophobic substrate, is formed on the glass slide;

[0022] S12, an aqueous solution (for example, nanometer microspheres are prepared into an aqueous solution) for forming a hydrophilic detection area is prepared, the aqueous solution is printed on the hydrophobic surface (such as the PDMS surface), and the medium (such as the nanometer microspheres) in the aqueous solution is deposited on the hydrophobic surface (such as the PDMS surface);

[0023] S13, the hydrophobic surface (such as the PDMS surface) is heated, and after the droplets are dried, the hydrophilic photonic crystal dots are self-assembled to form a hydrophilic detection area on the hydrophobic substrate;

[0024] In an embodiment of the present application, the mixed solution comprises monomers and cross-linking agents; further, the mass ratio of the monomers to the cross-linking agents is (5-20):1.

[0025] In an embodiment of the present application, the prepared mixed solution is spin-coated on the surface of a glass slide, the spin-coating speed is 1000-5000r, and the spin-coating time is 30-90s;

[0026] Preferably, the glass slide is heated at 75℃-85℃ for 12-20min;

[0027] For example, the glass slide is heated at 80℃ for 15min.

[0028] In one embodiment of the present application, the photonic crystal in the photonic crystal microchip (such as the medium in the aqueous solution) is selected from photonic crystals assembled by silica microspheres, photonic crystals assembled by titanium dioxide microspheres, photonic crystals assembled by zinc oxide microspheres, photonic crystals assembled by poly(styrene-methyl methacrylate-acrylamide) core-shell structure microspheres, photonic crystals assembled by poly(styrene-methyl methacrylate-acrylic acid) core-shell structure microspheres, and the like.

[0029] In one embodiment of the present application, the diameter of the microspheres is 150-300 nm, preferably 220-300 nm.

[0030] In one embodiment of the present application, the microspheres are prepared into an aqueous solution with a concentration of 5-20 wt%.

[0031] For example, 5 wt%, 10 wt%, 12 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, or any value between any two of the above-mentioned numerical ranges.

[0032] In one embodiment of the present application, the hydrophobic substrate of the photonic crystal microchip is selected from one or more of polydimethylsiloxane (PDMS), polystyrene, and polytetrafluoroethylene.

[0033] In one embodiment of the present application, the step S2 comprises:

[0034] S21, placing the chip in a substrate provided with a reaction groove, connecting the infusion pipeline to the reaction groove through a round hole punched on both sides of the substrate, one side of the pipeline inputting reaction reagents and acetonitrile cleaning solution into the reaction groove, and the other side of the pipeline collecting sample solution and waste liquid output from the reaction groove; covering the upper part with a cover plate to seal the reaction groove, so that the liquid enters the reaction groove through the pipeline and circulates on the surface of the entire chip in the form of a uniform liquid layer;

[0035] S22, after circulating each reaction reagent added with a fluorescent dye on the surface of the high-throughput DNA synthesis chip, circulating acetonitrile for cleaning;

[0036] S23, collecting acetonitrile cleaning liquid samples at several time points after the start of cleaning, preferably collecting 2-6 s of liquid each time;

[0037] S24, concentrating the collected liquid samples, and transferring the concentrated sample solution to be tested to the hydrophilic detection area on the surface of the photonic crystal microchip with wettability difference.

[0038] In one embodiment of the present application, the reaction reagent includes deprotection reagents, monomer reagents, capping reagents, and oxidation reagents used in the DNA synthesis process.

[0039] Preferably, the added fluorescent dye is selected from one or more of Rhodamine B, Rhodamine 6G, Nile Red and Fluorescein.

[0040] For example, the deprotection reagent is added at a concentration of 0.1 mM Rhodamine 6G, and the capping reagent is added at a concentration of 0.05 mM Nile Red.

[0041] In one embodiment of the present application, the specific collection time of the liquid sample in step S23 is selected from 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 65s, 70s, 75s, 80s, 85s, 90s, 95s, 100s, 105s, 110s, 115s, 120s, 125s, 130s, 135s, 140s, 145s, 150s, 155s, 160s, 165s, 170s, 175s, 180s, and the like, after the start of washing.

[0042] The length of each liquid sample collection is selected from 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, 11s, 12s, 13s, 14s, 15s.

[0043] In one embodiment of the present application, in step S3, the photonic crystal microchip is heated at 30-80°C; preferably 60-80°C.

[0044] The beneficial effects of the present application are:

[0045] 1) The present application provides a method for verifying the cleaning effect of a photonic crystal fluorescence-enhanced printed synthetic DNA chip. First, a photonic crystal microchip including a hydrophobic substrate and a hydrophilic detection region is prepared, then a chip testing platform is built, and a reaction cleaning liquid sample is collected for a specific time. The sample solution to be tested is added to the surface of the photonic crystal microchip with wettability difference, and after the sample solution is dried, the detection object is enriched in the hydrophilic detection region. The present application takes advantage of the fluorescence enhancement characteristics of photonic crystals to detect the fluorescence spectrum signal emitted by the detection object by fluorescence spectroscopy, and uses the change in fluorescence intensity to judge the cleaning effect of the chip.

[0046] 2) The present application analyzes the sample concentration by combining the fluorescence intensity-solution concentration change curve, determines whether there is a detection sample residue or the corresponding residual amount under the corresponding condition, and further realizes the detection of the detection object, solving the problem that the cleaning effect of the surface of the current high-throughput DNA synthesis chip is difficult to judge, thereby leading to low synthesis yield in the DNA synthesis process by chip method, and the difficulty in maintaining the synthesis stability and parallelism of different batches.

[0047] 3) The chip cleaning effect verification method of the application is also suitable for trace detection under extremely low concentration conditions. The wettability difference of the photonic crystal microchip surface enables low concentration sample enrichment, and with the help of the fluorescence enhancement characteristics of the photonic crystal, higher sensitivity than ordinary substrates such as glass is achieved, which is conducive to reducing the detection limit and realizing more accurate analysis under low concentration. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 A flowchart for verifying the cleaning effect of the photonic crystal microchip test platform;

[0049] Figure 2 A schematic diagram of the fluorescence detection results of the sample to be tested on the photonic crystal microchip and the glass substrate at 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s after the start of cleaning, respectively, each time collecting 2s of liquid. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0051] Embodiment 1

[0052] 1) Preparation of photonic crystal microchip:

[0053] Prepare PDMS mixed solution (monomer: crosslinking agent = 10:1 w / w), spin coat the PDMS mixed solution on the surface of the glass slide (rotation speed 2000r, time 60s), heat the glass slide at 80℃ for 15min to form a hydrophobic PDMS surface on the glass slide, i.e. a hydrophobic substrate. Prepare a 12wt% aqueous solution of silica microspheres with a diameter of about 180nm, and print it on the PDMS surface with a dispensing machine to deposit the nanoscale microspheres in the aqueous solution on the hydrophobic PDMS surface. Heat the hydrophobic PDMS surface to 60℃, and after the droplets are dried, self-assemble to form a hydrophilic photonic crystal dot on the hydrophobic PDMS substrate, which constitutes a hydrophilic detection area on the hydrophobic PDMS substrate.

[0054] 2) Build chip test platform: place the chip in the substrate with reaction grooves, connect the liquid pipeline to the reaction grooves through the round holes on both sides of the substrate, one side of the pipeline inputs reaction reagents and acetonitrile cleaning liquid into the reaction grooves, and the other side of the pipeline collects sample solutions and waste liquid output from the reaction grooves; cover the top with a cover plate to seal the reaction grooves, so that the liquid enters the reaction grooves through the pipeline and flows through the entire chip surface in the form of a uniform liquid layer.

[0055] 3) Chip surface deprotection reagent cleaning verification:

[0056] First, a series of gradient concentrations of fluorescent dye acetonitrile solution was prepared, and the solution concentration was 0.1 mM, 0.01 mM, 10 -4 mM, 10 -6 mM, 10 -8 mM, 10 -10 mM. The fluorescence intensity of different concentrations of fluorescent dye acetonitrile solution was measured by photonic crystal microchip, and the fluorescence intensity-solution concentration change curve was drawn.

[0057] In the deprotection reagent (deprotection reagent is a fixed reagent used in the deprotection step of DNA synthesis process, Hebei Dinaxingke Biological Technology Co., Ltd. Production item number R1005-4), 0.1 mM rhodamine 6G was added. The high-throughput DNA synthesis chip was fixed in the microchannel, and 0.1 mM rhodamine 6G deprotection reagent was added to the chip surface at a flow rate of 1-5 mL / min for 30 s, and then pure acetonitrile was washed at a flow rate of 5-20 mL / min.

[0058] As Figure 2 shown, the acetonitrile cleaning liquid samples were collected at 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s after the start of cleaning, respectively, and each time 2s of liquid was collected. The collected liquid sample was heated and concentrated to dryness, then 20 μL of acetonitrile was used to dissolve it, and 0.5 μL of the concentrated sample solution was removed and added to the hydrophilic detection area of the photonic crystal microchip prepared in Example 1 with different wettability on the surface. Heat the photonic crystal microchip at 60°C to dry the sample solution, and the test substance is enriched in the hydrophilic detection area; With the fluorescence enhancement characteristics of photonic crystal, the fluorescence spectrum signal emitted by the test substance rhodamine 6G in the hydrophilic detection area is detected by fluorescence spectroscopy.

[0059] 4) Use fluorescence intensity change to judge chip cleaning effect:

[0060] The fluorescence intensity of the sample received at different times was tested by photonic crystal microchip, and the fluorescence intensity-washing time change curve was drawn. Combined with the fluorescence intensity-solution concentration change curve, the concentration or concentration range of the liquid sample received at different washing times was analyzed; or the sample received at 0s after the start of cleaning was taken as the initial concentration, and whether there was residual or the corresponding residual amount after different washing times was calculated.

[0061] From Figure 2It can be seen that acetonitrile washing liquid samples are collected at 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, and 40s after the start of washing, respectively, and each time 2s of liquid is collected. The fluorescence detection results of the sample to be tested on the photonic crystal microchip and the glass substrate are detected. With the photonic crystal microchip as the substrate, a strong fluorescence signal can be detected at 0s. As the acetonitrile flushing time is prolonged, the fluorescence signal intensity is greatly reduced. No obvious fluorescence signal can be detected after 15s from the start of washing, and it is considered that the washing is completed.

[0062] The same detection means is used to detect the fluorescence signal on the ordinary glass substrate, and almost no obvious fluorescence signal can be detected, which cannot support the verification of the washing effect.

[0063] Example 2

[0064] 1) Preparation of photonic crystal microchip:

[0065] A PDMS mixed solution (monomer: crosslinking agent = 10:1 w / w) is spin-coated on the surface of a glass slide (2000r, 60s), and the glass slide is heated at 80°C for 15min to form a hydrophobic PDMS surface on the glass slide, i.e. a hydrophobic substrate. Poly(styrene-methacrylic acid-acrylamide) core-shell structure microspheres with a diameter of about 220nm are configured into a 15wt% aqueous solution, which is printed on the PDMS surface by a dispensing machine to deposit the nanoscale microspheres on the surface. The hydrophobic PDMS surface is heated at 80°C, and after the droplets are dried, the hydrophilic photonic crystal dots are self-assembled to form a hydrophilic detection area on the hydrophobic PDMS substrate.

[0066] 2) The chip is placed in a substrate provided with a reaction groove. The substrate is provided with a circular hole on both sides, and the liquid pipeline is connected to the reaction groove. One side of the pipeline inputs the reaction reagent and acetonitrile cleaning liquid into the reaction groove, and the other side of the pipeline collects the sample solution and waste liquid output from the reaction groove; the upper cover plate is covered to seal the reaction groove, so that the liquid enters the reaction groove through the pipeline and flows uniformly on the surface of the chip.

[0067] 3) Chip surface cap reagent cleaning verification:

[0068] First, a series of gradient concentrations of acetonitrile solutions of fluorescent dyes are prepared, and the solution concentrations are selected from 1-10 -10 mM, such as 0.1mM, 0.01mM, 10 -4 mM, 10 -6 mM… The fluorescence intensity of the acetonitrile solution of the fluorescent dye with different concentrations is measured by using the photonic crystal microchip, and the fluorescence intensity-solution concentration change curve is drawn.

[0069] Add 0.05 mM Nile red into the capping reagent (reagent required in the capping step of DNA synthesis process, produced by Hebei Dinaxingke Biological Technology Co., Ltd., product number R1002-4 / 1003-4). Fix the high-throughput DNA synthesis chip in the microfluidic channel, and add the capping reagent containing 0.05 mM Nile red at a flow rate of 1-5 mL / min for 30 s on the surface of the chip, and then wash with pure acetonitrile at a flow rate of 5-20 mL / min.

[0070] Collect acetonitrile washing liquid samples at 0 s, 5 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, and 40 s after the start of washing, respectively, and collect 6 s of liquid each time. Concentrate and evaporate the collected liquid samples, then dissolve 30 μL of acetonitrile, take 0.5 μL of the concentrated liquid sample, and drop it on the hydrophilic detection area of the photonic crystal microchip prepared in Example 2. Heat the photonic crystal microchip at 60°C to dry the droplet, and detect the fluorescence spectrum signal emitted by Nile red in the hydrophilic detection area by fluorescence spectroscopy.

[0071] 4) Determine the washing effect of the chip by using the change in fluorescence intensity:

[0072] Use the photonic crystal microchip to test the fluorescence intensity of samples received at different times, draw a fluorescence intensity-washing time change curve, and combine the fluorescence intensity-solution concentration change curve to analyze the concentration or concentration range of liquid samples received at different washing times; or take the sample received at 0 s after washing as the initial concentration, and calculate whether there is residual or the corresponding residual amount after different washing times.

[0073] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can make equivalent substitutions or changes to the technical solution and concept of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for verifying the cleaning effect of a printed synthetic DNA chip enhanced by photonic crystal fluorescence, characterized in that: The method comprises the following steps: S1, prepare the photonic crystal microchip to form a hydrophobic base and a hydrophilic detection area; S2, building a chip test platform, collecting the reaction cleaning liquid sample to be tested at a specific time, and dripping the sample solution to be tested onto the surface of the photonic crystal microchip; S3, heating the photonic crystal microchip, and after the sample solution dries, the analyte is concentrated in the hydrophilic detection area; S4, utilizing the fluorescence enhancement characteristics of the photonic crystal to detect the fluorescence spectrum signal emitted by the object to be detected by fluorescence spectroscopy, so as to detect the object to be detected; S5, using the change in fluorescence intensity to determine the cleaning effect of the chip; The step S2 comprises: S21, placing the chip in a substrate with a reaction groove, and connecting the infusion pipeline to the reaction groove through circular through holes on both sides of the substrate. One side of the pipeline inputs reaction reagents and acetonitrile cleaning solution into the reaction groove, and the other side of the pipeline collects the sample solution and waste liquid output from the reaction groove. Cover the reaction groove with a cover plate to seal it; S22, after flowing each reaction reagent with added fluorescent dye on the surface of the high-throughput DNA synthesis chip, flowing acetonitrile solution for washing; S23, starting to collect acetonitrile cleaning liquid samples at a certain time after the start of cleaning; S24, concentrating the collected liquid sample, removing the concentrated sample solution to be tested, and dripping it onto the hydrophilic detection area on the surface of the photonic crystal microchip with different wettability; The step of using the change in fluorescence intensity to judge the chip cleaning effect in step S5 includes: When a weak fluorescence signal is detected by the photonic crystal microchip, it is determined that the cleaning is not complete, and the concentration of the liquid sample at that time point or the residual amount of the liquid sample at 0 seconds after the start of cleaning is calculated; When no obvious fluorescent signal can be detected through the photonic crystal microchip, it is determined that the cleaning is complete and there is no residue.

2. The cleaning effect verification method according to claim 1, characterized in that: The step S5 further comprises: S51, prepare a series of gradient concentrations of fluorescent dye in acetonitrile or aqueous solution, the solution concentration is 1-10 -10 mM, using a photonic crystal microchip to measure the fluorescence intensity of fluorescent dye solutions with different concentrations, and draw a fluorescence intensity-solution concentration change curve; S52, using a photonic crystal microchip to test the fluorescence intensity of samples received at different times, plotting a fluorescence intensity-cleaning time change curve, combining the fluorescence intensity-solution concentration change curve to analyze the concentration or concentration range of the liquid samples received at different cleaning times; or taking the sample received at 0s after the start of cleaning as the initial concentration, calculate whether there is any residue or the corresponding residual amount after different cleaning times.

3. The cleaning effect verification method according to claim 1, characterized in that: The steps of preparing the photonic crystal microchip in step S1 include: A mixed solution for forming a hydrophobic base is prepared, the mixed solution is spin-coated on the surface of a glass slide, and the glass slide is heated for a certain period of time to form a hydrophobic surface on the glass slide, also known as a hydrophobic base; preparing an aqueous solution for forming a hydrophilic detection area, and printing the aqueous solution on a hydrophobic surface so that the medium in the aqueous solution is deposited on the hydrophobic surface; The hydrophobic surface is heated, and after the droplets dry, they self-assemble to form hydrophilic photonic crystal dots, forming a hydrophilic detection area on the hydrophobic substrate; The mixed solution includes a monomer and a cross-linking agent; the mass ratio of the monomer to the cross-linking agent is (5-20):1; The prepared mixed solution was spin-coated on the surface of the glass slide at a spin-coating speed of 1000-5000 r and a spin-coating time of 30-90 s.

4. The cleaning effect verification method according to any one of claims 1 to 3, characterized in that: The photonic crystals in the photonic crystal microchip include photonic crystals assembled from one or more of silica microspheres, titanium dioxide microspheres, zinc oxide microspheres, poly(styrene-methacrylic acid-acrylamide) core-shell microspheres, and poly(styrene-methyl methacrylate-acrylic acid) core-shell microspheres. The diameter of the microspheres is 150-300 nm; the microspheres are prepared into a 5-20 wt% aqueous solution.

5. The cleaning effect verification method according to claim 1, characterized in that: The hydrophobic substrate of the photonic crystal microchip is selected from one or more of polydimethylsiloxane, polystyrene and polytetrafluoroethylene.

6. The cleaning effect verification method according to claim 1, characterized in that: The reaction reagents include deprotection reagents, monomer reagents, capping reagents, and oxidation reagents used in the DNA synthesis process; The added fluorescent dye is selected from one or more of rhodamine B, rhodamine 6G, Nile red and fluorescein.

7. The cleaning effect verification method according to claim 1, characterized in that: In step S23, the specific collection time of the liquid sample is selected from 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 65s, 70s, 75s, 80s, 85s, 90s, 95s, 100s, 105s, 110s, 115s, 120s, 125s, 130s, 135s, 140s, 145s, 150s, 155s, 160s, 165s, 170s, 175s, and 180s after the start of cleaning; The duration of each liquid sample collection is selected from 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, 11s, 12s, 13, 14s, and 15s.

8. The cleaning effect verification method according to claim 1, characterized in that: In the step S3, the photonic crystal microchip is heated at 30-80°C.

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