Method for verifying cleaning effect of printed and synthesized DNA chip enhanced by photonic crystal fluorescence

Abstract

The invention discloses a photonic crystal fluorescence enhanced printing synthesis DNA chip cleaning effect verification method, which comprises: S1, preparing a photonic crystal microchip, and forming a hydrophobic substrate and a hydrophilic detection area; s2, building a chip test platform, collecting a reaction cleaning liquid to-be-tested sample within a specific time, and dropwise adding the to-be-tested sample to the surface of the photonic crystal microchip; s3, heating the photonic crystal microchip, and enriching the to-be-detected substance in the hydrophilic detection area after the sample solution is dried; s4, detecting the fluorescence spectrum signal emitted by the to-be-detected object through fluorescence spectrometry so as to realize detection of the to-be-detected object; and S5, judging the cleaning effect of the chip according to the change of the fluorescence intensity. According to the verification method, the chip cleaning effect can be judged according to the fluorescence intensity change, the method is also suitable for trace detection under the extremely low concentration condition, the sensitivity higher than that of a common substrate such as glass is achieved, the detection limit is reduced, and more accurate analysis under the low concentration condition is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of chip technology, and in particular relates to a method for verifying the cleaning effect of a printed synthetic DNA chip enhanced by photonic crystal fluorescence. Background Art

[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 a significant impact 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 chips, which directly affects the accuracy, coupling efficiency and cost of the synthesis. Since the entire DNA synthesis process involves multiple cycles of multiple reaction steps, and different reaction steps will affect each other, the cleaning process of the chip surface after each step is very important. If the cleaning is insufficient, the residual chemicals caused by inadequate cleaning may interfere with subsequent reactions, resulting in reduced reaction efficiency or even termination of the reaction, affecting the effectiveness and stability of the synthesis, limiting the length and load of the synthesis, and may also cause errors to accumulate and affect the quality of the synthetic product. If the cleaning is excessive, it may not only increase the time interval between the synthesis steps, thereby reducing the reaction efficiency, but also increase the time and cost of the synthesis process. Especially in a large number of synthesis processes, the use of large amounts of organic reagents and the generation of chemical waste will greatly increase the cost and environmental burden.

[0004] Therefore, determining the optimal cleaning method and time is not only beneficial for improving synthesis efficiency and quality, but also for achieving a low-cost and environmentally friendly synthesis process. However, due to the low content of chemical substances in the cleaning solution, accurate quantitative analysis is difficult, and how to effectively evaluate the cleaning effect is a challenge.

[0005] Currently commonly used detection methods, such as colorimetry, chemical titration, spectrophotometry, chromatography, and mass spectrometry, suffer from limitations in detection limit, sensitivity, and susceptibility to environmental influences, making it difficult to guarantee accurate detection of low-concentration solutions. Fluorescence detection technology, while offering relatively high sensitivity and interference resistance compared to other methods, is more suitable for detection under low-concentration conditions. However, it remains challenging to detect trace amounts at extremely low concentrations. Summary of the Invention

[0006] In response to the current problems of difficulty in judging the surface cleaning effect of high-throughput DNA synthesis chips, which leads to low synthesis yields and difficulty in maintaining synthesis stability and parallelism between different batches during chip-based DNA synthesis, the present invention provides a method for verifying the cleaning effect of printed synthetic DNA chips using photonic crystal fluorescence enhancement. By leveraging the fluorescence enhancement characteristics of photonic crystals, the fluorescence spectral signal emitted by the object to be detected is detected by fluorescence spectroscopy, and the chip cleaning effect is judged by the change in fluorescence intensity, providing support for determining the optimal cleaning method for the chip-based DNA synthesis process.

[0007] In order to achieve the above objectives, this application provides the following technical solutions: A method for verifying the cleaning effect of a printed synthetic DNA chip enhanced by photonic crystal fluorescence comprises the following steps: S1, prepare a 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 enriched 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.

[0008] In one embodiment of the present invention, in step S5, the step of judging the chip cleaning effect by using the change in fluorescence intensity 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.

[0009] In one embodiment of the present invention, in step S5, the step of judging the chip cleaning effect by using the change in fluorescence intensity includes: S51, prepare a series of gradient concentrations of fluorescent dye in acetonitrile or aqueous solution, the solution concentration is 1-10 -10 mM, such as 0.1mM, 0.01mM, 10 -4 mM, 10 -6 mM, 10 -8 mM, 10 -10 mM; the fluorescence intensity of fluorescent dye solutions with different concentrations was measured using a photonic crystal microchip, and the fluorescence intensity-solution concentration change curve was drawn; 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.

[0010] In one embodiment of the present invention, the step of preparing the photonic crystal microchip in step S1 includes: S11, preparing a mixed solution for forming a hydrophobic base (such as a PDMS mixed solution), spin-coating the mixed solution on the surface of a glass slide, and heating the glass slide for a certain period of time to form a hydrophobic surface (such as a PDMS surface) on the glass slide, also called a hydrophobic base; S12, preparing an aqueous solution for forming a hydrophilic detection area (for example, preparing nanospheres into an aqueous solution), printing the aqueous solution on a hydrophobic surface (such as a PDMS surface), so that the medium (such as nanospheres) in the aqueous solution is deposited on the hydrophobic surface (such as a PDMS surface); S13, heating the hydrophobic surface (such as PDMS surface), and after the droplets are dried, self-assembly to form hydrophilic photonic crystal dots, forming a hydrophilic detection area on the hydrophobic substrate; In one embodiment of the present invention, the mixed solution includes a monomer and a cross-linking agent; further, the mass ratio of the monomer to the cross-linking agent is (5-20):1.

[0011] In one embodiment of the present invention, the prepared mixed solution is spin-coated on the surface of a glass slide at a spin-coating speed of 1000-5000 r and a spin-coating time of 30-90 s; Preferably, the slide is heated at 75°C to 85°C for 12 to 20 minutes; For example, the slides were heated at 80°C for 15 minutes.

[0012] In one embodiment of the present invention, the photonic crystals in the photonic crystal microchip (such as the medium in the aqueous solution) are selected from photonic crystals assembled from silica microspheres, photonic crystals assembled from titanium dioxide microspheres, photonic crystals assembled from zinc oxide microspheres, photonic crystals assembled from microspheres with a core-shell structure of poly(styrene-methacrylic acid-acrylamide), photonic crystals assembled from microspheres with a core-shell structure of poly(styrene-methyl methacrylate-acrylic acid), etc.

[0013] In one embodiment of the present invention, the diameter of the microspheres is 150-300 nm, preferably 220-300 nm; In one embodiment of the present invention, the microspheres are formulated into a 5-20 wt % aqueous solution.

[0014] For example, 5wt%, 10wt%, 12wt%, 15wt%, 20wt%, 25wt%, 30wt% or any point between any two of the above numerical ranges.

[0015] In one embodiment of the present invention, the hydrophobic substrate of the photonic crystal microchip is selected from one or more of polydimethylsiloxane (PDMS), polystyrene, and polytetrafluoroethylene.

[0016] In one embodiment of the present invention, step S2 includes: 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; the reaction groove is sealed by covering the upper cover plate, so that the liquid enters the reaction groove through the pipeline and flows in a uniform liquid layer over the entire chip surface; S22, after flowing each reaction reagent with added fluorescent dye on the surface of the high-throughput DNA synthesis chip, acetonitrile is flowed for washing; S23, collecting acetonitrile cleaning liquid samples at a certain time after the start of cleaning, preferably collecting 2-6 seconds of liquid each time; S24, concentrating the collected liquid sample, taking out 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.

[0017] In one embodiment of the present invention, the reaction reagents include deprotection reagents, monomer reagents, capping reagents, and oxidizing reagents used in the DNA synthesis process; Preferably, the added fluorescent dye is selected from one or more of rhodamine B, rhodamine 6G, Nile red and fluorescein.

[0018] For example, a deprotection reagent of 0.1 mM Rhodamine 6G is added, and a capping reagent of 0.05 mM Nile Red is added.

[0019] In one embodiment of the present invention, 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, 180s, etc. 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.

[0020] In one embodiment of the present invention, in step S3, the photonic crystal microchip is heated at 30-80°C, preferably 60-80°C.

[0021] Beneficial effects of the present invention: 1) This invention provides a method for verifying the cleaning effectiveness of a printed synthetic DNA chip enhanced by photonic crystal fluorescence. First, a photonic crystal microchip comprising a hydrophobic base and a hydrophilic detection region is prepared. A chip testing platform is then constructed to collect reaction cleaning liquid samples at a specific time. The sample solution to be tested is then dripped onto the surface of the photonic crystal microchip, which has a differential wettability. After the sample solution dries, the target substance is concentrated in the hydrophilic detection region. Leveraging the fluorescence enhancement properties of photonic crystals, this method detects the fluorescence spectral signal emitted by the target substance through fluorescence spectroscopy, and uses changes in fluorescence intensity to determine the chip cleaning effectiveness.

[0022] 2) The present invention analyzes sample concentration by combining the fluorescence intensity-solution concentration curve to determine whether there is residual sample or the corresponding residual amount in this case, thereby realizing the detection of the object to be detected. This solves the problem of difficulty in judging the surface cleaning effect of current high-throughput DNA synthesis chips, which leads to low synthesis yield in chip-based DNA synthesis and difficulty in maintaining stability and parallelism of different batches.

[0023] 3) The chip cleaning effect verification method of the present invention is also applicable to trace detection under extremely low concentration conditions. The difference in wettability of the surface of the photonic crystal microchip enables the enrichment of low-concentration samples. At the same time, with the help of the fluorescence enhancement properties of the photonic crystal, it achieves higher sensitivity than ordinary substrates such as glass, which is conducive to reducing the detection limit and realizing more accurate analysis at low concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the process for verifying the cleaning effect of the photonic crystal microchip test platform; Figure 2 Schematic diagram of the fluorescence detection results of the samples on the photonic crystal microchip and glass slide substrate, respectively, with samples taken at 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, and 40s after the start of cleaning, collecting 2s of liquid each time. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0026] Example 1 1) Preparation of photonic crystal microchip: A PDMS mixture (monomer:crosslinker = 10:1 w / w) was prepared and spin-coated onto a glass slide (2000 rpm, 60 s). The slide was then heated at 80°C for 15 minutes to form a hydrophobic PDMS surface, effectively creating a hydrophobic substrate. Silica microspheres with a diameter of approximately 180 nm were prepared in a 12 wt% aqueous solution and printed onto the PDMS surface using a dispenser. The nanospheres in the aqueous solution were deposited onto the hydrophobic PDMS surface. The hydrophobic PDMS surface was then heated to 60°C. After the droplets dried, they self-assembled into hydrophilic photonic crystal dots, forming a hydrophilic detection region on the hydrophobic PDMS substrate.

[0027] 2) Build a chip testing platform: Place the chip in a substrate with a reaction groove. Drill circular through holes on both sides of the substrate to connect the infusion pipeline to the reaction groove. One side of the pipeline inputs reaction reagents and acetonitrile cleaning solution into the reaction groove, while 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 the reaction groove, allowing the liquid to enter the reaction groove through the pipeline and flow across the entire chip surface in a uniform liquid layer.

[0028] 3) Validation of chip surface deprotection reagent cleaning: First, a series of acetonitrile solutions of fluorescent dye with gradient concentrations were prepared, with the concentrations of 0.1 mM, 0.01 mM, 10 -4 mM, 10 -6 mM, 10 -8 mM, 10 -10 mM. The fluorescence intensity of acetonitrile solutions of different concentrations of fluorescent dye was measured using a photonic crystal microchip, and the fluorescence intensity-solution concentration change curve was drawn.

[0029] Add 0.1 mM rhodamine 6G to the deprotection reagent (the deprotection reagent is the immobilization reagent used in the deprotection step of DNA synthesis, produced by Hebei Dina Xingke Biotechnology Co., Ltd., catalog number R1005-4). A high-throughput DNA synthesis chip was fixed in a microfluidic channel. 0.1 mM rhodamine 6G deprotection reagent was flowed over the chip surface at 1-5 mL / min for 30 seconds, followed by washing with pure acetonitrile at 5-20 mL / min.

[0030] like Figure 2 As shown, acetonitrile cleaning liquid samples were collected at 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, and 40s after the start of cleaning, and 2s of liquid was collected each time. The collected liquid samples were heated, concentrated, and evaporated to dryness, and then dissolved in 20μL of acetonitrile. 0.5μL of the concentrated sample solution to be tested was pipetted and added dropwise to the hydrophilic detection area on the surface of the photonic crystal microchip with a surface wettability difference prepared in Example 1. The photonic crystal microchip was heated at 60°C to dry the sample solution, and the object to be detected was enriched in the hydrophilic detection area. With the help of the fluorescence enhancement characteristics of the photonic crystal, the fluorescence spectrum signal emitted by the object to be detected, Rhodamine 6G, in the hydrophilic detection area was detected by fluorescence spectroscopy.

[0031] 4) Use the change in fluorescence intensity to determine the cleaning effect of the chip: Use a photonic crystal microchip to test the fluorescence intensity of samples received at different times, draw a fluorescence intensity-cleaning 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 cleaning times; or use the sample received at 0s after the start of cleaning as the initial concentration to calculate whether there is any residue or the corresponding residual amount after different cleaning times.

[0032] from Figure 2 Acetonitrile wash liquid samples were collected at 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, and 40s after the start of the wash, with 2s of liquid collected each time. Fluorescence detection results of the samples on the photonic crystal microchip and glass slide substrates show that, using the photonic crystal microchip as the substrate, a strong fluorescence signal is detected in the sample collected at 0s. As the acetonitrile wash time increases, the fluorescence signal intensity decreases significantly. No significant fluorescence signal is detected from 15s after the start of the wash, indicating that the wash is complete.

[0033] When fluorescence signal detection is performed on an ordinary glass slide substrate using the same detection method, almost no obvious fluorescence signal can be detected, which cannot support the verification of the cleaning effect.

[0034] Example 2 1) Preparation of photonic crystal microchip: A PDMS mixture (monomer:crosslinker = 10:1 w / w) was spin-coated onto a glass slide (2000 rpm for 60 seconds). The slide was then heated at 80°C for 15 minutes to form a hydrophobic PDMS surface, also known as a hydrophobic substrate. A 15wt% aqueous solution of core-shell poly(styrene-methacrylic acid-acrylamide) microspheres with a diameter of approximately 220 nm were then printed onto the PDMS surface using a dispenser, depositing the nanospheres. The hydrophobic PDMS surface was then heated to 80°C. After drying, the droplets self-assembled into hydrophilic photonic crystal dots, forming the hydrophilic detection region on the hydrophobic PDMS substrate.

[0035] 2) The chip is placed in a substrate with a reaction groove. Circular through-holes are drilled on both sides of the substrate to connect the infusion pipeline to the reaction groove. One side of the pipeline inputs reaction reagents and acetonitrile cleaning solution into the reaction groove, while the other side of the pipeline collects the sample solution and waste liquid output from the reaction groove. A cover plate is placed on top to seal the reaction groove, allowing the liquid to enter the reaction groove through the pipeline and flow across the entire chip surface in a uniform liquid layer.

[0036] 3) Chip surface capping reagent cleaning verification: First, a series of acetonitrile solutions of fluorescent dye with gradient concentrations were prepared, with the concentrations of the solutions ranging from 1 to 10. -10 mM, such as 0.1mM, 0.01mM, 10 -4 mM, 10 -6 mM…. The fluorescence intensity of acetonitrile solutions of fluorescent dyes with different concentrations was measured using a photonic crystal microchip, and the fluorescence intensity-solution concentration curve was drawn.

[0037] Add 0.05 mM Nile Red to the capping reagent (required for the capping step in DNA synthesis, manufactured by Hebei Dinaxingke Biotechnology Co., Ltd., Cat. No. R1002-4 / 1003-4). A high-throughput DNA synthesis chip was mounted in a microfluidic channel. The capping reagent (0.05 mM Nile Red) was added to the chip surface at a flow rate of 1-5 mL / min for 30 seconds, followed by washing with pure acetonitrile at a flow rate of 5-20 mL / min.

[0038] Acetonitrile wash liquid samples were collected starting at 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, and 40s after the start of the wash, with 6s of liquid collected each time. The collected liquid samples were heated, concentrated, and evaporated to dryness. They were then dissolved in 30 μL of acetonitrile. 0.5 μL of the concentrated liquid sample was pipetted and dropwise added to the hydrophilic detection area of ​​the photonic crystal microchip prepared in Example 2. The photonic crystal microchip was heated at 60°C to dry the droplet. The fluorescence spectral signal emitted by Nile Red in the hydrophilic detection area was detected by fluorescence spectroscopy.

[0039] 4) Use the change in fluorescence intensity to determine the cleaning effect of the chip: Use a photonic crystal microchip to test the fluorescence intensity of samples received at different times, draw a fluorescence intensity-cleaning 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 cleaning times; or use the sample received at 0s after the start of cleaning as the initial concentration to calculate whether there is any residue or the corresponding residual amount after different cleaning times.

[0040] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solution and concept of the present application within the technical scope disclosed in the present application, and they should be covered by the scope of protection 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.

2. The cleaning effect verification method according to claim 1, characterized in that: 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.

3. The cleaning effect verification method according to claim 2, 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.

4. 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.

5. The cleaning effect verification method according to any one of claims 1 to 4, 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.

6. 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.

7. The cleaning effect verification method according to claim 1, characterized in that: 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, taking out 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.

8. The cleaning effect verification method according to claim 7, 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.

9. The cleaning effect verification method according to claim 7, 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.

10. 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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