Microarray chip, preparation method and application thereof

Abstract

The application belongs to the technical field of biology, and discloses a microarray chip, a preparation method and application thereof. The microarray chip has hydrophilic reaction holes and hydrophobic non-reaction areas, the depth of the reaction holes is 20-50 mu m, the etching depth is much higher than the depth of 3-10 mu m of traditional laser etching, the hydrophobic angle of the non-reaction areas is more than 110 degrees, and the chip surface is flat and smooth. The application adopts double photolithography and surface hydrophobization treatment, and can quickly prepare the microarray chip with multiple hydrophilic reaction holes and hydrophobic non-reaction areas, which meets the needs of large-scale industrial production; different patterned masks can be designed to obtain reaction holes with different shapes for different detection scenes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a microarray chip, its fabrication method, and its application. Background Technology

[0002] Hydrophilic-hydrophobic interleaved microarray chips are widely used in high-throughput biomolecule detection and drug screening, offering advantages such as small size and high experimental efficiency. While the hydrophilic and hydrophobic properties of the chip regions effectively prevent sample cross-contamination and improve the reliability of test results, this significantly increases the difficulty of fabricating the microarray chip. Currently, most hydrophilic-hydrophobic interleaved microarray chips are obtained by hydrophobizing followed by laser etching, which suffers from high technical costs and an uneven surface after etching. While the method of first using photolithography and then hydrophilizing each hole individually can solve the problem of uneven surface, it requires a significant amount of time (the more reaction holes there are, the more time is required), and the inconsistent hydrophilicity of each reaction hole due to the inconsistent time of the hydrophilicization process affects subsequent detection processes. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a microarray chip, its fabrication method and application. The present invention employs dual photolithography and surface hydrophobicization treatment to rapidly fabricate a microarray chip with reactive pores having consistent hydrophilicity and non-reactive regions having hydrophobicity.

[0004] A first aspect of the present invention is to provide a microarray chip having hydrophilic reactive pores and hydrophobic non-reactive regions, wherein the depth of the reactive pores is 20-50 μm and the hydrophobic angle of the non-reactive regions is 110° or more.

[0005] Preferably, the microarray chip has multiple reaction pores with consistent hydrophilicity.

[0006] Preferably, the reaction orifice is circular, and the diameter of the reaction orifice is 10 μm-1 mm. It is understood that the reaction orifice of the present invention can also be other shapes, such as elliptical reaction orifices, conical reaction orifices, polygonal reaction orifices, etc.

[0007] Preferably, the hydrophobic angle of the non-reactive region is 110-130°.

[0008] A second aspect of the present invention is to provide a method for fabricating the microarray chip, comprising the following steps:

[0009] A first photoresist layer is formed in the non-reactive region of the silicon dioxide chip;

[0010] The silicon dioxide in the reaction area of ​​the silicon dioxide chip is removed by a wet etching process to form reaction holes; the surface of the reaction holes is silicon dioxide, which is hydrophilic; while the non-reaction area is protected by the first photoresist layer and is not affected by the etching process.

[0011] The first photoresist layer is removed to obtain the target chip;

[0012] A second photoresist layer is formed on the surface of the reactive hole of the target chip;

[0013] The non-reactive regions of the target chip are hydrophobically treated; the reactive holes of the target chip are unaffected by the hydrophobic process due to the protection of the second photoresist layer.

[0014] The second photoresist layer is removed to obtain the microarray chip.

[0015] As a further improvement to the above scheme, a first photoresist layer is formed in the non-reactive region of the silicon dioxide chip. The specific process includes:

[0016] A positive photoresist layer is formed on the surface of the silicon dioxide chip;

[0017] A mask with a light-transmitting area corresponding to the reaction area is used to expose and develop the positive photoresist layer, forming a first photoresist layer in the non-reaction area of ​​the silicon dioxide chip.

[0018] Specifically, forming a first photoresist layer in a non-reactive region of a silicon dioxide chip includes the following steps:

[0019] Spin coating: Place the silicon dioxide chip on the spin coating stage, turn on the vacuum adsorption switch to ensure the chip is firmly on the spin coating stage, and uniformly coat the center of the surface of the silicon dioxide chip with positive photoresist. Spin coat at low speed for 5-20 minutes to spread the positive photoresist across the entire surface of the chip. Set the speed to 2000-5000 rpm and spin coat at high speed for 5-20 minutes to make the photoresist thickness uniform, with a thickness of 2-10 μm, forming a positive photoresist layer.

[0020] Pre-baking: Place the silicon dioxide chip with a positive photoresist layer on a hot plate at 90-100℃ and bake for 90-100 seconds;

[0021] Exposure: Place the silicon dioxide chip and the mask with the light-transmitting area corresponding to the reaction area into the photolithography machine, and position and align them according to the markings on the silicon dioxide chip and the mask. Set the ultraviolet wavelength to 350-450nm and expose for 5-30 minutes.

[0022] Development: Immerse the exposed silicon dioxide chip in the developer for 10-20 minutes to dissolve the exposed portion of the photoresist. After development, remove the silicon dioxide chip to form the first photoresist layer in the non-reactive area of ​​the silicon dioxide chip.

[0023] As a further improvement to the above solution, a wet etching process is used to remove the silicon dioxide in the reaction region of the silicon dioxide chip to form reaction holes. The specific process includes:

[0024] A silicon dioxide chip with the first photoresist layer formed on its non-reactive areas is placed in a hydrofluoric acid solution. The hydrofluoric acid solution removes the silicon dioxide from the reactive areas of the silicon dioxide chip, forming reactive holes. The non-reactive areas of the silicon dioxide chip are unaffected by the hydrofluoric acid solution due to the protection of the first photoresist layer, while the reactive areas are affected by the hydrofluoric acid solution, thus forming reactive holes.

[0025] Specifically, the silicon dioxide in the reaction region of the silicon dioxide chip is removed by a wet etching process to form reaction holes, including the following steps:

[0026] A silicon dioxide chip with the first photoresist layer formed in the non-reactive area is placed in a hydrofluoric acid solution with a mass concentration of 10-15% and etched for 0.5-3 hours. The silicon dioxide chip is then removed and cleaned with isopropanol 1-3 times. After etching, the silicon dioxide in the reactive area of ​​the silicon dioxide chip is removed according to the shape of the mask to form multiple regularly arranged reaction holes with a depth of 20-50 μm.

[0027] As a further improvement to the above solution, a second photoresist layer is formed on the surface of the reactive aperture of the target chip. The specific process includes:

[0028] A positive photoresist layer is formed on the surface of the target chip;

[0029] A mask with a non-transparent area corresponding to the reaction hole is used to expose and develop the positive photoresist layer, forming a second photoresist layer on the surface of the reaction hole of the target chip.

[0030] As a further improvement to the above solution, a second photoresist layer is formed on the surface of the reactive aperture of the target chip. The specific process includes:

[0031] A negative photoresist layer is formed on the surface of the target chip;

[0032] A mask with a light-transmitting area corresponding to the reaction hole is used to expose and develop the negative photoresist layer, forming a second photoresist layer on the surface of the reaction hole of the target chip.

[0033] As a further improvement to the above solution, the non-reactive regions of the target chip are hydrophobically treated, specifically including:

[0034] The target chip with the second photoresist layer forming the reactive holes is placed in a hydrophobic solvent, and the non-reactive areas of the target chip are hydrophobized by the hydrophobic solvent. The reactive holes are protected by the second photoresist layer and remain unaffected by the hydrophobic solvent, retaining their original hydrophilicity.

[0035] Specifically, the non-reactive regions of the target chip are hydrophobically treated, including the following steps:

[0036] A hydrophobicating agent is dissolved in a polar organic solvent to obtain a hydrophobic solvent. A catalyst is then added and the reaction is initiated for 5-6 hours. The target chip with the second photoresist layer formed on its reaction holes is then placed in the hydrophobic solvent and reacted at room temperature (20-27°C) for 7-8 hours. The chip is then removed and cured in an oven at 60-80°C for 1-3 hours. Finally, the chip is rinsed with isopropanol. The hydrophobicating agent includes perfluorodecyltrimethoxysilane or perfluorooctyltriethoxysilane; the polar organic solvent includes at least one of n-hexane, ethanol, isopropanol, and acetone; and the catalyst is acetic acid.

[0037] As a further improvement to the above scheme, before forming the first photoresist layer in the non-reactive region of the silicon dioxide chip, a coating process is also included on the silicon dioxide chip. The specific process includes: depositing a chromium film on the surface of the silicon dioxide chip using vacuum magnetron sputtering technology.

[0038] As a further improvement to the above solution, before the coating process, the silicon dioxide chip is activated and cleaned. The specific process includes: sequentially passing the silicon dioxide chip through acid cleaning, alkaline cleaning and QDR (Quick Drain Rinse) cleaning (a cleaning method that drains water from the tank within a few seconds and uses the rapid flushing force of water to clean impurities).

[0039] Specifically, the activation, cleaning, and coating process includes the following steps:

[0040] Activation and cleaning: Clean the silicon dioxide chip with fuming nitric acid for 5-20 minutes, then clean it with KOH (10%-30% KOH) heated to 60-80℃ for 10-20 minutes. Then place the silicon dioxide chip in an ultrasonic cleaner (450-550W) for QDR cleaning for 4-6 minutes and dry it.

[0041] Coating: A chromium film is deposited on the surface of an activated and cleaned silicon dioxide chip using vacuum magnetron sputtering technology. The target-substrate distance is set to 5-15 cm, the deposition time is 55-65 min, the sputtering current is 0.4-0.8 A, and the sputtering pressure is 0.4-0.8 V.

[0042] A third aspect of this invention is to provide the application of the microarray chip described herein in biomolecular detection and drug screening. Specifically, this includes, but is not limited to, its application in detecting nucleoside triphosphate molecules, proteins, or bacteria.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] The reactive holes on the microarray chip of this invention are uniform in size and have a depth of 20-50μm, which is much higher than the traditional laser etching depth (3-10μm). Traditional laser etching is prone to damaging the chip surface, and another disadvantage of traditional laser etching is that the surface is rough and not smooth after etching. The hydrophobic angle of the hydrophobic non-reactive region on the microarray chip of this invention is above 110°, and the chip surface is flat and smooth.

[0045] The fabrication method of the microarray chip of this invention employs dual photolithography and surface hydrophobicization treatment, which can rapidly produce a microarray chip with multiple hydrophilic reaction holes and hydrophobic non-reaction regions. The reaction holes with an etching depth of 20-50 μm can be etched in 0.5-3 hours, while traditional laser etching to a depth of 3-10 μm requires 5-10 hours. This significantly reduces the time consumption compared to traditional laser etching methods, making it suitable for large-scale industrial production. Furthermore, this invention can design different patterned masks to obtain reaction holes of different shapes for different detection scenarios. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the process of performing the first photolithography in Embodiment 1 of the present invention;

[0047] Figure 2 This is a schematic diagram of the process of performing the second photolithography in Embodiment 1 of the present invention;

[0048] Figure 3 This is a schematic diagram under a microscope after the second photolithography development in Embodiment 1 of the present invention;

[0049] Figure 4 This is a schematic diagram illustrating the effectiveness and stability of the neuraminidase based on a high-throughput microarray chip according to the present invention.

[0050] In the diagram, 100 is the silicon dioxide chip, 200 is the positive photoresist layer, 300 is the mask, 400 is the target chip, 500 is the negative photoresist layer, and 600 is the high-throughput microarray chip. Detailed Implementation

[0051] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0052] Unless otherwise specified, the raw materials or apparatus used in the following embodiments can be obtained from conventional commercial sources or by existing known methods.

[0053] In the following embodiments, SUN-120 positive photoresist and SUN-90i negative photoresist were purchased from Suzhou Yancai Micro-Nano Technology Co., Ltd.; the chromium removal solution was prepared by adding 50g of cerium ammonium nitrate, 12mL of HClO4, and water to a final volume of 300mL; and fuming nitric acid refers to nitric acid with a mass concentration of 90-97.5%.

[0054] Example 1

[0055] A method for fabricating a high-throughput microarray chip, employing dual photolithography, specifically includes the following steps:

[0056] 1. First photolithography step (hole etching)

[0057] Reference Figure 1 The chip undergoes its first photolithography process using positive photoresist, combined with a custom mask and HF etching technology, to process and etch 3000 regularly arranged, uniformly sized circular reaction holes. The specific steps are as follows:

[0058] 1.1 Chip Surface Activation and Cleaning: Prepare a clean 14-inch circular silicon dioxide chip 100. Clean the silicon dioxide chip 100 with fuming nitric acid for 10 minutes, then clean it with 25% KOH heated to 70°C for 10 minutes. Then place the silicon dioxide chip 100 in an ultrasonic cleaner (500W) for QDR cleaning for 5 minutes. After cleaning, place it on a spin coater to spin coat and remove water and dry.

[0059] 1.2 Chip Coating: A chromium film was deposited on the surface of the activated and cleaned silicon dioxide chip 100 using vacuum magnetron sputtering technology. The target-substrate distance was set to 9 cm, the deposition time to 60 min, the sputtering current to 0.6 A, and the sputtering pressure to 0.6 V.

[0060] 1.3 Spin Coating: Place the coated silicon dioxide chip 100 on the spin coater, turn on the vacuum suction switch to ensure the chip is firmly fixed on the spin coater, and uniformly coat the center of the silicon dioxide chip 100 with SUN-120 positive photoresist. Spin coat at a low speed of 1000 rpm for 10 minutes to spread the positive photoresist over the entire chip surface. Then, spin coat at a high speed of 3000 rpm for 10 minutes to achieve a uniform photoresist thickness of 5 μm, forming a positive photoresist layer 200 (see...). Figure 1 (as shown in (a));

[0061] 1.4 Pre-baking: Place the silicon dioxide chip 100 with the positive photoresist layer 200 on a hot plate at 100°C and bake for 90 seconds;

[0062] 1.5 Exposure: Place the baked silicon dioxide chip 100 and the mask 300 with a light-transmitting area corresponding to the reaction area into the photolithography machine. Align the silicon dioxide chip 100 with the markings on the mask, set the ultraviolet wavelength to 400nm, and expose for 10 minutes (see...). Figure 1 (b) shows the exposure path (the arrow represents the exposure path);

[0063] 1.6 Development: Immerse the exposed silicon dioxide chip 100 in the developing solution for 10 minutes to dissolve the exposed portion of the photoresist. After development, remove the silicon dioxide chip 100 (see...). Figure 1 (c) The chip was then cleaned three times with ethanol and isopropanol respectively. Then, an optical microscope was used to check that the exposed chip was fully developed and that the unexposed areas were not damaged. The developing solution was a mixture of isopropanol and methanol in a volume ratio of 30:70.

[0064] 1.7 Etching: The developed chip is placed in a 10% hydrofluoric acid solution and etched for 40 minutes. The silicon dioxide chip 100 is then removed and cleaned three times with isopropanol. After etching, the silicon dioxide in the reaction area of ​​the silicon dioxide chip 100 is removed according to the shape of the mask 300, forming 3000 regularly arranged circular reaction holes (see...). Figure 1 (d) The etching depth of each reaction hole was detected by a profilometer. The etching depth was 20±1μm. The morphology of each hole on the chip was observed under a microscope.

[0065] 1.8. Removal of Photoresist and Chromium Film: Remove residual photoresist and chromium film from the chip surface to expose the silicon dioxide layer. Then, immerse the chip sequentially in a photoresist remover (ethanol) and a chromium remover for 3 hours. After immersion, wash away the photoresist remover and chromium remover with isopropanol, rinse the chip with deionized water, and dry it to obtain the target chip 400 (see...). Figure 1(e) shows that the chip was completely de-adhesive / de-chromium under a microscope. The surfaces of the circular reactive holes and non-reactive areas of the target chip 400 are both made of silicon dioxide, which is hydrophilic.

[0066] 2. Second photolithography (hole protection)

[0067] Reference Figure 2 After the first photolithography, a second photolithography and hydrophobic process is performed using negative photoresist to achieve hydrophilic reactive holes and hydrophobic surfaces in non-reactive areas. The specific steps are as follows:

[0068] 2.1 Chip surface activation and cleaning: The target chip 400 was cleaned with fuming nitric acid for 10 min, and then cleaned with KOH of 25% mass concentration heated to 70℃ for 10 min. Then, the silicon dioxide chip 100 was placed in an ultrasonic cleaner (500W) for QDR cleaning for 5 min. After cleaning, it was placed on a spin coater for spin coating to remove water and drying.

[0069] 2.2 Spin Coating: Place the activated and cleaned target chip 400 on the spin coater, turn on the vacuum suction switch to ensure the chip is firmly fixed on the spin coater, and uniformly coat the center of the target chip 400 with SUN-90i negative photoresist. Spin coat at a low speed of 1000 rpm for 10 minutes to spread the negative photoresist across the entire chip surface. Set the spin coater to 3000 rpm and spin coat at a high speed for 10 minutes to achieve a uniform photoresist thickness of 5 μm, forming a negative photoresist layer 500 (see...). Figure 2 (as shown in (a));

[0070] 2.3 Pre-baking: Place the target chip 400 with the negative photoresist layer 500 on a hot plate at 100°C and bake for 90 seconds;

[0071] 2.4 Exposure: Place the baked target chip 400 and the mask 300 with the light-transmitting area corresponding to the reaction hole into the photolithography machine. Position and align the target chip 400 and the mask according to the markings on them. Set the ultraviolet wavelength to 400nm and expose for 10 minutes (see...). Figure 2 (b) shows the exposure path (the arrow represents the exposure path);

[0072] 2.5 Development: Immerse the exposed target chip 400 in the developer solution for 10 minutes to dissolve the non-exposed parts of the photoresist. After development is complete, remove the chip (see...). Figure 2 (c) is shown), and then cleaned three times each with ethanol and isopropanol. Then, an optical microscope is used to inspect the exposed chip for complete development, ensuring no damage to the exposed areas. All circular reactive holes on the chip surface are completely covered with the exposed photoresist, and the non-reactive areas on the chip surface are free of photoresist and are silicon dioxide surfaces (see [link]). Figure 3 As shown, Figure 3In this context, 'a' represents the chip, 'b' represents the reactive hole, and 'c' represents the photoresist; the developer is a mixture of isopropanol and methanol in a volume ratio of 30:70.

[0073] 2.6 Hydrophobication reaction: 0.64 g of perfluorodecyltrimethoxysilane was dissolved in 40 g of n-hexane, and then 10 mL of acetic acid aqueous solution (pH=3) was added. The reaction was carried out for 6 h and activated to obtain a reaction solution. The developed chip was immersed in the reaction solution and reacted at room temperature (25℃) for 8 h. The chip was then removed and cured in an oven at 60℃ for 2 h. Finally, the chip was rinsed with isopropanol.

[0074] 2.7 Photoresist Removal: Immerse the chip in the photoresist remover solution (ethanol) for 3 hours. After immersion, wash off the photoresist remover solution with isopropanol, then rinse the chip with deionized water, dry it, and observe under a microscope to ensure complete photoresist removal. The high-throughput microarray chip 600 is then obtained (see...). Figure 2 (d) is shown.

[0075] Example 2 (The difference between Example 2 and Example 1 is that a positive photoresist is used in the second photolithography process)

[0076] A method for fabricating a high-throughput microarray chip, employing dual photolithography, specifically includes the following steps:

[0077] 1. First photolithography step (hole etching)

[0078] The chip undergoes its first photolithography process using positive photoresist, combined with a custom mask and HF etching technology, to process and etch 3000 regularly arranged, uniformly sized circular reaction holes. The specific steps are as follows:

[0079] 1.1 Chip Surface Activation and Cleaning: Prepare a clean 14-inch round silicon dioxide chip. Clean the silicon dioxide chip with fuming nitric acid for 10 minutes, then clean it with 25% KOH heated to 70°C for 10 minutes. Then place the silicon dioxide chip in an ultrasonic cleaner (500W) for QDR cleaning for 5 minutes. After cleaning, place it on a spin coater to spin coat and remove water and dry.

[0080] 1.2 Chip Coating: A chromium film was deposited on the surface of an activated and cleaned silicon dioxide chip using vacuum magnetron sputtering technology. The target-substrate distance was set to 9 cm, the deposition time to 60 min, the sputtering current to 0.6 A, and the sputtering pressure to 0.6 V.

[0081] 1.3 Spin Coating: Place the coated silicon dioxide chip on the spin coating stage, turn on the vacuum adsorption switch to ensure the chip is firmly on the spin coating stage, and uniformly coat the center of the silicon dioxide chip surface with SUN-120 positive photoresist. Spin coat at a low speed of 1000 rpm for 10 minutes to spread the positive photoresist over the entire chip surface. Set the speed to 3000 rpm and spin coat at a high speed for 10 minutes to make the photoresist thickness uniform. The photoresist thickness is 5μm, forming a positive photoresist layer.

[0082] 1.4 Pre-baking: Place the silicon dioxide chip with the positive photoresist layer on a hot plate at 100°C and bake for 90 seconds;

[0083] 1.5 Exposure: Place the baked silicon dioxide chip and the mask with the light-transmitting area corresponding to the reaction area into the photolithography machine, and align them according to the markings on the silicon dioxide chip and the mask. Set the ultraviolet wavelength to 400nm and expose for 10min.

[0084] 1.6 Development: Immerse the exposed silicon dioxide chip in the developer for 10 minutes to dissolve the exposed photoresist. After development, remove the silicon dioxide chip and clean it three times with ethanol and isopropanol respectively. Then, use an optical microscope to check that the exposed chip is fully developed and that there is no damage in the non-exposed areas. The developer is a mixture of isopropanol and methanol in a volume ratio of 30:70.

[0085] 1.7 Etching: The developed chip was placed in a 10% hydrofluoric acid solution and etched for 40 minutes. The silicon dioxide chip was then removed and cleaned three times with isopropanol. After etching, the silicon dioxide in the reaction area was removed according to the shape of the mask to form 3000 regularly arranged circular reaction holes. The etching depth of each reaction hole was measured using a profilometer. The etching depth was 20±1μm. The morphology of each hole on the chip was observed under a microscope.

[0086] 1.8. Removal of Photoresist and Chromium Film: Residual photoresist and chromium film on the chip surface are removed to expose the silicon dioxide layer. The chip is then sequentially immersed in a photoresist remover (ethanol) and a chromium remover solution for 3 hours. After immersion, the photoresist remover and chromium remover solutions are washed away with isopropanol, followed by rinsing with deionized water and drying. The target chip is then obtained. Microscopic observation shows that the photoresist / chromium removal is complete. The surfaces of the circular reactive holes and non-reactive areas of the target chip are both composed of silicon dioxide, which is hydrophilic.

[0087] 2. Second photolithography (hole protection)

[0088] After the first photolithography step, a second photolithography and hydrophobic process is performed using positive photoresist to achieve hydrophilic reactive holes and hydrophobic surfaces in non-reactive areas. The specific steps are as follows:

[0089] 2.1 Chip surface activation and cleaning: The target chip was cleaned with fuming nitric acid for 10 min, then cleaned with 25% KOH heated to 70℃ for 10 min. Then the silicon dioxide chip was placed in an ultrasonic cleaner (500W) for QDR cleaning for 5 min. After cleaning, it was placed on a spin coater for spin coating to remove water and drying.

[0090] 2.2 Spin Coating: Place the activated and cleaned target chip on the spin coating stage, turn on the vacuum suction switch to ensure the chip is firmly on the spin coating stage, and uniformly coat the center of the target chip surface with SUN-120 positive photoresist. Spin coat at a low speed of 1000 rpm for 10 min to spread the positive photoresist over the entire chip surface. Set the speed to 3000 rpm and spin coat at a high speed for 10 min to make the photoresist thickness uniform. The photoresist thickness is 5 μm, forming a positive photoresist layer.

[0091] 2.3 Pre-baking: Place the target chip with the positive photoresist layer on a 100℃ hot plate and bake for 90 seconds;

[0092] 2.4 Exposure: Place the baked target chip and the mask with the non-transparent area corresponding to the reaction hole into the photolithography machine, and position and align them according to the markings on the target chip and the mask. Set the ultraviolet wavelength to 400nm and expose for 10min.

[0093] 2.5 Development: Immerse the exposed target chip in the developer solution for 10 minutes to dissolve the exposed portion of the photoresist. After development, remove the chip and clean it three times with ethanol and isopropanol respectively. Then, use an optical microscope to check that the exposed chip is fully developed and that there is no damage in the non-exposed areas. The developer solution is a mixture of isopropanol and methanol in a volume ratio of 30:70.

[0094] 2.6 Hydrophobication reaction: 0.64 g of perfluorodecyltrimethoxysilane was dissolved in 40 g of n-hexane, and then 10 mL of acetic acid aqueous solution (pH=3) was added. The reaction was carried out for 6 h and activated to obtain a reaction solution. The developed chip was immersed in the reaction solution and reacted at room temperature (25℃) for 8 h. The chip was then removed and cured in an oven at 60℃ for 2 h. Finally, the chip was rinsed with isopropanol.

[0095] 2.7 Photoresist Removal: Immerse the chip in photoresist remover solution (ethanol) for 3 hours. After immersion, wash off the photoresist remover solution with isopropanol, rinse the chip with deionized water, dry it, and observe under a microscope to ensure complete photoresist removal. This yields a high-throughput microarray chip.

[0096] Example 3 (The difference between Example 3 and Example 1 is the depth of the reaction pores)

[0097] A method for fabricating a high-throughput microarray chip, employing dual photolithography, specifically includes the following steps:

[0098] 1. First photolithography step (hole etching)

[0099] The chip undergoes its first photolithography process using positive photoresist, combined with a custom mask and HF etching technology, to process and etch 3000 regularly arranged, uniformly sized circular reaction holes. The specific steps are as follows:

[0100] 1.1 Chip Surface Activation and Cleaning: Prepare a clean 14-inch round silicon dioxide chip. Clean the silicon dioxide chip with fuming nitric acid for 10 minutes, then clean it with 25% KOH heated to 70°C for 10 minutes. Then place the silicon dioxide chip in an ultrasonic cleaner (500W) for QDR cleaning for 5 minutes. After cleaning, place it on a spin coater to spin coat and remove water and dry.

[0101] 1.2 Chip Coating: A chromium film was deposited on the surface of an activated and cleaned silicon dioxide chip using vacuum magnetron sputtering technology. The target-substrate distance was set to 9 cm, the deposition time to 60 min, the sputtering current to 0.6 A, and the sputtering pressure to 0.6 V.

[0102] 1.3 Spin Coating: Place the coated silicon dioxide chip on the spin coating stage, turn on the vacuum adsorption switch to ensure the chip is firmly on the spin coating stage, and uniformly coat the center of the silicon dioxide chip surface with SUN-120 positive photoresist. Spin coat at a low speed of 1000 rpm for 10 minutes to spread the positive photoresist over the entire chip surface. Set the speed to 3000 rpm and spin coat at a high speed for 10 minutes to make the photoresist thickness uniform. The photoresist thickness is 5μm, forming a positive photoresist layer.

[0103] 1.4 Pre-baking: Place the silicon dioxide chip with the positive photoresist layer on a hot plate at 100°C and bake for 90 seconds;

[0104] 1.5 Exposure: Place the baked silicon dioxide chip and the mask with the light-transmitting area corresponding to the reaction area into the photolithography machine, and align them according to the markings on the silicon dioxide chip and the mask. Set the ultraviolet wavelength to 400nm and expose for 10min.

[0105] 1.6 Development: Immerse the exposed silicon dioxide chip in the developer for 10 minutes to dissolve the exposed photoresist. After development, remove the silicon dioxide chip and clean it three times with ethanol and isopropanol respectively. Then, use an optical microscope to check that the exposed chip is fully developed and that there is no damage in the non-exposed areas. The developer is a mixture of isopropanol and methanol in a volume ratio of 30:70.

[0106] 1.7 Etching: The developed chip was placed in a 10% hydrofluoric acid solution and etched for 100 min. The silicon dioxide chip was then removed and cleaned three times with isopropanol. After etching, the silicon dioxide in the reaction area was removed according to the shape of the mask to form 3000 regularly arranged circular reaction holes. The etching depth of each reaction hole was measured using a profilometer. The etching depth was 50±1 μm. The morphology of each hole on the chip was observed under a microscope.

[0107] 1.8. Removal of Photoresist and Chromium Film: Residual photoresist and chromium film on the chip surface are removed to expose the silicon dioxide layer. The chip is then sequentially immersed in a photoresist remover (ethanol) and a chromium remover solution for 3 hours. After immersion, the photoresist remover and chromium remover solutions are washed away with isopropanol, followed by rinsing with deionized water and drying. The target chip is then obtained. Microscopic observation shows that the photoresist / chromium removal is complete. The surfaces of the circular reactive holes and non-reactive areas of the target chip are both composed of silicon dioxide, which is hydrophilic.

[0108] 2. Second photolithography (hole protection)

[0109] After the first photolithography step, a second photolithography and hydrophobic process is performed using negative photoresist to achieve hydrophilic reactive holes and hydrophobic surfaces in non-reactive areas. The specific steps are as follows:

[0110] 2.1 Chip surface activation and cleaning: The target chip was cleaned with fuming nitric acid for 10 min, then cleaned with 25% KOH heated to 70℃ for 10 min. Then the silicon dioxide chip was placed in an ultrasonic cleaner (500W) for QDR cleaning for 5 min. After cleaning, it was placed on a spin coater for spin coating to remove water and drying.

[0111] 2.2 Spin Coating: Place the activated and cleaned target chip on the spin coating stage, turn on the vacuum suction switch to ensure the chip is firmly on the spin coating stage, and uniformly coat the SUN-90i negative photoresist on the center of the target chip surface. Spin coat at a low speed of 1000 rpm for 10 min to spread the negative photoresist across the entire chip surface. Set the spin coating speed to 3000 rpm and spin coat at a high speed for 10 min to make the photoresist thickness uniform. The photoresist thickness is 5 μm, forming a negative photoresist layer.

[0112] 2.3 Pre-baking: Place the target chip with the negative photoresist layer on a 100℃ hot plate and bake for 90 seconds;

[0113] 2.4 Exposure: Place the baked target chip and the mask with the light-transmitting area corresponding to the reaction hole into the photolithography machine, and align them according to the markings on the target chip and the mask. Set the ultraviolet wavelength to 400nm and expose for 10min.

[0114] 2.5 Development: Immerse the exposed target chip in the developer solution for 10 minutes to dissolve the non-exposed parts of the photoresist. After development, remove the chip and clean it three times with ethanol and isopropanol respectively. Then check with an optical microscope to see if the exposed chip is fully developed and if the exposed area is undamaged. The developer solution is a mixture of isopropanol and methanol in a volume ratio of 30:70.

[0115] 2.6 Hydrophobication reaction: 0.64 g of perfluorodecyltrimethoxysilane was dissolved in 40 g of n-hexane, and then 10 mL of acetic acid aqueous solution (pH=3) was added. The reaction was carried out for 6 h and activated to obtain a reaction solution. The developed chip was immersed in the reaction solution and reacted at room temperature (25℃) for 8 h. The chip was then removed and cured in an oven at 60℃ for 2 h. Finally, the chip was rinsed with isopropanol.

[0116] 2.7 Photoresist Removal: Immerse the chip in photoresist remover solution (ethanol) for 3 hours. After immersion, wash off the photoresist remover solution with isopropanol, rinse the chip with deionized water, dry it, and observe under a microscope to ensure complete photoresist removal. This yields a high-throughput microarray chip.

[0117] Contact angle tests showed that the hydrophobic angle of the non-reactive region of the high-throughput microarray chips prepared in Examples 1-3 was approximately 130°.

[0118] The high-throughput microarray chip prepared in Example 1 was applied to the screening of neuraminidase inhibitors:

[0119] 1. Preparation of sample chips

[0120] A mixed control sample (63.6 μM oseltamivir, 764.4 μM hyperoside, 384.0 μM myricetin, 120 μM galantamine) was prepared and injected in 10 μL. After chromatographic separation, the separated fractions were collected into wells 1-300 of the high-throughput microarray chip prepared in Example 1 at a single-well collection time of 0.1 s. The high-throughput microarray chip loaded with the fractions was dried at 35 °C for 15 min to remove the solvent and was ready for use.

[0121] 2. Evaluation of Neuraminidase Inhibitory Activity Based on High-Throughput Microarray Chip

[0122] 10 nL of neuraminidase solution (20 mM Tris-HCl, pH 7.4, 0.125 mg / mL BSA) was added to each well of the high-throughput microarray chip. Then, 10 nL of 40 μM 4-MUNANA substrate solution was added to each well (final concentration 20 μM), and the mixture was incubated with shaking for 10 s. The enzyme-catalyzed reaction curves of each well were detected using a chip scanner over 60 min (excitation wavelength: 360 nm, emission wavelength: 450 nm).

[0123] 3. Data Processing

[0124] The dynamic fluorescence intensity of each reaction well was exported from the chip scanner, and a fluorescence growth curve was plotted. The slope of the linear segment from 2 to 20 min was calculated and divided by the slope of the linear segment of 10 control wells (without fractionation) to obtain the neuraminidase inhibition rate of that well. Combined with the residence time information of each well exported by the software, a time-(1-inhibition rate%) activity spectrum was plotted and integrated with the chromatogram to obtain a real-time matched high-resolution profile spectrum. This was repeated three times, and the results are as follows. Figure 4 As shown, Figure 4 Oseltamivir (a), hyperoside (b), myricetin (c).

[0125] 4. Result Verification

[0126] This experiment used a high-throughput microarray chip as a carrier to construct a highly sensitive method for evaluating neuraminidase activity. Oseltamivir, hyperoside, and myricetin all have inhibitory effects on neuraminidase, hence the corresponding inverted peaks appear in the activity spectrum. Furthermore, the reproducibility was good, and there was no significant cross-contamination at any reaction site.

[0127] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for fabricating a microarray chip, characterized in that, Includes the following steps: A first photoresist layer is formed in the non-reactive region of the silicon dioxide chip; The silicon dioxide in the reaction region of the silicon dioxide chip is removed by a wet etching process to form reaction holes; The first photoresist layer is removed to obtain the target chip; A second photoresist layer is formed on the surface of the reactive hole of the target chip; The non-reactive regions of the target chip are hydrophobically treated. The second photoresist layer is removed to obtain the microarray chip.

2. The preparation method according to claim 1, characterized in that, The microarray chip has hydrophilic reactive pores and hydrophobic non-reactive regions. The depth of the reactive pores is 20-50 μm, and the hydrophobic angle of the non-reactive regions is greater than 110°.

3. The preparation method according to claim 1, characterized in that, The first photoresist layer is formed in the non-reactive region of the silicon dioxide chip. The specific process includes: A positive photoresist layer is formed on the surface of the silicon dioxide chip; A mask with a light-transmitting area corresponding to the reaction area is used to expose and develop the positive photoresist layer, forming a first photoresist layer in the non-reaction area of ​​the silicon dioxide chip.

4. The preparation method according to claim 1, characterized in that, The silicon dioxide in the reaction region of the silicon dioxide chip is removed by a wet etching process to form reaction holes. The specific process includes: A silicon dioxide chip with the first photoresist layer formed in the non-reactive area is placed in a hydrofluoric acid solution. The hydrofluoric acid solution removes the silicon dioxide in the reactive area of ​​the silicon dioxide chip, forming a reactive hole.

5. The preparation method according to claim 1, characterized in that, A second photoresist layer is formed on the surface of the reactive aperture of the target chip, the specific process of which includes: A positive photoresist layer is formed on the surface of the target chip; A mask with a non-transparent area corresponding to the reaction hole is used to expose and develop the positive photoresist layer, forming a second photoresist layer on the surface of the reaction hole of the target chip.

6. The preparation method according to claim 1, characterized in that, A second photoresist layer is formed on the surface of the reactive aperture of the target chip, the specific process of which includes: A negative photoresist layer is formed on the surface of the target chip; A mask with a light-transmitting area corresponding to the reaction hole is used to expose and develop the negative photoresist layer, forming a second photoresist layer on the surface of the reaction hole of the target chip.

7. The preparation method according to claim 1, characterized in that, The non-reactive regions of the target chip are hydrophobically treated, specifically including: The target chip with the second photoresist layer formed on the reaction hole is placed in a hydrophobic solvent, and the non-reactive area of ​​the target chip is hydrophobized by the hydrophobic solvent.

8. The preparation method according to claim 1, characterized in that, Before forming the first photoresist layer in the non-reactive region of the silicon dioxide chip, the silicon dioxide chip is further subjected to a coating process, specifically including: depositing a chromium film on the surface of the silicon dioxide chip using vacuum magnetron sputtering technology.

9. The preparation method according to claim 8, characterized in that, Before the coating process, the silicon dioxide chip is activated and cleaned. The specific process includes: sequentially passing the silicon dioxide chip through acid cleaning, alkaline cleaning and QDR cleaning.

10. The application of the microarray chip prepared by the method of claim 1 in biomolecular detection and drug screening.

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