Method for preparing digital elisa micro‑well array

Abstract

A method for preparing a digital ELISA micro‑well array, comprising the steps of: cleaning and drying a quartz glass sheet, and performing a surface treatment; growing a chromium film on the surface of the treated quartz glass sheet substrate; applying a layer of a positive photoresist on the chromium film; performing photolithography by using a mask plate with an array pattern; immersing the sample in a cerium ammonium nitrate solution to etch the chromium film, and cleaning the etched chromium film with deionized water; and then performing reactive ion etching on the cleaned sample. The present invention further relates to a digital ELISA micro‑well array. In the preparation method, the quartz glass is used as a transparent substrate, and the micro‑well array can be illuminated from the back and side of the array to observe the filling of microspheres in the micro‑wells, thereby meeting the multi-directional requirements under different viewing conditions. The resulting micro‑wells have high precision, no edge effect, and uniform etching depth.

Description

A method for fabricating a digital ELISA micropore array Technical Field

[0001] This invention belongs to the field of micro-pore array technology, specifically relating to a method for preparing a digital ELISA micro-pore array. Background Technology

[0002] Digital ELISA technology proposes to continuously subdivide a uniformly dispersed solution, ultimately allowing each solute molecule to be distributed within a separate region. Therefore, utilizing an array of numerous micropores that accumulate to form a flyback pattern can improve the sensitivity of molecular detection, enabling the simultaneous detection of a large number of individual molecules. First, the target molecule specifically binds to an enzyme-labeled antibody loaded onto the surface of magnetic microspheres, ensuring that no more than one target molecule binds to each bead. Then, the target molecule is dispersed with the microspheres into individual chambers within the micropore array. Under the catalysis of the enzyme, positive molecules rapidly amplify to produce fluorescent molecules, and due to their sufficiently small size, a detectable concentration can be reached in a short time. Finally, digital technology is used to statistically analyze the fluorescent sites, allowing for the simultaneous processing of a large number of samples.

[0003] Existing micropore arrays typically use silicon as the substrate because silicon etching technology is relatively mature. However, due to the opacity of silicon, observation can only be performed using reflected light, and it is impossible to observe the filling of microspheres within the micropores using light from the back or side. Raphaela B. Liebherr et al. (Three-in-one enzyme assay based on single molecule detection in femtoliter arrays[J], Anal Bioanal Chem, 2015, 407:7443–7452) used quartz glass as the substrate and photoresist as a mask to fabricate a similar structure, but it still suffers from low precision, complex processes, and hazardous reagents. Therefore, it is necessary to find an efficient, rapid, accurate, and safe process to prepare transparent micropore arrays. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing a digital ELISA micropore array.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. A method for preparing a digital ELISA micropore array, comprising the following specific steps:

[0007] 1) After cleaning and drying the quartz glass slide, perform surface treatment with oxygen plasma;

[0008] 2) A metal film layer is grown on the surface of the treated quartz glass substrate, wherein the metal film layer is an aluminum film or a chromium film;

[0009] 3) A layer of positive photoresist is coated onto the metal film;

[0010] 4) Photolithography is performed using a mask with an array pattern;

[0011] 5) Then immerse the sample in cerium ammonium nitrate solution to etch the metal film layer, and rinse with deionized water after etching;

[0012] 6) After cleaning, the sample is subjected to reactive ion etching, and finally soaked in cerium ammonium nitrate solution to remove the metal film.

[0013] In the further described method for preparing the digital ELISA micropore array, the cleaning in step 1) is performed by ultrasonic cleaning in acetone, ethanol and deionized water in sequence.

[0014] Furthermore, in step 2), the film thickness is 50-200 nm.

[0015] Furthermore, the metal film layer is deposited using electron beam evaporation.

[0016] Furthermore, the membrane growth rate is 0.25 nm / s-5 nm / s.

[0017] Furthermore, the positive photoresist is AZ1500.

[0018] Further, in step 4), the photolithography exposure time is 3s, followed by immersion in 2.38% TMAH for 60s for development, then rinsing with deionized water for 30s, and heating on a 120°C hot plate for 2min.

[0019] Furthermore, in step 5), the etching time is 60s-100s.

[0020] Furthermore, reactive ion etching can employ one or more gases such as CHF3, C4F8, SF6, CH2F2, and Ar.

[0021] Preferably, reactive ion etching uses a mixture of CHF3 and Ar in a 40:15 ratio for etching.

[0022] 2. The digital ELISA micropore array obtained by any of the above preparation methods is also within the scope of protection of this invention.

[0023] The beneficial effects of this invention are as follows: This invention provides a digital ELISA micro-pore array prepared using quartz glass as a transparent substrate. Light can be shone from the back and sides of the array to observe the filling of microspheres within the micro-pores, meeting the multi-directional needs of different observation conditions. Existing technologies using photoresist as a mask for RIE have low durability and limited etching depth. In the preparation method provided by this invention, a chromium film is added between the substrate and the photoresist as a mask, resulting in high photolithography precision, easy cleaning, and high durability of reactive ion etching. Generally, to increase the adhesion of the photoresist to the substrate, pretreatment with silane coupling agents such as HMDS (hexamethyldisilazane) is required, and final cleaning with a piranha solution using heat is necessary. These steps are cumbersome and dangerous. This invention uses photoresist coated on the chromium film, avoiding photoresist contamination of the substrate. When photolithography is performed directly on the substrate and photoresist, the photolithography accuracy will be reduced if the photoresist is too thick, and the "edge effect" requires cleaning with acetone, which is a cumbersome process. In this invention, when etching the substrate and chromium film, the chromium film used is thin, has no edge effect, has high precision, and has a consistent etching depth. Attached Figure Description

[0024] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0025] Figure 1 is a schematic flowchart of the digital ELISA micropore array preparation method of the present invention.

[0026] Figure 2 shows a physical image of the digital ELISA micropore array product.

[0027] Figure 3 shows a micron-hole array under an optical microscope.

[0028] Figure 4 shows scanning electron microscope (SEM) images of the front (top) and cross-section (bottom) of the digital ELISA micropore array in Example 1.

[0029] Figure 5 shows a front view (top image) and a 3D microscope image (bottom image) of the digital ELISA micropore array in Example 1.

[0030] Figure 6 shows the excitation light of the digital ELISA micropore array loaded with fluorescent microspheres in Example 1 under ultraviolet light (A) and green light (B).

[0031] Figure 7 is a scanning electron microscope image of the cross-section of the digital ELISA micro-pore array after RIE etching for 120 min.

[0032] Figure 8 shows a schematic diagram of PDMS and the side of the flow channel.

[0033] Figure 9 is a top view of the PDMS and flow channel layout. Detailed Implementation

[0034] The preferred embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods not specified with specific conditions in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0038] The terms "contains," "includes," "has," and "contains" used in this article are all open-ended, meaning they include but are not limited to. All percentages used in this article refer to mass percentages.

[0039] The acetone used in the examples was analytical grade acetone, and the ethanol was anhydrous ethanol.

[0040] Example 1

[0041] This invention provides a method for preparing a digital ELISA micro-pore array, the process of which is shown in Figure 1. The specific operation steps are as follows:

[0042] 1. Immerse the quartz glass slide in acetone, ethanol and deionized water in turn and ultrasonically clean for 5-30 minutes each. After drying with nitrogen, perform surface treatment with oxygen plasma for more than 5 minutes at a pressure of 1000Pa-1200Pa.

[0043] 2. The substrate is placed in an electron beam evaporator, and a 100 nm thick chromium film is grown as a protective layer at a rate of 0.25 nm / s-5 nm / s. Reactive metals such as aluminum can also be used instead of chromium to achieve the technical problem to be solved in this invention. Experimental tests show that chromium is preferred because its etching rate is stable during subsequent etching, the etching solution has less impact on the photoresist, and its precision is higher than other metals. Multiple experimental tests have shown that a chromium film thickness of 50-200 nm is acceptable, with 100 nm being preferred. The thickness of the chromium film affects the etching precision; too thick a film requires a longer etching time, affecting the lateral etching precision; too thin a film cannot withstand the RIE and exposes the substrate.

[0044] 3. Add AZ1500 positive adhesive to the center of the chromium film, spin coat at 2000 rpm for 40 seconds, and heat on a 100℃ hot plate for 1 minute. The thickness of the positive adhesive is 0.2-1 μm.

[0045] 4. Photolithography is performed using a mask with an array pattern for 3 seconds. Then, the image is immersed in 2.38% TMAH (tetramethylammonium hydroxide) for 60 seconds, followed by rinsing with deionized water for 30 seconds and heating on a 120°C hot plate for 2 minutes.

[0046] 5. Immerse the sample in a pre-prepared cerium ammonium nitrate solution (80g cerium ammonium nitrate, 14ml glacial acetic acid, and water to 400ml) for 80 seconds to etch the chromium film, then rinse again with deionized water for 30 seconds. The immersion time in the cerium ammonium nitrate solution can be adjusted according to the thickness of the chromium film.

[0047] 6. Place the obtained sample in a RIE (Reactive Ion Etching) chamber and etch it for 90 minutes using a mixed gas of CHF3 and Ar at a flow rate ratio of 40 sccm:15 sccm. The power is 150-250W, and the etching time depends on the etching depth of the substrate. After etching, immerse the sample in a cerium ammonium nitrate solution until it becomes completely transparent, and then rinse it again with deionized water to obtain the digital ELISA micro-pore array. Figure 2 shows the physical image of the digital ELISA micro-pore array product prepared in this embodiment; Figure 3 shows the micro-pore array under an optical microscope; Figure 4 shows the scanning electron microscope images of the front (top) and cross-section (bottom) of the digital ELISA micro-pore array prepared in this embodiment. As can be seen from Figure 4, the etched micro-pores have no edge effect and high precision. Figure 5 shows the 3D microscope images of the front (top) and cross-section (bottom) of the digital ELISA micro-pore array. In the RIE, CHF3 can be replaced with C4F8, SF6, CH2F2, etc. Other ratios can be used, or multiple gases can be used in combination or alone without Ar.

[0048] 7. Steps 7-10 are the steps for preparing PDMS channels: Take another silicon wafer that has been cleaned according to step 1, drop SU8-2100 negative adhesive into the center of the silicon wafer, spin coat at 4000 rpm for 40s, and then soft bake on a 95℃ heating plate for 20min.

[0049] 8. The designed flow channel pattern is (3.1 x 4.1) mm. 2 The rectangle is formed by extending two right-angled triangles outward from the two widths as hypotenuses. The sample in step 7 is photolithographically etched with an exposure time of 10s. After that, it is baked at 95°C for 20min with a heating plate, and then immersed in propylene glycol methyl ether acetate (PGMEA) for 4min for development. After rinsing with acetone, it is heated at 150°C for 1min to harden the film.

[0050] 9. After thoroughly mixing the two-component polydimethylsilane (PDMS) at a base adhesive: curing agent ratio of 10:1 and degassing under vacuum, pour the mixture onto the sample pattern from step 8. Maintain a vacuum and heat at 120°C for half an hour to fully cure. Remove the mixture to obtain the PDMS-based flow channels. Use a 0.5mm diameter punch to drill a through-hole at the apex of each of the two triangles. This embodiment uses Dow Corning 184 two-component polydimethylsilane, but it can be replaced with other PDMS. Figure 8 shows a side view of the PDMS and flow channels, and Figure 9 shows a top view of the PDMS and flow channel layout.

[0051] 10. Immerse the micro-pore array from step 6 and the PDMS from step 9 in ethanol, ultrasonically clean for 15 min, dry with nitrogen, and then perform surface treatment with oxygen plasma for 5 min. After removal, quickly align and bond them, and place them in a 90℃ oven for 2 h and allow them to cool naturally in the oven.

[0052] 11. Draw up the well-vibrated fluorescent microsphere dispersion with a syringe, insert the needle into the triangular through-hole at one end, and slowly inject it to fill the entire channel. Place a strong magnet directly below the array and let it stand for 10 minutes. Then, inject about 1 ml of water with the syringe in the same way. After that, inject the fluorinated oil with the syringe until you see the liquid flowing out of the outlet in layers, which proves that the fluorinated oil has drained the excess water in the channel.

[0053] 12. Place the sample under a fluorescence microscope and take bright-field photographs and corresponding green-light excitation photographs and ultraviolet-light excitation photographs at the same location. As shown in Figure 6, the excitation light of the fluorescent microspheres under ultraviolet light (Figure 6A) and green light (Figure 6B) is shown.

[0054] Example 2

[0055] 1. Immerse the quartz glass slide in acetone, ethanol and deionized water respectively for ultrasonic cleaning for 15 minutes each, dry it with nitrogen gas and then perform surface treatment with oxygen plasma for 5 minutes.

[0056] 2. Place the substrate in an electron beam evaporator and grow a 100 nm thick chromium film as a protective layer at a speed of approximately 0.25 nm / s.

[0057] 3. Add AZ1500 positive adhesive to the center of the chromium film, spin coat at 2000 rpm for 40 seconds, and heat on a 100℃ hot plate for 1 minute.

[0058] 4. Photolithography is performed using a mask with an array pattern for 3 seconds. Then, the image is immersed in 2.38% TMAH (tetramethylammonium hydroxide) for 60 seconds, followed by rinsing with deionized water for 30 seconds and heating on a 120°C hot plate for 2 minutes.

[0059] 5. Immerse the sample in a pre-prepared cerium ammonium nitrate solution for 80 seconds to etch the chromium film, and then rinse again with deionized water for 30 seconds.

[0060] 6. Place the obtained sample in a RIE (Refined Iron Injection) and etch it for 120 minutes using a mixture of CHF3 and Ar in a 40:15 ratio at a power of 150-250W. After etching, immerse the sample in a cerium ammonium nitrate solution until it becomes completely transparent, and then rinse it thoroughly with deionized water.

[0061] Figure 7 shows a scanning electron microscope image of the cross-section of the digital ELISA micro-pore array after RIE etching for 120 min.

[0062] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a digital ELISA micropore array, characterized in that, The specific preparation steps include: 1) After cleaning and drying the quartz glass slide, perform surface treatment with oxygen plasma; 2) A metal film layer is grown on the surface of the treated quartz glass substrate, wherein the metal film layer is an aluminum film or a chromium film; 3) A layer of positive photoresist is coated onto the metal film; 4) Photolithography is performed using a mask with an array pattern; 5) Then immerse the sample in cerium ammonium nitrate solution to etch the metal film layer, and rinse with deionized water after etching; 6) After cleaning, the sample is subjected to reactive ion etching, and finally soaked in cerium ammonium nitrate solution to remove the metal film.

2. The method for fabricating a digital ELISA micropore array according to claim 1, characterized in that, Step 1) involves ultrasonic cleaning in sequence using acetone, ethanol, and deionized water.

3. The method for preparing a digital ELISA micropore array according to claim 1, characterized in that, In step 2), the film thickness is 50-200 nm.

4. The method for preparing a digital ELISA micropore array according to claim 1, characterized in that, The metal film layer is deposited by electron beam evaporation.

5. The method for preparing a digital ELISA micropore array according to claim 4, characterized in that, The membrane growth rate is 0.25 nm / s-5 nm / s.

6. The method for fabricating a digital ELISA micropore array according to claim 1, characterized in that, The positive photoresist is AZ1500.

7. The method for fabricating a digital ELISA micropore array according to claim 1, characterized in that, In step 4), the photolithography exposure time is 3s, followed by immersion in 2.38% TMAH for 60s, then rinsing with deionized water for 30s, and heating on a 120℃ hot plate for 2min.

8. The method for preparing a digital ELISA micropore array according to claim 1, characterized in that, In step 5), the etching time is 60s-100s.

9. The method for fabricating a digital ELISA micropore array according to claim 1, characterized in that, Reactive ion etching uses a mixture of CHF3 and Ar in a 40:15 ratio for etching.

10. A digital ELISA micropore array obtained by any one of the preparation methods of claims 1-9.

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