Preparation method of digital ELISA (enzyme-linked immuno sorbent assay) micron hole array
By performing oxygen plasma treatment, metal film layer growth and photoresist coating on quartz glass substrates, combined with reactive ion etching technology, the existing micropore array opacity and complex process problems are solved, and a high-precision and safe micropore array preparation method is achieved.
Patent Information
- Application Number
- CN202411489905.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-03-07
AI Technical Summary
The existing micropore arrays cannot be observed from the back or side due to the opaque substrate, and the filling of microspheres cannot be observed from the back or side. The process is complicated, the accuracy is low, and the reagents are dangerous.
Quartz glass is used as a transparent substrate, and the micropore array is gradually etched through oxygen plasma surface treatment, growth of metal film layers, coating positive photoresist and using mask plates with array patterns. Combined with reactive ion etching technology, micropore arrays are gradually etched.
The filling of microspheres in micropores is realized from the back and sides of the array to observe the filling of microspheres, which improves observation flexibility and accuracy, and reduces process complexity and reagent risk.
Smart Images
Figure CN119568987A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of micrometer hole arrays, and in particular relates to a method for preparing a digital ELISA micrometer hole array. Background Art
[0002] Digital ELISA technology proposes that the uniformly dispersed solution can be continuously subdivided, so that each solute molecule can be distributed in a separate area. Therefore, the sensitivity of molecular detection can be improved by using an array formed by a large number of micrometer holes with a large accumulation of femtoliters, so that a large number of single molecules can be detected at the same time. First, the target molecule is specifically bound to the enzyme-labeled antibody loaded on the surface of the magnetic microsphere, and the number of target molecules bound to each magnetic bead is ensured to be no more than one. Then, it is dispersed into independent chambers in the micrometer hole array with the microsphere. Under the catalysis of the enzyme, the positive molecules are rapidly amplified to produce fluorescent molecules, and because the volume is small enough, it can reach a detectable concentration in a short time. Finally, digital technology is used to count the sites showing fluorescence, so that a large number of samples can be processed at the same time.
[0003] Existing micrometer hole arrays usually use silicon as the substrate because the etching process of silicon is relatively mature. However, due to the opacity of silicon, it can only be observed using reflected light, and it is impossible to use back or side light to observe the filling of microspheres in the micrometer holes. 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 make a similar structure, but it still has problems such as low precision, complex process, and dangerous reagents. Therefore, it is necessary to find an efficient, fast, accurate and safe process to prepare transparent micrometer hole arrays. Summary of the invention
[0004] In view of this, an object of the present invention is to provide a method for preparing a digital ELISA micron well array.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] 1. A method for preparing a digital ELISA microwell array, the specific preparation steps comprising:
[0007] 1) After cleaning and drying the quartz glass sheet, the surface is treated with oxygen plasma;
[0008] 2) growing a metal film layer on the surface of the treated quartz glass substrate, wherein the metal film layer is an aluminum film or a chromium film;
[0009] 3) Apply a layer of positive photoresist on the metal film layer;
[0010] 4) using a mask with an array pattern to perform photolithography;
[0011] 5) Then, the sample is immersed in a cerium ammonium nitrate solution to etch the metal film layer, and then washed with deionized water;
[0012] 6) After cleaning, the sample is subjected to reactive ion etching and finally immersed in a cerium ammonium nitrate solution to remove the metal film layer.
[0013] In the method for preparing the digital ELISA microwell 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 by electron beam evaporation.
[0016] Furthermore, the film growth rate is 0.25 nm / s-5 nm / s.
[0017] Furthermore, the positive photoresist is AZ1500.
[0018] Furthermore, in step 4), the photolithography exposure time is 3 seconds, and then the film is immersed in TMAH with a concentration of 2.38% for development for 60 seconds, followed by rinsing with deionized water for 30 seconds, and heating on a 120° C. hot plate for 2 minutes.
[0019] Furthermore, in step 5), the etching time is 60s-100s.
[0020] Furthermore, reactive ion etching may use one or more gases such as CHF3, C4F8, SF6, CH2F2, Ar, etc.
[0021] Preferably, reactive ion etching is performed using a mixed gas of CHF3 and Ar in a ratio of 40:15.
[0022] 2. The digital ELISA microwell array obtained by any of the above preparation methods is also within the scope of protection of the present invention.
[0023] The beneficial effects of the present invention are as follows: the present invention provides a digital ELISA micrometer hole array prepared with quartz glass as a transparent substrate, which can be illuminated from the back and side of the array to observe the filling of microspheres in the micrometer holes, etc., to meet the multi-faceted needs of different observation conditions. In the prior art, the durability of RIE using photoresist as a mask is low and the etching depth is limited. In the preparation method provided by the present invention, a layer of chromium film is added to the substrate and the photoresist as a mask, the photolithography has high precision, is easy to clean, and has high durability for reactive ion etching. Generally, in order to increase the adhesion of the photoresist on the substrate, a silane coupling agent such as HMDS (hexamethyldisilazane) needs to be used for pretreatment, and finally it needs to be heated and cleaned with piranha solution, the steps are cumbersome and the risk level is high. The present invention adopts the method of coating photoresist on the chromium film to avoid the photoresist from contaminating the substrate. When photolithography is performed directly on a substrate plus a photoresist, the photolithography accuracy will be reduced due to the photoresist being too thick, and due to the existence of the "edge effect", acetone cleaning is required, and the steps are cumbersome. When etching is performed on a substrate + a chromium film, the chromium film used in the present invention is thin, has no edge effect, has high accuracy, and has a consistent etching depth. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:
[0025] Figure 1 The figure is a schematic diagram of the process of preparing the digital ELISA micron well array of the present invention.
[0026] Figure 2 This is a physical picture of the digital ELISA micron well array product.
[0027] Figure 3 This is a micrometer hole array under an optical microscope.
[0028] Figure 4 These are scanning electron microscope photos of the front (upper figure) and cross-section (lower figure) of the digital ELISA microwell array of Example 1.
[0029] Figure 5 The front side (upper figure) and the 3D microscope picture of the cross-section height of the digital ELISA microwell array of Example 1 (lower figure).
[0030] Figure 6 The digital ELISA microwell array of Example 1 is loaded with fluorescent microspheres and excited under ultraviolet light (A) and green light (B).
[0031] Figure 7 This is a scanning electron microscope photograph of the cross section of the digital ELISA microwell array after RIE etching for 120 minutes.
[0032] Figure 8 Schematic diagram of the side view of PDMS and flow channel.
[0033] Fig. 9 Top view of the PDMS and flow channel layout. DETAILED DESCRIPTION
[0034] The preferred embodiment of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods for which specific conditions are not specified in the embodiments are usually carried out under conventional conditions or under conditions 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 limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.
[0038] The terms "include", "comprising", "having", "containing", etc. used herein are all open terms, which means including but not limited to. The % used herein are all mass percentages.
[0039] The acetone used in the examples is analytical grade acetone, and the ethanol is anhydrous ethanol.
[0040] Example 1
[0041] The present invention provides a method for preparing a digital ELISA micron well array, and its flow chart is as follows: Figure 1 The specific steps are as follows:
[0042] 1. Soak the quartz glass sheet in acetone, ethanol and deionized water for ultrasonic cleaning for 5-30 minutes respectively, blow dry with nitrogen, and then use oxygen plasma for surface treatment for more than 5 minutes at a pressure of 1000Pa-1200Pa.
[0043] 2. Place the substrate in an electron beam evaporator and grow a 100nm thick chromium film as a protective layer at a speed of 0.25nm / s-5nm / s. Active metals such as aluminum can also replace chromium to achieve the technical problem to be solved by the present invention. After experimental testing, chromium is preferred. During subsequent etching, the etching rate of chromium is stable, the etching solution has little effect on the photoresist, and the accuracy is higher than other metals. In multiple experimental tests, it was found that the thickness of the chromium film can be 50-200nm, preferably 100nm. The thickness of the chromium film will affect the etching accuracy. If it is too thick, the etching time will be extended, affecting the lateral etching accuracy; if it is too thin, it cannot withstand RIE and the substrate will be exposed.
[0044] 3. Add AZ1500 positive photoresist to the center of the chrome film, spin-coat at 2000rpm for 40s, and heat on a 100℃ hot plate for 1min. The thickness of the positive photoresist is 0.2-1μm.
[0045] 4. The film was photolithographically exposed for 3 seconds using a mask with an array pattern, then immersed in 2.38% TMAH (tetramethylammonium hydroxide) for development for 60 seconds, then rinsed with deionized water for 30 seconds, and heated on a 120° C. hot plate for 2 minutes.
[0046] 5. Soak the sample in a pre-prepared ammonium cerium nitrate solution (80 g ammonium cerium nitrate, 14 ml glacial acetic acid, add water to 400 ml) for 80 seconds to etch the chromium film, and then rinse it again with deionized water for 30 seconds. The soaking time in the ammonium cerium nitrate solution can be adjusted according to the thickness of the chromium film.
[0047] 6. Place the obtained sample in RIE (reactive ion etching) and etch it for 90 minutes using a mixed gas of CHF3 and Ar at a flow rate ratio of 40sccm:15sccm, with a power of 150-250W. The etching time is determined according to the etching depth of the substrate. After etching, immerse the sample in an ammonium cerium nitrate solution until it is completely transparent, and then wash it again with deionized water to obtain a digital ELISA micron hole array. Figure 2 A physical picture of the digital ELISA microwell array product prepared in this example; Figure 3 This is a micrometer hole array under an optical microscope; Figure 4 The front (upper picture) and cross-section (lower picture) of the digital ELISA microwell array prepared in this example are shown in the scanning electron microscope photographs. Figure 4 It can be seen that the etched micron holes have no edge effect and high precision. Figure 5The front (above) and cross-sectional height 3D microscope photos of the digital ELISA microwell array (below). In RIE, CHF3 can be replaced by C4F8, SF6, CH2F2 and other gases, and other ratios can be used, and multiple combinations can be used or used alone without Ar.
[0048] 7. Steps 7-10 are steps for preparing PDMS flow channels: take another silicon wafer cleaned in step 1, drop SU8-2100 negative resist in the center of the silicon wafer, spin coat at 4000 rpm for 40 seconds, and soft bake on a 95°C hot plate for 20 minutes.
[0049] 8. Designed flow channel pattern is (3.1x4.1) mm 2 The sample in step 7 was photolithographically exposed for 10 seconds, and then post-baked on a hot plate at 95°C for 20 minutes. The sample was then immersed in propylene glycol methyl ether acetate (PGMEA) for development for 4 minutes. After being rinsed with acetone, the sample was heated on a hot plate at 150°C for 1 minute to harden the film.
[0050] 9. After fully mixing two-component polydimethylsilane (PDMS) in a ratio of base glue: curing agent = 10:1 and vacuum degassing, pour it on the sample pattern in step 8, heat it at 120°C for half an hour under vacuum to completely cure it, peel it off to obtain a PDMS-based flow channel, and use a 0.5mm diameter puncher to punch a through hole at the top of each of the two triangles. This embodiment uses Dow Corning 184 two-component polydimethylsilane, which can also be replaced by its PDMS. Figure 8 This is a side view of PDMS and flow channel. Fig. 9 Top view of the PDMS and flow channel layout.
[0051] 10. Soak the micropore array of step 6 and the PDMS of step 9 in ethanol, ultrasonically clean for 15 minutes, blow dry with nitrogen, and then use oxygen plasma to treat the surface for 5 minutes. After taking out, quickly align and fit, and put them in a 90°C oven for 2 hours and cool them naturally with the oven.
[0052] 11. Use a syringe to absorb the evenly vibrated dispersion of fluorescent beads, insert the needle from the triangular through hole at one end, and slowly inject it to fill the entire flow channel. Place a strong magnet directly below the array. After standing for 10 minutes, use a syringe to inject about 1 ml of water in the same way, and then use a syringe to inject fluorine oil until the liquid flowing out of the outlet is seen in layers, proving that the fluorine oil has drained the excess water in the flow channel.
[0053] 12. Place the sample under a fluorescence microscope and take bright field photos and corresponding green light excitation photos and ultraviolet light excitation photos of the same position. Figure 6 As shown, the fluorescent microspheres were loaded under ultraviolet light ( Figure 6Middle A) and green light ( Figure 6 B) Irradiation with excitation light.
[0054] Example 2
[0055] 1. Soak the quartz glass sheet in acetone, ethanol and deionized water for 15 minutes each, blow dry with nitrogen and then use oxygen plasma for surface treatment 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 rate of about 0.25 nm / s.
[0057] 3. Add AZ1500 positive photoresist to the center of the chrome film, spin coat at 2000 rpm for 40 seconds, and heat on a 100°C hot plate for 1 minute.
[0058] 4. The film was photolithographically exposed for 3 seconds using a mask with an array pattern, then immersed in 2.38% TMAH (tetramethylammonium hydroxide) for development for 60 seconds, then rinsed with deionized water for 30 seconds, and heated on a 120° C. hot plate for 2 minutes.
[0059] 5. Soak the sample in a pre-prepared ammonium cerium nitrate solution for 80 seconds to etch the chromium film, and then rinse it again with deionized water for 30 seconds.
[0060] 6. Place the obtained sample in RIE and etch it for 120 minutes using a mixture of CHF3 and Ar in a ratio of 40:15, with a power of 150-250 W. After etching, soak the sample in a cerium ammonium nitrate solution until it is completely transparent, and then rinse it again with deionized water.
[0061] like Figure 7 Shown is a scanning electron microscope image of a cross section of a digital ELISA microwell 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 rather than 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 in form and details without departing from the scope defined by the claims of the present invention.
Claims
1. A method for preparing a digital ELISA microwell array, characterized in that: The specific preparation steps include: 1) After cleaning and drying the quartz glass sheet, the surface is treated with oxygen plasma; 2) growing a metal film layer on the surface of the treated quartz glass substrate, wherein the metal film layer is an aluminum film or a chromium film; 3) Apply a layer of positive photoresist on the metal film layer; 4) using a mask with an array pattern to perform photolithography; 5) Then, the sample is immersed in a cerium ammonium nitrate solution to etch the metal film layer, and then washed with deionized water; 6) After cleaning, the sample is subjected to reactive ion etching and finally immersed in a cerium ammonium nitrate solution to remove the metal film layer.
2. The method for preparing a digital ELISA microwell array according to claim 1, characterized in that: The cleaning in step 1) is carried out by ultrasonic cleaning in acetone, ethanol and deionized water in sequence.
3. The method for preparing a digital ELISA microwell 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 microwell 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 microwell array according to claim 4, characterized in that: The film growth rate is 0.25nm / s-5nm / s.
6. The method for preparing a digital ELISA microwell array according to claim 1, characterized in that: The positive photoresist is AZ1500.
7. The method for preparing a digital ELISA microwell array according to claim 1, characterized in that: In step 4), the photolithography exposure time is 3 seconds, and then the film is immersed in TMAH with a concentration of 2.38% for development for 60 seconds, followed by rinsing with deionized water for 30 seconds and heating on a 120° C. hot plate for 2 minutes.
8. The method for preparing a digital ELISA microwell array according to claim 1, characterized in that: In step 5), the etching time is 60s-100s.
9. The method for preparing a digital ELISA microwell array according to claim 1, characterized in that: In reactive ion etching, a mixed gas of CHF3 and Ar in a ratio of 40:15 is used for etching.
10. A digital ELISA microwell array obtained by the preparation method according to any one of claims 1 to 9.