Preparation method and application of DNA nano array

Abstract

The invention relates to the technical field of DNA microarrays, and discloses a preparation method and application of a DNA nano array. The preparation method of the DNA nano array comprises the following steps: step 1, acquiring DNA ink, and then transferring the DNA ink to the edge of a substrate material; step 2, moving a flexible platform to enable a probe to be in contact with the DNA ink, then dropping the probe at a first preset position of the substrate material and then lifting the probe to obtain a first DNA nanodot; and 3, changing the target preset position of the substrate material, and repeating the step 2 to obtain the target DNA nanodot. By adopting the method disclosed by the invention, the DNA nano array of which the average diameter is less than or equal to 500 nm, the distance between adjacent points is less than or equal to 2000 nm and the average height is less than or equal to 50 nm can be prepared, the prepared DNA nano array has the characteristics of high density and high flux, meanwhile, the preparation process is simple, the cost is relatively low, and no other toxic chemical reagents are introduced.

Description

Technical Field

[0001] This invention relates to the field of DNA microarray technology, and in particular to a method for preparing and applying DNA nanoarrays. Background Technology

[0002] DNA microarrays are a new technology developed through the integration of nucleic acid hybridization technology and computer chip manufacturing technology. As a type of biochip, its principle is to integrate gene probes with known sequences onto a solid surface to achieve rapid detection of gene information. Compared with traditional methods for studying differential gene expression, it has advantages such as miniaturization, speed, accuracy, high sensitivity, and the ability to simultaneously detect large amounts of information on the same chip.

[0003] Common methods for preparing DNA microarrays include in-situ photolithography, photoacidification, and inkjet printing. However, the precision of DNA microarrays prepared by these methods is relatively low (micrometer level), making it difficult to meet the requirements of high density and high throughput. Furthermore, the preparation methods for these DNA microarrays are cumbersome and costly, and often require the introduction of various toxic chemicals (such as photoacid-producing agents), which can easily cause pollution.

[0004] Therefore, there is an urgent need to find a new method for preparing and applying DNA nanoarrays. Summary of the Invention

[0005] The first aspect of this invention is to provide a method for preparing DNA nanoarrays.

[0006] A second aspect of the present invention is to provide a DNA nanoarray.

[0007] A third aspect of the present invention is to provide a biochip.

[0008] The fourth aspect of the present invention is to provide a DNA nanoarray preparation device.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a method for preparing a DNA nanoarray, comprising the following steps: Step 1: Obtain DNA ink and then transfer it to the edge of the substrate material; Step 2: Move the flexible platform to make the probe contact the DNA ink, and then drop the probe at the first preset position on the substrate material and lift the probe to obtain the first DNA nanodot; Step 3: Change the target preset position of the substrate material and repeat step 2 to obtain the target DNA nanodots.

[0010] In some embodiments of the present invention, in step 1, the concentration of the DNA ink is 10-100 µM. For example, it can be 10, 20, 30, 50, 60, 80, or 100 µM.

[0011] In some embodiments of the present invention, in step 1, the DNA in the DNA ink is selected from at least one of natural DNA and artificially synthesized DNA.

[0012] In some embodiments of the present invention, the solvent of the DNA ink is selected from water, phosphate buffer, or physiological saline.

[0013] In some embodiments of the present invention, the volume of the DNA ink transfer is 10-1000 µL. For example, it can be 10, 50, 10, 200, 400, 500, 800 or 1000 µL, etc.

[0014] In some embodiments of the present invention, the DNA ink transfer is performed using at least one of a syringe, a pipette, and a dropper.

[0015] In some embodiments of the present invention, in step 1, the substrate material is selected from any one of mica sheet, polished silicon wafer, glass sheet or graphite.

[0016] In some embodiments of the present invention, the roughness of the substrate material is ≤5 nm. For example, it can be 5, 4, 3, 2, 1, 0.5, or 0.1 nm, etc.

[0017] In some embodiments of the present invention, the distance between the edge of the substrate material and the substrate material is 0-1000 µm. For example, it can be 0, 50, 100, 150, 200, 300, 500, 600, 800 or 1000 µm, etc.

[0018] In some embodiments of the present invention, step 2 further includes scanning the substrate material using an atomic force microscope before the probe contacts the DNA ink.

[0019] In some embodiments of the present invention, the scanning range is 1-10000 µm. 2 For example, it can be 1, 100, 200, 400, 800, 1000, 2000, 5000, 8000, or 10000 µm. 2 wait.

[0020] In some embodiments of the present invention, in step 2, the flexible platform moves within a range of 10-1000 µm. For example, it can be 10, 20, 50, 100, 200, 400, 800, or 1000 µm.

[0021] In some embodiments of the present invention, the probe contacts the DNA ink for 1-50 seconds. For example, it can be 1, 50, 10, 20, 30, 40, or 50 seconds.

[0022] In some embodiments of the present invention, the time for the needle to fall at the first preset position is 10-100 seconds. For example, it can be 10, 20, 30, 50, 80, or 100 seconds.

[0023] In some embodiments of the present invention, the needle is allowed to rest for 10-50 seconds before dropping at the first preset position. This resting period helps to improve stability and accuracy.

[0024] In some embodiments of the present invention, in step 3, the first preset position is marked as the origin; the x-coordinate X of the target preset position ranges from -1000 to 1000 µm, and the y-coordinate Y ranges from -1000 to 1000 µm.

[0025] In some embodiments of the present invention, the time for the needle to fall at the target preset position is 10-100 s.

[0026] In some embodiments of the present invention, the needle is left to stand still for 10-50 seconds before being dropped at the target preset position.

[0027] In some embodiments of the present invention, the spacing between adjacent DNA nanodots is ≤2000 nm. For example, it can be 1, 5, 10, 50, 100, 200, 500, 1000, 1500 or 2000 nm, etc.

[0028] In some embodiments of the present invention, the average diameter of the DNA nanodots is ≤500 nm.

[0029] In some embodiments of the present invention, the average height of the DNA nanodots is ≤50 nm.

[0030] The DNA nanodot preparation method of the present invention allows for control of the size and height of the DNA nanodots by utilizing the contact time between the probe dipped in DNA ink and the substrate material.

[0031] In some embodiments of the present invention, the DNA nanoarray comprises at least two DNA nanodots.

[0032] In some embodiments of the present invention, drying is further included after obtaining the target DNA nanodots. Preferably, the drying temperature is 25±5℃ and the drying time is 10-100 min.

[0033] In some embodiments of the present invention, after obtaining the DNA nanoarray, the method further includes using an atomic force microscope (AFM) in tapping mode, inserting a needle, and scanning to obtain an AFM image of the DNA nanoarray.

[0034] In a second aspect, the present invention provides a DNA nanoarray prepared using the method for preparing DNA nanoarrays described in the first aspect.

[0035] In some embodiments of the present invention, the spacing between adjacent DNA nanodots in the DNA nanoarray is ≤2000 nm. For example, it can be 1, 5, 10, 50, 100, 200, 500, 1000, 1500 or 2000 nm, etc.

[0036] In some embodiments of the present invention, the average diameter of the DNA nanodots in the DNA nanoarray is ≤500 nm.

[0037] In some embodiments of the present invention, the average height of the DNA nanodots in the DNA nanoarray is ≤50 nm.

[0038] In a third aspect, the present invention provides a biochip comprising the DNA nanoarray described in the second aspect.

[0039] In a fourth aspect, the present invention provides a DNA nanoarray fabrication apparatus, comprising a scanning device, a flexible platform, and a probe; The preparation apparatus operates the DNA nanoarray preparation method described in the first aspect.

[0040] In some embodiments of the present invention, the scanning device includes an atomic force microscope.

[0041] The method for preparing and applying the DNA nanoarray of the present invention has at least the following beneficial effects: The method of this invention can produce DNA nanoarrays with an average diameter of ≤500 nm, a spacing between adjacent dots of ≤2000 nm, and an average height of ≤50 nm. The prepared DNA nanoarrays have the characteristics of high density and high throughput. At the same time, the preparation process is simple, low in cost, and does not introduce other toxic chemical reagents.

[0042] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the DNA nanoarray fabrication device of the present invention.

[0044] Figure 2 This is a schematic diagram of the DNA nanoarray preparation process of the present invention.

[0045] Figure 3 The images show AFM scans of the substrate for preparing the DNA nanoarray according to this invention, with the left image being a planar view and the right image being a three-dimensional view.

[0046] Figure 4 The images show AFM scan images and relative height diagrams of the DNA nanodots of this invention, with the left image being the AFM scan image and the right image being the relative height characterization diagram.

[0047] Figure 5 The images show AFM scan images and relative height diagrams of the DNA nanoarray of the present invention, with the left image being the AFM scan image and the right image being the relative height characterization diagram. Detailed Implementation

[0048] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0049] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0050] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0051] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0052] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0053] In the description of this invention, the atomic force microscope was purchased from NT-MDT (NTMDT Spectrum Instruments, Moscow, Russia).

[0054] The AFM probe selected is a tapping mode AFM probe with an aluminum reflective coating (Tap190Al-G, BudgetSensors, Sofia, Bulgaria).

[0055] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] Inventive concept: The present invention aims to provide a method for preparing DNA nanoarrays and their applications.

[0057] refer to Figure 1 The apparatus for fabricating the DNA nanoarray of the present invention is shown, which mainly includes a scanning device, a flexible platform, and a probe. For fabricating the DNA nanoarray, a strategy combining an atomic force microscope (AFM), a probe, and a flexible nanoplatform is preferred.

[0058] Specifically, the modes of the atomic force microscope probe include contact mode and semicontact mode.

[0059] refer to Figure 2 The present invention illustrates the preparation process of the DNA nanoarray, which mainly consists of three steps: using an atomic force microscope (AFM) probe to pick up DNA solution, preparing the DNA nanoarray, and in-situ scanning of the DNA nanoarray.

[0060] In some embodiments of the present invention, the preparation method of the DNA nanoarray is as follows: S1. Dissolve DNA powder in a solvent to prepare DNA ink; S2. Transfer the DNA ink to the edge of the substrate material, move the flexible platform to allow the atomic force microscope probe to pick up the DNA ink, and then move it to the needle drop position (marked as the origin, coordinate 0, 0). Drop the needle onto the substrate material and then lift the needle to complete the preparation of a DNA nanodot. S3. Repeat step S2, changing the needle placement position (coordinates X, Y), to prepare multiple DNA nanodots, i.e., DNA nanoarrays.

[0061] In some specific embodiments, the DNA powder can be natural DNA or artificially synthesized DNA.

[0062] In some specific embodiments, the solvent is selected from at least one of water, phosphate buffer, or physiological saline. Ultrapure water is preferred.

[0063] In some specific embodiments, the concentration of the DNA ink is 10~100 µM.

[0064] In some specific embodiments, the substrate material can be a mica sheet, a polished silicon wafer, a glass sheet, or graphite, etc. Mica sheets are preferred.

[0065] In some specific embodiments, when the DNA ink is transferred to the edge of the substrate material, 10 to 1000 µL of DNA ink is transferred at a distance of 0 to 1000 µm from the substrate material.

[0066] Specifically, when transferring DNA ink to the edge of the substrate material, syringes, pipettes, or droppers can be used.

[0067] In some specific embodiments, the method further includes scanning the surface of the substrate material in contact mode using an atomic force microscope before using an atomic force microscope probe to pick up DNA ink.

[0068] Specifically, the scanning range is 1-10000 µm. 2 If the roughness is ≤5 nm, proceed to the subsequent DNA ink application step. Excessive roughness will interfere with the recognition of the DNA nanoarray.

[0069] In some specific implementations, the atomic force microscope probe dips into DNA ink in a contact mode.

[0070] In some specific embodiments, in S2, when the moving flexible platform causes the AFM probe to pick up DNA ink in contact mode, the time for the AFM probe to pick up DNA ink is 1-50 s. For example, it can be 10, 15, 20, 25, 30, 40, or 50 s, etc.

[0071] In some specific embodiments, in S2, after the flexible platform moves to allow the AFM probe to pick up DNA ink in contact mode, the movement range of the flexible platform is 10~1000 µm. For example, it can be 10, 20, 50, 80, 100, 150, 200, 400, 600, 800 or 1000 µm, etc.

[0072] In some specific implementations, during step S3, when changing the needle's position (coordinates X, Y), the value of X ranges from -1000 to 1000 µm, and the value of Y ranges from -1000 to 1000 µm. In some specific embodiments, after the DNA nanoarray is prepared, the process includes drying, adjusting the AFM to tapping mode, dropping the needle, scanning, and obtaining an AFM image of the DNA nanoarray.

[0073] Specifically, the drying temperature is 25±5℃ and the drying time is 10-100 min.

[0074] In some specific embodiments, the fabrication of DNA nanoarrays includes the following steps: S11. Dissolve the DNA powder in ultrapure water to prepare DNA ink with a concentration in the range of 10-100 µM. S12. Use a syringe to take 10-1000 µL of the DNA ink prepared in step S11 and transfer it to the edge of the mica sheet, 0-1000 µm away from the mica sheet; S13. Scan the mica sheet surface using AFM in contact mode, with a scanning range of 1-10000 µm. 2 If the roughness is ≤5nm, proceed to step S14; S14. The flexible platform moves so that the probe can pick up DNA ink in contact mode. The moving range of the flexible platform is 10-1000 µm, and the time for the probe to pick up DNA ink is 1-50 s. S15. Move the flexible platform again to return the probe to the first DNA nanodot writing position, and mark it as the origin position (i.e., the horizontal axis X is 0 and the vertical axis Y is 0). After the system stabilizes for 10-50 seconds, insert the probe at this position to deliver the DNA ink to the substrate surface. S16. Then wait 10-100 seconds before lifting the needle to complete the preparation of a DNA nanodot. S17. Change the x-coordinate X and y-coordinate Y values ​​of the target needle position. The value of X ranges from -1000 to 1000 µm, and the value of Y ranges from -1000 to 1000 µm. Repeat steps S14 to S16 to prepare a series of DNA nanodots, i.e. DNA nanoarrays. S18. After air-drying at room temperature for 10-100 min, switch AFM to tapping mode, drop the needle, and scan to obtain an AFM image of the DNA nanoarray.

[0075] Example: This embodiment provides a method for preparing a DNA nanoarray, specifically including the following:

[0076] 1. DNA Ink Preparation DNA powder with the base sequence “5'-GGATTATTGTTATAGCTCAACTCT-3' (SEQ ID NO.1)” was dissolved in ultrapure water to prepare a DNA ink with a concentration of 50 µM. Then, 20 µL of the DNA ink was transferred to the edge of a mica sheet, 800 µm away from the mica sheet, using a syringe.

[0077] 2. Basal scan The surface of the mica sheet was scanned in contact mode using an atomic force microscope (AFM), such as... Figure 3 As shown, specifically, the mica sheet is 10 µm long, 10 µm wide, and has a scanning range of 100 µm. 2 The roughness is 1.1 nm, which meets the requirements for the flatness of the substrate material, and subsequent steps can be carried out.

[0078] 3. Preparation of DNA Nanodots The flexible platform was moved to allow the AFM probe to pick up DNA ink from the edge of the mica sheet in contact mode. The platform moved 900 µm, and the probe was immersed in DNA ink for 10 s. The platform was then moved again to return the probe to the position with x-axis (X) and y-axis (Y) both at 0. After the system stabilized for 20 s, the probe was lowered into this position. After waiting 30 s, the probe was lifted, completing the preparation of one DNA nanodot.

[0079] After air-drying at room temperature for 20 minutes, switch the AFM to SemiContact mode, drop the needle, and press at 1 µm. 2 An in-plane scan was performed to obtain an AFM image of a DNA nanodot, as shown in the image. Figure 4 As shown, the DNA nanodot has a diameter of approximately 95 nm and a height of approximately 3.5 nm.

[0080] 4. Preparation of DNA Nanoarrays Repeat step 3 above, moving the flexible platform to allow the probe to pick up DNA ink in contact mode. The flexible platform moves 900 µm, and the probe is dipped in DNA ink for 10 s. Then, lift the probe and simultaneously move the flexible platform again to return the probe sequentially to positions with x-coordinates of 2, 4, 6, 8, 10, 12, or 14 (µm) and y-coordinate of 0. After the system stabilizes for 20 s, lower the probe to these positions. After waiting 30 s, lift the probe again. This completes the preparation of eight DNA nanodots, resulting in a DNA nanoarray.

[0081] After air-drying at room temperature for 40 minutes, switch the AFM to tapping mode, drop the needle, and tap at 400 µm. 2 An in-area scan can obtain an AFM image of an eight-point DNA nanoarray, as shown in the following figure. Figure 5 As shown, the average diameter of the DNA nanodot is approximately 500 nm, the average height is approximately 15 nm, and the average dot spacing is approximately 2000 nm.

[0082] In summary, this invention provides a method for preparing and applying DNA nanoarrays. Specifically, this invention employs a strategy combining atomic force microscopy (AFM), a probe, and a flexible nanoplatform. First, DNA powder is dissolved in ultrapure water to prepare DNA ink. Then, an appropriate amount of DNA ink is transferred to the edge of a mica sheet. The flexible platform is moved so that the probe picks up the DNA ink in contact mode. The flexible platform is then moved again to return the probe to a specific position. After the system stabilizes, the probe is inserted at that position, and after a period of time, it is lifted, completing the preparation of one DNA nanodot. By repeating the above process according to the preset dot matrix positions and changing the insertion position, a series of DNA nanodots can be prepared, thus obtaining a DNA nanoarray. Further, by switching AFM to semi-contact mode, inserting the probe, and scanning, an AFM image of the DNA nanoarray can be obtained. The method of this invention can prepare DNA nanoarrays with an average diameter ≤500 nm, an adjacent dot spacing ≤2000 nm, and an average height ≤50 nm. The prepared DNA nanoarrays exhibit high density and high throughput, and the preparation process is simple, low-cost, and does not introduce any other toxic chemical reagents.

[0083] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a DNA nanoarray, characterized in that, Includes the following steps: Step 1: Obtain DNA ink and then transfer it to the edge of the substrate material; Step 2: Move the flexible platform to make the probe contact the DNA ink, and then drop the probe at the first preset position on the substrate material and lift the probe to obtain the first DNA nanodot; Step 3: Change the target preset position of the substrate material and repeat step 2 to obtain the target DNA nanodots.

2. The preparation method according to claim 1, characterized in that, In step 1, the concentration of the DNA ink is 10-100 µM; And / or, the solvent of the DNA ink is selected from any one of water, phosphate buffer, or physiological saline; And / or, the volume of the DNA ink transferred is 10-1000 µL.

3. The preparation method according to claim 1 or 2, characterized in that, In step 1, the substrate material is selected from any one of mica sheet, polished silicon wafer, glass sheet or graphite; And / or, the distance between the edge of the substrate material and the substrate material is 0-1000 µm.

4. The preparation method according to claim 3, characterized in that, In step 2, before the probe contacts the DNA ink, the substrate material is scanned using an atomic force microscope. Preferably, the scanning range is 1-10000 µm. 2 ; Preferably, the roughness of the substrate material is ≤5 nm.

5. The preparation method according to claim 1, characterized in that, In step 2, the flexible platform moves within a range of 10-1000 µm; And / or, the probe contacts the DNA ink for 1-50 s; And / or, the time for the needle to fall at the first preset position is 10-100 s.

6. The preparation method according to claim 1, characterized in that, In step 3, the first preset position is marked as the origin; the x-coordinate X of the target preset position ranges from -1000 to 1000 µm, and the y-coordinate Y ranges from -1000 to 1000 µm.

7. The preparation method according to claim 6, characterized in that, The spacing between adjacent DNA nanodots is ≤2000nm; And / or, the average diameter of the DNA nanodots is ≤500 nm; And / or, the average height of the DNA nanodots is ≤50 nm.

8. A DNA nanoarray, characterized in that, The DNA nanoarray was prepared using the method described in any one of claims 1 to 7.

9. A biochip, characterized in that, It includes the DNA nanoarray as described in claim 8.

10. A DNA nanoarray fabrication device, characterized in that, Includes scanning device, flexible platform and probe; The preparation apparatus operates according to the method for preparing DNA nanoarrays as described in any one of claims 1 to 7.

Images