DNA Enzymatic Preparation Process and Its Device

Abstract

The present application provides a DNA enzymatic preparation process and a device thereof. The preparation process includes: providing a printing substrate, on which a plurality of wells are arranged, in the X direction, the distance between the center positions of two adjacent wells is L1, and in the Y direction, the distance between the center positions of two adjacent wells is L2; ​​a plurality of nozzles are arranged side by side through a rack, and the angle between the arrangement direction of the plurality of nozzles and the X direction can be adjusted, and the distance between the nozzle openings of two adjacent nozzles is L3, and L1 < L3, L2 < L3; the arrangement direction of the plurality of nozzles is made perpendicular or inclined to the Y direction, so that the ink can be sprayed into the well; after the nozzle moves along the X direction and passes through all the wells on the travel route, it moves along the Y direction to continue printing the wells. The DNA enzymatic preparation device includes a nozzle and a substrate mounting frame for carrying a printing substrate, and the preparation device is used to implement the aforementioned preparation process.

Description

Technical Field

[0001] This application relates to enzymatic or microbiological devices, and particularly to DNA enzymatic preparation processes and their devices. Background Art

[0002] DNA (deoxyribonucleic acid) synthesis is a key common technology in fields such as life science, biomedicine, materials science, and information storage. Currently, DNA synthesis mainly relies on chemical synthesis methods, whose synthesis length and cost have insurmountable limits and cannot meet the growing demands in the field of biofabrication. DNA enzymatic synthesis, on the other hand, has potential that chemical synthesis cannot match in terms of synthesis speed, length, efficiency, and cost, and has become a cutting-edge direction for the development of DNA synthesis technology. DNA enzymatic synthesis mainly involves two aspects: efficiency and accuracy.

[0003] The process of DNA enzymatic synthesis can include using an inkjet printing device to print bases into wells. The wells can be arranged in an array on the printing substrate for easy printing. The printing method can be point-to-point printing, that is, first controlling the print head to move to the well position, stopping the movement, then controlling the print head to eject ink, and then moving to the next well position. In this method, exemplarily, one print head can eject ink into at most two wells within one second, with a working frequency of 2 Hz (Hertz), resulting in low production efficiency.

[0004] In addition, DNA enzymatic synthesis is de novo synthesis, which requires non-template-dependent enzymes to catalyze DNA strand extension. During the synthesis process, multiple bases need to be printed into the wells. To avoid cross-contamination between print heads, non-contact printing is required. However, due to non-contact printing, there will be a certain positional deviation between droplet formation and the actual landing point, resulting in a decrease in accuracy.

[0005] In the context of pursuing high throughput, the density of wells is large, the distance between wells is small, and the distance between print heads is relatively large, which poses a greater challenge to efficiency and accuracy.

[0006] Therefore, there is still room for improvement in DNA enzymatic preparation processes and related devices. Summary of the Invention

[0007] To solve or alleviate at least one problem mentioned in the background art, this application provides a DNA enzymatic preparation process and its device.

[0008] The DNA enzymatic preparation process provided by the embodiments of the present application includes: providing a printing substrate, on which a plurality of wells are arranged in an array along the X direction and the Y direction. In the X direction, the distance between the central positions of two adjacent wells is L1, and in the Y direction, the distance between the central positions of two adjacent wells is L2; providing a frame and a plurality of nozzles, the plurality of nozzles are arranged side by side through the frame, the frame is configured to be rotatable so that the included angle between the arrangement direction of the plurality of nozzles and the X direction can be adjusted, the distance between the nozzle orifices of two adjacent nozzles is L3, and L1 < L3, L2 < L3; making the plurality of nozzles continuously travel in the X direction and controllably eject ink, so that the arrangement direction of the plurality of nozzles is perpendicular or inclined to the Y direction, so that the ink can be ejected into the wells; after the nozzles move along the X direction and pass all the wells on the travel route, move along the Y direction to be able to continue printing the wells.

[0009] In at least one embodiment, the arrangement direction of the plurality of nozzles is inclined to the Y direction, and the plurality of nozzles can respectively correspond to the positions of the plurality of wells.

[0010] In at least one embodiment, the nozzles arranged in the frame are arranged in multiple rows, and the plurality of nozzles can respectively correspond to the positions of the plurality of wells.

[0011] In at least one embodiment, in the X direction, the position deviation between the starting point of inkjet of the nozzle and the actual position where the ink lands on the printing substrate is measured, and inkjet starts in advance at the position deviation of the nozzle from the well.

[0012] In at least one embodiment, the well is formed in a waist shape, the dimension of the well along the X direction is A1, the dimension of the well along the Y direction is A2, A1 < A2, when the nozzle and the printing substrate are relatively stationary, the distance between the nozzle orifice and the printing substrate is A3, and the included angle between the perpendicular line of the nozzle orifice and the printing substrate and the connecting line between the nozzle orifice and the ink on the printing substrate is C, and it is set as: 2×A3×tan C ≤ A2 - A1.

[0013] In at least one embodiment, the printing parameters of the nozzle are set as: 500 μm ≤ A3 ≤ 1000 μm, C ≤ 10°, and the falling speed of the ink in the nozzle is controlled to be greater than 1.5 m / s.

[0014] In at least one embodiment, each nozzle ejects different types of ink.

[0015] In at least one embodiment, during the process that the nozzle moves from one end of the printing substrate to the other end in the X direction and during the process that the nozzle moves from the other end of the printing substrate to one end, printing can be performed.

[0016] In at least one embodiment, the movement relationship between the nozzle and the printing substrate is adjusted such that the nozzle is fixed and the printing substrate can move relative to the nozzle in the X direction and the Y direction.

[0017] The DNA enzymatic method preparation device provided by the embodiments of the present application includes a printing substrate, a substrate mounting rack for carrying the printing substrate, and a nozzle. The preparation device is used to implement the aforementioned DNA enzymatic method preparation process.

[0018] In the present application, a plurality of nozzles are arranged side by side through a frame. The arrangement direction of the plurality of nozzles can be adjusted by rotation, so that the distance between the wells (for example, in the Y direction) is less than the distance between the nozzles. In the case of pursuing high throughput, the ink ejection positions of each nozzle can respectively correspond to the wells, improving the printing efficiency. Description of the Drawings

[0019] Figure 1 Shows a schematic diagram of a flying printing.

[0020] Figure 2A Shows a schematic diagram of two ways of flying printing of the DNA enzymatic method preparation process according to the embodiments of the present application.

[0021] Figure 2B Shows a side view of the nozzle and the printing substrate of the DNA enzymatic method preparation process according to the embodiments of the present application.

[0022] Figure 3 Shows a schematic diagram of flying printing in which each nozzle ejects different kinds of inks in the DNA enzymatic method preparation process according to the embodiments of the present application.

[0023] Figure 4A Shows a schematic diagram of the nozzle moving along the X direction in the DNA enzymatic method preparation process according to the embodiments of the present application.

[0024] Figure 4B Shows a schematic diagram of the ink deflection angle when the nozzle and the printing substrate are relatively stationary in the DNA enzymatic method preparation process according to the embodiments of the present application.

[0025] Figure 5 Shows a comparison diagram of the ink landing points in the wells in the DNA enzymatic method preparation process according to the embodiments of the present application.

[0026] Description of the Reference Numerals

[0027] 100 Printing substrate; 200 Well; 300 Nozzle; 301 First nozzle; 302 Second nozzle; 303 Third nozzle; 304 Fourth nozzle; 310 Nozzle orifice; 400 Nozzle; 500 Printing substrate; 600 Well; 701 First nozzle; 702 Second nozzle. Detailed implementation mode

[0028] The exemplary implementation modes of the present application will be described below with reference to the accompanying drawings. It should be understood that these specific descriptions are only used to teach those skilled in the art how to implement the present application, rather than to exhaust all feasible ways of the present application, nor to limit the scope of the present application.

[0029] Figure 1 A schematic diagram of flying printing is shown. Among them, the nozzle 400 moves uniformly from one side of the printing substrate 500 to the other side along the X direction, and the nozzle 400 sprays ink into the well 600 (groove) at a constant frequency. Compared with the point-to-point printing method of first positioning, then inkjetting, and then shifting introduced in the background technology, flying printing can improve the printing frequency. For example, the droplet generation frequency of the nozzle 400 of Microfab can reach 30 kHz (kilohertz), and the droplet generation frequency of the nozzle 400 of Microdrop can reach 2 kHz. Therefore, flying printing can greatly improve the working efficiency compared with point-to-point printing.

[0030] See Figure 2A and Figure 2B , based on flying printing, the implementation mode of the present application provides a DNA enzymatic preparation process, which may include:

[0031] (S1) Provide a printing substrate 100, on which a plurality of wells 200 are arranged in an array along the X direction and the Y direction. In the X direction, the distance between the central positions of two adjacent wells 200 is L1. In the Y direction, the distance between the central positions of two adjacent wells 200 is L2.

[0032] (S2) Provide a plurality of nozzles 300 and a frame. The plurality of nozzles 300 are arranged side by side through the frame. The frame is configured to be rotatable so that the included angle between the arrangement direction of the plurality of nozzles 300 and the X direction can be adjusted. The distance between the nozzle orifices 310 of two adjacent nozzles 300 is L3, and L1 < L3, L2 < L3. It can be understood that each nozzle 300 has a nozzle orifice 310. The nozzle 300 in the present application can be in the form of a single nozzle.

[0033] (S3) Make the plurality of nozzles 300 continuously travel in the X direction, and the ink is ejected in a controlled manner. The arrangement direction of the plurality of nozzles 300 is perpendicular or inclined to the Y direction so that the ink can be sprayed into the well 200.

[0034] (S4) After the nozzle 300 moves in the X direction and waits for it to pass all the wells 200 on the travel route, it moves in the Y direction so as to be able to continue printing other wells 200.

[0035] For the printing substrate 100, exemplarily, the printing substrate 100 may be a chip, and a plurality of wells 200 are arranged in an array on the chip.

[0036] When synthesizing DNA, it is usually necessary to use a plurality of nozzles 300 to spray various inks (such as bases) into each well 200. Refer to Figure 2A , when a plurality of nozzles 400 are arranged in the Y direction, if L2 = L3 (refer to the example outlined by the dashed line on the left in Figure 2A ), after the nozzle 300 finishes printing a row of wells 200 in the X direction, each time it moves a distance of L2 in the Y direction, it can print the next row of wells 200.

[0037] When pursuing high-throughput DNA synthesis, the setting density of the wells 200 is relatively high, and the distance between the wells 600 is relatively close. Even the distance between the wells 600 in the X direction is less than the distance between the nozzles 300, that is, L2 < L3. In the case of, for example, L3 = 1.5×L2 (refer to the example outlined by the dashed line on the right in Figure 2A ), after the nozzle 300 finishes printing a row of wells 200 in the X direction, when moving in the Y direction, it needs to first move L2 in the Y direction to align the lowermost first nozzle 701 with the well 200. After printing a row of wells 200 in the X direction, it then moves 0.5L in the Y direction to align the second nozzle 702 with the well 200. The distance moved each time in the Y direction is inconsistent. When the first nozzle 701 is printing, the second nozzle 702 is in an idle state, and the efficiency is not high.

[0038] Refer to Figure 3 , in the first embodiment of the present application, the arrangement direction of the plurality of nozzles 300 can be inclined with respect to the Y direction, for example, inclined towards the lower right, and of course, it can also be inclined towards the lower left (not shown in the figure). Four kinds of inks can be applied to each well 200. In the case of L1 < L3, L2 < L3 (the density of the wells 200 is relatively large), the four nozzles 300 can work together. When the first nozzle 301 sprays the fourth row, the second nozzle 302 can simultaneously spray the third row, the third nozzle 303 can simultaneously spray the second row, and the fourth nozzle 304 can simultaneously spray the first row. Obviously, the efficiency is relatively high. At the same time, each time when switching in the Y direction, it only needs to move L2, and the control is simple.

[0039] Refer to Figure 3The nozzles 300 arranged in the bottom row in the present application. In the second embodiment of the present application, multiple nozzles 300 can be arranged along the X direction. By controlling the inkjet time, the ink ejected from each nozzle 300 can fall into the wells 200. And when it is necessary to move along the Y direction, each time when switching along the Y direction, it is only necessary to move by L2, and the control is simple.

[0040] See Figure 2A , in an embodiment of the present application, multiple nozzles 300 respectively correspond to the positions of multiple wells 200. Of course, the inkjet time of each nozzle 300 can be controlled separately so that the ink ejected from the nozzle 300 can fall into the well 200. Therefore, it is also allowed that, for example, three nozzles 300 correspond to three wells 200, and there is still a certain distance between one nozzle 300 and the nearest well 200.

[0041] In an embodiment of the present application, the nozzles 300 arranged in the frame can be arranged in multiple rows (not shown in the figure), and multiple nozzles 300 can respectively correspond to the positions of multiple wells 200. That is, compared with Figure 3 the row of nozzles 300 shown in, multiple rows of nozzles 300 can be set simultaneously to meet the situation of printing more types of ink and increase the printing efficiency.

[0042] See Figure 4A , in the X direction, when the nozzle 300 moves horizontally, there is a first position deviation in the horizontal direction between the inkjet starting point (where the nozzle orifice 310 is located) and the position of the ink on the printing substrate 100, and this first position deviation can be calculated. See Figure 4B , in the stationary state, the actual landing point of the ink will deviate from the vertical point, resulting in a second position deviation.

[0043] For the X direction, the position deviation (the sum of the first position deviation and the second position deviation) between the starting point of inkjet of the nozzle 300 and the position where the ink actually lands on the substrate 100 can be measured, and the inkjet can be started in advance at this position deviation of the nozzle 300 from the well 200 to eliminate this deviation. For example, the calibration amount can be confirmed by taking pictures. When there are multiple nozzles 300, the deviations of each nozzle 300 may also be different, so the calibration amount can be set separately for each nozzle 300.

[0044] For the Y direction, only the second position deviation needs to be considered. The applicant has found that the second position deviation is related to factors such as the type of ink, the nozzle 300, the printing height A3, the horizontal movement speed, the ambient temperature and humidity, etc. In order to enable the ink to smoothly enter the well 200, the size of the well 200 can be optimized.

[0045] In an embodiment of the present application, see Figure 5, the well 200 is formed in a kidney shape. The dimension of the well 200 in the X direction is A1, and the dimension of the well 200 in the Y direction is A2, where A1 < A2.

[0046] For A1, it can be set according to the inkjet mode of the printhead 300. For example, when the printhead 300 uses piezoelectric ceramic inkjet technology, A1 can be selected from 10 to 125 μm. When the printhead 300 uses solenoid valve inkjet, A1 can be selected from 125 to 500 μm. In other cases, 500 to 1000 μm can also be chosen. That is, corresponding dimensions can be selected according to different requirements.

[0047] In an embodiment of the present application, referring to Figure 4B , the distance between the nozzle orifice 310 of the printhead 300 and the printing substrate 100 is A3, and the angle between the perpendicular line B1 from the nozzle orifice 310 to the printing substrate 100 and the connecting line B2 between the nozzle orifice 310 and the printed ink is C, and this angle is also called the deflection angle C. Its parameters can be set such that 2×A3×tan C ≤ A2 - A1. Here, tan C represents the tangent function of the deflection angle C.

[0048] In an example, the present application sets the above parameters as: 500 μm ≤ A3 ≤ 1000 μm, C ≤ 10°, 160 μm ≤ A2 - A1 ≤ 350 μm, and the falling speed of the ink (droplet) is greater than 1.5 m / s.

[0049] Referring to Table 1 below, the present application has conducted experiments on relevant parameters, and the statistical results are as follows.

[0050] Table 1

[0051]

[0052] In an embodiment of the present application, the inkjet mode of the printhead 300 is one of inkjet through solenoid valve, inkjet through pump (micropump), inkjet through piezoelectric inkjet technology, and inkjet through electrohydrodynamic (EHD) inkjet. Exemplarily, for nL (nanoliter)-level droplet dispensing, solenoid valves or pumps can be used; for pL (picoliter)-level droplet dispensing, piezoelectric inkjet technology can be used; for fL (femtoliter)-level droplet dispensing, electrohydrodynamic (EHD) technology can be used. The synthesis of DNA enzymatic method requires setting multiple bases in a well 200, and the inkjet mode of the printhead 300 provided by the present application can achieve non-contact droplet dispensing, avoiding cross-contamination of the printhead 300.

[0053] In an embodiment of the present application, each printhead 300 is used to eject different types of ink, such as ejecting different bases.

[0054] In an embodiment of the present application, during the process of the print head 300 moving from one end of the printing substrate 100 to the other end in the X direction and during the process of moving from the other end of the printing substrate 100 to one end, printing can be performed, that is, bidirectional flying printing can be achieved.

[0055] In an embodiment of the present application, the movement relationship between the print head 300 and the printing substrate 100 can also be adjusted as follows: the print head 300 is fixed, and the printing substrate 100 can move relative to the print head 300 in the X direction and the Y direction.

[0056] The present application also provides a DNA enzymatic method preparation device, which includes a substrate mounting frame for carrying the printing substrate and a print head. The preparation device is used to implement the aforementioned DNA enzymatic method preparation process.

[0057] The above are the preferred embodiments of the present application. It should be noted that for those skilled in the art, without departing from the principle of the present application, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A DNA enzymatic preparation process, characterized in that: The preparation process comprises: A printing substrate (100) is provided, wherein a plurality of wells (200) are arranged in an array along an X direction and a Y direction on the printing substrate (100), wherein in the X direction, the distance between the center positions of two adjacent wells (200) is L1, and in the Y direction, the distance between the center positions of two adjacent wells (200) is L2; A rack and a plurality of nozzles (300) are provided, wherein the plurality of nozzles (300) are arranged side by side via the rack, and the rack is configured to be rotatable so that the angle between the arrangement direction of the plurality of nozzles (300) and the X direction can be adjusted, and the distance between the nozzle openings (310) of two adjacent nozzles (300) is L3, and L1<L3, and L2<L3; The plurality of nozzles (300) are caused to continuously move in the X direction and to eject ink in a controlled manner, so that the arrangement direction of the plurality of nozzles (300) is perpendicular or inclined to the Y direction, so that the ink can be ejected into the well (200); After the nozzle (300) moves along the X direction and passes through all the wells (200) on the path, it moves along the Y direction to continue printing on the wells (200). The well (200) is formed in a waist shape, the dimension of the well (200) along the X direction is A1, the dimension of the well (200) along the Y direction is A2, A1<A2, when the nozzle (300) and the printing substrate (100) are relatively stationary, the distance between the nozzle opening (310) and the printing substrate (100) is A3, and the angle between the perpendicular line (B1) between the nozzle opening (310) and the printing substrate (100) and the connecting line (B2) between the nozzle opening (310) and the ink on the printing substrate (100) is C, which is set to: 2×A3×tan C≤A2-A1.

2. The DNA enzymatic preparation process according to claim 1, characterized in that: The arrangement direction of the plurality of nozzles (300) is inclined to the Y direction, and the plurality of nozzles (300) can correspond to the positions of the plurality of traps (200) respectively.

3. The DNA enzymatic preparation process according to claim 1, characterized in that: The nozzles (300) arranged in the frame are arranged in multiple rows, and the multiple nozzles (300) can correspond to the positions of the multiple wells (200) respectively.

4. The DNA enzymatic preparation process according to claim 1, characterized in that: In the X direction, the position deviation between the position where the nozzle (300) starts to spray ink and the position where the ink actually lands on the printing substrate (100) is calculated, and the nozzle (300) starts to spray ink in advance at the position deviation from the well (200).

5. The DNA enzymatic preparation process according to claim 1, characterized in that: The printing parameters of the nozzle (300) are set to: 500 μm≤A3≤1000 μm, C≤10°, and the ink falling speed in the nozzle (300) is controlled to be greater than 1.5 m / s.

6. The DNA enzymatic preparation process according to claim 1, characterized in that: Each of the nozzles (300) sprays different types of ink.

7. The DNA enzymatic preparation process according to claim 1, characterized in that: Printing can be performed in the process of the nozzle (300) moving from one end of the printing substrate (100) to the other end, and in the process of moving from the other end of the printing substrate (100) to one end in the X direction.

8. The DNA enzymatic preparation process according to claim 1, characterized in that: The movement relationship between the nozzle (300) and the printing substrate (100) is adjusted so that the nozzle (300) is fixed and the printing substrate (100) can move in the X direction and the Y direction relative to the nozzle (300).

9. A DNA enzymatic preparation device, characterized in that: It comprises a printing substrate (100), a substrate mounting frame for carrying the printing substrate (100), and a nozzle (300), wherein the preparation device is used to implement the DNA enzymatic preparation process according to any one of claims 1 to 8.

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