PCR (Polymerase Chain Reaction) reagent drying method based on glass carrier and low-temperature vacuum and micro-fluidic chip

Abstract

The invention provides a PCR (Polymerase Chain Reaction) reagent drying method based on a glass carrier and low-temperature vacuum and a microfluidic chip, which comprises the following steps: laser modification: locally carrying out laser modification on glass subjected to coating hydrophobic treatment, and taking the part subjected to laser modification as an amplification reaction area; constant-temperature preheating: preheating the glass subjected to laser modification in a constant-temperature bath at 4-37 DEG C for 10-30 minutes; preparing a reagent: preparing a PCR reagent; dropwise adding a reagent: placing the preheated glass on a constant temperature table with the laser modified surface facing upwards, and dropwise adding the prepared PCR reagent in an amplification reaction area; vacuum drying: carrying out vacuum drying on the glass dropwise added with the PCR reagent under the conditions of-100kPa to-90kPa and 4 DEG C to 37 DEG C for 10s to 120s, and dropwise adding a liquid sealing protective agent after drying is completed, so as to prepare the micro-fluidic chip. The drying method is high in reagent drying efficiency, good in reagent performance, high in reagent uniformity, high in reagent processing efficiency and high in reagent drying repeatability.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a PCR reagent drying method based on a glass carrier and low-temperature vacuum, and a microfluidic chip. Background Art

[0002] POCT (Point-of-Care Testing) refers to point-of-care testing, which can quickly detect certain biochemical, immune or infectious indicators of patients at the medical site for timely diagnosis and treatment. It was originally used in only a few projects such as blood sugar and blood gas analysis, but has now been expanded to a wider range of fields, such as myocardial markers, tumor markers, bacterial or viral nucleic acids, etc. The advantages of POCT are that it is fast, simple and portable, which can reduce patient waiting time and improve medical efficiency. In order to meet the requirements of POCT, reagents are generally pre-stored in POCT products to reduce the time of manual operation during use.

[0003] POCT molecular diagnostic products often use PCR detection methods. PCR reagents are usually stored in advance by drying in a blast drying oven or freeze drying in a freeze dryer. Freeze drying reagents in freeze dryers are expensive, cumbersome to operate, and are extremely susceptible to moisture, requiring a demanding product processing environment. Although blast drying reagents reduces costs compared to freeze drying methods, the drying temperature and drying time are high. Drying a 1μL to 30μL reaction system requires 15 minutes to 120 minutes at a temperature of 4°C to 80°C. Long-term high-temperature drying affects the performance of the reagents.

[0004] Both of the above two commonly used methods have certain problems, so it is necessary to invent a method that is low in cost, simple to process, and can ensure the performance of the reagent. Summary of the invention

[0005] The first purpose of the present invention is to provide a PCR reagent drying method based on a glass carrier and low-temperature vacuum, which is used to solve the problems of high cost, complex processing and decreased reagent performance after drying in existing reagent drying methods.

[0006] The second object of the present invention is to provide a microfluidic chip, comprising a cover plate, wherein the cover plate is made of glass for reagent drying using the above-mentioned PCR reagent drying method based on a glass carrier and low-temperature vacuum.

[0007] In order to achieve the above first object, the present invention provides a PCR reagent drying method based on a glass carrier and low temperature vacuum, comprising the following steps:

[0008] (1) Preparation before drying:

[0009] Laser modification: laser modification is performed on a part of the glass that has been subjected to hydrophobic coating treatment. The part subjected to laser modification is the amplification reaction area.

[0010] Constant temperature preheating: preheat the laser-modified glass in a constant temperature bath at 4°C to 37°C for 10 to 30 minutes;

[0011] Prepare reagents: prepare PCR reagents;

[0012] (2) Drying

[0013] Add reagents: Place the preheated glass with the laser-modified surface facing upward on a constant temperature platform at 4°C to 37°C, and add the prepared PCR reagents to the amplification reaction area;

[0014] Vacuum drying: The glass with the PCR reagent added is vacuum dried at -100 kPa to -90 kPa and 4°C to 37°C for 10s to 120s.

[0015] It can be seen from the above scheme that the glass is laser modified before drying to enhance the hydrophilicity of the glass in the amplification reaction area, so that the glass can be naturally flattened after subsequent reagent dripping, and the reagent uniformity is high. The glass is preheated at a constant temperature, and the glass is also placed on a constant temperature table for subsequent operations to reduce the impact of room temperature on reagent drying, reduce batch differences, and the reagent drying is highly repeatable. Drying is carried out under vacuum drying at -100kPa to -90kPa and 4°C to 37°C for 10s to 120s. The reagent drying efficiency is high, and the drying is carried out at a low temperature, which has no effect on the reagent performance, and the reagent performance is good. At the same time, the reagent after drying is in a glassy state and has a certain moisture resistance. The drying method of the present invention does not require special equipment and instruments, has low cost, and is simple to operate.

[0016] A further solution is that the drying treatment further includes the step of dripping a protective agent, and the step of dripping the protective agent is performed after the step of vacuum drying. The step of dripping the protective agent is specifically as follows:

[0017] The vacuum-dried glass was placed on a constant temperature platform at 4°C to 37°C, and 2 μL to 15 μL of liquid sealing protective agent was added dropwise to the amplification reaction area.

[0018] It can be seen from the above scheme that after drying, a liquid sealing protective agent is dripped onto the reagent in the glassy state to isolate it from the air as much as possible.

[0019] A further solution is that the step of adding the protective agent is to place the vacuum-dried glass on a constant temperature table at 35° C. and to add 4 μL of the liquid sealing protective agent to the amplification reaction area.

[0020] As can be seen from the above scheme, adding a protective agent reduces the contact between the reagent and the air after the reagent is dried, reduces the influence of air and humidity on the reagent, and plays a liquid sealing effect. The amount of the protective agent added is moderate so that the protective agent can adhere to the glass pre-stored with the dried reagent, and the protective agent can be directly used for chip production after adding it.

[0021] A further solution is that the liquid seal protective agent is a liquid reagent insoluble in water, and the liquid seal protective agent contains mineral oil.

[0022] It can be seen from the above scheme that the above liquid sealing protective agent has no effect on the PCR reaction and can well isolate air and water.

[0023] A further solution is that the laser modification step specifically includes using a laser modification machine to perform laser modification on a part of the glass that has been subjected to the hydrophobic coating treatment.

[0024] As can be seen from the above scheme, by using a laser modification machine to modify the glass, there is no need to prepare additional special equipment, thus reducing cost expenditure. After modification, the amplification reaction area of ​​the glass becomes hydrophilic. On the one hand, the reagent drips onto the glass and is naturally flattened, ensuring the uniformity of the reagent while improving the processing efficiency without manual flattening. On the other hand, after flattening, the reagent has a large drying area, the water in the reagent is easier to evaporate, and the drying efficiency is high.

[0025] In a further embodiment, the PCR reagents include one or more of primers, probes, dNTPs, salts, buffers, drying protectants, enzymes and RNase inhibitors.

[0026] It can be seen from the above scheme that the reagent drying method of the present invention is applicable to multiple types of PCR reagents and has a wide range of applications.

[0027] A further solution is that the constant temperature preheating step is to preheat the laser modified glass in a constant temperature bath at 35° C. for 10 minutes.

[0028] A further solution is that the step of adding reagents is to place the preheated glass with the laser-modified surface facing upward on a constant temperature table at 35° C., and to add the prepared PCR reagents to the amplification reaction area.

[0029] It can be seen from the above scheme that treating the glass at a constant temperature can reduce the influence of room temperature on reagent drying, reduce the difference between batches of dried reagents, and improve the repeatability of reagent drying.

[0030] A further solution is that the vacuum drying step is to vacuum dry the glass to which the PCR reagent is added at -95 kPa and 35° C. for 40 seconds.

[0031] It can be seen from the above scheme that in a vacuum state or a low pressure state, the boiling point of water and other volatile components in the reagent is reduced, and they can be separated from the surface of the reagent at a relatively low temperature and pumped away by the vacuum pump, thus avoiding the influence of high temperature on the performance of the reagent.

[0032] To achieve the above second purpose, the present invention provides a microfluidic chip, including a cover plate, which is made of glass for reagent drying using a PCR reagent drying method based on a glass carrier and low-temperature vacuum according to any of the above schemes. DETAILED DESCRIPTION

[0033] The PCR reagent drying method based on glass carrier and low temperature vacuum provided in the embodiment of the present invention comprises the following steps:

[0034] (1) Preparation before drying:

[0035] Laser modification: laser modification is performed on a part of the glass that has been subjected to hydrophobic coating treatment. The part subjected to laser modification is the amplification reaction area.

[0036] Constant temperature preheating: preheat the laser-modified glass in a constant temperature bath at 4°C to 37°C for 10 to 30 minutes;

[0037] Prepare reagents: prepare PCR reagents;

[0038] (2) Drying

[0039] Add reagents: Place the preheated glass with the laser-modified surface facing upward on a constant temperature platform at 4°C to 37°C, and add the prepared PCR reagents to the amplification reaction area;

[0040] Vacuum drying: vacuum dry the glass with the PCR reagent added at -100 kPa to -90 kPa and 4°C to 37°C for 10s to 120s.

[0041] The drying process further includes the step of dripping a protective agent, which is performed after the vacuum drying step. The specific step of dripping a protective agent is as follows:

[0042] The vacuum-dried glass is placed on a thermostat at 4°C to 37°C, and 2 μL to 15 μL of a liquid seal protectant is dripped into the amplification reaction area. The liquid seal protectant is a liquid reagent insoluble in water, and contains mineral oil.

[0043] PCR reagents include one or more of primers, probes, dNTPs, salts, buffers, drying protectants, enzymes and RNase inhibitors.

[0044] The glass obtained by drying the reagent by the PCR reagent drying method of the embodiment of the present invention can be used to prepare the cover plate of the microfluidic chip.

[0045] The names and abbreviations of the detection methods, instruments and equipment involved in the embodiments and comparative examples of the present invention are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Technical personnel in the field can understand the conventional detection methods based on the names and apply the corresponding equipment, and implement them according to conventional conditions or conditions recommended by the manufacturer.

[0046] The various raw materials or reagents used in the examples and comparative examples of the present invention have no special restrictions on their sources, and are all conventional products that can be purchased commercially.

[0047] The present invention will be further described below with reference to the embodiments.

[0048] Example 1: PCR reagent drying method based on glass carrier and low temperature vacuum

[0049] The PCR reagent drying method of this embodiment specifically comprises the following steps:

[0050] (1) Preparation before drying:

[0051] Laser modification: laser modification is performed on a part of the glass that has been subjected to hydrophobic coating treatment. The part subjected to laser modification is the amplification reaction area.

[0052] Constant temperature preheating: Preheat the laser-modified glass in a constant temperature bath at 35°C for 10 minutes;

[0053] Prepare reagents: prepare PCR reagents. The PCR reagents in this embodiment include one or more of primers, probes, dNTPs, salts, buffers, drying protectants, enzymes and RNase inhibitors, which are any of the PCR reagents well known to those skilled in the art.

[0054] (2) Drying

[0055] Add reagents: Place the preheated glass with the laser-modified surface facing upward on a 35°C constant temperature platform, and add the prepared PCR reagents to the amplification reaction area;

[0056] Vacuum drying: The glass with PCR reagent added was vacuum dried at -95 kPa and 35 °C for 40 s;

[0057] Add protective agent: Place the vacuum-dried glass on a 35°C constant temperature platform, and add 4 μL of liquid seal protective agent to the amplification reaction area, wherein the liquid seal protective agent in this embodiment is a water-insoluble liquid reagent containing mineral oil.

[0058] The method of air drying specifically includes dropping the PCR reagent onto the glass, and air drying the reagent for 30 minutes in an air drying oven at 50°C and 1m / s. The glass with a glassy reagent formed on the surface after drying is placed on a 50°C constant temperature table, and 4μL of a liquid sealing protective agent is dripped into the glass amplification reaction area. The air-dried glass is used for the subsequent preparation of microfluidic chips. Among them, the PCR reagent in this comparative example is the same as the PCR reagent in Example 1, the glass is glass that has undergone the same laser modification and constant temperature preheating as in Example 1, and the liquid sealing protective agent is the same as the liquid sealing protective agent in Example 1.

[0059] The glass subjected to drying treatment in Example 1 and Comparative Example 1 was prepared into a cover plate of a microfluidic chip in the same manner, and further prepared into a microfluidic chip in the same manner.

[0060] PCR detection was performed on Example 1 and Comparative Example 1 respectively, and the PCR amplification program is as shown in Table 1 below:

[0061] Table 1

[0062]

[0063] The HEX channel template amount was set to 10,000 copies of the new coronavirus pseudovirus nucleic acid sample per reaction, and the microfluidic chips prepared in Example 1 and Comparative Example 1 were added respectively, and the control group 1 was set to be a microfluidic chip with a liquid reagent that was not dried and to which 10,000 copies of the new coronavirus pseudovirus nucleic acid sample per reaction had been added.

[0064] The amplification results are shown in Table 2 below:

[0065] Reagent Type Ct CV Example 1 28.16 1.21％ Comparative Example 1 30.71 2.09％ Control group 1 28.19 0.92％

[0066] After the above chip was subjected to PCR amplification reaction, the Ct value of the amplification reaction of the reagent in Example 1 was lower than that of the reagent in Comparative Example 1, indicating that the performance of the reagent in Example 1 was better than that of the reagent in Comparative Example 1, and the performance of the reagent in Example 1 was basically consistent with that of the reagent in Control Group 1.

[0067] In the process of drying the reagent, the lower the drying temperature and the shorter the drying time, the smaller the impact on the immediate performance of the reagent. In this embodiment 1, the vacuum drying time is only 40 seconds and the drying temperature is 35°C, while the blast drying time in comparative example 1 is 30 minutes and the drying temperature is 50°C.

[0068] Next, the PCR stability test of Example 1 was performed. The specific test method was as follows: the dried glass of Example 1 was made into a microfluidic chip, which was placed in an antistatic bag with a bag of desiccant inside for sealing. The chip was placed in a 4°C refrigerator and taken out for testing at 0 days, 7 days, 14 days, 21 days and 28 days respectively. The PCR amplification program is as shown in Table 3 below:

[0069] Table 3

[0070]

[0071]

[0072] The HEX channel template amount was set to 10,000 copies of the new coronavirus pseudovirus nucleic acid sample per reaction, and a chip made of vacuum-dried reagent was added. The control group 2 was a chip that was not dried and had liquid reagents with 10,000 copies of the new coronavirus pseudovirus nucleic acid sample added per reaction.

[0073] The amplification results are shown in Table 4 below:

[0074] Table 4

[0075] Number of days Example 1Ct Control group 2Ct Day 0 30.58 30.56 7 days 30.33 30.27 14 days 30.49 30.38 21 days 30.28 30.44 28 days 30.40 30.32

[0076] After the above chip was subjected to PCR amplification reaction, the difference in Ct values ​​of the amplification reaction of the reagent in Example 1 at 0 days, 7 days, 14 days, 21 days and 28 days was within 0.5, and the performance of the reagent in Example 1 was basically consistent with that of the reagent in Control Group 2, indicating that the reagent in Example 1 had relatively good stability.

[0077] It can be seen from the above experimental data that the PCR reagent drying method based on glass carrier and low-temperature vacuum in this embodiment performs laser modification on the glass before drying, so that the hydrophilicity of the glass in the amplification reaction area is enhanced, and the glass can be naturally flattened after subsequent reagent dripping, and the reagent uniformity is high. The glass is preheated at a constant temperature, and the glass is also placed on a constant temperature table for subsequent operations to reduce the influence of room temperature on reagent drying, and the reagent drying is highly repeatable. Drying is carried out under vacuum drying at -95kPa and 35°C for 40s, and the reagent drying efficiency is high. Drying is carried out at a low temperature, which has no effect on the performance of the reagent, and the reagent performance is good and the stability is good. At the same time, the reagent after drying is in a glassy state, and a liquid seal protective agent is added to give it a certain degree of moisture resistance. In addition, the drying method of the present invention does not require special equipment and instruments, has low cost, and is simple to operate.

[0078] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A PCR reagent drying method based on glass carrier and low temperature vacuum, characterized in that: The following steps are involved: (1) Preparation before drying: Laser modification: laser modification is performed on a part of the glass that has been subjected to hydrophobic coating treatment. The part subjected to laser modification is the amplification reaction area. Constant temperature preheating: preheat the laser-modified glass in a constant temperature bath at 4°C to 37°C for 10 to 30 minutes; Prepare reagents: prepare PCR reagents; (2) Drying Add reagents: Place the preheated glass with the laser-modified surface facing upward on a constant temperature platform at 4°C to 37°C, and add the prepared PCR reagents to the amplification reaction area; Vacuum drying: The glass with the PCR reagent added is vacuum dried at -100 kPa to -90 kPa and 4°C to 37°C for 10s to 120s.

2. A PCR reagent drying method based on a glass carrier and low temperature vacuum as claimed in claim 1, characterized in that: The drying process further includes the step of dripping a protective agent, which is performed after the vacuum drying step. The step of dripping a protective agent is specifically as follows: The vacuum-dried glass was placed on a constant temperature platform at 4°C to 37°C, and 2 μL to 15 μL of a liquid sealing protective agent was dripped into the amplification reaction area.

3. A PCR reagent drying method based on a glass carrier and low temperature vacuum as claimed in claim 2, characterized in that: The step of adding the protective agent is to place the vacuum-dried glass on a constant temperature platform at 35° C., and to add 4 μL of the liquid sealing protective agent to the amplification reaction area.

4. A PCR reagent drying method based on a glass carrier and low temperature vacuum as claimed in claim 2, characterized in that: The liquid seal protective agent is a liquid reagent insoluble in water, and the liquid seal protective agent contains mineral oil.

5. The method for drying PCR reagents based on a glass carrier and low-temperature vacuum as claimed in claim 1, characterized in that: The laser modification step specifically includes using a laser modification machine to perform laser modification on a part of the glass that has been subjected to the coating hydrophobic treatment.

6. A PCR reagent drying method based on a glass carrier and low temperature vacuum as claimed in claim 1, characterized in that: The PCR reagents include one or more of primers, probes, dNTPs, salts, buffers, drying protectants, enzymes and RNase inhibitors.

7. A PCR reagent drying method based on a glass carrier and low temperature vacuum as claimed in any one of claims 1 to 6, characterized in that: The step of constant temperature preheating is to preheat the laser-modified glass in a constant temperature bath at 35° C. for 10 minutes.

8. A PCR reagent drying method based on a glass carrier and low temperature vacuum as claimed in any one of claims 1 to 6, characterized in that: The step of adding reagents is to place the preheated glass with the laser-modified surface facing upward on a constant temperature table at 35° C., and to add the prepared PCR reagents to the amplification reaction area.

9. A PCR reagent drying method based on a glass carrier and low temperature vacuum as claimed in any one of claims 1 to 6, characterized in that: The vacuum drying step is to vacuum dry the glass to which the PCR reagent is added at -95 kPa and 35° C. for 40 seconds.

10. A microfluidic chip, comprising a cover plate, wherein the cover plate is made of glass for drying reagents using a PCR reagent drying method based on a glass carrier and low-temperature vacuum as described in any one of claims 1 to 9.

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