A microsphere freeze-drying process for microfluidic reagent trays

Abstract

The present invention provides a microsphere freeze-drying process for a microfluidic reagent tray, comprising the steps of preparing a reagent mixture, adding a mixture of mannitol, isopropyl alcohol, lactose, proline, and lysine at a 1:1 volume ratio to the reagent mixture, dripping the mixture into liquid nitrogen to form microspheres, and then freeze-drying the mixture to obtain freeze-dried microspheres. The microsphere freeze-drying process of the present invention achieves good stability, long shelf life, and high detection sensitivity and accuracy by more precisely controlling the reagents and operating parameters involved in the freeze-drying process.

Description

Technical Field

[0001] The present invention relates to the application field of microfluidic reagent discs, and in particular to a microsphere freeze-drying process for microfluidic reagent discs. Background Art

[0002] Microfluidic reagent discs primarily process or manipulate tiny fluids through microfluidics, enabling whole blood separation. They offer high integration, fast detection speed, high accuracy, and low cost. Furthermore, the disc design allows for parallel operation, enabling simultaneous microfluidic control and detection of multiple detection units. This facilitates automation and is suitable for multi-index or multi-sample testing, enabling medium- and high-throughput sample analysis.

[0003] Problems in any link during the operation of the reagent disc will affect the accuracy of the entire test, especially when the stability of the reagent microspheres is poor, which will directly affect the detection efficiency of the entire microfluidic reagent disc and also affect the sensitivity and accuracy of the final sample detection. Summary of the Invention

[0004] The present invention aims to provide a microsphere freeze-drying process for a microfluidic reagent tray. By more precisely controlling the reagents and operating parameters involved in the freeze-drying process, the resulting freeze-dried microspheres have good stability during subsequent use, a long shelf life, high detection sensitivity and accuracy, and strong resolubility, allowing them to dissolve quickly when encountering sample liquid.

[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0006] The present invention provides a microsphere freeze-drying process for a microfluidic reagent tray, comprising the following steps:

[0007] A reagent mixture was prepared, and a mixture of mannitol, isopropanol, lactose, proline and lysine was added at a volume ratio of 1:1 to the reagent mixture, and the mixture was dropped into liquid nitrogen to form microspheres, which were then freeze-dried to obtain freeze-dried microspheres.

[0008] Preferably, as a further feasible solution, the ingredients are, by mass, 7-10 parts of mannitol, 4-6 parts of isopropanol, 3-7 parts of lactose, 2-10 parts of proline, and 2-5 parts of lysine.

[0009] Preferably, as a further feasible solution, the ingredients are, by mass, 8-9 parts of mannitol, 4.5-5.5 parts of isopropanol, 4-6 parts of lactose, 4-8 parts of proline, and 3-4 parts of lysine.

[0010] Preferably, as a further feasible solution, the ingredients include 8 parts of mannitol, 5 parts of isopropanol, 5 parts of lactose, 6 parts of proline, and 3 parts of lysine.

[0011] In the microsphere freeze-drying process of the present invention, mannitol, isopropyl alcohol, lactose, proline and lysine are added to the actual mixed solution, thereby improving the stability of the active ingredient itself. In particular, the mutual matching of proline and lysine achieves a good antifreeze effect. The compounding of mannitol and isopropyl alcohol improves the stability of the active ingredient, plays a good protective role for the active ingredient itself, and prevents drastic changes in pH during the freezing process. The addition of lactose can enhance the stabilizing effect and prevent the active ingredient from undergoing structural changes during the freeze-drying process. Moreover, the addition of the above ingredients will not affect the detection function of the reagent itself.

[0012] Of course, through continuous practice, we discovered that the specific ingredients in the compound and the controlled dosage of each ingredient were optimized, especially the specific combinations of mannitol and isopropyl alcohol, and proline and lysine. Substituting other components did not produce such a good synergistic effect. The dosage was also continuously explored based on the stability of the microspheres during the specific operation process. In terms of the dosage of the reagent mixture, the mannitol and isopropyl alcohol combination can be relatively increased in mannitol, while the proline and lysine combination can be increased in proline. Following this trend, the stability, formability, and accuracy of subsequent testing of the freeze-dried microspheres can be significantly improved.

[0013] Preferably, as a further feasible solution, the freeze-drying procedure is carried out in multiple stages, wherein the first freeze-drying stage is carried out for 3-4 hours at a temperature of -35 to -50°C.

[0014] Preferably, as a further feasible solution, the freeze-drying procedure is carried out in three stages, the second freeze-drying stage is 7-8 hours, the temperature is -20 to -30°C, and the third freeze-drying stage is the drying stage, the time is 2-3 hours, and the temperature is 10-30°C.

[0015] Preferably, as a further feasible solution, the freeze-drying process is carried out under a vacuum degree of 50-150 ubar.

[0016] In order to improve the stability of the microspheres, the present invention implements procedural control over the entire freeze-drying process, which is divided into multiple stages. The freeze-drying temperature and time are adjusted in each stage. By precisely controlling the temperature and time, the active ingredients of the microspheres are not easily lost, cracked, or cracked during the freeze-drying process, and the microspheres have good formability.

[0017] Preferably, as a further feasible solution, bamboo vinegar and citric acid are added to the reagent mixture.

[0018] Preferably, as a further feasible solution, the amount of bamboo vinegar added is 0.1-0.5 parts by mass.

[0019] Preferably, as a further feasible solution, the amount of citric acid added is 0.3-0.7 parts by mass.

[0020] In the above scheme, the freeze-dried microspheres are dried under vacuum during the operation and are therefore less affected by oxygen. If the microspheres are taken out of the box and exposed to air, the oxygen in the air will immediately invade the gaps between the dried products, and some active groups will quickly combine with oxygen, causing irreversible effects on the products. To avoid the above situation, some products in the prior art use inert gases, such as nitrogen, for sealing, and some products use vacuum sealing. However, this method is not long-lasting. Once exposed to air, it will affect the quality of the microsphere reagent itself, and it is easy for some active ingredients in the reagent to be affected by oxygen when used, affecting the accuracy. The present invention adds bamboo vinegar and citric acid during the freeze-drying process, and through the synergistic effect of the two, the stability of the microspheres themselves is enhanced, and the antioxidant property thereof is improved. It is found that even if the microspheres are exposed to air, the quality of the microspheres themselves is not affected, thereby ensuring that the entire detection process is carried out accurately. Of course, after trial and error, it was found that the addition amounts of bamboo vinegar and citric acid do not need to be too large, and only a small amount is needed to produce the corresponding effect. Excessive addition not only affects the active components of the microspheres themselves, but is also not conducive to the subsequent storage of the freeze-dried microspheres. Therefore, the specific addition amount still needs to be controlled within an appropriate range.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The reagents and operating parameters involved in the microsphere freeze-drying process of the present invention are more precisely controlled, so that the obtained freeze-dried microspheres have good stability during subsequent use, long storage time, and high detection sensitivity and accuracy.

[0023] (2) The microspheres prepared by the microsphere freeze-drying process of the present invention have extremely strong resolubility and can dissolve quickly when encountering the sample liquid.

[0024] (3) The microspheres prepared by the freeze-drying process of the present invention are not easily affected by oxygen and thus affect the quality of the microspheres even if they are stored in an air atmosphere, which reduces the difficulty of storage and improves the stability of the microspheres themselves. DETAILED DESCRIPTION

[0025] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0026] Example 1

[0027] The freeze-drying method of microspheres in microfluidic reagent tray is as follows:

[0028] (1) Take the raw materials containing biological reagents out of the freezer and place them at room temperature to slowly thaw. Do not use heating to thaw to prevent the biological materials from breaking;

[0029] (2) Place the mixture in a blender for high-speed stirring, centrifuge at 4°C and 8000 rpm to collect the supernatant, discard the precipitate and other substances formed, magnetically stir for 20 minutes, and centrifuge at 4°C and 12000 rpm to collect the supernatant to obtain the biological reagent extract;

[0030] (3) Weigh 0.5 g of bamboo vinegar, 0.3 g of citric acid, 7 g of mannitol, 6 g of isopropyl alcohol, 3 g of lactose, 10 g of proline, 2 g of lysine, and 1000 ml of purified water, stir and dissolve them, and filter the resulting solution using a filter membrane;

[0031] (4) mixing the biological reagent extract formed in step (2) with the active ingredient in the solution of step (3) at a volume ratio of 1:1, dropping the mixture into liquid nitrogen to form microspheres, and freeze-drying the microspheres;

[0032] (5) The freeze-drying procedure was carried out in three stages: the first freeze-drying stage lasted 3 h at a temperature of −35°C, the second freeze-drying stage lasted 7 h at a temperature of −20°C, and the third freeze-drying stage was a drying stage lasting 2 h at a temperature of 30°C with a vacuum degree between 50 and 150 ubar;

[0033] (6) The freeze-dried microspheres obtained in the above steps are sealed and stored, and can be directly used in a microfluidic reagent plate for sample detection.

[0034] Example 2

[0035] The freeze-drying method of microspheres in microfluidic reagent tray is as follows:

[0036] (1) Take the raw materials containing biological reagents out of the freezer and place them at room temperature to slowly thaw. Do not use heating to thaw to prevent the biological materials from breaking;

[0037] (2) Place the mixture in a blender for high-speed stirring, centrifuge at 4°C and 8000 rpm to collect the supernatant, discard the precipitate and other substances formed, magnetically stir for 20 minutes, and centrifuge at 4°C and 12000 rpm to collect the supernatant to obtain the biological reagent extract;

[0038] (3) Weigh 0.1 g of bamboo vinegar, 0.7 g of citric acid, 10 g of mannitol, 4 g of isopropyl alcohol, 7 g of lactose, 2 g of proline, 5 g of lysine, and 1000 ml of purified water, stir and dissolve them, and filter the resulting solution using a filter membrane;

[0039] (4) mixing the biological reagent extract formed in step (2) with the active ingredient in the solution of step (3) at a volume ratio of 1:1, dropping the mixture into liquid nitrogen to form microspheres, and freeze-drying the microspheres;

[0040] (5) The freeze-drying procedure was carried out in three stages: the first freeze-drying stage lasted 4 h at a temperature of −50°C, the second freeze-drying stage lasted 8 h at a temperature of −30°C, and the third freeze-drying stage was a drying stage lasting 3 h at a temperature of 10°C with a vacuum degree between 50 and 150 ubar;

[0041] (6) The freeze-dried microspheres obtained in the above steps are sealed and stored, and can be directly used in a microfluidic reagent plate for sample detection.

[0042] Example 3

[0043] The freeze-drying method of microspheres in microfluidic reagent tray is as follows:

[0044] (1) Take the raw materials containing biological reagents out of the freezer and place them at room temperature to slowly thaw. Do not use heating to thaw to prevent the biological materials from breaking;

[0045] (2) Place the mixture in a blender for high-speed stirring, centrifuge at 4°C and 8000 rpm to collect the supernatant, discard the precipitate and other substances formed, magnetically stir for 20 minutes, and centrifuge at 4°C and 12000 rpm to collect the supernatant to obtain the biological reagent extract;

[0046] (3) Weigh 0.3 g of bamboo vinegar, 0.4 g of citric acid, 8 g of mannitol, 5.5 g of isopropyl alcohol, 4 g of lactose, 8 g of proline, 3 g of lysine, and 1000 ml of purified water, stir and dissolve them, and filter the resulting solution using a filter membrane;

[0047] (4) mixing the biological reagent extract formed in step (2) with the active ingredient in the solution of step (3) at a volume ratio of 1:1, dropping the mixture into liquid nitrogen to form microspheres, and freeze-drying the microspheres;

[0048] (5) The freeze-drying procedure was carried out in three stages: the first freeze-drying stage lasted 4 h at a temperature of −45°C, the second freeze-drying stage lasted 8 h at a temperature of −25°C, and the third freeze-drying stage was a drying stage lasting 3 h at a temperature of 20°C with a vacuum degree between 50 and 150 ubar;

[0049] (6) The freeze-dried microspheres obtained in the above steps are sealed and stored, and can be directly used in a microfluidic reagent plate for sample detection.

[0050] Example 4

[0051] The specific operation steps are consistent with those of Example 3, except that 0.4 g of bamboo vinegar, 0.5 g of citric acid, 9 g of mannitol, 4.5 g of isopropyl alcohol, 6 g of lactose, 4 g of proline, 4 g of lysine, and 1000 ml of purified water are stirred and dissolved.

[0052] Example 5

[0053] The specific operation steps are consistent with those of Example 3, except that 0.4 g of bamboo vinegar, 0.5 g of citric acid, 8 g of mannitol, 5 g of isopropyl alcohol, 5 g of lactose, 6 g of proline, 3 g of lysine, and 1000 ml of purified water are stirred and dissolved.

[0054] Example 6

[0055] The specific operation steps are consistent with Example 3, except that bamboo vinegar and citric acid are not added.

[0056] Example 7

[0057] The specific operation steps are the same as those in Example 3, except that 3 g of mannitol is used.

[0058] Example 8

[0059] The specific operation steps are the same as those in Example 3, except that 10 g of lysine is used.

[0060] Comparative Example 1

[0061] The specific operation steps were the same as those in Example 3, except that proline was replaced by leucine.

[0062] Comparative Example 2

[0063] The specific operation steps are the same as those in Example 3, except that isopropyl alcohol is replaced with ethylene glycol.

[0064] Example 9

[0065] The specific operation steps are consistent with those in Example 3, except that the freeze-drying procedure is divided into two stages. The first freeze-drying stage is carried out for 4 hours at a temperature of -45°C, and the second freeze-drying stage is carried out for 3 hours at a temperature of 20°C and a vacuum degree of 50-150 ubar.

[0066] Experimental Example 1

[0067] The stability of the microspheres of the above-mentioned embodiments and comparative examples was tested. The specific results are as follows. The stability was demonstrated by measuring the moisture absorption rate of the microspheres after being placed in air environments with different humidity for 24 hours, and the changes in their appearance after being exposed to air environments with different temperatures for 48 hours. The dry environment refers to an environment with humidity below 3%.

[0068] Table 1 Experimental results

[0069]

[0070]

[0071] From the data in Table 1 above, it can be seen that the microspheres prepared in each embodiment and comparative example are exposed to a humid air environment. Although the moisture absorption rate thereof tends to increase with the increase of the ambient humidity, the stability of the microspheres themselves is also different due to the different operating conditions of each embodiment and comparative example during the entire freeze-drying process. From the moisture absorption rate data, the optimal process is Example 3. In Example 3, since the added stabilizing components and the freeze-drying operating conditions are controlled to the best, the moisture absorption rate and the increasing trend of the moisture absorption rate are both low, indicating that the stability of the microspheres in this embodiment is better than that in the comparative example. The microspheres can still achieve good stability and antioxidant properties even when exposed to a humid air environment, and will not affect the accuracy and sensitivity of the test when subsequently used for sample detection on a microfluidic reagent disk. Compared with Example 3, although the operating conditions of Examples 1-2 and 4-5 are slightly different, the stability is still relatively good. Starting from Example 6, since the added bamboo vinegar and citric acid mainly complement each other for antioxidant effect, when these two substances are not added, the stability is significantly reduced. Therefore, it can be seen from the moisture absorption data that the moisture absorption rate of Example 6 increases very quickly. In the data of Example 7 and Example 8, the addition of alcohols has a certain dosage matching relationship. If the amount of mannitol added is smaller than that of isopropyl alcohol, the two cannot be well matched with each other, so the stability of its active ingredient is poor, and it is easily affected by the environment and the moisture absorption rate will rise relatively quickly. The addition of amino acids is mainly to achieve a good antifreeze effect. Similarly, there is a clear proportional relationship between lysine and proline. If the scheme of Example 8 is implemented, the antifreeze effect of the microspheres themselves will be poor, which will also affect the stability of the final microspheres. Comparative Examples 1 and 2 show that the effects of replacing the types of amino acids and alcohols with other types are not as expected, so it can be seen that fixed matching and specific ratios and dosages are important guarantees for the stability of the microspheres of the present invention. Finally, in Example 9, the freeze-drying operation process was not controlled to the optimal conditions, so the final moisture absorption rate was also affected.

[0072] From the changes in the appearance of the microspheres under different temperature conditions in the table, it can be seen that Example 3 is the best example. The active ingredients of other examples, especially Example 6, are affected by the air and the surface becomes relatively rough. Similarly, starting from Example 6, the stability of the microspheres is poor and they are affected to varying degrees.

[0073] In summary, it can be seen from the data in Table 1 above that the freeze-drying process of the present invention requires that all operating conditions be controlled within the optimal range in order to achieve good results.

[0074] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. It is therefore intended that the appended claims include all such changes and modifications that fall within the scope of the present invention.

Claims

1. A microsphere freeze-drying process for a microfluidic reagent tray, characterized in that: The steps include: A reagent mixture was prepared, and a mixture of mannitol, isopropanol, lactose, proline, and lysine was added at a volume ratio of 1:1 to the reagent mixture, and the mixture was dropped into liquid nitrogen to form microspheres, which were then freeze-dried to obtain freeze-dried microspheres; By mass, 8 parts of mannitol, 5 parts of isopropyl alcohol, 5 parts of lactose, 6 parts of proline, and 3 parts of lysine; The freeze-drying procedure is carried out in multiple stages, where the first freeze-drying stage takes 3-4 hours at a temperature of -35~-50°C; The freeze drying procedure is carried out in three stages: the second freeze drying stage lasts for 7-8 hours at a temperature of -20 to -30°C, the third freeze drying stage is the drying stage, lasts for 2-3 hours at a temperature of 10-30°C; Add bamboo vinegar and citric acid to the reagent mixture; The amount of bamboo vinegar added is 0.1-0.5 parts by mass; The added amount of the citric acid is 0.3-0.7 parts by mass.

2. The microsphere freeze-drying process according to claim 1, characterized in that: The freeze-drying process was carried out under a vacuum degree of 50-150 ubar.

Images