Magnetic bead preserving fluid as well as preparation method and application thereof

Abstract

The invention discloses a magnetic bead preserving fluid as well as a preparation method and application thereof. According to the magnetic bead preserving fluid, the dispersity of magnetic beads preserved at an extreme temperature is improved through the combined action of polyethylene glycol, sodium polycarboxylate and a surfactant. When the magnetic beads are preserved by using the magnetic bead preserving fluid disclosed by the invention, the extraction performance is not influenced and even the extraction efficiency is improved when the magnetic beads are preserved at normal temperature to 40 DEG C for a long time, and the nucleic acid recovery rate is relatively high when the magnetic beads are preserved at-20 DEG C to 50 DEG C.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular, to a magnetic bead preservation solution, a preparation method thereof, and an application thereof. Background Art

[0002] Silanol magnetic beads are a kind of magnetic microspheres widely used in biomedical applications. Their surfaces are covered with hydroxyl and silicon-based functional groups, and they have good biocompatibility and biosafety. Under certain conditions, these magnetic beads can specifically adsorb nucleic acids in large quantities, and can be desorbed when the conditions change. Therefore, they are widely used in the extraction and purification of nucleic acids.

[0003] Traditional silanol magnetic bead preservation solutions (such as purified water or basic buffer solutions) are prone to cause magnetic bead aggregation and surface group denaturation at low or high temperatures, affecting the nucleic acid binding efficiency. Therefore, the existing magnetic bead preservation solutions can usually only be stably preserved within the range of 4 - 28°C, and cannot meet the transportation and storage requirements under extreme environments. The disclosed preservation solutions applied to extreme environments improve stability by adding high molecular polymers (such as polyethylene glycol) or organic solvents (such as ethanol), but have the following limitations: too high a concentration of high molecular polymers may lead to an increase in viscosity, affecting the dispersibility of magnetic beads; the nucleic acid recovery rate decreases after long-term preservation; the required components are more, the preparation is more complicated, and the cost required for large-scale production is higher.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a magnetic bead preservation solution, a preparation method thereof, and an application thereof, and this magnetic bead preservation solution can be used for the long-term stable preservation of magnetic beads under extreme temperature conditions (-20°C to 50°C).

[0006] The present invention is implemented as follows:

[0007] In a first aspect, the present invention provides a magnetic bead preservation solution, which includes: a dispersant and a surfactant; wherein, the dispersant includes polyethylene glycol and polycarboxylate salt.

[0008] In some embodiments, the concentration of the polycarboxylate salt is 0.1% - 5% (v / v).

[0009] In some embodiments, the polyethylene glycol includes one or more of polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, or polyethylene glycol 800.

[0010] In some embodiments, the concentration of the polyethylene glycol is 1% - 20% (v / v).

[0011] In some embodiments, the surfactant is selected from at least one of Tween 20, Tween 40, Tween 60, Tween 80, SDS, Triton X100, Span 20, Span 40, Span 60, Span 80, and Nonidet (R) P-40.

[0012] In some embodiments, the surfactant is a combination of any one of Tween 20, Tween 40, Tween 60, Tween 80, or SDS and any one of Triton X100, Span 20, Span 40, Span 60, Span 80, or Nonidet (R) P-40, and the volume ratio of the two is 2:8 to 8:2.

[0013] In some embodiments, the concentration of the surfactant is 0.01% to 1% (v / v).

[0014] In some embodiments, the magnetic bead preservation solution further includes a preservative, which is Proclin 300 or sodium azide.

[0015] In some embodiments, the concentration of the preservative is 0.1% to 1% (m / v).

[0016] In a second aspect, the present invention provides a method for preparing the above magnetic bead preservation solution, which includes: adding polyethylene glycol, polycarboxylate salt, surfactant, and preservative to deionized water in proportion, mixing well, and then making up the volume to obtain the magnetic bead preservation solution.

[0017] In a third aspect, the present invention further provides a method for preserving magnetic beads, which includes: dispersing the magnetic beads in the above magnetic bead preservation solution.

[0018] In some embodiments, the preservation temperature of the magnetic beads is -20°C to 50°C.

[0019] The present invention has the following beneficial effects:

[0020] The present invention prepares a new magnetic bead preservation solution with polyethylene glycol, polycarboxylate salt, surfactant, and preservative as the main components. Among them, the dispersibility of the magnetic beads after preservation at extreme temperatures is improved by the combined action of polyethylene glycol, polycarboxylate salt, and surfactant. Using the magnetic bead preservation solution of the present invention to preserve magnetic beads can ensure that the extraction performance of the magnetic beads is not affected during long-term preservation at room temperature to 40°C, and even has the effect of improving the extraction efficiency. When preserved at -20°C to 50°C, it has a high nucleic acid recovery rate. Detailed embodiments

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0022] Based on the problems existing in the existing magnetic bead preservation solution, the present invention has obtained a new preservation solution by optimizing the components and ratios. This preservation solution can improve the problems such as the increase in viscosity caused by too high a concentration of high molecular polymers in the existing magnetic bead preservation solution, which affects the dispersibility of magnetic beads and the decrease in nucleic acid recovery rate after long-term storage. The components of this magnetic bead preservation solution mainly include a dispersant, a surfactant, and a preservative. Among them, the dispersant includes polyethylene glycol (PEG) and an aqueous dispersant - sodium polycarboxylate.

[0023] Polyethylene glycol is a high molecular polymer prepared by ring-opening polymerization of ethylene oxide and water or homopolymerization of ethylene glycol, and has a linear or branched structure; due to its good water solubility and compatibility with polar substances, it can effectively disperse and stabilize solid particles or droplets in a suspension, preventing aggregation and precipitation. In the present invention, PEG adsorbs on the surface of magnetic beads to form a hydrophilic protective layer, which prevents direct contact between magnetic beads through steric hindrance and avoids agglomeration (especially during long-term storage); at the same time, its presence can also slow down the pH fluctuation of the preservation solution (especially during long-term storage), avoiding precipitation caused by changes in the surface charge of magnetic beads.

[0024] The main function of sodium polycarboxylate is to disperse solid particles (such as pigment particles) into a liquid medium, making them evenly suspended and distributed without precipitation or agglomeration, thereby improving the color uniformity, transparency, and gloss of the product. It is currently commonly used in industries such as coatings, pigments, and inks, and has excellent dispersibility and stability. The present invention first uses it in the magnetic bead preservation solution. Sodium polycarboxylate adsorbs on the surface of magnetic beads, introducing ionized groups (such as carboxylate groups and sulfonate groups), making the surface of the magnetic beads mainly negatively charged. Since the surfaces of magnetic beads all carry the same charge, the magnetic beads repel each other and can be evenly dispersed in the reaction system, thereby improving the dispersibility of magnetic beads.

[0025] The combination of polyethylene glycol and sodium polycarboxylate can disassemble the formed soft agglomerates through wetting and electrostatic effects, synergistically improving the monodispersity of magnetic beads.

[0026] Specifically, the polyethylene glycol includes, but is not limited to, one or more of polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, or polyethylene glycol 800; preferably, the polyethylene glycol is polyethylene glycol 800.

[0027] The surfactant can reduce the surface tension of the magnetic bead preservation solution, enabling the magnetic beads to be evenly dispersed in the magnetic bead preservation solution, and thus improving the stability of the magnetic beads.

[0028] In the present invention, the surfactant is selected from at least one of Tween 20, Tween 40, Tween 60, Tween 80, SDS, Triton X100, Span 20, Span 40, Span 60, Span 80, and Nonidet (R) P-40.

[0029] More preferably, the surfactant is a combination of any one of Tween 20, Tween 40, Tween 60, Tween 80, or SDS and any one of Triton X100, Span 20, Span 40, Span 60, Span 80, or Nonidet (R) P-40. That is, the surfactant is composed of A + B, where A is any one of Tween 20, Tween 40, Tween 60, Tween 80, or SDS, and B is any one of Triton X100, Span 20, Span 40, Span 60, Span 80, or Nonidet (R) P-40. When the two reagents are used in combination, the volume ratio of A to B is 2:8 to 8:2, and more preferably, the volume ratio is 6:4.

[0030] Since the magnetic bead preservation solution contains an aqueous phase component and is prone to microbial growth, the preservative can effectively kill or inhibit these microorganisms.

[0031] Specifically, the preservative includes but is not limited to Proclin 300 and sodium azide, and can also be other conventional preservatives.

[0032] Based on the above components, the present invention optimizes the addition concentrations of the components, which are as follows:

[0033] Sodium polycarboxylate: 0.1% - 5% (v / v); polyethylene glycol: 0.01% - 20% (v / v); surfactant: 0.01% - 1% (v / v); preservative: 0.1% - 1% (m / v).

[0034] The corresponding preparation method of the above magnetic bead preservation solution is as follows:

[0035] (1) Add the raw materials to deionized water in proportion and stir evenly.

[0036] (2) After volume fixing, dispense and store in the dark.

[0037] The above magnetic bead preservation solution can be used to preserve magnetic beads. The preservation method is: Disperse the magnetic beads in the above magnetic bead preservation solution. Among them, the preservation temperature of the magnetic beads can reach -20°C to 50°C, and long-term stable preservation in extreme environments can be achieved.

[0038] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0039] The polyethylene glycol, surfactant and preservative used in the embodiments of the present invention are all derived from products sold by Sangon Biotechnology; sodium polycarboxylate (CAS: 62601-60-9) is derived from Shenzhen Yoshida Chemical with the item number SN-5040, or from Weifang Jintai Materials Factory with the item number WS-2650A.

[0040] Example 1

[0041] This embodiment is a magnetic bead preservation solution and a preparation method thereof, which are specifically as follows:

[0042] The components of magnetic bead storage solution are:

[0043] Component Concentration PEG 600 10%(v / v) Tween 20 and Triton X100 0.02%(v / v) Sodium polycarboxylate 1%(v / v) Proclin 300 0.02%(m / v)

[0044] The ratio of Tween 20 to Triton X100 is 6:4. The preparation method of the magnetic bead storage solution is as follows:

[0045] (1 Add the raw materials into deionized water according to the proportion and stir to mix well.

[0046] (2) After making up the volume, divide the mixture into smaller pieces and store in a dark place.

[0047] Example 2

[0048] This embodiment is a magnetic bead preservation solution and a preparation method thereof, which are specifically as follows:

[0049] The components of magnetic bead storage solution are:

[0050]

[0051]

[0052] The ratio of the two surfactants is 6:4. The preparation method of the magnetic bead preservation solution is as follows: (1 Add the raw materials into deionized water in proportion and stir to mix.

[0053] (2) After making up the volume, divide the mixture into smaller pieces and store in a dark place.

[0054] Example 3

[0055] This embodiment is a magnetic bead preservation solution and a preparation method thereof, which are specifically as follows:

[0056] The components of magnetic bead storage solution are:

[0057] Component Concentration PEG 800 15%(v / v) Tween 60 and Nonidet(R) P - 40 0.02%(v / v) Sodium polycarboxylate 1%(v / v) Proclin 300 0.01%(m / v)

[0058] The ratio of the two surfactants is 8:2. The preparation method of the magnetic bead preservation solution is as follows: (1) adding the raw materials into deionized water according to the proportion and stirring to mix well.

[0059] (2) After constant volume, aliquot and store in the dark.

[0060] Example 4

[0061] This example is a magnetic bead preservation solution and its preparation method, which are specifically as follows:

[0062] The components of the magnetic bead preservation solution are:

[0063] Component Concentration PEG 200 5%(v / v) Tween 20 and Span 80 0.02%(v / v) Sodium polycarboxylate 0.5%(v / v) Proclin 300 0.02%(m / v)

[0064] Among them, the ratio of the two surfactants is 2:8. The preparation method of the above magnetic bead preservation solution is:

[0065] (1) Add the raw materials to deionized water according to the ratio and stir evenly.

[0066] (2) After constant volume, aliquot and store in the dark.

[0067] Example 5

[0068] This example is a magnetic bead preservation solution and its preparation method, which are specifically as follows:

[0069] The components of the magnetic bead preservation solution are:

[0070] Component Concentration PEG 400 10%(v / v) SDS and Span 80 0.02%(v / v) Sodium polycarboxylate 0.1%(v / v) Proclin 300 0.02%(m / v)

[0071] Among them, the ratio of the two surfactants is 2:8. The preparation method of the above magnetic bead preservation solution is:

[0072] (1) Add the raw materials to deionized water according to the ratio and stir evenly.

[0073] (2) After constant volume, aliquot and store in the dark.

[0074] Experimental Example 1

[0075] Use the magnetic bead preservation solution of Example 1 for high-temperature stability verification, which is specifically as follows:

[0076] Disperse the magnetic beads in the preservation solution of Example 1 and store them in an environment of 4°C, 25°C, 40°C, and 55°C for 1 month and 6 months respectively, and then conduct tests on the extraction performance: Use the B518743 magnetic bead method DNA gel recovery kit sold by Sangon Biotech to extract nucleic acids from the engineering bacteria samples, and then measure the absorbance value and concentration of the extracted nucleic acids. The results are shown in Table 1:

[0077] Table 1 Stability results under high-temperature conditions

[0078]

[0079]

[0080] It can be seen from the results in Table 1 that under the same temperature conditions, the effects on the purity and concentration of nucleic acids after storage for 1 month and 6 months are relatively small, and it has high stability even under high temperature conditions of 40°C and 55°C.

[0081] The stability of the magnetic bead storage solution in Examples 2 - 5 under high temperature conditions is similar to that in Example 1.

[0082] Experimental Example 2

[0083] The magnetic bead storage solution in Example 1 was used for verification of low - temperature stability, as follows:

[0084] The magnetic beads were dispersed in the storage solution of Example 1 and stored for 1 month and 6 months respectively at 4°C, - 10°C and - 20°C environments, and then the extraction performance was tested: The nucleic acids of the engineering bacteria samples were extracted using the B518743 magnetic bead method DNA gel recovery kit sold by Sangon Biotech, and then the absorbance value and concentration of the extracted nucleic acids were measured. The results are shown in Table 2:

[0085] Table 2 Stability results under low - temperature conditions

[0086] Condition 260 / 280 260 / 230 Conc.(ng / ul) Magnetic beads for 1 month at 4℃ 1.98 2.02 141.50 Magnetic beads for 1 month at - 10℃ 1.91 1.97 143.47 Magnetic beads for 1 month at - 20℃ 1.96 1.95 137.65 Magnetic beads for 6 months at 4℃ 1.98 1.98 139.98 Magnetic beads for 6 months at - 10℃ 1.84 1.91 139.51 Magnetic beads for 6 months at - 20℃ 1.88 1.91 140.36

[0087] It can be seen from the results in Table 2 that under the same temperature conditions, the effects on the purity and concentration of nucleic acids after storage for 1 month and 6 months are relatively small, and it has high stability even under high temperature conditions of - 10°C and - 20°C.

[0088] The stability of the magnetic bead storage solution in Examples 2 - 5 under low - temperature conditions is similar to that in Example 1.

[0089] Experimental Example 3

[0090] The difference between the magnetic bead storage solution used in this experiment and that in Example 1 is that it does not contain the aqueous dispersant - sodium polycarboxylate. Its stability under low - temperature conditions was verified, and the experimental method was the same as that in Experimental Example 2. The results are shown in Table 3:

[0091] Table 3 Stability results of the magnetic bead storage solution without sodium polycarboxylate

[0092]

[0093]

[0094] Comparing the results of Table 1 and Table 3, it can be seen that under the same temperature and storage time conditions, the magnetic bead preservation solution without sodium polycarboxylate has a more obvious impact on nucleic acids, with greater changes in purity and concentration, indicating that the magnetic bead preservation solution in Example 1 has higher stability; from the purity data, it can also be seen that the magnetic bead preservation solution without sodium polycarboxylate also affects the extraction effect, and the purity of the obtained nucleic acid is lower than that in Example 1.

[0095] Experimental Example 4

[0096] The difference between the magnetic bead preservation solution used in this experiment and that in Example 1 is that the concentration of sodium polycarboxylate added is 8% (v / v). To verify its stability under low-temperature conditions, the experimental method is the same as that in Experimental Example 2. The results are shown in Table 4:

[0097] Table 4 Stability results of the magnetic bead preservation solution with excessive sodium polycarboxylate

[0098] Condition 260 / 280 260 / 230 Conc.(ng / ul) Magnetic beads for 1 month at 4℃ 1.77 1.48 143.18 Magnetic beads for 1 month at - 10℃ 1.81 1.41 138.07 Magnetic beads for 1 month at - 20℃ 1.76 1.35 139.79 Magnetic beads for 6 months at 4℃ 1.78 1.41 142.11 Magnetic beads for 6 months at - 10℃ 1.71 1.29 152.74 Magnetic beads for 6 months at - 20℃ 1.68 1.33 130.27

[0099] Comparing the results of Table 1 and Table 4, it can be seen that under the same temperature and storage time conditions, the magnetic bead preservation solution with excessive sodium polycarboxylate has an impact on the nucleic acid extraction effect, and the purity of the obtained nucleic acid is lower than that in Example 1.

[0100] Experimental Example 5

[0101] The difference between the magnetic bead preservation solution used in this experiment and that in Example 1 is that the aqueous dispersant added is a polyether-type aqueous dispersant. To verify its stability under low-temperature conditions, the experimental method is the same as that in Experimental Example 2. The results are shown in Table 5:

[0102] Table 5 Stability results of the magnetic bead preservation solution containing a polyether-type aqueous dispersant

[0103] Condition 260 / 280 260 / 230 Conc.(ng / ul) Magnetic beads for 1 month at 4℃ 1.79 1.08 73.18 Magnetic beads for 1 month at - 10℃ 1.83 1.21 68.07 Magnetic beads for 1 month at - 20℃ 1.80 1.05 69.79 Magnetic beads for 6 months at 4℃ 1.71 1.01 72.11 Magnetic beads for 6 months at - 10℃ 1.71 1.13 82.74 Magnetic beads for 6 months at - 20℃ 1.76 1.04 60.27

[0104] Comparing the results of Table 1 and Table 5, it can be seen that under the same temperature and storage time conditions, the magnetic bead preservation solution with excessive polycarboxylate sodium salt type dispersant has an impact on the nucleic acid extraction effect, and the purity and concentration of the obtained nucleic acid are lower than those in Example 1.

[0105] Experimental Example 6

[0106] Under the same experimental conditions, the stability effects of the magnetic bead preservation solution in Example 1 and the control magnetic bead preservation solution were compared, and the experimental method was the same as that in Experimental Example 2. Among them, the components of the control magnetic bead preservation solution refer to CN114480369B. The results are shown in Table 6:

[0107] Table 6 Stability results of the two preservation solutions

[0108] Condition 260 / 280 260 / 230 Conc.(ng / ul) Magnetic bead preservation solution of Example 1 for 1 month at 4℃ 1.97 1.91 140.40 Magnetic bead preservation solution of Example 1 for 1 month at - 10℃ 1.96 1.92 143.08 Magnetic bead preservation solution of Example 1 for 1 month at - 20℃ 1.97 1.98 139.27 Magnetic bead preservation solution of Example 1 for 6 months at 4℃ 1.88 1.94 142.11 Magnetic bead preservation solution of Example 1 for 6 months at - 10℃ 1.92 1.86 139.74 Magnetic bead preservation solution of Example 1 for 6 months at - 20℃ 1.98 1.86 141.27 Control magnetic bead preservation solution for 1 month at 4℃ 1.87 1.88 140.39 Control magnetic bead preservation solution for 1 month at - 10℃ 1.89 1.87 138.27 Control magnetic bead preservation solution for 1 month at - 20℃ 1.92 1.82 141.05 Control magnetic bead preservation solution for 6 months at 4℃ 1.84 1.68 114.55 Control magnetic bead preservation solution for 6 months at - 10℃ 1.88 1.63 94.90 Control magnetic bead preservation solution for 6 months at - 20℃ 1.82 1.64 146.83

[0109] It can be seen from the results in Table 6 that under the same temperature and time conditions, the stability effect of the magnetic bead preservation solution in Example 1 is better.

[0110] Experimental Example 7

[0111] With other conditions unchanged, different polyethylene glycols with the same dosage are used to prepare the magnetic bead preservation solution, and the comparison of their effects is as follows:

[0112] Table 7 Stability results of preservation solutions prepared with different types of polyethylene glycol

[0113] Condition 260 / 280 260 / 230 Conc.(ng / ul) Polyethylene glycol 200 1.88 1.96 141.88 Polyethylene glycol 400 1.87 1.99 142.57 Polyethylene glycol 600 1.89 2.05 143.83 Polyethylene glycol 800 1.88 2.01 142.61

[0114] It can be seen from the results in Table 7 that the preservation solutions prepared with polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, and polyethylene glycol 800 have little influence on the purity and concentration of nucleic acid extraction, and the preservation solutions prepared with these four types of polyethylene glycol can all achieve relatively good extraction effects.

[0115] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A magnetic bead preservation solution, characterized in that, Comprising: a dispersant and a surfactant; The dispersant includes polyethylene glycol and sodium polycarboxylate.

2. The magnetic bead preservation solution according to claim 1, wherein The concentration of the sodium polycarboxylate is 0.1% - 5% (v / v).

3. The magnetic bead preservation solution according to claim 1, wherein, The polyethylene glycol includes one or more of polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, or polyethylene glycol 800.

4. The magnetic bead preservation solution according to claim 3, characterized in that The concentration of the polyethylene glycol is 1% - 20% (v / v).

5. The magnetic bead preservation solution according to claim 1, characterized in that, The surfactant is selected from at least one of Tween 20, Tween 40, Tween 60, Tween 80, SDS, Triton X100, Span 20, Span 40, Span 60, Span 80, and Nonidet (R) P - 40; Preferably, the surfactant is a combination of any one of Tween 20, Tween 40, Tween 60, Tween 80, or SDS and any one of Triton X100, Span 20, Span 40, Span 60, Span 80, or Nonidet (R) P - 40, and the volume ratio of the two is 2:8 - 8:

2.

6. The magnetic bead preservation solution according to claim 5, wherein The concentration of the surfactant is 0.01% - 1% (v / v).

7. The magnetic bead preservation solution according to claim 1, wherein The magnetic bead storage solution further includes a preservative, which is Proclin 300 or sodium azide; Preferably, the concentration of the preservative is 0.1% - 1% (m / v).

8. The preparation method of the magnetic bead preservation solution according to any one of claims 1-7, characterized in that, Comprising: Adding polyethylene glycol, sodium polycarboxylate, surfactant, and preservative to deionized water in proportion, mixing well and then making up the volume to obtain the magnetic bead storage solution.

9. A method for storing magnetic beads, characterized in that, Comprising: Dispersing magnetic beads in the magnetic bead storage solution according to any one of claims 1 - 7.

10. The preservation method according to claim 9, characterized in that, The storage temperature of the magnetic beads is -20°C to 50°C.

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