CN113430251A - Concentration method of constant-temperature amplification reagent and integrated biochip - Google Patents
Concentration method of constant-temperature amplification reagent and integrated biochip Download PDFInfo
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- CN113430251A CN113430251A CN202110664282.5A CN202110664282A CN113430251A CN 113430251 A CN113430251 A CN 113430251A CN 202110664282 A CN202110664282 A CN 202110664282A CN 113430251 A CN113430251 A CN 113430251A
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Abstract
The application discloses a constant temperature amplification reagent concentration protection composition, a constant temperature amplification reagent concentration method and an integrated biochip, comprising the following steps: 1-8 parts of bovine serum albumin; 8-15 parts of mannitol; and 10-20 parts by weight of trehalose. The method can be widely applied to the fields of clinical diagnosis, agricultural detection, livestock detection, environmental detection and bioengineering.
Description
Technical Field
The application relates to the field of biochips, in particular to a constant-temperature amplification reagent concentration protection composition, a constant-temperature amplification reagent concentration method, a constant-temperature amplification reagent concentrated solution and an integrated biochip.
Background
The technology of a Lab-on-chip (Lab-on-chip) is a leading-edge technology in the field of diagnosis and detection at present, and can complete experiments with multiple functions on a tiny integrated biochip, thereby greatly reducing the requirements on personnel, operation and sites.
The integrated biochip needs to be pre-filled with a reagent (Lamp system) which can be used for constant temperature amplification, the volume of the constant temperature amplification reagent which is not processed is large, the whole detection space can be filled after the reagent is pre-filled, the filling process is not facilitated, meanwhile, the reagent also has large fluidity in the chip, and the reagent easily flows to other detection units or channels due to the vibration and the shaking of the chip and the siphoning phenomenon of each pipeline, so that the constant temperature amplification reagent needs to be concentrated or dried.
The general concentration method directly concentrates the sample or the liquid, which not only damages the performance of the sample or the liquid and causes the loss of key components, but also has more strict requirements on the concentration environment because the isothermal amplification reagent contains enzyme and biological raw materials, and the general concentration method does not protect the concentrated object and can not lead the liquid to reach the ideal viscosity degree, thereby not reaching the required state and directly influencing the performance and the result of the product or the experiment.
In addition, the isothermal amplification reagent is a bioactive substance, can be degraded, inactivated and deteriorated in the long-term storage process, and has extremely important stability.
Therefore, the constant temperature amplification reagents are generally processed by the freeze-drying method in the biomedical industry for storage, transportation and use, but the freeze-drying method has the following defects: 1) the freeze-drying time is long; 2) the processing environment requires low humidity; 3) is fragile and easy to absorb moisture; .
Disclosure of Invention
In view of the above-mentioned drawbacks, the present application provides a protective agent for isothermal amplification reagent concentration, which is mixed with an isothermal amplification reagent to ensure the performance of the isothermal amplification reagent to be protected to a great extent during the concentration process and to give the isothermal amplification reagent a certain viscosity.
The technical scheme is as follows: an isothermal amplification reagent concentrate protective composition comprising:
1-8 parts of bovine serum albumin;
8-15 parts of mannitol; and
10-20 parts of trehalose.
In one aspect, the present application also provides a method for concentrating isothermal amplification reagents.
The technical scheme is as follows: a method for concentrating isothermal amplification reagents comprises the following steps:
dissolving the constant-temperature amplification reagent concentrated protection composition in nuclease-free water to form a concentrated protection agent;
the concentrated protective agent and the constant-temperature amplification reagent form reagent liquid with the protective agent;
thirdly, transferring the reagent solution with the protective agent into a vacuum drying oven for vacuum drying and concentration, and forming the constant-temperature amplification reagent concentrated solution after vacuum drying and concentration.
The protective agent is mixed with the constant-temperature amplification reagent, and the performance of the constant-temperature amplification reagent is protected to a great extent in the concentration process through vacuum drying, and a certain viscosity is given to the concentrated solution.
Optionally, in the step three, the vacuum degree of vacuum drying is 10-1100mbar, the concentration temperature C is 10-60 ℃, and the concentration time is 1-1000 min.
Optionally, in the step three, the vacuum degree of vacuum drying is 40-80mbar, the concentration temperature C is 20-40 ℃, and the concentration time is 10-40 min.
Optionally, in the second step, the volume ratio of the concentrated protective agent to the isothermal amplification reagent is 1: 0.1-10.
Optionally, the volume ratio of the concentrated protective agent to the isothermal amplification reagent is 1: 0.5-5.
Optionally, in the third step, when the reagent solution with the protective agent is transferred into a vacuum drying oven for vacuum drying and concentration, the reagent solution is transferred into drying container vessels, and the volume of each drying container vessel is 10-1000 muL.
Optionally, each drying vessel has a volume of 50 μ L to 200 μ L.
In one aspect, the present application also provides a isothermal amplification reagent concentrate.
The technical scheme is as follows: the constant-temperature amplification reagent concentrated solution is characterized by being prepared by the method.
In one aspect, the present application also provides an integrated biochip.
The technical scheme is as follows: an integrated biochip using the isothermal amplification reagent concentrate of claim 9.
The invention principle and the beneficial effects are as follows:
the application establishes a method for concentrating the constant-temperature amplification reagent aiming at an integrated biochip, greatly protects the performance of the constant-temperature amplification reagent in the concentration process, keeps a certain viscous state to reduce the fluidity of the constant-temperature amplification reagent, and can quickly redissolve in the use process.
The application realizes that the constant temperature amplification reagent is pre-filled on the integrated micro-fluidic chip, increases the viscosity of the constant temperature amplification reagent and increases the stability of the constant temperature amplification reagent in the long-term storage process.
This application makes the volume of this reagent reduce through the concentration process, has realized the possibility on the pre-filled chip, has reduced the mobility of reagent again, has reduced the risk in depositing, transportation. In the frontier field of the life science field, a Lab-on-chip (Lab-on-chip) technology is used to perform experiments with multiple functions on a tiny biochip, so that tedious manual operation is replaced, the requirements on personnel, operation and sites are reduced, and the method can be widely applied to the fields of clinical diagnosis, agricultural detection, livestock detection, environmental detection and bioengineering.
Drawings
FIG. 1 is a graph showing the results of detection by a fluorometer in examples 2 and 3 of the present application and comparative example 1;
FIG. 2 is a test chart of a chip prepared from the isothermal amplification reagent concentrate formed in example 2;
FIG. 3 is a test chart of a chip prepared by freeze-drying the isothermal amplification reagent formed in comparative example 1.
Detailed Description
The present application will be further described with reference to the accompanying drawings.
In the present application, the isothermal amplification reagent is a PCR reagent, and the PCR reagent is a reagent for PCR, and in the present application, the PCR reagent is commercially available.
EXAMPLE 1 formulation of concentrated protectant
10g BSA, 20g mannitol and 20g trehalose were dissolved in 100mL nuclease-free water and mixed well.
Example 2
Mixing the concentrated protective agent of example 1 and a constant temperature amplification reagent to form a reagent solution with a protective agent.
Concentration and protection agent: the volume ratio of the isothermal amplification reagents is 1: 2.
And (2) concentrating:
the method comprises the steps of measuring reagent liquid with a protective agent in the process of using a pipettor to a drying container vessel, putting the drying container vessel into a vacuum drying oven for vacuum drying, and forming constant-temperature amplification reagent concentrated liquid after vacuum drying. The parameters adjusted by the vacuum drying oven are as follows: the vacuum degree D is 40mbar, the drying temperature C is 42 ℃, and the vacuum drying time T is 30 min.
A plurality of drying container vessels can be placed on a partition plate of the vacuum drying box, and each drying container vessel can be transferred into the reagent liquid with the protective agent in the process.
When the reagent solution with the protective agent in the second container moved into the drying container is 200 mu L/drying container, the volume of the constant temperature amplification reagent concentrated solution formed after drying is 100 mu L/drying container.
The constant temperature amplification reagent concentrated solution formed in the step is viscous and keeps certain fluidity, and can be moved to the detection space of the integrated chip through a liquid transfer device in the subsequent transportation process without siphoning to other channels.
The results are shown in Table 1 below.
Example 3
And (3) measuring the constant-temperature amplification reagent into a drying container vessel by using a pipettor, putting the drying container vessel into a vacuum drying oven for vacuum drying, and forming the constant-temperature amplification reagent concentrated solution after vacuum drying. The parameters adjusted by the vacuum drying oven are as follows: the vacuum degree D is 40mbar, the drying temperature C is 42 ℃, and the vacuum drying time T is 30 min.
A plurality of containers for drying can be placed on a partition plate of the vacuum drying box, and each container for drying can be transferred with a constant temperature amplification reagent.
When the volume of the isothermal amplification reagent transferred into the drying vessel was 200. mu.L/drying vessel, the volume of the isothermal amplification reagent concentrated liquid formed after drying was about 80. mu.L/drying vessel.
The results are shown in Table 1 below.
Comparative example 1.
And (3) measuring the isothermal amplification reagent into a drying container by using a pipette, and then putting the drying container into a freeze dryer to be freeze-dried by adopting the conventional freeze-drying process to form the isothermal amplification reagent freeze-dried, wherein the freeze-drying time is 36 hours.
The amount of the isothermal amplification reagent in the drying vessel was 200. mu.L per drying vessel, and the volume of the isothermal amplification reagent lyophilized was about 200. mu.L per drying vessel.
The results are shown in Table 1 below.
TABLE 1 comparison of the concentration results
The isothermal amplification reagent concentrates formed in the above examples 2 and 3 and the isothermal amplification reagent formed in the comparative example 1 were lyophilized for detection, each single concentration unit was reconstituted with water to a volume before concentration, 10. mu.L of each was pipetted into the prepared primer template mixture, and the real-time fluorescence quantitative analyzer was used for detection as shown in FIG. 1.
In FIG. 1, 1 is a graph of the isothermal amplification reagent concentrate of example 2, 2 is a graph of the isothermal amplification reagent concentrate of example 3, and 3 is a graph of lyophilization of the isothermal amplification reagent of comparative example 1.
The isothermal amplification reagent concentrated solutions formed in the above examples 2 and 3 and the isothermal amplification reagent freeze-dried solution formed in the comparative example 1 are used for producing and testing an integrated biochip, and the isothermal amplification reagent concentrated solution formed in the example 3 has a high liquid fluidity during the production of the integrated biochip, so that the liquid flows from the capillary channel to other detection units, and cannot be used for detecting the biochip. The constant temperature amplification reagent concentrated solution formed in example 2 and the constant temperature amplification reagent formed in comparative example 1 were lyophilized to successfully complete the production of the integrated biochip, and the results of the chip tests are shown in fig. 2 and 3, where fig. 2 is a chip test chart of the chip made of the constant temperature amplification reagent concentrated solution formed in example 2, and fig. 3 is a chip test chart of the chip made of the constant temperature amplification reagent lyophilized formed in comparative example 1.
As can be seen from FIG. 3, the chip prepared by freeze-drying the isothermal amplification reagent of comparative example 1 did not amplify normally.
In this application, unless otherwise specified, all are prior art.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. An isothermal amplification reagent concentrate protective composition comprising:
1-8 parts of bovine serum albumin;
8-15 parts of mannitol; and
10-20 parts of trehalose.
2. A method for concentrating isothermal amplification reagents comprises the following steps:
dissolving the constant-temperature amplification reagent concentration protection composition of claim 1 in nuclease-free water to form a concentration protection agent;
the concentrated protective agent and the constant-temperature amplification reagent form reagent liquid with the protective agent;
thirdly, transferring the reagent solution with the protective agent into a vacuum drying oven for vacuum drying and concentration, and forming a constant-temperature amplification reagent concentrated solution after vacuum drying and concentration;
the constant temperature amplification reagent concentrated solution is used for an integrated biochip.
3. The method for concentrating the isothermal amplification reagent according to claim 2, wherein in the step three, the vacuum degree of vacuum drying is 10-1100mbar, the concentration temperature C is 10-60 ℃, and the concentration time is 1-1000 min.
4. The method for concentrating isothermal amplification reagent according to claim 3, wherein vacuum drying is performed at a vacuum degree of 40-80mbar, a concentration temperature C of 20-40 ℃ and a concentration time of 10-40min in the sample.
5. The method for concentrating isothermal amplification reagents according to any one of claims 2 to 4, wherein the volume ratio of the concentrated protective agent to the isothermal amplification reagents in the second step is 1: 0.1-10.
6. The method for concentrating isothermal amplification reagent according to claim 5, wherein the volume ratio of the concentrated protective agent to the isothermal amplification reagent is 1: 0.5-5.
7. The method for concentrating isothermal amplification reagent according to any one of claims 2 to 6, wherein in the third step, when the reagent solution with the protective agent is transferred into a vacuum drying oven for vacuum drying and concentration, the reagent solution is transferred into drying vessels, and the volume of each drying vessel is 10 μ L to 1000 μ L.
8. The method for concentrating isothermal amplification reagent according to claim 7, wherein the volume of each drying container is 50 μ L to 200 μ L.
9. An isothermal amplification reagent concentrate prepared by the method of any one of claims 2 to 8.
10. An integrated biochip using the isothermal amplification reagent concentrate of claim 9.
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