Reaction reagent paraffin ball as well as preparation method and use method thereof

Abstract

The invention provides a reaction reagent paraffin ball as well as a preparation method and a use method thereof, the reaction reagent paraffin ball comprises a paraffin packaging shell, the paraffin packaging shell is prepared by mixing paraffin and magnetic beads, the paraffin packaging shell is solid, and the magnetic beads are dispersed in the solid paraffin; a reagent containing cavity is formed in the paraffin packaging shell, a reagent is contained in the reagent containing cavity, and the reagent is wrapped by the paraffin packaging shell. The magnetic beads comprise silicon-based magnetic beads. The preparation method comprises the following steps: S1, heating solid paraffin in a container to melt the solid paraffin into a liquid state, adding a proper amount of magnetic beads into the paraffin, and stirring and mixing the molten paraffin and the magnetic beads to prepare magnetic paraffin; s2, the first shell mold is coated with a release agent, first magnetic paraffin in a molten state is added into the first shell mold, and the first shell mold is taken out after being cooled and solidified; s3, placing a prepared reaction reagent on the solid first magnetic paraffin; s4, the reaction reagent is sealed and wrapped with second magnetic paraffin, and a reaction reagent paraffin ball is prepared. The device is good in storage effect and can move freely according to detection requirements.

Description

Technical Field

[0001] The invention relates to the field of microfluidic technology, and in particular to a reaction reagent paraffin ball and a preparation method and a use method thereof. Background Art

[0002] Microfluidic chips integrate sample preparation, reaction, detection and other operational units in chemical, biological, medical and other analytical processes onto a micron-scale carrier to automatically complete the entire analysis process.

[0003] The PCR reagents (PCR reaction system) currently used in microfluidic chips are mainly composed of basic elements such as primers, enzymes, dNTPs, templates, and Mg2+. The dried or freeze-dried PCR reagents need to be sealed and dried after drying or freeze-drying to prevent the PCR reagents from being affected by moisture and gradually inactivating biologically active components such as DNA polymerase and reverse transcriptase, resulting in performance degradation or even failure to use. PCR reagents are usually pre-stored in the reaction chamber in advance, and the upstream and downstream channels connected to the reaction chamber need to be sealed. The PCR reagents sealed and stored in the reaction chamber can only stay in the reaction chamber and cannot move to other pipes and cavities.

[0004] In addition, the dried or freeze-dried PCR reagents need to be dried or freeze-dried in situ. Although the freeze-dried PCR reagents can also be made into the form of freeze-dried beads to facilitate transfer to the microfluidic chip after freeze-drying, the PCR reagents made into freeze-dried beads still need to be sealed in the upstream and downstream channels connected to the reaction chamber after being placed in the microfluidic chip. The PCR reagents cannot be moved and released in the microfluidic chip according to actual needs, which greatly limits the design and channel layout of the microfluidic chip. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the first purpose of the present invention is to provide a reaction reagent paraffin ball with good storage effect and capable of being freely moved according to demand.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned reaction reagent paraffin balls.

[0007] The third object of the present invention is to provide a method for using the above-mentioned reaction reagent paraffin balls.

[0008] In order to achieve the above-mentioned first purpose, the reaction reagent paraffin ball provided by the present invention includes a paraffin encapsulation shell, the paraffin encapsulation shell is made of a mixture of paraffin and magnetic beads, the paraffin encapsulation shell is solid, and the magnetic beads are dispersed in the solid paraffin; a reagent containing cavity is formed in the paraffin encapsulation shell, the reagent containing cavity contains the reagent, and the reagent is wrapped by the paraffin encapsulation shell.

[0009] As can be seen from the above, using paraffin with magnetic beads to seal and wrap the reaction reagents can effectively improve the storage effect of the reaction reagents, prevent the reaction reagents from deteriorating or being contaminated, and at the same time facilitate the free movement of the reaction reagents in the microfluidic chip, thereby increasing the freedom of chip design.

[0010] In a further embodiment, the magnetic beads include silicon-based magnetic beads.

[0011] In order to achieve the above second purpose, the preparation method of the reaction reagent paraffin balls provided by the present invention comprises the following steps S1: heating solid paraffin in a container to melt it into a liquid state, adding an appropriate amount of magnetic beads to the paraffin, and stirring and mixing the melted paraffin and the magnetic beads to prepare magnetic paraffin;

[0012] S2: applying a release agent in the first shell mold, adding the first magnetic paraffin in a melted state into the first shell mold, and after cooling and solidification, taking out the first magnetic paraffin from the first shell mold;

[0013] S3: placing the prepared reaction reagent on the solid first magnetic paraffin;

[0014] S4: The reaction reagent is sealed and wrapped with a second magnetic paraffin to prepare a reaction reagent paraffin ball.

[0015] As can be seen from the above, wrapping the reaction reagent in the magnetic core paraffin reagent can effectively seal and store the reaction reagent, thereby preventing the reaction reagent from being damp or contaminated. The magnetic rod device of the external microfluidic detection device can freely move it to the specified position without sealing the upstream and downstream channels connected to the corresponding chamber, thereby improving the freedom of microfluidic chip design.

[0016] A further solution is that step S4 further includes dripping melted second magnetic paraffin wax around the reaction reagent in sequence until the reaction reagent is sealed to form reaction reagent paraffin wax balls.

[0017] A further solution is that it also includes a second shell mold, step S2 also includes coating a release agent in both the first shell mold and the second shell mold, adding the first magnetic paraffin in a melted state into the first shell mold, adding the second magnetic paraffin in a melted state into the second shell mold, and after cooling and solidification, taking out the first magnetic paraffin from the first shell mold for use, and taking out the second magnetic paraffin from the second shell mold for use; step S4 also includes covering the second magnetic paraffin on the first magnetic paraffin, sealing the reaction reagent between the first magnetic paraffin and the second magnetic paraffin, and sealing the connection between the first magnetic paraffin and the second magnetic paraffin with excess melted paraffin or excess melted magnetic paraffin to form a reaction reagent paraffin ball.

[0018] As can be seen from the above, the first magnetic paraffin wax and the second magnetic paraffin wax quickly cooperate to seal the PCR reagent, which can effectively improve the work efficiency and reduce the time the PCR reagent is exposed to the air.

[0019] A further solution is that step S3 also includes drying and purifying the reaction reagent, dripping the dried reaction reagent on the upper surface of the magnetic paraffin, and placing the first magnetic paraffin and the reaction reagent in a vacuum box for vacuum drying; or step S3 also includes making the reaction reagent into freeze-dried beads, placing the reaction reagent on the first magnetic paraffin, and placing the first magnetic paraffin and the reaction reagent in a vacuum box for vacuum drying.

[0020] As can be seen from the above, the purified reaction reagents can effectively improve the accuracy of the test results and reduce the impact of impurities. The reaction reagents made into freeze-dried beads can facilitate the sealing of the reagents, and the reaction reagents are vacuum dried before sealing. In this way, the sealed biological reaction reagents (such as PCR reaction reagents) can be stored at a low temperature of 4° or at room temperature.

[0021] A further solution is that the vacuum drying time is 10 min-60 min.

[0022] A further solution is that the release agent is white oil.

[0023] A further solution is that the number of first shell molds is at least two, and the two or more first shell molds are integrally formed; and / or the number of second shell molds is at least two, and the two or more second shell molds are integrally formed.

[0024] It can be seen from the above that multiple first shell molds and multiple second shell molds can effectively improve the processing efficiency and meet the needs of storing multiple PCR reagents.

[0025] In order to achieve the above-mentioned third purpose, the reagent usage method provided by the present invention is applied to any of the above-mentioned reaction reagent paraffin balls, and the usage method includes placing the reaction reagent paraffin balls in a microfluidic chip, moving the reaction reagent paraffin balls to the corresponding positions on the microfluidic chip through a magnetic rod device on a microfluidic detection device, heating the reaction reagent paraffin balls, and releasing the reaction reagent to react.

[0026] As can be seen from the above, this method can manipulate the prepared reaction reagent paraffin balls to move to any position for heating and release.

[0027] In summary, the method for preparing the reaction reagent paraffin balls of the present invention has good storage effect and can be freely moved according to demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of step S1 of Example 1 of the method for preparing the reaction reagent of the present invention.

[0029] Figure 2 This is a schematic diagram of step S2 of the method for preparing the reaction reagent of the present invention.

[0030] Figure 3 This is a schematic diagram of the first shell mold of an embodiment of the method for preparing a reaction reagent of the present invention.

[0031] Figure 4 It is a cross-sectional view of an embodiment of a reaction reagent paraffin ball of the present invention.

[0032] Figure 5 It is a curve diagram of the amplification results of the control group of the present invention.

[0033] Figure 6 It is a curve diagram of the amplification results of the dried PCR reagent in the paraffin shell mixed with magnetic beads of the present invention. DETAILED DESCRIPTION

[0034] See also Figure 4 The reaction reagent paraffin ball provided in this embodiment includes a paraffin encapsulation shell 11, which is made of a mixture of paraffin 2 and magnetic beads 3, the paraffin encapsulation shell 11 is solid, and the magnetic beads 3 are dispersed in the solid paraffin 2; a reagent containing cavity 10 is formed in the paraffin encapsulation shell 11, and a reagent 9 is contained in the reagent containing cavity 10, and the reagent 9 is wrapped by the paraffin encapsulation shell 11. Optionally, the magnetic beads 3 can be silicon-based magnetic beads.

[0035] See also Figures 1 to 4 The preparation method of the reaction reagent paraffin balls provided in this embodiment comprises the following steps: S1: placing a beaker 1 containing solid paraffin in a water bath for water bath heating to completely melt the paraffin 2 into a liquid state, adding an appropriate amount of magnetic beads 3 with a diameter of 0.5 mm, and stirring and mixing for 30-60 minutes to mix the magnetic beads 3 and the paraffin 2 evenly to make magnetic paraffin, and waiting for the magnetic paraffin to cool and solidify for use.

[0036] S2: Place the prepared first magnetic paraffin 5 in a water bath and heat it in a water bath so that the first magnetic paraffin 5 melts into a liquid state. A small amount of white oil is applied as a release agent in the prefabricated first shell mold 4. Use a pipette gun to drip the liquid first magnetic paraffin 5 into the first shell mold 4 to prepare a cylinder with a diameter of 10 mm and a thickness of 2 mm. After the first magnetic paraffin 5 is cooled and solidified, it is taken out of the first shell mold 4 for use. A receiving groove 12 is opened on the surface of the solid first magnetic paraffin 5. Alternatively, the first magnetic paraffin 5 can form a receiving groove 12 in the first shell mold 4.

[0037] S3: Dry and purify the prepared reaction reagent 9, drop the dried reaction reagent 9 on the plane of the solid first magnetic paraffin 5, and place the first magnetic paraffin 5 and the reaction reagent 9 in a vacuum drying oven for vacuum drying. The vacuum drying time is 10 minutes to 60 minutes, and the vacuum value in the vacuum drying oven is -98±1KPa. Take out the dried first magnetic paraffin 5 and the reaction reagent 9 for standby use.

[0038] Alternatively, the prepared reaction reagent 9 may be made into a freeze-dried bead form, and then there is no need to open the receiving groove 12 on the first magnetic paraffin 5 .

[0039] S4: Wrapping the dried first magnetic paraffin 5 containing the reaction reagent 9 with a second magnetic paraffin 6 shell, specifically, dripping the melted second magnetic paraffin 6 around the reaction reagent 9 in sequence, gradually wrapping and sealing the reaction reagent 9, and making a reaction reagent paraffin ball.

[0040] An optional solution is that step S2 further includes coating white oil as a release agent in the second shell mold (not shown in the figure), dripping the melted second magnetic paraffin 6 into the second shell mold, and taking it out from the second shell mold for standby after cooling and solidification. The first shell mold and the second shell mold have similar structures.

[0041] Step S4 also includes covering the second magnetic paraffin 6 on the first magnetic paraffin 5, sealing and wrapping the reaction reagent 9 between the first magnetic paraffin 5 and the second magnetic paraffin 6, and sealing the connection between the first magnetic paraffin 5 and the second magnetic paraffin 6 with excess melted paraffin or excess melted magnetic paraffin to form a reaction reagent paraffin ball.

[0042] Optionally, the number of the first shell molds 4 is at least two, and the two or more first shell molds 4 are integrally formed; and / or the number of the second shell molds is at least two, and the two or more second shell molds are integrally formed.

[0043] This embodiment also provides a method for using the above-mentioned reaction reagent paraffin balls, placing the prepared reaction reagent paraffin balls in a microfluidic chip, controlling the reaction reagent paraffin balls in the microfluidic chip to move to corresponding positions on the microfluidic chip through a magnetic rod device on a microfluidic detection device, and then raising the temperature to melt the reaction reagent paraffin balls to release the encapsulated reagent for reaction.

[0044] The temperature of the paraffin balls in the microfluidic chip can be increased by a heating wire arranged in the microfluidic chip or a heating structure on a microfluidic chip detection device.

[0045] See also Figure 5 and Figure 6, the drying reagent in the EP tube (centrifuge tube) is used as the control group, and the reaction reagent paraffin ball and the drying reagent are amplified in the PCR detector to compare the results. The fluorescence channel corresponding to the fluorescein of the primer used for amplification is the HEX channel:

[0046]

[0047] It can be seen from the above that when the PCR amplification reaction is carried out using the reaction reagent paraffin balls, the CT vertical value of the strong positive 7 and the CT value of the weak positive 8 produced are lower than those of the control group, which means that the gain produced by the PCR reaction using the reaction reagent paraffin balls is better than that of the control group.

[0048] In summary, it can be seen that the reaction reagent paraffin balls prepared by the reaction reagent preparation method of the present invention have good storage effect, and the stored reaction reagents can be freely moved according to demand.

[0049] Finally, it should be emphasized that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Reaction reagent paraffin ball, It is characterized in that include: A paraffin encapsulation shell, wherein the paraffin encapsulation shell is made by mixing paraffin and magnetic beads, the paraffin encapsulation shell is solid, and the magnetic beads are dispersed in the solid paraffin; A reagent containing cavity is formed in the paraffin packaging shell. The reagent containing cavity contains a reagent, and the reagent is wrapped by the paraffin packaging shell.

2. The reaction reagent paraffin ball according to claim 1, Features: The magnetic beads include silicon-based magnetic beads.

3. Preparation method of reaction reagent paraffin balls, It is characterized in that The preparation method comprises the following steps: S1: heating solid paraffin in a container to melt it into a liquid state, adding an appropriate amount of magnetic beads to the paraffin, and stirring and mixing the melted paraffin and the magnetic beads to prepare magnetic paraffin; S2: applying a release agent in the first shell mold, adding the first magnetic paraffin in a melted state into the first shell mold, and after cooling and solidification, taking out the first magnetic paraffin from the first shell mold; S3: placing the prepared reaction reagent on the first solid magnetic paraffin; S4: The reaction reagent is sealed and wrapped with a second magnetic paraffin to prepare the reaction reagent paraffin ball.

4. The method for preparing the reaction reagent paraffin balls according to claim 3, Features: Step S4 also includes dripping the melted second magnetic paraffin wax around the reaction reagent in sequence until the reaction reagent is sealed to form the reaction reagent paraffin wax balls.

5. The method for preparing the reaction reagent paraffin balls according to claim 3, Features: It also includes a second shell mold, and step S2 also includes coating a release agent on both the first shell mold and the second shell mold, adding the first magnetic paraffin in a melted state into the first shell mold, adding the second magnetic paraffin in a melted state into the second shell mold, and after cooling and solidification, taking out the first magnetic paraffin from the first shell mold for standby use, and taking out the second magnetic paraffin from the second shell mold for standby use; Step S4 also includes covering the second magnetic paraffin on the first magnetic paraffin, the reaction reagent is sealed and wrapped between the first magnetic paraffin and the second magnetic paraffin, and the connection between the first magnetic paraffin and the second magnetic paraffin is sealed with excess melted paraffin or excess melted magnetic paraffin to form the reaction reagent paraffin ball.

6. The method for preparing the reaction reagent paraffin balls according to any one of claims 3 to 5, Features: Step S3 also includes drying and purifying the reaction reagent, dripping the dried reaction reagent on the upper surface of the magnetic paraffin, and placing the first magnetic paraffin and the reaction reagent in a vacuum box for vacuum drying; Alternatively, step S3 further includes preparing the reaction reagent into freeze-dried beads, placing the reaction reagent on the first magnetic paraffin, and placing the first magnetic paraffin and the reaction reagent in a vacuum box for vacuum drying.

7. The method for preparing the reaction reagent paraffin balls according to claim 6, Features: The vacuum drying time in step S3 is 10 min-60 min.

8. The method for preparing the reaction reagent paraffin balls according to any one of claims 3 to 5, Features: The release agent is white oil.

9. The method for preparing the reaction reagent paraffin balls according to any one of claims 3 to 5, Features: The number of the first shell molds is at least two, and the two or more first shell molds are integrally formed; And / or the number of the second shell molds is at least two, and the two or more second shell molds are integrally formed.

10. A method for using the reaction reagent paraffin balls as claimed in claim 1 or 2, It is characterized in that The method includes: The reaction reagent paraffin balls are placed in a microfluidic chip, and the reaction reagent paraffin balls are moved to corresponding positions on the microfluidic chip by a magnetic rod device on a microfluidic detection device, and the reaction reagent paraffin balls are heated to release the reaction reagent for reaction.

Images