CN111748464B - Manufacturing method of digital PCR chip and digital PCR chip - Google Patents
Manufacturing method of digital PCR chip and digital PCR chip Download PDFInfo
- Publication number
- CN111748464B CN111748464B CN202010627422.7A CN202010627422A CN111748464B CN 111748464 B CN111748464 B CN 111748464B CN 202010627422 A CN202010627422 A CN 202010627422A CN 111748464 B CN111748464 B CN 111748464B
- Authority
- CN
- China
- Prior art keywords
- substrate
- microchannel
- microchannel plate
- reaction solution
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
Abstract
The invention provides a manufacturing method of a digital PCR chip and the digital PCR chip, wherein the manufacturing method comprises the following steps: bonding the first surface of the microchannel plate and the second surface of the first substrate such that a microchannel array is formed between the microchannel plate and the first substrate for fluid communication; injecting a reaction solution onto a third surface of the microchannel plate disposed opposite the first surface to fill the microchannel array with the reaction solution; the third surface is subjected to a sealing process to separate each microchannel of the microchannel array into separate reaction chambers from each other. The invention has the beneficial effects that: each microchannel of the microchannel plate is mutually separated into independent reaction chambers, namely, each microchannel of the microchannel plate is used as an independent reaction container, so that DNA molecules are better separated, the sensitivity is improved, and the detection accuracy of the concentration of a DNA molecule solution is improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a manufacturing method of a digital PCR chip and the digital PCR chip.
Background
Polymerase Chain Reaction (PCR) is an in vitro nucleic acid amplification technology, and is used for rapidly detecting or amplifying a sample with trace nucleic acid molecules, so that PCR can be widely used in genetic engineering fields such as gene detection and gene amplification at present, and plays a certain role in clinical medicine, environmental detection, food safety and the like.
However, the traditional PCR has certain limitations in quantitative detection or amplification, cannot accurately detect the concentration of nucleic acid molecules, and has the defects of sample waste, unstable amplification result and the like. In order to solve the problems of the conventional PCR, the prior art provides a digital PCR which plays an increasingly important role in the field of quantitative detection, wherein the basic principle of the digital PCR is to separate reaction solutions into tiny reaction vessels which are isolated from each other, and perform PCR reaction on a single DNA molecule in each tiny vessel, so as to accurately determine whether a DNA molecule to be detected exists in the vessel according to whether a fluorescence signal is generated. And recording the ratio of the number of containers generating the fluorescence signals to the total number of containers, and obtaining the accurate quantification of the DNA copy number according to the ratio because the DNA molecular distribution obeys Poisson distribution. In addition to the above-mentioned amplification of DNA molecules by PCR technology, the Loop-mediated isothermal amplification (LAMP) technology has also received great attention in recent years, and it is characterized by designing 4 specific primers for 6 regions of the target gene, and under the action of strand displacement DNA polymerase (Bst DNA polymerase, also can be directly written as Bst DNA polymerase), the amplification is performed at a constant temperature of 60-65 ℃, and compared with the traditional PCR, it does not need a complicated temperature raising and lowering process, the amplification multiple is larger, and it can realize higher sensitivity. However, the existing implementation methods of digital PCR include micro-droplet, micro-nano processing chip, etc., and have the challenges of difficult processing, small detection range, etc.
Disclosure of Invention
In view of the above problems in the prior art, a method for manufacturing a digital PCR chip and a digital PCR chip are provided.
The specific technical scheme is as follows:
a manufacturing method of a digital PCR chip specifically comprises the following steps:
bonding the first surface of the microchannel plate and the second surface of the first substrate such that a microchannel array is formed between the microchannel plate and the first substrate for fluid communication;
injecting a reaction solution onto a third surface of the microchannel plate disposed opposite the first surface to fill the microchannel array with the reaction solution;
the third surface is subjected to a sealing process to separate each microchannel of the microchannel array into separate reaction chambers from each other.
Preferably, the method for manufacturing a digital PCR chip, wherein the first surface of the microchannel plate and the second surface of the first substrate are bonded, so that a microchannel array for fluid communication is formed between the microchannel plate and the first substrate, specifically includes the following steps:
preparing a first substrate through a substrate mixed solution, and cleaning the prepared first substrate to obtain a cleaned first substrate;
and bonding the first surface of the microchannel plate and the second surface of the cleaned first substrate by plasma so as to form a microchannel array for fluid communication between the microchannel plate and the first substrate.
Preferably, the method for manufacturing a digital PCR chip, wherein a reaction solution is injected onto a third surface of the microchannel plate disposed opposite to the first surface, so that the microchannel array is filled with the reaction solution, specifically includes the steps of:
clinging the fourth surface of the second substrate to the third surface of the microchannel plate, and carrying out plasma treatment on the whole chip adhered with the first substrate, the microchannel plate and the second substrate;
taking the second substrate off the integrated chip, and dripping the reaction solution onto a third surface of the microchannel plate, which is opposite to the first surface, so that the reaction solution forms a hemispherical droplet chamber on the third surface of the microchannel plate;
and carrying out vacuum treatment on the microchannel plate so that the reaction solution in the hemispherical droplet chamber is injected into the microchannel array.
Preferably, the method for manufacturing a digital PCR chip, wherein the third surface is sealed to separate each microchannel in the microchannel array into independent reaction chambers, comprises the following steps:
removing the reaction solution above the third surface;
injecting a sealing liquid on the third surface;
and a second substrate is arranged on the third surface in a clinging manner, so that the second substrate seals the third surface to separate each microchannel in the microchannel array into independent reaction chambers.
Preferably, the method for manufacturing a digital PCR chip, wherein the method for manufacturing a reaction solution comprises the steps of:
performing first centrifugation treatment on each liquid forming a reaction mixed solution;
mixing each liquid subjected to the first centrifugal treatment according to a preset proportion to form a reaction mixed liquid;
and carrying out second centrifugation treatment on the reaction mixed solution to obtain a reaction solution.
The method also comprises a manufacturing method of the digital PCR chip, wherein the method specifically comprises the following steps:
bonding the first surface of the microchannel plate and the second surface of the first substrate such that a microchannel array is formed between the microchannel plate and the first substrate for fluid communication;
obtaining a second substrate, wherein a cavity is arranged between the first substrate and the second substrate, and a through hole for connecting a fifth surface of the second substrate with the cavity is formed at the position of each vertex angle of the cavity;
the third surface of the bonded microchannel plate and the fourth surface of the second substrate are oppositely arranged, a relative distance is arranged between the third surface and the fourth surface, and the fifth surface and the fourth surface are oppositely arranged on the second substrate;
injecting a reaction solution into the cavity through the through hole, so that the cavity and the micro-channel array are filled with the reaction solution;
and sealing the third surface and the through holes in the cavity to separate each microchannel in the microchannel array into independent reaction chambers.
Preferably, the method for manufacturing a digital PCR chip further comprises: two through holes on the same diagonal line are respectively used as a liquid inlet and a liquid outlet.
Preferably, the method for manufacturing a digital PCR chip, wherein the third surface and the through hole in the cavity are sealed to separate each microchannel in the microchannel array into independent reaction chambers, specifically includes the following steps:
injecting a sealing liquid into the cavity through the liquid inlet, and discharging the reaction solution above the third surface of the microchannel plate from the liquid outlet so as to mutually separate each microchannel in the microchannel array into independent reaction chambers;
a sealing member is disposed on each through hole such that the sealing member seals the cavity of the second substrate.
The device also comprises a digital PCR chip, wherein the digital PCR chip comprises a microchannel plate, a first substrate and a second substrate;
the microchannel plate is provided with a plurality of adjacently arranged microchannels, a first surface of the microchannel plate and a second surface of the first substrate are arranged in a bonding mode to form a microchannel array for fluid circulation between the microchannel plate and the first substrate, a reaction solution is arranged in the microchannel array, a third surface of the microchannel plate, opposite to the first surface, is in contact with a fourth surface of the second substrate, and a sealing liquid is filled between the third surface and the fourth surface.
Preferably, the digital PCR chip is characterized in that the reaction solution is a reaction mixed solution obtained by mixing and centrifuging a reaction buffer solution, a calcein solution, Bst DNA polymerase, deionized water, a plurality of primers and a DNA solution according to a preset proportion.
Preferably, the digital PCR chip, wherein a cavity is disposed between the first substrate and the second substrate, and the microchannel plate is disposed in the cavity.
Preferably, the digital PCR chip, wherein the cavity has a length, width and height greater than those of the microchannel plate.
Preferably, the second substrate is provided with a through hole on each vertex angle of the position of the cavity for communicating the fifth surface of the second substrate with the cavity;
wherein the fifth surface is disposed opposite the fourth surface.
The technical scheme has the following advantages or beneficial effects:
firstly, each microchannel of the microchannel plate is mutually divided into independent reaction chambers, namely, each microchannel of the microchannel plate is used as an independent reaction container, so that DNA molecules are better separated, the sensitivity is improved, and lower concentration of DNA molecule solution can be detected;
secondly, the operation is simple, so that the digital PCR chip is manufactured in large scale through a simple glass drawing process, and further, the digital PCR chip is applied to DNA detection in large scale.
Drawings
Embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying drawings. The drawings are, however, to be regarded as illustrative and explanatory only and are not restrictive of the scope of the invention.
FIG. 1 is a schematic structural view of a microchannel plate according to the present invention;
FIG. 2 is a schematic structural view of a microchannel of the present invention;
FIG. 3 is a diagram illustrating a first step of a method for fabricating a digital PCR chip according to a first embodiment of the present invention;
FIG. 4 is a schematic structural diagram of the digital PCR chip of the present invention after disassembly;
FIG. 5 is a schematic structural diagram of a second embodiment of the digital PCR chip of the present invention;
FIG. 6 is a plan view of a second embodiment of the digital PCR chip of the present invention when a reaction solution is dropped;
FIG. 7 is a front view of a second embodiment of the digital PCR chip of the present invention when a reaction solution is dropped;
FIG. 8 is a top view of a third embodiment of the digital PCR chip of the present invention;
FIG. 9 is a schematic diagram of a third embodiment of a digital PCR chip according to the present invention when a reaction solution is injected;
FIG. 10 is a schematic structural diagram of a third embodiment of a digital PCR chip according to the present invention when a sealing liquid is injected;
FIG. 11 is a diagram of the third step of the method for manufacturing a digital PCR chip according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
It should be noted that: in other embodiments, the steps of the corresponding methods are not necessarily performed in the order shown and described herein. In some other embodiments, the method may include more or fewer steps than those described herein. Moreover, a single step described in this specification may be broken down into multiple steps for description in other embodiments; multiple steps described in this specification may be combined into a single step in other embodiments.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
The first embodiment is as follows:
a method for manufacturing a digital PCR chip, as shown in FIG. 3, specifically comprises the following steps:
step a1, bonding a first surface 21 of a microchannel plate 1 (MCP) and a second surface 22 of a first substrate 2, so that a microchannel array for fluid communication is formed between the microchannel plate 1 and the first substrate 2;
a step a2 of injecting a reaction solution 4 on a third surface 23 of the microchannel plate 1 disposed opposite to the first surface 21 so that the microchannel array is filled with the reaction solution 4;
step a3, the third surface 23 is subjected to a sealing treatment to separate each microchannel 11 in the microchannel array into independent reaction chambers.
In the above-described embodiment, it is possible to separate each microchannel 11 of the microchannel plate 1 into independent reaction chambers by subjecting both the first surface 21 and the third surface 23 disposed opposite to the first surface 21 of the microchannel plate 1 to a sealing treatment, that is, to realize the use of each microchannel 11 of the microchannel plate 1 as a separate reaction vessel; therefore, the microchannel plate 1 obtained in the above embodiment has millions of reaction vessels, so that DNA molecules can be better separated, the sensitivity is improved, and lower concentration of DNA molecule solution can be detected.
In the above embodiment, the first substrate 2 is PDMS (polydimethylsiloxane), and since the basic composition of the microchannel plate 1 is glass and the biocompatibility of the microchannel plate 1 and the PDMS is good, the microchannel plate 1 and the PDMS can be bonded to form a microchannel array between the microchannel plate 1 and the PDMS for fluid flow.
In the above embodiment, the microchannel plate 1 and the first substrate 2 are bonded first; then directly injecting a reaction solution 4 from the upper surface of the microchannel plate 1 to fill the microchannel array with the reaction solution 4, finally removing the excess reaction solution 4, and sealing the third surface 23 to separate each microchannel 11 in the microchannel array into independent reaction chambers; the operation is simple, so that the digital PCR chip is manufactured in large scale through a simple glass drawing process, and further, the digital PCR chip is applied to DNA detection in large scale.
Further, in the above embodiment, the step a1 specifically includes the following steps:
step A11, preparing a first substrate 2 by a substrate mixed solution, and cleaning the prepared first substrate 2 to obtain a cleaned first substrate 2;
step a12, bonding the first surface 21 of the microchannel plate 1 and the second surface 22 of the cleaned first substrate 2 by plasma, so as to form a microchannel array between the microchannel plate 1 and the first substrate 2 for fluid communication.
In the above embodiment, first, the substrate mixture is prepared by mixing the curing agent (curing agent) and the base solution (base) at a ratio of 1:10, and then vacuum-treating the mixed solution to remove air bubbles in the solution, and using the solution from which air bubbles are removed as the substrate mixture;
then, adding the substrate mixed liquid on the surface of the glass sheet, curing the substrate mixed liquid at a preset temperature for a preset time, tearing the cured substrate mixed liquid off the surface of the glass sheet, performing edge adjustment on the torn cured substrate mixed liquid, and taking the adjusted cured substrate mixed liquid as a first substrate 2;
wherein the edge can be adjusted by cutting an irregular edge;
wherein, the preset temperature can be 80 ℃, and the preset time can be 1 hour;
subsequently, the first substrate 2 is cleaned, and the microchannel plate 1 may also be cleaned by the same method;
the cleaning method specifically comprises the following steps:
firstly, ultrasonically cleaning a first substrate 2 and/or a microchannel plate 1 by using acetone, wherein the cleaning time can be 5 min;
secondly, ultrasonically cleaning the first substrate 2 and/or the microchannel plate 1 by using isopropanol, wherein the cleaning time can be 10 min;
thirdly, ultrasonically cleaning the first substrate 2 and/or the microchannel plate 1 with deionized water (DI water), wherein the cleaning time may be 10 min;
fourth, the first substrate 2 and/or the microchannel plate 1 are blow-dried with nitrogen gas to complete the cleaning process.
Then, the first surface 21 of the microchannel plate 1 and the second surface 22 of the first substrate 2 are placed in a bonding manner in an operating instrument, so that the operating instrument bonds the first surface 21 of the microchannel plate 1 and the second surface 22 of the first substrate 2 by using plasma with power of 60W, a microchannel array for fluid circulation is formed between the microchannel plate 1 and the first substrate 2, and a digital nucleic acid detection chip of the microchannel plate 1 on the first substrate 2 is obtained.
Further, in the above embodiment, as shown in fig. 6 and 7, step a2 specifically includes the following steps:
step A21, clinging the fourth surface 24 of the second substrate 3 to the third surface 23 of the microchannel plate 1, and carrying out plasma treatment on the whole chip adhered with the first substrate 2, the microchannel plate 1 and the second substrate 3;
specifically, the second substrate 3 is tightly attached to the third surface 23 of the microchannel plate 1, and the chip, in which the first substrate 2, the microchannel plate 1 and the second substrate 3 are sequentially disposed, is placed in an operation instrument, so that the operation instrument processes the whole chip by using plasma with a power of 60W.
Step A22, taking the second substrate 3 off the whole chip, and dropping the reaction solution 4 onto the third surface 23 of the microchannel plate 1 opposite to the first surface 21, so that the reaction solution 4 forms a hemispherical droplet chamber on the third surface 23 of the microchannel plate 1;
specifically, the reaction solution 4 may be sucked up by using a pipette gun and dripped on the third surface 23 of the microchannel plate 1, and a hemispherical droplet chamber may be formed by using the difference in hydrophilicity and hydrophobicity between the surfaces of the microchannel plate 1 and the first substrate 2, as shown in fig. 6 and 7.
Step A23, carrying out vacuum treatment on the microchannel plate 1, so that the reaction solution 4 in the hemispherical droplet chamber is injected into the microchannel array, and the microchannel array is filled with the reaction solution 4;
specifically, the first substrate 2 provided with the microchannel plate 1 is placed in a vacuum pump for pumping, so that the reaction solution 4 is injected into the microchannel array, and the microchannel array is filled with the reaction solution 4, wherein the pumping time is 10min-15 min.
Further, in the above embodiment, the step a3 specifically includes the following steps:
step a31, removing the reaction solution 4 above the third surface 23;
step a31 may specifically include: removing the redundant reaction solution 4 above the third surface 23 of the microchannel plate 1 by using a PDMS stripping technology;
specifically, firstly, preparing a PDMS film with a first preset thickness and cleaning the PDMS film;
next, a PDMS film is attached over the third surface 23;
subsequently, the PDMS film over the third surface 23 was peeled off to remove the excess reaction solution 4.
Step a32, injecting a sealing liquid 5 on the third surface 23;
specifically, a pipette is used to suck the sealing liquid 5 and drop the sealing liquid onto the third surface 23 of the microchannel plate 1;
wherein the sealing liquid 5 may be mineral oil, so that each microchannel 11 in the microchannel array is isolated with mineral oil to prevent the reaction solution 4 from evaporating;
step a33, a second substrate 3 is disposed closely on the third surface 23, such that the second substrate 3 seals the third surface 23 to separate each microchannel 11 in the microchannel array into independent reaction chambers.
Further, in the above embodiment, the method for manufacturing the reaction solution 4 includes the steps of:
step C1, performing a first centrifugation treatment on each liquid forming a reaction mixed solution;
specifically, each of the liquids described above includes: reaction buffer solution (RM), calcein solution (FD), Bst DNA polymerase, Deionized Water (DW), six primers (including FIP, BIP, F3, B3, LF and LB) and DNA solution (the concentration is detected);
step C2, mixing each liquid subjected to the first centrifugation treatment according to a preset proportion to form a reaction mixed liquid;
specifically, 12.5. mu.L of reaction buffer, 1. mu.L of FD solution, 1. mu.L of Bst DNA polymerase, 2.5. mu.L of deionized water, and 25. mu.L of each of six primers and 2. mu.L of DNA solution were taken by a pipette and added to a test tube, and the test tube was shaken by a shaker to obtain a reaction mixture.
Step C3, the reaction mixture was subjected to a second centrifugation treatment to obtain a reaction solution 4.
Specifically, the test tube containing the reaction mixture is placed in a centrifuge to subject the reaction mixture to a second centrifugation treatment, thereby obtaining a LAMP system reaction solution 4.
Example two:
also provided is a digital PCR chip, as shown in FIGS. 4 and 5, comprising a microchannel plate 1, a first substrate 2 and a second substrate 3;
the microchannel plate 1 is provided with a plurality of adjacently arranged microchannels 11, a first surface 21 of the microchannel plate 1 and a second surface 22 of the first substrate 2 are bonded to form a microchannel array for fluid communication between the microchannel plate 1 and the first substrate 2, a reaction solution 4 is provided in the microchannel array, a third surface 23 of the microchannel plate 1 opposite to the first surface 21 is in contact with a fourth surface 24 of the second substrate 3, and a sealing liquid 5 is filled between the third surface 23 and the fourth surface 24.
Further, in the above-mentioned examples, the reaction solution 4 is a reaction mixture obtained by mixing and centrifuging a reaction buffer (RM), a calcein solution (FD), Bst DNA polymerase, Deionized Water (DW), six primers (including FIP, BIP, F3, B3, LF, LB), and a DNA solution (whose concentration is to be detected) in a predetermined ratio.
Specifically, a reaction buffer solution, a calcein solution, Bst DNA polymerase, deionized water, six primers (including FIP, BIP, F3, B3, LF and LB) and a liquid obtained after the DNA solution is centrifuged are obtained respectively;
then, 12.5. mu.L of the reaction buffer solution after centrifugation, 1. mu.L of FD solution, 1. mu.L of Bst DNA polymerase, 2.5. mu.L of deionized water, 1. mu.L of each of the six primers and 2. mu.L of the DNA solution were taken out by a pipette and added to a test tube in a total amount of 25. mu.L, and the test tube was shaken using a shaker to obtain a reaction mixture.
Further, in the above embodiment, as shown in fig. 1, the shape of the microchannel plate 1 may be self-set by a user, and may be, for example, a cylinder, a rectangular parallelepiped, or the like.
For example, the user may trim the microchannel plate 1.
Further, in the above-described embodiment, as shown in fig. 2, the cross section of the microchannel 11 is a regular circle or a regular hexagon.
Further, in the above embodiment, the number of the microchannels 11 in the microchannel array is 1 ten thousand to 500 ten thousand;
further, in the above-described embodiment, the diameter of the cross section of the microchannel 11 ranges from 1 μm to 100 μm;
further, in the above-described embodiment, the volume of the microchannel 11 ranges from 1pL to 1. mu.L;
further, in the above embodiment, the distance between two adjacent microchannels 11 is in the range of 1 μm to 100. mu.m.
Further, in the above embodiment, the sealing liquid 5 is mineral oil.
Further, in the above embodiment, the digital PCR chip may be placed in a PCR instrument, the temperature is set to 65 ℃, the reaction time is set to 50min, and the LAMP reaction is completed.
Example three:
a method for manufacturing a digital PCR chip, as shown in fig. 8-11, specifically comprising the steps of:
step B1, bonding the first surface 21 of the microchannel plate 1 and the second surface 22 of the first substrate 2, so that a microchannel array for fluid communication is formed between the microchannel plate 1 and the first substrate 2;
step B2, obtaining a second substrate 3, wherein a cavity 7 is arranged between the first substrate 2 and the second substrate 3, and a through hole 6 for connecting the fifth surface 25 of the second substrate 3 and the cavity 7 is arranged at the position of each vertex angle of the cavity 7;
step B3, oppositely arranging the third surface 23 of the bonded microchannel plate 1 and the fourth surface 24 of the second substrate 3, arranging a relative distance between the third surface 23 and the fourth surface 24, and oppositely arranging the fifth surface 25 and the fourth surface 24 on the second substrate 3;
step B4, injecting a reaction solution 4 into the cavity 7 through the through hole 6, so that the cavity 7 and the micro-channel array are filled with the reaction solution 4;
step B5, sealing the third surface 23 and the through-holes 6 in the cavity 7 to separate each microchannel 11 in the microchannel array into independent reaction chambers.
In the above embodiment, it is possible to separate each microchannel 11 of the microchannel plate 1 into independent reaction chambers by bonding the first surface 21 of the microchannel plate 1 and the second surface 22 of the first substrate 2, disposing the microchannel plate 1 in the cavity 7 between the first substrate 2 and the second substrate 3, and performing the sealing process on the third surface 23, which is carried by the microchannel 11 in the cavity 7, through the through-hole 6, and also performing the sealing process on the through-hole 6, that is, it is possible to realize the use of each microchannel 11 of the microchannel plate 1 as an individual reaction vessel; therefore, the microchannel plate 1 obtained in the above embodiment has millions of reaction vessels, so that DNA molecules can be better separated, the sensitivity is improved, and lower concentration of DNA molecule solution can be detected.
In the above embodiment, the first substrate 2 is PDMS, and since the basic composition of the microchannel plate 1 is glass and the biocompatibility of the microchannel plate 1 and the PDMS is good, the microchannel plate 1 and the PDMS may be bonded to form a microchannel array for fluid to flow between the microchannel plate 1 and the PDMS.
The operation of the embodiment is simple, and the digital PCR chip can be manufactured in large scale through a simple glass drawing process, so that the digital PCR chip can be applied to DNA detection in large scale.
Further, in the above embodiment, the step B1 specifically includes the following steps:
step B11, preparing a first substrate 2 by a substrate mixed solution, and cleaning the prepared first substrate 2 to obtain a cleaned first substrate 2;
step B12, bonding the first surface 21 of the microchannel plate 1 and the second surface 22 of the first substrate 2 by plasma, so as to form a microchannel array between the microchannel plate 1 and the first substrate 2 for fluid communication.
In the above embodiment, first, the substrate mixture is prepared by mixing the curing agent (curing agent) and the base solution (base) at a ratio of 1:10, and then vacuum-treating the mixed solution to remove air bubbles in the solution, and using the solution from which air bubbles are removed as the substrate mixture;
then, adding the substrate mixed liquid on the surface of the glass sheet, curing for a first time at a first temperature, tearing the cured substrate mixed liquid off the surface of the glass sheet, performing edge adjustment on the torn cured substrate mixed liquid, and taking the adjusted cured substrate mixed liquid as a first substrate 2;
wherein the edge can be adjusted by cutting an irregular edge;
wherein the first temperature may be 80 ℃ and the first time may be 1 hour;
subsequently, the first substrate 2 is cleaned, and the microchannel plate 1 may also be cleaned by the same method;
the cleaning method specifically comprises the following steps:
firstly, ultrasonically cleaning a first substrate 2 and/or a microchannel plate for 15min by using acetone;
secondly, ultrasonically cleaning the first substrate 2 and/or the microchannel plate for 110min by using isopropanol;
thirdly, ultrasonically cleaning the first substrate 2 and/or the microchannel plate by using deionized water (DI water) for 110 min;
fourth, the first substrate 2 and/or the microchannel plate 1 are blow-dried with nitrogen gas to complete the cleaning process.
Then, the first surface 21 of the microchannel plate 1 and the second surface 22 of the first substrate 2 are placed in a bonding manner in an operating instrument, so that the operating instrument bonds the first surface 21 of the microchannel plate 1 and the second surface 22 of the first substrate 2 by using plasma with power of 60W, a microchannel array for fluid circulation is formed between the microchannel plate 1 and the first substrate 2, and a digital nucleic acid detection chip of the microchannel plate 1 on the first substrate 2 is obtained.
Further, in the above embodiment, the step B2 specifically includes the following steps:
a step B21 of providing cavities 7 for accommodating microchannel plates 1 on the first substrate 2 and/or the second substrate 3;
specifically, the PDMS first substrate 2 and/or the second substrate 3 having one cavity 7 may be prepared by a reverse mold method;
here, only one cavity 7 may be provided on the single first substrate 2 or second substrate 3;
it is also possible to provide a cavity 7 between the first substrate 2 and the second substrate 3 by combining them, and after providing the cavity 7, perform the operation of step B1 on the first substrate 2.
Step B22, providing a through hole 6 connecting the fifth surface 25 of the second substrate 3 and the cavity 7 at the position of each top corner of the cavity 7;
specifically, a hole puncher may be used to punch holes at four corners of the second substrate 3 where the cavities 7 are located, the holes penetrating through the upper and lower surfaces of the second substrate 3, i.e., each through hole 6 links the fifth surface 25 of the second substrate 3 and the cavity 7.
Further, in the above embodiment, the method further includes: as shown in fig. 8, in step B6, two through holes 6 located on the same diagonal line are used as the liquid inlet 61 and the liquid outlet 62, respectively.
The above step B6 may be performed before step B4.
Further, in the above embodiment, as shown in fig. 9 and fig. 11, the step B4 specifically includes the following steps: the reaction solution 4 is injected into the cavity 7 through the inlet port 61 so that the cavity 7 and the microchannel array are filled with the reaction solution 4.
Specifically, a syringe is used for sucking the LAMP system reaction solution 4, and the reaction solution 4 is slowly injected from the liquid inlet 61 until the reaction solution 4 overflows from the liquid outlet 62 to complete sample injection. Wherein the sample injection time is 20min-30 min.
Further, in the above embodiment, as shown in fig. 10 and 11, step B5 specifically includes the following steps:
step B51, injecting a sealing liquid 5 into the cavity 7 through the liquid inlet 61, and discharging the reaction solution 4 above the third surface 23 of the microchannel plate 1 from the liquid outlet 62 to separate each microchannel 11 in the microchannel array into independent reaction chambers;
specifically, the sealing liquid 5 is slowly injected into the cavity 7 through the liquid inlet 61 by using an injector, the redundant reaction solution 4 in the cavity 7 is discharged from the liquid outlet 62, and the injection is stopped when the sealing liquid 5 overflows from the liquid outlet 62, so as to remove the redundant reaction solution 4 above the third surface 23 of the microchannel plate 1;
wherein, the sealing liquid 5 can be mineral oil.
Step B52, a sealing member is provided on each through hole 6 such that the sealing member seals the cavity 7 of the second substrate 3.
Specifically, each through-hole 6 may be sealed using a photo-curing adhesive.
Example four:
also provided is a digital PCR chip, as shown in FIGS. 5 and 8, comprising a microchannel plate 1, a first substrate 2 and a second substrate 3;
the microchannel plate 1 is provided with a plurality of adjacently arranged microchannels 11, a first surface 21 of the microchannel plate 1 and a second surface 22 of the first substrate 2 are bonded to form a microchannel array for fluid communication between the microchannel plate 1 and the first substrate 2, a reaction solution 4 is arranged in the microchannel array, a third surface 23 of the microchannel plate 1 opposite to the first surface 21 is in contact with a fourth surface 24 of the second substrate 3, and a sealing liquid 5 is filled between the third surface 23 and the fourth surface 24, wherein a cavity 7 is arranged between the first substrate 2 and the second substrate 3, and the microchannel plate 1 is arranged in the cavity 7.
Further, in the above embodiment, the second substrate 3 is provided with a through hole 6 communicating the fifth surface 25 of the second substrate 3 and the cavity 7 at each corner of the cavity 7;
as shown in fig. 4, the fifth surface 25 is disposed opposite to the fourth surface 24.
Further, in the above embodiment, the length, width and height of the cavity 7 are greater than those of the microchannel plate 1, so that a relative distance is provided between the third surface 23 and the fourth surface 24, and the sealing liquid 5 is filled between the third surface 23 and the fourth surface 24.
Further, in the above-mentioned examples, the reaction solution 4 is a reaction mixture obtained by mixing and centrifuging a reaction buffer (RM), a calcein solution (FD), Bst DNA polymerase, Deionized Water (DW), six primers (including FIP, BIP, F3, B3, LF, LB), and a DNA solution (whose concentration is to be detected) in a predetermined ratio.
Further, in the above embodiment, the preset ratio is: reaction buffer 12.5. mu.L, calcein solution 1. mu.L, Bst DNA polymerase 1. mu.L, deionized water 2.5. mu.L, each primer in a proportion of 1. mu. L, DNA solution 2. mu.L.
Specifically, a reaction buffer solution, a calcein solution, Bst DNA polymerase, deionized water, six primers (including FIP, BIP, F3, B3, LF and LB) and a liquid obtained after the DNA solution is centrifuged are obtained respectively;
then, 12.5. mu.L of the reaction buffer solution after centrifugation, 1. mu.L of FD solution, 1. mu.L of Bst DNA polymerase, 2.5. mu.L of deionized water, 1. mu.L of each of the six primers and 2. mu.L of the DNA solution were taken out by a pipette and added to a test tube in a total amount of 25. mu.L, and the test tube was shaken using a shaker to obtain a reaction mixture.
Further, in the above embodiment, the shape of the microchannel plate 1 may be self-set by a user, and may be, for example, a cylinder, a rectangular parallelepiped, or the like.
Further, in the above-described embodiment, the cross section of the microchannel 11 is a regular circle or a regular hexagon.
Further, in the above embodiment, the number of the microchannels 11 in the microchannel array is 1 ten thousand to 500 ten thousand;
further, in the above-described embodiment, the diameter of the cross section of the microchannel 11 ranges from 1 μm to 100 μm;
further, in the above-described embodiment, the volume of the microchannel 11 ranges from 1pL to 1. mu.L;
further, in the above embodiment, the distance between two adjacent microchannels 11 is in the range of 1 μm to 100. mu.m.
Further, in the above embodiment, the sealing liquid 5 is mineral oil.
Further, in the above embodiment, the digital PCR chip may be placed in a PCR instrument, the temperature is set to 65 ℃, the reaction time is set to 50min, and the LAMP reaction is completed.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (1)
1. A manufacturing method of a digital PCR chip is characterized by comprising the following steps:
bonding a first surface of a microchannel plate to a second surface of a first substrate such that a microchannel array is formed between the microchannel plate and the first substrate for fluid communication;
injecting a reaction solution on a third surface of the microchannel plate disposed opposite the first surface such that the microchannel array is filled with the reaction solution;
sealing the third surface to separate each microchannel of the microchannel array into separate reaction chambers;
the first substrate is PDMS, and the material for manufacturing the microchannel plate comprises glass;
the method for manufacturing the microchannel plate comprises the following steps of bonding a first surface of a microchannel plate and a second surface of a first substrate, so that a microchannel array for fluid circulation is formed between the microchannel plate and the first substrate, and specifically comprises the following steps:
preparing the first substrate through a substrate mixed solution, and cleaning the prepared first substrate to obtain the cleaned first substrate;
bonding the first surface of the microchannel plate and the second surface of the cleaned first substrate by plasma so as to form a microchannel array between the microchannel plate and the first substrate for fluid communication;
wherein the step of injecting a reaction solution onto a third surface of the microchannel plate disposed opposite to the first surface to fill the microchannel array with the reaction solution comprises:
clinging a fourth surface of a second substrate to the third surface of the microchannel plate, and carrying out plasma treatment on the whole chip attached with the first substrate, the microchannel plate and the second substrate;
removing the second substrate from the monolithic chip and dropping the reaction solution onto the third surface of the microchannel plate disposed opposite to the first surface such that the reaction solution forms a hemispherical droplet chamber on the third surface of the microchannel plate;
carrying out vacuum treatment on the microchannel plate so that the reaction solution in the hemispherical droplet chamber is injected into the microchannel array;
wherein the sealing treatment is performed on the third surface to separate each microchannel in the microchannel array into independent reaction chambers, and the method specifically comprises the following steps:
removing the reaction solution above the third surface;
injecting a sealing liquid on the third surface;
a second substrate is arranged on the third surface in a close fit manner, so that the second substrate seals the third surface to separate each microchannel in the microchannel array into independent reaction chambers;
the preparation method of the reaction solution comprises the following steps:
performing first centrifugation treatment on each liquid forming a reaction mixed solution;
mixing each liquid subjected to the first centrifugal treatment according to a preset proportion to form the reaction mixed liquid;
and carrying out second centrifugation treatment on the reaction mixed solution to obtain the reaction solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010627422.7A CN111748464B (en) | 2020-07-02 | 2020-07-02 | Manufacturing method of digital PCR chip and digital PCR chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010627422.7A CN111748464B (en) | 2020-07-02 | 2020-07-02 | Manufacturing method of digital PCR chip and digital PCR chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111748464A CN111748464A (en) | 2020-10-09 |
| CN111748464B true CN111748464B (en) | 2022-04-12 |
Family
ID=72678747
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010627422.7A Active CN111748464B (en) | 2020-07-02 | 2020-07-02 | Manufacturing method of digital PCR chip and digital PCR chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111748464B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001269196A (en) * | 2000-03-24 | 2001-10-02 | Hamamatsu Photonics Kk | Quantitative method for nucleic acid in test object and method for counting number of molecule of nucleic acid in test object |
| CN107287112A (en) * | 2017-08-03 | 2017-10-24 | 甘肃出入境检验检疫局检验检疫综合技术中心 | A kind of array digital pcr chip and its application method |
| CN108993621A (en) * | 2018-07-09 | 2018-12-14 | 浙江大学 | A kind of small room array micro-fluidic chip and method for digital enzyme linked immunosorbent detection |
| CN110241017A (en) * | 2019-05-07 | 2019-09-17 | 中国科学院苏州生物医学工程技术研究所 | Digitize biological detection chip and packaging fixture |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9981239B2 (en) * | 2015-04-21 | 2018-05-29 | Twist Bioscience Corporation | Devices and methods for oligonucleic acid library synthesis |
-
2020
- 2020-07-02 CN CN202010627422.7A patent/CN111748464B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001269196A (en) * | 2000-03-24 | 2001-10-02 | Hamamatsu Photonics Kk | Quantitative method for nucleic acid in test object and method for counting number of molecule of nucleic acid in test object |
| CN107287112A (en) * | 2017-08-03 | 2017-10-24 | 甘肃出入境检验检疫局检验检疫综合技术中心 | A kind of array digital pcr chip and its application method |
| CN108993621A (en) * | 2018-07-09 | 2018-12-14 | 浙江大学 | A kind of small room array micro-fluidic chip and method for digital enzyme linked immunosorbent detection |
| CN110241017A (en) * | 2019-05-07 | 2019-09-17 | 中国科学院苏州生物医学工程技术研究所 | Digitize biological detection chip and packaging fixture |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111748464A (en) | 2020-10-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9671317B2 (en) | Automatic injection device for microarray chip and automatic injection hybridization microarray chip | |
| CN103071548B (en) | A kind of passive delivery valveless type Single Molecule Detection chip and application | |
| EP2227690B1 (en) | The micro-chamber plate, manufacturing method thereof | |
| CN106755420A (en) | Digital pcr chip and method based on surfactant-modified PDMS | |
| JP6483118B2 (en) | Micro chamber plate | |
| CN111763612B (en) | Single cell gene detection chip and manufacturing method and detection method thereof | |
| US10022717B2 (en) | Method of manufacturing micro chamber plate with built-in sample and analytic micro chamber plate, analytic micro chamber plate and apparatus set for manufacturing analytic micro chamber plate with built-in sample | |
| JP3654481B2 (en) | Microreactor for biochemical reaction | |
| EP2237886A1 (en) | Microfluidic device | |
| US11674173B2 (en) | Operation method of multiplex slide plate device | |
| US20090197274A1 (en) | Biological sample reaction chip and biological sample reaction method | |
| CN104894106A (en) | High-integration equidistance equipartition nucleic acid amplification micro-fluidic chip and application | |
| KR20180062936A (en) | Methods for mixing fluids in microfluidic devices, and devices and systems therefor | |
| CN215906212U (en) | Nucleic acid amplification reactor | |
| US11478791B2 (en) | Flow control and processing cartridge | |
| US20120309084A1 (en) | Microchip for nucleic acid amplification reaction and a method of manufacturing the same | |
| CN111748464B (en) | Manufacturing method of digital PCR chip and digital PCR chip | |
| US20060057581A1 (en) | Microfabricated fluidic device for fragmentation | |
| KR20180064285A (en) | Analysis module and manufacturing method thereof | |
| CN113801925A (en) | Container for containing quantitative liquid drop PCR oil-in-water emulsion and use method thereof | |
| CN113275048B (en) | Microfluidic chip and application method thereof | |
| US20220283174A1 (en) | Systems and methods for generating droplets and performing digital analyses | |
| CN116057386A (en) | Fluid device and use thereof | |
| KR20190012690A (en) | Analysis module | |
| Kim et al. | Multi-DNA extraction chip based on an aluminum oxide membrane integrated into a PDMS microfluidic structure |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |