CN113462548A - Microfluidic chip for digital nucleic acid amplification and portable multi-channel detection system - Google Patents
Microfluidic chip for digital nucleic acid amplification and portable multi-channel detection system Download PDFInfo
- Publication number
- CN113462548A CN113462548A CN202110704613.3A CN202110704613A CN113462548A CN 113462548 A CN113462548 A CN 113462548A CN 202110704613 A CN202110704613 A CN 202110704613A CN 113462548 A CN113462548 A CN 113462548A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- liquid drop
- liquid
- microfluidic chip
- acid amplification
- 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.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- B01L3/502769—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 multiphase flow arrangements
- B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
-
- 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
Abstract
The invention discloses a micro-fluidic chip for digital nucleic acid amplification and a portable multi-channel detection system. The microfluidic chip comprises at least one reaction area arranged on a substrate, the reaction area comprises a droplet generation area, a droplet storage area and a waste liquid discharge area which are mutually connected, a step is arranged between the droplet generation area and the droplet storage area, and the outlet of the droplet generation area is higher than the inlet of the droplet storage area; the liquid drop storage area is originally filled with the continuous phase, the discrete phase is input into the liquid drop generation area, and liquid drops obtained by dispersion through step induction when flowing out from the outlet of the liquid drop generation area are directly collected by the liquid drop storage area and used for subsequent nucleic acid amplification and product detection. The invention can generate liquid drops manually without depending on a precise control device, integrates sample distribution, nucleic acid amplification and product detection, and effectively avoids the problems of complicated operation and pollution caused by liquid drop breakage due to liquid drop transfer; the multichannel optical signal detection can be realized, the detection efficiency is improved, and the accuracy of the detection result is improved.
Description
Technical Field
The invention relates to a micro-fluidic chip and a detection system based on the micro-fluidic chip in the technical field of biological detection, in particular to a micro-fluidic chip for digital nucleic acid amplification and a portable multi-channel detection system.
Background
For digital nucleic acid detection technology, three links of reaction solution digitization, nucleic acid amplification and micro-reaction unit detection are generally included. The digitization of the reaction solution means that the reaction solution is uniformly dispersed into thousands to millions of micro-reaction units, each of which may contain zero, one or more target DNA molecules in a poisson distribution. Nucleic Acid Amplification refers to Amplification of a target nucleic Acid molecule signal by placing each micro-Reaction unit in a suitable environment, and the adopted Amplification technology can be constant temperature Amplification technology such as Loop-mediated isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), nucleic Acid Sequence-dependent Amplification (NASBA) and the like in addition to conventional Polymerase Chain Reaction (PCR) Amplification technology. The detection of the micro-reaction unit means that the amplified micro-reaction unit is detected to determine whether or not each unit contains a target nucleic acid molecule. Detection of a micro-reaction unit is generally based on a fluorescence analysis method, and a unit containing a fluorescent signal is referred to as a positive reaction unit, and a reaction unit without a fluorescent signal is referred to as a negative reaction unit. According to the Poisson distribution principle, the number of nucleic acid molecules in the reaction solution to be detected can be calculated by only counting the number of the positive reaction units and the total number of the micro-reaction units, so that the absolute quantification of the target substance is realized.
At present, digital nucleic acid detection techniques can be broadly divided into two major categories, namely droplet-type digital nucleic acid detection and micro-reaction chamber digital nucleic acid detection, according to the structure type of reaction solution digitization. The drop-type digital nucleic acid detection platform is represented by a QX200 system of Bio-Rad company, and 3 devices are needed to complete the whole detection. Not only the detection time is long, but also the liquid drop damage and pollution can be caused in the process of transferring the liquid drop, and the accuracy of the detection result is influenced. In addition, the bulky equipment is difficult to integrate, and the field detection of the target object cannot be realized. Therefore, it is necessary to develop a new portable droplet digital nucleic acid detection system, which can improve the analysis efficiency and apply the technology to the field detection.
Disclosure of Invention
The invention aims to solve the problems that a droplet type digital nucleic acid detection system is poor in integration level, is easy to crack and pollute in the droplet transfer process, generally needs to rely on precise equipment to realize reaction liquid distribution and the like, and provides a microfluidic chip for digital nucleic acid amplification and a portable multi-channel detection system.
In order to achieve the above purpose, the technical scheme adopted by the invention mainly comprises:
a microfluidic chip for digital nucleic acid amplification:
the microfluidic chip comprises at least one reaction area arranged on a substrate, the reaction area comprises a liquid drop generation area and a liquid drop storage area which are mutually connected, a step is arranged between the liquid drop generation area and the liquid drop storage area, and the outlet of the liquid drop generation area is higher than the inlet of the liquid drop storage area; the liquid drop storage region is originally filled with a continuous phase oil phase, the discrete phase nucleic acid reaction solution is input into the liquid drop generation region, and liquid drops obtained by dispersion through step induction when flowing out from the outlet of the liquid drop generation region are directly collected by the liquid drop storage region and used for subsequent nucleic acid amplification and product detection.
The microfluidic chip generates liquid drops manually under the condition of simple control equipment even without control equipment, so that the whole system for amplifying and detecting the sample on the microfluidic chip is simpler and more convenient and is easy to integrate.
The liquid drop generating area comprises a sample inlet and a sample input channel, the sample input channel is a fork-shaped structure which is mainly formed by connecting a plurality of one-in-two channel structures, the sample input channel is only provided with one inlet end, the inlet end is connected to the sample inlet, the inlet end is communicated with a plurality of outlet ends through a plurality of cascaded one-in-two channel structures, and the plurality of outlet ends are communicated with the liquid drop storage area; and a step having a difference in height exists between the outlet end and the bottom surface of the inlet side of the droplet storage region.
The middle part of the area of the liquid drop storage area is provided with a plurality of supporting columns which are arranged in an array at intervals. The number, the shape and the position of the support columns are determined according to actual conditions.
The reaction zone also comprises a waste liquid discharge zone, the waste liquid discharge zone is positioned beside the liquid drop storage zone, the waste liquid discharge zone and the liquid drop storage zone are separated by a fence, micropores are formed on the fence, and the diameter of each micropore is smaller than that of the liquid drop in the liquid drop storage zone.
Inputting a nucleic acid reaction solution from an injection port, wherein the nucleic acid reaction solution comprises a primer (called FP) modified with a fluorescent group and a nucleotide sequence (called QP) modified with a corresponding quenching group and complementarily paired with a FP section, the nucleic acid reaction solution flows out from an outlet end of a sample input channel after passing through a crotch-shaped structure of the sample input channel, and forms liquid drops with uniform size and meeting the digital nucleic acid detection through the step-shaped induced Laplace pressure difference while flowing out from the outlet end, and the liquid drops are arranged in a liquid drop storage region in a continuous phase oil phase separation mode.
6. The microfluidic chip for digital nucleic acid amplification according to claim 1, wherein:
the nucleic acid reaction solution at least comprises two groups of amplification primer groups, each group of primers corresponds to different amplification target DNAs, each group of primers modified with fluorescent groups is modified with fluorescent groups with different emission wave bands, and a nucleotide sequence complementarily paired with the primers modified with the fluorescent groups is modified with corresponding quenching groups; when the amplification is not carried out, the primer modified with the fluorescent group is combined with the nucleotide sequence modified with the quenching group, and the signal of the fluorescent group is quenched according to the fluorescent energy resonance transfer principle; when the target DNA is amplified, the primer modified with the fluorescent group is separated from the nucleotide sequence modified with the quenching group, and the fluorescent group generates a fluorescent signal under the excitation of a corresponding excitation light source.
Secondly, a portable multi-channel optical detection system for digital nucleic acid amplification:
the device comprises a microfluidic chip, a temperature regulation module and an optical detection module; the bottom surface of the microfluidic chip is provided with a temperature control module which is used for heating and controlling the temperature of the microfluidic chip, so that a nucleic acid reaction solution in a liquid drop storage region is subjected to an amplification reaction, and a primer modified with a fluorescent group is separated from a nucleotide sequence modified with a quenching group; the optical detection module is positioned beside the upper part of the microfluidic chip and emits laser beams to the liquid drop storage area of the microfluidic chip, so that fluorescent groups on the inner surface of the liquid drops in the liquid drop storage area are excited by the laser to emit fluorescence and then are detected and received.
The temperature regulation and control module comprises a temperature measuring unit, a heating unit and a refrigerating unit, wherein the temperature measuring unit, the heating unit and the refrigerating unit are all arranged on the bottom surface of the substrate of the microfluidic chip, and the temperature measuring unit is respectively and electrically connected with the heating unit and the refrigerating unit; the temperature measuring unit monitors the temperature in real time to serve as the temperature of the liquid drops, the temperature of the liquid drops is fed back to the heating unit and the refrigerating unit, and whether refrigeration or heating is carried out or not is regulated so as to realize temperature regulation and control of nucleic acid amplification.
The optical detection module mainly comprises a light source and an optical detector, wherein the light source emits laser to irradiate each liquid drop in the liquid drop storage region, and a primer modified with a fluorescent group and a nucleotide sequence modified with a quenching group in the amplified liquid drop are separated; the fluorescent group on the primer modified with the fluorescent group is excited by laser to emit fluorescence, and the fluorescence is detected and received by the optical detector.
The optical detection module also comprises an excitation optical filter and an emission optical filter, wherein the excitation optical filter is arranged in front of the light source, and the emission optical filter is arranged in front of the optical detector; the light source emits laser, the laser is filtered by the excitation filter and then irradiates each liquid drop in the liquid drop storage region, and the fluorescence of the fluorescent group is detected and received by the optical detector after being filtered by the emission filter.
The invention has the beneficial effects that:
1. the invention realizes the micro-fluidic chip which can manually generate the liquid drops without depending on precise control equipment and carry out digital nucleic acid detection for the first time, integrates sample distribution, nucleic acid amplification and product detection on the same chip, and effectively avoids the problems of long detection time and liquid drop breakage and pollution caused by transferring the liquid drops in the liquid drop type digital nucleic acid detection.
2. The portable multi-channel optical detection system provided by the invention realizes multi-channel optical signal detection by utilizing the light source covering various fluorescent substance excitation wave bands and the multi-band-pass filter, improves the detection efficiency, can detect the end point and can detect in real time, and improves the accuracy of the detection result.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application.
Fig. 1 is a front view of a microfluidic chip provided in an embodiment of the present invention.
Fig. 2 is a schematic diagram of 3 different parallel ports in a microfluidic chip according to an embodiment of the present invention.
Fig. 3 is a schematic structural diagram of an optical detection module in the portable multi-channel fluorescence detection system according to an embodiment of the present invention.
FIG. 4 is a schematic structural diagram of a plurality of different optical detection modules according to an embodiment of the present invention.
Fig. 5 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention.
Fig. 6 is a schematic structural diagram of a step at an outlet end of a droplet generation region of a microfluidic chip according to an embodiment of the present invention.
Fig. 7 is a schematic diagram of a microfluidic chip for manually generating droplets according to an embodiment of the present invention.
FIG. 8 is a schematic diagram of the principle of LAMP amplification detection of nucleic acids provided by the embodiment of the present invention.
FIG. 9 is a graph showing the detection results of a droplet of single-plex digital nucleic acid amplification provided in an embodiment of the present invention.
FIG. 10 is a diagram showing the results of the double digital nucleic acid amplification droplet detection provided in the examples of the present invention.
In the figure: the device comprises a micro-fluidic chip 1, a reaction area 2, a liquid drop generating area 3, a liquid drop storage area 4, a sample inlet 5, a light source 6, an excitation filter 7, an emission filter 8, an optical detector 9, a support column 10, a sample outlet 11, a fence 12, a nucleic acid reaction liquid 13, liquid drops 14 and a waste liquid discharge area 15.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more clear, the present invention is further described below with reference to the accompanying drawings of the specification, but the present invention is not limited to the following embodiments.
The invention relates to a micro-fluidic chip for digital nucleic acid amplification and a portable multi-channel detection system, wherein the specific embodiment is that reaction liquid is dispersed into micro-reaction units on the micro-fluidic chip 1, and then the chip is placed in the portable multi-channel optical detection system for nucleic acid amplification, so that the end point detection can be carried out on an amplification product, and the real-time detection can also be carried out in the amplification process. And (3) obtaining the number of the positive reaction units and the total number of the reaction units according to detection, and realizing absolute quantitative detection of the target object according to the Poisson distribution principle.
As shown in fig. 1 and 5, the microfluidic chip 1 includes at least one reaction region 2 disposed on a substrate, the reaction region 2 includes three parts of a droplet generation region 3, a droplet storage region 4 and a waste liquid discharge region 15 connected to each other, a step is present between the droplet generation region 3 and the droplet storage region 4, and an outlet of the droplet generation region 3 is higher than an inlet of the droplet storage region 4; the droplet storage region 4 is originally filled with a continuous phase oil phase and can be pretreated by a surfactant, a discrete phase of the nucleic acid reaction liquid 13 is input into the droplet generation region 3, and droplets 14 obtained by dispersion through step induction when flowing out from the outlet of the droplet generation region 3 are directly collected by the droplet storage region 4 for subsequent nucleic acid amplification and product detection.
The droplet generation region 3 includes a sample inlet 5 and a sample input channel, as shown in fig. 2 (a), the sample input channel is a tree-fork structure formed by connecting a plurality of one-in-two channel structures, the sample input channel has only one inlet end, the inlet end is connected to the sample inlet 5, the inlet end is communicated with the droplet storage region 4 through a plurality of cascaded one-in-two channel structures and a plurality of outlet ends; the droplet 14 can be generated faster through such a sample input channel, increasing the rate of droplet 14 generation. And a step with a height difference exists between the outlet end and the bottom surface of the inlet side of the droplet storage region 4, as shown in fig. 6, when the discrete phase flowing out from the outlet end passes through a two-dimensional to three-dimensional space constraint abrupt step structure, because the curvature of the two-phase interface on the step is greater than the curvature of the two-phase interface in the droplet storage region 4, the pressure in the discrete phase on the step is greater than the pressure of the discrete phase in the droplet storage region 4, the laplace pressure difference is induced to form droplets 14 with uniform size, and the droplets 14 capable of meeting the digital nucleic acid detection are generated.
In specific implementation, the sample input channel may also be a divided channel or a plurality of separate channels, as shown in fig. 2 (b) and (c), respectively, and the sample inlet 5 is disposed at the inlet of the channel.
The liquid drop generating area 3 of the invention adopts a structure of a ladder, a rectangular micropipe and the like. The sample input channel in the droplet generation section 3 is designed to be a parallel port structure such as a bifurcation type structure composed of a one-to-two channel structure, a one-to-many parallel channel structure, a multi-channel input structure, and the like. As shown in fig. 2, the sample input channel can be designed as a fork type with a single inlet 5 (fig. 2a), a parallel channel structure with a single inlet 5 (fig. 2b), and a multi-inlet 5 input structure (fig. 2 c).
The micro-fluidic chip 1 is made of glass, silicon wafers, paper, high molecular polymers and other common micro-fluidic chips, wherein the high molecular polymers can be PDMS (polydimethylsiloxane), COC (cyclic olefin copolymer) and other materials.
When the microfluidic chip 1 is made of relatively soft materials such as PDMS, it is considered to add the supporting columns 10 in the droplet storage region 4 to prevent the storage region from collapsing due to a large-area cavity, and to add the corresponding fences 12 at the connection 15 between the droplet storage region 4 and the waste liquid discharge region to prevent the droplets 14 from leaving the droplet storage region 4 along with the continuous phase.
As shown in fig. 5, the droplet storage region 4 is provided with a plurality of support pillars 10 in the middle of the region, and the plurality of support pillars 10 are arranged in a spaced array.
The liquid drop storage area 4 is used for collecting liquid drops 14 after reaction liquid is dispersed, and the step height difference between the outlet end and the bottom surface of the inlet side of the liquid drop storage area 4 is smaller than 2 times of the diameter of the liquid drops 14, so that the liquid drops are prevented from being stacked into two layers to influence the detection of subsequent products; the droplet storage region 4 is large enough to allow a monolayer distribution of droplets 14 to be produced.
As shown in FIG. 5, the reaction zone 2 further comprises a waste liquid discharge zone 15, the waste liquid discharge zone 15 is located beside the droplet storage zone 4, the waste liquid discharge zone 15 and the droplet storage zone 4 are separated by a barrier 12, the barrier 12 is provided with micropores, and the diameter of the micropores is smaller than the diameter of the droplets 14 in the droplet storage zone 4, that is, smaller than the diameter of the droplets 14 dispersedly generated in the droplet generation zone 3. The size and number of the micro-holes in the barrier 12 depend on the diameter of the chip-generated droplet 14 and the width of the droplet storage region 4.
The nucleic acid reaction solution is input from the sample inlet 5, the nucleic acid reaction solution is an aqueous solution, the nucleic acid reaction solution comprises a primer (called FP) modified with a fluorescent group and a nucleotide sequence (called QP) modified with a corresponding quenching group and complementarily paired with the FP section, the nucleic acid reaction solution flows out from the outlet end of the sample input channel after passing through a fork-shaped structure of the sample input channel, and forms droplets 14 which are uniform in size and meet the requirement of digital nucleic acid detection by inducing Laplace pressure difference through a step shape while flowing out from the outlet end, and the droplets are separated by oil in the droplet storage region 4.
The nucleic acid reaction solution may further contain DNA, enzymes, dNTPs, etc.
The invention is also applicable to the expression of two amplification groups.
The nucleic acid reaction solution at least comprises two groups of amplification primer groups, each group of primers corresponds to different amplification target DNAs, and each group of FP primers is modified with fluorescent groups with different emission bands and QP is modified with corresponding quenching groups. When not amplified, FP is combined with corresponding QP, and the signal of the fluorescent group is quenched according to the fluorescent energy resonance transfer principle; when the target DNA is amplified, FP is separated from QP, and the fluorescent group generates a fluorescent signal under the excitation of a corresponding excitation light source.
The portable multi-channel optical detection system comprises a micro-fluidic chip, a temperature regulation module and an optical detection module; a temperature regulation and control module is arranged on the bottom surface of the microfluidic chip and is used for heating and controlling the temperature of the microfluidic chip, so that the nucleic acid reaction liquid in the liquid drop 14 in the liquid drop storage area 4 is subjected to amplification reaction, and FP is separated from QP; the optical detection module is positioned beside the upper part of the microfluidic chip and emits laser beams to the liquid drop storage area 4 of the microfluidic chip, so that fluorescent groups in the liquid drops 14 in the liquid drop storage area 4 are excited by the laser to emit fluorescence and then are detected and received.
The temperature regulation and control module comprises a temperature measuring unit, a heating unit and a refrigerating unit, wherein the temperature measuring unit, the heating unit and the refrigerating unit are all arranged on the bottom surface of the substrate of the microfluidic chip, and the temperature measuring unit is respectively and electrically connected with the heating unit and the refrigerating unit; the temperature measuring unit monitors the temperature in real time as the temperature of the liquid drop 14, feeds the temperature of the liquid drop 14 back to the heating unit and the refrigerating unit, and regulates and controls whether to refrigerate or heat so as to realize temperature regulation and control of nucleic acid amplification, so that not only can conventional PCR amplification be realized, but also isothermal amplification such as LAMP, RPA, NASBA and the like can be realized.
The position of the temperature regulation and control module is properly adjusted according to the optical detection module on the premise of not influencing the optical detection. Wherein, the heating unit can adopt Peltier elements, metal nano materials, infrared radiation sources and other materials; the refrigeration unit can be selected from a fan, a radiating fin and the like. The temperature regulation and control module can be realized by selecting a proper mode according to the adopted amplification technology, and if the PCR technology is adopted, the temperature change control is required to be realized; if a constant temperature amplification technology such as LAMP is adopted, only constant temperature control is needed.
As shown in fig. 3 and 4, the optical detection module mainly includes a light source 6 and an optical detector 9, the light source 6 emits laser light to irradiate each droplet 14 in the droplet storage region 4, FP and QP in the droplet 14 where amplification occurs are separated, fluorophores on FP are excited by the laser light to emit fluorescence, and the fluorescence is detected and received by the optical detector 9.
The optical detection module also comprises an excitation filter 7 and an emission filter 8, wherein the excitation filter 7 is arranged in front of the light source 6, and the emission filter 8 is arranged in front of the optical detector 9; laser emitted by the light source 6 is filtered by the excitation filter 7 and then irradiates each liquid drop 14 in the liquid drop storage region 4, and fluorescence of the fluorescent group is detected and received by the optical detector 9 after being filtered by the emission filter 8.
When a broadband light source or a multiband light source is selected, the half-peak width is larger, the optical filter 7 can be added in front of the light source 6 to reduce the light of irrelevant wave bands. The filter 7 is preferably a multi-band pass filter in order to facilitate system integration and reduce motion devices. A filter 8 can be added in front of the optical detector 9 to reduce the influence of the light source 6 on the detection result. Likewise, the filter 8 is preferably a multi-band pass filter. The schematic positional distribution of the light source 6 and the optical detector 9 is shown in fig. 4, which includes an orthogonal type fig. 4a, a straight type fig. 4b, a coaxial type fig. 4c, a side light type fig. 4d, and the like.
After the nucleic acid amplification reaction, the fluorescent substance bound to the nucleic acid amplification product substance is excited by the laser light to emit fluorescence, and the fluorescent substance alone not bound to the nucleic acid amplification substance is not excited by the laser light to emit fluorescence. Within different droplets 14, the FPs are separated from the QPs within the droplet 14 where target DNA amplification occurs, and the FPs and QPs within the droplet 14 where amplification does not occur remain bound, resulting in different intensities of fluorescence emitted by different droplets 14 excitations. The number of the liquid drops 14 larger than the brightness threshold is counted by the result obtained by detecting and receiving the optical detector 9, and the detection function of the microfluidic chip is realized.
According to the invention, a multi-band-pass filter is added in front of the light source 6 and/or the optical detector 9, so that multi-channel fluorescence signal detection is realized.
The light source 6 is an artificial light source capable of emitting multiple bands or wide bands, such as a white light LED (light emitting diode), a multicolor LED, or the like. The optical detector 9 is an optical imaging device, such as a CCD camera, a mobile phone with a photographing function, an optical microscope, or the like.
The optical detection module can not only detect a fluorescent signal, but also detect a nucleic acid amplification by-product, such as a isothermal amplification LAMP by-product white magnesium pyrophosphate precipitate.
And (3) placing the micro-fluidic chip 1 after generating the liquid drop 14 in a portable multi-channel optical detection system for nucleic acid amplification and product detection. For product detection, real-time detection or endpoint detection can be performed:
(1) real-time detection: in this way, in the amplification process, the light source 6 is turned on, the optical detector 9 is used for product detection at certain intervals, that is, the droplet storage region 2 on the microfluidic chip 1 is photographed, a group of optical images is obtained and then processed, the change of optical signals of each micro-reaction unit in the amplification process can be obtained, a curve of the change of the signals along with time can be drawn, and the number of positive reaction units and the total number of reaction units can be obtained, thereby realizing the absolute quantitative detection of target nucleic acid molecules.
(2) And (3) end point detection: in this way, the light source 6 is turned on only after the amplification is finished, the optical detector 9 is used for product detection, and the shot optical image is processed to obtain the number of positive reaction units and the total number of reaction units, so as to realize the absolute quantitative detection of the target nucleic acid molecules.
The content and implementation process of the present invention will be further described below by using a probe LAMP amplification technique (the principle is shown in FIG. 8) in combination with the microfluidic chip (shown in FIG. 5) and the portable detection system according to the embodiment of the present invention:
1) manufacturing a micro-fluidic chip: a microfluidic chip comprising two reaction regions shown in FIG. 5 is manufactured, a total of 64 outlet ends of a sample input channel are designed in a fork shape, a droplet generation region 3 is designed in a ladder shape, the height of the sample input channel is 25 μm, the height of a droplet storage region is 100 μm, an anti-collapse supporting column 10 is designed in the droplet storage region 4, and a fence 12 for preventing droplets 14 from flowing out along with waste liquid is designed at the outlet.
2) Preparation of reagents:
preparing corresponding reaction liquid according to a nucleic acid amplification technology selected actually, wherein in single detection, escherichia coli DNA is used as target DNA, and the nucleic acid reaction liquid contains a primer F-FIP for modifying an FAM fluorescent group and a nucleotide sequence QP modified with a corresponding quenching group; in the double detection, Escherichia coli DNA and phage DNA are used as target DNA, the nucleic acid reaction solution comprises two groups of primer groups, wherein the primer group corresponding to the Escherichia coli DNA is consistent with that in the single detection, and the primer group corresponding to the phage DNA comprises a primer F-FIP for modifying HEX fluorescent group and a nucleotide sequence QP for modifying corresponding quenching group.
② the continuous phase configured for generating droplets 14, typically an oil containing a certain proportion of surfactant, in this example 10% of 008-fluoro surfactant is used as continuous phase.
3) Droplet generation: the surface of the manufactured microfluidic chip is subjected to hydrophobic treatment, then the chip is filled with continuous phase, the sample is injected by manually pushing an injector without depending on a precision control device (as shown in figure 7), a certain volume of discrete phase reaction liquid is filled in a tube connecting the needle head of the injector and the sample inlet 5 on the chip, and when the piston of the injector is pushed, the liquid drop generating area 3 generates corresponding liquid drops 14 and the liquid drops are arranged in the liquid drop storage area 4 in a spontaneous single layer.
4) Nucleic acid amplification: the microfluidic chip with the generated droplet 14 is placed on a temperature control module to be heated, so that amplification of target molecules is realized, sample distribution, nucleic acid amplification and product detection are integrated by the chip, and the temperature of the droplet 14 in the chip is controlled to be about 65 ℃ by adopting an LAMP amplification technology in the embodiment.
5) And (3) product detection: in this embodiment, an end-point detection method is selected for detection of the amplification product, an orthogonal structure is adopted between the light source 6 and the optical detector 9, the light source 6 adopts a multi-color LED, the optical detector 9 adopts a portable microscope, and a multi-bandpass filter 8 is added in front of the optical detector 9, so that not only can single detection but also dual detection of the product be realized:
detecting the singleness: when only 1 test object is detected, fluorescent groups with a single waveband are used for detection, in the embodiment, a probe method for modifying FAM groups is used for detecting Escherichia coli DNA, the wavelength of excitation light is 486nm, the droplet 14 containing Escherichia coli DNA generates a fluorescent signal (green) under the excitation of an LED, the droplet 14 without DNA has no fluorescent signal, and the end point detection result is shown in FIG. 9.
Two-step detection: in this example, the detection of escherichia coli DNA and phage DNA was performed by a probe method for modifying FAM groups and HEX groups, respectively, and the excitation light wavelengths were 486nm and 535nm, respectively, the droplet 14 containing escherichia coli DNA generated a fluorescent signal (green) under excitation of a 486nm LED, the droplet 14 containing phage DNA generated a fluorescent signal (red) under excitation of a 535nm LED, and the droplet 14 without DNA did not generate a fluorescent signal, and the end point detection result is shown in fig. 10.
6) And counting the number of the liquid drops 14 with the fluorescence signal intensity larger than the brightness threshold value and the total number of the liquid drops 14 in the shot image, deducing the number of target nucleic acid molecules in the reaction solution according to Poisson distribution to obtain a detection result, and realizing absolute quantitative detection of the multi-target object.
7) And (3) removing the microfluidic chip, replacing the chip with a new chip, and repeating the steps 2) to 6) or finishing the detection.
The foregoing detailed description is intended to be illustrative of the invention and is not to be construed as limiting, since any modifications and variations of the invention are possible within the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A microfluidic chip for digital nucleic acid amplification, characterized in that: the microfluidic chip (1) comprises at least one reaction area (2) arranged on a substrate, the reaction area (2) comprises a liquid drop generation area (3) and a liquid drop storage area (4) which are connected with each other, a step is arranged between the liquid drop generation area (3) and the liquid drop storage area (4), and the outlet of the liquid drop generation area (3) is higher than the inlet of the liquid drop storage area (4); the liquid drop storage region (4) is originally filled with a continuous phase oil phase, the discrete phase nucleic acid reaction liquid (13) is input into the liquid drop generation region (3), and liquid drops (14) obtained by dispersion through step induction when flowing out from the outlet of the liquid drop generation region (3) are directly collected by the liquid drop storage region (4) and used for subsequent nucleic acid amplification and product detection.
2. The microfluidic chip for digital nucleic acid amplification according to claim 1, wherein: the liquid drop generating area (3) comprises a sample inlet (5) and a sample input channel, the sample input channel is a tree-fork type structure which is mainly formed by connecting a plurality of one-in-two channel structures, the sample input channel is only provided with one inlet end, the inlet end is connected to the sample inlet (5), the inlet end is communicated with a plurality of outlet ends through a plurality of cascaded one-in-two channel structures, and the plurality of outlet ends are communicated with the liquid drop storage area (4); and a step having a difference in height exists between the outlet end and the bottom surface of the inlet side of the droplet storage region (4).
3. The microfluidic chip for digital nucleic acid amplification according to claim 1, wherein: the middle part of the area where the liquid drop storage area (4) is located is provided with a plurality of supporting columns (10), and the supporting columns (10) are arranged in an array mode at intervals.
4. The microfluidic chip for digital nucleic acid amplification according to claim 1, wherein: the reaction zone (2) further comprises a waste liquid discharge zone (15), the waste liquid discharge zone (15) is located beside the liquid drop storage zone (4), the waste liquid discharge zone (15) and the liquid drop storage zone (4) are separated through a fence (12), micropores are formed in the fence (12), and the diameters of the micropores are smaller than the diameters of the liquid drops (14) in the liquid drop storage zone (4).
5. The microfluidic chip for digital nucleic acid amplification according to claim 1, wherein: the nucleic acid reaction solution is input from an injection port (5), the nucleic acid reaction solution comprises a primer modified with a fluorescent group and a nucleotide sequence modified with a corresponding quenching group and complementarily paired with the primer modified with the fluorescent group, the nucleic acid reaction solution flows out from an outlet end of a sample input channel after passing through a crotch-shaped structure of the sample input channel, and forms liquid drops (14) which are uniform in size and meet digital nucleic acid detection through step-shaped induced Laplace pressure difference while flowing out from the outlet end, and the liquid drops are arranged in a liquid drop storage region (4) in a continuous phase oil phase separation mode.
6. The microfluidic chip for digital nucleic acid amplification according to claim 1, wherein: the nucleic acid reaction solution at least comprises two groups of amplification primer groups, each group of primers corresponds to different amplification target DNAs, each group of primers modified with fluorescent groups is modified with fluorescent groups with different emission wave bands, and a nucleotide sequence complementarily paired with the primers modified with the fluorescent groups is modified with corresponding quenching groups; when the amplification is not carried out, the primer modified with the fluorescent group is combined with the nucleotide sequence modified with the quenching group, and the signal of the fluorescent group is quenched according to the fluorescent energy resonance transfer principle; when the target DNA is amplified, the primer modified with the fluorescent group is separated from the nucleotide sequence modified with the quenching group, and the fluorescent group generates a fluorescent signal under the excitation of a corresponding excitation light source.
7. A portable multi-channel optical detection system for digital nucleic acid amplification, characterized by:
comprises the microfluidic chip of any one of claims 1 to 6, a temperature regulation module and an optical detection module; the bottom surface of the microfluidic chip is provided with a temperature control module which is used for heating and controlling the temperature of the microfluidic chip, so that a nucleic acid reaction solution in a liquid drop (14) in a liquid drop storage area (4) is subjected to an amplification reaction, and a primer modified with a fluorescent group is separated from a nucleotide sequence modified with a quenching group; the optical detection module is positioned beside the upper part of the microfluidic chip and emits laser beams to the liquid drop storage area (4) of the microfluidic chip, so that fluorescent groups in the liquid drops (14) in the liquid drop storage area (4) are excited by laser to emit fluorescence and then are detected and received.
8. A portable multi-channel optical detection system for digital nucleic acid amplification according to claim 7, wherein: the temperature regulation and control module comprises a temperature measuring unit, a heating unit and a refrigerating unit, wherein the temperature measuring unit, the heating unit and the refrigerating unit are all arranged on the bottom surface of the substrate of the microfluidic chip, and the temperature measuring unit is respectively and electrically connected with the heating unit and the refrigerating unit; the temperature measuring unit monitors the temperature in real time to serve as the temperature of the liquid drops (14), the temperature of the liquid drops (14) is fed back to the heating unit and the refrigerating unit, and whether refrigeration or heating is carried out is regulated so as to realize temperature regulation of nucleic acid amplification.
9. A portable multi-channel optical detection system for digital nucleic acid amplification according to claim 7, wherein: the optical detection module mainly comprises a light source (6) and an optical detector (9), wherein the light source (6) emits laser to irradiate each liquid drop (14) in the liquid drop storage region (4), and primers modified with fluorescent groups and nucleotide sequences modified with quenching groups in the amplified liquid drops (14) are separated; the fluorescent group on the primer modified with the fluorescent group is excited by laser to emit fluorescence, and the fluorescence is detected and received by an optical detector (9).
The optical detection module further comprises an excitation filter (7) and an emission filter (8), wherein the excitation filter (7) is arranged in front of the light source (6), and the emission filter (8) is arranged in front of the optical detector (9); laser emitted by the light source (6) is filtered by the excitation filter (7) and then irradiates each liquid drop (14) in the liquid drop storage area (4), and fluorescence of the fluorescent group is detected and received by the optical detector (9) after being filtered by the emission filter (8).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110704613.3A CN113462548A (en) | 2021-06-24 | 2021-06-24 | Microfluidic chip for digital nucleic acid amplification and portable multi-channel detection system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110704613.3A CN113462548A (en) | 2021-06-24 | 2021-06-24 | Microfluidic chip for digital nucleic acid amplification and portable multi-channel detection system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113462548A true CN113462548A (en) | 2021-10-01 |
Family
ID=77872800
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110704613.3A Pending CN113462548A (en) | 2021-06-24 | 2021-06-24 | Microfluidic chip for digital nucleic acid amplification and portable multi-channel detection system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113462548A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114308155A (en) * | 2021-12-23 | 2022-04-12 | 广东工业大学 | Novel micro-fluidic chip and digital fluorescent quantitative PCR (polymerase chain reaction) amplification instrument |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106190774A (en) * | 2016-07-22 | 2016-12-07 | 深圳睿思生命科技有限公司 | For capturing the micro-fluidic chip of circulating tumor cell and capture thereof and authentication method |
| CN106434330A (en) * | 2016-10-09 | 2017-02-22 | 戴敬 | Absolute quantification type digital nucleic acid analytic system based on efficient liquid drop microreactor |
| CN112553063A (en) * | 2020-12-22 | 2021-03-26 | 苏州缔因安生物科技有限公司 | Integrated digital nucleic acid amplification chip based on micro-droplets and use method and application thereof |
-
2021
- 2021-06-24 CN CN202110704613.3A patent/CN113462548A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106190774A (en) * | 2016-07-22 | 2016-12-07 | 深圳睿思生命科技有限公司 | For capturing the micro-fluidic chip of circulating tumor cell and capture thereof and authentication method |
| CN106434330A (en) * | 2016-10-09 | 2017-02-22 | 戴敬 | Absolute quantification type digital nucleic acid analytic system based on efficient liquid drop microreactor |
| CN112553063A (en) * | 2020-12-22 | 2021-03-26 | 苏州缔因安生物科技有限公司 | Integrated digital nucleic acid amplification chip based on micro-droplets and use method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 连娇愿等: "协同流动对台阶乳化生成Janus 液滴的影响研究", 分析化学( FENXI HUAXUE) 研究报告, vol. 48, no. 1, pages 57 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114308155A (en) * | 2021-12-23 | 2022-04-12 | 广东工业大学 | Novel micro-fluidic chip and digital fluorescent quantitative PCR (polymerase chain reaction) amplification instrument |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11084039B2 (en) | Microfluidic analysis system | |
| US10814321B2 (en) | Method and molecular diagnostic device for detection, analysis and identification of genomic DNA | |
| US20220008928A1 (en) | Method of analysis | |
| US10279350B2 (en) | Method of generating droplets | |
| EP2209555B1 (en) | Detecting and mixing in a conduit in integrated bioanalysis systems | |
| CA3089411C (en) | Digital pcr detection apparatus, digital pcr quantitative detection method, multi-volume digital pcr quantitative analysis method, digital pcr detection method, nucleic acid detection microsphere, preparation method of nucleic acid detection microsphere, nucleic acid detection microsphere kit and high-throughput nucleic acid detection method | |
| CN112029653B (en) | Digital nucleic acid amplification detection method and integrated detection system based on CRISPR and Cas | |
| US11130128B2 (en) | Detection method for a target nucleic acid | |
| US10512910B2 (en) | Droplet-based analysis method | |
| CN1637407A (en) | Fluorescence detector for detecting microfluid | |
| CN113462548A (en) | Microfluidic chip for digital nucleic acid amplification and portable multi-channel detection system |
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 |