CN101928663B - Integrated fluidic chip device for digital nucleic acid amplification and application - Google Patents

Abstract

The invention provides an integrated flow channel chip device for digital nucleic acid amplification, which is composed of a vacuum system, a front pipeline, a buffer bottle, a rear pipeline, a capillary, a suction cup, a sealing layer and a channel layer, and the channel layer and the sealing layer After the layers are sealed, the integrated flow chip assembly is formed. The channel layer is engraved with channels, and is equipped with a sample outlet and a sample inlet. The channel is composed of a meandering main channel and a number of side chambers located on both sides of the main channel. The two ends are respectively connected to the sample outlet and the sample inlet. One end of the front pipeline is connected to the vacuum system, and the other end is connected to the buffer bottle. One end of the rear pipeline is connected to the buffer bottle, and the other end is connected to the capillary. The capillary passes through the suction cup and the integrated flow path. The chip components are connected. The invention is reasonable in design, miniaturized, portable, and easy to operate. It can distribute a small amount of liquid to thousands of independent small chambers within tens of seconds, reduce the complexity of chip production and use, and increase the experimental speed. It can be used in nucleic acid digital used in amplification.

Description

Integrated flow chip device and application for digital nucleic acid amplification

technical field

The invention belongs to detection devices in various fields such as life sciences, medicine, chemistry and engineering, and relates to an integrated flow chip device for digital nucleic acid amplification and its application.

Background technique

It has been proved that many human diseases are directly or indirectly related to genes, and genes are DNA (deoxyribonucleic acid) molecular fragments with genetic effects. Therefore, the detection and analysis of nucleic acid is not only widely used in medical fields such as genetic diseases, tumors and infectious diseases, but also plays an increasingly important role in forensic identification, food safety, archaeology and other fields. However, although nucleic acid research has achieved remarkable results, it is also facing more challenges. First of all, the growth and development of organisms are not determined by a single gene, but the result of the synergy of multiple genes, which is a very complicated process. High-throughput and rapid nucleic acid detection methods are urgently needed to study gene diversity and gene mutation or transformation. Secondly, the content of nucleic acid is very low, and it is difficult to directly detect it at present. Instead, polymerase chain reaction (PCR) technology is often used to amplify DNA in vitro until the concentration of target DNA is sufficient to be detected.

It has been more than 30 years since the invention of the polymerase chain reaction technology. During this period, the technology has been continuously developed. In recent years, the real-time quantitative PCR (real-time quantitative PCR) technology has realized the leap from qualitative to quantitative PCR. . The so-called real-time fluorescence quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally performing quantitative analysis on the template to be tested through the standard curve. However, this traditional quantitative PCR method also has its unavoidable shortcomings: ①The measurement point of fluorescent quantitative PCR is in the exponential phase of PCR amplification, and under different templates and different conditions, the number of cycles required for PCR amplification to reach equilibrium is uncertain. ②If the initial concentration of the target fragment is too low, it is often difficult to amplify to a detectable level. ③The amplification efficiency of the sample to be tested may be different from that of the standard sample.

In 1999, Vogelstein et al. developed a method called digital PCR (Digital PCR), which diluted the DNA template to be tested and loaded it into a multi-well plate, so that there was only one template molecule in every two or more wells, and carried out under optimized conditions. After conventional thermal cycling PCR reactions, hybridization with molecular beacons to detect specific amplification products, positive wells generate fluorescence. Compared with traditional fluorescent quantitative PCR, digital PCR quantifies the initial copy number of nucleic acid by directly counting the number of positive wells, and can achieve absolute quantitative determination in the true sense, so it is called digital PCR. In 2006, Ottesen et al. developed a microfluidic digital PCR chip that can accurately quantify multiple genes of bacteria. In the same year, Fluidigm Corporation of the United States developed a commercial digital array PCR chip, which uses a real-time PCR instrument for nucleic acid amplification and quantitative detection. This digital PCR chip involves a PDMS elastic microvalve prepared based on a multilayer soft lithography process. In this structure, the fluid passage is located in the lower PDMS layer and consists of many parallel small chambers connected in series; the gas passage is located in the lower PDMS layer. In the upper layer of PDMS, it consists of many channels perpendicular to the fluid channels. This valve is in an open state when there is no external force. When pressure (such as air pressure or water pressure) is applied to the gas passage, the PDMS film between the upper and lower layers expands downward, and the valve is in a closed state, so that the lower liquid flow passage is closed. Separated into several independent reaction chambers.

The above-mentioned digital PCR chip has broad application prospects in molecular detection of tumors, non-invasive prenatal diagnosis, genetic analysis of organisms, and diseases related to copy number variation. Dr. Blow, the editor of "Nature" and "Nature Method" magazines, wrote that digital PCR is a new frontier of PCR technology, which can conduct single-cell or even single-molecule research, and achieve absolute quantification through direct counting. However, the production of this digital PCR chip based on PDMS elastic microvalve involves the alignment and sealing of multi-layer structures, and the preparation process is long and complicated. Currently, commercialized chips and supporting instruments are very expensive. Therefore, from the perspective of large-scale application prospects of digital PCR chip technology, the current preparation method and application cost are unfavorable.

Contents of the invention

The object of the present invention is to provide a low-cost, easy-to-operate integrated flow chip device for digital nucleic acid amplification, which consists of a vacuum system, a front pipeline, a buffer bottle, a rear pipeline, a capillary, a suction cup, and a sealing layer and the channel layer, the channel layer and the sealing layer are sealed to form an integrated flow chip assembly, the suction cup is placed on the integrated flow chip assembly, the channel layer is engraved with channels, and is provided with a sample outlet and a sample inlet, The channel is composed of a meandering main channel and a number of side chambers located on both sides of the main channel. The two ends of the main channel are respectively connected to the sample port and the sample inlet. Polygonal, one end of the front pipeline is connected to the vacuum system, the other end is connected to the buffer bottle, one end of the rear pipeline is connected to the buffer bottle, and the other end is connected to the capillary, and the capillary passes through the suction cup to communicate with the integrated flow chip assembly.

The size of the main channel and the side chamber can be determined according to the needs. The width of the channel ranges from 1 μm to 1000 μm, the preferred width range is 50 to 1000 μm, and the best range is between 50 and 500 μm, such as 50 μm, 100 μm, 200 μm, 300 μm , 400 μm, 500 μm; the depth range of the channel 9 is between 1 μm and 1000 μm, the preferred range is between 1 μm and 500 μm, and the best range is between 10 μm and 100 μm, such as 10 μm, 20 μm, 30 μm, 50 μm, 75 μm, 100 μm ; The volume of the side chamber is in picoliter (pl) to nanoliter (nl) level.

The channel layer can be made of polymethyl methacrylate (PMMA), epoxy resin, polytetrafluoroethylene, polystyrene (PC), glass, PDMS and the like.

The channel layer can be made by different methods according to different materials, such as laser etching, chemical etching, photolithography, hot pressing, pouring and injection molding.

The material of the sealing layer is selected from heat-resistant transparent tape, PET film, fluorine film or a flat plate of the same material or a different material from the channel layer.

The channel layer and the sealing layer are sealed, and the sealing method can be bonded with heat-resistant transparent tape, thermal bonding, heat-resistant adhesive, or heat-pressed sealing, etc. according to different materials and applications; after sealing, use tape All holes are sealed for later use.

If the channel layer is made of non-transparent material, and the sealing layer is made of hard material, the sealing layer needs to drill small holes and small holes at the positions corresponding to the inlet and outlet of the channel layer in advance. Then seal it.

If the channel layer is made of a transparent material, holes can be directly drilled at the positions of the sample inlet and the sample outlet on it and then sealed with the sealing layer.

If the sealing layer is made of soft materials such as PET film, fluorine film or scotch tape, there is no need to punch holes in the sealing layer or the channel layer, and it is only necessary to use a needle to align the film or tape with the inlet and outlet. The position of the sample outlet can be punctured.

According to the material selected for the channel layer of the chip and the needs of the experiment, the back of the channel layer can be bonded to a material with a high thermal conductivity such as an aluminum sheet, a copper sheet or a silicon sheet.

The present invention also provides a method for digitally amplifying nucleic acid using the above device, the method comprising the following steps:

(1) Sample preparation:

If the nucleic acid amplification adopts the traditional PCR method, the template DNA is mixed with SYBR Premix EXTaq kit or Taqman probe kit;

If loop-mediated isothermal amplification is used for nucleic acid amplification, mix template DNA with LAMP reaction reagent and DNA fluorescent dye SYBR Green I;

(2) Puncture the small hole on the sealing layer or the scotch tape at the sample outlet 11 on the channel layer, place the suction cup on the hole, and then start vacuuming to make the vacuum degree reach 0-13332.2Pa , so that negative pressure can be used for sample injection and microfluidic distribution;

(3) According to the size of the main channel and the side chamber, add an appropriate amount of sample to the small hole on the chip sealing layer or the sample inlet on the channel layer, and puncture the adhesive tape here. Under the action of negative pressure, it enters the main channel and the small chambers on both sides. After the solution fills the main channel and the side chambers, it continues to evacuate until the liquid in the main channel is completely pumped out. At this time, the samples in the side chambers will not be pumped out but will remain. , so as to separate the sample into hundreds of picoliters to nanoliter chambers; rinse the main channel with a small amount of double distilled water;

(4) Introduce mineral oil into the main channel and make it fill the main channel, thereby separating thousands of side chambers; it is also possible to introduce low boiling point solutions, thermosetting plastics, etc. The outlet is blocked to prevent the solution in the side chamber from diffusing to the main channel after heating;

(5) seal the aperture and aperture on the sealing layer or the sample outlet and the inlet on the channel layer with curing glue or heat-resistant scotch tape;

(6) Put the integrated flow path chip assembly on the in-situ PCR instrument or a thermal cycle device similar to the in-situ PCR instrument to perform traditional PCR amplification; if the nucleic acid is subjected to loop-mediated isothermal amplification, the integrated flow path The road chip components are placed on a common hot plate and heated at 65 degrees for 30-60 minutes;

(7) Fluorescence imaging is performed on the amplified product, and the software counts the positive wells.

Advantages of the present invention:

1. Compared with the digital PCR carried out on the porous plate, the present invention utilizes the characteristics of chip miniaturization, and the consumption of reagents and samples is very little, which greatly reduces the experimental cost; in addition, the present invention only needs to vacuumize Negative pressure sampling can distribute a small amount of liquid to hundreds of independent small chambers within tens of seconds, which greatly improves the speed of the experiment. It fully embodies the development trend of miniaturization and portability of today's analytical equipment, and has great advantages in high throughput, low consumption and large-scale parallel processing. In addition, since the distribution of samples and reagents is completed inside the chip, without direct contact with the outside atmosphere, external contamination and cross-contamination can be effectively prevented.

2. The integrated flow chip in the present invention can choose different materials according to the needs of the experiment or different experimental conditions. Styrene (PC), polycarbonate (PC), epoxy resin, glass, etc. If positive pressure injection is used, polydimethylsiloxane (PDMS) can also be used.

3. Compared with the existing commercialized digital PCR chip, the present invention can distribute a small amount of liquid into thousands of independent small chambers without micro valves, which greatly reduces the complexity of making and using the chip.

4. The structure and operation of the present invention are simple, so no matter whether it is a chip or its required supporting equipment, its application cost will be greatly reduced compared with the current commercialized digital PCR chip based on PDMS elastic microvalve, which is the promotion of digital PCR technology. Provides a good platform with applications.

Description of drawings

Figure 1 is a schematic diagram of the device of the present invention.

Figure 2 is a partial enlarged view of the connection between the suction cup and the chip.

FIG. 3 is a schematic diagram of the chip channel layer.

Figure 4 is a working block diagram of the positive hole counting software.

Detailed ways

The present invention will be further described below in conjunction with drawings and embodiments.

Example 1

Referring to Fig. 1, Fig. 2 and Fig. 3, an integrated flow chip device for digital nucleic acid amplification consists of a vacuum system 1 (such as a vacuum pump), a front pipeline 2, a buffer bottle 3, a rear pipeline 4, and a capillary 5. The suction cup 6, the sealing layer 7 and the channel layer 8 are formed. The channel layer 8 and the sealing layer 7 are sealed to form an integrated flow path chip assembly. The suction cup 6 is placed on the integrated flow path chip assembly. The channel layer 8 is engraved with The channel and the sample inlet 12 and the sample outlet 11, the channel is composed of a meandering main channel 9 and a number of side chambers 10 located on both sides of the main channel 9, and the two ends of the main channel 9 are respectively connected to the sample outlet 11 and the sample inlet 12. One end of the front pipeline 2 is connected to the vacuum system 1, and the other end is connected to the buffer bottle 3. One end of the rear pipeline 4 is connected to the buffer bottle 3, and the other end is connected to the capillary 5. The capillary 5 passes through the suction cup 6 and communicates with the integrated flow chip assembly .

The channel layer 8 is made of black polymethyl methacrylate (PMMA) with a thickness of 500 μm and is made by laser etching; the main channel 9 has a width of 100 μm and a depth of 100 μm; the side chamber 10 is a square with a side length of 300 μm and a depth of 300 μm. The volume is 27nl; the cross section of the side chamber 10 can also be circular, trapezoidal or other polygonal.

The sealing layer 7 is made of heat-resistant transparent tape or PET film, fluorine film, transparent PMMA flat plate, etc. according to different applications:

If the sealing layer 7 is selected from hard materials such as PMMA flat plates, before sealing, it is necessary to drill small holes 13 and small holes 14 at positions corresponding to the sample outlet 11 of the channel layer 8 and the sample inlet 12, and then Carry out sealing, and seal all the holes with adhesive tape after successful sealing for future use;

However, if the sealing layer 7 is selected from soft materials such as PET film and fluorine film, then there is no need to punch holes in advance, and it is only necessary to puncture with a needle at the position corresponding to the sample outlet 11 and the sample inlet 12 during use. .

The sample outlet 11 is connected to the suction cup 6, the capillary 5, the rear pipeline 4, the buffer bottle 3, the front pipeline 2 and the vacuum system 1 through the corresponding small hole 13 on the chip sealing layer 7 (see Fig. 1, 2).

The vacuum system 1 draws the sample placed at the small hole 14 on the chip sealing layer 7 into the main channel 9 and the side chamber 10 by vacuuming, and after the sample is filled with the main channel 9 and the side chamber 10, continue to vacuumize, The liquid in the main channel 9 can be withdrawn while the liquid in the side chamber 10 remains, thereby separating the sample into hundreds or thousands of nanoliter chambers.

Example 2

Referring to Fig. 1, Fig. 2 and Fig. 3, with reference to Example 1, an integrated flow chip device for digital nucleic acid amplification, which consists of a vacuum system 1 (such as a vacuum pump), a front pipeline 2, a buffer bottle 3, a rear The pipeline 4, the capillary 5, the suction cup 6, the sealing layer 7 and the channel layer 8 are composed, and the channel layer 8 and the sealing layer 7 are sealed to form an integrated flow chip assembly, and the suction cup 6 is placed on the integrated flow chip assembly. The channel layer 8 is provided with a channel and a sample inlet 12 and a sample outlet 11. The channel is composed of a meandering main channel 9 and a number of side chambers 10 located on both sides of the main channel 9. The two ends of the main channel 9 are respectively connected to the sample Port 11 and injection port 12. One end of the front pipeline 2 is connected to the vacuum system 1, and the other end is connected to the buffer bottle 3. One end of the rear pipeline 4 is connected to the buffer bottle 3, and the other end is connected to the capillary 5. The capillary 5 passes through the suction cup 6 and communicates with the integrated flow chip assembly.

The channel layer 8 is made of epoxy resin by casting or by laser etching; the thickness of the channel layer 8 is 600 μm. The width of the main channel 9 is 50 μm or 100 μm, and the depth is 50 μm or 100 μm (is this possible?); The side chamber 10 is a square with a side length of 300 μm, a depth of 400 μm, and a volume of 36 nl. The side chamber 10 can also be trapezoidal or circular or polygons.

The chip sealing layer 7 can be heat-resistant transparent tape or PET film, fluorine film, polymer plate, etc. according to different applications:

If the sealing layer 7 is selected to be a hard material, it is necessary to drill small holes 13 and small holes 14 at positions corresponding to the sample outlet 11 and the sample inlet 12 of the channel layer 8 before sealing, and then perform sealing , after successful sealing, seal the hole with adhesive tape for future use;

However, if the sealing layer 7 is selected from soft materials such as PET film and fluorine film, then there is no need to punch holes in advance, and it is only necessary to puncture with a needle at the position corresponding to the sample outlet 11 and the sample inlet 12 during use. .

The sample outlet 11 is connected to the vacuum system 1 through the corresponding small hole 13 on the chip sealing layer 7, the suction cup 6, the capillary 5, the rear pipeline 4, the buffer bottle 3 and the front pipeline 2 (see Figures 1 and 2).

The vacuum system 1 draws the sample placed on the small hole 14 on the chip sealing layer 7 into the main channel 9 and the side chamber 10 by vacuuming, and when the sample is filled with the main channel 9 and the side chamber 10, continue to vacuumize, The liquid in the main channel 9 can be withdrawn while the liquid in the side chamber 10 remains, thereby separating the sample into hundreds or thousands of nanoliter chambers.

Example 3

Refer to FIG. 1 , FIG. 2 , and FIG. 3 for the structure of the integrated flow chip device for digital nucleic acid amplification in this embodiment, and refer to Embodiment 1. It consists of a vacuum system 1 (such as a vacuum pump), a front pipeline 2, a buffer bottle 3, a rear pipeline 4, a capillary 5, a suction cup 6, a sealing layer 7 and a channel layer 8, and the channel layer 8 is sealed with the sealing layer 7 Finally, the integrated flow chip assembly is formed. The suction cup 6 is placed on the integrated flow chip assembly. The channel layer 8 is provided with a channel, a sample inlet 12 and a sample outlet 11. The channel consists of a meandering main channel 9 and a channel located in the main channel. 9 consists of a number of side chambers 10 on both sides, the two ends of the main channel 9 are respectively connected to the sample port 11 and the sample inlet 12; one end of the front pipeline 2 is connected to the vacuum system 1, the other end is connected to the buffer bottle 3, and one end of the rear pipeline 4 It is connected with the buffer bottle 3, and the other end is connected with the capillary 5, and the capillary 5 passes through the suction cup 6 and communicates with the chip assembly.

The channel layer 8 is made of glass with a thickness of 1 mm, and is made by standard photolithography and etching techniques. The width of the main channel 9 is 30 μm, 50 μm, 100 μm; the depth of the main channel 9 is 20 μm, 30 μm, 50 μm; the cross section of the side chamber 10 is a square with a side length of 100 μm, 200 μm or 300 μm, and a depth of 50 μm, 100 μm, 200 μm; according to different widths and Etching depth, the volume of the side chamber 10 can be 500pl, 1nl, 2nl, 4.5nl, 8nl, 9nl, 18nl; the side chamber 10 can also be pentagonal, trapezoidal or circular;

After the channel layer 8 is made, the sample inlet 12 and the sample outlet 11 are opened with a drill bit with a diameter of 1mm, and then the channel surface is sealed with the chip sealing layer 7; The bonding is sealed with the channel layer 8; after the sealing is successful, the sample inlet 12 and the sample outlet 11 on the channel layer 8 are sealed for future use.

The sample outlet 11 is connected to the vacuum system 1 through the suction cup 6, the capillary 5, the rear pipeline 4, the buffer bottle 3 and the front pipeline 2; Draw into the main channel 9 and the side chamber 10, when the sample is full of the main channel 9 and the side chamber 10, continue vacuuming, the liquid in the main channel 9 can be drawn out, while the liquid in the side chamber 10 remains, thereby separating the sample into hundreds In chambers of thousands of picoliters or nanoliters.

Example 4 Digital-Loop Mediated Isothermal Amplification (Digital-Loop Mediated Isothermal Amplification, Digital-LAMP)

In this example, the device in Example 1 was used to inject samples, and subsequent digital loop-mediated isothermal amplification was performed.

Specific steps:

1. Design the channel structure, see Figure 3, and make a mask.

2. The chip channel layer 8 is made of 0.5mm PMMA, and the required channel structure is obtained by laser etching. The main channel 9 is 100 μm wide and 100 μm deep; the side chamber 10 is square in shape, with a side length of 300 μm and a depth of 0.5 mm. That is, all instant pass.

The size of the channel 9 and the side chamber 10 can be selected according to the needs, the width of the channel 9 varies from 10 μm to 1000 μm, and the depth is 10 μm to 1000 μm; the size of the side chamber 10 is nl to μl, and the side chamber can be square, circular or trapezoidal.

3. Glue the copper sheet with a thickness of 0.6mm-2mm to the back of the channel layer 8 of the chip.

The material bonded to the back of the channel layer 8 of the chip, in addition to the copper sheet, can also be nickel, silicon, etc., which have high thermal conductivity and are compatible with nucleic acid amplification reagents without treatment or after simple treatment.

4. Seal the channel surface of the chip channel layer 8 with heat-resistant transparent tape.

The chip channel layer 8 can also be sealed with PET film, fluorine film or with polymer plates such as drilled PMMA with glue or hot pressing. After sealing, use scotch tape to seal the perforated place for later use;

5. Use commercial λ-DNA as the reaction template to investigate the performance of the chip: Dilute the template DNA to an appropriate concentration and mix it with an appropriate amount of LAMP reaction reagent and DNA fluorescent dye SYBR Green I:

(1) Dilute 500ng/μL λ-DNA solution to 5pg/μL, 0.5pg/μL, 0.05pg/μL, 0.005pg/μL, and take 1μL each as DNA template for PCR reaction.

(2) Prepare the LAMP reaction mixture according to the 50uL system: 1 μL of λ-DNA template, 2 μL of primer FIP (40 μM), 2 μL of primer BIP (40 μM), 2 μL of primer F3 (5 μM), 2 μL of primer B3 (5 μM), dNTP (2.5 mM each ) 8 μL, MgSO ₄ (50 mM) 6 μL, betaine (5M) 10 μL, Bst enzyme 2 μL, Bstbuffer 5 μL, SYBR Green I 2 μL, ddH ₂ O 8 μL. LAMP primers are as follows:

F3: 5'GTTGGGAAGGGCGATCG 3'

B3: 5'ACTTTATGCTTCCGGCTCGTA 3'

FIP: 5'ACAACGTCGTGACTGGGAAAACCCTTTTTTGTGCGGGCCTCTTCGCTATTAC3'

BIP: 5'CGACTCTAGAGGATCCCCGGGTACTTTTTGTTGTTGTGTGGAATTGTGAGCGGAT 3'

6. Puncture the scotch tape corresponding to the sample outlet 11 on the chip sealing layer 7, place the suction cup 6 on the hole, and then start vacuuming to make the vacuum degree reach 0-13332.2Pa.

7. Add 50 μL of reaction mixture to the place corresponding to the injection port 12 on the chip sealing layer 7, and puncture the tape here. At this time, the solution will enter the main channel 9 and its two sides under the action of negative pressure. After the solution fills the main channel 9 and the side chamber 10, continue to evacuate until the liquid in the main channel 9 is completely pumped out. At this time, the sample in the side chamber 10 will not be drawn out but remains; a small amount of double distilled water rinses the main Channel 9.

8. Introduce mineral oil into the main channel 9 and fill it with main channel 9, thereby separating thousands of side chambers 10; it is also possible to load non-volatile liquids and thermosetting plastics into the main channel 9 for the purpose of In order to block the outlet of the side chamber 10 to prevent the solution in the side chamber 10 from diffusing to the main channel 9 after heating.

9. Use curing glue or heat-resistant transparent tape to seal the puncture of the tape.

10. Place the chip assembly on a hot plate and heat it at 65°C for 30 minutes to perform loop-mediated isothermal amplification of nucleic acid. Since the SYBR Green I fluorescent dye only binds to the minor groove of double-stranded DNA, when it binds to the double-stranded DNA, it emits 800-1000 times stronger fluorescence than the original one. Therefore, the side chamber 9 where the amplification reaction occurs will show green fluorescence, negative There will be no color change. Therefore, if nucleic acid molecules are distributed to a certain independent side chamber 10, the side chamber will produce green fluorescence after the reaction; if the concentration of nucleic acid is low enough, each side chamber 10 can be divided into one nucleic acid molecule at most, then, by Counting the positive wells allows accurate quantification of the amount of starting nucleic acid template.

11. After fluorescent imaging of the amplified product, count and analyze the positive wells with self-developed software.

Referring to Figure 4, the software consists of an adder, an original image storage unit, an image calculation unit, a detection standard selection unit, and a result storage unit. Original image storage part: save the data after the data image enters the processing software, and provide the original data in the intermediate result calculation and final result output; image calculation part: including key steps such as edge detection, threshold analysis, area selection, etc.; detection standard Selection part: Contains several different detection standards, which are executed in a certain order to meet the processing requirements of image data of different quality; Result storage part: used to save the data of each processing result, and after executing all detection standards , and output the results cumulatively.

Embodiment 5 digital polymerase chain reaction (Digital-PCR)

In this example, the device of Example 2 was used to inject samples, and the subsequent digital polymerase chain reaction was performed.

Specific steps:

1. Refer to Figure 3 to design the channel structure and make a mask;

2. The chip channel layer 8 is made of 0.6mm heat-resistant epoxy resin, and the required channel structure is obtained by laser etching. The main channel 9 is 100 μm wide and 100 μm deep; the side chamber 10 is square in shape, with a side length of 300 μm and a depth of 100 μm. 0.4mm.

The size of the channel 9 and the side chamber 10 can be selected according to the needs, the width of the channel 9 varies from 10 μm to 1000 μm, and the depth ranges from 10 μm to 1000 μm; the size of the side chamber 10 is from pl to nl, and the side chamber can be square, circular or trapezoidal.

3. Glue the aluminum plate with a thickness of 0.6 mm to 2 mm to the back of the chip channel layer 8 .

In addition to the aluminum plate, the material bonded to the back of the chip channel layer 8 can also be copper, nickel, silicon, etc., which have high thermal conductivity and are compatible with the nucleic acid amplification reagent without treatment or after simple treatment. Material.

4. Use heat-resistant fluorine film or PET film as the chip sealing layer 7, bonded with heat-resistant adhesive, and seal with the chip channel layer 8 by hot pressing;

5. Use commercial λ-DNA as the reaction template to investigate the performance of the chip: Dilute the template DNA to an appropriate concentration and mix it with an appropriate amount of fluorescent quantitative PCR reaction reagent:

1) Dilute 500ng/μL λ-DNA solution to 5pg/μL, 0.5pg/μL, 0.05pg/μL, 0.005pg/μL, and take 2μL each as DNA template for PCR reaction.

2) Use the SYBR PremixEX Taq kit for PCR amplification reaction, prepare the reaction solution according to the volume of 50 μL, SYBR Premix EX Taq (2×) 25 μL, PCR Forward Primer (10 μM) 1 μL, PCR Reverse Primer (10 μM) 1 μL, ddH ₂ O 21 μL, DNA template 2.0 μL. The reaction conditions were: pre-denaturation at 95°C for 30 sec, 5 sec at 95°C, and 30 sec at 60°C, a total of 40 cycles. The primer sequences of the λ-DNA used are as follows:

Upstream primer: 5'GATGAGTTCGTGTCCGTACAACTGG 3'

Downstream primer: 5'GGTTATCGAAATCAGCCACAGCGCC 3'

The PCR amplification reaction and detection can also be completed by using Taqman probes.

6. Puncture the film corresponding to the sample outlet 11 on the chip sealing layer 7, place the suction cup 6 there, and then start vacuuming to make the vacuum degree reach 0-13332.2Pa.

7. Add 50 μL of reaction mixture on the chip sealing layer 7 corresponding to the injection port 12, and puncture the film here. At this time, the solution will enter the main channel 9 and the channels on both sides under the action of negative pressure. Small chamber 10, after the solution fills the main channel 9 and side chamber 10, continue to evacuate until the liquid in the main channel 9 is completely pumped out. At this time, the sample in the side chamber 10 will not be drawn out and the surface remains; rinse the main channel with a small amount of double distilled water 9;

8. Load mineral oil into the main channel 9 and make it full of the main channel 9, thereby separating thousands of side chambers 10; it is also possible to load low boiling point liquids, thermosetting plastics, etc. into the main channel 9 for the purpose of This is to block the outlet of the side chamber 10 to prevent the solution in the side chamber 10 from diffusing to the main channel 9 after heating.

9. Seal the puncture of the film with curing glue or heat-resistant transparent tape.

10. Place the chip assembly on the in-situ PCR instrument, pre-denature at 95°C for 30 sec, 95°C for 5 sec, and 60°C for 30 sec, for a total of 40 cycles. Since the SYBR Green I fluorescent dye only binds to the minor groove of double-stranded DNA, when it binds to the double-stranded DNA, it emits 800-1000 times stronger fluorescence than the original one. Therefore, the side chamber 9 where the amplification reaction occurs will show green fluorescence, negative There will be no color change. Therefore, if nucleic acid molecules are distributed to a certain independent side chamber 10, the side chamber will produce green fluorescence after the reaction; Counting the positive wells allows accurate quantification of the amount of starting nucleic acid template.

11. After performing fluorescence imaging on the amplified product, count and analyze the positive wells by software, see Figure 4.

Claims (1)

1. An integrated flow chip device for digital nucleic acid amplification, consisting of a vacuum system (1), a front pipeline (2), a buffer bottle (3), a rear pipeline (4), a capillary (5), and a suction cup (6), the sealing layer (7) and the channel layer (8), the channel layer (8) and the sealing layer (7) are sealed to form an integrated flow chip assembly, and the suction cup (6) is placed on the integrated flow chip On the component, the channel layer (8) is engraved with a channel, and is provided with a sample outlet (11) and a sample inlet (12). The channel consists of a meandering main channel (9) and several The side chamber (10) is composed of the two ends of the main channel (9) respectively connected to the sample port (11) and the sample inlet (12). The cross section of the side chamber (10) is square, circular, trapezoidal or polygonal. One end of the pipeline (2) is connected to the vacuum system (1), the other end is connected to the buffer bottle (3), one end of the rear pipeline (4) is connected to the buffer bottle (3), the other end is connected to the capillary (5), and the capillary (5 ) through the suction cup (6) to communicate with the integrated flow chip assembly. 2. An integrated flow chip device for digital nucleic acid amplification according to claim 1, characterized in that the width of the main channel (9) is from 1 μm to 1000 μm, and the depth range is between 1 μm and 1000 μm , the volume of the side chamber (10) is at the picoliter to nanoliter level. 3. An integrated flow chip device for digital nucleic acid amplification according to claim 1, characterized in that the material of the channel layer (8) is polymethyl methacrylate, epoxy resin, polytetrafluoroethylene, One of polystyrene, glass or PDMS, the channel layer (8) is made by laser etching, chemical etching, photolithography, hot pressing, casting or injection molding. 4. An integrated flow chip device for digital nucleic acid amplification according to claim 1, characterized in that the sealing layer (7) is made of heat-resistant scotch tape, PET film, fluorine film or channel layer ( 8) Plates of the same material or different materials; 5. An integrated flow chip device for digital nucleic acid amplification according to claim 1, characterized in that the sealing method of the channel layer (8) and the sealing layer (7) is bonding with heat-resistant transparent tape, Thermal bonding, heat resistant adhesive bonding or thermocompression sealing. 6. An integrated flow chip device for digital nucleic acid amplification according to claim 5, characterized in that when the channel layer (8) is made of non-transparent material and the sealing layer (7) is made of hard material, the sealing The connection layer (7) needs to drill small holes (14) and small holes (13) at the positions corresponding to the inlet (12) and the outlet (11) in advance, and then seal them. Tape all holes closed for later use. 7. An integrated flow chip device for digital nucleic acid amplification according to claim 5, characterized in that, when the channel layer (8) is made of transparent materials, the inlet (12) and the outlet (11) ) directly drill holes and seal with the sealing layer 7, after sealing, seal all the holes with adhesive tape for future use. 8. An integrated flow chip device for digital nucleic acid amplification according to claim 5, characterized in that when the sealing layer (7) is made of soft materials, the sealing layer (7) or the channel layer (8 ) do not need to punch holes, just use a needle to pierce the film or tape corresponding to the position of the inlet (12) and the outlet (11). The soft material is PET film, fluorine film or Transparent glue. 9. An integrated flow chip device for digital nucleic acid amplification according to claim 1, characterized in that the back of the channel layer (8) is bonded to a material with a large thermal conductivity, and the material with a large thermal conductivity is selected from aluminum sheets, copper or silicon. 10. The application of an integrated flow chip device for digital nucleic acid amplification according to claim 1, characterized in that it is realized by the following steps: (1) Sample preparation: If the nucleic acid amplification adopts the traditional PCR method, the template DNA is mixed with SYBR Premix EX Taq Kit or Taqman Probe Kit; If loop-mediated isothermal amplification is used for nucleic acid amplification, mix template DNA with LAMP reaction reagent and DNA fluorescent dye SYBR Green I; (2) Puncture the small hole (13) on the sealing layer (7) or the scotch tape at the sample outlet (11) on the channel layer (8), and place the suction cup (6) on the hole , and then start vacuuming to make the vacuum degree reach 0-13332.2 Pa; (3) Add the sample at the small hole (14) on the chip sealing layer (7) or the sample inlet (12) on the channel layer (8), and puncture the tape here, at this time The solution will enter the main channel (9) and the small chambers (10) on both sides under the action of negative pressure. After the solution fills the main channel (9) and the side chambers (10), the vacuum will continue until the main channel (9) The liquid is completely pumped out, and the main channel (9) is rinsed with a small amount of double distilled water; (4) Introduce mineral oil into the main channel (9) and make it full of the main channel (9), thereby separating thousands of side chambers (10); or introduce a low boiling point solution into the main channel (9) or Thermosetting plastics to block the outlet of the side chamber (10); (5) Use curing glue or heat-resistant transparent tape to seal the small hole (13) and small hole (14) on the sealing layer (7) or the sample outlet (11) and sample inlet (11) on the channel layer (8). 12) sealing; (6) Place the integrated flow chip assembly on an in-situ PCR instrument or a thermal cycle device similar to an in-situ PCR instrument for traditional PCR amplification; if loop-mediated isothermal amplification is performed on nucleic acids, the integrated flow path Chip components are placed on a common hot plate and heated at 65 degrees for 30-60 minutes; (7) Perform fluorescence imaging on the amplified product, and count the positive wells by software. the

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