CN110835599A - Biological detection device and method based on microfluidic impact printing - Google Patents
Biological detection device and method based on microfluidic impact printing Download PDFInfo
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Abstract
The invention relates to the field of biochips, in particular to a biological detection device and a biological detection method based on microfluidic impact printing. The device comprises a microfluidic chip 2, a microfluidic printing platform 13 and a substrate 8. The invention has low material cost, can realize large-scale production, achieves the aim of one-time use, separates the chip from the actuator, does not need to be cleaned, and reduces the possibility of cross contamination. Meanwhile, the minimum working volume of the chip is only 2 microliters, the waste dead volume is in a submicron order, the consumption and waste of the reagent are less, and the method is particularly critical in rare target detection. The invention can realize flexible regulation and control of the volume of the liquid drop, and the detection range of the digital PCR system is greatly widened by changing the printing voltage, the liquid drop with the tunable volume generated by the nozzle of the microfluidic chip and the fixed-point printing times. The device provided by the invention is simple and is easy to combine with a flat PCR instrument and a fluorescence microscope which are commonly used in a biological laboratory to complete the whole digital PCR method.
Description
Technical Field
The invention relates to the field of biochips, in particular to a biological detection device and a biological detection method based on microfluidic impact printing.
Background
The Polymerase Chain Reaction (PCR), which has been the gold standard for gene detection since the appearance of 1983, is capable of replicating large amounts of target genes in a short time to amplify sample concentrations. Among them, conventional PCR techniques, such as agarose gel electrophoresis and real-time PCR, can only perform qualitative and relative detection. Digital PCR techniques provide absolute gene quantitation at the single molecule level, and do not require comparison to a standard curve. With digital PCR, the target molecule is extremely diluted and dispersed into different reaction chambers for single molecule amplification, so that copies of the target gene can be directly calculated by counting the positive reaction chambers, while this is also independent of amplification efficiency. Due to their unique absolute quantitative capabilities, high accuracy and sensitivity, digital PCR is finding increasingly widespread use in biological research, such as rare mutation detection, copy number variation, and next generation sequencing.
In 1999, Vogelstein et al implemented the earliest digital PCR system in multi-well plates. To address the inherent drawbacks of fewer reaction cells, sample reagent waste, and human manipulation errors, microfluidic technology has been developed for DNA amplification in droplets because of its unique advantage of being able to efficiently and automatically disperse small-volume droplets. A number of microfluidic-based droplet digital PCR platforms have been established and commercialized for single cell analysis, early cancer diagnosis and prenatal diagnosis. For example, Fluidigm and Thermal Fisher Scientific use integrated microfluidic chips and specially designed silicon array chips to physically isolate liquids into separate reaction chambers; bio-rad and Raindane Technologies use droplet microfluidics to produce large quantities of homogeneous water-in-oil microemulsions, with tens of thousands of parallel reactions taking place. However, these commercial digital PCR systems typically require complex chip fabrication or specialized external control of droplet generation with microvalves and pumps, and are typically equipped with specialized test equipment for signal reading, which is costly. Therefore, patent CN201210551846.5 proposes that multiple printing nozzles are used to alternately print to realize a droplet-in-oil array structure, and high-efficiency digital PCR amplification is realized, but such multiple nozzles are expensive and cannot be used at one time, so that cleaning is required when reagents are replaced, and the operation is complicated. In addition, such commercial jets require a large volume of reagent to be initially loaded and ultimately waste a large dead volume (volume that cannot be printed out), wasting expensive biological reagents and valuable biological samples. In summary, most of the methods use commercial printing nozzles, which have the risks of high use cost and cross contamination, and the methods have low flexibility in adjusting the volume of the droplets and cannot realize a wider dynamic detection range, so that it is necessary to develop a digital PCR system with simple operation, low cost and flexible and wide detection range, which is of great significance in clinical application of the digital PCR system.
Disclosure of Invention
In view of the above, the present invention provides a biological detection apparatus and method based on microfluidic impact printing. A device and a method for preparing digital PCR liquid drops and detecting DNA concentration based on microfluidic impact printing mainly solve the problems of complex manufacturing process, complex structure, high cost, pollution risk and inflexible adjustment of liquid drop volume in the existing digital PCR technology.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a biological detection device based on microfluidic impact printing, which comprises a microfluidic chip 2, a microfluidic printing platform 13 and a substrate 8;
the microfluidic chip 2 is loaded on the microfluidic printing platform 13;
the microfluidic printing platform 13 is further provided with a chip driver 14, the chip driver 14 is provided with a rigid extension piece 4, one end of the rigid extension piece 4, which is far away from the chip driver 14, is further provided with an impact head 5, and the chip driver 14 rotates the rigid extension piece 4 so as to change the relative position of the impact head 5 and the microfluidic chip 2;
the substrate 8 is arranged corresponding to the microfluidic chip 2 and is used for receiving liquid drops printed by the microfluidic chip 2;
further comprising one or more of an environmental control device (12), a programmable heating device (10) or a detection device (11).
In some embodiments of the present invention, the microfluidic chip 2 is fixed on the Z-axis, and the substrate 8 is fixed on the XY-plane of the moving platform, and the droplet alignment can be formed by moving the microfluidic chip 2, or by moving the substrate 8.
In some embodiments of the present invention, the microfluidic chip 2 includes a microfluidic channel 3 and a nozzle 15, the microfluidic channel 3 is communicated with the nozzle 15, and the nozzle 15 is disposed corresponding to and not in contact with the substrate 8.
In some embodiments of the invention, the diameter of the orifice 15 is 60 to 120 μm.
In some embodiments of the present invention, the surface of the substrate 8 is hydrophobically treated, and the surface contact angle of the substrate 8 is 60 ° to 100 °.
In some embodiments of the invention, the substrate 8 is further provided with a surrounding frame 7, the height of the surrounding frame 7 being smaller than the height of the droplets. In some embodiments of the invention, the biological testing device further comprises a cover sheet 9, and the enclosure frame 7 supports the cover sheet 9. The mineral oil can be covered firstly and then the transparent cover plate is covered, and the mineral oil can also be refilled by covering the transparent cover plate firstly. After the cover plate is covered, the liquid drops form a liquid bridge between the substrate and the cover plate, and the three form a sandwich structure. Therefore, the consistency of the fluorescence intensity of the liquid drop is improved, and the error caused by the volume change is reduced. In other embodiments, the height of the enclosure frame may not be less than the height of the liquid drop, no liquid bridge is formed, and the subsequent reaction and detection can still be performed.
In some embodiments of the invention, disposable microfluidic chips are used, where the minimum reagents required for the chip are in the microliter range and the dead volume is in the submicroliter range, and where disposable materials are used for both the chip and the substrate, no cleaning is required, reducing the possibility of cross-contamination.
In some embodiments of the invention, droplet generation may be generated from a single microfluidic channel 3, or may be generated in parallel from multiple microfluidic channels 3; the PCR system can be prepared in advance and injected into the microfluidic chip 2, and various reagents required by PCR reaction can be mixed on the chip by utilizing the microfluidic chip 2.
In some embodiments of the invention, the droplets may be of equal volume or of unequal volume; the droplet arrays with different volumes can be realized by changing the printing voltage of the microfluidic printing chip to 60-160V, the diameter of the nozzle 15 of the microfluidic chip 2 to 60-120 μm and the number of in-situ fixed-point printing times (the total volume of a droplet is times multiplied by the volume of a single droplet), so that the detection range can be expanded.
The invention also provides the application of the biological detection device in biological detection.
In some embodiments of the present invention, the liquid droplet can be used for digital PCR, and can also be used for other biological detection, and all detection samples (including nucleic acids, proteins, cells, microorganisms, etc.) in the biological field are within the protection scope of the present invention, and the present invention is not limited thereto.
In some embodiments of the invention, the environmental control device 12, may utilize increased ambient humidity to reduce droplet evaporation; the evaporation of the droplets can also be reduced by controlling the substrate temperature at 0-4 deg.C and gas (e.g., nitrogen, helium, etc.) protection. A programmable heating device 10 for heating droplets on a planar substrate; the PCR may be temperature-variable PCR or isothermal PCR. The detection device 11 may be a fluorescence detection device, and may utilize a fluorescence microscope with a splicing function, or a small animal imager, or a camera on which a small fluorescence lens is mounted for fluorescence droplet shooting.
The invention also provides a detection method based on the biological detection device, which comprises the following steps:
and 5, detecting the amplification product obtained in the step 4 by the detection device 11 to obtain a detection result.
In some embodiments of the present invention, the biological assay may be a nucleic acid assay, and may also be used for the assay of other biological samples, and all the assay samples (including nucleic acids, proteins, cells, microorganisms, etc.) in the biological field are within the scope of the present invention, and the present invention is not limited thereto.
The invention discloses a digital PCR liquid drop preparation and DNA concentration detection method based on microfluidic impact printing. A method for dividing a sample target on a planar substrate by utilizing a microfluidic chip printing technology to form a liquid drop array with a volume from picoliter to nanoliter, and then combining a flat heating system and a fluorescence detection system to complete the whole digital PCR is provided, and specifically the method comprises the following steps: the method comprises the steps of printing a liquid drop array on a modified flat substrate capable of fixing the liquid drop shape by a micro-fluidic impact printing method, changing the size of a liquid drop by changing the printing voltage of a micro-fluidic printing chip, changing the nozzle of the micro-fluidic printing chip and the fixed-point printing frequency, controlling the printing track and the imaging of the liquid drop by an XYZ three-axis moving platform, sealing by covering oil and another transparent substrate material, carrying out shooting and observation by a large-field-of-view fluorescence microscope after amplification by a flat PCR gene amplification instrument, and counting the Poisson distribution condition of positive liquid drops to finish the detection of a target sample. In the whole process, the reagent is only contacted with the disposable microfluidic printing chip and the disposable planar substrate, so that the low cost is realized, the reagent is saved, and the cross contamination of the reagent is avoided.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a flow diagram for droplet generation for a microfluidic impact printing platform according to the present invention;
FIG. 2 is a schematic diagram of the minimum working volume of the chip of the present invention;
FIG. 3 shows a schematic view of microfluidic impact printing and mobile station coordination according to the present invention;
FIG. 4 shows a flow chart of a preparation method in an embodiment of the present invention;
FIG. 5 shows a sandwich structure of the present invention, with the droplet sandwiched between two glasses;
FIG. 6 shows a schematic of a plate PCR amplification performed according to the present invention;
FIG. 7 shows a schematic view of a fluorescence detection system according to the invention;
FIG. 8 is a graph showing the comparison of fluorescence effects with and without a sandwich structure, wherein FIG. 8(A) is a graph showing the comparison of optical photographs of a droplet with and without a sandwich structure; FIG. 8(B) is a graph showing the comparison of the fluorescence effect of the droplets with and without the sandwich structure;
wherein, 1-reagent injector; 2-a microfluidic chip; 3-a microfluidic conduit; 4-a chip-driver upper rigid extension; 5-impact head; 6-liquid droplet; 7-enclosing a frame; 8-a substrate; 9-cover plate; 10-programmable heating means; 11-a fluorescence detection device; 12-an environmental control device; 13-a microfluidic printing platform; 14-a chip driver; 15-nozzle.
Detailed Description
The invention discloses a biological detection device and a biological detection method based on microfluidic impact printing, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a digital PCR liquid drop preparation and DNA concentration detection method based on microfluidic impact printing, which comprises the following steps:
the method comprises the following steps: filling a reagent into the disposable micro-fluidic chip 2;
step two: loading the microfluidic chip 2 on the microfluidic printing platform 13;
step three: controlling the microfluidic printing platform 13 to print micro droplets with the volume ranging from picoliter to nanoliter on the planar substrate 8 subjected to surface hydrophobic treatment to form a droplet array;
step four: covering the base 8 with mineral oil to cover the droplet array, and covering the transparent cover plate 9 to keep contact with the upper part of the droplets to ensure that the droplets are approximately cylindrical;
step five: placing the substrate 8 on a programmable heating device 10 for DNA amplification;
step six: and scanning fluorescence imaging is carried out on the amplified liquid drops, the number of the positive expression liquid drops is detected by a fluorescence detection device 11, and the concentration of the DNA to be detected is calculated according to the number of the positive expression liquid drops.
In some embodiments, droplet generation uses disposable microfluidic chips, the minimum reagents required for the chip are in the microliter range, the dead volume is in the submicroliter range, and the disposable materials used for the chip and the substrate do not need to be cleaned, thereby reducing the possibility of cross-contamination.
In some embodiments, droplet generation may occur from a single channel or from multiple channels in parallel; the PCR system can be prepared in advance and injected into the microfluidic printing chip, and various reagents required by PCR reaction can be mixed on the chip by utilizing the microfluidic printing chip.
In some embodiments, the droplets may be of equal volume or unequal volume; the liquid drop arrays with different volumes can be realized by changing the printing voltage of the microfluidic printing chip to be 60-160V, the nozzle of the microfluidic printing chip to be 60-120 micrometers and the in-situ fixed-point printing times (the total volume of the liquid drop is times multiplied by the volume of a single liquid drop), so that the detection range can be enlarged.
In some embodiments, the planar substrate surface is hydrophobically treated to change the substrate surface contact angle by about 60 ° to 100 °, preferably 80 °.
In some embodiments, a frame is bonded to the planar base for supporting the transparent cover sheet thereon, the frame having a height less than the droplet height. The mineral oil can be covered firstly and then the transparent cover plate is covered, and the mineral oil can also be refilled by covering the transparent cover plate firstly.
In some embodiments, an environmental control system is provided that can reduce droplet evaporation by increasing ambient humidity; the evaporation of the droplets can also be reduced by controlling the substrate temperature at 0-4 deg.C and gas (e.g., nitrogen, helium, etc.) protection.
In some embodiments, a programmable plate heating system is used to heat the droplets on the planar substrate; the PCR may be temperature-variable PCR or isothermal PCR.
In some embodiments, the microfluidic printing chip is fixed on the Z axis, the planar substrate is fixed on the XY plane of the moving platform, and the droplet arrangement can be formed by moving the printing chip and also can be formed by moving the substrate.
In some embodiments, the detection system is a fluorescence detection system, which may utilize a fluorescence microscope with a splicing function, a small animal imager, or a camera with a small fluorescence lens for fluorescence droplet shooting.
Compared with the prior art, the invention has the following beneficial effects:
the invention applies simple and convenient micro-fluidic impact printing technology, can realize large-scale production by utilizing the advantages of low material cost and simple manufacturing process, achieves the purpose of one-time use, separates a chip from an actuator, does not need cleaning, and reduces the possibility of cross contamination. Meanwhile, the minimum working volume of the chip is only 2 microliters, the waste dead volume is in a submicron order, the consumption and waste of the reagent are less, and the method is particularly critical in rare target detection.
The invention can realize the flexible regulation and control of the volume of the liquid drop, and the liquid drop with the tunable volume is generated by changing the printing voltage, the nozzle of the microfluidic printing chip and the fixed-point printing times, thereby greatly widening the detection range of the digital PCR system.
3, the microfluidic impact printing device is simple and is easy to combine with a flat PCR instrument and a fluorescence microscope which are common in a biological laboratory to finish the whole digital PCR method.
The raw materials and reagents used in the biological detection device and method based on microfluidic impact printing provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
as shown in FIG. 1, the digital PCR droplet preparation and DNA concentration detection method based on microfluidic impact printing mainly comprises a microfluidic printing head (including a microfluidic chip 2 and a microfluidic printing platform 13; the microfluidic printing platform 13 is also provided with a chip driver 14, the chip driver 14 is provided with a rigid extension piece 4, one end of the rigid extension piece 4 far away from the chip driver 14 is also provided with an impact head 5, and the relative position of the impact head 5 and the microfluidic chip 2 is changed by rotating the rigid extension piece 4 through the chip driver 14) and an XYZ moving table. The micro-fluidic chip is designed based on the micro-fluidic principle and comprises a three-layer structure: the elastic layer on upper strata, the supporting layer of middle pipeline layer and lower floor, elastic layer and pipeline layer can adopt macromolecular material, and the supporting layer can adopt macromolecular material or glass, and macromolecular material for example is PDMS (polydimethylsiloxane), and the chip size is 10 millimeters 16 millimeters 3 millimeters by 16 millimeters, and the pipeline width is 200 microns, can make different chip specifications as required in actual manufacturing process. The PCR reagent can be added into the microfluidic chip 2 through the injection 1, and can actually fill the microfluidic pipeline 3. The rigid extension piece 4 on the chip driver 14 knocks the cavity on the microfluidic chip 2 through the impact head 5 connected to the rigid extension piece, so that liquid in the cavity is sprayed out in the form of liquid drops 6, and the rigid extension piece 4 is of an elongated structure. As shown in fig. 2: the chip size is small and requires a small sample volume. In the printing process, the minimum working volume of the chip is only 2 microliters, the waste dead volume is in the submicron order, and the consumption and waste of the reagent are less. The voltage is controlled to be 60-160V, the diameter of the nozzle is 60-120 mu m, the volume change of the liquid drop of 1-20 nanoliters can be realized, and the in-situ fixed-point printing frequency N (V) can be changedGeneral assemblyNumber of times N × VSingle droplet) And the printing generation of the tunable liquid drop array is realized. The actuator (chip driver 14) of the microfluidic print head is separated from the disposable microfluidic chip 2 without cleaning and cross contamination. The periphery of the substrate 8 for receiving the liquid drop is formed by bonding high molecular materials, such as: PDMS, silica gel pad etc. form and enclose frame 7, and base 8 can fix the liquid drop array through surface hydrophobic treatment, has guaranteed that can not take place liquid drop removal in later stage oil covering process yetAnd fusion, after the oil completely covers the liquid drops, a transparent cover plate 9 is covered to stick the liquid drops to form a sandwich structure of the glass sheet-the liquid drops-the glass substrate, the liquid drop shape is changed from an approximate semicircle to an approximate cylinder, and the fluorescence contrast is improved. The transparent cover sheet can also be refilled with mineral oil. The liquid drops can be equal in volume or unequal in volume in the printing process; the liquid drop arrays with different volumes can be realized by changing the printing voltage of the microfluidic printing chip and the nozzle 15 and fixed-point printing times of the microfluidic chip 2, so that the detection range can be enlarged.
The droplet array is realized as shown in fig. 2, the microfluidic chip 2 is placed on an XYZ moving stage, the flat substrate 8 is fixed, and the droplet printing height and position are controlled, or the microfluidic chip 2 is placed on the Z moving stage, the flat substrate 8 is placed on the XY moving stage, and the droplet printing height and position are controlled, or the chip is placed on the XY moving stage, the flat substrate is placed on the Z moving stage, and the droplet printing height and position are controlled, or the chip is fixed, and the flat substrate is fixed on the XYZ moving stage, and the droplet printing height and position are controlled. A humidifier is needed in the whole test process, so that the working environment keeps high humidity, and liquid drops are prevented from evaporating.
After printing is completed, the droplet array is covered with mineral oil and covered with a cover sheet 9 to stick to the droplets to ensure that the droplet morphology is approximately cylindrical, as shown in FIG. 4, and then placed on a programmable heating system 10 for DNA amplification, as shown in FIG. 5. And finally, scanning fluorescence imaging is carried out on the amplified liquid drops, the number of the positive expression liquid drops is detected by the detection device 11, and the content of the DNA to be detected is calculated according to the number of the positive expression liquid drops, as shown in figure 6. Fig. 7 shows: the cylindrical droplet shape ensures better fluorescent consistency of the droplets. Heating is embodied as the flow chart of fig. 3, and the method comprises the following steps:
the method comprises the following steps: filling reagents into the disposable microfluidic printing chip, wherein the digital PCR reaction reagents comprise a detection sample and a PCR reaction system (reagents, probes, primers and the like);
step two: loading a microfluidic printing chip on a microfluidic printing platform, and setting a required printing mode;
step three: controlling a printing platform to print liquid drops on a planar substrate subjected to surface hydrophobic treatment to form a liquid drop array with a volume range from picoliter to nanoliter;
step four: covering mineral oil on the substrate, covering a transparent cover plate on the substrate to stick the liquid drops after the oil completely covers the liquid drops, and ensuring that the liquid drops are cylindrical;
step five: placing the substrate in a flat plate heating device for DNA amplification;
step six: and scanning fluorescence imaging is carried out on the amplified liquid drops, the number of the positive expression liquid drops is detected, and the content of the DNA to be detected is calculated according to the number of the positive expression liquid drops.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The biological detection device based on the microfluidic impact printing is characterized by comprising a microfluidic chip (2), a microfluidic printing platform (13) and a substrate (8);
the microfluidic chip (2) is loaded on the microfluidic printing platform (13);
the microfluidic printing platform (13) is further provided with a chip driver (14), the chip driver (14) is provided with a rigid extension piece (4), one end, far away from the chip driver (14), of the rigid extension piece (4) is further provided with an impact head (5), and the chip driver (14) rotates the rigid extension piece (4) so as to change the relative position of the impact head (5) and the microfluidic chip (2);
the substrate (8) is arranged corresponding to the microfluidic chip (2) and is used for receiving liquid drops printed by the microfluidic chip (2);
further comprising one or more of an environmental control device (12), a programmable heating device (10) or a detection device (11).
2. The biological detection device according to claim 1, wherein the microfluidic chip (2) comprises a microfluidic channel (3) and a nozzle (15), the microfluidic channel (3) is communicated with the nozzle (15), and the nozzle (15) is arranged corresponding to the substrate (8) and is not in contact with the substrate.
3. The bioassay device as set forth in claim 2, wherein said ejection opening (15) has a diameter of 60 to 120 μm.
4. A biological detection device as claimed in any one of claims 1 to 3, characterised in that the surface of the substrate (8) is hydrophobically treated, and the substrate (8) has a surface contact angle of 60 ° to 100 °.
5. The biodetection device as claimed in any one of claims 1 to 4, characterized in that the substrate (8) is further provided with an enclosure (7), the enclosure (7) having a height smaller than the height of the droplets.
6. The biological detection device according to claim 5, further comprising a cover sheet (9), the enclosure frame (7) supporting the cover sheet (9).
7. Use of a bioassay as claimed in any one of claims 1 to 6 in bioassays.
8. The detection method based on the bioassay device according to any one of claims 1 to 6, comprising the steps of:
step 1, adding a reagent into the microfluidic chip (2);
step 2, controlling the microfluidic printing platform (13), rotating the rigid extension piece (4) through the chip driver (14) so as to change the relative position of the impact head (5) and the microfluidic chip (2), knocking the microfluidic chip (2) to enable the reagent to be sprayed out in a droplet form, and printing the droplet on the substrate (8) to form a droplet array;
step 3, sealing the droplet array of the substrate (8) through the cover sheet (9);
step 4, placing the substrate (8) in the programmable heating device (10) for amplification;
and 5, detecting the amplification product obtained in the step 4 by the detection device (11) to obtain a detection result.
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