Application of crescent moon microspheres in digital polymerase chain reaction
By preparing crescent-shaped hydrogel microspheres, the balance between result stability and cost-effectiveness in existing digital PCR platforms was resolved. This enabled high-precision digital quantification without the need for microfluidic equipment, expanded the reaction space, improved quantification accuracy and stability, and reduced costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI TECH UNIV
- Filing Date
- 2023-09-25
- Publication Date
- 2026-06-26
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure HDA0004467937870000011
Abstract
Description
Technical Field
[0001] This application relates to the field of droplet microfluidic digital analysis and detection, and in particular to the application of a crescent-shaped microsphere in digital polymerase chain reaction. Background Technology
[0002] Digital analytical assays enhance the quantitative capabilities of diagnostic tests by lowering the detection limit and enabling absolute quantification of the target analyte. Digital polymerase chain reaction (D-PCR) has been shown to offer several quantitative advantages over real-time polymerase chain reaction (RT-PCR), including more accurate copy number quantification, lower detection limits, and better reproducibility.
[0003] To achieve digital quantification (DPCR) for polymerase chain reactions, the target analyte needs to be divided into compartments ranging from picoliters to nanoliters. After adjusting the size and number of these compartments based on the estimated DNA sample concentration, each compartment is assumed to contain one or zero nucleic acids. After the reaction, positive droplets containing the nucleic acid template are amplified and "lit up," while negative droplets without the template are not lit up. By statistically analyzing the proportion of positive droplets, combined with the Poisson quantification principle, absolute quantification of the target can be achieved. Currently, several digital PCR technologies have been established; however, many have key drawbacks. For example, many commercially available digital PCR reaction systems are based on microfluidic methods to generate water-in-oil droplets. While accurate and precise, the cost and space requirements of these platforms, as well as the need for skilled microfluidic system operation, pose a significant challenge for those outside the field.
[0004] Recent studies have shown that hydrogel microspheres can be used as templates for droplet generation to stably generate uniform droplets without requiring precise microfluidic droplet generators. However, in droplets prepared by emulsification based on spherical hydrogel microspheres, the hydrogel polymer matrix is repellent to macromolecular PCR reagents, such as DNA polymerase and template DNA. Therefore, in these droplets containing hydrogel microspheres, only a thin reaction volume shell is generated between the emulsion interface and the surface of the hydrogel microspheres for the DNA polymerization chain reaction. This limited reaction volume hinders volume-dependent Poisson loading of DNA entities, thus reducing quantitative accuracy and dynamic range. Furthermore, deformation of this thin shell can lead to relatively high reaction volume heterogeneity, which has been shown to reduce the precision and quantitative accuracy of digital PCR analysis. Therefore, current digital PCR platforms often struggle to balance result stability and cost-effectiveness. There is an urgent need to develop a low-cost, quantitatively stable, and precise digital microfluidic quantification platform that does not require microfluidic equipment or specialized personnel. Summary of the Invention
[0005] In view of the shortcomings of the prior art described above, and to solve the technical problems in the prior art, the inventors of this application propose an application of crescent-shaped microspheres in digital polymerase chain reaction (PCR). This application uses a two-phase aqueous method to prepare a crescent-shaped hydrogel structure with a special morphology. Because the cavity of the crescent-shaped structure can accommodate a certain volume of aqueous phase, it reduces the repulsive effect of microspheres and water molecules that occurs in pure spherical hydrogel microspheres, resulting in a larger space for PCR within the droplets formed based on the crescent structure. When the crescent-shaped microspheres are used in PCR, fluorescent chimeric dyes label positive droplets with targets and negative droplets without targets. By statistically analyzing the proportion of positive droplets and combining this with a Poisson distribution, a more accurate digital quantification of the initial sample can be obtained.
[0006] To achieve the above and other related objectives, the first aspect of this application provides a crescent-shaped microsphere, which is a concave sphere with a crescent-shaped cross-section. The diameter of the concave portion of the crescent-shaped microsphere is 10 to 200 μm, and the diameter of the sphere itself is 10 to 200 μm.
[0007] A second aspect of this application provides a method for preparing crescent-shaped microspheres, comprising the following steps:
[0008] 1) Provide a first solution and a second solution, mix them, and centrifuge to obtain two separate phases. The bottom solution is the second aqueous phase, and the top solution is the first aqueous phase.
[0009] 2) The first aqueous phase and the second aqueous phase are simultaneously sheared with the oil phase to obtain a two-phase droplet in which the first aqueous phase partially encapsulates the second aqueous phase;
[0010] 3) Irradiate the aqueous two-phase droplets to obtain gel-forming droplets in which the outer gel layer partially encapsulates the inner gel layer;
[0011] 4) The gel droplets were demulsified and the inner gel layer was dissolved to obtain crescent-shaped microspheres.
[0012] The third aspect of this application provides a crescent-shaped microsphere, which is prepared by the preparation method described in the second aspect.
[0013] A fourth aspect of this application provides a method for digital polymerase chain reaction, using the aforementioned crescent-shaped microspheres, comprising the following steps:
[0014] 1) The nucleic acid to be tested, crescent-shaped microspheres, and the first oil phase are mixed to obtain the first mixture;
[0015] 2) The crescent-shaped microspheres and the nucleic acid to be tested are fully encapsulated to obtain paired droplets;
[0016] 3) Replace the first oil phase in the paired droplets with the second oil phase and perform polymerase chain reaction amplification to obtain amplified droplets;
[0017] 4) Analyze the amplified droplets to obtain the quantification of the nucleic acid to be tested.
[0018] The fifth aspect of this application provides the aforementioned crescent-shaped microspheres, the aforementioned preparation method, or the application of the aforementioned method in digital polymerase chain reaction.
[0019] Compared with the prior art, the beneficial effects of this application are as follows:
[0020] 1. This application uses a two-phase aqueous method to prepare a crescent-shaped hydrogel structure with a special morphology. The cavity of the crescent-shaped structure can accommodate a certain volume of aqueous phase, thus reducing the repulsive effect of microspheres and water molecules that occurs in pure spherical hydrogel microspheres, and making the droplets formed based on the crescent structure have a larger space for polymerase chain reaction.
[0021] 2. This application uses a specific dextran hydrolase to treat the inner gel layer in the aqueous two-phase droplet, which makes it easier to obtain crescent-shaped microspheres with ideal shape.
[0022] 3. In the preparation process of the crescent-shaped microspheres of this application, the flow rate ratio of the first aqueous phase, the second aqueous phase and the oil phase, as well as the raw material concentration of the crescent-shaped microspheres, are adjusted to achieve precise control of the internal chamber volume of the crescent-shaped microspheres.
[0023] 4. When using crescent-shaped microspheres in polymerase chain reaction, the fluorescent chimeric dye will label positive droplets with targets and negative droplets without targets. By statistically analyzing the proportion of positive droplets and combining it with the Poisson distribution, a more accurate numerical quantification of the initial sample can be obtained.
[0024] 5. The digital polymerase chain reaction performed by this method is a microfluidic-free method, which gets rid of the constraints of chips and devices in traditional digital polymerase chain reactions, thus making it suitable for wider adoption. Attached Figure Description
[0025] Figure 1 A schematic diagram of a digital polymerase chain reaction (PCR) process in a hydrogel microchamber.
[0026] Figure 2 A schematic diagram of the fabrication of a chip from crescent-shaped gel microspheres, with a scale bar of 1000 μm.
[0027] Figure 3 This describes the preparation process of crescent-shaped gel microspheres. Figure 3 a is a two-phase aqueous droplet fabricated using a microfluidic chip. Figure 3 b represents the gel microspheres obtained after the gelation and demulsification of the aqueous two-phase droplets. Figure 3c represents crescent-shaped gel microspheres obtained by dissolving Dextran using Dextranase treatment; all scale bars are 200 μm.
[0028] Figure 4 Characterization of the particle size of crescent-shaped gel microspheres. Among them, Figure 4 a represents the statistical analysis of the particle sizes of the aqueous two-phase droplets and the crescent-shaped gel microspheres. Figure 4 b represents the statistical values of the internal core diameter and the overall external shell diameter of the crescent-shaped gel microspheres.
[0029] Figure 5 Fluorescent labeling characterization of crescent-shaped gel microspheres. FITC-dextran was added to the Dextran phase during microsphere preparation for fluorescent labeling of the prepared crescent-shaped microspheres; from left to right, the images show bright field, fluorescence field, and merge plots, with a scale bar of 50 μm.
[0030] Figure 6 For the preparation of crescent-shaped microsphere droplets at different Pipette times, among which, Figure 6 a represents the crescent-shaped microsphere droplets prepared by Pipette at 30 s. Figure 6 b represents the crescent-shaped microsphere droplets prepared by Pipette at 60 s. Figure 6 c represents the crescent-shaped microsphere droplets prepared at Pipette 120s. Figure 6 d represents the statistical values of droplet size prepared under three conditions. Figure 6 e represents the statistical value of droplets containing two or more crescent-shaped microspheres prepared under the three conditions, and the scale bar is 200 μm.
[0031] Figure 7 The results are from polymerase chain reaction (PCR). From top to bottom, they represent different test samples: no nucleic acid to be tested, 100 fg / μL nucleic acid to be tested, 1 pg / μL nucleic acid to be tested, and 10 pg / μL nucleic acid to be tested. Detailed Implementation
[0032] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.
[0033] The inventors of this application, through extensive research and exploration, discovered the application of a crescent-shaped microsphere in digital polymerase chain reaction, and completed this application based on this discovery.
[0034] The first aspect of this application provides a crescent-shaped microsphere, which is a concave sphere with a crescent-shaped cross-section. The size of the crescent-shaped microsphere can be adjusted according to specific experimental requirements. In some embodiments, the diameter of its internal core is 10-200 μm, specifically 50-55 μm, 55-60 μm, or 60-65 μm. The diameter of the shell of the crescent-shaped microsphere is 10-200 μm, specifically 70-75 μm, 75-80 μm, or 80-85 μm. In some embodiments of this application, the crescent-shaped microsphere can be selected in two sizes: ① the diameter of the core of the crescent-shaped microsphere is 25 μm, and the diameter of the shell of the crescent-shaped microsphere is 40 μm; ② the diameter of the core of the crescent-shaped microsphere is 50 μm, and the diameter of the shell of the crescent-shaped microsphere is 70 μm.
[0035] The crescent-shaped microspheres are formed into a gel by photocuring of raw materials. The raw materials include, but are not limited to, one or more combinations of polyethylene glycol diacrylate (PEGDA), dextran, tetra-arm PEGDA, gelatin, and tetra-arm PEGmaleimide. In a specific embodiment of this application, the molecular weight of the polyethylene glycol diacrylate (PEGDA) includes 575 and / or 8000. Based on the total volume of the crescent-shaped microspheres, the concentration of the PEGDA solution with a molecular weight of 575 is 3-10% (w / v), specifically, for example, 3-5% (w / v), 5-8% (w / v), or 8-10% (w / v). The concentration of the PEGDA solution with a molecular weight of 8000 is 3-10% (w / v), specifically, for example, 3-5% (w / v), 5-8% (w / v), or 8-10% (w / v). In a specific embodiment of this application, the molecular weight of the dextran is selected from 500,000. Based on the total volume of the crescent-shaped microspheres, the concentration of the dextran solution is 5.5-10% (w / v), specifically, for example, 5.5-6% (w / v), 6-8% (w / v), or 8-10% (w / v). The dextran can be, for example, dextran, which is commercially available. In some embodiments, photocuring can be carried out using a photoinitiator, such as LAP (lithium phenyl-2,4,6trimethylbenzoylphosphinate), TPO (diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide), photoinitiator 784 (bis(1-(2,4-difluorophenyl)-3-pyrrolithyl)titanium oxide), photoinitiator 819 (phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide), and other common photoinitiators.
[0036] In some embodiments, the crescent-shaped microspheres are prepared by partially encapsulating an inner gel with an outer gel, followed by dissolving the inner gel. The method for dissolving the inner gel includes one or more combinations of adding dextran hydrolase, adjusting the solution pH, and heating. Further, the solution pH is adjusted to 5-7; specifically, this can be achieved by first adding alkali, then acid, and washing repeatedly to adjust the pH to a suitable range. The heating temperature is 50-70 °C; specifically, it can be 50-55 °C, 55-65 °C, or 65-70 °C, etc. In a specific embodiment of this application, the raw material for the inner gel includes dextran. This application uses a specific dextran hydrolase to treat the inner gel, making it easier to obtain crescent-shaped microspheres with the desired shape. In the preparation of crescent-shaped microspheres, the concentration and flow rate of the raw materials determine that the circle formed by the two aqueous phases is an eccentric circle, which makes the crescent-shaped microspheres obtained after treatment with dextran hydrolase crescent-shaped. The cavity of the crescent-shaped structure can accommodate a certain volume of aqueous phase, thus reducing the repulsion effect of microsphere-water molecules that occurs in pure spherical hydrogel microspheres, and making the droplets formed based on the crescent structure have a larger space for polymerase chain reaction inside.
[0037] A second aspect of this application provides a method for preparing crescent-shaped microspheres, comprising the following steps:
[0038] 1) Provide a first solution and a second solution, mix them, and centrifuge to obtain two separate phases. The bottom solution is the second aqueous phase, and the top solution is the first aqueous phase.
[0039] 2) The first aqueous phase and the second aqueous phase are simultaneously sheared with the oil phase to obtain a two-phase droplet in which the first aqueous phase partially encapsulates the second aqueous phase;
[0040] 3) Irradiate the aqueous two-phase droplets to obtain gel-forming droplets in which the outer gel partially encapsulates the inner gel.
[0041] 4) The gel-forming liquid droplets were broken up and the inner gel layer was dissolved to obtain crescent-shaped microspheres.
[0042] In the preparation method provided in this application, step 1) refers to providing a first solution and a second solution, mixing them, and centrifuging to obtain two separate phases. The bottom layer solution is the second aqueous phase, and the top layer solution is the first aqueous phase. The solute in the first solution is selected from polyethylene glycol diacrylate, tetra-arm polyethylene glycol acrylate, gelatin, or tetra-arm polyethylene glycol maleimide. The solute in the second solution is selected from dextran. The second aqueous phase includes the second solution. The first aqueous phase includes the first solution and also includes a photoinitiator. Unless otherwise specified, the solutions in this application are aqueous solutions or other conventional solutions capable of dissolving the solutes in this application. The separation of the two phases after centrifugation is determined by the properties of the solutes in the first and second solutions themselves; they cannot be mixed, thus the stratification phenomenon can be clearly observed.
[0043] In one specific embodiment of this application, the first solution is a polyethylene glycol diacrylate (PEGDA) solution, and the second solution is a dextran solution. The two solutions are first mixed, then centrifuged to obtain two separate phases: the top layer is the PEGDA solution, and the bottom layer is the dextran solution. The centrifugation speed is 10000~12000 g, specifically, it can be 10000~11000 g, 11000~11500 g, or 11500~12000 g, etc. The centrifugation time is 10~15 min, specifically, it can be 10~12 min, 12~14 min, or 14~15 min, etc. A biomarker, such as a fluorescent marker, is added to the dextran solution to label the crescent-shaped microspheres, and the labeled droplets can be found under a subsequent fluorescence microscope. In some embodiments of this application, the biomarker can be, for example, commonly used fluorescent markers such as FITC, TET, FAM, Rhodamine, Cy3, and Cy5.
[0044] In step 1), the molecular weight of polyethylene glycol diacrylate (PEGDA) includes 575 and / or 8000. Based on the total volume of the crescent-shaped microspheres, the concentration of the PEGDA solution with a molecular weight of 575 is 3-10% (w / v), specifically, for example, 3-5% (w / v), 5-8% (w / v), or 8-10% (w / v). The concentration of the PEGDA solution with a molecular weight of 8000 is 3-10% (w / v), specifically, for example, 3-5% (w / v), 5-8% (w / v), or 8-10% (w / v). The molecular weight of the dextran is selected from 500,000. Based on the total volume of the crescent-shaped microspheres, the concentration of the dextran solution is 5.5–10% (w / v), specifically, for example, 5.5–6% (w / v), 6–8% (w / v), or 8–10% (w / v). The dextran can be, for example, dextran, which is commercially available.
[0045] In the preparation method provided in this application, a photoinitiator is added to the separated polyethylene glycol diacrylate solution to obtain a first aqueous phase. In some embodiments, the concentration of the photoinitiator solution is 0.2-1% (w / v) based on the total volume of the crescent-shaped microspheres, specifically, for example, 0.2-0.5% (w / v), 0.5-0.8% (w / v), or 0.8-1% (w / v). The photoinitiator can be, for example, common photoinitiators such as LAP (lithium phenyl-2,4,6trimethylbenzoylphosphinate), TPO (diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide), photoinitiator 784 (bis(1-(2,4-difluorophenyl)-3-pyrrolithyl)titanium oxide), and photoinitiator 819 (phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide).
[0046] In the preparation method provided in this application, step 2) refers to simultaneously shearing the first aqueous phase and the second aqueous phase with an oil phase to obtain a two-phase droplet in which the first aqueous phase partially encapsulates the second aqueous phase. In some embodiments, the oil phase is fluorinated oil; further, the fluorinated oil is HFE7500 fluorinated oil containing 2% (w / v) PEG-PFPE surfactant. During shearing, the flow rate of the oil phase is 200~5000 μL / h, the flow rate of the first aqueous phase is 100~1000 μL / h, and the flow rate of the second aqueous phase is 50~1500 μL / h. In a preferred embodiment of this application, when the flow rates of the oil phase, the first aqueous phase, and the second aqueous phase are 400 μL / h, 200 μL / h, and 100 μL / h, respectively, the size of the prepared crescent-shaped microspheres is: core 50 μm, shell 70 μm. In a preferred embodiment of this application, when the flow rates of the oil phase, the first aqueous phase, and the second aqueous phase are 400 μL / h, 100 μL / h, and 50 μL / h, respectively, the dimensions of the prepared crescent-shaped microspheres are: core 25 μm and shell 40 μm. During the preparation of the crescent-shaped microspheres in this application, the internal chamber volume of the crescent-shaped microspheres is precisely controlled by adjusting the flow rate ratio of the first aqueous phase, the second aqueous phase, and the oil phase, as well as the raw material concentration of the crescent-shaped microspheres. The aqueous two-phase droplet obtained in this application, where the first aqueous phase partially encapsulates the second aqueous phase, is determined by the surface tension between the first aqueous phase, the second aqueous phase, and the oil phase. When the first aqueous phase includes PEGDA and the second aqueous phase includes Dextran, three surface tensions are formed: γoil-pegda; γoil-dextran; and γpegda-dextran. When γoil-pegda < γpegda-dextran < γoil-dextran, an aqueous two-phase droplet where the first aqueous phase partially encapsulates the second aqueous phase is formed.
[0047] In the preparation method provided in this application, step 3) refers to irradiating the aqueous two-phase droplet with light to obtain a gelling droplet in which the outer gel partially encapsulates the inner gel. The irradiation time is 30~180s, specifically, it can be 30~60s, 60~100s, or 100~180s, etc. The irradiation light is ultraviolet light. In some embodiments, the irradiation condition is 365nm (20w) ultraviolet irradiation. The outer gel is obtained by irradiating the first aqueous phase, and the inner gel is obtained by irradiating the second aqueous phase. In some embodiments, the aqueous two-phase system (pegda + dextran) is a mutually doped state, and the inner dextran also contains some pegda, so it can also partially gel, forming a weakly cross-linked inner gel.
[0048] In the preparation method provided in this application, step 4) refers to demulsifying and dissolving the inner gel layer of the gel-forming droplets to obtain crescent-shaped microspheres. In some embodiments, demulsification is performed using a demulsifier, such as HFE7500 containing 20% (v / v) fluorinated polyol (PFO). In some embodiments, dissolving the inner gel layer can be achieved, for example, by adding dextran hydrolase, adjusting the solution pH, or raising the temperature. Further, the solution pH is adjusted to 5-7, specifically by first adding alkali, then adding acid, and washing multiple times to adjust the pH to a suitable range; the temperature is 50-70 °C, specifically 50-55 °C, 55-65 °C, or 65-70 °C, etc. In a specific embodiment of this application, the dextran hydrolase can be, for example, dextranase, which is commercially available. The enzyme treatment temperature is 55-60 °C, specifically 55-56 °C, 56-58 °C, or 58-60 °C, etc. The enzyme treatment time is 10-15 min, specifically 10-12 min, 12-14 min, or 14-15 min. This application uses a specific dextran hydrolase to remove the inner gel layer in the gel droplets, making it easier to obtain crescent-shaped microspheres. Optionally, a post-treatment step is also included: after demulsification, the oil phase of the gel droplets is washed, the first aqueous phase is treated with dextran hydrolase, and then washed again to obtain crescent-shaped microspheres. Washing can be performed, for example, with Dulbecco's phosphate-buffered saline (DPBS). In some embodiments, after washing the oil phase, the sample is resuspended in DPBS, and dextran hydrolase is added (approximately 1 / 10 the volume of the resuspended sample). After mixing, the sample is heated at 60 °C for 10 min to ensure complete degradation of the dextran.
[0049] A third aspect of this application provides a crescent-shaped microsphere, prepared by the aforementioned method. In the crescent-shaped microsphere, the cavity of the crescent-shaped structure can accommodate a certain volume of water phase, thus reducing the repulsive effect of microspheres and water molecules present in pure spherical hydrogel microspheres. This results in a larger space for polymerase chain reactions within the droplets formed based on the crescent structure.
[0050] A fourth aspect of this application provides a method for digital polymerase chain reaction, using the aforementioned crescent-shaped microspheres, comprising the following steps:
[0051] 1) The nucleic acid to be tested, crescent-shaped microspheres, and the first oil phase are mixed to obtain the first mixture;
[0052] 2) The crescent-shaped microspheres and the nucleic acid to be tested are fully encapsulated to obtain paired droplets;
[0053] 3) Replace the first oil phase in the paired droplets with the second oil phase and perform polymerase chain reaction amplification to obtain amplified droplets;
[0054] 4) Analyze the amplified droplets to obtain the quantification of the nucleic acid to be tested.
[0055] In the method provided in this application, step 1) refers to mixing the nucleic acid to be tested, crescent-shaped microspheres, and a first oil phase to obtain a first mixture. The first mixture also includes a mix reaction solution required for polymerase chain reaction, and / or a stabilizing solution; further, the raw material for the stabilizing solution is Tween and / or polyethylene glycol. The first oil phase is a fluorinated oil; further, the fluorinated oil is HFE-7500 containing PEG-PFPE. In a specific embodiment, the first oil phase is HFE-7500 containing 2% PEG-PFPE.
[0056] In some implementations, the mix reaction solution also includes primers.
[0057] In some embodiments, the mix reaction solution, stabilizing solution, and crescent-shaped microspheres are first mixed, incubated on ice to allow them to mix, then centrifuged to remove the supernatant, and then the nucleic acid to be tested is added to obtain the first mixture. The purpose of mixing the mix reaction solution, stabilizing solution, and crescent-shaped microspheres first is to ensure that the nucleic acid to be tested is not lost. The stabilizing solution serves to keep the droplets stable and reduce droplet fusion. The ratio of crescent-shaped microspheres to the template to be tested depends on the specific circumstances. In some embodiments, the diameter ratio of the test cells to the overall shell of the crescent-shaped microspheres is 1:2 to 3.5, specifically, it can be 1:2 to 2.5, 1:2.5 to 3, or 1:3 to 3.5, etc. In one specific embodiment, the diameter of the test cells is 20 μm, and the diameter of the overall shell of the crescent-shaped microspheres can be 40 μm or 70 μm.
[0058] In some implementations, after obtaining the first mixture in step 1), the oil phase can be removed as needed for the experiment, and the same oil phase can be added back. This step can be repeated several times to achieve sufficient pairing of droplets.
[0059] In the method provided in this application, step 2) refers to fully encapsulating the crescent-shaped microspheres and the nucleic acid to be tested to obtain paired droplets. Encapsulation refers to mixing the first mixture by blowing air for 30-120 seconds, specifically 30-40 seconds, 40-50 seconds, or 50-60 seconds, etc.
[0060] In the construction method provided in this application, step 3) refers to replacing the first oil phase in the paired droplets with a second oil phase and performing polymerase chain reaction (PCR) amplification to obtain amplified droplets. The second oil phase is fluorinated oil; further, the fluorinated oil is FC 40 containing PEG-PFPE. In one specific embodiment, the second oil phase is FC 40 containing 5% PEG-PFPE. Replacing the oil phase here is to make the droplets more stable during the PCR reaction. The PCR amplification is performed in a PCR instrument well-known to those skilled in the art, and the specific procedure is adjusted according to the nucleic acid to be tested.
[0061] In the method provided in this application, step 4) refers to analyzing the amplified droplets to obtain the quantification of the nucleic acid to be tested. The analysis includes fluorescence microscopy imaging of the amplified droplets. The fluorescent chimeric dye added to the dextran solution marks positive droplets with targets and negative droplets without targets, facilitating the statistical analysis of positive droplets. Combined with the Poisson distribution, a more accurate digital quantification of the initial sample can be obtained. The digital polymerase chain reaction performed in this method is a microfluidic-free method, eliminating the constraints of traditional digital polymerase chain reactions on chips and devices, thus making it suitable for wider adoption.
[0062] The fifth aspect of this application provides the aforementioned crescent-shaped microspheres, the aforementioned preparation method, or the application of the aforementioned method in digital polymerase chain reaction (PCR). When the crescent-shaped microspheres are used in PCR, fluorescent dyes label positive droplets containing the nucleic acid to be tested and negative droplets without the nucleic acid to be tested. By statistically analyzing the proportion of positive droplets and combining this with a Poisson distribution, a more accurate digital quantification of the initial sample can be obtained.
[0063] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.
[0064] Example 1
[0065] Design and fabrication of microfluidic chips
[0066] 1. First, use AutoCAD software to visualize the microfluidic chip ( Figure 2The design (as shown) is completed, and then the company manufactures a mask containing the corresponding pattern. The chip template is fabricated primarily by spin-coating SU-8 photoresist (Microchem) onto a 3-inch silicon wafer. The choice of SU-8 photoresist and the spin-coating speed are determined by the desired depth of the silicon wafer template. This application designs and fabricates a Coflow microfluidic chip with a depth of approximately 50 μm.
[0067] 2. Pour a coin-sized amount of SU-8 3025 onto the silicon wafer already placed on the spin coater, and spin coat at a speed of 1500 rpm / min. After spin coating, place the silicon wafer containing the photoresist on a 90 ℃ heating plate and heat for 20 minutes.
[0068] 3. Exposure was then performed under a 120 mW UV lamp (M365L2, Thorlabs). The exposure time was determined by the thickness of the photoresist; 10 μm depth corresponded to 50 s, therefore the exposure time was set to 250 s. After exposure, the silicon wafer was placed on a 90 °C heating plate and heated for 5 minutes until a pattern appeared on the chip.
[0069] 4. Next, place the silicon wafer in the developer solution for cleaning. After 10 minutes, remove it and clean it sequentially with clean developer, isopropanol, and ethanol. Then, dry the silicon wafer with a nitrogen gun to obtain a patterned silicon wafer mold. Next, mix the PDMS monomer (SYLGARD 184, Dow Corning) with the curing agent at a ratio of 10:1 (w:w) and pour the mixture onto the silicon wafer mold. Then, incubate the mold overnight at 60°C to allow the PDMS to fully cure.
[0070] 5. Peel the cured PDMS board off the silicon wafer, and then use a 0.7 mm punch to make small holes at the designated locations on the chip.
[0071] 6. After drilling the holes, place the chip and a clean glass plate in a plasma cleaning machine for treatment. After treatment, bond the chip and glass plate together to form a microfluidic chip. Then, treat the chip at 100°C for 30 minutes to enhance the bonding effect.
[0072] 7. Before the chip is used, the microfluidic channels need to be treated with aquapel (PPG Industries), a commercial hydrophobic agent, to make the channel surface hydrophobic. After hydrophobic treatment, the microfluidic chip is placed on a 90°C heating plate and heated for 10 minutes. After that, the microfluidic chip can be used to generate droplets.
[0073] Example 2
[0074] Preparation and characterization of crescent-shaped microspheres
[0075] 1. Dissolve PEGDA (MW 8k, Merck, CAS: 26570-48-9), PEGDA (MW 575, Merck, CAS: 26570-48-9), and Dextran (MW 500k, Yeasen, CAS: 9004-54-0) in DPBS in 1.5 mL centrifuge tubes to final concentrations of 3% (w / v), 3% (w / v), and 5.5% (w / v), respectively. Mix thoroughly by pipetting and quantify the final solution volume to 1 mL.
[0076] 2. Then, centrifuge at 12000 g for 10 min to allow for complete phase separation of the two phases;
[0077] 3. Transfer the top PEGDA phase to a clean centrifuge tube and add 0.1% (w / v) of the initiator LAP (lithium phenyl-2,4,6trimethylbenzoylphosphinate); transfer the bottom Dextran phase to another clean centrifuge tube and add FITC-dextran to the Dextran phase. Finally, green fluorescently labeled crescent-shaped microspheres can be obtained.
[0078] 4. The two phases were injected into the two aqueous phase inlets of the Coflow microfluidic chip prepared in Example 1, respectively. The oil phase inlet was fed with HFE7500 fluorinated oil containing 2% (w / v) PEG-PFPE surfactant. The flow rates were set to 400 μl / h for the oil phase, 200 μl / h for PEGDA, and 100 μl / h for Dextran to prepare the aqueous two-phase droplets. The resulting crescent-shaped microspheres had a core size of 50 μm and a shell size of 70 μm.
[0079] 5. After the droplets are prepared, they are placed under 365 nm (20 W) UV irradiation for 3 min to achieve PEGDA gelation;
[0080] 6. After the droplets gelled, demulsifier (20% (v / v) PFO in HFE7500) was added for demulsification. PFO was purchased from Sigma-Aldrich, Cat#370533, and HFE7500 was purchased from Sigma-Aldrich, Cat#370533. The droplets were washed three times with DPBS to remove excess oil phase.
[0081] 7. Finally, resuspend the sample in DPBS, add 1 / 10 volume of Dextranase, mix well, and heat in a 60 °C metal bath for 10 min.
[0082] 8. Wash three times with DPBS to obtain crescent-shaped gel microspheres.
[0083] The results are as follows Figures 3-6 As shown.
[0084] Example 3
[0085] Droplet digital PCR based on hydrogel crescent microspheres (process as follows) Figure 1 (As shown)
[0086] 1. Add approximately 100 μL of the crescent-shaped gel microspheres prepared in Example 2 to 150 μL of PCR mix system (containing primers and probes, sequences of which are shown in Table 3), and incubate at room temperature or on ice for 10 min to ensure thorough mixing of the crescent-shaped microspheres and PCR mix;
[0087] Centrifuge at 2,100 g for 1 min and discard the supernatant;
[0088] 3. Add 1 μl of the nucleic acid to be tested (sequences are shown in Table 3) to a centrifuge tube containing crescent-shaped microspheres, and gently tap the centrifuge tube to mix the crescent-shaped microspheres and the nucleic acid to be tested thoroughly;
[0089] 4. Add 100 μL of the first oil phase of HFE-7500 containing 2% PEG-PFPE, and then use a 200 μL pipette to pipette for 1 min to fully emulsify and form droplets, thereby achieving the encapsulation of nucleic acid templates;
[0090] 5. Let stand for 30 seconds, then remove the bottom first oil phase; add a new first oil phase containing 2% PEG-PFPE HFE-7500, and invert the centrifuge tube until the droplets and oil phase are fully mixed.
[0091] 6. Repeat step 5 twice, then let stand for 30 seconds and aspirate the bottom oil phase. Transfer the liquid to a low-adsorption PCR tube and add 30 μL of the second oil phase containing 5% PEG-PFPE in FC40.
[0092] 6. Place the PCR tube into the PCR instrument for amplification. The PCR formula is shown in Table 1 and the PCR program is shown in Table 2.
[0093] 7. After PCR, the droplets were removed for fluorescence microscopy imaging, and the positive droplets and their proportions were statistically analyzed.
[0094] The results are as follows Figure 7 As shown.
[0095] Table 1 Digital PCR Recipes
[0096] Microfluidics-free ddpcr system Unit / μL Crescent Beads 100 5x Taqpro ddpcr buffer 40 10x primer probe (primer: 9 μM, probe: 2 μM) 20 Taqpro HS DNA polymerase 2 10% triton 4 10% p188 4 <![CDATA[ddH2O]]> 30 Total Volume 200
[0097] Table 2 Digital PCR Procedure
[0098]
[0099] Table 3 DNA sequences used
[0100]
[0101] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.
Claims
1. A method for digital polymerase chain reaction, comprising the following steps: 1) The nucleic acid to be tested, crescent-shaped microspheres, and the first oil phase are mixed to obtain the first mixture; 2) The crescent-shaped microspheres and the nucleic acid to be tested are fully encapsulated to obtain paired droplets; 3) Replace the first oil phase in the paired droplets with the second oil phase and perform polymerase chain reaction amplification to obtain amplified droplets; 4) Analyze the amplification droplets to obtain the quantification of the nucleic acid to be tested; The crescent-shaped microsphere is a concave sphere with a crescent-shaped cross-section; the diameter of the concave part of the crescent-shaped microsphere is 10 to 200 μm; and the diameter of the sphere itself is 10 to 200 μm.
2. The method as described in claim 1, characterized in that, The crescent-shaped microspheres are formed into a gel by photocuring of raw materials, the raw materials including one or more of polyethylene glycol diacrylate, dextran, tetra-arm polyethylene glycol acrylate, gelatin, and tetra-arm polyethylene glycol maleimide.
3. The method as described in claim 1, characterized in that, The crescent-shaped microspheres are prepared by partially encapsulating an inner gel with an outer gel, and then dissolving the inner gel. The method for dissolving the inner gel includes one or more of the following: adding dextran hydrolase, adjusting the pH of the solution, and raising the temperature; further, the pH of the solution is adjusted to 5-7; and the temperature is raised to 50-70 ℃.
4. The method as described in claim 1, characterized in that, The crescent-shaped microspheres are prepared by a method comprising the following steps: 1) Provide a first solution and a second solution, mix them, and centrifuge to obtain two separate phases. The bottom solution is the second aqueous phase, and the top solution is the first aqueous phase. 2) The first aqueous phase and the second aqueous phase are simultaneously sheared with the oil phase to obtain a two-phase droplet in which the first aqueous phase partially encapsulates the second aqueous phase; 3) Irradiate the aqueous two-phase droplets to obtain gel-forming droplets in which the outer gel partially encapsulates the inner gel. 4) The gel droplets were demulsified and the inner gel layer was dissolved to obtain crescent-shaped microspheres.
5. The method as described in claim 4, characterized in that, In step 1) of the method for preparing crescent-shaped microspheres, the solute in the first solution is selected from polyethylene glycol diacrylate, tetra-arm polyethylene glycol acrylate, gelatin, or tetra-arm polyethylene glycol maleimide. And / or, in step 1), the solute in the second solution is selected from dextran; And / or, in step 1), the second aqueous phase comprises a second solution; And / or, in step 1), the first aqueous phase includes a first solution and also includes a photoinitiator; And / or, in step 2), the surface tension between the oil phase and the first aqueous phase is less than the surface tension between the first aqueous phase and the second aqueous phase, which is less than the surface tension between the oil phase and the second aqueous phase. And / or, in step 3), the outer gel is obtained by irradiation of the first aqueous phase; And / or, in step 3), the inner layer gel is obtained by irradiation of the second aqueous phase.
6. The method as described in claim 5, characterized in that, In step 1) of the method for preparing crescent-shaped microspheres, the molecular weight of the polyethylene glycol diacrylate includes 575 or 8000; further, based on the total volume of the crescent-shaped microspheres, the concentration of the polyethylene glycol diacrylate solution with a molecular weight of 575 is 3~10% (w / v), and the concentration of the polyethylene glycol diacrylate solution with a molecular weight of 8000 is 3~10% (w / v). And / or, in step 1) of the method for preparing crescent-shaped microspheres, the molecular weight of the dextran is selected from 500,000; further, based on the total volume of the crescent-shaped microspheres, the solution concentration of the dextran is 5.5~10% (w / v).
7. The method as described in claim 1, characterized in that, It also includes one or more of the following features: b1) In step 1), the first mixture also includes a mixed reaction solution required for polymerase chain reaction, and / or a stabilizing solution; further, the raw material of the stabilizing solution is Tween and / or polyethylene glycol; b2) In step 1), the first oil phase is fluorinated oil; further, the fluorinated oil is HFE-7500 containing PEG-PFPE; b3) In step 2), wrapping refers to mixing the first mixture by blowing; b4) In step 3), the second oil phase is fluorinated oil; further, the fluorinated oil is FC 40 containing PEG-PFPE; b5) In step 4), the analysis includes fluorescence microscopy imaging of the amplified droplets and statistical analysis of the positive droplets.
8. The application of the method as described in any one of claims 1 to 7, or the crescent-shaped microspheres used in the method as described in any one of claims 1 to 7, in digital polymerase chain reaction.