Microfluidic system for rapid extraction of miRNA

Abstract

The present application belongs to the field of micro-mechanical and micro-fluidic technology, and relates to a micro-fluidic system for rapidly extracting miRNA, which comprises a cell thermoelectric lysis module and a gel electrophoresis elution module. The cell thermoelectric lysis module uses high-frequency alternating voltage to generate Joule heat to cause cell thermoelectric lysis, and the cell products after lysis are used for gel electrophoresis. The gel electrophoresis elution module elutes miRNA by using a direct-current constant current, and completes the rapid separation process of miRNA. In the micro-fluidic system, a hydrogel is used as a separation layer and is processed and manufactured through a micro-processing manufacturing process. The micro-fluidic system integrates cell thermoelectric lysis and gel electrophoresis on a millimeter-level chip, shortens the time frame between the two steps, thereby protecting RNA from enzymatic degradation, and can extract miRNA in cells under the condition of small volume and low concentration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of micro-mechanical and micro-fluidic technology, and relates to a micro-fluidic system for rapid extraction of miRNA. BACKGROUND

[0002] For early cancer, surgical resection is the preferred treatment method. Therefore, early diagnosis of cancer is of great significance for subsequent treatment.

[0003] In recent years, there is an increasing trend in miniaturization and automation of diagnostic systems. The success of molecular methods such as real-time PCR and real-time NASBA greatly promotes this, and the sensitivity and low volume required for the reaction of these technologies make them the object of miniaturization methods, and attempts are made to create fully automated diagnostic cartridges. Current attempts to manufacture miniature diagnostic cartridges use a variety of cell lysis techniques, including electrical, mechanical, chemical and thermal techniques. At the same time, the purification method of nucleic acids is largely limited to solid-phase adsorption technology.

[0004] As an important regulatory molecule of life processes, miRNA has a very close relationship with the occurrence and development of tumors. miRNA changes gene expression by hybridizing with specific mRNA sites and is related to a variety of cell functions. The disorder of miRNA has been proven to play a key role in the pathogenesis of various cancers and chemotherapy resistance. Therefore, the measurement of the relative amount of miRNA extracted from cells can be used as a biomarker for tumor diagnosis and classification.

[0005] Traditional extraction of miRNA is usually derived from a three-step process. First, the cells are lysed by mechanical and / or chemical action. Then, the released miRNA is purified from cell debris and other co-released nucleic acids by adsorption to a silica surface, the most common form being a spin column or silica-coated magnetic beads. After release from the surface, specific miRNAs are detected by sequencing or amplification techniques such as dry-loop RT-qPCR. However, traditional miRNA extraction methods have low automation, are easy to contaminate samples and sometimes use toxic chemicals. Therefore, integrating miRNA extraction onto a microfluidic chip for automated detection can effectively reduce manual operation steps and improve automation. SUMMARY

[0006] The technical problem solved by the present application is that the traditional miRNA extraction and analysis method in the prior art has low efficiency, multiple purification steps, multiple manual operations, long time, easy sample pollution, and sometimes toxic solvents are used, and the extraction efficiency is low. At the same time, most macro purification systems require sample volumes in the milliliter range and a large number of manual handling and pipetting steps, and a large number of fluid operations are required for processing, resulting in complex driving protocols, hindering the miniaturization of the instrument. The present application provides a microfluidic system for rapid extraction of miRNA, which integrates cell thermoelectric lysis and gel electrophoresis on a millimeter-scale microfluidic chip.

[0007] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0008] The present application discloses a microfluidic system for rapid extraction of miRNA, wherein the microfluidic system comprises a first electrode region, a first hydrogel region, a lysis chamber region, a separation hydrogel region, an elution chamber region, a second hydrogel region and a second electrode region from one side to the other side of the microfluidic chip.

[0009] Specifically, the two ends of the first electrode fluid channel, the first hydrogel region, the separation hydrogel region, the second hydrogel region and the second electrode fluid channel are buffer cavities; one end of the lysis chamber region and the elution chamber region is provided with a buffer cavity; one end of the first electrode fluid channel is provided with a connecting hole for connecting the first electrode; one end of the second electrode fluid channel is provided with a connecting hole for connecting the second electrode; the buffer cavities are all provided with injection holes for injecting any one or a combination of several of the hydrogel, buffer and cell sample.

[0010] Specifically, the material of the first electrode is pure copper, graphite, brass or silver; the material of the second electrode is pure copper, graphite, brass or silver.

[0011] The specifications of the first electrode and the second electrode can be designed according to the specific size of the microfluidic chip, and are not fixed.

[0012] Specifically, the first hydrogel region, the separation hydrogel region and the second hydrogel region are injected with hydrogel; the lysis chamber region, the elution chamber region, the first electrode fluid channel and the second electrode fluid channel are injected with Tris-boric acid electrophoresis buffer.

[0013] Further, the present application provides the microfluidic system in the above for use in rapid extraction of miRNA in cells.

[0014] The microfluidic system for use in rapid extraction of miRNA in cells comprises the following steps:

[0015] (1) injecting a cell sample into the lysis chamber region, applying a high-frequency alternating voltage to the first electrode and the second electrode at both ends of the microfluidic system to lyse the cells, and generating cell lysate containing miRNA in the lysis chamber region;

[0016] (2) applying a direct current to the first electrode and the second electrode at both ends of the microfluidic system to make the miRNA in the cell lysate containing miRNA in the lysis chamber region migrate to the elution chamber region through the separation hydrogel region, i.e. to achieve rapid extraction of miRNA.

[0017] Specifically, in step (1), the high-frequency alternating voltage is 240-280 Vrms.

[0018] Specifically, in step (1), when the high-frequency alternating voltage is applied to the first electrode and the second electrode at both ends of the microfluidic system, the first electrode is an anode and the second electrode is a cathode.

[0019] In step (1), when the high-frequency alternating voltage is applied, the Joule heating generated by the alternating voltage in the liquid causes cell lysis.

[0020] Specifically, in step (2), the direct current is 120-200 mA, preferably 160 mA.

[0021] Specifically, in step (2), when the direct current is applied to the first electrode and the second electrode at both ends of the microfluidic system, the second electrode is an anode and the first electrode is a cathode.

[0022] Advantages:

[0023] The microfluidic system for rapid extraction of miRNA provided by the present application integrates thermal-electric lysis of cells and gel electrophoresis for miRNA extraction on a millimeter-scale chip, lyses cells by thermal-electric lysis, and purifies RNA using a gel electrophoresis purification step. Combining the two steps in an integrated microfluidic chip shortens the time frame between the two steps, thereby protecting RNA from enzymatic degradation. At the same time, miRNA in cells can be extracted in a small volume and at a low concentration, and by integrating electrophoresis actuation electrodes in a microfluidic chamber, the precise sample volume can be defined. BRIEF DESCRIPTION OF DRAWINGS

[0024] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings in which:

[0025] Figure 1 The structural schematic diagram of the microfluidic system of the present application is shown in the figure.

[0026] In the structural schematic diagram, the names of the parts are as follows: first electrode 1, first electrode fluid channel 2, first hydrogel region 3, lysis chamber region 4, separation hydrogel region 5, elution chamber region 6, second hydrogel region 7, second electrode fluid channel 8, second electrode 9, and buffer cavity 10.

[0027] Figure 2 The schematic diagram of cell lysis and miRNA gel electrophoresis of the present application is shown in the figure. DETAILED DESCRIPTION

[0028] The present application will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0029] The Tris-boric acid electrophoresis buffer (TBE) used in the embodiments of the present application has a pH of 8.2-8.4.

[0030] 10×TBE stock solution preparation method: dissolve Tris (tris-hydroxymethyl aminomethane) 108 g, boric acid 55 g, 40 mL 0.5M EDTA (ethylenediaminetetraacetic acid) in 600 mL of deionized water; then adjust the pH to 8.3, add deionized water to 1 L, and store at room temperature. When used, dilute 10 times, i.e. 1×TBE.

[0031] The Ordyl SY330 dry film resist used in the present application is produced by Elga Europe, Italy.

[0032] Example 1:

[0033] In the present application, the microfluidic chip is manufactured by plasma-enhanced chemical vapor deposition to deposit silicon nitride and patterned by reactive ion etching, and the microfluidic structure is patterned in the dry film resist.

[0034] In the above microfluidic chip, the specific manufacturing process can refer to Vulto, P., et al. Microfluidic channel fabrication in dry film resist for production and prototyping of hybrid chips. Lab Chip 5, 158-162 (2005), and the following process can be referred to:

[0035] (i) 1 pm low-stress silicon nitride was deposited on a Pyrex wafer (2 cm x 2 cm) using plasma-enhanced chemical vapour deposition (PECVD) followed by lift-off patterning; a second layer of 800 nm thick silicon nitride was then deposited and patterned by reactive ion etching (RIE). The microfluidic channels in the microfluidic system were then patterned in Ordyl SY330 dry film resist (Elga Europe, Italy), the first layer of Ordyl was laminated and exposed to 240 mJ-cm -2 to create the phase guides, three more layers of Ordyl were laminated and exposed with the same energy, followed by a post-exposure bake (PEB) at 85 °C for 1 min. The resist was then developed in a three-bath BMR developer (Elga Europe), increasing the cleanliness, followed by rinsing and drying. Finally, holes were drilled on the top wafer using a CNC platform and diamond drill bits.

[0036] (ii) The drilled and cleaned wafer was bonded to the Pyrex top substrate in a Süss Microtec SB6 wafer bonder at a pressure of 60 N-cm -2 and a temperature of 95 °C for 30 min, after wafer bonding, the cross-linking was finalized by a 1 min UV flood exposure, followed by a post-bake at 150 °C for 2 h.

[0037] The microfluidic chip was fabricated using multiple layers of dry film resist laminated together, thus creating a three-dimensional structure, forming phase guides without being completely enclosed, allowing cells to migrate across the phase guides created by the dry film resist, while the dry film resist has good biocompatibility and does not affect the activity of the cells.

[0038] Figure 1 The light grey areas on both sides of the first hydrogel region 3, the separation hydrogel region 5 and the second hydrogel region 7 are dry film resist, the dry film resist forms phase guides without being completely enclosed, so the various regions of the microfluidic system are interconnected. The microfluidic system is filled with nitrogen to form a nitrogen atmosphere, and then water is injected from the injection holes on the buffer cavities at one end of the first hydrogel region 3, the separation hydrogel region 5 and the second hydrogel region 7 to gel and dry. Subsequently, Tris-borate electrophoresis buffer is injected from the injection holes on the buffer cavities at one end of the lysis chamber region 4, the elution chamber region 6, the first electrode fluid channel 2 and the second electrode fluid channel 8, due to the gelation of the hydrogel, the Tris-borate electrophoresis buffer in the lysis chamber region 4, the elution chamber region 6, the first electrode fluid channel 2 and the second electrode fluid channel 8 do not mix with each other.

[0039] Example 2

[0040] AsFigure 1 As shown in the figure, a microfluidic system for rapid extraction of miRNA includes electrodes, a cell thermal-electric lysis module, and a gel electrophoresis elution module. The electrodes, the cell thermal-electric lysis module, and the gel electrophoresis elution module are integrated on a microfluidic chip, and the electrodes are on both sides of the microfluidic chip.

[0041] Figure 1 As shown in the figure, the microfluidic system in the figure sequentially includes, from one side of the microfluidic chip to the other side, a first electrode region, a first hydrogel region 3, a lysis chamber region 4, a separation hydrogel region 5, an elution chamber region 6, a second hydrogel region 7, and a second electrode region; the first electrode region includes a first electrode 1 and a first electrode fluid channel 2; the second electrode region includes a second electrode 9 and a second electrode fluid channel 8; the first electrode 1, the first electrode fluid channel 2, the first hydrogel region 3, the lysis chamber region 4, the separation hydrogel region 5, the elution chamber region 6, the second hydrogel region 7, the second electrode fluid channel 8, and the second electrode 9 are sequentially and adjacently connected.

[0042] In the figure, both ends of the first electrode fluid channel 2, the first hydrogel region 3, the separation hydrogel region 5, the second hydrogel region 7, and the second electrode fluid channel 8 are buffer cavities 10; one end of the lysis chamber region 4 and the elution chamber region 6 is provided with a buffer cavity 10; one end of the first electrode fluid channel 2 is provided with a connecting hole for connecting the first electrode 1; one end of the second electrode fluid channel 8 is provided with a connecting hole for connecting the second electrode 9; each of the buffer cavities 10 is provided with an injection hole for injecting hydrogel, buffer, or a cell sample; the buffer cavities 10 are convex circles; the first electrode 1 and the second electrode 9 are electrically connected to an external circuit; the first electrode 1 and the second electrode 9 are both made of silver.

[0043] Figure 1 In the figure, the dark gray solid region is silicon nitride, and the light gray region on both sides of the first hydrogel region 3, the separation hydrogel region 5, and the second hydrogel region 7 is Ordyl SY330 dry film resist, which can generate phase guidance to prevent the hydrogel from deviating from the region when being injected.

[0044] Example 3:

[0045] As shown in the figure, the figure shows a process diagram for rapid extraction of miRNA from a cell sample. Figure 2 Figure 2 ​Figure a) shows cells in a free state within the lysis chamber before a high-frequency AC voltage is applied. First, the microfluidic system is filled with nitrogen to create a nitrogen atmosphere before the cell sample is added. Then, hydrogel is injected through injection holes on the buffer chambers at one end of the first hydrogel region 3, the separation hydrogel region 5, and the second hydrogel region 7 for gelation and drying. Subsequently, 1×TEB buffer is injected through injection holes on the buffer chambers at one end of the lysis chamber region 4, the elution chamber region 6, the first electrode fluid channel 2, and the second electrode fluid channel 8. Due to the hydrogel gelation, the TEB buffer in these regions does not mix. Finally, the cell sample is injected through the injection hole on the buffer chamber at one end of the lysis chamber region 4.

[0046] like Figure 2 As shown, Figure 2 (b) illustrates cell lysis when a high-frequency alternating voltage is applied to the chip electrodes; Figure 2 (c) illustrates the gel electrophoresis phenomenon of miRNAs when a constant DC current is applied to the chip. An AC voltage of 240–280 Vrms is first applied to the electrodes at both ends of the chip (at this time, the first electrode 1 is the anode and the second electrode 9 is the cathode). Joule heating in the lysis chamber region 4 causes cell lysis, forming cells as shown in (c). Figure 2 As shown in b), cell lysis products containing miRNA are generated in the lysis chamber.

[0047] After thermoelectric lysis of cells, a constant DC current of 160mA is applied to the electrodes at both ends of the chip (at this time, the second electrode 9 is the anode and the first electrode 1 is the cathode). This allows the miRNA in the cell lysis products containing miRNA in lysis chamber region 4 to migrate through separation hydrogel region 5 to elution chamber region 6, completing the rapid separation and extraction process of miRNA. Figure 2 As shown in c).

[0048] The principle of gel electrophoresis: When molecules are placed in an electric field, they migrate towards the appropriate electrode at a certain speed. This migration speed of electrophoretic molecules under the influence of an electric field is called the electrophoretic mobility. It is directly proportional to the strength of the electric field and the net charge carried by the electrophoretic molecules themselves. That is, the stronger the electric field and the more net charge the electrophoretic molecules carry, the faster their migration speed, and vice versa. Because a non-reactive and stable supporting medium, such as a hydrogel, is used in electrophoresis, convective motion is reduced, so the electrophoretic mobility is inversely proportional to the coefficient of friction of the molecules. It is known that the coefficient of friction is a function of molecular size, polarity, and the viscosity of the medium. Therefore, based on differences in molecular size, composition, or shape, as well as the amount of net charge they carry, various components in a mixture of proteins or nucleic acids can be separated by electrophoresis.

[0049] The application provides a microfluidic system for rapidly extracting miRNA, and the method and approach for specifically implementing the technical solution are various. The above description is only the preferred embodiment of the application, and it should be pointed out that, for ordinary skilled in the art, some improvements and refinements can be made without departing from the principle of the application, and these improvements and refinements should also be regarded as the protection scope of the application. The components not explicitly described in the embodiment can be implemented by using the prior art.

Claims

1. A microfluidic system for rapid extraction of miRNA, characterized in that, The microfluidic system sequentially comprises a first electrode region, a first hydrogel region (3), a lysis chamber region (4), a separation hydrogel region (5), an elution chamber region (6), a second hydrogel region (7) and a second electrode region from one side to the other side of the microfluidic chip. The first electrode region comprises a first electrode (1) and a first electrode fluid channel (2); the second electrode region comprises a second electrode (9) and a second electrode fluid channel (8); the first electrode (1), the first electrode fluid channel (2), the first hydrogel region (3), the lysis chamber region (4), the separation hydrogel region (5), the elution chamber region (6), the second hydrogel region (7), the second electrode fluid channel (8) and the second electrode (9) are sequentially adjacent to each other; The first electrode fluid channel (2), the first hydrogel region (3), the separation hydrogel region (5), the second hydrogel region (7) and the second electrode fluid channel (8) have buffer cavities (10) at both ends; the lysis chamber region (4) and the elution chamber region (6) have buffer cavities (10) at one end; the first electrode fluid channel (2) has a connecting hole at one end for connecting the first electrode (1); the second electrode fluid channel (8) has a connecting hole at one end for connecting the second electrode (9); the buffer cavities (10) all have injection holes; 2. The microfluidic system of claim 1, wherein, The first hydrogel region (3), the separation hydrogel region (5) and the second hydrogel region (7) are injected with hydrogel; the lysis chamber region (4), the elution chamber region (6), the first electrode fluid channel (2) and the second electrode fluid channel (8) are injected with Tris-boric acid electrophoresis buffer. The first electrode (1) is made of pure copper, graphite, brass or silver; the second electrode (9) is made of pure copper, graphite, brass or silver.

4. Use according to claim 3, characterized in that, 3. The microfluidic system of any one of claims 1-2 for use in rapid extraction of miRNA in cells. The method comprises the following steps: (1) injecting a cell sample into the lysis chamber region (4), applying high-frequency alternating voltage to the first electrode (1) and the second electrode (9) at both ends of the microfluidic system to lyse the cells, and generating cell lysate containing miRNA in the lysis chamber region (4); 5. Use according to claim 4, characterized in that, (2) applying a direct current to the first electrode (1) and the second electrode (9) at both ends of the microfluidic system to make the miRNA in the cell lysate containing miRNA in the lysis chamber region (4) migrate through the separation hydrogel region (5) to the elution chamber region (6), i.e. to achieve rapid extraction of miRNA.

6. Use according to claim 4, characterized in that, In step (1), the high-frequency alternating voltage is 240-280 Vrms.

7. Use according to claim 4, characterized in that, In step (1), when high-frequency alternating voltage is applied to the first electrode (1) and the second electrode (9) at both ends of the microfluidic system, the first electrode (1) is the anode and the second electrode (9) is the cathode. In step (2), the direct current is 120-200 mA.

8. Use according to claim 4, characterized in that, In step (2), when a direct constant current is applied to the first electrode (1) and the second electrode (9) at two ends of the micro-fluidic system, the second electrode (9) is an anode, and the first electrode (1) is a cathode.

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