CN112697860A - Tumor cell exosome and nucleic acid detection chip thereof, and manufacturing and detection methods thereof - Google Patents
Tumor cell exosome and nucleic acid detection chip thereof, and manufacturing and detection methods thereof Download PDFInfo
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Abstract
Providing a tumor cell exosome and a nucleic acid detection chip thereof, wherein the tumor cell exosome comprises a glass substrate (1) and a PDMS sheet (2) which are mutually attached, and the PDMS sheet (2) is provided with a first through hole (3), a second through hole (4), a third through hole (5), a pit (6), an initial flow groove (7), a first flow groove (8) and a second flow groove (9) which are sequentially connected; the pit (6) forms a TEX capture detection area, and the third through hole (5) forms a microRNA capture detection area; three electrode systems for capturing and detecting TEX and microRNA are respectively sputtered in corresponding areas of the glass substrate (1); a first valve and a second valve which can close the flow passage section are arranged on the first flow passage section and the second flow passage section; integrating tumor cell exosomes and nucleic acid detection functions thereof; the invention also provides a manufacturing method and a detection method of the chip.
Description
The references of the related applications incorporate:
the patent application ZL 202010639672.2, entitled "Single cell temperature detection sensor and methods for making and detecting the same", filed by the applicant 2020-07-06, is incorporated by reference as if fully set forth herein.
The technical field is as follows:
the invention relates to the technical field of cell detection, in particular to a tumor cell exosome and a nucleic acid detection chip thereof, and a manufacturing and detection method thereof.
Background art:
early detection is critical in the control of cancer diseases. The cancer treatment capacity of China is not low, the equipment and diagnosis and treatment level of some large hospitals are close to those of developed countries, but the capacity of cancer prevention and control is very low.
The liquid biopsy becomes an ideal choice for tumor diagnosis and prognosis monitoring, and cancer markers in blood, namely exosomes, are micro-vesicles with a bilayer structure released in the cell growth process, and the diameters of the micro-vesicles are generally between 30 and 150 nm. Compared with normal cells, tumor cells produce more exosomes due to stimulation by the hypoxic environment. Exosomes are used as mediators of intercellular transport of substances and are involved in regulating a variety of physiopathological responses. The tumor exosomes are involved in the processes of tumor generation, angiogenesis and the like. In addition, Tumor-derived exosomes (TEX) may also release associated signaling molecules, preventing Tumor recognition and anti-Tumor processes of the immune system, etc. Exosomes also carry tumor cell genetic signature molecules and are present in larger numbers than circulating tumor cells. Therefore, clinically, TEX can be used as a stable and sensitive tumor marker, and the high-sensitivity detection of the TEX can be used for early diagnosis of cancer, real-time monitoring of curative effect and prognosis tracking.
Nucleic acid is the genetic material of organisms, including ribonucleic acid (RNA), and microRNA is called micro ribonucleic acid, and the microRNA is involved in the physiological activities of human bodies and plays an important role in the physiological activities. MicroRNA, also called miRNA, is a short sequence non-coding RNA, and is closely related to the formation and development of various cancers. miRNA can regulate the expression of tumor related genes at the level of posttranscriptional activity, and can play the role of inhibiting or promoting cancer. mirnas can be stably present in the blood in the form of exosome contents, protected from degradation by enzymes by exosome bilayer membrane structures. Compared with free miRNA in blood, the exosome can provide enriched and stable miRNA, and the reflected gene information is more accurate and reliable. Tumor cell-derived exosome miRNAs participate in various regulation by recognizing target mrnas, directing, silencing, compomer degrading mRNA, or preventing mRNA translation. Research shows that compared with the total amount of microRNA in serum, the method for quantitatively detecting the microRNA in the TEX can effectively improve the accuracy of tumor detection, and the microRNA of a tumor exosome is a valuable marker.
In order to accurately detect the TEX-miRNA, a high-purity exosome sample is obtained. Conventional exosome separation methods currently exist, such as an ultracentrifugation method, an ultrafiltration method, a precipitation method, an immunomagnetic bead method, and the like. The ultracentrifugation method has long treatment time, expensive instruments and equipment and high technical requirements, and the yield and purity of the exosome are not high, such as: patent document CN 211620485U. Precipitation methods allow for the rapid and simple isolation of exosomes, but this is also costly, highly fluctuating, and moderately high in yield and purity, as described in the patent literature: CN 111117949A. The immunomagnetic bead method utilizes the interaction between antigens (CD63, CD9 and the like) excessively expressed on the surface of an exosome membrane and a magnetic bead labeled antibody, and has high exosome purity and low yield, such as: patent document CN 111575228A. In summary, these conventional methods can obtain an enriched exosome sample to a certain extent, but have the problems of low exosome purity, low yield, high time and cost, and the like.
The currently disclosed tumor exosome microRNA separation technology and detection technology mainly have the following problems: 1) the detection steps are complicated and the cost is high. 2) The specificity detection of the tumor cell source exosome miRNA can not be carried out. 3) Low sensitivity and reliability.
The invention content is as follows:
the technical problem to be solved by the invention is to provide a tumor cell exosome and a nucleic acid detection chip thereof, which integrate the tumor cell exosome and the nucleic acid detection function thereof into a whole and solve the problems of complex structure, complex steps and the like of the tumor cell exosome and the nucleic acid detection device thereof in the prior art. The invention also provides a preparation method of the tumor cell exosome and the nucleic acid detection method thereof, and the tumor cell exosome and the nucleic acid detection method thereof realized by using the tumor cell exosome and the nucleic acid detection thereof.
In order to solve the technical problems, the tumor cell exosome and the nucleic acid detection chip thereof adopt the technical scheme that:
a tumor cell exosome and a nucleic acid detection chip thereof comprise a glass substrate and a PDMS sheet which are mutually attached, and are characterized in that the PDMS sheet is provided with a first through hole, a second through hole and a third through hole which penetrate through the PDMS sheet at intervals, a concave pit is arranged on the attachment surface of the PDMS sheet between the first through hole and the second through hole, an initial flow groove is arranged on the attachment surface of the PDMS sheet between the first through hole and the concave pit, a first flow groove is arranged between the concave pit and the second through hole, and a second flow groove is arranged between the second through hole and the third through hole and sequentially connected with the concave pit and the concave pit; after the PDMS sheet is attached to the glass substrate, a sample inlet is formed in the first through hole on the glass substrate, a TEX capture detection area is formed in the pit, an initial flow channel section, a first flow channel section and a second flow channel section through which a sample solution flows during detection are sequentially formed in the initial flow channel, the first flow channel and the second flow channel, a sample outlet is formed in the second through hole, and a microRNA capture detection area is formed in the third through hole; a TEX three-electrode system used for capturing and detecting TEX is sputtered in a region corresponding to the attachment of the TEX capture detection region on the attachment surface of the glass substrate, a microRNA three-electrode system used for capturing and detecting microRNA is sputtered in a region corresponding to the attachment of the microRNA capture detection region, the TEX three-electrode system comprises a TEX working electrode, a TEX reference electrode and a TEX counter electrode, and the microRNA three-electrode system comprises a microRNA working electrode, a microRNA reference electrode and a microRNA counter electrode; the first flow passage section is provided with a first valve which can close the flow passage section, and the second flow passage section is provided with a second valve which can close the flow passage section.
The following is a further scheme of the tumor cell exosome and the nucleic acid detection chip thereof of the invention:
a triangular column array is arranged on the initial flow passage section, the pit outlet shrinks in a V shape, and a cross column array is arranged on the TEX capture detection area.
The TEX counter electrode and the microRNA counter electrode are 1 shared counter electrode, and the shared counter electrode is positioned below the sample outlet.
The TEX working electrode comprises a circuitous section and a long straight strip which are repeatedly circuitously arranged in the TEX capture detection area, extend outwards to a junction block of the circuitous section, and the TEX reference electrode comprises a rectangular main block, a long straight strip which extends outwards to a junction block of the rectangular main block, and a junction block of the rectangular main block; the shared counter electrode comprises a circular main block, a long straight strip extending outwards to a wiring block of the main block, and a wiring block of the long straight strip; the microRNA working electrode comprises a circular main block, a long straight strip extending outwards to a junction block of the circular main block and the junction block of the circular main block, and the microRNA reference electrode extends outwards to the long straight strip of the junction block of the circular main block and the junction block of the circular main block.
The first valve and the second valve are arranged in a way that: the PDMS sheet is a film layer which is deformed to block the first flow channel section or the second flow channel section when meeting pressure at the positions of the first flow channel and the second flow channel, and closed cavities which can be filled with pressure gas are arranged on the first flow channel and the second flow channel.
The PDMS sheet is a sheet with the thickness of 140-160 μm, a PDMS reinforcing sheet is covered on the PDMS sheet in a fitting manner, and a first extending through hole and a second extending through hole are formed on the PDMS reinforcing sheet and correspond to the first through hole and the second through hole to respectively form a sample inlet and a sample outlet; the first valve and the second valve are arranged in a way that: the upper surface of the PDMS reinforcing sheet is respectively provided with a first vent hole and a second vent hole, the joint bottom surface of the PDMS reinforcing sheet is respectively provided with a first vent groove communicated with the first vent hole and a second vent groove communicated with the second vent hole at the positions of the first runner and the second runner as closed cavities, and the first vent groove and the second vent groove are separated from the first runner and the second runner of the PDMS sheet, so that the thicknesses of the bottoms of the first runner and the second runner form three-dimensional intersection with the first runner and the second runner.
And a third extending through hole is formed on the PDMS reinforcing sheet corresponding to the third through hole and is used as a liquid adding opening of the microRNA capture detection area.
The closed cavity is formed by the following steps: the PDMS groove blocks are arranged on the first flow groove and the second flow groove respectively, the vent groove with one end blocking the opening at the other end is formed below the PDMS groove blocks, the opening end is used for introducing pressure gas, the PDMS groove blocks are attached to the positions of the first flow groove and the second flow groove of the PDMS sheet, and the vent groove forms a closed cavity capable of introducing the pressure gas on the PDMS sheet.
In order to solve the technical problems, the method for manufacturing the tumor cell exosome and the nucleic acid detection chip thereof adopts the technical scheme that:
a tumor cell exosome and a method for manufacturing a nucleic acid detection chip thereof comprise the steps of manufacturing a PDMS sheet and a glass substrate which can be respectively carried out in sequence, and bonding the PDMS sheet and the glass substrate, wherein the manufacturing of the PDMS sheet comprises the manufacturing of a silicon substrate as a mould and the manufacturing of the PDMS sheet, and the method is characterized in that the manufacturing of the silicon substrate specifically comprises the following steps: adopting four-inch monocrystalline silicon as a substrate, carrying out thermal oxidation, and growing a silicon dioxide oxide layer with the thickness of 2 microns; spin-coating photoresist with the thickness of 2.5 μm according to the designed layout, performing primary photoetching, and removing the silicon dioxide layer by wet etching process; cleaning the silicon substrate by using sulfuric acid and hydrogen peroxide to remove the photoresist; spin-coating the photoresist again to a thickness of 1.4 μm, performing secondary photoetching, and forming silicon substrate molds with microstructures of different heights by using the rest microstructures;
the preparation process of the PDMS sheet is as follows: (1) preparation of PDMS mixture: weighing PDMS prepolymer and curing agent (weight ratio is 15: 1), mixing the PDMS prepolymer and the curing agent uniformly, and standing the mixture in a vacuum drier for 30min to remove most bubbles; (2) and reversing the mold: after the modulation of the PDMS mixture is finished, placing the inverted template on a horizontal table, pouring the modulated PDMS mixture, and standing for 30min to enable the inverted template structure to be filled with PDMS; (3) and curing: after standing, placing the mixture in an oven at 80 ℃ for heating for 1h, and after curing, stripping the mixture from the inverted template; punching at the position needing punching by adopting a puncher; (4) and storing the PDMS layer: adhering the upper surface and the lower surface of the PDMS layer by using a PCR (polymerase chain reaction) adhesive film, and preserving after packaging; (5) and hydrophilic treatment: placing the packaged chip in a plasma cleaning machine under a vacuum state for 2h, and irradiating for glow for 2 min; taking out the chip, dropwise adding a PEG (6-9) -siloxane and acetone mixed solution (v: v ═ 1:1) at a sample inlet, filling the whole chip pipeline with the mixed solution by utilizing the negative pressure action in the PDMS chip, and incubating for 1h at room temperature; and after the treatment is finished, the whole chip is flushed by ultrapure water, so that the surface of the micro-channel layer of the PDMS sheet is hydrophilic, and the adsorption of substances such as protein, nucleic acid and the like is reduced.
The manufacturing process of the glass substrate is as follows: and manufacturing each electrode metal layer on the glass substrate through photoetching and sputtering processes, and attaching the patterned PDMS sheet according to the alignment mark.
The bonding process of the PDMS sheet and the glass substrate comprises the steps of putting the PDMS sheet and the glass substrate with the binding surfaces facing upwards into a plasma cleaning machine for cleaning for 90s, quickly binding the PDMS sheet and the glass substrate together after taking out, and heating on a hot plate at 90 ℃ for 30min to enhance the binding degree of the PDMS sheet and the glass substrate.
After the bonding, adding a prepared nano gold particle reagent into the TEX capture detection area and the microRNA capture detection area, standing for 5 hours at 45 ℃, and after the liquid is evaporated, forming a layer of gold nano film with a specific superlattice structure by the electrode; then adding reagents required by modified antibodies and modified nucleic acid probes into the TEX capture detection area and the microRNA capture detection area respectively for fixing for 120 min; after aspirating the solution, washing away unbound capture probes with PBS solution; after finishing the modification, the excess PDMS layer is removed.
In order to solve the technical problems, the technical scheme adopted by the detection method of the tumor cell exosome and the nucleic acid thereof is as follows:
a tumor cell exosome and a nucleic acid detection method thereof are characterized in that the tumor cell exosome and the nucleic acid detection chip thereof according to any one of claims 1 to 7 are used, a specific test process is simulated, the tumor cell exosome and the nucleic acid detection chip thereof are tested and calibrated, a series of tumor cell exosome sample solutions with different concentrations, of which the exosome concentration value and the microRNA concentration value are known, are added, oxidation current values of a series of TEX working electrodes and oxidation current values of the microRNA working electrodes with different values are measured, a series of concentration gradient experiments are performed, a series of exosome concentrations and oxidation current values of the corresponding TEX working electrodes and oxidation current values of the corresponding microRNA working electrodes of the series of microRNA concentrations are obtained, a corresponding linear relation is established, and a linear relation between the oxidation current values of the TEX working electrodes and the exosome concentration values is obtained, and a linear relation between the oxidation current value of the microRNA working electrode and the oxidation current value of the microRNA working electrode.
Before detection, a PBS solution is used for washing the inner cavity of the whole chip; the specific test process is as follows:
firstly, carrying out exosome detection, and specifically comprising the following steps:
(1) and closing the second valve to enable the front half part of the chip to form an independent exosome detection area.
(2) And adding a sample solution to be detected into the sample inlet.
(3) And capturing exosomes in the sample solution by the biomolecules modified on the electrode through a biological specific reaction, so that all the exosomes are adsorbed on the surface of the TEX working electrode.
(4) Subsequently, a PBS rinse was added to rinse the chip channels.
(5) After washing, enzyme-labeled antibodies are added, and the antibodies are specifically combined with the exosomes to form a typical antibody-antigen-antibody sandwich structure.
(6) Adding an electrochemical indicator solution, measuring a current value by adopting a differential pulse voltammetry method, and converting a linear relation between the measured current value obtained by test calibration and an exosome concentration value to obtain an exosome concentration value;
then, carrying out MicroRNA detection, and specifically comprising the following steps:
(7) and applying low-voltage circulating square wave current on the exosome capturing electrode to crack the exosome so as to obtain the microRNA in the content of the exosome.
(8) And opening the second valve, and simultaneously applying pressure to the first through hole and the second through hole so that the lysate flows into the microRNA capture detection area.
(9) And after the lysate containing the microRNA flows into the microRNA capturing detection area, performing a biological specificity reaction on the aptamer modified on the microRNA working electrode in the area and the microRNA, and capturing the microRNA in the lysate to enable the microRNA to be adsorbed on the surface of the microRNA working electrode.
(10) After adsorption is finished, closing the first valve to enable the rear half part of the chip to form an independent microRNA capture detection area; adding a toluidine blue indicator into a microRNA capture detection zone, combining the indicator with microRNA, measuring a current value by adopting a differential pulse voltammetry, and converting a linear relation between the measured current value obtained by test calibration and the microRNA concentration value to obtain the microRNA concentration value.
The invention relates to a biological sensing detection method for integrating tumor cell exosome specificity capture, cracking and microRNA on-chip detection, which is constructed on the basis of a microelectrode and a fluid control microstructure. Its advantages are the following: (1) the microelectrode is combined with the fluid control microstructure, and the microelectrode is modified by utilizing the modification specificity capture probe, so that the high-efficiency and high-specificity enrichment and high-sensitivity detection of TEX-microRNA can be realized, and the detection efficiency and sensitivity are improved. (2) And detecting the microRNA through modification work of the electrode. The organic combination of biosensing and electrochemistry can excellently complete the detection task. (3) Two sets of three-electrode systems are simplified to form a five-electrode system, and the size and the cost are reduced. (4) The chip-level three-hole single-cavity structure can respectively represent exosome capture rate and microRNA content through the control of the double valves, so that the effect of one-chip multiple purposes is achieved, the cost is reduced, and the practicability and the commerciality are improved. In addition, the invention has the characteristics of batch manufacturing, low cost, high detection sensitivity and the like, and has important practical application value.
Drawings
FIG. 1 is a perspective view of the assembly of the tumor cell exosome and its nucleic acid detecting chip.
FIG. 2 is a schematic perspective view showing the separation state of the tumor cell exosome and the nucleic acid detecting chip PDMS sheet and the glass substrate.
Fig. 3 is a perspective view of a glass substrate.
Fig. 4 is a schematic perspective view of an exposed surface of a PDMS slab.
Fig. 5 is a schematic perspective view of a bonding surface of a PDMS sheet.
FIG. 6 is a schematic view of a triangular prism array and a cross-shaped prism array.
FIG. 7 is a schematic perspective view of an embodiment of the tumor cell exosome and the nucleic acid detecting chip according to the present invention.
FIG. 8 is a perspective view of an exposed surface of a PDMS-reinforced plate according to an embodiment.
FIG. 9 is a schematic perspective view of a bonding surface of a PDMS stiffener according to an embodiment.
FIG. 10 is a schematic perspective view of a tumor cell exosome and a nucleic acid detecting chip according to an embodiment of the present invention.
FIG. 11 is a perspective view of the second embodiment with the components separated.
FIG. 12 is a schematic diagram showing the cross-sectional shape change of each process in the process of preparing PDMS sheet.
FIG. 13 is a schematic view showing the change in the cross-sectional shape of each step in the process of manufacturing a glass substrate.
FIG. 14 is a schematic diagram of bonding a PDMS sheet to a glass substrate.
Fig. 12, 13, 14 are schematic and do not correspond to the actual cross-sectional shape of the PDMS sheet or glass substrate.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
The tumor cell exosome and the nucleic acid detection chip thereof comprise a glass substrate 1 and a PDMS sheet 2 which are mutually attached as shown in figures 1 and 2. As shown in fig. 4 and 5, the PDMS sheet 2 is provided with a first through hole 3, a second through hole 4, and a third through hole 5 penetrating through the PDMS sheet 2 at intervals. As shown in fig. 2 or fig. 5, a concave pit 6 is formed between the first through hole 3 and the second through hole 4 on the bonding surface of the PDMS sheet 2, an initial runner 7 is formed between the first through hole 3 and the concave pit 6 on the bonding surface of the PDMS sheet 2, a first runner 8 is formed between the concave pit 6 and the second through hole 4, and a second runner 9 is formed between the second through hole 4 and the third through hole 5, which are sequentially connected. As shown in fig. 1 and 2, after the PDMS sheet 2 is attached to the glass substrate 1, the first through hole 3 forms a sample inlet on the glass substrate 1, the pit 6 forms a TEX capture detection area, the initial flow channel 7, the first flow channel 8 and the second flow channel 9 sequentially form an initial flow channel section, a first flow channel section and a second flow channel section for flowing of a sample solution during detection, the second through hole 4 forms a sample outlet, and the third through hole 5 forms a microRNA capture detection area.
As shown in fig. 2 or fig. 3, a TEX three-electrode system for detecting TEX capture is sputtered in a region corresponding to the attachment of the TEX capture detection region on the attachment surface of the glass substrate 1, a microRNA three-electrode system for detecting microRNA capture is sputtered in a region corresponding to the attachment of the microRNA capture detection region, the TEX three-electrode system includes a TEX working electrode 10, a TEX reference electrode 11, and a TEX counter electrode, and the microRNA three-electrode system includes a microRNA working electrode 12, a microRNA reference electrode 13, and a microRNA counter electrode. In order to simplify the chip structure and save resources, as shown in fig. 2 or fig. 3, the TEX counter electrode and the microRNA counter electrode are 1 shared counter electrode 14, and the shared counter electrode 14 is located below the sample outlet.
As shown in fig. 2 or fig. 5, a first valve for closing the first flow path section is disposed on the first flow path section, and a second valve for closing the second flow path section is disposed on the second flow path section.
As shown in FIG. 2, a triangular prism array 25 is arranged on the initial flow passage section, the outlet of the pit 6 is contracted in a V shape, and a cross-shaped prism array 26 is arranged on the TEX capture detection area.
As shown in fig. 2 or fig. 3, the TEX working electrode 10 includes a meander section 15 and a long straight bar 16 extending outward to its terminal block 17 and its terminal block 17 repeatedly arranged in a meander shape at the TEX capturing detection area, and the TEX reference electrode 11 includes a rectangular main block 18 and a long straight bar 16 extending outward to its terminal block 17 and its terminal block 17; the shared counter electrode 14 comprises a circular main block 19 and long straight bars 16 extending outwards to its terminal block 17 and its terminal block 17; the microRNA working electrode 12 comprises a circular main block 19, a long straight strip 16 extending outwards to a junction block 17 of the circular main block, and the junction block 17 of the long straight strip 16, and the microRNA reference electrode 13 extends outwards to the junction block 17 of the microRNA reference electrode.
The first valve and the second valve are arranged as follows: the PDMS sheet 2 is a film layer which is deformed to block the first flow channel section or the second flow channel section when meeting pressure at the positions of the first flow channel 8 and the second flow channel 9, and closed cavities which can be filled with pressure gas are arranged on the first flow channel 8 and the second flow channel 9. Such valves are also known as membrane valves. The first valve and the second valve can be selectively arranged in the following 2 embodiments.
Example one
As shown in FIG. 7, the PDMS sheet 2 is a thin sheet with a thickness of 140 μm to 160 μm, and is covered with a PDMS reinforcement sheet 20. As shown in fig. 8 and 9, the PDMS reinforcing sheet 20 is provided with a first through hole 21 and a second through hole 22 corresponding to the first through hole 3 and the second through hole 4, respectively forming a sample inlet and a sample outlet. The first valve and the second valve are arranged as follows: as shown in fig. 8 and 9, the PDMS reinforcing sheet 20 is provided with a first vent hole 30 and a second vent hole 31 on the upper surface thereof, the bonded bottom surface of the PDMS reinforcing sheet 20 is provided with a first vent groove 23 communicated with the first vent hole 30 and a second vent groove 24 communicated with the second vent hole 31 at the positions of the first runner 8 and the second runner 9 as closed cavities, and the first vent groove 23 and the second vent groove 24 are three-dimensionally crossed with the first runner 8 and the second runner 9 via the groove bottom thicknesses of the first runner 8 and the second runner 9 of the PDMS sheet 2. As shown in fig. 8 and 9, the PDMS reinforcing sheet 20 is provided with a third through hole 27 corresponding to the third through hole 5, and serves as a filling port of the microRNA capture detection zone.
Example two
As shown in fig. 10 and 11, the closed cavity is formed by: the PDMS groove blocks 28 are respectively arranged on the first flow groove 8 and the second flow groove 9, the vent grooves 29 with one ends blocking the openings at the other ends are formed below the PDMS groove blocks 28, the open ends are used for introducing pressure gas, the PDMS groove blocks 28 are attached to the positions of the first flow groove 8 and the second flow groove 9 of the PDMS sheet 2, and the vent grooves 29 form closed cavities capable of introducing the pressure gas on the PDMS sheet 2. The PDMS sheet 2 is thinned at the positions of the first flow groove 8 and the second flow groove 9 until the PDMS sheet is deformed to a film layer which can block the first flow channel section or the second flow channel section under pressure, and the thickness of the film layer is 140-160 μm.
The preparation method of the tumor cell exosome and the nucleic acid detection chip thereof comprises the steps of respectively preparing the PDMS sheet 2 and the glass substrate 1 in sequence and bonding the two. The specific manufacturing process can also refer to ZL 202010639672.2 of the applicant 2020-07-06, patent application entitled "Single cell temperature detection sensor and manufacturing method and detection method thereof", which relates to the related description of the manufacturing process of PDMS sheet and glass substrate.
As shown in fig. 12, the manufacturing of the PDMS chip 2 first includes the manufacturing of the silicon substrate 32 as a mold thereof and the manufacturing of the PDMS chip 2, and the manufacturing of the silicon substrate 32 specifically includes the following steps: using four-inch single crystal silicon as a substrate, as shown in fig. 12-1, performing thermal oxidation to grow a 2 μm thick silicon dioxide 33 oxide layer, as shown in fig. 12-2; spin-coating a photoresist 34 with a thickness of 2.5 μm according to the designed layout, as shown in fig. 12-3, and performing a photolithography process, as shown in fig. 12-4; removing the silicon dioxide 33 layer by a wet etching process, as shown in fig. 12-5; cleaning the silicon substrate 32 using sulfuric acid and hydrogen peroxide to remove the photoresist 34, as shown in fig. 12-6; spin-coating photoresist 34 again to a thickness of 1.4 μm, as shown in FIGS. 12-7; performing a second photolithography, the remaining microstructures form a silicon substrate mold with microstructures of different heights, as shown in fig. 12-8.
The PDMS slab 2 was made as follows: (1) preparation of PDMS mixture: weighing PDMS prepolymer and curing agent (weight ratio is 15: 1), mixing the PDMS prepolymer and the curing agent uniformly, and standing the mixture in a vacuum drier for 30min to remove most bubbles; (2) and reversing the mold: after the modulation of the PDMS mixture is finished, placing the inverted template on a horizontal table, pouring the modulated PDMS mixture, and standing for 30min to enable the inverted template structure to be filled with PDMS, as shown in FIGS. 12-9; (3) and curing: after standing, placing the mixture in an oven at 80 ℃ for heating for 1h, and after curing, stripping the mixture from the inverted template, as shown in FIGS. 12-10; punching at the position needing punching by adopting a puncher; (4) and storing the PDMS layer: adhering the upper surface and the lower surface of the PDMS layer by using a PCR (polymerase chain reaction) adhesive film, and preserving after packaging; (5) and hydrophilic treatment: placing the packaged chip in a plasma cleaning machine under a vacuum state for 2h, and irradiating for glow for 2 min; taking out the chip, dropwise adding a PEG 6-9-siloxane and acetone mixed solution (v: v ═ 1:1) at a sample inlet, filling the whole chip pipeline with the mixed solution under the action of negative pressure in the PDMS sheet 2, and incubating at room temperature for 1 h; after the treatment is finished, the whole chip is washed by ultrapure water, so that the surface of the micro-channel layer of the PDMS sheet 2 is hydrophilic, and the adsorption of substances such as protein, nucleic acid and the like is reduced.
As shown in fig. 13, the glass substrate 1 is fabricated by forming a metal layer 35 for each electrode on a glass substrate by photolithography and sputtering. The specific process for manufacturing the glass substrate 1 can be also referred to ZL 202010639672.2 of the present applicant 2020-07-06, entitled "Single cell temperature detection sensor and method for making and detecting the same", which is related to the process for manufacturing the glass substrate.
The bonding process of the PDMS sheet 2 and the glass substrate 1 comprises putting the PDMS sheet 2 and the glass substrate 1 together with their bonding surfaces facing upwards into a plasma cleaning machine for cleaning for 90s, and rapidly bonding together after taking out, as shown in FIG. 14. Heating on a hot plate at 90 deg.C for 30min to enhance the bonding degree of the two.
After bonding, adding a prepared nano gold particle reagent into the TEX capture detection area and the microRNA capture detection area, standing for 5 hours at 45 ℃, and after the liquid is evaporated, forming a layer of gold nano film with a specific superlattice structure by the electrode; then adding reagents required by modified antibodies and modified nucleic acid probes into the TEX capture detection area and the microRNA capture detection area respectively for fixing for 120 min; after aspirating the solution, washing away unbound capture probes with PBS solution; after finishing the modification, the excess PDMS layer is removed.
The tumor cell exosome and the nucleic acid detection method thereof are used for simulating a specific test process, the tumor cell exosome and the nucleic acid detection chip thereof are tested and calibrated, a series of tumor cell exosome sample solutions with different concentrations, of which the exosome concentration value and the microRNA concentration value are known, are added, the oxidation current values of a series of TEX working electrodes 10 and the microRNA working electrodes 12 with different values are measured, a series of concentration gradient experiments are carried out, a series of exosome concentrations, the oxidation current values of the corresponding TEX working electrodes 10 and the oxidation current values of the corresponding microRNA working electrodes 12 of a series of microRNA concentrations are obtained, a corresponding linear relation is established, a linear relation between the oxidation current values of the TEX working electrodes 10 and the exosome concentration values is obtained, and a linear relation between the oxidation current values of the microRNA working electrodes 12 and the oxidation current values of the microRNA working electrodes 12 is obtained .
Before detection, a PBS solution is used for washing the inner cavity of the whole chip; the specific test process is as follows:
firstly, carrying out exosome detection, and specifically comprising the following steps:
(1) and closing the second valve to enable the front half part of the chip to form an independent exosome detection area.
(2) And adding a sample solution to be detected into the sample inlet.
(3) And the biomolecules modified on the electrode capture exosomes in the sample solution through a biospecific reaction, so that all the exosomes are adsorbed on the surface of the TEX working electrode 10.
(4) Subsequently, a PBS rinse was added to rinse the chip channels.
(5) After washing, enzyme-labeled antibodies are added, and the antibodies are specifically combined with the exosomes to form a typical antibody-antigen-antibody sandwich structure.
(6) Adding an electrochemical indicator solution, measuring a current value by adopting a differential pulse voltammetry method, and converting a linear relation between the measured current value obtained by test calibration and an exosome concentration value to obtain an exosome concentration value;
then, carrying out MicroRNA detection, and specifically comprising the following steps:
(7) and applying low-voltage circulating square wave current on the exosome capturing electrode to crack the exosome so as to obtain the microRNA in the content of the exosome.
(8) And opening the second valve, and simultaneously applying pressure to the first through hole and the second through hole so that the lysate flows into the microRNA capture detection area.
(9) And after the lysate containing the microRNA flows into the microRNA capture detection area, performing a biospecific reaction on the aptamer modified on the microRNA working electrode 12 in the area and the microRNA, and capturing the microRNA in the lysate to enable all the microRNA to be adsorbed on the surface of the microRNA working electrode 12. (10) After adsorption is finished, closing the first valve to enable the rear half part of the chip to form an independent microRNA capture detection area; adding a toluidine blue indicator into a microRNA capture detection zone, combining the indicator with microRNA, measuring a current value by adopting a differential pulse voltammetry, and converting a linear relation between the measured current value obtained by test calibration and the microRNA concentration value to obtain the microRNA concentration value.
Claims (10)
1. A tumor cell exosome and a nucleic acid detection chip thereof comprise a glass substrate (1) and a PDMS (polydimethylsiloxane) sheet (2) which are mutually attached, and are characterized in that the PDMS sheet (2) is provided with a first through hole (3), a second through hole (4) and a third through hole (5) which penetrate through the PDMS sheet (2) at intervals, a concave pit (6) is formed on the attaching surface of the PDMS sheet (2) between the first through hole (3) and the second through hole (4), an initial flow groove (7) is formed on the attaching surface of the PDMS sheet (2) between the first through hole (3) and the concave pit (6), a first flow groove (8) is formed between the concave pit (6) and the second through hole (4), and a second flow groove (9) is formed between the second through hole (4) and the third through hole (5) and is sequentially connected; after the PDMS sheet (2) is attached to the glass substrate (1), a sample inlet is formed in the first through hole (3) on the glass substrate (1), a TEX capture detection area is formed in the pit (6), an initial flow channel section, a first flow channel section and a second flow channel section through which a sample solution flows during detection are sequentially formed in the initial flow channel (7), the first flow channel (8) and the second flow channel (9), a sample outlet is formed in the second through hole (4), and a microRNA capture detection area is formed in the third through hole (5); a TEX three-electrode system for capturing and detecting TEX is sputtered in a region corresponding to the attachment of the TEX capture detection region on the attachment surface of the glass substrate (1), a microRNA three-electrode system for capturing and detecting microRNA is sputtered in a region corresponding to the attachment of the microRNA capture detection region, the TEX three-electrode system comprises a TEX working electrode (10), a TEX reference electrode (11) and a TEX counter electrode, and the microRNA three-electrode system comprises a microRNA working electrode (12), a microRNA reference electrode (13) and a microRNA counter electrode; the first flow passage section is provided with a first valve which can close the flow passage section, and the second flow passage section is provided with a second valve which can close the flow passage section.
2. The tumor cell exosome and nucleic acid detecting chip thereof according to claim 1, wherein a triangular prism array (25) is arranged on the initial flow channel section, the outlet of the pit (6) is contracted in a V shape, and a cross-shaped prism array (26) is arranged on the TEX capture detection area.
3. The tumor cell exosome and nucleic acid detecting chip according to claim 1, wherein the number of the TEX counter electrode and the microRNA counter electrode is 1, and the shared counter electrode (14) is located under the sample outlet.
4. The tumor cell exosome and its nucleic acid detecting chip according to claim 3, wherein said TEX working electrode (10) comprises a meander section (15) and a long straight strip (16) extending outward to its junction block (17) and its junction block (17) repeatedly arranged in a meander at a TEX capture detection region, said TEX reference electrode (11) comprises a rectangular main block (18) and a long straight strip (16) extending outward to its junction block (17) and its junction block (17); the shared counter electrode (14) comprises a circular main block (19) and a long straight strip (16) extending outwards to a junction block (17) thereof and the junction block (17) thereof; the microRNA working electrode (12) comprises a circular main block (19), a long straight strip (16) extending outwards to a junction block (17) of the circular main block, and the junction block (17) of the long straight strip, and the microRNA reference electrode (13) extends outwards to the long straight strip (16) of the junction block (17) of the microRNA working electrode and the junction block (17) of the microRNA working electrode.
5. The tumor cell exosome and nucleic acid detecting chip according to claim 1, wherein the first valve and the second valve are arranged in such a way that: the PDMS sheet (2) is a film layer which is deformed to block the first flow channel section or the second flow channel section when meeting pressure at the positions of the first flow channel (8) and the second flow channel (9), and a closed cavity which can be filled with pressure gas is arranged on the first flow channel (8) and the second flow channel (9).
6. The tumor cell exosome and the nucleic acid detection chip thereof according to claim 5, wherein the PDMS sheet (2) is a thin sheet with a thickness of 140 μm to 160 μm, a PDMS reinforcement sheet (20) is covered on the PDMS reinforcement sheet (20), and a first extending through hole (21) and a second extending through hole (22) are formed on the PDMS reinforcement sheet (20) corresponding to the first through hole (3) and the second through hole (4) to respectively form a sample inlet and a sample outlet; the first valve and the second valve are arranged in a way that: the PDMS reinforcing piece (20) is provided with a first vent hole (30) and a second vent hole (31) respectively, a first vent groove (23) communicated with the first vent hole (30) and a second vent groove (24) communicated with the second vent hole (31) are respectively arranged at the positions of the first flow groove (8) and the second flow groove (9) on the bottom surface of the PDMS reinforcing piece (20) and serve as closed cavities, and the first vent groove (23) and the second vent groove (24) are separated from the first flow groove (8) of the PDMS piece (2) and the groove bottom thickness of the second flow groove (9) and form three-dimensional intersection with the first flow groove (8) and the second flow groove (9).
7. The tumor cell exosome and the nucleic acid detecting chip thereof according to claim 5, wherein a third extended through hole (27) is formed on the PDMS reinforcing sheet (20) corresponding to the third through hole (5) and is used as a liquid adding port of the microRNA capture detecting area.
8. The tumor cell exosome and nucleic acid detecting chip according to claim 5, wherein said closed cavity is formed by: the PDMS groove blocks (28) are respectively arranged on the first groove (8) and the second groove (9), the vent grooves (29) with one ends blocking the openings at the other ends are formed below the PDMS groove blocks (28), the open ends are used for introducing pressure gas, the PDMS groove blocks (28) are attached to the positions of the first groove (8) and the second groove (9) of the PDMS sheet (2), and the vent grooves (29) form closed cavities capable of introducing the pressure gas on the PDMS sheet (2).
9. A tumor cell exosome and its nucleic acid detection chip preparation method, including PDMS sheet (2) preparation and glass substrate (1) preparation that can go on separately in order, and the bonding of the two, the preparation of PDMS sheet (2) includes the preparation of the silicon substrate (32) and preparation of PDMS sheet (2) as its mould at first, characterized by, the preparation of the silicon substrate (32) includes the following steps specifically: adopting four-inch monocrystalline silicon as a substrate, carrying out thermal oxidation, and growing a silicon dioxide (33) oxide layer with the thickness of 2 microns; spin-coating a photoresist (34) according to the designed layout, wherein the thickness of the photoresist is 2.5 mu m, carrying out primary photoetching, and removing a silicon dioxide (33) layer by adopting a wet etching process; cleaning the silicon substrate (32) by using sulfuric acid and hydrogen peroxide, and removing the photoresist (34); spin-coating photoresist (34) again to the thickness of 1.4 μm, performing secondary photoetching, and forming silicon substrate molds with microstructures of different heights by using the rest microstructures;
the preparation process of the PDMS sheet (2) is as follows: (1) preparation of PDMS mixture: weighing PDMS prepolymer and curing agent (weight ratio is 15: 1), mixing the PDMS prepolymer and the curing agent uniformly, and standing the mixture in a vacuum drier for 30min to remove most bubbles; (2) and reversing the mold: after the modulation of the PDMS mixture is finished, placing the inverted template on a horizontal table, pouring the modulated PDMS mixture, and standing for 30min to enable the inverted template structure to be filled with PDMS; (3) and curing: after standing, placing the mixture in an oven at 80 ℃ for heating for 1h, and after curing, stripping the mixture from the inverted template; punching at the position needing punching by adopting a puncher; (4) and storing the PDMS layer: adhering the upper surface and the lower surface of the PDMS layer by using a PCR (polymerase chain reaction) adhesive film, and preserving after packaging; (5) and hydrophilic treatment: placing the packaged chip in a plasma cleaning machine under a vacuum state for 2h, and irradiating for glow for 2 min; taking out the chip, dropwise adding a PEG (6-9) -siloxane and acetone mixed solution (v: v =1: 1) at a sample inlet, filling the whole chip pipeline with the mixed solution by utilizing the negative pressure action in the PDMS sheet (2), and incubating for 1h at room temperature; after the treatment is finished, the whole chip is flushed by ultrapure water, so that the surface of the micro-channel layer of the PDMS sheet (2) is hydrophilic, and the adsorption of substances such as protein, nucleic acid and the like is reduced;
the manufacturing process of the glass substrate (1) is as follows: manufacturing electrode metal (35) layers on a glass substrate through photoetching and sputtering processes, and attaching a graphical PDMS sheet (2) according to an alignment mark;
the bonding process of the PDMS sheet (2) and the glass substrate (1) comprises the steps of putting the bonding surfaces of the PDMS sheet (2) and the glass substrate (1) upwards together into a plasma cleaning machine for cleaning for 90s, taking out the bonding surfaces of the PDMS sheet and the glass substrate, rapidly bonding the bonding surfaces together, and heating the bonding surfaces on a hot plate at 90 ℃ for 30min to enhance the bonding degree of the PDMS sheet and the glass substrate;
after the bonding, adding a prepared nano gold particle reagent into the TEX capture detection area and the microRNA capture detection area, standing for 5 hours at 45 ℃, and after the liquid is evaporated, forming a layer of gold nano film with a specific superlattice structure by the electrode; then adding reagents required by modified antibodies and modified nucleic acid probes into the TEX capture detection area and the microRNA capture detection area respectively for fixing for 120 min; after aspirating the solution, washing away unbound capture probes with PBS solution; after finishing the modification, the excess PDMS layer is removed.
10. A tumor cell exosome and a nucleic acid detection method thereof are characterized in that the tumor cell exosome and the nucleic acid detection chip thereof according to any one of claims 1 to 7 are used, a specific test process is simulated, the tumor cell exosome and the nucleic acid detection chip thereof are tested and calibrated, a series of tumor cell exosome sample solutions with different concentrations, of which the exosome concentration value and the microRNA concentration value are known, are added, the oxidation current values of a series of TEX working electrodes (10) and the oxidation current values of the microRNA working electrodes (12) with different values are measured, a series of concentration gradient experiments are performed, a series of exosome concentrations and the oxidation current values of the corresponding TEX working electrodes (10) and the microRNA concentrations and the oxidation current values of the corresponding microRNA working electrodes (12) are obtained, a corresponding linear relation is established, and a linear relation between the oxidation current values of the TEX working electrodes (10) and the exosome concentration values is obtained, and a linear relation between the oxidation current value of the microRNA working electrode (12) and the oxidation current value of the microRNA working electrode (12);
before detection, a PBS solution is used for washing the inner cavity of the whole chip; the specific test process is as follows:
firstly, carrying out exosome detection, and specifically comprising the following steps:
(1) closing the second valve to form an independent exosome detection area on the front half part of the chip;
(2) adding a sample solution to be detected into the sample inlet;
(3) capturing exosomes in a sample solution by using the modified biomolecules on the electrodes through a biological specificity reaction, so that all exosomes are adsorbed on the surface of the TEX working electrode (10);
(4) then, adding PBS flushing liquid to flush the chip channel;
(5) after washing, adding an antibody with an enzyme label, and specifically combining the antibody with an exosome to form a typical antibody-antigen-antibody sandwich structure;
(6) adding an electrochemical indicator solution, measuring a current value by adopting a differential pulse voltammetry method, and converting a linear relation between the measured current value obtained by test calibration and an exosome concentration value to obtain an exosome concentration value;
then, carrying out MicroRNA detection, and specifically comprising the following steps:
(7) applying low-voltage circulating square wave current on the exosome capturing electrode to crack exosomes so as to obtain microRNA in the content of the exosomes;
(8) opening a second valve, and simultaneously applying pressure at the first through hole and the second through hole to enable the lysate to flow into the microRNA capture detection area;
(9) after lysate containing microRNA flows into a microRNA capturing detection area, an aptamer modified on a microRNA working electrode (12) in the area performs biospecific reaction with the microRNA, and the microRNA in the lysate is captured, so that all the microRNA is adsorbed on the surface of the microRNA working electrode (12);
(10) after adsorption is finished, closing the first valve to enable the rear half part of the chip to form an independent microRNA capture detection area; adding a toluidine blue indicator into a microRNA capture detection zone, combining the indicator with microRNA, measuring a current value by adopting a differential pulse voltammetry, and converting a linear relation between the measured current value obtained by test calibration and the microRNA concentration value to obtain the microRNA concentration value.
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