WO2023221023A1 - 基因检测基板、基因检测芯片及基因检测样本的制备方法 - Google Patents

基因检测基板、基因检测芯片及基因检测样本的制备方法 Download PDF

Info

Publication number
WO2023221023A1
WO2023221023A1 PCT/CN2022/093764 CN2022093764W WO2023221023A1 WO 2023221023 A1 WO2023221023 A1 WO 2023221023A1 CN 2022093764 W CN2022093764 W CN 2022093764W WO 2023221023 A1 WO2023221023 A1 WO 2023221023A1
Authority
WO
WIPO (PCT)
Prior art keywords
sub
gene detection
groove
trough
reaction
Prior art date
Application number
PCT/CN2022/093764
Other languages
English (en)
French (fr)
Inventor
牟芸青
刘浩男
刘祝凯
丁丁
Original Assignee
京东方科技集团股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 京东方科技集团股份有限公司 filed Critical 京东方科技集团股份有限公司
Priority to CN202280001263.7A priority Critical patent/CN117529546A/zh
Priority to PCT/CN2022/093764 priority patent/WO2023221023A1/zh
Publication of WO2023221023A1 publication Critical patent/WO2023221023A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • Embodiments of the present disclosure belong to the field of biomedical technology, and specifically relate to a method for preparing a genetic detection substrate, a genetic detection chip, and a genetic detection sample.
  • PCR polymerase chain reaction
  • DNA or deoxyribonucleic acid
  • DNA is one of the four biological macromolecules contained in biological cells. It carries the genetic information necessary for the synthesis of RNA and proteins. It is an essential biological macromolecule for the development and normal operation of organisms. molecular. RNA, or ribonucleic acid, is a carrier of genetic information that exists in biological cells and some viruses and viroids.
  • Embodiments of the present disclosure provide a method for preparing a genetic detection substrate, a genetic detection chip, and a genetic detection sample.
  • an embodiment of the present disclosure provides a gene detection substrate, which includes: a substrate;
  • a gene detection channel is opened in the base, and the opening of the gene detection channel is located on one side of the base;
  • the gene detection channel includes a sampling slot, a sample outlet slot and a flow channel slot, and the sampling slot, the flow channel slot and the sample outlet slot are connected in sequence and penetrate each other;
  • the flow channel groove includes a plurality of reaction holes and a plurality of flow channel structures
  • the plurality of reaction holes are spaced apart from each other, and any two adjacent reaction holes are connected through one of the flow channel structures;
  • the flow channel structure includes a first sub-trough, a second sub-trough and a third sub-trough, and the first sub-trough, the second sub-trough and the third sub-trough are connected and connected in sequence;
  • the width of the first sub-groove and the third sub-groove along the first direction is smaller than the width of the second sub-groove along the first direction;
  • the first direction is perpendicular to the arrangement direction of two adjacent reaction holes.
  • the depths of the first sub-trough and the third sub-trough are greater than the depth of the second sub-trough
  • the depths of the first sub-groove, the second sub-groove and the third sub-groove are respectively their dimensions along the thickness direction of the substrate.
  • the width of the second sub-groove along the first direction is smaller than the width of the reaction hole along the first direction.
  • the widths of the first sub-groove and the third sub-groove along the first direction are equal.
  • the depths of the first sub-trough and the third sub-trough are equal to the depth of the reaction well
  • the depth of the reaction hole is its dimension along the thickness direction of the substrate.
  • the plurality of reaction holes and the plurality of flow channel structures are arranged along a second direction; the second direction is perpendicular to the first direction;
  • the flow channel also includes a first end close to the sample inlet slot and a second end close to the sample outlet.
  • the flow channel further includes a first branch slot and a second branch slot.
  • the first The branch groove is connected between the sampling tank and the first reaction hole located at the first end, and communicates between the sampling tank and the first reaction hole;
  • the second branch groove is connected between the sample outlet and the second reaction hole located at the second end, and the first sub-trough is also connected between the second branch groove and the second reaction hole.
  • the groove and the second sub-trough, the second reaction hole, the first sub-trough, the second sub-trough and the second branch groove are arranged in sequence along the second direction;
  • the second branch groove, the second sub-trough and the first sub-trough connect the sample outlet and the second reaction well.
  • the orthographic shape of the reaction well on the substrate includes a circle
  • Orthographic shapes of the first sub-trough, the second sub-trough and the third sub-trough on the substrate include a rectangle.
  • the diameter of the orthographic projection of the reaction well on the substrate ranges from 68 to 88 ⁇ m
  • the depth of the reaction hole ranges from 70 to 90 ⁇ m
  • the depth of the second sub-groove ranges from 20 to 40 ⁇ m.
  • the width of the first sub-groove along the first direction ranges from 20 to 30 ⁇ m;
  • the width of the third sub-trough along the first direction ranges from 20 to 30 ⁇ m;
  • the width of the second sub-groove along the first direction ranges from 35 to 45 ⁇ m.
  • the length of the first sub-groove along the second direction ranges from 10 to 20 ⁇ m
  • the length range of the third sub-groove along the second direction is 10-20 ⁇ m;
  • the length of the second sub-groove along the second direction ranges from 25 to 35 ⁇ m.
  • the depths of the sampling slot, the sample outlet slot, the first branch slot, and the second branch slot are the same as the depths of the second sub-trough respectively;
  • the depths of the sample inlet groove, the sample outlet groove, the first branch groove, and the second branch groove are respectively their dimensions along the thickness direction of the substrate.
  • the number of flow channels is multiple;
  • a plurality of said flow channel grooves are arranged parallel to each other;
  • reaction holes in each of the flow channels are arranged at equal intervals
  • the reaction holes in the plurality of flow channels are arranged in an array.
  • the side panel of the base with the opening of the gene detection channel includes a first surface and a second surface; the first surface is the inner wall of the gene detection channel, and the first surface A hydrophilic layer is provided on the surface;
  • a hydrophobic layer is provided on the second surface.
  • the substrate is made of any material selected from the group consisting of polydimethylsiloxane, polymethyl methacrylate, and polycarbonate.
  • embodiments of the present disclosure also provide a gene detection chip, which includes the above-mentioned gene detection substrate.
  • it also includes an encapsulation film, which is paired with the gene detection substrate to encapsulate the gene detection channel in the gene detection substrate;
  • the area of the packaging film corresponding to the second sub-slot in the gene detection channel can be attached to and connected to the inner wall of the second sub-slot.
  • the encapsulation film includes double-sided adhesive film or ultraviolet curable adhesive film.
  • a plurality of the gene detection substrates are included, the plurality of gene detection substrates are spliced to each other, and the gene detection channels in the multiple gene detection substrates are isolated from each other.
  • embodiments of the present disclosure also provide a method for preparing a genetic detection sample, which includes: preparing a genetic detection substrate;
  • the area of the packaging film corresponding to the second sub-slot in the gene detection channel is attached to and connected to the inner wall of the second sub-slot.
  • preparing a gene detection substrate includes: using a patterning process to prepare the gene detection channel on the substrate.
  • Figure 1 is a schematic top view of a partial structure of a gene detection substrate in an embodiment of the present disclosure.
  • Figure 2 is a structural cross-sectional view along the AA' section line in Figure 1.
  • Figure 3 is a partial three-dimensional structural diagram of a gene detection substrate in an embodiment of the present disclosure.
  • Figure 4 is a three-dimensional structural diagram of a local fluid sample part in the gene detection substrate according to an embodiment of the present disclosure.
  • Figure 5 is a schematic cross-sectional view of the structure of a gene detection chip in an embodiment of the present disclosure.
  • Figure 6 is a schematic top view of the partial structure of a gene detection chip in an embodiment of the present disclosure.
  • Figure 7 is a schematic cross-sectional view of the partial structure of a gene detection sample prepared in an embodiment of the present disclosure.
  • Figure 8 is a schematic diagram of the sampling simulation effect of the genetic detection chip in the embodiment of the present disclosure.
  • digital PCR detection is to disperse DNA or RNA samples in a large number of micro-reaction units, and then perform single-molecule template PCR amplification, fluorescence detection and Statistical analysis achieves absolute quantification; it does not rely on standard curves and multiple gradient standards of known concentrations, and directly detects the original concentration of DNA or RNA samples in the sample. Because this detection method has better sensitivity and accuracy than traditional fluorescence quantitative PCR detection, digital PCR detection has quickly received widespread attention, especially in the detection of trace (i.e., tiny amounts) nucleic acid samples, detection of rare mutations in complex backgrounds, Its advantages in identifying nucleic acid copy number variations and small differences in gene expression have been generally recognized.
  • a PCR machine based on polymerase is actually a temperature control device that can control the denaturation temperature, renaturation temperature, and extension temperature well.
  • Microfluidic chip is one type of PCR instrument based on polymerase.
  • Microfluidic chip technology is a scientific technology whose main feature is the manipulation of fluids in micron-scale space. It has the ability to shrink the basic functions of biology, chemistry and other laboratories onto a chip of several square centimeters, so it is also called Lab-on-a-chip.
  • the mainstream form of microfluidic chip is mostly formed by a network of microchannels, with controllable fluids running through the entire system, to realize various functions in conventional chemical or biological laboratories.
  • microfluidic chips make digital PCR detection applications more convenient.
  • digital PCR detection methods and reagents on microfluidic chips, good compatibility and flexibility can be achieved.
  • PCR reagents It is divided into hundreds of independent nanoliter reaction units to perform digital PCR analysis, which improves the sensitivity and accuracy of detection.
  • the microfluidic chip realizes the injection and detection of the sample solution through the sample detection channel formed by etching on the substrate.
  • the sample detection channel includes an injection channel, a light outlet channel, and multiple micro-reaction units (such as micro-reaction wells) connected between the injection channel and the sample outlet channel. Adjacent micro-reaction units are connected by rectangles of the same depth and width.
  • the channel structure is connected; when the sample solution fills each micro-reaction unit, the sample detection channel is encapsulated through the packaging film, so that the micro-reaction units are separated from each other to form independent micro-chambers, so that the micro-reaction units in each micro-reaction unit can be measured. Samples are analyzed statistically.
  • the microfluidic chip with the above-mentioned structure in the public technology has problems such as insufficient micro-reaction well injection, residual bubbles in the micro-reaction wells, and gaps between adjacent micro-reaction wells after injection due to its small size and high processing difficulty.
  • the encapsulation effect is not good, and the connection channels between adjacent micro-reaction wells cannot be well separated, causing cross-contamination of reagents and samples between micro-reaction wells, affecting the detection effect, and reducing the precision and accuracy of the detection results. .
  • an embodiment of the present disclosure provides a gene detection substrate.
  • Figure 1 is a schematic top view of the partial structure of the gene detection substrate in the embodiment of the present disclosure
  • Figure 2 is a schematic top view along AA' in Figure 1 Structural cross-sectional view of the cutting line
  • Figure 3 is a partial three-dimensional structural schematic diagram of the gene detection substrate in the embodiment of the present disclosure; which includes: a substrate 1; a gene detection channel, which is opened in the substrate 1, and the opening of the gene detection channel is located on the base 1 On one side of the board; the genetic detection channel includes a sample inlet tank 2, a sample outlet tank 4 and a flow channel slot 3.
  • the sample inlet tank 2, the flow channel slot 3 and the sample outlet tank 4 are connected in sequence and penetrate each other;
  • the flow channel slot 3 includes A plurality of reaction holes 31 and a plurality of flow channel structures 32;
  • the plurality of reaction holes 31 are spaced apart from each other, and any two adjacent reaction holes 31 are connected through a flow channel structure 32;
  • the flow channel structure 32 includes a first sub-section
  • the groove 321, the second sub-groove 322 and the third sub-groove 323 are connected and connected in sequence; the edges of the first sub-groove 321 and the third sub-groove 323
  • the width s1 in the first direction Y is smaller than the width s2 of the second sub-groove 322 along the first direction Y;
  • the first direction Y is perpendicular to the arrangement direction of two adjacent reaction holes 31 .
  • the genetic detection substrate can be used to detect biological samples such as DNA (deoxyribonucleic acid) samples or RNA (ribonucleic acid) samples.
  • DNA sample or RNA sample is dispersed in a large number of reaction wells 31, and then single-molecule template PCR (polymerase chain reaction) amplification, fluorescence detection and Statistical analysis achieves absolute quantification; it does not rely on standard curves and multiple gradient standards of known concentrations, and directly detects the original concentration of DNA samples or RNA samples in the sample.
  • the widths of the first sub-groove 321 , the second sub-groove 322 and the third sub-groove 323 along the first direction Y are their respective slot sizes along the first direction Y.
  • the width s2 of the second sub-groove 322 along the first direction Y is smaller than the width s3 of the reaction hole 31 along the first direction Y.
  • the width of the reaction hole 31 along the first direction Y is the orifice size of the reaction hole 31 along the first direction Y.
  • the widths s1 of the first sub-groove 321 and the third sub-groove 323 along the first direction Y are equal.
  • the depth h1 of the first sub-groove 321 and the third sub-groove 323 is greater than the depth h2 of the second sub-groove 322;
  • the depth of 323 is respectively its dimension along the thickness direction of the substrate 1 .
  • the depths of the first sub-groove 321 , the second sub-groove 322 and the third sub-groove 323 are the dimensions from the surface of the groove bottom to the surface of the groove opening, that is, the depth of each of them into the base 1 .
  • the substrate 1 is a flat plate with a uniform thickness.
  • the depths of the first sub-groove 321 and the third sub-groove 323 are equal to the depth h3 of the reaction hole 31 ; the depth of the reaction hole 31 is its size along the thickness direction of the substrate 1 .
  • the biological sample to be tested in the gene detection channel in Figure 1; when the biological sample to be tested (usually a sample solution) is detected in the genetic detection channel, the biological sample to be tested is input into the sampling tank 2 ; Under the action of pressure, the biological sample to be tested flows from the inlet tank 2 into the flow channel tank 3; after the biological sample to be tested fills the flow channel tank 3, it will flow to the sample outlet tank 4; that is, the genetic detection channel can realize the biological sample to be tested of continuous sampling. Subsequently, at the end of the injection of the biological sample to be tested, the genetic detection channel is encapsulated with an encapsulation film.
  • the biological sample to be tested usually a sample solution
  • the second sub-trough 322 is used as a partition area to isolate the reaction hole 31 in subsequent packaging; for example, a film pressing method is used to make the groove wall of the second sub-trough 322 and the corresponding area of the packaging film adhere to each other, thereby achieving separation of flow channels. , the effect of partitioning between the reaction wells 31.
  • the first sub-trough 321 and the third sub-trough 323 serve as a reaction area together with the reaction hole 31, that is, the first sub-trough 321 and the third sub-trough 323 become the supplementary reaction area of the reaction hole 31; compared to In the disclosed technology, the micro-reaction well is used as a reaction chamber alone, which increases the volume of each reaction area. Appropriately increasing the volume of the reaction area is conducive to the performance and detection of digital polymerase chain reaction (i.e., dPCR reaction), thus The effectiveness of genetic detection substrates is improved.
  • the widths of the first sub-groove 321 , the second sub-groove 322 and the third sub-groove 323 along the first direction Y are designed to be narrow-wide-narrow respectively.
  • the depths of the second sub-trough 322 and the third sub-trough 323 are designed as deep-shallow-deep respectively. On the one hand, it can ensure the sampling efficiency of each reaction well 31 in the genetic detection substrate, and at the same time improve the injection efficiency of each reaction well 31.
  • the encapsulation effect can achieve sufficient isolation between the reaction wells 31 and improve the effectiveness of the encapsulation between the reaction wells 31; on the other hand, it can increase the volume of the reaction wells 31, that is, increase the volume of the effective reaction area, thereby improving the efficiency of gene detection. Precision and accuracy; in addition, the structural design of the genetic detection channel is relatively simple, easy to process, and convenient to package, which improves the packaging speed and efficiency.
  • multiple reaction wells 31 and multiple flow channel structures 32 are arranged along the second direction X; the second direction The first end of the slot 2 and the second end close to the sample outlet slot 4, the flow channel slot 3 also includes a first branch slot 33 and a second branch slot 34.
  • the first branch slot 33 is connected to the sampling slot 2 and is located at the first between the first reaction hole 311 at the second end, and connects the sampling tank 2 and the first reaction hole 311;
  • the second branch tank 34 is connected between the sample outlet 4 and the second reaction hole 312 at the second end,
  • a first sub-trough 321 and a second sub-trough 322 are also connected between the second branch tank 34 and the second reaction hole 312.
  • the second reaction hole 312, the first sub-trough 321, the second sub-trough 322 and the second branch tank are also connected. 34 are arranged sequentially along the second direction
  • the first sub-trough 321 and the second sub-trough 322 are effectively separated, thereby achieving independent packaging of the second reaction well 312 .
  • the orthographic projection shape of the reaction well 31 on the substrate 1 includes a circle; the orthographic projection shapes of the first sub-groove 321 , the second sub-groove 322 and the third sub-groove 323 on the substrate 1 Includes rectangles.
  • the cross-sectional shape of the reaction well 31 perpendicular to the second direction can achieve the shape.
  • the diameter of the orthographic projection of the reaction hole 31 on the substrate 1 ranges from 68 to 88 ⁇ m; the depth of the reaction hole 31 ranges from 70 to 90 ⁇ m; and the depth of the second sub-groove 322 ranges from 20 to 40 ⁇ m.
  • the diameter of the orthographic projection of the reaction hole 31 on the substrate 1 is 78 ⁇ m; the depth of the reaction hole 31 is 80 ⁇ m; and the depth of the second sub-groove 322 is 30 ⁇ m.
  • the width of the first sub-groove 321 along the first direction Y ranges from 20 to 30 ⁇ m; the width of the third sub-groove 323 along the first direction Y ranges from 20 to 30 ⁇ m; and the second sub-groove 322 extends along the first direction Y.
  • the width range in direction Y is 35 to 45 ⁇ m.
  • the width of the first sub-groove 321 along the first direction Y is 25 ⁇ m; the width of the third sub-groove 323 along the first direction Y is 25 ⁇ m; and the width of the second sub-groove 322 along the first direction Y is 40 ⁇ m. .
  • the size is designed such that the two ends of the second sub-groove 322 are respectively the first sub-groove 321 and the third sub-groove 323 with a narrow width.
  • the reaction hole 31 is directly connected to the reaction hole 31 instead of the wider second sub-trough 321 directly connected to the reaction hole 31; this helps the inner wall of the wider second sub-trough 322 to adhere to the packaging film during the actual packaging process.
  • the tight connection after sealing can reduce the leakage phenomenon after encapsulation in the area where the second sub-trough 322 is located, thereby avoiding crosstalk between the biological samples to be tested between the two adjacent reaction wells 31 during the detection process, and improving the detection precision and accuracy.
  • the length range of the first sub-groove 321 along the second direction X is 10-20 ⁇ m; the length range of the third sub-groove 323 along the second direction The length range of direction X is 25 to 35 ⁇ m.
  • the length of the first sub-groove 321 along the second direction X is 15 ⁇ m; the length of the third sub-groove 323 along the second direction X is 15 ⁇ m; and the length of the second sub-groove 322 along the second direction X is 32 ⁇ m. . In some embodiments, the distance between the centers of any two adjacent reaction wells 31 arranged along the second direction X on the substrate 1 is 140 ⁇ m.
  • the depths of the sampling slot 2, the sample outlet 4, the first branch slot 33, and the second branch slot 34 are the same as the depths of the second sub-trough 322 respectively; the sampling slot 2, the sample outlet 4, The depths of the first branch groove 33 and the second branch groove 34 are respectively their dimensions along the thickness direction of the substrate 1 .
  • 4 is a three-dimensional structural diagram of the local fluid sample part in the gene detection substrate according to the embodiment of the present disclosure. Such an arrangement can improve the sampling efficiency of the gene detection channel, and at the same time, the reaction well 31 can be filled up, and there is no need for liquid in the reaction well 31. Bubbles will be generated, which improves the effectiveness of sampling in the genetic detection channel and exhausting the reaction hole 31.
  • the number of flow channels 3 is multiple; the multiple flow channels 3 are arranged parallel to each other; the reaction holes 31 in any two adjacent flow channels 3 are on a straight line extending along the second direction X. Orthographic projections on the alternating distribution. With this arrangement, the area of the substrate 1 can be fully utilized when distributing the reaction holes 31 , so that a larger number of reaction holes 31 can be distributed on the effective utilization area of the substrate 1 , thereby improving the utilization rate of the substrate 1 .
  • reaction holes 31 in each flow channel 3 are arranged at equal intervals; the reaction holes 31 in multiple flow channels 3 are arranged in an array. Such an arrangement can also distribute a larger number of reaction wells 31 on the effective utilization area of the substrate 1, thereby improving the utilization rate of the substrate 1; at the same time, it can also improve the precision and accuracy of gene detection.
  • the sampling slot 2 includes a sampling hole 21 and a sampling slot 22.
  • the sampling hole 21 is located at one end of the sampling slot 22, and the sampling hole 21 and the sampling slot 22 are connected.
  • the sample outlet 4 includes a sample outlet 41 and an outlet channel 42.
  • the sample outlet 41 is located at one end of the outlet channel 42, and the sample outlet 41 and the outlet channel 42 are connected.
  • the plurality of flow channel grooves 3 are respectively connected between the inlet groove 22 and the outlet groove 42 and are connected with each other.
  • the entry groove 22 and the exit groove 42 respectively extend along the first direction Y.
  • the width of the inlet groove 22 and the outlet groove 42 along the second direction X is greater than the maximum width of the flow channel groove 3 along the first direction Y.
  • the orthographic projection shapes of the sampling hole 21 and the sampling hole 41 on the substrate 1 are both circular, and the diameter of the circle is larger than the maximum width of the flow channel groove 3 along the first direction Y. Referring to Figure 3, such an arrangement can improve the sampling efficiency of the gene detection channel, and at the same time, the reaction well 31 can be filled completely, and no bubbles will be generated in the reaction hole 31, which improves the sampling efficiency of the gene detection channel and the exhaust of the reaction hole 31. effectiveness.
  • the side plate of the substrate 1 with the opening of the gene detection channel includes a first surface and a second surface; the first surface is the inner wall of the gene detection channel, and a hydrophilic layer is provided on the first surface; A hydrophobic layer is provided on the two surfaces, that is, a hydrophobic layer is provided on the surface of the substrate 1 except for the surface of the gene detection channel. In this embodiment, a hydrophobic layer is provided on the surface of the area where the gene detection channel is not provided on the substrate 1 .
  • the length, width and depth of the reaction well 31, the first sub-groove 321, the second sub-groove 322 and the third sub-groove 323 are all in the micron level. Such an arrangement can utilize capillary force and surface tension to promote accurate loading of the biological sample solution to be tested into the reaction well 31 , thereby promoting the flow of the biological sample solution to be tested into the reaction well 31 and avoiding waste of the biological sample to be tested.
  • the substrate 1 is made of any one of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), and polycarbonate (PC plastic).
  • PDMS polydimethylsiloxane
  • PMMA polymethylmethacrylate
  • PC plastic polycarbonate
  • the gene detection substrate provided in the embodiment of the present disclosure is designed by setting the widths of the first sub-trough, the second sub-trough and the third sub-trough along the first direction to be narrow-wide-narrow respectively, and at the same time, the first sub-trough is provided.
  • the depths of the second sub-trough and the third sub-trough are designed as deep-shallow-deep respectively. On the one hand, it can ensure the sampling efficiency of each reaction well in the genetic detection substrate, and at the same time improve the packaging of each reaction well after injection.
  • the effect is to achieve sufficient isolation between reaction wells and improve the effectiveness of packaging between reaction wells; on the other hand, it can increase the volume of the reaction wells, that is, increase the volume of the effective reaction area, thereby improving the precision and accuracy of genetic detection;
  • the structural design of the gene detection channel is relatively simple, easy to process, and convenient to package, which improves the packaging speed and efficiency.
  • an embodiment of the present disclosure also provides a gene detection chip, which includes the gene detection substrate in the above embodiment.
  • FIG. 5 is a schematic cross-sectional view of the structure of a gene detection chip in an embodiment of the present disclosure; wherein, the gene detection chip also includes an encapsulation film 5 , which is aligned with the gene detection substrate 6 to seal the gene detection substrate 6
  • the gene detection channel in the gene detection channel is encapsulated; the area of the packaging film 5 corresponding to the second sub-groove 322 in the gene detection channel can be attached to and connected to the inner wall of the second sub-groove 322.
  • the separation between adjacent reaction wells 31 in the gene detection channel is realized, so that each reaction well 31 forms an independent reaction chamber for gene detection, statistics and analysis.
  • the encapsulation film 5 includes a double-sided adhesive film or a UV-curable adhesive film.
  • a film containing colloid on one side of the double-sided adhesive film or UV-curable adhesive film adheres to the inner wall of the second sub-trough 322 to achieve the effect of separating the flow channel and the reaction hole 31 .
  • the use of an adhesive film helps to ensure a tight connection between the packaging film 5 and the second sub-trough 322 after the corresponding surfaces are bonded, thereby reducing liquid leakage in the packaging area and crosstalk between the reaction holes 31 .
  • the packaging film 5 can be adhered to the inner wall of the second sub-trough 322 by pressing the film.
  • FIG. 6 is a schematic top view of the partial structure of a gene detection chip in an embodiment of the present disclosure; wherein, the gene detection chip includes multiple gene detection substrates 6 , the multiple gene detection substrates 6 are spliced to each other, and multiple gene detection substrates 6 are spliced together. The gene detection channels in the gene detection substrate 6 are isolated from each other.
  • one gene detection substrate 6 is used to analyze one biological sample to be tested, and multiple gene detection substrates 6 can be used to analyze different biological samples to be tested, thereby enabling simultaneous analysis of multiple organisms to be tested on one genetic detection chip. samples, thus improving the gene detection throughput of the genetic detection chip.
  • the gene detection chip provided in the embodiments of the present disclosure by using the above-mentioned gene detection substrate, can, on the one hand, ensure the sampling efficiency of each reaction well in the gene detection substrate, and at the same time improve the packaging effect after the injection of each reaction hole. It can achieve sufficient isolation between reaction wells and improve the effectiveness of packaging between reaction wells; on the other hand, it can increase the volume of reaction wells, that is, increase the volume of the effective reaction area, thereby improving the precision and accuracy of genetic detection.
  • embodiments of the present disclosure also provide a method for preparing a genetic detection sample, which includes: step S01: preparing a genetic detection substrate.
  • a patterning process is used to prepare a gene detection channel on the substrate.
  • Gene detection channels can be prepared on the substrate through exposure and development processes.
  • Step S02 Assemble the packaging film and the gene detection substrate to form a gene detection chip.
  • the encapsulation film and the side of the substrate where the gene detection channel is located are aligned and covered.
  • Step S03 Inject sample reagent into the injection slot of the gene detection channel in the gene detection chip.
  • sample reagent is injected into the injection hole of the injection tank by perfusion or dripping.
  • Sample reagents are usually in solution.
  • Step S04 When the sample reagents in the gene detection channel are filled, attach and connect the area of the packaging film corresponding to the second sub-slot in the gene detection channel to the inner wall of the second sub-slot.
  • the packaging film can be adhered to the inner wall of the second sub-trough by pressing the film.
  • Figure 7 is a schematic cross-sectional view of the partial structure of the gene detection sample prepared in the embodiment of the present disclosure
  • Figure 8 is a schematic diagram of the sampling simulation effect of the genetic detection chip in the embodiment of the present disclosure; wherein, for this The sampling effect of the genetic detection chip was simulated and verified using Ansys Fluent simulation software.
  • the grid-filled injection tank 2 and flow channel tank 3 are filled with sample reagents. There are no bubbles in 3; there is air in the diagonally filled sample outlet 4 (especially the sample outlet 41); the verification results show that the inside of the reaction hole 31 is filled with sample reagent 7 after the gene detection channel in the gene detection chip is injected. It is full, and no bubbles are generated in the reaction hole 31; which shows the effectiveness of the structural design of the gene detection channel in the injection and exhaust of the reaction hole 31.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

一种基因检测基板，其中，包括：基底（1）；基因检测通道，开设于所述基底中，且基因检测通道的开口位于基底（1）的一侧板面上：所述基因检测通道包括进样槽（2）、出样槽（4）和流道槽（3），所述进样槽（2）、所述流道槽（3），和所述出样槽（4）依次连接且彼此贯通；所述流道槽（3）包括多个反应孔（31）和多个流道结构（32）；所述多个反应孔（31）彼此间隔分布，且任意相邻的两个所述反应孔（31）之间通过一个所述流道结构（32）连通：所述流道结构（32）包括第一子槽（321）、第二子槽（322）和第三子槽（323），所述第一子槽（321）、所述第二子槽（323）和所述第三子槽（323）依次连接并贯通；所述第一子槽（321）和所述第三子槽（323）的沿第一方向Y的宽度s1小于所述第二子槽的（322）沿所述第一方向Y的宽度s2；所述第一方向Y垂直于相邻两个所述反应孔（31）的排布方向。

Description

基因检测基板、基因检测芯片及基因检测样本的制备方法 技术领域
本公开实施例属于生物医学技术领域，具体涉及一种基因检测基板、基因检测芯片及基因检测样本的制备方法。
背景技术
PCR(聚合酶链式反应)技术，是一种用于放大扩增特定的DNA片段的分子生物学技术，它可看作是生物体外的特殊DNA复制，PCR的最大特点是能将微量的DNA大幅增加。
DNA，即脱氧核糖核酸，是生物细胞内含有的四种生物大分子之一核酸的一种，携带有合成RNA和蛋白质所必需的遗传信息，是生物体发育和正常运作必不可少的生物大分子。RNA，即核糖核酸，存在于生物细胞以及部分病毒、类病毒中的遗传信息载体。
PCR技术被正式发明以来，经历了从终点法定性PCR检测到实时荧光相对定量PCR检测，再到绝对定量数字PCR检测的发展历程。
发明内容
本公开实施例提供一种基因检测基板、基因检测芯片及基因检测样本的制备方法。
第一方面，本公开实施例提供一种基因检测基板，其中，包括：基底；
基因检测通道，开设于所述基底中，且所述基因检测通道的开口位于所述基底的一侧板面上；
所述基因检测通道包括进样槽、出样槽和流道槽，所述进样槽、所述流道槽和所述出样槽依次连接且彼此贯通；
所述流道槽包括多个反应孔和多个流道结构；
所述多个反应孔彼此间隔分布，且任意相邻的两个所述反应孔之间通过一个所述流道结构连通；
所述流道结构包括第一子槽、第二子槽和第三子槽，所述第一子槽、所述第二子槽和所述第三子槽依次连接并贯通；
所述第一子槽和所述第三子槽的沿第一方向的宽度小于所述第二子槽的沿所述第一方向的宽度；
所述第一方向垂直于相邻两个所述反应孔的排布方向。
在一些实施例中，所述第一子槽和所述第三子槽的深度大于所述第二子槽的深度；
所述第一子槽、所述第二子槽和所述第三子槽的深度分别为其沿所述基底厚度方向的尺寸。
在一些实施例中，所述第二子槽沿所述第一方向的宽度小于所述反应孔沿所述第一方向的宽度。
在一些实施例中，所述第一子槽和所述第三子槽的沿所述第一方向的宽度相等。
在一些实施例中，所述第一子槽和所述第三子槽的深度等于所述反应孔的深度；
所述反应孔的深度为其沿所述基底厚度方向的尺寸。
在一些实施例中，所述多个反应孔和所述多个流道结构沿第二方向排布；所述第二方向垂直于所述第一方向；
所述流道槽还包括靠近所述进样槽的第一端和靠近所述出样槽的第二端，所述流道槽还包括第一支槽和第二支槽，所述第一支槽连接于所述进样槽和位于所述第一端的第一反应孔之间，并使所述进样槽与所述第一反应孔之间连通；
所述第二支槽连接于所述出样槽和位于所述第二端的第二反应孔之间，所述第二支槽与所述第二反应孔之间还连接有所述第一子槽和所述第二子槽，所述第二反应孔、所述第一子槽、所述第二子槽和所述第二支槽沿所述第二方向依次排布；
所述第二支槽、所述第二子槽和所述第一子槽使所述出样槽与所述第二反应孔之间连通。
在一些实施例中，所述反应孔在所述基底上的正投影形状包括圆形；
所述第一子槽、所述第二子槽和所述第三子槽在所述基底上的正投影形状包括矩形。
在一些实施例中，所述反应孔在所述基底上的正投影的直径范围为68～88μm；
所述反应孔的深度范围为70～90μm；
所述第二子槽的深度范围为20～40μm。
在一些实施例中，所述第一子槽沿所述第一方向的宽度范围为20～30μm；
所述第三子槽沿所述第一方向的宽度范围为20～30μm；
所述第二子槽沿所述第一方向的宽度范围为35～45μm。
在一些实施例中，所述第一子槽沿所述第二方向的长度范围为10～20μm；
所述第三子槽沿所述第二方向的长度范围为10～20μm；
所述第二子槽沿所述第二方向的长度范围为25～35μm。
在一些实施例中，所述进样槽、所述出样槽、所述第一支槽、所述第二支槽的深度与所述第二子槽的深度分别相同；
所述进样槽、所述出样槽、所述第一支槽、所述第二支槽的深度分别为其沿所述基底厚度方向的尺寸。
在一些实施例中，所述流道槽的数量为多条；
多条所述流道槽相互平行排布；
任意相邻两条所述流道槽中的所述反应孔在沿所述第二方向延伸的直线上的正投影交替分布。
在一些实施例中，每条所述流道槽中的所述反应孔等间隔排布；
多条所述流道槽中的所述反应孔排布呈阵列。
在一些实施例中，所述基底的开设有所述基因检测通道开口的一侧板面包括第一表面和第二表面；所述第一表面为所述基因检测通道的内壁，所述第一表面上设置有亲水层；
所述第二表面上设置有疏水层。
在一些实施例中，所述基底采用聚二甲基硅氧烷、聚甲基丙烯酸甲酯、聚碳酸酯中的任意一种材料。
第二方面，本公开实施例还提供一种基因检测芯片，其中，包括上述基因检测基板。
在一些实施例中，还包括封装膜，与所述基因检测基板对盒，以对所述基因检测基板中的基因检测通道进行封装；
所述封装膜的对应所述基因检测通道中第二子槽的区域能与所述第二子槽的内壁贴合并连接。
在一些实施例中，所述封装膜包括双面胶膜或者紫外固化胶膜。
在一些实施例中，包括多块所述基因检测基板，多块所述基因检测基板相互拼接，且多块所述基因检测基板中的基因检测通道相互隔断。
第三方面，本公开实施例还提供一种基因检测样本的制备方法，其中，包括：制备基因检测基板；
将封装膜与所述基因检测基板对盒，形成基因检测芯片；
向所述基因检测芯片中基因检测通道的进样槽注入样品试剂；
当所述基因检测通道中所述样品试剂注满时，将所述封装膜的对应所述基因检测通道中第二子槽的区域与所述第二子槽的内壁贴合并连接。
在一些实施例中，所述制备基因检测基板，包括：采用构图工艺在基底上制备所述基因检测通道。
附图说明
附图用来提供对本公开实施例的进一步理解，并且构成说明书的一部 分，与本公开实施例一起用于解释本公开，并不构成对本公开的限制。通过参考附图对详细示例实施例进行描述，以上和其它特征和优点对本领域技术人员将变得更加显而易见，在附图中：
图1为本公开实施例中基因检测基板的局部结构俯视示意图。
图2为沿图1中AA＇剖切线的结构剖视图。
图3为本公开实施例中基因检测基板的局部立体结构示意图。
图4为本公开实施例基因检测基板内局部流体样本部分的三维结构图。
图5为本公开实施例中基因检测芯片的结构剖视示意图。
图6为本公开实施例中基因检测芯片的局部结构俯视示意图。
图7为本公开实施例中制备完成的基因检测样本的局部结构剖视示意图。
图8为本公开实施例中基因检测芯片的进样仿真效果示意图。
具体实施方式
为使本领域技术人员更好地理解本公开实施例的技术方案，下面结合附图和具体实施方式对本公开实施例提供的一种基因检测基板、基因检测芯片及基因检测样本的制备方法作进一步详细描述。
在下文中将参考附图更充分地描述本公开实施例，但是所示的实施例可以以不同形式来体现，且不应当被解释为限于本公开阐述的实施例。反之，提供这些实施例的目的在于使本公开透彻和完整，并将使本领域技术人员充分理解本公开的范围。
本公开实施例不限于附图中所示的实施例，而是包括基于制造工艺而形成的配置的修改。因此，附图中例示的区具有示意性属性，并且图中所示区的形状例示了区的具体形状，但并不是旨在限制性的。
公开技术中，数字PCR检测是将DNA或RNA样本分散于大量的微反应单元中，然后对众多微反应单元内的靶序列(即样本的靶序列)进行单分子模板PCR扩增、荧光检测和统计学分析，实现绝对定量；不依赖于标准 曲线和已知浓度的多个梯度标准品，直接检测样品中DNA或RNA样本的原始浓度。由于这种检测方式具有比传统荧光定量PCR检测更加出色的灵敏度和精确性，所以数字PCR检测迅速得到广泛的关注，尤其在痕量(即微小量)核酸样本检测、复杂背景下稀有突变检测、核酸拷贝数变异和基因表达量微小差异鉴定方面表现出的优势已被普遍认可。
基于聚合酶制造的PCR仪实际就是一个温控设备，该温控设备能在变性温度，复性温度，延伸温度之间很好地进行控制。微流控芯片是基于聚合酶制造的PCR仪的其中一种。
微流控芯片技术是一种以微米尺度空间对流体进行操控为主要特征的科学技术，具有将生物、化学等实验室的基本功能微缩到一个几平方厘米芯片上的能力，因此又被称为芯片实验室(lab-on-a-chip)。公开技术中，主流形式的微流控芯片多由微通道形成网络，以可控流体贯穿整个系统，用以实现常规化学或生物等实验室的各种功能。
微流控芯片的出现使得数字PCR检测应用更加便捷，通过在微流控芯片上使用数字PCR检测方法与试剂，可以实现良好的兼容性与灵活性，如：在一张芯片上，将PCR试剂划分成数百个独立的纳升级反应单元，开展数字PCR分析，提高了检测的灵敏度和准确性。
公开技术中，微流控芯片通过在基板上刻蚀加工形成的样本检测通道实现对样本溶液的进样检测。样本检测通道包括进样通道、出光通道以及连接在进样通道和出样通道之间的多个微反应单元(如微反应孔)，相邻的微反应单元之间通过相同深度和宽度的矩形沟道结构连接；在样本溶液注满各个微反应单元时，通过封装膜对样本检测通道进行封装，使微反应单元之间相互隔断形成相互独立的微腔室，以便对各微反应单元中的样本进行分析统计。
但公开技术中上述结构的微流控芯片由于其体积微小、加工难度大等原因，存在微反应孔进样不充分、微反应孔内有气泡残留、进样后相邻微 反应孔之间的封装效果不好，不能很好地将相邻微反应孔之间的连接通道隔断分开，造成微反应孔之间的试剂和样品交叉污染，影响检测效果，降低检测结果的精度和准确度等问题。
针对目前存在的上述问题，第一方面，本公开实施例提供一种基因检测基板，参照图1，为本公开实施例中基因检测基板的局部结构俯视示意图；图2为沿图1中AA＇剖切线的结构剖视图；图3为本公开实施例中基因检测基板的局部立体结构示意图；其中，包括：基底1；基因检测通道，开设于基底1中，且基因检测通道的开口位于基底1的一侧板面上；基因检测通道包括进样槽2、出样槽4和流道槽3，进样槽2、流道槽3和出样槽4依次连接且彼此贯通；流道槽3包括多个反应孔31和多个流道结构32；多个反应孔31彼此间隔分布，且任意相邻的两个反应孔31之间通过一个流道结构32连通；流道结构32包括第一子槽321、第二子槽322和第三子槽323，第一子槽321、第二子槽322和第三子槽323依次连接并贯通；第一子槽321和第三子槽323的沿第一方向Y的宽度s1小于第二子槽322的沿第一方向Y的宽度s2；第一方向Y垂直于相邻两个反应孔31的排布方向。
其中，基因检测基板可用于检测DNA(脱氧核糖核酸)样本或者RNA(核糖核酸)样本等生物样本。将DNA样本或RNA样本分散于大量的反应孔31中，然后对众多反应孔31内的靶序列(即样本的靶序列)进行单分子模板PCR(聚合酶链式反应)扩增、荧光检测和统计学分析，实现绝对定量；不依赖于标准曲线和已知浓度的多个梯度标准品，直接检测样品中DNA样本或RNA样本的原始浓度。
在一些实施例中，第一子槽321、第二子槽322和第三子槽323的沿第一方向Y的宽度为其各自沿第一方向Y的槽口尺寸。
在一些实施例中，参照图1，第二子槽322沿第一方向Y的宽度s2小于反应孔31沿第一方向Y的宽度s3。其中，反应孔31沿第一方向Y的宽度为反应孔31沿第一方向Y的孔口尺寸。
在一些实施例中，参照图1，第一子槽321和第三子槽323的沿第一方向Y的宽度s1相等。
在一些实施例中，参照图2，第一子槽321和第三子槽323的深度h1大于第二子槽322的深度h2；第一子槽321、第二子槽322和第三子槽323的深度分别为其沿基底1厚度方向的尺寸。其中，第一子槽321、第二子槽322和第三子槽323的深度为其各自的槽底所在面至槽口所在面的尺寸，即其各自深入到基底1中的深度。在一些实施例中，基底1为一块厚度均匀的平板。
在一些实施例中，第一子槽321和第三子槽323的深度等于反应孔31的深度h3；反应孔31的深度为其沿基底1厚度方向的尺寸。
本实施例中，参照图1中待测生物样本在基因检测通道中的流动路线P；基因检测通道对待测生物样本(通常为样本溶液)进行检测时，将待测生物样本输入进样槽2；在压力作用下，待测生物样本由进样槽2流入流道槽3；在待测生物样本充满流道槽3之后会流至出样槽4；即基因检测通道能够实现待测生物样本的连续进样。后续在待测生物样本进样结束时，采用封装膜对基因检测通道进行封装。第二子槽322在后续封装中作为隔断反应孔31的隔断区；如：采用压膜的方式使得第二子槽322的槽壁与封装膜的对应区域相互粘连粘结，从而达到分隔流道、在反应孔31之间进行隔断的效果。同时，在封装结束后，第一子槽321和第三子槽323与反应孔31共同充当反应区，即第一子槽321和第三子槽323成为反应孔31的补充反应区；相比于公开技术中微反应孔单独作为反应腔室的方案，增大了每一个反应区的体积，适当增加反应区的体积有利于数字聚合酶链式反应(即dPCR反应)的进行和检测，从而提高了基因检测基板的有效性。
本实施例中，通过设置第一子槽321、第二子槽322和第三子槽323的沿第一方向Y的宽度分别为窄-宽-窄设计，同时设置第一子槽321、第二子槽322和第三子槽323的深度分别为深-浅-深设计，一方面，能够保证基 因检测基板中每个反应孔31的进样效率，同时改善每个反应孔31进样后的封装效果，实现反应孔31之间的充分隔断，提高反应孔31之间的封装有效性；另一方面，能够增加反应孔31的体积，即增加有效反应区的体积，从而提高基因检测的精度和准确度；另外，该基因检测通道的结构设计相对简单、易于实现加工，封装便捷，提高了封装速率和效率。
在一些实施例中，参照图1，多个反应孔31和多个流道结构32沿第二方向X排布；第二方向X垂直于第一方向Y；流道槽3还包括靠近进样槽2的第一端和靠近出样槽4的第二端，流道槽3还包括第一支槽33和第二支槽34，第一支槽33连接于进样槽2和位于第一端的第一反应孔311之间，并使进样槽2与第一反应孔311之间连通；第二支槽34连接于出样槽4和位于第二端的第二反应孔312之间，第二支槽34与第二反应孔312之间还连接有第一子槽321和第二子槽322，第二反应孔312、第一子槽321、第二子槽322和第二支槽34沿第二方向X依次排布；第二支槽34、第二子槽322和第一子槽321使出样槽4与第二反应孔312之间连通。
其中，通过在第二反应孔312与第二支槽34之间设置第一子槽321和第二子槽322，能在后续进样结束后基因检测通道的封装中，通过第二子槽322将第二反应孔312与第二支槽34之间进行有效隔断，从而实现对第二反应孔312的独立封装。
在一些实施例中，参照图1，反应孔31在基底1上的正投影形状包括圆形；第一子槽321、第二子槽322和第三子槽323在基底1上的正投影形状包括矩形。
在一些实施例中，反应孔31的垂直于第二方向X的截面形状为一边开口的矩形或者一边开口的倒梯形，具体形状为根据实际制备工艺(如构图工艺)能够实现的形状。第一子槽321、第二子槽322和第三子槽323的垂直于第二方向X的截面形状分别为一边开口的矩形或者一边开口的倒梯形，具体形状为根据实际制备工艺(如构图工艺)能够实现的形状。
在一些实施例中，反应孔31在基底1上的正投影的直径范围为68～88μm；反应孔31的深度范围为70～90μm；第二子槽322的深度范围为20～40μm。
在一些实施例中，反应孔31在基底1上的正投影的直径为78μm；反应孔31的深度为80μm；第二子槽322的深度为30μm。
在一些实施例中，第一子槽321沿第一方向Y的宽度范围为20～30μm；第三子槽323沿第一方向Y的宽度范围为20～30μm；第二子槽322沿第一方向Y的宽度范围为35～45μm。
在一些实施例中，第一子槽321沿第一方向Y的宽度为25μm；第三子槽323沿第一方向Y的宽度为25μm；第二子槽322沿第一方向Y的宽度为40μm。该尺寸设计使得第二子槽322的两端分别为宽度较窄的第一子槽321和第三子槽323，由宽度较窄的第一子槽321和第三子槽323与直径较大的反应孔31直接连接，而非由宽度较宽的第二子槽321直接与反应孔31连接；这在实际封装过程中有助于宽度较宽的第二子槽322的内壁与封装膜贴合后的紧密连接，减少第二子槽322所在区域封装后的漏液现象，从而避免检测过程中相邻两个反应孔31之间的待测生物样本发生串扰，提高检测精度和准确度。
在一些实施例中，第一子槽321沿第二方向X的长度范围为10～20μm；第三子槽323沿第二方向X的长度范围为10～20μm；第二子槽322沿第二方向X的长度范围为25～35μm。
在一些实施例中，第一子槽321沿第二方向X的长度为15μm；第三子槽323沿第二方向X的长度为15μm；第二子槽322沿第二方向X的长度为32μm。在一些实施例中，沿第二方向X排布的任意相邻两个反应孔31在基底1上正投影的中心之间的间距为140μm。
在一些实施例中，进样槽2、出样槽4、第一支槽33、第二支槽34的深度与第二子槽322的深度分别相同；进样槽2、出样槽4、第一支槽33、 第二支槽34的深度分别为其沿基底1厚度方向的尺寸。参照图4，为本公开实施例基因检测基板内局部流体样本部分的三维结构图，如此设置，能够提高基因检测通道的进样效率，同时能使反应孔31被填充满，反应孔31内不会产生气泡，提升了基因检测通道进样以及反应孔31排气的有效性。
在一些实施例中，流道槽3的数量为多条；多条流道槽3相互平行排布；任意相邻两条流道槽3中的反应孔31在沿第二方向X延伸的直线上的正投影交替分布。如此设置，能够在分布反应孔31时充分利用基底1的面积，使基底1的有效利用面积上分布更多数量的反应孔31，从而提高基底1的利用率。
在一些实施例中，每条流道槽3中的反应孔31等间隔排布；多条流道槽3中的反应孔31排布呈阵列。如此设置，同样能使基底1的有效利用面积上分布更多数量的反应孔31，提高基底1的利用率；同时还能提高基因检测的精度这准确度。
在一些实施例中，进样槽2包括进样孔21和进样子槽22，进样孔21位于进样子槽22的一端端部，且进样孔21和进样子槽22连通。出样槽4包括出样孔41和出样子槽42，出样孔41位于出样子槽42的一端端部，且出样孔41和出样子槽42连通。多条流道槽3分别连接于进样子槽22和出样子槽42之间且相互连通。进样子槽22和出样子槽42分别沿第一方向Y延伸。进样子槽22和出样子槽42的沿第二方向X的宽度大于流道槽3沿第一方向Y的最大宽度。进样孔21和出样孔41在基底1上的正投影形状均为圆形，圆形的直径大于流道槽3沿第一方向Y的最大宽度。参照图3，如此设置，能够提高基因检测通道的进样效率，同时能使反应孔31被填充满，反应孔31内不会产生气泡，提升了基因检测通道进样以及反应孔31排气的有效性。
在一些实施例中，基底1的开设有基因检测通道开口的一侧板面包括第一表面和第二表面；第一表面为基因检测通道的内壁，第一表面上设置 有亲水层；第二表面上设置有疏水层，即基底1的板面除基因检测通道表面以外的区域设置有疏水层。本实施例中，基底1上未设置有基因检测通道的区域表面设置有疏水层。反应孔31、第一子槽321、第二子槽322和第三子槽323的长度、宽度和深度均为微米级别的尺寸。如此设置，能够利用毛细力和表面张力等作用促进待测生物样本溶液向反应孔31内的精确加载，从而能够促进待测生物样本溶液流入反应孔31内，避免待测生物样本的浪费。
在一些实施例中，基底1采用聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、聚碳酸酯(PC塑料)中的任意一种材料。在基底1中开设基因检测通道通过曝光、显影即可实现制备，制备工艺简单，便于量产。
本公开实施例中所提供的基因检测基板，通过设置第一子槽、第二子槽和第三子槽的沿第一方向的宽度分别为窄-宽-窄设计，同时设置第一子槽、第二子槽和第三子槽的深度分别为深-浅-深设计，一方面，能够保证基因检测基板中每个反应孔的进样效率，同时改善每个反应孔进样后的封装效果，实现反应孔之间的充分隔断，提高反应孔之间的封装有效性；另一方面，能够增加反应孔的体积，即增加有效反应区的体积，从而提高基因检测的精度和准确度；另外，该基因检测通道的结构设计相对简单、易于实现加工，封装便捷，提高了封装速率和效率。
第二方面，本公开实施例还提供一种基因检测芯片，其中，包括上述实施例中的基因检测基板。
在一些实施例中，参照图5，为本公开实施例中基因检测芯片的结构剖视示意图；其中，基因检测芯片还包括封装膜5，与基因检测基板6对盒，以对基因检测基板6中的基因检测通道进行封装；封装膜5的对应基因检测通道中第二子槽322的区域能与第二子槽322的内壁贴合并连接。从而实现对基因检测通道中相邻反应孔31之间的隔断，使每个反应孔31形成独立的反应腔室，以便基因检测、统计和分析。
在一些实施例中，封装膜5包括双面胶膜或者紫外固化胶膜。双面胶膜或者紫外固化胶膜的一面含有胶质的薄膜与第二子槽322的内壁粘连，达到分隔流道、分离反应孔31的效果。采用胶膜有助于封装膜5与第二子槽322对应表面贴合后的紧密连接，减少封装区域漏液，反应孔31之间串扰。封装膜5可通过压膜的方式与第二子槽322的内壁粘连。
在一些实施例中，参照图6，为本公开实施例中基因检测芯片的局部结构俯视示意图；其中，基因检测芯片包括多块基因检测基板6，多块基因检测基板6相互拼接，且多块基因检测基板6中的基因检测通道相互隔断。
其中，一块基因检测基板6用于分析一个待测生物样本，多块基因检测基板6可分别用于分析不同的待测生物样本，从而实现在一张基因检测芯片上同时分析多个待测生物样本，从而提升了该基因检测芯片的基因检测通量。
本公开实施例中所提供的基因检测芯片，通过采用上述基因检测基板，一方面，能够保证基因检测基板中每个反应孔的进样效率，同时改善每个反应孔进样后的封装效果，实现反应孔之间的充分隔断，提高反应孔之间的封装有效性；另一方面，能够增加反应孔的体积，即增加有效反应区的体积，从而提高基因检测的精度和准确度。
第三方面，本公开实施例还提供一种基因检测样本的制备方法，其中，包括：步骤S01：制备基因检测基板。
该步骤中，采用构图工艺在基底上制备基因检测通道。通过曝光、显影工艺即可在基底上制备基因检测通道。
步骤S02：将封装膜与基因检测基板对盒，形成基因检测芯片。
该步骤中，封装膜与基底上基因检测通道所在侧进行对盒覆盖。
步骤S03：向基因检测芯片中基因检测通道的进样槽注入样品试剂。
该步骤中，通过灌注或滴注的方法向进样槽的进样孔注入样品试剂。样品试剂通常为溶液状态。
步骤S04：当基因检测通道中样品试剂注满时，将封装膜的对应基因检测通道中第二子槽的区域与第二子槽的内壁贴合并连接。
该步骤中，可通过压膜的方式使封装膜与第二子槽的内壁粘连粘结。
本实施例中，参照图7，为本公开实施例中制备完成的基因检测样本的局部结构剖视示意图；图8为本公开实施例中基因检测芯片的进样仿真效果示意图；其中，对该基因检测芯片的进样效果使用Ansys Fluent仿真软件进行了仿真验证，从图8中可见，网格填充的进样槽2和流道槽3内充满了样品试剂，进样槽2和流道槽3内没有气泡；斜纹填充的出样槽4(特别是出样孔41)内有空气存在；验证结果显示，该基因检测芯片中的基因检测通道进样后反应孔31内部被样品试剂7填充满，反应孔31内没有气泡产生；表明了该基因检测通道在进样及反应孔31排气上的结构设计有效性。
可以理解的是，以上实施方式仅仅是为了说明本公开的原理而采用的示例性实施方式，然而本公开并不局限于此。对于本领域内的普通技术人员而言，在不脱离本公开的精神和实质的情况下，可以做出各种变型和改进，这些变型和改进也视为本公开的保护范围。

Claims (21)

  1. 一种基因检测基板，其中，包括：基底；
    基因检测通道，开设于所述基底中，且所述基因检测通道的开口位于所述基底的一侧板面上；
    所述基因检测通道包括进样槽、出样槽和流道槽，所述进样槽、所述流道槽和所述出样槽依次连接且彼此贯通；
    所述流道槽包括多个反应孔和多个流道结构；
    所述多个反应孔彼此间隔分布，且任意相邻的两个所述反应孔之间通过一个所述流道结构连通；
    所述流道结构包括第一子槽、第二子槽和第三子槽，所述第一子槽、所述第二子槽和所述第三子槽依次连接并贯通；
    所述第一子槽和所述第三子槽的沿第一方向的宽度小于所述第二子槽的沿所述第一方向的宽度；
    所述第一方向垂直于相邻两个所述反应孔的排布方向。
  2. 根据权利要求1所述的基因检测基板，其中，所述第一子槽和所述第三子槽的深度大于所述第二子槽的深度；
    所述第一子槽、所述第二子槽和所述第三子槽的深度分别为其沿所述基底厚度方向的尺寸。
  3. 根据权利要求1或2所述的基因检测基板，其中，所述第二子槽沿所述第一方向的宽度小于所述反应孔沿所述第一方向的宽度。
  4. 根据权利要求1或2所述的基因检测基板，其中，所述第一子槽和所述第三子槽的沿所述第一方向的宽度相等。
  5. 根据权利要求2所述的基因检测基板，其中，所述第一子槽和所述第三子槽的深度等于所述反应孔的深度；
    所述反应孔的深度为其沿所述基底厚度方向的尺寸。
  6. 根据权利要求1所述的基因检测基板，其中，所述多个反应孔和所述多个流道结构沿第二方向排布；所述第二方向垂直于所述第一方向；
    所述流道槽还包括靠近所述进样槽的第一端和靠近所述出样槽的第二端，所述流道槽还包括第一支槽和第二支槽，所述第一支槽连接于所述进样槽和位于所述第一端的第一反应孔之间，并使所述进样槽与所述第一反应孔之间连通；
    所述第二支槽连接于所述出样槽和位于所述第二端的第二反应孔之间，所述第二支槽与所述第二反应孔之间还连接有所述第一子槽和所述第二子槽，所述第二反应孔、所述第一子槽、所述第二子槽和所述第二支槽沿所述第二方向依次排布；
    所述第二支槽、所述第二子槽和所述第一子槽使所述出样槽与所述第二反应孔之间连通。
  7. 根据权利要求1所述的基因检测基板，其中，所述反应孔在所述基底上的正投影形状包括圆形；
    所述第一子槽、所述第二子槽和所述第三子槽在所述基底上的正投影形状包括矩形。
  8. 根据权利要求7所述的基因检测基板，其中，所述反应孔在所述基底上的正投影的直径范围为68～88μm；
    所述反应孔的深度范围为70～90μm；
    所述第二子槽的深度范围为20～40μm。
  9. 根据权利要求1所述的基因检测基板，其中，所述第一子槽沿所述第一方向的宽度范围为20～30μm；
    所述第三子槽沿所述第一方向的宽度范围为20～30μm；
    所述第二子槽沿所述第一方向的宽度范围为35～45μm。
  10. 根据权利要求6所述的基因检测基板，其中，所述第一子槽沿所述第二方向的长度范围为10～20μm；
    所述第三子槽沿所述第二方向的长度范围为10～20μm；
    所述第二子槽沿所述第二方向的长度范围为25～35μm。
  11. 根据权利要求6所述的基因检测基板，其中，所述进样槽、所述出样槽、所述第一支槽、所述第二支槽的深度与所述第二子槽的深度分别相同；
    所述进样槽、所述出样槽、所述第一支槽、所述第二支槽的深度分别为其沿所述基底厚度方向的尺寸。
  12. 根据权利要求1所述的基因检测基板，其中，所述流道槽的数量为多条；
    多条所述流道槽相互平行排布；
    任意相邻两条所述流道槽中的所述反应孔在沿所述第二方向延伸的直线上的正投影交替分布。
  13. 根据权利要求12所述的基因检测基板，其中，每条所述流道槽中的所述反应孔等间隔排布；
    多条所述流道槽中的所述反应孔排布呈阵列。
  14. 根据权利要求书1所述的基因检测基板，其中，所述基底的开设有所述基因检测通道开口的一侧板面包括第一表面和第二表面；
    所述第一表面为所述基因检测通道的内壁，所述第一表面上设置有亲水层；
    所述第二表面上设置有疏水层。
  15. 根据权利要求1所述的基因检测基板，其中，所述基底采用聚二甲基硅氧烷、聚甲基丙烯酸甲酯、聚碳酸酯中的任意一种材料。
  16. 一种基因检测芯片，其中，包括权利要求1-15任意一项所述的基因检测基板。
  17. 根据权利要求16所述的基因检测芯片，其中，还包括封装膜，与所述基因检测基板对盒，以对所述基因检测基板中的基因检测通道进行封装；
    所述封装膜的对应所述基因检测通道中第二子槽的区域能与所述第二子槽的内壁贴合并连接。
  18. 根据权利要求17所述的基因检测芯片，其中，所述封装膜包括双面胶膜或者紫外固化胶膜。
  19. 根据权利要求16所述的基因检测芯片，其中，包括多块所述基因检测基板，多块所述基因检测基板相互拼接，且多块所述基因检测基板中的基因检测通道相互隔断。
  20. 一种基因检测样本的制备方法，其中，包括：制备基因检测基板；
    将封装膜与所述基因检测基板对盒，形成基因检测芯片；
    向所述基因检测芯片中基因检测通道的进样槽注入样品试剂；
    当所述基因检测通道中所述样品试剂注满时，将所述封装膜的对应所述基因检测通道中第二子槽的区域与所述第二子槽的内壁贴合并连接。
  21. 根据权利要求20所述的制备方法，其中，所述制备基因检测基板，包括：采用构图工艺在基底上制备所述基因检测通道。
PCT/CN2022/093764 2022-05-19 2022-05-19 基因检测基板、基因检测芯片及基因检测样本的制备方法 WO2023221023A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202280001263.7A CN117529546A (zh) 2022-05-19 2022-05-19 基因检测基板、基因检测芯片及基因检测样本的制备方法
PCT/CN2022/093764 WO2023221023A1 (zh) 2022-05-19 2022-05-19 基因检测基板、基因检测芯片及基因检测样本的制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/093764 WO2023221023A1 (zh) 2022-05-19 2022-05-19 基因检测基板、基因检测芯片及基因检测样本的制备方法

Publications (1)

Publication Number Publication Date
WO2023221023A1 true WO2023221023A1 (zh) 2023-11-23

Family

ID=88834252

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/093764 WO2023221023A1 (zh) 2022-05-19 2022-05-19 基因检测基板、基因检测芯片及基因检测样本的制备方法

Country Status (2)

Country Link
CN (1) CN117529546A (zh)
WO (1) WO2023221023A1 (zh)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006078475A (ja) * 2004-08-09 2006-03-23 Nsk Ltd 反応器及びその製造方法
JP2006223126A (ja) * 2005-02-15 2006-08-31 Shimadzu Corp 反応容器、遺伝子多型検出方法及び装置、並びに診断方法及び装置
JP2007136322A (ja) * 2005-11-17 2007-06-07 Konica Minolta Medical & Graphic Inc 反応物質同士の拡散および反応を効率化したマイクロリアクタ、およびそれを用いた反応方法
JP2011211946A (ja) * 2010-03-31 2011-10-27 Toppan Printing Co Ltd 反応チップ、反応チップの製造方法及び反応方法
JP2013051958A (ja) * 2011-08-05 2013-03-21 Toshiba Corp マルチ核酸増幅反応具
US20190217298A1 (en) * 2018-01-17 2019-07-18 Formulatrix, Inc. Microfluidic device with vented microchambers
CN110437978A (zh) * 2019-08-12 2019-11-12 北京航空航天大学 细菌总量、细菌孢子量的一体化的检测微流控芯片
WO2021038996A1 (ja) * 2019-08-29 2021-03-04 ファナック株式会社 細胞製造装置及びその製造方法
CN215757346U (zh) * 2021-08-30 2022-02-08 上海睿钰生物科技有限公司 一种微粒检测装置
CN114364786A (zh) * 2019-09-27 2022-04-15 富士胶片株式会社 容器及检测试剂盒

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006078475A (ja) * 2004-08-09 2006-03-23 Nsk Ltd 反応器及びその製造方法
JP2006223126A (ja) * 2005-02-15 2006-08-31 Shimadzu Corp 反応容器、遺伝子多型検出方法及び装置、並びに診断方法及び装置
JP2007136322A (ja) * 2005-11-17 2007-06-07 Konica Minolta Medical & Graphic Inc 反応物質同士の拡散および反応を効率化したマイクロリアクタ、およびそれを用いた反応方法
JP2011211946A (ja) * 2010-03-31 2011-10-27 Toppan Printing Co Ltd 反応チップ、反応チップの製造方法及び反応方法
JP2013051958A (ja) * 2011-08-05 2013-03-21 Toshiba Corp マルチ核酸増幅反応具
US20190217298A1 (en) * 2018-01-17 2019-07-18 Formulatrix, Inc. Microfluidic device with vented microchambers
CN110437978A (zh) * 2019-08-12 2019-11-12 北京航空航天大学 细菌总量、细菌孢子量的一体化的检测微流控芯片
WO2021038996A1 (ja) * 2019-08-29 2021-03-04 ファナック株式会社 細胞製造装置及びその製造方法
CN114364786A (zh) * 2019-09-27 2022-04-15 富士胶片株式会社 容器及检测试剂盒
CN215757346U (zh) * 2021-08-30 2022-02-08 上海睿钰生物科技有限公司 一种微粒检测装置

Also Published As

Publication number Publication date
CN117529546A (zh) 2024-02-06

Similar Documents

Publication Publication Date Title
EP2715357B1 (en) Microfluidic disc for use in with bead-based immunoassays
Ouellette A new wave of microfluidic devices
JP4141494B2 (ja) マイクロ分析測定装置及びそれを用いたマイクロ分析測定方法
US7476361B2 (en) Microfluidics devices and methods of diluting samples and reagents
US20020151078A1 (en) Microfluidics devices and methods for high throughput screening
US20090155125A1 (en) Microchip
US20200408752A1 (en) Fluidic system for performing assays
JP5137012B2 (ja) マイクロチップ
JP2004501360A (ja) ミクロ流体装置および高スループット・スクリーニングのための方法
US11123728B2 (en) Fast sample loading microfluidic reactor and system
WO2023221023A1 (zh) 基因检测基板、基因检测芯片及基因检测样本的制备方法
US20090291025A1 (en) Microchip And Method Of Using The Same
JP5137014B2 (ja) マイクロチップ
US6801875B1 (en) Methods, systems, and software for performing measurements
CN110736844A (zh) 一种心肌肌钙蛋白的检测方法
JP2013164268A (ja) マイクロチップ
CN111748464B (zh) 一种数字pcr芯片的制作方法和数字pcr芯片
CN208803052U (zh) 一种用于基因分型的多指标微流控芯片
JP2009281869A (ja) マイクロチップ
Tan et al. Droplet array with microfluidic concentration gradient (DA-MCG) for 2-dimensional reaction condition screening
Lutz Centrifugal microfluidic platforms for protein and nucleic acid analysis
Sedighi et al. 8 2-D Nanofluidic Bioarray
Wang et al. 563 Two-Dimensional Microfluidic Bioarray for Nucleic Acid Analysis
JP2014134393A (ja) マイクロチップ

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 202280001263.7

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22942065

Country of ref document: EP

Kind code of ref document: A1