阅读说明：本技术 具有集成处理及扩增显色功能的自驱动微流控芯片 (Self-driven micro-fluidic chip with integrated processing and amplification color development functions ) 是由 曹志 黄倢 董宣 邹培卓 万晓媛 李晨 于 2019-10-16 设计创作，主要内容包括：本发明属于微生物检测技术领域,尤其涉及一种具有集成处理及扩增显色功能的自驱动微流控芯片。所述芯片由若干病原检测单体组合而成；病原检测单体上各样液池由通道联通,所述通道通过遮蔽技术由纳米材料构建。本发明可以直接从组织裂解液中一步到位进行扩增,能有效降低样品检测成本,提高检测效率,保障检测准确性,从根本上增进对虾种业的疫病筛查能力。(The invention belongs to the technical field of microbial detection, and particularly relates to a self-driven micro-fluidic chip with integrated processing and amplification color development functions. The chip is formed by combining a plurality of pathogen detection monomers; the various liquid pools on the pathogen detection monomer are communicated by a channel, and the channel is constructed by a nano material through a shielding technology. The invention can directly amplify from the tissue lysate in one step, effectively reduce the sample detection cost, improve the detection efficiency, ensure the detection accuracy and fundamentally improve the epidemic disease screening capability of the shrimp breeding industry.)

1. A self-driven micro-fluidic chip with integrated processing and amplification color development functions is characterized in that: is formed by combining a plurality of pathogen detection monomers; the various liquid pools on the pathogen detection monomer are communicated by a channel, and the channel is constructed by a nano material through a shielding technology.

2. The self-driven microfluidic chip with integrated processing and color amplification functions as claimed in claim 1, wherein: the sample liquid pool comprises a sample adding pool, a nucleic acid denaturation pool, an amplification detection pool and an overflow pool which are sequentially arranged according to the liquid flow direction; a nucleic acid extraction channel is arranged between the sample adding pool and the nucleic acid denaturation pool.

3. The self-driven microfluidic chip with integrated processing and color amplification functions as claimed in claim 2, wherein: the surface of the nucleic acid extraction channel structure is subjected to molecular modification by utilizing a solid phase reversible immobilization principle.

4. The self-driven microfluidic chip with integrated processing and color amplification functions as claimed in claim 2 or 3, wherein: the chip is divided into an upper layer and a lower layer, all channels, the nucleic acid denaturation pool, the amplification detection pool and the overflow pool are arranged on the lower layer of the chip, and the sample adding pool penetrates through the upper layer and the lower layer of the chip.

5. The self-driven microfluidic chip with integrated processing and color amplification functions as claimed in claim 2 or 3, wherein: the amplification detection tanks are provided with a plurality of amplification detection tanks, are symmetrically distributed in a radial circular shape around the nucleic acid denaturation tank according to the center, and the periphery of the amplification detection tanks is provided with an overflow tank.

6. The self-driven microfluidic chip with integrated processing and color amplification functions as claimed in claim 2 or 3, wherein: the chip is rectangular and has the size of 25 multiplied by 80 mm; the thickness of the upper layer and the lower layer of the chip is 2.0 mm; the width of the channel is 0.1mm, and the depth is 0.1 mm; the diameter of the nucleic acid denaturation tank is 2.0mm, and the depth is 1.0 mm; the diameter of the amplification detection pool is 1.0mm, and the depth is 1.0 mm; the diameter of the overflow pool is 1.1mm, and the depth is 1.0 mm; the diameter of the sample adding pool is 2.0mm, and the depth is 2.0 mm; the chip is made of polymethyl methacrylate.

7. The self-driven microfluidic chip with integrated processing and color amplification functions as claimed in any one of claims 1 to 3, wherein: the nano materials for constructing the channel are super-hydrophilic ZXL-CQS nano self-cleaning liquid and super-hydrophobic ZXL-CSS nano self-cleaning liquid.

8. A method for preparing the self-driven microfluidic chip with integrated processing and amplification color development functions as claimed in claim 1, which is characterized by comprising the following steps: designing, processing and assembling a die according to the chip structure diagram; pouring polymethyl methacrylate; cleaning the chip after curing; carrying out molecular modification on the surface of a channel structure of a lab-on-a-chip; constructing a chromatography channel of nucleic acid; pre-embedding a detection reagent into the detection pool by utilizing a freeze-drying process; the upper layer and the lower layer of the chip are assembled by low-temperature bonding or adhesion.

9. The use of the self-driven microfluidic chip with integrated processing and color amplification functions as claimed in claim 1, wherein: the method is used for detecting pathogens in prawn samples.

Technical Field

The invention belongs to the technical field of microbial detection, and particularly relates to a self-driven micro-fluidic chip with integrated processing and amplification color development functions.

Background

From the current situation and trend of disease prevention and control development of prawn cultivation in China, the technology upgrade and structure adjustment of prawn cultivation industry in China are the key points to be guided by biological security, and the development of biological security work needs a Point-of-care testing (POCT) technology with larger sample quantity, more pathogens, accuracy, sensitivity and low cost. The ideal POCT analysis integrates sample preparation, amplification and detection, and can get rid of the limitation of detection environment. Polymerase Chain Reaction (PCR) is the standard for high sensitivity molecular detection at present, but is not suitable for fast and portable field operation; as it typically involves laboratory equipment that requires an external power source (e.g., thermocyclers and centrifuges), several hours of assay time, multiple manual sample preparation steps, and a trained technician. Therefore, it is very important to convert the centralized laboratory test into the miniaturized and practical research of nucleic acid detection which is ubiquitous in the field culture environment.

The first option for miniaturization and practicality is to reduce the complexity of the machinery required. Fully automated integrated Real-time PCR instruments have been commercialized (e.g., GeneXpert, Cepheid); however, the expensive detection cost of high precision instruments remains an obstacle for large scale adoption. The Nucleic Acid Isothermal Amplification Technology (NAIAT) is very suitable for POCT because of the advantages of constancy of reaction temperature, simple and rapid detection procedure, elimination of high-precision instruments and the like. The development results of the microfluidic chip technology are injecting new vitality into the medical detection field, and the conventional laboratory technology of nucleic acid detection can be completely integrated into a very small chip, so that the analysis target of 'Sample to answer' is realized. The high-efficiency integration of the microfluidic chip technology and the NAIAT is expected to bring revolutionary breakthrough to the development of low-cost and portable rapid detection technology of nucleic acid (RNA and DNA).

The option for further miniaturization and practicality is to simplify the overall process from sample input to result output. The problem to be solved is how to realize the self-driving of the microfluid by using a very simple microfluid chip structure without any external equipment driving; secondly, there is a need for a method that can concentrate reagents into highly defined dot blots suitable for microwells, so that multiple reactions can be initiated directly in microwells without cross-contamination.

Disclosure of Invention

The invention aims to solve the technical problem that a pathogen detection kit based on nucleic acid amplification and a high-throughput detection chip can not process samples, although various thermal cycle amplification or isothermal amplification technologies have a lot of new developments in recent years, the extraction technology of an amplification template is always lack of more convenient and faster breakthrough, and becomes a bottleneck influencing the application of various detection kits or chips based on nucleic acid amplification technologies. The existing various nucleic acid extraction kits have high cost and complex extraction operation, depend on certain instruments and equipment, are mainly suitable for scientific research work, can be used for thousands of detection reactions after one-time extraction of nucleic acid, and are too wasted when used for ultramicro detection of mass samples. At present, a chip laboratory develops a nucleic acid extraction technology on a microfluidic chip, but external force driving and precise flow channel design are needed, so that high cost and a complex operation process still exist, and the application of the chip laboratory in on-site instant detection is limited.

In order to solve the problems, a self-driven micro-fluidic chip laboratory integrating nucleic acid extraction and constant-temperature amplification functions is provided, amplification can be directly carried out in one step from tissue lysate, the sample detection cost can be effectively reduced, the detection efficiency is improved, the detection accuracy is guaranteed, and the epidemic disease screening capability of the shrimp breeding industry is fundamentally improved. The technology adopts a plurality of nano materials to shield and spray a microfluidic self-driven channel, carries out molecular modification on the surface of the microfluidic channel structure, establishes a chromatography channel for nucleic acid extraction of the channel, and combines buffer solution component optimization and local precise temperature control processing of the channel to realize the nucleic acid extraction function of the microfluidic self-driven chip. The amplification reaction is indicated by direct color development of the amplification product, and the method is visual, simple and convenient; fixing the required reagent in a detection pool of the chip by a pre-embedding technology; the sample automatically flows into the detection pool through the nucleic acid extraction channel by self-driven microfluidics, so that the operation flow is greatly simplified; the detection of various pathogens is realized through the accurate design of the branches of the multi-path detection pool; a plurality of repeating units are designed in a lab-on-a-chip to realize the detection capability of multiple samples.

In order to achieve the purpose, the invention is realized by the following technical scheme: a self-driven micro-fluidic chip with integrated processing and amplification color development functions is formed by combining a plurality of pathogen detection monomers; the various liquid pools on the pathogen detection monomer are communicated by a channel, the channel is constructed by a nano material through a shielding technology, the interaction between the fluid and the surface of the chip is adjusted by screening a modified material, and the self-driving of the fluid is realized by utilizing the capillary action of microfluid in combination with the symmetrical design of the fluid channel, so that the micro-droplets of the sample can automatically flow through each channel and the sample liquid pool.

Further, as shown in fig. 1 and fig. 2, the self-driven microfluidic chip may be provided with a 7 pathogen detection monomer structure with 6 sample combinations, and the design of multiple repeating units may realize the detection capability for multiple samples. Wherein, each sample liquid pool with a monomer structure comprises a sample adding pool 1, a nucleic acid denaturation pool 2, an amplification detection pool 3 and an overflow pool 4 which are sequentially arranged according to the liquid flow direction; a nucleic acid extraction channel 5 is arranged between the sample adding pool 1 and the nucleic acid denaturation pool 2. The capillary action of microfluid is utilized to realize self-driving, so that the sample micro-droplets in the sample adding pool automatically flow through the nucleic acid extraction channel to enter the nucleic acid denaturation pool, then enter the amplification detection pool through the channel, and the redundant liquid can enter the overflow pool; a multi-branch nucleic acid isothermal amplification detection pool is designed behind a nucleic acid extraction channel of the chip, a nucleic acid isothermal amplification reaction system, a target gene specific primer, an immobilized enzyme preparation, a freeze-drying protective agent and an amplification reaction indicator are added into the amplification detection pool, and a self-driven microfluidic chip laboratory with integrated functions of nucleic acid extraction, nucleic acid amplification, amplification reaction color development and the like is established.

Further, the surface of the structure of the nucleic acid extraction channel 5 is subjected to molecular modification by using the Solid-phase reversible immobilization (SPRI) principle. The extraction and purification of the sample nucleic acid can be realized by the surface carboxylation of the micro-channel after the molecular modification and the combination of the binding buffer solution with the optimal proportion.

Furthermore, the chip is divided into an upper layer and a lower layer, all the channels, the nucleic acid denaturation pool, the amplification detection pool and the overflow pool are arranged on the lower layer of the chip, so that the super-hydrophilic channel and the nucleic acid denaturation channel are constructed in advance, and a detection reagent is pre-embedded in the amplification detection pool; the sample adding pool penetrates through the upper layer and the lower layer of the chip.

Furthermore, a plurality of amplification detection pools 3 are arranged, are symmetrically distributed in a radial circular shape around the nucleic acid denaturation pool 2 according to the center, and the periphery of the amplification detection pools is provided with an overflow pool. The detection of various pathogens can be realized through the accurate design of the branches of the multi-path detection pool.

Further, as shown in fig. 3, the chip is rectangular and has a size of 25 × 80 mm; the thickness of the upper layer and the lower layer of the chip is 2.0 mm; the width of the channel is 0.1mm, and the depth is 0.1 mm; the diameter of the nucleic acid denaturation tank is 2.0mm, and the depth is 1.0 mm; the diameter of the amplification detection pool is 1.0mm, and the depth is 1.0 mm; the diameter of the overflow pool is 1.1mm, and the depth is 1.0 mm; the diameter of the sample adding pool is 2.0mm, and the depth is 2.0 mm; the material of the chip is polymethyl methacrylate (PMMA). The larger the size, the more volume of the detection system is needed, and the cost is high; the smaller the size is, the lower the cost is, but the manufacturing difficulty is correspondingly increased; the dimensions defined by the invention are the minimum dimensions that can be achieved under the conditions of the prior art, and also the minimum final reaction volume that can be detected.

Furthermore, the nano materials for constructing the channel are super-hydrophilic ZXL-CQS nano self-cleaning liquid and super-hydrophobic ZXL-CSS nano self-cleaning liquid. Both of these are materials that can optimize the effect of channel surface superhydrophilic or superhydrophobic, resulting in the best quality chip.

A preparation method of the self-driven microfluidic chip comprises the following steps: designing, processing and assembling a die according to the structure diagram of the chip, and pouring a poly-ACuring methyl acrylate, and cleaning the chip; carrying out molecular modification on the surface of a channel structure of a lab-on-a-chip, namely, spraying and painting a super-hydrophobic nano material on an upper substrate and spraying and painting a super-hydrophilic nano material on a lower substrate; construction of chromatographic channels for nucleic acids, i.e.subjecting microfluidic channels to amino groups (NH) on the surface at room temperature₂-) modification; pre-embedding a detection reagent into the detection pool by utilizing a freeze-drying process; the upper layer and the lower layer of the chip are assembled by low-temperature bonding or adhesion.

Further, the detection reagent embedded in the detection pool comprises a conventional reagent for isothermal amplification of nucleic acid, a specific primer and a freeze-drying protective agent; the freeze-drying protective agent comprises trehalose and bovine serum albumin, wherein the mass concentration of the trehalose in the detection reagent before freeze-drying is 0.1-0.15 g/mL, and the mass concentration of the bovine serum albumin is 0.01-0.02 g/mL.

The invention also provides an application of the self-driven microfluidic detection chip, which is used for detecting pathogens in prawn samples.

Wherein, the 7 pathogens of the prawn comprise 5 DNA viruses of the prawn, namely prawn White Spot Syndrome Virus (WSSV), Infectious subcutaneous and hematopoietic necrosis virus (IHHNV), decapod iridescent virus1 (DIV 1), Acute hepatopancreas necrosis disease (AHPND), and prawn Enterosporidium (EHP);

2 kinds of prawn RNA viruses are Taura Syndrome Virus (TSV) and Covert Mortality Nodavirus (CMNV).

Compared with the prior art, the invention has the following advantages:

1) the invention applies the nanometer modified material to the modification of the chip fluid self-driven channel for the first time, provides the nucleic acid chromatography extraction technology of the channel surface molecule modification, and provides a simple and convenient solution for the problem of nucleic acid extraction of the self-driven micro-fluidic chip; the interaction between the fluid and the chip surface is adjusted by screening the modified material, and the fluid is self-driven by combining the symmetrical design of the fluid channel, so that the fluid gets rid of the problems such as a centrifuge and an injection pump, and a precise fluid channel with expensive research and development and production cost is not needed.

2) The optimal micro-fluidic chip reagent pre-embedding process screened by the invention greatly simplifies the detection operation process, so that multiple reactions can be directly started in micropores without cross contamination.

3) The invention realizes multifunctional organic integration of a self-driven microfluidic technology, a channel chromatography technology for sample nucleic acid extraction, a reagent pre-embedding freeze-drying technology, an enzyme immobilization technology, an ultramicro NAIAT technology and an amplification reaction real-time color development technology in a lab-on-a-chip, develops the self-driven integrated microfluidic field instant low-cost detection lab-on-a-chip, directly carries out simple, rapid, sensitive, specific and synchronous instant detection on various pathogens from prawn tissue samples, and is expected to become a practical low-cost, portable and integrated high-sensitivity nucleic acid detection technology.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.

FIG. 1 is a schematic structural diagram of a pathogen detection monomer of the present invention 7;

FIG. 2 is a diagram of a combination of the samples of the present invention 6;

FIG. 3 is a schematic structural diagram of a self-driven microfluidic chip in example 1;

in the figure, a sample adding pool 1, a nucleic acid denaturation pool 2, an amplification detection pool 3, an overflow pool 4 and a nucleic acid extraction channel 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the specific techniques or conditions are not indicated in the examples, and the techniques or conditions are described in the literature in the field or according to the product specification; the reagents and materials, both of which are analytically pure reagents, are commercially available without specific reference. The adopted solution is prepared by deionized water for sterilizing and inactivating degradation enzyme.

Equipment for chip injection molding technology, Sumitomo Heavy industries, Ltd., JAPAN, SE180 DU-C360.

Model LY0-0.5 Dongfulong freeze dryer, available from Shanghai Dongfulong science and technology Co.

The super-hydrophilic ZXL-CQS nano self-cleaning solution and the super-hydrophobic ZXL-CSS nano self-cleaning solution are purchased from Rioyangzi Xilai environmental protection science and technology Limited company.

The Kafft K-303 shadowless adhesive transparent acrylic adhesive is purchased from Higashi-Tech Co., Ltd.

The Litopenaeus vannamei tissues with positive WSSV, IHHNV, DIV1, AHPND, EHP, TSV and CMNV and Specific Pathogen Free (SPF) are preserved and provided by the seawater culture disease control key laboratory of the yellow sea aquaculture institute of China department of Aquaculture research.

The positive plasmids of 7 prawn DNA pathogenes and RNA pathogenes are constructed by selecting related sequence segments reported by GenBack and adopting an artificial gene synthesis method to prepare (Takara, Dalian). Cloning the specific nucleic acid fragments of the 7 prawn DNA or RNA pathogens to a pMD18-T vector, respectively constructing 7 recombinant plasmids of pMD18-WSSV, pMD18-IHHNV, pMD18-DIV1, pMD18-AHPND, pMD18-EHP, pMD18-TSV and pMD18-CMNV, and sequencing the constructed plasmids by engineering bioengineering (Shanghai) corporation Limited to determine positive clones.

Bst 2.0DNA polymerase was purchased from NEB along with 10 XBst buffer and 100mM MgSO4(# M0538L).

RTx reverse transcriptase (# M0380L), purchased from NEB.

5M Betaine (non-hydrochloride salt): betaine (C5H11NO2, molecular weight 117.15) was purchased and the reagents were analyzed neat. 117.15g of betaine was weighed, dissolved in 100mL of RNase-free water, adjusted to pH 8.0. + -. 0.2 with 1M HCl on a special pH meter, and added with RNase-free water to a volume of 200 mL. Subpackaging and freezing at-20 ℃.

350mM MnCl 2: anhydrous manganese chloride (MnCl2, MW 125.91) or tetrahydrate manganese chloride (MnCl 2.4h 2O, MW197.91) were purchased and analyzed pure. 4.407g of anhydrous manganese chloride or 6.927g of tetrahydrate manganese chloride is weighed and dissolved in 80mL of RNase-free water, the volume is determined to be 100mL, the obtained product is subpackaged by 1 mL/piece and frozen at-20 ℃.

3.5mM CaClein: calcein (C30H26N2O13, MW 622.55) or sodium calcein (Na2C30H26N2O13, MW 666.5) was purchased and analyzed pure. 43.58mg of calcein was weighed out and added to 6mL of DMSO, and after dissolution, approximately 14mL of RNase-free water was added to make 20mL, or 46.66mg of sodium calcein was weighed out and dissolved in 20mL of RNase-free water. The prepared solution is subpackaged to 0.5 mL/piece and frozen at-20 ℃.

SEMP liquid: 1mL of 1M Tris-HCl (pH8.0), 10mL of 700mM EDTA, 10mL of 10% SDS, 0.5mL of mercaptoethanol, 20mL of equilibrated phenol, and 100mL of double distilled water were prepared and stored at 4 ℃.

The specific primers for 7 prawn DNA pathogens and RNA pathogens are synthesized by the generation of Biotechnology engineering (Shanghai) GmbH. The sequences of the primers specific to the above 7 pathogens are specifically shown in table 1:

TABLE 1 prawn pathogen specific primer sequences