CN114252602A - Micro-fluidic chip, detection system based on micro-fluidic chip and detection method of bacteria - Google Patents
Micro-fluidic chip, detection system based on micro-fluidic chip and detection method of bacteria Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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Abstract
The invention discloses a micro-fluidic chip, a detection system based on the micro-fluidic chip and a detection method of bacteria, wherein the micro-fluidic chip is composed of a substrate and a cover plate which are bonded together, the cover plate is provided with a mixing micro-channel and a reaction micro-channel, the mixing micro-channel and the reaction micro-channel are mutually connected, a micro-column array is arranged in the reaction micro-channel, and furthermore, a hairpin-shaped oligonucleotide 1 is modified on the micro-column. The detection method of the invention is to initiate two hairpin-shaped oligonucleotides (H1, H2) to perform a catalysis hairpin self-assembly reaction through the competitive binding of pathogenic bacteria, the reaction not only amplifies the concentration of the pathogenic bacteria, but also fixes horseradish peroxidase on a micro-column array, so as to catalyze luminol and hydrogen peroxide to perform a chemiluminescence reaction, and the pathogenic bacteria are quantified through the chemical reaction signal intensity. The invention has good specificity, low detection limit and wide linear range, and provides more accurate data for quantitatively detecting the food-borne pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of microfluidic chips, and particularly relates to a microfluidic chip, a detection system based on the microfluidic chip and a detection method of bacteria, in particular to a microfluidic chip for pathogenic bacteria, a detection system based on the microfluidic chip and a detection method of pathogenic bacteria.
Background
Food-borne pathogenic bacteria are pathogenic bacteria that can cause food poisoning or are food borne vehicles. Pathogenic bacteria directly or indirectly pollute food and water sources, can cause livestock and poultry infectious diseases or human intestinal infectious diseases and food poisoning, and is one of the roots of food safety problems. Common food-borne pathogenic bacteria mainly comprise pathogenic Escherichia coli, salmonella, staphylococcus aureus, Listeria monocytogenes and the like. Escherichia coli O157: h7(E.coil O157: H7) is one of the main food-borne pathogenic bacteria, is a representative strain of enterohemorrhagic Escherichia coli (EHEC), has extremely low infection dosage, a latency period of 3-10 days and a disease course of 2-9 days, can cause Hemorrhagic Colitis (HC), usually acute abdominal pain and watery diarrhea, hemorrhagic diarrhea appears after several days, some patients can develop Hemolytic Uremic Syndrome (HUS), Thrombotic Thrombocytopenic Purpura (TTP) and the like, and severe patients can cause death. Food poisoning caused by food-borne pathogenic bacteria brings heavy burden to public health and also brings serious threat to the life health of people. Therefore, a limited amount of pathogenic bacteria in food products will help to prevent and control the occurrence and spread of such events. According to the restriction of pathogenic bacteria in national food Standard for food safety, the restriction of acceptable levels of Escherichia coli O157: H7, Salmonella and Listeria monocytogenes is 0, except that the restriction of acceptable levels of Staphylococcus aureus is 100CFU/g (mL). The limiting amount of food-borne pathogenic bacteria must depend on an accurate and rapid detection method, so that it is very important to detect the food-borne pathogenic bacteria by adopting an effective method.
Currently, methods for detecting food-borne pathogenic bacteria can be divided into indirect and direct methods. The indirect method mainly detects the secretion of food-borne pathogenic bacteria such as exotoxin, exotoxin and nucleic acid, indirectly obtains whether the pathogenic bacteria exist, and usually needs complex sample pretreatment. The direct method is to directly carry out quantitative detection on the number or the concentration of food-borne pathogenic bacteria in a sample, and comprises a plate counting method and a biosensor method. The plate counting method is a gold standard for detecting food-borne pathogenic bacteria, has high accuracy and reliability, needs professional technicians to culture and pre-enrich samples for a long time, has detection time of 2-3 days, and consumes a large amount of time, manpower and material resources.
The micro-fluidic chip technology is a technology that biological or chemical experiments (such as reaction preparation, analysis and purification, detection and analysis of samples and the like) are miniaturized and integrated on a chip with micro-or even nano-scale micro-channels, and a complex analysis process is completed by accurately controlling fluid. The micro-fluidic chip technology has the advantages of small sample and reagent consumption, capability of detecting multiple samples in parallel, high flux, high analysis speed, easiness in integration and automation and the like, so that the micro-fluidic chip technology is widely applied to the field of analysis and detection. More and more researches and developments are being made on methods for detecting food-borne pathogenic bacteria based on the microfluidic core, such as colorimetric methods, fluorescence methods, electrochemical methods, surface raman scattering methods, surface plasmon resonance methods, lateral flow strip and the like. These methods have certain disadvantages, including low sensitivity of colorimetric method and fluorescence method, poor reproducibility of electrochemical method, and complex and expensive signal detection instrument required for surface raman scattering method and surface plasmon resonance method. Therefore, a food-borne pathogenic bacteria detection method with high detection sensitivity and simple and cheap signal detection instrument needs to be developed.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a micro-fluidic chip, a detection system based on the micro-fluidic chip and a detection method of bacteria based on a catalytic hairpin self-assembly and a chemiluminescence method.
Chemiluminescence is a method for converting a concentration signal of a detection target into chemiluminescence intensity, and the main signal generation mechanism is based on the chemiluminescence reaction of an enzyme catalyzed substrate and an oxidant (such as a luminol-hydrogen peroxide system). Chemiluminescence methods rely on chemical reaction of trace analytes to produce chemiluminescence and are therefore highly sensitive. In addition, an excitation light source is not needed, so that the interference of light scattering is avoided, the instrument and the operation of the chemiluminescence method are simple, the calibration range is wide, and the miniaturized applicability provides great potential for the detection of food-borne pathogenic bacteria.
The basic principle of Catalytic Hairpin Assembly (CHA) is that two carefully designed hairpin-shaped oligonucleotides and one chain-shaped oligonucleotide change the structure of naked nucleic acid which can be complemented by sequence by using the 'foothold' on the stem-loop structure through the base complementary pairing principle and the action of topological reaction kinetics, thereby completing the self-assembly and de-assembly process between oligonucleotides. The net result of this reaction is the production of a hybridized strand of two hairpin oligonucleotides, immobilization of the hybridized strand on a substrate and generation of an analytical signal by introduction of specific groups in the hairpin oligonucleotides. Since the chain oligonucleotide is repeatedly used a plurality of times, the signal of the concentration of the target analyte can be amplified by the above reaction as long as the chain oligonucleotide is associated with the target analyte.
The specific technical scheme of the invention is as follows:
the invention provides a micro-fluidic chip, which consists of a substrate and a cover plate which are bonded together, wherein the cover plate is provided with a mixing micro-channel and a reaction micro-channel, the mixing micro-channel and the reaction micro-channel are mutually connected, and a micro-column array is arranged in the reaction micro-channel.
Further, the mixing microchannel is a snake-shaped mixing microchannel, and the reaction microchannel is a boat-shaped reaction microchannel;
the boat-shaped reaction microchannel comprises two boat heads and a boat body positioned between the two boat heads, wherein one boat head is used as an inlet, and the other boat head is used as an outlet;
the mixing microchannel comprises an inlet and an outlet;
preferably, the mixing microchannel comprises 2 inlets;
preferably, the mixing microchannel outlet is interconnected with the bow inlet of the boat-shaped reaction microchannel;
preferably, the reaction microchannel has a length of 16mm and a width of 3mm, and a streamline design with a gradually expanding inlet and a gradually contracting outlet, so that dead angles are avoided, and mixed reagents can be distributed more uniformly after entering the ship-shaped channel;
preferably, the serpentine mixing microchannel has a width of 200 μm, a depth of 150 μm, and a length of 20cm, and the narrow elongated feature thereof allows the chemiluminescent reagents to be mixed sufficiently in a greater degree in terms of fluid mechanics, enables a higher chemiluminescent signal to be generated rapidly, and the mixing channel is folded into a serpentine shape until the space-saving effect is achieved.
Preferably, the diameter of the microcolumn is 200 μm, the depth is 150 μm, the space between the microcolumns is 200 μm, the internal surface area of the boat-shaped reaction microchannel is increased, more space is provided for the modification of the internal surface, the collision probability of the priming strand catalyzing the hairpin self-assembly reaction with the microcolumn can be increased to a greater extent in the aspect of fluid mechanics, and the binding efficiency of the priming strand and the hairpin oligonucleotide 1 is increased;
preferably, the cover plate is made of Polydimethylsiloxane (PDMS), which has strong biocompatibility, easy surface modification and good light transmittance and is suitable for chemiluminescence detection.
Further, the microcolumn is modified with hairpin oligonucleotide 1(H1), and the hairpin oligonucleotide 1 is used for catalyzing hairpin self-assembly reaction; preferably, the H1 is fluorescently labeled to verify successful modification of H1, and the official experiment uses H1 without fluorescent label.
Preferably, the microcolumn is modified with streptavidin, and the hairpin oligonucleotide 1 is modified on the microcolumn by binding of biotin and streptavidin.
The invention provides a detection system based on a micro-fluidic chip, which comprises the micro-fluidic chip, a sample introduction unit and a detection and analysis unit, wherein the sample introduction unit is communicated with an inlet of a micro-fluidic chip mixing micro-channel;
preferably, the detection and analysis unit comprises a PMT detector and an ultra-weak chemiluminescence analyzer, and a light inlet of the PMT detector is matched with the reaction microchannel;
preferably, the sample introduction unit comprises a micro-injection pump, a conduit and an injector, the micro-fluidic chip can be connected with the injector through the conduit, the micro-injection pump accurately controls the flow and the flow rate, and the chemiluminescent reagent is automatically filled after programming;
preferably, the detection system further comprises a waste liquid pool communicated with the outlet of the microfluidic chip.
The third aspect of the invention provides a detection kit based on a microfluidic chip, which comprises the microfluidic chip, a hybrid chain (I/Apt) formed by a specific aptamer (Apt) of pathogenic bacteria and a priming chain (I), hairpin-shaped oligonucleotide 2(H2), horseradish peroxidase (HRP) modified gold nanoparticles (H2-AuNP-HRP), luminol and hydrogen peroxide;
the specific aptamer, the priming strand, the hairpin oligonucleotide 1 and the hairpin oligonucleotide 2 of the pathogenic bacterium satisfy the following condition (1) or (2);
(1) the specific aptamer of the pathogenic bacteria has m basic groups, the 3' end of the specific aptamer of the pathogenic bacteria and an initiating chain have n complementary pairing basic groups, wherein n/m is 1/4-1/3;
the 5 'end of the priming chain is matched with 6-10 naked basic groups at the 3' end of the hairpin oligonucleotide 1;
the 5 'end of the hairpin oligonucleotide 1 and the 3' end of the hairpin oligonucleotide 2 are subjected to complementary pairing of 6-10 naked basic groups;
preferably, the 5' end of the hairpin oligonucleotide 1 is modified with biotin; the 5' end of the hairpin-shaped oligonucleotide 2 is marked by sulfydryl, amino acid residue sulfydryl is arranged on horseradish peroxidase, and the hairpin-shaped oligonucleotide 2 and the horseradish peroxidase are modified on the gold nanoparticles through gold-sulfur bonds to form hairpin-shaped oligonucleotide 2 and horseradish peroxidase modified gold nanoparticles;
(2) the specific aptamer of the pathogenic bacteria has m basic groups, the 5' end of the specific aptamer of the pathogenic bacteria and an initiating chain have n complementary pairing basic groups, wherein n/m is 1/4-1/3;
the 3 'end of the priming chain is matched with 6-10 naked basic groups at the 5' end of the hairpin-shaped oligonucleotide 1;
the 3 'end of the hairpin oligonucleotide 1 and the 5' end of the hairpin oligonucleotide 2 are subjected to complementary pairing of 6-10 naked bases;
preferably, the 3' end of the hairpin oligonucleotide 1 is modified with biotin; the 3' end of the hairpin-shaped oligonucleotide 2 is marked by sulfydryl, amino acid residue sulfydryl is arranged on horseradish peroxidase, and the hairpin-shaped oligonucleotide 2 and the horseradish peroxidase are modified on the gold nanoparticles through gold-sulfur bonds to form the hairpin-shaped oligonucleotide 2 and the horseradish peroxidase modified gold nanoparticles.
Preferably, the hairpin oligonucleotide 2(H2) is fluorescently labeled to verify successful modification of the hairpin oligonucleotide 1(H1) and the formal experiment uses aptamer 2 without fluorescent label.
Preferably, the horseradish peroxidase has a characteristic absorption peak at 420nm, and the horseradish peroxidase is verified by the existence of the characteristic absorption peak at 420nm before and after modification of the gold nanoparticles.
Further, the pathogenic bacterium is escherichia coli O157: h7, the sequence of the specific aptamer of the pathogenic bacterium is shown as SEQ ID NO.1, the sequence of the priming strand is shown as SEQ ID NO.2, the sequence of the hairpin oligonucleotide 1 is shown as SEQ ID NO.3, and the sequence of the hairpin oligonucleotide 2 is shown as SEQ ID NO. 4.
The invention provides the application of the microfluidic chip, the microfluidic chip-based detection system or the microfluidic chip-based detection kit in pathogenic bacteria detection;
preferably, the pathogenic bacteria are food-borne pathogenic bacteria.
Taking the example that the 3' end of the specific aptamer of the pathogenic bacteria and the priming strand have a plurality of complementary paired bases, the principle of the pathogenic bacteria detection carried out by the invention is as follows: pathogenic bacteria can bind to specific aptamers (Apt) of pathogenic bacteria in said hybrid strand (I/Apt) thereby competing against the initiating strand (I) that catalyzes the hairpin self-assembly reaction. The priming strand (I) catalyzing the self-assembly reaction of the hairpin can trigger the self-assembly of the hairpin oligonucleotide 1(H1) and the hairpin oligonucleotide 2(H2) to form the hybrid strand (H1/H2), specifically, the 5 'end of the priming strand (I) is paired with the naked base of the 3' end of the hairpin oligonucleotide 1(H1) to thereby trigger the development of the clamp oligonucleotide 1(H1), the 5 'end of the opened hairpin oligonucleotide 1(H1) is paired with the naked base of the 3' end of the hairpin oligonucleotide 2(H2) to thereby trigger the development of the clamp oligonucleotide 2(H2), the hybrid strand (H1/H2) is formed as the hairpin oligonucleotide 2(H2) is bound to the hairpin oligonucleotide 1(H1), the priming strand (I) is competed, and the competitive priming strand (I) continues to trigger the development of the clamp oligonucleotide 1(H1), this was repeated until all hairpin oligonucleotide 2(H2) was bound to hairpin oligonucleotide 1 (H1). As hairpin oligonucleotide 2(H2) was bound to hairpin oligonucleotide 1(H1) to form a hybrid chain (H1/H2), which was immobilized on the micropillar array, gold nanoparticles (AuNP) and horseradish peroxidase (HRP) modified thereon were also immobilized on the micropillar array. The horseradish peroxidase can generate chemiluminescence reaction under catalysis to generate chemiluminescence signals, so that detection of pathogenic bacteria is realized.
The invention provides a bacteria detection method based on a microfluidic chip, which is characterized by comprising the following steps:
uniformly mixing a sample to be detected, a specific aptamer of pathogenic bacteria and a hybrid chain formed by an initiation chain and the hairpin-shaped oligonucleotide 2, injecting the mixture into a reaction microchannel of the microfluidic chip, standing, and carrying out a catalytic hairpin self-assembly reaction initiated by competitive binding of the pathogenic bacteria;
washing a reaction microchannel of the microfluidic chip by using a buffer solution, and collecting a background signal of the reaction microchannel;
respectively injecting luminol and hydrogen peroxide into a mixed micro-channel of the micro-fluidic chip, mixing in the mixed micro-channel, injecting into a reaction micro-channel, and carrying out a chemiluminescent reaction under the catalysis of horseradish peroxidase;
collecting a chemiluminescence atlas of the reaction microchannel, and judging the existence and concentration of pathogenic bacteria in a sample to be detected by using the chemiluminescence atlas.
Further, the bacteria detection method based on the microfluidic chip further comprises the steps of taking pathogenic bacteria to be detected as a standard sample, collecting a background signal and a chemiluminescence spectrum by adopting the detection method, drawing a standard curve by taking the concentration of the standard sample as a horizontal coordinate and the peak value of the chemiluminescence spectrum as a vertical coordinate, and performing linear fitting to obtain a standard equation;
preferably, the peak value of the chemiluminescence spectrum of the sample to be detected is substituted into a standard equation to obtain the concentration of pathogenic bacteria in the sample to be detected.
Further, the buffer solution is Tris-HCl buffer solution;
the volume ratio of the luminol to the hydrogen peroxide is 1: 1;
preferably, the volume of the luminol and the hydrogen peroxide is 10 μ L;
preferably, the luminol and hydrogen peroxide are injected into the mixing microchannel at a flow rate of 10 μ L/min;
preferably, the pathogenic bacteria are food-borne pathogenic bacteria.
Further, collecting a background signal and a chemiluminescence map by using an ultra-weak chemiluminescence analyzer;
preferably, after the ultra-weak chemiluminescence analyzer is preheated for 30min, a background signal is collected;
preferably, the chemiluminescence spectra are collected within 900s from the start of luminol and hydrogen peroxide injection.
The invention has the beneficial effects that:
1. when the micro-fluidic chip based on the catalysis hairpin self-assembly (CHA) and the chemiluminescence biosensing is used for detecting the food-borne pathogenic bacteria, compared with the traditional food-borne pathogenic bacteria detection method, the micro-fluidic chip has the advantages of low consumption of required samples and reagents, high automation degree and high detection speed.
Further, the narrow and elongated features of the serpentine mixing microchannel allow for greater degree of mixing of the chemiluminescent reagents in fluid mechanics, enabling rapid generation of higher chemiluminescent signals, and folding the mixing channel into a serpentine shape can be achieved up to space saving. The ship-shaped reaction micro-channel adopts the streamline design of gradually expanding at the inlet and gradually reducing at the outlet, thereby avoiding the generation of dead angles and ensuring that the mixed reagent can be more uniformly distributed after entering the ship-shaped channel. The diameter of the micro-column array is 200 μm, the depth is 150 μm, the micro-column spacing is 200 μm, the internal surface area of the boat-shaped reaction micro-channel is improved, more space is provided for the modification of the internal surface, the collision probability of the initiating chain (I) for catalyzing the hairpin self-assembly reaction and the micro-column can be increased to a greater extent in the fluid mechanics, and the combination efficiency of the initiating chain (I) and the hairpin-shaped oligonucleotide 1(H1) is increased. The micro-fluidic chip is made of Polydimethylsiloxane (PDMS), has good biocompatibility and strong light transmittance, and is suitable for detecting biomarkers.
2. According to the detection method of the pathogenic bacteria, two hairpin-shaped oligonucleotides (H1, H2) are triggered to generate a catalytic hairpin self-assembly reaction (CHA) through competitive binding of the pathogenic bacteria, wherein the hairpin-shaped oligonucleotide 1(H1) is modified on a micro-column array in a ship-shaped reaction micro-channel of a microfluidic chip, the hairpin-shaped oligonucleotide 2(H2) and horseradish peroxidase (HRP) are jointly modified on the surface of a gold nanoparticle, the catalytic hairpin self-assembly reaction (CHA) not only amplifies the concentration of the pathogenic bacteria, but also fixes the horseradish peroxidase (HRP) on the micro-column array, further catalyzes luminol and hydrogen peroxide to generate a chemiluminescence reaction, and the pathogenic bacteria are quantified through the intensity of chemical reaction signals. The method has good specificity, low detection limit and wide linear range, and provides more accurate data for quantitatively detecting pathogenic bacteria. The method has an important application prospect in the field of food-borne pathogenic bacteria detection. In addition, the invention can detect the concentration of the food-borne pathogenic bacteria in the sample in a short time, thereby greatly improving the efficiency of quantitatively detecting the food-borne pathogenic bacteria. And the micro-fluidic chip platform has small reagent dosage, can be integrated and automated, greatly reduces the material cost and the labor cost for quantitatively detecting the food-borne pathogenic bacteria, provides a new technical platform for detecting the food-borne pathogenic bacteria, and has better food safety detection application prospect.
Drawings
Fig. 1 is a perspective view of a microfluidic chip of example 1; 1-snake-shaped mixing micro-channel, 2-ship-shaped reaction micro-channel and 3-micro-column array;
FIG. 2 is a cover slip channel mask sizing diagram of example 2;
FIG. 3 is a diagram showing the results of microchannel modification in example 2;
FIG. 4 is a microfluidic chip-based detection system of example 3; a-micro injection pump, b-injector, c-PTFE conduit, d-micro flow control chip, e-waste liquid pool, f-PMT detector, g-ultra-weak chemiluminescence analyzer.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1: structure of micro-fluidic chip
The present embodiment provides a microfluidic chip, and the three-dimensional structure of the microfluidic chip is shown in fig. 1. The microfluidic chip consists of a substrate and a cover plate bonded together. In this embodiment, the substrate is made of glass, and the cover is made of Polydimethylsiloxane (PDMS). The cover plate is provided with a snake-shaped mixing micro-channel 1 and a boat-shaped reaction micro-channel 2, and micro-column arrays 3 are uniformly distributed in the boat-shaped reaction micro-channel 2. The boat-shaped reaction microchannel 2 comprises two boat heads and a boat body positioned between the two boat heads, one boat head is used as an inlet, and the other boat head is used as an outlet. The mixing microchannel comprises 2 inlets and 1 outlet, wherein the 2 inlets are respectively used as the inlets of the luminol and the hydrogen peroxide. The outlet of the mixing microchannel is connected with the bow inlet of the boat-shaped reaction microchannel.
The serpentine mixing microchannel 1 has the characteristics of 200 μm width, 150 μm depth and 20cm length, and the narrow and prolonged narrow mixing channels, and has the advantages of rapidly generating higher chemiluminescent signals and folding the mixing channel into a serpentine shape to play a role in saving space.
The length of the boat-shaped reaction micro-channel 2 is 16mm, the width is 3mm, and the streamline design of gradual expansion at the inlet and gradual reduction at the outlet avoids the generation of dead angles, so that the mixed reagent can be distributed more uniformly after entering the boat-shaped channel.
The diameter of the micro-column array 3 of the chip is 200 μm, the depth is 150 μm, the micro-column spacing is 200 μm, the internal surface area of the boat-shaped micro-channel is increased, and more space is provided for the modification of the internal surface.
Furthermore, the microcolumn is modified with hairpin oligonucleotide 1(H1) for catalysis of hairpin self-assembly reaction. The microcolumn was modified with streptavidin, and hairpin oligonucleotide 1(H1) was modified on the microcolumn by binding of biotin and streptavidin.
Example 2: preparation of microfluidic chip
The silicon wafer is firstly treated by ethanol ultrasound for 5 minutes, then treated by deionized water ultrasound for 5 minutes, and heated on a heating plate for 120 minutes after three times.
And (3) coating a thin layer of SU-82050 negative photoresist on the silicon wafer in a spinning mode, and controlling the rotating speed to enable the thickness of the photoresist layer to be about 50 mu m. The mask having the structure of fig. 2 was attached to a silicon wafer, and the photoresist was exposed to ultraviolet rays through the photomask, and the unexposed portions were dissolved in a developing solution. The SU-8 structure protruding on the surface of the silicon chip is used as a male mold of the PDMS cover plate. The silicon male mold was then silanized overnight.
PDMS prepolymer (monomer/curing agent blend: 10/1) was cast onto a silicon male mold and vacuum degassed, then placed in a 75 ℃ oven to cure for about 2 hours, and then the PDMS was peeled off the male mold to form a PDMS coversheet. The PDMS coverslip was cut and perforated.
And putting the glass substrate and the PDMS cover plate into an oxygen plasma vacuum tube, vacuumizing for 90 seconds, turning on a high-frequency power supply, taking out the substrate and the cover plate after 90 seconds, and immediately bonding.
After bonding, the channels were treated with 4% (v/v) MPTS in absolute ethanol at room temperature and left to stand for 30 min. Washing with absolute ethyl alcohol repeatedly for three times or completely washing. After washing, the chip is dried in an oven at 100 ℃ for 1 h.
The chips were treated with a freshly prepared 0.01. mu. mol/mL GMBS solution in absolute ethanol at room temperature and allowed to stand for 30 min. Washing with absolute ethyl alcohol repeatedly for three times or completely washing. After washing, the chip is dried in an oven at 100 ℃ for 1 h.
The channel was filled with a 10. mu.g/mL streptavidin in PBS at room temperature, left to stand for 1H, after which a biotin-labeled hairpin oligonucleotide 1(H1) solution at a concentration of 1. mu.M was introduced and incubated at room temperature for 30min, followed by washing with PBS buffer to remove unbound hairpin oligonucleotide 1 (H1). After the modification, the chip was stored at 4 ℃ for further use.
In a specific embodiment, the successful modification of hairpin oligonucleotide 1(H1) was verified by modifying the microchannel with biotin-labeled hairpin oligonucleotide 1(H1) modified with a FAM group, and washing with PBS buffer removed unbound capture aptamer. A photograph of the microchannel was taken using a fluorescence microscope, as shown in fig. 3.
Example 3: detection system based on micro-fluidic chip
The embodiment provides a detection system based on a microfluidic chip, which comprises a microfluidic chip d, a sample introduction unit and a detection and analysis unit in embodiment 1, wherein the sample introduction unit is communicated with an inlet of a micro-channel of the microfluidic chip.
In a specific embodiment, the detection and analysis unit comprises a PMT detector f and an ultra-weak chemiluminescence analyzer g. The micro-fluidic chip is matched with a clamping groove (25 × 25mm) in a box of the PMT detector in size, the boat-shaped reaction micro-channel is just matched with the light inlet position of the PMT detector after the micro-fluidic chip is placed, and a generated chemiluminescence signal is collected by the PMT and analyzed and processed by the ultra-weak chemiluminescence analyzer g.
The sample introduction unit comprises a micro-injection pump a, a PTFE conduit c and an injector b, the micro-fluidic chip d can be connected with the injector b through the PTFE conduit c, the flow and the flow rate are accurately controlled through the micro-injection pump a, and the chemiluminescent reagent is automatically filled after programming.
The outlet of the micro-fluidic chip needs to be connected with a waste liquid pool e through a PTFE (polytetrafluoroethylene) guide pipe, so that the liquid at the outlet is prevented from damaging a PMT (photomultiplier tube) detector.
Example 4: detection kit based on micro-fluidic chip
Comprises the microfluidic chip described in example 1, a hybrid chain formed by a specific aptamer of pathogenic bacteria and a priming chain, hairpin-shaped oligonucleotide 2, horseradish peroxidase-modified gold nanoparticles, luminol and hydrogen peroxide.
The 3' end of the specific aptamer of the pathogenic bacterium and the priming strand are provided with a plurality of complementary paired bases, wherein the number of the complementary paired bases accounts for about 1/3 of the number of the bases of the specific aptamer sequence, the specific aptamer of the pathogenic bacterium and the priming strand form a hybrid strand (I/Apt), and the pathogenic bacterium can be combined to the specific aptamer (Apt) of the pathogenic bacterium in the hybrid strand (I/Apt), so that the priming strand (I) catalyzing hairpin self-assembly reaction is competed.
The 5 'end of the priming strand is paired with the naked base at the 3' end of the hairpin oligonucleotide 1. The 5 'end of the hairpin oligonucleotide 1 is complementarily paired with the naked base at the 3' end of the hairpin oligonucleotide 2. The initiating strand (I) that catalyzes the hairpin self-assembly reaction can initiate the self-assembly of hairpin oligonucleotide 1(H1) and hairpin oligonucleotide 2(H2) to form a hybrid strand (H1/H2). Specifically, the 5 'end of the priming strand (I) and the naked base at the 3' end of the hairpin oligonucleotide 1(H1) are paired with each other to thereby develop the hairpin oligonucleotide 1(H1), the 5 'end of the opened hairpin oligonucleotide 1(H1) and the naked base at the 3' end of the hairpin oligonucleotide 2(H2) are complementarily paired to thereby develop the hairpin oligonucleotide 2(H2), the priming strand (I) is competed as the hairpin oligonucleotide 2(H2) binds to the hairpin oligonucleotide 1(H1) to form the hybrid strand (H1/H2), and the competed priming strand (I) continues to develop the clamp oligonucleotide 1(H1), and so on until all the hairpin oligonucleotides 2(H2) bind to the hairpin oligonucleotide 1 (H1).
As hairpin oligonucleotide 2(H2) was bound to hairpin oligonucleotide 1(H1) to form a hybrid chain (H1/H2), which was immobilized on the micropillar array, gold nanoparticles (AuNP) and horseradish peroxidase (HRP) modified thereon were also immobilized on the micropillar array. The horseradish peroxidase can generate chemiluminescence reaction under catalysis to generate chemiluminescence signals, so that detection of pathogenic bacteria is realized.
Example 4: bacteria detection method based on micro-fluidic chip
Detection of escherichia coli O157 using the microfluidic chip prepared in example 2: H7. the method comprises the following specific steps:
(1) a sample to be detected, a compound formed by a specific aptamer of Escherichia coli O157: H7 and a CHA initiation chain, hairpin-shaped oligonucleotide 2(H2) and horse radish peroxidase modified gold nanoparticles are uniformly mixed, injected into a boat-shaped reaction microchannel of a microfluidic chip and kept stand to generate catalytic hairpin self-assembly reaction triggered by competitive binding of Escherichia coli O157: H7.
(2) And washing the ship-shaped reaction microchannel of the microfluidic chip three times by using Tris-HCl buffer solution.
(3) And (3) opening the ultra-weak chemiluminescence analyzer and analysis software, preheating for 30min, and collecting background signals.
(4) Placing a micro-fluidic chip in a clamping groove in a box with a PMT detector, respectively sucking luminol and hydrogen peroxide by two injectors, connecting the micro-fluidic chip with a PTFE (polytetrafluoroethylene) catheter, pouring 10 mu L of luminol and 10 mu L of hydrogen peroxide into the micro-fluidic chip at a flow rate of 10 mu L/min by a precise micro-injector, mixing the luminol and the hydrogen peroxide by a mixing micro-channel, flowing into a boat-shaped reaction micro-channel, and carrying out chemiluminescent reaction under the catalysis of horseradish peroxidase.
(5) The chemiluminescence spectra were collected within 900s from the start of the perfusion of the chemiluminescent reagent.
(6) Collecting 500, 10 according to the above steps (1) - (5)3，104，105，106，106，107，108Chemiluminescence spectra of CFU/mL Escherichia coli O157: H7 standard samples.
(7) And (3) taking the concentration of the Escherichia coli O157: H7 standard sample as an abscissa, taking the peak value of the chemiluminescence spectrum as an ordinate to draw a standard curve, and performing linear fitting to obtain a standard equation.
(8) And (4) collecting the chemiluminescence spectra of the samples to be detected according to the steps (1) to (5), and substituting the peak values of the chemiluminescence spectra into the standard equation in the step (7) to obtain the concentration of the Escherichia coli O157: H7 in the samples to be detected.
The nucleotide sequences of the Escherichia coli O157: H7-specific aptamer, the priming strand, hairpin oligonucleotide 1 and hairpin oligonucleotide 2 in this example are shown in the following table. The 5 'end of hairpin oligonucleotide 1 was modified with biotin and the 5' end of hairpin oligonucleotide 2 was labeled with thiol.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> microfluidic chip, detection system based on microfluidic chip and detection method of bacteria
<130> CP121011279C
<160> 4
<170> PatentIn version 3.3
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atacgggagc caacaccacg catc 24
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ttttttttca acaccacgca tccaaagtca aagtgatgcg tggtgttggc tcccgtat 58
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Claims (10)
1. The microfluidic chip is characterized by consisting of a substrate and a cover plate which are bonded together, wherein a mixing micro-channel and a reaction micro-channel are arranged on the cover plate, the mixing micro-channel and the reaction micro-channel are mutually connected, and a micro-column array is arranged in the reaction micro-channel.
2. The microfluidic chip according to claim 1, wherein the mixing microchannel is a serpentine mixing microchannel, and the reaction microchannel is a boat-shaped reaction microchannel;
the boat-shaped reaction microchannel comprises two boat heads and a boat body positioned between the two boat heads, wherein one boat head is used as an inlet, and the other boat head is used as an outlet;
the mixing microchannel comprises an inlet and an outlet;
preferably, the mixing microchannel comprises 2 inlets;
preferably, the mixing microchannel outlet is interconnected with the bow inlet of the boat-shaped reaction microchannel;
preferably, the reaction microchannel has a length of 16mm and a width of 3 mm;
preferably, the serpentine mixing microchannel has a width of 200 μm, a depth of 150 μm, and a length of 20 cm;
preferably, the diameter of the micro-column is 200 μm, the depth is 150 μm, and the micro-column pitch is 200 μm;
preferably, the material of the cover sheet is polydimethylsiloxane.
3. The microfluidic chip according to claim 1, wherein the microcolumns are modified with hairpin-shaped oligonucleotides 1, and the hairpin-shaped oligonucleotides 1 are used for catalyzing hairpin self-assembly reaction;
preferably, the microcolumn is modified with streptavidin, and the hairpin oligonucleotide 1 is modified on the microcolumn by binding of biotin and streptavidin.
4. A detection system based on a microfluidic chip, which comprises the microfluidic chip of any one of claims 1 to 3, a sample introduction unit and a detection and analysis unit, wherein the sample introduction unit is communicated with an inlet of a mixing microchannel of the microfluidic chip;
preferably, the detection and analysis unit comprises a PMT detector and an ultra-weak chemiluminescence analyzer, and a light inlet of the PMT detector is matched with the reaction microchannel;
preferably, the sample introduction unit comprises a micro-injection pump, a conduit and a syringe;
preferably, the detection system further comprises a waste liquid pool communicated with the outlet of the microfluidic chip.
5. A detection kit based on a microfluidic chip, which comprises the microfluidic chip of any one of claims 1 to 3, a hybrid chain formed by a specific aptamer of a pathogenic bacterium and a priming chain, hairpin-shaped oligonucleotide 2, horseradish peroxidase-modified gold nanoparticles, luminol and hydrogen peroxide;
the specific aptamer, the priming strand, the hairpin oligonucleotide 1 and the hairpin oligonucleotide 2 of the pathogenic bacterium satisfy the following condition (1) or (2);
(1) the specific aptamer of the pathogenic bacteria has m basic groups, the 3' end of the specific aptamer of the pathogenic bacteria and an initiating chain have n complementary pairing basic groups, wherein n/m is 1/4-1/3;
the 5 'end of the priming chain is matched with 6-10 naked basic groups at the 3' end of the hairpin oligonucleotide 1;
the 5 'end of the hairpin oligonucleotide 1 and the 3' end of the hairpin oligonucleotide 2 are subjected to complementary pairing of 6-10 naked basic groups;
preferably, the 5' end of the hairpin oligonucleotide 1 is modified with biotin; the 5' end of the hairpin-shaped oligonucleotide 2 is marked by sulfydryl, amino acid residue sulfydryl is arranged on horseradish peroxidase, and the hairpin-shaped oligonucleotide 2 and the horseradish peroxidase are modified on the gold nanoparticles through gold-sulfur bonds to form hairpin-shaped oligonucleotide 2 and horseradish peroxidase modified gold nanoparticles;
(2) the specific aptamer of the pathogenic bacteria has m basic groups, the 5' end of the specific aptamer of the pathogenic bacteria and an initiating chain have n complementary pairing basic groups, wherein n/m is 1/4-1/3;
the 3 'end of the priming chain is matched with 6-10 naked basic groups at the 5' end of the hairpin-shaped oligonucleotide 1;
the 3 'end of the hairpin oligonucleotide 1 and the 5' end of the hairpin oligonucleotide 2 are subjected to complementary pairing of 6-10 naked bases;
preferably, the 3' end of the hairpin oligonucleotide 1 is modified with biotin; the 3' end of the hairpin-shaped oligonucleotide 2 is marked by sulfydryl, amino acid residue sulfydryl is arranged on horseradish peroxidase, and the hairpin-shaped oligonucleotide 2 and the horseradish peroxidase are modified on the gold nanoparticles through gold-sulfur bonds to form the hairpin-shaped oligonucleotide 2 and the horseradish peroxidase modified gold nanoparticles.
6. The microfluidic chip-based detection kit according to claim 5, wherein the pathogenic bacterium is Escherichia coli O157: h7, the sequence of the specific aptamer of the pathogenic bacterium is shown as SEQ ID NO.1, the sequence of the priming strand is shown as SEQ ID NO.2, the sequence of the hairpin oligonucleotide 1 is shown as SEQ ID NO.3, and the sequence of the hairpin oligonucleotide 2 is shown as SEQ ID NO. 4.
7. Use of a microfluidic chip according to any one of claims 1 to 3, a microfluidic chip-based detection system according to claim 4 or a microfluidic chip-based detection kit according to claim 5 or 6 for the detection of pathogenic bacteria;
preferably, the pathogenic bacteria are food-borne pathogenic bacteria.
8. A bacteria detection method based on a microfluidic chip is characterized by comprising the following steps:
uniformly mixing a sample to be detected, a specific aptamer of pathogenic bacteria and a hybrid chain formed by an initiating chain and the hairpin-shaped oligonucleotide 2, injecting the mixture into a reaction microchannel of the microfluidic chip in claim 1, standing, and carrying out a catalytic hairpin self-assembly reaction initiated by competitive binding of the pathogenic bacteria;
washing a reaction microchannel of the microfluidic chip by using a buffer solution, and collecting a background signal of the reaction microchannel;
respectively injecting luminol and hydrogen peroxide into a mixing microchannel of the microfluidic chip as claimed in claim 1, mixing in the mixing microchannel, injecting into a reaction microchannel, and carrying out a chemiluminescent reaction under the catalysis of horseradish peroxidase;
collecting a chemiluminescence atlas of the reaction microchannel, and judging the existence and concentration of pathogenic bacteria in a sample to be detected by using the chemiluminescence atlas.
9. The detection method according to claim 8, further comprising collecting a background signal and a chemiluminescence spectrum by using the pathogenic bacteria to be detected as a standard sample according to the detection method according to claim 8, drawing a standard curve by using the concentration of the standard sample as an abscissa and the peak value of the chemiluminescence spectrum as an ordinate, and performing linear fitting to obtain a standard equation;
preferably, the peak value of the chemiluminescence spectrum of the sample to be detected is substituted into a standard equation to obtain the concentration of pathogenic bacteria in the sample to be detected.
10. The assay of claim 8, wherein the buffer solution is a Tris-HCl buffer solution;
the volume ratio of the luminol to the hydrogen peroxide is 1: 1;
preferably, the volume of the luminol and the hydrogen peroxide is 10 μ L;
preferably, the luminol and hydrogen peroxide are injected into the mixing microchannel at a flow rate of 10 μ L/min;
preferably, the pathogenic bacteria are food-borne pathogenic bacteria;
preferably, a super-weak chemiluminescence analyzer is adopted to collect background signals and chemiluminescence spectrums;
preferably, after the ultra-weak chemiluminescence analyzer is preheated for 30min, a background signal is collected;
preferably, the chemiluminescence spectra are collected within 900s from the start of luminol and hydrogen peroxide injection.
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| PCT/CN2022/080095 WO2023115717A1 (en) | 2021-12-22 | 2022-03-10 | Microfluidic chip, microfluidic chip-based detection system, and method for detecting bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115353968A (en) * | 2022-10-20 | 2022-11-18 | 湖南冠牧生物科技有限公司 | Rapid nucleic acid detection micro-fluidic chip, nucleic acid detection system and method |
Citations (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005001123A1 (en) * | 2003-06-30 | 2005-01-06 | Tsinghua University | Dna chip based genetic typing |
| CN101643701A (en) * | 2009-07-23 | 2010-02-10 | 清华大学 | Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells |
| CN201628717U (en) * | 2010-02-09 | 2010-11-10 | 中国人民解放军第三军医大学 | Microfluidic chip used for detecting pathogenic microorganisms |
| US20130053252A1 (en) * | 2009-09-25 | 2013-02-28 | President & Fellows Of Harvard College | Nucleic acid amplification and sequencing by synthesis with fluorogenic nucleotides |
| WO2013063366A2 (en) * | 2011-10-27 | 2013-05-02 | Stc.Unm | Methods for screening viral like particles and identifying neutralizing epitopes and related vaccines, constructs, and libraries |
| CN104955959A (en) * | 2012-11-02 | 2015-09-30 | 生命技术公司 | Novel compositions, methods and kits for enhancing PCR specificity |
| CN105648070A (en) * | 2016-02-25 | 2016-06-08 | 青岛科技大学 | Method for detecting nucleic acid or cells based on enzymatic cycle amplification and nano-particle reinforced SPR (surface plasmon resonance) |
| WO2016177808A1 (en) * | 2015-05-07 | 2016-11-10 | Paris Sciences Et Lettres - Quartier Latin | Formation of hairpins in situ using force-induced strand invasion |
| WO2017189525A1 (en) * | 2016-04-25 | 2017-11-02 | President And Fellows Of Harvard College | Hybridization chain reaction methods for in situ molecular detection |
| CN107400623A (en) * | 2016-05-20 | 2017-11-28 | 益善生物技术股份有限公司 | Circulating tumor cell automatic capture micro-fluidic chip and its automatic capture method |
| US20170362640A1 (en) * | 2016-06-16 | 2017-12-21 | Life Technologies Corporation | Novel compositions, methods and kits for microorganism detection |
| US20180251825A1 (en) * | 2017-02-02 | 2018-09-06 | New York Genome Center Inc. | Methods and compositions for identifying or quantifying targets in a biological sample |
| CN110095608A (en) * | 2019-04-12 | 2019-08-06 | 南方医科大学南方医院 | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly |
| WO2020218831A1 (en) * | 2019-04-22 | 2020-10-29 | 포항공과대학교 산학협력단 | Novel probe set for isothermal one-pot reaction, and uses thereof |
| US20200354767A1 (en) * | 2017-11-16 | 2020-11-12 | The Regents Of The University Of Colorado, A Body Corporate | Methods to measure functional heterogeneity among single cells |
| CN112011435A (en) * | 2020-09-11 | 2020-12-01 | 徐州工程学院 | Microfluidic system for accurately capturing circulating tumor cells and preparation method thereof |
| WO2021074087A1 (en) * | 2019-10-16 | 2021-04-22 | Illumina, Inc. | Systems and methods for detecting multiple analytes |
| CN112964767A (en) * | 2021-03-19 | 2021-06-15 | 济南大学 | Application of photothermal effect-photoelectric biological sensing paper chip in microRNA-141 detection |
| CN113820487A (en) * | 2020-06-18 | 2021-12-21 | 海宁先进半导体与智能技术研究院 | Micro-fluidic chip for cancer marker detection and preparation method thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101900723B (en) * | 2009-05-27 | 2013-05-08 | 中国科学技术大学 | Application of nano-gold directly bonded with luminol in immunoassay |
| CN206715966U (en) * | 2017-04-28 | 2017-12-08 | 刘玲 | A kind of bioanalysis micro-fluidic detection device |
| CN111804356B (en) * | 2020-07-16 | 2021-11-09 | 清华大学 | Microfluidic chip and preparation method thereof, microfluidic device and detection method of pathogenic bacteria |
| CN214668024U (en) * | 2021-01-15 | 2021-11-09 | 南京大学 | Microfluidic sample pretreatment chip |
| CN113533719B (en) * | 2021-06-18 | 2023-08-22 | 清华大学 | Detection method of food-borne pathogenic bacteria based on microfluidic chip |
-
2021
- 2021-12-22 CN CN202111582058.8A patent/CN114252602B/en active Active
-
2022
- 2022-03-10 WO PCT/CN2022/080095 patent/WO2023115717A1/en unknown
Patent Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005001123A1 (en) * | 2003-06-30 | 2005-01-06 | Tsinghua University | Dna chip based genetic typing |
| CN101643701A (en) * | 2009-07-23 | 2010-02-10 | 清华大学 | Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells |
| US20130053252A1 (en) * | 2009-09-25 | 2013-02-28 | President & Fellows Of Harvard College | Nucleic acid amplification and sequencing by synthesis with fluorogenic nucleotides |
| CN201628717U (en) * | 2010-02-09 | 2010-11-10 | 中国人民解放军第三军医大学 | Microfluidic chip used for detecting pathogenic microorganisms |
| WO2013063366A2 (en) * | 2011-10-27 | 2013-05-02 | Stc.Unm | Methods for screening viral like particles and identifying neutralizing epitopes and related vaccines, constructs, and libraries |
| CN104955959A (en) * | 2012-11-02 | 2015-09-30 | 生命技术公司 | Novel compositions, methods and kits for enhancing PCR specificity |
| WO2016177808A1 (en) * | 2015-05-07 | 2016-11-10 | Paris Sciences Et Lettres - Quartier Latin | Formation of hairpins in situ using force-induced strand invasion |
| CN105648070A (en) * | 2016-02-25 | 2016-06-08 | 青岛科技大学 | Method for detecting nucleic acid or cells based on enzymatic cycle amplification and nano-particle reinforced SPR (surface plasmon resonance) |
| WO2017189525A1 (en) * | 2016-04-25 | 2017-11-02 | President And Fellows Of Harvard College | Hybridization chain reaction methods for in situ molecular detection |
| CN107400623A (en) * | 2016-05-20 | 2017-11-28 | 益善生物技术股份有限公司 | Circulating tumor cell automatic capture micro-fluidic chip and its automatic capture method |
| US20170362640A1 (en) * | 2016-06-16 | 2017-12-21 | Life Technologies Corporation | Novel compositions, methods and kits for microorganism detection |
| US20180251825A1 (en) * | 2017-02-02 | 2018-09-06 | New York Genome Center Inc. | Methods and compositions for identifying or quantifying targets in a biological sample |
| US20200354767A1 (en) * | 2017-11-16 | 2020-11-12 | The Regents Of The University Of Colorado, A Body Corporate | Methods to measure functional heterogeneity among single cells |
| CN110095608A (en) * | 2019-04-12 | 2019-08-06 | 南方医科大学南方医院 | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly |
| WO2020218831A1 (en) * | 2019-04-22 | 2020-10-29 | 포항공과대학교 산학협력단 | Novel probe set for isothermal one-pot reaction, and uses thereof |
| WO2021074087A1 (en) * | 2019-10-16 | 2021-04-22 | Illumina, Inc. | Systems and methods for detecting multiple analytes |
| CN115176028A (en) * | 2019-10-16 | 2022-10-11 | 伊鲁米纳公司 | Systems and methods for detecting multiple analytes |
| CN113820487A (en) * | 2020-06-18 | 2021-12-21 | 海宁先进半导体与智能技术研究院 | Micro-fluidic chip for cancer marker detection and preparation method thereof |
| CN112011435A (en) * | 2020-09-11 | 2020-12-01 | 徐州工程学院 | Microfluidic system for accurately capturing circulating tumor cells and preparation method thereof |
| CN112964767A (en) * | 2021-03-19 | 2021-06-15 | 济南大学 | Application of photothermal effect-photoelectric biological sensing paper chip in microRNA-141 detection |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115353968A (en) * | 2022-10-20 | 2022-11-18 | 湖南冠牧生物科技有限公司 | Rapid nucleic acid detection micro-fluidic chip, nucleic acid detection system and method |
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