High-throughput staphylococcus aureus enterotoxin microfluidic chip detection method based on chain amplification and enhanced fluorescence and application

Abstract

The present application relates to a kind of high-throughput staphylococcus aureus enterotoxin microfluidic chip detection method and application based on chain quantity amplification enhanced fluorescence.The microfluidic chip is provided with fluorescence sensor, and the fluorescence sensor includes identification probe and signal probe;The identification probe is composed of magnetic nanoparticle, nucleic acid aptamer H1 of target to be measured and target aptamer complementary chain H2, and the signal probe is composed of nanomaterial with signal amplification function and three oligonucleotides H3, H4, H5.The present application combines MNPs-ssDNA-AuNPs sandwich structure system and microfluidic chip, realizes detection signal amplification using the excellent superparamagnetic property of MNPs and the high loading capacity of AuNPs, constructs high-throughput, high-sensitivity fluorescence sensing technology for SEA, SEB and SEC1 detection, simplifies experimental process, and builds the rapid, efficient detection platform of multiple pollutants in food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of rapid analysis and detection technology, and particularly relates to a high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amplification and enhanced fluorescence, a detection method thereof and application. BACKGROUND

[0002] Toxins pose a great threat to human health and food safety. At present, many analysis methods have been developed to detect toxins in food and environment. Commonly used methods for detecting toxins can be divided into five categories: the first category is molecular biology methods, such as polymerase chain reaction (PCR), real-time quantitative polymerase chain reaction (RT-PCR / qPCR), and loop-mediated isothermal amplification (LAMP); the second category is immunological methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and chemiluminescence immunoassay (CLIA); the third category is quantitative detection methods, such as liquid chromatography / mass spectrometry (LC-MS / MS) and the like; the fourth category is electrochemical and colorimetric fluorescence sensor methods (biosensors).

[0003] Traditional biological assay methods are high in cost, time-consuming, and single in detection target. For example, surface plasmon resonance and surface-enhanced Raman spectroscopy require special equipment and involve complex procedures; fluorescence and chemiluminescence techniques require pretreatment and are time-consuming; LC-MS / MS methods usually have good sensitivity and high specificity, but the equipment for detection is expensive and not convenient to carry; and antibody-based immunological methods as recognition molecules are often limited by high price and poor stability. Therefore, it is urgent to establish a method for detecting toxins in biological fluids with high accuracy, sensitivity, specificity, throughput, reliability, and rapidity at a trace concentration. SUMMARY

[0004] In view of this, in order to make up for the deficiency of the prior art, the purpose of the present application is to provide a high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amplification and enhanced fluorescence and a detection method. The present application combines a fluorescence sensor with a microfluidic chip, which can amplify the signal and detect samples in high throughput, thereby improving the sensitivity and speed of the toxin detection method. The method established by the present application has the advantages of naked-eye visualization, low detection limit, wide linear range, multiple detection targets, simple operation, wide application prospect, and the like, and can meet the demand for high-throughput and high-sensitivity rapid detection of toxins.

[0005] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amplification and enhanced fluorescence (referred to as a fluorescence microfluidic chip), wherein a fluorescence sensor is arranged in the microfluidic chip, and the fluorescence sensor comprises a recognition probe and a signal probe.

[0006] The recognition probe is composed of a magnetic nanoparticle, a nucleic acid aptamer H1 of a target to be detected, and a target aptamer complementary chain H2, wherein the nucleic acid aptamer H1 is connected with the magnetic nanoparticle, and a part of H2 is complementary to H1.

[0007] The signal probe is composed of a nanomaterial with signal amplification function and three oligonucleotides H3, H4, and H5, wherein H3 is connected with the nanomaterial with signal amplification function, a part of H4 is complementary to at least part of H3, another part is complementary to part of H2, and H5 is complementary to at least part of H3 and is modified with a fluorescent group at the 5' end.

[0008] In the present application, the term "fluorescence sensor" is a device that converts a specific molecular recognition event into a detectable fluorescence signal, and realizes sensitive detection of a target by analyzing the visualized signal. The fluorescence sensor comprises a biomolecular recognition element and a fluorescence signal generator. In the present application, the term "nucleic acid aptamer" is a single-stranded oligonucleotide that specifically recognizes a target.

[0009] In the present application, the term "microfluidic" is a scientific technology for manipulating or processing micro-liters or nano-liters of small volumes of fluid. With the help of a microfluidic chip, reaction reagents can be saved, high-throughput detection can be realized, and the volume and size of the equipment can be reduced, thereby reducing the detection cost.

[0010] In some embodiments, the number of nucleotides of H1 is 20-100. In a specific embodiment of the present application, the sequence of H1 is as shown in at least one of SEQ ID NO: 1-3. When a single target is detected, one H1 sequence for the target can be used, and when multiple targets are detected simultaneously, multiple H1 sequences for the respective targets can be used.

[0011] In some embodiments, H1 has a modification group that can connect to a magnetic nanoparticle. In a specific embodiment of the present application, H1 is modified with biotin.

[0012] According to the present application, a portion of H2 is complementary to H1, thus the number of nucleotides of H2 is more than that of H1, in one embodiment, the number of nucleotides of H2 that is complementary to H1 is 10-50. In one embodiment, the number of nucleotides of H2 that is complementary to H4 is 4-15. In a specific embodiment of the present application, the sequence of H2 is as shown in at least one of SEQ ID NO: 4-6. Likewise, the number of types of H2 depends on the number of targets to be detected.

[0013] In one embodiment, H3 is a sequence composed of the same base, such as a sequence composed entirely of A, a sequence composed of T, a sequence composed of C, a sequence composed of G. In one embodiment, H3 is a sequence composed entirely of A.

[0014] The number of nucleotides of H3 is not limited, as long as the detection function of the present application can be achieved. In one embodiment, the number of nucleotides of H3 is 5-20, preferably 5-15, more preferably 8-12. In a specific embodiment of the present application, the sequence of H3 is as shown in SEQ ID NO: 7.

[0015] In one embodiment, H3 has a modification group that can connect to a nanomaterial with signal amplification function. In a specific embodiment of the present application, the 3' end of H3 is modified with a thiol group.

[0016] In one embodiment, H4 is selected from a sequence composed of nucleotides complementary to H3, a portion of H4 is completely complementary to H3. Among them, the number of nucleotides of H4 is at least 4 more than the number of nucleotides of H3, in one embodiment, at least 5, in one embodiment at least 10, further preferably 10-15. In a specific embodiment of the present application, the sequence of H4 is as shown in SEQ ID NO: 8.

[0017] In some embodiments, H5 has a sequence that is completely complementary to H3. In a specific embodiment of the present application, the sequence of H5 is as shown in SEQ ID NO: 9.

[0018] In one embodiment, the fluorescent group modified on H5 can be selected from 6-FAM, 6-HEX, 6-TET, 6-ROX, 6-TAMRA, 6-JOE, Cy3 or Cy5. In a specific embodiment of the present application, the 5' end of H5 is modified with 6-FAM.

[0019] The fluorescence sensor of the present invention requires control over the proportion of certain sequences. Preferably, the molar ratio of H1 to H2 is 1:5 to 1:200. The molar ratio of H4 to H5 is 1:1 to 1:10000, preferably 1:80 to 120, and most preferably 1:100.

[0020] In one embodiment, the molar ratio of the identification probe to the signal probe is 1:0.5 to 1:2.

[0021] In this invention, "magnetic nanoparticles" are paramagnetic nanoparticles. These magnetic nanoparticles include, but are not limited to, nanomaterials composed of magnetic elements such as iron, nickel, cobalt, chromium, manganese, gadolinium, and their compounds. In some embodiments, the magnetic nanoparticles are nanomaterials composed of iron and its compounds. In some embodiments, the magnetic nanoparticles are Fe3O4, γ-Fe3O4, or FeC. O In a specific embodiment of the present invention, the magnetic nanoparticles are Fe3O4.

[0022] In one embodiment, the magnetic nanoparticles have a modifying group capable of linking H1. Commonly used materials for surface modification of magnetic nanoparticles can be categorized into organic molecules and inorganic materials. Organic molecule modification can be further divided into coupling agent modification and surfactant modification. Typically, the purpose of coupling agent modification is to introduce reactive groups, such as -NH2, -COOH, -SH, and streptavidin, onto the surface of the nanoparticles for subsequent linking of biomolecules. In a specific embodiment of the present invention, the magnetic nanoparticles are modified with streptavidin, which can be linked to a biotin-modified DNA sequence. In one embodiment, H1 has a modification capable of linking to magnetic nanoparticles. In a specific embodiment of the present invention, H1 is modified with biotin, which can be linked to the streptavidin-modified magnetic nanoparticles.

[0023] In this invention, the nanomaterials with signal amplification functions include, but are not limited to, metal nanomaterials, quantum dots, and carbon nanomaterials. Metal nanomaterials may include gold nanomaterials and / or silver nanomaterials. In one embodiment, the metal nanomaterial is a gold nanomaterial. In a specific embodiment of this invention, the metal nanomaterial is a gold nanoparticle. Gold has a strong chemical bond with thiol groups; therefore, gold modification is beneficial for modifying the surface of nanoparticles with biomolecules in one embodiment. In a specific embodiment of this invention, the 3' end of H3 is modified with a thiol group, which can be linked to the gold nanomaterial.

[0024] In one embodiment, the method for preparing the fluorescence sensor includes: mixing and incubating a complex of nanomaterials with signal amplification function and DNA sequences H3, H4, and H5 with a complex of magnetic nanoparticles and nucleic acid aptamers H1 and aptamer complementary strands H2.

[0025] In one embodiment, the amount of H1, H2, H3, H4, H5 is independently 10-100 μL, and the concentration is independently 0.1-100 μmol / L.

[0026] In one embodiment, the preparation method of the fluorescent sensor comprises adding AuNPs-H3-H4-H5 complex solution to the MNPs-H1-H2 solution after incubation, and incubating for a period of time to form MNPs-H1-H2-H3-H4-H5-AuNPs complex (referred to as MNPs-ssDNA-AuNPs).

[0027] In one embodiment, the incubation time of MNPs-H1-H2 and AuNPs-H3-H4-H5 complex solution is 5-25 min. In a preferred embodiment, the incubation time of MNPs-H1-H2 and AuNPs-H3-H4-H5 complex solution is 18-22 min, and most preferably 20 min.

[0028] In one embodiment, the nano-material with signal amplification function is gold nanoparticles AuNPs, and the preparation method comprises the following steps:

[0029] (1) Mix HAuCl4 and deionized water, and heat and stir;

[0030] (2) When the solution is refluxed, add sodium citrate to obtain AuNPs solution.

[0031] In one embodiment, the preparation method of the complex of nano-material with signal amplification function and DNA sequences H3, H4, H5 comprises the following steps:

[0032] (1) Mix AuNPs solution and thiol-modified H3, and then freeze incubate to obtain AuNPs-H3;

[0033] (2) Mix H4 and H5 modified with a fluorescent group, and then mix with AuNPs-H3 solution to obtain AuNPs-H3-H4-H5.

[0034] In one embodiment, the ratio of H4 to H5 is 1:1-1:10000. In a preferred embodiment, the ratio of H4 to H5 is 1:100.

[0035] In one embodiment, the AuNPs in step (1) are gold nanoparticles with an average particle size of 8-14 nm. In one embodiment, the AuNPs in step (1) are gold nanoparticles with a lattice fringe width of 0.235 nm. In one embodiment, the amount of the thiol-modified DNA sequence H3 used in step (1) is 100 pmol / L, 10-20 pL.

[0036] In one embodiment, the conditions of the freeze incubation in step (1) are -20- -80 °C for 1-2 h. In one embodiment, the amount of the DNA sequence H4 used in step (2) is 1 pmol / L, 10-20 pL. In one embodiment, the concentration of the FAM fluorescent group-modified DNA sequence H5 used in step (2) is 1-10000 pmol / L.

[0037] In one embodiment, the method for preparing the complex of the magnetic nanoparticles and the aptamer H1 and the aptamer complementary strand H2 comprises the following steps:

[0038] (1) mixing a solution of the biotin-labeled aptamer H1 and a solution of the streptavidin surface-modified magnetic nanoparticles to obtain aptamer-functionalized magnetic nanoparticles MNPs-H1;

[0039] (2) mixing the MNPs-H1 suspension obtained in step (1) and the target aptamer complementary strand H2 to obtain the MNPs-H1-H2 complex.

[0040] In one embodiment, the streptavidin surface-modified magnetic nanoparticles in step (1) are magnetic nanoparticles with an average particle size of 200-480 nm. In one embodiment, the amount of the streptavidin surface-modified magnetic nanoparticles used in step (1) is 100 pL, 10 mg / mL. In one embodiment, the biotin-labeled aptamer sequence H1 in step (1) is dissolved with Buffer I, which is a buffer solution containing 10 mmol / L Tris-HCl, 1 mol / L NaCl, 1 mmol / L EDTA, and 0.1% Tween-20.

[0041] In one embodiment, the concentration of the biotin-labeled aptamer sequence H1 in step (1) is 0.1-4 pmol / L. In one embodiment, the amount of the MNPs-H1 used in step (2) is 100 pL, and the concentration is 0.2-1.4 mg / mL.

[0042] In one embodiment, the microfluidic chip is provided with a sample injection area, a liquid channel area, a reaction area and a detection area; the sample injection area is communicated with the reaction area through the liquid channel area, and the detection area is provided in the same number as the reaction area and correspondingly arranged; the signal probe of the fluorescent sensor is arranged in the sample injection area; and the identification probe of the fluorescent sensor is arranged in the reaction area.

[0043] In one embodiment, the sample injection area, the reaction area and the detection area are multiple and in the form of circular holes, and each sample injection area is communicated with multiple reaction areas through multiple liquid channel areas.

[0044] In one embodiment, the number of the sample injection areas is two and symmetrically arranged close to the center of the chip; the multiple liquid channel areas are equal in length and radially distributed, one end of each liquid channel area is communicated with the sample injection area, and the other end is communicated with the reaction area; and each detection area is arranged outside the corresponding reaction area. The arrangement of two groups of symmetric sample injection area-liquid channel area-reaction area-detection area can realize the simultaneous detection of the control group and multiple target groups.

[0045] In one embodiment, more preferably, the diameter of the sample injection area is 1-3 mm, the depth is 7-9 mm, the distance between the two sample injection areas is 8-9 mm, the diameter of the circular hole of the reaction area and the detection area is independently 5-10 mm, and the depth is 7-9 mm, and the distance between the reaction area and the detection area is 3-5 mm.

[0046] In one embodiment, the microfluidic chip is made of a chip prepared from a silicon wafer.

[0047] The second aspect of the present application provides a preparation method of a high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amplification enhanced fluorescence, comprising the following steps:

[0048] (1) The microfluidic chip is prepared by using a photolithography method;

[0049] (2) The identification probe of the fluorescent sensor is added to the reaction area of the microfluidic chip, and the signal probe is added to the sample injection area of the microfluidic chip.

[0050] In one embodiment, the method comprises the following steps:

[0051] (1) Design of the microfluidic chip: composed of four parts of a sample injection area, a liquid channel area, a reaction area and a detection area;

[0052] (2) Microfluidic chip manufacturing: after plasma processing silicon wafer, add photoresist, uniform glue, bake; after exposure, develop, clean and dry, then heat, add trimethylchlorosilane solution to prevent adhesion; pour dimethylsiloxane and curing agent mixture into the mold, bake and cure, peel off, cut and punch according to design requirements; place the dimethylsiloxane channel surface and the glass to be bonded into the plasma cleaning machine, press and bond, and bake to form.

[0053] The third aspect of the application provides a high-throughput Staphylococcus aureus enterotoxin microfluidic chip detection method based on chain amplification of fluorescence enhancement, which is carried out by using the microfluidic chip of the application and includes the following steps:

[0054] (1) Mix the sample to be tested with the recognition probe of the fluorescence sensor, and then add the signal probe and incubate;

[0055] (2) After a period of mixed incubation, perform magnetic separation, remove supernatant, redissolve, DNA double-strand dissociation, again magnetic separation, and then take the supernatant and detect fluorescence;

[0056] (3) Substitute the fluorescence value obtained in step (2) into the toxin standard curve to calculate the concentration of the toxin in the sample to be tested; the toxin standard curve is established by measuring a series of toxin standard solutions by the method of steps (1) and (2);

[0057] In one embodiment, the molar ratio of the recognition probe and the signal probe is 1:0.5-1:2.

[0058] In one embodiment, step (2) includes naked-eye visual and / or fluorescence spectrum analysis of the fluorescence intensity of the supernatant. For example, the fluorescence intensity of the supernatant at an excitation wavelength of 470 nm and an emission wavelength range of 500-700 nm is measured and analyzed.

[0059] According to a preferred embodiment, the detection method includes the following steps:

[0060] (1) Add the sample to be tested into the sample inlet area of the fluorescence microfluidic chip; make the sample to be tested enter different reaction areas via the liquid channel area and mix with the recognition probe in the reaction area;

[0061] (2) Then add the signal probe solution into the sample inlet area of the microfluidic chip, so that the signal probe enters different reaction areas via the liquid channel area and is mixed and incubated;

[0062] (3) After a period of mixed incubation, perform magnetic separation, remove supernatant, redissolve, DNA double-strand dissociation, again magnetic separation, and then take the supernatant and detect fluorescence;

[0063] (4) the fluorescence value obtained in step (3) is substituted into the toxin standard curve to calculate the concentration of the toxin in the sample to be measured; the toxin standard curve is established by measuring a series of toxin standard solutions by the method of steps (1)-(3).

[0064] In one embodiment, the incubation reaction in step (2) is performed for 5-30 min. In a specific embodiment of the present application, the incubation reaction in step (2) is performed for 15 min.

[0065] In one embodiment, the fluorescent microfluidic chip is as shown in Figure 13A The method comprises the following steps:

[0066] (1) preloading the aptamer functionalized magnetic nanoparticles MNPs-H1 and the target aptamer complementary chain H2 in the reaction zone, magnetic separation, discarding the supernatant, adding the sample to be measured and the AuNPs-H3-H4-H5 complex in the right sample inlet zone, adding the AuNPs-H3-H4-H5 complex in the left sample inlet zone, and incubating the right sample inlet zone to obtain the MNPs-ssDNA-AuNPs complex;

[0067] (2) remelting and mixing the MNPs-ssDNA-AuNPs complex obtained in step (1), magnetic separation, taking the supernatant to the detection zone for fluorescence imaging, and analyzing the fluorescence value.

[0068] The fourth aspect of the present application provides any one of the following applications of the microfluidic chip:

[0069] (i) application in detecting toxins;

[0070] (ii) application in preparing products for detecting diseases caused by toxins.

[0071] In the present application, the toxin is a naturally occurring organic poison produced by the metabolic activity of living cells or organisms. The toxin is especially in the form of protein or binding protein, which can cause serious damage to humans, animals and plants. The toxin can include fungal toxins, bacterial toxins, plant and animal toxins, and in a specific embodiment, the toxin can be a bacterial toxin, a fungal toxin. Among them, the bacterial toxin includes but is not limited to Staphylococcus aureus enterotoxin, diphtheria toxin, tetanus toxin, botulinum toxin, cholera enterotoxin and Pseudomonas exotoxin A. In some embodiments, the bacterial toxin is selected from Staphylococcus aureus enterotoxin. In a specific embodiment of the present application, the Staphylococcus aureus enterotoxin includes SEA, SEB and SEC1. Among them, the fungal toxin can be secreted by Aspergillus (mainly secreting aflatoxin, ochratoxin, etc.), Penicillium (mainly secreting citrinin, etc.), Claviceps (mainly secreting ergot), Fusarium. In some embodiments, the fungal toxin is secreted by Aspergillus and Fusarium. In some embodiments, the fungal toxin is AFB1, ZEN, OTA, FB1.

[0072] The present application combines the fluorescent aptamer sensor with the microfluidic chip, based on the "sandwich" sandwich structure system of AuNPs and MNPs combination, to construct the high-throughput detection technology of SEA, SEB and SEC1 in food. The principle is as follows Figure 18As shown, the streptavidin modified MNPs are combined with the biotin modified aptamer sequence H1, and in the presence of the target, the aptamer complementary strand H2 competes with the target for binding to the aptamer. Secondly, the thiol labeled DNA sequence H3 is combined with the AuNPs through gold thiol bonds, and different proportions of H4 and the H5 sequence labeled with a FAM fluorescent group are base-paired with the H3 sequence, and the chain amplification effect of the AuNPs is used to achieve fluorescence signal enhancement. Finally, the MNPs-ssDNA complex and the AuNPs-ssDNA complex are mixed and incubated to form a MNPs-ssDNA-AuNPs sandwich structure system. The microfluidic chip is designed with four regions, namely the sample inlet region, the liquid channel region, the reaction region and the detection region. After preloading the aptamer functionalized magnetic beads in the reaction region, magnetic separation is performed, the left sample inlet region is added with buffer, the right sample inlet region is added with the target toxin, and the left and right sample inlet regions are added with the AuNPs labeled with a fluorescent group. The DNA double-stranded structure is dissociated by heating. A large amount of fluorescently labeled signal molecules are separated out by using the excellent superparamagnetic property of the MNPs, and the supernatant is transferred to the detection region for fluorescence imaging analysis. In one specific embodiment of the present application, each three numbers of the fluorescent microfluidic chip are preloaded with a recognition probe for one SE, and each toxin can be detected three times simultaneously in a single chip, and the chip allows simultaneous detection of three toxins and analysis of the fluorescence values. In addition, the microfluidic chip can complete the detection process of all samples through simple manual operation, and the fluorescence values can be analyzed by Image J software. The chip detection system can perform high-throughput detection on three SEs, and is a portable, low-cost and simple-to-operate quantitative analysis and detection technology.

[0073] Compared with the prior art, the advantages and beneficial effects of the present application are as follows:

[0074] The present application prepares a fluorescent sensor for high-throughput rapid detection of toxins, which comprises a signal probe AuNPs-H3-H4-H5 and a recognition probe MNPs-H1-H2. The fluorescent sensor is combined with a microfluidic chip to establish a method for sensitive and rapid detection of toxins, which has the advantages of naked eye visualization, multiple detection targets, low detection limit, wide linear range, high detection sensitivity, high detection accuracy, short detection time, simple operation, and the like, and can meet the demand for high sensitivity and on-site rapid detection of toxins, and is a method for intuitive and high-sensitivity detection of toxins.

[0075] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0076] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

[0077] Figure 1A and 1B are TEM images of AuNPs nanoparticles and AuNPs-H3-H4-H5, respectively.

[0078] Figure 2A and 2B are particle size distribution diagrams of AuNPs nanoparticles and AuNPs-H3-H4-H5, respectively.

[0079] Figure 3 is a high-resolution TEM image of AuNPs nanoparticles.

[0080] Figure 4 are UV-Vis absorption spectra of AuNPs and AuNPs-H3-H4-H5.

[0081] Figure 5A and 5B are result diagrams of fluorescence signal amplification performance, including fluorescence emission spectrum curves and fluorescence intensity values of AuNPs, AuNPs-H3-H4-H5 and AuNPs-H3-H4-H5 heated at 95℃ (AuNPs-ssDNA is AuNPs-H3-H4-H5).

[0082] Figure 6A and 6B are SEM images of MNPs under different fields of view.

[0083] Figure 7A and 7B are SEM images of MNPs-H1-H2 under different fields of view.

[0084] Figure 8A and 8B are particle size distribution diagrams of MNPs and MNPs-H1-H2, respectively.

[0085] Figure 9 is a diagram of H2 probe concentration before and after detecting the target aptamer complementary strand H2 connected aptamer functionalized magnetic nanoparticles MNPs-H1.

[0086] Figure 10 is a Zeta potential diagram of AuNPs, AuNPs-H3-H4-H5, MNPs, MNPs-H1, MNPs-H1-H2 and MNPs-ssDNA-AuNPs.

[0087] Figure 11A and 11B are hydration diameter distribution diagrams of AuNPs, AuNPs-H3-H4-H5, and MNPs, MNPs-H1, MNPs-H1-H2, MNPs-ssDNA-AuNPs, respectively.

[0088] Figure 12 shows the optimization results of the SEs fluorescence detection system, wherein Figure A is a result graph of the concentration optimization of MNPs and biotin-labeled aptamer sequence H1; Figure B is a result graph of the concentration optimization of aptamer functionalized magnetic nanoparticles MNPs-H1; Figure C is a result graph of the reaction time optimization of aptamer functionalized magnetic nanoparticles MNPs-H1 and aptamer complementary chain H2 connection; Figure D is a result graph of the reaction time optimization of MNPs-H1-H2 and AuNPs-H3-H4-H5; Figure E is a result graph of the ratio optimization of DNA sequence H4 and DNA sequence H5; and Figure F is a result graph of the pH value optimization of TAE buffer.

[0089] Figure 13A and 13B are respectively a plan design graph and a physical graph of the microfluidic chip.

[0090] Figure 14 shows the optimization results of the reaction time of the microfluidic chip.

[0091] Figure 15 is the aliquot evaluation results of the reaction area of the microfluidic chip, wherein Figure A is a microfluidic chip image; Figure B is a quantitative analysis graph of the solution volume of the reaction area; and Figure C shows the mixing performance of the fluorescein isothiocyanate and sulforhodamine B solution in the microfluidic chip.

[0092] Figure 16A and 16B are performance analysis of the microfluidic chip combined with the MNPs-ssDNA-AuNPs fluorescence sensor, wherein Figure A is a fluorescence imaging graph; and Figure B is a fluorescence intensity graph.

[0093] Figure 17 is a result graph of the establishment of the SEs standard curve by the fluorescence detection mode.

[0094] Figure 18 is a principle diagram of the high-throughput fluorescence sensing detection method for detecting SEs.

[0095] Figure 19 is a result graph of the specific detection of SEs by the high-throughput fluorescence sensing detection method. DETAILED DESCRIPTION

[0096] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0097] The sequence information of the oligonucleotide sequences used in the examples is shown in Table 1.

[0098] Table 1 Oligonucleotide sequences

[0099]

[0100] Note: H1 modified with biotin-TEG (3'); H3 modified with SH C6 (3'); H5 modified with 6-FAM (5').

[0101] Example 1 Preparation of signal probe AuNPs-H3-H4-H5 and recognition probe MNPs-H1

[0102] 1. Preparation of AuNPs

[0103] (1) After the round-bottom flask was completely immersed with a mixture of HNO3 and HC1 (V HNO3 :V HCl =1:3), it was thoroughly rinsed with deionized water and kept dry before use.

[0104] (2) A mixture of 3 mL of HAuCl4 with a concentration of 5 mg / mL and 147 mL of deionized water was added to a clean round-bottom flask, which was heated at 140℃ in an oil bath and stirred vigorously.

[0105] (3) When the solution was refluxed vigorously, sodium citrate (4.05 mL, 1%) was quickly added to the flask and boiled for another 15 min. The color of the mixed solution changed from yellow to purple and then to deep burgundy. The stirring was continued until the solution cooled to room temperature, and the AuNPs solution was obtained.

[0106] (4) The UV absorption spectrum of the AuNPs solution prepared above was measured in the range of 200-900 nm, and the solution was stored in a refrigerator at -4℃ for standby.

[0107] 2. Preparation of signal probe AuNPs-H3-H4-H5 complex

[0108] (1) The thiol-labeled DNA probe H3 was connected to AuNPs through gold-thiol bonds. Specifically, 1 mL of AuNPs solution and 10 μL of thiol-modified DNA sequence H3 with a concentration of 100 μmol / L were mixed thoroughly and then placed in a refrigerator at -20℃ for incubation for more than 2 h.

[0109] (2) The incubated AuNPs-H3 solution was thawed at room temperature, and the mixed solution was washed with 1 mL of TAE buffer with a concentration of 0.01 mol / L at 4℃ and 13000 r / min for 15 min for 3 times. After washing, the mixed solution was resuspended.

[0110] (3) Mix 10 μL of DNA sequence H4 and 10 μL of H5 sequence modified by FAM fluorescent group, and then add them to the AuNPs-H3 solution. After vortex mixing, the mixture is incubated at room temperature for 20 min in a constant temperature mixing instrument, and the rotation speed is set to 500 r / min.

[0111] (4) The incubated AuNPs-H3-H4-H5 complex solution is placed in a high-speed refrigerated centrifuge at 4°C, 13000 r / min, and centrifuged with TAE buffer for 3 times, 15 min each time.

[0112] (5) Finally, the washed AuNPs-H3-H4-H5 complex is resuspended with TAE buffer, and the fluorescence intensity of the above prepared solution is measured in the range of 500-700 nm.

[0113] 3. Preparation of recognition probe MNPs-H1

[0114] (1) After the magnetic microsphere MNPs solution is placed on a shaker and shaken for 20 s, 100 μL of magnetic microspheres with a concentration of 10 mg / mL is taken into a centrifuge tube. The centrifuge tube is placed on a magnetic stand for 1 min (this operation is referred to as magnetic separation), and then the supernatant is removed with a pipette.

[0115] (2) 1 mL of Buffer I (10 mmol / L Tris-HCl, 1 mol / L NaCl, 1 mmol / L EDTA, 0.1% Tween-20) solution is added to the centrifuge tube, which is shaken and mixed thoroughly, and then the supernatant is removed after magnetic separation.

[0116] (3) After the centrifuge tube is removed, the SEA aptamer sequence H1 dissolved in Buffer I is added, and the magnetic beads are shaken and mixed thoroughly. The centrifuge tube is placed in a vortex mixing instrument and incubated at room temperature for 30 min.

[0117] (4) The incubated solution is placed on a magnetic stand, and the supernatant is removed and transferred to a new centrifuge tube after magnetic separation. Repeat step (2) for 3 times.

[0118] (5) Finally, the incubated MNPs-H1 solution is resuspended with TAE buffer.

[0119] Example 2 Characterization of signal probe AuNPs-H3-H4-H5 and recognition probe MNPs-H1

[0120] 1. Characterization and analysis of AuNPs and AuNPs-H3-H4-H5

[0121] Using a transmission electron microscope, it is observed that the AuNPs have good dispersibility and uniform overall particle size.Figure 1A ), the average diameter was about 11.38 nm ( Figure 2A , Table 1). Figure 1B The TEM image of AuNPs-H3-H4-H5 was still shown good dispersity, the average diameter was about 22.33 nm ( Figure 2B ). The lattice fringe spacing of AuNPs was observed to be 0.235 nm by high-resolution transmission electron microscopy image, which was in good agreement with the interplanar spacing of Au (III) crystal face ( Figure 3 ).

[0122] 2. Feasibility analysis of signal probe AuNPs-H3-H4-H5 fluorescence detection

[0123] Compared with AuNPs, the highest ultraviolet absorption peak position of DNA modified AuNPs was red shifted, the absorbance wavelength was 526 nm and 520 nm, respectively ( Figure 4 ). In addition, the fluorescence intensity of DNA modified AuNPs changed slightly, and the fluorescence intensity increased significantly after heating, from 601 to 8150, the relative intensity increased by 83.2 times ( Figure 5A and 5B ), which confirmed that AuNPs-H3-H4-H5 was successfully labeled by FAM fluorescent molecules, and highlighted the significant fluorescence signal amplification characteristics of AuNPs, indicating that it had the potential to be used as an efficient quencher for subsequent target detection. The excellent fluorescence signal amplification performance of AuNPs-H3-H4-H5 could be attributed to the multiple modification of DNA probes. First, the thiol-labeled DNA probe H3 was combined with AuNPs through gold thiol bond. Second, different proportions of H4 and H5 sequences labeled with FAM fluorescent groups were base-paired with H3 sequences. In summary, the chain volume amplification effect of AuNPs was used to achieve fluorescence signal enhancement.

[0124] 3. Characterization and analysis of MNPs, MNPs-ssDNA and MNPs-ssDNA-AuNPs complex

[0125] SEM images showed the spherical morphology of MNPs with different proportions ( Figure 6A and 6B ) and functionalized MNPs with different proportions ( Figure 7A and 7B ), the average particle size was 314.97 nm and 355.75 nm, respectively ( Figure 8A and 8B , Table 2). After the DNA probe H2 of SEA, SEB and SEC1 was hybridized with the aptamer functionalized magnetic nanoparticles MNPs-H1, the concentration of DNA probe H2 decreased from 16.7 ng / μL to 17.0 ng / μL ( Figure 9). The Zeta potential analysis of AuNPs-H3-H4-H5 showed that its Zeta potential value was -33.10 mV, which was significantly lower than that of AuNPs (-27.63 mV) Figure 10 ). MNPs, MNPs-H1, MNPs-H1-H2 and MNPs-ssDNA-AuNPs complexes showed similar trends, i.e. with the increase of one DNA sequence, the hydrodynamic diameter increased (Figure 11), and the negative potential increased. These results confirmed that the DNA probes successfully modified AuNPs and MNPs, thereby synthesizing MNPs-ssDNA-AuNPs for subsequent experiments.

[0126] Table 2 Particle size and zeta potential values of six samples

[0127]

[0128] Example 3 Target detection based on novel fluorescent aptamer sensor

[0129] (1) Signal probe AuNPs-H3-H4-H5 and recognition probe MNPs-H1 for SEA were prepared according to the method of Example 1.

[0130] (2) 100 μΐ, of magnetic bead aptamer complex (MNPs-H1) was magnetically separated, and the supernatant was discarded. Then, 50 μΐ, of a mixed solution of SEA at a concentration of 100 ng / mL and SEA aptamer complementary strand H2 at a concentration of 20 μιηοΙ / L was added, and the mixture was incubated at room temperature for a period of time.

[0131] (3) After incubation, 100 μΐ, of the prepared AuNPs-H3-H4-H5 complex solution was added and incubated for a period of time to form MNPs-ssDNA-AuNPs complex.

[0132] (4) After magnetic separation, the unbound AuNPs-H3-H4-H5 complex was removed, and 100 μΐ, of TE solution was added for reconstitution. The mixture was then placed in a constant temperature mixer and heated at 95°C for 5 min to dissociate the double-stranded DNA sequence. After magnetic separation, the supernatant was collected and placed in a fluorescence spectrophotometer to measure the fluorescence intensity.

[0133] 4. Construction of novel fluorescent aptamer sensor of MNPs-ssDNA-AuNPs system

[0134] (1) 100 μΐ, of magnetic bead aptamer complex (MNPs-H1) was magnetically separated, and the supernatant was discarded. Then, 50 μΐ, of a mixed solution of SEA aptamer complementary strand H2 at a concentration of 20 μιηοΙ / L was added, and the mixture was incubated at room temperature for a period of time.

[0135] (2) After the incubation is completed, 100 μL of the solution of the AuNPs-H3-H4-H5 complex prepared in the foregoing is added and incubated for a period of time to form the MNPs-ssDNA-AuNPs complex.

[0136] The method for constructing the fluorescent aptamer sensor for detecting SEB and SEC1 is the same as above, except that the aptamer H1 and the aptamer complementary strand H2 corresponding to SEB and SEC1 are used.

[0137] Example 4 Optimization of experimental conditions

[0138] In order to improve the detection performance of the biosensor, the experimental parameters such as the aptamer concentration, the functionalized magnetic bead concentration, the reaction time, and the AuNPs probe ratio are optimized.

[0139] 1. MNPs and biotin-labeled aptamer sequence H1 concentration optimization

[0140] (1) Experimental method

[0141] The aptamer concentration on the MNPs is crucial to the entire system. According to the preparation steps of the MNPs-captured aptamer probe, the aptamer sequence H1 (SEA) with concentrations of 0.1, 0.25, 0.5, 1, 2, 3, and 4 µmol / L is selected to be incubated with the magnetic microsphere solution at room temperature for 30 min, and the supernatant is separated by magnetic separation. The concentration of the supernatant and the original solution of the aptamer is detected by using an ultramicro spectrophotometer, and the optimal concentration is determined according to the difference in the aptamer concentration before and after the reaction. The SEB and SEC1 aptamer concentration determination is the same as above.

[0142] (2) Experimental results

[0143] As shown in Figure 12A , when the aptamer concentration is 1 µM, the difference between the initial aptamer concentration and the supernatant aptamer concentration after connecting the magnetic beads reaches the maximum. In order to ensure complete reaction, 2 µM is selected as the optimal concentration of the magnetic bead and aptamer reaction.

[0144] 2. Aptamer-functionalized magnetic nanoparticles MNPs-H1 concentration optimization

[0145] (1) Experimental method

[0146] According to the preparation steps of the MNPs-captured aptamer probe, the magnetic microsphere concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg / mL are selected to detect the fluorescence intensity of the entire reaction system, and the change in the fluorescence intensity difference with the increase of the functionalized magnetic bead concentration is observed.

[0147] (2) Experimental results

[0148] AsFigure 12B It is shown that the fluorescence intensity reaches the highest when the concentration of functionalized magnetic beads is 1 mg / mL, so 1 mg / mL is considered as the best reaction concentration.

[0149] 3. Optimization of reaction time for the connection between aptamer functionalized magnetic nanoparticles MNPs-H1 and aptamer complementary chain H2

[0150] (1) Experimental method

[0151] According to the detection steps of the MNPs-H1-H2 system, the time for the target to compete with the aptamer for binding is controlled to be 5, 10, 15, 20, and 25 min, and the influence of different reaction times on the MNPs-H1-H2 system is observed.

[0152] (2) Experimental results

[0153] As Figure 12C The results show that the reaction between the aptamer functionalized magnetic nanoparticles MNPs-H1 and the aptamer complementary chain H2 is complete at 20 min, and the fluorescence intensity no longer changes, so 20 min is selected as the best reaction time.

[0154] 4. Optimization of reaction time for MNPs-H1-H2 and AuNPs-H3-H4-H5

[0155] (1) Experimental method

[0156] Reaction time is a key factor affecting DNA interaction. If the time is too short, the reaction may not be complete. The reaction time for the combination of the MNPs-H1-H2 complex and the AuNPs-H3-H4-H5 complex is controlled to be 5, 10, 15, 20, and 25 min, and the influence of different reaction times on the MNPs-ssDNA-AuNPs detection system is observed.

[0157] (2) Experimental results

[0158] As Figure 12D The results show that the reaction between the MNPs-H1-H2 complex and the AuNPs-H3-H4-H5 complex is complete at 20 min, and the fluorescence intensity no longer changes, so 20 min is selected as the best reaction time.

[0159] 5. Optimization of the ratio of DNA sequence H4 to DNA sequence H5

[0160] (1) Experimental method

[0161] In the experiment, the probe H4 was designed as the important sequence connecting the MNPs-ssDNA complex and the AuNPs-ssDNA complex, and the FAM fluorescent group labeled probe H5 was as important as the signal response index of the fluorescent sensor. Through the metal enhanced fluorescence effect of AuNPs, the pairing of H3 and H4, H5 can realize chain amplification to enhance the fluorescence signal, therefore, it is necessary to optimize the proportion of H4 and H5. According to the preparation steps of the AuNPs fluorescence signal probe, the molar ratio of DNA sequences H4 and H5 was selected as 1:1, 1:10, 1:100, 1:1000, 1:10000, and the fluorescence intensity of the AuNPs fluorescence signal probe solution in the range of 500-700 nm was measured.

[0162] (2) Experimental results

[0163] Results as shown in Figure 12E When the sequence H4:H5 is 1:100, the fluorescence intensity change value AF reaches the peak value, which is the optimal ratio of modification on AuNPs.

[0164] 6, TAE buffer pH optimization

[0165] (1) Experimental method

[0166] The pH value of the TAE buffer was controlled to be 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 respectively, and other conditions were unchanged. The fluorescence images at different pH values were collected, and the fluorescence intensity was observed after analyzing the fluorescence value.

[0167] (2) Experimental results

[0168] The fluorescence response intensity of different pH buffers was measured, and the results are shown in Figure 12F The fluorescence value is the highest when the pH value is 7.5, and the subsequent experiments are carried out under the above optimized conditions.

[0169] 7, Microfluidic chip reaction time optimization

[0170] (1) Experimental method

[0171] According to the microfluidic chip (Figure 13) combined with the fluorescence sensor detection steps, the reaction time in the chip was controlled to be 5, 10, 15, 20, 25, and 30 min respectively, and other conditions were unchanged. The fluorescence images at different time points were collected, and the fluorescence intensity was observed after analyzing the fluorescence value.

[0172] (2) Experimental results

[0173] As shown in Figure 14As shown, the fluorescence response intensity values of the microfluidic chip at different reaction times were determined. In the presence of the target, the fluorescence intensity gradually weakened over time, and conversely, in the absence of the target, the fluorescence signal did not change significantly. The curve showed a steady state at 15 min, and therefore 15 min was selected as the optimal reaction time for the solution in the reaction zone of the chip.

[0174] Example 5. Design and fabrication of a microfluidic chip

[0175] (1) Design of a microfluidic chip: The model of the microfluidic chip designed in the present application is shown in FIG. 1 and consists of four parts: a sample injection zone, a liquid channel zone, a reaction zone, and a detection zone. The sample injection zone is two circular holes with a diameter of 1 mm and a depth of 8 mm, and the distance between the two sample injection zones is 8.5 mm. The reaction zone and the detection zone are each composed of 18 circular holes with a diameter of 6 mm and a depth of 8 mm, and the distance between each reaction zone and the corresponding detection zone is 4 mm. Figure 13A

[0176] (2) Fabrication of a microfluidic chip: The silicon wafer was ultrasonically cleaned with isopropyl alcohol for 20 min and then dried. After plasma treatment of the silicon wafer, an appropriate amount of photoresist was added and placed on the spin coater. After spin coating, the wafer was baked on a hot plate (65°C for 1 min and then 95°C for 60 min). The wafer was then exposed to light in a photoetching machine (10 mw, 20 s) and developed for 20 min. After isopropyl alcohol cleaning and drying, the wafer was placed on a 150°C hot plate for 5 min, and 100 µL of trimethylchlorosilane solution was added to the sealed container for anti-adhesion. A mixture of dimethylsiloxane and curing agent (10:1) was poured into the mold, which was baked in an oven at 85-90°C for 1 h to allow it to solidify. The product was then peeled off, cut and punched according to the design requirements. Finally, the dimethylsiloxane channel surface and the glass to be bonded were placed in the plasma cleaning machine for about 1 min, gently pressed together, and then placed in an 80°C oven for 30 min to form the chip, as shown in FIG. 2. Figure 13B

[0177] Example 6. Evaluation of the aliquotting ability of a microfluidic chip

[0178] (1) Experimental method

[0179] Evaluation of the aliquotting ability of the chip: 1.35 mL of red complexing colorant was taken and injected into the center holes of the left and right sample injection zones, and the volume of the complexing colorant at each outlet of the reaction zone was measured.

[0180] Simulation of the mixing of the solution in the reaction zone: 50 µL of fluorescein isothiocyanate with a concentration of 10 µmol / L was preloaded into the circular holes numbered 1-18 in the reaction zone, and 450 µL of sulfonyl rhodamine B solution with a concentration of 3 µmol / L was injected from the left and right sample injection zones.

[0181] ​​Capture fluorescence images: After 5 min, the reaction zone solution was pipetted into the wells numbered 19~36, and the fluorescence images of the green, red and mixed channels of each outlet were captured under the fluorescence inverted microscope.

[0182] (2) Experimental results

[0183] A series of experiments were performed to evaluate the performance of the microfluidic chip, which can transport liquid from the center hole to the reaction zone along the microchannel through the sample injection zone. To verify the aliquotting ability of the microfluidic chip sample injection zone liquid into the reaction zone, an equal amount of red colorant was injected into the center hole, and the dye volume in each well of the reaction zone was measured using a precision syringe. As shown in Figure 15A , the solution can be evenly distributed in the 18 reaction zone wells, and the dye volume is about 150 µL ( Figure 15B ), with a relative standard deviation RSD of 1.2%. This result proves that the chip has good aliquotting ability, which can ensure that the liquid in the reaction zone is evenly distributed in each well. To simulate the mixing of the solution during the process from the sample injection zone to the reaction zone, the fluorescence images of the red, green and mixed channels were captured under the fluorescence inverted microscope after mixing sulfonyl rhodamine B and fluorescein isothiocyanate. As shown in Figure 15C , the fluorescence images of red sulfonyl rhodamine B and green fluorescein isothiocyanate in the mixed channel detection zone well are uniformly distributed yellow images, which verifies the combination of fluorescein isothiocyanate and sulfonyl rhodamine B and the full mixing of the solution in the microfluidic chip.

[0184] Example 7 Multi-channel detection based on microfluidic chip combined with fluorescence sensor

[0185] (1) Experimental method

[0186] (i) The reaction zones numbered 1~3, 16~18 were preloaded with 100 µL of SEA aptamer functionalized magnetic bead coupled H2 (SEA recognition probe), the reaction zones numbered 4~6, 13~15 were preloaded with 100 µL of SEB aptamer functionalized magnetic bead coupled H2 (SEB recognition probe), and the reaction zones numbered 7~9, 10~12 were preloaded with 100 µL of SEC1 aptamer functionalized magnetic bead coupled H2 (SEC1 recognition probe). The supernatant was discarded after magnetic separation.

[0187] (ii) (Left channel) The blank group added 900 µL of TAE buffer solution to the center hole of the sample injection zone, reacted for a certain period of time, discarded the supernatant solution after magnetic separation, and added FAM fluorescence group labeled AuNPs solution to the sample injection zone. After a period of time, the supernatant solution was discarded after magnetic separation.

[0188] (iii) (right channel) 900 μL of SEA, SEB and SEC1 mixed solution was added to the center hole of the sample area, reacted for a certain time, the supernatant solution was removed by magnetic separation, and the FAM fluorescent group labeled AuNPs solution was added to the sample area, and after a period of time, the supernatant solution was removed by magnetic separation.

[0189] (iv) 900 μL of TAE buffer was added to the left and right sample areas to resuspend the MNPs-ssDNA-AuNPs complex, and the metal bath was heated at 95°C for 5 min, then magnetic separation was performed, and the supernatant solution in the reaction area was taken and placed in the round hole of the detection area numbered 19-36, and the round hole was imaged using a fluorescence inverted microscope.

[0190] (v) ImageJ and Origin2021 were used for further analysis of the images, and the fluorescence values of each reaction hole were quantitatively measured, the images were loaded into the ImageJ software, and the processing steps were: "image", "color", "segmentation channel", "select green channel" and "green value analysis".

[0191] (2) Experimental results

[0192] To verify whether the combination of microfluidic chip and fluorescence sensor system can be imaged in the chip, MNPs-H1-H2 (SEA recognition probe) was preloaded into the reaction areas numbered 1-3, 16-18, MNPs-H1-H2 (SEB recognition probe) was preloaded into the reaction areas numbered 4-6, 13-15, and MNPs-H1-H2 (SEC1 recognition probe) was preloaded into the reaction areas numbered 7-9, 10-12, TAE buffer was added to the left sample area, and SEA, SEB and SEC1 were added to the right sample area. Fluorescent group labeled AuNPs solution was added to the center sample hole on the left and right sides, and after the reaction was completed, the liquid was removed to the detection area. As shown in Figure 16A , each round hole on the left side showed similar fluorescence intensity, and the fluorescence brightness of the right detection area was significantly weaker than that of the left blank group, and the brightness change of the fluorescence image could be observed intuitively. Figure 16B is the fluorescence value after processing by ImageJ software, the fluorescence values of the detection areas numbered 19-27 all fluctuate around 60 with a small change range, and the fluorescence images of the detection areas numbered 28-36 all fluctuate around 120. These results all prove that the microfluidic chip can simultaneously detect SEA, SEB and SEC1, and can detect three parallel samples at a time, further proving that the chip device realizes unbiased solution distribution and excellent mixed solution state. The present application simplifies the experimental process, can successfully realize the characteristics of high-efficiency detection of multiple target substances by fluorescence sensor and microfluidic chip, and lays a foundation for subsequent multi-channel detection of toxins using the chip.

[0193] Example 8. Establishment of standard curve for high-throughput fluorescent sensing detection method

[0194] (1) Experimental method

[0195] The supernatant was removed after magnetic separation after preloading of MNPs-H1-H2 in the microfluidic chip as in Example 7. 900 μL of TAE buffer solution was added to the left channel blank group into the sample injection area, and 300 μL of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 fg / mL of SEA, SEB and SEC1 solution was added to the right channel into the sample injection area, respectively. After 15 min, the supernatant was discarded after magnetic separation, and FAM fluorescent group labeled AuNPs solution was added to the left and right sample injection areas. After incubation for 15 min, the supernatant was discarded after magnetic separation. Finally, TAE buffer was added to resuspend the complex, and after heating at 95℃, magnetic separation was performed. The supernatant in the reaction area was taken and placed in the detection area round hole, and fluorescence imaging and quantitative analysis were performed.

[0196] (2) Experimental results

[0197] Based on the optimization of the above experimental parameters, the present application evaluated the relationship between the three Staphylococcus aureus enterotoxins (SEA, SEB and SEC1) and the fluorescence intensity, from Figure 17 It can be seen that the fluorescence value decreases with the increase of the concentration of the toxin, and the detection range of SEA, SEB and SEC1 is good, showing a good linear relationship in the range of 10 2 ~10 7 For SEA, the linear regression equation y = 151.364x-16.596 (linear regression coefficient R 2 = 0.9951) was obtained by fitting calculation. For SEB, the linear regression equation y = 135.458x-15.371 (R 2 = 0.9977) was obtained by fitting calculation. For SEC1, the formula y = 139.326x-13.172 (R 2 = 0.9975) was obtained. The detection limit (LOD, 3N / S) of SEA, SEB and SEC1 was 9.19 fg / mL, 9.67 fg / mL and 11.66 fg / mL, respectively. N is the standard deviation of the detection of 15 blank solutions, and S is the slope of the calibration curve. Compared with other detection methods (Table 3), the fluorescence aptamer sensor provided by the present application has lower detection limit and wider detection range.

[0198] Table 3 Performance comparison of different methods for detecting SEs

[0199]

[0200] In summary, the microfluidic chip established by the application realizes rapid and sensitive detection by using fluorescence imaging technology, has low detection limit, wide detection range, and can realize multi-channel detection of three toxins at the same time, thereby providing a new method for the application of multi-toxin detection.

[0201] Example 9 Specificity detection of high-throughput fluorescence sensing detection method

[0202] To further verify the specificity of the method, the microfluidic chip designed by the application is used to detect other common biological toxins, including AFB1, ZEN, OTA and FB1.

[0203] 1. Experimental method

[0204] After the microfluidic chip is preloaded with MNPs-H1-H2 complex and magnetic separation, the supernatant is removed, 900 μL of TAE buffer solution is added to the center hole of the sample inlet area of the left channel blank group, 300 μL of SEA, SEB and SEC1 solution with a concentration of 10 6 fg / mL is added to the sample inlet area of the right channel, after the supernatant is removed, the AuNPs solution labeled with FAM fluorescent group is added to the sample inlet area, after incubation, the supernatant is removed, and finally the TAE buffer is added to resuspend the complex, after heating at 95℃, magnetic separation is performed, the supernatant in the reaction area is taken and placed in the detection area, and fluorescence imaging and quantitative analysis are performed. To explore the specificity, the target is replaced with AFB1, ZEN, OTA and FB1 with the same concentration, and other conditions remain unchanged, and the fluorescence value change of different target substances is observed.

[0205] 2. Experimental results

[0206] As Figure 19 shown, under the same concentration, three SEs (SEA, SEB and SEC1) show obvious fluorescence image changes, the fluorescence value is obviously reduced, and the image is dark, while other biological toxins AFB1, ZEN, OTA and FB1 do not cause fluorescence value changes, and the image is bright, which shows that the aptamer has excellent selectivity, and further verifies that the method established by the application has good specificity.

[0207] Example 10 Detection of SEs in actual samples

[0208] In order to evaluate the accuracy and practicability of the high-throughput fluorescence sensing detection method developed by the application in real sample detection, common food samples, milk, are selected, and recovery rate experiments are carried out by adding different concentrations of SEA, SEB and SEC1 standard in milk.

[0209] 1. Experimental method

[0210] (1) Pretreatment of milk samples: The milk samples purchased from a local supermarket were treated: centrifuged at 7000 r / min for 20 min at room temperature to remove the upper fat and separate and collect the supernatant.

[0211] (2) Determination of recovery rate: Different concentrations of SEA, SEB and SEC1 (0, 1, 10, 100 pg / mL) were added to the samples to prepare standard addition samples of different pollution levels, and the recovery rates of the samples SEA, SEB and SEC1 were calculated respectively, and the calculation method was: recovery rate (%) = (C 加标后 -C 样品 ) / C 标准品 x 100%, wherein C 加标后 is the detection concentration after adding SEA, SEB and SEC1 standard to the sample, C 样品 is the detection concentration before adding SEA, SEB and SEC1 standard to the sample, and C 标准品 is the concentration of the added standard sample, and each concentration was repeated 3 times.

[0212] 2. Experimental results

[0213] Different concentrations (1, 10, 100 pg / mL) of the target were added to the pretreated milk, and the fluorescence intensity was detected. As shown in Table 4, the average recovery rates of SEA, SEB and SEC1 in milk were 92.89%~109.00%, 90.00%~107.35% and 89.00%~110.40% respectively, and the relative standard deviation RSD was less than 5%. These results show that the detection method proposed by the present application can effectively and accurately detect SEs in real samples, and has good application potential in food samples.

[0214] Table 4 Recovery rate of SEs added in milk samples

[0215]

[0216] Note: - indicates that the data does not exist.

[0217] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence, characterized in that, The microfluidic chip is provided with a fluorescence sensor, and the fluorescence sensor comprises a recognition probe and a signal probe; The recognition probe is composed of magnetic nanoparticles, nucleic acid aptamer H1 of the target to be detected and target aptamer complementary chain H2, wherein the nucleic acid aptamer H1 is connected with the magnetic nanoparticles, and a part of H2 is complementary to H1; The signal probe is composed of a nanomaterial with signal amplification function and three oligonucleotides H3, H4 and H5, wherein H3 is connected with the nanomaterial with signal amplification function, a part of H4 is complementary to at least part of H3, and another part is complementary to part of H2, and H5 is complementary to at least part of H3 and is modified with a fluorescent group at the 5' end; The sequence of H1 is shown in at least one of SEQ ID NO: 1-3; The sequence of H2 is shown in at least one of SEQ ID NO: 4-6; The sequence of H3 is shown in SEQ ID NO: 7; The sequence of H4 is shown in SEQ ID NO: 8; The sequence of H5 is shown in SEQ ID NO:

9.

2. The high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amount amplification enhanced fluorescence according to claim 1, wherein H1 has a modification group capable of connecting with the magnetic nanoparticles; The molar ratio of H1 to H2 is 1:5-1:

200.

3. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 2, characterized in that, H1 is modified with biotin.

4. The high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amount amplification enhanced fluorescence according to claim 1, wherein H3 has a modification group capable of connecting with the nanomaterial with signal amplification function; The fluorescent group is selected from 6-FAM, 6-HEX, 6-TET, 6-ROX, 6-TAMRA, 6-JOE, Cy3 or Cy5; The molar ratio of H4 to H5 is 1:1-1:10000.

5. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 4, characterized in that, The 3' end of H3 is modified with a thiol group.

6. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 4, wherein, The molar ratio of H4 to H5 is 1:80-120.

7. The high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amount amplification enhanced fluorescence according to claim 1, wherein The magnetic nanoparticles are selected from magnetic elements iron, nickel, cobalt, chromium, manganese, gadolinium and their compounds; The magnetic nanoparticles have a modification group capable of connecting with H1; The nanomaterial with signal amplification function is selected from at least one of metal nanomaterial, quantum dot and carbon nanomaterial; The molar ratio of the recognition probe to the signal probe is 1:0.5-1:

2.

8. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 7, characterized in that, The magnetic nanoparticles are Fe3O4.

9. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 7, characterized in that, The surface of the magnetic nanoparticles is modified with streptavidin.

10. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 7, characterized in that, The metal nanomaterial is selected from gold nanomaterial and / or silver nanomaterial.

11. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 10, characterized in that, The metal nanomaterial is gold nanoparticles.

12. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to any one of claims 1-11, characterized in that, The preparation method of the fluorescence sensor comprises: mixing and incubating a complex of the nanomaterial with signal amplification function and DNA sequences H3, H4 and H5 with a complex of magnetic nanoparticles and nucleic acid aptamer H1 and aptamer complementary chain H2.

13. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 12, characterized in that, The incubation time is 5-25 min.

14. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 13, characterized in that, The incubation time is 18-22 min.

15. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 12, wherein, The nanomaterial with signal amplification function is gold nanoparticles AuNPs, and the preparation method comprises the following steps: (1) mix HAuCl4 and deionized water, and heat and stir; (2) when the solution is refluxed, add sodium citrate to obtain an AuNPs solution.

16. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 12, wherein, The preparation method of the complex of the nanomaterial with signal amplification function and the DNA sequences H3, H4 and H5 comprises the following steps: (1) mix the AuNPs solution and the thiol-modified H3, and then freeze and incubate to obtain AuNPs-H3; (2) mix H4 and the fluorescent group-modified H5, and then mix with the AuNPs-H3 solution to obtain AuNPs-H3-H4-H5.

17. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence of claim 12, wherein, The preparation method of the complex of the magnetic nanoparticles and the aptamer H1 and the aptamer complementary strand H2 comprises the following steps: (1) mix the magnetic nanoparticle resuspension solution modified with streptavidin and the solution of the biotin-labeled aptamer H1 to obtain aptamer-functionalized magnetic nanoparticles MNPs-H1; (2) mix the MNPs-H1 resuspension solution obtained in step (1) and the target aptamer complementary strand H2 to obtain the MNPs-H1-H2 complex.

18. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 17, characterized in that, The concentration of the magnetic nanoparticles in the magnetic nanoparticle resuspension solution is 0.2-1.4 mg / mL; and the concentration of H1 in the solution of the aptamer H1 is 0.1-4 µmol / L.

19. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to any one of claims 1-11, characterized in that, The microfluidic chip is provided with a sample injection area, a liquid channel area, a reaction area and a detection area; The sample injection area is communicated with the reaction area through the liquid channel area, and the detection area is provided in the same number as the reaction area and correspondingly arranged; The signal probe of the fluorescent sensor is arranged in the sample injection area; The recognition probe of the fluorescent sensor is arranged in the reaction area.

20. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence according to claim 19, wherein, The sample injection area, the reaction area and the detection area are all multiple and in the form of circular holes, and each sample injection area is communicated with multiple reaction areas through multiple liquid channel areas.

21. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence of claim 20, wherein, The number of the sample injection areas is two, and the two sample injection areas are symmetrically arranged close to the center of the chip; the multiple liquid channel areas are equal in length and radially distributed, one end of each liquid channel area is communicated with the sample injection area, and the other end is communicated with the reaction area; and each detection area is arranged on the outer side of the corresponding reaction area.

22. The high-throughput S. aureus enterotoxin microfluidic chip based on chain amplification-enhanced fluorescence of claim 21, wherein, The diameter of the sample injection area is 1-3 mm, the depth is 7-9 mm, the distance between the two sample injection areas is 8-9 mm, the diameters of the circular holes of the reaction area and the detection area are independently 5-10 mm, the depths are 7-9 mm, and the distance between the reaction area and the detection area is 3-5 mm; The microfluidic chip is made of a chip prepared from a silicon wafer.

23. The method for preparing a high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amplification enhanced fluorescence according to any one of claims 1-22, characterized in that, The method comprises the following steps: (1) preparing a microfluidic chip by using a photolithography method; (2) adding the recognition probe of the fluorescent sensor to the reaction area of the microfluidic chip, and adding the signal probe to the sample injection area of the microfluidic chip.

24. A high-throughput Staphylococcus aureus enterotoxin microfluidic chip detection method based on chain amplification enhanced fluorescence for non-disease diagnosis and treatment purposes, characterized in that, The high-throughput Staphylococcus aureus enterotoxin microfluidic chip based on chain amplification and enhanced fluorescence is used, and the method comprises the following steps: (1) mixing the sample to be tested with the recognition probe of the fluorescent sensor, and then adding the signal probe to mix and incubate; (3) the fluorescence value obtained in step (2) is substituted into the toxin standard curve to calculate the concentration of the toxin in the sample to be measured; the toxin standard curve is established by measuring a series of toxin standard solutions by the method of steps (1) and (2). The method comprises the following steps:

25. The detection method of claim 24, wherein, (1) the sample to be measured is added to the sample inlet area of the microfluidic chip, so that the sample to be measured enters different reaction areas via the liquid channel area and mixes with the recognition probe in the reaction area; (2) then the signal probe solution is added to the sample inlet area of the microfluidic chip, so that the signal probe enters different reaction areas via the liquid channel area and is mixed and incubated; (3) after a period of mixed incubation, magnetic separation is performed, the supernatant is removed, the sample is redissolved, the DNA double strands are dissociated, magnetic separation is performed again, and the supernatant is sucked into the detection area for fluorescence detection; (4) the fluorescence value obtained in step (3) is substituted into the toxin standard curve to calculate the concentration of the toxin in the sample to be measured; the toxin standard curve is established by measuring a series of toxin standard solutions by the method of steps (1)-(3). The toxin is at least one selected from the group consisting of fungal toxins, bacterial toxins, plant and animal toxins.

26. The detection method of claim 25, wherein, The bacterial toxin is at least one selected from the group consisting of staphylococcal enterotoxin, diphtheria toxin, tetanus toxin, botulinum toxin, cholera enterotoxin and Pseudomonas aeruginosa exotoxin A.

27. The method of claim 26, wherein, The staphylococcal enterotoxin is at least one selected from the group consisting of SEA, SEB and SEC1.

28. The method of claim 27, wherein, 29. Any one of the high-throughput staphylococcal enterotoxin microfluidic chips based on chain amplification enhanced fluorescence according to any one of claims 1-22 for any one of the following applications: (i) application in detecting toxins for non-disease diagnosis and treatment purposes; (ii) application in preparing products for detecting diseases caused by toxins. The toxin is at least one selected from the group consisting of fungal toxins, bacterial toxins, plant and animal toxins.

30. The use according to claim 29, wherein The bacterial toxin is at least one selected from the group consisting of staphylococcal enterotoxin, diphtheria toxin, tetanus toxin, botulinum toxin, cholera enterotoxin and Pseudomonas aeruginosa exotoxin A.

31. The use according to claim 30, wherein The staphylococcal enterotoxin is at least one selected from the group consisting of SEA, SEB and SEC1.

32. The use according to claim 31, characterized in that, ​

Images