CN116804673B - Pathogenic bacteria side-stream chromatography detection method integrating multivalent aptamer and multifunctional nano-enzyme - Google Patents
Pathogenic bacteria side-stream chromatography detection method integrating multivalent aptamer and multifunctional nano-enzyme Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a pathogenic bacteria lateral flow chromatography detection method integrating multivalent aptamer and multifunctional nano-enzyme, in particular relates to a detection method and a product for detecting pathogenic bacteria by using multivalent aptamer and multifunctional nano-composite material development test strips, and belongs to the field of biological detection. According to the invention, through constructing the multivalent aptamer based on HCR amplification, the affinity ratio of the aptamer and a target is greatly improved, and the affinity ratio is increased by 8 times as high as that of a conventional aptamer; by means of sum Fe 3 O 4 The excellent peroxidase activity of the MOF@PtPd nano enzyme improves the combination time of the test strip for detecting pathogenic bacteria by 3 times, and can be used for detecting other pathogenic bacteria by changing special aptamer, so that a rapid, sensitive and low-cost test strip detection method is constructed, and the detection method has huge application potential.
Description
Technical Field
The invention relates to a pathogenic bacteria lateral flow chromatography detection method integrating multivalent aptamer and multifunctional nano-enzyme, in particular to a detection method and a product for detecting pathogenic bacteria by using a multivalent aptamer and multifunctional nano-composite material development test strip, belonging to the field of biological detection.
Background
The side-stream immunoassay (LFIA) based on antigen-antibody specific recognition is one of the most widely used technologies for instantly detecting pathogenic bacteria, and has the advantages of simplicity, rapidness, visualization, no need of complex instruments and the like. However, the screening process for antibodies is complex and time consuming, and suffers from large batch-to-batch variation, high storage requirements, and the like. The single-stranded oligonucleotide ligand-aptamer screened in vitro through exponential enrichment (SELEX) has the advantages of better stability, lower cost and easier synthesis compared with an antibody. Therefore, aptamer-based lateral flow assay (Apt-LFA) has attracted considerable interest to researchers. Although aptamer related studies have progressed rapidly, there are certain limitations to the use of aptamers in the detection of pathogenic bacteria due to non-ideal binding affinities.
A multivalent aptamer (multi-Apt) system of multiple aptamers that can bind to more than one binding site or receptor on a target. Compared with monomer aptamer (mono-Apt), the synergistic effect of multi-Apt can effectively reduce the entropy loss of aptamer and target binding, thus make the aptamer have stronger binding affinity, higher specificity, higher stability. Currently, a number of methods have been developed to construct multivalent aptamers, including the use of inorganic nanomaterials, polymeric nanoparticles, liposomes, and DNA nanostructures as scaffolds, combining multiple aptamers into multivalent systems. Among these methods, hybrid Chain Reaction (HCR) is an isothermal, enzyme-free nucleic acid amplification strategy, a convenient and efficient method of constructing DNA scaffolds.
Conventional colloidal gold nanoparticles (Au NPs) have been widely used for signaling probes on LFA strips. However, the detection sensitivity of conventional LFAs is often limited, and it is difficult to meet the detection requirements of pathogenic bacteria in food. Therefore, the aptamer and the novel nano probe are adopted to develop the pathogenic bacteria LFA method and test paper with stronger identification capability and high sensitivity, and the method and the test paper have important application value.
Disclosure of Invention
[ technical problem ]
The existing detection method for the aptamer pathogenic bacteria has the problem that the affinity of the aptamer and the pathogenic bacteria is low. The conventional colloidal gold LFA has low sensitivity in detecting pathogenic bacteria, and is often combined with a plurality of nucleic acid amplification signal amplification methods so as to ensure that the detection time is long in order to meet the requirements of food monitoring. The above-mentioned shortcomings severely limit the development of aptamer lateral chromatography (LFA) papers for pathogen detection.
Technical scheme
Carboxyl-functionalized Fe used in the present invention 3 O 4 Nanoparticles are synthesized by solvothermal reaction. Fe (Fe) 3 O 4 @MOF@PtPd,Fe 3 O 4 @MOF@Pt,Fe 3 O 4 Preparation of nanoparticles @ MOF @ Pd platinum-palladium nanoparticles (PtPdNPs), platinum nanoparticles (PtNPs) and palladium nanoparticles (PdNPs) solutions were first prepared separately and added dropwise to 2.0-3.0 mL of Fe under vigorous stirring 3 O 4 N, N-Dimethylformamide (DMF) at MOF (10-12 mg/mL). The mixed solution is stirred, the sample is recovered by a magnetic field, and the mixed solution is washed, dried in vacuum and the like.
The invention adopts Fe 3 O 4 The probe is prepared by modifying vancomycin and cDNAc under the action of Bovine Serum Albumin (BSA), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in Fe 3 O 4 Probe Fe formed on the surface of @ MOF @ PtPd 3 O 4 The universal pathogen detection method is constructed by spraying SA on the test line (T line) of the NC membrane and SA-Bio-DNAc on the control line (C line) of the NC membrane. According to the detection method, SA sprayed on the T line and SA-Bio-DNAc sprayed on the C line are not required to be changed, and another gram bacterium can be detected by replacing a specific aptamer, so that convenience is provided for application expansion in the future.
The invention is based on mHCR to construct multivalent aptamer (mHCR-multi-Apt), two metastable biotinylated hairpin DNA (H1 and H2) are prepared, wherein H1 with a suspension structure is hybridized with biotinylated mono-Apt with an extension chain (Sa-Bio-exopt) through a linker, and a multi-branched DNA bracket is rich in biotin, so that the multi-branched DNA bracket is beneficial to acting with streptavidin on a test strip T line to be effectively captured.
In the invention, mHCR-multi-Apt and magnetic nano enzyme Fe are based 3 O 4 The test strip detection method (MA-MN LFA) of the @ MOF @ PtPd consists of two parts, wherein one is a target recognition system and mainly comprises a probe Fe 3 O 4 Target pair of MOF@PtPd@VAN\cDNAc and multivalent aptamer mHCR-multi-AptThe bacteria are specifically identified and captured, the other is an LFA signal output system, in the presence of target bacteria, the target bacteria form a sandwich complex through Vancomycin (VAN) and mHCR-multi-Apt double identification on the probe, and the sandwich complex is further captured by SA on T line through biotin on mHCR-multi-Apt, so that the probe Fe is captured 3 O 4 The @ MOF @ PtPd @ VAN\cDNAc is fixed to the T-line. In the absence of target bacteria, the probe is captured only on line C.
Fe in the invention 3 O 4 The magnetic properties of the @ MOF @ PtPd nanocomposite are a key feature in achieving separation of pathogenic bacteria from complex substrate samples. Fe (Fe) 3 O 4 The @ MOF @ PtPd nanocomposite has stronger superparamagnetism, and can show rapid reaction within 22 seconds under an external magnetic field, so that the magnetic separation of target bacteria in a complex sample is ensured.
The rapid detection characteristic of the invention is embodied on the response rate of the nanocomposite probe to the target bacteria. The aptamer needs to be incubated with the target bacteria for 60min or more to be fully combined, so that a better signal response is achieved, but the mHCR-multi-Apt can achieve the effect of incubating the aptamer for 60min even more than the conventional aptamer only for 15 min.
It is a first object of the present invention to provide multivalent aptamers based on multi-branched HCR (mHCR) (mHCR-multi-Apt), including biotinylated aptamer mono-Apt, priming and biotinylated hairpin chains H1, H2.
In one embodiment, the multivalent aptamer is prepared by combining a priming strand with a hairpin strand in a concentration ratio of 1: 20-1: 5, adding the aptamer to react for a certain time after mixing and incubating.
In one embodiment, the incubation is performed at 30-37℃for 1-2 h and the reaction is performed at 30-37℃for 0.5-1 h.
In one embodiment, the nucleotide sequence of the priming strand is shown in SEQ ID NO.5 and the nucleotide sequences of the hairpin strands H1 and H2 are shown in SEQ ID NO.3 and SEQ ID NO. 4.
In one embodiment, the biotinylated aptamer mono-Apt consists of an extension strand Sa-Bio-exppt and an aptamer Sa-Apt, the extension strand Sa-Bio-exppt being attached to the 5' end of the aptamer Sa-Apt; the nucleotide sequence of the Sa-Apt is shown as SEQ ID NO.1, and the nucleotide sequence of the Sa-Bio-expt is shown as SEQ ID NO. 6.
In one embodiment, the nucleotide sequence of the biotinylated aptamer mono-Apt is shown in SEQ ID No. 13.
A second object of the present invention is to provide a MA-MN LFA sensor comprising the above multivalent aptamer, a signaling probe Fe 3 O 4 @MOF@PtPd@VAN\cDNAc, chromogenic reagent and streptavidin-biotin-DNAc.
In one embodiment, the signaling probe Fe 3 O 4 The @ MOF @ PtPd @ VAN/cDNAc is made of Fe 3 O 4 The nanoparticle is composed of a core, MIL-100 (Fe) and PtPdNPs as shell layers, and vancomycin and cDNAc are modified on the surface.
In one embodiment, the Fe 3 O 4 The particle size of the nano particles is 235+/-12 nm, and the particle size of the platinum-palladium nanocubes (PtPdNPs) is 5-10 nm.
In one embodiment, the MIL-100 (Fe) can retain Fe as a spacer layer 3 O 4 The magnetic property of the nano particles can be used as an adsorption layer to load PtPdNPs, and MIL-100 (Fe) has high peroxidase activity, so that the catalytic performance of the nano enzyme is improved.
In one embodiment, the signaling probe Fe 3 O 4 The cDNAc on the @ MOF @ PtPd @ VAN\cDNAc is complementary to DNAc in streptavidin-biotin-DNAc.
In one embodiment, the nucleotide sequence of the DNAc is shown in SEQ ID No. 2; the nucleotide sequence of the cDNAc is shown as SEQ ID NO. 12.
In one embodiment, the MA-MN LFA sensor comprises a sample pad, a gold-labeled pad, an NC membrane, an absorbent pad and a PVC base plate; the sample pad, the gold mark pad, the NC film and the absorption pad are sequentially adhered to the PVC bottom plate; 2-4 mg/mL of Streptavidin (SA) and 83-85 mu mol/L of streptavidin-biotin-DNAc (SA-Bio-DNAc) are sprayed on the T line and the C line of the NC film respectively, and the NC film is dried for 4-5 h at 37-40 ℃, wherein the distance between the T line and the C line is 5-8 mm.
In one embodiment, the length of the overlapping part between the sample pad and the PVC gum back is 1-2 mm, and the sample pad is arranged above the PVC gum back; the length of the overlapping part between the sample pad and the NC film is 1-2 mm, and the NC film is arranged above the sample pad; the length of the overlapping part between the NC film and the absorption pad is 1-3 mm, and the absorption pad is arranged above the NC film.
In one embodiment, the detection of the MA-MN LFA sensor is based on a sandwich mode, multivalent aptamer, signaling probe Fe 3 O 4 Incubating the @ MOF @ PtPd @ VAN/cDNAc and a sample to be tested in an operation buffer solution at a pH of 7.4 and a temperature of 37-50 ℃ for 15-30 min, dripping the mixed solution onto a sample pad, and firmly capturing biotin on mHCR-multi-Apt by SA on T line when migrating to the T line, thereby capturing a signal probe Fe 3 O 4 The @ MOF @ PtPd @ VAN\cDNAc is immobilized on the T-line. Excessive signal probes are captured by DNAc on the C line through cDNAc, chromatography is carried out for 5-10 min, 0.3-1.0 mu L of developing solution is dripped on the T line and the C line of the test strip, the color is developed for 50s-120s, and magnetic nano enzyme Fe on the signal probes 3 O 4 The @ MOF @ PtPd can effectively catalyze H under acidic conditions 2 O 2 Hydroxyl radicals (·oh) are generated, and colorless DAB is further oxidized to brown product and deposited on the test strip, amplifying LFA colorimetric signal. Reading the result after 90-120 s, performing qualitative analysis by naked eyes or taking a picture of a test strip by using a smart phone, obtaining a gray value by imageJ processing, and quantitatively judging the target bacteria by a TURN ON signal starting mode, wherein the test result is negative only when the C line is developed; and the color development of the T line and the C line is positive. The concentration of the target bacteria is positively correlated with the signal intensity of the T line.
The third object of the present invention is to provide a method for preparing the MA-MN LFA sensor, which comprises the following specific steps:
(1) Preparation of multivalent aptamers:
initiating chain and hairpin chain are mixed according to a concentration ratio of 1: 20-1: 5, mixing and incubating for 1-2 h at 30-37 ℃, and then adding an aptamer for reacting for 0.5-1 h at 30-37 ℃;
(2) Preparation of Signal Probe Fe 3 O 4 @MOF@PtPd@VAN\cDNAc：
Platinum palladium nanoparticle PtPd NPs are loaded on Fe 3 O 4 Obtaining Fe on the surface of the @ MOF 3 O 4 @MOF@PtPd particles, and in Fe 3 O 4 Modified vancomycin and cDNAc on the surface of the particles of the @ MOF @ PtPd;
(3) Assembly of test strips
The sample pad, NC film and absorbent pad were sequentially attached to a PVC plastic backing, each overlapped by 2mm, 2mg/mL SA and 83. Mu. Mol/L SA-Bio-DNAc were sprayed on the T line and C line of the NC film, respectively, with a three-dimensional spray point platform, and dried at 30 to 37℃for 4 hours, with a distance of 5mm between the T line and C line.
In one embodiment, the nucleotide sequence of the cDNAc is set forth in SEQ ID No. 12.
A fourth object of the present invention is to provide a method for rapidly detecting pathogenic bacteria, comprising the steps of: probe the signal probe Fe 3 O 4 Mixing the @ MOF @ PtPd @ VAN/cDNAc, the multivalent aptamer and the sample to be tested, performing incubation at the pH of 7.4 and 30-37 ℃ for 10-30 min in an operation buffer solution, removing unbound pathogenic bacteria in the sample to be tested by magnetic separation, re-suspending the precipitate in the operation buffer solution, taking 50-100 mu L of precipitate, dripping the precipitate onto a sample pad of the sensor, and dripping a color development solution at T and C lines of a test strip after 5-10 min.
In one embodiment, the running buffer comprises 4 XSSC, 0.5% Tween-20, pH 7.4.
In one embodiment, the color development liquid comprises 4mmol/L DAB, 3mol/L H 2 O 2 And 0.2mol/L NaAc buffer solution (pH 3.5).
In one embodiment, the signaling probe Fe 3 O 4 The @ MOF @ PtPd @ VAN/cDNAc is made of Fe 3 O 4 The nanoparticle is composed of a core, MIL-100 (Fe) and PtPdNPs as shell layers, and vancomycin and cDNAc are modified on the surface.
In one embodiment, the Fe 3 O 4 The particle size of the nano particles is 235+/-12 nm, and the particle size of the PtPdNPs is 5-10 nm.
In one embodiment, the MIL-100 (Fe) can retain Fe as a spacer layer 3 O 4 The magnetic property of the nano particles can be used as an adsorption layer to load PtPdNPs, and MIL-100 (Fe) has high peroxidase activity, so that the catalytic performance of the nano enzyme is improved.
In one embodiment, the nucleotide sequence of the cDNAc is set forth in SEQ ID No. 12.
The beneficial effects are that:
(1) The invention discloses a rapid, high-sensitivity and high-specificity pathogen detection method based on multifunctional complex enzyme and multivalent aptamer. The method has the advantages of sensitivity, rapidness (about 30min in total time consumption) and the like for detecting staphylococcus aureus, and can be widely used for detecting various gram-positive bacteria.
(2) The detection method provided by the invention realizes high-sensitivity colorimetric detection of pathogenic bacteria (staphylococcus aureus).
(3) In the design of the multivalent aptamer of the detector, the aptamer is immobilized on the HCR bracket, so that the affinity of the aptamer to staphylococcus aureus is greatly improved, the reaction kinetics is improved, and the aptamer is combined with a probe with high recognition and enrichment capability to form a test strip detection product.
(4) The invention introduces Fe 3 O 4 The signal amplification of the aptamer test strip detection product is realized by using the excellent nano enzyme activity of the MOF@PtPd@VAN/cDNAc as a signal probe, so that the detection sensitivity is remarkably improved, and the detection linear range is widened.
Drawings
FIG. 1 (A) a schematic structural assembly diagram of mHCR-multi-Apt; (B) schematic representation of detection of target bacteria by MA-MN LFA;
FIG. 2 (A) Fe 3 O 4 Schematic diagram of synthesis and modification process of @ MOF @ PtPd; (B) Fe (Fe) 3 O 4 、(C)Fe 3 O 4 @MOF and (D) Fe 3 O 4 SEM image of @ mof @ ptpd; (E) Fe (Fe) 3 O 4 、(F)Fe 3 O 4 @MOF and (G) Fe 3 O 4 TEM @ MOF @ PtPd;
FIG. 3 (A) Fe 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 FTIR spectrum of @ MOF @ PtPd and (B) Fe 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 Ultraviolet visible absorption spectrum of @ MOF @ PtPd; (C) Fe (Fe) 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 The Zeta potential of @ MOF @ PtPd; (D) Fe (Fe) 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 Hysteresis loop of @ MOF @ PtPd; (E) Fe (Fe) 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 Photo collected using external magnetic field at mof@ptpd;
FIG. 4 (A) Fe 3 O 4 、(C)Fe 3 O 4 @MOF、(E)Fe 3 O 4 @MOF@Pd、(G)Fe 3 O 4 @MOF@Pt and (I) Fe 3 O 4 Photographs of aqueous suspensions of @ MOF @ PtPd and at H 2 O 2 Photographs of a reaction solution of catalytic oxidation TMB (1 mmol/L) in the presence of (0.1 mmol/L); (B) Fe (Fe) 3 O 4 、(D)Fe 3 O 4 @MOF、(F)Fe 3 O 4 @MOF@Pd、(H)Fe 3 O 4 @MOF@Pt and (J) Fe 3 O 4 The corresponding absorbance values at 450nm and 650nm before and after the catalyst TMB is catalyzed by the @ MOF @ PtPd;
FIG. 5 characterizes the construction of a multi-Apt by agarose gel electrophoresis: lanes 1-7 are respectively: h1 H2, a priming strand, h1+h2, a priming strand +h1+h2, an aptamer, a priming strand +h1+h2+ aptamer;
FIG. 6 (A) shows gel electrophoresis results of hairpin and trigger chains in different ratios; (B) Influence of the ratio of hairpin to priming strand on multivalent aptamer performance; (C) influence of different length linker on test strip signal;
FIG. 7 (A) saturation binding curve of mono-Apt (B) saturation binding curve of multi-Apt;
FIG. 8 (A) visualization of the corresponding effects of multi-Apt and mono-Apt with Staphylococcus aureus; (B) Response time of multi-Apt and mono-Apt to staphylococcus aureus;
FIG. 9 (A) MA-MN LFA test strip used for catalyzing front-back reaction by nano-enzyme; (B) MA-MN LFA detects the linear range of staphylococcus aureus; (C) specificity of the MA-MN LFA strip; (D) labeling experiments;
FIG. 10 is a schematic diagram of the mHCR-multi-Apt structure;
FIG. 11 optimization of pH (B) temperature catalyzed by Fe3O4@MOF@PtPd; (C) storage stability of fe3o4@mof@ptpd (n=3);
optimization of the MA-MN LFA method of fig. 12: (a) optimizing the concentration of DAB in the color development solution; (B) H in the color development liquid 2 O 2 Optimizing the concentration; (C) optimization of signaling probe volume; (D) optimization of aptamer concentration; (E) Optimization of LFA sensor run time before catalytic color development; (F) storage stability of LFA (n=3).
Detailed Description
Embodiment one: fe (Fe) 3 O 4 Preparation of @ MOF @ PtPd and evaluation of Performance
1.Fe 3 O 4 Synthesis and identification of the @ MOF @ PtPd composite (FIG. 2)
(1) Carboxyl functional Fe 3 O 4 Preparation of nanoparticles
Synthesis of carboxyl-functionalized Fe by solvothermal reaction 3 O 4 The specific steps of the nanoparticle are as follows:
0.8g of FeCl 3 ·6H 2 O was dissolved in 40mL of ethylene glycol, and the mixture was stirred well to obtain a transparent dispersion. Then, 2g of anhydrous NH 4 OAc (ammonia acetate) and 0.5g EDTA-2Na (disodium tetraacetate oxalate) were added sequentially to the dispersion. After vigorous ultrasonic stirring for 0.5h, until it became homogeneous, it was then transferred to a teflon-lined stainless steel autoclave for reaction at 200 ℃ for 12h. Washing the obtained product with deionized water and ethanol for 3 times, and vacuum drying at 60deg.C for 12 hr to obtain carboxyl functional Fe 3 O 4 And (3) nanoparticles.
(2)Fe 3 O 4 Preparation of MOF nanoparticles
Fe synthesized by one-pot assembly procedure 3 O 4 The specific steps of @ MOF are as follows:
50mg of carboxyl-functionalized Fe prepared in step (1) 3 O 4 Nanoparticles dispersed in 4mLFeCl 3 ·6H 2 To the O (ferric chloride hexahydrate) ethanol solution, 4mL of 10mmol/L H btc (trimesic acid) ethanol solution was then added and stirring was continued for 10min. After stirring at 70 ℃ for 2h, the product was isolated and collected by external magnetic field and washed three times with ethanol. Then the product is added with FeCl with the same amount again 3 ·6H 2 O ethanol solution and H3btc ethanol solution, the process was repeated 5 times. Finally, recovering the sample by an external magnetic field, washing with ethanol three times, and vacuum drying at 60 ℃ for 12 hours to prepare Fe 3 O 4 @mof nanoparticles.
(3) Preparation of platinum palladium nanocubes
Preparation of PtPdNPs:
1mLNa 2 PdCl 4 (sodium tetrachloropalladate) (20 mmol/L), 1mLH 2 PtCl 6 ·6H 2 O (chloroplatinic acid hexahydrate) (20 mmol/L), potassium iodide (KI) (83 mg) and polyvinylpyrrolidone (PVP) (160 mg) were mixed together with 10mL of DMF. The mixture was sonicated for about 2min. The resulting homogeneous mixture was heated at 130 ℃ for 5h, then cooled to room temperature and the precipitate was washed 3 times with acetone. Resuspended in DMF and stored at 4 ℃.
(4)Fe 3 O 4 Preparation of @ MOF @ PtPd
Fe prepared in the step (2) 3 O 4 20mg of MOF nanoparticles were mixed with 2ml of PtPdNPs resuspended in DMF from step (3), stirred at room temperature for two hours, the samples recovered by external magnetic field and washed with ethanol, dried in vacuo for 12 hours.
Further characterization of Fe using Fourier IR Spectroscopy 3 O 4 The structure of the @ MOF @ PtPd composite is shown in FIG. 3A at 592cm -1 Is due to Fe 3 O 4 Stretching vibration of Fe-O in the composite material shows that Fe exists in the composite material 3 O 4 The method comprises the steps of carrying out a first treatment on the surface of the The radical is 1579cm -1 And 1382cm -1 A nearby vibration band, indicating the presence of a dicarboxylic acid salt, which can impart Fe 3 O 4 The excellent dispersibility and stability of the @ MOF @ PtPd can also provide active sites for the fixation of other substances, thereby facilitating the subsequent surface modification. The ultraviolet-visible absorption spectrum (FIG. 3B) shows that Fe 3 O 4 Has a typical absorption peak around 450nm when PtPdNPs of the platinum-palladium nano particles are loaded on Fe 3 O 4 The absorption peak disappears and a new absorption peak appears at 640 nm. Together, these results demonstrate successful synthesis of Fe 3 O 4 @MOF@PtPd。
Fe 3 O 4 @MOF@Pt and Fe 3 O 4 The preparation method of the@MOF@Pd is the same as the step (4), and PtPdNPs prepared by taking hexahydrated chloroplatinic acid as a raw material according to a nanoparticle conventional method and PdNPs prepared by taking tetrachloropalladate potassium as a raw material according to a nanoparticle conventional method are replaced.
2.Fe 3 O 4 Magnetic separation effect of @ MOF @ PtPd
(1) As shown in FIG. 3C, fe 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 The zeta potential of @ MOF @ PtPd was-24.5 mV, -39.1mV and-28.3 mV, respectively.
(2) According to the typical criterion of nano dispersion stability of zeta potential values, the potential values within 20-30 indicate better stability of the nanoparticles. Fe (Fe) 3 O 4 The zeta potential of @ MOF @ PtPd was-28.3 mV, indicating Fe 3 O 4 The @ MOF @ PtPd has sufficient electrostatic repulsive force to secure its dispersion stability.
(3) The hysteresis curve (fig. 3D) shows a typical curve of superparamagnetic nanomaterials, wherein Fe 3 O 4 @MOF and Fe 3 O 4 Saturation magnetization (Ms) of @ MOF @ PtPd was 51.3emu/g and 47.6emu/g, respectively, relative to Fe 3 O 4 Ms (68.4 emu/g) is low, indicating that MIL-100 (Fe) and PtPdNPs vs. Fe 3 O 4 Has a certain shielding effect.
(4) As shown in FIG. 3E, fe under shielding effect 3 O 4 The magnetic properties of @ MOF @ PtPd are still very strong and can exhibit a fast response within 22s under an external magnetic field, thereby ensuring magnetic separation of target bacteria in complex samples.
3.Fe 3 O 4 Peroxidase Activity of @ MOF @ PtPd
The signal amplifying capacity of a nanocomposite with nanoenzyme properties is generally determined by its peroxidase activity, while comparing Fe 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 @MOF@PtPd at H 2 O 2 In the presence of TMB (3, 3', 5' -tetramethylbenzidine).
The Fe was changed at a fixed concentration of hydrogen peroxide (0.1 mmol/L) and TMB (1 mmol/L) 3 O 4 、Fe 3 O 4 @MOF and Fe 3 O 4 Particle concentration of @ MOF @ PtPd (0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 2000, 5000 and 10000 fmol/L), absorbance values at 450nm and 600nm before and after incubation at 37℃for 1 hour were measured for catalytic TMB (3, 3', 5' -tetramethylbenzidine).
The results show that by recording the absorbance at 450nm, fe, without catalytic reaction 3 O 4 Nanoparticle (FIG. 4A), fe 3 O 4 @MOF (FIG. 4C) and Fe 3 O 4 The lowest detection concentrations of @ MOF @ PtPd (FIG. 4I) were each 500fmol/L. After adding H 2 O 2 After TMB, by recording the absorbance at 650nm, it was found that the occurrence of LOD (minimum detection limit) of three nanomaterials was reduced to different degrees, respectively 100fmol/L, 25fmol/L, and 0.75fmol/L, which all achieved signal amplification to different degrees. Compared with the prior to catalytic amplification, the multifunctional Fe 3 O 4 The @ MOF @ PtPd nano-enzyme showed about 667-fold signal amplification.
By TMB and H respectively 2 O 2 Using Fe as a substrate 3 O 4 Catalytic reaction of @ MOF @ PtPd at 37℃for 1h, monitoring the change in absorbance of the solution at 650nm, wherein Fe 3 O 4 The @ MOF @ PtPd concentration was 5pmol/L and reacted in 200. Mu.L of 0.2mol/L NaAc buffer (pH 3.5, 37 ℃).
First, H of a fixed concentration 2 O 2 (1 mmol/L) and TMB (20, 40, 80, 160, 320, 480, 640 and 960. Mu. Mol/L) at different concentrations were used for kinetic analysis with TMB as substrate.
Also, the process of the present invention is,fixed concentrations of TMB (1 mmol/L) and different concentrations of H 2 O 2 (2, 4, 6, 10, 15, 20, 25 and 30 mmol/L) in H 2 O 2 Is a kinetic analysis of the substrate.
FIGS. 4E, 4G and 4I compare Fe 3 O 4 @MOF@Pt、Fe 3 O 4 @MOF@Pd and Fe 3 O 4 Catalytic activity of three nano materials of @ MOF @ PtPd, and the result shows that Fe 3 O 4 @MOF@Pt、Fe 3 O 4 @MOF@Pd and Fe 3 O 4 LOD of the three nano materials of @ MOF @ PtPd are respectively 5fmol/L, 7.5fmol/L and 0.75fmol/L. The results prove that the synergistic effect of PtPd bimetallic of PtPdNPs can indeed improve the catalytic activity of nano-enzyme.
4. Stability of nanoenzyme
The stability of the nano-enzyme plays a key role in the accuracy and repeatability of the MA-MN LFA sensor. Nano enzyme Fe prepared in step 1 3 O 4 After storage at 4℃for 1-30 days @ PDA @ PtPd, the mixture was removed and TMB and H were immobilized 2 O 2 The concentration of (1 mmol/L) and the catalytic temperature were 37℃and the catalytic time was 1h, and the absorbance of the solution at 650nm was monitored, wherein Fe 3 O 4 The @ MOF @ PtPd concentration was 5pmol/L and reacted in 200. Mu.L of 0.2mol/L NaAc buffer (pH 3.5, 37 ℃ C.) with the results shown in FIG. 11C, fe 3 O 4 The @ PDA @ PtPd remained active at day 30, indicating good long term storage stability.
Embodiment two: construction of multivalent aptamers
1) Construction of multivalent aptamers
Before the reaction, all DNA sequences (priming strand, H1, H2 and mono-Apt) were buffered with BB buffer (BB buffer comprising 50mmol/L Tris-HCl (pH 7.4), 100mmol/L NaCl, 5mmol/L KCl and 1mmol/L MgCl) 2 ) Dissolve to 100. Mu. Mol/L, denature at 95℃for 5-10 min, then immediately cool on ice for 10-15 min to form a stable hairpin structure (FIG. 10). Subsequently, the initiating chain (final concentration of 0.1. Mu. Mol/L) was mixed with H1 (final concentration of 1. Mu. Mol/L) and H2 (final concentration of 1. Mu. Mol/L) in BB buffer, and incubated at 37℃for 1 to 2 hours to form a stable HCR product. Thereafter, it willMono-Apt (final concentration 1.4. Mu. Mol/L) was added to the HCR product and incubated at 37℃for 0.5h to construct mHCR-multi-Apt. For comparison with monovalent aptamers, the concentration of aptamer added to the HCR product is used to represent the concentration of multivalent aptamer.
Taking the case of an aptamer of staphylococcus aureus, the formation of multivalent aptamers was characterized by agarose gel electrophoresis. As shown in FIG. 5, lane 5 forms long-chain DNA, confirming the production of HCR product. When the aptamer was added to the HCR product, no aptamer band was present in lane 7, indicating that the aptamer was assembled on HCR, forming the mHCR-multi-Apt product (lane 7).
2) Optimization of multivalent aptamers
1. Selection of ratio of hairpin to initiating strand
The number of identical aptamers grafted onto a multi-Apt scaffold is called the multi-Apt valency. Which can affect the binding affinity of multi-Apt. In order to design an effective multi-Apt, the present invention evaluates the effect of valency on multi-Apt binding affinity.
The valence of mHCR-multi-Apt is controlled by controlling the hairpin DNA and the priming strand in different concentration ratios, namely the step of constructing the multivalent aptamer is the same as the step 1, the concentration of the priming strand is controlled to be 0.1 mu mol/L, and the concentration ratio of the hairpin DNA to the priming strand is adjusted to be 1:1, 1.25:1, 1.5:1, 2:1, 3:1, 5:1, 10:1, 20:1, 50:1 and 100:1. FIG. 6A shows the HCR product of hairpin DNA and priming strand at different concentration ratios. The electropherograms show that the average length of HCR product is inversely proportional to the ratio of the concentrations of the initiating chains.
Visual characterization was performed with the constructed MA-MN LFA (FIG. 6B), showing that as the hairpin to priming strand ratio increased from 1:1 to 100:1, the signal rapidly increased to a maximum and then decreased with further increases in ratio.
In this example the optimal ratio of hairpin to priming strand can be chosen to be 10:1.
Linker Length selection
linker is a sequence on H1 complementary to the extended strand of the aptamer (Sa-Bio-expt), and the steric flexibility of the aptamer is improved by optimizing the length of linker that the aptamer is attached to the HCR product in order to minimize conformational barriers during the interaction of multi-Apt with the target molecule.
As shown in FIG. 6C, a linker having a length ranging from 20 to 28 bp was designed, and as a result, it was found that the MA-MN LFA detected the maximum signal of Staphylococcus aureus when the length of the linker was 25bp, i.e., (20+5). The nucleotide sequence of (20+5) is shown as SEQ ID NO. 9.
3. Optimized mHCR-Multi-Apt evaluation
After successful synthesis of mHCR-multi-Apt (principle referring to FIG. 1), the affinity of the optimized mHCR-multi-Apt was further evaluated using a flow cytometer.
Firstly, taking a plurality of 1mL staphylococcus aureus culture solution in logarithmic phase, centrifuging for 5min at 4500r/min at 4 ℃, discarding the culture medium, and then continuously washing twice with 1 XBB buffer solution. The multivalent aptamer solution or monovalent aptamer solution (0, 25, 50, 100, 150, 200, and 300 nmol/L) prepared in step 2 was added to the washed pellet, resuspended, and incubated at 37℃for 45min before flow cytometry analysis. Data were processed using GraphPadPrism 8 software and the dissociation constants of the aptamers were calculated.
As shown in FIG. 7, the Kd values of mHCR-multi-Apt and mono-Apt were 16.77nmol/L and 135.9nmol/L, respectively. Thus, the binding affinity of the aptamer is increased 8-fold due to multivalent binding.
Oligonucleotide strand sequences used in Table 1
Embodiment III: preparation of lateral chromatography test strip based on multivalent aptamer
1. Preparation of magnetic nano enzyme marked VAN and cDNAc probes
2mL of Fe prepared in example 1 3 O 4 @MOF@PtThe Pd suspension (10 mg/mL) was washed 3 times with PBS (0.01 mol/L, pH 7.4) and resuspended in PBS containing 5.8mg EDC,6.5mgNHS. After 1h of activation, PBS was washed 3 times and dispersed in 8mL of PBS containing 24mg BSA. After 2h, BSA-modified Fe was collected by external magnetic field 3 O 4 @MOF@PtPd, and after washing 3 times with PBS, dispersed in PBS.
To prepare the probe Fe 3 O 4 @MOF@PtPd@VAN\cDNAc, 10mg/mL of VAN was first activated with 5mg/mL of EDC and 5.0mg/mL of NHS in 1.0mL of PBS for 10min at room temperature to give a mixture A. Simultaneously 20. Mu.L (100. Mu. Mol/L) cDNAc was activated with 5mg/mL EDC and 5mg/mL NHS in 1mL PBS for 10min at room temperature to give mixture B. Adding the mixed solution A and the mixed solution B to the BSA modified Fe 3 O 4 In @ MOF @ PtPd, the reaction was carried out for 6h at 37℃with continuous shaking at 180r/min, washed 3 times with PBS, and dispersed in 2mL of PBS containing 10% sucrose and 0.25% Tween-20. The probes were stored at 4 ℃ for further use.
2. Preparation of test paper strip
Four materials were used to prepare a lateral flow device, namely a sample pad, NC membrane and absorbent pad, sequentially attached to a PVC gum back, each 2mm overlap. 2mg/mL SA and 83. Mu. Mol/LSA-Bio-DNAc were sprayed on the test line (T line) and the control line (C line) of the NC film, respectively, using a three-dimensional spray point platform, and dried at 37℃for 4 hours, the distance between the T line and the C line being 5mm. Finally, the strips were cut to 4mm width and stored at 4 ℃.
3. Establishment of detection method based on LFA sensor
Taking several 1mL culture solutions of bacteria to be tested in logarithmic phase, centrifuging at 4500r/min at 4deg.C for 5min, discarding culture medium, washing with 1×BB buffer solution twice, and re-suspending to 1×10 6 Then, 6.0. Mu.L of Fe prepared in step 1 was added 3 O 4 @MOF@PtPd@VAN\cDNAc (0.5 mg/mL) and mHCR-multi-Apt (40 nmol/L) prepared in example 2 were performed in running buffer (pH 7.4) in a total volume of 200. Mu.L. After incubation at 37℃for 20min, unbound bacteria were removed by magnetic separation and the pellet was resuspended in 200. Mu.L of running buffer (4 XSSC, 0.5% Tween-20, pH 7.4). 70.0. Mu.L of the sample dropped into the lateral flow chromatography device was takenAfter 8min on the pad, 0.8. Mu.L of the chromogenic solution (4 mmol/L DAB, 3mol/L H) was added dropwise to the T and C lines of the test strip 2 O 2 And 0.2mol/L NaAc buffer solution (pH 3.5)).
4. The experimental conditions of the LFA are optimized:
(1) Concentration of DAB (3, 3' -diaminobenzidine) in the color development solution;
under the condition that other conditions are not changed (the temperature is 37 ℃, the pH is 7.4, the probe concentration is 0.5mg/mL, the probe adding volume is 6 mu L, the mHCR-multi-Apt concentration is 40nmol/L, the time before the catalytic color development is 8min, and the H in the color development liquid 2 O 2 The concentration of the pathogen is 1 multiplied by 10, and the pH of the running buffer solution is 7.4 6 ) The signal intensity is judged by the color development of the test strip by only changing the concentration (1-10 mmol/L) of DAB in the color development liquid.
(2) H in the color development liquid 2 O 2 Is a concentration of (2);
in the specific mode, in the same step (1), only H in the color development liquid is changed 2 O 2 Concentration (0.5-5 mol/L).
(3) The volume of the signaling probe;
in the same manner as in step (1), only the volume (1-10. Mu.L) of the signaling probe is changed.
(4) Concentration of the aptamer;
in the specific manner, in the same manner as in the step (1), only the concentration (10-60 nmol/L) of the aptamer was changed.
(5) The LFA sensor run time before catalytic color development. And LFA sensor stability was evaluated.
In the specific manner, the method is the same as the step (1), and only the time (1-10 min) of the LFA sensor before the catalytic color development is changed
As shown in FIGS. 12A-E, the concentration of DAB in the color development liquid was (1) 4mmol/L as analyzed by single factor experiment; (2) H 2 O 2 The concentration of (2) is 3mol/L; (3) the volume of the probe used was 6. Mu.L; (4) the concentration of the aptamer is 40nmol/L; (5) performing catalytic color development after the LFA sensor is operated for 8 min. As shown in fig. 12F, the LFA sensor showed no significant drop in output signal at day 90, indicating good long-term storage stability.
(6) Optimization of detection reaction conditions (pH, temperature)
pH value: the detection method is the same as that in the step 3, the pH of the prepared buffer solution is changed to change the pH (2.5-8), and the result shows that Fe 3 O 4 The activity of @ MOF @ PtPd was maximum at pH3.5 (FIG. 11A).
Next, optimizing the reaction temperature, changing the catalytic temperature (4-70 ℃ C.), and the result shows that Fe is present when the temperature is between 37 ℃ and 50 ℃ C 3 O 4 The catalytic activities @ MOF @ PtPd were all at a higher level with 45℃being the optimum temperature (FIG. 11B).
5. Assessment of binding kinetics to target
According to the detection method of the step 3, staphylococcus aureus is respectively incubated with the immobilized monovalent aptamer and the multivalent aptamer for different time (0-90 min), then the staphylococcus aureus is dripped on a test strip, and a chromogenic signal is recorded.
The binding kinetics to the target was briefly assessed by comparing the response times of mHCR-multi-Apt and mono-Apt in practical applications. As shown in FIG. 8, mono-Apt requires about 60 minutes to achieve optimal signal response, whereas mHCR-multi-Apt signal reaches a relatively high level after 15 minutes of reaction and reaches a maximum at 20 minutes. Thus, mHCR-multi-Apt can provide higher binding affinity and fast kinetics, which will greatly increase the efficiency of pathogen detection.
Embodiment four: detection of staphylococcus aureus
(1) The nano enzyme improves the detection sensitivity
Taking a plurality of 1mL of bacterial culture solution in logarithmic phase, respectively centrifuging in 1.5mL centrifuge tube at 4500r/min for 5min at 4deg.C, discarding the culture medium, washing with 1 XBB buffer solution twice, and adding 6.0 μl Fe prepared in example 3 3 O 4 MOF@PtPd@VAN\cDNAc (0.5 mg/mL) was added to mHCR-multi-Apt (40 nmol/L) and suspensions of Staphylococcus aureus at various concentrations (0, 1X 10) 1 、5×10 1 、1×10 2 、5×10 2 、1×10 3 、5×10 3 、1×10 4 、5×10 4 、1×10 5 、1×10 6 、1×10 7 And 1X 10 8 CFU/mL) was performed in running buffer (pH 7.4) in a total volume of 200. Mu.L. After incubation at 37℃for 20min, unbound Staphylococcus aureus was removed by magnetic separation and the pellet was resuspended in 200. Mu.L of running buffer (4 XSSC, 0.5% Tween-20, pH 7.4). 70.0 μl was dropped onto the sample pad of the lateral flow device prepared in example 3, and after 8min, 0.8 μl of the color development liquid was dropped at the T and C lines of the test strip, and the reaction was catalyzed for 90s. Then, a picture of the test strip is taken with a smart phone, and ImageJ processing obtains a gray value.
As a result, as shown in fig. 9A, the signal intensity of T line gradually increased with the increase of staphylococcus aureus concentration before and after catalysis. The linear range of MA-MN LFA detection of staphylococcus aureus before and after nano enzyme catalysis is 5 multiplied by 10 respectively 2 CFU/mL～1×10 8 CFU/mL (pre-catalysis, y=0.1306 x-0.2722, r 2 =0.987) and 1×10 1 CFU/mL～1×10 5 CFU/mL (after catalysis, y=0.2322x+0.0754, r 2 =0.976), LOD values of 97CFU/mL and 2CFU/mL, respectively (fig. 9B), demonstrating that MA-MN LFA strip detection method passed through nano-enzyme Fe 3 O 4 After catalysis of @ MOF @ PtPd, the detection sensitivity is improved by 42 times compared with that before catalysis.
(2) Specificity of MA-MN LFA test strip
The specificity of the MA-MN LFA test strip constructed in example 3 was tested using Listeria monocytogenes, escherichia coli, salmonella typhimurium, vibrio parahaemolyticus and Shigella flexneri, respectively, and the bacterial concentration was set to 1X 10 6 CFU/mL, the result is shown in figure 9C, and the MA-MN LFA test strip provided by the invention has good specificity.
(3) The practical application capacity of the test strip is evaluated by adopting a classical labeling recovery test:
the recovery rate was tested using a milk simulation sample, the steps were: adding staphylococcus aureus at different concentrations into milk, and adding 6.0 mu LFe 3 O 4 @MOF@PtPd@VAN\cDNAc (0.5 mg/mL) and mHCR-multi-Apt (40 nmol/L) prepared in example 2 were performed in running buffer (pH 7.4) in a total volume of 200. Mu.L. After incubation at 37℃for 20min, unbound bacteria were removed by magnetic separation,and the pellet was resuspended in 200. Mu.L of running buffer (4 XSSC, 0.5% Tween-20, pH 7.4). 70.0 mu L of the color development liquid (4 mmol/L DAB, 3 mol/LH) is dripped on a sample pad of a lateral flow chromatography device, and after 8min, 0.8 mu L of the color development liquid (4 mmol/L DAB, 3 mol/LH) is dripped on T and C lines of a test strip 2 O 2 And 0.2mol/L NaAc buffer solution (pH 3.5)).
The results are shown in Table 2, and the MA-MN LFA test strip detection method shows excellent recovery rate (96.9% -103.4%) and a relative standard deviation of less than 6.7%. FIG. 9D further shows that the amount of Staphylococcus aureus detected in milk using MA-MN LFA is comparable to the amount of Staphylococcus aureus added. Therefore, the MA-MN LFA test strip detection method has good accuracy and reproducibility for detecting staphylococcus aureus in a real sample.
Table 2 detection of staphylococcus aureus in milk samples (n=3)
Note that: a is not detected; rsd= (s.d./mean) ×100%, each data represents an average of three replicates.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A MA-MN lateral chromatography sensor is characterized in that the sensor comprises a multivalent aptamer and a signaling probe Fe 3 O 4 @MOF@PtPd@VAN\cDNAc, chromogenic reagent and streptavidin-biotin-DNAc;
the signal probe Fe 3 O 4 The @ MOF @ PtPd @ VAN/cDNAc is made of Fe 3 O 4 The nano particles are composed of cores, MIL-100 (Fe) and platinum-palladium nano cubic PtPd NPs as shell layers, and vancomycin VAN and cDNAc are modified on the surfaces;
the multivalent aptamer comprises a biotinylated aptamer, a priming strand, and a biotinylated hairpin strand, the biotinylated hairpin strand comprising biotinylated H1 and biotinylated H2;
the nucleotide sequence of the initiation chain is shown as SEQ ID NO. 5; the nucleotide sequence of H1 is shown as SEQ ID NO.3, and the nucleotide sequence of H2 is shown as SEQ ID NO. 4; the nucleotide sequence of the aptamer in the biotinylated aptamer is shown as SEQ ID NO. 13; the nucleotide sequence of DNAc is shown as SEQ ID NO. 2; the nucleotide sequence of the cDNAc is shown in SEQ ID NO. 12;
the method for preparing the multivalent aptamer is to mix a priming strand and a biotinylated hairpin strand according to a concentration ratio of 1: 20-1: 5, mixing the mixture and incubating the mixture for 1 to 2 hours at the temperature of between 30 and 37 ℃, and then adding the biotinylated aptamer to react for 0.5 to 1 hour at the temperature of between 30 and 37 ℃;
the MA-MN lateral chromatography sensor is a test strip, and the test strip comprises a sample pad, an NC film, an absorption pad and a PVC gum back; the PVC adhesive back is sequentially stuck with a sample pad, an NC film and an absorption pad; 2-4 mg/mL of streptavidin and 83-85 mu mol/L of streptavidin-biotin-DNAc are sprayed on a test line and a control line of the NC film respectively, and the NC film is dried for 4-5 hours at 37-40 ℃, and the distance between the test line and the control line is 5-8 mm.
2. The MA-MN lateral-chromatography sensor of claim 1, wherein the Fe 3 O 4 The particle size of the nano particles is 235+/-12 nm, and the particle size of the platinum-palladium nano cubic PtPd NPs is 5-10 nm.
3. A method of making the MA-MN lateral-chromatography sensor of claim 1 or 2, characterized by the specific steps of:
(1) Preparation of multivalent aptamers:
the ratio of the initiator chain to biotinylated hairpin chain was 1: 20-1: 5, mixing the mixture and incubating the mixture for 1 to 2 hours at the temperature of between 30 and 37 ℃, and then adding the biotinylated aptamer to react for 0.5 to 1 hour at the temperature of between 30 and 37 ℃;
(2) Preparation of Signal Probe Fe 3 O 4 @MOF@PtPd@VAN\cDNAc：
Platinum palladium nanocube PtPd NPs are loaded on Fe 3 O 4 Obtaining Fe on the surface of the @ MOF 3 O 4 @MOF@PtPd particles, and in Fe 3 O 4 Modified vancomycin VAN and cDNAc on the surface of the particles of the @ MOF @ PtPd;
(3) Assembly of test strips
Sequentially attaching a sample pad, an NC film and an absorption pad on a PVC rubber back, wherein each pad is overlapped by 2mm, respectively spraying 2mg/mL streptavidin and 83 mu mol/L streptavidin-biotin-DNAc on a test line and a control line of the NC film by using a three-dimensional spray point platform, and drying at 30-37 ℃ for 4 hours, wherein the distance between the test line and the control line is 5mm;
the nucleotide sequence of the initiation chain is shown as SEQ ID NO. 5; the nucleotide sequence of H1 is shown as SEQ ID NO.3, and the nucleotide sequence of H2 is shown as SEQ ID NO. 4; the nucleotide sequence of the aptamer in the biotinylated aptamer is shown as SEQ ID NO. 13; the nucleotide sequence of DNAc is shown as SEQ ID NO. 2; the nucleotide sequence of the cDNAc is shown as SEQ ID NO. 12.
4. A method for rapidly detecting pathogenic bacteria for non-disease diagnostic purposes, wherein the method is to detect pathogenic bacteria using the MA-MN lateral chromatography sensor of claim 1 or 2.
5. The method according to claim 4, characterized in that the method comprises the following specific steps: probe the signal probe Fe 3 O 4 Mixing the @ MOF @ PtPd @ VAN/cDNAc, the multivalent aptamer and the sample to be tested, incubating in an operation buffer solution for 10-30 min at 30-37 ℃, removing unbound pathogenic bacteria in the sample to be tested by magnetic separation, suspending the precipitate in the operation buffer solution again, taking 50-100 mu L of precipitate, dripping the precipitate onto a sample pad of the sensor, and dripping a color development solution at a test line and a control line of a test strip after 5-10 min.
6. The method according to claim 5, wherein the color-developing solution comprises 4mmol/L DAB, 3mol/L H 2 O 2 And 0.2mol/LNaAc buffer solution, pH 3.5.
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