Bimodal biosensor detection kit for detecting escherichia coli and preparation method of bimodal biosensor detection kit

Abstract

The invention particularly relates to a bimodal biosensor detection kit for detecting escherichia coli and a preparation method, and belongs to the technical field of food safety detection. The bimodal biosensor detection kit is composed of reticular targeted magnetic beads and a biosensor detection reagent. A biosensor detection reagent is a copper metal doped carbon dot solution modified by an aptamer, and the aptamer is an aminated aptamer. The dual signals provided by the dual-mode sensor can be mutually calibrated, accidental errors of single signal output are avoided, and advantage detection modes can be selected according to different scenes; a reticular PGA structure is used as an intermediate to coat ferroferric oxide magnetic beads, and a high-load reticular structure provides more binding sites for a recognition element, so that the capture efficiency of a magnetic separation technology is further enhanced; the bimetallic targeted modified carbon dots driven by the enhanced magnetic separation technology have good detection performance when being used for detecting escherichia coli O157: H7, and the efficiency of capturing low-concentration target bacteria in a food matrix can reach 90% or above.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety detection, and particularly relates to a dual-mode biosensor detection kit for detecting Escherichia coli. BACKGROUND

[0002] In recent years, food safety problems caused by Escherichia coli O157:H7 are increasing, which can cause fever, vomiting, severe dehydration, hemorrhagic diarrhea and other symptoms in the human body, and has strong infectivity. It is of great significance to pay attention to and quickly detect Escherichia coli O157:H7 in food for the development of food industry and the protection of consumer rights. The traditional detection method of Escherichia coli O157:H7 mainly includes pre-enrichment, separation, biochemical identification and other steps, and is considered as the gold standard for detection due to its high accuracy. However, the traditional culture method is time-consuming, labor-intensive, complex, and it takes 48h-7d to detect a batch of samples, the number of identified microorganisms is limited, and professional operators are needed. Similarly, the molecular detection method needs large instruments and professional operators, and the immunological detection method needs expensive recognition elements and has low accuracy. Although the biosensor based on the new recognition mode has good performance in detection, most of them exist in the form of single mode. The existing single signal detection strategy such as fluorescence, colorimetric or Raman detection technology has good sensitivity and accuracy. However, accidental false positive / negative results may lead to standardization difficulties, limiting its wide application.

[0003] In recent years, considerable progress has been made in discovering new nanomaterials with intrinsic peroxidase-like catalytic activity and finding effective strategies to design intrinsic peroxidase properties. Among them, carbon-based nanozymes as a new type of catalyst have attracted widespread attention due to their simple synthesis steps and environmental friendliness. However, there are certain limitations in the high cost of recognition elements and the low binding efficiency of magnetic beads. In order to further solve the problem of sample matrix interference, PEG-mediated ampicillin functionalized magnetic beads were used to modify the surface of magnetic nanoparticles with biological ligands, which has been proved to improve dispersion and achieve effective bacterial capture. Mannose functionalized magnetic beads selectively improve the efficiency of bacterial separation, while helping to form stable monodisperse suspensions, improving the sample pretreatment effect. In addition, polyethyleneimine and hyperbranched polyether polyol were covalently attached to the gold shell of magnetic beads to prepare a new type of nanoparticle, which showed excellent stability in complex biological systems. Some studies also linked bovine serum albumin and polyethylene glycol 2k to magnetic beads to form dandelion-like biomimetic magnetic nanoparticles. However, the excess of positive charge groups in the intermediate can destroy the stability of the magnetic nanoparticles. Therefore, developing a stable, low-cost, efficient magnetic separation strategy combined with multifunctional sensitive signals can not only improve the detection sensitivity of foodborne pathogens, but also enhance the detection accuracy. SUMMARY

[0004] In order to realize high sensitivity, low cost and stable rapid detection of Escherichia coli in food matrix, the application provides a dual-mode biosensor detection kit for detecting Escherichia coli, and simultaneously provides a preparation method of the dual-mode biosensor detection kit.

[0005] The dual-mode biosensor detection kit for detecting Escherichia coli is composed of reticular targeting magnetic beads and a biosensor detection agent, the mass of the reticular targeting magnetic beads is 55 μg, and the volume of the biosensor detection agent is 200 μL; The reticular targeting magnetic beads are γ-polyglutamic acid modified ferroferric oxide reticular targeting magnetic beads; The biosensor detection agent is an aptamer modified copper metal doped carbon dot solution; The aptamer is an aminated aptamer, the DNA sequence of the aminated aptamer is shown in SEQ ID No: 1, and the 5' end of the aminated aptamer is modified with an amino group; The DNA sequence of the aminated aptamer shown in SEQ ID No: 1 is: 5'-NH2- (CH2) 6-CCG GAC GCTTAT GCC TTG CCA TCT ACA GAG CAG GT TGA CGG-3'.

[0006] The preparation method of the dual-mode biosensor detection kit for detecting Escherichia coli is as follows: (1) Preparation of reticular targeting magnetic beads (1.1) 0.1 mL of ferroferric oxide magnetic beads with a mass volume concentration of 10 mg / mL is incubated with 5% bovine serum albumin overnight, the supernatant is removed by magnetic separation, and the precipitate is left to obtain a first compound; (1.2) 1.71 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and 0.63 mg of N-hydroxysuccinimide are added to the first compound, and stirring is performed in a vertical stirrer for 1 h to obtain a second compound; (1.3) 10 mg of γ-polyglutamic acid, 1.0 mL of sterile phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.4 are added to the second compound, and rotation mixing reaction is performed for 4 h, magnetic separation is performed, the supernatant is removed, and the precipitate is left to obtain reticular magnetic beads; (1.4) 1 mL of the phosphate buffer solution, 5 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and 6 mg of N-hydroxysuccinimide are added to the reticular magnetic beads, and reaction is performed for 2 h to obtain γ-polyglutamic acid modified reticular magnetic beads; (1.5) The 4-aminophenyl-alpha-d-mannopyranoside with a mass concentration of 20 mg / mL was added to the gamma-polyglutamic acid modified reticular magnetic beads and reacted for 4 h to synthesize reticular targeted magnetic beads, i.e., gamma-polyglutamic acid modified ferroferric oxide reticular targeted magnetic beads; (2) Preparation of copper metal doped carbon dot freeze-dried powder (2.1) 30 mL of ultrapure water was taken and 3.2 g of citric acid, 0.5 g of cysteine, and 0.1 g of copper chloride were added and stirred uniformly, and ultrasonic treatment was performed for 30 min to obtain a first pretreatment solution; (2.2) The first pretreatment solution was transferred to a polytetrafluoroethylene sealed autoclave, and reacted at 180°C for 6 h, and then naturally cooled to room temperature to obtain a second pretreatment solution; (2.3) The second pretreatment solution was placed in a centrifuge and centrifuged at a speed of 10,000 x g for 15 min, and the supernatant was taken and freeze-dried to obtain copper metal doped carbon dot freeze-dried powder; ultrapure water was added to obtain a copper metal doped carbon dot solution with a concentration of 1.5 mg / mL, which was stored at 4°C for standby; (3) Preparation of a biosensor detection reagent 500 μL of the copper metal doped carbon dot solution was added to 800 μL of aminylated aptamer with a molar concentration of 1 μM, 0.46 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide, and 0.17 mg of N-hydroxysuccinimide, and incubated at a speed of 180 rpm for 2.5 h to obtain an aminylated aptamer modified copper metal doped carbon dot solution, i.e., a biosensor detection reagent, which was stored at 4°C for standby.

[0007] The dual-mode biosensor detection reagent kit is used for a detection method for detecting Escherichia coli, and the specific detection operation steps are as follows: (1) Preparation of a sample to be detected 1 mL of commercially available milk was taken, and 5000 ×g centrifuged for 5 min, and the supernatant was discarded, and 1 mL of sterile phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.4 was used to resuspend to prepare a mixed solution, and then 3.25 x 10 4 cfu / mL of Escherichia coli O157:H7 solution was added, the mixed solution was allowed to stand for 5 min, the supernatant was taken, and a sample to be detected was obtained; (2) Detection of Escherichia coli (2.1) 200 μL of the biosensor detection reagent was taken and added to 1 mL of the sample to be detected and 55 μg of the reticular targeted magnetic beads, and incubated at 37°C for 35 min to obtain a complex solution; (2.2) The complex solution was subjected to magnetic separation and placed on a magnetic stand for 1.5 min, and the supernatant solution was removed to obtain a precipitate; (2.3) Resuspend the precipitate with 1.2 mL of the phosphate buffer solution to obtain a test solution; (2.4) Take 50 μL of the test solution and add it to 450 μL of the phosphate buffer solution, and stand for 5 min, and then measure the fluorescence intensity at 435 nm; (2.5) Take another 50 μL of the test solution, add 50 μL of a 1.25 mM hydrogen peroxide solution, 50 μL of a 0.5 mM 3,3',5,5'-tetramethylbenzidine solution, and 350 μL of an acetic acid-sodium acetate buffer solution, and react under the condition of 35°C and light shielding for 15 min to obtain a reaction solution; (3) Calculate the test result Take 200 μL of the reaction solution to detect the fluorescence intensity value, and the fluorescence intensity value greater than 0.137 is positive; through a standard curve 1 with the fluorescence intensity value of the reaction solution at 435 nm as the vertical coordinate Y and the logarithmic value of the E. coli O157:H7 bacterial liquid concentration (cfu / mL) as the horizontal coordinate X, and through a detection equation: Y=0.12043X+0.01297, the correlation coefficient is 0.97579, the concentration of E. coli O157:H7 in the test solution is calculated, and the detection is completed; Take another 200 μL of the reaction solution to measure the ultraviolet absorption intensity at 654 nm, and the colorimetric mode detection value greater than 0.294 is positive; through a standard curve 2 with the ultraviolet absorption intensity value at 654 nm as the vertical coordinate Y and the logarithmic value of the E. coli O157:H7 bacterial liquid concentration (cfu / mL) as the horizontal coordinate X, and through a detection equation: Y=0.14528X+0.0031, the correlation coefficient is 0.9919, the concentration of E. coli O157:H7 in the test solution is calculated, and the target bacterial concentration obtained by fluorescence is calibrated.

[0008] The beneficial technical effects of the present application are embodied in the following aspects: (1) The dual-mode sensor developed by the present application provides dual signals, which not only can support the accuracy and reproducibility of the method, effectively avoid accidental errors of single signal output detection, but also can select the advantage detection mode according to different scenes, and provide a convenient detection method. The doping of copper metal elements also makes the modified carbon dots have more efficient peroxide enzyme catalytic activity, and the dual-mode efficient signal output lays a good foundation for the trace sensitive detection of pathogenic bacteria.

[0009] (2) It has been verified that the detection kit of the present application has a high linear fitting degree, and the correlation coefficient is as high as 0.97579, and presents a low detection limit, and the linear response range is 10 1 -10 7 cfu / mL and 10 2 -10 7cfu / mL, the detection limit of the current existing technology for E. coli O157:H7 is generally more than 10 2 cfu / mL; at the same time, the detection time of the traditional technology is 4h-7d, while the detection time of the present application can be controlled within 1h and has good specificity.

[0010] (3) The present application further improves the capture efficiency of traditional magnetic beads. By coating the ferroferric oxide magnetic beads with a reticular gamma-polyglutamic acid structure as an intermediate, the high-load ferroferric oxide reticular magnetic bead structure modified by gamma-polyglutamic acid provides more binding sites for recognition elements, further enhancing the capture efficiency of the magnetic separation technology. The efficiency of capturing low-concentration target bacteria in food matrices can reach more than 90%, and has good application prospects.

[0011] (4) It has been verified that the present application also has excellent tolerance in the application of actual sample examples 3-5. The colorimetric mode and the fluorescent mode have good selectivity in drinking water, milk and sausage, proving that the sensing platform also shows excellent specificity even in actual samples. Further, when the amino-modified aptamer modified copper metal-doped carbon dots detection reagent is compared with real-time fluorescent quantitative PCR, the detection results have no obvious difference, and there is no need for complex specific nucleic acid extraction process, and the operation is more convenient. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is a schematic diagram of the detection method of the present application.

[0013] Figure 2 It is a transmission electron microscope image of the reticular targeted magnetic beads prepared in the present application.

[0014] Figure 3 It is a potential characterization image of the reticular magnetic beads, gamma-polyglutamic acid modified magnetic beads and reticular targeted magnetic beads prepared in the present application.

[0015] Figure 4 It is a transmission electron microscope image of the copper metal-doped carbon dots prepared in the present application.

[0016] Figure 5 It is a potential characterization image of the copper metal-doped carbon dots and the aptamer-modified copper metal-doped carbon dots prepared in the present application.

[0017] Figure 6 It is a fluorescence spectrum image of different concentrations of E. coli O157:H7 and the corresponding standard curve image in the present application.

[0018] Figure 7 It is a UV absorption image of different concentrations of E. coli O157:H7 and the corresponding standard curve image in the present application. DETAILED DESCRIPTION

[0019] The application will be further described below with reference to the examples, but is not limited to the examples.

[0020] Unless otherwise defined, the technical and scientific terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which this application belongs.

[0021] The test reagent consumables used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods; the quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged; the % in the following examples, unless otherwise specified, are mass percent.

[0022] In the following examples, 10x enzyme digestion buffer was purchased from Dalian Baobi Engineering Co., Ltd.; hydrogen peroxide solution was purchased from Shanghai Reagent Co., Ltd.; 3,3',5,5'-tetramethylbenzidine and gamma-polyglutamic acid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; phosphate buffer solution and 5% bovine serum albumin were purchased from Beijing Solabio Technology Co., Ltd.; citric acid, DL-cysteine and amino-modified nucleic acid aptamer were purchased from Shanghai Sangon Biological Engineering Co., Ltd.; carboxylated Fe3O4 was purchased from Tianjin Baisi Chromatography Technology Development Center; copper chloride was purchased from Shanghai Merck Chemical Technology Co., Ltd.; 4-aminophenyl-alpha-d-mannopyranoside was purchased from Shanghai Yuanye Biological Technology Co., Ltd.; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were purchased from Sigma-Aldrich Company, USA.

[0023] The number of E. coli O157:H7 used in the application is ATCC 43895 (Guangdong Microbial Institute is a national preservation agency), which is provided by Guangdong Microbial Institute.

[0024] The instruments and equipment, raw materials and reagents or method steps used in the application are all ensured to be processed under sterile conditions.

[0025] The instruments and equipment, raw materials and reagents or method steps not mentioned in the application belong to the conventional or commonly known technical methods for those skilled in the art, which are not described in detail in the application. Example 1

[0026] The preparation operation steps of the bimodal biosensor detection kit for detecting E. coli are as follows: (1) Preparation of reticular targeting magnetic beads (1.1) 0.1 mL of 10 mg / mL mass volume concentration of ferric oxide magnetic beads was incubated with 5% bovine serum albumin overnight, the supernatant was removed by magnetic separation, and the precipitate was left to obtain the first compound.

[0027] (1.2) 1.71 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.63 mg of N-hydroxysuccinimide were added to the first complex, stirred in a vertical stirrer for 1 h to obtain a second complex.

[0028] (1.3) 10 mg of γ-polyglutamic acid, 1.0 mL of sterile phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.4 were added to the second complex, and the reaction was rotated and mixed for 4 h, magnetically separated, the supernatant was removed, and the precipitate was left to obtain a reticular magnetic bead.

[0029] (1.4) 1 mL of the phosphate buffer solution, 5 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 6 mg of N-hydroxysuccinimide were added to the reticular magnetic bead and reacted for 2 h to obtain a γ-polyglutamic acid modified reticular magnetic bead.

[0030] (1.5) 4-amino phenyl-α-d-mannopyranoside with a mass volume concentration of 20 mg / mL was added to the γ-polyglutamic acid modified reticular magnetic bead and reacted for 4 h to synthesize a reticular targeted magnetic bead, a γ-polyglutamic acid modified ferroferric oxide reticular targeted magnetic bead.

[0031] See FIG. A in Figure 1 for a synthesis principle diagram of the reticular targeted magnetic bead; see FIG. B in Figure 2 for a transmission electron microscope diagram of the reticular targeted magnetic bead; and see FIG. C in Figure 3 for a Zeta potential characterization diagram of the reticular magnetic bead, the γ-polyglutamic acid modified reticular magnetic bead and the reticular targeted magnetic bead.

[0032] (2) Preparation of copper metal doped carbon dot lyophilized powder (2.1) 30 mL of ultrapure water was taken and 3.2 g of citric acid, 0.5 g of cysteine and 0.1 g of copper chloride were added, stirred uniformly, and then ultrasonically treated for 30 min to obtain a first pretreatment solution.

[0033] (2.2) The first pretreatment solution was transferred to a polytetrafluoroethylene sealed autoclave and reacted at 180°C for 6 h. After natural cooling to room temperature, a second pretreatment solution was obtained.

[0034] (2.3) The second pretreatment solution was placed in a centrifuge and centrifuged at a speed of 10,000 x g for 15 min. The supernatant was taken and freeze-dried to obtain copper metal doped carbon dot lyophilized powder. Ultrapure water was added to obtain a copper metal doped carbon dot solution with a concentration of 1.5 mg / mL, which was stored at 4°C for standby use.

[0035] See FIG. B in Figure 1 for a synthesis principle diagram of the copper metal doped carbon dot; and see FIG. C in Figure 4Figure 1 is a transmission electron microscope image of copper metal-doped carbon dots; see Example 1 Figure 5 Figure 2 is a potential characterization chart of copper metal-doped carbon dots and aptamer-modified copper metal-doped carbon dots.

[0036] (3) Preparation of a biosensor detection reagent 500 μL of copper metal-doped carbon dot solution was added to 800 μL of amino-modified aptamer with a molar concentration of 1 μM, 0.46 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and 0.17 mg of N-hydroxysuccinimide, and incubated at a rotation speed of 180 rpm for 2.5 h to obtain an amino-modified aptamer-modified copper metal-doped carbon dot solution, i.e., a biosensor detection reagent, which was stored at 4°C for standby.

[0037] The DNA sequence of the amino-modified aptamer is 5'-NH2-(CH2)6-CCG GAC GCT TAT GCC TTG CCA TCT ACA GAG CAG GT TGA CGG-3'.

[0038] In the dual-mode biosensor detection reagent kit, the mass of the reticular targeting magnetic beads is 55 μg, and the volume of the biosensor detection reagent is 200 μL, and they are separately sealed and packaged. Example 2

[0039] Establishment of a detection equation for detecting E. coli (1) 1 mL of E. coli O157:H7 stock solution cultured in LB broth for 12 h to the late logarithmic growth phase was taken and transferred into a sterilized centrifuge tube, and centrifuged at 5000 rpm for 5 min to obtain a bacterial suspension. ×g The supernatant was discarded, and the bacterial suspension was resuspended in 1 mL of sterile phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.4 to obtain a bacterial suspension; the bacterial suspension was gradiently diluted with sterile phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.4 to prepare E. coli O157:H7 solutions with concentrations of 0, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cfu / mL; another sterile phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.4 was used as a blank control solution, and the concentration of E. coli O157:H7 in the blank control solution was 0 cfu / mL.

[0040] (2) 200 μL of the blank control solution and the E. coli O157:H7 solutions with concentrations of 10 1 , 10 2 , 10 3 , 104 10 5 10 6 10 7 10 8 In a CFU / mL E. coli O157:H7 solution, 200 μL of aptamer-modified copper-doped carbon dot solution and 55 μg of mesh-targeted magnetic beads were added respectively. After incubation at 37 °C for 35 min, nine composite solutions were obtained.

[0041] (3) The nine composite solutions were magnetically separated. After standing on a magnetic rack for 1.5 min, the supernatant was removed to obtain nine different precipitates.

[0042] (4) Each of the nine precipitates was resuspended and mixed with 1.2 mL of phosphate buffer solution to obtain nine different test solutions.

[0043] (5) First, take 50 μL of each of the above nine test solutions and add them to 450 μL of 1× phosphate buffer solution. After standing for 5 min, measure the fluorescence intensity at 435 nm.

[0044] (6) Take nine 50 μL test solutions respectively, add 50 μL of 1.25 mM hydrogen peroxide solution, 50 μL of 0.5 mM 3,3′,5,5′-tetramethylbenzidine and 350 μL of acetate-sodium acetate buffer solution, and react at 35 °C in the dark for 15 min to obtain reaction solutions.

[0045] The copper-doped carbon dot solution modified with mesh-targeted magnetic beads and aptamers was prepared in Example 1.

[0046] (7) Observation revealed that it contained a concentration of 10 1 10 2 10 3 10 4 10 5 10 6 10 7 The fluorescence intensity values ​​of the reaction solutions of *E. coli* O157:H7 at cfu / mL were all greater than 0.137. (See [reference needed]). Figure 6 In Figure A, the UV absorption spectra of different concentrations of *E. coli* O157:H7 are shown. The fluorescence intensity values ​​at 435 nm for seven 200 μL reaction solutions were selected as the ordinate (Y), and the logarithm of the *E. coli* O157:H7 bacterial concentration (cfu / mL) was plotted as the abscissa (X). The detection equation was calculated as: Y = 0.12043X + 0.01297, with a correlation coefficient of 0.97579. (See [reference needed]). Figure 6 In the figure, B represents the linear equation graph of the colorimetric mode; additionally, it was observed that it contains a concentration of 10...2 10 3 10 4 10 5 10 6 10 7 The reaction solution of *E. coli* O157:H7 with cfu / mL showed UV absorbance values ​​greater than 0.294. (See [reference needed]). Figure 7 In Figure A, the fluorescence spectra of different concentrations of *E. coli* O157:H7 are shown. The UV absorption intensity values ​​at 654 nm for the six reaction solutions were selected as the ordinate (Y), and the logarithm of the *E. coli* O157:H7 bacterial concentration (cfu / mL) was plotted as the abscissa (X). The detection equation was calculated as: Y = 0.14528X + 0.0031, with a correlation coefficient of 0.9919. (See Figure A for details.) Figure 7 In the figure, B represents the linear equation diagram of the fluorescence mode. Example 3

[0047] Application of the dual-modal biosensor detection kit prepared in Example 1 in the detection of Escherichia coli O157:H7 content in milk (1) Sample processing Take 1 mL of commercially available milk and mix it with 5000 ml of water. ×g Centrifuge for 5 min, discard the supernatant, and resuspend in 1 mL of 0.1 M, pH 7.4 sterile phosphate buffer to prepare a mixture. Then, add 3.25 × 10⁻⁶ ppm of the solution. 4 CFU / mL Escherichia coli O157:H7 solution was prepared. The spiked mixture was allowed to stand for 5 minutes, and the supernatant was collected to obtain the test solution.

[0048] (2) Detection (2.1) Take 200 μL of biosensor detection reagent, add 1 mL of test solution and 55 μg of mesh targeting magnetic beads, and incubate at 37 °C for 35 min to obtain composite solution.

[0049] (2.2) Perform magnetic separation on the composite solution. After standing on a magnetic rack for 1.5 min, remove the supernatant solution to obtain the precipitate.

[0050] (2.3) The precipitate was resuspended in 1.2 mL of the phosphate buffer solution and mixed well to obtain the test solution.

[0051] (2.4) Take 50 μL of the above test solution and add it to 450 μL of the phosphate buffer solution. Let it stand for 5 min and measure its fluorescence intensity at 435 nm.

[0052] (2.5) Take another 50 μL of the test solution, add 50 μL of 1.25 mM hydrogen peroxide solution, 50 μL of 0.5 mM 3,3',5,5'-tetramethylbenzidine and 350 μL of acetic acid-sodium acetate buffer solution, and react at 35°C for 15 min in the dark to obtain a reaction solution.

[0053] (3) Calculation of the detection result (3.1) It is observed that the fluorescence intensity value I of 200 μL of the reaction solution at 435 nm is read and substituted into the standard curve 1, and the detection equation is Y=0.12043X+0.01297, wherein X is the logarithmic value of the concentration of the E. coli O157:H7 solution, and Y is I, which is the fluorescence intensity value at 435 nm when the test solution is detected by the present application. The concentration of the E. coli O157:H7 in the test solution is calculated, and the detection is completed. In addition, the ultraviolet absorption intensity value A of the reaction solution at 654 nm is read and substituted into the standard curve 2, and the detection equation is Y=0.14528X+0.0031, wherein X is the logarithmic value of the concentration of the E. coli O157:H7 solution, and Y is I, which is the ultraviolet absorption intensity value at 654 nm when the test solution is detected by the present application. The concentration of the E. coli O157:H7 in the test solution is calculated, and is calibrated with the target bacterial concentration obtained by fluorescence.

[0054] The detection equation is obtained from Example 2.

[0055] (3.2) 200 μL of the reaction solution is taken into a cuvette, and the fluorescence value at 435 nm is measured to be 0.552, which is substituted into the detection equation 1 to calculate that the concentration of the E. coli O157:H7 in the measured object is 2.97x10 4 cfu / mL; and another 200 μL of the reaction solution is taken, and the ultraviolet absorption intensity at 654 nm is measured to be 0.652, which indicates that the concentration of the E. coli O157:H7 in the measured object is 2.95x10 4 cfu / mL. Example 4

[0056] Application of the bimodal biosensor detection kit prepared in Example 1 in detecting the content of E. coli O157:H7 in ham (1) Sample processing The ham to be detected is cut into small pieces of 25 g, mixed with 25 mL of phosphate buffer solution with a pH value of 7.4 and a concentration of 0.1 M in a sterilized bag, and homogenized by a homogenizer for 2 min to prepare a mixed solution, and obtain a test solution.

[0057] (2) Detection (2.1) Take 200 μL of biosensor detection reagent, add 1 mL of test solution and 55 μg of mesh targeting magnetic beads respectively, and incubate at 37℃ for 35 min to obtain composite solution.

[0058] The aptamer-modified copper metal-doped carbon dot solution and the mesh-targeted magnetic beads were obtained from Example 1.

[0059] (2.2) Perform magnetic separation on the composite solution. After standing on a magnetic rack for 1.5 min, remove the supernatant solution to obtain the precipitate.

[0060] (2.3) The precipitate was resuspended in 1.2 mL of the phosphate buffer solution and mixed well to obtain the test solution.

[0061] (2.4) Take 50 μL of the above test solution and add it to 450 μL of the phosphate buffer solution, let it stand for 5 min, and measure its fluorescence intensity at 435 nm.

[0062] (2.5) Take another 50 μL of the test solution, add 50 μL of 1.25 mM hydrogen peroxide solution, 50 μL of 0.5 mM 3,3′,5,5′-tetramethylbenzidine and 350 μL of acetate-sodium acetate buffer solution, and react at 35 °C in the dark for 15 min to obtain the reaction solution.

[0063] (3) Calculate the test results (3.1) Observation revealed that the fluorescence intensity value I, which showed a peak at 435 nm in 200 μL of the reaction solution, was substituted into standard curve 1, and the detection equation was: Y = 0.12043X + 0.01297. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the fluorescence intensity value at 435 nm when the test solution was detected by this invention. The concentration of Escherichia coli O157:H7 in the test solution was calculated, and the detection was completed. In addition, the ultraviolet absorption intensity value A of the reaction solution at 654 nm was read and substituted into standard curve 2, and the detection equation was: Y = 0.14528X + 0.0031. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the ultraviolet absorption intensity value at 654 nm when the test solution was detected by this invention. The concentration of Escherichia coli O157:H7 in the test solution was calculated and calibrated with the target bacterial concentration obtained from the fluorescence.

[0064] The detection equation was obtained from Example 2.

[0065] (3.2) Observation revealed that the fluorescence intensity and ultraviolet absorption intensity values ​​of the reaction solution were 0.130 and 0.289, respectively, indicating that Escherichia coli O157:H7 was not detected in the analyte. Example 5

[0066] Application of the dual-modal biosensor detection kit prepared in Example 1 in the detection of Escherichia coli O157:H7 content in drinking water (1) Sample processing Drinking water was used directly as the test solution without any treatment.

[0067] (2) Detection (2.1) Take 200 μL of biosensor detection reagent, add 1 mL of test solution and 55 μg of mesh targeting magnetic beads respectively, and incubate at 37℃ for 35 min to obtain composite solution.

[0068] The aptamer-modified copper metal-doped carbon dot solution and the mesh-targeted magnetic beads were obtained from Example 1.

[0069] (2.2) Perform magnetic separation on the composite solution. After standing on a magnetic rack for 1.5 min, remove the supernatant solution to obtain the precipitate.

[0070] (2.3) The precipitate was resuspended in 1.2 mL of sterile phosphate buffer solution with a concentration of 0.1 M and a pH of 7.4 and mixed well to obtain the test solution.

[0071] (2.4) Take 50 μL of the above test solution and add it to 450 μL of the phosphate buffer solution, let it stand for 5 min, and measure its fluorescence intensity at 435 nm.

[0072] (2.5) Take another 50 μL of the test solution, add 50 μL of 1.25 mM hydrogen peroxide solution, 50 μL of 0.5 mM 3,3′,5,5′-tetramethylbenzidine and 350 μL of acetate-sodium acetate buffer solution, and react at 35 °C in the dark for 15 min to obtain the reaction solution.

[0073] (3) Calculate the test results (3.1) Observation revealed that the fluorescence intensity value I, which showed a peak at 435 nm in 200 μL of the reaction solution, was substituted into standard curve 1, and the detection equation was: Y = 0.12043X + 0.01297. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the fluorescence intensity value at 435 nm when the test solution was detected by this invention. The concentration of Escherichia coli O157:H7 in the test solution was calculated, and the detection was completed. In addition, the ultraviolet absorption intensity value A of the reaction solution at 654 nm was read and substituted into standard curve 2, and the detection equation was: Y = 0.14528X + 0.0031. In the equation, X is the logarithm of the concentration of Escherichia coli O157:H7, Y is I, and I is the ultraviolet absorption intensity value at 654 nm when the test solution was detected by this invention. The concentration of Escherichia coli O157:H7 in the test solution was calculated and calibrated with the target bacterial concentration obtained from the fluorescence.

[0074] The detection equation was obtained from Example 2.

[0075] (3.2) Observation showed that the fluorescence intensity and ultraviolet absorption intensity values ​​of the reaction solution were 0.131 and 0.287, respectively, indicating that Escherichia coli O157:H7 was not detected in the analyte.

[0076] Those skilled in the art will readily understand that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dual-modal biosensor detection kit for detecting Escherichia coli, characterized in that: The dual-modal biosensor detection kit consists of mesh-targeted magnetic beads and biosensor detection reagents, with the mesh-targeted magnetic beads weighing 55 μg and the biosensor detection reagents having a volume of 200 μL. The mesh-targeting magnetic beads are γ-polyglutamic acid-modified iron oxide mesh-targeting magnetic beads; The biosensor detection reagent is an aptamer-modified copper-doped carbon dot solution. The aptamer is an aminated aptamer, and the DNA sequence of the aminated aptamer is shown in SEQ ID No:

1. The 5' end of the aminated aptamer is modified with an amino group.

2. The method for preparing the dual-modal biosensor detection reagent for detecting Escherichia coli as described in claim 1, characterized in that, The preparation steps are as follows: (1) Preparation of mesh-like targeted magnetic beads (1.1) Take 0.1 mL of ferric oxide magnetic beads with a mass-volume concentration of 10 mg / mL and incubate with 5% bovine serum albumin overnight. Remove the supernatant by magnetic separation, leaving the precipitate to obtain the first complex. (1.2) 1.71 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.63 mg of N-hydroxysuccinimide were added to the first complex and stirred in a vertical stirrer for 1 h to obtain the second complex; (1.3) Add 10 mg of γ-polyglutamic acid and 1.0 mL of sterile phosphate buffer solution with a concentration of 0.1 M and a pH of 7.4 to the second complex, rotate and mix for 4 h, perform magnetic separation, remove the supernatant, leave the precipitate, and obtain network magnetic beads. (1.4) Add 1 mL of the phosphate buffer solution, 5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide to the reticulated magnetic beads and react for 2 h to obtain γ-polyglutamic acid modified reticulated magnetic beads. (1.5) 4-aminophenyl-α-d-mannopyranoside with a mass-volume concentration of 20 mg / mL was added to the γ-polyglutamic acid modified mesh magnetic beads and reacted for 4 h to synthesize the mesh targeting magnetic beads, namely γ-polyglutamic acid modified iron oxide mesh targeting magnetic beads. (2) Preparation of copper metal-doped carbon dot lyophilized powder (2.1) Take 30 mL of ultrapure water, add 3.2 g of citric acid, 0.5 g of cysteine ​​and 0.1 g of copper chloride, stir evenly, and sonicate for 30 min to obtain the first pretreatment solution; (2.2) The first pretreatment solution was transferred to a polytetrafluoroethylene sealed autoclave and reacted at 180°C for 6 hours. After it cooled naturally to room temperature, the second pretreatment solution was obtained. (2.3) The second pretreatment solution was placed in a centrifuge and centrifuged at 10000×g for 15 min. The supernatant was taken and freeze-dried to obtain copper metal-doped carbon dot lyophilized powder. Ultrapure water was added to obtain a copper metal-doped carbon dot solution with a concentration of 1.5 mg / mL, which was stored at 4℃ for later use. (3) Preparation of biosensor detection reagents 500 μL of copper-doped carbon dot solution was added to 800 μL of 1 μM aminated aptamer, 0.46 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 0.17 mg of N-hydroxysuccinimide. The mixture was incubated at 180 rpm for 2.5 h to obtain an aminated aptamer-modified copper-doped carbon dot solution, which is the biosensor detection reagent. The solution was stored at 4 °C for later use.

3. The detection method for Escherichia coli using the dual-modal biosensor detection kit of claim 1, characterized in that, The specific testing procedures are as follows: (1) Preparation of the test solution Take 1 mL of commercially available milk and mix it with 5000 ml of water. ×g Centrifuge for 5 min, discard the supernatant, and resuspend in 1 mL of 0.1 M, pH 7.4 sterile phosphate buffer to prepare a mixture. Then, add 3.25 × 10⁻⁶ ppm of the solution. 4 CFU / mL Escherichia coli O157:H7 solution, let the spiked mixture stand for 5 min, take the supernatant to obtain the test solution; (2) Detection of Escherichia coli (2.1) Take 200 μL of biosensor detection reagent, add 1 mL of test solution and 55 μg of mesh targeting magnetic beads respectively, and incubate at 37℃ for 35 min to obtain composite solution; (2.2) Perform magnetic separation on the composite solution, place it on a magnetic rack and let it stand for 1.5 min, remove the supernatant solution and obtain the precipitate; (2.3) Resuspend the precipitate in 1.2 mL of the phosphate buffer solution and mix well to obtain the test solution; (2.4) Take 50 μL of the above test solution and add it to 450 μL of the phosphate buffer solution, let it stand for 5 min, and measure its fluorescence intensity at 435 nm; (2.5) Take another 50 μL of the test solution, add 50 μL of 1.25 mM hydrogen peroxide solution, 50 μL of 0.5 mM 3,3′,5,5′-tetramethylbenzidine and 350 μL of acetate-sodium acetate buffer solution, and react at 35 °C in the dark for 15 min to obtain the reaction solution; (3) Calculate the test result Take 200 μL of the reaction solution and detect its fluorescence intensity value. A fluorescence intensity value greater than 0.137 is considered positive. A standard curve is constructed with the fluorescence intensity value of the reaction solution at 435 nm as the ordinate (Y) and the logarithm of the E. coli O157:H7 bacterial concentration (cfu / mL) as the abscissa (X). The concentration of E. coli O157:H7 in the test solution is calculated using the detection equation: Y = 0.12043X + 0.01297, with a correlation coefficient of 0.97579. This completes the detection. Another 200 μL of reaction solution was taken and its UV absorption intensity at 654 nm was measured. A colorimetric modal detection value greater than 0.294 was considered positive. A standard curve was constructed with the UV absorption intensity at 654 nm as the ordinate (Y) and the logarithm of the E. coli O157:H7 bacterial concentration (cfu / mL) as the abscissa (X). The concentration of E. coli O157:H7 in the test solution was calculated using the detection equation: Y = 0.14528X + 0.0031, with a correlation coefficient of 0.9919. The result was then calibrated with the target bacterial concentration obtained from fluorescence.

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