Preparation and application of a UiO-66-NH2@Au-MPBA nanoprobe

Abstract

The application discloses a kind of preparation and application of UiO-66-NH2@Au- MPBA nano probe, first preparation UiO-66-NH2, then preparation UiO-66-NH2@Au, finally UiO-66-NH2@Au, deionized water and 4-mercapto phenylboric acid solution are mixed, and stirring reaction is carried out at room temperature;After stirring, the supernatant is removed by centrifugal separation, and UiO-66-NH2@Au-MPBA precipitate is obtained, which is washed with ethanol and deionized water, and is stored in 4 DEG C refrigerator for standby use again dispersed in deionized water.The UiO-66-NH2@Au-MPBA nano probe prepared by the application has superior bacterial capture capacity and good peroxidase-like catalytic activity.The nano probe is applied to vertical flow immunoassay platform and combined with smart phone, and under the assistance of color signal converter software of smart phone, standard curve between ΔB / G value and E. coli O157:H7 concentration is drawn, and simple, low-cost, on-site rapid detection of E. coli O157:H7 is realized E. coli . E. coli .

Description

Technical Field

[0001] This invention relates to nanomaterials, specifically the preparation and application of a borate-functionalized UiO-66-NH2@Au nanoprobe (UiO-66-NH2@Au-MPBA). Background Technology

[0002] Escherichia coli O157:H7 (E. coli O157:H7) is a foodborne pathogen that produces Shiga toxin. It is primarily transmitted through the consumption of contaminated food and water, posing a serious threat to human health and public health safety. Traditional standard bacterial detection methods, including the MPN counting method and plate count method, while accurate and reliable, suffer from drawbacks such as time-consuming processes, reliance on specialized technicians and large instruments. These methods are unsuitable for use in remote areas or regions with limited resources, and cannot provide real-time on-site testing of bacterial levels in drinking water. Therefore, early, on-site, and immediate detection of E. coli O157:H7 is crucial to limiting the spread of foodborne illnesses.

[0003] In recent years, the immunochromatographic test strip (ICTS) method has received widespread attention in the field of point-of-care testing for pathogens due to its advantages such as low cost, simplicity, speed, and portability. However, this method has some shortcomings:

[0004] 1. The test area on the NC membrane is relatively small, resulting in weak detection signal intensity when analyzing low-concentration samples, which makes direct visual analysis of the measurement results difficult.

[0005] 2. Nanoprobes used to identify targets require modification with capture antibodies, which is costly and susceptible to unstable environments, making it difficult to preserve the probes and thus affecting the stability of the detection signal.

[0006] Therefore, there is an urgent need to develop a nanoprobe that can identify target bacteria without the need for labeling capture antibodies and to improve the enrichment method of target bacteria in order to enhance the detection signal intensity.

[0007] In recent years, vertical flow immunoassay (VFIA) has received widespread attention and is gradually becoming an alternative to ICTS methods, offering significant advantages in sample purification and enrichment. However, VFIA, which relies solely on visual readings, cannot guarantee the sensitivity and accuracy of the test results, and some VFIA methods that depend on specialized equipment cannot perform real-time quantitative analysis in resource-scarce areas. Due to the portability and widespread availability of smartphones, along with their powerful data recording and processing capabilities, they hold immense potential in the field of quantitative analysis. Therefore, combining smartphones with VFIA methods shows great promise for real-time quantitative analysis in the field.

[0008] Currently, a related patent for pathogen detection using a syringe has been disclosed: CN 116879165 A, "A Novel Rapid Pathogen Detection Method Based on Gold Nanostar Syringes." This method functionalizes 4-mercaptoboric acid (BA) onto gold nanostars (AuNSs), yielding BA-AuNSs nanoparticle probes with excellent SERS and photothermal properties. A syringe laboratory (LIS) device is constructed using paper and BA-AuNSs, distinguishing different types of bacteria through colorimetry, photothermal analysis, and SERS signals. This method minimizes sample pretreatment and expands the analyzable sample volume, enabling rapid and highly specific identification of common foodborne pathogens, and can be used for on-site detection of foodborne pathogens. However, this method cannot provide real-time quantitative analysis of the concentration of specific pathogens.

[0009] CN 114354917 A discloses a target vertical flow array immunoassay device and method. The method includes a microporous carrier membrane with an array region and coated probes attached; gold-labeled probes (metal nanoparticles connected to Raman dyes and labeled probes modified on the surface of the metal nanoparticles); mixing the sample to be analyzed with a solution of the gold-labeled probes and injecting the mixture into the array region to obtain the sample to be analyzed; and performing Raman spectroscopy on the sample to be analyzed. This method can enhance the detection signal and achieve highly sensitive, real-time detection of target biomarkers. However, this method requires large-scale instruments and equipment, and cannot meet the needs of on-site real-time detection. Summary of the Invention

[0010] This invention addresses the shortcomings of existing technologies by providing a boric acid-functionalized UiO-66-NH2@Au nanoprobe (UiO-66-NH2@Au-MPBA), its preparation method, and its applications. The UiO-66-NH2@Au-MPBA nanoprobe prepared by this invention exhibits superior bacterial capture ability and good peroxidase-like catalytic activity. Applying this probe to a vertical flow immunoassay platform and integrating it with a smartphone enables on-site, real-time detection of E. coli O157:H7.

[0011] The first objective of this invention is to provide a method for preparing UiO-66-NH2@Au-MPBA nanoprobes, comprising the following steps:

[0012] (1) Preparation of UiO-66-NH2 includes the following steps:

[0013] (1.1) 2-Aminoterephthalic acid and zirconium tetrachloride (ZrCl4) were added to N,N-dimethylformamide (DMF), and then acetic acid (CH2COOH) was added dropwise under continuous stirring. After ultrasonic treatment and stirring at room temperature, the homogeneous mixture was transferred to the inner lining of a polytetrafluoroethylene reactor and reacted at high temperature.

[0014] (1.2) After cooling the reaction solution to room temperature, the precipitate was collected by centrifugation and washed with DMF. After washing, it was dried continuously at high temperature to obtain a pale yellow solid UiO-66-NH2.

[0015] (2) Preparation of UiO-66-NH2@Au includes the following steps:

[0016] (2.1) UiO-66-NH2 was ultrasonically dispersed in deionized water, and HAuCl4 was added and mixed. The solution turned light red. Then, NaBH4 solution was added dropwise under an ice-water bath, and the reaction was continued with stirring. The solution turned dark red, indicating that UiO-66-NH2@Au was successfully prepared.

[0017] (2.2) Centrifuge the dark red UiO-66-NH2@Au solution to remove the supernatant, wash with deionized water, redisperse in deionized water, and store in a refrigerator for later use;

[0018] (3) Preparation of UiO-66-NH2@Au-MPBA nanoprobes, including the following steps:

[0019] (3.1) Mix UiO-66-NH2@Au, deionized water and 4-mercaptophenylboronic acid (4-MPBA) solution and stir to react at room temperature;

[0020] To prepare a 4-MPBA solution, simply weigh out a certain amount of 4-MPBA solid and dissolve it in ethanol (CH3CH2OH).

[0021] (3.2) After stirring the reaction, the solution was centrifuged to remove the supernatant, and the UiO-66-NH2@Au-MPBA precipitate was obtained. It was washed with ethanol and deionized water, redispersed in deionized water, and stored in a refrigerator for later use.

[0022] Furthermore, in step (1.1), the molar ratio of 2-aminoterephthalic acid to ZrCl4 is 1:1;

[0023] The volume ratio of DMF to acetic acid solvent is 9–10:1;

[0024] The reactor temperature is 80–120℃, and the reaction time is 12–24 h.

[0025] In step (1.2), after washing, the product is dried continuously at 60–100°C for 12–24 hours.

[0026] Further, in step (2.1), the mass-to-volume ratio of UiO-66-NH2 to deionized water is 1:1, and the concentration of UiO-66-NH2 is 1.0 mg / mL;

[0027] The mass ratio of UiO-66-NH2 to HAuCl4 is 1:10, and the concentration of HAuCl4 is 1.0 wt%.

[0028] The molar ratio of HAuCl4 to NaBH4 is 3:500, and the concentration of NaBH4 is 100 mmol / L.

[0029] Furthermore, in step (3.1), the volume ratio of deionized water to UiO-66-NH2@Au is 9:1;

[0030] The volume ratio of UiO-66-NH2@Au to 4-MPBA is 1:2;

[0031] The mass-to-volume ratio of 4-MPBA to ethanol is 3:200;

[0032] The stirring reaction time is 12–24 hours.

[0033] The second objective of this invention is to apply the UiO-66-NH2@Au-MPBA nanoprobe to a vertical flow immunoassay platform and combine it with a smartphone to achieve on-site, real-time detection of E. coli O157:H7.

[0034] Furthermore, the UiO-66-NH2@Au-MPBA nanoprobe was used to identify E. coli O157:H7. The bacterial culture process included the following steps:

[0035] (A) After adding a single colony of E. coli O157:H7 to the culture medium for culture, take an appropriate amount of the cultured bacterial solution, wash it with sterile water, disperse the bacterial precipitate with Tris-HCl buffer to obtain the bacterial stock solution, and gradually dilute the bacterial stock solution to E. coli O157:H7 standard solutions of different concentrations.

[0036] (B) The UiO-66-NH2@Au-MPBA nanoprobe was transferred to E.coli O157:H7 standard solutions of different concentrations, mixed and incubated to obtain the UiO-66-NH2@Au-MPBA-E.coli O157:H7 complex;

[0037] The volume ratio of UiO-66-NH2@Au-MPBA to E.coli O157:H7 is 1:20.

[0038] Furthermore, the preparation of the immunofilter membrane in the vertical flow immunoassay platform specifically includes the following steps:

[0039] (a) The nitrocellulose microporous membrane (NC membrane) was washed with Na2CO3-NaHCO3 buffer, and E. coli O157:H7 monoclonal antibody (E. coli-Ab) was added and incubated at 4°C.

[0040] (b) After incubation, wash with PBST (PBS buffer containing Tween-20), vacuum dry at 25–40°C, and store the obtained immunofilter membrane at 4°C for later use.

[0041] By placing the immunofilter membrane in a removable filter head and tightening the filter head, a vertical flow immunoassay platform is formed. The prepared immunofilter membrane plays a crucial role in this platform.

[0042] Furthermore, a vertical flow immunoassay platform was constructed using the prepared UiO-66-NH2@Au-MPBA nanoprobes for the detection of E. coli O157:H7. The detection method includes the following steps:

[0043] Use tweezers to pick up an immunofiltration membrane and place it between the two rubber rings of the detachable filter head. After tightening the filter head, use a 1mL medical syringe to slowly pass deionized water through the filter membrane to wet it. Then, rinse the membrane 1 to 3 times with Tris-HCl buffer containing Tween-20.

[0044] Next, use a syringe to aspirate the UiO-66-NH2@Au-MPBA-E.coli O157:H7 complex obtained from incubation and rinse. Repeat 1 to 3 times. The antibody on the filter membrane specifically binds to the bacteria. Finally, aspirate Tris-HCl buffer containing Tween-20 and rinse the filter membrane 2 to 3 times to remove material that has not bound to the bacteria.

[0045] The filter membrane was removed with tweezers and transferred to a centrifuge tube. Tris-HCl buffer was added, followed by 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2. The mixture was allowed to develop color for 10–30 min. The resulting mixture was then transferred to a 96-well plate, and the optical density (OD) value was measured using a microplate reader. A standard curve was constructed based on the change in OD value at 652 nm and different concentrations of E. coli O157:H7. Images were recorded using a mobile device, and the RGB values ​​of the experimental images were analyzed using a color signal converter app. Furthermore, based on the change in B / G value relative to the blank group, the ΔB / G value of the colorimetric vertical flow immunoassay platform for different concentrations of target bacteria was calculated, and a standard curve was plotted between the ΔB / G value and the concentration of E. coli O157:H7.

[0046] Based on the standard curve, the concentration of E. coli O157:H7 in spiked environmental water samples was detected to achieve quantitative detection.

[0047] Furthermore, in the detection method, the molar concentration ratio of TMB to H2O2 is 1:10.

[0048] This invention first prepares UiO-66-NH2@Au-MPBA as a nanoprobe, which possesses superior bacterial capture ability and good peroxidase-like catalytic activity. A label-free, portable, colorimetric vertical flow immunoassay platform based on boric acid-functionalized UiO-66-NH2@Au is constructed. Through the extrusive force of a medical syringe filter, the material-bacterial complex rapidly binds to and effectively enriches antibodies on the NC membrane, eliminating the need for incubation time. A mixture of TMB and H2O2 is then added to generate a colorimetric signal. With the assistance of color signal converter software on a smartphone, a standard curve is plotted between the ΔB / G value and the E. coli O157:H7 concentration, achieving simple, low-cost, and rapid on-site detection of E. coli O157:H7.

[0049] The vertical flow immunoassay platform of this invention avoids the cumbersome antibody modification process, resulting in lower cost, easier storage, and wider application. Because the bacterial surface is rich in glycolipids with cis-diol structures, bacteria can effectively bind to boric acid groups, allowing the material to reversibly bind to bacteria via covalent bonds to form a material-bacteria complex. Furthermore, thanks to the material's excellent catalytic activity and the superior enrichment effect of the vertical flow immunoassay platform, it overcomes the low signal intensity of traditional immunochromatographic test strips, reduces sample pretreatment steps, and achieves specific and sensitive detection of E. coli O157:H7. This invention combines the colorimetric signal of the vertical flow immunoassay platform with a smartphone, providing a new approach for real-time bacterial detection in the field of environmental monitoring. Attached Figure Description

[0050] Figure 1 The sensitivity test results of the vertical flow immunoassay platform prepared for this example in detecting E. coli O157:H7 are shown in the figure.

[0051] in, Figure 1 (A) is the UV-Vis absorption spectrum of different concentrations of E. coli O157:H7 detected by the vertical flow immunoassay platform based on UiO-66-NH2@Au-MPBA;

[0052] Figure 1 (B)ΔOD 652 nm The linear relationship between the value and the logarithmic value of E. coli O157:H7 concentration;

[0053] Figure 1 (C) Linear relationship between ΔB / G values ​​of digital photographs and logarithmic values ​​of E. coli O157:H7 concentration;

[0054] Figure 1 (D) Photographs and colorimetric cards showing the color changes of the solution corresponding to the changes in the concentration of E. coli O157:H7;

[0055] Figure 2 The figure shows the specificity test results of the vertical flow immunoassay platform prepared for the example for detecting E. coli O157:H7. Detailed Implementation

[0056] The present invention will be further described below with reference to the embodiments and accompanying drawings, but this is not intended to limit the scope of the invention.

[0057] Example 1

[0058] A method for preparing a UiO-66-NH2@Au-MPBA nanoprobe includes the following steps:

[0059] (1) Preparation of UiO-66-NH2 includes the following steps:

[0060] (1.1) 1.0 mmol of 2-aminoterephthalic acid and 1.0 mmol of ZrCl4 were added to 30 mL of DMF, and then 3.3 mL of acetic acid was added dropwise under continuous stirring. The mixture was sonicated for 10 min, stirred at room temperature for 20 min, and then the homogeneous mixture was transferred to the liner of a polytetrafluoroethylene reactor and reacted at 120 °C for 24 h.

[0061] (1.2) After cooling the reaction solution to room temperature, centrifuge at 10,000 rpm for 5 min to collect the precipitate, wash the precipitate 3 times with DMF, and then dry it continuously at 100℃ for 12 h to obtain a pale yellow solid UiO-66-NH2.

[0062] (2) Preparation of UiO-66-NH2@Au includes the following steps:

[0063] (2.1) Disperse 5 mL of UiO-66-NH2 with a concentration of 1.0 mg / mL in 5 mL of deionized water by ultrasonication, add 124 μL of 1.0 wt% HAuCl4 and mix for 5 min. The solution turns light red.

[0064] Then, 500 μL of 100 mmol / L NaBH4 was added dropwise under an ice-water bath, and the reaction was continued to be stirred for 10 min. The solution turned dark red, indicating that UiO-66-NH2@Au was successfully prepared.

[0065] (2.2) Centrifuge the deep red UiO-66-NH2@Au solution at 10000 rpm for 5 min, remove the supernatant, wash it 3 times with deionized water, redisperse it in 1.0 mL of deionized water, and store it in a refrigerator at 4℃ for later use.

[0066] (3) Preparation of UiO-66-NH2@Au-MPBA nanoprobes, including the following steps:

[0067] (3.1) Mix 200 μL UiO-66-NH2@Au, 1.8 mL deionized water and 400 μL of 15 μg / mL 4-mercaptophenylboronic acid solution (4-MPBA solution, ethanol as solvent), and stir the mixture at room temperature for 12 h.

[0068] (3.2) After stirring the reaction, the solution is centrifuged to remove the supernatant, and the UiO-66-NH2@Au-MPBA precipitate is obtained. It is washed 2 to 3 times with ethanol and deionized water, redispersed in deionized water, and stored in a refrigerator at 4°C for later use.

[0069] Example 2

[0070] The UiO-66-NH2@Au-MPBA nanoprobe prepared in Example 1 was used to identify E. coli O157:H7. The bacterial culture process was as follows:

[0071] The purchased E. coli O157:H7 strain was inoculated onto LB agar medium using the streak plate method and cultured at 37°C for 16 hours. One to three single colonies obtained from the culture were scraped into 10 mL of LB broth and cultured at 37°C for 16 hours.

[0072] Take an appropriate amount of bacterial culture, centrifuge at 5000 rpm for 5 min to collect the bacterial precipitate, wash the precipitate three times with sterile water, and redisperse the bacteria with Tris-HCl (10 mmol / L, pH 7.4) buffer, adjusting the bacterial concentration to 2.08 × 10⁻⁶.8 CFU / mL;

[0073] The bacterial suspension was serially diluted with Tris-HCl (10 mmol / L, pH 7.4) buffer to a final concentration of 2.08 × 10⁻⁶. 7 2.08×10 6 2.08×10 5 2.08×10 4 2.08×10 3 CFU / mL concentration;

[0074] Take 1000 μL of bacterial suspensions of various concentration gradients and place them in centrifuge tubes with 50 μL of UiO-66-NH2@Au-MPBA. Incubate at 37℃ and 1000 rpm for 30 min to obtain the UiO-66-NH2@Au-MPBA-E.coli O157:H7 complex.

[0075] Example 3

[0076] Preparation of immunofiltration membranes in a vertical flow immunoassay platform:

[0077] Nitrocellulose microporous membrane (NC membrane, diameter 13 mm, pore size 2 μm) was washed three times with Na2CO3-NaHCO3 (100 mmol / L, pH 9.6), and 12.5 μg / mL E. coli-Ab was added and incubated at 4 °C for 12 h.

[0078] After incubation, the membrane was washed three times with 3.0 mL PBST (10 mmol / L, pH 7.4, PBS buffer containing 0.05% Tween-20), and then vacuum dried at 25 °C for 12 h. The obtained immunofilter membrane was stored and sealed at 4 °C for later use.

[0079] Example 4

[0080] A vertical flow immunoassay platform was constructed, and the bacterial culture process was the same as in Example 2, while the preparation of the immunofilter membrane was the same as in Example 3.

[0081] The constructed vertical flow immunoassay platform was used to assess the sensitivity of E. coli O157:H7.

[0082] Testing procedure: Use tweezers to pick up an immunofilter membrane and place it between the two rubber rings of the detachable filter head. After tightening the filter head, use a 1mL medical syringe to draw 1.0mL of deionized water and slowly pass it through the filter membrane to wet it. Then, draw 1.0mL of Tris-HCl (10mmol / L, pH 7.4) buffer containing 0.05% Tween-20 and rinse once.

[0083] Next, the incubated bacterial-material mixture was incubated and rinsed for 2 minutes using a syringe, and this was repeated twice. The antibodies on the filter membrane specifically bind to the bacteria. Finally, 1.0 mL of Tris-HCl (10 mmol / L, pH 7.4) buffer containing 0.05% Tween-20 was used to rinse the filter membrane twice to remove any material that had not bound to the bacteria.

[0084] Remove the filter membrane with forceps and transfer it to a 5.0 mL centrifuge tube. Add 800 μL of Tris-HCl buffer (10 mmol / L, pH 7.4), then add 100 μL of 10 mmol / L TMB and 100 μL of 100 mmol / L H2O2. Incubate at 37 °C and 1000 rpm for 20 min to develop the color. Transfer 200 μL of the resulting mixture to a 96-well plate and measure the OD value at 652 nm using a microplate reader. 652 nm They used their smartphones to record images.

[0085] Based on the change in OD value at 652nm (ΔOD) 652 nm OD of the experimental group 652 nm OD values ​​and blank groups 652 nm A standard curve was constructed using the difference in values ​​(E. coli O157:H7) and different concentrations of E. coli O157:H7. The concentration of E. coli O157:H7 was between 2.08 × 10⁻⁶. 3 ~2.08×10 7 Within the CFU / mL range, ΔOD 652 nm The concentrations of E. coli O157:H7 showed a linear relationship with the logarithmic value. The linear regression equation was y = 0.1242x - 0.2508 (R²). 2 =0.9921, n=3), where y represents ΔOD 652 nm The value, x, represents the logarithm of the E. coli O157:H7 concentration. The limit of detection (LOD) is 1.32 × 10⁻⁶. 2 CFU / mL (3σ / K, where σ is the OD measured in 11 blank samples) 652 nm The standard deviation; K is the slope of the linear equation.

[0086] In addition, the RGB values ​​of the experimental images were read and analyzed using a mobile app (color signal converter). Based on the change in B / G values ​​relative to the control group, the ΔB / G value (the difference between the B / G values ​​of the experimental group and the control group) for different concentrations of target bacteria could be calculated using the colorimetric vertical flow immunoassay platform. A standard curve was plotted between the ΔB / G value and the concentration of E. coli O157:H7. (The last sentence appears to be incomplete and possibly refers to a different plot.) 3 ~2.08×10 7 Within the CFU / mL range of E. coli O157:H7 concentration, the ΔB / G value increases linearly with increasing logarithmic value of E. coli O157:H7 concentration. The linear regression equation is y = 0.04425x - 0.2508 (R²). 2 =0.9812, n=3), where y represents the ΔB / G value and x represents the logarithmic value of the E. coli O157:H7 concentration. LOD is 1.37×10 2 CFU / mL (3σ / K, where σ is the standard deviation of the B / G values ​​measured in 11 blank samples; K is the slope of the linear equation).

[0087] Reference Figure 1 The variation of OD value at 625 nm for different concentrations of E. coli O157:H7 in the examples, ΔOD 652 nm The linear relationship between the ΔB / G value and the logarithmic value of E. coli O157:H7 concentration, the linear relationship between the ΔB / G value of digital photographs and the logarithmic value of E. coli O157:H7 concentration, and the corresponding color changes of the solution with changes in E. coli O157:H7 concentration and the corresponding colorimetric cards. From Figure 1 (A) As the concentration of E. coli O157:H7 increases, the OD value at 625 nm gradually increases. Figure 1 (B) Display ΔOD 652 nm The values ​​showed a linear relationship with the logarithmic values ​​of E. coli O157:H7 concentration, with a linear range of 2.08 × 10⁻⁶. 3 ~2.08×10 7 CFU / mL. Figure 1 (C) The results show a linear relationship between the ΔB / G value and the logarithm of the E. coli O157:H7 concentration, with a linear range of 2.08 × 10⁻⁶. 3 ~2.08×10 7 CFU / mL. Figure 1 (D) Displays a colorimetric card for detecting changes in E. coli O157:H7 concentration obtained from photos taken with a smartphone. The sensitivity of the direct visual colorimetric method is 2.08 × 10⁻⁶. 3 CFU / mL.

[0088] The prepared vertical flow immunoassay platform was used to detect the specificity of E. coli O157:H7.

[0089] Detection Procedure: Four pathogens were selected as interfering bacteria for the study, including *Escherichia coli* (ATCC 25922), *Salmonella typhimurium*, *Staphylococcus aureus*, and *Bacillus subtilis*. The concentration of *E. coli* O157:H7 was 2.08 × 10⁻⁶. 6 CFU / mL, the concentration of other interfering bacteria was 2.08 × 10⁻⁶. 7 CFU / mL. Take 1000 μL of bacterial suspension and 50 μL of UiO-66-NH2@Au-MPBA and place them in centrifuge tubes. Incubate at 37℃ and 1000 rpm for 30 min to obtain bacterial-material complex.

[0090] Use a 1mL medical syringe to slowly pass 1.0mL of deionized water through the filter membrane to wet it, and then rinse once with 1.0mL of Tris-HCl (10mmol / L, pH 7.4) buffer containing 0.05% Tween-20.

[0091] Next, the incubated bacterial-material mixture was incubated and rinsed for 2 minutes using a syringe, and this was repeated twice. The antibodies on the filter membrane specifically bound to the target bacteria. Finally, 1.0 mL of Tris-HCl (10 mmol / L, pH 7.4) buffer containing 0.05% Tween-20 was used to rinse the filter membrane twice to remove any material that had not bound to the bacteria.

[0092] Remove the filter membrane with forceps and transfer it to a 5.0 mL centrifuge tube. Add 800 μL of Tris-HCl buffer (10 mmol / L, pH 7.4), then add 100 μL of 10 mmol / L TMB and 100 μL of 100 mmol / L H2O2. Incubate at 37 °C and 1000 rpm for 20 min to develop the color. Transfer 200 μL of the resulting mixture to a 96-well plate and measure the OD value at 652 nm using a microplate reader. 652 nm They used their smartphones to record images.

[0093] Reference Figure 2In the examples, compared with the blank control group (Tris-HCl buffer), other interfering bacteria did not produce obvious signals, and only the addition of E. coli O157:H7 resulted in a significant catalytic signal. This indicates that the influence of other bacteria on the present invention is negligible in practical applications.

[0094] The prepared vertical flow immunoassay platform was used for the practical application of detecting E. coli O157:H7;

[0095] In this example, water from Shanhu Lake in Guilin was used as a simulated sample for testing. The following are the results and specific procedures for detecting *E. coli* O157:H7 in the water sample. The sample was serially diluted with Tris-HCl buffer (10 mmol / L, pH 7.4) to achieve a bacterial concentration of 2.08 × 10⁻⁶. 7 2.08×10 5 2.08×10 3 CFU / mL. Impurities in the collected water samples were removed using a 0.22 μm filter. The treated water samples were then mixed with the prepared bacterial suspension at a 1:1 volume ratio to prepare a series of E. coli O157:H7 spiked samples, which served as positive samples. No bacterial suspension was added; the treated water samples were used as negative samples.

[0096] 1000 μL of actual sample and 50 μL of UiO-66-NH2@Au-MPBA were placed in centrifuge tubes and incubated at 37 °C and 1000 rpm for 30 min to obtain the bacteria-material complex.

[0097] Use a 1mL medical syringe to slowly pass 1.0mL of deionized water through the filter membrane to wet it, and then rinse once with 1.0mL of Tris-HCl (10mmol / L, pH 7.4) buffer containing 0.05% Tween-20.

[0098] Next, the incubated bacterial-material mixture was incubated and rinsed for 2 minutes using a syringe, and this was repeated twice. The antibodies on the filter membrane specifically bound to the target bacteria. Finally, 1.0 mL of Tris-HCl (10 mmol / L, pH 7.4) buffer containing 0.05% Tween-20 was used to rinse the filter membrane twice to remove any material that had not bound to the bacteria.

[0099] Remove the filter membrane with forceps and transfer it to a 5.0 mL centrifuge tube. Add 800 μL of Tris-HCl buffer (10 mmol / L, pH 7.4), then add 100 μL of 10 mmol / L TMB and 100 μL of 100 mmol / L H2O2. Incubate at 37 °C and 1000 rpm for 20 min to develop the color. Transfer 200 μL of the resulting mixture to a 96-well plate and measure the OD value at 652 nm using a microplate reader. 652 nm They used their smartphones to record images.

[0100] Table 1. Recovery rates of E. coli O157:H7 in spiked environmental water samples detected using the VFIA platform based on UiO-66-NH2@Au-MPBA (n=3)

[0101]

[0102] As shown in Table 1, the spiked recoveries of this method in Shanhu Lake water ranged from 92.9% to 109.5%, with relative standard deviations ranging from 2.99% to 7.09%. The detection results for E. coli O157:H7 were roughly the same in both types of simulated environmental water samples, indicating that this invention has good accuracy and applicability in environmental water sample detection.

Claims

1. A method for preparing a UiO-66-NH2@Au-MPBA nanoprobe, characterized in that, The steps include the following: (1) Preparation of UiO-66-NH2 includes the following steps: (1.1) Add 2-aminoterephthalic acid and ZrCl4 to DMF, then add acetic acid dropwise under continuous stirring, sonicate, stir at room temperature, transfer the homogeneous mixture to the inner lining of a polytetrafluoroethylene reactor, and react at high temperature; (1.2) After cooling the reaction solution to room temperature, the precipitate was collected by centrifugation and washed with DMF. After washing, it was dried continuously at high temperature to obtain a pale yellow solid UiO-66-NH2. (2) Preparation of UiO-66-NH2@Au includes the following steps: (2.1) UiO-66-NH2 was ultrasonically dispersed in deionized water, and HAuCl4 was added and mixed. The solution turned light red. Then, NaBH4 solution was added dropwise under an ice-water bath, and the reaction was continued with stirring. The solution turned dark red, indicating that UiO-66-NH2@Au was successfully prepared. ( 2.2) Centrifuge the dark red UiO-66-NH2@Au solution to remove the supernatant, wash with deionized water, redisperse in deionized water, and store in a refrigerator for later use; (3) Preparation of UiO-66-NH2@Au-MPBA nanoprobes, including the following steps: (3.1) Mix UiO-66-NH2@Au, deionized water and 4-MPBA solution, and stir the mixture at room temperature. To prepare a 4-MPBA solution, weigh out a certain amount of 4-MPBA solid and dissolve it in ethanol. (3.2) After stirring the reaction, the solution was centrifuged to remove the supernatant, and the UiO-66-NH2@Au-MPBA precipitate was obtained. It was washed with ethanol and deionized water, redispersed in deionized water, and stored in a refrigerator for later use.

2. The preparation method of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 1, characterized in that: In step (1.1), the molar ratio of 2-aminoterephthalic acid to ZrCl4 is 1:1; The volume ratio of DMF to acetic acid solvent is 9–10:1; The reactor temperature is 80–120℃, and the reaction time is 12–24 h. In step (1.2), after washing, the product is dried continuously at 60–100°C for 12–24 hours.

3. The preparation method of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 1, characterized in that: In step (2.1), the mass-to-volume ratio of UiO-66-NH2 to deionized water is 1:1, and the concentration of UiO-66-NH2 is 1.0 mg / mL; the mass ratio of UiO-66-NH2 to HAuCl4 is 1:10, and the concentration of HAuCl4 is 1.0 wt%. The molar ratio of HAuCl4 to NaBH4 is 3:500, and the concentration of NaBH4 is 100 mmol / L.

4. The preparation method of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 1, characterized in that: In step (3.1), the volume ratio of deionized water to UiO-66-NH2@Au is 9:1; The volume ratio of UiO-66-NH2@Au to 4-MPBA is 1:2; The mass-to-volume ratio of 4-MPBA to ethanol is 3:200; The stirring reaction time is 12–24 hours.

5. The UiO-66-NH2@Au-MPBA nanoprobe prepared by the preparation method according to any one of claims 1-4.

6. The application of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 5, characterized in that: The UiO-66-NH2@Au-MPBA nanoprobe was applied to a vertical flow immunoassay platform and combined with a smartphone to enable on-site, real-time detection of E. coli O157:H7.

7. The application of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 6, characterized in that: The detection of E. coli O157:H7 using a UiO-66-NH2@Au-MPBA nanoprobe involved the following bacterial culture steps: (A) After adding a single colony of E. coli O157:H7 to the culture medium for culture, take an appropriate amount of the cultured bacterial solution, wash it with sterile water, disperse the bacterial precipitate with Tris-HCl buffer to obtain the bacterial stock solution, and gradually dilute the bacterial stock solution to E. coli O157:H7 standard solutions of different concentrations. (B) The UiO-66-NH2@Au-MPBA nanoprobe was transferred to E.coli O157:H7 standard solutions of different concentrations, mixed and incubated to obtain the UiO-66-NH2@Au-MPBA-E.coli O157:H7 complex.

8. The application of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 7, characterized in that: In step (B), the volume ratio of UiO-66-NH2@Au-MPBA and E.coli O157:H7 is 1:

20.

9. The application of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 6, characterized in that: The preparation of the immunofiltration membrane in the vertical flow immunoassay platform includes the following steps: (a) The nitrocellulose microporous membrane was washed with Na2CO3-NaHCO3 buffer, E. coli O157:H7 monoclonal antibody was added, and the membrane was incubated at 4°C. (b) After incubation, wash with PBS buffer containing Tween-20, vacuum dry at 25–40°C, and store the obtained immunofilter membrane at 4°C for later use.

10. The application of the UiO-66-NH2@Au-MPBA nanoprobe according to claim 9, characterized in that, A vertical flow immunoassay platform was constructed using the prepared UiO-66-NH2@Au-MPBA nanoprobes to detect E. coli O157:H7. The detection method includes the following steps: Use tweezers to pick up an immunofiltration membrane and place it between the two rubber rings of the detachable filter head. After tightening the filter head, use a 1mL medical syringe to slowly pass deionized water through the filter membrane to wet it. Then, rinse the membrane 1 to 3 times with Tris-HCl buffer containing Tween-20. Next, use a syringe to aspirate the UiO-66-NH2@Au-MPBA-E.coli O157:H7 complex obtained from incubation and rinse. Repeat 1 to 3 times. The antibody on the filter membrane specifically binds to the bacteria. Finally, aspirate Tris-HCl buffer containing Tween-20 and rinse the filter membrane 2 to 3 times to remove material that has not bound to the bacteria. Remove the filter membrane with tweezers and transfer it to a centrifuge tube. Add Tris-HCl buffer, then add TMB and H2O2. Catalyze and develop the color for 10–30 min. Transfer the catalyzed and developed mixture to a 96-well plate and measure the optical density (OD) value using a microplate reader. Construct a standard curve based on the OD value change at 652 nm and different concentrations of E. coli O157:H7. Record images using a mobile device, take photos with a smartphone, and analyze the RGB values ​​of the experimental images using a color signal converter app. In addition, based on the change in B / G value relative to the blank group, the ΔB / G value of the colorimetric vertical flow immunoassay platform for different concentrations of target bacteria was calculated, and a standard curve between the ΔB / G value and the concentration of E. coli O157:H7 was plotted. Based on the standard curve, the concentration of E. coli O157:H7 in spiked environmental water samples was detected to achieve quantitative detection.

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