A rapid and accurate detection method for pathogens based on lanthanide MOFs and paper needles

Abstract

The present invention belongs to the field of biological detection technology, and in particular relates to a rapid and accurate detection method for pathogens based on lanthanide MOF and paper needles. The present invention constructs a fluorescent biosensor by combining LnMOF with an aptamer (Apt-LnMOF) that specifically captures target pathogens. A needle-shaped paper-based device (paper needle) is used as a visual signal output carrier, and qualitative or quantitative analysis is performed by the diffusion distance and red fluorescence intensity (R value) of Apt-LnMOF at the paper-based interface. Apt-LnMOF is used as a signal label for a visualization detection strategy to improve the sensitivity and response speed of the visualization detection method for pathogens; the dual signal coupled with the diffusion distance and R value of Apt-LnMOF at the paper-based interface is used to visualize the result output, thereby improving the accuracy of the detection method. The present invention uses paper needles as signal output carriers, which improves the convenience of operation for non-professionals.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a method for rapid and accurate detection of pathogens based on lanthanide MOF and paper needles. Background Art

[0002] Vibrio parahaemolyticus (VP), a Gram-negative, halophilic bacterium, is a major cause of human gastroenteritis, including diarrhea, headache, vomiting, nausea, abdominal cramps, and low-grade fever. It is also a major source of biosafety concerns in seafood. Currently, methods used to detect V. parahaemolyticus include traditional methods, PCR, LAMP, gene chips, ELISA, and UPT-LF. Some of these methods suffer from shortcomings such as slow detection, high cost, complex instrumentation, and poor sensitivity. Therefore, establishing rapid and accurate detection methods is crucial for detecting seafood-related safety issues.

[0003] Paper-based devices can meet the needs of non-professionals for pathogen detection in the wild or in resource-limited areas. Lateral flow test strips, one of the most mature paper-based devices, have been successfully used for rapid detection of targets such as pathogens. However, their further development has been hampered by low sensitivity, strict particle size requirements for signal tags (generally less than 100–200 nm), and reliance on a single signal, which can lead to inaccurate results.

[0004] Using the diffusion distance of the signal tag at the paper interface as a visual signal output method can solve the problem of signal tag particle limitations in lateral flow test strips. Among them, the self-fluorescent lanthanide MOF (LnMOF) can smoothly diffuse a certain distance at the paper-based interface due to its moderate particle size. In addition, LnMOF has the advantages of high signal intensity and fast response speed, which can improve the detection sensitivity and speed of paper-based devices to a certain extent. Therefore, the development of a method suitable for VP detection based on lanthanide MOF and paper-based devices will not only improve the accuracy of pathogen detection of paper-based devices, but also improve the convenience of operation for non-professionals. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the present invention provides a method for rapid and accurate detection of pathogens based on lanthanide MOF (LnMOF) and paper needles. By combining LnMOF with a nucleic acid aptamer (specifically capturing target pathogens) (Apt-LnMOF) as a fluorescent biosensor and using absorbent paper as a visual signal output carrier, a needle-shaped paper-based device (paper needle) is constructed. The distance signal can be directly observed by the naked eye for qualitative analysis, and the R value can be quantitatively analyzed using a smartphone. The accuracy of pathogen detection by the paper-based device can also be further improved by combining the diffusion distance and color intensity signals.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] The first aspect of the present invention provides a method for detecting pathogens for non-disease diagnosis and treatment purposes based on lanthanide MOFs and paper needles, comprising the following steps:

[0008] S1. Prepare a lanthanide MOF (LnMOF) solution: dissolve zirconium tetrachloride, europium nitrate hexahydrate, 2,6-naphthalene dicarboxylic acid, and 2-fluorobenzoic acid in an organic solvent, heat-treat at 110-120° C. for 40-60 h, collect the product, wash it, and redissolve it;

[0009] S2. Preparation of paper needle device: trimming absorbent paper into a T-shaped device;

[0010] S3. Pathogen detection: Mix the pathogen sample solution, the nucleic acid aptamer (Apt) solution that specifically captures the target pathogen, and the lanthanide MOF solution. After standing for 20-30 minutes, insert the needle end of the paper needle device into the mixed solution for signal output. Then, observe the diffusion distance of the fluorescent band through fluorescence excitation. A shorter diffusion distance compared to the negative control is a positive result.

[0011] Preferably, the pathogen is Vibrio parahaemolyticus (VP), and the nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO: 1.

[0012] Preferably, the concentration of pathogens can also be calculated by the red fluorescence intensity R.

[0013] This invention uses Vibrio parahaemolyticus (VP) as an example to demonstrate the performance of the method of the invention in performing rapid and accurate visual analysis of pathogens. Figure 1As shown, the basic principle of the assay is that VP specifically binds to the aptamer on the Apt-LnMOF surface (positive), causing Apt-LnMOF aggregation and shortening its diffusion distance at the paper-needle interface. Conversely, in the absence of VP (negative), Apt-LnMOF remains dispersed, allowing it to diffuse over a longer distance at the paper-needle interface. Notably, in positive reactions, some non-aggregated Apt-LnMOF remains, and this portion also diffuses over a longer distance (near the absorbent pad of the paper-needle device). Since the red fluorescence (R value) displayed near the absorbent pad varies with VP concentration, this phenomenon provides the prerequisite for collecting a secondary signal. For signal acquisition, the paper needles, which have absorbed the positive and negative reaction mixtures, are exposed to 254nm UV light to generate fluorescence signals. Qualitative and quantitative analysis can then be performed by visual observation (observing the fluorescence diffusion distance) or by photographing with a smartphone (quantitatively reading the R value).

[0014] The detection method constructed in this invention is very simple to operate, requiring only a single step of mixing the sample and reagents at room temperature. After a 20–30 minute static reaction, the paper needle device quickly outputs the visual results. This detection strategy is well-suited for non-professionals to operate in the field, making it possible for pathogen visualization detection technology to truly move beyond the laboratory.

[0015] More preferably, the nucleic acid aptamer is an amino-modified nucleic acid aptamer with the sequence of 5'-NH2-TCTAAAAATGGGCAA AGA AACAGTGACTCGTTGAGATACT-3'.

[0016] Preferably, the dual signal N coupled with the diffusion distance D and the red fluorescence intensity R Dual -P Dual Output as visualization result: N Dual -P Dual The calculation of refers to formula (2)-(4):

[0017] ΔR=normalized R Negative –normalized R Positive (2);

[0018] ΔD=normalized D Negative –normalized D Positive (3);

[0019] N Dual -P Dual =(normalized D Negative+normalized R Negative )–(normalized D Positive +

[0020] normalized R Positive )(4);

[0021] Among them, normalized R Negative is the normalized R value of the negative control, normalized R Positive is the normalized R value of the positive sample, normalized D Negative is the normalized distance of the negative control, normalized D Positive is the normalized distance of positive samples.

[0022] A positive result is a positive value greater than 0, and a negative result is a negative value of 0 or less than 0. In actual testing, in order to make the test results more reliable, a result value greater than 0.5 is usually considered positive (greater than or equal to the value corresponding to the lowest VP detection concentration).

[0023] During quantitative analysis, the mobile phone software first displays the RGB values ​​of the selected area (only the R value) as the second signal. The resulting R value and the distance value observed by the naked eye (the first signal) are substituted into the calculation formula (4). The resulting value is then substituted into the working curve equation to calculate the corresponding bacterial concentration.

[0024] Preferably, in S3, the concentration of the aptamer solution is 0.4-0.6 μM, the concentration of the lanthanide MOF solution is 8-9 mg / mL, and the volume ratio of the pathogen sample solution, the aptamer solution, and the lanthanide MOF solution is 20:10:15.

[0025] Preferably, the mass ratios of zirconium tetrachloride, europium nitrate hexahydrate, 2,6-naphthalene dicarboxylic acid and 2-fluorobenzoic acid in S1 are 2-3, 10-15: 6-7: 30-35.

[0026] Preferably, the organic solvent in S1 is a mixed aqueous solution of DMF and HNO3, and the volume ratio of water, DMF and HNO3 is 3-4:14-15:0.2-0.5.

[0027] Preferably, the paper needle area of ​​the paper needle device in S1 is 2 mm wide and 3 cm long, and the absorbent pad is a square with a side length of 1 cm.

[0028] Preferably, before use, the paper needle device is first placed in a PBS buffer solution containing 1% BSA and soaked for 40-50 minutes, and then soaked in water for 1-3 minutes to perform antifouling modification on the paper needle device.

[0029] Preferably, the solvent of the nucleic acid aptamer solution is 100 mM pH=7.5 PBS, and the solvent of the lanthanide MOF solution is DMF.

[0030] A second aspect of the present invention provides a kit for pathogen detection, comprising a lanthanide MOF solution, a nucleic acid aptamer solution for specifically capturing target pathogens, and a paper needle device. The preparation of the lanthanide MOF solution and paper needle device is described in steps S1 and S2 of the first aspect.

[0031] Preferably, the nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO: 1.

[0032] More preferably, the nucleic acid aptamer is an amino-modified nucleic acid aptamer with the sequence of 5'-NH2-TCTAAAAATGGGCAA AGA AACAGTGACTCGTTGAGATACT-3'.

[0033] Preferably, the method for using the kit is: mixing a pathogen sample solution to be tested, a nucleic acid aptamer solution that specifically captures the target pathogen, and a lanthanide MOF (LnMOF) solution, allowing the reaction to proceed for 20-30 minutes, then inserting the needle end of the paper needle device into the mixed solution for signal output, and then observing the diffusion distance of the fluorescent band through fluorescence excitation. A shorter diffusion distance than that of the negative control is a positive result.

[0034] More preferably, the dual signal N coupled with the diffusion distance D and the red fluorescence intensity R Dual -P Dual Output as visualization result: N Dual -P Dual The calculation of refers to formula (2)-(4):

[0035] ΔR=normalized R Negative –normalized R Positive (2);

[0036] ΔD=normalized D Negative –normalized D Positive (3);

[0037] N Dual -P Dual =(normalized D Negative +normalized R Negative )–(normalized D Positive +

[0038] normalized R Positive )(4);

[0039] Among them, normalized R Negative is the normalized R value of the negative control, normalized R Positive is the normalized R value of the positive sample, normalized D Negative is the normalized distance of the negative control, normalized D Positive is the normalized distance of positive samples.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention discloses a method for detecting pathogens based on lanthanide MOF and paper needles. A fluorescent biosensor is constructed by combining LnMOF with a nucleic acid aptamer (specifically capturing target pathogens) through a covalent bond (Apt-LnMOF). Then, a T-shaped absorbent paper is used as a visual signal output carrier. By optimizing the geometric shape and size of the absorbent paper, a needle-shaped paper-based device (paper needle) is constructed. During detection, a pathogen sample solution, a nucleic acid aptamer (Apt) solution that specifically captures target pathogens, and a lanthanide MOF solution are mixed. After standing for reaction, the needle-shaped end of the paper needle device is inserted into the mixed solution for signal output. Finally, qualitative or quantitative analysis is performed based on the diffusion distance and red fluorescence intensity (R value) of the Apt-LnMOF at the paper-based interface. On the one hand, the lanthanide MOF (Apt-LnMOF) with an aptamer modified on the surface is used as a signal label for the pathogen visualization detection strategy, and the excellent fluorescence luminescence properties of the lanthanide MOF are utilized to improve the sensitivity and response speed of the detection method. On the other hand, the combination of the diffusion distance of Apt-LnMOF at the paper-based interface and the red fluorescence intensity (R value) as a visual signal output mode allows for qualitative analysis of the distance signal by direct visual observation and quantitative analysis of the R value using a smartphone. Furthermore, the R value and distance signal can be coupled to improve the accuracy of the test results. This invention uses a self-made paper needle device as a dual-signal output carrier, making the results easy to read. The combination of a one-step reaction and a self-priming paper needle simplifies the operation steps, making it easier for non-professionals to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of VP dual signal visualization analysis based on Apt-LnMOF and paper needle device;

[0043] Figure 2 Characterization of LnMOF structure: (A) and (B) characterized by SEM and TEM, respectively, (C) excitation and emission spectra of LnMOF;

[0044] Figure 3 Schematic diagram of the production process of needle-shaped paper-based devices;

[0045] Figure 4 Schematic diagram of the one-step VP detection process based on Apt-LnMOF and paper needles;

[0046] Figure 5 Feasibility verification results of the lanthanide MOF and paper needle detection method (A); Differences in positive and negative detection signals with and without covalent binding of the aptamer to LnMOF (B); Schematic diagram of the dual-signal acquisition process (C); Comparison of positive and negative results based on single and dual signals (D);

[0047] Figure 6 is the single signal ΔR(A), ΔD(B) and the dual signal N Dual -P Dual (C) Sensitivity comparison;

[0048] Figure 7 Sensitivity evaluation results in standard samples and actual samples: (A) and (B) are the actual analysis results and calibration curves at different VP concentrations in standard samples, respectively; (C) and (D) are the detection sensitivity analysis results in actual samples corresponding to (A) and (B), respectively;

[0049] Figure 8 The specificity and stability evaluation results of the lanthanide MOF and paper needle detection method are as follows: (A) is the specificity and stability evaluation results of the lanthanide MOF and paper needle detection method at the same concentration (10 5 The selectivity of the method (Mix = VP + Sal + E. coli + Sta) was evaluated by detecting VP and other bacteria at 4% CFU / mL. (B) and (C) are the stability evaluations of LnMOF after storage for 7-35 days. DETAILED DESCRIPTION

[0050] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0051] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0052] Example 1 Establishment of a pathogen detection method based on lanthanide MOF and paper needles

[0053] (1) Synthesis of LnMOF:

[0054] 0.021g of zirconium tetrachloride (ZrCl4), 0.1208g of europium nitrate hexahydrate (Eu(NO3)3·6H2O), 0.0626g of 2,6-naphthalenedicarboxylic acid (2,6-H2NDC), and 0.3247g of 2-fluorobenzoic acid (2-FBA) were weighed separately. H2O (3.335mL), DMF (14.665mL), and HNO3 (0.4mL) were then added to the mixture, mixed thoroughly, and sonicated for 5 minutes. The mixture was sealed in a Teflon-lined stainless steel autoclave and heat-treated at 115°C for 50 hours before cooling to room temperature. The resulting product was collected by centrifugation at 9000rpm for 5 minutes, washed twice with 5mL of acetone and 5mL of DMF, and then dispersed in 10mL of DMF (concentration: 8.3mg / mL).

[0055] like Figure 2 As shown in Figures 2A and 2B, the synthesized LnMOF was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. The characterization results show that the prepared LnMOF is spherical with a uniform particle size distribution and a diameter of approximately 200 nm. Figure 2 C shows the excitation and emission spectra of LnMOF. The excitation spectrum shows a broad band with a maximum band around 320 nm, which can be attributed to the π–π* transition of the 2,6-H2NDC ligand. Under 320 nm excitation, the emission spectrum shows two distinguishable emission bands: the ligand emission peak is at 415 nm, and the characteristic narrow spectral lines are at 579, 594, 618, 651, and 698 nm, corresponding to 5 D0→ 7 F J (J=0–4). Eu dominates the emission spectrum 3+ The band confirms the energy transfer from the ligand to Eu 3+ Efficient transfer of ions.

[0056] (2) In the production of paper needle devices:

[0057] Use a paper cutter to cut the absorbent paper into 1×4cm pieces, and then use scissors to further trim the paper into a "T" shaped device ( Figure 3, also known as a paper needle device). The paper needle area of ​​the device is 2 mm wide and 3 cm long, and the absorbent pad is a square with a side length of 1 cm. The needle-shaped portion of the device is designed to absorb the detection solution and output the signal (the diffusion distance of Apt-LnMOF at the paper needle interface and the R value), while the square area is used to provide sufficient capillary siphon force and store waste liquid. Subsequently, the paper needle device was modified to prevent fouling. The paper needle device was placed in a PBS buffer containing 1% BSA for 45 minutes, and then soaked in pure water for 1–3 minutes. Finally, the paper needle device was removed and placed in a drying oven to fully dry. After that, it was placed in a sealed bag and stored at room temperature for future use.

[0058] (3) Detection of pathogens:

[0059] In the process of VP detection, it is only necessary to simply mix the sample to be tested with the reaction reagent in one step, and then a visual signal output can be obtained on the paper needle. Figure 4 As shown, the specific operation process is as follows: 10 μL of VP diluted with 100 mM pH = 7.5 PBS (Vibrio parahaemolyticus powder was purchased from China Industrial Microbiological Culture Collection (CICC, No. 21617)), 5 μL of amino-modified VP nucleic acid aptamer diluted to 0.5 μM with 100 mM pH = 7.5 PBS (purchased from Sangon Biotech (Shanghai) Co., Ltd., sequence 5'-NH2-TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGA TACT-3') and 7.5 μL of LnMOF (dispersed in DMF, concentration of 8.3 mg / mL) were added to a 200 μL centrifuge tube, and the reaction was allowed to stand at room temperature (about 25°C) for 20-30 min. The needle tip of the paper needle device is then inserted into a reaction centrifuge tube for signal output. (After the sample is completely absorbed, 30 μL of 70% DMF-containing PBS buffer is added to the centrifuge tube. The needle tip spontaneously absorbs liquid to promote further diffusion of the sample.) The sample diffuses a certain distance on the needle interface through the capillary siphon action of the paper needle.

[0060] Output of test results: The device is placed under ultraviolet light (254nm) for fluorescence excitation, which can produce bright fluorescent stripes (the fluorescent signal of a positive result is shorter, and the fluorescent signal of a negative result is longer). The results can be quickly and qualitatively analyzed directly by the naked eye (observing the length of the fluorescent stripe). In addition, the camera function and color analysis software (RGB analysis) of a smartphone (such as the Xiaomi 11 smartphone) can be used to quantitatively analyze the results (R values ​​in the range of 0-255 can be output). It is worth noting that different mobile phones have different photo-taking effects, which may lead to large errors in the results.

[0061] Example 2 Feasibility Verification of Pathogen Detection Method Based on Lanthanide MOF and Paper Needles

[0062] To demonstrate the feasibility of the method, we first explored the differences in fluorescence signals under different reagent combinations. The output of the fluorescence signal was represented by the diffusion distance of LnMOF at the paper-needle interface. Figure 5 Figure A shows the fluorescence signals diffused on paper needles from four different combinations. The treatment without LnMOF (the first combination, including aptamer and VP) exhibited no red fluorescence signal, while pure LnMOF (the second combination) exhibited the longest red fluorescence band. The negative treatment (the third combination, including aptamer and LnMOF) exhibited a longer red fluorescence signal. Notably, in the negative treatment, electrostatic adsorption or π–π interaction between LnMOF and the aptamer occurred, resulting in a slightly shorter diffusion distance for LnMOF in the negative treatment than for pure LnMOF. The shorter red fluorescence distance in the positive treatment (the fourth combination, including aptamer, VP, and LnMOF) is attributed to the specific binding of VP to the aptamer coupled to the LnMOF surface (via covalent bonding), leading to LnMOF aggregation. A significant difference in red fluorescence distance was observed between the negative and positive treatments. These results demonstrate the feasibility of this method.

[0063] In order to explore the influence of the binding mode between the aptamer and LnMOF on the test results, the present invention compares the positive and negative test results after the aptamer and LnMOF are bound by non-covalent bonds (electrostatic interaction) and covalent bonds. Figure 5 B compares the positive and negative results for VP detection after covalently coupling the unmodified (non-covalent) and amino-modified (covalent) aptamers to LnMOF. The results show that the difference between the negative and positive diffusion distances for the amino-modified aptamer and LnMOF is greater than the difference between the negative and positive diffusion distances for the unmodified aptamer and LnMOF combination. These results suggest that the stronger covalent coupling of the aptamer to LnMOF (resulting in a shorter positive diffusion distance) ultimately leads to a greater difference between the positive and negative diffusion distances. Therefore, amino-modified aptamers covalently coupled to LnMOF are more suitable for this detection strategy.

[0064] In the detection methods that have been reported so far that use distance as a signal output, the sample is usually dripped onto the interface of a material such as a paper base in a horizontal loading mode, and then the capillary force is used to make the sample move spontaneously to produce a distance change. The biggest disadvantage of these reported distance-based detection methods is that the operator is difficult to control the dripping speed of the sample, resulting in uneven diffusion of the sample on the paper interface. This uneven diffusion phenomenon will eventually affect the accuracy of the test results. In the present invention, a vertical self-absorption mode based on a paper needle device is adopted to absorb and separate the sample ( Figure 5C, left figure), realizes the automatic output of the distance signal (without human interference), which undoubtedly improves the accuracy of the test results. In addition, although the reported visual detection method based on the output of the distance signal is simple to operate and the results are more intuitive, there is usually a problem of poor repeatability of the test results. One of the reasons for the above problems is that these methods are generally based on a single signal (such as distance or RGB value) to judge the results. In order to solve this problem, the present invention introduces the red signal of LnMOF (R value in RGB) as the second signal on the basis of retaining the distance signal. The above two signals are coupled to jointly improve the accuracy of the test results. The detailed acquisition process of the dual signal is shown in the figure. Figure 5 C (right image). In summary, Signal 1 (distance) is determined by naked eye observation, while Signal 2 (R value) is output by taking a photo with a smartphone and using the R value analysis function of the built-in software.

[0065] In order to compare the sensing performance based on single signal and dual signal, the present invention compares the sensitivity and accuracy in single signal (pure distance and R value) and dual signal (distance and R value coupled) modes respectively. Figure 5 The illustration in D shows the comparison of positive and negative signals based on signal 1 (distance), signal 2 (R value), and dual signal (coupled signals 1 and 2). In terms of data output, the present invention normalizes the distance and R value to facilitate comparison and coupling between the two. Among them, the normalized distance value and the normalized R value are respectively used as single signal output values. The sum of the normalized distance and the normalized R value is the dual-signal output value (Dual-signal, defined by formula (1)):

[0066] Dual-signal(Distance&R Value)=normalized Distance+normalized R value(1).

[0067] Data analysis showed that compared with single-signal output methods based on distance or R value, the coupled dual-signal strategy had smaller errors and a larger difference between negative and positive signals. Specifically, the standard deviation (SD) of the dual-signal strategy accounted for 21.8% of the total mean (the average SD percentage of positive and negative), which was lower than the standard deviation of the total mean based on distance (51.7%) and R value (52.8%), respectively. Furthermore, the difference between negative and positive signals based on the dual-signal strategy (1.5) was much larger than the difference between negative and positive signals based on distance (0.74) and R value (0.76). Therefore, these results indicate that the signal output method based on the coupling of dual signals can improve the accuracy and sensitivity of the detection method to a certain extent.

[0068] In terms of sensor performance evaluation, the present invention compares the sensor performance based on single signal output and dual signal output in standard samples. Figure 6 As shown, the change of R value (ΔR, see formula (2)) is selected respectively ( Figure 6 A), the change of LnMOF diffusion distance (ΔD, see formula (3)) ( Figure 6 B) and dual signal phase coupling (N Dual -P Dual , defined by formula (4))( Figure 6 C) signal output mode to compare the VP detection sensitivity. By comparison, it was found that the slope of the calibration curve based on the dual signal and R 2 The slope and R of the calibration curve are significantly better than those based on a single signal. 2 Therefore, the present invention speculates that the result output strategy based on dual signal coupling can effectively improve the accuracy of the detection method. Dual -P Dual Used as a dual-signal output mode for subsequent detection performance evaluation.

[0069] ΔR=normalized R Negative –normalized R Positive (2);

[0070] ΔD=normalized D Negative –normalized D Positive (3);

[0071] N Dual -P Dual =(normalized D Negative +normalized R Negative )–(normalized D Positive +

[0072] normalized R Positive )(4).

[0073] Among them, normalized R Negative is the normalized R value of the negative control, normalized R Positive is the normalized R value of the positive sample, normalized D Negative is the normalized distance of the negative control, normalized D Positive is the normalized distance of positive samples.

[0074] A positive result is a positive value greater than 0, and a negative result is a negative value of 0 or less than 0. In actual testing, in order to make the test results more reliable, a result value greater than 0.5 is usually considered positive (greater than or equal to the value corresponding to the lowest VP detection concentration).

[0075] In quantitative analysis, first use the mobile phone software to display the RGB value of the selected area (only the R value) as the second signal. Substitute the resulting R value and the distance value observed by the naked eye (the first signal) into the calculation formula (4), and then substitute the obtained value into the working curve equation (such as the subsequent Figure 7 B or Figure 7 D) and calculate the corresponding bacterial concentration.

[0076] Example 3 Verification of the stability, specificity and accuracy of the pathogen detection method based on lanthanide MOF and paper needles

[0077] like Figure 7 As shown, the present invention adopts a dual-signal phase coupled output mode and compares the method in the standard sample (PBS diluted VP) ( Figure 7 A and B) and real samples (shrimp homogenate diluted VP, which was prepared by first grinding commercially available shrimp into a paste using a mortar and pestle, then diluting the ground shrimp 10-fold with sterile PBS and mixing evenly, then filtering the mixture with sterile gauze to collect the shrimp homogenate for diluting VP) ( Figure 7 C and D) for VP detection. The results showed that this method has good sensitivity to VP detection in both standard samples and actual samples. 2 –10 7 The working curves of VP detection showed good linearity and high sensitivity. The lowest detection concentration of VP based on naked eye observation can reach 10 2 The detection performance of this method in real samples is comparable to that in standard samples, indicating that the sensing strategy has good resistance to interference from complex components.

[0078] Subsequently, the present invention evaluated the specificity of the method. Figure 8 The specificity test results of A showed that only pure VP samples and mixed bacterial samples containing VP [including VP, Escherichia coli (E.coli), Salmonella (Sal) and Staphylococcus aureus (Sta); all bacterial concentrations were 10 5 The results were published in the journal Nature Communications. The results were published in the Journal of Industrial Microbiology ... Figure 8B and C show the bar graphs of the absolute positive and negative values ​​and the difference between the negative and positive values ​​for VP detection after LnMOF storage for different periods of time, respectively. Analysis of the stability evaluation results revealed that LnMOF maintained excellent sensing stability even after storage for over a month, with the difference between the negative and positive detection signals remaining at a high level (the difference between the negative and positive values ​​remained at approximately 88.5% of the initial value). These performance evaluation results demonstrate the promising potential for practical application of this method.

[0079] The present invention also evaluated the accuracy of the detection method with reference to the classic plate culture method (GB 4789.7-2013). First, 100 samples (including 50 positive samples and 50 negative samples) were identified based on the plate culture method, and then these 100 samples were tested using the method of the present invention. The results showed that there were 4 false negative results in the analysis of 50 positive samples, and 5 false positive results in the analysis of 50 negative samples. As shown in Table 1, it was calculated that the sensitivity, specificity and accuracy of this method in actual sample analysis were 92%, 90% and 91% respectively.

[0080] In summary, the present invention utilizes Apt-LnMOF as a signal tag in a visualization detection strategy, improving the sensitivity and response speed of the pathogen visualization detection method. The dual signal output of the visualization results, coupled with the diffusion distance of Apt-LnMOF at the paper-based interface and the red fluorescence intensity (R value), enhances the accuracy of the detection method. The present invention utilizes a paper needle device as a signal output carrier, making it more convenient for non-experts to operate.

[0081] Table 1 Evaluation results of the accuracy of VP detection in actual samples using the plate method as a reference

[0082]

[0083] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A method for detecting pathogens for non-disease diagnosis and treatment purposes based on lanthanide MOFs and paper needles, characterized in that: The following steps are involved: S1. Prepare lanthanide MOF solution: dissolve zirconium tetrachloride, europium nitrate hexahydrate, 2,6-naphthalene dicarboxylic acid and 2-fluorobenzoic acid in an organic solvent, heat treat at 110-120°C for 40-60h, collect the product, wash and redissolve it; S2. Preparation of paper needle device: trimming absorbent paper into a "T"-shaped device; S3. Pathogen detection: Mix the pathogen sample solution, the nucleic acid aptamer solution that specifically captures the target pathogen, and the lanthanide MOF solution. After standing for 20-30 minutes, insert the needle end of the paper needle device into the mixed solution for signal output. Then, observe the diffusion distance D of the fluorescent strip through fluorescence excitation. A shorter diffusion distance D indicates a positive result. The pathogen concentration is calculated based on the red fluorescence intensity R. The dual signal N coupled with the diffusion distance D and the red fluorescence intensity R is used. Dual -P Dual Output as visualization result: N Dual -P Dual The calculation of refers to formula (2)-(4): ΔR=normalizedR Negative –normalizedR Positive (2); ΔD=normalizedD Negative –normalizedD Positive (3); N Dual -P Dual =(normalized D Negative +normalized R Negative )–(normalized D Positive +normalizedR Positive )(4); The pathogen is Vibrio parahaemolyticus, and the nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO:

1.

2. The method for detecting pathogens for non-disease diagnosis and treatment purposes based on lanthanide MOF and paper needles according to claim 1, characterized in that: The nucleic acid aptamer is an amino-modified nucleic acid aptamer, and the sequence is 5'-NH2-TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3'.

3. The method for detecting pathogens for non-disease diagnosis and treatment purposes based on lanthanide MOF and paper needles according to claim 1, characterized in that: In S3, the concentration of the aptamer solution is 0.4-0.6 μM, the concentration of the lanthanide MOF solution is 8-9 mg / mL, and the volume ratio of the pathogen sample solution, the aptamer solution, and the lanthanide MOF solution is 20:10:

15.

4. The method for detecting pathogens for non-disease diagnosis and treatment purposes based on lanthanide MOF and paper needles according to claim 1, characterized in that: The mass ratio of zirconium tetrachloride, europium nitrate hexahydrate, 2,6-naphthalene dicarboxylic acid and 2-fluorobenzoic acid in S1 is 2-3:10-15:6-7:30-35.

5. The method for detecting pathogens for non-disease diagnosis and treatment purposes based on lanthanide MOF and paper needles according to claim 1, characterized in that: The organic solvent in S1 is a mixed aqueous solution of DMF and HNO3, and the volume ratio of water, DMF and HNO3 is 3-4:14-15:0.2-0.5.

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