A lateral flow test strip for ultra-high sensitive detection of pathogenic bacteria and application thereof

Abstract

This invention belongs to the field of medical testing technology, specifically relating to a lateral flow test strip for ultra-high sensitivity detection of pathogens and its application. The lateral flow test strip provided by this invention uses polydopamine nanoparticles modified with bacterial antibodies as signal probes. Multiple detection lines are set on the NC membrane, and the detection lines are obtained by scribing with a bacterial antibody solution as ink. This invention replaces Au NPs in classic LFIAs with PDA NPs. Relying on the high extinction coefficient, affinity for bacteria, and stability of antibody binding of PDA NPs, a two-order-of-magnitude improvement in sensitivity is achieved when using LFIAs to detect bacteria. Simultaneously, utilizing the high extinction coefficient of PDA NPs, their signals can be identified and statistically analyzed by ImageJ software even when invisible to the naked eye. Combined with multiple test strips, this improves target capture efficiency, thereby achieving highly sensitive detection of pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a lateral flow test strip for ultra-sensitive detection of pathogens and its application. Background Technology

[0002] Lateral flow assay strips (LFIAs) are a reliable, immediate, and low-cost method for the rapid detection of pathogens without the need for trained personnel or basic infrastructure. However, the sensitivity of currently available commercially available LFIAs for pathogens often falls below 100 CFU / mL, failing to meet national standards and limiting their application in the detection of foodborne pathogens. This is because current commercially available LFIAs primarily use colloidal gold as the signal source, and colloidal gold has a low extinction coefficient, making it invisible to the naked eye at low concentrations. Furthermore, the low capture efficiency of the detection line also significantly impacts the detection sensitivity of LFIAs.

[0003] Existing methods for improving the sensitivity of LFIAs mainly focus on altering their output signal, such as combining surface-enhanced Raman spectroscopy (SERS) and fluorescence techniques. While this method effectively improves sensitivity, it also requires additional equipment, which contradicts the original intention of LFIAs to be independent of other specialized instruments. Colloidal gold immunochromatographic test strips, with their advantages of being rapid and convenient, requiring no special instruments, providing results that can be interpreted visually, and exhibiting high sensitivity and specificity, have become one of the most rapid and sensitive immunoassay techniques available today and are widely used in the detection of pathogens.

[0004] Colloidal gold immunochromatographic test strips are 0.3 cm wide strips with a base plate. From one end to the bottom, the base plate has a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad attached in a stepped fashion, with each part overlapping. Typically, the gold-labeled pad is coated with antibody-labeled colloidal gold, and the nitrocellulose membrane has an antibody-coated detection line (a) and a control line (b) composed of goat anti-mouse IgG. The detection principle utilizes the capillary action formed by the absorbent pad. The antigen to be detected, placed on the sample pad, first binds to the colloidal gold-labeled antibody on the gold-labeled pad and continues to move forward along the nitrocellulose membrane (NC membrane). When it reaches the antibody-coated detection line (a), the antibody captures it, and as the reaction proceeds, it continuously accumulates to a visible level. Excess colloidal gold-labeled antibody continues to move forward, reaching the control line (b) containing goat anti-mouse IgG, where it is captured and continuously accumulated. Therefore, if the sample is positive, colored bands will appear on both the test line and the control line; if the sample is negative, a colored band of the same color will only appear on the control line. However, colloidal gold immunochromatographic test strips still suffer from low detection sensitivity, especially for larger bacteria, where the detection limit is high, thus limiting their application. Therefore, developing LFIAs with high sensitivity for pathogen detection holds significant application potential. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides a multi-detection-line lateral flow test strip based on polydopamine nanoparticles. Polydopamine has a high affinity for bacteria, which can effectively improve the binding efficiency. At the same time, polydopamine has a high extinction coefficient, which ultimately improves the detection sensitivity. In addition, the introduction of multiple detection lines effectively improves the capture efficiency of pathogenic bacteria, and combined with ImageJ software analysis, the detection sensitivity is further improved.

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

[0007] The first aspect of the present invention provides a lateral flow test strip, wherein the lateral flow test strip uses a 0.2-4 mg / mL solution of polydopamine nanoparticles modified with bacterial antibodies as a signal probe, and the NC membrane of the test strip is provided with 3-7 detection lines, wherein the detection lines are obtained by scribing with a 0.2-4 mg / mL solution of bacterial antibodies as ink.

[0008] Polydopamine nanoparticles (PDA NPs) are non-toxic, biocompatible black nanoparticles with broad application prospects. PDA NPs possess various functional groups (such as hydroxyl and amino groups), enabling them to be used for antibody modification. Simultaneously, PDA NPs exhibit good bioaffinity and a certain degree of bacterial recognition ability, effectively improving the probe's ability to capture targets. More importantly, compared to colloidal gold (Au NPs), PDA NPs have a higher extinction coefficient, making them easier to observe with the naked eye. Therefore, using PDA NPs as a signal source to replace Au NPs not only allows for more stable antibody modification but also, relying on the material's inherent affinity for bacteria, effectively improves the binding efficiency of signal probes to targets. Furthermore, the introduction of multiple lines helps improve target binding efficiency, and with the assistance of ImageJ software, signals invisible to the naked eye can be output, thereby improving detection sensitivity.

[0009] The combination of a high extinction coefficient material and multiple detection lines enables ultra-high sensitivity detection of lateral flow test strips. This invention reveals that classic colloidal gold at low concentrations cannot be captured by ImageJ software. However, when replaced with polydopamine nanoparticles, a material with a high extinction coefficient, although low concentrations of polydopamine nanoparticles are not visible to the naked eye, ImageJ can capture their signal. Combining multiple detection lines improves the capture efficiency of pathogens, ultimately achieving ultra-high sensitivity detection of pathogens through grayscale value analysis.

[0010] Preferably, the NC membrane of the lateral flow test strip has 5 detection lines, which are obtained by scribing with a solution of 0.5-2 mg / mL bacterial antibody as ink.

[0011] Preferably, the method for preparing the polydopamine nanoparticles modified with bacterial antibodies includes the following steps:

[0012] S1. Prepare a 1-4 mM dopamine hydrochloride solution with Tris-HCl buffer, and synthesize polydopamine nanoparticles by shaking in a shaker (100-200 rpm / min) at 20-45℃ for 6-48 h.

[0013] S2. Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in PBS buffer, then add PDA NPs and bacterial antibodies to prepare PDA NPs modified with bacterial antibodies (PDA-AbNPs).

[0014] This invention utilizes a dopamine self-polymerization method to prepare PDA NPs. These PDA NPs contain abundant amino functional groups, and antibodies can be covalently modified onto them via an EDC / NHS reaction for target capture. The modified PDA NPs replace traditional colloidal gold in the detection of bacteria using LFIAs (Laminated Fluorescent Inhalation Imaging). The high extinction coefficient of the PDA NPs effectively improves the detection sensitivity of LFIAs for bacteria.

[0015] More preferably, before use, the polydopamine nanoparticles in step S1 are centrifuged to remove the supernatant and then resuspended in water.

[0016] More preferably, the concentration of the Tris-HCl buffer in step S1 is 0.01-0.1M and the pH is 8.5; the concentration of the PBS buffer in step S2 is 0.01-0.1M and the pH is 6.0.

[0017] More preferably, the ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, polydopamine nanoparticles and bacterial antibodies is 100:140:26:4-6.

[0018] The second aspect of the present invention provides the application of the lateral flow test strip described in the first aspect in bacterial detection.

[0019] The LFIAs provided by this invention replace Au NPs in classic LFIAs with PDA NPs. PDA NPs have a high extinction coefficient and high affinity for bacteria, effectively improving binding efficiency and detection sensitivity. Furthermore, these LFIAs are multi-line LFIAs. Due to the large size of bacteria, their capture efficiency on the detection lines of LFIAs is too low. The application of multiple test lines effectively ensures the capture efficiency of the target analytes, thus guaranteeing that all target analytes can be captured. Compared with traditional single-line test strips, multi-line test strips can capture more target analytes, resulting in higher signals under ImageJ software analysis, effectively improving detection sensitivity. Simultaneously, PDA NPs with high extinction coefficients can still be recognized by ImageJ at concentrations where color changes are not visible to the naked eye, breaking through the detection limit of LFIAs without additional instruments.

[0020] Preferably, the bacteria include Escherichia coli.

[0021] Modified PDA NPs were used to replace traditional colloidal gold for LFIAs detection of E. coli. The high extinction coefficient of PDA NPs effectively improved the detection sensitivity of LFIAs for E. coli, with a visual detection range of 10. 2 CFU / mL-10 7 CFU / mL; at the same time, the introduction of multiple detection lines effectively improved the capture efficiency of E. coli, and combined with Image J software analysis, the detection sensitivity was further improved (10 CFU / mL).

[0022] The third aspect of this invention provides a method for detecting bacteria, specifically: using the lateral flow test strip described in the first aspect, firstly, a sample is dropped onto the sample pad, and after reacting for more than 10 minutes, the test line is allowed to fully capture the target. Then, polydopamine nanoparticles modified with bacterial antibodies (PDA-Ab NPs) are added and reacted for more than 5 minutes. Next, PBS buffer is added so that all the polydopamine nanoparticles flow through the NC membrane and bind to the target. Then, the detection results are observed.

[0023] Preferably, the grayscale values ​​of the detection results are analyzed using ImageJ software to calculate the bacterial concentration. For detection results that are invisible to the naked eye, ImageJ software can further improve the detection limit and enable quantitative analysis of bacteria.

[0024] More preferably, the detection limit of the method for bacteria is 10 CFU / mL.

[0025] The multi-detection-line lateral flow test strip based on polydopamine nanoparticles provided by this invention uses polydopamine nanoparticles instead of colloidal gold, effectively improving the binding ability with target pathogenic bacteria, and the signal is more easily observed with the naked eye, thus improving the visual detection sensitivity (10). 2 The detection efficiency of pathogens was improved by introducing multiple detection lines (CFU / mL), and the detection sensitivity was further improved by combining ImageJ software analysis (10 CFU / mL).

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] This invention discloses a lateral flow test strip. The lateral flow test strip uses polydopamine nanoparticles modified with bacterial antibodies as signal probes. Multiple detection lines are formed on its NC membrane by scribing with a bacterial antibody solution as ink. This invention replaces Au NPs in classic LFIAs with PDA NPs. Relying on the high extinction coefficient, affinity for bacteria, and stability of antibody binding of PDA NPs, it achieves a two-order-of-magnitude improvement in sensitivity when detecting bacteria using LFIAs. Simultaneously, utilizing the high extinction coefficient of PDA NPs, their signals can be identified and statistically analyzed by ImageJ software even when invisible to the naked eye. Furthermore, the combination of multiple test strips improves target capture efficiency, thereby achieving highly sensitive detection of pathogenic bacteria. Attached Figure Description

[0028] Figure 1 The process of synthesizing PDA NPs and their modified antibodies (a); scanning electron micrographs of PDA NPs (b, d) and PDA-Ab (c, e); particle size distribution of PDA NPs (f); and zeta potential detection results of PDA NPs and PDA-Ab (g).

[0029] Figure 2 The preparation process (a), detection principle (b), and positive / negative comparison diagram (c) of PDA-based multi-strip lateral flow test strips are shown.

[0030] Figure 3 Visible light absorption of PDA NPs and Au NPs (a), storage stability at room temperature (b); relationship between PDA NP concentration and absorbance (c); relationship between Au NP concentration and absorbance (d); photographs (f) of PDA NPs and Au NPs at different concentrations dropped onto NC film and grayscale values ​​(e) obtained from Image J statistics.

[0031] Figure 4The detection results of PDA NPs-based LFIAs and Au NPs-based LFIAs for Escherichia coli are shown in the following figures: (a) gray value statistics; (b) detection curve; (c, d) schematic diagrams of detection of Escherichia coli at various concentrations: gray value detection results of PDA NPs-based LFIAs (c) and Au NPs-based LFIAs (d).

[0032] Figure 5 The number of test lines required to detect 10 CFU / mL of E. coli using grayscale values ​​as the output signal.

[0033] Figure 6 The results of PDA-based LFIAs on actual samples are as follows: (ab) is a milk sample; (cd) is an orange juice sample; (ef) is a tomato juice sample. Detailed Implementation

[0034] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0036] Example 1: Preparation and Detection Performance Verification of Multi-Detection Line PDA NPs-Based LFIAs

[0037] The preparation of these LFIAs includes the following steps:

[0038] (1) First, prepare 0.05M Tris-HCl (pH 8.5) buffer solution, then use it to prepare 2mM dopamine hydrochloride solution, and place it in a shaker (100-200rpm / min) at 20-45℃ for 24h (or place it at room temperature) to obtain polydopamine nanoparticles.

[0039] (2) The mixture from step (1) was centrifuged at 8000 rpm to remove the supernatant, and then the polydopamine nanoparticles (0.13 mg / mL) were resuspended in water and stored in a refrigerator at 4°C for later use.

[0040] (3) Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in PBS buffer (0.01M) at pH 6.0. Then add PDA NPs and E. coli antibody [E. coli O157:H7 antibody, purchased from Fitzgerald Co., Ltd (North Acton, MA, USA)]. The concentrations of EDC, NHS, PDA NPs, and E. coli antibody were 0.5 mg / mL, 0.7 mg / mL, 0.13 mg / mL, and 5 mg / mL, respectively, to prepare PDA NPs modified with E. coli antibody (PDA-Ab NPs). The preparation and modification process of PDA NPs is as follows: Figure 1 As shown in figure a. Characterization of PDA NPs confirmed that the antibody was successfully modified onto the PDA NPs. Figure 1 g), and will not affect the morphology of PDA NPs ( Figure 1 bf).

[0041] (4) Using a 1 mg / mL solution of E. coli antibody as ink, draw 5 detection lines on the NC membrane with a pen, with each detection line spaced 1 mm apart. Then use the NC membrane to assemble LFIAs, and use the PDA-Ab NPs prepared in step (3) as signal probes to obtain PDA-based E. coli LFIAs.

[0042] Figure 2 'a' represents the composition of LFIAs, with 5 detection lines. Figure 2 b elucidates the mechanism and detection procedure for detecting *E. coli* using the prepared LFIAs. The first step involves adding a sample (typically 20 μL) to the sample pad and reacting for 10 minutes to allow the test line to fully capture the target. Next, 15 μL of PDA-Ab NPs (1 mg / mL) is added and reacted for 5 minutes. Finally, 20 μL of PBS buffer is added to ensure all PDA-Ab NPs flow through the NC membrane and bind to the target. The entire detection process can be completed within 30 minutes. The results are not only visually apparent but can also be analyzed using ImageJ software to calculate the *E. coli* concentration and improve the detection limit. Figure 2 c represents the detection result of LFIAs.

[0043] For the first time, the extinction coefficients of PDA NPs and Au NPs were compared. Concentration-absorbance standard curves for the two nanoparticles (PDA NPs and Au NPs) were prepared, and the extinction coefficients were calculated according to Beer–Lambert's law: A = log I0 / I = εC l (where A is the absorbance, ε is the extinction coefficient, C is the concentration, and l is the optical path length). Figure 3 The extinction coefficients of PDA NPs and Au NPs were calculated to be 37715 L / (g·cm) and 4685 L / (g·cm) respectively, indicating that PDA NPs are more easily observed by the naked eye than Au NPs. To further verify the above statement, a mixture of PDA NPs and Au NPs at the same concentration (0.13 mg / mL) was subjected to UV full-spectrum detection, and the results are as follows. Figure 3 As shown in Figure a, in the visible light region, the absorbance of PDA NPs is much greater than that of Au NPs, which is consistent with the results of the extinction coefficient detection. Furthermore, comparing the stability of the two reveals that PDA NPs are more stable at room temperature, making them more suitable for long-distance transportation. The results are as follows... Figure 3 As shown in b. Finally, the visual concentrations of PDA NPs and Au NPs on the NC membrane were analyzed and compared, and the results are shown in... Figure 3 As shown in f, a color change can still be observed on the NC membrane at a concentration of 0.01 mg / mL for PDA NPs, while it becomes unobservable at concentrations below 0.5 mg / mL for Au NPs. This indicates that PDA NPs, due to their high extinction coefficient, are more easily observed on the NC membrane than Au NPs. Therefore, replacing Au NPs with PDA NPs can effectively improve the sensitivity of LFIAs. Meanwhile, for... Figure 3 The data for f were analyzed for grayscale values ​​using ImageJ software, and the results are as follows: Figure 3 As shown in e, even when the concentration of PDA NPs is as low as 0.01 mg / mL, making the change imperceptible to the naked eye, Image J software can still effectively capture its grayscale information and output the signal, which is beneficial for further improving the detection sensitivity of LFIAs. However, the grayscale signal of low-concentration Au NPs cannot be effectively captured, so Image J cannot be used to improve its sensitivity.

[0044] Secondly, the sensitivity of PDA NPs-based LFIAs and Au NPs-based LFIAs in detecting Escherichia coli was compared, and the results are as follows: Figure 4 As shown. From Figure 4 It can be observed that the detection limit of classical Au NPs-based LFIAs is only 10. 4 CFU / mL, while the detection limit of PDA NPs-based LFIAs can reach 10. 2 CFU / mL. Statistical analysis of the grayscale values ​​of the detection line revealed that the detection limit of PDA NPs-based LFIAs can reach 10 CFU / mL, while the detection limit is within the range of 10 CFU / mL-10 CFU / mL. 7 CFU / mL exhibits good linearity. In contrast, AuNPs-based LFIAs can only detect 10... 4 -107 CFU / mL of bacteria. Furthermore, the number of test lines when 10 CFU / mL of E. coli was detected was investigated. Figure 5 The results showed that four detection lines were sufficient to achieve the desired detection effect.

[0045] Finally, PDA-based LFIAs were used to detect real samples (milk, orange juice, tomato juice), and the results were as follows: Figure 6 As shown. Among them Figure 6 ab represents the detection results of E. coli in milk. The results show that this detection method can effectively detect 10... 2 The detection limit for E. coli was CFU / mL. However, when testing orange juice, the detection limit was only 10. 5 CFU / mL Figure 6 The detection limit (CD) is lower because orange juice has a slightly acidic pH, which significantly affects the binding of antibodies to E. coli, leading to a decrease in detection sensitivity. When detecting tomato juice, the detection limit also decreases, reaching only 10. 3 CFU / mL Figure 6 ef).

[0046] In summary, polydopamine exhibits a higher extinction coefficient compared to colloidal gold, making it easier to observe with the naked eye. Replacing colloidal gold with polydopamine effectively improves the sensitivity of LFIAs. Furthermore, low-concentration polydopamine signals can be captured by ImageJ software, and combining multiple bands can further enhance the sensitivity of LFIAs. Therefore, multi-band LFIAs based on polydopamine nanoparticles can achieve ultra-high sensitivity detection of pathogens.

[0047] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A lateral flow test strip, characterized in that, The lateral flow test strip uses a 0.2-4 mg / mL solution of polydopamine nanoparticles modified with bacterial antibodies as a signal probe. The NC membrane of the test strip has 3-7 detection lines, which are obtained by scribing with a 0.2-4 mg / mL solution of bacterial antibodies as ink.

2. The lateral flow test strip according to claim 1, characterized in that, The method for preparing the polydopamine nanoparticles modified with bacterial antibodies includes the following steps: S1. Prepare a 1-4 mM dopamine hydrochloride solution using Tris-HCl buffer, and synthesize polydopamine nanoparticles by shaking at 20-45℃ for 6-48 h. The shaking speed is 100-200 rpm / min. S2. Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in PBS buffer, then add PDA NPs and bacterial antibodies to prepare polydopamine nanoparticles modified with bacterial antibodies.

3. A lateral flow test strip according to claim 2, characterized in that, Before use, the polydopamine nanoparticles from step S1 are centrifuged to remove the supernatant and then resuspended in water.

4. A lateral flow test strip according to claim 2, characterized in that, The concentration of Tris-HCl buffer in step S1 is 0.01-0.1 M and the pH is 8.5; the concentration of PBS buffer in step S2 is 0.01-0.1 M and the pH is 6.

0.

5. A lateral flow test strip according to claim 2, characterized in that, The ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, polydopamine nanoparticles, and bacterial antibodies is 100:140:26:4-6.

6. The use of the lateral flow test strip according to any one of claims 1-5 in bacterial detection for non-disease diagnostic purposes.

7. The application according to claim 6, characterized in that, The bacteria include Escherichia coli.

8. A method for detecting bacteria for non-disease diagnostic purposes, characterized in that, Using the lateral flow test strip according to any one of claims 1-5, first drop the sample onto the sample pad, react for more than 10 minutes to allow the test line to fully capture the target, then add polydopamine nanoparticles modified with bacterial antibodies and react for more than 5 minutes, then add PBS buffer to allow all the polydopamine nanoparticles to flow through the NC membrane and bind to the target, and then observe the detection results.

9. A method for detecting bacteria for non-disease diagnostic purposes according to claim 8, characterized in that, The concentration of bacteria was calculated by analyzing the grayscale values ​​of the detection results using ImageJ software.

10. A method for detecting bacteria for non-disease diagnostic purposes according to claim 8, characterized in that, The detection limit for bacteria by the method is 10 CFU / mL.

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