Integrated microneedle array device for detecting pathogenic bacteria on solid surface and application

Abstract

The invention discloses an integrated microneedle array device for detecting pathogenic bacteria on a solid surface and application, and belongs to the technical field of analysis and detection. The preparation method comprises the following steps: adding a mixed solution of polyethylene glycol diacrylate, methacrylated hyaluronic acid and a photoinitiator into a microneedle array mold, and then embedding a response type probe which is fixed by magnetic beads and is used for detecting escherichia coli; and irradiating a mold loaded with the mixed solution under an ultraviolet lamp for 10-60 seconds, then demolding to prepare a microneedle array for detecting bacteria, then dropwise adding a pullulan solution containing lysozyme on the surfaces of microneedles of the prepared microneedle array, and naturally air-drying to obtain the integrated microneedle array. The microneedle array device integrates the processes of bacterial lysis, active sampling, bacterial detection and the like, avoids the complex and time-consuming operation process of a traditional method, has the analysis performance of portability, simplicity, rapidness, low cost and the like, and has potential application value for analysis of various complex solid surfaces.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to an integrated microneedle array device and application for detecting pathogenic bacteria on a solid surface. Background Art

[0002] Bacterial infections have become the culprit for endangering the global public health system and pose a great threat to the environment in which humans live. According to data provided by the World Health Organization, nearly 10% of the world's population becomes ill each year due to eating contaminated food, and foodborne pathogens cause about 600 million illnesses and 420,000 deaths each year. Diseases caused by the most common foodborne pathogens such as Escherichia coli and Salmonella have become a major problem in the field of public health. They are mostly environmental hosts and spread through eating uncooked or contaminated food. Bacteria and their toxins are the most important pathogens in foodborne diseases. They usually spread quickly. Once infected, they lead to food poisoning with high morbidity and mortality, causing serious harm to human health and environmental safety.

[0003] The development of pathogen detection technology in solid media (especially solid surfaces) is limited by specific sampling methods (contact plate method or wiping method), and the detection process is scattered, time-consuming, and increases the risk of sample contamination. In order to make up for the shortcomings of pathogen detection on solid surfaces, people have been focusing on exploring integrated in-situ monitoring biosensor methods. Constructing a simple, rapid and low-cost Escherichia coli detection method to achieve instant detection of solid media foodborne pathogens and reduce the negative impact of foodborne diseases on public health is of great significance for preventing the occurrence of diseases and protecting human health.

[0004] Microneedle arrays are devices composed of micron-sized (<1000 μm in length) needle tips. They are small in size and large in specific surface area and have been widely used in the study of minimally invasive sensing of biomarkers. These devices have the advantage of feeding back target information of target sites in situ (applied to the desired location) or ex situ (analyzed after removal), and may be a reasonable method for the detection of pathogenic bacteria on solid surfaces. In the past few decades, different types of microneedle arrays have been explored and given different functions. For example, dissolving microneedles can dissolve the polymers that form the needle structure and release the embedded drugs, the inherent hydrophilicity of hydrogel microneedles enables them to swell in liquids to passively extract the targets therein, and porous microneedles load large particulate matter to prevent them from leaking. Therefore, by cleverly designing different reagents to be loaded into different areas of the microneedles, they can have the ability to sample, react, and analyze in situ. This can avoid the contamination of samples by the environment during the detection process. In addition, the microneedle, as an independent microreaction unit, can provide up to 100 sensing results in a single test, and averaging the large amount of data obtained can further reduce the error of manual operation, which is particularly important for analyzing heterogeneous solid surfaces. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide an integrated microneedle array device and application for detecting pathogenic bacteria on solid surfaces. The present invention designs a simple and portable microneedle array device that integrates processes such as bacterial lysis, active sampling and bacterial detection, which can realize the detection of pathogenic bacteria on solid surfaces. Compared with traditional methods for detecting pathogenic bacteria on solid surfaces, the integrated microneedle array device described in the present invention is simple to operate, does not require time-consuming operation by professionals, and only requires a few microliters of sample to complete the detection, which has potential application value for the detection of pathogenic bacteria on solid surfaces.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides an integrated microneedle array device, comprising a microneedle array layer for detecting bacteria and a bacterial lysis layer, wherein the bacterial lysis layer is a polysaccharide layer covering the outside of the microneedle array layer, the microneedle array layer contains a response probe for detecting Escherichia coli fixed with magnetic beads, and the bacterial lysis layer contains lysozyme.

[0008] Furthermore, the material of the microneedle array layer includes polyethylene glycol diacrylate (PEGDA) and methacrylated hyaluronic acid (MeHA); the material of the bacteria lysis layer includes pullulan.

[0009] Furthermore, the structure of the magnetic bead-fixed responsive probe for detecting Escherichia coli is shown in SEQ ID NO:3.

[0010] Furthermore, the shapes of the microneedle array layer and the bacteria lysis layer include cones.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned integrated microneedle array device, comprising the following steps: adding a mixture of polyethylene glycol diacrylate, methacrylated hyaluronic acid and a photoinitiator into a PDMS microneedle array mold, allowing the mixture to enter the mold by centrifugation, adding a response probe for detecting Escherichia coli fixed by magnetic beads to the mixture, and then using magnetic attraction to load the response probe for detecting Escherichia coli fixed by magnetic beads in the mixture into the interior of the microneedles; irradiating the PDMS mold containing the mixed solution under ultraviolet light for 10 to 60 seconds and then demolding to prepare a microneedle array for detecting bacteria, and then dripping a pullulan solution containing lysozyme on the surface of the microneedles of the prepared microneedle array, and after natural air drying, a pullulan layer is covered on the surface of the microneedle array to obtain an integrated microneedle array device.

[0012] Furthermore, the mass ratio of polyethylene glycol diacrylate to methacrylated hyaluronic acid is 30:1 to 5:1.

[0013] Furthermore, the photoinitiator is LAP, and the added amount is 0.1wt% to 1wt% of the total mass of polyethylene glycol diacrylate and methacrylated hyaluronic acid.

[0014] Furthermore, the final concentration of the pullulan solution is 5wt% to 10wt%, and the lysozyme content is 100 to 500 μg / mL.

[0015] In a third aspect, the present invention provides an application of the above-mentioned integrated microneedle array device in detecting pathogenic bacteria on a solid surface.

[0016] Furthermore, the application comprises the following steps:

[0017] (1) Adding a buffer solution dropwise onto the solid surface to be tested;

[0018] (2) placing the microneedle array of the integrated microneedle array device on the solid surface of step (1) to carry out a reaction;

[0019] (3) After the reaction is completed, the fluorescence intensity of the microneedle array of the integrated microneedle array device is detected, and the fluorescence intensity is scanned and quantified using Image J software to achieve the detection of pathogenic bacteria on the solid surface.

[0020] Furthermore, the pathogenic bacteria include Escherichia coli.

[0021] Furthermore, the buffer solution in step (1) comprises: 10 mM Tri-HCl, 1 mM EDTA, 1 M NaCl, 0.01%-0.1% Tween-20, pH=7.5.

[0022] Furthermore, the bacterial content in the buffer solution in step (1) is 10 3 ~10 9 CFU / mL.

[0023] Furthermore, the reaction time in step (2) is 30 to 90 minutes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention designs a simple and portable microneedle array device that integrates bacterial lysis, active sampling and bacterial detection, which can detect pathogenic bacteria on solid surfaces. Compared with the traditional method of detecting pathogenic bacteria on solid surfaces, the integrated microneedle array device described in the present invention is simple to operate, does not require time-consuming operation by professionals, and only requires a few microliters of sample to complete the detection, which has potential application value in the detection of pathogenic bacteria on solid surfaces. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart for preparing the integrated microneedle array device in Example 1.

[0027] Figure 2 This is a schematic diagram of the detection principle of the integrated microneedle array device in Example 2.

[0028] Figure 3 This is a feasibility analysis diagram of the integrated microneedle array in Example 3 for detecting pathogenic bacteria on solid surfaces.

[0029] Figure 4 This is a kinetic analysis diagram of the integrated microneedle array detecting pathogenic bacteria on a solid surface in Example 4.

[0030] Figure 5 This is an analysis diagram of testing pathogenic bacteria on different solid surfaces using the integrated microneedle array device of the present invention in Example 5. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the specific implementation methods of the present invention are further described in detail below in conjunction with embodiments.

[0032] Table 1: Nucleic acid sequences used in the present invention

[0033]

[0034]

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] The reaction buffer used in the following examples: 10 mM Tri-HCl, 1 mM EDTA, 1 M NaCl, 0.01%-0.1% Tween-20, pH=7.5.

[0038] Example 1 Preparation of integrated microneedle array device

[0039] Polyethylene glycol diacrylate (PEGDA) and methacrylated hyaluronic acid (MeHA) were mixed together in a mass ratio of 13:1. After being fully mixed, 0.5wt% photoinitiator (LAP, dissolved in 1mL water) of the total mass of polyethylene glycol diacrylate and methacrylated hyaluronic acid was added, and a mixed solution for preparing microneedles was obtained after being fully mixed. 600μL of the mixed solution for preparing microneedles was added to the PDMS microneedle array mold, and centrifuged for 3min in each direction of four different directions at a speed of 3500r / min to ensure the needle rate. The response probe (SEQ ID NO:3) for detecting Escherichia coli fixed by magnetic beads was added to the mixed solution of the mold, and then the response probe (SEQ ID NO:3) for detecting Escherichia coli fixed by magnetic beads in the mixed solution was loaded into the microneedle interior by magnetic attraction. The residual mixture on the surface of the mold was sucked off, and the mold was demoulded after irradiation under ultraviolet light for 30s to make a detection layer microneedle array. We dripped 8 wt% pullulan solution containing lysozyme onto the microneedles of the prepared detection layer microneedle array. After natural air drying, a thin layer of pullulan was covered on the surface of the microneedle array. The integrated microneedle array was prepared. The preparation process of the integrated microneedle array device is as follows: Figure 1 shown.

[0040] Example 2 Detection principle of integrated microneedle array device

[0041] When the integrated microneedle array device meets water, the bacteria lyse the pullulan sugar layer of the microneedle array, dissolving and releasing lysozyme, which contacts the pathogenic bacteria on the solid surface to achieve the lysis of the pathogenic bacteria. Then the microneedle array of the bacterial detection layer absorbs water and swells to actively sample. After the internal DNAzyme recognizes the target substance, it cuts the RNA base A in SEQ ID NO:2 with a fluorescent group and converts the chemical signal into a fluorescent signal. The fluorescent signal is analyzed using an inverted fluorescence microscope. The principle of the integrated microneedle array device is as follows: Figure 2 shown.

[0042] Example 3 Feasibility analysis of integrated microneedle array detection of pathogenic bacteria on solid surfaces

[0043] (1) Take 10 μL of E. coli culture stored at -80℃ and inoculate it into 5 mL of LB (Luria-Bertani) liquid medium. Incubate it in a shaker at 37℃ overnight. Take 100 μL of the culture solution and inoculate it into 100 mL of LB liquid medium. Incubate it in a shaker at 37℃. When the absorbance value OD 600 When it reaches 1, take 1 mL of the bacterial solution and centrifuge it at 4°C for 10 min. The relative centrifugal force of the centrifuge is set to 11000g. Remove the supernatant to obtain Escherichia coli.

[0044] (2) Add 200 μL of reaction buffer to the E. coli obtained by centrifugation and resuspend the resultant to prepare an E. coli suspension.

[0045] (3) 100 μL of E. coli suspension and 100 μL of reaction buffer were added to the solid surface respectively, and the integrated microneedle array device prepared in Example 1 was placed therein respectively. After reacting for 1 h, the reaction was terminated, and an inverted fluorescence microscope was used to take a picture to detect the fluorescence intensity. The fluorescence intensity of the reaction area was scanned and quantified using Image J software. The feasibility analysis results are shown in FIG. Figure 3 shown.

[0046] Example 4: Dynamic analysis of pathogenic bacteria detected on solid surfaces using integrated microneedle arrays

[0047] 100 μL of E. coli suspension was added dropwise to the solid surface, and the integrated microneedle array device prepared in Example 1 was placed therein. The reaction was performed for 0, 3.75, 7.5, 15, 30, 60 and 120 minutes, respectively. The device was photographed using an inverted fluorescence microscope to detect the fluorescence intensity. The fluorescence intensity of the reaction area was scanned and quantified using Image J software to obtain kinetic data, as shown in FIG. Figure 4 shown.

[0048] Example 5: Analysis of pathogenic bacteria on different solid surfaces tested using integrated microneedle array devices

[0049] In order to study the applicability of the integrated microneedle array in actual samples, we took 100 μL of 10 7 , 10 6 and 10 5 The surface of fruits and vegetables was "contaminated" with a suspension of CFU / mL Escherichia coli, and then the contaminated parts were detected using the integrated microneedle array prepared in Example 1. An inverted fluorescence microscope was used to take photos to detect the fluorescence intensity, and the fluorescence intensity of the reaction area was scanned and quantified using Image J software. The results are shown in FIG. Figure 5 As shown. It can be seen that the functionalized microneedle array constructed by the present invention can accurately identify Escherichia coli on different solid surfaces, and the analysis results are not affected by the complex matrix of the solid surface.

Claims

1. An integrated microneedle array device, characterized in that: The integrated microneedle array device includes a microneedle array layer for detecting bacteria and a bacterial lysis layer. The bacterial lysis layer is a polysaccharide layer covering the outside of the microneedle array layer. The microneedle array layer contains a response probe for detecting Escherichia coli fixed with magnetic beads, and the bacterial lysis layer contains lysozyme.

2. The integrated microneedle array device according to claim 1, characterized in that: The material of the microneedle array layer includes polyethylene glycol diacrylate (PEGDA) and methacrylated hyaluronic acid (MeHA); the material of the bacteria lysis layer includes pullulan; the shapes of the microneedle array layer and the bacteria lysis layer include cones.

3. The integrated microneedle array device according to claim 1, characterized in that: The structure of the magnetic bead-fixed responsive probe for detecting Escherichia coli is shown in SEQ ID NO:

3.

4. The method for preparing the integrated microneedle array device according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: adding a mixed solution of polyethylene glycol diacrylate, methacrylated hyaluronic acid and a photoinitiator into a PDMS microneedle array mold, allowing the mixed solution to enter the mold by centrifugation, adding a response probe for detecting Escherichia coli fixed by magnetic beads into the mixed solution, and then using magnetic attraction to load the response probe for detecting Escherichia coli fixed by magnetic beads in the mixed solution into the interior of the microneedles; irradiating the PDMS mold carrying the mixed solution under an ultraviolet lamp for 10 to 60 seconds and then demoulding to prepare a microneedle array for detecting bacteria, and then dripping a pullulan solution containing lysozyme on the surface of the microneedles of the prepared microneedle array, and after natural air drying, a pullulan layer is covered on the surface of the microneedle array to obtain an integrated microneedle array device.

5. The preparation method according to claim 4, characterized in that: The mass ratio of polyethylene glycol diacrylate to methacrylated hyaluronic acid is 30:1-5:1; the photoinitiator is LAP, and the added amount is 0.1wt%-1wt% of the total mass of polyethylene glycol diacrylate and methacrylated hyaluronic acid.

6. The preparation method according to claim 4, characterized in that: The final concentration of the pullulan solution is 5wt%-10wt%, and the lysozyme content is 100-500μg / mL.

7. Use of the integrated microneedle array device according to any one of claims 1 to 3, characterized in that: Application in detecting pathogenic bacteria on solid surfaces.

8. The use according to claim 7, characterized in that: The application comprises the following steps: (1) Adding a buffer solution dropwise onto the solid surface to be tested; (2) placing the microneedle array of the integrated microneedle array device on the solid surface of step (1) to carry out a reaction; (3) After the reaction is completed, the fluorescence intensity of the microneedle array of the integrated microneedle array device is detected, and the fluorescence intensity is scanned and quantified using Image J software to achieve the detection of pathogenic bacteria on the solid surface.

9. The use according to claim 7, characterized in that: The pathogenic bacteria include Escherichia coli.

10. The use according to claim 8, characterized in that: The bacterial content in the buffer described in step (1) is 10 3 ~10 9 CFU / mL; the reaction time in step (2) is 30 to 90 min.

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