Bacteria detection method based on urchin-shaped magnetic microbeads and application thereof

Abstract

The invention provides a bacteria detection method based on sea-urchin-shaped magnetic microbeads and application, the sea-urchin-shaped magnetic microbeads enrich bacteria dispersed in a sample through electrostatic adsorption and magnetic separation, and cell walls and cell membranes of the bacteria are destroyed by spines on the surfaces of the sea-urchin-shaped magnetic microbeads, so that fluorescent dye enters a bacterial cell structure, and the bacteria are detected. The fluorescent probe can be applied to detection of bacteria. According to the bacterium detection method based on the sea urchin-shaped magnetic microbeads and the application thereof, centrifugation or marking is not needed in the whole process, and the method is simple, rapid, environmentally friendly and suitable for field application detection of rapid bacterium detection.

Description

Technical Field

[0001] This invention relates to a bacterial detection method based on sea urchin-shaped magnetic microbeads and its application, and is applied in the field of bacterial detection technology. Background Technology

[0002] Bacterial infections are currently a major cause of global health losses. Timely detection of pathogens and interruption of their transmission routes are crucial for reducing the risk of bacterial infections and protecting public health. Developing rapid and economical on-site bacterial detection technologies can not only help regulatory authorities improve detection coverage, shorten response times, and enhance regulatory efficiency, but also provide strong support for home food safety testing, further ensuring microbial safety for individuals.

[0003] There are various methods for bacterial detection, among which culture is the gold standard, offering high specificity and applicability to a wide range of samples. However, its detection cycle is long (3-7 days), requiring strict aseptic operation and culture condition control by professional technicians, making it difficult to efficiently process large batches of samples. To achieve more efficient detection, many (semi-)automated detection devices, such as flow cytometers, gas chromatographs, microplate readers, Fourier transform near-infrared spectroscopy, and Raman spectroscopy, have been applied to bacterial analysis. Although these technologies can perform high-throughput, rapid, and multi-parameter accurate identification of bacteria, their reliance on large-scale equipment makes them unsuitable for on-site testing.

[0004] Fluorescence detection technology is widely used for rapid on-site detection due to its sensitivity and simplicity. To further improve detection sensitivity, many bacterial detection techniques typically detect high concentrations of bacterial metabolites, such as ATP and cysteine ​​(Cys). However, these small molecule metabolites are widely distributed in nature, leading to insufficient specificity in these detection methods. Gene amplification-based bacterial detection methods offer high sensitivity and specificity, but they usually require extraction of the bacterial genome and strict temperature control during amplification to achieve high amplification efficiency. This operational complexity limits the on-site application of bacterial detection.

[0005] In summary, existing technologies still have limitations in terms of material stability, ease of operation, and testing efficiency, making it difficult to meet the comprehensive needs of rapid on-site testing for high efficiency, convenience, and accuracy.

[0006] Therefore, it is necessary to design a new bacterial detection method based on sea urchin-shaped magnetic microbeads and its application to overcome the above problems. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a bacterial detection method based on sea urchin-shaped magnetic microbeads and its application. The entire process requires no centrifugation or labeling, is simple and fast, environmentally friendly, and suitable for on-site rapid bacterial detection applications.

[0008] This invention is implemented as follows: This invention provides a bacterial detection application based on sea urchin-shaped magnetic microbeads. The sea urchin-shaped magnetic microbeads enrich dispersed bacteria in the sample through electrostatic adsorption and magnetic separation. The spikes on the surface of the sea urchin-shaped magnetic microbeads disrupt the cell walls and cell membranes of the bacteria, allowing fluorescent dyes to enter the bacterial cell structure and emit fluorescent signals, which can then be used for bacterial detection.

[0009] Furthermore, the sea urchin-shaped magnetic microspheres are sea urchin-shaped Fe3O4@ZnO microspheres.

[0010] The present invention also provides a bacterial detection method based on sea urchin-shaped magnetic microbeads, comprising the following steps: Step 1: Add sea urchin-shaped magnetic microbeads to the bacterial sample and mix them evenly; Step 2: Use a magnetic rack to enrich the mixture obtained in Step 1, remove the supernatant, and wash the lower layer multiple times to obtain the bacterial enrichment product. Step 3: Add propidium iodide (PI) dye to the above bacterial enrichment product, mix thoroughly, and then add the mixture to an ELISA plate; Step 4: Place the ELISA plate into the ELISA reader to detect the fluorescence signal, and you can obtain the quantitative detection results.

[0011] Furthermore, in step two, the lower layer material is washed multiple times using a phosphate buffer solution.

[0012] Furthermore, in step three, the enzyme-labeled plate is a 96-well enzyme-labeled plate.

[0013] Furthermore, in step four, the excitation wavelength is 535 nm.

[0014] The present invention has the following beneficial effects: The bacterial detection method based on sea urchin-shaped magnetic microbeads provided by this invention efficiently enriches dispersed bacteria in samples through electrostatic adsorption and magnetic separation. The spikes on the surface of the sea urchin-shaped magnetic microbeads disrupt the bacterial cell walls / membranes, allowing fluorescent dyes to rapidly enter the bacterial cell structure and emit fluorescent signals. Utilizing the physical disruption of bacterial structures by the sea urchin-shaped magnetic microbeads and the inherent bactericidal properties of zinc oxide, this method can efficiently kill bacteria and prevent secondary contamination of the sample. The method requires no centrifugation or labeling throughout the process, is simple, rapid, and environmentally friendly, and is suitable for on-site applications of rapid bacterial detection. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 A schematic diagram illustrating the principle of the bacterial detection method based on sea urchin-shaped magnetic microbeads provided in this embodiment of the invention; Figure 2 This is a schematic diagram of fluorescence spectroscopy detection results provided in an embodiment of the present invention; Figure 3 This is a schematic diagram of the confocal microscopy imaging results provided in an embodiment of the present invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] like Figure 1 This invention provides a bacterial detection method and application based on sea urchin-shaped magnetic microbeads. The sea urchin-shaped magnetic microbeads are sea urchin-shaped Fe3O4@ZnO microbeads, abbreviated as SMMB. Their sea urchin-like morphology gives them a high surface area, and the abundant positive charge on their surface allows them to efficiently adsorb bacteria through electrostatic interactions. The sea urchin-shaped magnetic microbeads have a sea urchin-like spike structure, which can disrupt the integrity of the bacterial cell wall / cell membrane structure; this allows nucleic acid dyes to enter the cell through the disrupted bacterial membrane, bind to its nucleic acid, and then emit a fluorescent signal.

[0019] Propidium iodide (PI) dye is a fluorescent dye that selectively binds to the double helix structure of DNA. Its fluorescence in the free state is very weak, but it significantly increases after embedding into the DNA double helix. However, it cannot penetrate the bacterial cell membrane to stain the DNA of living cells and is typically only used for fluorescent staining dead bacteria. After sea urchin-shaped microbeads disrupt the bacterial cell membrane, PI dye can diffuse into the cell and bind to the DNA, emitting fluorescence and producing a detectable signal.

[0020] The present invention also provides a bacterial detection method based on sea urchin-shaped magnetic microbeads, comprising the following steps: Step 1: Add sea urchin-shaped magnetic microbeads to the bacterial sample and mix them evenly; Step 2: Enrich the mixture obtained in Step 1 using a magnetic rack, remove the supernatant, and wash the lower layer multiple times with phosphate-buffered saline (PBS) solution to obtain the bacterial enrichment product; Step 3: Add propidium iodide (PI) dye to the above bacterial enrichment product, mix evenly, and then add an ELISA plate; the ELISA plate is a 96-well ELISA plate.

[0021] Step 4: Place the ELISA plate into the ELISA reader to detect the fluorescence signal. The excitation wavelength is 535 nm; the quantitative detection result can then be obtained.

[0022] The above method is verified through several embodiments below: Fluorescence intensity verification: Equal amounts of bacteria were aliquoted into three centrifuge tubes, and equal volumes of 1×PBS buffer, conventional Fe3O4 magnetic beads (MMB), and sea urchin-shaped magnetic microbeads (SMMB) were added to each tube. After thorough mixing, equal volumes of PI dye were added. The results of the ELISA reader detection are as follows: Figure 2 As shown, MMB and SMMB themselves do not have fluorescent properties, while the bacterial group (E. coli) exhibits some autofluorescence. MMB treatment led to a certain increase in fluorescence signal, while SMMB treatment led to a significantly stronger fluorescence signal enhancement. This indicates that the fluorescence signal in the SMMB-treated group mainly comes from the effect of the urchin-like zinc oxide shell of SMMB, proving that this method can achieve efficient fluorescence detection while enriching bacteria.

[0023] Confocal imaging verification: The above-treated sample was observed under a confocal microscope, and the results are as follows. Figure 3 As shown, because the negative charge on the surface of MMB repels the negative charge on the surface of bacteria, it cannot adsorb bacteria or destroy the bacterial cell membrane structure. Therefore, the MMB-treated group did not show bacterial enrichment ability or PI staining fluorescence signal. In contrast, the SMMB-treated group showed obvious bacterial enrichment, and the enriched bacteria could be stained by PI and emit obvious fluorescence signals, which verified the disruptive effect of the spike structure on the cell membrane and the effectiveness of fluorescence detection.

[0024] In summary, the bacterial detection method based on sea urchin-shaped magnetic microbeads provided by this invention efficiently enriches dispersed bacteria in samples through electrostatic adsorption and magnetic separation. The spikes on the surface of the sea urchin-shaped magnetic microbeads disrupt the bacterial cell walls / membranes, allowing fluorescent dyes to rapidly enter the bacterial cell structure and emit fluorescent signals. Utilizing the physical disruption of bacterial structures by the sea urchin-shaped magnetic microbeads and the inherent bactericidal properties of zinc oxide, this method can efficiently kill bacteria and prevent secondary contamination of the sample. The method requires no centrifugation or labeling throughout the process, is simple, rapid, and environmentally friendly, making it suitable for rapid on-site bacterial detection applications.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A sea urchin-like magnetic microbead based bacterial detection application, characterized by: The urchin-like magnetic microbeads enrich the dispersed bacteria in the sample through electrostatic adsorption and magnetic separation, break the cell wall and cell membrane of the bacteria by the spikes on the surface of the urchin-like magnetic microbeads, make the fluorescent dye enter the bacterial cell structure, and emit a fluorescent signal, so that the urchin-like magnetic microbeads can be applied to the detection of bacteria.

2. The sea urchin-like magnetic microbead-based bacterial detection method according to claim 1, wherein: The urchin-like magnetic microbeads are urchin-like Fe3O4@ZnO microbeads.

3. A method for detecting bacteria based on sea urchin-like magnetic microbeads, characterized by, The method comprises the following steps: step one, adding urchin-like magnetic microbeads to a bacterial sample and uniformly mixing them; Step two, enriching the mixture obtained in step one using a magnetic stand, removing the supernatant, and repeatedly washing the lower layer to obtain a bacterial enrichment product; Step three, adding propidium iodide dye to the bacterial enrichment product, uniformly mixing them, and then adding an enzyme-labeled plate; Step four, placing the enzyme-labeled plate into an enzyme-labeled instrument to detect the fluorescent signal, so that quantitative detection results can be obtained.

4. The sea urchin-like magnetic microbead-based bacterial detection method according to claim 3, wherein the magnetic microbead is a magnetic microbead having a diameter of 0.1 to 10 μm. In step two, the lower layer is repeatedly washed using a phosphate buffered saline solution.

5. The sea urchin-like magnetic microbead-based bacterial detection method according to claim 3, wherein the magnetic microbead is a magnetic microbead having a diameter of 0.1 to 10 μm. In step three, the enzyme-labeled plate is a 96-well enzyme-labeled plate.

6. The sea urchin-like magnetic microbead-based bacterial detection method according to claim 3, wherein the magnetic microbead is a magnetic microbead having a diameter of 0.1 to 10 μm. In step four, the excitation wavelength is 535 nm.

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