A method for detecting bacteria using solid-state pore nanotechnology

Abstract

The present invention discloses a method for detecting bacteria using solid-state pore nanotechnology, and relates to the field of bacterial detection of pathogenic microorganisms in food detection and control. First, a bacterial sample in the form of a black precipitate is obtained by bacterial enrichment; then, the silanized silicon nitride solid-state pore is modified, and finally, the bacterial detection is performed, that is, a detection buffer is added to the above-mentioned silanized silicon nitride solid-state pore, and the enriched bacterial sample is added, and the bacterial through-hole signal is detected at a voltage of +150mV. The present invention proposes a streptavidin Fe3O4 magnetic bead method for enriching bacteria, and a method for modifying silicon nitride solid-state pores using silanization, using modified magnetic beads to quickly enrich samples in a short time, and using the designed and modified solid-state nanopores to achieve rapid detection of bacteria, saving detection time and reducing manual operations.

Description

Technical Field

[0001] The present invention belongs to the field of bacteria detection of pathogenic microorganisms in food detection and control, and specifically relates to a method for detecting bacteria using solid-state pore nanotechnology. Background Art

[0002] Food safety has always been a topic of widespread concern. Pathogenic bacteria contamination in food can have extremely serious consequences. Therefore, safety monitoring of pathogenic bacteria contamination in food is very important. However, since samples for food safety monitoring have complex background matrices and low bacterial content, efficient identification and detection of bacteria are extremely difficult.

[0003] Studies have shown that the surface of magnetic beads is easy to modify and can specifically adsorb to the sample to be tested, removing inhibitory substances in the sample solution, such as organic solvents, detergents, metal ions and fuels. Biomagnetic beads can be made superparamagnetic through a special synthesis process, so they can be automated through instruments to meet the needs of database construction for large-scale sample extraction. In addition, the synthesis of nano-biomagnetic beads uses low-cost inorganic and organic raw materials and is widely used.

[0004] Patent "CN104459125A" discloses a method for rapid detection of Gram-negative and Gram-positive bacteria. The method couples nanomagnetic beads with nucleic acid aptamers to obtain positive and invisible bacteria oxygen-enriched magnetic beads, respectively, which are mixed with the test samples, incubated and washed, and then fluorescently labeled secondary antibodies or enzyme-labeled secondary antibodies are added to incubate and wash to determine the results.

[0005] However, although the above-mentioned traditional methods such as antibody modification or aptamer modification have the specificity of separation testing, when the bacterial species in the sample is unclear or the total bacteria in the sample need to be enriched, the above-mentioned traditional methods are prone to missed detection, and the excess magnetic beads are also likely to interfere with subsequent detection. At the same time, these methods also have limitations such as high cost of antibodies or aptamers, frequent and complicated operations, and low stability of the test system.

[0006] In fact, in the field of disease detection, biological nanopore technology is widely used in the detection of molecules such as nucleic acids and proteins. As a single-molecule detection technology, nanopore has developed rapidly in the fields of gene sequencing and biomedical testing in recent years due to its advantages of label-free, high-throughput, PCR-free amplification and simple operation. Based on this, the present invention utilizes the advantages of solid-state nanopores in detecting pathogens through holes and proposes a method for rapid detection of bacteria after enriching pathogens. Summary of the invention

[0007] In view of the fact that traditional separation detection is prone to missed detection and excess magnetic beads are likely to interfere with subsequent detection, these methods also have defects and problems such as high cost of antibodies or aptamers, frequent and complicated operations, long time consumption and low stability of the test system, the present invention provides a method for detecting bacteria using solid-state pore nanotechnology.

[0008] The solution adopted by the present invention to solve the technical problem is: a method for detecting bacteria using solid-state pore nanotechnology, comprising the following steps:

[0009] S1. Bacterial enrichment: First, 5-200 μg / mL of the sample to be tested is mixed with magnetic beads to obtain a suspension; the suspension is incubated at an oscillation speed of 110-180 rpm and a temperature of 27-39° C. for 1-2 hours; then magnetic separation is performed for 5-10 minutes; the magnetic beads after magnetic separation are slowly resuspended in a corresponding buffer to obtain a magnetic bead resuspension;

[0010] The magnetic bead resuspension and the gel solution are mixed in a centrifuge tube and subjected to magnetic separation for 10-15 minutes; the black precipitate after separation is the enriched bacterial sample;

[0011] S2. Modification of solid-state pores in silanized silicon nitride:

[0012] First, clean the silicon nitride nanopores with anhydrous ethanol and isopropanol for 3 to 5 minutes; then clean with Milli-Q water for 3 to 5 minutes; then blow dry with dry nitrogen; then clean with H2 / Ar mixed gas for 3 to 8 minutes to obtain hydroxylated silicon nitride nanopores; apply silane solution to the hydroxylated silicon nitride solid pores and treat with nitrogen for 30-50 minutes; finally, clean with isopropanol for 3 to 5 minutes and blow dry with nitrogen to obtain silanized silicon nitride nanopores;

[0013] S3. Bacteria detection: Add 1-3 mL of detection buffer into the above-mentioned silanized silicon nitride solid-state wells, and add 5-15 μL of enriched bacteria sample, and detect bacteria crossing the well signal at a voltage of +150 mV.

[0014] Furthermore, the magnetic beads in S1 use Fe3O4 magnetic beads modified with hydroxyl, carboxyl or streptavidin, and the particle size is 100-300 nm.

[0015] Furthermore, if carboxyl-modified magnetic beads are used, a phosphate buffer solution with a pH of 3.0 to 6.5 is selected; if streptavidin-modified magnetic beads are used, a phosphate buffer solution with a pH of 4.5 to 7.0 is selected; and if hydroxyl-modified magnetic beads are used, a Tirs buffer solution with a pH of 7.5 to 9.0 is selected.

[0016] Furthermore, the gel solution in S1 is prepared by mixing polyethylene glycol 6000, gelatin and double distilled water in a mass ratio of 0.1:1.2:1.1.

[0017] Furthermore, the magnetic bead resuspension and the gel solution are mixed in a centrifuge tube at a volume ratio of 0.5:1.0.

[0018] Furthermore, the detection buffer in S3 is a mixture of 0.6M KCl, 15mM HEPES, 2mM EDTA and 5mM ATP.

[0019] Further, 100 μg / mL of the sample to be tested was mixed with streptavidin-modified Fe3O4 magnetic beads with a particle size of 200 nm, and the magnetic beads were resuspended with 100 μL of PBS (phosphate buffer) with a pH of 6.0 to obtain a magnetic bead resuspension.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a method for detecting bacteria using solid-state pore nanotechnology. After the bacteria are enriched by a streptavidin Fe3O4 magnetic bead method, the bacterial concentration is improved; and the present invention can efficiently enrich the target bacteria using streptavidin-modified Fe3O4 magnetic beads. This method can be used to separate and enrich bacteria and reduce interference from other impurities.

[0022] The present invention designs a method for modifying silicon nitride solid-state pores using silanization. The silicon nitride solid-state pores obtained by the modification method can achieve silicon nitride nanopores with specific affinity, which helps to improve the sensitivity and selectivity of bacterial detection; the nanopores modified by silanization change the active groups on the surface to affect the conductivity, thereby improving the sensitivity for detecting specific molecules and increasing the speed at which molecules pass through the pores, thereby achieving rapid detection of bacteria.

[0023] The method of the present invention combines magnetic bead enrichment, solid-state pore modification and bacterial detection technology, and can perform large-scale detection of bacteria rapidly, accurately and at low cost. It has the characteristics of high efficiency, sensitivity and specificity, and the concentration of bacteria after enrichment is improved, so that pathogenic microorganisms in food can be accurately and rapidly detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of bacteria passing through the modified solid-state nanopore.

[0025] Figure 2 Schematic diagram of bacteria perforation signal in Example 1. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0027] See also Figure 1 and Figure 2 The present invention provides a technical solution for a method of detecting bacteria using solid-state nanopore technology. The present invention uses the advantage of solid-state nanopores in detecting bacteria through holes to achieve rapid detection of bacteria after enrichment. That is, when the analyte to be tested passes through the nanoscale pores, it will cause changes in the ion current passing through the pores. The physicochemical properties of the analyte can be characterized by detecting and analyzing the current change signal. Example

[0028] This embodiment provides a method for detecting bacteria using solid-state pore nanotechnology, which specifically includes the following steps:

[0029] 1. Enrichment of bacteria using magnetic beads:

[0030] (1) Mix 100 μg / mL of the sample to be tested and streptavidin-modified Fe3O4 magnetic beads with a particle size of 200 nm, shake and mix to obtain a suspension; incubate on a shaker under the following conditions: shaking speed of 120 rpm, temperature of 30°C, and time of 2 h; then perform magnetic separation on a magnetic stand for 5 minutes; remove the supernatant, and resuspend the magnetic beads with 100 μL of PBS (phosphate buffer) with a pH of 6.0 to obtain a magnetic bead resuspension;

[0031] (2) placing the prepared gel solution in a 1.5 ml centrifuge tube, wherein the gel solution is made of polyethylene glycol 6000, gelatin and double distilled water in a mass ratio of 0.1:1.2:1.1, wherein the mass fraction of polyethylene glycol in this embodiment is 20%, the amount added is 500 μL, and the remaining components are mixed and added in proportion; and adding 100 μL of the above magnetic bead suspension to the centrifuge tube, followed by magnetic separation on a magnetic stand; the separation time is 10 minutes, and the black precipitate on the magnetic stand after separation is the enriched bacterial sample;

[0032] 2. Modification of solid-state pores: using silanization-modified silicon nitride solid-state pores. The modification process includes:

[0033] (1) First, clean the silicon nitride nanopores (pore size 200 nm) with anhydrous ethanol and isopropanol for 3 min, then clean them with Milli-Q water for 3 min, and finally blow dry with dry nitrogen;

[0034] (2) placing the cleaned solid nanopore in a plasma cleaning machine and cleaning it with a H2 / Ar mixed gas for 5 minutes to obtain a hydroxylated silicon nitride nanopore;

[0035] (3) Applying the silane solution to the hydroxylated silicon nitride nanopores and treating with nitrogen for 30 minutes to complete the alkylation modification of the silicon nitride nanopores;

[0036] (4) Finally, the silanized silicon nitride nanopores are obtained by washing with isopropanol and drying with nitrogen.

[0037] 3. Bacteria detection: First, prepare the detection buffer, which is a mixture of the following components: 0.6M KCl, 15mM HEPES, 2mM EDTA and 5mM ATP.

[0038] Then, 1 mL of detection buffer was added to the above-mentioned silanized silicon nitride solid-state wells, and 10 μL of the enriched bacterial sample was added, and the bacterial through-hole signal was detected at a voltage of +150 mV. The schematic diagram of the bacterial through-hole signal detected by the method of this embodiment is shown in FIG. Figure 1 shown.

[0039] Specifically, the enriched bacterial solution to be tested is added to the positive electrode chamber of the electrolytic cell in the modified solid-state nanopore sequencing device; a voltage of 150 mV is applied between the positive and negative electrodes of the electrolytic cell to allow the bacteria to be tested to pass through the solid-state nanopore channel on the solid-state nanopore film, see Figure 2 ; The solid-state nanopore film is arranged between the positive electrode and the negative electrode of the electrolytic cell to separate the electrolytic cell into a positive electrode chamber and a negative electrode chamber, and captures the current change signal caused by the nucleic acid to be tested passing through the solid-state nanopore channel; the captured ion current change signal is analyzed to determine the bacteria to be tested.

[0040] The method for detecting bacteria using solid-state pore nanotechnology provided by the present invention combines magnetic bead enrichment, solid-state pore modification and bacteria detection technology, can rapidly, accurately and low-costly perform large-scale detection of bacteria, has the characteristics of high efficiency, sensitivity and specificity, and the concentration of bacteria after enrichment is improved, so that rapid detection of pathogenic microorganisms in food can be accurately performed. Example

[0041] This embodiment provides a method for detecting bacteria using solid-state pore nanotechnology, which specifically includes the following steps:

[0042] 1. Enrichment of bacteria using magnetic beads:

[0043] (1) Mix 200 μg / mL of the sample to be tested and streptavidin-modified Fe3O4 magnetic beads with a particle size of 100 nm, shake and mix to obtain a suspension; incubate on a shaker, and the incubation conditions are: shaking speed of 150 rpm, temperature of 33°C, and time of 1.5 h; then perform magnetic separation on a magnetic stand for 5 minutes; remove the supernatant, and resuspend the magnetic beads with 150 μL of PBS (phosphate buffer) with a pH of 7.0 to obtain a magnetic bead resuspension;

[0044] (2) placing the prepared gel solution in a 1.5 ml centrifuge tube, wherein the gel solution is made of polyethylene glycol 6000, gelatin and double distilled water in a mass ratio of 0.1:1.2:1.1; in this embodiment, the mass fraction of polyethylene glycol is 20%, the amount added is 300 μL, and the remaining components are mixed and added in proportion; and 150 μL of the above-mentioned magnetic bead suspension is added to the centrifuge tube, and then magnetic separation is performed on a magnetic rack; the separation time is 10 minutes, and the black precipitate on the magnetic rack after separation is the enriched bacterial sample;

[0045] 2. Modification of solid-state pores: using silanization-modified silicon nitride solid-state pores. The modification process includes:

[0046] (1) First, clean the silicon nitride nanopores (pore size 200 nm) with anhydrous ethanol and isopropanol for 5 min, then clean them with Milli-Q water for 5 min, and finally blow dry with dry nitrogen;

[0047] (2) placing the cleaned solid nanopore in a plasma cleaning machine and cleaning it with a H2 / Ar mixed gas for 6 minutes to obtain a hydroxylated silicon nitride nanopore;

[0048] (3) Applying the silane solution to the hydroxylated silicon nitride nanopores and treating with nitrogen for 50 minutes to complete the alkylation modification of the silicon nitride nanopores;

[0049] (4) Finally, the silanized silicon nitride nanopores are obtained by washing with isopropanol and drying with nitrogen.

[0050] 3. Bacteria detection: Add 1.5 mL of detection buffer into the above-mentioned silanized silicon nitride solid-state wells, and add 15 μL of enriched bacteria sample, and detect the bacteria through-hole signal at a voltage of +150 mV. Example

[0051] This embodiment provides a method for detecting bacteria using solid-state pore nanotechnology, which specifically includes the following steps:

[0052] 1. Enrichment of bacteria using magnetic beads:

[0053] (1) Mix 100 μg / mL of the sample to be tested and carboxyl-modified Fe3O4 magnetic beads with a particle size of 200 nm, shake and mix to obtain a suspension; incubate on a shaker under the following conditions: shaking speed of 120 rpm, temperature of 30°C, and time of 2 h; then perform magnetic separation on a magnetic stand for 5 minutes; remove the supernatant, and resuspend the magnetic beads with 100 μL of PBS (phosphate buffer) with a pH of 6.0 to obtain a magnetic bead resuspension;

[0054] (2) Place the prepared gel solution in a 1.5 ml centrifuge tube and add 100 μL of the above magnetic bead suspension, followed by magnetic separation on a magnetic rack; the separation time is 10 minutes. The black precipitate on the magnetic rack after separation is the enriched bacterial sample.

[0055] 2. Modification of solid-state pores, using silanization-modified silicon nitride solid-state pores, the specific steps are the same as those in the above-mentioned embodiment 1.

[0056] 3. Bacteria detection: Add 1 mL of detection buffer into the above-mentioned silanized silicon nitride solid-state wells, and add 10 μL of enriched bacteria sample, and detect the bacteria through-hole signal at a voltage of +150 mV. Example

[0057] This embodiment provides a method for detecting bacteria using solid-state pore nanotechnology, wherein when enriching bacteria, 100 μg / mL of the sample to be detected is mixed with Fe3O4 magnetic beads modified with hydroxyl groups, with a particle size of 200 nm, and the magnetic beads are resuspended with 100 μL of Tirs buffer with a pH of 8.0 to obtain a magnetic bead resuspension, and other conditions are the same as in Example 1.

[0058] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting bacteria using solid-state pore nanotechnology, Features: The following steps are involved: S1. Bacterial enrichment: First, 5-200 μg / mL of the sample to be tested is mixed with magnetic beads to obtain a suspension; the suspension is incubated at an oscillation speed of 110-180 rpm and a temperature of 27-39° C. for 1-2 hours; then magnetic separation is performed for 5-10 minutes; the magnetic beads after magnetic separation are slowly resuspended in a corresponding buffer to obtain a magnetic bead resuspension; The magnetic bead resuspension and the gel solution are mixed in a centrifuge tube and subjected to magnetic separation for 10 to 15 minutes; the black precipitate after separation is the enriched bacterial sample; The magnetic beads are modified with hydroxyl, carboxyl or streptavidin 3 O 4 The magnetic beads have a particle size of 100 to 200 nm. The corresponding buffer for the carboxyl-modified magnetic beads is a phosphate buffer with a pH of 6.0; the corresponding buffer for the streptavidin-modified magnetic beads is a phosphate buffer with a pH of 6.0 to 7.0; the corresponding buffer for the hydroxyl-modified magnetic beads is a Tirs buffer with a pH of 8.0; The gel solution is prepared by mixing polyethylene glycol 6000, gelatin and double distilled water in a mass ratio of 0.1:1.2:1.1; S2. Modification of solid-state pores in silanized silicon nitride: First, clean the silicon nitride nanopores with anhydrous ethanol and isopropanol for 3 to 5 minutes; then clean them with Milli-Q water for 3 to 5 minutes; then dry them with dry nitrogen; and then rinse them with H 2 / Ar mixed gas cleaning for 3 to 8 minutes to obtain hydroxylated silicon nitride nanopores; applying silane solution to the hydroxylated silicon nitride solid pores and treating with nitrogen for 30-50 minutes; finally, cleaning with isopropanol for 3 to 5 minutes and then drying with nitrogen to obtain silanized silicon nitride nanopores; S3, bacterial detection: 1-3 mL of detection buffer is added to the silanized silicon nitride nanopore, and 5-15 μL of enriched bacterial sample is added, and a voltage of 150 mV is applied to allow the bacteria to be tested to pass through the solid nanopore channel on the silanized silicon nitride nanopore film. The silanized silicon nitride nanopore film is arranged between the positive and negative electrodes of the electrolytic cell to separate the electrolytic cell into a positive electrode chamber and a negative electrode chamber, and the current change signal caused by the bacteria to be tested passing through the solid nanopore channel on the silanized silicon nitride nanopore film is captured, thereby determining the bacteria to be tested; the detection buffer in S3 is a mixture of 0.6 M KCl, 15 mM HEPES, 2 mM EDTA and 5 mM ATP.

2. The method for detecting bacteria using solid-state pore nanotechnology according to claim 1, Features: The magnetic bead resuspension and the gel solution were mixed in a centrifuge tube at a volume ratio of 0.5:1.

0.

3. The method for detecting bacteria using solid-state pore nanotechnology according to claim 1, Features: 100 μg / mL of the sample to be tested and streptavidin-modified Fe 3 O 4 The magnetic beads were mixed to a particle size of 200 nm, and the magnetic beads were resuspended in 100 μL of PBS with a pH of 6.0 to obtain a magnetic bead resuspension solution.

Images