A probe for detecting bacteria in the air and its preparation method and application

Abstract

The present invention provides a probe for detecting bacteria in the air, and its preparation method and application. The probe includes Au-Fe3O4 nanoparticles and an identification medium bound to the surface of the Au-Fe3O4 nanoparticles, the identification medium including a mixture of 5(6)-carboxyfluorescein and 3-(aminomethyl)phenyl boronate hydrochloride, and at least one of the products obtained by including the two as reactants. When using the probe, the air is sampled, maltose is added for incubation, a solution that may contain bacteria after incubation is taken, and the obtained solution is then mixed with the probe. The fluorescent signal displayed by the probe can be used to obtain the corresponding bacterial content in the air. That is, the bacterial content in the air can be effectively obtained by the probe. At the same time, the probe is simple to prepare and easy to use, which is conducive to promoting the development of air bacteria detection technology.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a probe for detecting bacteria in the air, a preparation method thereof, and an application thereof. Background Art

[0002] Airborne bacteria have a significant impact on human health, industrial production, and environmental hygiene. In medical settings, such as hospital operating rooms and wards, airborne bacteria can cause serious complications such as postoperative infections, increasing medical risks and costs. In food processing and storage environments, bacterial contamination can cause food spoilage and decay, leading to food safety incidents, harming consumer health, and damaging corporate reputations. In public spaces, such as schools, offices, and shopping malls, the spread of bacteria can also trigger the spread of various infectious diseases, impacting people's lives and work.

[0003] Traditional methods for detecting bacteria in the air have many limitations. As a classic detection method, the plate culture method is simple and intuitive in principle, but the operation is extremely cumbersome and requires multiple steps such as sampling, inoculation, culture, and colony counting. The entire detection cycle often takes several days, which is difficult to meet the needs of rapid detection. The liquid culture method is also time-consuming and is easily interfered by various factors during the bacteria counting process, resulting in inaccurate counts. Although the PCR technology based on molecular biology has high sensitivity and specificity and can detect low concentrations of bacteria, this technology has strict requirements on experimental equipment and requires expensive instruments such as professional PCR amplifiers. In addition, the experimental operation requires professional technicians to strictly follow the procedures, and the detection cost is high. It is not suitable for large-scale routine testing and on-site rapid testing. Therefore, the development of a simple, rapid, sensitive and low-cost method for detecting bacteria in the air has become an urgent problem to be solved. Summary of the Invention

[0004] To address these challenges, the present invention provides a probe for airborne bacteria detection, its preparation method, and its application. This probe enables efficient, accurate, and rapid detection of airborne bacteria, while offering the advantages of ease of operation and low cost. This approach addresses the needs for airborne bacteria detection in diverse fields and promotes the development and application of airborne bacteria detection technology.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A probe for detecting bacteria in the air comprises Au-Fe3O4 nanoparticles and an identification medium bound to the surface of the Au-Fe3O4 nanoparticles, wherein the identification medium comprises a mixture of 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride, and at least one of products obtained by comprising the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylborate hydrochloride as reactants.

[0007] In some embodiments, the identification medium includes a mixture of the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylborate hydrochloride, and the molar ratio of the 5(6)-carboxyfluorescein to the 3-(aminomethyl)phenylborate hydrochloride is 1:(0.8-1.2).

[0008] In some embodiments, the identification medium includes the product obtained by reacting the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylborate hydrochloride, and the molar ratio of the 5(6)-carboxyfluorescein to the 3-(aminomethyl)phenylborate hydrochloride is 1:(1.5-2.5).

[0009] In some embodiments, the Au-Fe3O4 nanoparticles have a particle size of 20 nm to 50 nm.

[0010] In some embodiments, the recognition medium is bound to the surface of the Au-Fe3O4 nanoparticles by chemical bonding or physical adsorption.

[0011] A method for preparing the probe for detecting bacteria in the air comprises the following steps:

[0012] preparing the Au-Fe3O4 nanoparticles;

[0013] The recognition medium is combined with the surface of the Au-Fe3O4 nanoparticles.

[0014] In some embodiments, the Au-Fe3O4 nanoparticles are prepared by a co-precipitation method.

[0015] In some embodiments, the identification medium comprises a mixture of the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylborate hydrochloride;

[0016] Binding the identification medium to the surface of the Au-Fe3O4 nanoparticles comprises:

[0017] The 5(6)-carboxyfluorescein, the 3-(aminomethyl)phenylborate hydrochloride and the Au-Fe3O4 nanoparticles are mixed in a buffer solution for reaction.

[0018] In some embodiments, the identification medium includes a product obtained by reacting the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylborate hydrochloride;

[0019] The preparation of the identification medium comprises: performing an amidation reaction or a peptide formation reaction of the amino group and the carboxyl group with the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylboronic acid hydrochloride, and purifying the mixture;

[0020] Binding the identification medium to the surface of the Au-Fe3O4 nanoparticles comprises:

[0021] The recognition medium and the Au-Fe3O4 nanoparticles are mixed and reacted in a buffer solution.

[0022] An application of the probe for detecting bacteria in air in detecting bacteria in air comprises the following steps:

[0023] Air is sampled using a culture medium; maltose is added to the sampled culture medium for incubation, the incubated product is subjected to solid-liquid separation, the liquid phase obtained by the solid-liquid separation is mixed with the probe, and the bacterial content in the sampled air is obtained through the fluorescent signal displayed by the probe.

[0024] Beneficial effects

[0025] The probe of the present invention can realize efficient, accurate and rapid detection of bacterial content in the air, and has the advantages of simple operation and low cost, so as to meet the needs of air bacteria detection in different fields and promote the development and application of air bacteria detection technology. DETAILED DESCRIPTION

[0026] One embodiment of the present invention provides a probe for detecting bacteria in the air. The probe includes Au-Fe3O4 nanoparticles and an identification medium bound to the surface of the Au-Fe3O4 nanoparticles, wherein the identification medium includes a mixture of 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride, and at least one of products obtained by reacting the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylborate hydrochloride. The probe can achieve efficient, accurate, and rapid detection of the bacterial content in the air, while having the advantages of simple operation and low cost, so as to meet the needs of different fields for air bacteria detection and promote the development and application of air bacteria detection technology.

[0027] In some embodiments, the identification medium comprises a mixture of 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylboronic acid hydrochloride, and the molar ratio of 5(6)-carboxyfluorescein to 3-(aminomethyl)phenylboronic acid hydrochloride is 1:(0.8-1.2). When the identification medium comprises a mixture of 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylboronic acid hydrochloride, the molar ratio of 5(6)-carboxyfluorescein to 3-(aminomethyl)phenylboronic acid hydrochloride can be appropriately selected within the range of 1:(0.8-1.2). For example, the molar ratio of 5(6)-carboxyfluorescein to 3-(aminomethyl)phenylboronic acid hydrochloride can be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, etc.

[0028] In some embodiments, the identification medium includes a product obtained by reacting 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylboronic acid hydrochloride as reactants, and the molar ratio of the 5(6)-carboxyfluorescein to the 3-(aminomethyl)phenylboronic acid hydrochloride is 1:(1.5-2.5). When the identification medium includes a product obtained by reacting 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylboronic acid hydrochloride as reactants, the molar ratio of the 5(6)-carboxyfluorescein to the 3-(aminomethyl)phenylboronic acid hydrochloride can be 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, etc.

[0029] In some embodiments, the Au-Fe3O4 nanoparticles have a particle size of 20 nm to 50 nm. This particle size range provides an appropriate number of binding sites for the recognition medium, promoting the binding of the recognition medium to the Au-Fe3O4 nanoparticles. Alternatively, the Au-Fe3O4 nanoparticles may have a particle size of 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, etc.

[0030] In some embodiments, the recognition medium is bound to the surface of the Au-Fe3O4 nanoparticles by chemical bonding or physical adsorption. Chemical bonding or physical adsorption can stably bind the recognition medium to the surface of the Au-Fe3O4 nanoparticles, allowing the probe to maintain a stable structure.

[0031] One embodiment of the present invention provides a method for preparing a probe for airborne bacteria detection, comprising the steps of: preparing Au-Fe₃O₄ nanoparticles; and binding a recognition medium to the surface of the Au-Fe₃O₄ nanoparticles. This method is simple and easy to implement, and can produce a probe with excellent performance without requiring complex processes or expensive equipment.

[0032] In some embodiments, the Au-Fe3O4 nanoparticles are prepared by a coprecipitation method. Optionally, the preparation of the Au-Fe3O4 nanoparticles includes the following steps: weighing ferric chloride (FeCl3) and chloroauric acid (HAuCl4), dissolving them in deionized water, and stirring them thoroughly until completely dissolved to prepare a mixed metal salt solution. Under stirring conditions, the mixed solution is added dropwise to a pre-prepared sodium hydroxide (NaOH) solution at a slow and steady rate. During the dropwise addition process, the temperature of the reaction system is controlled at 60°C using a thermostat, and the pH value of the solution is monitored in real time to maintain the stability of the reaction conditions. The reaction time is 2 hours, during which stirring is maintained to ensure that the reaction proceeds fully. After the reaction is completed, a black precipitate is generated, which is transferred to a centrifuge tube and centrifuged and washed multiple times with deionized water (e.g., 5 minutes each time at 8000 rpm) until a small amount of washing solution is taken and no white precipitate is generated after the addition of silver nitrate solution, indicating that the chloride ions have been completely removed. Finally, the washed precipitate was transferred to a drying dish and dried in an 80°C oven for 12 hours to obtain Au-Fe3O4 nanoparticles. The particle size of the prepared nanoparticles was determined to be approximately 30 nm using a particle size analyzer.

[0033] In some embodiments, the recognition medium comprises a mixture of 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride. Binding the recognition medium to the surface of the Au-Fe3O4 nanoparticles comprises: mixing the 5(6)-carboxyfluorescein, the 3-(aminomethyl)phenylborate hydrochloride, and the Au-Fe3O4 nanoparticles in a buffer solution.

[0034] Optionally, the buffer solution is phosphate buffered saline (PBS, pH=7.4).

[0035] Further optionally, the identification medium is bound to the surface of the Au-Fe3O4 nanoparticles, comprising: weighing 5(6)-carboxyfluorescein and 3-(aminomethyl)phenyl boron hydrochloride in a molar ratio of 1:(0.8-1.2), dissolving the two in a phosphate buffer solution (PBS, pH=7.4), and ultrasonically treating the solution for 30 minutes using an ultrasonic oscillator to fully disperse the substances in the solution. Then, the Au-Fe3O4 nanoparticles are slowly added to the above solution, and stirred for 4 hours at room temperature to allow the identification medium to fully react and bind to the surface of the Au-Fe3O4 nanoparticles. After the reaction is completed, the reaction solution is transferred to a centrifuge, centrifuged at 10,000 rpm for 15 minutes, and the bottom precipitate is collected. The precipitate is washed three times with PBS solution to remove unbound 5(6)-carboxyfluorescein and 3-(aminomethyl)phenyl boron hydrochloride and other impurities, thereby obtaining a probe for detecting bacteria in the air.

[0036] In some embodiments, the recognition medium includes a product obtained by reacting 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylboronic acid hydrochloride. The preparation of the recognition medium includes: performing an amidation reaction or a peptide formation reaction on the amino and carboxyl groups of the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylboronic acid hydrochloride, and purifying the mixture. Binding the recognition medium to the surface of the Au-Fe3O4 nanoparticles includes: mixing the recognition medium and the Au-Fe3O4 nanoparticles in a buffer solution.

[0037] Optionally, the identification medium includes a product obtained by reacting 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride as reactants. The preparation of the identification medium includes: step (1) adding 5(6)-carboxyfluorescein to a phosphate buffer solution with a pH of 4.5 to 7.2, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and stirring at 25°C to 35°C for 10 to 30 minutes; step (2) adding 3-(aminomethyl)phenylborate hydrochloride and adjusting the pH of the reaction system to 7 to 8, stirring and reacting for 2 to 5 hours; step (3) purifying the product.

[0038] Optionally, the method for purifying the product in step (3) is purification through a non-polar solid phase extraction column, the non-polar solid phase extraction column is a C18 extraction column; the elution solvent is a gradient solvent of dimethyl sulfoxide and n-hexane or a gradient solvent of dimethyl sulfoxide and n-pentane.

[0039] The molar ratio of 5(6)-carboxyfluorescein to 3-(aminomethyl)phenylboronic acid hydrochloride is 1:(1.5-2.5). The amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 40 mg / mL to 80 mg / mL of the reaction solution, and the amount of N-hydroxysuccinimide is 40 mg / mL to 80 mg / mL of the reaction solution.

[0040] Another embodiment of the present invention provides an application of the probe for detecting bacteria in the air in detecting bacteria in the air, comprising the following steps: sampling the air using a culture medium; adding maltose to the sampled culture medium for incubation, performing solid-liquid separation on the incubated product, mixing the liquid phase obtained by the solid-liquid separation with the probe, and obtaining the bacterial content in the sampled air through the fluorescent signal displayed by the probe.

[0041] Optionally, sampling the air using a culture medium includes: using an impact method, using a suitable air sampling device (such as an Anderson sampler) to sample the air, setting the sampling flow rate (such as 28.3L / min) and sampling time (such as 5min to 15min) according to different detection environments and requirements, and collecting the bacteria in the air onto the culture medium. The natural sedimentation method can also be used, and the time is 5 minutes.

[0042] Adding maltose to the culture medium after sampling and incubating includes transferring the culture medium after sampling to a PBS solution containing a certain concentration of maltose (e.g., 1%) and incubating at an appropriate temperature (e.g., 37°C) for a certain period of time (e.g., 30 minutes). Maltose can specifically interact with specific structures on the bacterial surface, promoting the release of bacteria from the sampling medium into the solution, thereby improving the detectability of the bacteria.

[0043] The incubated product is subjected to solid-liquid separation, the liquid phase obtained by the solid-liquid separation is mixed with the probe, and the bacterial content in the sampled air is obtained through the fluorescent signal displayed by the probe, including: taking 1 ml of the liquid phase obtained by solid-liquid separation, adding an appropriate amount (such as 100 μl) of the probe prepared by the present invention, mixing thoroughly, and detecting the fluorescent signal on a fluorescence spectrophotometer. Because certain components on the bacterial surface (such as polysaccharides, etc.) will specifically interact with the recognition medium on the probe surface, this interaction will cause the fluorescent signal of the probe to change. The bacterial content in the air can be obtained by using a pre-established standard curve of fluorescence signal and bacterial content.

[0044] Example 1

[0045] (1) Preparation of Au-Fe3O4 nanoparticles: Weigh ferric chloride (FeCl3) and chloroauric acid (HAuCl4), dissolve them in deionized water, and stir thoroughly until completely dissolved to prepare a mixed metal salt solution. Under stirring conditions, the mixed solution is added dropwise to the pre-prepared NaOH solution at a slow and steady rate. During the dropwise addition process, the temperature of the reaction system is controlled at 60°C using a thermostat, and the pH value of the solution is monitored in real time to maintain the stability of the reaction conditions. The reaction time is 2 hours, and the stirring state is always maintained during the reaction to ensure that the reaction proceeds fully. After the reaction is completed, a black precipitate is generated. The precipitate is transferred to a centrifuge tube and washed by centrifugation with deionized water for multiple times, each time for 5 minutes at a speed of 8000 rpm, until a small amount of washing liquid is taken and no white precipitate is generated after the addition of silver nitrate solution, indicating that the chloride ions have been completely removed. Finally, the washed precipitate is transferred to a drying dish and dried in a constant temperature oven at 80°C for 12 hours to obtain Au-Fe3O4 nanoparticles. Detected by a particle size analyzer, the particle size of the prepared nanoparticles was about 30 nm.

[0046] (2) Preparation of probes for airborne bacteria detection: 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride were weighed in a molar ratio of 1:1, dissolved in a phosphate buffer solution at pH = 7.4, and ultrasonicated for 30 minutes using an ultrasonic oscillator to fully disperse the substances in the solution. Then, Au-Fe3O4 nanoparticles were slowly added to the above solution and stirred for 4 hours at room temperature to allow the recognition medium to fully react and bind to the surface of the Au-Fe3O4 nanoparticles. After the reaction was completed, the reaction solution was transferred to a centrifuge and centrifuged at 10,000 rpm for 15 minutes to collect the bottom precipitate. The precipitate was washed three times with PBS solution to remove unbound 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride and other impurities, and finally a probe for airborne bacteria detection was obtained.

[0047] (3) The application of the probe for detecting bacteria in the air in the detection of bacteria in the air includes the following steps: using the impact method, using an impact air sampler based on the Anderson principle to sample the air, setting the sampling flow rate to 28.3L / min and the sampling time to 15min, and collecting the bacteria in the air onto the culture medium. The sampled culture medium is transferred to a PBS solution containing 1% maltose by mass concentration and incubated at a constant temperature of 37°C for 30min. The incubated product is subjected to solid-liquid separation, 1ml of the liquid phase obtained by solid-liquid separation is taken, 100μl of the probe prepared in this embodiment is added, and after thorough mixing, the fluorescence signal is detected on a fluorescence spectrophotometer. The deviation of the test probe for the detection of Staphylococcus aureus at different concentrations is tested. The results are shown in Table 1.

[0048] Example 2

[0049] Compared with Example 1, the difference of this embodiment is that the preparation method of the probe is different. The preparation method of the probe includes: step (1) adding 5(6)-carboxyfluorescein to a phosphate buffer with a pH of 4.8, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and stirring at 30°C for 20 minutes; the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 50 mg / mL, and the amount of N-hydroxysuccinimide is 50 mg / mL. Step (2) adding 3-(aminomethyl)phenylborate hydrochloride and adjusting the pH of the reaction system to 7.5, stirring and reacting for 3 hours; the molar ratio of 5(6)-carboxyfluorescein to 3-(aminomethyl)phenylborate hydrochloride is 1:2.2. Step (3) purifying the product by a non-polar solid phase extraction column to obtain a solution containing a recognition medium (elution phase). Then, Au-Fe3O4 nanoparticles were slowly added to the above solution and stirred at room temperature for 4 hours to allow the recognition medium to fully react and bind to the surface of the Au-Fe3O4 nanoparticles. After the reaction was completed, the reaction solution was transferred to a centrifuge and centrifuged at 10,000 rpm for 15 minutes to collect the bottom precipitate. The precipitate was washed three times with PBS solution to remove unbound 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride and other impurities, ultimately obtaining a probe for detecting bacteria in the air.

[0050] Comparative Example 1

[0051] Compared with Example 1, the difference of this comparative example is that the identification medium in Example 1 is directly used (not combined with Au-Fe3O4 nanoparticles) to detect bacteria in the air.

[0052] Comparative Example 2

[0053] Compared with Example 2, the difference of this comparative example is that the identification medium in Example 2 is directly used (not combined with Au-Fe3O4 nanoparticles) to detect bacteria in the air.

[0054] Comparative Example 3

[0055] Compared with Example 1, the difference of this comparative example is that only 5(6)-carboxyfluorescein is used as the recognition medium, and after combining with Au-Fe3O4 nanoparticles, a probe is obtained, and then the probe is used to detect bacteria in the air.

[0056] Comparative Example 4

[0057] The air was tested for bacteria using the traditional plate culture method.

[0058] Table 1

[0059]

[0060]

[0061] In Table 1, relative error = absolute value of the difference between the set concentration of Staphylococcus aureus and the test value / set concentration of Staphylococcus aureus × 100%.

[0062] As can be seen from Table 1, the embodiment has a smaller relative error than the comparative example, indicating that the probe in the embodiment has a higher accuracy in detecting bacteria in the air.

[0063] 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 and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0064] For those skilled in the art, according to the concept of the present invention, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A probe for detecting Staphylococcus aureus in the air, characterized in that: The invention comprises Au-Fe3O4 nanoparticles and an identification medium bound to the surface of the Au-Fe3O4 nanoparticles, wherein the identification medium is a mixture of 5(6)-carboxyfluorescein and 3-(aminomethyl)phenylborate hydrochloride.

2. The probe for detecting Staphylococcus aureus in the air according to claim 1, wherein The molar ratio of the 5(6)-carboxyfluorescein to the 3-(aminomethyl)phenylborate hydrochloride is 1:(0.8-1.2).

3. The probe for detecting Staphylococcus aureus in the air according to claim 1, wherein: The particle size of the Au-Fe3O4 nanoparticles is 20nm to 50nm.

4. The probe for detecting Staphylococcus aureus in the air according to any one of claims 1 to 3, characterized in that: The identification medium is combined with the surface of the Au-Fe3O4 nanoparticles by chemical bonding or physical adsorption.

5. A method for preparing a probe for detecting Staphylococcus aureus in the air according to any one of claims 1 to 4, characterized in that: The steps include: preparing the Au-Fe3O4 nanoparticles; The recognition medium is combined with the surface of the Au-Fe3O4 nanoparticles.

6. The method for preparing a probe for detecting Staphylococcus aureus in air according to claim 5, wherein: The Au-Fe3O4 nanoparticles are prepared by a co-precipitation method.

7. The method for preparing a probe for detecting Staphylococcus aureus in the air according to any one of claims 5 to 6, characterized in that: The identification medium is a mixture of the 5(6)-carboxyfluorescein and the 3-(aminomethyl)phenylborate hydrochloride; Binding the identification medium to the surface of the Au-Fe3O4 nanoparticles comprises: The 5(6)-carboxyfluorescein, the 3-(aminomethyl)phenylborate hydrochloride and the Au-Fe3O4 nanoparticles are mixed in a buffer solution for reaction.

8. Use of the probe for detecting Staphylococcus aureus in the air according to any one of claims 1 to 4 in detecting Staphylococcus aureus in the air, characterized in that: The following steps are involved: Air sampling was performed using culture medium; Maltose is added to the culture medium after sampling and incubated, and the incubated product is subjected to solid-liquid separation. The liquid phase obtained by the solid-liquid separation is mixed with the probe, and the content of Staphylococcus aureus in the sampled air is obtained through the fluorescent signal displayed by the probe.

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