Detection method of pseudomonas aeruginosa

Abstract

The invention provides a detection method of pseudomonas aeruginosa, and belongs to the technical field of biological detection. The method comprises the following steps: combining carboxymethyl chitosan with a specific aptamer of pseudomonas aeruginosa to form a chitosan-aptamer compound; mixing the chitosan-aptamer compound with a carbon nano tube-gold nano solution, and enabling the chitosan-aptamer compound to be adsorbed on the surface of a carbon nano tube-gold nano substance, so as to obtain a compound adsorption substance; mixing a to-be-detected sample with the composite adsorption substance to obtain a to-be-detected mixture; and qualitatively or quantitatively detecting the mixture to be detected to obtain a detection result. According to the method, the used raw materials are low in cost and wide in source, the cell entry efficiency is improved, DNA of the pseudomonas aeruginosa does not need to be extracted, the bacteria can be directly detected, the detection time is shortened, and the method is a rapid, economical and convenient method for detecting the pseudomonas aeruginosa.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection method for Pseudomonas aeruginosa. Background Art

[0002] In the field of modern medicine and public health, the monitoring and diagnosis of pathogenic microorganisms are of great significance. Pseudomonas aeruginosa, a non-fermenting Gram-negative bacillus widely distributed in the natural environment, is commonly found in water sources, air, and the skin and respiratory tract of organisms. Due to its common pathogenicity in hospitals and its potential threat to food safety, effective monitoring and rapid diagnosis have become an urgent task. Especially in the context of clinical medicine and public health safety, rapid and accurate detection of Pseudomonas aeruginosa has important application value.

[0003] Nevertheless, current Pseudomonas aeruginosa detection technologies mainly rely on traditional microbiological methods, including pure culture and biochemical identification. These methods usually take a long time to complete, for example, pure culture takes several days to a week, and the biochemical identification process is also more complicated and time-consuming. In addition, the sensitivity of these traditional methods is usually limited, which may lead to low-level bacterial infections being overlooked, thus affecting the final diagnosis and treatment decisions.

[0004] The main drawbacks of the existing technology are that it is cumbersome to operate and has low detection efficiency. In emergency situations, such as severe infections or food safety incidents, rapid diagnosis is particularly critical. However, due to the long detection time of the existing technology, it cannot meet the demand for rapid response. In addition, the low sensitivity of the existing method often leads to missed detection of low-concentration Pseudomonas aeruginosa, which may lead to serious consequences in clinical and public health monitoring.

[0005] In summary, the existing Pseudomonas aeruginosa detection technology has obvious deficiencies in terms of rapidity, sensitivity, and ease of operation. These limitations not only increase the difficulty of clinical diagnosis and treatment, but also increase the risk of public health monitoring. Therefore, the development of a new technology that can achieve efficient, rapid and highly sensitive detection is crucial to improving the ability of disease prevention, control and food safety supervision. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for detecting Pseudomonas aeruginosa, comprising:

[0007] Carboxymethyl chitosan is combined with a specific aptamer of Pseudomonas aeruginosa to form a chitosan-aptamer complex;

[0008] The chitosan-aptamer complex is mixed with a carbon nanotube-gold nano-solution, so that the chitosan-aptamer complex is adsorbed on the surface of the carbon nanotube-gold nano-substance to obtain a composite adsorbed substance;

[0009] Mixing the sample to be tested with the composite adsorbent to obtain a mixture to be tested;

[0010] Performing qualitative or quantitative detection on the test mixture for Pseudomonas aeruginosa to obtain a detection result;

[0011] Preferably, there is an amino modification in the nucleotide sequence of the specific aptamer;

[0012] Preferably, one end of the nucleotide sequence of the specific aptamer has an amino modification.

[0013] Preferably, the carboxymethyl chitosan is O,N-carboxymethyl chitosan.

[0014] Preferably, the specific aptamer sequence is a cross-linked ring structure.

[0015] Preferably, the nucleotide sequence of the specific aptamer is as shown in SEQ ID NO.1; and the 5' end of the nucleotide sequence is connected with a thiol modification group. Preferably, in the chitosan-aptamer complex, the mass ratio of the carboxymethyl chitosan to the specific aptamer is (3-5):1.

[0016] Preferably, before combining carboxymethyl chitosan with a specific aptamer of Pseudomonas aeruginosa to form a chitosan-aptamer complex, the method further comprises:

[0017] Activating the carboxymethyl chitosan;

[0018] Preferably, the activation treatment comprises:

[0019] Adding N-hydroxysuccinimide to the carboxymethyl chitosan;

[0020] While the mixture was stirred, 1-ethyl-3-(dimethylaminopropyl)carbodiimide was added to complete activation.

[0021] Preferably, the mixture is stirred and 1-ethyl-3-(dimethylaminopropyl)carbodiimide is added to complete the activation, which is carried out in an ice-water bath.

[0022] Preferably, in the activation treatment, the mass ratio of the carboxymethyl chitosan, the N-hydroxysuccinimide and the 1-ethyl-3-(dimethylaminopropyl)carbodiimide is (0.28-0.32):(0.15-0.18):(0.24-0.28).

[0023] Preferably, in the step of mixing the chitosan-aptamer complex with the carbon nanotube-gold nano-solution to allow the chitosan-aptamer complex to be adsorbed on the surface of the carbon nanotube-gold nano-matter, the chitosan-aptamer complex is added to the carbon nanotube-gold nano-solution and then mixed and then subjected to a first incubation treatment;

[0024] Preferably, the first incubation treatment lasts for 20 to 35 hours.

[0025] Preferably, in the step of mixing the sample to be tested with the composite adsorbent, the sample to be tested and the composite adsorbent are mixed, followed by a second incubation treatment, and then an inorganic salt is added;

[0026] Preferably, the second incubation treatment lasts for 20 to 30 minutes.

[0027] The invention provides a method for detecting Pseudomonas aeruginosa, comprising: combining carboxymethyl chitosan with a specific aptamer of Pseudomonas aeruginosa to form a chitosan-aptamer complex; wherein the nucleotide sequence of the specific aptamer is amino-modified; mixing the chitosan-aptamer complex with a carbon nanotube-gold nano solution, allowing the chitosan-aptamer complex to be adsorbed on the surface of the carbon nanotube-gold nano material to obtain a composite adsorbed material; mixing a sample to be tested with the composite adsorbed material to obtain a mixture to be tested; and performing qualitative or quantitative detection on the mixture to be tested for Pseudomonas aeruginosa to obtain a detection result.

[0028] The present invention has the following beneficial effects:

[0029] 1. The present invention uses carboxymethyl chitosan as a carrier for the first time, combines it with a Pseudomonas aeruginosa-specific aptamer, and uses a gold nanosolution as a visualization indicator. The designed biosensor-type detection system combines many advantages such as high efficiency, short detection time, and low cost. It also has the advantages of high specificity and visualization of detection results. The detection results can be directly qualitatively observed by the naked eye, and Pseudomonas aeruginosa can be detected specifically. In addition, the sample pretreatment process is reduced, and the bacterial solution can be directly detected.

[0030] 2. The present invention uses carboxymethyl chitosan as a transport carrier and utilizes its surface charge to carry the aptamer-gold nanocomplex into cells, thereby solving the problem of low cell entry efficiency, thereby achieving direct detection of Pseudomonas aeruginosa bacterial liquid without extracting DNA.

[0031] 3. The carbon nanotube-gold nanoparticle solution used in the present invention changes color in a high-salt solution, making the detection result visible. The selected specific aptamer has a wide source and low cost, which greatly improves the economic benefits of the method, can specifically identify Pseudomonas aeruginosa, and can perform quantitative detection of Pseudomonas aeruginosa.

[0032] In summary, the raw materials used in the present invention are low in cost and widely available, and the cell entry efficiency is improved. There is no need to extract the DNA of Pseudomonas aeruginosa, and the bacteria themselves can be directly detected, which shortens the detection time. The method is a fast, economical and convenient method for detecting Pseudomonas aeruginosa, and can be widely used for the detection of Pseudomonas aeruginosa in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the process of the detection method of Pseudomonas aeruginosa in the present invention;

[0034] Figure 2 This is a reaction principle diagram of carboxymethyl chitosan, specific aptamer and carbon nanotube-gold nanosolution in an embodiment of the detection method of Pseudomonas aeruginosa of the present invention;

[0035] Figure 3 This is a schematic diagram of the detection effect of the reaction system on Pseudomonas aeruginosa at different incubation times in Example 2 of the detection method of Pseudomonas aeruginosa of the present invention.

[0036] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] refer to Figure 1 In the embodiments of the present application, a method for detecting Pseudomonas aeruginosa is provided, comprising:

[0039] Step S1, combining carboxymethyl chitosan with a specific aptamer of Pseudomonas aeruginosa to form a chitosan-aptamer complex;

[0040] The above steps involve combining carboxymethyl chitosan with a specific aptamer of Pseudomonas aeruginosa.

[0041] Among them, the specific aptamer is a specially designed short-chain nucleic acid that can bind to a specific target molecule (Pseudomonas aeruginosa) with high specificity.

[0042] It should be noted that the specific aptamer can be an oligonucleotide sequence with high specificity and high affinity for the target screened by SELEX technology. Aptamers have many advantages, such as easy synthesis, high stability, good repeatability, low price, and a wide range of aptamer targets, which can specifically bind to cells, viruses, toxins, proteins and vitamins. Due to these advantages of aptamers, the application range of aptamers has become wider and wider in recent years. In terms of detection, since aptamers can be conjugated with a variety of bacteria, toxins, etc. to form complexes, thereby realizing their detection, this is a fast and convenient detection method, and has good sensitivity and selectivity. Compared with traditional detection methods, the detection cycle is short and the process is simple.

[0043] It should be noted that carboxymethyl chitosan is a derivative of chitosan, which is modified on the basis of chitosan, and the carboxymethyl group is substituted on the hydroxyl group or the amino group. By changing the preparation conditions, O-carboxymethyl chitosan, N-carboxymethyl chitosan or O,N-carboxymethyl chitosan can be formed according to the different positions of the substituents.

[0044] Carboxymethyl chitosan is a sodium salt of carboxylic acid, so it is water-soluble. The introduction of carboxymethyl groups changes the structure of chitosan itself, thereby enhancing its water solubility. Carboxymethyl chitosan has stable properties.

[0045] By combining the aptamer (with a specific amino modification) with carboxymethyl chitosan, a complex is formed. This modification enhances the stability and affinity of the aptamer. Upon combination, the chitosan-aptamer complex formed will be used for target bacteria detection in the subsequent steps.

[0046] This step provides high selectivity through the use of specific aptamers, ensuring that only the target microorganisms are captured and detected.

[0047] Step S2, mixing the chitosan-aptamer complex with the carbon nanotube-gold nanomaterial solution, so that the chitosan-aptamer complex is adsorbed on the surface of the carbon nanotube-gold nanomaterial to obtain a composite adsorbed material;

[0048] The above steps involve mixing the chitosan-aptamer complex with the carbon nanotube-gold nanoparticle solution, and the chitosan-aptamer complex is adsorbed onto the surface of the carbon nanotube and the gold nanoparticle to form a composite adsorbent.

[0049] The composite adsorbent has a larger surface area and higher surface activity, which helps to improve the efficiency of capturing microorganisms.

[0050] The combined use of carbon nanotubes and gold nanoparticles can improve conductivity and chemical stability, making the final detection signal stronger and more stable.

[0051] It should be noted that gold nanoparticles are microparticles with a particle size of generally less than 15nm. Gold nanoparticles have a stable structure and a small particle size. They can be combined with many biological macromolecules without changing their biological activity. They can be used for drug treatment in the human body and are a substance with good optical and electrochemical properties.

[0052] It should be noted that carbon nanotubes have good electrical conductivity and mechanical strength, large specific surface area and good stability. When combined with carbon nanotubes for biological detection, they can effectively overcome the disadvantage of easy aggregation of gold nanoparticles. The large specific surface area exposes more active sites, allowing more targets to gather around the carbon nanotubes. The high contact efficiency increases the labeling amount and conductivity of biological molecules, achieves signal amplification and improves detection sensitivity.

[0053] Step S3, mixing the sample to be tested with the composite adsorbent to obtain a mixture to be tested;

[0054] As mentioned above, the sample to be tested (the sample that may contain Pseudomonas aeruginosa and needs to be tested) is mixed with the composite adsorption substance. If Pseudomonas aeruginosa exists in the sample to be tested, it will be captured by the composite adsorption substance.

[0055] The mixture to be tested after mixing contains the captured Pseudomonas aeruginosa, ready for the next step of detection. This step can effectively separate Pseudomonas aeruginosa from complex samples, improving the accuracy of subsequent detection.

[0056] Step S4, performing qualitative or quantitative detection on the mixture to be tested for Pseudomonas aeruginosa to obtain a detection result;

[0057] In the above, the test mixture is subjected to qualitative or quantitative detection of Pseudomonas aeruginosa.

[0058] The qualitative or quantitative analysis of microorganisms can be performed by electrochemical, spectroscopy or biosensor methods. For example, UV-visible spectrophotometry is used for quantitative analysis.

[0059] In this step, specific data on the presence and concentration of P. aeruginosa in the sample can be obtained. The final detection step is fast, sensitive and provides quantitative information, which helps to respond quickly and make decisions.

[0060] In summary, reference Figure 2 In the embodiments of the present application, the following technical principle is used: the specific aptamer sequence of Pseudomonas aeruginosa can specifically recognize Pseudomonas aeruginosa and stably bind to it. The gold nanoparticles can bind to the aptamer through electrostatic interaction to form a targeted complex that can specifically detect Pseudomonas aeruginosa.

[0061] The reason for combining carboxymethyl chitosan with the complex is that the surface of the complex formed by the aptamer and the carbon nanotube / gold nanoparticles is negatively charged, which makes it difficult to cross the cell membrane with negative surface charge and is easily degraded by nucleases, so it cannot enter the cell by itself and needs an ideal transport carrier to carry it into the cell. Therefore, the positively charged chitosan-aptamer complex and the negatively charged gold nanoparticles are used to solve the problem of low cell entry efficiency. The selected carboxymethyl chitosan has a large number of carboxyl and amino groups, and the carboxyl groups are combined with the amino-modified aptamer, and the aptamer is then combined with the carbon nanotube / gold nanoparticle by electrostatic action. The entire material is positively charged due to the amino groups on the carboxymethyl chitosan, which can improve the cell entry efficiency of the material and realize the direct detection of the bacterial solution without extracting DNA.

[0062] In summary, in the embodiments of the present application, carboxymethyl chitosan is used as a carrier for the first time, combined with a Pseudomonas aeruginosa-specific aptamer, and a gold nanosolution is used as a visualization indicator. The designed biosensor-type detection system combines many advantages such as high efficiency, short detection time, and low cost. It also has the advantages of high specificity and visualization of detection results. The detection results can be directly qualitatively observed by the naked eye, and Pseudomonas aeruginosa can be detected specifically. The sample pretreatment process is reduced, and the bacterial solution can be directly detected.

[0063] Since the specific aptamer in the embodiment of the present application is specific to the target bacteria (Pseudomonas aeruginosa), targeted detection can be performed. The characteristic of gold nanometers to produce color changes under saline solution makes it possible to combine aptamer technology with gold nanometers for visual detection, but the aptamer itself is an oligonucleotide, which is difficult to cross the cell membrane with negative surface charge, and the cell entry rate is very low. The existing method is to extract the DNA of Pseudomonas aeruginosa and then detect it. In the embodiment of the present application, carboxymethyl chitosan is used as a transport carrier, and its surface charge is used to carry the aptamer-gold nanocomplex into the cell to solve the problem of low cell entry efficiency, so that the bacterial solution of Pseudomonas aeruginosa can be directly detected without extracting DNA.

[0064] In the examples of the present application, the carbon nanotube-gold nanoparticle solution used changes color in a high-salt solution, making the detection results visible. The selected specific aptamers are widely available and low in cost, which greatly improves the economic benefits of the method, can specifically identify Pseudomonas aeruginosa, and can perform quantitative detection of Pseudomonas aeruginosa.

[0065] In summary, the raw materials used in the embodiments of the present application are low in cost and widely available, and the cell entry efficiency is improved. There is no need to extract the DNA of Pseudomonas aeruginosa, and the bacteria themselves can be directly detected, which shortens the detection time. It is a fast, economical and convenient method for detecting Pseudomonas aeruginosa, which can be widely used for the detection of Pseudomonas aeruginosa in food.

[0066] Furthermore, there is an amino modification in the nucleotide sequence of the specific aptamer;

[0067] Furthermore, one end of the nucleotide sequence of the specific aptamer is amino-modified.

[0068] As mentioned above, the nucleotide sequence of the specific aptamer has an amino modification, which can enhance the chemical stability and biological stability of the aptamer. The nucleotide sequence is easily degraded by nucleases, and the amino modification can protect the aptamer from such degradation to a certain extent, thereby extending its life in biological samples or complex environments.

[0069] Amino modification can change the spatial structure of the aptamer, helping it to better bind to the target molecule (such as the specific molecular marker of Pseudomonas aeruginosa). This structural adjustment can enhance the affinity of the aptamer to the target, thereby improving the sensitivity and specificity of the detection.

[0070] The amino-modified end of the aptamer provides a convenient chemical functional group that can be used for further functionalization. For example, the amino group can be linked to other molecules (such as fluorescent markers, enzyme markers or other biomolecules) through covalent or non-covalent bonds for signal amplification or to establish more complex detection systems.

[0071] Therefore, by introducing an amino modification at one end of the aptamer, it is not only possible to enhance its binding efficiency with the target molecule, but also to provide more flexibility and functionality in the development and application of detection technology. This modification is one of the key technologies to achieve efficient and accurate biological detection.

[0072] Furthermore, the carboxymethyl chitosan is O,N-carboxymethyl chitosan.

[0073] As mentioned above, O,N-carboxymethyl chitosan refers to a specific type of carboxymethyl chitosan, in which the carboxymethyl group (-COOCH 3 ) are introduced into the oxygen atoms (O) and nitrogen atoms (N) of the chitosan molecules.

[0074] It should be noted that chitosan itself is composed of natural polysaccharides, mainly composed of glucosamine units linked by β-(1→4). In its natural form, the molecular structure of chitosan contains many amino groups (-NH 2 ) and hydroxyl group (-OH).

[0075] As mentioned above, compared with O-carboxymethyl chitosan and N-carboxymethyl chitosan, the carboxylmethyl groups of O,N-carboxymethyl chitosan are substituted on both -OH and -NH, which can introduce more carboxylmethyl groups, facilitating better combination with amino-modified aptamers, and the introduction of carboxylmethyl groups improves the water solubility of carboxymethyl chitosan.

[0076] Furthermore, the specific aptamer sequence is a cross-linked ring structure.

[0077] As mentioned above, the specific aptamer sequence is a cross-linked ring structure, which means that the molecular structure of the aptamer forms a ring structure through chemical cross-linking, which is a stable, closed-loop molecular structure.

[0078] The circular structure enables the aptamer to maintain its functionality under a variety of test conditions and is suitable for application in complex samples. Due to the high specificity of the circular aptamer, the background noise can be greatly reduced and the detection sensitivity can be improved. The circular aptamer does not require frequent replacement and recalibration and is suitable for portable or on-site rapid detection equipment.

[0079] In summary, the cross-linked ring structure of the specific aptamer provides an efficient, stable and highly specific biorecognition method for the detection of Pseudomonas aeruginosa, significantly improving the practicality and accuracy of the detection technology.

[0080] Furthermore, the nucleotide sequence of the specific aptamer is shown in SEQ ID NO.1; and the 5' end of the nucleotide sequence is connected with a thiol modification group.

[0081] Specifically, the nucleotide sequence of the specific aptamer is:

[0082] 5'-SH-CCCCCGTTGCTTCGCTTTTCCTTTCGCTTTTGTTCGTTTCG TCCCTGCTTCCTTTCTTG-3'.

[0083] As mentioned above, in the nucleotide sequence of the specific aptamer, the 5' end has a thiol (SH) modification. In aptamer technology, the thiol modification at the 5' end allows the aptamer to be fixed on gold nanoparticles or other surfaces through covalent bonds, which is particularly important for constructing stable biosensors or other biological diagnostic devices.

[0084] Furthermore, in the chitosan-aptamer complex, the mass ratio of the carboxymethyl chitosan to the specific aptamer is (3-5): 1. For example, it can be 3:1, 4:1, 5:1, etc.

[0085] Furthermore, before the step S1, combining carboxymethyl chitosan with the specific aptamer of Pseudomonas aeruginosa to form a chitosan-aptamer complex, the step further includes:

[0086] Step S5, activating the carboxymethyl chitosan.

[0087] The above steps involve activating carboxymethyl chitosan, which is an important pre-treatment step for preparing chitosan-aptamer complexes. The purpose of the activation treatment is to improve the reaction efficiency and binding strength between carboxymethyl chitosan and specific aptamers, thereby ensuring the stability and functionality of the complex.

[0088] Furthermore, the step S5, activation treatment, includes:

[0089] (1) First step reaction: Step S51, adding N-hydroxysuccinimide to the carboxymethyl chitosan;

[0090] In the above, N-hydroxysuccinimide (NHS) is added. NHS is an activating agent that can react with the carboxyl group in carboxymethyl chitosan to form an active ester intermediate. This active ester intermediate is more likely to react with nitrogen-containing compounds (such as the amino group in the aptamer) to achieve the formation of a covalent bond.

[0091] (2) Second step reaction: Step S52, while stirring the mixture, add 1-ethyl-3-(dimethylaminopropyl)carbodiimide to complete activation.

[0092] In the above, 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) is added, wherein EDC is a carbodiimide coupling reagent used to promote the cross-linking reaction between carboxyl groups and amino groups. With the help of NHS, EDC can more effectively connect the carboxyl groups in carboxymethyl chitosan with the amino groups in the aptamer to form a stable amide bond.

[0093] The purpose of stirring the mixture is to ensure that carboxymethyl chitosan, NHS and EDC are fully mixed and to improve the reaction uniformity and efficiency.

[0094] Before carboxymethyl chitosan is combined with the specific aptamer sequence, the carboxymethyl chitosan is first treated as follows to activate the carboxyl group. The mechanism is as follows: -NH 2 It undergoes an acid-base neutralization reaction with the carboxyl group, and the hydroxyl group in the carboxyl group loses hydrogen ions and becomes activated. NHS is usually used in combination with EDC to improve the reaction efficiency.

[0095] Through the above activation treatment, the reactivity of carboxymethyl chitosan is significantly improved, and it can react more effectively with the amino group in the aptamer, thereby accelerating and strengthening the binding between the two. The amide bond formed after the activation treatment is a very stable chemical bond, which makes the final chitosan-aptamer complex stable in various environments, especially in biological samples and complex detection environments. The stronger binding ensures that the aptamer is not easy to fall off during the detection process, which directly enhances the sensitivity and repeatability of the detection, reduces background interference, and improves the specificity of the detection.

[0096] Furthermore, in step S52, 1-ethyl-3-(dimethylaminopropyl)carbodiimide is added to the mixture while stirring to complete activation, which is actually carried out in an ice water bath.

[0097] As mentioned above, using an ice water bath during a chemical reaction can control the reaction temperature and prevent excessively fast reaction rates from causing side reactions or incomplete reactions, thereby ensuring higher yields and product quality.

[0098] Furthermore, in the step S5, during the activation treatment, the mass ratio of the carboxymethyl chitosan, the N-hydroxysuccinimide and the 1-ethyl-3-(dimethylaminopropyl)carbodiimide is (0.28-0.32):(0.15-0.18):(0.24-0.28).

[0099] Among the above three components, according to the mass fraction, the proportion of carboxymethyl chitosan can be 0.28, 0.30, 0.31, 0.32, etc.; the proportion of N-hydroxysuccinimide can be 0.15, 0.16, 0.17, 0.18, etc.; the proportion of 1-ethyl-3-(dimethylaminopropyl)carbodiimide can be 0.24, 0.25, 0.26, 0.27, 0.28, etc.

[0100] Furthermore, in the step S2, mixing the chitosan-aptamer complex with the carbon nanotube-gold nano-solution to allow the chitosan-aptamer complex to be adsorbed on the surface of the carbon nanotube-gold nano-matter, the chitosan-aptamer complex is added to the carbon nanotube-gold nano-solution and then mixed and then subjected to a first incubation treatment;

[0101] Furthermore, the first incubation treatment time is 20 hours to 35 hours.

[0102] As mentioned above, the first incubation treatment is used in the Pseudomonas aeruginosa detection method to ensure sufficient interaction and binding between the chitosan-aptamer complex and the carbon nanotube-gold nanosolution.

[0103] First, when the chitosan-aptamer complex is added to a solution containing carbon nanotubes and gold nanoparticles, sufficient time is required to ensure that the complex can be completely and evenly adsorbed to the surface of the nanomaterials. This adsorption is achieved through physical adsorption and possible chemical bonds (such as the combination of thiol groups with gold).

[0104] Secondly, through incubation, the binding between the chitosan-aptamer complex and the nanomaterial is more stable, which helps to provide a more stable signal output in the subsequent detection steps. Gold nanoparticles play an important role in improving electrochemical signals and enhancing fluorescence.

[0105] Third, long incubation time helps to form uniformly covered nanocomposites, which is key to achieve highly sensitive and reproducible detection.

[0106] Furthermore, in the step S3, in the step of mixing the sample to be tested with the composite adsorbent, the sample to be tested and the composite adsorbent are mixed, followed by a second incubation treatment, and then an inorganic salt is added;

[0107] Furthermore, the second incubation treatment time is 20 minutes to 30 minutes.

[0108] The second incubation treatment mentioned above is mainly aimed at ensuring that the Pseudomonas aeruginosa in the sample to be tested can effectively react with the composite adsorbed substance adsorbed on the surface of the carbon nanotube-gold nanomaterial by the chitosan-aptamer complex.

[0109] During the incubation process, the Pseudomonas aeruginosa in the sample to be tested has enough time to interact and bind with the aptamer. Aptamers are highly specific and can accurately identify and capture target microorganisms. Incubation makes the interaction between the microorganism and the aptamer more stable, and enhances the stability of the complex through possible biochemical reactions or physical adsorption. Incubation ensures that every possible Pseudomonas aeruginosa cell can be detected, thereby improving the sensitivity of the detection method.

[0110] As mentioned above, the addition of inorganic salts can be used to adjust the ionic strength and pH of the reaction mixture, thereby optimizing the binding environment between the aptamer and the target cell. Inorganic salts are sometimes used to reduce nonspecific adsorption and enhance specific binding, especially when using aptamers, inorganic salts can help the aptamer recognize the target more specifically.

[0111] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0112] Embodiment 1:

[0113] The method for detecting Pseudomonas aeruginosa in this embodiment specifically comprises the following steps:

[0114] (1) Preparation of carbon nanotube-gold nanoparticle solution: carbon nanotubes and HAuCl 4 The solutions were mixed, heated in microwave for 1-3 minutes, and then sodium citrate solution was added and mixed. After microwave irradiation for 4-6 minutes, the suspension was cooled to room temperature to obtain a suspension, which was centrifuged and filtered to obtain a carbon nanotube-gold nanoparticle solution.

[0115] (2) Preparation of chitosan-aptamer complex: 0.3 g of carboxymethyl chitosan (purchased from BELLANCOM LIFESCIENCESDEP, 100 g / bottle) was placed in a 50 mL beaker, 10 mL of deionized water was added, and the solution was stirred with a glass rod to obtain a carboxymethyl chitosan solution with a mass concentration of 30 mg / mL.

[0116] Then, 0.17 g of N-hydroxysuccinimide (NHS) was added and stirred for 15 min. Then, an aqueous solution of 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) (0.26 g of EDC was dissolved in 5 mL of deionized water in a 15 mL centrifuge tube) was slowly added dropwise in an ice-water bath stirrer at 2°C and stirred at 800 r / min.

[0117] After the addition was completed for 10 minutes, the reaction was continued under stirring in an ice-water bath for 1 hour to fully activate the carboxyl groups of the carboxymethyl chitosan.

[0118] The specific aptamer of Pseudomonas aeruginosa (purchased from Sangon Biotech (Shanghai) Co., Ltd., with amino modification at the 5' end) was added to the above solution at a mass ratio of carboxymethyl chitosan to aptamer of 4:1 and treated for 1 hour to obtain the chitosan-aptamer complex.

[0119] (3) Preparation of composite adsorbent: Take 1 mL of homemade nanogold solution in a 1.5 mL EP tube, centrifuge at 4°C and 10,000 r / min for 10 min, discard the supernatant, and resuspend. Add 100 μL of the prepared chitosan-aptamer complex to the above carbon nanotube / nanogold solution, mix well, and incubate in a shaker at 37°C for 30 h. In order to remove possible excess aptamer, the above mixture was centrifuged at 4°C and 10,000 r / min for 10 min, and the supernatant was discarded to finally obtain carboxymethyl chitosan-aptamer-carbon nanotube / nanogold material, which was stored at 4°C in the dark for future use.

[0120] (4) Pseudomonas aeruginosa sample detection: Take 9 1.5 mL EP tubes, add 100 μL of the prepared composite adsorbent, add Pseudomonas aeruginosa bacterial solution, and dilute 6 times to 1:10, 1:10 respectively. 2 , 1:10 3 , 1:10 4 , 1:10 5 and 1:10 6 , (bacterial concentration is 10cfu / mL, 10 2 cfu / mL, 10 3 cfu / mL, 10 4 cfu / mL, 10 5 cfu / mL, 10 6 cfu / mL) and incubated at 37°C for 30 min.

[0121] 1.5 mol / L NaCl solution was added to aggregate the released gold nanoparticles, and the absorbance A value at the maximum absorption wavelength was detected using a UV-visible spectrophotometer.

[0122] Table 1. Test data of Pseudomonas aeruginosa samples at various concentrations

[0123] Concentration (cfu / mL) 10 <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 ]]> Absorbance (A value) 0.137 0.211 0.441 0.779 1.105 1.624

[0124] According to the above test data, the standard curve is y=0.0014×x+0.0135; R=0.999.

[0125] For unknown sample A, the same serial dilution ratio as the standard curve was used to obtain 6 samples, and the same processing method as the standard was used to obtain the test data corresponding to the samples.

[0126] Table 2. Test data of unknown sample A at various concentrations

[0127] Preparation concentration (cfu / mL) 10 <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 ]]> Absorbance (A value) 0.150 0.235 0.365 0.615 0.880 1.450 Calculated concentration (cfu / mL) 87.500 150.000 255.000 432.500 625.000 1025.000

[0128] It can be seen that the unknown sample A contains Pseudomonas aeruginosa, and the corresponding concentration can be measured to obtain a quantitative result.

[0129] Embodiment 2:

[0130] The method for detecting Pseudomonas aeruginosa in this embodiment specifically comprises the following steps:

[0131] (1) Preparation of carbon nanotube-gold nanoparticle solution: After the CNTs are ultrasonically treated, 1.65 mL of 1% chloroauric acid is taken into a 250 mL conical flask, 38.35 mL of water is added, and the conical flask is placed on a magnetic heating stirrer for reflux in the dark (a condenser is connected to the conical flask), a stirrer is placed in the conical flask, and magnetic stirring is performed at the same time. When the solution boils, 6.4 mL of 38.8 mmol / L trisodium citrate solution is immediately added, and reflux in the dark is continued, heating and stirring are continued, and boiling is maintained for 15 minutes. Remove the heat source and stir until cooled to obtain a wine-red dispersed CNTs / AuNPs composite nanogold solution. After filtering the gold nanoparticle solution with a 0.22 μm needle-type water-soluble filter, place it in a 125 mL brown ground-mouth reagent bottle, wrap it with aluminum foil, and store it in the dark at 4°C for later use. After the CNTs are ultrasonically treated, HAuCl 4 The solution is mixed evenly and then reduced with a reducing agent to obtain a carbon nanotube-gold nanoparticle solution.

[0132] (2) Preparation of chitosan-aptamer complex: 0.3 g of carboxymethyl chitosan (purchased from BELLANCOM LIFESCIENCESDEP, 100 g / bottle) was placed in a 50 mL beaker, 10 mL of deionized water was added, and a carboxymethyl chitosan solution with a mass concentration of 30 mg / mL was obtained by stirring with a glass rod. 0.17 g of N-hydroxysuccinimide (NHS) was added to the above solution, and stirred for 15 min. Then, an aqueous solution of 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) (0.26 g of EDC was dissolved in a 15 mL centrifuge tube and 5 mL of deionized water was added) was slowly dripped into the solution in an ice-water bath at 2°C at 800 r / min. After the addition was completed for 10 min, the reaction was continued under stirring for 1 h in an ice-water bath to fully activate the carboxyl groups of the carboxymethyl chitosan. The specific aptamer of Pseudomonas aeruginosa (purchased from Sangon Biotech (Shanghai) Co., Ltd., with amino modification at the 5' end) was added to the above solution at a mass ratio of carboxymethyl chitosan to aptamer of 4:1 and treated for 1 hour to obtain the chitosan-aptamer complex.

[0133] (3) Preparation of composite adsorbent: 1 mL of self-made carbon nanotube / nano-gold solution was placed in a 1.5 mL EP tube, centrifuged at 4°C and 10,000 r / min for 10 min, the supernatant was discarded, and the mixture was resuspended. 100 μL of the prepared chitosan-aptamer complex was added to the carbon nanotube / nano-gold solution, mixed well, and incubated in a 37°C shaker for 12 h, 18 h, 24 h, 30 h, 36 h, and 42 h, respectively. In order to remove possible excess aptamer, the above mixture was centrifuged at 4°C and 10,000 r / min for 10 min, the supernatant was discarded, and finally the carboxymethyl chitosan-aptamer-carbon nanotube / nano-gold material was obtained, which was stored at 4°C in the dark for future use.

[0134] (4) Pseudomonas aeruginosa sample detection: Take 9 1.5 mL EP tubes, add 100 μL of the prepared composite adsorbent, add Pseudomonas aeruginosa bacterial solution, and dilute 6 times to 1:10, 1:10 respectively. 2 , 1:10 3 , 1:10 4 , 1:10 5 and 1:10 6 , incubate at 37°C for 30 min.

[0135] 1.5 mol / L NaCl solution was added to aggregate the released gold nanoparticles, and the absorbance A value at the maximum absorption wavelength was detected by UV-visible spectrophotometer, and a standard curve was drawn for quantitative determination.

[0136] Table 3. Test data of Pseudomonas aeruginosa samples at various concentrations

[0137] Concentration (cfu / mL) 10 <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 ]]> Absorbance (A value) 0.130 0.218 0.369 0.752 1.018 1.578

[0138] According to the above test data, the standard curve is y=0.03220×x+0.513; R=0.999.

[0139] For unknown sample B, the same serial dilution ratio as the standard curve was used to obtain 6 samples, and the same processing method as the standard was used to obtain the test data corresponding to the samples.

[0140] Table 4. Test data of unknown sample A at various concentrations

[0141] Preparation concentration (cfu / mL) 10 <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 ]]> Absorbance (A value) 0.10 Calculated concentration (cfu / mL) 87.500 150.000 255.000 432.500 625.000 1025.000

[0142] It can be seen that the unknown sample A contains Pseudomonas aeruginosa, and the corresponding concentration can be measured to obtain a quantitative result.

[0143] The reaction system obtained in this example has the following detection effect on the DNA sequence of Pseudomonas aeruginosa: Figure 2 As shown by Figure 3 It can be seen that when the incubation time is 30h, the detection effect is best.

[0144] Embodiment 3:

[0145] The method for detecting Pseudomonas aeruginosa in this embodiment specifically comprises the following steps:

[0146] (1) Preparation of carbon nanotube-gold nanoparticle solution: 1.65 mL of 1% chloroauric acid was taken into a 250 mL conical flask, 38.35 mL of water was added, and the conical flask was placed on a magnetic heating stirrer for reflux in the dark (a condenser was connected to the conical flask), a stirring bar was placed in the conical flask, and magnetic stirring was performed at the same time. When the solution boiled, 6.4 mL of 38.8 mmol / L trisodium citrate solution was immediately added, and reflux in the dark was continued, heating and stirring were continued, and boiling was maintained for 15 min. The heat source was removed and stirred until cooled to obtain a wine-red dispersed nanogold solution. After filtering the gold nanoparticle solution with a 0.22 μm needle-type water-soluble filter, it was placed in a 125 mL brown ground-mouth reagent bottle, wrapped with aluminum foil, and stored in the dark at 4°C for later use. After the CNTs were ultrasonically treated, HAuCl was added 4 The solution is mixed evenly and then reduced with a reducing agent to obtain a carbon nanotube-gold nanoparticle solution.

[0147] (2) Preparation of chitosan-aptamer complex: 0.3 g of carboxymethyl chitosan (purchased from BELLANCOM LIFESCIENCESDEP, 100 g / bottle) was placed in a 50 mL beaker, 10 mL of deionized water was added, and a carboxymethyl chitosan solution with a mass concentration of 30 mg / mL was obtained by stirring with a glass rod. 0.17 g of N-hydroxysuccinimide (NHS) was added to the above solution, and stirred for 15 min. Then, an aqueous solution of 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) (0.26 g of EDC was dissolved in a 15 mL centrifuge tube and 5 mL of deionized water was added) was slowly dripped into the solution in an ice-water bath at 2°C at 800 r / min. After the addition was completed for 10 min, the reaction was continued under stirring for 1 h in an ice-water bath to fully activate the carboxyl groups of the carboxymethyl chitosan. The specific aptamer of Pseudomonas aeruginosa (purchased from Sangon Biotech (Shanghai) Co., Ltd., with amino modification at the 5' end) was added to the above solution at a mass ratio of carboxymethyl chitosan to aptamer of 4:1 and treated for 1 hour to obtain the chitosan-aptamer complex.

[0148] (3) Preparation of composite adsorbent: Take 1 mL of the homemade carbon nanotube / nano-gold solution in a 1.5 mL EP tube, centrifuge at 4°C and 10,000 r / min for 10 min, discard the supernatant, and resuspend. Add 100 μL of the prepared chitosan-aptamer complex to the above nano-gold solution, mix well, and incubate in a shaker at 37°C for 30 h. In order to remove possible excess aptamers, the above mixture was centrifuged at 4°C and 10,000 r / min for 10 min, and the supernatant was discarded to finally obtain the carboxymethyl chitosan-aptamer-carbon nanotube / nano-gold material, which was stored at 4°C in the dark for future use.

[0149] (4) Pseudomonas aeruginosa sample detection: Take 9 1.5 mL EP tubes, add 100 μL of the prepared composite adsorbent, add Pseudomonas aeruginosa bacterial solution, and dilute 6 times to 1:10, 1:10 respectively. 2 , 1:10 3 , 1:10 4 , 1:10 5 and 1:10 6 , incubate at 37°C for 30 min.

[0150] 1.5 mol / L NaCl solution was added to aggregate the released gold nanoparticles, and the absorbance A value at the maximum absorption wavelength was detected by UV-visible spectrophotometer, and a standard curve was drawn for quantitative determination.

[0151] Table 5. Test data of Pseudomonas aeruginosa samples at various concentrations

[0152] Concentration (cfu / mL) 10 <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 ]]> Absorbance (A value) 0.131 0.178 0.227 0.281 0.341 0.407

[0153] According to the above test data, the standard curve is y=0.03218×x+0.135; R=0.999.

[0154] For unknown sample C, the same serial dilution ratio as the standard curve was used to obtain 6 samples, and the same processing method as the standard was used to obtain the test data corresponding to the sample.

[0155] Table 6. Test data of unknown sample A at various concentrations

[0156] Preparation concentration (cfu / mL) 10 <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 <!-- 10 -->]]> Absorbance (A value) 0.100 0.178 0.227 0.281 0.341 0.407 Calculated concentration (cfu / mL) 2.55 4.98 6.41 8.03 10.02 12.03

[0157] It can be seen that the unknown sample C contains Pseudomonas aeruginosa, and the corresponding concentration can be measured to obtain a quantitative result.

[0158] The above are preferred implementation modes and corresponding embodiments of the present invention. It should be pointed out that, for those skilled in the art, without departing from the creative concept of the present invention, several modifications and improvements may be made, including but not limited to the adjustment of proportions, processes, dosages and reaction vessels, which all fall within the scope of protection of the present invention.

Claims

1. A method for detecting Pseudomonas aeruginosa, characterized in that: include: Carboxymethyl chitosan is combined with a specific aptamer of Pseudomonas aeruginosa to form a chitosan-aptamer complex; The chitosan-aptamer complex is mixed with a carbon nanotube-gold nano-solution, so that the chitosan-aptamer complex is adsorbed on the surface of the carbon nanotube-gold nano-substance to obtain a composite adsorbed substance; Mixing the sample to be tested with the composite adsorbent to obtain a mixture to be tested; Performing qualitative or quantitative detection on the test mixture for Pseudomonas aeruginosa to obtain a detection result; Preferably, there is an amino modification in the nucleotide sequence of the specific aptamer; Preferably, one end of the nucleotide sequence of the specific aptamer has an amino modification.

2. The method for detecting Pseudomonas aeruginosa according to claim 1, characterized in that: The carboxymethyl chitosan is O,N-carboxymethyl chitosan.

3. The method for detecting Pseudomonas aeruginosa according to claim 1, characterized in that: The specific aptamer sequence is a cross-linked ring structure.

4. The method for detecting Pseudomonas aeruginosa according to claim 1, characterized in that: The nucleotide sequence of the specific aptamer is shown in SEQ ID NO.1; and a thiol modification group is connected to the 5' end of the nucleotide sequence.

5. The method for detecting Pseudomonas aeruginosa according to claim 1, characterized in that: In the chitosan-aptamer complex, the mass ratio of the carboxymethyl chitosan to the specific aptamer is (3-5):

1.

6. The method for detecting Pseudomonas aeruginosa according to claim 1, characterized in that: Before combining the carboxymethyl chitosan with the specific aptamer of Pseudomonas aeruginosa to form the chitosan-aptamer complex, the method further comprises: Activating the carboxymethyl chitosan; Preferably, the activation treatment comprises: Adding N-hydroxysuccinimide to the carboxymethyl chitosan; While the mixture was stirred, 1-ethyl-3-(dimethylaminopropyl)carbodiimide was added to complete activation.

7. The method for detecting Pseudomonas aeruginosa according to claim 6, characterized in that: While the mixture is being stirred, 1-ethyl-3-(dimethylaminopropyl)carbodiimide is added to complete activation, which is carried out in an ice-water bath.

8. The method for detecting Pseudomonas aeruginosa according to claim 6, characterized in that: In the activation treatment, the mass ratio of the carboxymethyl chitosan, the N-hydroxysuccinimide and the 1-ethyl-3-(dimethylaminopropyl)carbodiimide is (0.28-0.32):(0.15-0.18):(0.24-0.28).

9. The method for detecting Pseudomonas aeruginosa according to claim 1, characterized in that: In the step of mixing the chitosan-aptamer complex with the carbon nanotube-gold nano-solution to allow the chitosan-aptamer complex to be adsorbed on the surface of the carbon nanotube-gold nano-matter, the chitosan-aptamer complex is added to the carbon nanotube-gold nano-solution and then mixed and then subjected to a first incubation treatment; Preferably, the first incubation treatment lasts for 20 to 35 hours.

10. The method for detecting Pseudomonas aeruginosa according to claim 1, characterized in that: In the step of mixing the sample to be tested with the composite adsorbent, the sample to be tested and the composite adsorbent are mixed, followed by a second incubation treatment, and then an inorganic salt is added; Preferably, the second incubation treatment lasts for 20 to 30 minutes.

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