POCT type bacterial visual detection hydrogel and application thereof

Abstract

The application discloses POCT type bacterial visual detection hydrogel and application thereof, and belongs to the technical field of bacterial detection and detection result visualization. The POCT type bacterial visual detection hydrogel provided by the application comprises a hydrogel sensor and a hydrogel color developer. The POCT type bacterial visual detection hydrogel provided by the application can obtain a detection result in 30-60 min in an actual detection process, the detection limit is lower than 5 CFU / mL, and laboratory equipment and professional experiment personnel are not needed. The POCT type bacterial visual detection hydrogel is very suitable for application scenes, such as clinical treatment, outdoor activities and family, which need on-site detection, and can realize rapid and sensitive monitoring of bacteria.

Description

Technical Field

[0001] This invention belongs to the field of bacterial detection and visualization of detection results, and particularly relates to a POCT-type bacterial visualization detection hydrogel and its application. Background Technology

[0002] Bacterial pathogens are important targets for detection and identification in various fields such as medicine, food safety, public health, and security. Early diagnosis and treatment will effectively reduce the mortality rate caused by bacterial infections and are crucial for food security, environmental improvement, and disease prevention.

[0003] Currently, bacterial detection and diagnosis are primarily performed using laboratory-based methods. Traditional bacterial culture is widely considered the "gold standard" for clinical diagnosis of bacterial diseases, but it suffers from drawbacks such as its time-consuming nature and the inability of certain bacteria to grow on ordinary culture dishes. For example, in cases of bacterial meningitis, the longer the diagnostic time, the higher the probability of death or severe disabling sequelae (such as deafness). Current neuroimaging and post-culture staining techniques are very time-consuming, delaying crucial antibiotic treatment. Therefore, there is an urgent clinical need for rapid and accurate detection of bacteria in their native state or in an uncultured environment. Subsequent immunological methods, such as enzyme-linked immunosorbent assays (ELISA), are faster but complex to perform, with antibody quality affecting results. They also require large equipment and specialized personnel, and have limited detection efficiency. Molecular biological methods, such as polymerase chain reaction (PCR), offer high sensitivity, but require pre-selected gene probes to correctly pair with the target bacterial sequence. Incorrect pairing can lead to false positives, and genetically mutated strains may evade correct probe matching. Furthermore, PCR-based methods cannot distinguish between live and dead bacteria, which may be a potential limitation to the future development of PCR.

[0004] In recent years, many point-of-care testing (POCT) methods for detecting bacteria have been developed, including fluorescence, electrochemical, and colorimetric detection technologies. These methods are independent of laboratories, require no specialized medical personnel, and effectively save time on sample processing and transfer, thus improving diagnostic efficiency. Among them, color signals, as a simple and reliable direct readout analysis signal in POCT, are widely used in the construction of visual sensor analysis methods. Visual signals based on color changes mainly rely on the specific physical or chemical changes caused by the recognition of the target analyte by the sensing substrate (receptor), producing a color change signal that is recognizable to the naked eye. However, the detection limits of current colorimetric POCT technologies are generally higher than 10. 3The concentration of bacteria is limited to CFU / mL, and the color change range is small, making accurate quantification of bacterial concentration difficult. Furthermore, most POTC methods focus only on a single bacterial species (such as E. coli, Salmonella, etc.), but in practical applications, the absence of a particular bacterium in a clinical sample (or food sample) is insufficient to determine the absence of infection (bacterial contamination). Therefore, we need a broader range of detection methods, such as detecting Gram-negative or Gram-positive bacteria in samples. This is essential for guiding clinical medication: Gram-positive bacteria are more sensitive to penicillin-type drugs, while Gram-negative bacteria are not. Gram-positive bacteria are not sensitive to streptomycin and tetracycline-type drugs, while Gram-negative bacteria are.

[0005] In summary, developing new POCT-based bacterial detection technologies and methods with a wider range of detection targets and lower detection limits to achieve efficient and rapid identification and detection of low concentrations of bacteria has become a pressing issue that needs to be addressed. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a POCT-type bacterial visualization detection hydrogel and its application that can quickly and instantly detect bacteria with low detection limits and a wide range of detection targets.

[0007] To achieve the above objectives, in a first aspect of the present invention, the present invention provides a POCT-type bacterial visualization detection hydrogel, the hydrogel comprising a hydrogel sensor and a hydrogel chromogenic device;

[0008] The hydrogel sensor is prepared by the following steps:

[0009] An aqueous solution of bacterial extracellular hydrolytic enzyme substrate was mixed with an aqueous solution of chlorophenol red β-D-galactopyranoside to obtain a loading solution; then the loading solution was mixed with an aqueous solution of sodium alginate to obtain a hydrogel precursor solution; then the hydrogel precursor solution was subjected to a gelation reaction to obtain a hydrogel sensor.

[0010] The preparation of the hydrogel colorimeter agent includes the following steps:

[0011] After preheating the agarose aqueous solution, it is mixed with the β-galactosidase aqueous solution and stirred magnetically or mechanically under water bath heating conditions. Then, it is poured into a mold and cooled to obtain a hydrogel colorimeter.

[0012] The POCT-based bacterial visualization detection hydrogel provided by this invention includes a hydrogel sensor and a hydrogel chromogenic device. The hydrogel sensor releases different concentrations of chlorophenol red β-D-galactopyranoside (CPRG) based on the concentration or type of the test sample (bacterial culture). Subsequently, CPRG reacts with substances in the hydrogel chromogenic device to produce a colorimetric reaction, thereby detecting bacteria based on the color change. This invention relies on bacterial extracellular enzymes secreted by live bacteria for detection; therefore, the detection result represents the concentration of live bacteria in the sample, which has significant clinical value. Furthermore, the inventors have verified that the detection limit of the POCT-based bacterial visualization detection hydrogel provided by this invention is below 5 CFU / mL, far lower than existing detection limits, and the entire detection process can be completed within 30-60 minutes, meaning the detection time is short and meets the requirements for rapid and immediate detection.

[0013] The preparation method provided by this invention first involves loading a bacterial extracellular enzyme substrate with CPRG, followed by preparing a hydrogel precursor by combining it with sodium alginate aqueous solution, and then obtaining a hydrogel sensor through a gelation reaction. Subsequently, an agarose aqueous solution and a β-galactosidase aqueous solution are loaded together to obtain a hydrogel chromogenic device. Finally, the hydrogel sensor and hydrogel chromogenic device are combined to obtain a POCT-type bacterial visualization detection hydrogel. Specifically, in actual detection using the hydrogel provided by this invention, the hydrogel sensor can be responsively degraded by bacteria to release CPRG. Because different types (Gram-negative or Gram-positive bacteria) and different concentrations of bacteria produce different concentrations of bacterial extracellular enzymes, the concentration of CPRG released by the hydrogel sensor also varies. Different concentrations of CPRG react with β-galactosidase in the hydrogel chromogenic device to produce different color changes, thus achieving bacterial detection.

[0014] As a preferred embodiment of the POCT-type bacterial visualization detection hydrogel of the present invention, in the preparation of the hydrogel sensor: the concentration of the bacterial extracellular hydrolytic enzyme substrate aqueous solution is 5-10 wt%; the concentration of the chlorophenol red β-D-galactopyranoside aqueous solution is 0.05-2 mg / mL; and the volume ratio of the bacterial extracellular hydrolytic enzyme substrate aqueous solution to the chlorophenol red β-D-galactopyranoside aqueous solution is (1-5):1.

[0015] In a preferred embodiment of the POCT-type bacterial visualization detection hydrogel of the present invention, the concentration of sodium alginate aqueous solution is 1-5 wt% in the preparation of the hydrogel sensor; the volume ratio of the loading solution to the sodium alginate aqueous solution is 1:(2-4).

[0016] The inventors discovered that when raw materials within the above concentration range and the above raw material volume ratio range are used in the preparation of hydrogel sensors, the prepared hydrogels are more sensitive to bacterial extracellular enzymes in subsequent applications, and the stability of the prepared hydrogels is also superior.

[0017] As a preferred embodiment of the POCT-type bacterial visualization detection hydrogel of the present invention, the gelation reaction is to add an aqueous solution of calcium chloride to the hydrogel precursor solution, keep the two solutions in layers, and then let it stand at 25-37°C for 0.5-2 hours.

[0018] The inventors discovered that using a calcium chloride aqueous solution for gelation, and at the aforementioned gelation temperature and time, yields a hydrogel sensor and a hydrogel further formed from it with superior overall performance.

[0019] As a preferred embodiment of the POCT-type bacterial visualization detection hydrogel of the present invention, the bacterial extracellular hydrolytic enzyme substrate is at least one of collagen, gelatin, silk fibroin, hyaluronic acid, nucleic acid, cellulose, starch or N-acetylgalactoside.

[0020] Preferably, the molecular weight of the bacterial extracellular hydrolase substrate is 1-150 kDa.

[0021] Preferably, the bacterial extracellular hydrolytic enzyme substrate is a mixture of bacterial extracellular hydrolytic enzyme substrates with different molecular weights.

[0022] The inventors discovered that when bacterial extracellular enzymes of different molecular weights are combined with substrates, they become more sensitive to bacterial extracellular enzymes, thereby further reducing the detection limit and shortening the detection time.

[0023] As a preferred embodiment of the POCT-type bacterial visualization detection hydrogel of the present invention, in the preparation of the hydrogel sensor: the temperature for mixing and stirring the aqueous solution of bacterial extracellular hydrolytic enzyme substrate with the aqueous solution of chlorophenol red β-D-galactopyranoside is room temperature; the temperature for mixing and reacting the loading solution with the aqueous solution of sodium alginate is 40-60℃.

[0024] Preferably, the aqueous solution of the bacterial extracellular hydrolytic enzyme substrate and the aqueous solution of chlorophenol red β-D-galactopyranoside are mixed and stirred for 0.5-2 hours; the endpoint is determined when the solution is homogeneous and transparent with no insoluble matter or flocculent matter.

[0025] Preferably, the reaction time between the loading solution and the sodium alginate aqueous solution is 0.5-2 hours; the endpoint is determined when the solution has a uniform color and does not separate into layers after standing for 12 hours.

[0026] As a preferred embodiment of the POCT-type bacterial visualization detection hydrogel of the present invention, in the preparation of the hydrogel chromogenic device: the concentration of the agarose aqueous solution is 5-20 g / L; the concentration of the β-galactosidase aqueous solution is 5-1000 μg / mL; and the volume ratio of the agarose aqueous solution to the β-galactosidase aqueous solution is (1-5):1.

[0027] As a preferred embodiment of the POCT-type bacterial visualization detection hydrogel of the present invention, in the preparation of the hydrogel chromogenic device: the temperature of the agarose aqueous solution preheating is 37-50℃; the temperature of the mixture of the preheated agarose aqueous solution and the β-galactosidase aqueous solution for heating reaction is >37℃, and the time is 5-10min; the temperature after cooling is 4-25℃.

[0028] The inventors discovered that when raw materials within the above concentration range and the above raw material volume ratio range are used in the preparation of the hydrogel colorimeter, the prepared hydrogel is more sensitive to bacterial extracellular enzymes in subsequent applications, and the obtained hydrogel has better stability.

[0029] In a second aspect, the present invention also provides the application of the POCT-type bacterial visualization detection hydrogel in bacterial detection.

[0030] As a preferred embodiment of the application described in this invention, the application method is to integrate the hydrogel sensor and the hydrogel chromogenic device in the POCT-type bacterial visualization detection hydrogel into the cavity respectively, wherein the hydrogel sensor and the hydrogel chromogenic device are placed in two independent cavities. After the sample to be tested is added to the hydrogel sensor cavity and reacted for 30 minutes, the sample to be tested is then added to the hydrogel chromogenic device cavity and reacted for 30-60 minutes to complete the bacterial detection.

[0031] Preferably, the cavity is a commercially available 6-well plate, 12-well plate, or 24-well plate, or can be custom-made from metal, ceramic, or polydimethylsiloxane (PDMS) materials.

[0032] The POCT-type bacterial visualization detection hydrogel provided by this invention is simple to operate in actual testing, has a low detection limit, and provides fast detection results, enabling real-time and accurate detection.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention discloses a point-of-care testing (POCT) hydrogel for visual bacterial detection. The POCT hydrogel contains a hydrogel sensor and a hydrogel chromogenic device. The hydrogel chromogenic device is prepared by carrying β-galactosidase on an agarose polymer, which can react with CPRG to produce a colorimetric reaction. The RGB values ​​of the resulting hydrogel color are linearly correlated with the bacterial concentration, thus different bacterial concentrations correspond to different colors, enabling visual detection of bacterial concentration. Furthermore, the hydrogel provided by this invention can produce detection results within 30-60 minutes during actual testing, with a detection limit below 5 CFU / mL. It does not require laboratory equipment or professional personnel, making it highly suitable for clinical, outdoor, and home applications requiring on-site testing, enabling rapid and sensitive bacterial monitoring. Simultaneously, the preparation method of the POCT hydrogel provided by this invention is simple, requiring no complex equipment, which is beneficial for large-scale industrial production. Attached Figure Description

[0035] Figure 1 A linear relationship between the concentration of extracellular hydrolytic enzymes (taking hyaluronidase as an example) produced by different types of bacteria (Gram-negative and Gram-positive bacteria) and bacterial concentration.

[0036] Figure 2 The results of co-incubating different concentrations of different types of bacteria with the hydrogel sensor mentioned in this invention for 30 minutes are shown in the following figures:

[0037] A is a schematic diagram of the co-incubation of the hydrogel sensor and the bacterial solution;

[0038] B is a macroscopic morphological diagram of the hydrogel sensor after co-incubation with different types and concentrations of bacterial solutions;

[0039] C is a graph showing the CPRG content released after the hydrogel sensor was co-incubated with different types and concentrations of bacterial solutions;

[0040] Figure 3 The image shows the result of incubating the liquid with the hydrogel sensor for 30 minutes, followed by co-incubation with the hydrogel developer for another 30 minutes:

[0041] A shows the final color change results of the hydrogel chromogenic device caused by different types and concentrations of bacterial solutions;

[0042] B is a standard color chart drawn based on the results in A;

[0043] C is a standard curve plotted based on the results in A, showing the relationship between bacterial concentration and the RGB values ​​of the hydrogel colorimeter.

[0044] Figure 4 The figure shows the results of optimizing the incubation time of the hydrogel developer.

[0045] Figure 5 The results of the "clinical gold standard" testing method are shown in the image.

[0046] Figure 6 This is a reaction flow diagram of the present invention. Detailed Implementation

[0047] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0048] Example 1

[0049] This invention provides a POCT-based bacterial visualization detection hydrogel, the hydrogel comprising a hydrogel sensor and a hydrogel colorimeter;

[0050] The method for preparing the hydrogel sensor includes the following steps:

[0051] S1: Mix two volumes of 10wt% bacterial extracellular hydrolytic enzyme substrate aqueous solution (analytical grade hyaluronic acid with molecular weights of 7kDa and 150kDa, dissolved in deionized water at a mass ratio of 1:1) with one volume of 1mg / mL CPRG aqueous solution at room temperature, and use a magnetic stirrer to carry out the loading reaction until the solution is homogeneous and transparent without insoluble matter and flocculent matter, to obtain the loading solution;

[0052] S2: Mix 3 parts by volume of the loading solution with 6 parts by volume of 3wt% sodium alginate aqueous solution, and react at 50°C for 1 hour until the system is uniform in color and does not separate into layers after standing for 12 hours; to obtain the hydrogel precursor solution.

[0053] S3: Next, slowly add 2 mol / L calcium chloride aqueous solution to the hydrogel precursor solution, keep the two solutions in layers, and let stand at 30°C for 2 hours to carry out the gelation reaction to obtain the hydrogel sensor.

[0054] The preparation of the hydrogel chromogen includes the following steps:

[0055] One volume of 15 g / L agarose aqueous solution (analytical grade agarose) was preheated to 37°C and then mixed with one volume of 100 μg / mL β-galactosidase aqueous solution (chromatographic grade β-galactosidase). The mixture was heated and stirred for 10 min, maintaining the temperature above 37°C, specifically 40°C. After thorough mixing, the mixture was injected into a 48-well plate and cooled to 25°C to obtain a hydrogel chromogenic device.

[0056] Example 2

[0057] This invention provides a POCT-based bacterial visualization detection hydrogel, the hydrogel comprising a hydrogel sensor and a hydrogel colorimeter;

[0058] The hydrogel sensor is prepared by the following steps:

[0059] S1: Mix 2 volumes of 6.67 wt% bacterial extracellular hydrolase substrate aqueous solution (analytical grade gelatin dissolved in deionized water) with 1 volume of 1 mg / mL CPRG aqueous solution at room temperature, and load the reaction for 2 hours using a magnetic stirrer until the solution is homogeneous, transparent and free of insoluble matter and flocculent matter, to obtain the loading solution.

[0060] S2: Mix 3 parts by volume of the loading solution with 9 parts by volume of 5 wt% sodium alginate aqueous solution, and react at 40°C for 2 h until the system is uniform in color and does not separate into layers after standing for 12 h; to obtain the hydrogel precursor solution.

[0061] S3: Next, slowly add 1.5 mol / L calcium chloride aqueous solution to the hydrogel precursor solution, keep the two solutions in layers, and let stand at 25°C for 2 hours to carry out the gelation reaction to obtain the hydrogel sensor.

[0062] The preparation of the hydrogel chromogen includes the following steps:

[0063] Two volumes of 10 g / L agarose aqueous solution (analytical grade agarose) were preheated to 40°C and then mixed with one volume of 1000 μg / mL β-galactosidase aqueous solution (chromatographic grade β-galactosidase). The mixture was heated and reacted for 10 min, maintaining the temperature above 37°C, specifically 45°C, during the reaction. After the reaction was completed, the temperature was lowered to 25°C to obtain the hydrogel colorimeter.

[0064] Example 3

[0065] This invention provides a POCT-based bacterial visualization detection hydrogel, the hydrogel comprising a hydrogel sensor and a hydrogel colorimeter;

[0066] The hydrogel sensor is prepared by the following steps:

[0067] S1: Mix 3 volumes of 5 wt% bacterial extracellular hydrolytic enzyme substrate aqueous solution (analytical grade gelatin and hyaluronic acid dissolved in deionized water at a mass ratio of 1:1 to prepare a gelatin-hyaluronic acid mixed solution) with 1 volume of 2 mg / mL CPRG aqueous solution at room temperature, and carry out the loading reaction using a magnetic stirrer until the solution is homogeneous, transparent and free of insoluble matter and flocculent matter, to obtain the loading solution;

[0068] S2: Mix 3 parts by volume of the loading solution with 6 parts by volume of 1 wt% sodium alginate aqueous solution, and react at 60°C for 1 h until the system is uniform in color and does not separate into layers after standing for 12 h; to obtain the hydrogel precursor solution.

[0069] S3: Next, slowly add 2 mol / L calcium chloride aqueous solution to the hydrogel precursor solution, keep the two solutions in layers, and let stand at 37°C for 1 hour to carry out the gelation reaction to obtain the hydrogel sensor.

[0070] The preparation of the hydrogel chromogen includes the following steps:

[0071] Three volumes of 5 g / L agarose aqueous solution (analytical grade agarose) were preheated to 40°C and then mixed with one volume of 10 μg / mL β-galactosidase aqueous solution (chromatographic grade β-galactosidase). The mixture was heated and reacted for 10 min, maintaining the temperature above 37°C, specifically 50°C, during the reaction. After the reaction was completed, the temperature was lowered to 25°C to obtain the hydrogel colorimeter.

[0072] Example of effect 1

[0073] The theoretical basis for the prepared hydrogel is verified by the effect examples of this invention, specifically including the following steps:

[0074] First, commercially available extracellular hydrolase solutions with concentrations of 2, 4, 6, 8, 10, 15, and 20 mg / mL were prepared and mixed with a substrate solution of 2 mg / mL at a volume ratio of 1:1. The solutions were then incubated at 37°C for 30 min, and the concentration of the products generated after substrate degradation was measured. The functional relationship between "extracellular hydrolase concentration and product concentration" was obtained.

[0075] Secondly, preparations were made with a concentration of 10... 2 10 4 10 6 10 8 Staphylococcus aureus (representing Gram-positive bacteria) and Escherichia coli (representing Gram-negative bacteria) at CFU / mL were mixed with a substrate solution at a volume ratio of 2 mg / mL at a 1:1 ratio. The mixture was then incubated at 37°C for 30 min. The concentration of the products generated after substrate degradation was then measured. Substituting this concentration into the functional relationship equation of "hyaluronidase concentration - reducing sugar concentration after reaction" obtained in the first step, the concentration of extracellular hydrolases produced by different bacterial concentrations can be obtained.

[0076] The results obtained are as follows Figure 1 As stated, from Figure 1It can be seen that the concentration of extracellular hydrolytic enzymes secreted by bacteria is directly proportional to the bacterial concentration, showing a good linear relationship. Furthermore, different types of bacteria (Gram-negative and Gram-positive bacteria) have different enzyme production capabilities. Therefore, the type and concentration of bacteria can be further deduced by detecting the concentration of extracellular hydrolytic enzymes secreted by bacteria.

[0077] Example 2

[0078] The effectiveness of this invention is demonstrated by the following example: the hydrogel sensor prepared in Example 1 was placed in a 48-well plate, and samples were prepared at concentrations of 10... 2 10 4 10 6 10 8 Staphylococcus aureus (representing Gram-positive bacteria) and Escherichia coli (representing Gram-negative bacteria) at CFU / mL were added slowly to 48-well plates, covering the hydrogel sensor. The plates were then incubated at 37°C for 30 min. The plates were then removed to observe the reaction between the hydrogel sensor and different types and concentrations of bacterial solutions. The incubated bacterial solutions were also aspirated to determine the CPRG release from the hydrogel after bacterial degradation.

[0079] The results obtained are as follows Figure 2 As shown, A is a schematic diagram of the co-incubation of the hydrogel sensor with bacterial solution; B is a macroscopic morphological diagram of the hydrogel sensor after co-incubation with bacterial solutions of different types and concentrations; C is a diagram showing the content of CPRG released after co-incubation of the hydrogel sensor with bacterial solutions of different types and concentrations. Figure 2 It can be seen that the extracellular hydrolytic enzymes secreted by different types and concentrations of bacteria can cause different degrees of degradation of the hydrogel sensor and further release different concentrations of CPRG; and the linear relationship between the amount of CPRG released and the bacterial concentration is good, and the CPRG concentration can be used as an indicator for detecting bacterial concentration.

[0080] Example 3

[0081] The effectiveness of this invention is verified by incubating the liquid with the hydrogel sensor for 30 minutes and then co-incubating it with the hydrogel effector for another 30 minutes. Specifically, the hydrogel prepared in Example 1 was integrated into a 48-well plate, with the hydrogel sensor and colorimeter placed in two separate wells. Concentrations of 5, 10, 50, and 10 were prepared respectively. 2 500, 10 3 5000, 10 4 50000, 10 5The plate contained CFU / mL of Staphylococcus aureus (representing Gram-positive bacteria) and Escherichia coli (representing Gram-negative bacteria). Step 1: Slowly add 400 μL of the above bacterial solution to the wells containing the hydrogel sensor, then incubate the 48-well plate at 37°C for 30 min. Step 2: Transfer the incubated liquid to the wells containing the hydrogel developer.

[0082] The results obtained are as follows Figure 3 As shown, A represents the final color change results of the hydrogel chromogenic device caused by different types and concentrations of bacterial solutions; B represents the standard color chart plotted based on the results in A; when detecting bacterial solutions of unknown concentration, the bacterial concentration can be determined by comparing the color of the hydrogel chromogenic device with the standard color chart; C represents the standard curve plotted based on the results in A, showing the relationship between bacterial concentration and the RGB values ​​of the hydrogel chromogenic device color; when detecting bacterial solutions of unknown concentration, the bacterial concentration can be calculated by substituting the RGB values ​​of the hydrogel chromogenic device color into the standard curve plotted in the figure; further... Figure 3 It can be seen that different concentrations of bacteria degrade the hydrogel sensor, releasing CPRG; this further causes a color change in the hydrogel effector. Furthermore, from... Figure 3 As can be seen, different types of bacteria exhibit different color schemes. Gram-positive bacteria show a purple hue (the R value of the hydrogel effector after color change is greater than the B value), while Gram-negative bacteria show a pink hue (the R value of the hydrogel effector after color change is less than the B value). Furthermore, the color intensity caused by different concentrations of bacteria varies noticeably to the naked eye. Figure 3 It is known that this hydrogel detection system can detect bacterial concentrations as low as 5 CFU / mL.

[0083] Example of effect 4

[0084] The present invention records the detection time for different concentrations and types of bacteria in an example; the verification steps for the detection time of different bacteria are as follows:

[0085] 1. The hydrogel prepared in Example 1 is integrated into a 48-well plate, wherein the hydrogel chromogenic device and the hydrogel chromogenic device are placed in two separate wells respectively;

[0086] 2. Incubate the hydrogel sensor with 400 μL of bacterial cultures of different types and concentrations for 30 min;

[0087] 3. The incubated liquid was transferred to the well of the hydrogel developer for co-incubation for different times, and the color changes were recorded by taking pictures.

[0088] The results obtained are as follows Figure 4 As shown; from Figure 4 As can be seen, when detecting bacterial solutions of different concentrations and types (Staphylococcus aureus as a representative of Gram-positive bacteria, and Escherichia coli as a representative of Gram-negative bacteria), the hydrogel sensor gradually darkens in color over the first 30 minutes. However, after 30 minutes, the color of the hydrogel sensor stops changing; that is, no further color change occurs after 30 minutes and 40 minutes of incubation. This indicates that the endpoint of the color change reaction is reached within 30 minutes. Therefore, in practical applications, a reaction time of at least 30 minutes is sufficient to obtain results. Furthermore, to ensure detection efficiency, we recommend that the color development time should not exceed 60 minutes.

[0089] Example 5

[0090] The effectiveness of this invention was verified by integrating the hydrogel prepared in Example 1 into a 48-well plate, with the hydrogel sensor and chromogenic device placed in two separate wells. The hydrogel sensor of this invention was then compared with the current clinical gold standard detection method, the "culture method". Specifically, a dermatologist used a cotton swab to collect body fluid from a suspected infected area on the patient's skin, then sealed it in a sterile empty test tube and sent it to the laboratory. The laboratory technician used the cotton swab to inoculate the solid culture medium using the "four-zone streak method", and then placed the solid culture medium in an incubator for 24-72 hours.

[0091] The reaction flow diagram of the present invention is as follows: Figure 6 As shown; the results of the "clinical gold standard" testing method are as follows: Figure 5 As shown, from Figure 5 As can be seen, the "clinical gold standard" requires 24-48 hours to obtain test results, so the present invention has a significant advantage in effectiveness. Secondly, the "clinical gold standard" method can only report the number of bacteria in the test sample as "+", "++", "+++", and "++++" based on the results of the "four-zone line" and cannot give an accurate bacterial concentration. In comparison, the present invention has a significantly broadened detection range.

[0092] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An application of a POCT-type bacterial visualization detection hydrogel for non-disease diagnostic purposes in bacterial detection, characterized in that, The hydrogel includes a hydrogel sensor and a hydrogel colorimeter; The hydrogel sensor is prepared by the following steps: A mixture of bacterial extracellular hydrolytic enzyme substrate aqueous solution and chlorophenol red β-D-galactopyranoside aqueous solution was stirred until the solution was uniform in color, transparent and free of agglomerates or flocculent matter, to obtain a loading solution; then the loading solution was mixed with sodium alginate aqueous solution to obtain a hydrogel precursor solution; then the hydrogel precursor solution was subjected to a gelation reaction to obtain a hydrogel sensor. The preparation of the hydrogel colorimeter includes the following steps: The agarose aqueous solution is preheated and mixed with β-galactosidase aqueous solution. The mixture is then stirred magnetically or mechanically under water bath heating conditions, and then poured into a mold. After cooling, a hydrogel colorimeter can be obtained. The gelation reaction involves adding an aqueous solution of calcium chloride to the hydrogel precursor solution, maintaining the two solutions in separate layers, and then allowing it to stand at 25-37°C for 0.5-2 hours. The substrate of the bacterial extracellular hydrolytic enzyme is at least one of gelatin and hyaluronic acid; The application involves integrating the hydrogel sensor and hydrogel chromogenic device of a POCT-type bacterial visualization detection hydrogel into separate chambers. The hydrogel sensor and hydrogel chromogenic device are placed in two independent chambers. The sample to be tested is added to the hydrogel sensor chamber and reacted for 30 minutes, and then the sample to be tested is added to the hydrogel chromogenic device chamber and reacted for 30-60 minutes to complete the bacterial detection.

2. The application according to claim 1, characterized in that, In the fabrication of the hydrogel sensor: The concentration of the bacterial extracellular hydrolytic enzyme substrate in aqueous solution is 5-10 wt%; The concentration of chlorophenol red β-D-galactopyranoside in aqueous solution is 0.05-2 mg / mL; The volume ratio of the bacterial extracellular hydrolytic enzyme substrate aqueous solution to the chlorophenol red β-D-galactopyranoside aqueous solution was (1-5):

1.

3. The application according to claim 1, characterized in that, In the fabrication of the hydrogel sensor: The concentration of sodium alginate aqueous solution is 1-5 wt%; The volume ratio of the loading solution to the sodium alginate aqueous solution is 1:(2-4).

4. The application according to claim 1, characterized in that, In the fabrication of the hydrogel sensor: The aqueous solution of bacterial extracellular hydrolytic enzyme substrate was mixed and stirred at room temperature with an aqueous solution of chlorophenol red β-D-galactopyranoside. The loading solution is mixed with sodium alginate aqueous solution and heated in a water bath at a temperature of 40-60℃.

5. The application according to claim 1, characterized in that, In the preparation of the hydrogel colorimeter: The concentration of agarose aqueous solution is 5-20 g / L; The concentration of β-galactosidase in aqueous solution is 5-1000 μg / mL; The volume ratio of agarose aqueous solution to β-galactosidase aqueous solution is (1-5):

1.

6. The application according to claim 1, characterized in that, In the preparation of the hydrogel colorimeter: The preheating temperature of the agarose aqueous solution is 37-50℃; The agarose aqueous solution was preheated and then mixed with the β-galactosidase aqueous solution and heated to a temperature >37℃ for 5-10 min. The temperature after cooling is 4-25℃.

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