A bacteriophage and beta-galactosidase co-modified detection stirring rod, and a preparation method and application thereof

Abstract

The application discloses a kind of based on bacteriophage and beta-galactosidase co-modification detection stirring rod and its preparation method and application, belong to analytical detection technical field, mainly for the field detection of pseudomonas aeruginosa ATCC 15442 type.The detection stirring rod of the application uses wooden fiber stick as substrate, and the surface of substrate is modified with poly diallyl dimethyl ammonium chloride, and bacteriophage and beta-galactosidase are co-modified on poly diallyl dimethyl ammonium chloride;Bacteriophage is immobilized by the dual effect of covalent binding and electrostatic adsorption, and beta-galactosidase is immobilized by electrostatic adsorption.The stirring rod of the application integrates enrichment-detection function, and the total detection time is less than or equal to 6 min, the detection limit is as low as 4 CFU / ml, the specificity is strong and there is no cross reaction, the cost is low, the operation is simple, the recovery rate of real sample detection reaches 83%-118%, and it is suitable for multi-scene on-site rapid detection demand.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a detection stirring rod based on co-modification of bacteriophage and β-galactosidase, its preparation method and application. Background Technology

[0002] Bacterial infections are a significant threat to global health, ranging in severity from localized skin infections to life-threatening systemic diseases like sepsis. *Pseudomonas aeruginosa*, a typical opportunistic pathogen, is often found in low concentrations in lakes, tap water outlets, and bottled water production facilities. It exhibits strong drug resistance and can cause serious or even fatal infections in certain high-risk populations. Currently, detection methods for *Pseudomonas aeruginosa* primarily rely on traditional bacterial culture methods and techniques based on molecular biology and immunology (such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA)). However, these methods also have limitations. Traditional methods require significant time and labor investment and are susceptible to environmental influences. PCR requires specialized testing equipment and is cumbersome and time-consuming. ELISA requires expensive antibodies, has high detection limits, and is prone to false results.

[0003] Currently, there is still a lack of rapid on-site detection methods for the conditionally pathogenic bacterium Pseudomonas aeruginosa. Summary of the Invention

[0004] In view of this, the present invention provides a pre-concentrated stirring rod modified with β-galactosidase (β-gal) and phage-directed modification for trace enrichment detection of Pseudomonas aeruginosa, achieving detection on the rod itself. In short, β-gal exhibits excellent enzymatic catalytic properties, rapidly catalyzing the substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) to produce a yellow color, and also decomposing 4-methyl-7-oxocoumarin-β-D-galactopyranoside (MUG) to produce fluorescence, enabling a dual-mode detection. In the presence of Pseudomonas aeruginosa, the enzyme's active site is shielded, thus significantly reducing its catalytic properties. The detection time is within 6 minutes, and the limit of detection is 12 CFU / ml. Results from real samples show no significant difference between this method and the spiked results, demonstrating broad application prospects in the detection of environmental pathogens.

[0005] This invention designs a portable colorimetric biosensor (detection stirring rod), such as... Figure 6As shown, woody fiber rods co-modified with bacteriophage and β-galactosidase (β-gal) were used for on-site identification of *Pseudomonas aeruginosa* strain ATCC 15442. β-gal, a purified enzyme derived from *Escherichia coli*, preferentially hydrolyzes lactose and exhibits excellent enzymatic catalytic properties. It catalyzes the substrate ONPG (o-nitrophenyl-β-D-galactopyranoside) to produce a yellow color at 420 nm and decomposes MUG (4-methyl-7-oxocoumarin-β-D-galactopyranoside) to produce fluorescence at 423 nm. The stirring rod was modified with polydiallyldimethylammonium chloride (PDDA), and the bacteriophage was modified using EDC / NHS to activate the carboxyl group in the bacteriophage head. The carboxyl group in the bacteriophage head then covalently binds to the amino group on PDDA. Meanwhile, PDDA is positively charged and the phage head is negatively charged, which can be physically modified through electrostatic adsorption, achieving dual physical and chemical modification. β-gal is also negatively charged, so physical modification of β-gal can be achieved while preserving the stability of the natural enzyme's catalytic properties.

[0006] Mechanistically, enzyme catalysis is like a rapid and intense rainstorm, while the binding of bacteriophages to target bacteria is like opening an umbrella. The higher the concentration of target bacteria, the larger the umbrella, and the more significant the signal drop. However, the addition of other bacteria did not significantly change the A420nm and F423nm values ​​of the reaction system. Therefore, this portable colorimetric biosensor was successfully constructed, enabling on-site trace detection of target bacteria based on the high specificity of bacteriophage recognition.

[0007] Specifically, in a first aspect, the present invention provides a detection stirring rod based on phage and β-galactosidase co-modification for rapid detection of Pseudomonas aeruginosa, the detection stirring rod comprising: The substrate is a wood fiber rod; The substrate surface is modified with polydiallyldimethylammonium chloride; The polydiallyl dimethylammonium chloride was co-modified with bacteriophage and β-galactosidase; The bacteriophage is modified onto polydiallyldimethylammonium chloride through a combination of covalent binding and electrostatic adsorption, and the β-galactosidase is modified onto polydiallyldimethylammonium chloride through electrostatic adsorption.

[0008] Furthermore, the bacteriophage is ATCC 15442 type Pseudomonas aeruginosa bacteriophage.

[0009] In a second aspect, the present invention provides a method for preparing the above-described detection stirring rod, comprising the following steps: (1) The wood fiber rod was immersed in a polydiallyldimethylammonium chloride solution and stirred and incubated, and then dried under vacuum to obtain a stirring rod modified with polydiallyldimethylammonium chloride. (2) After ultrafiltration of the phage, the pH was adjusted, and after activation by EDC and NHS, it was incubated with the stirring rod of step (1) to obtain the phage-modified stirring rod; (3) After washing the stirring rod from step (2), immerse it in β-galactosidase solution and incubate it by shaking. After washing, immerse it in BSA solution to seal it, and the detection stirring rod is obtained.

[0010] Furthermore, in step (1), the stirring incubation temperature is 20~30℃ and the time is 1~3h; the vacuum drying temperature is 35~40℃ and the time is 0.5~1.5h.

[0011] Further, in step (2), the phage is adjusted to pH 5.0-6.0 with EMS buffer, the concentration of EDC and NHS is 0.5-1.5 mmol / L, the activation time of EDC is 10-30 min and the activation time of NHS is 20-40 min, and the pH is adjusted to 6-8 with PBS after activation; the incubation temperature of the phage and the stirring rod is 20-30℃ and the time is 2-4 h.

[0012] Furthermore, in step (3), the incubation conditions for the β-galactosidase solution are 20~28℃ with shaking incubation for 1~3h; after incubation, it is washed with deionized water.

[0013] Furthermore, in step (3), the concentration of the BSA solution is 0.5~1.5% (w / v), and the sealing conditions are incubation at 2~8℃ for 12~24h.

[0014] In a third aspect, the present invention provides the application of the detection stirring rod described in any of the preceding claims in the detection of Pseudomonas aeruginosa.

[0015] Furthermore, during the detection process, the enrichment time of Pseudomonas aeruginosa on the detection stirring rod is 1-3 min, and the color development / fluorescence reaction time of the substrate is 1-3 min.

[0016] Furthermore, the detection stirring rod is used for rapid on-site detection of Pseudomonas aeruginosa in samples, including vegetable garden samples, medical wastewater, hospital wastewater, tap water, natural drinking water, and bottled mineral water.

[0017] In a fourth aspect, the present invention provides a rapid on-site detection kit for Pseudomonas aeruginosa, comprising a detection stirring rod based on phage and β-galactosidase co-modification as described in any one of the preceding claims, and further comprising a detection substrate and a buffer solution; wherein the detection substrate comprises o-nitrophenyl-β-D-galactopyranoside (ONPG) and 4-methyl-7-oxocoumarin-β-D-galactopyranoside (MUG).

[0018] The beneficial effects of this invention include at least the following: (1) The stirring rod provided by the present invention has a fast detection speed (total time ≤ 6 min), which is far superior to traditional PCR, ELISA and other methods; the detection limit is as low as 4 CFU / ml, which can efficiently capture trace amounts of Pseudomonas aeruginosa (ATCC 15442 type); relying on the high specificity recognition of bacteriophage, it only responds to the target bacteria and has no cross-reaction to other strains and other types of Pseudomonas aeruginosa, and has both colorimetric + fluorescence dual-mode detection, with high visualization.

[0019] (2) The detection rod provided by the present invention is easy to operate and integrates the "enrichment-detection" function. It does not require complex pretreatment and professional equipment. It is inexpensive and the reagents used (ONPG, MUG, etc.) are easy to obtain, avoiding the use of expensive antibodies in ELISA. It is highly stable, the phage is resistant to extreme temperatures and pH, the β-galactosidase catalytic activity is stable, and the non-specific adsorption is reduced by BSA blocking. The detection recovery rate of real samples such as vegetable field samples, hospital wastewater, and drinking water reaches 83%-118%, and the accuracy is reliable, which is suitable for the on-site detection needs of multiple scenarios. Attached Figure Description

[0020] Figure 1 Images show the characterization of the stir bar and bacteria. (A) SEM of the unmodified stir bar. (B) SEM of the modified stir bar. (C) A single bacteriophage. (D) The initial binding behavior of bacteriophage with bacteria ATCC 15442. (E) The binding behavior of bacteriophage with bacteria ATCC 15442 after a period of time. (F) Dead and live bacteria captured on the stir bar. (G) Fully live bacteriophages and attenuated and inactivated plaque experiments. Figure 2 Construction and validation of a phage-modified stir bar for bacterial detection. (A) UV-Vis spectra of three reaction systems. (B) Comparison of the ONPG-β-gal chromogenic system before and after bacterial and sterile detection. (C) Feasibility analysis under fluorescence imaging. (D) Fluorescence imaging of phage cyto staining and fluorescence imaging of the stir bar after phage modification staining. (E) Plaque presentation of different bacteria on a stir bar modified with a specific ATCC15442 phage.

[0021] Figure 3Kinetic characterization of the β-gal modified sensor and fluorescence spectra of phage staining. (A) Steady-state kinetic analysis of the β-gal-based colorimetric fluorescence sensor glycoside hydrolase: (a) The Michaelis-Menten curve (left) and (b) The Lineweaver-Burk plot (right). (B) Steady-state kinetic analysis of the β-gal-modified stir bar: (a) The Michaelis-Menten curve (left) and (b) The Lineweaver-Burk plot (right). (C) Fluorescence spectra of phage cytosyl staining. (D) Staining of live and dead bacteria killed by phage at different time points.

[0022] Figure 4 Optimization of reaction conditions for a β-gal and phage-based detection system. (A) Optimization of enzyme activity. (B) Optimization of phage concentration. (C) Optimization of native enzyme color development temperature. (D) Whether to use BSA blocking. (E) Optimization of BSA blocking concentration. (F) Optimization of color development time. (G) Optimization of enrichment time.

[0023] Figure 5 Performance analysis of colorimetric-fluorescence dual-mode sensor for detecting *Pseudomonas aeruginosa*. (A) ONPG colorimetric system for different concentrations of *P. aeruginosa*. (B) Linear fitting between absorbance of the colorimetric system at 420 nm and the logarithm of *P. aeruginosa* concentration. (C) MUG fluorescence system for different concentrations of *P. aeruginosa*. (D) Linear fitting between fluorescence system at 423 nm and the logarithm of *P. aeruginosa* concentration. (E) Using a portable colorimetric sensor to capture 10... 1 CFU / ml-10 7 Bioluminescent imaging of Pseudomonas aeruginosa at CFU / ml. (F) The anti-interference capability of the colorimetric sensor is determined by the difference in absorbance before and after the presence of bacteria.

[0024] Figure 6 To illustrate the detection principle, construction, and application of a portable sensor. (A): The colorimetric mechanism of enzyme-induced probe detection. (B): The fabrication process of a β-gal-based portable colorimetric sensor and its colorimetric detection of Pseudomonas aeruginosa. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0027] The reagents and instruments used in this invention are as follows: β-galactosidase (β-gal) was purchased from Shanghai Shifeng Biotechnology Co., Ltd. (Shanghai, China); o-nitrobenzene-β-D-galactopyranoside (ONPG) was purchased from Nanjing Dulai Biotechnology Co., Ltd. (Nanjing, Jiangsu, China); N-hydroxysuccinimide (NHS) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China); 4-methyl-7-oxocoumarin-β-D-galactopyranoside (MUG), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), polydienedimethylammonium chloride solution (PDDA wt% aqueous solution), bovine serum albumin (BSA), and phosphate buffer were purchased from Shanghai Aladdin Information Technology Co., Ltd. (Shanghai, China); dimethyl sulfoxide (DMSO) was purchased from YuanYe Biotechnology (Shanghai, China); LB broth was purchased from Guangdong Huankai Microbial Technology Co., Ltd. (Guangzhou, China); agar powder was purchased from Beijing Coollabo Technology Co., Ltd.; and Pseudomonas aeruginosa (ATCC) was also purchased. 15442) and its bacteriophage were obtained from the Guangdong Provincial Microbial Culture Collection Center.

[0028] The ultraviolet spectrophotometer was purchased from Shimadzu Corporation (Kyoto, Japan), the fluorescence spectrophotometer from Hitachi High-Tech Corporation (Ibaraki Prefecture, Japan), the animal in vivo imaging system from Shanghai Tianneng Life Science Co., Ltd. (Shanghai, China), the microplate spectrophotometer from Bio-Rad Life Science Research Co., Ltd. (Shanghai, China), the fully automated gel imaging analysis system from Beijing Saizhi Venture Technology Co., Ltd. (Beijing, China), the fluorescence microscope from Guangzhou Mingmei Optoelectronic Technology Co., Ltd. (Guangzhou, China), the vacuum drying oven and intelligent dark box type three-in-one ultraviolet spectrophotometer from Shanghai Jinpeng Analytical Instruments Co., Ltd. (Shanghai, China), and the benchtop high-speed centrifuge from Hunan Kecheng Instrument Equipment Co., Ltd. (Changsha, Hunan).

[0029] The following specific embodiments illustrate the solution proposed in this invention: Example 1 The experimental methods include: (1) Pathogen culture Pseudomonas aeruginosa (ATCC 15442) was incubated overnight in LB medium at 37°C with shaking at 180 rpm. Bacterial concentration was determined using a standard counting method. The bacteria were diluted in several different 10-fold incremental dilutions, and 100 μL of each dilution was coated onto LB agar plates and incubated at 37°C for 24 hours. The number of colonies formed in each petri dish was recorded, and the total bacterial count per milliliter of the original solution was calculated based on the dilution. Cell counts are expressed as CFU / mL.

[0030] (2) Culture of bacteriophages Phage culture was performed using the double-layer plate method. The semi-solid culture medium was melted and dispensed into 3 mL portions, which were then incubated in a 53°C water bath for later use (Step 1). The cultured bacteria were diluted in the corresponding 3 mL of culture medium until the medium became slightly turbid, and set aside (Step 2). The phage stock solution was serially diluted 10-fold to prepare multiple phage solutions of different concentrations, and set aside (Step 3). 100 μL each of the bacteria and phage prepared in Steps 1 and 2 were added to the dispensed semi-solid culture medium, gently shaken to mix, and then poured onto a solid plate, spreading it evenly. After solidification, the plate was inverted and incubated at 35°C for 12 hours (Step 4). The density of phage plaques on the culture dish depends on the phage concentration. The semi-solid culture medium containing bacteriophages was scraped from the petri dish using a spreader, and then an appropriate amount of physiological saline was added. The scraped mixture was centrifuged at 8000 rpm for 30 min, and then the liquid suspension was passed through a 0.22 μm disposable filter. The filtered liquid is a freshly purified bacteriophage suspension. At least two more purifications are required to obtain the desired bacteriophage suspension. For plaque experiments using stir bar-modified bacteriophages, simply replace the original bacteriophage solution with a bacteriophage-modified stir bar.

[0031] (3) Synthesis of phage-modified stir bar Cellulose sticks were immersed in polydiallyldimethylammonium chloride (PDDA, 2 ml, 2%) and stirred at 25°C for 2 hours to fix the PDDA. The sticks were then removed and vacuum-dried at 37°C for 1 hour. After ultrafiltration of the phages, the pH was adjusted to 5.5 using EMS buffer solution, followed by activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 1 mmol / L, 2 ml) for 20 min, and then activated with N-hydroxysuccinimide (NHS, 1 mmol / L, 2 ml) for 30 min. After ultrafiltration, the pH was adjusted to 7 using PBS. The sticks were washed once with deionized water, drained, and incubated with the phage suspension (1.0 × 10¹¹ PFU / mL, 2 mL) at 25°C for 2–4 hours.

[0032] (4) Synthesis of stir bar co-modified with β-gal and bacteriophage After removing the modified phage stir bar, wash it several times with deionized water, drain it, and place it in β-gal solution (2U, 2ml). Incubate it at 23°C and 155rpm for 2 hours. Remove the stir bar, wash it with deionized water, and incubate it overnight at 4°C with bovine serum albumin (BSA, 1%, 2ml).

[0033] (5) Pseudomonas aeruginosa ATCC 15442 in the standard sample During the detection process, the prepared stirring rod was incubated in the prepared ONPG (0.001 g / ml) substrate for 2 min as a blank control. Then, the stirring rod was washed multiple times with deionized water and placed in a 2 ml standard sample solution. It was enriched with different concentrations of ATCC 15442 at 25℃ and 155 rpm for 2 min. After washing three times with deionized water, it was incubated in ONPG solution for 2 min, and the bacterial concentration was determined by measuring the change in absorbance before and after incubation. Similarly, the substrate was replaced with 4-methyl-7-oxocoumarin-β-D-galactopyranoside (MUG). The MUG solution was prepared at a concentration of 1 mmol / L, and the total volume of the reaction buffer PBS was 2 ml (pH 7.4, containing 30% dimethyl sulfoxide). The above steps were repeated.

[0034] (6) Detection of real samples Samples were collected from vegetable gardens near the Second People's Hospital of Guangdong Province, hospital wastewater, and medical wastewater from relevant departments. Local tap water and bottled mineral water from Qiandao Lake were also collected. These samples demonstrated the practicality of the proposed detection method. For vegetable garden samples containing sand and hospital wastewater, solids were removed by centrifugation at 5000 rpm for 5 minutes. Then, each sample was spiked with Pseudomonas aeruginosa at concentrations of 102 CFU / ml and 105 CFU / ml for detection.

[0035] (7) Determination of catalytic kinetic parameters ONPG substrate concentration gradients were set (0.3, 0.15, 0.075, 0.0375, 0.01875, 0.009, and 0.0047 M). The reaction was detected within 5 minutes under optimal conditions, and the initial reaction rate was recorded. Kinetic parameters (Kp) were established. m and V max The value is calculated based on the Michaelis-Menten equation. max It is the maximum reaction rate, K. mis the Michaelis constant, [s] is the substrate concentration, and V is the initial reaction rate.

[0036] (8) Validation of the phage adsorption stirring rod Take 50 μL of phage stock solution of the same concentration from two 0.5 mL centrifuge tubes (Step 1). Incubate the PDDA-modified and EDC / NHS-activated stir bar in one of the phage stock solutions for 30 min (Step 2). Dilute 10 μL of CYTO-13 working solution with 990 μL of PBS buffer and stain using a 1:1 volume ratio of working solution to phage. Incubate at room temperature in the dark for 60 min, then aspirate the supernatant and add PBS to the original volume (Step 3). Then, take pictures using a gel imaging system and scan the spectrum with a fluorescence spectrophotometer. Similarly, stain the phage stock solution of the same concentration again using the above steps, incubate the modified stir bar in the stained phage for 30 min, and then take pictures of the stir bar using an animal in vivo imaging system.

[0037] (9) Phage staining The phage stock solution before and after stir bar modification was fluorescently stained and photographed using a fluorescence microscope. The staining steps are as follows: Dilute 10 μL of CYTO-13 working solution with 990 μL of PBS buffer, then add 50 μL of the phage solution to a 1.5 mL centrifuge tube and mix thoroughly. Incubate at room temperature for 60 min. Then remove most of the liquid from the tube and add 100 μL of PBS buffer, mixing thoroughly again. Take another 1 mL of the bacterial suspension that has been cultured for 6-8 hours, centrifuge at 13000 rpm for 1 min, remove the supernatant, and resuspend the bacterial pellet in an equal volume of PBS buffer. This washing process needs to be repeated twice. Pipette 50 μL of the bacterial suspension into the above centrifuge tube, mix well, and incubate at 37°C for 10 min. After incubation, take 10 μL, spread it on a glass slide, let it dry, and observe it under a fluorescence microscope.

[0038] The experimental results are as follows: (1) Characterization of stir bar co-modified with β-gal and bacteriophage By modifying the stir bar with bacteriophage, its ability to enrich bacteria and specifically capture bacteria is obtained. Then, by modifying it with β-gal and blocking it with 1% BSA, its detection performance as a signal element is achieved. The stir bar with PDDA adsorption and modified enzyme and bacteriophage exhibits a more compact planar structure. Figure 1B), while the control group showed a rougher appearance (Figure 1A). Furthermore, plaque experiments confirmed that the ATCC 15442 phage is a warm-type phage, thus it can capture both live and dead bacteria in a short time. Dead bacteria exhibit loss of integrity, shrinkage, and perforation, while live bacteria remain intact in a rod-like shape (Figure 1F). To clearly understand the binding behavior of phages and bacteria, transmission electron microscopy (TEM) revealed the tadpole-like structure of the phage, approximately 280 nm in length, including the phage capsid and tail structure. Figure 1 C). As shown in Figures 1D and 1F, after a period of time, several tadpole-shaped bacteriophages adhered tightly to the bacteria, demonstrating the specificity of the phage's binding to bacteria. The phage plaques (Figure 1E) resulting from the modification of the stir bar by different active phages showed a gradual decline in their ability to kill the target bacteria on a macroscopic scale.

[0039] (2) Feasibility of detecting Pseudomonas aeruginosa The catalytic cleavage ability of β-gal on the substrate ONPG was evaluated. Under neutral conditions at room temperature, β-gal rapidly catalyzed ONPG and MUG, producing a strong colorimetric fluorescence signal (Figures 2A and 2C), while no color change was observed in the control group. Therefore, the detection capability of the portable colorimetric fluorescence sensor based on β-gal was verified. The results showed that without the addition of P. aeruginosa, this β-gal sensor could rapidly catalyze the substrate to develop color and had a significant colorimetric fluorescence signal. However, after adding P. aeruginosa, the catalytic efficiency decreased and the reaction system value decreased significantly. Therefore, this portable colorimetric fluorescence biosensor can be used to detect P. aeruginosa (Figures 2B and 2C). Simultaneously, the bioactivity of ATCC 15442 phage directionally modified onto the stir bar was verified through on-site testing. Cyto fluorescence staining and imaging of the phage stock solution revealed a strong fluorescence signal. However, after directional modification of the stir bar, the signal of the phage stock solution was significantly weakened, and fluorescence with a certain signal was observed on the stir bar. Therefore, ATCC 15442 phage was successfully modified onto the stir bar (Figure 2D). Similarly, cyto fluorescence staining of the phage stock solution and imaging using a fluorescence microscope (Figure 3B) showed a significant change in fluorescence intensity after phage modification of the stir bar. The modification of bacteriophages was also verified in the plaque assay using a stir bar. Plaque assays were performed on stir bars co-modified with Pseudomonas aeruginosa ATCC 15442 phage and β-gal, using different strains and serotypes of Pseudomonas aeruginosa, including Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus, Salmonella, Listeria, Escherichia coli CMCC 44484, and Pseudomonas aeruginosa ATCC 27853. The results showed that only plates containing Pseudomonas aeruginosa ATCC 15442 had obvious plaques, while other strains and serotypes showed almost no plaques. Figure 2 E). This demonstrates that this portable colorimetric / fluorescent biosensor based on β-gal exhibits phage activity.

[0040] (3) Mechanism exploration Given that bacteriophages can specifically capture this type of pathogen, portable biosensors with glycoside hydrolase activity have the potential to significantly and specifically detect *Pseudomonas aeruginosa*, thus contributing to further elucidation of its fundamental mechanisms. (Based on supplementary materials) Figure 4 F) indicates that this natural enzyme achieves a significant catalytic effect within 2 minutes, with a noticeable color change. Enzyme kinetic data can intuitively reflect its catalytic ability towards the substrate, V max This reflects the enzyme's catalytic rate on the substrate when the substrate concentration is sufficiently high and in excess, while K... mThis represents the substrate concentration at which the enzyme's rate reaches half. The lower the substrate concentration, the stronger the enzyme's affinity for the substrate. Here, Km is 0.26 M / L, and V... max It is 0.48 (10⁻³ mol / L S⁻¹). Figure 3 A) Both demonstrate that β-gal possesses excellent catalytic activity. Furthermore, when β-gal is modified on the stir bar, the amount of adsorbed enzyme increases significantly, and its catalytic rate Vm also accelerates significantly to 4.46 (10⁻³ mol / L S⁻¹), k m The value is 0.05M ( Figure 3 B) can catalyze the color development of more substrates, while also enhancing its detection sensitivity when shielding signal sites. Cyto fluorescence staining of the phage stock solution before and after modification of the stir bar was performed (Figure 3C). The number of phages adsorbed on the stir bar was determined by analyzing the differences in spectral peaks, and this number was calculated using the following formula.

[0041] Dphage=(A0-A) / A0×Cphage×V / (S×N) Formula 1 Where Dphage is the adsorption density of phage per unit area of ​​the stir bar; A0 and A are the FL intensity before and after phage modification of the stir bar, respectively; Cphage and V are the initial concentration and volume of the phage suspension, respectively; S and N are the surface area of ​​the phage and the stir bar, and the number of stir bar, respectively. The initial FL intensity of the phage suspension (1 mL, 10T) is... 9 The PFU / mL concentration was 9764. After incubation with a stir bar, it decreased to 5845. The contact surface area was approximately 0.628 cm². 2 From Formula 1, we can obtain Dphage = 1.28 × 10 8 PFU / ml. Simultaneously, the bactericidal effect of the phage was assessed by staining for both live and dead bacteria. Within 10 minutes, it showed virtually no killing effect on the target bacteria; therefore, this live phage can be used as a probe for detecting live bacteria, while the attenuated phage can be used as a probe for detecting total bacteria. Figure 3 D).

[0042] These results demonstrate that the β-gal-based portable colorimetric sensor exhibits good selectivity and excellent enzyme catalytic effect against ATCC 15442 Pseudomonas aeruginosa.

[0043] (4) Optimization of detection conditions Enzyme activity is crucial to the sensitivity of the entire detection system. Here, enzymes with different activities were used to modify the stir bar (Figure 4A). Higher enzyme activity resulted in higher catalytic efficiency, faster catalytic speed, and a more significant increase in absorbance. However, once the enzyme modification reached a certain level, the catalytic effect on the substrate plateaued, and absorbance remained relatively stable thereafter. The amount of phage modification directly affects the capture efficiency of the target bacteria. Stirring bars were incubated with phages of different concentration gradients, and the difference in absorbance before and after phage capture of bacteria was observed. Figure 4 (B) As the phage concentration increases, the absorbance continuously increases. When the phage concentration reaches 10⁸ PFU / ml, the phage-bound bacteria's shielding effect on β-gal reaches its optimum, after which the absorbance changes little. Since the natural enzyme is extremely sensitive to temperature, colorimetric conditions at different temperatures were compared (Figure 4C). The absorbance gradually decreases after 25℃. At 37℃, because this exceeds the optimal temperature for phage-bound bacteria, the absorbance decreases to some extent. With further increases in temperature, the enzyme gradually becomes inactive. Because the wooden stirring rod readily produces non-specific adsorption, it needs to be sealed with BSA. Figure 4 Compared to a stir bar modified only with β-gal, the non-specific adsorption caused by the difference in absorbance before and after BSA blocking was significantly reduced (A0-A). Optimization with different concentrations of BSA (Figure 4E) revealed that excessively high BSA concentrations would compete with β-gal, leading to decreased catalytic efficiency, while insufficient concentrations would result in incomplete blocking, non-specific adsorption, and reduced target bacterial shielding. 1% BSA showed the best effect, while low concentrations of 0.001%, 0.01%, 0.1%, and 0.5% BSA showed little change in absorbance, indicating no competitive reaction and insufficient blocking, thus reducing the specific shielding effect of bacteria on the sensor's catalytic performance. Time optimization was also performed, including color development time and enrichment time. Figure 4 For both F and 4G, 2 minutes yielded the best results. A blank control was added with a 2-minute enrichment time, bringing the total time to within 6 minutes. Beyond 2 minutes, the bacterial specificity decreased because the target bacteria concentration was relatively low compared to the bacteriophage. Prolonged enrichment caused the bacteriophage to detach from the stir bar, reducing the specific enrichment of the target bacteria and thus decreasing signal shielding. This method achieves rapid detection of pathogenic bacteria within 6 minutes, shorter than qPCR (87 minutes) and the single-tube detection platform combining RPA and CRISPR / Cas12a (80 minutes). This method is based on polythiophene-functionalized TiC2T. xAn electrochemical capacitive biosensor based on TiO2 nanorod composite material was developed (40 min), followed by a colorimetric method based on a 3D-printed integrated operable device (40 min). Therefore, the β-gal-modified portable colorimetric biosensor represents a faster biosensing platform.

[0044] (5) Testing of detection performance As the concentration of *Pseudomonas aeruginosa* increased, the color of the ONPG reaction system gradually changed from yellow to light yellow, and the fluorescence imaging fully reflected the signal intensity changes of the MUG system. The values ​​of the A420nm and F423nm reaction systems gradually decreased (Figures 4B and 4D), suggesting that when the test bacteria were added, the phage's capture of *Pseudomonas aeruginosa* masked the enzyme's catalytic site. The detection range of *Pseudomonas aeruginosa* was 10... 1 — 10 5 With a detection limit as low as 4 CFU / ml, the portable colorimetric biosensor proposed in this invention has a lower LOD than other detection methods for Pseudomonas aeruginosa and even other bacteria, such as phage-modified hydrogels assembled in 3D printing equipment and used in a pressure colorimetric "two-in-one" platform (1.5 × 10³ CFU / ml). A microfluidic platform combined with a phage receptor-binding protein device (10⁴ CFU / ml). A phage-modified magnetic bead and enzyme-activated ALE platform (24.5 CFU / ml). In summary, the method proposed in this invention is more advantageous for rapid detection at low concentrations. The binding specificity of phage receptor-binding protein (RBP) to bacterial host surface terpenic acid (WTA) is the main determinant of host recognition and adsorption processes. Furthermore, bioluminescent imaging validation was also performed. Figure 5 E) Bacterial bioluminescence is a set of enzymatic reactions encoded by specific genes. Essentially, it involves the direct conversion of chemical energy generated by cellular respiration and metabolism into visible light energy through enzymatic catalysis. This utilizes ATP and luciferin released during bacterial life activities, catalyzed by O2 and luciferase, to induce luciferin to emit light. Bacterial solutions at concentration gradients from 10¹ CFU / ml to 10⁵ CFU / ml were enriched and bioluminescently imaged. The changes in bioluminescent signals followed a concentration gradient, thus demonstrating the extent to which the stirring rod could capture bacterial solutions of different concentrations.

[0045] To test the specificity of this assay for *Pseudomonas aeruginosa*, absorbance values ​​for different strains were measured, including *Salmonella*, *Staphylococcus aureus*, *Escherichia coli*, *Listeria*, and different serotypes of *Pseudomonas aeruginosa*, including ATCC 9027 and ATCC 27853. A concentration of 1 × 10⁴ CFU / ml was used for each strain. Figure 5F) shows the color change detected by colorimetric assay. No significant difference in absorbance was observed before and after slime mold in solutions of other strains and serotypes. In contrast, there was a significant change in absorbance only in solutions containing Pseudomonas aeruginosa ATCC 15442. Plaque assays of different strains and serotypes also confirmed this (Fig. 2E). Only Pseudomonas aeruginosa ATCC 15442 was captured and lysed.

[0046] (6) Detection of actual samples To further evaluate the detection performance of this colorimetric sensor, samples commonly found in the environment containing *Pseudomonas aeruginosa* were evaluated, including: vegetable gardens near hospitals, hospital wastewater, medical wastewater, tap water, and bottled mineral water from Qiandao Lake. The calculated average recoveries ranged from 83% to 118% (Table 1), indicating that the system of this invention has high accuracy and reliability in colorimetric and fluorescence detection of real samples. Furthermore, compared to the expensive equipment and kits required by traditional detection methods (such as PCR and ELISA), the other chemical reagents in the β-gal-ONPG and β-gal-MUG colorimetric systems (such as ONPG and MUG) are inexpensive and readily available. Therefore, the β-gal-ONPG and β-gal-MUG colorimetric systems proposed in this invention are an economical, efficient, and practical tool for detecting live *Staphylococcus aureus* in practical food applications.

[0047] Table 1 Recovery rates of different samples

[0048] In summary, the colorimetric sensor based on β-gal and phage-directed modification achieves highly sensitive, selective, and rapid detection of *Pseudomonas aeruginosa* strain ATCC 15442. β-gal exhibits excellent catalytic cleavage effects on ONPG and MUG. Stable modification of the stir bar by phage and β-gal was achieved through PDDA-directed adhesion and EDC / NHS chemical cross-linking. Due to the pre-concentration effect of the stir bar, the sensor demonstrates high sensitivity to changes in the colorimetric signal generated by the binding of the target bacteria and phage. This signal change is attributed to the shielding of the active sites on the stir bar by the target bacteria. The limit of detection (LOD) for *P. aeruginosa* strain ATCC 15442 is only 4 CFU / ml, and the detection time is within 6 minutes. Compared to most other detection methods, the detection procedure is also simpler. Overall, this simple and low-cost method can be used for the real-time detection of *P. aeruginosa* strain ATCC 15442, thereby ensuring food and environmental hygiene.

[0049] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0050] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. 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 spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A detection stirring rod based on co-modification of bacteriophage and β-galactosidase, characterized in that, include: The substrate is a wood fiber rod; The substrate surface is modified with polydiallyldimethylammonium chloride; The polydiallyl dimethylammonium chloride was co-modified with bacteriophage and β-galactosidase; The bacteriophage is modified onto polydiallyldimethylammonium chloride through a combination of covalent binding and electrostatic adsorption, and the β-galactosidase is modified onto polydiallyldimethylammonium chloride through electrostatic adsorption.

2. The detection stirring rod according to claim 1, characterized in that, The bacteriophage is ATCC 15442 type Pseudomonas aeruginosa bacteriophage.

3. A method for preparing the detection stirring rod according to any one of claims 1 to 2, characterized in that, Includes the following steps: (1) The wood fiber rod was immersed in a polydiallyldimethylammonium chloride solution and stirred and incubated, and then dried under vacuum to obtain a stirring rod modified with polydiallyldimethylammonium chloride. (2) After ultrafiltration of the phage, the pH was adjusted, and after activation by EDC and NHS, it was incubated with the stirring rod of step (1) to obtain the phage-modified stirring rod; (3) After washing the stirring rod from step (2), immerse it in β-galactosidase solution and incubate it by shaking. After washing, immerse it in BSA solution to seal it, and the detection stirring rod is obtained.

4. The preparation method according to claim 3, characterized in that, In step (1), the stirring incubation temperature is 20~30℃ and the time is 1~3h; the vacuum drying temperature is 35~40℃ and the time is 0.5~1.5h.

5. The preparation method according to claim 3, characterized in that, In step (2), the phage is adjusted to pH 5.0-6.0 with EMS buffer, and the concentrations of EDC and NHS are 0.5-1.5 mmol / L. The activation time of EDC is 10-30 min and the activation time of NHS is 20-40 min. After activation, the pH is adjusted to 6-8 with PBS. The incubation temperature of the phage and the stirring rod is 20-30℃ and the time is 2-4 h.

6. The preparation method according to claim 3, characterized in that, In step (3), the incubation conditions for the β-galactosidase solution are 20-28℃ with shaking incubation for 1-3 hours; after incubation, the solution is washed with deionized water.

7. The preparation method according to claim 3, characterized in that, In step (3), the concentration of the BSA solution is 0.5-1.5%, and the sealing conditions are 2-8℃ for incubation for 12-24 hours.

8. The use of the detection stirring rod according to any one of claims 1 to 2 in the detection of Pseudomonas aeruginosa.

9. The application according to claim 8, characterized in that, During the detection process, the enrichment time of Pseudomonas aeruginosa on the detection stirring rod is 1-3 min, and the color development / fluorescence reaction time of the substrate is 1-3 min.

10. The application according to claim 8, characterized in that, The detection stirring rod is used for rapid on-site detection of Pseudomonas aeruginosa in samples, including vegetable garden samples, medical wastewater, hospital wastewater, tap water, natural drinking water, and bottled mineral water.

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