An additive for improving the sensitivity of a fluorescent enzyme substrate method

Abstract

This invention relates to the field of biodetection technology and discloses an additive for improving the sensitivity of fluorescent enzyme substrate methods. The additive is an amphiphilic block copolymer. The amphiphilic block copolymer comprises hydrophilic and hydrophobic segments linked by covalent bonds. The hydrophilic segments are selected from one or more combinations of polyethylene glycol, polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, and their quaternary ammonium salt modified derivatives. The hydrophobic segments are selected from one or more combinations of polylactic acid, polycaprolactone, polylactide-glycolic acid, polystyrene, polyethylene, polymethyl methacrylate, and their derivatives. This invention significantly improves the fluorescence quantum efficiency of fluorescent groups, represented by 4-methylumbelliferone, in aqueous solutions, and can significantly enhance detection sensitivity when used in biochemical reactions involving the enzymatic digestion of fluorescent substrates.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to an additive that improves the sensitivity of the fluorescent enzyme substrate method. Background Technology

[0002] Fluorescent enzyme substrate assays are a commonly used detection technique in the field of biological detection. Due to their high specificity and ease of operation, they are widely used in microbial detection, enzyme activity analysis, and other scenarios. The core principle is to utilize the specific cleavage of a fluorescent substrate by a fluorescent enzyme, causing the substrate to release a fluorescent group. Qualitative and quantitative analysis of the target microorganism or enzyme is achieved by detecting the intensity of the fluorescence signal.

[0003] However, existing fluorescent enzyme substrate methods have significant limitations in practical applications: the enzyme cleavage products of fluorescent substrates are usually hydrophobic, easily aggregated in aqueous solutions, leading to nonradiative energy decay. They are also susceptible to quenching or degradation in aqueous environments, resulting in low fluorescence quantum efficiency and insufficient fluorescence signal intensity. Furthermore, the limited solubility of hydrophobic fluorescent substrates in aqueous phases and insufficient loading sites further restrict detection sensitivity. These problems lead to long detection times, high false-negative rates (easily missing weakly positive samples), and poor quantitative accuracy in existing fluorescent enzyme substrate methods, making it difficult to meet the demands for high sensitivity, rapid detection, and precise quantification in practical applications.

[0004] To address the aforementioned issues, there is an urgent need in this field to develop a technical solution that can effectively improve the sensitivity of fluorescent enzyme substrate detection, shorten detection time, and improve quantitative accuracy. Summary of the Invention

[0005] In view of this, the present invention provides an additive to improve the sensitivity of the luciferase substrate method, aiming to solve the above-mentioned problems.

[0006] On the one hand, the present invention provides an additive for improving the sensitivity of the luciferase substrate method, wherein the additive is an amphiphilic block copolymer; The amphiphilic block copolymer comprises hydrophilic segments and hydrophobic segments connected by covalent bonds; The hydrophilic segment is selected from any one or a combination of polyethylene glycol, polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, and their quaternary ammonium salt modified derivatives; The hydrophobic segment is selected from one or more combinations of polylactic acid, polycaprolactone, polylactide-glycolic acid, polystyrene, polyethylene, polymethyl methacrylate and their derivatives.

[0007] Preferably, the amphiphilic block copolymer is an unmodified amphiphilic block copolymer or a quaternary ammonium salt modified amphiphilic block copolymer; The quaternary ammonium salt modified amphiphilic block copolymer was prepared by the following method: Ion exchange resin was mixed with acetylthiocholine iodide for adsorption to obtain a resin loaded with a modifier. The unmodified amphiphilic block copolymer was dissolved in an organic solvent, and the resin loaded with the modifier and the initiator were added. The temperature was then raised to carry out the grafting reaction. After the reaction was completed, rotary evaporation was performed to obtain a quaternary ammonium salt modified amphiphilic block copolymer.

[0008] Preferably, the structure of the amphiphilic block copolymer is a diblock, triblock, or multiblock structure; preferably, it is a diblock copolymer.

[0009] Preferably, the number-average molecular weight of the amphiphilic block copolymer is in the range of 1,000 to 200,000 Daltons; and the weight percentage of the hydrophilic segment in the amphiphilic block copolymer is 5% to 60%.

[0010] In another aspect, the present invention also provides a composition for improving the sensitivity of the luciferase substrate method, comprising a luciferase substrate and the above-mentioned additives, as well as a selective inhibitor and / or a basic component of a microbial culture medium; The selective inhibitor is selected from one or more of bile salts, sodium dodecyl sulfate, sodium azide, and high-concentration sodium chloride; the basic component of the microbial culture medium is selected from one or more of buffer salts and peptone.

[0011] Preferably, the fluorescent enzyme substrate is selected from one or more of the following: 4-methylumbelliferone-β-D-glucoside, 4-methylumbelliferone-β-D-glucuronide, 4-methylumbelliferone-β-D-galactoside, 4-methylumbelliferone-α-D-glucoside, 4-methylumbelliferone phosphate, fluorescein 2-β-D-galactopyranoside, halogen-β-D-galactopyranoside, halogen-β-D-glucoside, fluorescein di(β-D-galactopyranoside), and chlorophenol red-β-D-galactopyranoside.

[0012] On the other hand, the present invention also provides a method for improving the sensitivity of the luciferase substrate method, comprising the following steps: S1: The above-mentioned luciferase substrate and amphiphilic block copolymer are mixed in a solvent to form a complex containing the luciferase substrate; S2: React the complex with the luciferase in the sample to be tested, and detect the fluorescence signal generated by the reaction; Under the same detection conditions, the fluorescence signal intensity generated by the reaction of the complex with the luciferase is higher than that generated by the reaction of the luciferase substrate that has not formed the complex with the luciferase.

[0013] Preferably, in step S1, the solvent is a buffer solution or an organic solvent; The buffer solution is selected from one of carbonate buffer and phosphate buffer, and the pH range of the buffer solution is 7.0-10.0; The organic solvent is selected from one or more of acetone and N,N'-dimethylformamide; The mixing temperature of the luciferase substrate and the amphiphilic block copolymer is 25-37°C, and the mixing time is 5-120 minutes. Preferably, in step S2, the reaction includes selectively culturing the target microorganism in the sample to be tested, causing the fluorescent enzyme expressed by the microorganism to undergo an enzymatic reaction with the complex, and the reaction temperature is 37-44.5℃; during the reaction, the fluorescence intensity change is continuously monitored by a fluorescence detection device, and the excitation wavelength and emission wavelength of the fluorescence detection device are adjusted according to the type of fluorescent enzyme substrate, wherein the excitation wavelength corresponding to 4-methylumbelliferous ketone substrate is set to 360-365nm and the emission wavelength is set to 405-440nm; the excitation wavelength corresponding to halogenated substrate is set to 570nm and the emission wavelength is set to 585nm.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention develops a novel hydrophobic-hydrophilic block copolymer encapsulation technology, which significantly improves the fluorescence quantum efficiency of fluorescent groups, represented by 4-methylumbelliferone, in aqueous solution, and can significantly enhance detection sensitivity when used in biochemical reactions involving enzymatic digestion of fluorescent substrates.

[0015] Hydrophilic-hydrophobic block copolymers are typically composed of hydrophilic blocks (such as polyethylene glycol, polyethylene oxide, and polyethylene oxide) and hydrophobic blocks (such as polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), and polycaprolactone (PCL)) linked by covalent bonds. Their structures include diblock (AB type), triblock (ABA, BAB type), multiblock, and grafted polymers. The mechanism of action involves the hydrophobic segments forming the core and the hydrophilic segments forming the shell. This structure encapsulates the hydrophobic fluorescent product released after enzymatic cleavage, restricting its molecular motion, reducing non-radiative energy dissipation, and protecting it from quenching or degradation in the aqueous environment, thereby significantly enhancing the fluorescence signal. The hydrophilic blocks, through surface hydrophilization, improve the dispersibility and stability of the entire system in the aqueous phase, increase the loading capacity of the fluorescent substrate, and further enhance the saturated fluorescence intensity of the fluorescent molecules in water. Based on this microenvironment regulation, the fluorescence quantum yield of 4-methylumbelliferone can be increased by approximately 3 times, resulting in a significantly enhanced fluorescence signal.

[0016] To further optimize the fluorescence enhancement effect, this invention also introduces quaternary ammonium groups into the block copolymer. Quaternary ammonium groups can interact electrostatically and hydrophobically with fluorescent molecules such as 4-methylumbelliferone and fluorescein, enhancing their positioning and binding stability within the hydrophobic core. This further restricts the rotational and vibrational degrees of freedom of the fluorescent molecules, thereby reducing the probability of nonradiative transitions and improving fluorescence quantum efficiency. This modification, while maintaining the original hydrophilic-hydrophobic microregion structure, further strengthens the confinement protection of the fluorescent products, resulting in a further improvement in the fluorescence signal.

[0017] Based on the aforementioned synergistic mechanism, the enhanced fluorescence signal in this system allows the system to reach the detection threshold more quickly, significantly shortening the positive reporting time and thus substantially improving detection speed. Furthermore, the introduction of block polymers improves detection sensitivity, particularly for weakly positive samples, where the detection rate can be significantly increased. In addition, the linear relationship between fluorescence intensity and bacterial count / enzyme activity is more significant (R² > 0.99), which is beneficial for achieving accurate quantification.

[0018] This invention provides an efficient and stable technical platform for fluorescence enhancement and enzyme-catalyzed reaction detection through the synergistic effect of hydrophobic-hydrophilic block copolymer encapsulation and quaternary ammonium group modification. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The curve showing the effect of the block polymer on the enzyme-fluorescent substrate method provided in the embodiments of the present invention; Figure 2 This is a comparison chart showing the effect of adding block polymer to water samples with different bacterial concentrations on improving the positive reporting time, provided in an embodiment of the present invention. Figure 3 The quaternary ammonium modified block copolymer enhanced fluorescence substrate method provided in this embodiment of the invention is limited to the detection of Enterococcus faecalis. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Experimental Example 1: Polystyrene-block-polyacrylic acid copolymer shortens the detection time of heat-resistant coliforms. 1.1 Experimental Materials and Instruments Strains: Thermostable coliform standard strain ATCC 25922; Additive: PS-b-PAA diblock copolymer, number average molecular weight Mn=15000, of which hydrophilic segment polyacrylic acid Mn=4500 and hydrophobic segment polystyrene Mn=10500; Fluorescent enzyme substrate: 4-methylumbelliferone-β-D-galactoside (4-MUGal), chromatographic grade; Basic culture medium system: Each 1L of culture medium contains 20.0g peptone, 2.5g dipotassium hydrogen phosphate, 1.0g potassium dihydrogen phosphate, 5.0g sodium chloride, 0.5g bile salt No. 3, and 0.1g 4-MUGal. Make up to volume with deionized water, adjust the pH to 7.2±0.2, and autoclave at 121℃ for 15min for later use. Instruments: Level 2 biosafety cabinet, fully automatic high-pressure steam sterilizer, constant temperature incubator, real-time fluorescence quantitative detector, microbial plate counting agar.

[0024] 1.2 Test Methods Preparation of bacterial culture: Thermotolerant standard strains of coliform bacteria were inoculated into nutrient broth and incubated at 37°C for 18 hours to obtain activated bacterial culture; the activated bacterial culture was then serially diluted 10-fold with physiological saline to prepare 10... 0 Three gradients of bacterial suspensions were prepared: 10¹, 10², and CFU / 100mL. The actual concentration of the bacterial suspensions was confirmed by plate counting.

[0025] Experimental Groups: Experimental group: PS-b-PAA copolymer stock solution was added to culture medium for sterilization, cooled down and then 1 / 100 volume of 100× enzyme substrate was added. The solution was then dispensed into sterile culture bottles with stoppers, and 100 mL of culture medium was added to each bottle to make the final polymer concentration in the system 100 ppm. The solution was then mixed and set aside. Control group: Take sterile culture tubes with plugs, add 100 mL of the above sterile culture medium to each tube, replace the polymer stock solution with an equal volume of sterile physiological saline, and keep the other components completely consistent with the experimental group; Blank control group: Take a sterile culture tube with a stopper, add 9 mL of the above sterile culture medium to each tube, do not inoculate with bacterial solution, and only add an equal volume of sterile physiological saline to remove system contamination.

[0026] Inoculation and culture: For each gradient of bacterial suspension in both the experimental and control groups, three replicates were set up. 1 mL of the corresponding gradient bacterial suspension was inoculated into each culture tube to achieve a final bacterial concentration of 10⁻⁶. 0 -10 2 CFU / 100mL; after mixing all culture tubes, they were placed in a constant temperature incubator at 44.5℃ and the fluorescence signal was monitored throughout the process using a real-time fluorescence quantitative detector. The excitation wavelength was set to 365nm and the emission wavelength was set to 405nm. The fluorescence value was read every 5 minutes and monitored continuously for 18h.

[0027] Judgment criteria: The positive threshold was defined as the fluorescence value exceeding three times the standard deviation of the fluorescence value of the blank control group. The time for each group to reach the positive threshold (positive reporting time) was recorded. After 18 hours of incubation, the positive detection rate of different concentrations of bacterial solutions in each group was calculated, and the difference in fluorescence signal intensity between the experimental group and the control group was compared.

[0028] 1.4 Experimental Results and Analysis See Figure 1 and Figure 2 As shown, the time to report a positive result was significantly shorter in the experimental group compared to the control group at the same bacterial concentration. Among them, 10... 2 With a high concentration of CFU / 100mL bacterial culture, the positive reporting time for the control group was approximately 600 minutes, while the positive reporting time for the experimental group was comparable. 1 In a medium-concentration bacterial culture with CFU / 100mL, the positive reporting time for the control group was approximately 600 minutes, while for the experimental group it was approximately 700 minutes; 10 0 In the control group, the positive detection threshold was not reached within the detection time of CFU / 100mL weak positive bacterial solution, while in the experimental group, a positive detection was achieved in 920min.

[0029] Comparison of fluorescence signal intensity: After 24 hours of cultivation, at the same bacterial concentration, the fluorescence signal intensity of the experimental group was 2-2.4 times higher than that of the control group, which is consistent with the fluorescence quantum yield enhancement effect described in this invention.

[0030] Detection rate comparison: within a 1040-minute culture period, 10 0 In the CFU / mL weakly positive bacterial suspension, the positive detection rate was 0% in the control group and 100% in the experimental group. In the 10¹CFU / mL low-concentration bacterial suspension, the positive detection rate was 66.7% in the control group and 100% in the experimental group, significantly reducing the false negative rate of weakly positive samples. Furthermore, the low concentration in the control group could not significantly distinguish between different concentration gradients, while this was not the case in the experimental group.

[0031] See Figure 3As shown, this experiment verified that the PS-b-PAA amphiphilic block copolymer can significantly enhance the detection signal of the fluorescent enzyme substrate method, greatly shorten the detection positive time of thermotolerant coliforms, and show significant differences in incubation time for different concentration gradients. This provides a technical basis for quantitative correlation between incubation time and inoculation concentration. In addition, the experimental group significantly improved the detection rate of weak positive samples and improved the detection sensitivity.

[0032] Experimental Example 2: Polystyrene-block-polyacrylic acid copolymer shortens the detection time for Escherichia coli 2.1 Experimental Objective The synergistic effect of PS-b-PAA amphiphilic block copolymer additive in the selective culture detection system of Escherichia coli was verified, and its effect on shortening the positive detection time of Escherichia coli and enhancing the fluorescence signal was investigated.

[0033] 2.2 Experimental Materials and Instruments Strains: Escherichia coli standard strain ATCC25922; Additives: PS-b-PAA diblock copolymer as described in Example 1; Fluorescent enzyme substrate: 4-methylumbelliferone-β-D-glucuronide (4-MUG), chromatographic grade; Basic culture medium system: Each 1L of culture medium contains 20.0g peptone, 2.5g dipotassium hydrogen phosphate, 1.0g potassium dihydrogen phosphate, 5.0g sodium chloride, 0.2g sodium dodecyl sulfate (SDS), and 0.1g 4-MUG. Make up to volume with deionized water, adjust pH to 7.0±0.2, and autoclave at 121℃ for 15min for later use. Instruments: Same as in Experiment 1.

[0034] 2.3 Test Methods Preparation of bacterial culture: Same as in Experiment 1, 10 0 Escherichia coli bacterial suspensions with three gradients of 10, 10², and CFU / 100mL were prepared, and plate counts were used to confirm the actual concentration.

[0035] Experimental Groups: Experimental group: Take sterile, stoppered culture tubes, add 9 mL of the above-mentioned sterile culture medium to each tube, add PS-b-PAA copolymer stock solution to make the final polymer concentration in the system 20 ppm, mix well and set aside; (same as Experimental Example 1) Control group: Take sterile culture tubes with plugs, add 9 mL of the above sterile culture medium to each tube, replace the polymer stock solution with an equal volume of sterile physiological saline, and keep the other components completely consistent with the experimental group; Blank control group: Same as in Experiment 1, excluding system contamination.

[0036] Inoculation and culture: Three parallel samples were set up for each gradient of bacterial culture in the experimental group and the control group. 1 mL of the corresponding gradient bacterial culture was inoculated into each tube, mixed well and incubated at 44.5℃. The fluorescence signal was monitored in real time with an excitation wavelength of 365 nm and an emission wavelength of 405 nm. Data was read every 30 minutes and monitored continuously for 24 hours. The criteria for positive reporting were the same as in Experiment 1.

[0037] 2.4 Experimental Results and Analysis Comparison of positive reporting time: Under the same bacterial concentration, the positive reporting time in the experimental group was significantly shorter than that in the control group. 4 For high-concentration bacterial suspensions (CFU / mL), the positive reporting time was 6 hours in the control group and only 3 hours in the experimental group, a 50% reduction. For medium-concentration bacterial suspensions (10²CFU / mL), the positive reporting time was 13 hours in the control group and only 7 hours in the experimental group, a 46.2% reduction. 0 CFU / mL weakly positive bacterial suspension: the control group did not report any positive results within 24 hours, while the experimental group achieved positive detection within 20 hours.

[0038] Comparison of fluorescence signal intensity: After 24 hours of culture, at the same bacterial concentration, the fluorescence signal intensity of the experimental group was 2.8-3.2 times higher than that of the control group, achieving a significant fluorescence enhancement effect.

[0039] Detection rate comparison: within a 24-hour culture period, 10 0 The detection rate of CFU / mL weakly positive bacterial solution was 0% in the control group and 100% in the experimental group; the detection rate of 10¹CFU / mL low concentration bacterial solution was 50% in the control group and 100% in the experimental group, effectively reducing the risk of false negatives.

[0040] Experimental Example 3: Polyethylene-block-polyethylene glycol copolymer shortens the detection time of heat-resistant coliforms. 3.1 Experimental Objective To verify the synergistic effect of polyethylene-block-polyethylene glycol (PE-b-PEG) amphiphilic block copolymer additives combined with halogenated fluorescent substrates in a thermostable coliform detection system, and to investigate its effect on shortening the detection positive time and enhancing the fluorescence signal.

[0041] 3.2 Experimental Materials and Instruments Strains: Thermostable coliform standard strain ATCC25922; Additive: PE-b-PEG diblock copolymer, number average molecular weight Mn=12000, of which the hydrophilic segment polyethylene glycol Mn=3600 and the hydrophobic segment polyethylene Mn=8400; Fluorescent enzyme substrate: halogen-β-D-galactopyranoside, chromatographic grade; Basic culture medium system: Each 1L of culture medium contains 20.0g peptone, 2.5g dipotassium hydrogen phosphate, 1.0g potassium dihydrogen phosphate, 5.0g sodium chloride, 0.5g bile salt No. 3, and 0.08g halogen-β-D-galactopyranoside. The medium is brought to a final volume with deionized water, the pH is adjusted to 7.2±0.2, and the medium is autoclaved at 121℃ for 15min before use. Instruments: Same as in Experiment 1.

[0042] 3.3 Test Methods Preparation of bacterial culture: Same as in Experiment 1, 10 0 10¹, 10², 10³, 10 4 10 5 Six gradients of thermostable coliform cultures were prepared using CFU / mL, and plate counts were used to confirm the actual concentration.

[0043] Experimental Groups: Experimental group: Take sterile, stoppered culture tubes, add 9 mL of the above-mentioned sterile culture medium to each tube, add PE-b-PEG copolymer stock solution to make the final polymer concentration in the system 500 ppm, mix well and set aside; (same as Experiment 1) Control group: Take sterile culture tubes with plugs, add 9 mL of the above sterile culture medium to each tube, replace the polymer stock solution with an equal volume of sterile physiological saline, and keep the other components completely consistent with the experimental group; Blank control group: Same as in Experiment 1, excluding system contamination.

[0044] Inoculation and culture: Three parallel samples were set up for each gradient of bacterial culture in the experimental group and the control group. 1 mL of the corresponding gradient bacterial culture was inoculated into each tube, mixed well and incubated at 44.5℃. The excitation wavelength was set to 570 nm and the emission wavelength was set to 585 nm. The fluorescence signal was monitored in real time, and the data was read every 30 minutes for 24 hours. The criteria for positive reporting were the same as those in Experiment 1.

[0045] 3.4 Experimental Results and Analysis Comparison of positive reporting time: Under the same bacterial concentration, the positive reporting time in the experimental group was significantly shorter than that in the control group. 4 For high-concentration bacterial suspensions (CFU / mL), the positive reporting time was 5 hours in the control group and only 2 hours in the experimental group, a reduction of 60%. For medium-concentration bacterial suspensions (10²CFU / mL), the positive reporting time was 11 hours in the control group and only 5.5 hours in the experimental group, a reduction of 50%. 0 CFU / mL weakly positive bacterial suspension: the control group did not report positive results within 24 hours, while the experimental group could achieve positive detection within 17 hours.

[0046] Comparison of fluorescence signal intensity: After 24 hours of culture, at the same bacterial concentration, the fluorescence signal intensity of the experimental group was 3.3-3.7 times higher than that of the control group, showing a significant fluorescence enhancement effect.

[0047] Detection rate comparison: within a 24-hour culture period, 10 0 The detection rate of CFU / mL weakly positive bacterial suspension was 0% in the control group and 100% in the experimental group; the detection rate of 10¹CFU / mL low concentration bacterial suspension was 66.7% in the control group and 100% in the experimental group.

[0048] This experimental example verifies that the PE-b-PEG amphiphilic block copolymer still has excellent synergistic effects in the detection system of halogenated fluorescent substrates, and expands the compatibility of the additives of this invention with different types of fluorescent enzyme substrates.

[0049] Experimental Example 4: Polyethylene glycol-block-polylactide-glycolic acid copolymer improves the sensitivity of enzyme activity detection 4.1 Experimental Objective The fluorescence enhancement effect of polyethylene glycol-block-polylactide-glycol (PEG-b-PLGA) amphiphilic block copolymer additive in the alkaline phosphatase (ALP) pure enzyme activity detection system was verified, and its effect on improving detection sensitivity and quantitative accuracy was investigated to determine the optimal concentration of the additive.

[0050] 4.2 Experimental Materials and Instruments Enzyme preparation: Bovine small intestinal alkaline phosphatase (ALP), specific activity ≥1000U / mg, commercially available standard; Additive: PEG-b-PLGA diblock copolymer, number average molecular weight Mn=18000, of which the hydrophilic segment polyethylene glycol Mn=5400 and the hydrophobic segment PLGA (LA:GA=75:25) Mn=12600; Fluorescent enzyme substrate: 4-methylumbelliferyl ketone phosphate (4-MUP), chromatographic grade; Buffer system: 100mM pH 9.8 carbonate buffer, freshly prepared and used immediately; Instruments: Full-wavelength multi-functional fluorescent microplate reader, constant temperature metal bath, magnetic stirrer, volumetric flasks, black opaque 96-well microplate.

[0051] 4.3 Test Methods Preparation of polymer stock solution: Accurately weigh PEG-b-PLGA copolymer and dissolve it in acetone to prepare a 10 g / L polymer acetone solution; slowly add the solution dropwise to a 100 mM pH 9.8 carbonate buffer solution under vigorous stirring. After the addition is complete, gently stir overnight at room temperature to allow the acetone to evaporate completely, and a clear polymer stock solution with a concentration of 1000 ppm is obtained. Store at 4°C in the dark for later use.

[0052] Reagent preparation: Prepare a 100 mg / L 4-MUP substrate solution using carbonate buffer, and use immediately; serially dilute the ALP standard with carbonate buffer to prepare five ALP working solutions with concentrations of 0.1, 1, 10, 50, and 100 mU / mL, and store on ice for later use.

[0053] Basic synergistic effect verification test: Experimental group: In a black 96-well plate, add 150 μL of 100 mM pH 9.8 carbonate buffer, 20 μL of the above polymer stock solution, and 20 μL of 100 mg / L 4-MUP substrate solution to each well in sequence, mix well, and pre-incubate at 37°C for 5 min. Control group: In a black 96-well plate, each well was replaced with an equal volume of carbonate buffer instead of polymer stock solution. All other components and the order of sample addition were exactly the same as in the experimental group. The plate was pre-incubated at 37°C for 5 min. Reaction initiation and detection: After pre-incubation, quickly add 10 μL of 10 mU / mL ALP working solution to each well to start the enzymatic reaction, with a total system volume of 200 μL; immediately place the microplate in a 37℃ constant temperature fluorescence microplate reader and continuously monitor the changes in fluorescence intensity. The excitation wavelength is 360 nm and the emission wavelength is 440 nm. Data is read every 30 seconds and monitored continuously for 30 minutes.

[0054] Optimal concentration screening test: Six gradients were set for the final polymer concentrations of 0.01, 0.05, 0.1, 0.2, 0.5, and 1.0 mg / mL. The other system components and reaction conditions were completely consistent with the above test. The ALP working solution concentration was fixed at 1 mU / mL. After 30 minutes of reaction, the fluorescence value was read. The optimal concentration of the polymer was determined by the fluorescence enhancement factor.

[0055] Sensitivity and quantitative accuracy verification test: The experimental group was set up with the optimal concentration and the control group was set up with the polymer-free system. ALP working solutions with gradients of 0.1, 1, 10, 50 and 100 mU / mL were detected respectively. After 30 minutes of reaction, the fluorescence value was read and the fluorescence intensity-ALP activity standard curve was plotted. The limit of detection and linear correlation coefficient R² of the two groups were compared to evaluate the quantitative accuracy.

[0056] 4.4 Experimental Results and Analysis Basic fluorescence enhancement effect: After 30 minutes of reaction, the fluorescence signal intensity of the experimental group was 3.2 times higher than that of the control group, and the fluorescence signal growth rate of the experimental group was significantly higher than that of the control group throughout the process, which confirms that the PEG-b-PLGA copolymer can significantly enhance the fluorescence signal of the 4-MUP-ALP enzymatic reaction system.

[0057] Optimal concentration results: Within the concentration range of 0.01-0.2 mg / mL, the fluorescence enhancement factor of the polymer increased with increasing concentration, reaching a peak at 0.2 mg / mL with a fluorescence enhancement factor of 3.6 times; after the concentration exceeded 0.2 mg / mL, the enhancement effect decreased slightly, and 0.2 mg / mL was determined to be the optimal concentration of the polymer for use in this system.

[0058] Sensitivity enhancement: The limit of detection for ALP in the control group was 1 mU / mL, while the limit of detection in the experimental group reached 0.1 mU / mL, representing a 10-fold increase in detection sensitivity and achieving highly sensitive detection of low-activity ALP.

[0059] Improved quantitative accuracy: The linear correlation coefficient R² of the standard curve of fluorescence intensity-ALP activity in the control group was 0.978, while that in the experimental group was 0.9992. The linear relationship was significantly optimized, greatly improving the accuracy of enzyme activity quantification.

[0060] This experimental example verifies the excellent synergistic effect of PEG-b-PLGA copolymer in pure enzyme activity detection system, which can significantly improve detection sensitivity and quantitative accuracy, and confirms the applicability of the technical solution of the present invention in enzyme activity analysis scenarios.

[0061] Experimental Example 5: Preparation of Quaternary Ammonium Salt Modified Polystyrene-Block-Polyacrylic Acid Copolymer and Validation of its Synergistic Effect on Enterococcus faecalis Detection 5.1 Experimental Objective A quaternary ammonium salt-modified PS-b-PAA amphiphilic block copolymer was prepared. The synergistic effect of the modified copolymer in the Enterococcus faecalis luciferase substrate detection system was verified, and the synergistic optimization effect of quaternary ammonium group modification on fluorescence signal enhancement and detection positive time reduction was investigated.

[0062] 5.2 Experimental Materials and Instruments Strains: Enterococcus faecalis standard strain ATCC29212; Raw materials: PS-b-PAA diblock copolymer (same as in Experiment 1), D201 type macroporous anion exchange resin, acetylthiocholine iodide, N,N'-dimethylformamide (DMF), and azobisisobutyronitrile (AIBN), all of which are commercially available analytical grade. Fluorescent enzyme substrate: 4-methylumbelliferone-β-D-galactoside (4-MUGal), chromatographic grade; Basic culture medium system: Each 1L of culture medium contains 20.0g peptone, 2.5g dipotassium hydrogen phosphate, 1.0g potassium dihydrogen phosphate, 50.0g sodium chloride, 0.1g sodium azide, and 0.1g 4-MUGal. Make up to volume with deionized water, adjust the pH to 7.2±0.2, and autoclave at 121℃ for 15min for later use. Instruments: Same as in Experiment 1, except for a rotary evaporator, an oil bath constant temperature reaction vessel, and a vacuum drying oven.

[0063] 5.3 Test Methods Preparation of quaternary ammonium salt modified PS-b-PAA copolymer: Modifier loading: Accurately weigh 1g of D201 type ion exchange resin, place it in an Erlenmeyer flask, add 1g of acetylthiocholine iodide and 20mL of anhydrous ethanol, seal and shake at room temperature overnight for adsorption, filter and wash the resin 3 times with anhydrous ethanol, and vacuum dry to obtain modified resin loaded with acetylthiocholine iodide. Grafting reaction: Dissolve 100g of PS-b-PAA copolymer in 1LDMF. After complete dissolution, add the resin with the above-mentioned loaded modifier and 1g of AIBN initiator. Stir evenly and then heat to 70℃ and react at a constant temperature for 4h. Post-processing: After the reaction was completed, the mixture was cooled to room temperature, filtered to remove the resin, and the filtrate was evaporated at 70°C to remove the DMF solvent, yielding a solid product. The solid product was washed three times with anhydrous ethanol and dried under vacuum to constant weight to obtain the quaternary ammonium salt modified PS-b-PAA amphiphilic block copolymer, which was then sealed and stored at 4°C for later use.

[0064] Preparation of bacterial suspension: Enterococcus faecalis standard strains were inoculated into brain and heart broth and incubated at 37°C for 18 hours to obtain activated bacterial suspension; the activated bacterial suspension was then serially diluted 10-fold with physiological saline to prepare 10... 0 10¹, 10², 10³, 10 4 10 5 Six CFU / mL bacterial solutions were prepared, and plate counting was used to confirm the actual concentration.

[0065] Experimental Groups: Modified polymer experimental group: Take a sterile culture tube with a stopper, add 9 mL of the above sterile culture medium to each tube, add the quaternary ammonium salt modified PS-b-PAA copolymer stock solution to make the final polymer concentration in the system 50 ppm, mix well and set aside; Unmodified polymer control group: Take sterile plugged culture tubes, add 9 mL of the above sterile culture medium to each tube, add unmodified PS-b-PAA copolymer stock solution to make the final polymer concentration in the system 50 ppm, and the other components are completely consistent with the experimental group; Blank control group: Take a sterile culture tube with a stopper, add 9 mL of the above sterile culture medium to each tube, replace the polymer stock solution with an equal volume of sterile physiological saline, and the remaining components are completely consistent with the experimental group. Contaminated blank group: Same as in Experiment 1, but without bacterial inoculation to eliminate system contamination.

[0066] Inoculation and culture: Three parallel samples were set up for each gradient of bacterial solution in each group. 1 mL of the corresponding gradient bacterial solution was inoculated into each tube, mixed well, and incubated at 44.5℃. Fluorescence signal was monitored in real time with an excitation wavelength of 365 nm and an emission wavelength of 405 nm. The criteria for positive reporting were the same as in Experiment 1.

[0067] 5.4 Experimental Results and Analysis Comparison of positive reporting time: Under the same bacterial concentration, the positive reporting time of the modified polymer experimental group was significantly shorter than that of the unmodified control group and the blank control group. 4 For high-concentration bacterial suspensions (CFU / mL), the positive reporting time was 7 hours for the blank control group, 4 hours for the unmodified control group, and only 2 hours for the modified polymer experimental group, representing a 71.4% reduction compared to the blank control group and a 50% reduction compared to the unmodified control group. For medium-concentration bacterial suspensions (10²CFU / mL), the positive reporting time was 14 hours for the blank control group, 8 hours for the unmodified control group, and only 5 hours for the modified polymer experimental group. 0 CFU / mL weakly positive bacterial suspension: no positive results were reported in the blank control group within 24 hours, positive results were reported in the unmodified control group within 22 hours, and positive results were achieved in the modified polymer experimental group within 16 hours.

[0068] Comparison of fluorescence signal intensity: After 24 hours of cultivation, at the same bacterial concentration, the fluorescence signal intensity of the unmodified control group was 3.0 times higher than that of the blank control group, and the fluorescence signal intensity of the modified polymer experimental group was 4.5 times higher than that of the blank control group and 50% higher than that of the unmodified control group. This confirms that quaternary ammonium group modification can further enhance the fluorescence enhancement effect, which is consistent with the electrostatic-hydrophobic synergistic enhancement mechanism described in this invention.

[0069] Detection rate comparison: within a 24-hour culture period, 10 0 The detection rate of CFU / mL weak positive bacterial suspension was 0% in the blank control group, 66.7% in the unmodified control group, and 100% in the modified polymer experimental group; the detection rate of 10¹CFU / mL low concentration bacterial suspension was 33.3% in the blank control group, 100% in the unmodified control group, and 100% in the modified polymer experimental group within 12 hours.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the present invention.

Claims

1. An additive for improving the sensitivity of the luciferase substrate method, characterized in that, The additive is an amphiphilic block copolymer; The amphiphilic block copolymer comprises hydrophilic segments and hydrophobic segments connected by covalent bonds; The hydrophilic segment is selected from any one or a combination of polyethylene glycol, polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, and their quaternary ammonium salt modified derivatives; The hydrophobic segment is selected from one or more combinations of polylactic acid, polycaprolactone, polylactide-glycolic acid, polystyrene, polyethylene, polymethyl methacrylate and their derivatives.

2. The additive for improving the sensitivity of the luciferase substrate method according to claim 1, characterized in that, The amphiphilic block copolymer is an unmodified amphiphilic block copolymer or a quaternary ammonium salt modified amphiphilic block copolymer; The quaternary ammonium salt modified amphiphilic block copolymer was prepared by the following method: Ion exchange resin was mixed with acetylthiocholine iodide for adsorption to obtain a resin loaded with a modifier. The unmodified amphiphilic block copolymer was dissolved in an organic solvent, and the resin loaded with the modifier and the initiator were added. The temperature was then raised to carry out the grafting reaction. After the reaction was completed, rotary evaporation was performed to obtain a quaternary ammonium salt modified amphiphilic block copolymer.

3. The additive for improving the sensitivity of the luciferase substrate method according to claim 1, characterized in that, The structure of the amphiphilic block copolymer is a diblock, triblock, or multiblock structure; preferably a diblock copolymer.

4. The additive for improving the sensitivity of the luciferase substrate method according to claim 1, characterized in that, The number-average molecular weight of the amphiphilic block copolymer ranges from 1,000 to 200,000 Daltons; the weight percentage of the hydrophilic segment in the amphiphilic block copolymer is 30% to 70%.

5. A composition for improving the sensitivity of a luciferase substrate assay, characterized in that, Includes a luciferase substrate and the additives described in any one of claims 1-4, as well as a selective inhibitor and / or a basic component of a microbial culture medium; The selective inhibitor is selected from one or more of bile salts, sodium dodecyl sulfate, sodium azide, and high-concentration sodium chloride; the basic component of the microbial culture medium is selected from one or more of buffer salts and peptone.

6. The composition for improving the sensitivity of the luciferase substrate method according to claim 5, characterized in that, The luciferase substrate is selected from one or more of the following: 4-methylumbelliferone-β-D-glucoside, 4-methylumbelliferone-β-D-glucuronide, 4-methylumbelliferone-β-D-galactoside, 4-methylumbelliferone-α-D-glucoside, 4-methylumbelliferone phosphate, luciferin 2-β-D-galactopyranoside, halogen-β-D-galactopyranoside, halogen-β-D-glucoside, luciferin di(β-D-galactopyranoside), and chlorophenol red-β-D-galactopyranoside.

7. A method for improving the sensitivity of a fluorescent enzyme substrate method, characterized in that, Includes the following steps: S1: The luciferase substrate and the amphiphilic block copolymer described in claim 5 or 6 are mixed in a solvent to form a complex containing the luciferase substrate; S2: React the complex with the luciferase in the sample to be tested, and detect the fluorescence signal generated by the reaction; Under the same detection conditions, the fluorescence signal intensity generated by the reaction of the complex with the luciferase is higher than that generated by the reaction of the luciferase substrate that has not formed the complex with the luciferase.

8. The method for improving the sensitivity of the luciferase substrate method according to claim 7, characterized in that, In step S1, the solvent is a buffer solution or an organic solvent; The buffer solution is selected from one of carbonate buffer and phosphate buffer, and the pH range of the buffer solution is 7.0-10.0; The organic solvent is selected from one or more of acetone and N,N'-dimethylformamide; The mixing temperature of the luciferase substrate and the amphiphilic block copolymer is 25-37°C, and the mixing time is 5-120 minutes.

9. The method for improving the sensitivity of the luciferase substrate method according to claim 8, characterized in that, In step S2, the reaction includes selectively culturing the target microorganism in the sample to be tested, causing the fluorescent enzyme expressed by the microorganism to undergo an enzymatic reaction with the complex, and the reaction temperature is 37-44.5℃. During the reaction, the fluorescence intensity change is continuously monitored by a fluorescence detection device. The excitation wavelength and emission wavelength of the fluorescence detection device are adjusted according to the type of fluorescent enzyme substrate. Specifically, the excitation wavelength for 4-methylumbelliferous ketone substrates is set to 360-365nm, and the emission wavelength is set to 405-440nm; the excitation wavelength for halogenated substrates is set to 570nm, and the emission wavelength is set to 585nm.

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