A method for detecting acid phosphatase by constructing a ratiometric fluorescence sensor based on bifunctional BSA-MnO2 QDs

Abstract

This invention discloses a method for detecting acid phosphatase by constructing a ratiometric fluorescence sensor based on bifunctional BSA-MnO2QDs, comprising: synthesizing bifunctional BSA-MnO2QDs; incubating with AAP, different concentrations of ACP, BSA-MnO2QDs, and OPD; measuring the fluorescence emission curve of the sample to obtain the fluorescence ratio F1 / F2 at 438 nm and 570 nm; establishing an ACP standard curve with the ACP concentration as the x-axis and the fluorescence ratio F1 / F2 as the y-axis; measuring the fluorescence ratio F1 / F2 of the sample to be tested and substituting it into the ACP standard curve to obtain the ACP concentration. The ratiometric fluorescence sensor constructed by this invention based on BSA-MnO2QDs for detecting ACP has a linear range of 0.1–1.5 mU / mL and a detection limit of 0.038 mU / mL.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection and relates to a method for detecting acid phosphatase by constructing a ratio fluorescence sensor based on bifunctional BSA-MnO2 QDs. More specifically, it relates to a method for detecting acid phosphatase by a high-sensitivity ratio fluorescence sensor based on high-oxidase-activity BSA-MnO2 QDs. Background Technology

[0002] Fluorescence analysis boasts advantages such as high sensitivity, simple operation, and rapid signal response, making it one of the most commonly used detection methods. However, many fluorescence methods employ a single-wavelength output mode, making them susceptible to interference from the microenvironment and excitation source fluctuations, leading to large systematic errors, inaccurate results, and low reproducibility. To address these shortcomings, ratiometric fluorescence has emerged. Ratiometric fluorescence utilizes the ratio of dual-wavelength signal outputs to effectively eliminate interference from the external environment, ensuring that the results are solely related to the analyte itself, thus making the method more accurate. Based on different modes and mechanisms, ratiometric fluorescence methods include fluorescence resonance energy transfer (FRET), internal filtering effect (IFE), and photoinduced electron transfer (PET). Depending on the mode and mechanism, single-signal fluctuation and dual-signal fluctuation ratiometric fluorescence methods can be constructed; compared to the former, the latter exhibits a lower background signal. Furthermore, its inverse dual-signal changes result in higher sensitivity. Ratiometric fluorescence also requires the introduction of various fluorescent materials, including quantum dots, nanoclusters, carbon dots, fluorescent dyes, and upconversion fluorescent materials. Among these, quantum dots play an indispensable role in the development of ratiometric fluorescence.

[0003] Quantum dots, or semiconductor nanocrystals, are primarily composed of Group II and Group VI transition elements and have become an important component of sensing, imaging, and optical devices over the past few decades. Quantum dots possess a wide excitation wavelength range and a narrow emission wavelength range. Therefore, by adjusting the excitation wavelength, the autofluorescence of biological samples can be minimized, improving resolution and sensitivity. Quantum dots exhibit strong resistance to photobleaching and high photochemical stability, approximately 100 times that of ordinary fluorescent dyes. Furthermore, quantum dots have a longer fluorescence lifetime, reaching tens of nanoseconds, much longer than the few nanoseconds of typical organic fluorescent dyes. Thus, after a few nanoseconds of photoexcitation, most of the autofluorescence background has decayed, but the fluorescence of the quantum dots remains, resulting in a fluorescence signal without background interference. In addition, individual quantum dots possess good water solubility, low toxicity, and good biocompatibility. Due to these unique optical properties, quantum dots can replace organic fluorescent dyes as ideal fluorescent probes.

[0004] Nanozymes are nanomaterials with catalytic activity similar to natural enzymes. Nanozymes possess advantages such as high stability, simple preparation, high cost-effectiveness, and easy storage, and have gradually become a substitute for natural enzymes. Nanozymes exhibit high activity and ease of modification, and have wide applications in fields such as biosensing, environmental protection, antibacterial, antitumor, and cell protection. Since the discovery of the intrinsic peroxidase activity of Fe3O4 nanoparticles, researchers have been striving to explore nanomaterials with similar natural enzyme activity. To date, a large number of nanomaterials with enzyme activity have been discovered, including metal oxides, noble metals, and carbon-based nanomaterials. However, most nanomaterials only provide a single enzyme activity. With the development of science and technology, researchers have begun to focus on the research of multifunctional nanozymes. For example, manganese dioxide nanosheets have been found to simultaneously possess intrinsic oxidase, peroxidase, catalase, and superoxide dismutase activities. Modifying the surface of Fe3O4 nanoparticles with platinum nanoparticles gives them both magnetic and peroxidase properties. Surface modification of carbonyl groups can yield carbon nanotube quantum dots with ultra-high peroxidase activity. Compared with single-enzyme-active nanomaterials, multifunctional nanozymes combine their own properties and enzyme activity, making them a novel type of biosensing probe for the future field of real-time diagnostics.

[0005] Acid phosphatase (ACP) is a hydrolase that catalyzes the hydrolysis of phosphate monoesters to inorganic phosphate under acidic conditions. It is mainly found in the prostate, liver and spleen, cells, and bone. Serum ACP is highly expressed when these organs are diseased, such as in prostate cancer with bone metastasis, primary or metastatic bone tumors, leukemia, breast cancer, hepatitis, cirrhosis, and hemolytic diseases. Therefore, ACP can serve as an essential biomarker for auxiliary diagnosis and prognosis, and its analysis is necessary. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing detection methods, such as low sensitivity, long detection time, high cost, and cumbersome procedures. A novel fluorescent nanozyme BSA-MnO2 QDs was synthesized, and a ratiometric fluorescence sensing system was established using BSA-MnO2 QDs. The sensitive fluorescent sensor based on the high oxidase activity BSA-MnO2 QDs was used to detect ACP. This method can detect ACP simply, quickly, intuitively, and with high sensitivity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A bifunctional BSA-MnO2 quantum dots (BSA-MnO2 QDs) are prepared by using manganese chloride tetrahydrate (MnCl2·4H2O) as raw material, hydrogen peroxide as oxidant to synthesize MnO2 nanosheets, and then using MnO2 (MnO2) nanosheets as precursor and BSA as etchant via a hydrothermal method.

[0009] Specifically, the bifunctional BSA-MnO2 QDs are prepared by the following method:

[0010] Step (a): Dissolve 0.1–1 g of MnCl2·4H2O in 10–30 mL of ultrapure water to obtain a manganese chloride solution; under vigorous stirring, add 1–5 mL of 30% hydrogen peroxide to the manganese chloride solution, react at room temperature for 10–15 h, centrifuge the reaction solution, wash the precipitate with ethanol and ultrapure water in sequence, vacuum dry, grind, and obtain MnO2 nanosheets;

[0011] Step (b): Add 10-40 mg of MnO2 nanosheets to 20 mL of BSA aqueous solution with a concentration of 0.25-3 mg / mL, sonicate to dissolve, react at 120 °C for 8-10 h, centrifuge, discard the precipitate, dialyze the supernatant using a dialysis bag with a molecular weight cutoff of 3500 Da to remove impurities, and vacuum dry to obtain BSA-MnO2 quantum dots.

[0012] In step (a), preferably, 0.4 g of MnCl2·4H2O is dissolved in 20 mL of ultrapure water.

[0013] Preferably, 2 mL of 30% hydrogen peroxide is used for every 0.4 g MnCl2·4H2O.

[0014] Preferably, the reaction time is 12 hours.

[0015] The centrifugation speed is 12000 rpm, and the centrifugation time is 3 to 10 minutes, preferably 5 minutes;

[0016] In step (b), preferably, 20 mg of MnO2 nanosheets are added to 20 mL of a BSA aqueous solution with a concentration of 1 mg / mL.

[0017] The centrifugation speed is 12000 rpm, and the centrifugation time is 10-30 min, preferably 20 min.

[0018] A method for detecting acid phosphatase by constructing a ratiometric fluorescence sensor based on bifunctional BSA-MnO2 QDs includes the following steps:

[0019] Step (a) Synthesis of MnO2 nanosheets: MnO2 nanosheets were synthesized using manganese chloride tetrahydrate (MnCl2·4H2O) as raw material and hydrogen peroxide as oxidant;

[0020] Step (b) Synthesis of bifunctional nanocomposite material BSA-MnO2 QDs: BSA-MnO2 quantum dots (BSA-MnO2 QDs) were prepared by hydrothermal method using MnO2 nanosheets as precursor and BSA as etchant.

[0021] Step (c) Construction of an acid phosphatase ratio fluorescence sensor: Add trisodium 2-phosphate-L-ascorbate (AAP) solution and acid phosphatase solutions of different concentrations to an acetate-sodium acetate buffer solution, incubate at 30-40℃ for 10-40 min, then add phosphate buffer containing o-phenylenediamine (OPD) and BSA-MnO2 QDs, incubate at 30-40℃ for 1-10 min to obtain the detection system; measure the fluorescence emission curve of the detection system under 380 nm excitation light to obtain the fluorescence ratio F1 / F2 at 438 nm and 570 nm; establish an ACP standard curve with ACP concentration as the x-axis and fluorescence ratio F1 / F2 as the y-axis;

[0022] Step (d), Sample detection: The fluorescence ratio F1 / F2 of the test sample with unknown acid phosphatase concentration was measured according to step (c), and substituted into the ACP standard curve of step (c) to obtain the acid phosphatase concentration in the test sample.

[0023] In step (c), the 2-phosphate-L-ascorbic acid trisodium solution is prepared using ultrapure water or acetate-sodium acetate buffer solution as the solvent.

[0024] The acid phosphatase solution is prepared using ultrapure water or acetate-sodium acetate buffer solution as the solvent.

[0025] The acetate-sodium acetate buffer solution is an acetate-sodium acetate buffer solution with a pH of 3.0 to 5.0, preferably an acetate-sodium acetate buffer solution with a pH of 4.0.

[0026] Preferably, after adding trisodium 2-phosphate-L-ascorbate and acid phosphatase to the acetate-sodium acetate buffer, the incubation temperature is 35°C and the incubation time is 30 min.

[0027] The phosphate buffer containing o-phenylenediamine and BSA-MnO2 QDs is obtained by adding o-phenylenediamine solution and BSA-MnO2 QDs solution to the phosphate buffer.

[0028] The o-phenylenediamine solution was prepared using ultrapure water or phosphate buffer as a solvent.

[0029] The BSA-MnO2 QDs are prepared using ultrapure water or phosphate buffer as solvents.

[0030] The phosphate buffer solution is a pH 6-8 phosphate buffer solution, preferably a pH 7.5 phosphate buffer solution.

[0031] Preferably, after adding phosphate buffer containing o-phenylenediamine (OPD) and BSA-MnO2 QDs, the incubation temperature is 35°C and the incubation time is 5 minutes.

[0032] The volume ratio of the acetate-sodium acetate buffer, the trisodium 2-phosphate-L-ascorbate solution, the phosphatase solution, and the phosphate buffer containing o-phenylenediamine and BSA-MnO2 QDs is 40:5:5:450.

[0033] In the detection system, the final concentration of trisodium 2-phosphate-L-ascorbate is 5–15 mM, preferably 10 mM; the final concentration of BSA-MnO2 QDs is 1–10 μg / mL, preferably 5 μg / mL; the final concentration of OPD is 1–5 mM, preferably 2 mM; and the concentration of acid phosphatase is 0.1–1.5 mU / mL, specifically selected from 0.1, 0.25, 0.5, 1, and 1.5 mU / mL.

[0034] Specifically, an acid phosphatase ratio fluorescence sensor was constructed as follows: 50 μL of OPD solution and 50 μL of BSA-MnO2 QDs solution were added to 350 μL of phosphate buffer to obtain a phosphate buffer containing OPD and BSA-MnO2 QDs; 5 μL of trisodium 2-phosphate-L-ascorbate solution and 5 μL of acid phosphatase solutions of different concentrations were added to 40 μL of acetate-sodium acetate buffer, and the mixture was incubated. Then, 450 μL of phosphate buffer containing OPD and BSA-MnO2 QDs was added, and the mixture was incubated again to obtain the detection system.

[0035] In step (d), specifically, sample detection: add trisodium 2-phosphate-L-ascorbate solution and the sample to be tested with an unknown concentration of acid phosphatase to the acetate-sodium acetate buffer, incubate at 30-40℃ for 10-40 min, then add phosphate buffer containing o-phenylenediamine and BSA-MnO2 QDs, incubate at 30-40℃ for 1-10 min to obtain the detection system; measure the fluorescence emission curve of the detection system under 380 nm excitation light, and obtain the fluorescence ratio F1 / F2 of the fluorescence intensity at 438 nm and 570 nm. Substitute the fluorescence ratio F1 / F2 into the ACP standard curve in step (c) to obtain the concentration of acid phosphatase in the sample to be tested.

[0036] The sample to be tested was selected from serum.

[0037] When the sample to be tested is serum, mix the serum with 10% trichloroacetic acid at a volume ratio of 1:1, cool in an ice bath for 10 minutes, centrifuge, and collect the supernatant.

[0038] The detection mechanism of the method of the present invention ( Figure 1 BSA-MnO2 QDs possess oxidase activity and can catalyze the formation of superoxide radicals (O2 free radicals) from free O2. ·- Based on the oxidase-like activity of BSA-MnO2 QDs, a ratiometric fluorescence sensor for the quantitative determination of ACP was established. In the absence of ACP, the oxidase-like activity of BSA-MnO2 QDs cannot be inhibited by AAP, thus catalytically oxidizing OPD to 2,3-diaminophenazine (DAP). The abundant catalytic product DAP undergoes an intermolecular emission reaction (IFE) with the BSA-MnO2 QDs quantum dots, leading to a decrease in the fluorescence intensity of the BSA-MnO2 QDs quantum dots at 438 nm. Conversely, in the presence of ACP, ACP hydrolyzes AAP to generate AA, thereby inhibiting the oxidase activity of BSA-MnO2 QDs, resulting in a decrease in DAP yield. Therefore, the IFE is also inhibited, and the fluorescence intensity of BSA-MnO2 QDs at 438 nm recovers.

[0039] Compared with the prior art, the present invention has the following significant advantages:

[0040] 1. This invention prepares a novel "two-in-one" multifunctional BSA-MnO2 quantum dot, which has excellent fluorescence properties and high oxidase-like activity.

[0041] 2. This invention constructs a highly sensitive and selective ratiometric fluorescence sensor based on BSA-MnO2 quantum dots for the detection of acid phosphatase, with a linear range of 0.1–1.5 mU / mL and a detection limit of 0.038 mU / mL.

[0042] 3. The ratio fluorescence sensor constructed in this invention can be successfully applied to the determination of acid phosphatase in human serum samples. Attached Figure Description

[0043] Figure 1 Mechanism for quantitative determination of ACP using a ratio fluorescence sensor.

[0044] Figure 2Characterization of the supernatant obtained at different reaction times; where a is the TEM image of MnO2 nanosheets, b is the TEM image of the supernatant obtained after 4 h of reaction, c is the TEM image of the supernatant obtained after 6 h of reaction, d is the TEM image of the supernatant containing BSA-MnO2 quantum dots obtained after 8 h of reaction, e is the XPS narrow scan spectrum of Mn 2p in BSA-MnO2 quantum dots, and f is the UV-Vis absorption curve of MnO2 nanosheets (black curve) and BSA-MnO2 quantum dots (red curve).

[0045] Figure 3 Fluorescence emission curves (A) and ACP standard curves of the detection systems with different concentrations of ACP constructed in Example 3.

[0046] Figure 4 This is a comparison chart of the selectivity of the detection system constructed in Example 3 for ACP.

[0047] Figure 5 The results show the effect of different pH values ​​on the fluorescence performance of BSA-MnO2 quantum dots.

[0048] Figure 6 The results show the photostability of BSA-MnO2 quantum dots. Detailed Implementation

[0049] The technical solution of the present invention will be described in more detail with reference to the accompanying drawings and specific embodiments. Although the following are preferred embodiments of the present invention, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0050] The inventors used fluorescence methods to study the properties of BSA-MnO2 quantum dots, which exhibit strong oxidase activity. The reactions involved incubating BSA-MnO2 quantum dots, OPD, and different concentrations of ACP, measuring the fluorescence curves of the samples, and establishing a standard curve based on the fluorescence ratio.

[0051] Example 1

[0052] Step (a) Synthesis of MnO2 nanosheets: 0.4 g of MnCl2·4H2O was dissolved in 20 mL of ultrapure water to obtain a manganese chloride solution; under vigorous stirring, 2 mL of 30% hydrogen peroxide was added to the manganese chloride solution, and the reaction was carried out at room temperature for 12 h to obtain a brown solution. The solution was centrifuged at 12000 rpm for 5 min, and the precipitate was washed successively with anhydrous ethanol and ultrapure water until the supernatant was transparent. The obtained black lumps were vacuum dried and then ground into fine powder in a mortar to obtain MnO2 nanosheets. Figure 2 a);

[0053] Step (b): Add 20 mg of MnO2 nanosheets to 20 mL of BSA aqueous solution with a concentration of 1 mg / mL and dissolve by sonication; transfer the mixed solution to a reaction vessel and react at 120 °C for 4, 6, 8 and 10 h respectively. Centrifuge the resulting suspension at 12000 rpm for 20 min, discard the bottom precipitate, and characterize the supernatant.

[0054] Depend on Figure 2 As can be seen, the TEM images of the products obtained after 4, 6, and 8 hours of reaction are as follows: Figure 2 As shown in b, c, and d, when the reaction time is 4 and 6 hours, the product is nanorods. When the reaction reaches 8 hours, the product changes from the nanorod state to the quantum dot state, indicating that BSA-MnO2 quantum dots were obtained. The average particle size of BSA-MnO2 quantum dots is 6.44 nm.

[0055] Figure 2 The two peaks in e are attributed to two characteristic peaks of the Mn element. Figure 2 f represents the UV absorption spectra of MnO2 nanosheets and BSA-MnO2 quantum dots, both of which demonstrate the successful synthesis of BSA-MnO2 quantum dots.

[0056] Example 2

[0057] The preparation method of MnO2 nanosheets is described in step (a) of Example 1; the preparation method of BSA-MnO2 quantum dots is described in step (b) of Example 1. During the synthesis process, only the concentration of BSAs aqueous solution was adjusted to 0.25, 0.5 and 1 mg / mL, and the reaction time was 8 h for all of them, and BSA-MnO2 quantum dots were obtained in all cases.

[0058] The quantum yield was calculated using quinine sulfate quantum dots as a standard, and the relative quantum number y (Yu = Ys·Fu / Fs·as / Au) of BSA-MnO2 quantum dots at an excitation wavelength of 310 nm was determined. Table 1 shows that the yield of BSA-MnO2 QDs increases with increasing BSA concentration, reaching 10.7% when the BSA concentration is 1 mg / mL.

[0059] To quantify the catalytic efficiency and affinity for TMB, steady-state kinetic parameters, including the Michaelis-Menten constant (Km) and the maximum initial velocity Vmax, were calculated using a double reciprocal function (1 / V = (Km / Vmax) × (1 / [S]) + 1 / Vmax). 100 μL of BSA-MnO2 quantum dot solution (50 μg / mL) and 100 μL of 10 mM TMB were added to pH 4 acetate-sodium acetate buffer, and the absorbance was measured after 10 minutes of reaction. Table 1 shows that the Km of BSA-MnO2 QDs increases with increasing BSA concentration. This phenomenon is attributed to the inhibitory effect of the sulfhydryl groups in bovine serum albumin on oxidase activity.

[0060] Taking into account both the fluorescence properties and enzyme activity of BSA-MnO2 quantum dots, 0.5 mg / mL was selected as the optimal concentration of BSA for the synthesis of BSA-MnO2 quantum dots.

[0061] Table 1. Kinetic parameters (Km and Vmax) and yield of BSA-MnO2 quantum dots synthesized with different BSA concentrations

[0062]

[0063] Example 3

[0064] A method for detecting acid phosphatase using a ratiometric fluorescence sensor constructed from bifunctional BSA-MnO2 quantum dots includes the following steps:

[0065] Step (a) Synthesis of MnO2 nanosheets: 0.4 g of MnCl2·4H2O was dissolved in 20 mL of ultrapure water to obtain a manganese chloride solution; under vigorous stirring, 2 mL of 30% hydrogen peroxide was added to the manganese chloride solution, and the reaction was carried out at room temperature for 12 h to obtain a brown solution. The solution was centrifuged at 12000 rpm for 5 min, and the precipitate was washed with anhydrous ethanol and ultrapure water until the supernatant was transparent. The obtained black block was vacuum dried and then ground into fine powder in a mortar to obtain MnO2 nanosheets.

[0066] Step (b): Using MnO2 nanosheets as a precursor, BSA-MnO2 quantum dots (BSA-MnO2 QDs) were prepared by hydrothermal method: 20 mg of MnO2 nanosheets were added to 20 mL of BSA aqueous solution with a concentration of 1 mg / mL and dissolved by ultrasonication; the mixed solution was transferred to a reaction vessel and reacted at 120 °C for 8 h; the resulting suspension was centrifuged at 12000 rpm for 20 min, the bottom precipitate was discarded, and the supernatant was dialyzed with a dialysis bag with a molecular weight cutoff of 3500 Da to remove impurities from the supernatant; the dialysate (BSA-MnO2 quantum dot concentration of 50 μg / mL) was vacuum dried to obtain BSA-MnO2 quantum dots;

[0067] Step (c) Construction of an acid phosphatase ratio fluorescence sensor: Trisodium 2-phosphate-L-ascorbate (AAP) solution, acid phosphatase solutions of different concentrations, OPD solution, and BSA-MnO2 QDs solution (concentration 50 μg / mL) were prepared using ultrapure water. 50 μL of OPD solution and 50 μL of BSA-MnO2 QDs solution were added to 350 μL of phosphate buffer (pH 7.5, 5 mM) to obtain a phosphate buffer containing OPD and BSA-MnO2 QDs. 5 μL of trisodium 2-phosphate-L-ascorbate solution and 5 μL of acid phosphatase solutions of different concentrations were added to 40 μL of pH 4.0 acetate-sodium acetate buffer. The mixture was incubated at 35°C for 30 min, and then 450 μL of a solution containing OPD and BSA-MnO2 was added. The phosphate buffer of QDs was incubated at 35°C for 5 minutes to obtain the detection system. The final concentrations of AAP in the detection system were 10 mM, acid phosphatase concentrations were 0.1, 0.25, 0.5, 1, and 1.5 mU / mL, OPD concentration was 2 mM, and BSA-MnO2 QDs concentration was 5 μg / mL. The fluorescence emission curve of the detection system was measured under 380 nm excitation light. Figure 3 The output signal fluorescence ratio F1 / F2 is the ratio of the intensities of the two peaks at 438 nm and 570 nm; an ACP standard curve is constructed with the concentration of ACP on the x-axis and the fluorescence ratio F1 / F2 on the y-axis. Figure 3 B): Y = 1.26X + 0.6285, R 2 =0.9949.

[0068] Step (d), Sample detection: The fluorescence ratio of the sample to be tested with unknown acid phosphatase concentration was measured according to step (c), and substituted into the hydrogen peroxide standard curve in step (c) to obtain the acid phosphatase concentration in the sample to be tested.

[0069] Example 4

[0070] Investigating the selectivity of ratio fluorescence sensors for ACP

[0071] The selectivity of the ratiometric fluorescence sensor was investigated using common interfering enzymes such as lysozyme, horseradish peroxidase (HRP), bovine serum albumin (BSA), trypsase, glucose oxidase (GOx), and alkaline phosphatase (ALP).

[0072] Trisodium 2-phosphate-L-ascorbate (AAP) solution, acid phosphatase solution, interferon enzyme solution, OPD solution, and BSA-MnO2 QDs solution (concentration 50 μg / mL) were prepared using ultrapure water. 50 μL of OPD solution and 50 μL of BSA-MnO2 QDs solution were added to 350 μL of phosphate buffer (pH 7.5, 5 mM) to obtain phosphate buffer containing OPD and BSA-MnO2 QDs. Add 5 μL of trisodium 2-phosphate-L-ascorbate solution and 5 μL of interferon solution or acid phosphatase solution to 40 μL of pH 4.0 acetate-sodium acetate buffer. Incubate at 35℃ for 30 min. Then add 450 μL of phosphate buffer containing OPD and BSA-MnO2 QDs and incubate at 35℃ for 5 min to obtain the detection system. In the detection system, the final concentration of AAP is 10 mM, the final concentration of interferon or acid phosphatase is 10 mU / mL, the final concentration of OPD is 2 mM, and the final concentration of BSA-MnO2 QDs is 5 μg / mL. Measure the fluorescence emission curve of the detection system under 380 nm excitation light and record the fluorescence value.

[0073] See results Figure 3 Compared with the blank control and other interfering enzymes, ACP showed a significant signal response, indicating that the detection method based on BSA-MnO2 quantum dots has high selectivity and can distinguish ACP from other non-targets.

[0074] Example 5

[0075] Investigating the effect of different pH values ​​on the fluorescence properties of BSA-MnO2 quantum dots

[0076] 50 μL of BSA-MnO2 quantum dot solution (50 μg / mL, prepared with ultrapure water) was added to 450 μL of phosphate buffer solutions with pH values ​​of 3, 4, 5, 6, 6.4, 7, 8, and 9, respectively. The mixture was thoroughly mixed, and the fluorescence spectra of the BSA-MnO2 quantum dots were measured.

[0077] See results Figure 4The results showed that the fluorescence intensity of BSA-MnO2 quantum dots at 438 nm was pH-dependent, increasing with increasing pH. The fluorescence intensity reached its maximum at pH = 7 and pH = 8. Therefore, we ultimately chose pH 7.5 as the optimal reaction pH.

[0078] Example 6

[0079] Investigating the photostability of BSA-MnO2 quantum dots

[0080] The BSA-MnO2 quantum dot solution (50 μg / mL, prepared with ultrapure water) was irradiated under a 365 nm UV lamp for 120 minutes. The fluorescence emission curve of the detection system was measured and the fluorescence value was recorded under 380 nm excitation light to investigate the change in fluorescence intensity of BSA-MnO2 quantum dots at 438 nm.

[0081] See results Figure 5 As can be seen, the fluorescence intensity of BSA-MnO2 quantum dots did not change significantly, indicating that they have good optical stability and resistance to photobleaching.

[0082] Example 7

[0083] Table 2 shows a comparison of the analytical performance of the method of this invention with other reported detection methods.

[0084] Table 2. Comparison of the ACP detection performance of the method of the present invention with other reported detection methods.

[0085]

[0086] References:

[0087] [1] Y.Chen, Z.Wang,

[0088] [2]Z.Qian,L.Chai,Q.Zhou,Y.Huang,C.Tang,J.Chen and H.Feng,ReversibleFluorescent Nanoswitch Based on Carbon Quantum Dots Nanoassembly for Real-Time Acid Phosphatase Activity Monitoring,ANAL CHEM 14(2015)7332-7339.

[0089] [3]Y.Huang,H.Feng,W.Liu,Y.Zhou,C.Tang,H.Ao,M.Zhao,G.Chen,J.Chen andZ.Qian,Luminescent Aggregated Copper Nanoclusters Nanoswitch Controlled byHydrophobic Interaction for Real-Time Monitoring of Acid PhosphataseActivity,ANAL CHEM 23(2016)11575-11583.

[0090] [4]Z.Zhu,X.Lin,L.Wu,C.Zhao,Y.Zheng,A.Liu,L.Lin and X.Lin,“Switch-On”fluorescent nanosensor based on nitrogen-doped carbon dots-MnO2nanocomposites for probing the activity of acid phosphatase,Sensors andActuators B:Chemical(2018)609-615.

[0091] [5]X.Yan,C.Xia,B.Chen,Y.F.Li,P.F.Gao and C.Z.Huang,Enzyme ActivityTriggered Blocking of Plasmon Resonance Energy Transfer for Highly SelectiveDetection of Acid Phosphatase,ANAL CHEM 2(2020)2130-2135.

[0092] [6]Y. Xu, B. Li, L. Xiao, J. Ouyang, S. Sun and Y. Pang, A colorimetric and near-infrared fluorescent probe with high sensitivity and selectivity for acid phosphatase and inhibitor screening, CHEM COMMUN 63(2014)8677.

[0093] [7]H. H. Deng, X. L. Lin, Y. H. Liu, K. L. Li, Q. Q. Zhuang, H. P. Peng, A. L. Liu, X. H. Xia and W. Chen, Chitosan-stabilized platinum nanoparticles as effective oxidase mimics for colorimetric detection of acid phosphatase, NANOSCALE 29(2017)10292-10300.

[0094] [8]X. Gan, F. Qiu, B. Jiang, R. Yuan and Y. Xiang, Convenient and highly sensitive electrochemical biosensor for monitoring acid phosphatase activity, Sensors and Actuators B: Chemical(2021)129483.

[0095] [9]Z. Lin, X. Zhang, S. Liu, L. Zheng, Y. Bu, H. Deng, R. Chen, H. Peng, X. Lin and W. Chen, Colorimetric acid phosphatase sensor based on MoO3 nanozyme, ANALCHIMACTA(2020)162-168.

[0096] Application Example 1

[0097] Serum samples from healthy individuals were analyzed. Serum was mixed with 10% trichloroacetic acid at a volume ratio of 1:1, cooled in an ice bath for 10 minutes to obtain protein precipitation, centrifuged at 12000 rpm for 15 minutes, and the supernatant was used as the test sample. ACP standards of 0, 1.5, 2.0, and 2.5 mU / mL were added to the test sample, and the fluorescence signal was measured according to the method in Example 3. The fluorescence ratio F1 / F2 was obtained and substituted into the ACP standard curve established in Example 3 to obtain the concentration of ACP standards in the sample. Each sample was measured three times, and the average value was taken. The RSD and recovery rate were calculated, as shown in Table 3.

[0098] Table 3. ACP recovery rate in whole blood (n=3)

[0099]

[0100] The embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for detecting acid phosphatase using a ratiometric fluorescence sensor constructed based on bifunctional BSA-MnO2 QDs, characterized in that: Includes the following steps: Step (a) Synthesis of MnO2 nanosheets: MnO2 nanosheets were synthesized using manganese chloride tetrahydrate as raw material and hydrogen peroxide as oxidant; Step (b) Synthesis of bifunctional BSA-MnO2QDs: BSA-MnO2QDs were prepared by hydrothermal method using MnO2 nanosheets as precursor and BSA as etchant. Step (c) Construction of an acid phosphatase ratio fluorescence sensor: Add trisodium 2-phosphate-L-ascorbate solution and acid phosphatase solutions of different concentrations to an acetate-sodium acetate buffer solution, incubate at 30-40 °C for 10-40 min, then add phosphate buffer containing o-phenylenediamine and BSA-MnO2 QDs, and incubate at 30-40 °C for 1-10 min to obtain the detection system; measure the fluorescence emission curve of the detection system under 380 nm excitation light to obtain the fluorescence ratio F1 / F2 at 438 nm and 570 nm; establish an ACP standard curve with ACP concentration as the x-axis and fluorescence ratio F1 / F2 as the y-axis; Step (d), Sample detection: The fluorescence ratio F1 / F2 of the test sample with unknown acid phosphatase concentration was measured according to step (c), and substituted into the ACP standard curve of step (c) to obtain the acid phosphatase concentration in the test sample.

2. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1, characterized in that: In step (a), 0.1–1 g of manganese chloride tetrahydrate is dissolved in 10–30 mL of ultrapure water to obtain a manganese chloride solution; under stirring, 1–5 mL of 30% hydrogen peroxide is added to the manganese chloride solution, and the reaction is carried out at room temperature for 10–15 h. The reaction solution is centrifuged, and the precipitate is washed successively with ethanol and ultrapure water, vacuum dried, and ground to obtain MnO2 nanosheets.

3. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1 or 2, wherein in step (a), 0.4 g of manganese chloride tetrahydrate is dissolved in 20 mL of ultrapure water; 2 mL of 30% hydrogen peroxide is used for every 0.4 g of manganese chloride tetrahydrate; and in step (b), 20 mg of MnO2 nanosheets are added to 20 mL of a 1 mg / mL BSA aqueous solution.

4. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1, characterized in that: In step (b), 10–40 mg of MnO2 nanosheets were added to 20 mL of BSA aqueous solution with a concentration of 0.25–3 mg / mL, dissolved by sonication, reacted at 120 °C for 8–10 h, centrifuged, the precipitate was discarded, the supernatant was dialyzed with a dialysis bag with a molecular weight cutoff of 3500 Da to remove impurities, and vacuum dried to obtain BSA-MnO2 quantum dots.

5. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1, characterized in that: In step (c), the 2-phosphate-L-ascorbic acid trisodium solution is prepared using ultrapure water or acetate-sodium acetate buffer as the solvent; the acid phosphatase solution is prepared using ultrapure water or acetate-sodium acetate buffer as the solvent.

6. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1 or 5, characterized in that: In step (c), the acetate-sodium acetate buffer solution is an acetate-sodium acetate buffer solution with a pH of 3.0 to 5.

0.

7. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 6, characterized in that: In step (c), the acetate-sodium acetate buffer solution is a pH 4.0 acetate-sodium acetate buffer solution.

8. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1, characterized in that: In step (c), after adding trisodium 2-phosphate-L-ascorbate and acid phosphatase to the acetate-sodium acetate buffer, the incubation temperature is 35 °C and the incubation time is 30 min; after adding phosphate buffer containing o-phenylenediamine and BSA-MnO2 QDs, the incubation temperature is 35 °C and the incubation time is 5 min.

9. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1, characterized in that: In step (c), the phosphate buffer containing o-phenylenediamine and BSA-MnO2 QDs is obtained by adding o-phenylenediamine solution and BSA-MnO2 QDs solution to phosphate buffer; the o-phenylenediamine solution is prepared using ultrapure water or phosphate buffer as solvent; the BSA-MnO2 QDs are prepared using ultrapure water or phosphate buffer as solvent.

10. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1 or 9, characterized in that: In step (c), the phosphate buffer solution is a pH 6-8 phosphate buffer solution.

11. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 10, characterized in that: In step (c), the phosphate buffer is a pH 7.5 phosphate buffer.

12. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 1, characterized in that: In step (c), the final concentration of trisodium 2-phosphate-L-ascorbate in the detection system is 5–15 mM, the final concentration of BSA-MnO2QDs is 1–10 μg / mL, the final concentration of OPD is 1–5 mM, and the concentration of acid phosphatase is 0.1–1.5 mU / mL.

13. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 12, characterized in that: In step (c), the final concentration of trisodium 2-phosphate-L-ascorbic acid in the detection system is 10 mM.

14. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2 QDs according to claim 12, characterized in that: In step (c), the final concentration of BSA-MnO2QDs in the detection system is 5 μg / mL.

15. The method for detecting acid phosphatase based on a ratiometric fluorescence sensor constructed using bifunctional BSA-MnO2QDs according to claim 12, characterized in that: In step (c), the final concentration of OPD in the detection system is 2 mM.

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