A ratiometric fluorescence analysis method for determining alkaline phosphatase activity in samples based on the inner filter effect

Abstract

The present invention provides a ratio fluorescence analysis method for measuring alkaline phosphatase activity in a sample based on an inner filter effect, which belongs to the field of enzyme activity detection technology. It includes: mixing a sample with an incubation buffer, incubating, and standing to obtain a static solution; mixing the static solution with a quantum dot solution to obtain a mixed solution, exciting with 310nm, measuring the fluorescence intensity of the mixed solution at 405nm and 585nm, obtaining a fluorescence intensity ratio of 585nm to 405nm, defined as R, defining the fluorescence intensity ratio of a blank sample as R0, and obtaining R / R0; substituting R / R0 into the formula to obtain alkaline phosphatase activity in the sample. The ratio fluorescence analysis method based on the inner filter effect proposed by the present invention has the obvious advantages of label-free detection, no need for sample pretreatment, and low biological matrix interference, and provides a simple, sensitive, selective, and low-sample consumption new method for detecting alkaline phosphatase activity in complex biological matrices.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme activity detection, and in particular relates to a ratiometric fluorescence analysis method for measuring alkaline phosphatase activity in a sample based on an inner filter effect. Background Art

[0002] Alkaline phosphatase (ALP) is an important phosphomonoesterase found in most human tissues, including serum. Abnormal changes in ALP activity are associated with hepatobiliary diseases, bone diseases, kidney diseases, and cancer. Therefore, convenient, sensitive, and highly selective ALP activity detection is crucial for disease diagnosis. Colorimetry, electrochemistry, and fluorescence analysis have been used for ALP activity detection. Each of these methods has its advantages, but they all have some unavoidable problems, such as the low sensitivity of colorimetry and the complex principle design and chemical modification required by electrochemical methods. Fluorescence methods are simple and sensitive and are widely used in enzyme activity analysis and immunoassays. However, conventional fluorescence methods are often limited by the poor photostability and water insolubility of organic small molecule dyes. In addition, the influence of the biological matrix on the fluorophore cannot be ignored.

[0003] In recent years, quantum dots (QDs) have garnered significant attention for their exceptional photostability, good water solubility, and bright fluorescence. As a novel fluorescent material, they are widely used in the detection of drugs, biomarkers, and viruses. In most fluorescence detection methods involving quantum dots, the enhancement or quenching of the fluorescence signal relies on the interaction between the quantum dots and the target or other intermediate molecules. An effective approach to achieving this specific interaction is to conjugate the QDs with recognition tools such as molecular imprints, aptamers, and antibodies. However, such conjugation can easily lead to QD aggregation or a decrease in luminescence intensity. In practice, constructing high-quality quantum dots functionalized with recognition tools remains a significant challenge. Furthermore, the fluorescence signal is susceptible to environmental factors and sample injection errors, resulting in large fluctuations in the results of traditional fluorescence assays and poor stability and reproducibility. The inner filter effect (IFE) refers to the phenomenon in which the absorption spectrum of an absorbing species in a system overlaps with the excitation or emission spectrum of a fluorophore, leading to quenching of the fluorophore through competitive absorption of the excitation or emission light. The inner filter effect, which only requires spectral overlap and does not require any interaction between the absorbing species and the fluorophore, is an important approach for label-free detection. Furthermore, because quantum dots have a broad excitation spectrum, the choice of light-absorbing substances and the design of detection methods are more flexible when using quantum dots as fluorophores. In recent years, fluorescence analysis methods based on quantum dots and the inner filter effect have been applied to the detection of pesticides, therapeutic drugs, and biomarkers.

[0004] Fluorescence analysis has been previously reported for alkaline phosphatase activity detection. However, significant interference from biological matrices on single-wavelength fluorescence signals limits the application of these methods in complex biological matrices such as serum. In some studies, measures such as sample dilution or target extraction are often employed to reduce matrix interference. However, sample dilution also dilutes the target, requiring the method to be sufficiently sensitive. Furthermore, the tedious sample pretreatment process increases operator workload and can also reduce the accuracy of the test results. Unlike single-wavelength fluorescence analysis, ratiometric fluorescence analysis uses the ratio of fluorescence intensities at multiple wavelengths as the detection signal. In some cases, fluorescence intensities at multiple wavelengths exhibit the same trend in the presence of interfering substances. While fluorescence intensity at a single wavelength is significantly interfered with, the ratio of fluorescence intensities at multiple wavelengths is largely unaffected. Ratiometric fluorescence analysis has become an important approach for reducing matrix interference and has been applied to the detection of both large and small molecules in biological matrices. Therefore, ratiometric fluorescence analysis based on quantum dots and the inner filter effect is a promising method for highly selective, sensitive, and simple detection of alkaline phosphatase activity in biological matrices. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a ratiometric fluorescence analysis method for determining alkaline phosphatase activity in samples based on the inner filter effect. The ratiometric fluorescence analysis method based on the inner filter effect proposed in the present invention has the obvious advantages of label-free detection, no need for sample pretreatment, and low biological matrix interference. It provides a new method for the detection of alkaline phosphatase activity in complex biological matrices that is simple, sensitive, selective, and consumes less sample.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a ratiometric fluorescence analysis method for measuring alkaline phosphatase activity in a sample based on the inner filter effect, comprising the following steps:

[0008] 1) mixing the sample with an incubation buffer, incubating, and allowing to stand to obtain a standing solution;

[0009] The incubation buffer comprises: 50 mM diethanolamine, 1 mM MgCl2 and 200-1000 μM disodium p-nitrophenyl phosphate, with a pH value of 7.5-11.0;

[0010] The incubation temperature is 4 to 45° C., and the incubation time is 15 to 120 minutes;

[0011] 2) mixing the static solution obtained in step 1) with the quantum dot solution to obtain a mixed solution, exciting the solution at 310 nm, and measuring the fluorescence intensity of the mixed solution at 405 nm and 585 nm to obtain a fluorescence intensity ratio of 585 nm to 405 nm, defined as R, and the fluorescence intensity ratio of the blank sample is defined as R0, to obtain R / R0;

[0012] The quantum dot solution includes: 10 mg / ml Mn:ZnS and 0.1 mg / ml ZnSe@ZnS;

[0013] 3) Substituting the R / R0 obtained in step 2) into the following formula to obtain the alkaline phosphatase activity in the sample;

[0014] When the sample is an ALP solution, the formula is: R / R0=0.03688C ALP +0.7513, R 2 =0.9969, where C ALP The unit is U / L;

[0015] When the sample is serum, the formula is: R / R0=0.02085C ALP +1.098, R 2 =0.9907, where C ALP Unit: U / L;

[0016] When the sample is HepG2 cell lysate, the formula is: R / R0=0.0597C HepG2 +0.9932, R 2 =0.9983 where C HepG2 Unit: pieces / ml.

[0017] Preferably, the preparation method of Mn:ZnS comprises the following steps:

[0018] A. Mix zinc stearate, stearic acid, and 1-octadecene at 140° C. to obtain a zinc stock solution;

[0019] B. Mix zinc stearate, manganese stearate, sulfur powder, oleylamine and 1-octadecene at 100° C. to obtain a clear solution;

[0020] C. Mixing the zinc stock solution obtained in step A with the clarified solution obtained in step B at 250° C. for 30 min, filtering the obtained mixture, washing it, and drying the obtained precipitate to obtain oil-soluble Mn:ZnS;

[0021] D. mixing the oil-soluble Mn:ZnS obtained in step C with chloroform and 3-mercaptopropionic acid, performing sonication, and centrifuging to obtain a precipitate;

[0022] E. Mixing the precipitate obtained in step D with a tetramethylammonium hydroxide solution, ultrasonicating, and centrifuging to obtain a supernatant. Adjusting the pH value of the supernatant to 7-8 and then vacuum drying to obtain water-soluble Mn:ZnS, i.e., obtaining Mn:ZnS.

[0023] Preferably, in step A, the volume ratio of the moles of zinc stearate, the moles of stearic acid, and the moles of 1-octadecene is 10 mmol:10 mmol:10 ml;

[0024] In step B, the volume ratio of the moles of zinc stearate, the moles of manganese stearate, the moles of sulfur powder, the moles of oleylamine and the moles of 1-octadecene is 1 mmol:0.05 mmol:5 mmol:7.5 mmol:50 ml;

[0025] The volume ratio of the zinc stock solution to the clarified solution in step C is 1:5.

[0026] Preferably, in step D, the ratio of the mass of the oil-soluble Mn:ZnS to the volume of chloroform and the volume of 3-mercaptopropionic acid is 1.5 g:10 ml:1.6 ml;

[0027] The ultrasonic time is 30min;

[0028] The centrifugal conditions include: centrifugal force of 2500g and centrifugal time of 2 minutes.

[0029] Preferably, the volume ratio of the mass of the precipitate in step E to the tetramethylammonium hydroxide solution is 1.5 g:10 ml;

[0030] The ultrasonic time is 5 minutes;

[0031] The centrifugal conditions include: centrifugal force of 2500g, centrifugal time of 2min;

[0032] The vacuum drying temperature is 50°C.

[0033] Preferably, the preparation method of ZnSe@ZnS comprises the following steps:

[0034] a. Mix selenium powder, sodium borohydride and deionized water, stir, place in an ice bath, and collect the supernatant;

[0035] b. Mix zinc sulfate, reduced glutathione, and deionized water, adjust the pH to 10.5, and then add the supernatant obtained in step a, stir, and reflux in sequence to obtain a ZnSe quantum dot solution;

[0036] c. The ZnSe quantum dot solution obtained in step b was mixed with zinc sulfate and 3-mercaptopropionic acid, and the pH value was adjusted to 10.5. The mixture was stirred and refluxed to obtain a ZnSe@ZnS solution, which was mixed with anhydrous ethanol and centrifuged to obtain a precipitate of ZnSe@ZnS.

[0037] Preferably, in step a, the volume ratio of the molar ratio of selenium powder, the molar ratio of sodium borohydride and deionized water is 0.1 mmol:0.6 mmol:1 ml; the stirring time is 40 min; and the ice bath time is 10 min.

[0038] In step b, the volume ratio of the molar amount of zinc sulfate to the molar amount of reduced glutathione and deionized water is 0.4 mmol:0.5 mmol:100 ml; the stirring time is 30 min; and the reflux time is 1.5 h.

[0039] Preferably, in step c, the volume ratio of the ZnSe quantum dot solution to the molar ratio of zinc sulfate and 3-mercaptopropionic acid is 20 ml:0.1 mmol:26 μl;

[0040] The stirring time is 1h;

[0041] The reflux time is 1h;

[0042] The centrifugal conditions include: centrifugal force of 2500 g, and centrifugal time of 30 min.

[0043] Preferably, the content of disodium p-nitrophenylphosphate in the incubation buffer is 600 μM, and the pH value of the incubation buffer is 10.0;

[0044] The incubation temperature was 37° C., and the incubation time was 75 min.

[0045] The principle of the method provided by the present invention is as follows:

[0046] The present invention proposes a ratiometric fluorescence analysis method based on the inner filter effect and two quantum dots (ZnSe@ZnS and Mn:ZnS) for simple, highly selective and highly sensitive detection of alkaline phosphatase activity in biological matrices. First, due to the high overlap of the excitation spectra of disodium p-nitrophenyl phosphate (PNPP) and the two quantum dots, both quantum dots are quenched by the inner filter effect of PNPP under irradiation with 310nm excitation light. In the presence of alkaline phosphatase (ALP), PNPP is catalytically hydrolyzed to p-nitrophenol (PNP), the absorption band of which red-shifts from 310nm to 405nm. With the consumption of PNPP, the fluorescence intensity of Mn:ZnS quantum dots at 585nm gradually increases as the inner filter effect is lifted. At the same time, the absorption band of PNP overlaps with the emission peak of ZnSe@ZnS. As the concentration of PNP increases, the fluorescence intensity of ZnSe@ZnS quantum dots at 405nm gradually decreases as the inner filter effect is enhanced. The fluorescence intensity ratio at 585 nm and 405 nm (F 585 / F 405 ) increases with increasing alkaline phosphatase activity and exhibits good linearity over the range of 4 to 96 U / L, with a detection limit as low as 0.57 U / L. This method also demonstrates good applicability in serum and HepG2 cell lysates. Furthermore, the performance of this method in real samples was compared with that of a commercial alkaline phosphatase assay kit, demonstrating similar results between the two methods, demonstrating its potential for clinical sample testing.

[0047] The beneficial effects of the present invention are:

[0048] (1) The present invention has high sensitivity and good quantitative performance for alkaline phosphatase activity detection.

[0049] Under the optimal conditions, alkaline phosphatase solution samples with different activities were tested. As the activity of the ALP sample increased, the fluorescence intensity of the incubation system near 585nm increased significantly, while the fluorescence intensity near 405nm gradually decreased, which is consistent with the design principle. The ratio fluorescence signal R / R0 showed a positive correlation with the alkaline phosphatase activity of the sample ( Figure 3 In the range of 4-96 U / L, R / R0 and alkaline phosphatase activity showed good linearity (R / R0 = 0.03688C ALP +0.7513,R 2 =0.9969). Using the 3σ / k principle, the detection limit can be as low as 0.57 U / L. Previous studies have shown that the serum ALP activity range for normal adults is 20 to 140 U / L, making this method highly sensitive enough to meet clinical testing requirements.

[0050] (2) The present invention has good selectivity for the detection of alkaline phosphatase

[0051] To evaluate the selectivity of this protocol, common substances in clinical samples were used as interfering samples, and the alkaline phosphatase sample was replaced by the interfering sample solution. The detection was performed in the same manner as the alkaline phosphatase detection. Common metal ions and common amino acids in serum had little effect on the ratio fluorescence signal R / R0, which was close to that of the control group ( Figure 3 The 100U / L alkaline phosphatase sample resulted in a significant increase in R / R0, indicating that the detection method has good selectivity for common small molecules such as metal ions, amino acids and glucose in serum samples.

[0052] In addition, within the scope of investigation, all macromolecules had almost no effect on the detection signal, which was highly consistent with the results of the control group ( Figure 3 Importantly, interference from the serum matrix was further investigated using heat-inactivated human serum (endogenous ALP is inactivated by treatment at 65°C for 10 minutes). The results showed that even undiluted inactivated serum did not significantly alter the ratiometric fluorescence signal, R / R0, and the results were very close to those of the control group. These results demonstrate that this method has excellent selectivity for proteins and is virtually unaffected by interference from the serum matrix.

[0053] (3) This protocol is reliable and can be used not only for serum sample testing but also for alkaline phosphatase activity analysis in cell lysates and tumor cell detection. It is easy to operate and, as it is virtually unaffected by the biological matrix, requires no sample pretreatment, greatly simplifying the testing process. The test results are accurate and precise.

[0054] The test results further showed that even if the ALP sample matrix was replaced by inactivated human serum from buffer, the ratio fluorescence signal R / R0 still had a good linear relationship in the range of 4 to 96 U / L (R / R0 = 0.02085C ALP +1.098,R 2 =0.9907); Furthermore, the serum of three healthy volunteers was used as test samples, and the method of this protocol and the commercial kit method were used for detection, and the results showed that the overall trend of the detection results of the two methods was consistent ( Figure 4 In addition, spike recovery experiments using commercial human serum showed recoveries ranging from 109.8% to 116.7%, with RSDs ≤ 2.9% (Table 3). These results demonstrate that the proposed method can be used to detect alkaline phosphatase activity in real serum samples with high accuracy and precision, and its overall performance is comparable to that of commercial kits.

[0055] In addition, the method proposed in the present invention can also be used to detect alkaline phosphatase activity in cell lysates. HepG2 suspensions with different cell densities were chemically lysed according to the lysis method proposed in this protocol and then detected according to the method proposed in this protocol. The ratio fluorescence signal R / R0 and cell density were significantly correlated in the range of 0.2×10 5 ~3.2×10 5 cells / ml range is good (R / R0=0.05927C HepG2 +0.9932, R 2 =0.9983). Figure 4 Figure D shows that only after chemical lysis of the HepG2 suspension does the ratiometric fluorescence signal R / R0 increase. The signal decreases again after heat inactivation or treatment with the alkaline phosphatase inhibitor sodium orthovanadate, demonstrating the feasibility of the chemical lysis method employed in this invention. In this way, considering the sample volume used is only 20 μl, the number of HepG2 cells that can be detected can be as low as 400. This demonstrates that the proposed method is not only applicable to alkaline phosphatase detection in serum samples, but also has the potential for quantitative detection of tumor cells.

[0056] (4) The nanomaterials used in the present invention are low in toxicity, environmentally friendly, and have a safe operation process.

[0057] Unlike traditional quantum dots that contain highly toxic components such as cadmium, mercury, and tellurium, the ZnSe@ZnS and Mn:ZnS quantum dots used in this invention do not contain highly toxic elements, are environmentally friendly, and pose less potential harm to operators. In the method of the present invention, the final concentration of Mn:ZnS quantum dots is 2.5mg / ml, and the final concentration of ZnSe@ZnS quantum dots is 25μg / ml. According to experimental results, at these concentrations, both quantum dots have little effect on HepG2 cell viability, suggesting that both quantum dots have low biological toxicity and a safer operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Characterization of the morphology, structure, and optical properties of ZnSe@ZnS QDs and Mn:ZnS QDs, including (AC) TEM images and SEAD images of oil-soluble Mn:ZnS QDs, water-soluble Mn:ZnS QDs, and ZnSe@ZnS QDs; (DE) EDS spectra of oil-soluble Mn:ZnS QDs and water-soluble ZnSe@ZnS; (FG) Fluorescence spectra and UV-visible absorption spectra of water-soluble Mn:ZnS QDs and ZnSe@ZnS QDs, with the insets showing photos under 254 nm UV light and fluorescent light.

[0059] Figure 2Figure 3 Biocompatibility and surface group characterization of ZnSe@ZnS QDs and Mn:ZnS QDs, including (A) FTIR spectra of ZnSe@ZnS QDs and Mn:ZnS QDs; (B-C) Cell viability of HepG2 cells after incubation in culture medium containing different concentrations of Mn:ZnS QDs or ZnSe@ZnS QDs for 24 h; (D) Fluorescence intensity of ZnSe@ZnS QDs and Mn:ZnS QDs after incubation at room temperature for 1 h after adding the following substances: (a) without addition of other substances; (b) with addition of 600 μM PNPP; (c) with addition of 600 μM PNPP and 100 U / L alkaline phosphatase; (E) fluorescence intensity of Mn:ZnS QDs (c) and ZnSe@ZnS Overlap of the excitation spectrum of QDs (b) and the absorption spectrum of PNPP (a); (F) Absorption spectra of ALP with different activities (0, 16, 32, 64, 96 U / L) in the detection system; (G) Overlap of the emission spectra of ZnSe@ZnS QDs (b) and Mn:ZnS QDs (d) with the absorption spectra of PNPN (a) and PNP (c);

[0060] Figure 3 To optimize the ALP detection conditions and verify the ALP solution sample detection method, (AD) the effects of incubation buffer pH, incubation temperature, PNPP concentration, and incubation time on the detection signal R / R0; R and R0 are the ratios of the fluorescence intensity at 585 nm and 405 nm in the system when ALP is present and absent, respectively (F 585 / F 405 ); (E) Fluorescence spectra of the incubation system in the presence of ALP with different activities (0, 4, 8, 16, 32, 64, 96 U / L); (F) Relationship between the ratio fluorescence signal R / R0 and ALP activity in the range of 0-256 U / L, and the inset shows the linear relationship between the two in the range of 4-96 U / L; (G) Selectivity of the ratio fluorescence analysis method for small molecule interfering substances, among which Na + , K + 、SO4 2- The concentration of Ca is 100 mM, 2+ Mg 2+ The concentration was 1 mM, Zn 2+ 、Fe 3+ 、Mn 2+The concentration was 5 μM, the concentrations of glutathione and all amino acids were 1 mM, the concentration of glucose was 10 mM, and the ALP activity was 100 U / L. (H) Selectivity of the ratiometric fluorescence assay for macromolecular interfering substances: the concentrations of trypsin, pepsin, proteinase K, and bovine serum albumin (BSA) were 1 mg / ml, the activity of thrombin was 100,000 U / L, and the inactivated human serum was prepared from normal human serum by heating it in a water bath at 65°C for 10 minutes. The ALP activity was 100 U / L.

[0061] Figure 4 The results of human serum and HepG2 lysate samples are shown, where (A) is the linear relationship between the ratio fluorescence signal R / R0 and the ALP activity in spiked inactivated human serum; R and R0 are the ratios of the fluorescence intensity at 585 nm and 405 nm in the system in the presence and absence of ALP (F 585 / F 405 ); (B) Detection results of serum samples from three adult volunteers using the commercial kit and the ratiometric fluorescence analysis method established by the present invention; (C) Linear relationship between the ratiometric fluorescence signal R / R0 and the density of HepG2 cells in the cell suspension; (D) HepG2 cell suspension (3.2×10 5 The changes of the ratio fluorescence signal R / R0 of HepG2 lysate (1×10 5 The changes of the ratio fluorescence signal R / R0 after treatment with different concentrations of the ALP inhibitor sodium orthovanadate were analyzed. The cell lysate was incubated with sodium orthovanadate solution at 4°C for 30 min before detection.

[0062] Figure 5 Schematic diagram of the alkaline phosphatase detection principle based on the inner filter effect. DETAILED DESCRIPTION

[0063] The present invention provides a ratiometric fluorescence analysis method for measuring alkaline phosphatase activity in a sample based on the inner filter effect, comprising the following steps:

[0064] 1) mixing the sample with an incubation buffer, incubating, and allowing to stand to obtain a standing solution;

[0065] The incubation buffer comprises: 50 mM diethanolamine, 1 mM MgCl2 and 200-1000 μM disodium p-nitrophenyl phosphate, with a pH value of 7.5-11.0;

[0066] The incubation temperature is 4 to 45° C., and the incubation time is 15 to 120 minutes;

[0067] 2) mixing the static solution obtained in step 1) with the quantum dot solution to obtain a mixed solution, exciting the solution at 310 nm, and measuring the fluorescence intensity of the mixed solution at 405 nm and 585 nm to obtain a fluorescence intensity ratio of 585 nm to 405 nm, defined as R, and the fluorescence intensity ratio of the blank sample is defined as R0, to obtain R / R0;

[0068] The quantum dot solution includes: 10 mg / ml Mn:ZnS and 0.1 mg / ml ZnSe@ZnS;

[0069] 3) Substituting the R / R0 obtained in step 2) into the following formula to obtain the alkaline phosphatase activity in the sample;

[0070] When the sample is an ALP solution, the formula is: R / R0=0.03688C ALP +0.7513, R 2 =0.9969, where C ALP The unit is U / L;

[0071] When the sample is serum, the formula is: R / R0=0.02085C ALP +1.098, R 2 =0.9907, where C ALP Unit: U / L;

[0072] When the sample is HepG2 cell lysate, the formula is: R / R0=0.0597C HepG2 +0.9932, R 2 =0.9983 where C HepG2 Unit: pieces / ml.

[0073] The present invention mixes a sample with an incubation buffer, incubates, and allows the mixture to stand to obtain a standing solution; the incubation buffer comprises: 50mM diethanolamine, 1mM MgCl2 and 200-1000μM disodium p-nitrophenyl phosphate, and the pH value is 7.5-11.0; the incubation temperature is 4-45°C, and the incubation time is 15-120min.

[0074] In the present invention, the content of disodium p-nitrophenylphosphate in the incubation buffer is preferably 600 μM, and the pH of the incubation buffer is preferably 10.0; the incubation temperature is preferably 37°C, and the incubation time is preferably 75 minutes. In the present invention, the sample comprises an ALP solution and / or serum and / or a HepG2 cell lysate. The present invention does not specifically limit the processing methods for the ALP solution, serum, and HepG2 cell lysate; those skilled in the art can follow conventional procedures. In the present invention, the resting time is preferably 10 minutes.

[0075] The method comprises mixing the obtained static liquid with a quantum dot solution to obtain a mixed solution, exciting the mixed solution at 310 nm, measuring the fluorescence intensity of the mixed solution at 405 nm and 585 nm, obtaining a fluorescence intensity ratio of 585 nm to 405 nm, which is defined as R, and defining the fluorescence intensity ratio of a blank sample as R0 to obtain R / R0. The quantum dot solution comprises 10 mg / ml Mn:ZnS and 0.1 mg / ml ZnSe@ZnS.

[0076] In the present invention, the preparation method of Mn:ZnS preferably comprises the following steps:

[0077] A. Mix zinc stearate, stearic acid, and 1-octadecene at 140° C. to obtain a zinc stock solution;

[0078] B. Mix zinc stearate, manganese stearate, sulfur powder, oleylamine and 1-octadecene at 100° C. to obtain a clear solution;

[0079] C. Mixing the zinc stock solution obtained in step A with the clarified solution obtained in step B at 250° C. for 30 min, filtering the obtained mixture, washing it, and drying the obtained precipitate to obtain oil-soluble Mn:ZnS;

[0080] D. mixing the oil-soluble Mn:ZnS obtained in step C with chloroform and 3-mercaptopropionic acid, performing sonication, and centrifuging to obtain a precipitate;

[0081] E. Mixing the precipitate obtained in step D with a tetramethylammonium hydroxide solution, ultrasonicating, and centrifuging to obtain a supernatant. Adjusting the pH value of the supernatant to 7-8 and then vacuum drying to obtain water-soluble Mn:ZnS, i.e., obtaining Mn:ZnS.

[0082] The present invention preferably mixes zinc stearate, stearic acid, and 1-octadecene at 140°C to obtain a zinc stock solution. In the present invention, the volume ratio of the moles of zinc stearate, the moles of stearic acid, and the moles of 1-octadecene is preferably 10 mmol:10 mmol:10 ml. In the present invention, the volume ratio of the moles of zinc stearate, the moles of manganese stearate, the moles of sulfur powder, the moles of oleylamine, and the moles of 1-octadecene is preferably 1 mmol:0.05 mmol:5 mmol:7.5 mmol:50 ml. In the present invention, the volume ratio of the zinc stock solution to the clarified solution is preferably 1:5. In the present invention, the volume ratio of the mass of the oil-soluble Mn:ZnS to the volume of chloroform and 3-mercaptopropionic acid is preferably 1.5 g:10 ml:1.6 ml. In the present invention, the ultrasonication time is preferably 30 minutes. In the present invention, the centrifugal conditions preferably include a centrifugal force of 2500 g and a centrifugation time of 2 minutes. In the present invention, the volume ratio of the precipitate mass to the tetramethylammonium hydroxide solution is preferably 1.5 g:10 ml. In the present invention, the ultrasonication time is preferably 5 minutes. In the present invention, the centrifugation conditions preferably include: a centrifugal force of 2500 g and a centrifugation time of 2 minutes. In the present invention, the vacuum drying temperature is preferably 50°C.

[0083] In the present invention, the preparation method of ZnSe@ZnS preferably includes the following steps:

[0084] a. Mix selenium powder, sodium borohydride and deionized water, stir, place in an ice bath, and collect the supernatant;

[0085] b. Mix zinc sulfate, reduced glutathione, and deionized water, adjust the pH to 10.5, and then add the supernatant obtained in step a, stir, and reflux in sequence to obtain a ZnSe quantum dot solution;

[0086] c. The ZnSe quantum dot solution obtained in step b was mixed with zinc sulfate and 3-mercaptopropionic acid, and the pH value was adjusted to 10.5. The mixture was stirred and refluxed to obtain a ZnSe@ZnS solution, which was mixed with anhydrous ethanol and centrifuged to obtain a precipitate of ZnSe@ZnS.

[0087] In the present invention, the molar ratio of the selenium powder, the molar ratio of sodium borohydride, and the volume ratio of deionized water is preferably 0.1mmol:0.6mmol:1ml; the stirring time is 40min; and the ice bath time is 10min. In the present invention, the molar ratio of zinc sulfate to the molar ratio of reduced glutathione and the volume ratio of deionized water is preferably 0.4mmol:0.5mmol:100ml. In the present invention, the stirring time is preferably 30min. In the present invention, the reflux time is preferably 1.5h. In the present invention, the volume ratio of the ZnSe quantum dot solution to the molar ratio of zinc sulfate and the volume ratio of 3-mercaptopropionic acid is preferably 20ml:0.1mmol:26μl. In the present invention, the stirring time is preferably 1h. In the present invention, the reflux time is preferably 1h. In the present invention, the centrifugal conditions preferably include: a centrifugal force of 2500g and a centrifugation time of 30min.

[0088] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0089] Example 1

[0090] (1) Preparation of water-soluble Mn:ZnS quantum dots

[0091] In order to prepare high-quality Mn:ZnS quantum dots, oil-soluble Mn:ZnS quantum dots modified with oleylamine were first synthesized in 1-octadecene, and then ligand exchange was carried out in chloroform to obtain water-soluble Mn:ZnS quantum dots modified with 3-mercaptopropionic acid on the surface.

[0092] To a 50ml beaker, add zinc stearate (10mmol), stearic acid (10mmol), and 1-octadecene (10ml). Preheat to 140°C to obtain a zinc stock solution, which should be kept at 140°C until ready for use. To a 100ml four-necked flask, add zinc stearate (1mmol), manganese stearate (0.05mmol), sulfur powder (5mmol), oleylamine (7.5mmol), and 1-octadecene (50ml). Heat to 100°C to obtain a clear solution, and bubble nitrogen through for 10 minutes. Seal the reaction system and continue bubbling nitrogen for 20 minutes to fully remove air. Rapidly heat to 270°C for 5 minutes, then cool to 250°C and inject the zinc stock solution all at once. Maintain at 250°C for 30 minutes. Stop heating and cool to room temperature. The reaction mixture was filtered and washed three times with n-hexane to remove 1-octadecene. The precipitate was dried at 40°C and ground into a fine powder. 100 ml of n-hexane was added and sonicated for 5 minutes. The precipitate was then filtered and repeatedly extracted with n-hexane until the filtrate showed almost no fluorescence. The combined filtrates were concentrated by rotary evaporation and dried at 65°C to obtain a brown solid, which was the oil-soluble Mn:ZnS quantum dots.

[0093] The phase transfer of oil-soluble Mn:ZnS quantum dots was achieved using a ligand exchange strategy. 1.5 g of oil-soluble Mn:ZnS was dissolved in 10 ml of chloroform. 3-Mercaptopropionic acid (1.6 ml) was added with vigorous stirring, followed by sonication for 30 minutes and centrifugation at 2500 g for 2 minutes. The supernatant was discarded, and the precipitate was washed three times with chloroform to remove excess 3-mercaptopropionic acid. 10 ml of 1 M tetramethylammonium hydroxide solution was added to the precipitate, sonicated for 5 minutes, and centrifuged at 2500 g for 2 minutes. The supernatant was collected. The precipitate was repeatedly extracted with tetramethylammonium hydroxide solution until the supernatant showed little fluorescence. The combined supernatants were adjusted to a pH of 7-8 with concentrated hydrochloric acid, concentrated by rotary evaporation, and dried under vacuum at 50°C overnight to obtain water-soluble Mn:ZnS quantum dots.

[0094] (2) Preparation of water-soluble ZnSe@ZnS quantum dots

[0095] First, selenium powder (0.1 mmol) and sodium borohydride (0.6 mmol) were added to a 25 ml three-necked flask. Deionized water (1 ml) was then added and nitrogen was rapidly introduced. The mixture was stirred at room temperature for 40 minutes to obtain a colorless, transparent solution. The mixture was then placed in an ice bath for 10 minutes. The supernatant was the NaHSe solution. Zinc sulfate heptahydrate (0.4 mmol) and reduced glutathione (0.5 mmol) were added to a 250 ml three-necked flask. Deionized water (100 ml) was added and the pH was adjusted to 10.5 with 1 M sodium hydroxide solution. The NaHSe solution was then added and the mixture was vigorously stirred for 30 minutes. The mixture was then refluxed for 1.5 hours to obtain a ZnSe quantum dot solution. Zinc sulfate heptahydrate (0.1 mmol) and 3-mercaptopropionic acid (26 μl) were added to 20 ml of the ZnSe quantum dot solution. The pH was adjusted to 10.5 with 1 M sodium hydroxide solution. The mixture was vigorously stirred for 1 hour and refluxed for 1 hour to obtain a ZnSe@ZnS solution. The ZnSe@ZnS solution was concentrated by rotary evaporation to 1 / 3 of the original volume, and three times the volume of anhydrous ethanol was added. The precipitate was collected by centrifugation at 2500 g for 30 min, and then dried in a vacuum at 50°C to obtain ZnSe@ZnS quantum dots.

[0096] (3) Ratio fluorescence detection of alkaline phosphatase based on inner filter effect

[0097] In order to obtain the strongest detection signal, the pH of the incubation buffer, incubation temperature, PNPP concentration, and incubation time were investigated.

[0098] Incubation buffer pH investigation: The incubation temperature was 25°C, the PNPP concentration was 400 μM, and the incubation time was 60 min. The incubation buffer pH values ​​were 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, and 11.0. Three parallel experimental groups were set for each pH condition.

[0099] Incubation temperature investigation: The incubation buffer pH was 10.0, the PNPP concentration was 400 μM, and the incubation time was 60 min. The incubation temperatures were 4°C, 25°C, 37°C, and 45°C, respectively. Three parallel experimental groups were set up for each temperature condition.

[0100] PNPP concentration investigation: Incubation temperature was 37°C, incubation buffer pH was 10.0, and incubation time was 60 min. PNPP concentrations were 0, 200, 400, 600, 800, and 1000 μM. Three parallel experimental groups were set up for each PNPP concentration condition.

[0101] Incubation time study: The incubation temperature was 37°C, the incubation buffer pH was 10.0, and the PNPP concentration was 600 μM. The incubation times were 0, 15, 30, 45, 60, 75, 90, 105, and 120 min. Three parallel experimental groups were set up for each incubation time condition.

[0102] The assay procedure for conditional analysis was as follows: First, 20 μl of ALP samples of varying concentrations or a blank sample was added to a 96-well plate. Then, 130 μl of incubation buffer (50 mM diethanolamine, varying pH values, containing 1 mM MgCl2 and varying PNPP concentrations) was added and mixed by pipetting. The reaction mixture was incubated at different temperatures for varying times. After standing at room temperature for 10 minutes, 50 μl of a quantum dot solution (containing 10 mg / ml Mn:ZnS and 0.1 mg / ml ZnSe@ZnS) was added and mixed by pipetting. Fluorescence intensity at 405 nm and 585 nm was measured with excitation at 310 nm.

[0103] The alkaline phosphatase activity assay of solution samples based on the IFE effect was performed using the following optimal method. First, 20 μl of ALP samples with different activities (0, 4, 8, 16, 32, 64, 96, 128, 192, 256 U / L) or blank samples were added to a 96-well plate, followed by 130 μl of incubation buffer (50 mM diethanolamine, pH = 10.0, containing 1 mM MgCl2, 600 μM PNPP) and pipetting to mix. The reaction solution was incubated at 37°C for 75 minutes. After standing at room temperature for 10 minutes, 50 μl of quantum dot solution (containing 10 mg / ml Mn:ZnS and 0.1 mg / ml ZnSe@ZnS) was added and pipetting to mix. The fluorescence intensity at 405 nm and 585 nm was measured with excitation at 310 nm.

[0104] The preparation method of the standard curve is as follows: the ratio of the fluorescence intensity at 585 nm to that at 405 nm (F 585 / F 405) was defined as R, the corresponding value of the blank sample was defined as R0, and R / R0 was used as the detection signal related to alkaline phosphatase activity. The test data were processed using Graphpad Prism 5.0 software, and regression analysis was performed on sample alkaline phosphatase activity using R / R0, with 1 / x as the weight (where x is alkaline phosphatase activity).

[0105] When conducting sensitivity investigation, 6 blank samples were measured under the above optimal conditions. 585 / F 405 The R value of the corresponding sample was taken as the mean of the R values ​​of the six samples as R0, and the R / R0 ratio of the six blank samples was obtained. The standard deviation (SD) of the R / R0 of the six blank samples was taken as σ, and the slope of the standard curve equation was taken as k. The value of 3σ / k was defined as the method detection limit.

[0106] result:

[0107] Characterization of ZnSe@ZnS QDs and Mn:ZnS QDs

[0108] TEM images showed that the oil-soluble and water-soluble Mn:ZnS quantum dots were spherical particles with sizes of 3.68±0.78nm ( Figure 1 A) and 4.49±0.73nm( Figure 1 Middle B), SEAD image shows that the crystal form of Mn:ZnS quantum dots does not change during ligand exchange and phase transition. Figure 1 The C in the middle shows that the ZnSe@ZnS quantum dots are spherical particles with a particle size of 2.93±0.50nm. The EDS spectrum of Mn:ZnS quantum dots shows the characteristic spectral lines of manganese ( Figure 1 (D) shows that manganese has been successfully doped into the ZnS lattice.

[0109] The optical properties of quantum dots are Figure 1 Medium F and Figure 1 As shown in Figure G, the maximum emission peaks of Mn:ZnS quantum dots and ZnSe@ZnS quantum dots are 585nm and 385nm respectively. The absorption spectrum shows that ZnSe@ZnS quantum dots have an exciton absorption peak around 360nm ( Figure 1 As can be seen from Tables 1 and 2, the relative fluorescence quantum yields of Mn:ZnS quantum dots and ZnSe@ZnS quantum dots are 27.8% and 24.6%, respectively, indicating that the synthesized quantum dots have bright fluorescence, which helps to obtain a stronger detection signal.

[0110] Table 1 Calculation of fluorescence quantum yield of Mn:ZnS QDs

[0111]

[0112] Table 2 Calculation of fluorescence quantum yield of ZnSe@ZnS QDs

[0113]

[0114] The FTIR spectrum of oil-soluble Mn:ZnS QDs is at 2850 cm -1 、2918cm -1 and 721cm -1 There is an obvious absorption peak at Figure 2 A) are attributed to the symmetric and asymmetric stretching vibrations and bending vibrations of the methylene group. The bending vibration of the methylene group indicates that there is a long carbon chain of oleylamine (carbon number > 4) on the surface of the oil-soluble Mn:ZnS quantum dots. However, the water-soluble Mn:ZnS quantum dots do not have the above peaks. In addition, 1732cm -1 Although the peak at is not obvious, it still indicates the presence of carbonyl groups. The above results show that 3-mercaptopropionic acid has successfully replaced oleylamine on the surface of oil-soluble Mn:ZnS QDs. In addition, the migration of orange-yellow fluorescence from the chloroform layer to the water layer also confirms the occurrence of phase transfer.

[0115] FTIR spectrum of ZnSe@ZnS quantum dots Figure 2 As shown in A, 1604cm -1 and 1398cm -1 The peak at 1531 cm is attributed to the asymmetric and symmetric stretching vibrations of C=O in the ionized carboxyl group. -1 and 1307cm -1 The peak at is attributed to the bending vibration of the NH bond and the symmetric stretching vibration of the CN bond in the amide bond. These results indicate that 3-mercaptopropionic acid and glutathione actually coexist on the ZnSe@ZnS surface.

[0116] In addition, ZnSe@ZnS QDs and Mn:ZnS QDs have good biocompatibility. Even when the concentrations of ZnSe@ZnS QDs and Mn:ZnS QDs reach 25 μg / ml ( Figure 2 C) and 1.5mg / ml ( Figure 2 No significant cytotoxicity was observed in the sample (B). Low-toxicity quantum dots not only pose less potential risk to operators but are also more environmentally friendly.

[0117] Feasibility analysis results of ALP detection:

[0118] First, the feasibility of IFE-based ALP detection was analyzed. The fluorescence of ZnSe@ZnS QDs and Mn:ZnS QDs was sharply quenched in the presence of 600 μM PNPP ( Figure 2D), this is because there is extensive overlap between the absorption spectrum of PNPP and the excitation spectra of the two quantum dots ( Figure 2 Middle E). Figure 2 The c line in D shows that the fluorescence of Mn:ZnS QDs is restored to a certain extent in the presence of ALP. This is because PNPP is hydrolyzed into PNP in the presence of ALP, resulting in a red shift in the absorption spectrum of the reaction system ( Figure 2 F). Therefore, the IFE produced by PNPP on Mn:ZnS QDs gradually decreases with the hydrolysis of PNPP, and the absorption band of PNP no longer overlaps with the excitation wavelength of Mn:ZnS QDs. At the same time, the fluorescence intensity of ZnSe@ZnS quantum dots further decreases. Figure 2 Lines b and c in Figure 5 clearly show that there is a high degree of overlap between the PNP absorption and emission spectra of ZnSe@ZnS QDs, which leads to a strong IFE of PNP on ZnSe@ZnS QDs and further quenching of ZnSe@ZnS QDs.

[0119] Briefly, when ALP catalyzes the hydrolysis of PNPP, the fluorescence intensities of Mn:ZnS QDs and ZnSe@ZnS QDs are opposite, resulting in a ratiometric fluorescence signal (F 585 / F 405 ) increases, thereby correlating the ratiometric fluorescence signal with ALP activity.

[0120] ALP activity ratio fluorescence detection results based on the inner filter effect:

[0121] In order to obtain higher sensitivity, the pH of the incubation buffer, incubation temperature, PNPP concentration and incubation time were investigated. Since the activity of alkaline phosphatase is greatly affected by pH and temperature, the pH of the incubation buffer and the incubation temperature were first investigated. When the pH of the incubation buffer was 10.0 ( Figure 3 A), when the incubation temperature is 37℃ ( Figure 3 (B) The ratiometric fluorescence signal (R / R0) reaches its maximum. Previous reports suggest that an alkaline environment provides sufficient hydroxide ions for substrate dephosphorylation, which helps enhance ALP activity. Therefore, at optimal pH and temperature, the ratiometric fluorescence signal increases due to enhanced ALP activity.

[0122] The effect of PNPP concentration in the incubation buffer on the ratio fluorescence signal is relatively complex. On the one hand, when the substrate (PNPP) concentration is too low, the dephosphorylation reaction is slow, so the detection signal is weak within the limited incubation time; on the other hand, when the PNPP concentration is too high, due to the low PNPP consumption rate, the PNPP concentration is still high after the incubation, which causes ZnSe@ZnSQDs and Mn:ZnS QDs to be completely quenched after the incubation, and no changes in the ratio fluorescence signal can be observed. Figure 3 As shown in Figure C, with the increase of PNPP concentration, the ratio fluorescence signal R / R0 value gradually increases. When the PNPP concentration reaches 600μM, R / R0 reaches the maximum value. Therefore, in the subsequent detection, the PNPP concentration is selected to be 600μM. Finally, in order to shorten the analysis cycle and take into account a strong detection signal, the incubation time was investigated. Figure 3 As shown in Figure D, the R / R0 value gradually increases with the extension of incubation time. When the incubation time is greater than 75 minutes, the change range of R / R0 decreases significantly, so 75 minutes is selected as the optimal incubation time.

[0123] In vitro detection of alkaline phosphatase activity was performed under optimal conditions (incubation buffer pH 10.0, incubation temperature 37°C, PNPP concentration 600 μM, incubation time 75 min). Figure 3 As shown in Figure E, with the increase of ALP activity, the fluorescence intensity of the detection system at 585nm gradually increased, while the fluorescence intensity at 405nm gradually decreased. The ratio fluorescence signal (R / R0) and ALP activity had a good linear relationship in the range of 4 to 96 U / L (Fig. 4F, R / R0 = 0.03688C ALP +0.7513,R 2 =0.9969), with a limit of detection of 0.57 U / L (3σ / k). The limit of detection (LOD) of the established ratiometric fluorescence assay is comparable to or lower than previously reported methods (Table 4). According to literature, the normal level of ALP activity in human serum is 20-140 U / L. The sensitivity of the established ratiometric fluorescence assay is fully sufficient for the determination of ALP activity in human serum.

[0124] Selective results of ratiometric fluorescence analysis:

[0125] In order to verify the selectivity of the established ratiometric fluorescence analysis method for ALP activity detection, the inorganic ions (Na + , K + , Ca 2+ Mg 2+ 、Fe 3+ 、Zn 2+ 、Mn 2+ 、SO42- ), amino acids (Lys, His, Arg, Trp, Cys, Met, Ser), glucose, GSH and proteins (trypsin, pepsin, bovine serum albumin, proteinase K, thrombin). Figure 3 As shown in Figure G, most of the small molecules investigated in the experiment did not cause a significant increase in the ratio fluorescence signal R / R0. Moreover, the effect of all proteins investigated in the experiment on R / R0 was negligible ( Figure 3 In addition, we also studied the effect of inactivated human serum on the ratio fluorescence signal. The results showed that the detection signal of inactivated human serum was close to that of the control group ( Figure 3 The above experimental results show that this method has good selectivity and has great potential for detecting alkaline phosphatase activity in human serum.

[0126] Example 2

[0127] Human serum sample preparation and serum sample testing Phosphatase activity detection:

[0128] Human serum was prepared as follows: whole blood was allowed to rest at room temperature for 1 hour and then centrifuged at 1500 g for 10 minutes. The pale yellow supernatant was serum. The collected serum was stored at -20°C until testing.

[0129] The detection method of serum samples was the same as the best method of Example 1, except that the ALP solution sample was replaced with an equal volume of serum sample.

[0130] The result is:

[0131] like Figure 4 As shown in A, in spiked inactivated human serum, the ratio fluorescence signal R / R0 has a good linear relationship with the spiked ALP activity (4-96 U / L, R 2 =0.9907). To explore the applicability of this detection method in the detection of ALP activity in clinical samples, serum samples from three adult volunteers were tested using a commercial kit and the ratiometric fluorescence analysis method established by the present invention. Figure 4 As can be seen from Figure B, the results obtained by the two methods are close.

[0132] To further validate the accuracy of ratiometric fluorescence analysis for the detection of ALP activity in human serum, spiked human serum samples were tested. The experimental results showed that the average spiked serum recovery rates for these samples ranged from 109.8% to 116.7% (Table 3). These results demonstrate that the ratiometric fluorescence analysis method developed in this invention is capable of accurately measuring alkaline phosphatase activity in clinical samples and has great potential for clinical application in the detection of ALP activity.

[0133] Table 3 Spiked human serum sample recovery (Means ± SD, n = 6)

[0134]

[0135] Example 3

[0136] Preparation of HepG2 cell lysate and detection of alkaline phosphatase activity in HepG2 cell lysate:

[0137] HepG2 cell lysate was prepared as follows. HepG2 cells were cultured in RPMI1640 medium supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum at 37°C in the presence of 5% carbon dioxide. HepG2 cultures were digested with trypsin-EDTA for 2 minutes, resuspended in 10 mM HEPES buffer (pH 7.4, containing 137 mM NaCl, 3 mM KCl), and centrifuged at 1000 g for 5 minutes. HepG2 cells were harvested, redispersed in 10 mM HEPES buffer (pH 7.4, containing 137 mM NaCl, 3 mM KCl), and diluted to the desired cell density in HEPES buffer. 20 μl of lysis buffer (10 mM HEPES, pH = 7.4, containing 137 mM NaCl, 3 mM KCl, 0.2% Triton X-100) was added to 100 μl of HepG2 cell suspension, mixed, and incubated at 4°C for 30 min to obtain HepG2 cell lysate.

[0138] The method for detecting alkaline phosphatase in HepG2 cell lysate was the same as the optimal method in Example 1, except that the ALP solution was replaced with an equal volume of HepG2 cell lysate.

[0139] ALP activity test results in HepG2 lysate:

[0140] According to literature reports, upregulation of ALP activity often occurs in certain tumor cells including HepG2. Therefore, we used HepG2 cells to evaluate the performance of the established ratiometric fluorescence assay for detecting ALP activity in tumor cell lysates.

[0141] Depend on Figure 4 As shown in Figure C, the ratio fluorescence signal R / R0 increases strictly with the increase of HepG2 cell density. 5 ~3.2×10 5 Good linearity within the range of cells / ml (R 2=0.9983). Considering the low sample volume (20 μl), this method can detect HepG2 cells as low as 400 cells. To eliminate matrix interference, unlysed HepG2 cell suspension and inactivated HepG2 cell lysate samples were tested under the same conditions. The results showed that only the ratio fluorescence signal (R / R0) of the non-heat-inactivated HepG2 lysate increased. Compared with the control group, the signal of the unlysed HepG2 suspension and the inactivated lysate did not change much ( Figure 4 In addition, as the concentration of Na3VO4 (a classic alkaline phosphatase inhibitor) increased, the signal of HepG2 lysate treated with Na3VO4 also decreased significantly ( Figure 4 The above experimental results indicate that it is ALP, rather than the matrix, that causes the increase in R / R0 in HepG2 lysate, and also demonstrate that the established ratiometric fluorescence analysis method can be applied to the detection of liver cancer cells.

[0142] The present invention establishes an IFE-based ratiometric fluorescence analysis method for determining ALP activity in biological samples, in which Mn:ZnS QDs and ZnSe@ZnS QDs can respond to changes in PNPP and PNP concentrations, and the ratiometric fluorescence signal (R / R0) increases with the increase of ALP activity. This method has high sensitivity, a detection limit of 0.57U / L, and low sample consumption (20μl). It is worth mentioning that this method has excellent specificity when determining alkaline ALP activity in biological matrices, the detection process is simple, and no pretreatment of the sample is required. In the detection of actual samples, the detection performance of the ratiometric fluorescence analysis method is close to that of the commercial kit, and the spike recovery rate for serum samples is 109.8% to 116.7%. In addition, the method has also been successfully applied to the determination of ALP activity in HepG2 cells, and there is a good linear relationship between the detection signal and the number of HepG2 cells, which is expected to be used for early diagnosis of tumors. In summary, the IFE-based ratiometric fluorescence analysis method developed by the present invention will provide a novel strategy for detecting ALP activity in complex biological matrices.

[0143] Table 4 Performance comparison of different analytical methods for alkaline phosphatase activity detection

[0144]

[0145]

[0146] The literature sources in Table 4 are:

[0147] [1] Cai, M., Ding, C., Wang, F., Ye, M., Zhang, C., Xian, Y., 2019. Biosens. Bioelectron. 137, 148–153.

[0148] [2] Huang,

[0149] [3]Li,Y.,Xie,R.,Pang,X.,Zhou,Z.,Xu,H.,Gu,B.,Wu,C.,Li,H.,Zhang,Y.,2019.Talanta 205.120143.

[0150] [4]Mao, G., Zhang, Q., Yang, Y., Ji, X., He, Z., 2019. Anal. Chim. Acta.1047, 208–213.

[0151] [5]Qi, W., Fu, Y., Zhao, M., He, H., Tian, ​​X., Hu, L., Zhang, Y., 2020. Anal. Chim. Acta.1097,71–77.

[0152] [6] Sun, W., Han, X., Qu, F., Kong, RM, Zhao, Z., 2021. Analyst 146, 2862–2870.

[0153] [7]Tong, X., Zhu, Y., Tong, C., Shi, S., Long, R., Guo, Y., 2021. Anal.Chim.Acta.1178.338829.

[0154] [8] Wang, WX, Jiang, WL, Guo, H., Li, Y., Li, C. Y., 2021. Chem. Commun. 57, 480–483.

[0155] [9] Zhang, X., Ren, C., Hu, F., Gao, Y., Wang, Z., Li, H., Liu, J., Liu, B., Yang, C., 2020. Anal. Chem. 92, 5185–5190.

[0156]

[10] Zhang, YJ, Guo, L., Chen, S., Yu, YL, Wang, JH, 2020. Anal.Chim.Acta.1108,54–60.

[0157]

[11] Zhao, M., Gao, Y., Ye, S., Ding, J., Wang, A., Li, P., Shi, H., 2019. Analyst144, 6262–6269.

[0158]

[12] Zhu, Y., Tong, X., Wei, Q., Cai, G., Cao, Y., Tong, C., Shi, S., Wang, F., 2022. Biosens. Bioelectron. 196.113691.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A ratiometric fluorescence analysis method for determining alkaline phosphatase activity in a sample based on the inner filter effect, characterized in that: The following steps are involved: 1) mixing the sample with an incubation buffer, incubating, and allowing to stand to obtain a standing solution; The incubation buffer comprises: 50 mM diethanolamine, 1 mM MgCl2 and 200-1000 μM disodium p-nitrophenyl phosphate, with a pH value of 7.5-11.0; The incubation temperature is 4 to 45° C., and the incubation time is 15 to 120 minutes; 2) mixing the static solution obtained in step 1) with the quantum dot solution to obtain a mixed solution, exciting the solution at 310 nm, and measuring the fluorescence intensity of the mixed solution at 405 nm and 585 nm to obtain a fluorescence intensity ratio of 585 nm to 405 nm, defined as R, and the fluorescence intensity ratio of the blank sample is defined as R0, to obtain R / R0; The quantum dot solution includes: 10 mg / ml Mn:ZnS and 0.1 mg / ml ZnSe@ZnS; 3) Substituting the R / R0 obtained in step 2) into the following formula to obtain the alkaline phosphatase activity in the sample; When the sample is alkaline phosphatase ALP solution, the formula is: R / R0=0.03688C ALP +0.7513, R 2 =0.9969, where C ALP The unit is U / L; When the sample is serum, the formula is: R / R0=0.02085C ALP +1.098, R 2 =0.9907, where C ALP Unit: U / L; When the sample is HepG2 cell lysate, the formula is: R / R0=0.0597C HepG2 +0.9932, R 2 =0.9983 where C HepG2 Unit: pieces / ml.

2. The ratiometric fluorescence analysis method according to claim 1, wherein The preparation method of Mn:ZnS comprises the following steps: A. Mix zinc stearate, stearic acid, and 1-octadecene at 140° C. to obtain a zinc stock solution; B. Mix zinc stearate, manganese stearate, sulfur powder, oleylamine and 1-octadecene at 100° C. to obtain a clear solution; C. Mixing the zinc stock solution obtained in step A with the clarified solution obtained in step B at 250° C. for 30 min, filtering the obtained mixture, washing it, and drying the obtained precipitate to obtain oil-soluble Mn:ZnS; D. mixing the oil-soluble Mn:ZnS obtained in step C with chloroform and 3-mercaptopropionic acid, performing sonication, and centrifuging to obtain a precipitate; E. Mixing the precipitate obtained in step D with a tetramethylammonium hydroxide solution, ultrasonicating, and centrifuging to obtain a supernatant. Adjusting the pH value of the supernatant to 7-8 and then vacuum drying to obtain water-soluble Mn:ZnS, i.e., obtaining Mn:ZnS.

3. The ratiometric fluorescence analysis method according to claim 2, wherein In step A, the volume ratio of the moles of zinc stearate, the moles of stearic acid, and the moles of 1-octadecene is 10 mmol:10 mmol:10 ml; In step B, the volume ratio of the moles of zinc stearate, the moles of manganese stearate, the moles of sulfur powder, the moles of oleylamine and the moles of 1-octadecene is 1 mmol:0.05 mmol:5 mmol:7.5 mmol:50 ml; The volume ratio of the zinc stock solution to the clarified solution in step C is 1:

5.

4. The ratiometric fluorescence analysis method according to claim 2, wherein In step D, the ratio of the mass of the oil-soluble Mn:ZnS to the volume of chloroform and the volume of 3-mercaptopropionic acid is 1.5 g:10 ml:1.6 ml; The ultrasonic time is 30min; The centrifugal conditions include: centrifugal force of 2500g and centrifugal time of 2 minutes.

5. The ratiometric fluorescence analysis method according to claim 2, wherein The volume ratio of the precipitate in step E to the tetramethylammonium hydroxide solution is 1.5 g:10 ml; The ultrasonic time is 5 minutes; The centrifugal conditions include: centrifugal force of 2500g, centrifugal time of 2min; The vacuum drying temperature is 50°C.

6. The ratiometric fluorescence analysis method according to claim 2, wherein The preparation method of ZnSe@ZnS comprises the following steps: a. Mix selenium powder, sodium borohydride and deionized water, stir, place in an ice bath, and collect the supernatant; b. Mix zinc sulfate, reduced glutathione, and deionized water, adjust the pH to 10.5, and then add the supernatant obtained in step a, stir, and reflux in sequence to obtain a ZnSe quantum dot solution; c. The ZnSe quantum dot solution obtained in step b was mixed with zinc sulfate and 3-mercaptopropionic acid, and the pH value was adjusted to 10.

5. The mixture was stirred and refluxed to obtain a ZnSe@ZnS solution, which was mixed with anhydrous ethanol and centrifuged to obtain a precipitate of ZnSe@ZnS.

7. The ratiometric fluorescence analysis method according to claim 6, characterized in that The volume ratio of the molar ratio of selenium powder, the molar ratio of sodium borohydride and deionized water in step a is 0.1 mmol:0.6 mmol:1 ml; the stirring time is 40 min; and the ice bath time is 10 min. In step b, the volume ratio of the molar amount of zinc sulfate to the molar amount of reduced glutathione and deionized water is 0.4 mmol:0.5 mmol:100 ml; the stirring time is 30 min; and the reflux time is 1.5 h.

8. The ratiometric fluorescence analysis method according to claim 6, wherein In step c, the volume ratio of the ZnSe quantum dot solution to the molar ratio of zinc sulfate and 3-mercaptopropionic acid is 20 ml:0.1 mmol:26 μl; The stirring time is 1h; The reflux time is 1h; The centrifugal conditions include: centrifugal force of 2500 g, and centrifugal time of 30 min.

9. The ratiometric fluorescence analysis method according to claim 1, wherein The content of disodium p-nitrophenyl phosphate in the incubation buffer is 600 μM, and the pH value of the incubation buffer is 10.0; The incubation temperature was 37° C., and the incubation time was 75 min.

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