Bi-color nano probe for detecting alkaline phosphatase and preparation and detection methods thereof

Abstract

The invention discloses a double-color nanoprobe for detecting alkaline phosphatase as well as a preparation method and a detection method of the double-color nanoprobe. The bicolor nano-probe is mainly formed by self-assembly of guanylic acid, a terbium ion compound and a water-soluble carboxyl functionalized quantum dot at room temperature under a dark condition; the mass ratio of the guanylic acid to the terbium ions to the water-soluble carboxyl functionalized quantum dots is (750000 to 800000): (750000 to 800000): 1; the bicolor nanoprobe and a sample solution containing alkaline phosphatase are uniformly mixed, after a reaction in a dark place at 37 DEG C, the concentration of the alkaline phosphatase is obtained from a fluorescence emission peak value at 546 nm under the excitation wavelength of 254 nm, and the lowest concentration of the alkaline phosphatase in the detection method is 0.3874 U / L. The bicolor nanoprobe is simple in synthesis mode and mild in synthesis condition, the generated color contrast signal is visible to naked eyes, and the bicolor nanoprobe has the advantages of being simple, rapid and sensitive.

Description

Technical Field

[0001] The invention belongs to the field of rapid detection methods for alkaline phosphatase, and specifically relates to a dual-color nanoprobe for alkaline phosphatase detection and a preparation and detection method thereof. Background Art

[0002] Milk is rich in nutrients and high-quality protein, and is an important part of the human diet. Cow mastitis is a tissue resistance inflammatory response disease caused by infection of the original microorganisms of the cow's mammary gland. Cow mastitis is very harmful to the dairy farming industry, which can easily lead to reduced milk production in dairy cows. Pathogenic microorganisms and bacteria in the cows seriously pollute the quality of dairy products. Cows infected with mastitis will also increase the risk of human infection and secondary infection in cows. In actual breeding, farmers usually choose antibiotics to treat cow mastitis. However, irrational use of antibiotics can easily lead to antibiotic residues, and also cause antibiotics to remain in milk, affecting human health. Therefore, early diagnosis of mastitis is of great significance for preventing and treating cow mastitis and reducing the use of antibiotics.

[0003] Studies have shown that alkaline phosphatase (ALP) can be used as an important indicator for diagnosing early bovine mastitis. Alkaline phosphatase is an enzyme that hydrolyzes phosphate groups and is naturally produced by somatic cells such as mammary cells. The current industry standard method for detecting alkaline phosphatase activity is the phenol catalysis method. Although this method has high sensitivity, it has the problem of complicated operation steps. In recent years, the fluorescence method has attracted much attention due to its advantages such as high sensitivity, simple operation, and significant phenomena. At present, many fluorescent materials have been developed for the development of detection systems, but the complex synthesis method and high cost limit the application scope of these fluorescent materials. Due to the shortcomings of the above-mentioned standard detection methods, researchers at home and abroad have developed many methods for rapid detection of alkaline phosphatase, such as electrochemical method, colorimetric method and surface Raman scattering method. These methods have high sensitivity and simplified operation steps to a certain extent, but they have high operation requirements and insufficient stability, and cannot meet the needs of rapid detection of alkaline phosphatase. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention proposes a dual-color nanoprobe for alkaline phosphatase detection and its preparation and detection method. 3+ ) as the metal central node, guanosine monophosphate (GMP) and water-soluble carboxylated quantum dots as ligands, and self-assembled through coordination between the metal central node and the organic ligands to form a dual-color nanoprobe. This dual-color nanoprobe has dual-emission fluorescence and significant fluorescence intensity, and has a rapid "turn off" fluorescence response to alkaline phosphatase.

[0005] The technical solution adopted by the present invention is as follows:

[0006] 1. A dual-color nanoprobe for alkaline phosphatase detection

[0007] The dual-color nanoprobe is mainly formed by self-assembly of guanylic acid, terbium ion compound and water-soluble carboxyl functionalized quantum dots at room temperature.

[0008] Specifically, the terbium ion compound is used to provide terbium ions. The terbium ions serve as the central atom of the dual-color nanoprobe, guanylic acid and water-soluble carboxyl functionalized quantum dots serve as ligands of the dual-color nanoprobe, and the terbium ions are connected to the phosphate group or carboxyl group of guanylic acid and the carboxyl group of the water-soluble carboxyl functionalized quantum dots through coordination bonds.

[0009] Preferably, the molar ratio of the guanylate, the terbium ions and the water-soluble carboxyl functionalized quantum dots is 750,000-800,000:750,000-800,000:1.

[0010] Preferably, the water-soluble carboxyl functionalized quantum dots are QD 625 .

[0011] Preferably, the terbium ion compound is terbium nitrate hexahydrate Tb(NO3)3·6H2O.

[0012] Preferably, the particle size of the dual-color nanoprobe is nanometer-scale, and the particle size range of the dual-color nanoprobe is most preferably 30 mm to 50 mm.

[0013] 2. Preparation method of a dual-color nanoprobe for alkaline phosphatase detection

[0014] The specific process of the preparation method is:

[0015] A guanylate solution, a Hepes buffer solution with a pH of 9.0 to 9.2, a water-soluble carboxyl functionalized quantum dot solution and a terbium ion compound solution are mixed in order at room temperature, vortexed vigorously and stirred in the dark for reaction, and after the reaction, the precipitate is collected by filtration or centrifugation and washed to obtain the dual-color nanoprobe.

[0016] Specifically, the concentration of the guanylate solution is 10 to 20 mmol / L;

[0017] Preferably, the concentration of terbium ions in the terbium ion compound solution is 10 to 20 mmol / L;

[0018] Preferably, the concentration of the water-soluble carboxyl functionalized quantum dot solution is 7 to 9 μmol / L;

[0019] Preferably, the concentration of the Hepes buffer solution is 1 mol / L.

[0020] Preferably, the light-shielded stirring reaction time is 0.8 to 1.5 hours.

[0021] It should be noted that during the preparation process, the guanylate solution needs to be added dropwise.

[0022] During the preparation process, water-soluble carboxyl functionalized quantum dots and terbium ions Tb 3+ After stirring and reacting with guanosine acid at pH 9.0-9.2, the two-color nanoprobe is self-assembled.

[0023] The volume ratio of the guanylate solution, the Hepes buffer solution, the water-soluble carboxyl functionalized quantum dot solution and the terbium ion compound solution is 1800-2000:400:3:1800-2000.

[0024] 3. A method for detecting alkaline phosphatase using a dual-color nanoprobe

[0025] The following steps are involved:

[0026] 1) Mixing a dual-color nanoprobe solution with a concentration of 0.6 to 1 mg / mL, a Hepes buffer solution with a concentration of 1 mol / L and a pH of 7.4 to 8.0, and water in a volume ratio of preferably 3:1:5 to obtain a detection solution;

[0027] 2) adding the sample solution containing alkaline phosphatase to the detection solution to obtain a sample detection solution, shaking and mixing, and reacting the sample detection solution at 35-40° C. in the dark for 35-45 minutes; the volume ratio of the sample solution to the detection solution is preferably 1:9;

[0028] 3) Setting the excitation wavelength, detecting and obtaining the fluorescence emission peak of the sample test solution at 546 nm;

[0029] The excitation wavelength is selected from 250 to 280 nm;

[0030] The fluorescence emission peak of the dual-color nanoprobe at 546 nm after reacting with alkaline phosphatase in the sample detection solution is positively correlated with the concentration of alkaline phosphatase;

[0031] 4) Substitute the peak value of the fluorescence emission of the sample test solution at 546 nm into the pre-drawn linear regression curve to calculate the concentration of alkaline phosphatase in the sample solution.

[0032] The process of establishing the linear regression curve is specifically as follows: reacting the dual-color nanoprobe with multiple alkaline phosphatase standard solutions of different concentrations and known concentrations, detecting the fluorescence emission peak value of each sample detection solution at 546nm, and fitting the fluorescence emission peak value of each sample detection solution at 546nm with the corresponding alkaline phosphatase standard solution concentration value to obtain a linear regression curve.

[0033] Preferably, the sample solution is bovine serum, and the concentration range of alkaline phosphatase in the sample solution is 0.3874-40 U / L.

[0034] In the present invention, unless otherwise specified, the solvent of all solutions is water.

[0035] In the present invention, unless otherwise specified, room temperature refers to a temperature of 25±5°C.

[0036] Beneficial effects of the present invention:

[0037] 1. The present invention utilizes terbium ions, guanylate and water-soluble carboxylated quantum dots QD 625 A composite coordination polymer nanomaterial was self-assembled and prepared at room temperature and in the dark as a dual-color nanoprobe. The dual-color nanoprobe has yellow-green dual-emission fluorescence and a significant fluorescence response to alkaline phosphatase. The detection results can be judged by the naked eye, and it has good sensitivity, specificity and anti-interference ability for detecting alkaline phosphatase.

[0038] 2. The dual-color nanoprobe in the present invention can be prepared using low-concentration quantum dots, terbium ions and guanylate solution, which effectively reduces costs, and the preparation process is simple and the conditions are mild; the process of detecting alkaline phosphatase using the dual-color nanoprobe is simple to operate, and only requires mixing and incubation reactions, without other separation, enrichment, washing and other steps, and an ELISA instrument can be used for high-throughput, rapid, simple and convenient detection of alkaline phosphatase.

[0039] 3. When the dual-color nanoprobe of the present invention detects alkaline phosphatase, it shows a good linear relationship in the activity range of 2 to 40 U / L, and the minimum detection limit is 0.3874 U / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the principle of dual-color nanoprobe for detecting alkaline phosphatase;

[0041] Figure 2 The two-color nanoprobe and its raw material are shown in the figure and fluorescence spectrum at an excitation wavelength of 254 nm; (A) is the figure; (B) is the fluorescence spectrum;

[0042] Figure 3The figures are the physical picture and fluorescence spectrum of the dual-color nanoprobe before and after the reaction with alkaline phosphatase at an excitation wavelength of 254 nm; (A) is the physical picture; (B) is the fluorescence spectrum;

[0043] Figure 4 The fluorescence spectra and linear fitting curves of the dual-color nanoprobe for detecting alkaline phosphatases of different activities; (A) is the fluorescence spectrum, (B) is the linear fitting curve in the low activity range, and (C) is the linear fitting curve in the high activity range. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0045] The present invention provides a dual-color nanoprobe for alkaline phosphatase detection. The dual-color nanoprobe of the present invention is a hybrid material formed by self-assembly of a metal central node and an organic ligand through coordination. The particle size of the dual-color nanoprobe of the present invention is nanometer-scale. The metal central node is a terbium ion Tb 3+ , organic ligands include guanylate and water-soluble carboxylated quantum dots. This dual-color nanoprobe belongs to lanthanide metal coordination polymers, has yellow-green dual emission fluorescence, and has the advantages of large Stokes shift, high quantum yield and high photochemical stability, mild synthesis conditions, and dual emission peaks. This dual-color nanoprobe has a significant fluorescence response to alkaline phosphatase, and the test results can be judged by the naked eye, and has good sensitivity, specificity and anti-interference ability for the detection of alkaline phosphatase.

[0046] The present invention also provides a method for preparing the above-mentioned dual-color nanoprobe. The method uses terbium ions, guanylate and water-soluble carboxylated quantum dots QD 625 Self-assembly of dual-color nanoprobe QDs at room temperature and in the dark 625 @Tb / GMP, the preparation steps are simple and the preparation conditions are mild.

[0047] The present invention also provides a method for rapid detection of alkaline phosphatase using the above-mentioned dual-color nanoprobe. This detection method only requires mixing and incubation reaction, without other separation, enrichment, washing and other steps, and is simple to operate. This detection method can also use an ELISA instrument for high-throughput, rapid, simple and convenient detection. This detection method is applicable to the alkaline phosphatase activity range of 2 to 40 U / L, and the detection limit is 0.3874 U / L.

[0048] The dual-color nanoprobe provided by the present invention has a "turn off" fluorescence response to alkaline phosphatase. Its working principle is as follows Figure 1 As shown. Figure 1 It can be seen that the quantum dots QD with water-soluble carboxyl groups625 、Tb 3+ Mixed with guanylate, and stirred continuously at room temperature, a composite coordination polymer nanomaterial QD with yellow-green fluorescence was formed through self-assembly. 625 @Tb / GMP, that is, a dual-color nanoprobe. The prepared dual-color nanoprobe has dual-emission fluorescence characteristics under an excitation wavelength of 254nm. The dual-emission fluorescence characteristic refers to the dual-color nanoprobe having fluorescence emission peaks at 546nm and 625nm. Alkaline phosphatase exists in cow serum. After the dual-color nanoprobe is mixed with alkaline phosphatase, alkaline phosphatase will hydrolyze the phosphate group of the dual-color nanoprobe, weakening the fluorescence of the dual-color nanoprobe at 546nm, and the green fluorescence of the dual-color nanoprobe is weakened, while the quantum dot QD 625 The red fluorescence still exists, thus producing a significant hue change from green to red, thereby achieving visual and quantitative detection of alkaline phosphatase.

[0049] The specific embodiments of the present invention are as follows:

[0050] Example

[0051] This example prepares a dual-color nanoprobe QD with dual-emission fluorescence 625 @Tb / GMP. The molecular formula of the dual-color nanoprobe obtained in this example is TbCdSeZnSC 10 H 12 N5Na2O8P. The water-soluble carboxylated quantum dots QD in this embodiment 625 It was obtained through commercial means.

[0052] In this embodiment, the preparation process of the dual-color nanoprobe includes the following steps:

[0053] S1) Prepare a 10 mmol / L Tb(NO3)3·6H2O aqueous solution, a 1 mol / L Hepes buffer solution with a pH of 9.0, a 10 mmol / L guanylate aqueous solution, and an 8 μM water-soluble carboxylated quantum dot QD 625 The solution is ready for use;

[0054] S2) Take a 5 mL centrifuge tube and add 1.8 mL of guanosine acid aqueous solution, 400 μL of Hepes buffer solution, 3 μL of QD 625 and 1.8 mL of Tb(NO3)3·6H2O aqueous solution to obtain a first solution;

[0055] S3) vortexing the first solution in step S2) to mix evenly, and stirring the mixture in the dark at room temperature for 1 h to obtain a second solution;

[0056] S4) centrifuging the second solution in step S3) at 10000 rpm for 10 min, removing the supernatant to obtain a yellow precipitate;

[0057] S5) re-dissolving the yellow precipitate obtained in step S4) with ultrapure water, sonicating for 10 min, centrifuging at 10000 rpm for 10 min, removing the supernatant, and obtaining a yellow precipitate;

[0058] S6) Repeat step S5) three times to wash the yellow precipitate three times again, and obtain a light yellow precipitate after washing, which is the dual-color nanoprobe QD 625 @Tb / GMP.

[0059] Finally, the obtained dual-color nanoprobe QD 625 @Tb / GMP was dispersed in 1 mL of ultrapure water to obtain a dual-color nanoprobe solution with a mass fraction of 1 wt%, which was stored at 4°C away from light.

[0060] Comparative Example

[0061] In this comparative example, a 10 mmol / L guanylate solution and a 10 mmol / L Tb(NO3)3·6H2O solution were mixed at room temperature in a volume mixing ratio of 1:1 to prepare a fluorescent complex Tb / GMP.

[0062] In order to verify the fluorescence response effect of the dual-color nanoprobe to alkaline phosphatase and the detection effect of the dual-color nanoprobe on bovine serum samples, the present invention conducted the following test on the response effect of the dual-color nanoprobe prepared in Example 1 to alkaline phosphatase. The test process and results are as follows:

[0063] Test a. Dual-color nanoprobe QD 625 @Tb / GMP conducts real-life photography and fluorescence emission spectrum characterization

[0064] a1) Tb(NO3)3·6H2O aqueous solution (Tb in the figure) 3+ ), guanylate aqueous solution (GMP in the figure), QD 625 Aqueous solution, fluorescent complex Tb / GMP aqueous solution prepared in comparative example, and dual-color nanoprobe QD prepared in example 625 @Tb / GMP aqueous solution was added to 5mL test tubes respectively. The actual image was taken under 254nm UV light. The results are as follows Figure 2 As shown in (A), under the irradiation of ultraviolet light at 254nm, Tb(NO3)3·6H2O aqueous solution (Tb in the figure) 3+ ) and guanosine acid aqueous solution (GMP in the figure) have no fluorescence, QD 625 The aqueous solution shows bright red fluorescence, Tb / GMP shows green fluorescence, and the dual-color nanoprobe QD 625@Tb / GMP exhibits yellow-green fluorescence.

[0065] a2) The five materials in step a1) were mixed by oscillation, taken out from the test tubes with a pipette, and added to the sample wells of a 96-well ELISA plate suitable for measuring fluorescence spectra. The excitation wavelength of the ELISA plate was set to 254 nm, the emission spectrum collection range was set to 300-700 nm, the step size was set to 2 nm, and the fluorescence emission spectra of the five materials were obtained by scanning. The results are shown in FIG. Figure 2 As shown in (B), QD 625 There is a characteristic peak at 625nm, Tb / GMP has characteristic peaks at 488nm, 546nm and 620nm, and QD 625 @Tb / GMP has fluorescence emission peaks at 488nm, 546nm and 625nm, which shows that QD 625 @Tb / GMP has Tb / GMP and QD 625 characteristic fluorescence peaks.

[0066] Test b: Dual-color nanoprobe QD 625 @Tb / GMP detects alkaline phosphatase for feasibility verification

[0067] b1) preparing a Hepes buffer solution with a concentration of 1 mol / L and a pH of 8.0 for later use;

[0068] b2) Add 50 μL of H2O and 10 μL of Hepes buffer solution to a 200 μL test tube, and then add 30 μL of 1% QD 625 @Tb / GMP solution and 10 μL alkaline phosphatase solution were gently mixed and reacted at 37°C in the dark for 40 minutes;

[0069] b3) After the reaction is completed, use a pipette to take 90 μL of the sample solution from the sample solution reacted in step b2), add the sample solution to the sample wells of a 96-well ELISA plate suitable for measuring fluorescence spectra, set the excitation wavelength of the ELISA instrument to 254 nm, set the emission spectrum collection range to 300-700 nm, set the step size to 2 nm, and scan the fluorescence emission spectrum.

[0070] Test results:

[0071] like Figure 3 As shown in (A), alkaline phosphatase and dual-color nanoprobe QD 625 After Tb / GMP mixing, the yellow-green fluorescence (left) turns into dark red fluorescence (right) because alkaline phosphatase hydrolyzes the dual-color nanoprobe QD 625 @The phosphate group of Tb / GMP quenches the green fluorescence of Tb / GMP, QD 625 The fluorescence retention of Figure 3 As shown in (B), the dual-color nanoprobe QD 625 After the mixed reaction of @Tb / GMP and alkaline phosphatase, the characteristic peak fluorescence intensities at 488nm, 546nm and 625nm decreased significantly.

[0072] The test results of this test confirmed that QD 625 @Tb / GMP is used to test the feasibility of alkaline phosphatase.

[0073] Test c. Using QD 625 @Tb / GMP dual-color nanoprobe for rapid detection of alkaline phosphatase in water

[0074] c1) Preparation of the dual-color nanoprobe QD prepared in the above example 625 @Tb / GMP aqueous solution, concentration 0.8mg / mL;

[0075] c2) In a 200 μL test tube, add 50 μL of H2O and 10 μL of 1 mol / L pH 8.0 Hepes buffer solution, then add 30 μL of QD 625 @Tb / GMP aqueous solution, add 10mL of alkaline phosphatase standard sample solution containing different concentrations, shake and mix, and stand at 37℃ in the dark for 40min;

[0076] c3) After the reaction is completed, 90 mL of the sample solution prepared in c2) is taken with a pipette and added to the sample wells of a 96-well ELISA plate suitable for measuring fluorescence spectra. The excitation wavelength of the ELISA plate reader is set to 254 nm, the emission spectrum collection range is set to 300-700 nm, the step size is set to 2 nm, and the fluorescence emission spectrum is scanned.

[0077] According to the process from step c1) to step c3) above, the sample solution containing the same concentration of alkaline phosphatase was measured in parallel three times.

[0078] Test results:

[0079] like Figure 4 As shown in (A), as the detection concentration of alkaline phosphatase increases from 0 to 80 U / L (from low to high, 0, 2, 4, 6 / 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80 U / L), QD 625 The peak values ​​of fluorescence emission of @Tb / GMP at 488nm, 546nm and 625nm gradually decrease.

[0080] like Figure 4 (B) and Figure 4 As shown in (C), QD625 @The fluorescence intensity of the Tb / GMP dual-color nanoprobe at 546 nm was linearly fitted with the alkaline phosphatase concentration. It can be seen that when the alkaline phosphatase concentration is in the range of 0 to 12 U / L and 14 to 40 U / L, the peak value of the fluorescence emission at 546 nm is linearly related to the alkaline phosphatase concentration.

[0081] The linear regression equation between the alkaline phosphatase concentration 0-12U / L and the emission peak value was obtained as y=2.8889x+5.0068, and the linear correlation coefficient R 2 is 0.9985;

[0082] The linear regression equation between the alkaline phosphatase concentration of 14-40U / L and the emission peak value is y=1.8962x+19.975, and the linear correlation coefficient is R 2 The linear range of detection is 0 to 40 U / L.

[0083] The detection limit of alkaline phosphatase detected by the method of the present invention is 0.3874 U / L.

[0084] Test d, QD 625 @Tb / GMP dual-color nanoprobe for detecting alkaline phosphatase with actual sample addition and recovery experiment

[0085] d1) A certain concentration of alkaline phosphatase was added to the cow blood sample, and the three levels of added concentration were 1, 5 and 10 U / L respectively.

[0086] d2) For the cow blood sample, centrifuge the cow blood at 3000 rpm for 5 min, and take out the supernatant to obtain the cow serum;

[0087] d3) treating the cow blood samples obtained in step d1) according to the process in step d2) to obtain cow serum samples with different standard concentrations of alkaline phosphatase (1, 5 and 10 U / L) for later use.

[0088] d4) Add 50 μL of H2O, 10 μL of 1 mol / L Hepes buffer solution at pH 8.0, and 30 μL of 0.8 mg / mL QD to multiple centrifuge tubes with a volume of 200 μL. 625 @Tb / GMP, then add 10μL of cow serum samples of different concentrations to each centrifuge tube, mix well, and place all centrifuge tubes at 37°C to react for 40 minutes in the dark. Use a pipette to take 90μL of each of the above reaction solutions and add them to the sample wells of the black 96-well ELISA plate. Set the excitation wavelength of the ELISA reader to 254nm, the emission spectrum acquisition range to 400-700nm, and scan the fluorescence emission spectrum.

[0089] d5) According to the process of steps d1) to d4) above, the alkaline phosphatase samples containing the same added concentration are measured in parallel three times.

[0090] d6) Use the following formula to calculate the spike recovery rate.

[0091]

[0092] In the formula, K0, K1, and K2 represent the blank measurement value, spiked amount, and spiked sample measurement value, respectively.

[0093] Test results:

[0094] Combining the results in test c with the formula to calculate the recovery rate and relative standard deviation, the results are as follows:

[0095]

[0096] Note: The spike concentration is the concentration calculated after adding a known amount of alkaline phosphatase to the sample; the detected concentration is the concentration detected in the spiked sample.

[0097] The test results of this addition recovery experiment further demonstrated the reliability of the method of the present invention.

[0098] Test e, QD 625 @Comparison of authenticity of Tb / GMP dual-color nanoprobe in detecting alkaline phosphatase.

[0099] e1) collecting blood samples from healthy cows and cows with mastitis at local pastures; repeating step d2) in Example 6 to obtain serum samples from healthy cows and cows with mastitis;

[0100] e2) purchasing a commercial alkaline phosphatase detection kit, detecting alkaline phosphatase in the serum of healthy cows and cows with mastitis according to the steps in the kit instructions, and calculating the test results according to the alkaline phosphatase calculation formula corresponding to the kit;

[0101] e3) Repeating step d6) in test d to detect alkaline phosphatase in the serum of healthy cows and cows suffering from mastitis using the method of the present invention.

[0102] The test results were calculated by combining the test results with the standard curve obtained from test b.

[0103] The test results are shown in the table below:

[0104]

[0105] As can be seen from the above table, the results of the method of the present invention for detecting the bovine serum samples are consistent with the results of the commercial kit. Therefore, the method of the present invention can be used to quickly detect the prevalence of mastitis in dairy cows.

[0106] In summary, the dual-color nanoprobe provided by the present invention is formed by self-assembly through coordination, the preparation process is simple, less raw materials are required, and the cost is low. The dual-color nanoprobe of the present invention belongs to a lanthanide metal coordination polymer, and has yellow-green dual emission fluorescence. After the dual-color nanoprobe of the present invention reacts with alkaline phosphatase, a significant hue change from green to red can occur, and the detection result can be judged by naked eye observation, and the detection of alkaline phosphatase has good sensitivity, specificity and anti-interference ability.

[0107] The present invention has been described in detail above. For those skilled in the art, it is clear that the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experiments.

[0108] Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in the application and are made with conventional techniques known in the art.

Claims

1. A dual-color nanoprobe for alkaline phosphatase detection, characterized in that: The dual-color nanoprobe is mainly formed by self-assembly of guanylic acid, terbium ion compound and water-soluble carboxyl functionalized quantum dots at room temperature.

2. The dual-color nanoprobe for alkaline phosphatase detection according to claim 1, characterized in that: The terbium ion compound is used to provide terbium ions, and the amount ratio of the guanylic acid, terbium ions and water-soluble carboxyl functionalized quantum dots is 750,000-800,000:750,000-800,000:

1.

3. The dual-color nanoprobe for alkaline phosphatase detection according to claim 1, characterized in that: The water-soluble carboxyl functionalized quantum dots are QD 625 , the terbium ion compound is terbium nitrate hexahydrate.

4. The dual-color nanoprobe for alkaline phosphatase detection according to claim 1, characterized in that: The terbium ion serves as the central atom of the dual-color nanoprobe, and guanylic acid and water-soluble carboxyl functionalized quantum dots serve as ligands of the dual-color nanoprobe. The terbium ion is connected to the phosphate or carboxyl of guanylic acid and the carboxyl of the water-soluble carboxyl functionalized quantum dots through coordination bonds.

5. The dual-color nanoprobe for alkaline phosphatase detection according to claim 1, characterized in that: The particle size range of the dual-color nanoprobe is 30 mm to 50 mm.

6. A method for preparing a dual-color nanoprobe according to any one of claims 1 to 5, characterized in that: The specific process of the preparation method is: A guanylate solution, a buffer solution with a pH of 9.0 to 9.2, a water-soluble carboxyl functionalized quantum dot solution and a terbium ion compound solution are mixed in order at room temperature, vortexed and stirred in the dark for reaction, and after the reaction, the precipitate is collected by filtration or centrifugation, and the dual-color nanoprobe is obtained after washing; The concentration of the guanylate solution is 10-20 mmol / L, the concentration of terbium ions in the terbium ion compound solution is 10-20 mmol / L, the concentration of the water-soluble carboxyl functionalized quantum dot solution is 7-9 μmol / L, and the concentration of the buffer solution is 1 mol / L.

7. The preparation method according to claim 6, characterized in that: The volume ratio of the guanylate solution, the buffer solution, the water-soluble carboxyl functionalized quantum dot solution and the terbium ion compound solution is 1800-2000:400:3:1800-2000.

8. A method for rapidly detecting alkaline phosphatase using the dual-color nanoprobe according to any one of claims 1 to 5 or the dual-color nanoprobe prepared by the method according to any one of claims 6 to 7, characterized in that: The following steps are involved: 1) mixing the dual-color nanoprobe solution, a buffer solution with a pH of 7.4 to 8.0, and water to obtain a detection solution; 2) adding the sample solution containing alkaline phosphatase to the detection solution to obtain a sample detection solution, oscillating and mixing, and reacting the sample detection solution in the dark for 35 to 50 minutes; 3) setting an excitation wavelength to detect and obtain the fluorescence emission peak of the sample test solution at 546 nm; the excitation wavelength is selected from 250 to 280 nm; 4) Substitute the peak value of the fluorescence emission of the sample test solution at 546 nm into the pre-drawn linear regression curve to calculate the concentration of alkaline phosphatase in the sample solution.

9. The method for rapid detection of alkaline phosphatase according to claim 8, characterized in that: The process of establishing the linear regression curve is specifically as follows: reacting the dual-color nanoprobe with multiple alkaline phosphatase standard solutions of different concentrations and known concentrations, detecting the fluorescence emission peak value of each sample detection solution at 546nm, and fitting the fluorescence emission peak value of each sample detection solution at 546nm with the corresponding alkaline phosphatase standard solution concentration value to obtain a linear regression curve.

10. The method for rapid detection of alkaline phosphatase according to claim 8, characterized in that: The sample solution is cow serum, and the concentration range of alkaline phosphatase in the sample solution is 0.3874-40 U / L.

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