CN113354703B - Rare earth metal-based enzyme response type probe and preparation method and application thereof - Google Patents

Rare earth metal-based enzyme response type probe and preparation method and application thereof Download PDF

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CN113354703B
CN113354703B CN202010140295.8A CN202010140295A CN113354703B CN 113354703 B CN113354703 B CN 113354703B CN 202010140295 A CN202010140295 A CN 202010140295A CN 113354703 B CN113354703 B CN 113354703B
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CN113354703A (en
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� 刘
陈学元
杨颖婕
张云钦
李幸俊
高航
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Fujian Institute of Research on the Structure of Matter of CAS
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Abstract

The invention discloses an enzyme response type probe based on rare earth fluorescence resonance energy transfer, a preparation method thereof and application thereof in alkaline phosphatase detection. The enzyme response type probe utilizes supermolecule self-aggregation of rare earth sensitized ions, rare earth activated ions and phospholipid molecules, effectively shortens the energy transmission distance between the rare earth sensitized ions and the activated ions, successfully constructs the nano probe with good fluorescence characteristic, and realizes the specificity and high sensitivity detection of alkaline phosphatase. The enzyme response type probe provided by the invention is simple and quick to prepare, safe and nontoxic, and can effectively avoid interference in a biological complex system. The detection process can realize the detection of the concentration of alkaline phosphatase in serum or whole blood by simply mixing the nano probe with a sample to be detected, has the advantages of simplicity, sensitivity, good specificity, economy, practicability and the like, and provides a simple and efficient novel method and corresponding technical support for the detection of alkaline phosphatase in a complex system.

Description

Rare earth metal-based enzyme response type probe and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological detection, in particular to an enzyme response type probe based on rare earth metal, a preparation method and application thereof.
Background
Enzymes are catalysts of organism metabolism and play an important role in maintaining the normal operation of various biochemical and physiological reactions of organisms. With the further knowledge of enzymes, biological enzymes have been found to be closely related to cellular activity, pathological reactions, disease occurrence, etc., and most of the disease occurrence is related to enzyme deficiency or synthesis failure. Therefore, the enzyme has an indication effect in disease prevention, early diagnosis, drug reaction and the like. Alkaline phosphatase is an important enzyme disease marker, and is closely related to the occurrence and development of serious diseases such as cancer, bone diseases, liver and gall diseases, diabetes and the like. The concentration of alkaline phosphatase is the most direct evidence for the diagnosis of related diseases, and is helpful for tracking the clinical treatment effect, and is convenient for the alleviation and treatment of the disease. Therefore, the realization of high-sensitivity detection of the alkaline phosphatase marker has extremely important significance for establishing a precise health condition assessment system, preventing diseases, early warning, diagnosing and the like.
The existing detection methods for alkaline phosphatase mainly comprise an electrochemical method, a surface enhanced Raman spectroscopy method, a fluorescence analysis method and the like. Among them, the fluorescence analysis method is widely used because of its advantages of low requirement on instruments, simple operation, high sensitivity, good selectivity, etc. However, the problems of poor photochemical stability, high toxicity, high cost, long time consumption and the like of organic dyes, quantum dots, metal organic framework materials and the like used in the traditional fluorescence analysis method cannot be avoided, and the method cannot realize the rapid and accurate direct detection of the concentration of alkaline phosphatase.
Compared with traditional organic dyes, quantum dots, metal organic frame materials and the like, the rare earth down-conversion luminescent material has the unique advantage of long-life fluorescence emission, so that the short-life nonspecific fluorescence and stray light interference from the inside of a sample to be detected and an instrument can be effectively removed by controlling proper delay time and data acquisition time. In addition, the rare earth down-conversion luminescence spectrum band is narrow, which is helpful for reducing background and improving resolution; the emission spectrum is positioned in the visible light region, and the intensity of emitted light can be directly observed by naked eyes; the fluorescent dye has excellent stability, can be stored for standby for a long time, and overcomes the problems of organic dye photobleaching, quantum dot photoflash and the like. Many advantages make the rare earth fluorescent probe very suitable for directly detecting the concentration of alkaline phosphatase in a complex biological system. The fluorescent detection method based on rare earth resonance energy transfer is developed and used for accurately detecting the alkaline phosphatase level in a complex system, achieves the purpose of convenient, accurate and economic detection, and is important and significant in the invention.
Disclosure of Invention
In order to improve the above problems, the present invention provides a rare earth metal-based enzyme-responsive probe comprising a rare earth metal ion group and a phospholipid molecule.
According to an embodiment of the present invention, the rare earth metal ion group may be an ion group composed of two or more rare earth ions selected from lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), yttrium (Y), and scandium (Sc); such as cerium-terbium (Ce-Tb), cerium-europium (Ce-Eu), cerium-dysprosium (Ce-Dy), terbium-europium (Tb-Eu), cerium-neodymium (Ce-Nd), cerium-gadolinium (Ce-Gd), cerium-erbium (Ce-Er), gadolinium-europium (Gd-Eu), gadolinium-terbium (Gd-Tb), gadolinium-dysprosium (Gd-Dy), gadolinium-terbium-europium (Gd-Tb-Eu) or cerium-ytterbium-erbium (Ce-Yb-Er) ion groups, preferably cerium-terbium ion groups.
According to an embodiment of the present invention, the phospholipid molecule may be a phospholipid bond-containing molecule, and the phospholipid bond-containing molecule may be one, two or more of Adenosine Triphosphate (ATP), adenosine Diphosphate (ADP), adenosine Monophosphate (AMP), guanosine Triphosphate (GTP), guanosine Diphosphate (GDP), guanosine Monophosphate (GMP), and ATP is exemplified.
The invention provides a preparation method of the rare earth metal-based enzyme response probe, which comprises the following steps:
1) Dissolving a combination of rare earth metal salts in a buffer solution to prepare a rare earth ion solution;
2) Dissolving phospholipid molecules in a buffer solution to prepare a phospholipid molecule solution;
3) Mixing the rare earth ion solution in the step 1) with the phospholipid molecule solution in the step (2) to obtain the rare earth metal-based enzyme response probe.
According to the present invention, in step 1), the combination of rare earth metal salts may be a combination of two or more rare earth metal salts capable of providing the above-mentioned rare earth ion group; the rare earth metal salt may be nitrate, chloride, acetate, sulfate, perchlorate, or salt hydrate thereof, etc. of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, yttrium, or scandium. For example, the rare earth metal salt may be a trivalent nitrate of a rare earth metal or a hydrate thereof (e.g., hexahydrate, pentahydrate, tetrahydrate, tri-hydrate, dihydrate, monohydrate, etc.), such as Ce (NO 3 ) 3 Or a hydrate thereof (e.g. Ce (NO) 3 ) 3 ·6H 2 O)、Tb(NO 3 ) 3 Or a hydrate thereof (e.g. Tb (NO) 3 ) 3 ·6H 2 O)、La(NO 3 ) 3 、Pr(NO 3 ) 3 、Nd(NO 3 ) 3 、Sm(NO 3 ) 3 、Eu(NO 3 ) 3 、Gd(NO 3 ) 3 、Yb(NO 3 ) 3 Etc.; chlorides of rare earth metals or their hydrates (e.g. heptahydrate, hexahydrate, pentahydrate, tetrahydrate, trihydrate, dihydrate, monohydrate, etc.), e.g. LaCl 3 Or a hydrate thereof (e.g. LaCl) 3 ·7H 2 O)、CeCl 3 Or a hydrate thereof (e.g. CeCl) 3 ·7H 2 O)、PrCl 3 Or a hydrate thereof (e.g. PrCl 3 ·6H 2 O)、TbCl 3 Or a hydrate thereof (e.g. TbCl 3 ·6H 2 O)、DyCl 3 Or a hydrate thereof (e.g. DyCl) 3 ·6H 2 O); can be sulfate or its hydrate (such as heptahydrate, hexahydrate, pentahydrate, tetrahydrate, trihydrate, dihydrate, monohydrate, etc.), such as Y (SO) 4 ) 2 Or a hydrate thereof, ti (SO) 4 ) 2 Or a hydrate thereof, ce (SO) 4 ) 2 Or a hydrate thereof (e.g. Ce (SO) 4 ) 2 ·4H 2 O), and the like.
According to the invention, in step 1), the total concentration of the rare earth ion solution may be 0.5-32mM; for example, 1-30mM,1.5-28mM,2-25mM,2.5-20mM,3-18mM, for example, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM.
In the context of the present invention, the "total concentration of rare earth metal ion solution" refers to the sum of the concentrations of two or more rare earth ions present in the solution. For example, when the rare earth metal-based enzyme-responsive probe employs a cerium-terbium ion group, the total concentration of the rare earth metal ion solution refers to Ce present in the solution 3+ And Tb 3+ The sum of the concentrations of the ions.
According to the present invention, in step 1), when the rare earth metal-based enzyme-responsive probe employs an ion group composed of two rare earth ions, such as cerium-terbium (Ce-Tb), cerium-europium (Ce-Eu), cerium-dysprosium (Ce-Dy), terbium-europium (Tb-Eu), cerium-neodymium (Ce-Nd), cerium-gadolinium (Ce-Gd), cerium-erbium (Ce-Er), gadolinium-europium (Gd-Eu), gadolinium-terbium (Gd-Tb), gadolinium-dysprosium (Gd-Dy), the molar ratio of the two rare earth ions in the rare earth ion solution, that is, the molar ratio of the former rare earth ion to the latter rare earth ion in the ion group may be 1:10 to 10:1; for example, 1:9-9:1,1:8-8:1,1:7-7:1,1:6-6:1,1:5-5:1,1:4-4:1, e.g., 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1; specifically, ce 3+ And Tb 3+ May be in a molar ratio of 1:10 to 10:1; for example, 1:9-9:1,1:8-8:1,1:7-7:1,1:6-6:1,1:5-5:1,1:4-4:1, for example, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. When the rare earth metal-based enzyme-responsive probe employs an ion group composed of three rare earth ions, such as gadolinium-terbium-europium (Gd-Tb-Eu) or cerium-ytterbium-erbium (Ce-Yb-Er) ion group, the molar ratio of the three rare earth ions in the rare earth ion solution, i.e., the molar ratio of the former rare earth ion to the intermediate rare earth ion to the latter rare earth ion, may be (1-10): 10-1): 1-10, for example, (1-9): 9-1): 1-9, (1-8): 8-1): 1-8, (1-7): 7-1): 1-7,(1-6): (6-1): (1-6), (1-5): (5-1): (1-5), (1-4): (4-1): (1-4), (1-3): (3-1): (1-3), such as 1:1:1,1:2:1,2:1:1,1:1:2.
According to the present invention, in step 2), the buffer solution may be N-methyl-D-glucosamine (MEG) buffer, 2-amino-2-methyl-1-propanol (AMP) buffer, tris-HCl buffer; exemplary is Tris-HCl buffer; the pH of the buffer may be 7.0-9.0, preferably pH 9.0.
According to the invention, in step 2), the concentration of the phospholipid molecule solution may be 0.5-32mM; for example, 1-30mM,1.5-28mM,2-25mM, e.g., 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM.
According to the invention, the solvent used to formulate the solution in step 1) and step 2) may be water.
According to the present invention, in step 3), the volume ratio of the rare earth ion solution to the phospholipid molecule solution may be 1:5 to 5:1; for example, 1:4-4:1, 1:3-3:1, 1:2-2:1, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1.
The invention provides the use of the rare earth metal-based enzyme-responsive probes for the detection of alkaline phosphatase.
The invention also provides an alkaline phosphatase detection method, which comprises the following steps:
1) Mixing the rare earth metal-based enzyme response probe with a buffer solution to obtain a probe solution;
2) Mixing alkaline phosphatase with a buffer solution to obtain an alkaline phosphatase solution;
3) Mixing the probe solution in the step 1) with the alkaline phosphatase solution in the step 2), oscillating at constant temperature, measuring the fluorescence intensity of the alkaline phosphatase in the sample to be measured, and calculating the concentration of the alkaline phosphatase in the sample to be measured according to a standard curve;
according to an embodiment of the invention, the temperature of the constant temperature oscillation may be 25-40 ℃, for example 30-39 ℃,35-38 ℃,37 ℃; the time of the constant temperature oscillation may be 0.5 to 5 hours, for example 3 hours.
According to an embodiment of the invention, in step 1), the concentration of the probe solution is selected from 0.5-32mM; for example, 1-30mM,1.5-28mM,2-25mM, e.g., 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM.
According to the invention, in step 2), the alkaline phosphatase solution may have a concentration of 0-5000U/L; for example 3000, 2500, 2000, 1500, 1250, 1000, 750, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 0U/L;
in the present invention, the "cerium-terbium" ion group is exemplified as Ce which allows transition with f-d electric dipole by being coordinated to phospholipid molecules to draw a distance from each other 3+ As a sensitizing ion, tb is sensitized 3+ Realizes the high-efficiency energy transfer process from cerium ion to terbium ion. In the sensitization of ion Ce 3+ Ce by cross relaxation under excitation of excitation light of (c) 3+ Transmitting energy to Tb 3+ Make Tb 3+ Non-radiative transition of high level electrons to 5 D 4 Thereby respectively radiating and transitioning to 7 F 6 ， 7 F 5 ， 7 F 4 Make Tb 3+ The fluorescence emission signal of (2) is significantly enhanced. Further studies have found that the presence of alkaline phosphatase can effectively cleave the phospholipid bond in ATP, thereby causing the cerium-terbium distance in the probe to become distant, resulting in fluorescence quenching. The content of alkaline phosphatase is positively correlated with the fluorescence quenching effect, so that qualitative and quantitative detection of alkaline phosphatase can be achieved by the probe by utilizing the property.
Advantageous effects
The rare earth metal-based enzyme response probe has the following technical effects:
(1) The probe designed by the invention can effectively shorten the energy transmission distance between the rare earth sensitized ions and the rare earth activated ions by utilizing the strong coordination effect between phospholipid molecules and the rare earth ions, thereby realizing the effective enhancement of the luminescence of the rare earth activated ions. The probe has excellent enzyme response of fluorescent signals and can effectively improve detection sensitivity.
(2) Compared with the traditional fluorescence analysis method, the detection method of the probe design of the invention avoids the defects of high cost, low stability, high toxicity and the like of materials such as organic dye, quantum dot, metal organic framework and the like, realizes the detection of the object to be detected by the probe formed by phospholipid molecules and rare earth ions, and has the advantages of environmental protection, low cost and the like.
(3) The invention overcomes the interference of background fluorescence, stray light and the like in a biological complex system by utilizing the long fluorescence lifetime characteristic of rare earth ions, can be used for detecting alkaline phosphatase or phospholipid related substances, further realizes the detection of the alkaline phosphatase or phospholipid related substances in serum or whole blood samples, has the advantages of simple operation, good anti-interference performance, rapidness, sensitivity, low cost, wide application range and the like, can provide theoretical basis and technical support for real-time monitoring of enzyme disease markers in the complex biological system, and has a certain clinical application prospect.
Drawings
FIG. 1 is a schematic diagram of the principle of the enzyme-responsive rare earth metal probe for ALP detection;
FIG. 2 shows the physicochemical characterization result of the enzyme-responsive probe of preparation example 1;
FIG. 3 shows the ALP concentration-dependent response curves of (a) fluorescence spectra and (b) fluorescence intensities of the mixed solution after the addition of alkaline phosphatase at different concentrations as described in example 1;
FIG. 4 shows the results of the specificity verification of alkaline phosphatase by the enzyme-responsive probes described in example 3;
FIG. 5 shows the detection of alkaline phosphatase in serum as described in example 3: (a) fluorescence contrast in serum steady state and time resolution mode, (b) fluorescence spectrum of mixed solution after adding alkaline phosphatase with different concentrations, and (c) and (d) ALP concentration dependent response curve of fluorescence intensity;
FIG. 6 shows the result of comparing the fluorescence intensity of the dual-ion probe of preparation example 1 with that of the single-ion probe of control preparation example.
Terminology and definitions
"phospholipid bond" in the present invention means phosphoric acid H 3 PO 4 A chemical bond formed after one, two or three hydroxyl groups are removed and hydrogen on the hydroxyl groups are removed from one, two or more phosphoric acid molecules or other hydroxyl-containing molecules; the hydroxyl-containing molecule may be adenosine, guanosine, uridine, xanthosine, inosine, cytidine, thymidine.
"phospholipid molecule" in the present invention means a molecule comprising a phospholipid linkage, which may be a phosphoglycoside comprising a phospholipid linkage, which may be Adenosine Triphosphate (ATP), adenosine Diphosphate (ADP), adenosine Monophosphate (AMP), guanosine Triphosphate (GTP), guanosine Diphosphate (GDP), guanosine Monophosphate (GMP).
Detailed Description
The invention is described in further detail below with reference to the drawings and examples. It should be understood that the scope of the present invention is not limited to the following examples. Various changes and modifications to the present invention will become apparent to those skilled in the art upon reading the disclosure herein, and such equivalents are intended to fall within the scope of the invention as defined by the appended claims.
Unless otherwise indicated, the starting materials or reagents employed in the examples were all commercially available or may be prepared by known methods.
Instrument: the model of the apparatus for detecting the transmission electron microscope image is JEM-2010 manufactured by JEOL company; the instrument for detecting the fluorescent signal is a fluorescent enzyme-labeled instrument with the model number of Synergy 4 manufactured by BioTek company.
Preparation example 1: synthesis of rare earth metal-based enzyme-responsive probes (zwitterionic probes)
The probe is prepared by a room temperature stirring method, and takes rare earth nitrate and ATP compound as raw materials, and the preparation method is as follows:
(1) The same amount of Ce (NO) was weighed out separately 3 ) 3 ·6H 2 O、Tb(NO 3 ) 3 ·6H 2 O is dissolved in Tris-HCl aqueous solution with pH value of 9.0 to prepare rare earth ion solution with total concentration of rare earth ions of 4 mM;
(2) ATP is weighed and dissolved in Tris-HCl aqueous solution to prepare ATP solution with the concentration of 2mM;
(3) Taking 1mL of the rare earth ion solution in the step (1), uniformly mixing, dripping into 1mL of the ATP solution in the step (2), and stirring at room temperature for reacting for one minute.
The characterization result of the prepared probe (also called as a double-ion probe) is shown in fig. 2, wherein (a) and (b) are transmission electron microscope images of the double-ion probe; (c) is an XRD diffraction pattern of the dual ion probe; (d) X-ray photoelectron spectroscopy for a dual ion probe; (e) is the infrared absorption spectrum of the double ion probe. From the results, it can be seen that the prepared ATP-Ce/Tb double ion probe has a size of 20-50nm and is in an amorphous state (amorphous state).
Control preparation example: synthesis of rare earth metal-based enzyme-responsive probes (single-ion probes)
Preparation of ATP-Ce single ion probe
(1) Preparation of Ce (NO) at a concentration of 4mM 3 ) 3 ·6H 2 O rare earth ion solution (Tris-HCl, ph=9.0);
(2) ATP is weighed and dissolved in Tris-HCl aqueous solution to prepare ATP solution with the concentration of 2mM;
(3) Slowly dripping 2mL of the rare earth ion solution in the step (1) into 1mL of the ATP solution in the step (2), and stirring at room temperature for reacting for one minute;
(4) Centrifugal cleaning, dispersing the precipitate in ultrapure water again, and preserving at low temperature for standby.
Preparation of ATP-Tb single ion probe
(1) Preparation of Tb (NO) at a concentration of 4mM 3 ) 3 ·6H 2 O rare earth ion solution (Tris-HCl, ph=9.0);
(2) ATP is weighed and dissolved in Tris-HCl aqueous solution to prepare ATP solution with the concentration of 2mM;
(3) Slowly dripping 2mL of the rare earth ion solution in the step (1) into 1mL of the ATP solution in the step (2), and stirring at room temperature for reacting for one minute;
(4) Centrifugal cleaning, dispersing the precipitate in ultrapure water again, and preserving at low temperature for standby.
FIG. 6 shows the fluorescence intensity contrast of a dual ion probe versus a single ion probe. From this figure it is evident that the fluorescence intensity of the ATP-Ce/Tb probe is significantly enhanced compared to that of the single ion probes (ATP-Ce and ATP-Tb). The single ion luminescence is weak, and the double ion system greatly improves the luminescence intensity by using sensitization, so that the double ion system has higher detection sensitivity.
Example 1: detection of alkaline phosphatase concentration
(1) Using polystyrene 96-well plate as carrier, adding 7 rows of 100 μL probe solution prepared in preparation example 1 into the prepared microwells, wherein each row has concentration of 32, 16, 8, 4, 2, 1, 0.5mM, and each row sequentially adds 100 μL alkaline phosphatase (ALP) water solution with different concentration, and the concentration is 3000, 2500, 2000, 1500, 1250, 1000, 750, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 0U/L; placing the mixture at a constant temperature of 37 ℃ for shaking for 3 hours, respectively measuring the fluorescence intensity of 7 rows and 15 columns of mixed solutions, and calculating the fluorescence quenching efficiency of each group of mixed solutions; the probe concentration corresponding to the mixed solution was 4mM when the fluorescence quenching efficiency was maximized, and Tb was measured by the concentration of alkaline phosphatase in the mixed solution 3+ The concentration-dependent curve of alkaline phosphatase can be obtained by plotting the fluorescence intensity of (a) as shown in FIG. 3.
FIG. 3a shows that the detection system established by the invention has response to alkaline phosphatase in a certain concentration range, and the higher the concentration of alkaline phosphatase is, the weaker the fluorescence intensity of the corresponding mixed liquor is. FIG. 3b shows that the fluorescence intensity shows a good linear relationship with the concentration of alkaline phosphatase in a certain concentration range.
The results of FIGS. 3a and 3b show that the detection method of the present embodiment can realize the detection of the alkaline phosphatase concentration.
Example 2: assay specificity verification for alkaline phosphatase to be assayed
(1) Reagents, instruments, probes required for the experiment were as in example 1.
(2) The experiment selects common interferents in blood: arginine, glycine, biotin, citric acid, lactose, bovine Serum Albumin (BSA), glucose Oxidase (GOD), cholinesterase, metal ion (K) + 、Ca 2+ 、Cl - 、Na + 、Mg 2+ )。
(3) After the detection well plates were set in groups in the 96 well plates, 100. Mu.L of the above-mentioned aqueous solution of the interfering substance at a concentration of 100mg/mL and 100. Mu.L of the aqueous solution of alkaline phosphatase at a concentration of 100U/L were added to 100. Mu.L of Tris-HCl buffer solution of pH=9.0 containing a probe (final concentration of 4 mM), the 96 well plates were subjected to a constant temperature shaking reaction at 37℃for 3 hours, and the fluorescence intensities of the respective groups of mixed solutions were measured in an enzyme-labeled instrument, with the corresponding fluorescence intensity values shown in FIG. 4.
As can be seen from the bar graph of FIG. 4, only alkaline phosphatase is capable of significantly quenching fluorescence, while the influence of the rest of interferents on fluorescence intensity is insignificant, indicating that the probe has good specificity for alkaline phosphatase detection, and the influence of the interferents can be avoided in practical detection.
Example 3: investigation of alkaline phosphatase recovery in Complex System samples
1. The 96-well plate settings and instruments required for the experiment were the same as in example 1, and the probe solutions required for the experiment were the same as in example 1.
2. The model matrix for complex system detection is a serum and whole blood sample of a healthy person.
For the detection of alkaline phosphatase concentration in serum or whole blood samples, the feasibility of the alkaline phosphatase concentration is verified by using a labeling recovery rate experiment, and the specific operation steps are as follows:
taking two healthy human serum samples and one healthy human whole blood sample (in which alkaline phosphatase has been inactivated) subjected to protein inactivation by high temperature treatment, diluting both the serum samples and the whole blood sample 50 times with Tris-HCl buffer solution (ph=9.0), and adding a certain amount of alkaline phosphatase to each as a matrix; to the preset microwells, 100. Mu.L of a probe solution having a concentration of 4mM was added, 50. Mu.L of an alkaline phosphatase solution diluted with a serum sample and a whole blood sample was added, and then the solution to be tested was fixed to a volume of 200. Mu.L with a Tris-HCl buffer solution (pH=9.0). The concentration of the probe in each of the mixed solutions was 2mM, and the alkaline phosphatase concentrations were 50U/L, 100U/L and 200U/L, respectively. After the reaction is carried out for 3 hours at the constant temperature of 37 ℃, the mixture is placed in an enzyme-labeled instrument to measure the fluorescence intensity of the mixed liquid at 550nm, and then the standard curve is substituted to calculate the alkaline phosphatase content after the serum and the whole blood sample are added, and the sample adding recovery rate is calculated. The specific results are shown in Table 1 in detail, and the results show that the sample recovery rate values of the serum sample and the whole blood sample are in a reasonable range, so that the detection system of the embodiment has good precision and reproducibility.
TABLE 1 detection results of alkaline phosphatase in example 3

Claims (11)

1. Use of a rare earth metal-based enzyme-responsive probe for detecting alkaline phosphatase, the probe comprising a rare earth metal ion group and a phospholipid molecule;
the rare earth metal ion group is a cerium-terbium ion group;
the phospholipid molecule is a molecule containing a phospholipid bond, and the molecule containing a phospholipid bond is one, two or more of Adenosine Triphosphate (ATP), adenosine Diphosphate (ADP), adenosine Monophosphate (AMP), guanosine Triphosphate (GTP), guanosine Diphosphate (GDP) and Guanosine Monophosphate (GMP);
the use is not for the diagnosis and/or treatment of diseases.
2. The use according to claim 1, wherein the method for preparing the enzyme-responsive probe comprises:
1) Dissolving a combination of rare earth metal salts in a buffer solution to prepare a rare earth ion solution;
2) Dissolving phospholipid molecules in a buffer solution to prepare a phospholipid molecule solution;
3) Mixing the rare earth ion solution in the step 1) with the phospholipid molecule solution in the step (2) to obtain the rare earth metal-based enzyme response probe.
3. Use according to claim 2, characterized in that in step 1) the rare earth metal salt is a nitrate, chloride, acetate, sulfate, perchlorate or its salt hydrate of cerium, terbium.
4. The use according to claim 2, wherein in step 1) the total concentration of the rare earth ion solution is between 0.5 and 32 and mM.
5. The use according to claim 2, wherein in step 1), when the rare earth metal-based enzyme-responsive probe employs an ion set of two rare earth ions, the molar ratio of the two rare earth ions in the rare earth ion solution is 1:10-10:1.
6. The use according to claim 2, wherein in step 2) the buffer solution is N-methyl-D-glucamine (MEG) buffer, 2-amino-2-methyl-1-propanol (AMP) buffer, tris-hydroxymethyl aminomethane-hydrochloric acid (Tris-HCl) buffer; the pH of the buffer is 7.0-9.0.
7. The use according to claim 2, wherein in step 2) the concentration of the phospholipid molecule solution is between 0.5 and 32 and mM.
8. The use according to claim 2, wherein the solvent used for the preparation of the solution in step 1) and step 2) is water.
9. The use according to claim 2, characterized in that in step 3) the rare earth ion solution is mixed with the phospholipid molecule solution in a volume ratio of 1:5-5:1.
10. A method for detecting alkaline phosphatase, comprising the steps of:
1) Mixing the probe in the use according to any one of claims 1-9 with a buffer to obtain a probe solution;
2) Mixing alkaline phosphatase with a buffer solution to obtain an alkaline phosphatase solution;
3) Mixing the probe solution in the step 1) with the alkaline phosphatase solution in the step 2), oscillating at constant temperature, measuring the fluorescence intensity of the alkaline phosphatase in the sample to be measured, and calculating the concentration of the alkaline phosphatase in the sample to be measured according to a standard curve;
the detection method is not aimed at diagnosis and/or treatment of the disease.
11. The method according to claim 10, wherein the temperature of the constant temperature oscillation is 25-40 ℃;
and/or, in step 1), the concentration of the probe solution is selected from 0.5-32mM;
and/or, in the step 2), the concentration of the alkaline phosphatase solution is 0-5000U/L.
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