CN115326770A - Time-resolved fluorescent rare earth probe with renal clearance function and preparation method thereof - Google Patents
Time-resolved fluorescent rare earth probe with renal clearance function and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a renal-clearing time-resolved fluorescence rare earth probe and a preparation method thereof. The rare earth ion complex has long-life fluorescence, can overcome biological autofluorescence background interference by using a time resolution technology, and can realize early diagnosis and development monitoring of acute kidney injury of a living body by evaluating the kidney clearing condition through urine fluorescence analysis based on the characteristics of high-sensitivity time resolution fluorescence and kidney clearing; in addition, the rare earth ion complex can be embedded in a degradable silicon sphere to construct an activatable intelligent nano probe, and the activatable intelligent nano probe releases the kidney-clearing rare earth ion complex under the specific activation of active oxygen, so that the high-sensitivity urine diagnosis of living liver injury is realized. The rare earth fluorescent probe has the advantages of no background fluorescence, high sensitivity, good biocompatibility and the like, and can analyze in-vivo disease information in real time through in-vitro urine detection.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to preparation of a kidney-clearing rare earth complex probe and application thereof in diagnosis of living diseases.
Background
Organ damage caused by drugs is an important problem in current clinical disease treatment and drug use. For example, acute Kidney Injury (AKI) and pharmaceutical liver injury (DILI) have high morbidity and mortality. Currently, clinical diagnostic methods for AKI and DILI rely primarily on serological indicators, such as measurement of serum creatinine and blood urea nitrogen for diagnosis of levels of AKI, glutamic-pyruvic transaminase, etc. for diagnosis of DILI. However, this serological method is generally insensitive and cannot achieve early and accurate diagnosis of organ damage and monitoring of disease progression. Inulin and inulin have been developed in recent years, since Glomerular Filtration Rate (GFR) directly reflects the impaired status of glomeruli and the impaired level of overall renal function 99m Tc-DTPA and other markers are used to assess GFR and renal injury diagnosis. However, this method has the disadvantages of relatively complicated evaluation process, low sensitivity, high cost, and radioactivity. Although some current studies utilize in vivo fluorescence imaging to realize early diagnosis of liver injury in mice, in vivo optical imaging still faces the problems of limited penetration depth, large interference and the like. Therefore, it is of great practical significance to develop new methods for early diagnosis of organ injury in vivo with high sensitivity and specificity.
Rare earth ion (Ln) 3+ ) Due to its unique electronic configuration, it has excellent luminescence properties, such as narrow emission peak, high quantum yield, large Stokes shift, long fluorescence lifetime, and the like. Especially Eu 3+ ，Tb 3+ ，Sm 3+ ，Dy 3+ The fluorescence lifetime of the chelate can be from tens of nanoseconds to milliseconds, which is far more than other nonspecific fluorescence background (nanosecond fluorescence lifetime), and the rare earth ion complex can be effectively cleared by the kidney. Currently, time-resolved fluoroimmunoassay (TRFIA) based on rare earth complexesCompared with other immunoassay methods, including Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), the technology overcomes the defects of radioactive pollution, unstable enzyme substrate, low sensitivity and the like. Especially the fluorescence immunoassay method (DELFIA) of the dissolution enhanced rare earth compound, is recognized as one of the most sensitive commercial bioassay methods. DELFIA can completely eliminate background noise of scattered light and other short-lived biological auto-fluorescence using time-resolved (TR) techniques, thereby providing higher sensitivity than conventional steady-state fluorescence immunoassay. Therefore, the time-resolved fluorescent rare earth probe with the renal function being removable is constructed, high-sensitivity early diagnosis and development monitoring of in-vivo serious diseases such as organ injury are realized through in-vitro urine analysis, and the method has wide application prospects in the fields of clinical disease diagnosis, precise medical research and the like.
Disclosure of Invention
The invention aims to provide a preparation method of a time-resolved fluorescence rare earth probe which is simple to prepare, good in water solubility, high in biological safety and high in kidney clearing efficiency, can be used for in-vitro optical urinalysis and an application of the time-resolved fluorescence rare earth probe in the aspect of in-vivo disease diagnosis.
In order to realize the purpose, the following technical scheme is adopted:
the invention provides a preparation method of a renal-removable time-resolved fluorescence rare earth probe, which comprises a rare earth complex probe formed by rare earth ions and ligands with long-life fluorescence life characteristics and a rare earth complex biomarker activatable intelligent nanoprobe prepared on the basis of the rare earth complex probe.
(1) The preparation method of the rare earth complex probe comprises the following steps:
s1, adding a ligand into deionized water, heating and stirring;
and S2, dissolving a rare earth salt compound in deionized water, slowly adding a rare earth salt aqueous solution into the suspension obtained in the step S1 according to a certain proportion, continuously heating and stirring until the mixed solution is clear and transparent, and adjusting the pH value with a NaOH solution to obtain the rare earth complex probe.
In step S1, the ligand used may be one or more of diethyltriaminepentaacetic acid (DTPA), 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), 1,4, 7-triazacyclononane-N, N', N "-triacetic acid (NOTA), preferably DTPA; in the step S1, in order to fully mix the ligand and be more beneficial to the subsequent reaction, the ligand is heated in a water bath at 50 ℃ and is kept warm for 20 minutes.
In step S2, the rare earth salt is rare earth ion Eu with long-life fluorescence lifetime characteristic 3+ 、Tb 3+ 、Sm 3+ And Dy 3+ Acetate, hydrochloride, sulfate or nitrate, preferably nitrate, such as Eu (NO) 3 ) 3 ，Tb(NO 3 ) 3 ，Sm(NO 3 ) 3 ，Dy(NO 3 ) 3 One or more of; the molar ratio of the ligand to the rare earth ions is 1 (1-1.2), preferably 1.
In the step S2, heating and stirring in a water bath at 50 ℃ until the solution is clear and transparent so as to complete the reaction; the concentration of NaOH solution used was 0.4M and the pH adjusted to 6.8-7.2 to stabilize the product.
(2) The preparation method of the rare earth complex biomarker activatable intelligent nanoprobe comprises the following steps:
s1, adding the rare earth complex probe into a mixed solution of ethanol and strong ammonia water, and stirring to precipitate the rare earth complex probe;
s2, uniformly mixing Tetraethoxysilane (TEOS) and bis- [3- (triethoxysilyl) propyl ] -tetrasulfide BTEPDS (CAS No. 40372-72-3) in proportion, slowly dripping the mixture into the suspension obtained in the S1, stirring for 12-24 h at room temperature, and carrying out centrifugal washing to obtain white precipitate;
and S3, re-dispersing the white precipitate obtained in the step S2 in deionized water, adding the white precipitate into a distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) solution, stirring overnight, and centrifuging and washing to obtain the rare earth complex biomarker activatable intelligent nanoprobe.
In step S1, a rare earth complex probe is usedPreferably Eu-DTPA, in particular,adding 500 μ L of 40mM Eu-DTPA into a mixed solution composed of 600 μ L of 30wt% ammonia water and 12ml of anhydrous ethanol, and stirring for 20min to fully precipitate;
in the step S2, the volumes of TEOS and BTEPDS are respectively 360 mu L and 240 mu L, the TEOS and the BTEPDS are uniformly mixed, slowly added into the suspension obtained in the step S1 in a dropwise manner at the speed of 30 mu L/min, stirred and reacted for 12-24 h at room temperature, and centrifuged to obtain a precipitate, and the obtained precipitate is washed by ethanol for 1 time and then by water for 3 times to obtain a white precipitate.
In step S3, the white precipitate obtained in step S2 is re-dispersed in deionized water, added into DSPE-PEG2000 solution with the concentration of 10mg/ml, stirred overnight, and centrifuged and washed to obtain the activatable intelligent nano-probe marked by the rare earth complex biomarker, which is marked as Eu-DTPA @ SiO 2 /DSPE-PEG2000。
The obtained rare earth complex probe has good water solubility and high safety, and can be effectively removed by the kidney and discharged with urine; under the action of the antenna ligand, fluorescence can be emitted, and the fluorescence intensity of the fluorescence has correlation with the content of the rare earth probe in urine; in addition, the probe can be embedded in a degradable silicon sphere to construct an activatable intelligent nano probe, and the activatable intelligent nano probe releases a renal clearance rare earth complex under the specific activation of active oxygen, so that the high-sensitivity urine diagnosis of living liver injury is realized.
The second purpose of the invention is to provide the application of the time-resolved fluorescent rare earth probe with renal clearance in diagnosing drug-induced acute renal injury and liver injury. In a particular embodiment, the drug-induced acute kidney injury is cisplatin-induced acute kidney injury and the liver injury is acetaminophen-induced drug-induced liver injury.
The invention has the advantages that:
(1) The preparation steps of the fluorescent probe are environment-friendly and simple, the water solubility is good, the biological safety is high, and the kidney clearing efficiency is high;
(2) The prepared fluorescent probe can be discharged with urine through renal clearance and can act with rare earth dissolved fluorescence enhancement liquid, so that a fluorescence signal is amplified by million times, and the clearance rate of the probe is quantified through a time-resolved fluorescence analysis technology, so that early diagnosis and development monitoring of living organ diseases are realized.
Drawings
FIG. 1 is an infrared spectrum of a fluorescent probe Eu-DTPA of a renal clearance rare earth complex prepared in example 1 of the present invention.
FIG. 2 shows the excitation spectrum and fluorescence emission spectrum of the renal clearance rare earth complex fluorescent probe Eu-DTPA in example 1 of the present invention.
FIG. 3 is a fluorescence plot of urine collected at various time points under 365nm UV lamp excitation in example 3 of the present invention.
FIG. 4 is a graph of steady state and time resolved fluorescence spectra of probe Eu-DTPA in urine.
FIG. 5 is a graph of Eu-DTPA time-resolved fluorescence intensity in urine as a function of concentration.
FIG. 6 is a fluorescence image of urine under 365nm UV light at different time points of a control group of healthy mice and an experimental group of kidney injury mice collected in example 7 of the present invention.
FIG. 7 is a histogram of Eu-DTPA time-resolved fluorescence intensity in urine of healthy mice in the control group and kidney-injured mice in the experimental group collected at different time points in example 7 of the present invention.
FIG. 8 is Eu-DTPA @ SiO obtained in example 8 of the present invention 2 Topographic map of/DSPE-PEG 2000 (upper right corner) and X-ray spectral analysis.
FIG. 9 shows Eu-DTPA @ SiO in example 9 of the present invention 2 DSPE-PEG2000 vs. ONOO - The responsiveness of (1).
FIG. 10 is a histogram of Eu-DTPA time-resolved fluorescence intensity in urine collected 6h after injection of the probe in example 10 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples. However, those skilled in the art will appreciate that the scope of the present invention is not limited to the following examples. In light of the present disclosure, those skilled in the art will recognize that many variations and modifications may be made to the embodiments described above without departing from the spirit and scope of the present invention.
Example 1: and (3) preparing a kidney-clearing rare earth complex fluorescent probe Eu-DTPA. The method comprises the following specific steps:
weighing diethyl triaminePutting pentaacetic acid (DTPA) into a single-neck round-bottom flask, dispersing in 5ml of deionized water, heating in a water bath at 50 ℃ and stirring for 20min; then weighing rare earth nitric acid compound selected from Eu (NO) 3 ) 3 ·6H 2 Dissolving the rare earth ion aqueous solution by using 500 mu L of deionized water, gradually and slowly dripping the rare earth ion aqueous solution into the DTPA mixed solution (the molar ratio of DTPA to Eu is 1; adjusting the pH value to 6.8-7.2 with 0.4M NaOH solution, and freeze-drying the solution to obtain corresponding white solid powder. The infrared spectrum is shown in FIG. 1 (see FIG. 1).
Example 2: the Eu-DTPA prepared in example 1 was subjected to optical property testing. The method comprises the following specific steps:
the Eu-DTPA powder prepared in example 1 was dissolved in an aqueous solution to give a final concentration of 1mM. A volume of Eu-DTPA solution was added to a volume of fluorescence enhancing solution (15. Mu.M. Beta. -NTA, 50. Mu.M TOPO,1% glacial acetic acid, 0.1% Triton-X100) at a ratio of Eu-DTPA aqueous solution to fluorescence enhancing solution of 1. The optical property of the rare earth complex fluorescent probe Eu-DTPA is measured, the rare earth complex fluorescent probe Eu-DTPA has strong absorption in a range of 340-360 nm in fluorescence enhancement solution, and a fluorescence absorption peak at 614 +/-5 nm can be observed by further exciting with exciting light at 340nm (see figure 2).
Example 3: renal clearance analysis was performed on Eu-DTPA prepared in example 1. The method comprises the following specific steps:
the Eu-DTPA powder prepared in example 1 was dissolved in an aqueous solution to give a final concentration of 1mM. 200 μ LEu-DTPA solution was injected into mice tail vein, urine was collected at different time points and urine volume was recorded. A volume of the collected urine at different time points and a volume of fluorescence enhancing fluid (15. Mu.M. Beta. -NTA, 50. Mu.M TOPO,1% glacial acetic acid, 0.1% Triton-X100) were added to a 96-well plate at a volume ratio of urine to fluorescence enhancing fluid of 1. The fluorescence of Eu-DTPA in urine can be observed at each time point by irradiating with 365nm ultraviolet lamp. The rare earth complex fluorescent probe Eu-DTPA can observe obvious fluorescence 1-2 h after intravenous injection, and almost no fluorescence can be observed after 4h (see figure 3). The phenomenon proves that the rare earth complex fluorescent probe Eu-DTPA provided by the invention can be effectively cleared by the kidney in a short time, and the clearing condition of the probe can be observed conveniently and intuitively through naked eyes.
Example 4: the urine collected in example 3 was subjected to steady state and time resolved fluorescence tests. The method comprises the following specific steps:
mu.L of the urine collected in 1 hour in example 3 was collected, 190. Mu.L of fluorescence enhancing solution (15. Mu.M. Beta. -NTA, 50. Mu.M OPO,1% glacial acetic acid, 0.1% Triton-X100) was added to the 96-well plate, and incubated at room temperature for 30min. Placing the pore plate on an enzyme labeling instrument, setting the excitation wavelength to be 340nm and the delay time to be 0 mu s, and scanning a fluorescence signal within the range of 450-725 nm; further, setting the excitation wavelength to be 340nm and the delay time to be 100 mus, and scanning the fluorescence signal within the range of 450-725 nm to obtain the steady-state and time-resolved fluorescence spectrum of the rare earth complex fluorescent probe Eu-DTPA in the urine (see figure 4). The phenomenon can prove that the detection method provided by the invention can effectively shield the background fluorescence of urine, thereby improving the detection sensitivity.
Example 5: and quantifying the Eu-DTPA fluorescent probe in the urine by a time-resolved fluoroimmunoassay method. The method comprises the following specific steps:
preparing a series of Eu-DTPA aqueous solutions with different concentrations, and mixing the Eu-DTPA aqueous solutions with different concentrations according to the Eu-DTPA aqueous solution/blank urine =1/9 volume ratio to obtain a series of Eu-DTPA solutions with different concentrations in urine, so as to simulate a urine sample containing Eu-DTPA discharged in vivo. The samples and the enhancing solution were incubated in a 96-well plate for 30min at a sample/enhancing solution =1/9 volume ratio, to obtain a series of standard samples (wherein the content of Eu-DTPA is 0.005,0.01,0.02,0.04,0.08,0.2,0.4,0.8,4,8, 20 μ M in this order). And (3) placing the ELISA plate containing the standard sample on an ELISA reader, setting the excitation wavelength to be 340nm and the delay time to be 100 mu s, and measuring the fluorescence intensity of the emission wavelength to be 614nm to obtain a Eu-DTPA concentration-fluorescence intensity diagram in urine (see figure 5). And the Eu-DTPA fluorescent probe contained in the urine can be quantitatively analyzed according to the concentration-fluorescence standard curve.
Example 6: establishing a mouse drug-induced acute kidney injury model. The method comprises the following specific steps:
BALB/C nude mice were selected and randomly divided into two groups:
(1) Blank group (n = 3): gavage was continued for two weeks with 0.9% saline (0.1 mL/10 g) and a single injection of saline (0.1 mL/10 g) was given intraperitoneally on day 15.
(2) Experimental group (n = 3): continuous gavage with 0.9% saline (0.1 mL/10 g) was performed for two weeks with a single intraperitoneal injection of cisplatin (20 mg/kg) on day 15.
Example 7: in vitro urinalysis to assess drug-induced acute kidney injury. The method comprises the following specific steps:
the Eu-DTPA powder prepared in example 1 was dissolved in an aqueous solution to give a final concentration of 1mM. 200 mu LEu-DTPA aqueous solution was injected into the two groups of mice of example 6 separately in the tail vein, and urine was collected from the blank group of healthy mice and the experimental group of acute kidney injury mice at different time points and the urine volume was recorded. A volume of the collected urine at different time points and a volume of fluorescence enhancing fluid (15. Mu.M. Beta. -NTA, 50. Mu.M TOPO,1% glacial acetic acid, 0.1% Triton-X100) were added to a 96-well plate at a volume ratio of urine to fluorescence enhancing fluid of 1. The fluorescence of the Eu-DTPA probe in urine and the trend thereof were observed at each time point by irradiation with a 365nm UV lamp (see FIG. 6). It can be seen that a large amount of Eu-DTPA probes in the bodies of the healthy mice in the blank group are discharged within a time period of 1-2 hours, and almost discharged after 4 hours; and strong fluorescence can still be observed in the mice with the kidney injury in the experimental group after the mice lasts for 24 hours, which indicates that Eu-DTPA probe still remains in vivo and indicates that the kidney function is injured. Further, an elisa plate containing two groups of mouse urine samples is placed on an elisa instrument, the excitation wavelength is set to be 340nm, the delay time is 100 mus, the fluorescence intensity of the emission wavelength at 614nm is measured, the change of the urine fluorescence intensity of the two groups of mice with time at different time points is obtained (refer to fig. 7), and the content of the Eu-DTPA fluorescent probe in the urine at different time points can be estimated by combining the concentration-fluorescence standard curve of the example 5, so that the kidney clearing capacity of the mice is estimated.
Example 8: eu-DTPA @ SiO 2 Preparation of/DSPE-PEG 2000. The method comprises the following specific steps:
adding 500 μ L of 40mM Eu-DTPA into a mixture of 12ml ethanol and 600 μ L of 30wt% ammonia water, and stirring at room temperature for 20min to precipitate completely; mixing 360 μ L TEOS and 240 μ L BTEPDS, slowly adding dropwise into the mixture at 30 μ L/min, stirring at room temperature for 24 hr, centrifuging to obtain white precipitate, washing with ethanol for 1 time, washing with water for 3 times, dispersing the precipitate in 10mL deionized water, adding into 10mL10 mg/mL DSPE-PEG2000 solution, stirring overnight, centrifuging and washing for 2 times to obtain Eu-DTPA @ SiO 2 2 The morphology and X-ray energy spectrum analysis (EDS) of the/DSPE-PEG 2000 are shown in FIG. 8 (see FIG. 8), the particle size is about 180nm as seen in TEM inset, and the Eu loading ratio determined by EDS analysis is 0.18% (w/w).
Example 9: eu-DTPA @ SiO 2 The responsiveness of/DSPE-PEG 2000 to active oxygen is verified. The method comprises the following specific steps:
1mL of Eu-DTPA @ SiO prepared in example 8 was taken 2 the/DSPE-PEG 2000 was mixed with 0. Mu.M and 20. Mu.M ONOO, respectively - After incubation at 37 ℃ for 4h, the supernatant was centrifuged. A volume of supernatant and a volume of fluorescence enhancing fluid (15. Mu.M. Beta. -NTA, 50. Mu.M TOPO,1% glacial acetic acid, 0.1% Triton-X100) were taken, and the supernatant and fluorescence enhancing fluid were added together at a volume ratio of 1:19 to a 96-well plate, and incubated at room temperature for 30min. Placing the microplate containing two groups of samples on a microplate reader, setting excitation wavelength at 340nm and delay time at 100 mus, and measuring fluorescence intensity of emission wavelength at 614nm (refer to FIG. 9), thereby showing that the rare earth complex biomarker can be activated by using the intelligent nanoprobe Eu-DTPA @ SiO 2 the/DSPE-PEG 2000 has better response performance to active oxygen.
Example 10: in vitro urinalysis to assess drug-induced liver injury. The method comprises the following specific steps:
s1, liver injury model construction
BALB/C nude mice were selected and randomly divided into two groups:
(1) Blank group (n = 3): after grain breaking for 12h, injecting normal saline (0.1 mL/10 g) into the abdominal cavity;
(2) Experimental group (n = 3): after the diet is cut off for 12 hours, paracetamol (500 mg/kg) is injected into the abdominal cavity.
S2, evaluating drug-induced liver injury by in-vitro urine analysis
200 μ L of Eu-DTPA @ SiO prepared in example 8 was taken 2 the/DSPE-PEG 2000 tail vein injection is carried out in the two groups of mice, the urine of a blank group of healthy mice and an experimental group of liver injury mice is collected after 6 hours, and the urine volume is recorded. A volume of the collected urine and a volume of fluorescence enhancing fluid (15. Mu.M. Beta. -NTA, 50. Mu.M TOPO,1% glacial acetic acid, 0.1% Triton-X100) were added to a 96-well plate at a volume ratio of urine to fluorescence enhancing fluid of 1.19, and incubated at room temperature for 30min. Placing an ELISA plate containing two groups of mouse urine samples on an ELISA reader, setting excitation wavelength at 340nm and delay time at 100 mus, and measuring fluorescence intensity of emission wavelength at 614nm to obtain time-resolved fluorescence intensity of the two groups of mouse urine (refer to FIG. 10). It can be seen that the fluorescence intensity of the urine in the liver injury group is obviously higher than that of the healthy control group, so that the level of active oxygen in the liver can be indirectly evaluated by comparing the fluorescence intensity of the urine, and the purpose of diagnosing the liver injury is further achieved.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (9)
1. The time-resolved fluorescent rare earth probe with the renal clearance function is characterized by comprising a rare earth complex probe formed by rare earth ions and ligands, wherein the rare earth complex probe has long-life fluorescence life characteristics;
the preparation method of the rare earth complex probe comprises the following steps:
s1, adding a ligand into deionized water, heating and stirring;
and S2, dissolving a rare earth salt compound in deionized water, slowly adding a rare earth salt aqueous solution into the turbid liquid obtained in the step S1 according to a certain proportion, continuously heating and stirring until the mixed liquid is clear and transparent, and adjusting the pH value with a NaOH solution to obtain the rare earth complex probe.
2. The renally erasable time resolved fluorescent rare earth probe of claim 1, wherein in step S1 the ligand is one or more of diethyltriaminepentaacetic acid, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, 1,4, 7-triazacyclononane-N, N', N "-triacetic acid.
3. The renal-removable time-resolved fluorescent rare earth probe according to claim 1, wherein the heating temperature in step S1 is 50 ℃ and the incubation time is 20 minutes.
4. The renal cleanable time-resolved fluorescent rare earth probe according to claim 1, wherein in step S2, the rare earth salt compound is rare earth ion Eu having long-life fluorescence lifetime characteristics 3+ 、Tb 3+ 、Sm 3+ And Dy 3+ Acetate, hydrochloride, sulfate or nitrate of one or more of (a); the molar ratio of the ligand to the rare earth ion is 1 (1 to 1.2).
5. The renal cleanable time-resolved fluorescent rare earth probe according to claim 1, wherein the heating temperature in step S2 is 50 ℃; the concentration of the NaOH solution is 0.4M, and the pH value is adjusted to be 6.8-7.2, so that the product is stable.
6. The renal-removable time-resolved fluorogenic rare earth probe of claim 1, further comprising a rare earth complex biomarker activatable smart nanoprobe prepared based on the rare earth complex probe;
the preparation method of the rare earth complex biomarker activatable intelligent nanoprobe comprises the following steps:
(1) Adding the rare earth complex probe into a mixed solution of ethanol and concentrated ammonia water, and stirring to precipitate the rare earth complex probe;
(2) Uniformly mixing tetraethoxysilane TEOS and bis- [3- (triethoxysilyl) propyl ] -tetrasulfide BTEPDS in proportion, slowly dripping the mixture into the suspension obtained in the step (1), stirring for 12-24 h at room temperature, and centrifuging and washing to obtain white precipitate;
(3) And (3) re-dispersing the white precipitate obtained in the step (2) in deionized water, adding the deionized water into the DSPE-PEG2000 solution, stirring overnight, and centrifugally washing to obtain the rare earth complex biomarker activatable intelligent nanoprobe.
7. The renal-cleanable time-resolved fluorescent rare earth probe according to claim 6, wherein the operation of the step (1) is carried out by adding 500. Mu.L of 40mM rare earth complex probe to a mixture of 600. Mu.L of 30wt% aqueous ammonia and 12ml of anhydrous ethanol, and stirring for 20min to sufficiently precipitate the probe.
8. The time-resolved fluorescent rare-earth probe capable of being cleared by the kidney of claim 6, wherein the specific operation of the step (2) is to take 360 μ L of TEOS and 240 μ L of BTEPDS to be mixed uniformly, slowly drop-add the mixture into the suspension obtained in the step (1) at a speed of 30 μ L/min, stir and react for 12 to 24 hours at room temperature, centrifuge to obtain a precipitate, wash the precipitate with ethanol for 1 time, and wash the precipitate with water for 3 times to obtain a white precipitate.
9. The renal-removable time-resolved fluorescent rare-earth probe according to claim 6, wherein the step (3) is specifically performed by re-dispersing the white precipitate obtained in the step (2) in 10ml of deionized water, adding the deionized water to a DSPE-PEG2000 solution of 10mg/ml according to the volume of 1/1, stirring, after reacting overnight, centrifuging to remove the excessive DSPE-PEG2000, and centrifuging and washing with deionized water for 2 times to obtain the rare-earth complex biomarker activatable intelligent nanoprobe.
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