CN117233372A - AIE fluorescent probe, preparation method and application for simultaneously detecting two antigens of LNCaP cells - Google Patents
AIE fluorescent probe, preparation method and application for simultaneously detecting two antigens of LNCaP cells Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses an AIE fluorescent probe, a preparation method and application for simultaneously detecting two antigens of LNCaP cells, wherein the probe simultaneously analyzes and tracks the two antigens of PSA and PSMA, realizes fluorescence imaging and detection of prostate cancer cells, and overcomes the limitation of single type biomarker detection. The method of the invention can quantify the concentration of PSA and PSMA, the linear range of PSA is 0.0001-0.1 mug/mL (R 2 = 0.99153), the limit of detection (LOD) was 6.18pg/mL. The linear range of PSMA is 0.0001-0.1. Mu.g/mL (R 2 =0.99), the limit of detection (LOD) was 8.79pg/mL. The method can provide a new way for early screening and diagnosis of cancers.
Description
Technical Field
The invention belongs to the technical field of biological analytical chemistry, and relates to a specific probe for tumor detection, in particular to an AIE fluorescent probe, a preparation method and application of two antigens for simultaneously detecting LNCaP cells.
Background
Prostate Specific Antigen (PSA), a gold standard for diagnosing and prognosticating prostate cancer progression, is a specific glycoprotein secreted in human serum at normal levels below 4ng/mL -1 . Since the early 90 s of the 20 th century, the number of early diagnosed patients has increased dramatically since the screening for prostate cancer using PSA testing. However, the specific decrease in total PSA detection requires a large number of unnecessary biopsies and over-treatments, resulting in excessive detection of painless cancer. A long-term goal of prostate cancer detection is to develop screening methods with higher specificity, so that clinically significant and non-significant prostate cancers can be distinguished. Recently, there have been studies reporting platforms for PSA detection using fluorescence, photoelectrochemistry, surface enhanced raman scattering, enzyme linked immunoassay and electrochemical techniques. In the research process, people gradually recognize that the accuracy of a single PSA detection result is insufficient, and in order to improve the accuracy and the specificity of cancer detection, a plurality of markers are selected for simultaneous detection. In recent years, prostate Specific Membrane Antigen (PSMA), which is one of the markers of prostate cancer, has been a membrane protein that is more excellent than PSA. PSMA is not readily detected in the patient's blood and is highly expressed in most tumor cells. PSMA can be obtained with higher quality images due to lower background radioactivity by binding to a radiolabeled antibody ligand. Clinically, the radioimmunoassay is used for diagnosing the pathogenesis of the prostate cancer, and the discovery of the radioimmunoassay has important significance for early detection.
Fluorescent organic materials are widely used in biosensing, bioimaging, chemical sensors, and the like. However, conventional organic compounds have reduced luminous efficiency due to aggregation quenching (ACQ), and aggregation-induced emission (AIE) phenomenon has been contrary to its performance. Tetraphenyl ethylene (TPE) is one of the typical representatives of AIE because of its simple synthesis and easy functionalization. TPE has a structure in which 4 phenyl groups are attached to the double bond of the central olefin. When TPE molecules are dispersed in a nonpolar solvent, the phenyl group in the excited state becomes very active with the solvent interacting olefinic double bonds, resulting in non-radiative deactivation of the excited state molecules. Due to limited intramolecular rotation, free rotation of phenyl groups is hindered during aggregation, and the efficiency of non-radiative transition of the excited state energy is reduced, so that the excited state molecules must return to the ground state in a radiative transition mode, and fluorescence is remarkably enhanced. Compared with the traditional fluorescent probe, TPE has low background, no damage and stable fluorescent signal in situ, and becomes an important tool for biological detection and imaging
Aptamers and antibodies are of great interest as specific binders to antigens. Because the antibody is expensive and easy to degrade, the aptamer has the advantages of low price, easy synthesis, easy modification, high selectivity and stability, and replaces the antibody. In addition, the aptamer can easily modify functional groups, such as fluorophores, at the end. Fluorophores have advantages in detecting and imaging antigen, protein, and antibody specific binding. Therefore, aptamers are of great interest in bioanalytical detection.
Disclosure of Invention
The technical problems to be solved are as follows: in order to overcome the limitation of single type biomarker detection in the prior art, a double-functional aggregation-induced emission (AIE) fluorescent probe capable of simultaneously detecting two prostate cancer antigens is obtained, so that more accurate detection of prostate cancer is realized. In view of this, the invention provides AIE fluorescent probes, methods of preparation, and applications for simultaneous detection of two antigens of LNCaP cells.
The technical scheme is as follows: a method of preparing an AIE fluorescent probe, the method comprising the steps of:
s1, preparation of amino artificial cells
Liposome components 1, 2-distearoyl-SN-glycerol-3-phosphorylcholine (DSPC) and dipalmitoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG 2000-NH) 2 ) Mixing according to the mass ratio of 1:2-2:1, fully dissolving in chloroform, freeze-drying to remove the solvent, and then dissolving in BIn alcohol, in N 2 Extruding through a membrane filter under the pressure of gas;
s2, synthetic Artificial cell @ Prostate Specific Membrane Antigen (PSMA)
Adding carbodiimide (EDC) into the artificial cell solution prepared in the step S1, incubating in a water bath at 37 ℃, then adding N-hydroxysuccinimide (NHS), mixing, adding a buffer solution with pH of 7 to activate carboxyl, finally adding PSMA and carboxyl for coupling, and incubating overnight;
s3, synthesizing AIE material
With tetra-4-hydroxy-styrene (TPE- (OH) 4 ) Adding tetrahydrofuran as a solvent, potassium carbonate and ethyl bromoacetate into tetrahydrofuran according to a mass ratio of 3:4 to form a mixed solution, and carrying out reflux reaction on the monomer in the mixed solution to obtain an ester group through dehydrobromination; then adding tetrahydrofuran and water in a volume ratio of 1:1 under the alkaline condition of sodium hydroxide, refluxing, and hydrolyzing ester groups into carboxyl groups to obtain AIE material tetra-4-carboxystyrene (TPE- (COOH) 4 )；
S4, synthesizing dual-function AIE probe
Adding PSMAL and PSA-Aptamer into EDC/NHS solution, reacting at room temperature, removing unreacted substances by an ultrafiltration tube, adding AIE material prepared by S3, washing with pure water and PBS solution with pH of 7.4 after low-temperature reaction, removing unconjugated PSMAL and PSA-Aptamer to obtain a dual-function AIE probe, re-suspending the probe in the PBS solution, and storing at low temperature of 4 ℃.
Preferably, the molar ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) in S2 is 1:1 to 4:1, and the concentration of PSMA in the system is 0.0001 to 1. Mu.g/mL.
Preferably, the reflux reaction time for dehydrobromination to form ester groups in S3 is 24-72 hours, and the reflux reaction time for hydrolyzing ester groups to carboxyl groups is 3-8 hours.
Preferably, the concentration of PSMAL in the S4 system is 75-100 mug/mL, and the concentration of PSA-Aptamer in the system is 75-100 mug/mL; the concentration of the EDC/NHS solution is 5-10 mg/mL, wherein the mass ratio of EDC to NHS is 1:1; the low-temperature reaction is carried out for 6 to 10 hours at the temperature of 4 ℃; the concentration of the probe resuspended in PBS solution is 1-3 mg/mL.
The AIE fluorescent probe obtained by any one of the above methods.
The use of the AIE fluorescent probe described above for simultaneous detection of Prostate Specific Membrane Antigen (PSMA) and Prostate Specific Antigen (PSA).
Preferably, the AIE fluorescent probe has a limit of detection (LOD) for PSA of 6.18p g/mL and for PSMA of 8.79pg/mL.
Preferably, the detection comprises fluorescence detection and cell imaging analysis.
Preferably, the fluorescence detection method comprises the following steps: adding AIE fluorescent probes into PSMA and PSA, wherein the concentration of the AIE fluorescent probes is 10-30 mu M, and incubating in a water bath at 25 ℃ for 1-3 h; the quenching concentration ratio of PSA-Aptamer to DNA-BHQ2 is 1:2-2:1. Among them, sequence information of PSA-Aptamer and DNA-BHQ2 is shown in Table 1:
TABLE 1 nucleic acid strand according to the invention
Preferably, the method of cell imaging analysis is as follows: adding AIE fluorescent probes, PSA or PSMA into human prostate cancer cells (LNCaP), incubating in a water bath at 25 ℃, and observing specific fluorescent images of the two antigens by using an inverted fluorescent microscope; wherein the addition concentration of the PSA is 0.001-0.1 mug/mL, the PSMA emits red light, the addition concentration of the PSMA is 0.001-0.1 mug/mL, and the PSMA emits blue light; the concentration of AIE fluorescent probe is 1-10. Mu.M.
The principle of the method of the invention is as follows:
the AIE fluorescent probe is used as a substrate, and an aptamer PSA-Aptamer of a free antigen PSA and an antibody PSMAL of a membrane antigen PSMA are modified by EDC and NHS coupling agents. First, a fluorophore Cy3 is modified at the PSA-Aptamer end, and a quencher is modified at the complementary strand end, whereby fluorescence is quenched. In the presence of the target PSA, PSA-Aptamer competes for capture of the target and fluorescence is restored for quantitative detection of the PSA content.
PSMA was modified on the surface of artificial cells using the compound covalent-amide bond approach. Artificial cell-modified antigens were used to mimic prostate cancer cells.
Because the prostate cancer antigen is highly expressed in the prostate cancer, after the membrane surface antigen PSMA is specifically captured by PSMAL on the functionalized AIE probe, the AIE fluorescent probe is aggregated, so that the enhancement of the membrane surface fluorescent signal is realized. By simultaneous fluorescence analysis detection of different emission spectra Cy3 (480 nm) and AIE (570 nm), the content of two prostate cancer antigens can be detected simultaneously.
The beneficial effects are that: the method disclosed by the invention is based on the characteristics of easy modification and good biological imaging of the AIE material, can be used for detecting the prostate cancer markers, realizes fluorescence imaging and detection of prostate cancer cells, can realize high-sensitivity analysis, and accurately monitors the prostate cancer. The method can provide a new way for early screening and diagnosis of cancers.
Drawings
FIG. 1 is an SEM image of an aAIE material of example 1 at a scale of 10 μm; SEM images of the baiie material at 5 μm scale; the cAIE material had DMF/H at fw of 10% 2 SEM images formed in the O mixture; dAIE material with 90% of DMF/H in fw 2 DMF/H of SEM image formed in O mixture 2 Agglomerates formed in the O mixture;
FIG. 2 is a TEM image of (a) liposomes of example 1 (scale bar, 100 nm); (b) liposome TEM image (scale bar, 0.5 μm);
FIG. 3 is an inverted micrograph of fluorescence generated by liposomes and liposome modified PSMA of example 1 (inverted micrograph of fluorescence generated by liposomes and PSMA incubated with rhodamine B and indocyanine green dyes, respectively, and combination of both incubation);
FIG. 4 is a gel electrophoresis chart of the strategy of (a) in example 1, (b) a fluorescence spectrum chart of the capture of the target PSA by the PSA-aptamer (3 '-Cy 3) +PSA-cDNA (5' -BHQ 2);
FIG. 5 is a graph of the optimization of example 1 under different experimental conditions. (a) The concentration ratio of the modified BHQ2 DNA to Apatm-Cy 3 is from 1:3 to 3:1; (b) incubation time from 1h to 3h; (c) the concentration of AIE is from 5. Mu.M to 50. Mu.M;
FIG. 6 is a graph showing fluorescence spectra of (a) PSA assays of different concentrations in example 1; (b) a calibration plot and a linear range plot of fluorescence intensity RFU;
FIG. 7 is a graph showing fluorescence spectra of (a) PSMA at various concentrations in example 1; (b) calibration plot and linear range of fluorescence intensity RFU;
FIG. 8 is a graph showing the specificity of example 1 for various proteins;
FIG. 9 is a fluorescence micrograph of LNCaP cells incubated with AIE probe, PSA for 4h, in example 1;
FIG. 10 is a fluorescence micrograph of LNCaP cells incubated with AIE probe, PSMA for 4h in example 1;
FIG. 11 is a schematic representation of the aggregation-induced emission probe protocol for fluorescence detection and cell imaging of prostate cancer antigens of example 1 mechanism (a) synthetic bifunctional AIE probes; (b) the artificial cells mimic prostate cancer fluorescent detection antigen; (c) a cell imaging process.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1
The preparation method of the AIE fluorescent probe and the application of the AIE fluorescent probe in simultaneously detecting two antigens of LNCaP cells comprise the following steps:
(1) Synthesis of aminated artificial cells
Liposome preparation Liposome Components 1, 2-distearoyl-SN-glycero-3-phosphorylcholine (DSPC) and dipalmitoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG 2000-NH) 2 ) Mixing at equal mass ratio, dissolving in chloroform, and removing solvent with freeze dryer. Then dissolve it in ethanol solution at N 2 The mixture was extruded 4 times under gas pressure through a membrane filter (pore size 200 nm). At this stage, an aqueous solution of nonfunctionalized artificial cells was obtained. As shown in the TEM image of the liposome of FIG. 2, the liposome has a diameter of about 150nm and a uniform and stable size, indicating successful synthesis of the aminated artificial cell
(2) Synthetic Artificial cell @ PSMA
Carbodiimide (EDC) was added to the centrifuge tube containing the artificial cell solution, incubated in a 37℃water bath for 15min, and N-hydroxysuccinimide (NHS) was added to activate the carboxyl groups by mixing at a molar ratio of 4:1 and adding to a buffer solution at pH 7. Finally PSMA was added to the mixture, coupled to carboxyl groups and incubated overnight. As shown in FIG. 3, an image of the artificial cells was observed using an inverted fluorescence microscope (magnification 40X). The artificial cells were stained with rhodamine B dye, and then PSMA was stained with indocyanine green dye. In bright field, an image of the artificial cells can be observed, and red artificial cells can be observed under a green excitation fluorescent field, and green PSMA can also be observed under a blue excitation fluorescent field. In the pooled field, localized areas can be seen to turn yellow, indicating successful modification of PSMA onto artificial cell membranes, further confirming successful synthesis of artificial cell-modified membrane antigens.
(3) Synthetic AIE materials
Tetra-4-hydroxy-styrene (TPE- (OH) 4 ) The solvent is tetrahydrofuran, the mass ratio of potassium carbonate to ethyl bromoacetate is 3:4, the mixture is refluxed for 72 hours, and the dehydrobromination is carried out to form ester groups. Adding sodium hydroxide, refluxing under alkaline condition with tetrahydrofuran and water volume ratio of 1:1 for 4h, and hydrolyzing ester group into carboxyl to obtain final product tetra-4-carboxystyrene-ethylene (TPE- (COOH) 4 ). As shown in the Scanning Electron Microscope (SEM) images of different volume fractions fw=10% and 90% of water in DMF/water solution for the AIE material of fig. 1a, b and for the AIE material of fig. 1c, d. SEM images showed different aggregation forms of AIE, reflecting the activity of AIE. The AIE material is amorphous in the absence of dissolution and assumes an irregular sheet form. SEM images showed higher crystallinity when fw=0%, indicating good dissolution of AIE in DMF solution. In contrast, SEM images of the amorphous form showed poor solubility of AIE in fw=90% DMF/water, demonstrating that AIE exists in an aggregated state.
(4) Synthetic bifunctional AIE probes
First, PSMAL (75-100. Mu.g/mL) and PSA-Aptamer (75-100. Mu.g/mL) were added to EDC/NHS solution (mass ratio 1:1), reacted at room temperature for 30min, and unreacted materials were removed by ultrafiltration tube. AIE material was then added to the solution and reacted at 4℃for 8h, which was washed several times with pure water and PBS solution (pH 7.4), uncoupled PSMAL and PSA-Aptamer were removed, and the final product AIE probe was resuspended in PBS solution. The prepared capture probes and detection probes were stored at 4 ℃.
(5) Gel electrophoresis analysis
Agarose gel electrophoresis experiments verify whether aptamer capture of the target can proceed normally. The method comprises the following steps: first, all DNA probes (1-Complementary probe, 2-PSA-Aptamer, 3-Complementary probe +PSA-Aptamer) were first prepared as a solution. 1 and 2 were added to a centrifuge tube and reacted at 37℃for 2 hours to form Complementary probe + PSA-Aptamer. Agarose gel was prepared using 1×tae buffer and stain was added. The ratio of DNA loading buffer to reaction solution was 1:5, agarose gel was run at 85V for 50 minutes, and agarose gel was imaged with a gel imaging system. From the agarose gel electrophoresis chart of the reaction in FIG. 4 (a), it was evident that the fourth lane was the complex product of the second lane and the third lane by qualitative analysis of lanes 2 (Complementary probe), 3 (PSA-Aptamer) and 4 (Complementary probe +PSA-Aptamer) in agarose gel electrophoresis. In the fluorescence graph of fig. 4 (b), it can be seen that the quenched Cy3 aptamer fluorescence is significantly enhanced upon capture of the target antigen PSA, which also demonstrates the specific recognition of the aptamer with the target.
(6) Fluorescence detection procedure
And adding the prepared AIE probe into the PSA and PSMA, incubating for 3 hours at the water bath temperature of 25 ℃, and carrying out fluorescence detection. In order to reduce the detection limit and improve the sensitivity of the method for detecting the antigen, experimental conditions are optimized, and firstly, the quenching concentration ratio (PSa-Aptamer and DNA-BHQ 2) is optimized, wherein the PSa-Aptamer and the Aptag-Cy 3 have the same sequence and are different identifications of the same substance, and the PSa-Aptamer can be seen from fig. 5 (a): DNA-BHQ2 greater than 1:2, the fluorescence intensity reaches the plateau. Thus 2. Mu.M DNA-BHQ2 and 1. Mu.M PSA-Aptamer were chosen as optimal concentration ratios. Next, the quenching time was optimized as shown in FIG. 5 (b), 2. Mu.M DNA-BHQ2 and 1. Mu.M PSA-Aptamer were selected, incubated at 25℃in a water bath, and fluorescence was performed for 1 to 3 hoursAnd (5) detecting. With increasing time, the fluorescence intensity was lower and lower, indicating that the two strands were perfectly base-paired at 2.5 hours, with 2.5 hours being the optimal time. Finally, AIE concentration is optimized, which concentration affects sensitive detection of PSMA. If the concentration is too low, the effect of fluorescence enhancement cannot be obtained. If the concentration is too high, the background value is too high, which makes it impossible to quantitatively detect PSMA. As shown in FIG. 5 (c), the fluorescence intensity reached plateau at 10. Mu.M, with increasing concentration, and 10. Mu.M was the optimal AIE concentration. After the most favorable reaction conditions were determined, the sensitivity and selectivity of the analytical method were investigated. FIG. 6 (a) is a graph of fluorescence spectra of PSA assays at different concentrations, with a linear trend. From fig. 6 (b), it can be seen that the Relative Fluorescence Unit (RFU) value is positively correlated with the target PSA concentration. Has good linear dynamic range in the range of 0.0001-0.1 mug/mL, the regression equation is y=233.39563x+1216.8714, the regression coefficient (R 2 ) 0.99153. The detection Limit (LOD) calculated from the 3-fold background noise standard deviation divided by the slope was 6.18pg/mL. Fig. 7 (a) is a graph of fluorescence spectra of PSMA detection at different concentrations, with a certain linear trend. From FIG. 7 (b), it can be seen that the Relative Fluorescence Unit (RFU) value is positively correlated with the target PSMA concentration, showing good linearity over the range of 0.0001 to 0.1 μg/mL, with regression equation y=161.37593x+832.94092 (R) 2 =0.99). The LOD calculated from the 3-fold background noise standard deviation divided by the slope was 8.79pg/mL. Within this linear range, its Relative Standard Deviation (RSD) is less than 5%. As can be seen from FIG. 8, the fluorescence intensity is significantly lower than that of the target antigen, although the interfering protein concentration is as high as 100 times (50. Mu.g/mL) the PSA, PAMA concentration (0.5. Mu.g/mL). It can be seen that the ligands used have good specificity for the antigen.
(7) Cell imaging analysis procedure
To study the effect of PSA and PSMA on fluorescence imaging in prostate cancer cells (LNCaP cells), the effect of PSA (first biomarker) on LNCaP cells was studied by standard addition labeling. As shown in FIG. 9, varying concentrations of PSA were added to LNCaP cells, and recovery of fluorescence was achieved after capture of PSA after AIE probe-modified PSA-Aptamer had been quenched by a quencher. As the concentration of PSA increases, the red free fluorescence intensity of PSA-Aptamer increases, indicating that AIE probes can achieve specific fluorescence imaging of prostate cancer marker PSA. Meanwhile, in the same way, the effect of PSMA (second biomarker) on LNCaP cells was investigated. As shown in FIG. 10, various concentrations of PSMA were added to LNCaP cells, and AIE probe-modified PSMAL specifically captured PSMA, resulting in blue fluorescence, whose membrane surface fluorescence intensity increased with increasing PSMA content.
Claims (10)
- A method of preparing an aie fluorescent probe, the method comprising the steps of:s1, preparation of amino artificial cellsLiposome components 1, 2-distearoyl-SN-glycerol-3-phosphorylcholine (DSPC) and dipalmitoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG 2000-NH) 2 ) Mixing according to the mass ratio of 1:2-2:1, fully dissolving in chloroform, freeze-drying to remove the solvent, then dissolving in ethanol, and adding the mixture into N 2 Extruding through a membrane filter under the pressure of gas;s2, synthetic Artificial cell @ Prostate Specific Membrane Antigen (PSMA)Adding carbodiimide (EDC) into the artificial cell solution prepared in the step S1, incubating in a water bath at 37 ℃, then adding N-hydroxysuccinimide (NHS), mixing, adding a buffer solution with pH of 7 to activate carboxyl, finally adding PSMA and carboxyl for coupling, and incubating overnight;s3, synthesizing AIE materialWith tetra-4-hydroxy-styrene (TPE- (OH) 4 ) The method comprises the steps of taking tetrahydrofuran as a solvent, dissolving potassium carbonate and ethyl bromoacetate in the tetrahydrofuran according to a mass ratio of 3:4 to form a mixed solution, carrying out reflux reaction on the monomer in the mixed solution, and dehydrobrominating to form an ester group; then refluxing under alkaline conditions of sodium hydroxide, tetrahydrofuran and water, and hydrolyzing the ester group into carboxyl to obtain AIE material tetra-4-carboxystyrene (TPE- (COOH) 4 )；S4, synthesizing dual-function AIE probeAdding a Prostate Specific Membrane Antigen (PSMAL) and a prostate specific antigen aptamer (PSA-Aptamer) into EDC/NHS solution, reacting at room temperature, removing unreacted substances by an ultrafiltration tube, then adding an AIE material prepared by S3, washing with pure water and a PBS solution with pH of 7.4 after low-temperature reaction, removing unconjugated PSMAL and PSA-Aptamer to obtain a bifunctional AIE probe, re-suspending the probe in the PBS solution, and storing at a low temperature of 4 ℃; wherein the sequence of PSA-Aptamer is SEQ ID NO.1.
- 2. The method for preparing AIE fluorescent probe according to claim 1, wherein the molar ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) in S2 is 1:1-4:1, and the concentration of PSMA in the system is 0.0001-1. Mu.g/mL.
- 3. The method for preparing an AIE fluorescent probe according to claim 1, wherein the reflux reaction time for dehydrobromination to form an ester group in S3 is 24 to 72 hours, and the reflux reaction time for hydrolysis of the ester group to a carboxyl group is 3 to 8 hours.
- 4. The method for preparing AIE fluorescent probe according to claim 1, wherein the concentration of PSMAL in S4 is 75-100. Mu.g/mL, and the concentration of PSA-Aptamer in S4 is 75-100. Mu.g/mL; the concentration of the EDC/NHS solution is 5-10 mg/mL, wherein the mass ratio of EDC to NHS is 1:1; the low-temperature reaction is carried out for 6 to 10 hours at the temperature of 4 ℃; the concentration of the probe resuspended in PBS solution is 1-3 mg/mL.
- 5. An AIE fluorescent probe prepared by the method of any one of claims 1 to 4.
- 6. Use of the AIE fluorescent probe of claim 5 for simultaneous detection of Prostate Specific Membrane Antigen (PSMA) and Prostate Specific Antigen (PSA).
- 7. The use of claim 6, wherein the AIE fluorescent probe has a limit of detection (LOD) for PSA of 6.18p g/mL and PSMA of 8.79pg/mL.
- 8. The use of claim 6, wherein the detection comprises fluorescence detection and cell imaging analysis.
- 9. The use according to claim 8, wherein the fluorescence detection method is: adding AIE fluorescent probes into PSMA and PSA, wherein the concentration of the AIE fluorescent probes is 10-30 mu M, and incubating in a water bath at 25 ℃ for 1-3 h; the quenching concentration ratio of PSA-Aptamer to DNA-BHQ2 is 1:2-2:1; wherein the sequence of DNA-BHQ2 is SEQ ID NO.2.
- 10. The use according to claim 8, wherein the method of cell imaging analysis is: adding AIE fluorescent probes, PSA or PSMA into human prostate cancer cells (LNCaP), incubating in a water bath at 25 ℃, and observing specific fluorescent images of the two antigens by using an inverted fluorescent microscope; wherein the addition concentration of the PSA is 0.001-0.1 mug/mL, the PSMA emits red light, the addition concentration of the PSMA is 0.001-0.1 mug/mL, and the PSMA emits blue light; the concentration of AIE fluorescent probe is 1-10. Mu.M.
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