A fluorescent aptamer sensor and method for detecting prostate specific antigen

Abstract

The application discloses a fluorescent aptamer sensor and a method for detecting prostate specific antigen, wherein the fluorescent aptamer sensor comprises a solvent, and 5-carboxyltetramethylrhodamine labeled aptamer sequences and quencher PCN-223 dispersed in the solvent. The fluorescent aptamer sensor provided by the application has excellent selectivity and sensitivity in the detection of prostate specific antigen, and has a good application prospect.

Description

Technical Field

[0001] This application relates to the field of sensor technology, and in particular to a fluorescent aptamer sensor and method for detecting prostate-specific antigen. Background Technology

[0002] Prostate cancer is the most common malignant tumor in men and a leading cause of cancer death in men. Prostate-specific antigen (PSA), located within the prostate gland, specifically reflects the condition of the prostate. Generally, when the concentration of PAS in human serum exceeds 10 ng / mL, it indicates a significantly increased risk of prostate cancer. Therefore, developing highly sensitive PAS detection methods is crucial for the early diagnosis of prostate cancer. However, in real-world samples, the concentration of prostate-specific antigen is very low, making it difficult to achieve highly sensitive and selective detection.

[0003] Currently, commonly used methods for detecting prostate-specific antigen (PSA) include electrochemiluminescence, liquid chromatography, photoelectrochemical methods, surface-enhanced Raman spectroscopy (SERS), and electrochemical aptamer sensors. However, these methods suffer from poor selectivity, low sensitivity, and cumbersome operation, which affect the detection results of PSA. Summary of the Invention

[0004] To address the aforementioned technical issues, this application provides a fluorescent aptamer sensor and method for detecting prostate-specific antigen (PSA), which can detect the content of PSA in a sample with high sensitivity and high selectivity.

[0005] The fluorescent aptamer sensor of this application includes a solvent, and a 5-carboxytetramethylrhodamine-labeled aptamer and quencher PCN-223 dispersed in the solvent.

[0006] PCN-223 is a Zr-MOF material composed of Zr6 metal clusters and tetrakis(4-carboxyphenyl)porphyrin organic ligands, with 12 linkage nodes. PCN-223 has carboxyl, phenyl, and porphyrin groups, a large specific surface area, and good biocompatibility.

[0007] like Figure 1 The schematic diagram shown illustrates the principle of a fluorescent aptamer sensor using PCN-223 as a quencher for detecting prostate-specific antigen (PSA): The TAMRA-labeled aptamer adsorbs onto PCN-223 through π-π stacking and hydrogen bonding. The fluorescence is quenched due to the photoinduced electron transfer process between TAMRA and PCN-223. When PSA is added, it binds to the TAMRA-labeled aptamer to form a complex, causing the aptamer to move away from the PCN-223 surface, thereby restoring the fluorescence.

[0008] Optionally, the nucleic acid sequence of the aptamer is:

[0009] 5-AATTAAAGCTCGCCATCAAATAGC-3'.

[0010] Optionally, the molar ratio of the 5-carboxytetramethylrhodamine-labeled aptamer sequence to the quencher PCN-223 is 1:164–1974.

[0011] Optionally, the concentration of the 5-carboxytetramethylrhodamine-labeled aptamer sequence is 50 nM.

[0012] Optionally, the concentration of the quencher PCN-223 solution is 0.025–0.2 mg / mL.

[0013] Optionally, the solvent is a phosphate buffer solution, such as a 10 mM phosphate buffer solution with a pH of 7.4.

[0014] This application also provides a method for detecting prostate-specific antigen using the aforementioned fluorescent aptamer sensor.

[0015] Optionally, the method includes the following steps:

[0016] A solution of 5-carboxytetramethylrhodamine-labeled aptamer sequence and a solution of prostate-specific antigen were prepared separately, and the two solutions were mixed for the first incubation.

[0017] Add quencher solution for a second incubation;

[0018] After incubation, the fluorescence intensity of the system was measured.

[0019] Optionally, the temperature of the first incubation is 30–60°C, and the time is 5–50 min; the purpose of the first incubation is to allow PSA to bind to the TAMRA aptamer.

[0020] The second incubation is carried out at a temperature of 30–60°C for 5–20 min; the purpose of the second incubation is to quench the fluorescence of the TAMRA aptamer with PCN-223.

[0021] Optionally, the method includes:

[0022] Establish prostate-specific antigen concentration C PSA The standard curve of fluorescence recovery response value ΔF, wherein the fluorescence recovery response value ΔF = F - F0, where F0 is the fluorescence intensity at 582 nm of the system without prostate-specific antigen, and F is the fluorescence intensity at 582 nm of the system with prostate-specific antigen.

[0023] The fluorescence intensity F of the sample to be detected is detected, and the content of PSA in the sample to be detected is calculated according to the relationship equation of the standard curve.

[0024] Compared with the prior art, the fluorescence aptamer sensor constructed based on PCN-223 has excellent selectivity and sensitivity to PSA, can efficiently detect the content of PSA in the sample to be detected, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The principle diagram of the fluorescence aptamer sensor of the application for detecting PSA;

[0026] Figure 2 The fluorescence emission spectrum diagram of (a) TAMRA aptamer, (b) PCN-223+TAMRA aptamer+PSA, (c) PCN-223+TAMRA aptamer, (d) PCN-223, wherein the concentration of PCN-223 is 0.1 mg / mL, the concentration of TAMRA aptamer is 50 nM, and the concentration of PSA is 20 ng / mL;

[0027] Figure 3 A is the fluorescence change diagram of the fluorescence aptamer sensor prepared by different concentrations of PCN-223 for detecting PAS, and the PCN-223 concentrations from top to bottom are a (0 mg / mL), b (0.025 mg / mL), c (0.05 mg / mL), d (0.075 mg / mL), e (0.1 mg / mL), f (0.2 mg / mL), and g (0.3 mg / mL);

[0028] Figure 3 B is the relationship diagram of PCN-223 concentration and detection fluorescence intensity, and the PCN-223 concentrations from top to bottom are a (0 mg / mL), b (0.025 mg / mL), c (0.05 mg / mL), d (0.075 mg / mL), e (0.1 mg / mL), f (0.2 mg / mL), and g (0.3 mg / mL);

[0029] Figure 3 C is the influence diagram of incubation time on fluorescence change;

[0030] Figure 3 D is the visible absorption spectrum diagram of (a) PCN-223+TAMRA aptamer, (b) PCN-223, and (c) TAMRA aptamer;

[0031] Figure 4 The relationship diagram of incubation time and fluorescence recovery response value ΔF;

[0032] Figure 5The figure of the quenching time of PCN-223 and the fluorescence recovery response value ΔF;

[0033] Figure 6 The figure of the incubation temperature and the fluorescence recovery response value ΔF;

[0034] Figure 7 The figure of the pH and the fluorescence recovery response value ΔF;

[0035] Figure 8 The figure of different kinds of quenchers and the fluorescence recovery response value ΔF;

[0036] Figure 9 A is the fluorescence emission spectrum of the fluorescence aptamer sensor under different concentrations of PAS, from top to bottom, the concentrations are a (0 ng / mL), b (0.1 ng / mL), c (1 ng / mL), d (4 ng / mL), e (8 ng / mL), f (10 ng / mL), g (16 ng / mL), h (20 ng / mL), i (24 ng / mL);

[0037] Figure 9 B is the standard curve of the fluorescence recovery response value ΔF and the concentration of PAS;

[0038] Figure 10 A is the fluorescence recovery response of the fluorescence aptamer sensor for detecting PSA, CEA, AFP, CA125, HSA and BSA; wherein the concentrations of PSA and other interferents are 20 ng / mL respectively.

[0039] Figure 10 B is the influence diagram of the fluorescence recovery response of the fluorescence aptamer sensor for PSA in the presence of 3 times CEA, AFP, CA125, HSA and BSA, wherein the concentration of PSA is 20 ng / mL. DETAILED DESCRIPTION

[0040] The technical solutions described in the present application will be further described below in conjunction with specific embodiments, but the present application is not limited to this.

[0041] The reagents used below are all commercially available, wherein the prostate specific antigen (PSA) is purchased from Shanghai Lingchao New Material Co., Ltd. The carbohydrate antigen 125 (CA125), alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) are purchased from Beijing Boao Sun Biological Technology Co., Ltd. The human serum albumin (HSA) and bovine serum albumin (BSA) are purchased from Beijing Bailingwei Technology Co., Ltd. The 5-carboxy tetramethyl rhodamine (TAMRA) dye-labeled aptamer (5-TAMRA-AATTAAAGCTCGCCATCAAATAGC-3’) is synthesized by Shanghai Shengong Biological Engineering Co., Ltd.

[0042] Detection process of PSA in Example 1

[0043] 10 μL TAMRA (50 nM) aptamer was incubated with 80 μL PSA solution (20 ng / mL) at 37 °C for 30 min; then, 20 μL 1 mg / mL PCN-223 solution was added into the above solution, and PBS was added to 200 μL, and incubated at 37 °C for 15 min; finally, the sample was put into the fluorescence spectrometer to monitor the change of fluorescence spectrum.

[0044] All experiments were carried out in 200 μL 10 mM phosphate buffered saline solution (PBS, pH 7.4). The fluorescence measurement parameters were set as follows: excitation wavelength was 545 nm, excitation and emission slit width was 10 nm, and photomultiplier voltage was set to 750 V.

[0045] Reference is made to Figure 2 The fluorescence intensity of TAMRA aptamer in PBS was strong at 582 nm, and PCN-223 had no fluorescence emission at 560-600 nm, indicating that PCN-223 could effectively quench the fluorescence of TAMRA aptamer. When PSA was added into the mixture of TAMRA aptamer and PCN-223, the fluorescence was enhanced due to the specific binding of PSA to TAMRA aptamer, thereby moving away from PCN-223. Therefore, it is feasible to construct a fluorescence aptamer sensor with PCN-223 as a quencher for detecting PSA.

[0046] Optimal concentration of quencher in Example 2

[0047] 10 μL TAMRA (50 nM) aptamer was incubated with 80 μL PSA solution (20 ng / mL) at 37 °C for 30 min; then, different volumes of 1 mg / mL PCN-223 (0, 0.025, 0.05, 0.075, 0.1, 0.2, 0.3 mg / mL) were added into the above solution, and PBS was added to 200 μL, and incubated at 37 °C for 15 min; finally, the sample was put into the fluorescence spectrometer to monitor the change of fluorescence spectrum.

[0048] As shown in Figure 3 A, Figure 3 B, the fluorescence of TAMRA aptamer decreased with the increase of PCN-223 concentration, and when the concentration of PCN-223 was 0.1 mg / mL, there was a significant fluorescence recovery response, while when the concentration of PCN-223 was 0.2 and 0.3 mg / mL, the fluorescence recovery response was low. This may be because in high concentration of PCN-223, the complex formed by PSA binding to the aptamer is difficult to leave the surface of PCN-223. Therefore, 0.1 mg / mL is the optimal concentration of PCN-223.

[0049] The change of fluorescence quenching degree with time was also studied, as shown in Figure 3 As shown in Fig. C, the fluorescence intensity of TAMRA aptamer decreased rapidly with time after the addition of PCN-223, and the fluorescence intensity reached the minimum after 5 min and tended to be stable. The results showed that PCN-223 had a high fluorescence quenching effect. The fluorescence quenching mechanism of TAMRA aptamer and PCN-223 may be related to the photo-induced electron transfer between TAMRA aptamer and PCN-223. PCN-223 has carboxyl, benzene and porphyrin groups, which can have strong interaction with TAMRA aptamer through π-π stacking and hydrogen bonding. PCN-223 accepts the electron of photo-excited TAMRA dye and quenches its fluorescence.

[0050] According to the ultraviolet absorption spectrum, the interaction between TAMRA aptamer and PCN-223 was confirmed, as shown in Figure 3 As shown in Fig. D, TAMRA aptamer, TAMRA aptamer added with PCN-223 and PCN-223 respectively appeared visible absorption peaks at 559, 562 and 566 nm. The differences in these spectra indicated that there was a strong interaction between TAMRA aptamer and PCN-223.

[0051] Example 3 Incubation time

[0052] 10 μL of TAMRA (50 nM) aptamer was incubated with 80 μL of PSA solution (20 ng / mL) at 37°C for different time (5-50 min); then, 20 μL of 1 mg / mL PCN-223 solution was added to the above solution, and PBS was added to 200 μL, and the incubation was continued at 37°C for 15 min; finally, the sample was placed in a fluorescence spectrometer to monitor the change of fluorescence spectrum.

[0053] The calculation formula of ΔF (fluorescence recovery response) value is: ΔF = F-F0, wherein F0 is the fluorescence intensity of TAMRA aptamer-PCN-223 at 582 nm in the absence of PSA, and F is the fluorescence intensity of TAMRA aptamer-PCN-223 under the action of PSA.

[0054] As shown in Figure 4 Fig. E, the fluorescence recovery response gradually increased and reached the maximum at 30 min, which may be related to the increase of the concentration of TAMRA aptamer combined with PSA; the fluorescence recovery response decreased after 30 min. It may be due to the change of the structure of TAMRA aptamer combined with PSA caused by the extension of incubation time, thereby leading to the decrease of fluorescence recovery response. Therefore, 30 min was selected as the incubation time.

[0055] Example 4 PCN-223 quenching time

[0056] 10 μL TAMRA (50 nM) aptamer was incubated with 80 μL PSA solution (20 ng / mL) at 37 °C for 30 min; then, 20 μL 1 mg / mL PCN-223 solution was added into the above solution, and diluted to 200 μL with PBS, and incubated at 37 °C for different time (5-20 min); finally, the sample was put into the fluorescence spectrometer, and the change of fluorescence spectrum was monitored.

[0057] As shown in Figure 4, the fluorescence recovery response increased significantly with the increase of quenching time within the first 15 min after the addition of PCN-223, and then tended to be stable after 20 min. Therefore, 15 min was selected as the quenching time. Figure 5

[0058] Example 5 Incubation temperature

[0059] 10 μL TAMRA (50 nM) aptamer was incubated with 80 μL PSA solution (20 ng / mL) at 37 °C (or 30, 45, 60 °C) for 30 min; then, 20 μL 1 mg / mL PCN-223 solution was added into the above solution, and diluted to 200 μL with PBS, and incubated at 37 °C (or 30, 45, 60 °C) for 15 min; finally, the sample was put into the fluorescence spectrometer, and the change of fluorescence spectrum was monitored.

[0060] As shown in Figure 6, the fluorescence recovery response was the highest at 37 °C. Figure 6

[0061] Example 6 pH

[0062] 10 μL TAMRA (50 nM) aptamer was incubated with 80 μL PSA solution (20 ng / mL) at 37 °C (or 30, 45, 60 °C) for 30 min; then, 20 μL 1 mg / mL PCN-223 solution was added into the above solution, and diluted to 200 μL with PBS, and incubated at 37 °C (or 30, 45, 60 °C) for 15 min; finally, the sample was put into the fluorescence spectrometer, and the change of fluorescence spectrum was monitored. The whole experiment was carried out in PBS with pH = 6.5 (or pH = 7.0, 7.4, 8.0, 8.5).

[0063] As shown in Figure 8, the fluorescence recovery response was the highest at pH 7.4. Figure 7

[0064] Comparative Examples 1-3 Comparison of different MOF quenchers

[0065] ​​​The same as example 1, with UiO-66-NH2, ZIF-67, Ni3(HITP)2 as quenching agent, respectively. Among them, PCN-223, ZIF-67, UiO-66-NH2 are purchased from Shanghai Keshu Chemical Technology Co., Ltd.; the synthesis method of Ni3(HITP)2 refers to the method reported before [1].

[0066] As shown in Figure 8 , the fluorescence recovery response of the fluorescence aptamer sensor with PCN-223 as quenching agent to detect PSA is the highest, and the fluorescence recovery response of the fluorescence aptamer sensor with Ni3(HITP)2 as quenching agent to detect PSA is the lowest. The fluorescence recovery response of the fluorescence aptamer sensor based on PCN-223 is 16.1, 7.1 and 24.6 times that of UiO-66-NH2, ZIF-67 and Ni3(HITP)2 fluorescence quenching agent, respectively.

[0067] Test example

[0068] (1) Standard curve, detection limit

[0069] 10 μL TAMRA (50 nM) aptamer was incubated with different volumes of 50 ng / mL PSA solution (0-24 ng / mL) at 37°C for 30 minutes; then, 20 μL of 1 mg / mL PCN-223 was added to the above solution, and the volume was made to 200 μL with PBS, and the incubation was continued at 37°C for 15 minutes; finally, the sample was placed in a fluorescence spectrometer to monitor the change of fluorescence spectrum. All experiments were carried out in 200 μL of 10 mM phosphate buffered saline solution (PBS, pH 7.4). The fluorescence measurement parameters were set as follows: excitation wavelength was 545 nm, excitation and emission slit width was 10 nm, and photomultiplier tube voltage was set to 750 V.

[0070] The calculation formula of fluorescence recovery response value ΔF is: ΔF = F-F0, wherein F0 is the fluorescence intensity of TAMRA aptamer-PCN-223 at 582 nm in the absence of PSA, and F is the fluorescence intensity of TAMRA aptamer-PCN-223 under the action of PSA.

[0071] Figure 9 A shows the fluorescence emission spectrum of the fluorescence aptamer sensor in the presence and absence of PSA. As shown in Figure 9 B, within the range of 0.1-24 ng / mL of PSA concentration, the fluorescence aptamer sensor shows a good linear relationship. The linear equation is ΔF = 50.40C PSA +59.25 (R 2= 0.9958), the limit of detection of PSA was 0.05 ng / mL (S / N = 3). Compared with other methods for detecting PSA, the results are shown in Table 1, which indicates that the fluorescent aptamer sensor constructed in the present application has a lower detection limit.

[0072] Table 1 Comparison of the present method with other methods for detecting PSA

[0073]

[0074] References:

[0075] [1] X. Lei, Z. Deng, Y. Zeng, S. Huang, Y. Yang, H. Wang, L. Guo, L. Li, A novel composite of conductive metal organic framework and molecularly imprinted poly(ionic liquid) for highly sensitive electrochemical detection of bisphenol A, Sensor. Actuator. B Chem. 339 (2021) 129885.

[0076] [2] W. Argoubi, A. Sanchez, C. Parrado, N. Raouafi, R. Villalonga, Label-free electrochemical aptasensing platform based on mesoporous silica thin film for the detection of prostate specific antigen, Sensor. Actuator. B Chem. 255 (2018) 309-315.

[0077] [3] R. Wang, W. Liu, A. Wang, Y. Xue, L. Wu, J. Feng, A new label-free electrochemical immunosensor based on dendritic core-shell AuPd@Au nanocrystals for highly sensitive detection of prostate specific antigen, Biosens. Bioelectron. 99 (2018) 458-463.

[0078] [4] A. Ouhibi, A. Raouafi, N. Lorrain, M. Guendouz, N. Raouafi, A. Moadhen, Functionalized SERS substrate based on silicon nanowires for rapid detection of prostate specific antigen, Sensor. Actuator. B Chem. 330 (2021) 129352.

[0079] [5] J. Chen, X. Li, X. Yu, W. Zhou, Q. Wang, Determination of prostate-specific antigen via the assembly of a two-dimensional nanoplatform, J Nanopart Res 24 (2022) 88.

[0080] [6] F. Qu, Y. Ding, X. Lv, L. Xia, J. You, W. Han, Emissions of terbium metal-organic frameworks modulated by dispersive / agglomerated gold nanoparticles for the construction of prostate-specific antigen biosensor, Anal. Bioanal. Chem. 411 (2019) 3979-3988.

[0081] [7] B. Fang, J. An, B. Liu, Y. Zhao, Hybridization induced fluorescence enhanced DNA-Ag nanocluster / aptamer probe for detection of prostate-specific antigen, Colloids Surf. B 175 (2019) 358-364.

[0082] (2) Selectivity

[0083] To evaluate the selectivity of the fluorescent aptamer sensor for detecting PSA, selectivity experiments were performed in the presence of interfering substances such as PSA, CEA, AFP, CA125, HSA, and BSA. As shown in Fig. 6, the fluorescent aptamer sensor had high selectivity for PSA, and the fluorescence intensity of the sensor was not affected by the presence of other interfering substances. Figure 10As shown in Figure A, PSA has the highest fluorescence recovery response, while the fluorescence response of other interferents is very low.

[0084] The fluorescence response of the fluorescent aptamer sensor to PSA in the presence of each interferent was further studied. The results are shown in Figure B. Figure 10 As shown in Figure B, it was found that 3-fold CEA, AFP, CA125, HAS and BSA had no interference with PSA detection, respectively. Therefore, it was shown that the fluorescent aptamer sensor of the present application has good selectivity.

[0085] (3) Actual sample determination

[0086] Human serum samples were provided by Jiaxing Hospital of Traditional Chinese Medicine. The human serum samples were stored at -20°C, diluted to 1% with PBS before use, and then different concentrations of PSA were added to the 1% human serum samples.

[0087] The results are shown in Table 2. The average recovery rate of the spiked samples was 98.43-104.69%, and the relative standard deviation (RSD) was 2.52-5.64%. The results showed that the fluorescent aptamer sensor was suitable for determining PSA in human serum.

[0088] Table 2 Determination of PSA in human serum by the fluorescent aptamer sensor of the present application

[0089]

[0090] The above description of the embodiments is to facilitate the understanding and use of the present application by those of ordinary skill in the art, and those of ordinary skill in the art can also make appropriate changes and modifications to the above embodiments. Therefore, the present application is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present application should also fall within the protection scope of the claims of the present application.

Claims

1. A fluorescent aptamer sensor for detecting prostate specific antigen, characterized by, The solvent, and 5-carboxy tetramethyl rhodamine labeled aptamer and quencher PCN-223 dispersed in the solvent; The nucleic acid sequence of the aptamer is: 5- AATTAAAGCTCGCCATCAAATAGC-3'; The molar ratio of the 5-carboxy tetramethyl rhodamine labeled aptamer and the quencher PCN-223 is 1:164~1974; The concentration of the quencher PCN-223 solution is 0.025~0.3 mg / mL.

2. The fluorescent aptamer sensor of claim 1, wherein, The concentration of the 5-carboxy tetramethyl rhodamine labeled aptamer sequence is 50nM.

3. A method of detecting prostate specific antigen, characterized by, The fluorescent aptamer sensor of claim 1 is used.

4. The method of claim 3, wherein, The method comprises the following steps: 5-carboxy tetramethyl rhodamine labeled aptamer sequence solution and prostate specific antigen solution are respectively prepared, and the two solutions are mixed to carry out the first incubation; The quencher solution is added to carry out the second incubation; After the incubation, the fluorescence intensity of the system is measured.

5. The method of claim 4, wherein, The temperature of the first incubation is 30~60℃, and the time is 5~50 min; The temperature of the second incubation is 30~60℃, and the time is 5~20 min.

6. The method of claim 4, wherein, The method comprises the following steps: establishing the concentration of prostate specific antigen C PSA with the standard curve of the fluorescence recovery response value ΔF = F - F0, wherein F0 is the fluorescence intensity at 582 nm of the system without the addition of prostate specific antigen and F is the fluorescence intensity at 582 nm of the system with the addition of prostate specific antigen; The fluorescence intensity F0 and F of the sample to be tested are detected, and the ∆F is calculated, and the content of the prostate specific antigen in the sample to be tested is calculated according to the relationship equation of the standard curve.

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