阅读说明：本技术 一种检测***特异性抗原的光谱分析中荧光传感器的构建方法及其应用 (Construction method and application of fluorescence sensor in spectral analysis for detecting prostate specific antigen ) 是由 赵媛 郑芳杰 施丽霞 于 2019-09-12 设计创作，主要内容包括：本发明提供了一种检测前列腺特异性抗原的光谱分析中荧光传感器的构建方法,属于光谱分析技术领域。本发明主要内容包括ZGO:Mo/PSA-A溶液和Au@Ag@SiO<Sub>2</Sub>/PSA-C的制备,并利用ZGO:Mo/PSA-A可特异性识别前列腺特异性抗原,Au@Ag@SiO<Sub>2</Sub>/PSA-C可以与检测探针杂交从而猝灭探针的发光,从而构建一种检测前列腺特异性抗原的光谱分析中荧光传感器。该荧光传感器用于前列腺特异性抗原的检测。本发明构建的荧光传感器具有较高的准确度以及灵敏度。(The invention provides a construction method of a fluorescence sensor in spectral analysis for detecting prostate specific antigen, belonging to the technical field of spectral analysis. The main contents of the invention comprise ZGO, Mo/PSA-A solution and Au @ Ag @ SiO 2 Preparation of/PSA-C and utilizes ZGO, Mo/PSA-A to specifically recognize prostate specific antigen, Au @ Ag @ SiO 2 the/PSA-C can be hybridized with a detection probe to quench the luminescence of the probe, so that a fluorescence sensor in the spectral analysis for detecting the prostate specific antigen is constructed. The fluorescence sensor is used for detecting prostate specific antigen. The fluorescence sensor constructed by the invention has higher accuracy and sensitivity.)

1. A method of constructing a fluorescence sensor for spectroscopic analysis for detection of prostate specific antigen, said method comprising the steps of:

(1) preparation of ZGO Mo/PSA-A solution:

(ii) reacting Zn (NO)₃)₂、Na₂MoO₄Adding the solid into 400 mu L of concentrated HNO₃Mixing the solution, stirring, and addingAdding water and mixing uniformly to obtain a colorless transparent solution 1;

② weighing GeO₂Stirring with NaOH to dissolve in water, and stirring for 7-8h to obtain Na₂GeO₃A solution;

thirdly, Na obtained in the step II₂GeO₃Dropwise adding the solution into the solution 1 obtained in the step I, uniformly mixing, adjusting the pH value of the solution to 7-10 by using ammonia water, stirring for 1-1.5h, then transferring into a polytetrafluoroethylene hydrothermal reaction kettle, heating for 4-4.5h at the temperature of 220-225 ℃, and heating at the rate of 2-2.5 ℃/min; after the reaction is finished, carrying out solid-liquid separation to obtain a precipitate, dispersing the precipitate in an NaOH aqueous solution again, stirring at room temperature for 12-13h, then centrifugally collecting the solid precipitate, dispersing the obtained solid precipitate in N, N-dimethylformamide, dropwise adding 3-aminopropyltriethoxysilane APTES, heating in a water bath at the temperature of 80-85 ℃ for reaction for 24-25h, and carrying out solid-liquid separation after the reaction is finished to obtain aminated ZGO, Mo NRs;

adding prostate specific antigen aptamer PSA-A into PBS buffer solution, stirring and mixing uniformly, adding mixed solution EDC/NHS of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, activating for 30-60min, adding aminated ZGO, namely Mo NRs obtained in the step (iii), oscillating and reacting for 5-5.5h at room temperature, carrying out solid-liquid separation to obtain a solid phase, washing the solid phase with water, and ultrasonically dispersing the solid phase in the PBS buffer solution to obtain a ZGO, namely Mo/PSA-A solution, wherein the concentration of the obtained solution is 1-1.2 mg/mL;

(2)[email protected]@SiO₂preparation of/PSA-C:

adding the Au @ Ag NPs solution into an ethanol water solution, stirring uniformly at room temperature, sequentially adding ammonia water and a 10% ethyl orthosilicate solution, and stirring for reacting for 3-3.5 h; after the reaction is finished, carrying out solid-liquid separation to obtain Au @ Ag @ SiO₂NPs, and washing with water at least three times, followed by re-dispersion in water to give a solution with a particle concentration of 0.1-0.2 nM; mixing Au @ Ag @ SiO₂Adding the NPs solution into a Tris-boric acid buffer TBE solution, mixing and stirring uniformly, adding a prostate specific antigen aptamer complementary chain PSA-C solution, mixing uniformly, incubating at room temperature for 12-12.5h, performing solid-liquid separation to obtain a solid phase substance, and mixing the solid phase substance with the solutionWashing the substance with water for more than three times, and finally dispersing the substance in a PBS buffer solution to obtain Au @ Ag @ SiO₂A PSA-C solution, the concentration of the obtained solution being 0.1-0.2 nM;

(3) preparation of a fluorescence sensor:

adding the solution of ZGO, Mo/PSA-A obtained in the step (1) into a PBS buffer solution, and adding the solution of Au @ Ag @ SiO @ obtained in the step (2)₂Performing oscillation incubation on the solution of/PSA-C at room temperature for 2-2.5h, performing solid-liquid separation to obtain solid substances, and re-dispersing the solid substances in a PBS buffer solution to obtain a mixed solution 2; respectively adding a series of prepared prostate specific antigen aptamer PSA standard solutions with concentration gradients into the mixed solution 2, and performing shaking incubation on a shaking table for 5-6 h; irradiating by using an ultraviolet lamp, finally testing the luminescence spectrum and the luminescence intensity of each group of solution by using a fluorescence spectrometer, and obtaining a standard curve by using the concentration logarithm of the prostate specific antigen aptamer PSA as a horizontal coordinate and the luminescence recovery intensity of the detection probe ZGO, Mo/PSA-A as a vertical coordinate through the fluorescence spectrum determination of a series of concentrations.

2. The method according to claim 1, wherein the concentration of the concentrated nitric acid in step (1): is 15.8 to 16.2mol/L, and Zn (NO) is present therein₃)₂、Na₂MoO₄The dosage ratio of the concentrated nitric acid to the water is as follows: 2-2.2mmol, 0.005-0.006mmol, 300-400. mu.L, 11-12 mL.

3. The method according to claim 1, wherein the solid GeO in step (1) (-), (ii) is₂The mass ratio of the sodium hydroxide to NaOH is as follows: 1.3-1.6:1.5-1.8.

4. The method according to claim 1, wherein Na is present in step (1) —₂GeO₃The volume ratio of the solution to the solution 1 is: 1.5-2:11.3-12.4.

5. The method according to claim 1, wherein the PBS buffer solution in the step (1), (3) has a concentration of 10mM, a pH of: 7.4-7.5.

6. The method for constructing a concrete structure according to claim 1, wherein the amount ratio of the absolute ethanol to the water in the ethanol aqueous solution in the step (2) is as follows: 0.4-0.5: 3.0-3.2; the concentration of Au @ Ag NPs is 1-1.2 nM; wherein the volume ratio of the Au @ Ag NPs solution, the ethanol aqueous solution, the ammonia water and the 10% TEOS solution is as follows: 200-250 μ L, 3400-3700 μ L, 285-300 μ L, 9-42 μ L.

7. The construction method according to claim 1, wherein the fluorescence sensor is prepared in step (3) by the following steps:

adding the solution of ZGO, Mo/PSA-A obtained in the step (1) into a PBS buffer solution, and adding the solution of Au @ Ag @ SiO @ obtained in the step (2)₂Performing oscillation incubation on the solution of/PSA-C at room temperature for 2-2.5h, performing solid-liquid separation to obtain solid substances, and re-dispersing the solid substances in PBS buffer solution to obtain mixed solution 2; preparing a series of prostate specific antigen aptamer PSA standard solutions with equal volumes and different concentration gradients, wherein the concentration range of the standard solutions is 0pg/mL-1 mu g/mL, taking 5-12 concentration gradient values, respectively adding the standard solutions into the mixed solution 2, and performing shaking incubation on a shaking table for 5-6 hours; irradiating by using a 254nm ultraviolet lamp, finally testing the luminescence spectrum and the luminescence intensity of each group of solution by using a fluorescence spectrometer, and obtaining a standard curve by taking the concentration logarithm of the prostate specific antigen aptamer PSA as a horizontal coordinate and the luminescence recovery degree of the detection probe ZGO, Mo/PSA-A as a vertical coordinate through the measurement of a series of fluorescence spectra of concentrations.

8. The method of claim 7, wherein the ZGO is a Mo/PSA-A solution, Au @ Ag @ SiO @₂The volume ratio of the/PSA-C solution to the PBS buffer solution is as follows: 10-15:40-45:100-150.

9. The method according to claim 1 or 7, wherein the luminescence recovery intensity of the detection probe ZGO/PSA-A is equal to the difference between the luminescence intensity F in the presence of the detection probe for prostate specific antigen aptamer PSA with a different concentration gradient and the luminescence intensity F0 in the absence of the detection probe for prostate specific antigen aptamer PSA.

10. A fluorescence sensor for spectroscopic analysis for detection of prostate specific antigen produced by the construction method according to claim 1, wherein said fluorescence sensor is used for detection of prostate specific antigen PSA.

Technical Field

The invention belongs to the technical field of spectral analysis, and particularly relates to a technology for detecting prostate specific antigen.

Background

Prostate Specific Antigen (PSA) is a tumor marker secreted by prostate epithelial cells to leak from pathological tissues into the vascular system, and is present in serum at a level effective to reflect the occurrence of prostate disease. At present, PSA is a specific tumor marker most commonly used for diagnosing prostate cancer and judging the recurrence of cancer after treatment. It is now generally accepted that the threshold for diagnosis of prostate disease is that the serum PSA level is below 4ng/mL, and that prostate disease is likely to occur when the level is above this level.

The accurate and sensitive detection of PSA content is of great importance in the early diagnosis of cancer and the monitoring of cancer recurrence after treatment, which requires the development of a sensor or detection method capable of accurately, sensitively and specifically detecting PSA content. Wu et al developed a microcantilever array-based method for determining PSA content. In recent years, some researches have been carried out to design a portable electrochemical sensor or fluorescent sensor for detecting the content of PSA in biological samples such as serum by using a glassy carbon electrode and an electroactive nano material or a fluorescent nano material. Although these methods can be used for detection of PSA, there are disadvantages such as poor reproducibility, complicated preparation process, and inability to completely eliminate background interference of biological samples, which require further improvement.

The long afterglow material is a substance which can absorb energy when excited light is irradiated and can continuously emit light after excitation is stopped, and plays an important role in the fields of biosensing analysis, biological imaging, tumor treatment and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a construction method and application of a fluorescence sensor in spectral analysis for detecting prostate specific antigen. The fluorescence sensor constructed by the invention has good stability, high sensitivity and good reproducibility, and has wide application prospect in the aspect of detecting the content of tumor markers.

The technical scheme of the invention is as follows:

a method of constructing a fluorescence sensor for spectroscopic analysis for detection of prostate specific antigen, said method of construction comprising the steps of:

(1) preparation of ZGO Mo/PSA-A solution:

(ii) reacting Zn (NO)₃)₂、Na₂MoO₄Adding the solid into 400 mu L of concentrated HNO₃Uniformly mixing and stirring the solution, adding water, and uniformly mixing to obtain a colorless and transparent solution 1;

② weighing GeO₂Stirring with NaOH to dissolve in water, and stirring for 7-8h to obtain Na₂GeO₃A solution;

thirdly, Na obtained in the step II₂GeO₃Dropwise adding the solution into the solution 1 obtained in the step I, uniformly mixing, adjusting the pH value of the solution to 7-10 by using ammonia water, stirring for 1-1.5h, then transferring into a polytetrafluoroethylene hydrothermal reaction kettle, heating for 4-4.5h at the temperature of 220-225 ℃, and heating at the rate of 2-2.5 ℃/min; after the reaction is finished, carrying out solid-liquid separation to obtain a precipitate, dispersing the precipitate in an NaOH aqueous solution again, stirring at room temperature for 12-13h, then centrifugally collecting the solid precipitate, dispersing the obtained solid precipitate in N, N-dimethylformamide, dropwise adding 3-aminopropyltriethoxysilane APTES, heating in a water bath at the temperature of 80-85 ℃ for reaction for 24-25h, and carrying out solid-liquid separation after the reaction is finished to obtain aminated ZGO, Mo NRs;

adding prostate specific antigen aptamer PSA-A into PBS buffer solution, stirring and mixing uniformly, adding mixed solution EDC/NHS of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, activating for 30-60min, adding aminated ZGO, namely Mo NRs obtained in the third step, oscillating and reacting for 5-5.5h at room temperature, carrying out solid-liquid separation to obtain a solid phase, washing the solid phase with water, and ultrasonically dispersing the solid phase in the PBS buffer solution to obtain a ZGO, namely Mo/PSA-A solution, wherein the concentration of the obtained solution is 1-1.2 mg/mL;

(2)[email protected]@SiO₂preparation of/PSA-C:

adding the Au @ Ag NPs solution into an ethanol water solution, stirring uniformly at room temperature, sequentially adding ammonia water and a 10% ethyl orthosilicate solution, and stirring for reacting for 3-3.5 h; after the reaction is finished, carrying out solid-liquid separation to obtain Au @ Ag @ SiO₂NPs, and washing with water at least three times, followed by re-dispersion in water to give a solution with a particle concentration of 0.1-0.2 nM; mixing Au @ Ag @ SiO₂Adding the NPs solution into a Tris-boric acid buffer TBE solution, mixing and stirring uniformly, adding a prostate specific antigen aptamer complementary chain PSA-C solution, mixing uniformly, incubating at room temperature for 12-12.5h, and reactingAfter the reaction is finished, solid-liquid separation is carried out to obtain solid-phase substances, the solid-phase substances are washed for more than three times by water and finally dispersed in PBS buffer solution to obtain Au @ Ag @ SiO₂A PSA-C solution, the concentration of the obtained solution being 0.1-0.2 nM;

(3) preparation of a fluorescence sensor:

adding the solution of ZGO, Mo/PSA-A obtained in the step (1) into a PBS buffer solution, and adding the solution of Au @ Ag @ SiO @ obtained in the step (2)₂Performing oscillation incubation on the solution of/PSA-C at room temperature for 2-2.5h, performing solid-liquid separation to obtain solid substances, and re-dispersing the solid substances in a PBS buffer solution to obtain a mixed solution 2; respectively adding a series of prepared prostate specific antigen aptamer PSA standard solutions with concentration gradients into the mixed solution 2, and performing shaking incubation on a shaking table for 5-6 h; irradiating by using an ultraviolet lamp, finally testing the luminescence spectrum and the luminescence intensity of each group of solution by using a fluorescence spectrometer, respectively testing each group of solution for 3 times, and obtaining a standard curve which takes the concentration logarithm of the prostate specific antigen aptamer PSA as a horizontal coordinate and the luminescence recovery intensity of the detection probe ZGO, Mo/PSA-A as a vertical coordinate through the fluorescence spectrum measurement of a series of concentrations.

The concentration of the concentrated nitric acid in the step (1) is 15.8-16.2mol/L, wherein Zn (NO)₃)₂、Na₂MoO₄The dosage ratio of the concentrated nitric acid to the water is as follows: 2-2.2mmol, 0.005-0.006mmol, 300-400. mu.L, 11-12 mL.

Step (1) and solid GeO₂The mass ratio of the sodium hydroxide to NaOH is as follows: 1.3-1.6:1.5-1.8.

4. The method according to claim 1, wherein Na is present in step (1) —₂GeO₃The volume ratio of the solution to the solution 1 is: 1.5-2:11.3-12.4.

In the step (1), the concentration of the PBS buffer solution in the step (3) is 10mM, and the pH value is as follows: 7.4-7.5.

The dosage ratio of the absolute ethyl alcohol to the water in the ethyl alcohol water solution in the step (2) is as follows: 0.4-0.5:3.0-3.2, and the concentration of Au @ Ag NPs is 1-1.2nM, wherein the volume ratio of the Au @ Ag NPs solution, the ethanol aqueous solution, the ammonia water and the 10% TEOS solution is as follows: 200-250 μ L, 3400-3700 μ L, 285-300 μ L, 9-42 μ L.

The preparation of the fluorescence sensor in the step (3) comprises the following specific steps:

adding the solution of ZGO, Mo/PSA-A obtained in the step (1) into a PBS buffer solution, and adding the solution of Au @ Ag @ SiO @ obtained in the step (2)₂Performing oscillation incubation on the solution of/PSA-C at room temperature for 2-2.5h, performing solid-liquid separation to obtain solid substances, and re-dispersing the solid substances in PBS buffer solution to obtain mixed solution 2; preparing a series of prostate specific antigen aptamer PSA standard solutions with equal volumes and different concentration gradients, wherein the concentration range of the standard solutions is 0pg/mL-1 mu g/mL, taking 5-12 concentration gradient values, respectively adding the standard solutions into the mixed solution 2, and performing shaking incubation on a shaking table for 5-6 hours; irradiating by using a 254nm ultraviolet lamp, finally testing the luminescence spectrum and the luminescence intensity of each group of solution by using a fluorescence spectrometer, and obtaining a standard curve by taking the concentration logarithm of the prostate specific antigen aptamer PSA as a horizontal coordinate and the luminescence recovery degree of the detection probe ZGO, Mo/PSA-A as a vertical coordinate through the measurement of a series of fluorescence spectra of concentrations.

The ZGO is Mo/PSA-A solution, Au @ Ag @ SiO₂The volume ratio of the/PSA-C solution to the PBS buffer solution is as follows: 10-15:40-45:100-150.

The luminescence recovery intensity of the detection probe ZGO, Mo/PSA-A is equal to the difference between the luminescence intensity F when the detection probe with prostate specific antigen aptamer PSA with different concentration gradients exists and the luminescence intensity F0 when the detection probe with prostate specific antigen aptamer PSA does not exist.

The fluorescence sensor is applied to detecting prostate specific antigen PSA.

The beneficial technical effects of the invention are as follows:

the detection method of the prostate specific antigen provided by the invention has high sensitivity and specificity, and when the content of the prostate specific antigen in an actual sample is detected, the interference of autofluorescence, light scattering and the like of other substances in a biological matrix can be completely avoided, so that the detection result also has high accuracy and sensitivity.

The invention dopes molybdenum element in zinc germanate (Zn)₂GeO₄) Prepared in a matrix withThe blue-emitting long-afterglow nanorod is marked as ZGO and Mo NRs, and the adjustment of the luminous intensity and the decay time of the ZGO and the Mo NRs is realized by changing the pH value. Meanwhile, ZGO NRs with high luminous intensity and long decay time are screened out and used for preparing a nano probe for detecting PSA. Au @ Ag @ SiO₂The preparation method of the NPs comprises the steps of coating a silicon dioxide shell layer on the surface of the Au @ Ag NPs, etching the Ag shell and reducing the Ag shell layer into smaller Ag nano particles, and the structure not only improves the defect that the Au @ Ag NPs are easy to gather in a solution, but also ensures the quenching capability of the Au @ Ag NPs. We absorbed the spectrum in the blue wavelength region of Au @ Ag @ SiO₂NPs serve as energy receptors to construct a sensor based on a FRET principle, and sensitive detection of PSA without interference of autofluorescence signals is realized.

The detection probe capable of specifically recognizing the prostate specific antigen is prepared by coupling the long-afterglow nanorod ZGO and Mo NRs capable of continuously emitting blue light and an aptamer PSA-A as a luminescent material. Selecting Au @ Ag @ SiO with absorption spectrum in blue light range₂NPs construct a luminescent receptor, and an aptamer complementary chain PSA-C and Au @ Ag @ SiO are subjected to the action of an Ag-SH covalent bond₂The NPs are coupled so that they can hybridize to the detection probes to quench the luminescence of the probes. When no prostate specific antigen exists, the detection probe long afterglow nano rod ZGO, Mo NRs and Au @ Ag @ SiO₂NPs are connected together through hybridization of nucleic acid chains, and the luminescent signals of ZGO and Mo NRs are quenched; when prostate specific antigen exists, the aptamer chain on the detection probe ZGO: Mo NRs is specifically combined with the prostate specific antigen so as to be combined with Au @ Ag @ SiO₂NPs are separated, so that the luminescence of the nano-rods is recovered, and a spectral analysis method capable of sensitively detecting the prostate specific antigen is designed according to the luminescence of the nano-rods. Can effectively eliminate the interference of autofluorescence and realize the accurate detection of the content of prostate specific antigen in the detected sample.

The detection method of the prostate specific antigen provided by the invention has high sensitivity and specificity, and when the content of the prostate specific antigen in an actual sample is detected, the interference of autofluorescence, light scattering and the like of other substances in a biological matrix can be completely avoided, so that the detection result also has high accuracy and sensitivity.

Drawings

FIG. 1 is a schematic diagram of an experiment for detecting prostate specific antigen in the present invention;

FIG. 2 is a transmission electron microscope image of the long afterglow nanorods ZGO and Mo NRs prepared in example 2;

FIG. 3 is Au @ Ag @ SiO prepared in example 3₂Transmission electron microscopy images of NPs;

FIG. 4 is a graph of the luminescence spectrum and the linear relationship of the sensor in the presence of different concentrations of prostate specific antigen from example 3.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples.