CN112129819A - Construction method and application of specific electrochemical sensor for tumor marker detection - Google Patents

Abstract

The invention provides a preparation method of Ru nano particles, researches the electrochemical activity of the Ru nano particles, constructs a sensor for detecting a tumor marker by using an electrochemical signal of the Ru nano particles, and belongs to the field of electrochemical application. The invention mainly discloses that the Ru nano material has a unique electrochemical oxidation peak; the change of the electrochemical oxidation peak of the Ru nano material under the illumination condition is explored, and an electrochemical sensor for detecting a tumor marker is designed by utilizing the characteristic. The method is simple and easy to implement, and has high detection sensitivity.

Description

Construction method and application of specific electrochemical sensor for tumor marker detection

technical field

The invention belongs to the technical field of electrochemical analysis, in particular to a construction method and application of a specific electrochemical sensor for tumor marker detection.

Background technique

Electrochemical redox active probes are the basis for the construction of stable electrochemical biosensors, and are the key to rapid detection electrode development and clinical applications. Compared with electroactive organic molecules, electroactive nanomaterials have unique electronic properties and fast electron transfer efficiency without additional carrier loading, and can obtain electrochemical redox signals with tunable intensity and selectable peak positions by adjusting the size, composition and structure. , to meet the specific needs of biological applications. However, the research on electroactive nanomaterials themselves as signal labels is still in its infancy, with only a few reports. And the research on electroactive metal nanomaterials mainly focuses on Ag, Cu ₂ O and so on. Therefore, it is urgent to develop a new electroactive nanoprobe with unique electrochemical response to expand the application of nanoparticles in the field of electrochemistry.

Among transition metals, Ru has excellent properties of high selectivity, corrosion resistance, and high temperature resistance. And its d electron orbital is not filled, and it has good electrical activity. Therefore, in recent years, Ru nanomaterials have been widely used in various fields of photochemistry and electrochemistry (such as photocatalytic nitrogen fixation, electrocatalytic hydrogen evolution, electrocatalytic oxygen evolution, etc.). However, there are few reports on whether Ru nanomaterials can use light energy to change their electrochemical signals. Through this type of research, the potential relationship between light energy and electrical energy in nanomaterials can be explored, and potential development prospects can be provided for the combination of these two energy sources. Therefore, it is necessary to further explore the intrinsic link between the electrochemical signal of nanomaterials and light energy. In addition, the use of light energy to enhance the electrochemical signal further enhances the signal intensity of the electroactive nanoprobe, which is more conducive to the construction of a sensor for detecting tumor markers and broadens its application field.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the applicant of the present invention provides a construction method and application of a specific electrochemical sensor for tumor marker detection. The present invention reveals for the first time that Ru nanomaterials have their unique electrochemical oxidation peaks and can be used as novel electroactive nanometer probes. It was found that the electrochemical oxidation peak of Ru nanomaterials changed under illumination and the reasons were explored. In addition, an electrochemical sensor for the detection of tumor markers was constructed by utilizing the feature of light-enhanced electrochemical oxidation peaks.

The technical scheme of the present invention is as follows:

A construction method of a specific electrochemical sensor for tumor marker detection, the specific construction method is:

S1: Mix Ru NPs and tumor marker aptamer solution in Tris-boronic acid TBE buffer solution, mix and incubate at room temperature for 11-13 hours, then separate the solid-liquid to take the solid phase and re-disperse the solid phase in water to obtain Apt- Ru NPs solution;

S2: The Apt-Ru NPs solution obtained in S1 was mixed and reacted with the GO/Fe ₃ O ₄ NSs solution, and then the magnetic substances in the solution were separated from the reaction solution by magnetic adsorption, and the magnetic substances were re-dissolved in water to obtain Ru NPs @GO/Fe ₃ O ₄ NSs solution;

S3: Use alumina polishing powder to polish the magnetic glassy carbon electrode MGCE. After polishing, rinse the electrode surface with water and ethanol, keep the electrode surface dry, and then grind the Ru NPs@GO/Fe ₃ obtained in step S2. The O ₄ NSs solution was added dropwise to the polished MGCE and left at room temperature for 1-2 h to obtain Ru NPs@GO/Fe ₃ O ₄ NSs electrode. After standing for 10-50 min, the electrode was rinsed and illuminated to detect the electrochemical signal of the Ru NPs@GO/Fe ₃ O ₄ NSs electrode after illumination, and record the electrochemical response of the Ru NPs@GO/Fe ₃ O ₄ NSs electrode. This detects the PSA solution concentration.

Further, the molar ratio of Ru NPs and tumor marker aptamers in S1 was 1:80-1:120.

Further, the tumor marker in S1 includes one of PSA, CEA or HER2.

Further, the molar ratio of Apt-Ru NPs to GO/Fe ₃ O ₄ NSs in S2 is 2:1-4:1.

Further, the conditions for the mixed reaction of the Apt-Ru NPs solution and the GO/Fe ₃ O ₄ NSs solution in S2 are as follows: maintaining at 36-38 °C for 0.5-1.5 h.

Further, the illumination conditions in S3 are as follows: using yellow light with a wavelength of 550-650 nm as a light source to irradiate the electrode to be tested, and the irradiation time is 10-40 min.

Further, an application of a specific electrochemical sensor for tumor marker detection, the detection is not for the purpose of disease treatment and prevention.

The beneficial technical effects of the present invention are:

The present invention discovers for the first time that the Ru nanomaterial has its unique and strong electrochemical oxidation peak when the potential is around 0.8V (compared to the Ag/AgCl electrode). Compared with conventional electroactive metal nanomaterials, it can be used as a novel electroactive nanoprobe, providing a basis for the construction of electrochemical biosensors in the future.

Based on the phenomenon that the electrochemical oxidation peak of Ru nanomaterials is enhanced after illumination, it is found that the reason for its strong electrochemical signal is that a large number of electrons are generated in Ru NPs under the excitation of localized surface plasmon resonance (LSPR). hole pair. The generated photogenerated electrons flow from Ru NPs to the external circuit, while the remaining holes promote the oxidation of Ru NPs due to their oxidizing effect, resulting in enhanced electrochemical signals after illumination.

The detection mechanism of the invention is as follows: firstly, the aptamer of the tumor marker is connected with the prepared Ru NPs by using the thiol group to form Apt-Ru NPs. The Apt-Ru NPs were then assembled with GO/Fe ₃ O ₄ NSs through the π-π stacking interaction between aptamers and graphene to form Ru NPs@GO/Fe ₃ O ₄ NSs. Since the formed Ru NPs@GO/Fe ₃ O ₄ NSs are magnetic and can be directly adsorbed on MGCE, no additional modification operations are required on the electrodes. Due to the existence of Ru NPs, the electrodes adsorbed with nanomaterials have excellent electrochemical signals under illumination. When the electrode is in contact with the target, due to the excellent binding force between the aptamer and the tumor marker, the Ru NPs will fall off the surface of the electrode, and the previously strong electrochemical signal will be weakened. An electrochemical sensor for the specific detection of tumor markers was constructed according to this principle.

Compared with the traditional research on Ru nanomaterials, the traditional use of Ru nanomaterials is only applied in the field of photochemistry or electrochemistry, so it is subject to certain limitations. The present invention explores the intrinsic relationship between the electrochemical signal of Ru nanomaterials and light energy, and builds an electrochemical sensor that specifically detects tumor markers based on the relationship between the two, which is the mutual combination of light energy and electrical energy. And applications provide potential development prospects.

Description of drawings

Figure 1 shows the DPV electrochemical signal response of Ru nanomaterials in Example 2 under dark and light conditions.

Fig. 2 is a graph showing the detection results of PSA by the specific electrochemical sensor prepared in Example 2 after illumination, in which Fig. A shows the DPV of Ru NPs@GO/Fe ₃ O ₄ NSs electrode reacted with different concentrations of PSA. Electrochemical signal response. Panel B represents the standard curve between the DPV electrochemical signal of Ru NPs@GO/Fe ₃ O ₄ NSs electrode and the logarithm of PSA concentration.

3 is a graph showing the detection results of the electrochemical sensor prepared in Example 2 on positive serum containing different PSA concentrations, and the results of evaluating the accuracy of the electrochemical sensor constructed in Example 2 for PSA detection.

Detailed ways

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

Example 1:

The construction method of a ratiometric electrochemical sensor for tumor marker detection specifically includes the following steps:

(1) Preparation of Ru/glassy carbon electrode:

Using alumina polishing powder with a particle size of 0.02 μm, after mixing with ultrapure water, the glassy carbon electrode with a diameter of 2mm was polished and ground, and then ultrasonically washed with ultrapure water and ethanol in turn, and dried with ultrapure nitrogen. 2 μL of RuNPs solution was evenly coated on the electrode surface and placed at room temperature for 1 h to obtain a Ru/glassy carbon electrode;

(2) Electrochemical activity detection of Ru/glassy carbon electrode under dark and light conditions:

Using a three-electrode system, the Ru/glassy carbon electrode obtained in the previous step was placed in a PBS buffer (volume 2 mL, pH 7.0, concentration 0.005 mol/L). Under dark and light conditions (wherein the light conditions are: using yellow light with a wavelength of 550 nm as the light source, irradiating the electrode to be tested, and the irradiation time is 10 min), the electrochemical response of Ru NPs in the test data is detected and recorded by DPV. , where the abscissa is the potential range under the test conditions, and the ordinate is the current value corresponding to the oxidation peak of Ru NPs.

(3) Application of Ru/glassy carbon electrode in the detection of PSA

1) Preparation of Apt-Ru NPs solution:

The Ru NPs and PSA aptamer solution were mixed in Tris-boronic acid (TBE) buffer solution in a molar ratio of 1:80 (volume 100 μL, concentration 1 mM, pH value 8.2), and after mixing for 11 h at room temperature, the ligation The Ru NPs with aptamers were purified by centrifugation and then redissolved in ultrapure water to obtain Apt-Ru NPs solution.

2) Construction of electrochemical sensor:

The GO/Fe ₃ O ₄ NSs and the Apt-Ru NPs solution obtained in the previous step were mixed in a molar ratio of 1:2, and kept at 36 °C for 0.5 h. Then, the nanomaterials combined by π-π stacking in the mixed solution were redissolved in ultrapure water by magnetic adsorption to obtain Ru NPs@GO/Fe ₃ O ₄ NSs solution. After that, the alumina polishing powder with a particle size of 0.02 μm was mixed with ultrapure water, and the magnetic glassy carbon electrode (MGCE) with a diameter of 2mm was polished and ground, and then ultrasonically washed with ultrapure water and ethanol in turn. After drying with pure nitrogen, 2 μL of Ru NPs@GO/Fe ₃ O ₄ NSs solution was uniformly coated on the electrode surface and left at room temperature for 1 h to obtain Ru NPs@GO/Fe ₃ O ₄ NSs electrode. After the electrode was dried, a series of PSA standard solutions (8 μL) with different concentrations were dropped on the surface of the electrode. After 10 min at 36°C, the electrode was rinsed. The electrochemical response of Ru NPs@GO/Fe ₃ O ₄ NSs electrode after irradiation was detected and recorded by DPV, the abscissa is the logarithm of PSA concentration, and the ordinate is the oxidation peak of Ru NPs@GO/Fe ₃ O ₄ NSs electrode The peak intensity of the Ru NPs@GO/Fe ₃ O ₄ NSs electrode was established to establish a standard curve between the peak intensity of the oxidation peak and the logarithm of the PSA concentration.

Example 2:

The construction method of a ratiometric electrochemical sensor for tumor marker detection specifically includes the following steps:

(1) Preparation of Ru/glassy carbon electrode:

Using alumina polishing powder with a particle size of 0.05μm, after mixing with ultrapure water, the glassy carbon electrode with a diameter of 4mm was polished and ground, and then ultrasonically washed with ultrapure water and ethanol in turn, and dried with ultrapure nitrogen. 5 μL of RuNPs solution was evenly coated on the electrode surface and placed at room temperature for 1.5 h to obtain Ru/glassy carbon electrode;

(2) Electrochemical activity detection of Ru/glassy carbon electrode under dark and light conditions:

Using a three-electrode system, the Ru/glassy carbon electrode obtained in the previous step was placed in a PBS buffer (volume 3 mL, pH 7.4, concentration 0.01 mol/L). Under dark and light conditions (wherein the light conditions are: using yellow light with a wavelength of 600 nm as the light source, irradiating the electrode to be tested, and the irradiation time is 25 min), the electrochemical response of Ru NPs in the test data is detected and recorded by DPV. , where the abscissa is the potential range under the test conditions, and the ordinate is the current value corresponding to the oxidation peak of Ru NPs. The results are shown in Figure 1.

(3) Application of Ru/glassy carbon electrode in the detection of PSA

1) Preparation of Apt-Ru NPs solution:

The Ru NPs and PSA aptamer solution were mixed in Tris-boronic acid (TBE) buffer solution in a molar ratio of 1:100 (volume 200 μL, concentration 5 mM, pH value 8.3), and after mixing at room temperature for 12 h, the ligation The Ru NPs with aptamers were purified by centrifugation and then redissolved in ultrapure water to obtain Apt-Ru NPs solution.

2) Construction of electrochemical sensor:

The GO/Fe ₃ O ₄ NSs and the Apt-Ru NPs solution obtained in the previous step were mixed in a molar ratio of 1:3 and kept at 37 °C for 1 h. Then, the nanomaterials combined by π-π stacking in the mixed solution were redissolved in ultrapure water by magnetic adsorption to obtain Ru NPs@GO/Fe ₃ O ₄ NSs solution. After that, the alumina polishing powder with a particle size of 0.05 μm was mixed with ultrapure water, and the magnetic glassy carbon electrode (MGCE) with a diameter of 4mm was polished and ground, and then ultrasonically washed with ultrapure water and ethanol in turn, and ultrapure water was used. After drying with pure nitrogen, 5 μL of Ru NPs@GO/Fe ₃ O ₄ NSs solution was uniformly coated on the electrode surface and left at room temperature for 1.5 h to obtain Ru NPs@GO/Fe ₃ O ₄ NSs electrode. After the electrode was dried, a series of PSA standard solutions (10 μL) with different concentrations were dropped on the surface of the electrode. After 30min at 37°C, the electrode was rinsed. The electrochemical response of Ru NPs@GO/Fe ₃ O ₄ NSs electrode after irradiation was detected and recorded by DPV, the abscissa is the logarithm of PSA concentration, and the ordinate is the oxidation peak of Ru NPs@GO/Fe ₃ O ₄ NSs electrode peak intensity, the results are shown in Figure 2(A). In addition, a standard curve between the oxidation peak intensity of Ru NPs@GO/Fe ₃ O ₄ NSs electrode and the logarithmic value of PSA concentration was established, and the results are shown in Fig. 2(B).

Example 3:

The construction method of a ratiometric electrochemical sensor for tumor marker detection specifically includes the following steps:

(1) Preparation of Ru/glassy carbon electrode:

Using alumina polishing powder with a particle size of 0.08μm, after mixing with ultrapure water, the glassy carbon electrode with a diameter of 6mm was polished and ground, and then ultrasonically washed with ultrapure water and ethanol in turn, and dried with ultrapure nitrogen. 8 μL of RuNPs solution was evenly coated on the electrode surface and left at room temperature for 2 h to obtain a Ru/glassy carbon electrode;

(2) Electrochemical activity detection of Ru/glassy carbon electrode under dark and light conditions:

Using a three-electrode system, the Ru/glassy carbon electrode obtained in the previous step was placed in a PBS buffer (volume 4 mL, pH 7.5, concentration 0.015 mol/L). Under dark and light conditions (wherein the light conditions are: using yellow light with a wavelength of 650 nm as the light source, irradiating the electrode to be tested, and the irradiation time is 40 min), the electrochemical response of Ru NPs in the test data is detected and recorded by DPV. , where the abscissa is the potential range under the test conditions, and the ordinate is the current value corresponding to the oxidation peak of Ru NPs.

(3) Application of Ru/glassy carbon electrode in the detection of PSA

1) Preparation of Apt-Ru NPs solution:

The Ru NPs and the PSA aptamer solution were mixed in Tris-boronic acid (TBE) buffer solution (300 μL in volume, 10 mM, pH 8.4) in a molar ratio of 1:120. The Ru NPs with aptamers were purified by centrifugation and then redissolved in ultrapure water to obtain Apt-Ru NPs solution.

2) Construction of electrochemical sensor:

The GO/Fe ₃ O ₄ NSs and the Apt-Ru NPs solution obtained in the previous step were mixed in a molar ratio of 1:4, and kept at 38 °C for 1.5 h. Then, the nanomaterials combined by π-π stacking in the mixed solution were redissolved in ultrapure water by magnetic adsorption to obtain Ru NPs@GO/Fe ₃ O ₄ NSs solution. After that, the alumina polishing powder with a particle size of 0.08 μm was mixed with ultrapure water, and the magnetic glassy carbon electrode (MGCE) with a diameter of 6mm was polished and ground, and then ultrasonically washed with ultrapure water and ethanol in turn, and ultrapure water was used. After drying with pure nitrogen, 8 μL of Ru NPs@GO/Fe ₃ O ₄ NSs solution was uniformly coated on the electrode surface and left at room temperature for 2 h to obtain Ru NPs@GO/Fe ₃ O ₄ NSs electrode. After the electrode was dried, a series of PSA standard solutions (12 μL) with different concentrations were dropped on the surface of the electrode. After 50min at 38°C, the electrode was rinsed. The electrochemical response of Ru NPs@GO/Fe ₃ O ₄ NSs electrode after irradiation was detected and recorded by DPV, the abscissa is the logarithm of PSA concentration, and the ordinate is the oxidation peak of Ru NPs@GO/Fe ₃ O ₄ NSs electrode The peak intensity of the Ru NPs@GO/Fe ₃ O ₄ NSs electrode was established to establish a standard curve between the peak intensity of the oxidation peak and the logarithm of the PSA concentration.

test case

Accuracy determination

Take 3 mL of serum from positive patients and purify by centrifugation first. The obtained solid phase was then redissolved in PBS solution (volume 3 mL, concentration 0.1 M, pH=7.4). The PSA content in the solution was 1600 pg/mL as measured by a chemiluminescence immunoassay. Then dilute the solution or add PSA standard solutions of different concentrations respectively to obtain sample No. 1 (with PSA content of 100 pg/mL), sample No. 2 (with PSA content of 300 pg/mL), and sample No. 3 (with PSA content of 1000 pg). /mL), sample No. 4 (PSA content was 4000 pg/mL) and sample No. 5 (PSA content was 8000 pg/mL). Then, 10 μL of different samples were added dropwise to the Ru NPs@GO/Fe ₃ O ₄ NSs electrode prepared in Example 2, and incubated at 37 °C for 30 min. After that, the surface of the electrode was washed, and the Ru NPs@GO was detected and recorded. The electrochemical signal of /Fe ₃ O ₄ NSs electrode to obtain the content of PSA in different positive serum samples, as shown in Figure 3. The recovery rate of PSA in the positive serum was finally detected to be 98.2% to 101.8% (the recovery rate of sample 1 was 99.8%, the recovery rate of sample 2 was 101.7%, the recovery rate of sample 3 was 101.8%, and the recovery rate of sample 4 was 101.8%. 98.2%, the recovery rate of sample No. 5 was 98.6%). It can be seen from the recovery rate that the ratio electrochemical sensor constructed in Example 2 has good accuracy for the determination of PSA.

Claims (7)

1. the construction method of the specific electrochemical sensor of tumor marker detection, is characterized in that, described concrete construction method is: S1: Mix Ru NPs and tumor marker aptamer solution in Tris-boronic acid TBE buffer solution, mix and incubate at room temperature for 11-13 hours, then separate the solid-liquid to take the solid phase and re-disperse the solid phase in water to obtain Apt- Ru NPs solution; S2: The Apt-Ru NPs solution obtained in S1 was mixed and reacted with the GO/Fe ₃ O ₄ NSs solution, and then the magnetic substances in the solution were separated from the reaction solution by magnetic adsorption, and the magnetic substances were re-dissolved in water to obtain Ru NPs @GO/Fe ₃ O ₄ NSs solution; S3: Use alumina polishing powder to polish the magnetic glassy carbon electrode MGCE. After polishing, rinse the electrode surface with water and ethanol, keep the electrode surface dry, and then grind the Ru NPs@GO/Fe ₃ obtained in step S2. The O ₄ NSs solution was added dropwise to the polished MGCE and left at room temperature for 1-2 h to obtain Ru NPs@GO/Fe ₃ O ₄ NSs electrode. After standing for 10-50 min, the electrode was rinsed and illuminated to detect the electrochemical signal of the Ru NPs@GO/Fe ₃ O ₄ NSs electrode after illumination, and record the electrochemical response of the Ru NPs@GO/Fe ₃ O ₄ NSs electrode. This detects the PSA solution concentration. 2 . The method for constructing a specific electrochemical sensor for tumor marker detection according to claim 1 , wherein the molar ratio of Ru NPs and tumor marker aptamers in S1 is 1:80-1:120. 3 . 3 . The method for constructing a specific electrochemical sensor for tumor marker detection according to claim 1 , wherein the tumor marker in S1 comprises one of PSA, CEA or HER2. 4 . 4. The method for constructing a specific electrochemical sensor for tumor marker detection according to claim 1, wherein the molar ratio of Apt-Ru NPs to GO/Fe ₃ O ₄ NSs in S2 is 2:1-4 :1. 5. The method for constructing a specific electrochemical sensor for tumor marker detection according to claim 1, wherein the conditions for the mixed reaction of the Apt-Ru NPs solution and the GO/Fe ₃ O ₄ NSs solution in S2 are: Keep at 36～38℃ for 0.5～1.5h. 6 . The method for constructing a specific electrochemical sensor for tumor marker detection according to claim 1 , wherein the illumination conditions in S3 are: using yellow light with a wavelength of 550-650 nm as a light source, irradiating the to-be-measured Electrodes, the irradiation time is 10 ~ 40min. 7. An application of the specific electrochemical sensor for tumor marker detection according to any one of claims 1-6, wherein the detection is not for the purpose of disease treatment and prevention.

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