CN112129819B - Construction method and application of specific electrochemical sensor for detecting tumor marker - Google Patents
Construction method and application of specific electrochemical sensor for detecting tumor marker Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of electrochemical analysis, and particularly relates to a construction method and application of a specific electrochemical sensor for detecting a tumor marker.
Background
The electrochemical oxidation-reduction active probe is the basis for constructing a stable electrochemical biosensor and is the key for the research and development and clinical application of a rapid detection electrode. Compared with electroactive organic molecules, the electroactive nano material has unique electronic characteristics and high electron transfer efficiency, does not need extra carrier load, obtains electrochemical redox signals with adjustable strength and selectable peak positions by regulating and controlling the size, the composition and the structure, and meets the specific requirements of biological application. However, the research of the electroactive nanomaterial itself as a signal label is still in the initial stage, and only a few reports are available. Moreover, the research on the electroactive metal nano-materials is mainly focused on Ag, Cu2O, and the like. Therefore, there is a need to develop a novel electroactive nanoprobe with unique electrochemical response to expand the application of nanoparticles in the electrochemical field.
Among transition metals, Ru has excellent characteristics of high selectivity, corrosion resistance and high temperature resistance. And the d electron orbit is not filled, so that the device has good electrical activity. Therefore, in recent years, Ru nanomaterials are widely used in various fields of photochemistry and electrochemistry (e.g., photocatalytic nitrogen fixation, electrocatalytic hydrogen evolution, electrocatalytic oxygen evolution, etc.). However, there are few reports on the study on whether Ru nanomaterials can change their electrochemical signals by light energy. The research can explore the potential relation between the light energy and the electric energy on the nanometer material, and provide potential development prospect for the mutual combination of the two energy sources. Therefore, the intrinsic relationship between the electrochemical signal and the optical energy of the nanomaterial needs to be further explored. In addition, the electrochemical signal is enhanced by applying the light energy, the signal intensity of the electroactive nanoprobe is further improved, the construction of a sensor for detecting a tumor marker is facilitated, and the application field of the sensor is widened.
Disclosure of Invention
In order to solve the problems in the prior art, the applicant of the present invention provides a construction method and an application of a specific electrochemical sensor for detecting a tumor marker. The invention discloses that the Ru nano material has a unique electrochemical oxidation peak for the first time and can be used as a novel electroactive nano probe. The electrochemical oxidation peak of the Ru nano material is found to change under illumination and the reason of the change is researched. In addition, the characteristic that the electrochemical oxidation peak is enhanced by utilizing illumination is utilized to construct an electrochemical sensor for detecting a tumor marker.
The technical scheme of the invention is as follows:
the construction method of the specific electrochemical sensor for detecting the tumor marker comprises the following steps:
s1: mixing the Ru NPs and the tumor marker aptamer solution in a Tris-TBE buffer solution, incubating for 11-13 h at normal temperature, performing solid-liquid separation to obtain a solid phase, and re-dispersing the solid phase in water to obtain an Apt-Ru NPs solution;
s2: mixing Apt-Ru NPs solution obtained in S1 with GO/Fe3O4NSs solution is mixed and reacted, then magnetic adsorption is utilized to separate magnetic substances in the solution from reaction solution, and the magnetic substances are re-dissolved in water to obtain Ru NPs @ GO/Fe3O4(ii) a NSs solution;
s3: polishing and grinding the MGCE by using alumina polishing powder, and after the MGCE is polished to be clean, using water and ethanol to polish the surface of the electrodeRinsing the surface to keep the electrode surface dry, and then adding the Ru NPs @ GO/Fe obtained in step S23O4The NSs solution is dripped on MGCE after polishing, and the mixture is placed at room temperature for 1-2h to finally obtain Ru NPs @ GO/Fe3O4NSs electrode, after the electrode surface is dried, adding PSA solution on the electrode surface, placing for 10-50 min at 36-38 ℃, then washing and illuminating the electrode, and detecting Ru NPs @ GO/Fe after illumination3O4Electrochemical signal of NSs electrode, and recording Ru NPs @ GO/Fe3O4The electrochemical response of the NSs electrode is detected, so that the concentration of the PSA solution is detected.
Furthermore, the molar ratio of the Ru NPs to the tumor marker aptamer in S1 is 1: 80-1: 120.
Further, the tumor marker in S1 includes one of PSA, CEA, or HER 2.
Further, Apt-Ru NPs and GO/Fe in S23O4The NSs molar ratio is 2: 1-4: 1.
Further, Apt-Ru NPs solution and GO/Fe in S23O4The NSs solution mixing reaction conditions are as follows: keeping the temperature at 36-38 ℃ for 0.5-1.5 h.
Further, in S3, the illumination conditions are: yellow light with the wavelength of 550-650 nm is used as a light source to irradiate the electrode to be measured, and the irradiation time is 10-40 min.
Furthermore, the application of the specific electrochemical sensor for detecting the tumor marker is not aimed at treating and preventing diseases.
The beneficial technical effects of the invention are as follows:
the invention discovers for the first time that the Ru nano material has a unique and strong electrochemical oxidation peak when the potential is about 0.8V (compared with an Ag/AgCl electrode). Compared with the conventional electroactive metal nano material, the electroactive metal nano material can be used as a novel electroactive nano probe, and provides a foundation for constructing an electrochemical biosensor in the future.
Based on the phenomenon that the electrochemical oxidation peak of the Ru nano material is enhanced after illumination, the reason that the Ru nano material has a strong electrochemical signal is found through research and is due to the fact that a large number of electron-hole pairs are generated in Ru NPs under the excitation of Local Surface Plasmon Resonance (LSPR). The generated photogenerated electrons flow to an external circuit from the Ru NPs, and the left holes have oxidation action, so that the oxidation of the Ru NPs is promoted, and the electrochemical signal of the Ru NPs is enhanced 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 utilizing sulfydryl to form Apt-Ru NPs. Then Apt-Ru NPs and GO/Fe are subjected to pi-pi accumulation effect between the aptamer and the graphene3O4NSs assembled together to form Ru NPs @ GO/Fe3O4NSs. Due to the formation of Ru NPs @ GO/Fe3O4The NSs have magnetism and can be directly adsorbed on the MGCE, so that additional modification operation on the electrode is not needed. Due to the existence of Ru NPs, the electrode adsorbed with the nano material has excellent electrochemical signals under the condition of illumination. When the electrode is contacted with a target object, due to the excellent binding force between the aptamer and the tumor marker, the Ru NPs can fall off from the surface of the electrode, and the previous strong electrochemical signal is weakened accordingly. An electrochemical sensor for specifically detecting a tumor marker was constructed according to this principle.
Compared with the traditional research on the Ru nano material, the traditional research only uses the Ru nano material in the photochemical or electrochemical field, thereby being limited to a certain extent. The invention explores the internal relation between the electrochemical signal and the light energy of the Ru nano material, constructs the electrochemical sensor for specifically detecting the tumor marker based on the relation between the electrochemical signal and the light energy, and provides a potential development prospect for the mutual combination and application of the light energy and the electric energy.
Drawings
FIG. 1 shows DPV electrochemical signal response of Ru nano-materials in example 2 under dark and light conditions.
FIG. 2 is a graph showing the detection results of the specific electrochemical sensor prepared in example 2 on PSA after light irradiation, wherein A represents Ru NPs @ GO/Fe3O4After NSs electrode reacts with PSA with different concentrations, DPV electrochemical signal response condition of the NSs electrode is shown in B diagram of Ru NPs @ GO/Fe3O4Standard curve between DPV electrochemical signal and PSA concentration log for NSs electrodes.
Fig. 3 is a graph showing the results of the electrochemical sensor prepared in example 2 detecting positive sera with different PSA concentrations, and the electrochemical sensor constructed in example 2 was evaluated for the accuracy of PSA detection.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
Example 1:
the construction method of the tumor marker detection ratio electrochemical sensor specifically comprises the following steps:
(1) preparation of Ru/glassy carbon electrode:
mixing alumina polishing powder with the particle size of 0.02 mu m with ultrapure water, polishing and grinding a glassy carbon electrode with the diameter of 2mm, then sequentially ultrasonically washing with the ultrapure water and ethanol, blowing the ultrapure nitrogen for standby, uniformly coating 2 mu L of Ru NPs solution on the surface of the electrode, and standing for 1h at room temperature to obtain a Ru/glassy carbon electrode;
(2) and (3) detecting the electrochemical activity of the Ru/glassy carbon electrode under dark and light conditions:
the Ru/glassy carbon electrode obtained in the previous step was placed in PBS buffer (2 mL in volume, 7.0 pH, 0.005mol/L) using a three-electrode system. And respectively detecting and recording the electrochemical response condition of the Ru NPs in the test data by DPV under dark and light conditions (wherein the light condition is that yellow light with the wavelength of 550nm is used as a light source to irradiate the electrode to be tested for 10min), wherein the abscissa is the potential range under the test condition, and the ordinate is the current value corresponding to the oxidation peak of the Ru NPs.
(3) Application of Ru/glassy carbon electrode in detection of PSA (pressure swing adsorption)
1) Preparation of Apt-Ru NPs solution:
mixing the Ru NPs and the PSA aptamer solution in a molar ratio of 1:80 in a Tris-boric acid (TBE) buffer solution (the volume is 100 mu L, the concentration is 1mM, and the pH value is 8.2), mixing at normal temperature for 11h, then carrying out centrifugal purification on the Ru NPs connected with the aptamer, and then re-dissolving the Ru NPs in ultrapure water to obtain an Apt-Ru NPs solution.
2) Construction of the electrochemical sensor:
mixing GO/Fe3O4NSs and the Apt-Ru NPs solution obtained in the previous step are mixed according to the molar ratio of 1:2, and the mixture is kept at 36 ℃ for 0.5 h. Then, dissolving the nanometer material combined by pi-pi accumulation in the mixed solution into ultrapure water again by using magnetic adsorption to obtain Ru NPs @ GO/Fe3O4NSs solution. Then mixing alumina polishing powder with the particle size of 0.02 mu m with ultrapure water, polishing and grinding a Magnetic Glassy Carbon Electrode (MGCE) with the diameter of 2mm, then sequentially ultrasonically washing with the ultrapure water and ethanol, blowing with the ultrapure nitrogen for standby application, and drying 2 mu L of Ru NPs @ GO/Fe3O4The NSs solution is uniformly coated on the surface of the electrode and placed for 1h at room temperature to obtain Ru NPs @ GO/Fe3O4NSs electrode. After the electrode was dried, a series of PSA standard solutions (8 μ L) with different concentrations were added dropwise to the electrode surface. After standing at 36 ℃ for 10min, the electrodes were rinsed. Detecting and recording Ru NPs @ GO/Fe after illumination through DPV3O4The electrochemical response of the NSs electrode is shown by the logarithm of PSA concentration on the abscissa and Ru NPs @ GO/Fe on the ordinate3O4Establishing Ru NPs @ GO/Fe by using oxidation peak intensity of NSs electrode3O4Standard curve between oxidation peak intensity and PSA concentration log for NSs electrodes.
Example 2:
the construction method of the tumor marker detection ratio electrochemical sensor specifically comprises the following steps:
(1) preparation of Ru/glassy carbon electrode:
mixing alumina polishing powder with the particle size of 0.05 mu m with ultrapure water, polishing and grinding a glassy carbon electrode with the diameter of 4mm, then sequentially ultrasonically washing with the ultrapure water and ethanol, blowing the ultrapure nitrogen for standby, uniformly coating 5 mu L of Ru NPs solution on the surface of the electrode, and standing at room temperature for 1.5h to obtain a Ru/glassy carbon electrode;
(2) and (3) detecting the electrochemical activity of the Ru/glassy carbon electrode under dark and light conditions:
the Ru/glassy carbon electrode obtained in the previous step was placed in PBS buffer (volume 3mL, pH 7.4, concentration 0.01mol/L) using a three-electrode system. Under dark and light conditions (wherein the light condition is that yellow light with the wavelength of 600nm is used as a light source to irradiate the electrode to be tested for 25min), the electrochemical response condition of the Ru NPs in the test data is detected and recorded by DPV, wherein the abscissa is the potential range under the test condition, and the ordinate is the current value corresponding to the oxidation peak of the Ru NPs, and the result is shown in figure 1.
(3) Application of Ru/glassy carbon electrode in detection of PSA (pressure swing adsorption)
1) Preparation of Apt-Ru NPs solution:
mixing the Ru NPs and the PSA aptamer solution in a molar ratio of 1:100 in a Tris-boric acid (TBE) buffer solution (the volume is 200 mu L, the concentration is 5mM, and the pH value is 8.3), mixing at normal temperature for 12h, then carrying out centrifugal purification on the Ru NPs connected with the aptamer, and then re-dissolving the Ru NPs in ultrapure water to obtain an Apt-Ru NPs solution.
2) Construction of the electrochemical sensor:
mixing GO/Fe3O4NSs and the Apt-Ru NPs solution obtained in the previous step are mixed according to the molar ratio of 1:3, and the mixture is kept at 37 ℃ for 1 h. Then, dissolving the nanometer material combined by pi-pi accumulation in the mixed solution into ultrapure water again by using magnetic adsorption to obtain Ru NPs @ GO/Fe3O4NSs solution. Then mixing alumina polishing powder with the particle size of 0.05 mu m with ultrapure water, polishing and grinding a Magnetic Glassy Carbon Electrode (MGCE) with the diameter of 4mm, then sequentially ultrasonically washing with the ultrapure water and ethanol, blowing with the ultrapure nitrogen for standby, and drying 5 mu L of Ru NPs @ GO/Fe3O4The NSs solution is uniformly coated on the surface of the electrode and is placed for 1.5h at room temperature to obtain Ru NPs @ GO/Fe3O4NSs electrode. After the electrode was dried, a series of PSA standard solutions (10 μ L) with different concentrations were added dropwise to the electrode surface. After standing at 37 ℃ for 30min, the electrodes were rinsed. Detecting and recording Ru NPs @ GO/Fe after illumination through DPV3O4Electrochemical response of NSs electrodesThe abscissa is the logarithm of the PSA concentration and the ordinate is the Ru NPs @ GO/Fe3O4The oxidation peak intensity of the NSs electrode is shown in fig. 2 (a). In addition, Ru NPs @ GO/Fe is established3O4The results of a standard curve of oxidation peak intensity of NSs electrode versus PSA concentration log are shown in fig. 2 (B).
Example 3:
the construction method of the tumor marker detection ratio electrochemical sensor specifically comprises the following steps:
(1) preparation of Ru/glassy carbon electrode:
mixing alumina polishing powder with the particle size of 0.08 mu m with ultrapure water, polishing and grinding a glassy carbon electrode with the diameter of 6mm, then sequentially ultrasonically washing with the ultrapure water and ethanol, blowing the ultrapure nitrogen for standby, uniformly coating 8 mu L of Ru NPs solution on the surface of the electrode, and standing for 2 hours at room temperature to obtain a Ru/glassy carbon electrode;
(2) and (3) detecting the electrochemical activity of the Ru/glassy carbon electrode under dark and light conditions:
the Ru/glassy carbon electrode obtained in the previous step was placed in PBS buffer (volume 4mL, pH 7.5, concentration 0.015mol/L) using a three-electrode system. And respectively detecting and recording the electrochemical response condition of the Ru NPs in the test data by DPV under dark and light conditions (wherein the light condition is that yellow light with the wavelength of 650nm is used as a light source to irradiate the electrode to be tested, and the irradiation time is 40min), wherein the abscissa is the potential range under the test condition, and the ordinate is the current value corresponding to the oxidation peak of the Ru NPs.
(3) Application of Ru/glassy carbon electrode in detection of PSA (pressure swing adsorption)
1) Preparation of Apt-Ru NPs solution:
mixing the Ru NPs and the PSA aptamer solution in a Tris-boric acid (TBE) buffer solution (the volume is 300 mu L, the concentration is 10mM, and the pH value is 8.4) according to a molar ratio of 1:120, mixing the mixture at normal temperature for 13h, then carrying out centrifugal purification on the Ru NPs connected with the aptamer, and then re-dissolving the mixture in ultrapure water to obtain an Apt-Ru NPs solution.
2) Construction of the electrochemical sensor:
mixing GO/Fe3O4NSs and the Apt-Ru NPs solution obtained in the previous step are mixed according to the molar ratio of 1:4, and the mixture is kept at 38 ℃ for 1.5 h. Then, dissolving the nanometer material combined by pi-pi accumulation in the mixed solution into ultrapure water again by using magnetic adsorption to obtain Ru NPs @ GO/Fe3O4NSs solution. Then mixing alumina polishing powder with the particle size of 0.08 mu m with ultrapure water, polishing and grinding a Magnetic Glassy Carbon Electrode (MGCE) with the diameter of 6mm, then sequentially ultrasonically washing with the ultrapure water and ethanol, blowing with the ultrapure nitrogen for standby application, and drying 8 mu L of Ru NPs @ GO/Fe3O4The NSs solution is uniformly coated on the surface of the electrode and placed for 2 hours at room temperature to obtain Ru NPs @ GO/Fe3O4NSs electrode. After the electrode was dried, a series of PSA standard solutions (12 μ L) with different concentrations were added dropwise to the electrode surface. After standing at 38 ℃ for 50min, the electrodes were rinsed. Detecting and recording Ru NPs @ GO/Fe after illumination through DPV3O4The electrochemical response of the NSs electrode is shown by the logarithm of PSA concentration on the abscissa and Ru NPs @ GO/Fe on the ordinate3O4Establishing Ru NPs @ GO/Fe by using oxidation peak intensity of NSs electrode3O4Standard curve between oxidation peak intensity and PSA concentration log for NSs electrodes.
Test example
Determination of accuracy
Taking 3mL of serum of a positive patient, and carrying out centrifugal purification firstly. The solid phase obtained was then redissolved in PBS solution (volume 3mL, concentration 0.1M, pH 7.4). The PSA content of the solution was 1600pg/mL by chemiluminescence immunoassay. Then diluting the solution or respectively adding PSA standard solutions with different concentrations to obtain a sample No. 1 (PSA content of 100pg/mL), a sample No. 2 (PSA content of 300pg/mL), a sample No. 3 (PSA content of 1000pg/mL), a sample No. 4 (PSA content of 4000pg/mL) and a sample No. 5 (PSA content of 8000 pg/mL). Then 10. mu.L of each of the different samples was added dropwise to the Ru NPs @ GO/Fe prepared in example 23O4NSs electrode, incubating for 30min at 37 ℃, then washing the electrode surface, detecting and recording Ru NPs @ GO/Fe3O4The electrochemical signal of the NSs electrode, and thus the PSA content of the different positive serum samples obtained, are shown in fig. 3. Finally, the recovery rate of PSA in the positive serum is detected to be 98.2-101.8% (the recovery rate of sample No. 1 is 99.8%, the recovery rate of sample No. 2 is 101.7%, the recovery rate of sample No. 3 is 101.8%, the recovery rate of sample No. 4 is 98.2%, and the recovery rate of sample No. 5 is 98.6%). From the recovery rate, the ratiometric electrochemical sensor constructed in example 2 had good accuracy for the determination of PSA.
Claims (7)
1. The construction method of the specific electrochemical sensor for detecting the tumor marker is characterized by comprising the following steps:
s1: mixing the Ru NPs and the tumor marker aptamer solution in a Tris-TBE buffer solution, incubating for 11-13 h at normal temperature, performing solid-liquid separation to obtain a solid phase, and re-dispersing the solid phase in water to obtain an Apt-Ru NPs solution;
s2: mixing Apt-Ru NPs solution obtained in S1 with GO/Fe3O4NSs solution is mixed and reacted, then magnetic adsorption is utilized to separate magnetic substances in the solution from reaction solution, and the magnetic substances are re-dissolved in water to obtain Ru NPs @ GO/Fe3O4(ii) a NSs solution;
s3: polishing and grinding the MGCE by using alumina polishing powder, rinsing the surface of the electrode by using water and ethanol after the MGCE is polished to be clean, keeping the surface of the electrode dry, and then performing the step S2 to obtain Ru NPs @ GO/Fe3O4The NSs solution is dripped on MGCE after polishing, and the mixture is placed at room temperature for 1-2h to finally obtain Ru NPs @ GO/Fe3O4NSs electrode, dripping tumor marker aptamer solution on the surface of the electrode after the surface of the electrode is dried, standing at 36-38 ℃ for 10-50 min, washing the electrode and illuminating, and detecting Ru NPs @ GO/Fe after illumination3O4Electrochemical signal of NSs electrode, and recording Ru NPs @ GO/Fe3O4The electrochemical response condition of the NSs electrode is used for detecting the concentration of the tumor marker aptamer solution.
2. The method for constructing a specific electrochemical sensor for detecting a tumor marker according to claim 1, wherein the molar ratio of Ru NPs to the tumor marker aptamer in S1 is 1: 80-1: 120.
3. The method for constructing a specific electrochemical sensor for detecting tumor markers according to claim 1, wherein the tumor marker in S1 comprises one of PSA, CEA or HER 2.
4. The method of claim 1, wherein Apt-Ru NPs and GO/Fe are detected in S23O4The NSs molar ratio is 2: 1-4: 1.
5. The method of claim 1, wherein the Apt-Ru NPs solution in S2 is mixed with GO/Fe3O4The NSs solution mixing reaction conditions are as follows: keeping the temperature at 36-38 ℃ for 0.5-1.5 h.
6. The method of claim 1, wherein the illumination conditions in S3 are as follows: yellow light with the wavelength of 550-650 nm is used as a light source to irradiate the electrode to be measured, and the irradiation time is 10-40 min.
7. Use of a specific electrochemical sensor for the detection of a tumor marker as defined in any one of claims 1 to 6, which is not for the purpose of disease treatment or diagnosis.
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