Application of porous gold electrode in field of electrochemical luminescence sensing

Abstract

The invention belongs to the technical field of electrochemical luminescence sensors, and particularly relates to application of a porous electrode in an electrochemical luminescence sensor and the electrochemical luminescence sensor. The electrochemical luminescence sensor is prepared by taking the porous electrode as a working electrode, the prepared porous electrode has a relatively large specific surface area, and compared with an electrode with a smooth surface, the porous electrode can accelerate redox current and improve accessibility of an analyte. Due to the large surface area and the high volume ratio, the porous electrode can enhance the interaction between the metal surface and biomolecules, so that the sensitivity is remarkably improved, and the application range of the electrochemical luminescence sensor is expanded. The test result of the embodiment shows that compared with the traditional planar gold substrate, the ECL intensity based on [Ru (bpy) 3] < 2 + > is improved by more than 80 times, which indicates that the electrochemical luminescence sensor has stronger optical response, so that the electrochemical luminescence sensor prepared from the porous electrode is more effective in ultrasensitive biosensing application.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemiluminescence sensors, and in particular relates to the application of a porous electrode in an electrochemiluminescence sensor and an electrochemiluminescence sensor. Background Art

[0002] Electrochemiluminescence (ECL) sensors are an important biosensing technology that converts chemical energy into light signals, providing a sensitive and efficient method for the quantitative detection of target molecules. ECL is a unique optical sensing method that produces light through a chemical reaction with the electric field in a solution. Currently, a challenge facing ECL sensors is their limited ability to detect low-abundance molecules (such as miRNAs and neurodegenerative biomarkers), which affects the accuracy of their detection results and limits their scope of application. Improving the detection sensitivity of ECL sensors is a technical problem that urgently needs to be solved. Summary of the Invention

[0003] In view of this, the present invention provides an application of a porous electrode in an electrochemiluminescence sensor and an electrochemiluminescence sensor. Using the porous electrode as a working electrode to prepare an electrochemiluminescence sensor greatly improves the detection sensitivity of the electrochemiluminescence sensor and expands its application range.

[0004] In order to solve the above technical problems, the present invention provides an application of a porous electrode in an electrochemiluminescence sensor, wherein the porous electrode serves as a working electrode of the electrochemiluminescence sensor.

[0005] Preferably, the porous electrode comprises a substrate and a gold layer having a porous structure attached to the surface of the substrate.

[0006] Preferably, the substrate comprises a silicon wafer, a titanium layer and a gold plane layer stacked in sequence, and the gold layer containing the pore structure is in direct contact with the gold plane layer.

[0007] Preferably, the thickness of the silicon wafer is 0.8 to 1.2 mm, and the thickness of the titanium layer is 18 to 22 nm.

[0008] Preferably, the thickness of the gold plane layer is 148-152 nm.

[0009] Preferably, the thickness of the gold layer containing the pore structure is 0.18 to 0.22 μm.

[0010] Preferably, the average pore diameter of the pore structure in the gold layer containing the pore structure is 30 to 60 nm.

[0011] Preferably, the porosity of the gold layer containing the porous structure is 48-52%.

[0012] Preferably, the method for preparing the porous electrode comprises the following steps:

[0013] dissolving a polystyrene-block-polyethylene oxide diblock copolymer in an organic solvent to obtain a first solution;

[0014] adding a swelling agent, ethanol, an aqueous solution of tetrachloroauric acid and water to the first solution in sequence to obtain a gold precursor solution;

[0015] A titanium layer and a gold plane layer are sequentially arranged on the surface of the substrate, and then a gold precursor solution is electro-deposited on the surface of the gold plane layer to form a gold layer containing a pore structure, thereby obtaining the porous electrode.

[0016] The present invention also provides an electrochemiluminescence sensor, wherein the working electrode of the electrochemiluminescence sensor is the porous electrode.

[0017] The present invention uses a porous electrode as a working electrode to prepare an electrochemiluminescence sensor. The porous electrode has a large specific surface area, which accelerates the conduction rate of the redox current and improves the accessibility of the analyte compared to an electrode with a smooth surface. Due to the large surface area and high volume ratio, the porous electrode can enhance the interaction between the metal surface and the biological molecules, thereby significantly improving the sensitivity and expanding the application range of the electrochemiluminescence sensor. The test results of the embodiment show that compared with the traditional planar gold substrate, the ECL intensity of the planar electrode based on the present invention is increased by more than 80 times, which shows that it has a stronger optical response, making the electrochemiluminescence sensor prepared by the porous electrode more effective in ultra-sensitive biosensing applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The SEM images of the porous electrodes prepared in Examples 1 to 4 are shown;

[0019] Figure 2 is a pore size distribution curve diagram of the porous electrodes prepared in Examples 1 to 4;

[0020] Figure 3 The XPS spectra of the porous electrode prepared in Example 3, wherein a is the XPS wide scan spectrum and b is the high resolution scan spectrum;

[0021] Figure 4 The XRD spectrum of the porous electrode prepared in Example 3, wherein d is the enlarged spectrum of 30-80° in c;

[0022] Figure 5 The results of emission spectroscopy-electrochemical characterization of the electrodes of Examples 1 to 6 and Comparative Example 1 are shown in Figure 1, where a is a cyclic voltammogram and b is a graph showing the [Ru(bpy)3] measured at 1.5 V using an electrode. 3+a is a graph showing the change of ECL intensity over time, c is a chronopotentiometry curve at 1.5 V, and d is an ECL emission spectrum of various electrodes at 1.5 V using the potential-static method;

[0023] Figure 6 The electrochemiluminescence results of the electrodes of Examples 1 to 6 are shown in FIG. DETAILED DESCRIPTION

[0024] The present invention provides an application of a porous electrode in an electrochemiluminescence sensor, wherein the porous electrode serves as a working electrode of the electrochemiluminescence sensor.

[0025] As a specific embodiment of the present invention, the porous electrode may include a substrate and a gold layer containing a porous structure attached to the surface of the substrate. As a specific embodiment of the present invention, the substrate may include a silicon wafer, a titanium layer, and a gold planar layer stacked in sequence, with the gold layer containing the porous structure directly contacting the gold planar layer; the thickness of the silicon wafer may be 0.8 to 1.2 mm, specifically 1 mm; the thickness of the titanium layer may be 18 to 22 nm, specifically 20 nm; and the thickness of the gold planar layer may be 148 to 152 nm, specifically 150 nm.

[0026] As a specific embodiment of the present invention, the thickness of the gold layer containing a pore structure can be 0.18 to 0.22 μm, specifically 0.2 μm; the average pore diameter of the pore structure in the gold layer containing a pore structure can be 30 to 60 nm, specifically 30 nm, 40 nm, 50 nm, 60 nm, 70 nm or 80 nm; the porosity of the gold layer containing a pore structure can be 48 to 52%, specifically 50%.

[0027] As a specific embodiment of the present invention, the method for preparing the porous electrode may include the following steps:

[0028] dissolving a polystyrene-block-polyethylene oxide diblock copolymer in an organic solvent to obtain a first solution;

[0029] adding a swelling agent, ethanol, an aqueous solution of tetrachloroauric acid and water to the first solution in sequence to obtain a gold precursor solution;

[0030] A titanium layer and a gold plane are sequentially arranged on the surface of the substrate, and then a gold precursor solution is electro-deposited on the surface of the gold plane layer to form a gold layer containing a pore structure, thereby obtaining the porous electrode.

[0031] In the present invention, unless otherwise specified, all materials are commercially available products of conventional analytical grade.

[0032] The present invention dissolves a polystyrene-block-polyethylene oxide diblock copolymer in an organic solvent to obtain a first solution. As a specific embodiment of the present invention, the organic solvent may include tetrahydrofuran; the polystyrene-block-polyethylene oxide diblock copolymer may be PS18000-b-PEO7500; the volume ratio of the mass of the polystyrene-block-polyethylene oxide diblock copolymer to the organic solvent may be 0.03g:8~10mL, and may be specifically 0.03g:9mL. As a specific embodiment of the present invention, the dissolution may be under stirring, the stirring temperature may be 35~45°C, and may be specifically 40°C; the stirring may be magnetic stirring. The present invention may provide the required stirring temperature by a water bath.

[0033] After obtaining the first solution, the present invention sequentially adds a swelling agent, ethanol, an aqueous solution of tetrachloroauric acid, and water to the first solution to obtain a gold precursor solution. As a specific embodiment of the present invention, the swelling agent may include triisopropylbenzene (TIPBz), and the volume ratio of triisopropylbenzene to the aqueous solution of tetrachloroauric acid may be 20 to 120 μL:3mL, specifically 20 μL:3mL, 40 μL:3mL, 60 μL:3mL, 80 μL:3mL, 100 μL:3mL, or 120 μL:3mL. The present invention adjusts the pore size of the pore structure by adjusting the amount of the swelling agent. When the volume ratio of triisopropylbenzene to the tetrachloroauric acid aqueous solution is 20 μL:3 mL, 40 μL:3 mL, 60 μL:3 mL, 80 μL:3 mL, 100 μL:3 mL, or 120 μL:3 mL, the resulting pore diameter is 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, or 80 nm, respectively. As a specific embodiment of the present invention, the molar concentration of the tetrachloroauric acid aqueous solution can be 38 to 42 mmol / L, specifically 40 mmol / L; the water can be deionized water. As a specific embodiment of the present invention, the volume ratio of the first solution to ethanol can be 30:4 to 5, specifically 30:4.5; the volume ratio of the first solution to the tetrachloroauric acid aqueous solution can be 10:00.8 to 1.2, specifically 10:1; and the volume ratio of the first solution to water can be 30:7 to 8, specifically 30:7.5. The present invention has no special requirements on the rate of the dripping, as long as it can be added dropwise.

[0034] As a specific embodiment of the present invention, after the dropwise addition is completed, the following step may be further included: stirring the mixed system after the dropwise addition; the stirring time may be 25 to 35 minutes, specifically 25 minutes, 28 minutes, 30 minutes or 35 minutes; the stirring temperature may be room temperature, and the room temperature may be 20 to 35 degrees Celsius, or 25 to 30 degrees Celsius. In the present invention, micelles can be formed after adding an expander, ethanol, an aqueous solution of tetrachloroauric acid and water to the first solution, and the micelles can be evenly dispersed in the system after stirring; the micelles are used as a directing agent for the pore size of the pore structure in the gold layer containing the pore structure. The present invention has no special requirements for the stirring speed, as long as the micelles can be evenly dispersed. In the present invention, the gold precursor solution is yellow and transparent.

[0035] After obtaining the gold precursor solution, the present invention sequentially arranges a titanium layer and a gold plane layer on the surface of the substrate, and then electro-deposits the gold precursor solution on the surface of the gold plane layer to form a gold layer containing a pore structure, thereby obtaining the porous electrode. As a specific embodiment of the present invention, the substrate can be a silicon wafer, and the silicon wafer can be a single-sided polished silicon wafer. The present invention arranges a titanium layer on the polished surface. As a specific embodiment of the present invention, before sequentially arranging the titanium layer on the substrate surface, the present invention can further include: cleaning the substrate and then drying it; the cleaning can be ultrasonic cleaning in acetone and isopropanol followed by water washing, and the time for ultrasonic cleaning in acetone can be 8 to 12 minutes, and can be specifically 10 minutes; the time for ultrasonic cleaning in isopropanol can be 8 to 12 minutes, and can be specifically 10 minutes; the water used for water washing can be deionized water, and the present invention can remove residual acetone or isopropanol on the surface of the substrate through water washing. As a specific embodiment of the present invention, the drying can be drying with nitrogen blowing.

[0036] As a specific embodiment of the present invention, the method of setting the titanium layer on the surface of the substrate can be electron beam evaporation physical deposition, and the method of depositing the gold plane layer on the surface of the titanium layer can be electron beam evaporation physical deposition; the present invention has no special requirements for the electron beam evaporation physical deposition, and the conventional methods in the field can be used.

[0037] As a specific embodiment of the present invention, a three-electrode system can be used to electrodeposit a gold precursor solution on a gold plane layer; the reference electrode of the three-electrode system is an Ag / AgCl electrode, the counter electrode is a Pt mesh, and the working electrode is a substrate containing a titanium layer and a gold plane layer.

[0038] The present invention also provides an electrochemiluminescence sensor, wherein the working electrode of the electrochemiluminescence sensor is a porous electrode. The present invention has no particular limitation on the preparation method of the electrochemiluminescence sensor, and conventional methods in the art can be used.

[0039] The electrochemiluminescence sensor provided by the present invention can be used as a cancer and neurodegenerative disease detection device. As a specific embodiment of the present invention, the gain ratio when using the electrochemiluminescence sensor to detect cancer or neurodegenerative disease biomarkers is about 80 times.

[0040] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] 0.03 g of polystyrene-block-polyethylene oxide diblock copolymer (PS18000-b-PEO7500) was dissolved (with magnetic stirring in a 40° C. water bath) in 9 mL of tetrahydrofuran to obtain a first solution.

[0043] 20 μL of triisopropylbenzene (swelling agent), 4.5 mL of ethanol, 3 mL of a 40 mmol / L aqueous solution of tetrachloroauric acid, and 7.5 mL of deionized water were sequentially added dropwise to the first solution, and the mixture was stirred at 30° C. for 30 min to obtain a yellow transparent gold precursor solution.

[0044] A single-side polished 1mm thick silicon wafer was used as the substrate. The substrate was ultrasonically cleaned in acetone and isopropanol for 10 minutes, rinsed with deionized water, and blown dry with nitrogen. A 20nm thick titanium layer was deposited on the polished surface of the substrate using electron beam evaporation physical deposition. A 150nm thick gold planar layer was deposited on the surface of the titanium layer using electron beam evaporation physical deposition.

[0045] A three-electrode system was used to electrodeposit a gold precursor solution on a gold flat layer to form a gold layer with a porous structure (average pore size of 30 nm), thereby obtaining a porous electrode. The three-electrode system consisted of an Ag / AgCl reference electrode, a Pt mesh counter electrode, and a substrate containing a titanium layer and a gold flat layer.

[0046] Example 2

[0047] A porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added dropwise was 40 μL; and the average pore diameter of the pore structure in the prepared porous electrode was 40 nm.

[0048] Example 3

[0049] A porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added dropwise was 60 μL; and the average pore diameter of the pore structure in the prepared porous electrode was 50 nm.

[0050] Example 4

[0051] A porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added dropwise was 80 μL; and the average pore diameter of the pore structure in the prepared porous electrode was 60 nm.

[0052] Example 5

[0053] A porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added dropwise was 100 μL; and the average pore diameter of the pore structure in the prepared porous electrode was 70 nm.

[0054] Example 6

[0055] A porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added dropwise was 120 μL; and the average pore diameter of the pore structure in the prepared porous electrode was 80 nm.

[0056] Comparative Example 1

[0057] An electrode was prepared according to the method of Example 1, except that the gold layer containing the porous structure was not deposited on the gold plane layer. The specific steps were as follows:

[0058] A single-side polished silicon wafer with a thickness of 1 mm was used as a substrate. The substrate was ultrasonically cleaned in acetone and isopropanol for 10 minutes, rinsed with deionized water, and blown dry with nitrogen. A titanium layer with a thickness of 20 nm was deposited on the polished surface of the substrate by electron beam evaporation physical deposition. A gold planar layer with a thickness of 150 nm was deposited on the surface of the titanium layer by electron beam evaporation physical deposition to obtain an electrode.

[0059] The surface of the gold layer containing the porous structure in the porous electrodes prepared in Examples 1 to 4 was examined by scanning electron microscopy to obtain SEM images, as shown in FIG. Figure 1 shown.

[0060] The average pore size of the porous structure in the gold layer containing the porous structure in the porous electrodes prepared in Examples 1 to 4 was measured according to the following method; it was determined by desorption of nitrogen isotherms (Micromeritics ASAP 2020plus HD88); the pore volume at different pore sizes was calculated based on the relationship between adsorption potential and pressure using the Barrett-Joyner-Halenda (BJH) method. The final pore size distribution curve was generated by plotting the desorption pore volume as a function of pore size, as shown in Figure 1. Figure 2 shown.

[0061] Combine Figure 1 and Figure 2 It can be seen that the porous electrode provided by the present invention contains a pore structure, and the average pore diameter is 30 to 80 nm.

[0062] The porous electrode prepared in Example 3 was subjected to X-ray photoelectron spectroscopy to obtain an XPS spectrum, as shown in FIG. Figure 3 As shown, a is the XPS wide scan spectrum, b is the high resolution scan spectrum. The porous electrode prepared in Example 3 was subjected to X-ray diffraction detection to obtain an XRD spectrum, as shown in FIG. Figure 4 As shown, d is the enlarged spectrum of 30~80° in c.

[0063] Wide-scan XPS spectroscopy confirmed the presence of gold; significant peaks were observed at Au4f, Au4d, and Au4p, which are characteristic of metallic gold. Small peaks of O1s and C1s were also observed, which are due to contamination caused by oxidation and atmospheric exposure, as well as surface adsorption. The spectrum of Au4f showed peaks of 4f7 / 2 and 4f5 / 2 at 83.5 and 87.2 eV, respectively, separated by 3.7 eV due to spin-orbit coupling, confirming the presence of metallic gold (Au(0)).

[0064] The two main peaks in the XRD spectrum are located near 40° and 70°, respectively. These peaks are attributed to the reflection characteristics of the face-centered cubic (FCC) structure of gold. The peak near 40° corresponds to the (111) face of gold, while the peak near 70° corresponds to the (400) face of gold.

[0065] The porous gold prepared in Examples 1 to 6 and the flat gold electrode of Comparative Example 1 were subjected to emission spectroscopy-electrochemical characterization using a standard three-electrode system (counter electrode: Pt, reference electrode: Ag / AgCl). The results are as follows: Figure 5 As shown, where a is the molar concentration of 200 μmol / L [Ru(bpy)3] 3+ At a scan rate of 100 mV s -1 The results of cyclic voltammetry test of the electrode under the conditions of b are shown in Fig. 2. b is the determination of [Ru(bpy)3] at 1.5V using the electrode 3+ The ECL intensity of each electrode is shown in Table 1. c is the IT curve measured using the chronoamperometry method. d is the ECL spectrum of porous gold electrodes with different pore sizes measured using the potentiostatic method at 1.5 V. The specific test results are listed in Table 1.

[0066] Table 1 Electrochemical properties of electrodes of Examples 1 to 6 and Comparative Example 1

[0067]

[0068] Figure 5 As can be seen in Figure a, the oxidation peak appears at about 1.3 V. As the pore size increases from 30 nm to 50 nm, the oxidation current increases; however, as the pore size further increases to above 50 nm, the oxidation current decreases.

[0069] Because the oxidation current intensity reflects the reaction sites and surface area of ​​the electrode, and smaller pore size is associated with higher surface area. Figure 5 Figure b shows the temporal evolution of ECL intensity using different electrodes under the same CV conditions. The ECL intensity reaches its maximum when the average pore size is 50 nm, representing an over 80-fold increase compared to the flat electrode. The ECL intensity is relatively stable, but after several CV cycles, the ECL intensity of the porous gold electrode decreases by approximately 20%. Figure 5 Figure c is the chronopotentiometry curve at 1.5 V. As can be seen from the figure, the oxidation current of the porous gold electrode is higher than that of the flat gold electrode, and the maximum current intensity is obtained when the pore diameter is 50 nm. Figure 5 Figure d shows the ECL emission spectra of various electrodes at 1.5 V using the potentiostatic method. The emission peak is observed at ~597 nm, with a full-width at half-maximum (FWHM) of approximately 80 nm. All porous gold electrodes exhibit higher ECL intensities compared to flat electrodes, with the maximum intensity observed when the pore size is approximately 50 nm.

[0070] Figure 6 The results of spectroelectrochemical detection of the electrodes of Examples 1 to 6 are shown. The enhancement rate using CV is much higher than that of the static potential scan (e.g., 80-fold enhancement vs. 7-fold enhancement). This may indicate that the reactants are present in the pores at a high concentration at the beginning of the reaction, but rapidly decrease after the oxidation reaction, and insufficient mass transfer inside and outside the pores leads to limited resupply in the pores. The ECL intensity of the electrochemiluminescence sensor prepared using the porous electrode is related to the pore size in the porous electrode; the present invention uses different amounts of triisopropylbenzene (TIPBz) to well control the pore size. Compared with the traditional planar gold electrode, the ECL intensity of the porous gold electrode is significantly increased by more than 80 times, with high sensitivity and obvious biosensing advantages. The enhancement of the ECL intensity of the porous electrode is mainly attributed to the increase in surface area and the optical coupling of the plasma substrate. The limiting factor for enhanced luminescence is that at a smaller pore size below 50 nm, the reactants cannot diffuse significantly, thereby reducing the ECL intensity. The maximum ECL intensity is observed at a pore diameter of approximately 50 nm, indicating that at a pore diameter of 50 nm, reactants can easily enter and exit the pore and that the pore size is small enough to increase the surface area, resulting in the observed peak in ECL intensity. The analysis also showed that the ECL intensity and current follow different trends, indicating that ECL intensity depends on current, as current is related to active probes, and active probes are related to surface area.

[0071] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of a porous electrode in an electrochemiluminescence sensor, characterized in that: The porous electrode serves as a working electrode of an electrochemiluminescence sensor.

2. The application according to claim 1, characterized in that The porous electrode comprises a substrate and a gold layer containing a pore structure and attached to the surface of the substrate.

3. The application according to claim 2, characterized in that: The substrate comprises a silicon wafer, a titanium layer and a gold plane layer stacked in sequence, and the gold layer containing the hole structure is in direct contact with the gold plane layer.

4. The application according to claim 3, characterized in that The thickness of the silicon wafer is 0.8-1.2 mm, and the thickness of the titanium layer is 18-22 nm.

5. The application according to claim 3, characterized in that: The thickness of the gold plane layer is 148-152 nm.

6. The use according to claim 2 or 3, characterized in that: The thickness of the gold layer containing the pore structure is 0.18 to 0.22 μm.

7. The application according to claim 6, characterized in that The average pore diameter of the pore structure in the gold layer containing the pore structure is 30 to 60 nm.

8. The use according to claim 6, characterized in that The porosity of the gold layer containing the pore structure is 48-52%.

9. The use according to claim 1, 2, 3, 4, 7 or 8, characterized in that: The method for preparing the porous electrode comprises the following steps: dissolving a polystyrene-block-polyethylene oxide diblock copolymer in an organic solvent to obtain a first solution; adding a swelling agent, ethanol, an aqueous solution of tetrachloroauric acid and water to the first solution in sequence to obtain a gold precursor solution; A titanium layer and a gold plane layer are sequentially arranged on the surface of the substrate, and then a gold precursor solution is electro-deposited on the surface of the gold plane layer to form a gold layer containing a pore structure, thereby obtaining the porous electrode.

10. An electrochemiluminescence sensor, characterized in that The working electrode of the electrochemiluminescence sensor is the porous electrode.

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