Application of a porous gold electrode in electrochemiluminescence sensing field

Abstract

This invention belongs to the field of electrochemiluminescence sensor technology, specifically relating to the application of porous electrodes in electrochemiluminescence sensors and electrochemiluminescence sensors themselves. This invention uses porous electrodes as working electrodes to prepare electrochemiluminescence sensors. The prepared porous electrodes have a large specific surface area, which accelerates the redox current and improves the accessibility of analytes compared to electrodes with smooth surfaces. Due to the large surface area and high volume ratio, the porous electrodes can enhance the interaction between the metal surface and biomolecules, thereby significantly improving sensitivity and expanding the application range of electrochemiluminescence sensors. Example test results show that, compared with traditional planar gold substrates, this invention, based on [Ru(bpy]... )3 ] 2+ The ECL intensity was increased by more than 80 times, indicating that it has a stronger optical response, making the electrochemiluminescence sensor prepared by porous electrodes more effective in ultrasensitive biosensing applications.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence sensor technology, specifically relating to the application of porous electrodes in electrochemiluminescence sensors and electrochemiluminescence sensors. Background Technology

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

[0003] In view of this, the present invention provides the application of porous electrodes in electrochemiluminescence sensors and electrochemiluminescence sensors. The use of porous electrodes as working electrodes to prepare electrochemiluminescence sensors significantly improves the detection sensitivity of electrochemiluminescence sensors and expands their application range.

[0004] To address the aforementioned technical problems, this invention provides the application of porous electrodes in electrochemiluminescence sensors, wherein the porous electrodes serve as the working electrodes of the electrochemiluminescence sensors.

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

[0006] Preferably, the substrate comprises a silicon wafer, a titanium layer, and a gold planar layer stacked sequentially, wherein the gold layer containing a porous structure is in direct contact with the gold planar 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 porous structure is 0.18–0.22 μm.

[0010] Preferably, the average pore size of the pore structure in the gold layer containing the pore structure is 30-60 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 includes the following steps:

[0013] The polystyrene-block-polyethylene oxide diblock copolymer was dissolved in an organic solvent to obtain the first solution;

[0014] A swelling agent, ethanol, tetrachloroauric acid aqueous solution and water were added dropwise to the first solution in sequence to obtain a gold precursor solution;

[0015] A titanium layer and a gold planar layer are sequentially deposited on the surface of a substrate. Then, a gold precursor solution is electrodeposited on the surface of the gold planar layer to form a gold layer with a porous 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] This invention utilizes porous electrodes as working electrodes to fabricate an electrochemiluminescence (ECL) sensor. Porous electrodes possess a large specific surface area, which, compared to smooth-surfaced electrodes, accelerates the conduction rate of redox current and improves analyte accessibility. Due to their larger surface area and higher volume ratio, porous electrodes enhance the interaction between the metal surface and biomolecules, thereby significantly improving sensitivity and expanding the application range of ECL sensors. Test results from the examples show that, compared to traditional planar gold substrates, the ECL intensity of this invention, based on planar electrodes, is more than 80 times higher, indicating a stronger optical response. This makes the ECL sensor fabricated using porous electrodes more effective in ultrasensitive biosensing applications. Attached Figure Description

[0018] Figure 1 SEM images of the porous electrodes prepared in Examples 1-4;

[0019] Figure 2 The figures show the pore size distribution curves of the porous electrodes prepared in Examples 1-4.

[0020] Figure 3 The XPS spectra of the porous electrode prepared in Example 3 are shown, where a is a wide scan XPS spectrum and b is a high-resolution scan spectrum.

[0021] Figure 4 The image shows the XRD pattern of the porous electrode prepared in Example 3, where d is the magnified pattern of c at 30-80°.

[0022] Figure 5 The figures show the emission spectroscopic-electrochemical characterization results of the electrodes used in Examples 1-6 and Comparative Example 1, where a is a cyclic voltammogram and b is the result of [Ru(bpy)3] measured using the electrode at 1.5 V. 3+The graph shows the ECL intensity changing over time, where c is the chronopotential curve at 1.5V and d is the ECL emission spectrum of various electrodes at 1.5V using the potential-static method.

[0023] Figure 6 The images show the electrochemiluminescence results of the electrodes in Examples 1-6. Detailed Implementation

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

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

[0026] In one specific embodiment of the present invention, the thickness of the gold layer containing the porous structure can be 0.18–0.22 μm, specifically 0.2 μm; the average pore size of the porous structure in the gold layer containing the porous structure can be 30–60 nm, specifically 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, or 80 nm; and the porosity of the gold layer containing the porous structure can be 48–52%, specifically 50%.

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

[0028] The polystyrene-block-polyethylene oxide diblock copolymer was dissolved in an organic solvent to obtain the first solution;

[0029] A swelling agent, ethanol, tetrachloroauric acid aqueous solution and water were added dropwise to the first solution in sequence to obtain a gold precursor solution;

[0030] A titanium layer and a gold plane are sequentially deposited on the surface of a substrate. Then, a gold precursor solution is electrodeposited on the surface of the gold plane layer to form a gold layer with a porous structure, thus obtaining the porous electrode.

[0031] In this invention, unless otherwise specified, all materials are commercially available analytical grade products.

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

[0033] After obtaining the first solution, the present invention sequentially adds a swelling agent, ethanol, tetrachloroauric acid aqueous solution, 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 tetrachloroauric acid aqueous solution may be 20–120 μL:3 mL, specifically 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. This invention adjusts the pore size of the pore structure by regulating the amount of expanding agent. When the volume ratio of triisopropylbenzene to 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 sizes are 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, or 80 nm, respectively. In one specific embodiment of this invention, the molar concentration of the tetrachloroauric acid aqueous solution can be 38–42 mmol / L, specifically 40 mmol / L; the water can be deionized water. In another specific embodiment of this invention, the volume ratio of the first solution to ethanol can be 30:4–5, specifically 30:4.5; the volume ratio of the first solution to the tetrachloroauric acid aqueous solution can be 10:0.8–1.2, specifically 10:1; the volume ratio of the first solution to water can be 30:7–8, specifically 30:7.5. The present invention does not have any special requirements on the dripping rate, as long as it can be dripped.

[0034] In one specific embodiment of the present invention, after the addition is completed, the process may further include: stirring the mixed system after addition; the stirring time may be 25-35 minutes, specifically 25 minutes, 28 minutes, 30 minutes, or 35 minutes; the stirring temperature may be room temperature, specifically 20-35°C, or 25-30°C. The present invention forms micelles by adding a swelling agent, ethanol, tetrachloroauric acid aqueous solution, and water to the first solution. Stirring allows the micelles to be uniformly dispersed in the system; the micelles serve as a guiding agent for the pore size of the porous structure in the gold layer. The present invention does not have special requirements for the stirring speed, as long as it ensures uniform dispersion of the micelles. In the present invention, the gold precursor solution is yellow and transparent.

[0035] After obtaining the gold precursor solution, the present invention sequentially deposits a titanium layer and a gold planar layer on the substrate surface, and then electrodeposits the gold precursor solution on the gold planar layer surface to form a gold layer with a porous structure, thereby obtaining the porous electrode. In one specific embodiment of the present invention, the substrate can be a silicon wafer, which can be a single-sided polished silicon wafer, and the present invention deposits a titanium layer on the polished surface. In another specific embodiment of the present invention, before sequentially depositing the titanium layer on the substrate surface, the method may further include: cleaning and drying the substrate; the cleaning may be sequential ultrasonic cleaning in acetone and isopropanol followed by water rinsing, the ultrasonic cleaning time in acetone being 8-12 minutes, specifically 10 minutes; the ultrasonic cleaning time in isopropanol being 8-12 minutes, specifically 10 minutes; the water used for rinsing may be deionized water, and the present invention can remove residual acetone or isopropanol from the substrate surface through water rinsing. In another specific embodiment of the present invention, the drying may be nitrogen drying.

[0036] As a specific embodiment of the present invention, the titanium layer on the substrate surface can be formed by electron beam evaporation physical deposition, and the gold planar layer deposited on the titanium layer surface can also be formed by electron beam evaporation physical deposition; the present invention has no special requirements for the electron beam evaporation physical deposition, and conventional methods in the art 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] This invention also provides an electrochemiluminescence sensor, wherein the working electrode of the electrochemiluminescence sensor is a porous electrode. This invention does not impose any particular limitation on the preparation method of the electrochemiluminescence sensor; conventional methods in the art can be used.

[0039] The electrochemiluminescence sensor provided by this invention can be used as a detection device for cancer and neurodegenerative diseases. In one specific embodiment of this invention, the gain ratio is approximately 80 times when using the electrochemiluminescence sensor to detect biomarkers of cancer or neurodegenerative diseases.

[0040] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to 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 the first solution.

[0043] Add 20 μL of triisopropylbenzene (expansion agent), 4.5 mL of ethanol, 3 mL of tetrachloroauric acid aqueous solution with a molar concentration of 40 mmol / L and 7.5 mL of deionized water dropwise to the first solution, and stir at 30 °C for 30 min to obtain a yellow transparent gold precursor solution.

[0044] Using a silicon wafer with a single-sided polished thickness of 1 mm as the substrate, the substrate was ultrasonically cleaned in acetone and isopropanol for 10 min, rinsed with deionized water, and dried 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.

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

[0046] Example 2

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

[0048] Example 3

[0049] The porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added was 60 μL; the average pore size of the porous electrode was 50 nm.

[0050] Example 4

[0051] The porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added was 80 μL; the average pore size of the porous electrode was 60 nm.

[0052] Example 5

[0053] The porous electrode was prepared according to the method of Example 1, except that the volume of triisopropylbenzene added was 100 μL; the average pore size of the porous electrode was 70 nm.

[0054] Example 6

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

[0056] Comparative Example 1

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

[0058] Using a silicon wafer with a single-sided polished thickness of 1 mm as the substrate, the substrate was ultrasonically cleaned in acetone and isopropanol for 10 min, rinsed with deionized water, and dried 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 the electrode.

[0059] The surface of the gold layer containing the porous structure in the porous electrodes prepared in Examples 1-4 was examined by scanning electron microscopy (SEM), and SEM images were obtained, as shown below. Figure 1 As shown.

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

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

[0062] The porous electrode prepared in Example 3 was subjected to X-ray photoelectron spectroscopy (XPS) to obtain the XPS spectrum, as shown below. Figure 3 As shown, a is the XPS wide scan spectrum, and b is the high-resolution scan spectrum. X-ray diffraction was performed on the porous electrode prepared in Example 3 to obtain the XRD pattern, as shown below. Figure 4 As shown, d is the magnified spectrum of c from 30° to 80°.

[0063] XPS broad-scan spectra confirmed the presence of gold; significant peaks were observed at Au4f, Au4d, and Au4p, characteristic of metallic gold. Small peaks at O1s and C1s were also observed, likely due to contamination from oxidation, atmospheric exposure, and surface adsorption. The Au4f spectrum showed peaks at 83.5 and 87.2 eV for 4f7 / 2 and 4f5 / 2, respectively, with a spacing of 3.7 eV between them due to spin-orbit coupling, confirming the presence of metallic gold Au(0).

[0064] The two main peaks in the XRD pattern 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) facet of gold, while the peak near 70° corresponds to the (400) facet of gold.

[0065] The porous gold electrodes prepared in Examples 1–6 and the flat gold electrode of Comparative Example 1 were characterized by emission spectroscopy-electrochemical analysis using a standard three-electrode system (counter electrode: Pt, reference electrode: Ag / AgCl). The results are as follows: Figure 5 As shown, where 'a' represents the molar concentration of [Ru(bpy)3] at 200 μmol / L. 3+ At a scan rate of 100 mV / s -1 The graph shows the results of cyclic voltammetry testing of the electrode under the specified conditions. b represents the measurement of [Ru(bpy)3] using the electrode at 1.5V. 3+ The ECL intensity is shown in Table 1. c represents the it curves of each electrode measured using the chronoamperometry method. d represents the ECL spectra of porous gold electrodes with different pore sizes measured using the potentiostatic method at 1.5V. Specific detection results are listed in Table 1.

[0066] Table 1. Electrochemical performance of electrodes from Examples 1-6 and Comparative Example 1

[0067]

[0068] Figure 5 As can be seen in Figure a, the oxidation peak appears at approximately 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] This is because the oxidation current intensity reflects the reaction sites and surface area of ​​the electrode, and smaller pore size is associated with a higher surface area. Figure 5 Figure b shows the trend of ECL intensity over time when using different electrodes under the same CV conditions. The ECL intensity reaches its maximum when the average pore size is 50 nm, with a signal enhancement of more than 80 times compared to the planar electrode. The ECL intensity is relatively stable, but after several CV cycles, the ECL intensity of the porous gold electrode decreases by about 20%. Figure 5 In the figure, c represents the chronopotential curve at 1.5V. It can be seen from the figure that the oxidation current of the porous gold electrode is higher than that of the flat gold electrode, with the maximum current intensity obtained when the pore size is 50nm. Figure 5 In the figure, d represents the ECL emission spectra of various electrodes at 1.5 V using the potentiometric-static 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 intensity compared to flat electrodes, with the maximum intensity observed at a pore size of approximately 50 nm.

[0070] Figure 6 The results show the spectroelectrochemical detection of electrodes in Examples 1-6. The enhancement rate using CV is significantly higher than that of potentiometric scanning (e.g., 80-fold enhancement vs. 7-fold enhancement). This may indicate that the reactants are present in high concentrations within the pores at the start of the reaction, but decrease rapidly after oxidation, and insufficient mass transfer between the inside and outside of the pores results in limited replenishment within the pores. The ECL intensity of the electrochemiluminescence sensor prepared using porous electrodes is related to the pore size in the porous electrode; the present invention effectively controls the pore size using different amounts of triisopropylbenzene (TIPBz). Compared with conventional planar gold electrodes, the ECL intensity of porous gold electrodes is significantly increased by more than 80 times, exhibiting high sensitivity and obvious biosensing advantages. The enhancement of the ECL intensity of porous electrodes is mainly attributed to the increased surface area and light coupling with the plasmonic substrate. The limiting factor for enhanced luminescence is mainly that reactants cannot diffuse significantly at smaller pore sizes below 50 nm, thus reducing the ECL intensity. The maximum ECL intensity was observed at a pore size of approximately 50 nm, indicating that reactants can easily enter and exit the pores at this size, and that the pore size is small enough to increase the surface area, thus leading to the observed peak ECL intensity. The analysis also showed that the ECL intensity and current follow different trends, suggesting that the ECL intensity depends on the current, as the current is related to the active probe, and the active probe is related to the surface area.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of porous electrodes in electrochemiluminescence sensors, characterized in that, The porous electrode serves as the working electrode of the electrochemiluminescence sensor. The porous electrode includes a substrate and a gold layer with a porous structure attached to the surface of the substrate; The substrate comprises a silicon wafer, a titanium layer, and a gold planar layer stacked sequentially, wherein the gold layer containing a porous structure is in direct contact with the gold planar layer; the thickness of the silicon wafer is 0.8~1.2 mm, the thickness of the titanium layer is 18~22 nm, and the thickness of the gold planar layer is 148~152 nm. The thickness of the gold layer containing the porous structure is 0.18~0.22μm; the average pore size of the porous structure in the gold layer containing the porous structure is 30~60nm; The method for preparing the porous electrode includes the following steps: The polystyrene-block-polyethylene oxide diblock copolymer was dissolved in an organic solvent to obtain the first solution; A swelling agent, ethanol, tetrachloroauric acid aqueous solution and water were added dropwise to the first solution in sequence to obtain a gold precursor solution; A titanium layer and a gold planar layer are sequentially deposited on the surface of a substrate silicon wafer. Then, a gold precursor solution is electrodeposited on the surface of the gold planar layer to form a gold layer with a porous structure, thereby obtaining the porous electrode.

2. The application according to claim 1, characterized in that, The porosity of the gold layer containing the porous structure is 48-52%.

3. An electrochemiluminescence sensor, characterized in that, The working electrode of the electrochemiluminescence sensor is a porous electrode; the porous electrode includes a substrate and a gold layer with a porous structure attached to the surface of the substrate; The substrate comprises a silicon wafer, a titanium layer, and a gold planar layer stacked sequentially, wherein the gold layer containing a porous structure is in direct contact with the gold planar layer; the thickness of the silicon wafer is 0.8~1.2 mm, the thickness of the titanium layer is 18~22 nm, and the thickness of the gold planar layer is 148~152 nm. The thickness of the gold layer containing the porous structure is 0.18~0.22μm; the average pore size of the porous structure in the gold layer containing the porous structure is 30~60nm; The method for preparing the porous electrode includes the following steps: The polystyrene-block-polyethylene oxide diblock copolymer was dissolved in an organic solvent to obtain the first solution; A swelling agent, ethanol, tetrachloroauric acid aqueous solution and water were added dropwise to the first solution in sequence to obtain a gold precursor solution; A titanium layer and a gold planar layer are sequentially deposited on the surface of a substrate silicon wafer. Then, a gold precursor solution is electrodeposited on the surface of the gold planar layer to form a gold layer with a porous structure, thereby obtaining the porous electrode.

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