A hyaluronidase detection method based on double potential ratio electrochemiluminescence

Abstract

The application relates to the technical field of electrochemical luminescence detection, and discloses a hyaluronidase detection method based on double-potential ratio electrochemical luminescence, which comprises the following steps: uniformly mixing cyclohexane, n-hexanol and triton X-100 in a serum bottle, adding Ru(bpy)3 2+ solution, and then performing magnetic stirring and centrifugal collection of Ru-SiO2 NP precipitates; the electrode after enzymolysis is used as a working electrode, a saturated Ag / AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode to perform electrochemical luminescence detection. Traditional ECL sensors are mostly dependent on a single signal output mode, are easily affected by factors during the detection process, and result in the generation of false positive or false negative results. The application realizes the ultra-low background detection of HAase through the calculation of high / low potential ECL signal ratios, and effectively avoids the generation of false negative and false positive results.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence detection technology, and specifically relates to a method for detecting hyaluronidase based on dual-potential ratio electrochemiluminescence. Background Technology

[0002] Hyaluronidase (HAase), a class of hydrolases that can degrade hyaluronic acid in the extracellular matrix, has been shown to be closely related to the occurrence, invasion and metastasis of various malignant tumors such as bladder cancer, pancreatic cancer and ovarian cancer due to its abnormally high expression. Therefore, it has become a highly promising biomarker for tumor diagnosis and prognostic assessment.

[0003] Electrochemiluminescence (ECL) sensing technology has been widely used in fields such as disease biomarker analysis, food safety testing, and environmental monitoring due to its advantages such as high sensitivity, wide linear range, and low background.

[0004] However, traditional ECL sensors mostly rely on a single signal output mode, making them susceptible to environmental interference, probe concentration variations, and electrode conditions during detection, leading to false positive or false negative results. To overcome these limitations, ratiometric ECL sensors have emerged, effectively improving detection reliability through dual-signal self-calibration. Currently reported ratiometric ECL sensors mostly employ two different luminescent materials or co-reactants, or rely on resonant energy transfer effects to construct a dual-signal system. These sensors are complex in design and construction, requiring repeated reactions, processing, and cleaning of the working electrode to link multiple ECL signal probes, further increasing the complexity of sensor fabrication. Summary of the Invention

[0005] In view of the above situation, the main objective of the present invention is to propose a hyaluronidase detection method based on dual-potential ratio electrochemiluminescence to solve the above-mentioned technical problems.

[0006] This invention proposes a method for detecting hyaluronidase based on dual-potential ratio electrochemiluminescence, the method comprising the following steps: Step 1: Mix cyclohexane, n-hexanol and Triton X-100 in a serum bottle until homogeneous. Add ruthenium bipyridine solution and stir magnetically. After stirring, add ammonia and tetraethyl silicate sequentially and stir at room temperature. After the reaction is complete, add acetone to break the emulsion and centrifuge to collect the Ru-SiO2NPs precipitate. Wash the Ru-SiO2NPs precipitate with deionized water and ethanol by cross-centrifugation to obtain Ru-SiO2NPs. Step 2: Pre-treat the gold electrode to obtain a pre-treated gold electrode; Step 3: Immerse the pretreated gold electrode in a 4-aminobenzylthiophenol solution to react and obtain the modified gold electrode; rinse the surface of the modified gold electrode with anhydrous ethanol to remove the physically adsorbed free molecules, and rinse with ultrapure water to obtain the 4-ATP modified gold electrode. Step 4: Hyaluronic acid, ultrapure water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are mixed and incubated in a metal bath to activate the carboxyl groups and obtain a mixed solution; the mixed solution is dropped onto the surface of the 4-ATP modified gold electrode and transferred to an electrically heated drying oven for reaction; after the reaction, it is soaked in ultrapure water to obtain a hydrogel modified gold electrode; Step 5: Immerse the hydrogel-modified gold electrode in the test sample containing hyaluronidase to carry out an enzymatic hydrolysis reaction, causing the hydrogel to degrade and obtain the enzymatically hydrolyzed electrode. Use the enzymatically hydrolyzed electrode as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode. Insert them together into an electrochemiluminescence detection cell containing buffer solution, Ru-SiO2NPs, ultrapure water, and tri-n-propylamine. Detection is performed using electrochemiluminescence. Record the electrochemiluminescence signal intensity at high and low potentials. Calculate the ratio of the electrochemiluminescence signal intensity at high potential to that at low potential to achieve quantitative analysis of hyaluronidase and obtain the detection results.

[0007] Beneficial effects: 1. This invention combines the spatial barrier effect of a single signal probe, silica nanoparticles (Ru-SiO2NPs), with a hyaluronic acid hydrogel. By utilizing hyaluronidase (HAase) to specifically hydrolyze the hydrogel and regulate the distance between the Ru-SiO2NPs and the electrode surface, the same signal probe can switch its ECL emission path between high potential (1.05V) and low potential (0.8V), thus constructing a dual-potential ratiometric ECL sensor based on a single signal probe. Compared to traditional ratiometric sensors that rely on dual probes or energy transfer, this invention offers a simpler sensor design and fewer construction steps, significantly reducing the complexity of sensor fabrication.

[0008] 2. This invention utilizes in-situ modification of the electrode surface with hyaluronic acid hydrogel to form an effective spatial barrier layer in the absence of HAase, confining Ru-SiO2NPs beyond the effective interaction distance (3.1µm) of tri-n-propylamine (TPrA) radicals, resulting in a weak background signal only at low potentials. In the presence of HAase, the hydrogel is specifically enzymatically hydrolyzed, allowing Ru-SiO2NPs to approach the electrode surface and trigger strong ECL emission at high potentials. By calculating the high / low potential ECL signal ratio, ultra-low background detection of HAase is achieved, effectively avoiding false negatives and false positives.

[0009] 3. This invention achieves optimal sensor response performance by optimizing key parameters in sensor construction, including the concentrations of 4-aminothiophenol (4-ATP), sodium hyaluronate (HA), Ru-SiO2 NPs, TPRA dosage, and HAase reaction time. Experimental results show that under optimal conditions, the HAase concentration exhibits a good linear relationship with the ECL signal ratio in the range of 0.5-80 U / mL, with a detection limit as low as 0.00128 U / mL. This is significantly superior to existing HAase detection methods such as fluorescence, electrochemiluminescence, and surface-enhanced Raman scattering, demonstrating higher detection sensitivity and a wider linear detection range.

[0010] 4. This invention combines a single signal probe with a ratio detection mode, retaining the advantages of ratio sensors such as strong anti-interference capability and high detection reliability, while overcoming the complex design of traditional ratio sensors that require the introduction of dual emitters or energy transfer pairs. This detection method not only provides a simple and efficient new technical means for the accurate detection of HAase, but also provides a completely new research paradigm for the innovative design of ratio ECL sensors.

[0011] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by means of embodiments of the invention. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating the detection principle of the present invention; wherein, Figure 1 In the diagram, A represents the in-situ formation of the gel film on the electrode surface; Figure 1 B in the diagram represents the switching principle of the emission potential of the ECL system excited by the target HAase and the change in the intensity of the ECL signal at high and low potentials. Figure 2 This is an experimental diagram illustrating the feasibility analysis of the present invention; wherein, Figure 2 In the image, A represents the TEM image of Ru-SiO2 NPs; Figure 2 B in the graph represents the change in ECL signal intensity before and after the addition of the target substance; Figure 3 Graph showing the optimization of 4-ATP concentration; Figure 4 Optimization diagram for HA concentration; Figure 5 Figure showing the optimized concentration of Ru-SiO2 NPs; Figure 6 A graph showing the optimization of TPRA usage; Figure 7 The graph shows the optimal reaction time for HAase. Figure 8 A graph showing the linear relationship between HAase concentration and the ratio of high / low potential ECL signals; Figure 9 This is a comparison diagram of the linear range between the present invention and representative prior art; Figure 10 This is a comparison chart of the detection limits of the present invention and representative prior art. Detailed Implementation

[0013] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0014] These and other aspects of the embodiments of the present invention will become clear from the following description and accompanying drawings. In these descriptions and drawings, some specific embodiments of the present invention are specifically disclosed to illustrate some ways of implementing the principles of the embodiments of the present invention; however, it should be understood that the scope of the embodiments of the present invention is not limited thereto.

[0015] Example 1: This embodiment 1 provides a hyaluronidase detection method based on dual-potential ratio electrochemiluminescence, the method comprising the following steps: Step 1: Mix 12 mL of cyclohexane, 3.0 mL of n-hexanol, and 3.0 mL of Triton X-100 in a serum bottle until homogeneous. Add 1 mL of 10.0 mM ruthenium bipyridine solution and stir magnetically for 28 min. After stirring magnetically, add 150 μL of ammonia and 250 μL of tetraethyl silicate sequentially, and stir at room temperature for 23 h. After stirring, add 12 mL of acetone to break the emulsion, and centrifuge at 6500 rpm to collect the Ru-SiO2NPs precipitate. Wash the Ru-SiO2NPs precipitate four times with deionized water and ethanol by alternating centrifugation at speeds of 3500 rpm, 6000 rpm, 7000 rpm, and 9000 rpm, respectively, for 8 min each time, to obtain Ru-SiO2NPs.

[0016] Step 2: Place 0.3 μm polishing powder (Al2O3) on a polishing disc, add ultrapure water and stir well. Hold the gold electrode vertically and move it slowly on the polishing disc for 2 minutes. Rinse the polished gold electrode surface with ultrapure water, place it in a centrifuge tube containing ultrapure water, and ultrasonically clean it for 1 minute. Prepare a 3 mM K3[Fe(CN)6] solution and a 1 M KCl solution, and mix them in a 1:1 ratio to obtain a mixed solution of K3[Fe(CN)6] and KCl. Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the rinsed gold electrode as the working electrode, perform calibration in the mixed solution of K3[Fe(CN)6] and KCl, controlling the peak potential difference in the cyclic voltammogram to be less than 80 mV to obtain the calibrated gold electrode. Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the calibrated gold electrode as the working electrode, perform calibration in a 0.25 M... The gold electrode was activated in H2SO4 solution and then rinsed with ultrapure water with a resistivity greater than 18 MΩ·cm to obtain a pretreated gold electrode.

[0017] Step 3: Immerse the pretreated gold electrode in a 4-aminothiophenol solution with anhydrous ethanol as the solvent, so that the concentration of the 4-aminothiophenol solution is 2.0 mM, and react at room temperature for 11 h to obtain the modified gold electrode; rinse the surface of the modified gold electrode with anhydrous ethanol to remove the physically adsorbed free molecules, and rinse with ultrapure water to obtain the 4-ATP modified gold electrode.

[0018] Step 4: Mix 18 mg / mL hyaluronic acid, ultrapure water, 4.5 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1.1 mg / mL N-hydroxysuccinimide (NHS), and incubate in a metal bath at 37 °C and 400 rpm for 10 min to activate the carboxyl groups and obtain a mixed solution. Drop the mixed solution onto the surface of the 4-ATP modified gold electrode, transfer it to an electric heating drying oven at 37 °C and react for 1 h. After the reaction, soak it in ultrapure water for 1 min to remove excess hydrogel and obtain the hydrogel modified gold electrode.

[0019] Step 5: Immerse the hydrogel-modified gold electrode in the test sample containing hyaluronidase and incubate it in a metal bath at 37℃ and 0 rpm for 1 h to carry out enzymatic hydrolysis, thereby degrading the hydrogel and obtaining the enzymatically hydrolyzed electrode. Use the enzymatically hydrolyzed electrode as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, and insert them together into an electrochemiluminescence detection cell containing 19.5 mM PBS buffer, 1.4 mg / mL Ru-SiO2 NPs, ultrapure water, and 15 μL tri-n-propylamine. Set the high voltage to 600 V and use electrochemiluminescence method for detection. Measure and record the data every 1.5 min. Record the electrochemiluminescence signal intensity at the high potential of 1.05 V and the low potential of 0.8 V, and calculate the ratio of the two to achieve quantitative analysis of hyaluronidase.

[0020] It should be noted that the ultrapure water used in steps 1 to 5 is the same, and its resistivity is greater than 18 MΩ·cm.

[0021] Example 2 This embodiment 2 provides a hyaluronidase detection method based on dual-potential ratio electrochemiluminescence, the method comprising the following steps: 15 mL of cyclohexane, 3.6 mL of n-hexanol, and 3.5 mL of Triton X-100 were mixed thoroughly in a serum bottle. 1 mL of 10.0 mM ruthenium bipyridine solution was added, and the mixture was magnetically stirred for 30 min. After magnetic stirring, 180 μL of ammonia and 300 μL of tetraethyl silicate were added sequentially, and the mixture was stirred at room temperature for 24 h. After stirring, 15 mL of acetone was added to break the emulsion, and the Ru-SiO2NPs precipitate was collected by centrifugation at 7000 rpm. The Ru-SiO2NPs precipitate was washed four times by alternating centrifugation with deionized water and ethanol at speeds of 4000 rpm, 6500 rpm, 7500 rpm, and 10000 rpm, respectively, with each centrifugation and washing time being 10 min, to obtain Ru-SiO2NPs.

[0022] Step 2: Place 0.3 μm polishing powder on a polishing disc, add ultrapure water and stir well. Hold the gold electrode vertically and move it slowly on the polishing disc for 2.5 min. Rinse the polished gold electrode surface with ultrapure water, place it in a centrifuge tube containing ultrapure water, and ultrasonically clean for 1.5 min. Prepare a 3 mM K3[Fe(CN)6] solution and a 1 M KCl solution, and mix them in a 1:1 ratio to obtain a mixed solution of K3[Fe(CN)6] and KCl. Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the rinsed gold electrode as the working electrode, perform calibration in the mixed solution of K3[Fe(CN)6] and KCl, controlling the peak potential difference in the cyclic voltammogram to be less than 80 mV to obtain a calibrated gold electrode. Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the calibrated gold electrode as the working electrode, perform an activation reaction in a 0.6 M H2SO4 solution, and use a resistivity greater than 18 The gold electrode was rinsed with ultrapure water at MΩ·cm to obtain a pretreated gold electrode.

[0023] Step 3: Immerse the pretreated gold electrode in a 4-aminothiophenol solution with anhydrous ethanol as the solvent, so that the concentration of the 4-aminothiophenol solution is 2.5 mM, and react at room temperature for 12 h to obtain the modified gold electrode; rinse the surface of the modified gold electrode with anhydrous ethanol to remove the physically adsorbed free molecules, and rinse with ultrapure water to obtain the 4-ATP modified gold electrode.

[0024] Step 4: Mix 20 mg / mL hyaluronic acid, ultrapure water, 5.0 mg / mL EDC, and 1.2 mg / mL NHS, and incubate in a metal bath at 37°C and 500 rpm for 15 min to activate the carboxyl groups and obtain a mixed solution. Add the mixed solution dropwise to the surface of the 4-ATP modified gold electrode, transfer it to a 37°C electric heating drying oven and react for 1.5 h. After the reaction, soak it in ultrapure water for 2 min to remove excess hydrogel and obtain the hydrogel modified gold electrode.

[0025] Step 5: Immerse the hydrogel-modified gold electrode in the test sample containing hyaluronidase and incubate it in a metal bath at 37℃ and 250rpm for 1.5h for enzymatic hydrolysis to degrade the hydrogel and obtain the enzymatically hydrolyzed electrode. Use the enzymatically hydrolyzed electrode as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, and insert them together into an electrochemiluminescence detection cell containing 20mM PBS buffer, 1.5mg / mL Ru-SiO2NPs, ultrapure water, and 15μL tri-n-propylamine. Set the high voltage to 600V and use electrochemiluminescence method for detection. Measure and record the data every 2.0min. Record the electrochemiluminescence signal intensity at the high potential of 1.05V and the low potential of 0.8V, and calculate the ratio of the two to achieve quantitative analysis of hyaluronidase.

[0026] Example 3 This embodiment 3 provides a hyaluronidase detection method based on dual-potential ratio electrochemiluminescence, the method comprising the following steps: Step 1: Mix 18 mL of cyclohexane, 4.2 mL of n-hexanol, and 4.0 mL of Triton X-100 in a serum bottle until homogeneous. Add 1 mL of 10.0 mM ruthenium bipyridine solution and stir magnetically for 32 min. After stirring magnetically, add 210 μL of ammonia and 350 μL of tetraethyl silicate sequentially, and stir at room temperature for 25 h. After stirring, add 18 mL of acetone to break the emulsion, and centrifuge at 7500 rpm to collect the Ru-SiO2NPs precipitate. Wash the Ru-SiO2NPs precipitate four times with deionized water and ethanol by alternating centrifugation at speeds of 4500 rpm, 7000 rpm, 8000 rpm, and 11000 rpm, respectively, for 12 min each time, to obtain Ru-SiO2NPs.

[0027] Step 2: Place 0.3 μm polishing powder on a polishing disc, add ultrapure water and stir well. Hold the gold electrode vertically and move it slowly on the polishing disc for 3 minutes. Rinse the polished gold electrode surface with ultrapure water, place it in a centrifuge tube containing ultrapure water, and ultrasonically clean for 2 minutes. Prepare a 3 mM K3[Fe(CN)6] solution and a 1 M KCl solution, and mix them in a 1:1 ratio to obtain a mixed solution of K3[Fe(CN)6] and KCl. Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the rinsed gold electrode as the working electrode, perform calibration in the mixed solution of K3[Fe(CN)6] and KCl, controlling the peak potential difference in the cyclic voltammogram to be less than 80 mV to obtain a calibrated gold electrode. Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the calibrated gold electrode as the working electrode, perform calibration in a 1 M KCl mixed solution. The gold electrode was activated in H2SO4 solution and then rinsed with ultrapure water with a resistivity greater than 18 MΩ·cm to obtain a pretreated gold electrode.

[0028] Step 3: Immerse the pretreated gold electrode in a 4-aminothiophenol solution with anhydrous ethanol as the solvent, so that the concentration of the 4-aminothiophenol solution is 3.0 mM, and react at room temperature for 13 h to obtain the modified gold electrode; rinse the surface of the modified gold electrode with anhydrous ethanol to remove the physically adsorbed free molecules, and rinse with ultrapure water to obtain the 4-ATP modified gold electrode.

[0029] Step 4: Mix 22 mg / mL hyaluronic acid, ultrapure water, 5.5 mg / mL EDC and 1.3 mg / mL NHS, and incubate in a metal bath at 37℃ and 600 rpm for 20 min to activate the carboxyl groups and obtain a mixed solution. Add the mixed solution dropwise to the surface of the 4-ATP modified gold electrode, transfer it to a 37℃ electric heating drying oven and react for 2 h. After the reaction, soak it in ultrapure water for 3 min to remove excess hydrogel and obtain the hydrogel modified gold electrode.

[0030] Step 5: Immerse the hydrogel-modified gold electrode in the test sample containing hyaluronidase and incubate it in a metal bath at 37℃ and 500rpm for 2 hours to carry out enzymatic hydrolysis, thereby degrading the hydrogel and obtaining the enzymatically hydrolyzed electrode. Use the enzymatically hydrolyzed electrode as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, and insert them together into an electrochemiluminescence detection cell containing 20.5mM PBS buffer, 1.6mg / mL Ru-SiO2NPs, ultrapure water, and 15μL tri-n-propylamine. Set the high voltage to 600V and use electrochemiluminescence method for detection. Measure and record the data every 2.5 minutes. Record the electrochemiluminescence signal intensity at the high potential of 1.05V and the low potential of 0.8V, and calculate the ratio of the two to achieve quantitative analysis of hyaluronidase.

[0031] To verify the effectiveness of this invention, experimental analysis was conducted; in Figure 1 Figure A illustrates the in-situ generation process of the gel film on the electrode surface. First, 4-ATP is self-assembled and modified onto the gold electrode surface using gold-sulfur bonding (Au–S). Then, carboxyl-activated HA is added, causing it to undergo an amidation reaction with the amino groups in the 4-ATP molecules, thereby forming a stable HA hydrogel film at the electrode interface. Figure 1 Figure B illustrates the switching principle of the emission potential of the ECL system excited by the target HAase and the schematic diagram of the changes in ECL signal intensity at high and low potentials. ECL signal probes Ru-SiO2NPs are used, with TPrA selected as a co-reactant. Because TPrA is a small molecule, it can freely penetrate the HA gel film and reach the electrode surface. However, Ru-SiO2NPs, due to their larger particle size, have difficulty approaching the electrode and participating in interfacial electron transfer when the gel film is intact. Therefore, in the absence of HAase, the system mainly generates ECL signals along the low-potential emission path.

[0032] When the target HAase is present, it can specifically degrade the HA gel film on the electrode surface, disrupting its steric barrier effect on the nanoprobe and allowing Ru-SiO2NPs to approach and contact the electrode surface. At this point, Ru-SiO2NPs and TPRA can jointly participate in the interfacial electrochemical reaction, causing the system to switch from the original low-potential luminescence mode to a high-potential redox luminescence mode, thereby achieving a controllable conversion of the ECL luminescence potential and thus enabling the detection of HAase.

[0033] exist Figure 2 In section A, this invention characterizes the synthesized Ru-SiO2NPs and verifies the feasibility of the proposed method. Under a 100nm scale, the prepared particles are generally approximately spherical, with good dispersion and relatively uniform size, and no obvious large-area agglomeration was observed. This indicates that the reverse microemulsion method can stably obtain Ru-SiO2NPs with relatively uniform morphology, providing a foundation for the subsequent construction of stable ECL signal probes. Figure 2 Section B presents the ECL-potential response curves under two conditions: without HAase and with HAase (80 U / mL). Without HAase, the system exhibits significant luminescence primarily in the low potential region (0.8-0.95 V), while showing only a weak response in the high potential region (1.05-1.3 V). Upon addition of HAase, the ECL signal in the high potential region is significantly enhanced, while only a weak background signal is retained in the low potential region. These results demonstrate that HAase can alter the spatial distance and interfacial reaction environment between Ru-SiO2 NPs and the electrode surface by enzymatically hydrolyzing the hyaluronic acid hydrogel on the electrode surface, thereby achieving a transformation from weak luminescence at low potential to strong luminescence at high potential using the same ECL signal probe. This verifies the feasibility of the dual-potential ratio detection strategy of this invention.

[0034] To optimize the performance of this dual-potential ratio ECL sensor, several key conditions were adjusted, including 4-ATP concentration, HA concentration, Ru-SiO2NPs concentration, TPrA dosage, and HAase reaction time.

[0035] exist Figure 3In the graph, the horizontal axis represents the 4-ATP concentration (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mM), and the vertical axis represents the ECL signal ratio (1.05 V / 0.8 V). The graphs show the results under HAase-free and HAase-containing (80 U / mL) conditions, with the black line representing the ECL signal ratio with HAase / without HAase. As the 4-ATP modification concentration increases from 1.0 mM to 2.5 mM, the relative ratio shown by the red line continuously increases, reaching its maximum at 2.5 mM. When the 4-ATP concentration continues to increase to 3.0-4.0 mM, this ratio gradually decreases. This result indicates that too low a 4-ATP modification density is detrimental to the subsequent stable construction of the interface, while too high a modification density may lead to increased interface accumulation, restricted electron transport, or increased non-specific background, thereby weakening the distinction between high and low potential signals. Therefore, 2.5 mM can achieve a better balance between the stability of interface modification and the ratio signal amplification effect, so it was chosen as the optimal modification concentration for 4-ATP.

[0036] HA concentration is a key factor affecting the density and spatial barrier capacity of hydrogel networks; in Figure 4 In the graph, the horizontal axis represents HA concentration (4, 6, 8, 10, 12 mg / mL), and the left and right vertical axes represent the ECL signal ratios (1.05V / 0.8V) with and without HAase (80 U / mL). The black line represents the ECL signal ratio with HAase / without HAase. With increasing HA concentration, the ECL high / low potential ratio in the HA-free group generally decreased, while the ECL high / low potential ratio in the HAase (80 U / mL) group first increased and then decreased, reaching its maximum at 10 mg / mL. Simultaneously, the relative ratio shown by the red line also peaked at 10 mg / mL, indicating that the background inhibition effect and the signal release effect after HAase enzymatic hydrolysis were most significant at this point. Therefore, when the HA concentration is too low, the hydrogel network is relatively loose, making it difficult to effectively restrict the interface approach of Ru-SiO2NPs; when the HA concentration is too high, it may lead to insufficient enzymatic hydrolysis or hindered mass transfer. After comprehensive comparison, 10 mg / mL was selected as the optimal HA concentration.

[0037] exist Figure 5In the figure, the horizontal axis represents the concentration of Ru-SiO2NPs. The ECL signal ratio (1.05V / 0.8V) was compared between the concentrations of Ru-SiO2NPs without and with HAase (80 U / mL), and the black line represents the ECL signal ratio with HAase / ECL signal ratio without HAase. It can be seen that as the concentration of Ru-SiO2NPs increases, the relative ratio of the system first increases and then decreases, reaching a maximum at 1.5 mg / mL. This result indicates that when the concentration of Ru-SiO2NPs is too low, the number of effective luminescent centers in the system is insufficient, making it difficult to form sufficient signal difference; while when the concentration is too high, it may cause interparticle self-aggregation, local self-quenching, or limited diffusion / mass transfer, thus reducing the ratio output. Therefore, 1.5 mg / mL is determined to be the optimal Ru-SiO2NPs concentration, balancing luminescence intensity and ratio resolution.

[0038] exist Figure 6 In the graph, the horizontal axis represents the amount of TPrA used (5, 10, 15, 20, 25, 30 μL), and the left and right vertical axes represent the ECL signal ratios (1.05V / 0.8V) with and without HAase (80 U / mL), respectively. The black line represents the ECL signal ratio with HAase / ECL signal ratio without HAase. With increasing TPrA dosage, the high / low potential ratio of ECL under both HAase-containing and HAase-free conditions initially increases and then decreases, with the relative ratio shown by the black line reaching its maximum at 15 μL. This indicates that TPrA, as a co-reactant, cannot fully drive Ru(bpy)3 when its dosage is too low. 2+ The relevant ECL reaction is important, but adding too much TPR may introduce additional background, side reactions, or system instability, thereby reducing the effectiveness of the dual potential ratio differentiation. Therefore, 15 μL was selected as the optimal amount of TPR.

[0039] The reaction time between HAase and hydrogel directly determines the degree of enzymatic hydrolysis of the hydrogel and further affects the ECL high / low potential signal ratio. Figure 7 In the graph, the horizontal axis represents the HAase reaction time (0.25, 0.5, 0.75, 1, 1.5, 2, 2.5 h), and the vertical axis represents the ECL signal ratio (1.05V / 0.8V). The graphs show the results under conditions without HAase and with HAase (80 U / mL), with the black line representing the relative ratio. It can be seen that as the reaction time increases, the relative ratio shown by the black line continuously increases, and the increase gradually slows down after 1.5 h. Especially at 1 h, the system has obtained a high ECL ratio signal, which meets the detection requirements; if the reaction time is further extended, although the signal still increases, the increase is significantly reduced. Considering the balance between detection timeliness and signal output, this invention ultimately selects 1 h as the optimal HAase reaction time.

[0040] To further characterize the analytical performance of this dual-potential ratiometric ECL sensor, different concentrations of the target analyte HAase in the range of 0.5–80 U / mL were detected. Figure 8 In Figure A, the horizontal axis represents the HAase concentration (U / mL), and the vertical axis represents the ECL signal ratio (1.05V / 0.8V). As the HAase concentration increases, the high / low potential ratio of the system gradually increases, indicating that more HAase participating in hydrogel hydrolysis can further enhance high-potential luminescence and reduce low-potential background interference. Figure 8 In section B, the ECL signal ratio and lg(C) are further given. HAase The linear relationship between ECL high / low potential ratio (ΔA) and HAase concentration was investigated. Results showed a good linear correlation within the range of 0.5-80 U / mL, with the regression equation being ΔA = 28.19lgC. HAase The correlation coefficient was +9.93 and 0.9834. Based on S / N=3, the detection limit was calculated to be 0.00128 U / mL. These results demonstrate that the dual-potential ratio ECL sensor based on a single ECL signal probe constructed in this invention possesses a wide linear detection range, high sensitivity, and good quantitative analysis capability.

[0041] Combination Figures 2 to 8 The experimental results show that the technical effect of this invention does not stem from the simple superposition of conventional dual probes, dual luminescent materials, or additional reference signals, but rather from the synergistic design of "a single Ru-SiO2 NPs signal probe + hyaluronic acid hydrogel spatial barrier + dual-potential readout". Figure 2 In section B, it was first demonstrated that the same ECL system can undergo a switchable response from weak luminescence at low potential to strong luminescence at high potential before and after the action of HAase. Figures 3 to 7 Further, it was shown that the concentrations of 4-ATP, HA, Ru-SiO2NPs, TprA dosage, and HAase reaction time all had a significant impact on the difference between high and low potential signals, and there was a clear optimal parameter window. Figure 8 This demonstrates that under optimal conditions, this method can achieve stable quantitative detection in the range of 0.5-80 U / mL, and obtain a detection limit of 0.00128 U / mL.

[0042] therefore, Figures 2 to 8This directly demonstrates that the technical concept of achieving dual-potential ratio output in a single-signal probe system by adjusting the spatial distance between the electrode interfaces and the ECL reaction path has verifiable technical effects. Compared to conventional ratiometric ECL schemes that rely on dual labeling, dual luminescence centers, or energy transfer mechanisms, this invention is simpler in system construction, has lower detection background, and provides more intuitive result interpretation. It also improves the accuracy and stability of HAase detection, thus providing strong support for the inventiveness of this invention.

[0043] To further illustrate the performance advantages of the present invention compared to existing technologies, HAase detection methods disclosed in published literature were selected as comparative examples for horizontal comparison. The selected comparative examples include: Comparative Example 1, fluorescence method; Comparative Example 2, electrochemiluminescence method-1; Comparative Example 3, electrochemiluminescence method-2; Comparative Example 4, surface-enhanced Raman scattering method; Comparative Example 5, gravimetric reading method; Comparative Example 6, length reading method; Comparative Example 7, time reading method; Comparative Example 8, multicolor method-1; Comparative Example 9, multicolor method-2. It should be noted that the following comparisons use the linear range and detection limit data reported in published literature to illustrate the performance advantages of the present invention in HAase detection. While the platform conditions, instrument models, and testing systems in the relevant literature are not entirely the same, their published data can still serve as a valid reference for evaluating the technical effectiveness of the present invention. The test results are shown in Table 1 below. Table 1: Comparison of HAase detection performance between existing technologies and the present invention

[0044] For easier and more intuitive comparison, in Figure 9 and Figure 10 Five representative prior art methods were selected as comparative examples in the accompanying drawings. Specifically, C1 corresponds to Example 1 (fluorescence method), C2 to Example 2 (electrochemiluminescence method-1), C3 to Example 3 (electrochemiluminescence method-2), C4 to Example 4 (surface-enhanced Raman scattering method), and C5 to Example 5 (gravimetric reading method). Purple data represents the present invention. Figure 9 and Figure 10In this invention, continuous linear detection is achieved within the range of 0.5-80 U / mL. Regarding the lower limit of linearity, this invention is the same as Comparative Example 1, both starting detection from 0.5 U / mL. However, this invention increases the upper limit of linearity from 37.5 U / mL to 80 U / mL. Compared to Comparative Examples 2 and 3, this invention not only reduces the lower limit of detection from 2.0 U / mL to 0.5 U / mL, but also increases the upper limit of linearity from 40 U / mL and 60 U / mL to 80 U / mL, respectively. Compared to Comparative Example 4, this invention significantly reduces the lower limit of detection from 5 U / mL to 0.5 U / mL, while simultaneously increasing the upper limit of linearity from 70 U / mL to 80 U / mL. For Comparative Example 5, although published literature reports a minimum linear threshold of 0.2 U / mL, its linearity is exhibited in two intervals: 0.2-10 U / mL and 1-60 U / mL, which are piecewise linear ranges. In contrast, this invention achieves continuous single-interval linear detection of 0.5-80 U / mL, thus offering advantages in terms of detection interval continuity, upper limit extension capability, and convenience for practical quantitative analysis. Furthermore, comparing Comparative Examples 6 to 9 in Table 1, it can be seen that the upper limit of linearity of this invention is also higher than that of the length reading method, time reading method, and the 0.6-40 U / mL multicolor method. Compared to the 5-80 U / mL multicolor method, this invention further lowers the lower limit of detection while maintaining a similar upper limit of linearity.

[0045] In Table 1 and Figure 10 In this invention, the detection limit is 0.00128 U / mL, which is significantly lower than that of all previously disclosed technologies. Figure 10 Taking the representative prior art as an example, the detection limits of the present invention are only about 1 / 234 of Comparative Example 1, about 1 / 258 of Comparative Example 2, about 1 / 1562 of Comparative Example 3, about 1 / 1328 of Comparative Example 4, and about 1 / 156 of Comparative Example 5, respectively. Further, combining all the data in Table 1, it can be seen that the detection limit of the present invention is also superior to 0.2 U / mL of Comparative Example 6, 0.23 U / mL of Comparative Example 7, 1.98 U / mL of Comparative Example 8, and 0.3 U / mL of Comparative Example 9. This indicates that the present invention has stronger signal resolution and higher sensitivity in the detection of low concentrations of HAase.

[0046] The comparative results above show that this invention not only significantly outperforms existing technologies in terms of detection limit, but also combines the advantages of a lower detection threshold, a higher detection upper limit, and continuous single-interval output in terms of linear range. This performance improvement does not stem from a simple replacement of existing methods, but rather from the synergistic design of this invention through a single Ru-SiO2 NPs signal probe, hyaluronic acid hydrogel spatial barrier, and dual-potential ratio readout, enabling the system to achieve higher signal amplification efficiency and a more stable quantitative response while maintaining low background. Therefore, in Table 1 and... Figure 9, Figure 10 The comparative data further demonstrates, from the perspectives of detection range, detection limit, and output method, that the present invention has outstanding substantive features and significant progress compared to the disclosed prior art.

[0047] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for detecting hyaluronidase based on dual-potential ratio electrochemiluminescence, characterized in that, The method includes the following steps: Step 1: Mix cyclohexane, n-hexanol and Triton X-100 in a serum bottle until homogeneous. Add ruthenium bipyridine solution and stir magnetically. After stirring, add ammonia and tetraethyl silicate sequentially and stir at room temperature. After the reaction is complete, add acetone to break the emulsion and centrifuge to collect the Ru-SiO2NPs precipitate. Wash the Ru-SiO2NPs precipitate with deionized water and ethanol by cross-centrifugation to obtain Ru-SiO2NPs. Step 2: Pre-treat the gold electrode to obtain a pre-treated gold electrode; Step 3: Immerse the pretreated gold electrode in a 4-aminobenzylthiophenol solution to react and obtain the modified gold electrode; rinse the surface of the modified gold electrode with anhydrous ethanol to remove the physically adsorbed free molecules, and rinse with ultrapure water to obtain the 4-ATP modified gold electrode. Step 4: Hyaluronic acid, ultrapure water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are mixed and incubated in a metal bath to activate the carboxyl groups and obtain a mixed solution; the mixed solution is dropped onto the surface of the 4-ATP modified gold electrode and transferred to an electrically heated drying oven for reaction; after the reaction, it is soaked in ultrapure water to obtain a hydrogel modified gold electrode; Step 5: Immerse the hydrogel-modified gold electrode in the test sample containing hyaluronidase to carry out an enzymatic hydrolysis reaction, thereby degrading the hydrogel and obtaining the enzymatically hydrolyzed electrode. The enzymatically digested electrode was used as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode. All three electrodes were inserted into an electrochemiluminescence detection cell containing buffer solution, Ru-SiO2NPs, ultrapure water, and tri-n-propylamine. Electrochemiluminescence was used for detection. The electrochemiluminescence signal intensities at high and low potentials were recorded. By calculating the ratio of the electrochemiluminescence signal intensity at high to low potentials, quantitative analysis of hyaluronidase was achieved, yielding the detection results.

2. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 1, characterized in that, In step 1, during the process of obtaining Ru-SiO2NPs, 12-18 mL of cyclohexane, 3.0-4.2 mL of n-hexanol, 3.0-4.0 mL of Triton X-100, 1 mL of ruthenium bipyridine solution with a concentration of 10.0 mM, and magnetic stirring time of 28-32 min were used. The amount of ammonia water is 150-210 μL, the amount of tetraethyl silicate is 250-350 μL, and the reaction time is 23-25 ​​h with stirring at room temperature; the amount of acetone for demulsification is 12-18 mL, and the centrifugation speed is 6500-7500 rpm. The total number of times the centrifugation washing was performed was 4, with the centrifugation speeds for each centrifugation being 3500-4500 rpm, 6000-7000 rpm, 7000-8000 rpm, and 9000-11000 rpm, respectively, and the centrifugation washing time for each time being 8-12 minutes.

3. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 1, characterized in that, Step 2, the pretreatment of the gold electrode, specifically includes the following steps: Place polishing powder on the polishing disc, then add ultrapure water and stir well. Hold the gold electrode vertically and move it slowly on the polishing disc to polish the gold electrode and obtain a polished gold electrode. Rinse the polished gold electrode surface with ultrapure water, then place it into a centrifuge tube containing ultrapure water, and place the centrifuge tube in an ultrasonic water bath for ultrasonic cleaning to remove the alumina slurry, thus obtaining the rinsed gold electrode. Prepare and mix K3[Fe(CN)6] solution and KCl solution to obtain a mixed solution of K3[Fe(CN)6] and KCl; Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and a rinsed gold electrode as the working electrode, calibration was performed in a mixed solution of K3[Fe(CN)6] and KCl. The peak potential difference in the cyclic voltammogram was controlled to obtain the calibrated gold electrode. Using a saturated Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and a calibrated gold electrode as the working electrode, an activation reaction was carried out in H2SO4 solution, and the gold electrode was rinsed with ultrapure water to obtain a pretreated gold electrode.

4. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 3, characterized in that, In the process of obtaining the pretreated gold electrode, the polishing powder is 0.3 μm, the gold electrode is polished for 2-3 min, the centrifuge tube is placed in an ultrasonic water bath for ultrasonic cleaning for 1-2 min, the K3[Fe(CN)6] solution is 3 mM, the KCl solution is 1 M, the mixing ratio of K3[Fe(CN)6] solution and KCl solution is 1:1, the peak potential difference in the cyclic voltammogram is controlled to be <80 mV, the H2SO4 solution is 0.25-1 M, and the resistivity of the ultrapure water used is >18 MΩ·cm.

5. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 1, characterized in that, In step 3, during the process of obtaining the gold electrode modified with 4-ATP, 4-aminothiophenol was dissolved in anhydrous ethanol to make the concentration of 4-aminothiophenol 2.0-3.0 mM, and the reaction was carried out at room temperature for 11-13 h.

6. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 1, characterized in that, In step 4, during the process of obtaining the hydrogel-modified gold electrode, the concentrations of hyaluronic acid (18-22 mg / mL), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.5-5.5 mg / mL), and N-hydroxysuccinimide (1.1-1.3 mg / mL) were incubated in a metal bath at 37°C and 400-600 rpm for 10-20 min; the reaction was then carried out in an electric heating drying oven at 37°C for 1-2 h, followed by immersion in ultrapure water for 1-3 min, with the resistivity of the ultrapure water being >18 MΩ·cm.

7. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 1, characterized in that, In step 5, during the process of obtaining the detection results, the enzymatic digestion reaction conditions are as follows: incubation at 37℃ and 0-500rpm in a metal bath for 1-2 hours; the parameters of the electrochemiluminescence analyzer used in the electrochemiluminescence method are as follows: high voltage set at 600V; buffer solution at 19.5-20.5mM PBS Buffer; Ru-SiO2NPs at 1.4-1.6mg / mL; and the electrochemiluminescence signal intensity at high and low potentials is recorded every 1.5-2.5 minutes.

8. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 7, characterized in that, In step 5, as the HAase concentration increased, the high / low potential ratio of the system gradually increased, indicating that more HAase participated in the hydrogel hydrolysis, further enhancing high-potential luminescence and reducing low-potential background interference. The corresponding regression equation is ΔA = 28.19lgC. HAase +9.93, with a correlation coefficient of 0.9834; Where ΔA represents the ECL high / low potential ratio, and lg represents the logarithm of the HAase concentration.

9. The hyaluronidase detection method based on dual-potential ratio electrochemiluminescence according to claim 8, characterized in that, The detection limit of the test results is 0.00128 U / mL.

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