Method for detecting myocardial injury marker CK-MB through electrogenerated chemiluminescence-photoacoustic dual-mode sensor

Abstract

The invention discloses a method for detecting a myocardial injury marker CK-MB by using an electrogenerated chemiluminescence-photoacoustic dual-mode sensor. The method comprises the following steps: preparing a difunctional nano-enzyme o < x >-CNOs; preparing an HNTs (at) luminol (at) PDDA composite material; the preparation method comprises the following steps: preparing an o < x >-CNOs (at) aptamer compound; constructing a dual-mode sensor and carrying out detection; and measuring the sample. The detection method is simple in step, high in sensitivity, short in detection time and low in cost, the ox-CNOs are synthesized by adopting a hydrothermal method, an electrogenerated chemiluminescence signal can be reduced, a photoacoustic signal can be provided by utilizing laccase to simulate enzyme properties, an HNTs (at) luminol (at) PDDA composite material is synthesized by utilizing an electrostatic adsorption method by utilizing the tubular structure and the inside-positive and outside-negative electrical property of the HNTs, and the electrochemical performance of the composite material is improved. The HNTs are used as a co-reaction accelerant in an electrogenerated chemiluminescence system, so that the sensitivity of an electrogenerated chemiluminescence mode is improved.

Description

Technical Field

[0001] This invention relates to the field of CK-MB analysis and detection technology, and in particular to a method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor. Background Technology

[0002] Natural enzymes are highly efficient biocatalysts, selectively catalyzing biological reactions under optimal conditions. They exhibit high activity and substrate specificity, making them an indispensable part of molecular biology and the pharmaceutical industry. However, natural enzymes suffer from inherent limitations, such as variability, cumbersome preparation, difficulty in recycling, and high cost, which significantly restrict their practical applications. Engineered nanomaterials with enzyme-mimicking catalytic properties offer an alternative platform to overcome these limitations. They possess advantages such as ease of large-scale synthesis, stability under harsh conditions, tunable catalytic activity, recyclability, long-term storage, and lower cost. Various nanomaterials have been studied due to their intrinsic mimicry of catalase, peroxidase, oxidase, haloperoxidase, and superoxide dismutase activities in biological media. These innovative biomaterials pave the way for powerful and improved platforms in fields such as biosensing, disease diagnosis, biomedical development, biocatalysis, and pollutant removal.

[0003] Electrochemiluminescence (ECL) is an important biosensing technology that has attracted much attention due to its unique characteristics. However, most ECL biosensors are based on a single signal change, which can lead to inaccurate detection results due to the inherent limitations of traditional single-signal output. Therefore, multimodal systems have become a hot topic in recent years because of their advantages in improving the accuracy and specificity of sensing platforms.

[0004] According to a WHO survey, myocardial infarction is currently the leading cause of death worldwide. When a patient has an acute myocardial infarction (AMI), symptoms include shortness of breath, nausea, fainting, cold sweats, and fatigue. If AMI is not recognized promptly, the patient may experience heart failure, arrhythmias, cardiogenic shock, and cardiac arrest. Therefore, early diagnosis of AMI is crucial. Diagnosis of AMI requires testing for myocardial injury markers.

[0005] Existing methods for detecting myocardial injury markers suffer from drawbacks such as low sensitivity, time-consuming detection, high cost, and cumbersome procedures. Summary of the Invention

[0006] The purpose of this invention is to provide a technical solution for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor, addressing the shortcomings of existing technologies. This detection method is simple, highly sensitive, time-efficient, and low-cost. By synthesizing a dual-signal laccase mimic, ox-CNOs, using a hydrothermal method, not only can the electrochemiluminescence signal be reduced, but the photoacoustic signal can also be provided by utilizing the enzyme-mimicking properties of laccase. Simultaneously, by utilizing the tubular structure and positive inner and negative outer charge of HNTs, an HNTs@luminol@PDDA composite material is synthesized using an electrostatic adsorption method. HNTs act as a co-reaction promoter in the electrochemiluminescence system, further enhancing the sensitivity of the electrochemiluminescence mode.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor, characterized by comprising the following steps: Step 1: Prepare bifunctional nanozymes ox-CNOs; Step 2: Prepare HNTs@luminol@PDDA composite material; Step 3: Prepare the ox-CNOs@aptamer complex; Step 4: Construct a dual-mode sensor and perform detection: First, polish and rinse the glassy carbon electrode. Then, drop-coat the HNTs@luminol@PDDA composite material onto the surface of the glassy carbon electrode. After drying, drop-add the ox-CNOs@aptamer composite material. After incubation, rinse. Then, drop-add the CK-MB solution onto the surface of the glassy carbon electrode. After incubation, collect the detached ox-CNOs@aptamer composite material. React the collected material with p-phenylenediamine solution and drop-add it onto a paper model. Detect the signal intensity using an electrochemiluminescence system and a photoacoustic system, respectively. Plot the logarithm of the CK-MB solution concentration on the x-axis and the electrochemiluminescence signal intensity and photoacoustic signal intensity on the y-axis, respectively, to establish the electrochemiluminescence standard curve and the photoacoustic standard curve of the CK-MB solution. Step 5, Sample Measurement: Measure the electrochemiluminescence signal intensity and photoacoustic signal intensity of the sample to be tested as in Step 4. Substitute the electrochemiluminescence signal intensity and photoacoustic signal intensity into the electrochemiluminescence standard curve and photoacoustic standard curve, respectively, to obtain the CK-MB solution concentration in the sample to be tested.

[0008] This detection method is simple, highly sensitive, time-efficient, and low-cost. By using a hydrothermal method to synthesize a dual-signal laccase mimic, ox-CNOs, it can not only reduce the electrochemiluminescence signal but also provide a photoacoustic signal by utilizing the enzyme-mimicking properties of laccase. Simultaneously, by utilizing the tubular structure and positive inner and negative outer charge of HNTs, an HNTs@luminol@PDDA composite material is synthesized by electrostatic adsorption. HNTs act as a co-reaction promoter in the electrochemiluminescence system, further enhancing the sensitivity of the electrochemiluminescence mode.

[0009] Furthermore, the preparation of bifunctional nanozymes ox-CNOs in step 1 specifically includes the following steps: first, HNO3 and H2SO4 are mixed at a volume ratio of 1:1 to 1:5, and then added to the CNOs solution. The mixture is then heated in a water bath at a temperature of 80 to 100°C and stirred for 5 to 10 hours. After centrifugation and washing, the pH is adjusted to 7 to obtain the ox-CNOs solution.

[0010] Preferably, the volume ratio of HNO3 to H2SO4 is 1:3, the water bath heating temperature is 90℃, and the reaction time is 7h.

[0011] Furthermore, the preparation of the HNTs@luminol@PDDA composite material in step 2 specifically includes the following steps: adding HNTs to deionized water, sonicating for 1 hour, then adding luminol solution, with a volume ratio of HNTs to luminol of 100:3 to 10:3, centrifuging to remove unadsorbed luminol, adding PDDA and deionized water, with a volume ratio of HNTs to PDDA of 400:1 to 50:1, and shaking overnight to obtain the HNTs@luminol@PDDA composite material.

[0012] Preferably, the volume ratio of HNTs to luminol is 10:1, the volume ratio of HNTs to PDDA is 100:1, and the mass-volume concentration of PDDA is 0.5%.

[0013] Preferably, the centrifugation speed is 5000-10000 r / min.

[0014] Furthermore, the preparation of the ox-CNOs@aptamer complex in step 3 specifically includes the following steps: mixing the ox-CNOs solution with EDC / NHS at a molar ratio of 4:1, adding aptamer, with a volume ratio of ox-CNOs to aptamer of 1:16 to 1:2, stirring overnight, adding bovine serum albumin (BSA) to block non-specific sites, centrifuging, adding IXTE buffer to the precipitate, and obtaining the ox-CNOs@aptamer complex; Preferably, the concentration of ox-CNOs is 1 mg / mL, and the volume ratio of ox-CNOs to EDC / NHS is 14:1.

[0015] Preferably, the volume ratio of ox-CNOs to aptamer is 5:2.

[0016] Preferably, the concentration of aptamer is 10 μM, the concentration of EDC is 20 mM, and the concentration of NHS is 5 mM.

[0017] Preferably, the mass-volume concentration of bovine serum albumin (BSA) is 0.5%, and the volume ratio of ox-CNOs to bovine serum albumin (BSA) is 1:2.

[0018] Preferably, the amount of HNTs@luminol@PDDA composite material, ox-CNOs@aptamer composite material and CK-MB solution added in step 4 is 5 μL.

[0019] Preferably, the incubation time for both the ox-CNOs@aptamer complex and the CK-MB solution in step 4 is 20–60 min.

[0020] Preferably, the concentration of the CK-MB solution in step 4 is 10. -4 ng / mL~10 3 ng / mL, and diluted with PBS solution at pH 7.4.

[0021] Furthermore, the electrochemiluminescence detection in step 4 was performed in 2 mL of PBS and 2 μL of 0.1 M hydrogen peroxide solution, with a photomultiplier tube voltage of 600 V and a scanning voltage range of 0–0.9 V.

[0022] Furthermore, the light source of the photoacoustic system in step 4 is an 808nm near-infrared laser light source with a laser power of 0.9W to 1.5W.

[0023] The present invention, by adopting the above-described technical solution, has the following beneficial effects: 1. The detection method of the present invention is simple in steps, highly sensitive, short in detection time, and low in cost. By using a hydrothermal method to synthesize a dual-signal laccase mimic ox-CNOs, it can not only reduce the electrochemiluminescence signal, but also provide a photoacoustic signal by utilizing the enzyme properties of laccase. At the same time, by utilizing the tubular structure and positive inner and negative outer charge of HNTs, HNTs@luminol@PDDA composite material is synthesized by electrostatic adsorption. HNTs, as a co-reaction promoter in the electrochemiluminescence system, are introduced into the electrochemiluminescence system to further improve the sensitivity of the electrochemiluminescence mode.

[0024] 2. This invention synthesizes an ox-CNOs mimic enzyme with high laccase activity, and the ox-CNOs exhibit good activity and stability.

[0025] 3. This invention constructs an electrochemiluminescence-photoacoustic dual-mode sensor, which has higher accuracy and can collect two signals to measure the incidence of false positive and false negative results. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings: Figure 1 This is a flowchart of a method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to the present invention; Figure 2 This is a schematic diagram of the principle of CK-MB detection based on the ox-CNOs electrochemiluminescence-photoacoustic dual-mode sensor in this invention; Figure 3 This is a schematic diagram of the electrochemical signal intensity of the detection system constructed in Example 1 of the present invention with different concentrations of CK-MB added; Figure 4 The standard curves were constructed by electrochemical method using different concentrations of CK-MB in the detection system constructed in Example 1 of the present invention. Figure 5 This is a schematic diagram showing the photoacoustic signal intensity of CK-MB with different concentrations added to the detection system constructed in Embodiment 1 of the present invention. Figure 6 The standard curves were constructed by photoacoustic method using different concentrations of CK-MB in the detection system constructed in Example 1 of the present invention. Figure 7 This is a schematic diagram of the results of the detection system in Embodiment 2 of the present invention; Figure 8 This is a schematic diagram of the results of the detection system in Embodiment 3 of the present invention. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0029] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion.

[0030] The Chinese translations of the English terms in this invention are as follows:

[0031] The carbon nanotubes (CNOs) of this invention, also known as multilayer fullerenes, are composed of several stacked sp2 graphite sheets and have attracted widespread attention due to their unique advantages. Compared with other carbon materials such as nanotubes and graphene, CNOs are more attractive because of their larger specific surface area, regular spherical shape, small size, and chemical stability.

[0032] Aptamer refers to a nucleic acid aptamer, which is a short oligonucleotide sequence or polypeptide obtained through in vitro screening that can bind to a corresponding ligand with high affinity and strong specificity. The aptamer used in this invention is a specific aptamer for CK-MB. The aptamer contains an amino group at its 5' end, and the ox-CNOs surface has a large number of carboxyl groups. Under the activation of EDC / NHS, the amino and carboxyl groups connect to form ox-CNOs@aptamer. CK-MB can specifically bind to the aptamer through complementary base pairing, and the binding force of complementary base pairing is greater than the electrostatic force of electrostatic adsorption.

[0033] Luminol is an electrochemiluminescent luminescent material. When a composite material is dropped onto the surface of a glassy carbon electrode, luminol can be fixed onto the glassy carbon electrode surface, thereby improving the luminescence efficiency of luminol.

[0034] PDDA, or poly(diallyldimethylammonium chloride), is a linear cationic polyelectrolyte that has been reported to be attractive for the surface functionalization of nanomaterials. It can also act as a stabilizer, dispersant, and linker in nanocomposites. Its positive surface charge can provide additional active sites and facilitate the adsorption of electrostatically attracted analytes.

[0035] like Figure 1 and Figure 2 As shown, this invention provides a method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor, comprising the following steps: Step 1: Prepare bifunctional nanozymes ox-CNOs; The preparation of bifunctional nanozymes ox-CNOs specifically includes the following steps: First, HNO3 and H2SO4 are mixed at a volume ratio of 1:1 to 1:5, and then added to 10 mL of a CNOs solution. The mixture is then heated in a water bath at 80–100 °C and stirred for 5–10 h to oxidize the CNOs solution. After centrifugation and washing, the pH of the oxidized CNOs solution is adjusted to 7 to obtain the ox-CNOs solution.

[0036] Preferably, the volume ratio of HNO3 to H2SO4 is 1:3, the water bath heating temperature is 90℃, and the reaction time is 7h.

[0037] By introducing ox-CNOs with excellent laccase activity as signal tags for aptamers, it can not only reduce the ECL signal to provide a signal "on-off-on" sensor, but also introduce photoacoustic signal output.

[0038] Step 2: Prepare HNTs@luminol@PDDA composite material; The preparation of HNTs@luminol@PDDA composite material includes the following steps: 1 mg of pure halloysite nanotubes (HNTs) powder is added to 1 mL of deionized water, and then luminol solution is added after sonication for 1 h. The volume ratio of HNTs to luminol is 100:3 to 10:3. The mixture is shaken for 12 h, and then centrifuged at 5000 to 10000 r / min for about 5 to 20 min to remove unadsorbed luminol.

[0039] Next, PDDA (0.5%, w / v) and 1 mL of deionized water were added to the mixture. The volume ratio of HNTs to PDDA was 400:1 to 50:1. After shaking overnight, the HNTs@luminol@PDDA composite material was obtained.

[0040] Preferably, the volume ratio of HNTs to luminol is 10:1, the volume ratio of HNTs to PDDA is 100:1, and the mass-volume concentration of PDDA is 0.5%.

[0041] Halloysite nanotubes (HNTs) were introduced as a co-reaction promoter in the luminol-H₂O₂ECL system. HNTs are hollow tubular structures composed of outer silicon-oxygen tetrahedra and inner aluminum-oxygen octahedra. Their unique property of carrying a positive charge inside and a negative charge outside provides favorable conditions for the electrostatic adsorption of different substances. Utilizing this property, luminol was adsorbed inside the HNTs lumen, and PDDA was adsorbed outside, synthesizing HNTs@luminol@PDDA.

[0042] Step 3: Prepare the ox-CNOs@aptamer complex; The preparation of the ox-CNOs@aptamer complex specifically includes the following steps: mixing a 1 mg / mL ox-CNOs solution with EDC / NHS at a molar ratio of 4:1, wherein the concentration of EDC is 20 mM and the concentration of NHS is 5 mM. Preferably, the volume ratio of ox-CNOs to EDC / NHS is 14:1.

[0043] Next, aptamer is added to the mixture, with a volume ratio of ox-CNOs to aptamer of 1:16 to 1:2, preferably 5:2. Preferably, the concentration of aptamer is 10 μM.

[0044] After stirring overnight, bovine serum albumin (BSA) was added to block nonspecific sites, and the mixture was incubated at 4°C for 1 hour. The complex was centrifuged at 8000 rpm for 10 minutes, and then IXTE buffer was added to the precipitate to obtain the ox-CNOs@aptamer complex. This product was stored at 4°C for further use.

[0045] IXTE buffer is prepared by combining Tris hydrochloric acid buffer and EDTA solution.

[0046] Preferably, the mass-volume concentration of bovine serum albumin (BSA) is 0.5%, and the volume ratio of ox-CNOs to bovine serum albumin (BSA) is 1:2.

[0047] Step 4: Construct a dual-mode sensor and perform detection: A bare glassy carbon electrode with a clean surface can be obtained by polishing with 0.05 μM Al2O3 slurry, and then the surface of the glassy carbon electrode is rinsed three times with deionized water and ethanol in sequence.

[0048] The HNTs@luminol@PDDA composite material was then drop-coated onto the surface of the glassy carbon electrode and dried in an oven. After that, the ox-CNOs@aptamer composite material was added and incubated at 4°C for 20–60 min to reduce the ECL signal and provide connection sites for the next step. Then, the electrode was rinsed to remove unconnected ox-CNOs@aptamer composite material.

[0049] Then the concentration is 10 -4 ng / mL~10 3 A ng / mL CK-MB solution was dropped onto the surface of a glassy carbon electrode and incubated at 4 °C for 20–60 min. The CK-MB solution was tightly bound to aptamer. Due to the weakening electrostatic attraction between the HNTs@luminol@PDDA composite and the ox-CNOs@aptamer composite, the ox-CNOs@aptamer composite detached from the glassy carbon electrode.

[0050] Preferably, the drop volume of HNTs@luminol@PDDA composite material, ox-CNOs@aptamer composite material and CK-MB solution is 5 μL.

[0051] The concentration of the CK-MB solution is 10. -4 ng / mL~10 3 The concentration was ng / mL, and diluted with PBS solution at pH 7.4 to a specific concentration of 10. -4 ng / mL, 10 -3 ng / mL, 10 -2 ng / mL, 10 -1 ng / mL, 1 ng / mL, 10 ng / mL, 10 2 ng / mL, 10 3 ng / mL.

[0052] After incubation, the detached ox-CNOs@aptamer complex was collected for further photoacoustic signal detection.

[0053] The collected material was reacted with a 40mM p-phenylenediamine solution. After 30 minutes, the solution turned brown. A circular paper model with a diameter of 8mm was made using a tablet press. The solution that had changed color in the previous step was dropped onto the paper model. After drying at 40℃, it was used for photoacoustic detection.

[0054] ECL, EIS, and CV were performed in a three-electrode system. A modified glassy carbon electrode was used as the working electrode, a platinum electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode. All ECL measurements were performed in 0.1M PBS (pH=8.0) containing 2 μL of 0.1M H₂O₂. The potential scan range for the photomultiplier tube (PTM) and CV was 0 to 0.6 V, with a scan rate of 0.01 V / s. -1 The photomultiplier tube voltage measured by electrochemiluminescence was 600V. EIS testing was performed using 0.1mM KCl and 5mM [Fe(CN)6]. 3- / 4- The experiment was conducted in a 10 mL mixed solution. A standard electrochemiluminescence curve of CK-MB was established with the logarithm of the CK-MB concentration as the x-axis and the electrochemiluminescence signal intensity as the y-axis.

[0055] The photoacoustic detection system consists of three parts: an audio acquisition device, a sealed cavity, and a near-infrared (NIR) light source (wavelength 808nm). The laser power ranges from 0.9W to 1.5W. When the prepared paper model is irradiated with near-infrared light, thermal expansion generates an acoustic signal, which is accurately recorded by specialized software (REW). A photoacoustic standard curve for CK-MB is established with the logarithm of CK-MB concentration as the x-axis and the photoacoustic signal intensity as the y-axis.

[0056] Before incubation, the carboxyl groups of ox-CNOs are activated and amidated with amino groups on the aptamer to form ox-CNO@aptamer. The active sites of ox-CNOs@aptamer are then blocked with BSA, followed by modification on the glassy carbon electrode surface. Due to the negative charge of the aptamer surface and the positive charge of the PDDA surface, HNTs@luminol@PDDA and ox-CNOs@aptamer are firmly bound by electrostatic adsorption. In the absence of CK-MB, the aptamer exhibits an open-chain structure. When CK-MB falls onto the glassy carbon electrode surface, based on the principle of complementary base pairing, CK-MB binds with ox-CNOs@aptamer to form a rigid hairpin structure. Due to the reduced electrostatic force, the rigid hairpin structure separates from the glassy carbon electrode surface and is collected for photoacoustic detection. The higher the CK-MB content, the more ECL signal is recovered, and the stronger the photoacoustic signal generated by the fallen rigid hairpin structure. Due to the different transduction mechanisms, the photoacoustic signal and ECL signal are output independently, thus providing good calibration capabilities for both signals.

[0057] Step 5, Sample Measurement: Measure the electrochemiluminescence signal intensity and photoacoustic signal intensity of the sample to be tested as in Step 4. Substitute the electrochemiluminescence signal intensity and photoacoustic signal intensity into the electrochemiluminescence standard curve and photoacoustic standard curve, respectively, to obtain the CK-MB solution concentration in the sample to be tested.

[0058] The sample to be tested was a human serum sample diluted 100 times with PBS.

[0059] Based on the different electrochemiluminescence and photoacoustic signal intensities corresponding to different CK-MB concentrations: the electrochemiluminescence signal increases with increasing CK-MB concentration; under 808nm near-infrared laser irradiation, the photoacoustic signal intensity increases with increasing CK-MB concentration.

[0060] This detection method is simple, highly sensitive, time-efficient, and low-cost. By using a hydrothermal method to synthesize a dual-signal laccase mimic, ox-CNOs, it can not only reduce the electrochemiluminescence signal but also provide a photoacoustic signal by utilizing the enzyme-mimicking properties of laccase. Simultaneously, by utilizing the tubular structure and positive inner and negative outer charge of HNTs, an HNTs@luminol@PDDA composite material is synthesized by electrostatic adsorption. HNTs act as a co-reaction promoter in the electrochemiluminescence system, further enhancing the sensitivity of the electrochemiluminescence mode. Example

[0061] A method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor includes the following steps: Step 1: A mixture of HNO3 and H2SO4 in a 1:3 ratio is added to 10 mL of a CNOs solution. The mixture is then stirred vigorously at 90°C for 7 hours to oxidize the CNOs. Afterwards, the solution is centrifuged and washed, and the pH of the oxidized CNOs solution is adjusted to 7.

[0062] Step 2: Add 1 mg of pure halloysite nanotubes (HNTs) powder to 1 mL of deionized water, then sonicate for 1 hour and add 100 μL of luminol solution. Shake the mixture vigorously for 12 hours, then centrifuge at 8000 rpm for about 10 minutes to remove unadsorbed luminol. Next, add 10 μL of PDDA (0.5%, w / v) and 1 mL of deionized water to the mixture, shake again, and incubate overnight to prepare the HNTs@luminol@PDDA mixed solution.

[0063] Step 3: Add 1120 μL of EDC / NHS in a 4:1 ratio to a 1 mg / mL ox-CNOs solution. Next, add the aptamer to the mixture and stir overnight. The amino group of the aptamer and the hydroxyl group of the ox-CNOs bind strongly together. Subsequently, 1120 μL of 0.5 wt% bovine serum albumin (BSA) is added dropwise to the mixture to block nonspecific binding sites, and the mixture is incubated at 4 °C for 1 h. The complex is centrifuged at 8000 rpm for 10 min, and then 560 μL of IXTE buffer is added to the precipitate to obtain 1 mg / mL ox-CNO@aptamer (aptamer concentration of 4 μM). This product is stored at 4 °C for further use.

[0064] Step 4: First, a bare glassy carbon electrode with a clean surface is obtained by polishing with a 0.05 μm Al2O3 slurry. Then, the electrode surface is rinsed three times sequentially with deionized water and ethanol. 5 μL of HNTs@luminol@PDDA is dropped onto the electrode surface and dried in an oven. Subsequently, 5 μL of ox-CNOs@aptamer is added and incubated at 4 °C for 40 minutes to reduce the ECL signal and provide binding sites for the next step. Rinsing is then performed to remove unbound ox-CNOs@aptamer molecules. Finally, different concentrations of CK-MB are dropped onto the electrode surface and incubated at 4 °C for 40 minutes. CK-MB binds tightly to the aptamer, and due to the disappearance of the electrostatic attraction between HNTs@luminol@PDDA and ox-CNOs@aptamer, ox-CNOs@aptamer detaches from the electrode. The detached material is collected for the next step of photoacoustic signal detection. Add 40 mM p-phenylenediamine solution to the material collected in the previous step, and after 30 minutes, the solution turns brown. Use a tablet press to make a circular paper model with a diameter of 8 mm, then drop the discolored solution from the previous step onto the paper model, dry it at 40°C, and then use it for photoacoustic detection.

[0065] ECL, EIS, and CV were performed in a three-electrode system. A modified glassy carbon electrode was used as the working electrode, a platinum electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode. All ECL measurements were performed in 0.1M PBS (pH=8.0) containing 2 μL of 0.1M H₂O₂. The potential scan range for the photomultiplier tube (PTM) and CV was 0 to 0.9 V, with a scan rate of 0.01 V / s. -1 EIS testing was performed using 0.1 mM KCl and 5 mM [Fe(CN)6]. 3- / 4- The experiment was conducted in a 10 mL mixed solution. A standard electrochemiluminescence (ECL) curve for CK-MB was established, with the logarithm of CK-MB concentration as the x-axis and the electrochemiluminescence signal intensity as the y-axis: ECL = 1741.7lgC + 20075 R. 2 =0.9902 ( Figure 3 and Figure 4 ).

[0066] The photoacoustic detection system consists of three parts: an audio acquisition device, a sealed cavity, and a near-infrared (NIR) light source (wavelength 808nm). When the prepared paper model is irradiated with near-infrared light, thermal expansion generates an acoustic signal, which is accurately recorded by specialized software (REW). A photoacoustic standard curve for CK-MB is established with the logarithm of CK-MB concentration as the x-axis and the photoacoustic signal intensity as the y-axis: IPA = 1.992lgC + 44.025 R. 2 =0.9986( Figure 5 and Figure 6 ).

[0067] Step 5: Measure the electrochemiluminescence intensity and photoacoustic signal intensity of the sample to be tested according to Step 4. Substitute the electrochemiluminescence intensity into the CK-MB electrochemiluminescence signal standard curve of Step 4 to obtain the CK-MB concentration in the sample to be tested, or substitute the photoacoustic signal intensity value into the CK-MB photoacoustic standard curve of Step 4 to obtain the CK-MB concentration in the sample to be tested. Example

[0068] Based on steps 1, 2, 3, 4, and 5 of Example 1, a dual-mode sensor was constructed. The test samples were changed to a blank control group, 10 ng / mL of alpha-fetoprotein, 10 ng / mL of prostate-specific antigen, and 10 ng / mL of carcinoembryonic antigen. The effects of other components present in serum on the sensor were investigated.

[0069] The results show that Figure 7 This dual-mode sensor is not easily affected by the above four conditions and has strong anti-interference capabilities. Example

[0070] Based on steps 1, 2, 3, 4 and 5 of Example 1, a dual-mode sensor was constructed. The sample to be tested was changed to CK-MB with a concentration of 1 ng / mL, and 6 sets of parallel experiments were conducted.

[0071] The results show that Figure 8 The dual-mode sensor has strong repeatability and can be reproduced in six different experimental groups.

[0072] Application Example 1 A dual-mode sensor was constructed based on steps 1, 2, 3, 4, and 5 of Example 1. Using the standard addition method, 0.05 ng / mL, 0.5 ng / mL, and 5 ng / mL of CK-MB standard samples were added to serum samples, respectively. Electrochemiluminescence and photoacoustic signals were measured according to the method of Example 1. The CK-MB standard curve established in Example 1 was substituted to obtain the CK-MB concentration in the samples. Each sample was measured three times, and the average value was taken. The RSD and recovery rate are shown in Table 1.

[0073]

[0074] Table 1. CK-MB spiked recoveries in actual samples (n=3) The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to achieve substantially the same technical effect are all covered within the protection scope of the present invention.

Claims

1. A method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor, characterized in that... Includes the following steps: Step 1: Prepare bifunctional nanozymes ox-CNOs; Step 2: Prepare HNTs@luminol@PDDA composite material; Step 3: Prepare the ox-CNOs@aptamer complex; Step 4: Construct a dual-mode sensor and perform detection: First, polish and rinse the glassy carbon electrode. Then, drop-coat the HNTs@luminol@PDDA composite material onto the surface of the glassy carbon electrode. After drying, drop-add the ox-CNOs@aptamer composite material. After incubation, rinse. Then, drop-add the CK-MB solution onto the surface of the glassy carbon electrode. After incubation, collect the detached ox-CNOs@aptamer composite material. React the collected material with p-phenylenediamine solution and drop-add it onto a paper model. Detect the signal intensity using an electrochemiluminescence system and a photoacoustic system, respectively. Plot the logarithm of the CK-MB solution concentration on the x-axis and the electrochemiluminescence signal intensity and photoacoustic signal intensity on the y-axis, respectively, to establish the electrochemiluminescence standard curve and the photoacoustic standard curve of the CK-MB solution. Step 5, Sample Measurement: Measure the electrochemiluminescence signal intensity and photoacoustic signal intensity of the sample to be tested as in Step 4. Substitute the electrochemiluminescence signal intensity and photoacoustic signal intensity into the electrochemiluminescence standard curve and photoacoustic standard curve, respectively, to obtain the CK-MB solution concentration in the sample to be tested.

2. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: The preparation of bifunctional nanozyme ox-CNOs in step 1 specifically includes the following steps: First, HNO3 and H2SO4 are mixed at a volume ratio of 1:1 to 1:5, and then added to the CNOs solution. The mixture is then heated in a water bath at a temperature of 80 to 100°C and stirred for 5 to 10 hours. After centrifugation and washing, the pH is adjusted to 7 to obtain the ox-CNOs solution.

3. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 2, characterized in that: The volume ratio of HNO3 to H2SO4 is 1:3, the water bath heating temperature is 90℃, and the reaction time is 7h.

4. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: The preparation of the HNTs@luminol@PDDA composite material in step 2 specifically includes the following steps: adding HNTs to deionized water, sonicating for 1 hour, then adding luminol solution, with a volume ratio of HNTs to luminol of 100:3 to 10:3, centrifuging to remove unadsorbed luminol, adding PDDA and deionized water, with a volume ratio of HNTs to PDDA of 400:1 to 50:1, and shaking overnight to obtain the HNTs@luminol@PDDA composite material.

5. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 4, characterized in that: The volume ratio of HNTs to luminol is 10:1, the volume ratio of HNTs to PDDA is 100:1, and the mass-volume concentration of PDDA is 0.5%.

6. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 4, characterized in that: The centrifuge speed is 5000-10000 r / min.

7. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: The preparation of the ox-CNOs@aptamer complex in step 3 specifically includes the following steps: mixing the ox-CNOs solution with EDC / NHS at a molar ratio of 4:1, adding aptamer, with a volume ratio of ox-CNOs to aptamer of 1:16 to 1:2, stirring overnight, adding bovine serum albumin (BSA) to block non-specific sites, centrifuging, adding IXTE buffer to the precipitate, and obtaining the ox-CNOs@aptamer complex.

8. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 7, characterized in that: The concentration of the ox-CNOs is 1 mg / mL, and the volume ratio of the ox-CNOs to the EDC / NHS is 14:

1.

9. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 7, characterized in that: The volume ratio of the ox-CNOs to the aptamer is 5:

2.

10. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 7, characterized in that: The concentration of aptamer is 10 μM, the concentration of EDC is 20 mM, and the concentration of NHS is 5 mM.

11. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 7, characterized in that: The bovine serum albumin (BSA) has a mass-volume concentration of 0.5%, and the volume ratio of ox-CNOs to BSA is 1:

2.

12. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: In step 4, the amount of HNTs@luminol@PDDA composite material, ox-CNOs@aptamer composite material, and CK-MB solution added is 5 μL.

13. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: The incubation time for both the ox-CNOs@aptamer complex and the CK-MB solution in step 4 is 20–60 min.

14. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: The concentration of the CK-MB solution in step 4 is 10. -4 ng / mL~10 3 ng / mL, specifically a concentration of 10 -4 ng / mL, 10 -3 ng / mL, 10 -2 ng / mL, 10 -1 ng / mL, 1 ng / mL, 10 ng / mL, 10 2 ng / mL, 10 3 ng / mL, and diluted with PBS solution at pH 7.

4.

15. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: The electrochemiluminescence detection in step 4 was performed in 2 mL of PBS and 2 μL of 0.1 M hydrogen peroxide solution, with a photomultiplier tube voltage of 600 V and a scanning voltage range of 0–0.6 V.

16. The method for detecting the myocardial injury biomarker CK-MB using an electrochemiluminescence-photoacoustic dual-mode sensor according to claim 1, characterized in that: The light source of the photoacoustic system in step 4 is an 808nm near-infrared laser light source with a laser power of 0.9W to 1.5W.

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