An immunosensor for detecting cardiac troponin I and a preparation method, use method and application thereof

Abstract

The application discloses a cardiac troponin I detection immunosensor and a preparation method, use method and application thereof, and belongs to the technical field of biomedical sensing. The cardiac troponin I detection immunosensor comprises a solid-phase capture interface for fixing a sample to be detected and a liquid signal amplification probe for amplifying a detection signal; the solid-phase capture interface comprises platinum single-atom-nitrogen-doped carbon composite material modified on the surface of an electrode; the liquid signal amplification probe comprises gold nanoparticle-MXene composite material; the application further discloses a preparation method, use method and application of the cardiac troponin I detection immunosensor. The cardiac troponin I detection immunosensor has high specificity and sensitivity, can realize rapid detection of cardiac troponin I contained in saliva, and can rapidly and preliminarily diagnose and risk stratify various cardiac pathological states.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical sensing technology, and particularly relates to a cardiac troponin I detection immunosensor, a preparation method, a use method and an application thereof. BACKGROUND

[0002] Acute myocardial infarction (AMI) is the main culprit of global cardiovascular disease morbidity and mortality. Myocardial injury is a typical pathological feature of AMI, which is manifested by elevated circulating cardiac troponin I (cTnI) levels. As a result, cTnI has become a specific biomarker for diagnosing AMI and evaluating its prognosis. In clinical practice, the gold standard for diagnosing AMI is the quantitative detection of high-sensitivity cTnI in serum. However, this detection method has obvious limitations. On the one hand, serum cTnI quantitative detection requires professional hospital equipment and trained professionals to operate; on the other hand, the detection result usually cannot be obtained until several hours after the onset of AMI. This critical time delay often causes patients to miss the golden treatment window, resulting in irreversible physical damage to millions of AMI patients worldwide.

[0003] In order to seize the golden treatment window, there is an urgent need for a rapid, non-invasive, simple-to-operate and highly sensitive detection method. Electrochemical biosensing technology has become a promising diagnostic platform in the current and future disease detection field due to its low cost, convenient operation, integrability, miniaturization and rapid response. Using this technology, advanced sensing devices for monitoring various physiological markers or biophysical parameters have been widely developed. In particular, this technology has broad application prospects in the non-invasive screening of biomarkers in body fluids such as saliva, tears, urine and sweat, and studies have shown that its diagnostic value is comparable to blood detection, which creates conditions for rapid evaluation of cTnI levels in AMI patients. In fact, emerging evidence suggests that there is a strong correlation between cTnI concentrations in serum and saliva during myocardial injury, especially during AMI.

[0004] However, the electrochemical biosensors currently designed for detecting cTnI also have some problems. Its sensitivity is usually in the nanogram range, which makes it unable to accurately quantify the pathologically relevant cTnI concentration in saliva (which is in the picogram or even femtogram level during AMI). This key limitation largely limits current advanced sensors to only quantitatively detecting cTnI in serum. However, serum detection methods are inherently invasive and require time-consuming and complex sample processing protocols. In addition, such sensors usually require milliliter-level sample volumes to work properly, and often require mandatory pretreatment steps such as centrifugation or dilution before detection. Considering the difficulty of quickly collecting such a large amount of saliva, this constitutes an obstacle in practical applications.

[0005] How to develop an immunosensor for non-invasive rapid detection of saliva cTnI that can accurately quantify the cTnI level in saliva, achieve a sub-femtogram detection limit, and shorten the diagnostic time is one of the problems that needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an immunosensor for detecting cardiac troponin I, its preparation method, usage method and application. This immunosensor for detecting cardiac troponin I has high specificity and sensitivity, and can realize the rapid detection of cardiac troponin I contained in saliva, so as to quickly perform preliminary triage and risk stratification of various cardiac pathological states.

[0007] The technical solution adopted to achieve the above objectives is to provide an immunosensor for detecting cardiac troponin I, including a solid-phase capture interface for immobilizing the sample to be tested and a liquid-phase signal amplification probe for amplifying the detection signal.

[0008] The solid-phase trapping interface includes a platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface; the liquid-phase signal amplification probe includes a gold nanoparticle-MXene composite material.

[0009] Preferably, in the platinum single-atom-nitrogen-doped carbon composite material, platinum is coordinated with nitrogen in a single-atom form to form a Pt-N4 structure.

[0010] More preferably, the platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface is prepared by the following steps:

[0011] (1) A precursor was prepared by stirring a reaction using 2-methylimidazole and zinc salt as raw materials;

[0012] (2) The precursor is carbonized under a protective atmosphere to obtain a two-dimensional nitrogen-doped carbon support;

[0013] (3) Load a two-dimensional nitrogen-doped carbon support onto the electrode, and then load platinum single atoms onto the two-dimensional nitrogen-doped carbon support by electrochemical deposition to obtain a platinum single atom-nitrogen-doped carbon composite material modified on the electrode surface.

[0014] More preferably, in step (1), the zinc salt is zinc acetate dihydrate; the mass ratio of 2-methylimidazolium to zinc salt is (2~3):(0.6~0.7); the stirring reaction is carried out at room temperature for 22~26 h; in step (2), the heating rate of the carbonization treatment is 4~6℃ / min, the temperature is 850~950℃, and the time is 4~6 h.

[0015] More preferably, the mass ratio of 2-methylimidazole to zinc salt is 2.5:0.65; the stirring reaction is carried out at room temperature for 24 h; the heating rate of carbonization treatment in step (2) is 5℃ / min, the temperature is 900℃, and the time is 5 h.

[0016] More preferably, step (1) includes the following steps: weigh 2-methylimidazole and zinc acetate dihydrate in a mass ratio of 2.5:0.65, and then dissolve them in water to obtain 2-methylimidazole solution and zinc acetate dihydrate solution; mix the 2-methylimidazole solution and zinc acetate dihydrate solution and react at room temperature for 24 h, filter, wash and dry to obtain the precursor.

[0017] More preferably, the protective atmosphere in step (2) is nitrogen.

[0018] More preferably, step (3) includes the following steps: preparing a two-dimensional nitrogen-doped carbon support as a catalyst ink and loading it onto an electrode and drying it; then using KOH and H2PtCl6 aqueous solution as electrolyte, Ag / AgCl electrode as reference electrode, and platinum wire as counter electrode, performing electrochemical deposition at -1.0~-1.2 V to obtain a platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface.

[0019] More preferably, the electrode is a screen-printed electrode.

[0020] Preferably, the gold nanoparticle-MXene composite material is prepared through the following steps:

[0021] (1) Monolayer MXene was prepared by chemical exfoliation;

[0022] (2) The surface of monolayer MXene was modified with polyallylamine hydrochloride solution to obtain MXene with a positive charge on the surface;

[0023] (3) Mix positively charged MXene with a gold-containing solution, and grow gold nanoparticles on the MXene surface by adding formaldehyde in situ to obtain a gold nanoparticle-MXene composite material.

[0024] More preferably, the chemical stripping method in step (1) includes the following steps: dispersing multilayer MXene in water and sonicating for 1 hour, then centrifuging at 3500 rpm for 1 hour, collecting the supernatant and drying it under vacuum to obtain a single layer of MXene.

[0025] More preferably, step (2) includes the following steps: preparing monolayer MXene into a monolayer MXene nanosheet suspension and adding it to a polyallylamine hydrochloride solution for sonication, then stirring and reacting at room temperature, and obtaining positively charged MXene after centrifugation and washing.

[0026] More preferably, the mass ratio of monolayer MXene to polyallylamine hydrochloride is 1:6; the stirring reaction time is 4 h; the centrifugation speed is 14800 rpm and the time is 10 min; and the washing is done with deionized water.

[0027] More preferably, step (3) includes the following steps:

[0028] S1. After dispersing tetramethylphosphoric acid and NaOH evenly in water, add tetrachloroauric acid solution and stir overnight to obtain gold seed solution;

[0029] S2. Add potassium carbonate and tetrachloroauric acid solution to water to react and age to obtain gold growth solution;

[0030] S3. Add positively charged MXene to the gold seed solution and sonicate, then perform the first stirring reaction. After the reaction is complete, wash, then add gold growth solution and perform the second stirring reaction to obtain a mixture.

[0031] S4. Add formaldehyde to the mixture, centrifuge and wash to obtain the gold nanoparticle-MXene composite material.

[0032] More preferably, the volume ratio of tetramethylhydroxyphosphoric acid, NaOH and tetrachloroauric acid solution in S1 is 0.012:0.25:2; the mass fraction of tetramethylhydroxyphosphoric acid is 80%, the molar concentration of NaOH is 2 M, the mass fraction of tetrachloroauric acid solution is 1%, and the stirring temperature is 25°C.

[0033] More preferably, the ratio of potassium carbonate, tetrachloroauric acid solution and water in S2 is 10 mg:1.5 mL:100 mL; the reaction time is 10 min; and the aging time is 24 h.

[0034] More preferably, the sonication time in S3 is 30 min; the first stirring reaction is carried out at room temperature for 4 h; and the second stirring reaction takes 2 h.

[0035] More preferably, the formaldehyde volume concentration in S4 is 29%; the centrifugation speed is 14800 rpm and the time is 20 min.

[0036] This invention also provides a method for preparing the above-mentioned cardiac troponin I detection immunosensor, comprising the following steps:

[0037] (1) Aminosilane was used to modify the platinum single-atom-nitrogen-doped carbon composite material on the electrode surface, and then the capture antibody was immobilized and the non-specific active sites were sealed to obtain a solid-phase capture interface for immobilizing the sample to be tested.

[0038] (2) The detection antibody is immobilized on the surface of the gold nanoparticle-MXene composite material and the non-specific active sites are sealed to obtain a liquid phase signal amplification probe for amplifying the detection signal.

[0039] Preferably, in step (1), the aminosilane is (3-aminopropyl)triethoxysilane; the amination modification time is 6 h; and the capture antibody is capture antibody Ab1.

[0040] Preferably, the detection antibody in step (2) is detection antibody Ab2.

[0041] Preferably, bovine serum albumin is used to seal nonspecific active sites.

[0042] The beneficial effects of the above-mentioned technical solution in this invention are as follows: After the platinum single-atom-nitrogen-doped carbon composite material on the electrode surface is aminated with (3-aminopropyl)triethoxysilane, Ab1 is effectively immobilized through the interaction of Pt-NH2 bonds with the carboxyl groups of Ab1, forming a 2D-Pt SA-Ab1 immunocomplex, which can be used to immobilize the sample to be tested. At the same time, the thiol groups exposed on the surface of the zero-valent gold nanoparticles in the gold nanoparticle-MXene composite material interact with the cysteine ​​residues or free thiol groups in the Fc region of the antibody to form an Au NPs / MXene-Ab2 immunocomplex, which can be used to amplify the detection signal as a liquid phase signal amplification probe.

[0043] This invention also provides a method of using the above-mentioned cardiac troponin I detection immunosensor, comprising the following steps:

[0044] The sample to be tested is mixed and incubated with the solid-phase capture interface, and then a liquid-phase signal amplification probe is added and mixed and incubated to form a sandwich immune complex. A time-ampere current response is generated by catalyzing the H2O2 reduction reaction to achieve quantitative detection of cardiac troponin I in the sample to be tested.

[0045] The beneficial effects of the above technical solution adopted in this invention are as follows: The detection principle of the cardiac troponin I detection immunosensor in this invention is that cTnI in the sample to be tested binds to Ab1 on the solid-phase capture interface, and then binds to Ab2 fixed on AuNPs / MXene to form a sandwich-type immune complex; subsequently, a time-ampere (it) current response is generated through the catalytic H2O2 reduction reaction to realize the quantitative detection of saliva cTnI.

[0046] Preferably, the sample to be tested is a saliva sample.

[0047] The present invention also provides the application of the above-mentioned cardiac troponin I detection immunosensor in the preparation of cardiac troponin I detection kits.

[0048] This invention also provides the application of a cardiac troponin I detection immunosensor in the preparation of a cardiac pathological monitoring device.

[0049] Preferably, the cardiac pathologies include acute myocardial infarction, myocarditis, and rheumatic heart disease.

[0050] The present invention has the following beneficial effects:

[0051] (1) The platinum single-atom-nitrogen-doped carbon composite material used to fix the sample in the solid-phase capture interface of the present invention is prepared by electrodeposition, which can effectively prevent the migration and aggregation of Pt atoms, retain the inherent electrochemical properties of the two-dimensional structure, have high conductivity and abundant Pt-NH2 bonds, realize efficient and directional fixation of Ab1, and improve detection sensitivity and operational stability.

[0052] (2) The gold nanoparticle-MXene composite material in the liquid phase signal amplification probe used to amplify the detection signal in this invention is prepared by modifying a single layer of MXene with polyallylamine hydrochloride and growing gold nanoparticles in situ. It has a large electroactive area and excellent charge transfer kinetics, and provides abundant anchoring sites for zero-valent Au NPs, realizing efficient fixation of Ab2 and significantly enhancing the electrochemical signal.

[0053] (3) This invention successfully constructed a sandwich-type amperometric immunosensor for saliva cTnI detection for the first time. This sensor has excellent distinguishing ability (AUC=1.0) between saliva cTnI concentrations of patients with different heart diseases (AMI, myocarditis, RHD) and healthy individuals, and saliva cTnI concentration is highly correlated with serum cTnI concentration, realizing rapid, non-invasive detection and triage of various cardiac pathological states such as AMI, myocarditis, and rheumatic heart disease; at the same time, the immunosensor of this invention has ultra-high sensitivity (detection limit as low as 0.28 fg / mL). -1 ) and a wide linear range (1 fg mL) -1 Up to 200 ng / mL -1 It can accurately quantify the cTnI level in the saliva of healthy individuals and patients with heart disease.

[0054] (4) The detection method of the present invention does not require complex sample pretreatment, only 2 μL of saliva sample is required, and the analysis can be completed within 23 minutes from sample collection, which greatly shortens the diagnosis time and saves valuable time for the early diagnosis and intervention of acute myocardial infarction. Attached Figure Description

[0055] Figure 1 The preparation process and working mechanism diagram of the immunosensor for detecting cardiac troponin I;

[0056] Figure 2This is a flowchart illustrating the preparation process of platinum single-atom-nitrogen-doped carbon composite materials.

[0057] Figure 3 The image shows the XRD pattern of a platinum single-atom-nitrogen-doped carbon composite material.

[0058] Figure 4 SEM image of a two-dimensional nitrogen-doped carbon support;

[0059] Figure 5 TEM image of a two-dimensional nitrogen-doped carbon support;

[0060] Figure 6 Selected area electron diffraction pattern of a two-dimensional nitrogen-doped carbon support;

[0061] Figure 7 TEM image of a platinum single-atom-nitrogen-doped carbon composite material;

[0062] Figure 8 The image shows the elemental distribution and EDX spectrum of a platinum single-atom-nitrogen-doped carbon composite material; where (a) is the C elemental distribution; (b) is the N elemental distribution; (c) is the Pt elemental distribution; and (d) is the EDX spectrum.

[0063] Figure 9 High-angle annular dark-field-scanning transmission electron microscope image of platinum single-atom-nitrogen-doped carbon composite material at 1 µm;

[0064] Figure 10 High-angle annular dark-field-scanning transmission electron microscope image of platinum single-atom-nitrogen-doped carbon composite material at 5 nm;

[0065] Figure 11 XPS full spectrum scan of platinum single-atom-nitrogen-doped carbon composite material;

[0066] Figure 12 The energy dispersive spectroscopy (EDS) spectra of platinum single-atom-nitrogen-doped carbon composite material and two-dimensional nitrogen-doped carbon support are shown below. Among them, (a) is the C 1s spectrum of the two-dimensional nitrogen-doped carbon support; (b) is the C 1s spectrum of the platinum single-atom-nitrogen-doped carbon composite material; (c) is the N 1s spectrum; and (d) is the Pt 4f spectrum of the platinum single-atom-nitrogen-doped carbon composite material.

[0067] Figure 13 This is a flowchart illustrating the preparation process of gold nanoparticle-MXene composite materials.

[0068] Figure 14 TEM image of a single-layer MXene material;

[0069] Figure 15 TEM image of gold nanoparticle-MXene composite material;

[0070] Figure 16 This is a high-resolution transmission electron microscope image of a single layer of MXene;

[0071] Figure 17 High-resolution transmission electron microscope image of gold nanoparticle-MXene composite material;

[0072] Figure 18 Selected area electron diffraction pattern of gold nanoparticle-MXene composite material;

[0073] Figure 19 XRD patterns of multilayer MXne, single-layer MXene, and gold nanoparticle-MXene composites;

[0074] Figure 20 XPS full spectrum image of gold nanoparticle-MXene composite material and its corresponding high-resolution Ti 2p and Au 4f energy spectra;

[0075] Figure 21 The images show the elemental distribution and EDS spectrum of monolayer MXene; where (a) is the TEM image of monolayer MXene; (b) is the C elemental distribution; (c) is the Ti elemental distribution; and (d) is the EDS spectrum.

[0076] Figure 22 The image shows the elemental distribution and EDS spectrum of the gold nanoparticle-MXene composite material; where (a) is the TEM image of the gold nanoparticle-MXene composite material; (b) is the Ti elemental distribution; (c) is the Au elemental distribution; and (d) is the EDS spectrum.

[0077] Figure 23 Schematic diagram of the electrochemical testing mechanism of the immunosensor for detecting cardiac troponin I;

[0078] Figure 24 Linear fitting plots of the it response current of saliva immunosensors prepared with PBS solutions of different cTnI concentrations to their steady-state current and corresponding cTnI concentrations; where (a) is a current-time curve; (b) is a magnified view of region A in (a); and (c) is a linear fitting plot.

[0079] Figure 25 The results of cTnI level detection in saliva and a data point plot of five groups of subjects;

[0080] Figure 26 The results of cTnI level detection in saliva and box plots of five groups of subjects;

[0081] Figure 27 This is a diagram showing the diagnostic power of salivary cTnI in distinguishing between patients with heart disease and healthy controls.

[0082] Figure 28 A graph showing the correlation between saliva and serum cTnI levels in patients with acute myocardial infarction;

[0083] Figure 29 A graph showing the correlation between saliva and serum cTnI levels in patients with myocarditis.

[0084] Figure 30 A graph showing the correlation between saliva and serum cTnI levels in patients with rheumatic heart disease. Detailed Implementation

[0085] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0086] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0087] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0088] Example 1: Immunosensor for Detecting Cardiac Troponin I and its Preparation Method

[0089] An immunosensor for detecting cardiac troponin I includes a solid-phase capture interface for immobilizing the sample to be tested and a liquid-phase signal amplification probe for amplifying the detection signal.

[0090] The solid-phase trapping interface includes a platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface; the liquid-phase signal amplification probe includes a gold nanoparticle-MXene composite material.

[0091] The preparation process and working mechanism of the cardiac troponin I detection immunosensor in this embodiment are as follows: Figure 1 As shown.

[0092] In the platinum single-atom-nitrogen-doped carbon composite material, platinum coordinates with nitrogen in a single-atom form to form a Pt-N4 structure.

[0093] The platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface was prepared through the following steps:

[0094] (1) Dissolve 2.5 g of 2-methylimidazole in 100 mL of deionized water to obtain a 2-methylimidazole solution; dissolve 0.65 g of zinc acetate dihydrate in 100 mL of deionized water to obtain a zinc acetate dihydrate solution; mix the 2-methylimidazole solution and the zinc acetate dihydrate solution and stir at room temperature for 24 h. After the reaction is complete, collect the product by filtration, wash with deionized water, and dry at 60 °C overnight to obtain the precursor (2D-ZIF-8).

[0095] (2) Place 100 mg of 2D-ZIF-8 in a tube furnace and heat it at a rate of 5 °C / min under N2 flow. Heat it to 900 °C and hold it for 5 h. Then cool it naturally to room temperature to obtain a two-dimensional nitrogen-doped carbon support (2D-NC).

[0096] (3) Mix 20 μL of 5 wt% Nafion with 750 μL of ethanol and 250 μL of deionized water, then add 10 mg of 2D-NC powder, and sonicate for 30 min to obtain a uniform suspension to obtain catalyst ink; then load 2 μL of catalyst ink onto a screen-printed electrode with a diameter of 4 mm and dry at room temperature; then use a CHI760F electrochemical workstation in a three-electrode system with 0.1 M KOH and 10 mg L -1 H₂PtCl₆ aqueous solution was used as the electrolyte, Ag / AgCl (3 M KCl) electrode was used as the reference electrode, and platinum wire was used as the counter electrode for Pt electrochemical deposition. The electrochemical deposition potential range was -1.0 to -1.2 V (vs. Ag / AgCl), and the scan rate was 4 mV s. -1 After 40 consecutive cycles, a platinum single-atom-nitrogen-doped carbon composite material (2D-Pt SA) modified on the electrode surface was obtained.

[0097] The preparation process of platinum single-atom-nitrogen-doped carbon composite material is as follows: Figure 2 As shown.

[0098] The gold nanoparticle-MXene composite material was prepared through the following steps:

[0099] (1) Disperse 100 mg of multilayer MXene in deionized water and sonicate for 1 h, then centrifuge at 3500 rpm for 1 h, collect the supernatant and dry it under vacuum to obtain monolayer MXene;

[0100] (2) 2 mL of monolayer MXene nanosheet suspension (1 mg / mL) -1 Add 4 mL of polyallylamine hydrochloride solution (3 mg / mL) -1Mix and sonicate for 30 min, then stir at room temperature for 4 h, centrifuge at 14800 rpm for 10 min, and wash three times with deionized water to remove free polyallylamine hydrochloride, forming MXene (MXene-PAH composite material) with a positively charged surface.

[0101] (3) Add 12 μL of 80% tetramethylhydroxyphosphoric acid and 0.25 mL of 2 M NaOH to 45 mL of deionized water and stir for 5 min. Then add 2 mL of 1% HAuCl4 solution. The solution immediately turns dark brown. Transfer it to a light-proof container and stir overnight at 25°C to obtain the gold seed solution.

[0102] (4) Add 10 mg K2CO3 to 100 mL of deionized water containing 1.5 mL 1% HAuCl4 and stir. The solution turns colorless within 10 min and is aged for 24 h to obtain gold growth solution.

[0103] (5) Add positively charged MXene to 10 mL of gold seed solution and sonicate for 30 min. Then, carry out the first stirring reaction at room temperature for 4 h. After the reaction, wash twice with deionized water and add it to the same volume of aged gold growth solution for the second stirring reaction for 2 h to obtain a mixture.

[0104] (6) Under continuous stirring, add 29% HCHO solution dropwise over 1 h until the solution turns brownish-blue; then centrifuge at 14800 rpm for 20 min and collect the precipitate, wash with deionized water 3 times to obtain gold nanoparticle-MXene composite material (Au NPs / MXene nanocomposite material).

[0105] The preparation process of gold nanoparticle-MXene composite material is as follows: Figure 13 As shown.

[0106] This embodiment also provides a method for preparing the above-mentioned cardiac troponin I detection immunosensor, including the following steps:

[0107] (1) Add 10 μL of APTES to 700 μL of ethanol and stir for 30 min to obtain an aminosilane solution; then add 2 μL of the aminosilane solution dropwise to a 2D-Pt SA-modified screen-printed electrode and incubate for 6 h to obtain aminated 2D-Pt SA, forming Pt-NH2 bonds; then add 2 μL of capture antibody Ab1 solution (20 μg mL) dropwise to obtain amino-modified 2D-Pt SA, forming Pt-NH2 bonds; -1The capture antibody Ab1 was immobilized using 2 μL of 1 wt% BSA solution; after immobilization, non-specific active sites were blocked using 2 μL of 1 wt% BSA solution; after each modification step, the electrode was gently rinsed with PBS buffer (pH 7.4), and finally stored at 4°C to obtain the solid-phase capture interface (2D-Pt SA-Ab1) for immobilizing the test sample.

[0108] (2) Disperse Au NPs / MXene in 2 mL of detection antibody Ab2 solution (20 μg / mL) -1 The sample was placed in a solution and incubated overnight at 4°C with shaking. Then, 1 wt% BSA solution was added and incubated for 4 h to block non-specific binding sites. PBS buffer (pH=7.4) was added and the sample was centrifuged 3 times to obtain a liquid-phase signal amplification probe (Au NPs / MXene-Ab2) for amplifying the detection signal.

[0109] Example 2: Method of using the immunosensor for detecting cardiac troponin I

[0110] The method for using the immunosensor for cardiac troponin I detection includes the following steps:

[0111] (1) Add 2.0 μL of saliva sample containing cardiac troponin I to 2D-Pt SA-Ab1 and incubate for 10 min; then add 2 μL of Au NPs / MXene-Ab2 and incubate for 10 min. Wash away unbound material with PBS buffer (pH=7.4) to form sandwich immune complex.

[0112] (2) Using an electrochemical workstation, the time-amperometry (it) method was used to detect the cardiac troponin I in a PBS solution (pH=7.4) containing 5.0 mM H2O2. The working electrode was a sandwich immune complex, the counter electrode was a platinum wire, and the reference electrode was Ag / AgCl (3 MKCl). The working potential was -0.4 V, and the current-time curve was recorded to realize the quantitative detection of cardiac troponin I in the test sample.

[0113] The electrochemical testing mechanism of the immunosensor for cardiac troponin I detection is as follows: Figure 23 As shown.

[0114] Example 3 Morphology and property characterization of two-dimensional nitrogen-doped carbon support and platinum single-atom-nitrogen-doped carbon composite material

[0115] The morphology and properties of two-dimensional nitrogen-doped carbon supports and platinum single-atom-nitrogen-doped carbon composites were characterized. Surface composition and chemical state were analyzed using X-ray photoelectron spectroscopy (XPS, Thermo Fisher K-Alpha, USA), and the crystal structure of the catalyst was characterized using X-ray diffraction (XRD, Rigaku Ultima IV, Japan). Sample morphology was observed using scanning electron microscopy (SEM, ZEISS Sigma 360, Germany) and transmission electron microscopy (TEM, Thermo Fisher Talos F200X, USA). Elemental analysis of the nanomaterials was performed using energy-dispersive spectroscopy (EDS, ULTIM MAX 40, UK). High-resolution TEM (HRTEM), selected area electron diffraction (SAED), and high-angle annular dark-field scanning TEM (HAADF-STEM) images were acquired at 200 keV using a JEOL JEM-ARM200F NEO ARM (Japan) to analyze the sample morphology and structural characteristics. Results are as follows: Figures 3-12 As shown.

[0116] like Figure 3 As shown, in the XRD pattern of the two-dimensional nitrogen-doped carbon support, only two broad peaks were observed at 26.2° and 44.0°. These peaks are typical diffraction features of graphitic carbon, indicating that the carbonization process of the zeolite imidazoline framework was successfully completed. The peak at the lower 2θ angle corresponds to the amorphous phase, which plays a crucial role in the platinum single-atom-nitrogen-doped carbon composite material, constituting the most active sites. This characteristic is precisely the superior advantage of ZIF-8 as a precursor for the synthesis of various single-atom catalysts. The diffraction peaks at 39.8° and 46.2° correspond to the characteristic crystallographic planes of platinum nanoparticles (PDF number 04-0802). Figure 4 and Figure 5 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images show that the two-dimensional nitrogen-doped carbon support exhibits a distinct layered structure. Figure 6 The SAED image only shows a diffuse halo in the central region, confirming that the platinum species did not crystallize significantly on the two-dimensional nitrogen-doped carbon support substrate. Figure 7 TEM images show that the morphology of the platinum single-atom-nitrogen-doped carbon composite material is basically consistent with that of 2D-NC. From... Figure 8 (All scales are 1 µm) and Figure 9 As can be seen from the (platinum atoms are marked in a dot form) diagram, platinum single atoms are uniformly distributed in the two-dimensional nitrogen-doped carbon support; the elemental distribution diagram further shows that carbon, nitrogen and platinum elements are uniformly distributed. Figure 10Platinum atoms were labeled with pink circles. HAADF-STEM imaging revealed atomically dispersed platinum atoms within the two-dimensional nitrogen-doped carbon support, appearing as bright spots stably anchored on the substrate. The platinum, nitrogen, and carbon elemental composition of the 2D-Pt SA was characterized using XPS. Figure 11 and Figure 12 As can be seen, compared with the two-dimensional nitrogen-doped carbon support, the introduction of platinum single atoms resulted in a platinum signal in the XPS full spectrum of the platinum single-atom-nitrogen-doped carbon composite material, with a content of approximately 0.02% as determined by quantitative analysis. The C 1s peak of the platinum single-atom-nitrogen-doped carbon composite material can be decomposed into three peaks: C=O (289.9 eV), CN (285.5 eV), and CC (284.7 eV). In contrast, the C 1s spectrum of the two-dimensional nitrogen-doped carbon support shows three peaks: C=O (288.6 eV), CN (286.1 eV), and CC (284.8 eV). The observed increase in C=O bonding energy is attributed to the withdrawal of electrons from the carbon substrate by the platinum single atom, leading to a decrease in the electron density around the carbonyl carbon. Conversely, the decrease in CN bonding energy stems from the donation of lone pairs of electrons from nitrogen atoms to platinum atoms, thereby weakening the electron-withdrawing effect of nitrogen on adjacent carbon atoms. High-resolution N 1s spectra of platinum single-atom-nitrogen-doped carbon composites show peaks at 400.8, 399.7, and 398.4 eV, corresponding to graphitic nitrogen, pyrrole nitrogen, and pyridine nitrogen, respectively. In contrast, the N 1s peaks of the two-dimensional nitrogen-doped carbon support appear at 401.9, 399.2, and 396.8 eV. Compared to the two-dimensional nitrogen-doped carbon support, the peak positions of pyridine nitrogen and pyrrole nitrogen in the platinum single-atom-nitrogen-doped carbon composites are shifted to higher binding energies, indicating that nitrogen lone pair electrons transfer charge to the empty orbitals of platinum atoms, thereby promoting the formation of Pt-N coordination bonds and accelerating electron transfer. In the platinum single-atom-nitrogen-doped carbon composites, platinum single atoms are mainly in the +2 oxidation state, as evidenced by the Pt 4f peaks at 72.4 and 75.7 eV. 7 / 2 and 4f 5 / 2 The bimodal distribution was confirmed. Pt 4f at 70.9 and 74.2 eV... 7 / 2 With 4f 5 / 2 Peak belongs to Pt 0 Pt 4f at 72.5 and 76.1 eV 7 / 2 With 4f 5 / 2 The peak may originate from Pt 2+ High oxidation state.

[0117] Example 4: Characterization of the properties of gold nanoparticle-MXene composite material

[0118] The morphology and properties of monolayer Mxene and gold nanoparticle-MXene composites were characterized. X-ray photoelectron spectroscopy (XPS, Thermo Fisher K-Alpha, USA) was used to analyze surface composition and chemical state; X-ray diffraction (XRD, Rigaku Ultima IV, Japan) was used to characterize the catalyst crystal structure; transmission electron microscopy (TEM, Thermo Fisher Talos F200X, USA) was used to observe sample morphology; and energy-dispersive spectroscopy (EDS, ULTIM MAX 40, UK) was used for elemental analysis of the nanomaterials. High-resolution TEM (HRTEM) and selected area electron diffraction (SAED) were performed at 200 keV using a JEOL JEM-ARM200F NEO ARM (Japan) to analyze sample morphology and structural characteristics. Results are as follows: Figures 14-22 As shown.

[0119] To verify the successful preparation of monolayer MXene nanosheets and gold nanoparticle / MXene nanocomposites, transmission electron microscopy (TEM) was used for characterization, and the results are as follows: Figures 14-15 As shown. From Figures 14-15 As can be seen, the synthesized monolayer MXene has a smooth and wrinkled surface, exhibiting excellent flexibility and providing ideal loading sites for gold nanoparticles. Compared with MXene nanosheets, the gold nanoparticle-MXene composite also maintains a planar structure. However, a large number of gold nanoparticles fixed on the surface of the monolayer MXene are clearly visible, indicating that the gold nanoparticle-MXene composite has been successfully prepared. The particle size distribution of the gold nanoparticles ranges from 3 to 60 nm, with an average particle size of 27.3 nm.

[0120] Figure 16 and Figure 17 The middle part consists of lattice fringes of monolayer MXene and lattice fringes of gold nanoparticle-MXene composite material; Figure 16 and 17 The HRTEM images clearly show diffraction spacings of 0.251 nm corresponding to the (006) crystal plane of MXene and 0.205 nm corresponding to the (002) crystal plane; furthermore, a crystal plane spacing of 0.198 nm corresponds to the (200) crystal plane of the gold nanoparticles. Meanwhile, the SAED electron diffraction pattern of the selected region shows the typical hexagonal symmetry of MXene. Figure 18 ); This shows that the (006) crystal plane of MXene and the (220) crystal plane of gold coexist, further confirming the successful synthesis of gold nanoparticle-MXene nanocomposite material.

[0121] To further elucidate the structural composition of the synthesized monolayer MXene and gold nanoparticle-MXene nanocomposite material, XRD and XPS were used for characterization. Figure 19 As can be seen, both monolayer and multilayer MXene exhibit typical MXene (002) diffraction peaks; however, when gold nanoparticles are fixed on the surface of monolayer MXene, the intensity of the (002) diffraction peak is significantly weakened, while the (004) peak shows a significant enhancement. This change originates from the epitaxial orientation of the MXene layers induced by the gold nanoparticles. This structure suppresses scattering from the (002) basal plane while enhancing the diffraction signal of the (004) orientation. Further analysis of the XPS full-spectrum scan of the gold nanoparticle-MXene pattern reveals… Figure 20 As can be seen from the data, the composite material is mainly composed of titanium, carbon, oxygen, and gold. The peaks at 458.3 and 464.1 eV correspond to the 2p phase of titanium, respectively. 3 / 2 and 2p 1 / 2 The two characteristic gold 4f peaks at energy levels 83.6 and 87.3 eV correspond to the 4f energy of gold, respectively. 7 / 2 and 4f 5 / 2 The energy level, with a peak spacing of 3.6 eV for the Au 4f doublet, confirms that the gold nanoparticles are in a zero-valence state. Furthermore, energy-dispersive X-ray spectroscopy (EDS) analysis further confirms the elemental composition and uniform distribution of the monolayer MXene material and the gold nanoparticle-MXene nanocomposite material. Figure 21 and Figure 22 , Figure 21 The medium scale is 100 nm. Figure 22 The medium scale is 500 nm.

[0122] Example 5: Determination of the detection limit of an immunosensor for cardiac troponin I detection

[0123] (1) Different concentrations of cTnI solutions were prepared using PBS buffer. The concentrations of the cTnI solutions were 0.1 fg / mL. -1 1 fg mL -1 10 fg mL -1 100 fg mL -1 1 pg mL -1 10 pg mL -1 100 pg mL -1 1 ng mL -1 10 ng mL -1 100 ng mL -1 200 ng mL -1 and 500 ng mL -1 ;

[0124] (2) Add 2.0 μL of cTnI solution of different concentrations to 2D-Pt SA-Ab1 and incubate for 10 min; then add 2 μL of Au NPs / MXene-Ab2 and incubate for 10 min. Wash away unbound material with PBS buffer (pH=7.4) to form sandwich immune complex.

[0125] (3) Using an electrochemical workstation, the time-amperometry (it) method was employed to detect the cTnI in a PBS solution (pH=7.4) containing 5.0 mM H2O2. The working electrode was a sandwich immune complex, the counter electrode was a platinum wire, and the reference electrode was Ag / AgCl (3 MKCl). A working potential of -0.4 V was applied, and the current-time curve was recorded. The corresponding cTnI concentration was linearly fitted, and the results are as follows: Figure 24 As shown. From Figures 24-25 As can be seen from this, the steady-state current value is related to the logarithm (lg c) of the cTnI concentration within 1 fg mL. -1 Up to 200 ng / mL -1 It exhibits a linear relationship within the range, with a detection limit (LOD) as low as 0.28 fg / mL. -1 .

[0126] Example 6 Clinical Saliva Sample Detection

[0127] 1. Saliva samples were collected from healthy subjects before bedtime (Group I, n=6), healthy subjects 8 hours after sleep (Group II, n=6), patients diagnosed with acute myocardial infarction (Group III, n=6), patients diagnosed with myocarditis (Group IV, n=6), and patients diagnosed with rheumatic heart disease (RHD) (Group V, n=6). Immunosensors were prepared according to the steps in Examples 1-2 above and used for detection. The distribution of saliva cTnI concentration in each group was detected, and the results are as follows: Figures 25-26 As shown. Figure 27 Groups labeled with Roman numerals (i-iv) represent statistically significantly different homogeneous subsets (Dunn-Bonferroni test, P<0.001); numerical labels reflect the order of cTnI: acute myocardial infarction (i) > myocarditis (ii) > rheumatic heart disease (iii) > healthy control group (iv), focusing on specific measured concentrations (i.e. data points). Figure 27The bottom whiskers of the box represent the minimum value, the top whiskers represent the maximum value, and the dots represent the mean value. The data displayed is the interquartile range (IQR), defined as the difference between the 75th and 25th percentiles, representing the middle 50% of the data range and reflecting the variability of the central distribution, minimizing the influence of extreme values. Groups labeled with Roman numerals (i-iv) represent statistically significantly different homogeneous subsets (Dunn-Bonferroni test, P<0.001). Numerical labels reflect the rank of cTnI: acute myocardial infarction (i) > myocarditis (ii) > rheumatic heart disease (iii) > healthy control group (iv). Further analysis of the diagnostic efficacy of salivary cTnI in distinguishing between heart disease and healthy controls yielded the following results: Figures 25-27 As shown. ROC (Receiver Operating Characteristic) curve analysis demonstrates the diagnostic discrimination achieved by salivary cTnI concentration, with AUC being Area Under Curve. Specific comparison results are as follows: (i) healthy control group vs. all heart disease groups (AUC=1.000, P<0.0001), (ii) healthy control group vs. acute myocardial infarction (AUC=1.000, P<0.001), (iii) healthy control group vs. myocarditis (AUC=1.000, P<0.001), (iv) healthy control group vs. rheumatic heart disease (AUC=1.000, P<0.001), (v) acute myocardial infarction vs. non-acute myocardial infarction cases (AUC=1.000, P<0.001), (vi) myocarditis vs. rheumatic heart disease (AUC=1.000, P<0.01). Statistical significance for all pairwise comparisons was determined using the Mann-Whitney U test (all pairwise comparisons P<0.01). The gray dashed line represents random classification (AUC=0.5). Figures 28-30 The enlarged view within the black dashed box is a magnified view of the ROC curve. For easier observation, the curve has been manually offset, and all AUC values ​​are 1. ​ As can be seen, the concentration of salivary cTnI is significantly elevated in patients with heart disease, and there are significant differences among different disease groups (AMI > myocarditis > RHD > healthy control group). It is highly correlated with serum cTnI concentration (Pearson correlation coefficient ρ > 0.984). ROC curve analysis shows that the AUC of the discrimination between all groups is 1.0, indicating that the cardiac troponin I detection immunosensor prepared in this invention has perfect diagnostic accuracy.

[0128] 2. Correlation analysis was performed on the saliva and serum cTnI levels of patients diagnosed with acute myocardial infarction, myocarditis, and rheumatic heart disease, respectively. The correlation coefficient ρ was obtained through Pearson correlation analysis. The results are as follows: ​As shown, Pearson correlation analysis revealed a high correlation between salivary cTnI concentrations and serum cTnI concentrations in patients with acute myocardial infarction (ρ=0.997), myocarditis (ρ=0.995), and rheumatic heart disease (ρ=0.984). These strong correlations highlight the potential of salivary cTnI as a non-invasive biomarker for monitoring various cardiac diseases. This high correlation may be attributed to the configuration of the sandwich immunoassay and the high specificity of the anti-cTnI antibody—which ensures accurate target recognition and maintains signal integrity.

[0129] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.

Claims

1. An immunosensor for detecting cardiac troponin I, characterized in that, It includes a solid-phase trapping interface for immobilizing the sample and a liquid-phase signal amplification probe for amplifying the detection signal; The solid-phase trapping interface comprises a platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface; the liquid-phase signal amplification probe comprises a gold nanoparticle-MXene composite material. In the platinum single-atom-nitrogen-doped carbon composite material, platinum is coordinated with nitrogen in single-atom form to form a Pt-N4 structure. The platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface was prepared through the following steps: (1) A precursor was prepared by stirring a reaction using 2-methylimidazole and zinc salt as raw materials; (2) The precursor is carbonized under a protective atmosphere to obtain a two-dimensional nitrogen-doped carbon support; (3) Load a two-dimensional nitrogen-doped carbon support onto the electrode, and then load platinum single atoms onto the two-dimensional nitrogen-doped carbon support by electrochemical deposition to obtain a platinum single atom-nitrogen-doped carbon composite material modified on the electrode surface. The gold nanoparticle-MXene composite material was prepared through the following steps: 1) Monolayer MXene was prepared by chemical exfoliation; 2) The surface of monolayer MXene was modified with polyallylamine hydrochloride solution to obtain MXene with a positively charged surface; 3) Mix positively charged MXene with a gold-containing solution, and grow gold nanoparticles on the MXene surface by adding formaldehyde in situ to reduce it, thus obtaining a gold nanoparticle-MXene composite material.

2. The cardiac troponin I detection immunosensor as described in claim 1, characterized in that, In step (1), the zinc salt is zinc acetate dihydrate; the mass ratio of 2-methylimidazolium to zinc salt is (2~3):(0.6~0.7); the stirring reaction is carried out at room temperature for 22~26 h; in step (2), the heating rate of carbonization treatment is 4~6℃ / min, the temperature is 850~950℃, and the time is 4~6 h.

3. The method for preparing the cardiac troponin I detection immunosensor according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Aminosilane is used to modify the platinum single-atom-nitrogen-doped carbon composite material modified on the electrode surface, and then the capture antibody is immobilized and the non-specific active sites are sealed to obtain a solid-phase capture interface for immobilizing the sample to be tested. S2. The detection antibody is immobilized on the surface of the gold nanoparticle-MXene composite material and the non-specific active sites are sealed to obtain a liquid phase signal amplification probe for amplifying the detection signal.

4. The method of using the cardiac troponin I detection immunosensor according to any one of claims 1 to 2 for non-diagnostic purposes, characterized in that, Includes the following steps: The sample to be tested is mixed and incubated with the solid-phase capture interface, and then a liquid-phase signal amplification probe is added and mixed and incubated to form a sandwich immune complex. A time-ampere current response is generated by catalyzing the H2O2 reduction reaction to achieve quantitative detection of cardiac troponin I in the sample to be tested.

5. The method of using the cardiac troponin I detection immunosensor as described in claim 4 for non-diagnostic purposes, characterized in that, The sample to be tested is a saliva sample.

6. The use of the cardiac troponin I detection immunosensor according to any one of claims 1 to 2 in the preparation of a cardiac troponin I detection kit.

7. The application of the cardiac troponin I detection immunosensor according to any one of claims 1 to 2 in the preparation of a cardiac pathological monitoring device.

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