Biosensor for simultaneously detecting cardiac troponin I and interleukin 6 as well as construction method and detection method of biosensor

Abstract

The biosensor for simultaneously detecting cardiac troponin I and interleukin 6 comprises an indium tin oxide electrode, and two sensing areas with independent functions, namely an area I and an area II, are integrated on a single indium tin oxide electrode; the area I is modified with a cardiac troponin I binding aptamer, rhodamine B and a cadmium sulfide and bismuth tungstate composite material, and is used for detecting the cardiac troponin I aptamer through photoelectrochemical and colorimetric-fluorescence double signals; the region II is modified with an interleukin-6 binding aptamer and methylene blue silicon dioxide and is used for detecting interleukin-6 through photoelectrochemical and electrochemical double signals. The sensor and the detection method are good in accuracy and reliability.

Description

Technical Field

[0001] This invention relates to a biosensor for simultaneously detecting cardiac troponin I and interleukin-6, as well as its construction and detection methods, belonging to the field of chemical technology. Background Technology

[0002] Cardiac troponin I (cTnI) is a regulatory protein expressed exclusively in cardiomyocytes and is a crucial component of the troponin complex that regulates myocardial contraction. Under normal physiological conditions, cardiac troponin I is undetectable in the blood. However, any event that impairs the integrity of the cardiomyocyte membrane (most commonly myocardial ischemia in acute myocardial infarction) will lead to its release into the circulation. This release follows a well-defined kinetic pattern: levels begin to rise within hours of infarction, peak at 12-24 hours, and remain elevated for 7-10 days. Due to its high cardiac specificity, direct correlation with myocardial necrosis, and diagnostic kinetics, cardiac troponin I has become the clinical gold standard for assessing myocardial injury, especially acute myocardial infarction.12 Utilizing these properties, various methods for detecting cardiac troponin I have been developed over the past decade, ranging from single-modality to advanced dual-modality strategies. However, the detection of a single biomarker (such as detecting only cardiac troponin I) is inherently insufficient to capture the full complexity of the pathophysiology of cardiovascular disease. Biomarker release patterns are highly dynamic and heterogeneous. Single biomarker detection only provides a fragmented, static "snapshot" of the highly dynamic disease process, failing to explain the complex interactions between multiple biological pathways driving cardiovascular disease progression. Therefore, exploring multiplex detection platforms is crucial for more accurate diagnosis and risk stratification. These platforms aim to capture the multidimensional characteristics of cardiovascular disease pathophysiology by simultaneously measuring a set of complementary biomarkers. The prognostic value of inflammatory markers such as interleukin-6 (IL-6) is increasingly recognized. IL-6 is a pro-inflammatory cytokine that is rapidly upregulated during myocardial ischemia, reflecting the severity of systemic inflammatory responses. Elevated IL-6 levels after acute myocardial infarction are closely associated with an increased risk of heart failure, re-infarction, and long-term mortality. In light of this, the 2022 European Society of Cardiology (ESC) guidelines for the management of acute coronary syndromes explicitly emphasize the complementary value of combining structural biomarkers (such as cardiac troponin I to confirm myocardial injury) and inflammatory biomarkers (such as interleukin-6 to assess prognostic risk) for comprehensive risk stratification and personalized treatment planning in patients. Therefore, simultaneous detection of cardiac troponin I and interleukin-6 is crucial for the accurate diagnosis and risk stratification of acute myocardial infarction. While traditional sensing platforms (including single-analyte detection (single-mode or dual-mode) and multi-analyte detection based on a single signal modality) have made significant progress, they still face key limitations, including insufficient reliability, lack of cross-validation results, and difficulty in revealing the complex interactions between multiple biomarkers in clinical samples. In contrast, multi-modal, multi-analyte sensing platforms offer a more reliable alternative.By integrating multiple detection modes for various analytes, it not only improves accuracy through built-in signal verification (effectively reducing false positives and false negatives), but also supports multidimensional correlation analysis between biomarkers. Summary of the Invention

[0003] The purpose of this invention is to provide a biosensor for simultaneously detecting cardiac troponin I and interleukin-6, as well as its construction and detection methods.

[0004] To achieve the objectives of this invention, the following technical solution is adopted: a biosensor for simultaneously detecting cardiac troponin I and interleukin-6, comprising an indium tin oxide electrode, wherein two functionally independent sensing regions, region I and region II, are integrated on a single indium tin oxide electrode; region I is modified with a cardiac troponin I binding aptamer and a composite material of rhodamine B and cadmium sulfide@bismuth tungstate, for detecting cardiac troponin I by photoelectrochemical and colorimetric-fluorescence dual signals; region II is modified with an interleukin-6 binding aptamer and methylene blue@silica, for detecting interleukin-6 by photoelectrochemical and electrochemical dual signals; the cardiac troponin I binding aptamer is an amino-modified cardiac troponin I aptamer with the sequence CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-NH2; the interleukin-6 binding aptamer is an amino-modified interleukin-6 aptamer with the sequence ACCACCTTCCAACGCTCGTATTGTCAGTCTTTAGT-NH2.

[0005] A method for constructing a dual-channel, dual-mode biosensor for detecting cardiac troponin I and interleukin 6, for constructing the biosensor for detecting cardiac troponin I and interleukin 6 as described in claim 1, comprising the following steps: S1: Pattern the ultrasonically cleaned indium tin oxide glass slide with insulating adhesive to create two regions, I and II, with a diameter of 4 mm; S2: Region I was used for cardiac troponin I detection: The electrode was modified by drop-coating 20 μL of a first aqueous solution mixture, which contained 2 mg / mL cadmium sulfide@bismuth tungstate, 20 μM rhodamine B, 0.1 wt% chitosan, and 0.01 wt% perfluorosulfonic acid resin; then dried. Region II was used for interleukin-6 detection: The electrode was modified by drop-coating 20 μL of a second aqueous solution mixture, which contained 4 mg / mL methylene blue@silica, 0.1 wt% chitosan, and 0.01 wt% perfluorosulfonic acid resin; then dried. S3: Functionalization before testing: Region I was crosslinked with 1.0 wt% glutaraldehyde at 37°C for 1 hour; then, 1.0 μM of amino-modified cardiac troponin I aptamer was fixed by glutaraldehyde-aptamer condensation at 37°C for 1 hour; then, it was blocked with 1.0 wt% bovine serum albumin at 37°C for 0.5 hours; this is then ready for use in capturing the target analyte, cardiac troponin I, during detection. Region II was cross-linked with 1.0 wt% glutaraldehyde at room temperature for 1 hour; then, 1.0 μM amino-modified interleukin-6 aptamer was fixed by glutaraldehyde-aptamer condensation at 37°C for 1 hour; then, it was blocked with 1.0 wt% bovine serum albumin at 37°C for 0.5 hours; it can then be used to capture the target analyte interleukin-6 during detection.

[0006] A dual-channel, dual-mode method for detecting cardiac troponin I and interleukin-6 was developed. The biosensor for detecting cardiac troponin I and interleukin-6, constructed using the aforementioned method, was used as follows: Photoelectrochemical response measurement simultaneously detects cardiac troponin I and interleukin-6: A three-electrode system was used. The modified indium tin oxide electrode was used as the working electrode, the silver / silver chloride electrode as the reference electrode, and the platinum wire electrode as the counter electrode; the electrolyte was a 0.1 M phosphate buffer solution with pH 7.4 containing 0.1 M ascorbic acid; the bias voltage was 0 V; and the light source was a flashlight with wavelengths of 450 nm and 650 nm. Electrochemical response measurement of interleukin-6: A three-electrode system was used for electrochemical response measurement. The modified indium tin oxide electrode was used as the working electrode, the silver / silver chloride electrode as the reference electrode, and the platinum wire electrode as the counter electrode; a 0.1 M phosphate buffer solution with pH 7.4, deoxygenated by nitrogen, was used as the electrolyte solution; the differential pulse voltammetric response of methylene blue in region I was monitored in the range of -0.6 V to 0 V, with a sampling width of 0.0167 s. Colorimetric-fluorescence assay for cardiac troponin I: photographs of the region I electrode were taken under a 365 nm UV light source; RGB analysis was then performed.

[0007] Furthermore, the synthesis method of cadmium sulfide@bismuth tungstate composite material is as follows: 0.06 g of bismuth tungstate nanoflowers are dispersed in 5 mL of deionized water and ultrasonically treated for 10 minutes to form a uniform suspension; then 0.024 g of cadmium nitrate tetrahydrate and 0.042 g of sodium sulfide nonahydrate are added to the bismuth tungstate nanoflower suspension, and the mixture is stirred continuously for 6 hours; the product is collected by centrifugation, washed with deionized water, and dried at 50°C for 6 hours to obtain yellow cadmium sulfide@bismuth tungstate composite powder; The synthesis method of bismuth tungstate nanoflowers is as follows: 2 mmol of bismuth nitrate pentahydrate and 1 mmol of sodium tungstate dihydrate are added to 30 mL of ultrapure water and stirred at 500 rpm for 1 hour at room temperature until homogeneous; then the mixture is poured into a 50 mL polytetrafluoroethylene-lined autoclave and reacted at 150 °C for 12 hours. Finally, the product is cooled to room temperature, washed three times alternately with ethanol and ultrapure water, and dried using a freeze dryer.

[0008] Furthermore, the synthesis method of methylene blue@silica is as follows: 1) To prepare silica, 9 mL of concentrated ammonia (26 wt%) and 24.75 mL of deionized water were mixed into 16.25 mL of ethanol to obtain solution A; then 4.5 mL of tetraethyl orthosilicate and 45.5 mL of ethanol were mixed to obtain solution B; solution B was added dropwise to solution A, and the mixture was stirred at 500 rpm for 3 hours at room temperature; the product was washed three times alternately with ethanol and deionized water, and then freeze-dried to obtain silica; 2) Synthesis of methylene blue@silica: 0.008 g of silica was dissolved in 1 mL of deionized water and sonicated. 1 mL of 500 μM methylene blue solution was added to the above solution, and the mixture was reacted in a shaker for 30 minutes. After centrifugation, methylene blue@silica was obtained.

[0009] Furthermore, the detection method described herein has a detection limit of less than or equal to 0.86 pg / mL for myotropin I and a detection limit of less than 0.65 pg / mL for interleukin-6.

[0010] The positive and beneficial technical effects of this invention are: the sensor and detection method have good accuracy and reliability, which will be described in detail with reference to specific implementation methods. Attached Figure Description

[0011] Figure 1 This is a SEM image of bismuth tungstate nanoflowers.

[0012] Figure 2This is a SEM image of cadmium sulfide@bismuth tungstate.

[0013] Figure 3 This is a comparison of the XRD patterns of cadmium sulfide, bismuth tungstate, and cadmium sulfide@bismuth tungstate.

[0014] Figure 4 This is a zeta potential diagram of Rhodamine B / Cadmium sulfide@bismuth tungstate.

[0015] Figure 5 This is a fluorescence absorption diagram of the bismuth tungstate and cadmium sulfide@bismuth tungstate composite material.

[0016] Figure 6 This is a fluorescence comparison image of Rhodamine B / CdS@bismuth indium tin oxide electrode (B) and CdS@bismuth indium tin oxide electrode (A) under 365 nm illumination.

[0017] Figure 7 This is a graph showing the changes in photoelectric signals during the preparation of the RhB / CdS@Bi2WO6 composite material. In the graph: a. Bi2WO6NFs (nanoflowers); b. CdS@Bi2WO6; c. RhB / CdS@Bi2WO6).

[0018] Figure 8 This is a graph showing the change in fluorescence signal (R / G ratio) during the preparation of the RhB / CdS@Bi2WO6 composite material. In the graph: a. Bi2WO6NFs; b. CdS@Bi2WO6; c. RhB / CdS@Bi2WO6).

[0019] Figure 9 It is the ultraviolet-visible (UV-Vis) absorption spectrum of silica and methylene blue@silica.

[0020] Figure 10 This is a graph showing the changes in photoelectrochemical (PEC) signals during the preparation of methylene blue@silica composite materials. (a. SiO2, b. MB@SiO2).

[0021] Figure 11 This is a graph showing the changes in electrochemical (EC) signals during the preparation of methylene blue@silica composite materials. (a. SiO2, b. MB@SiO2).

[0022] Figure 12 This is a graph showing the photoelectrochemical (PEC) response of cardiac troponin I in region I under 450 nm excitation as a function of concentration; (concentrations a–h correspond to: 0, 0.001, 0.01, 0.1, 1, 10, 20, 50 ng / mL) -1 ).

[0023] Figure 13 This is a graph showing the linear relationship between the signal and the concentration of cardiac troponin I.

[0024] Figure 14 A schematic diagram of the linear relationship between the R / G ratio and cardiac troponin I concentration in colorimetric-fluorescence (CF) mode.

[0025] Figure 15 This is a graph showing the change in photoelectrochemical (PEC) response as a function of concentration in the detection of interleukin-6 (IL-6) in Region II; (concentrations a–h correspond to: 0, 0.001, 0.01, 0.1, 1, 10, 20, 50 ng / mL) -1 ).

[0026] Figure 16 This is a graph showing the electrochemical (EC) response as a function of concentration in the detection of interleukin-6 (IL-6) in Region II; (concentrations a–h correspond to: 0, 0.001, 0.01, 0.1, 1, 10, 20, 50 ng / mL) -1 ).

[0027] Figure 17 This is a graph showing the linear relationship between electrochemical (EC) response and concentration in the detection of interleukin-6 (IL-6) in Region II.

[0028] Figure 18 This is one of the selective schematic diagrams for the detection of cardiac troponin I (cTnI) (photoelectric detection).

[0029] Figure 19 This is the second schematic diagram of the selectivity of cardiac troponin I (cTnI) detection (fluorescence detection).

[0030] Figure 20 This is one of the selective schematic diagrams for interleukin-6 detection (photoelectric detection).

[0031] Figure 21 This is the second schematic diagram of the selectivity of interleukin-6 detection (electrochemistry). Detailed Implementation

[0032] To more fully explain the implementation of the present invention, implementation examples are provided. These implementation examples are merely illustrative of the present invention and do not limit the scope of the present invention.

[0033] 1. Synthesis of cadmium sulfide@bismuth tungstate composite material:

[0034] Bismuth tungstate nanoflowers (Bi₂WO₆NFs) were synthesized via a typical hydrothermal reaction. First, 2 mmol of bismuth nitrate pentahydrate and 1 mmol of sodium tungstate dihydrate were added to 30 mL of ultrapure water and stirred at 500 rpm for 1 hour at room temperature until homogeneous. The mixture was then poured into a 50 mL PTFE-lined autoclave and reacted at 150 °C for 12 hours. Finally, the product was cooled to room temperature, washed three times alternately with ethanol and ultrapure water, and then dried using a freeze dryer.

[0035] Synthesis of cadmium sulfide@bismuth tungstate composite material: 0.06 g of bismuth tungstate nanoflowers were dispersed in 5 mL of deionized water and sonicated for 10 minutes to form a homogeneous suspension. Subsequently, 0.024 g of cadmium nitrate tetrahydrate and 0.042 g of sodium sulfide nonahydrate were added to the bismuth tungstate nanoflower suspension, and the mixture was stirred continuously for 6 hours. The product was collected by centrifugation, washed with deionized water, and dried at 50 °C for 6 hours to obtain a yellow cadmium sulfide@bismuth tungstate composite powder for later use. A homogeneous suspension of cadmium sulfide@bismuth tungstate at 2 mg / mL was prepared in a 0.01 wt% perfluorosulfonic acid resin aqueous solution. Therefore, Rhodamine B can be fixed on the surface of cadmium sulfide@bismuth tungstate by electrostatic adsorption.

[0036] like Figure 1 As shown, pure bismuth tungstate nanoflowers exhibit a multi-layered flower-like morphology with an average diameter of approximately 800 nm, possessing a high specific surface area, which is beneficial for subsequent loading. After the introduction of cadmium sulfide (Figure 2), the composite particle size increases and the surface roughness increases, indicating successful cadmium sulfide modification. Figure 3 The XRD patterns of cadmium sulfide, bismuth tungstate, and cadmium sulfide@bismuth tungstate were compared (Figure 1). The diffraction peaks of pure cadmium sulfide matched the cubic phase (JCPDS 89-0440), while those of bismuth tungstate corresponded to the orthorhombic phase (JCPDS 39-0256). The composite material retained all the bismuth tungstate peaks, with an additional weak peak at 28.12°, attributed to the cadmium sulfide (101) crystal plane, indicating successful loading of cadmium sulfide without altering the host crystal structure.

[0037] Cadmium sulfide@bismuth tungstate is an excellent photoelectrochemical material. Combined with Rhodamine B (a fluorescent dye), the hybrid system possesses both optical and fluorescent properties. As shown in Figure 4, pristine bismuth tungstate exhibits almost no absorption in the visible light region (>400 nm), significantly limiting its visible light utilization efficiency. After loading with cadmium sulfide, the light absorption of the cadmium sulfide@bismuth tungstate composite is significantly enhanced. Its absorption intensity in the visible spectrum increases, and the absorption edge shows a noticeable redshift. This extended absorption indicates its excellent visible light trapping ability, mainly attributed to the strategic addition of cadmium sulfide as a highly efficient photosensitizer.

[0038] Based on the zeta potential shown in Figure 4, the Rhodamine B / Cd sulfide@bismuth tungstate hybrid system can be prepared via electrostatic interactions. Cadmium sulfide@bismuth tungstate is an excellent photoelectrochemical material; combined with Rhodamine B (a fluorescent dye), the hybrid system possesses both optical and fluorescent properties. As shown in Figure 5, pristine bismuth tungstate exhibits almost no absorption in the visible light region (>400 nm), significantly limiting its visible light utilization efficiency. After loading with cadmium sulfide, the light absorption of the cadmium sulfide@bismuth tungstate composite material is significantly enhanced. Its absorption intensity in the visible spectrum increases, and the absorption edge undergoes a significant redshift. This extended absorption indicates its excellent visible light trapping ability, mainly attributed to the strategic addition of cadmium sulfide as a highly efficient photosensitizer. As shown in Figure 6, under 365 nm illumination, the Rhodamine B / Cd sulfide@bismuth tungstate indium tin oxide electrode exhibits significant fluorescence emission, while the cadmium sulfide@bismuth tungstate indium tin oxide electrode shows no fluorescence, demonstrating the fluorescent properties of Rhodamine B / Cd sulfide@bismuth tungstate. Figure 7 shows the changes in photoelectrochemical and colorimetric-fluorescence signal (R / G ratio) during the preparation of Rhodamine B / cadmium sulfide@bismuth tungstate composite materials. Figure 8 As shown. In summary, these results demonstrate that Rhodamine B / CdSulphide@bismuth tungstate can be used as a feasible photoelectrochemical / colorimetric-fluorescence dual-performance material to construct a dual-modal (photoelectrochemical / colorimetric-fluorescence) sensing interface in Region I.

[0039] 2. Synthesis of methylene blue@silica:

[0040] Synthesis of silica: First, 9 mL of concentrated ammonia (26 wt%) and 24.75 mL of deionized water were mixed with 16.25 mL of ethanol to obtain solution A. Then, 4.5 mL of tetraethyl orthosilicate and 45.5 mL of ethanol were mixed to obtain solution B. Solution B was added dropwise to solution A, and the mixture was stirred at 500 rpm for 3 hours at room temperature. The product was washed three times alternately with ethanol and deionized water, and then lyophilized using a freeze dryer. For the synthesis of methylene blue@silica, 0.008 g of silica was dissolved in 1 mL of deionized water and sonicated. 1 mL of 500 μM methylene blue solution was added to the above solution, and the mixture was reacted in a shaker for 30 minutes, then centrifuged for later use.

[0041] Figure 9 shows the UV-Vis absorption spectra of pure silica and methylene blue@silica. Pure silica exhibits negligible absorption in the visible region, while methylene blue@silica shows a distinct characteristic absorption peak at approximately 650 nm. This optical property indicates that methylene blue@silica can serve as a red light-sensitive element, generating a photoelectric response under specific red light excitation (corresponding to the 650 nm absorption peak), laying a crucial optical foundation for achieving red light-driven photoelectric signal output in Region II. The changes in photoelectrochemical (PEC) and electrochemical (EC) signals during the preparation of the methylene blue@silica composite material are shown below. Figure 10 , Figure 11 As shown, the photoelectrochemical and electrochemical responses of pristine silica are negligible, with the signal almost overlapping with the background. After loading with methylene blue, both signals are significantly enhanced, consistent with the material characterization results. The observed signal enhancement stems from the high loading capacity of the silica support and the excellent photoelectrochemical response and redox activity of methylene blue itself, which together contribute to sensitive dual-mode detection. This property enables Region II to simultaneously output photoelectrochemical and electrochemical signals.

[0042] 3. Construction of biosensors:

[0043] An ultrasonically cleaned indium tin oxide glass slide (0.7 cm × 3.3 cm) was patterned with insulating adhesive to define two working areas with a diameter of 4 mm. The preparation steps for each area are as follows: Region I (for cardiac troponin I detection): The electrode was modified by drop-coating 20 μL of a mixture containing cadmium sulfide@bismuth tungstate (2 mg / mL), 20 μM rhodamine B (RhB), chitosan (CS, 0.1 wt%), and perfluorosulfonic acid resin (0.01 wt%), followed by drying. Subsequent functionalization steps included: (1) crosslinking with 1.0 wt% glutaraldehyde (37°C, 1 h); (2) immobilization of aminated cardiac troponin I aptamers by glutaraldehyde-aptamer condensation (1.0 μM, 37°C, 1 h); (3) blocking with 1.0 wt% bovine serum albumin (37°C, 0.5 h); (4) capturing the cardiac troponin I target at detection time (1 h); washing with deionized water after each step.

[0044] Region II (for interleukin-6 detection): The electrode was modified by drop-coating 20 μL of a mixture containing methylene blue@silica (4 mg / mL), chitosan (CS, 0.1 wt%), and perfluorosulfonic acid resin (0.01 wt%), followed by drying. Subsequent functionalization steps included: (1) crosslinking with 1.0 wt% glutaraldehyde (room temperature, 1 h); (2) immobilization of aminated interleukin-6 aptamer by glutaraldehyde-aptamer condensation (1.0 μM, 37°C, 1 h); (3) blocking with 1.0 wt% bovine serum albumin (37°C, 0.5 h); and (4) capture of interleukin-6 at detection (room temperature, 1 h). Each step was followed by washing with deionized water.

[0045] 4. Simultaneous multimodal detection of cardiac troponin I and interleukin-6

[0046] 1) A three-electrode system was used to measure the photoelectrochemical response. The modified indium tin oxide electrode was the working electrode, the silver / silver chloride electrode was the reference electrode, and the platinum wire electrode was the counter electrode. The electrolyte was a 0.1 M phosphate buffer solution containing 0.1 M ascorbic acid (pH 7.4). The bias voltage was 0 V, and the light source was a flashlight with wavelengths of 450 nm and 650 nm.

[0047] 2) Electrochemical Testing: Electrochemical response measurements were performed using a three-electrode system. The modified indium tin oxide electrode served as the working electrode, the silver / silver chloride electrode as the reference electrode, and the platinum wire electrode as the counter electrode. A 0.1 M phosphate buffer solution (pH 7.4) deoxygenated with nitrogen was used as the electrolyte solution. The differential pulse voltammetry (DPV) response of methylene blue in region I was monitored within the range of -0.6 V to 0 V, with a sampling width of 0.0167 s.

[0048] 3) Colorimetric-fluorescence test: A photograph of the I electrode area was taken using a mobile phone (camera) under a 365 nm ultraviolet light source. Subsequently, RGB analysis was performed using the intelligent software Adobe Illustrator.

[0049] Multiple signals and multiple detection Dual-mode detection of cardiac troponin I (cTnI) in Region I showed that the photoelectrochemical (PEC) response to 450 nm excitation decreased in a concentration-dependent manner (Figure 12). The signal was highly significantly linearly correlated with cardiac troponin I concentration in the range of 1 pg / mL to 50 ng / mL (R0). 2 =0.9959)( Figure 12The calculated limit of detection (LOD) was 0.27 pg / mL (signal-to-noise ratio S / N = 3). In colorimetric-fluorescence (CF) mode, the R / G ratio decreased in a dependent manner with increasing cardiac troponin I concentration, exhibiting good linearity in the range of 10 pg / mL to 50 ng / mL (R... 2 =0.9963), and the limit of detection was 0.86 pg / mL (Figure 14).

[0050] In the detection of interleukin-6 (IL-6) in Region II, both photoelectrochemical and electrochemical (EC) modes exhibited a signal-off response. The linear range of the photoelectrochemical mode was 1 pg / mL to 20 ng / mL, with a detection limit of 0.57 pg / mL (Figure 15); the linear range of the electrochemical mode was similar (1 pg / mL to 20 ng / mL), with a detection limit of 0.65 pg / mL. Figure 16 , Figure 17 These results further confirm the practical feasibility of using this sensor to read out complementary signals and simultaneously quantify cardiac troponin I and interleukin-6.

[0051] Selective validation of biosensors: In this application, selectivity was evaluated using 10 μg / mL cytochrome C (Cyt C), 1 μg / mL human immunoglobulin (IgG), 50 ng / mL human cardiac myoglobin (Myo), 500 pg / mL interleukin-17 (IL-17), 500 pg / mL interleukin-23 (IL-23), 1 ng / mL cardiac troponin I, 1 ng / mL interleukin-6, and a mixed solution containing all of these substances. The prepared electrodes were stored in a sealed environment for a certain period to test their corresponding storage stability. Serum from both healthy individuals and patients was provided by the First Affiliated Hospital of Guangzhou Medical University. Patient serum was used for actual sample testing. All serum was diluted 1:10 before use. Comparative data for interleukin-6 were provided by the hospital and measured using a Roche e801 chemiluminescence analyzer. Cardiac troponin I was measured using an enzyme-linked immunosorbent assay (ELISA) kit. The research submitted in this application follows the principles of the Declaration of Helsinki and has been approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University, with the approval number ES-2024-217-02.

[0052] To comprehensively evaluate the practical application value of biosensors, their selectivity was systematically studied. For the detection of cardiac troponin I (cTnI), potential interfering agents such as cytochrome C (Cyt C), human immunoglobulin (IgG), human myoglobin (Myo), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), cTnI alone, and mixtures of these substances were investigated. (See Figure 18 and...) Figure 1 As shown in Figure 9, the photocurrent change of cardiac troponin I in photoelectrochemical (PEC) sensing mode was minimal (<5%) upon contact with the six interfering substances. Notably, the platform exhibited almost identical photocurrent responses to cardiac troponin I alone and to the mixture of cardiac troponin I and interfering substances, with a signal deviation of less than 3%. Similarly, in colorimetric-fluorescence (CF) mode, the R / G ratio of individual interfering substances remained at the background level (0.05 ± 0.01), while both cardiac troponin I alone and the mixture of cardiac troponin I and interfering substances showed consistent responses (0.82 ± 0.04). These results confirm that the sensor possesses excellent selectivity when detecting cardiac troponin I in region I using a photoelectrochemical / colorimetric-fluorescence dual-mode approach.

[0053] For the specificity assessment of interleukin-6, cytochrome C, human immunoglobulin, human cardiac myoglobin, N-terminal pro-B-type brain natriuretic peptide (NT-proBNP), interleukin-17, interleukin-23, interleukin-6 alone, and mixtures of these substances were selected as interfering agents. Figures 20 and 21 show that: (1) the signal changes caused by the six individual interfering agents were minimal (only <8% of the target analyte response); (2) the sample containing only interleukin-6 produced a significant signal; and (3) the signal produced by the mixture was almost identical to that of interleukin-6 alone. The signal changes between individual detection and mixed detection were within 5%, indicating that the sensor has excellent selectivity when using photoelectrochemical / electrochemical dual-mode detection of interleukin-6 in Region II.

[0054] Analysis of the construction principles of the sensing interfaces in Region I and Region II: The construction of the Region I sensing platform began with the deposition of cadmium sulfide@bismuth tungstate (CdS@Bi2WO6) on an indium tin oxide (ITO) electrode. Subsequently, a rhodamine B (RhB)-chitosan (CS) hybrid layer was assembled via electrostatic interaction with the negatively charged cadmium sulfide@bismuth tungstate. To immobilize the amino-modified cardiac troponin I (cTnI) specific aptamer, the interface was further functionalized via glutaraldehyde (GA)-mediated crosslinking. The bifunctional aldehyde group of glutaraldehyde forms Schiff base bonds with the primary amino groups on chitosan and the aptamer, ensuring aptamer stability and directional immobilization while retaining bioaffinity. Therefore, by integrating the photoelectrochemical activity of cadmium sulfide@bismuth tungstate under 450 nm irradiation and the fluorescence signal of rhodamine B under 365 nm irradiation, a dual-mode aptamer sensing platform for cardiac troponin I detection was constructed.

[0055] In Region II, the sensing interface was constructed by immobilizing the interleukin-6 (IL-6) aptamer (AptIL-6) on a composite membrane of methylene blue@silica (MB@SiO2), perfluorosulfonic acid resin (Nafion), and chitosan via glutaraldehyde crosslinking. The specific binding of interleukin-6 to the aptamer triggers a conformational change. Utilizing the dual-mode activity of the methylene blue@silica probe (exhibiting photoelectrochemical behavior under 650 nm irradiation and intrinsic electrochemical behavior in the absence of light), this binding event was converted into a detectable signal.

[0056] The experimental reagents and instruments involved in this application are detailed below: Bismuth nitrate pentahydrate (Bi(NO3)3・5H2O, ≥99%), Rhodamine B (RhB, 99%), chitosan (CS), glutaraldehyde (GA), and ascorbic acid (AA, >99%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Sodium tungstate dihydrate (Na2WO6・2H2O, ≥98%) and perfluorosulfonic acid resin (Nafion, 20 wt%) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Ammonia and tetraethyl orthosilicate (TEOS, 98%) were purchased from Chengdu Kelon Chemical Co., Ltd. and Sigma-Aldrich (Shanghai) Trading Co., Ltd., respectively. All DNA sequences were purchased from Shanghai Sangon Biotech Co., Ltd., and the sequences are as follows: Amino-modified cardiac troponin I aptamer: CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-NH2 Amino-modified interleukin-6 aptamer: ACCACCTTCCAACGCTCGTATTGTCAGTCTTTAGT-NH2 Scanning electron microscopy (SEM) images were acquired using a JSM-7610F field emission scanning electron microscope (JEC Ltd., Tokyo). Ultraviolet-visible (UV-vis) spectra were determined using a NanoDrop OneC micro-spectrophotometer (Thermo Fisher Scientific, California, USA). X-ray diffraction (XRD) patterns were obtained using a Rigaku Ultima IV diffractometer (Rigaku Ltd., Tokyo). Photoelectrochemical and electrochemical measurements were performed on a CHI830e electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., Shanghai). 450 nm, 650 nm, and 365 nm LED flashlights were used as light sources for photoelectrochemical and colorimetric experiments, respectively. Electrochemical impedance spectroscopy (EIS) was performed using a Gamry Reference 600 potentiostat (Gamry Instruments, Warminster, Pennsylvania, USA). X-ray photoelectron spectroscopy (XPS) was performed by Thermo Fisher Scientific - ESCALAB Xi+ (Thermo Fisher Scientific, California, USA).

[0057] After a detailed description of the embodiments of the present invention, those skilled in the art will clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned patent applications. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention, and the present invention is not limited to the embodiments of the examples given in the specification.

Claims

1. A biosensor for simultaneously detecting cardiac troponin I and interleukin-6, comprising an indium tin oxide electrode, characterized in that: Two functionally independent sensing regions, Region I and Region II, are integrated on a single indium tin oxide electrode. Region I is modified with a cardiac troponin I binding aptamer and a composite material of rhodamine B and cadmium sulfide@bismuth tungstate for the detection of cardiac troponin I by photoelectrochemical and colorimetric-fluorescence dual signals. Region II is modified with an interleukin-6 aptamer and methylene blue@silica for the detection of interleukin-6 via photoelectrochemical and electrochemical dual signals; the cardiac troponin I binding aptamer is an amino-modified cardiac troponin I aptamer with the sequence CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-NH2; the interleukin-6 aptamer is an amino-modified interleukin-6 aptamer with the sequence ACCACCTTCCAACGCTCGTATTGTCAGTCTTTAGT-NH2.

2. A method for constructing a biosensor for simultaneously detecting cardiac troponin I and interleukin 6, used to construct the biosensor for simultaneously detecting cardiac troponin I and interleukin 6 as described in claim 1, characterized in that... Includes the following steps: S1: Pattern the ultrasonically cleaned indium tin oxide glass slide with insulating adhesive to create two regions, I and II, with a diameter of 4 mm; S2: Region I is used for cardiac troponin I detection: The electrode is modified by drop-coating 20 μL of a first aqueous solution mixture, which contains 2 mg / mL cadmium sulfide@bismuth tungstate, 20 μM rhodamine B, 0.1 wt% chitosan, and 0.01 wt% perfluorosulfonic acid resin; then dried. Region II is used for interleukin-6 detection: The electrode is modified by drop-coating 20 μL of a second aqueous solution mixture, which contains 4 mg / mL methylene blue@silica, 0.1 wt% chitosan, and 0.01 wt% perfluorosulfonic acid resin; then dried. S3: Functionalization before testing: Region I was cross-linked with 1.0 wt% glutaraldehyde at 37°C for 1 hour; then, 1.0 μM of amino-modified cardiac troponin I aptamer was fixed by glutaraldehyde-aptamer condensation at 37°C for 1 hour; then, it was blocked with 1.0 wt% bovine serum albumin at 37°C for 0.5 hours; this is then ready for use in capturing the target analyte, cardiac troponin I, during detection. Region II was cross-linked with 1.0 wt% glutaraldehyde at room temperature for 1 hour; then, 1.0 μM amino-modified interleukin-6 aptamer was fixed by glutaraldehyde-aptamer condensation at 37°C for 1 hour; then, it was blocked with 1.0 wt% bovine serum albumin at 37°C for 0.5 hours; it can then be used to capture the target analyte interleukin-6 during detection.

3. A method for simultaneously detecting cardiac troponin I and interleukin-6, using the biosensor for simultaneously detecting cardiac troponin I and interleukin-6 constructed according to claim 2, characterized in that... The detection method is as follows: Photoelectrochemical response measurement simultaneously detects cardiac troponin I and interleukin-6: A three-electrode system was used. The modified indium tin oxide electrode was used as the working electrode, the silver / silver chloride electrode as the reference electrode, and the platinum wire electrode as the counter electrode; the electrolyte was a 0.1 M phosphate buffer solution with pH 7.4 containing 0.1 M ascorbic acid; the bias voltage was 0 V; and the light source was a flashlight with wavelengths of 450 nm and 650 nm. Electrochemical response measurement of interleukin-6: A three-electrode system was used for electrochemical response measurement. The modified indium tin oxide electrode was used as the working electrode, the silver / silver chloride electrode as the reference electrode, and the platinum wire electrode as the counter electrode. A 0.1 M phosphate buffer solution with pH 7.4, after nitrogen deoxygenation, was used as the electrolyte solution; the differential pulse voltammetric response of methylene blue in region I was monitored in the range of -0.6 V to 0 V, with a sampling width of 0.0167 s. Colorimetric-fluorescence assay for cardiac troponin I: a photograph of region I electrode was taken under a 365 nm UV light source; RGB analysis was then performed.

4. The biosensor for simultaneously detecting cardiac troponin I and interleukin-6 according to claim 1, characterized in that: The synthesis method of cadmium sulfide@bismuth tungstate composite material is as follows: 0.06 g of bismuth tungstate nanoflowers are dispersed in 5 mL of deionized water and ultrasonically treated for 10 minutes to form a uniform suspension; Subsequently, 0.024 g of cadmium nitrate tetrahydrate and 0.042 g of sodium sulfide nonahydrate were added to the bismuth tungstate nanoflower suspension, and the mixture was stirred continuously for 6 hours. The product was collected by centrifugation, washed with deionized water, and dried at 50°C for 6 hours to obtain yellow cadmium sulfide@bismuth tungstate composite powder. The synthesis method of bismuth tungstate nanoflowers is as follows: 2 mmol of bismuth nitrate pentahydrate and 1 mmol of sodium tungstate dihydrate are added to 30 mL of ultrapure water and stirred at 500 rpm for 1 hour at room temperature until homogeneous; then the mixture is poured into a 50 mL polytetrafluoroethylene-lined autoclave and reacted at 150 °C for 12 hours. Finally, the product is cooled to room temperature, washed three times alternately with ethanol and ultrapure water, and dried using a freeze dryer.

5. The biosensor for simultaneously detecting cardiac troponin I and interleukin-6 according to claim 1, characterized in that: The synthesis method of methylene blue@silica is as follows: 1) To prepare silica, 9 mL of concentrated ammonia (26 wt%) and 24.75 mL of deionized water were mixed in 16.25 mL of ethanol to obtain solution A; then 4.5 mL of tetraethyl orthosilicate and 45.5 mL of ethanol were mixed to obtain solution B; solution B was added dropwise to solution A, and the mixture was stirred at 500 rpm for 3 hours at room temperature; the product was washed three times alternately with ethanol and deionized water, and then freeze-dried to obtain silica; 2) Synthesis of methylene blue@silica: 0.008 g of silica was dissolved in 1 mL of deionized water and sonicated. 1 mL of 500 μM methylene blue solution was added to the above solution, and the mixture was reacted in a shaker for 30 minutes. After centrifugation, methylene blue@silica was obtained.

6. The method for simultaneous detection of cardiac troponin I and interleukin-6 according to claim 3, characterized in that: The detection method described herein has a detection limit of less than or equal to 0.86 pg / mL for myotropin I and a detection limit of less than 0.65 pg / mL for interleukin-6.

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