Construction of photoelectrochemical-multimodal synergistic monitoring platform for brain natriuretic peptide precursor and cardiac troponin-i and detection method thereof

Abstract

The application discloses a kind of for brain natriuretic peptide precursor and electrochemical multimodal synergistic monitoring platform of troponin-I photoelectricity construction and detection method, belong to biomedical detection technical field.Sensor platform includes ITO glass substrate, in ITO glass substrate region I and region II, respectively with the sensor for NT-proBNP and cTnI detection;Sensor for NT-proBNP detection includes mesoporous silica nanosphere attached on ITO, on silica nanosphere, there is mercapto acetic acid coated cadmium sulfide quantum dots, on cadmium sulfide quantum dots, there is NT-proBNP detection probe connection;Sensor for cTnI detection includes mesoporous silica nanosphere attached on ITO, on silica nanosphere, there is gold nanoparticle, on gold nanoparticle, there is cTnI detection probe coupling.The application can realize multi-mode multi-analyte detection.

Description

Technical Field

[0001] This invention specifically relates to the construction and detection method of a photoelectric-electrochemical multimodal synergistic monitoring platform for brain natriuretic peptide precursors and cardiac troponin-I, belonging to the field of biomedical sensor detection technology. Background Technology

[0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide. It often presents with acute, nonspecific symptoms, making accurate differential diagnosis crucial. For example, the treatment regimens for acute heart failure (HF) and myocardial infarction (AMI) differ significantly, requiring targeted intervention. N-terminal pro-B-type natriuretic peptide (NT-proBNP, reflecting myocardial wall pressure) is currently used as a biomarker for detecting HF, while cardiac troponin I (cTnI, reflecting myocardial injury) is a biomarker for detecting AMI. Currently, these biomarkers are commonly used in medicine using methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunochromatographic assay (ICA), and electrochemiluminescent immunoassay (ECLIA). However, these methods suffer from long analysis times, cross-reactivity, and may not meet clinically required threshold levels in some cases. Furthermore, single detection modalities have limitations in sensitivity, selectivity, and stability. Moreover, CVD has complex pathogenesis, and relying on a single biomarker often fails to provide complete clinical information, making it difficult to meet the needs of accurate diagnosis. The rapidly developing photoelectrochemical (PEC) detection technology, developed from electrochemical (EC) technology, demonstrates significant application value in the field of biological detection and analysis due to its high sensitivity, low background signal, and rapid response. Therefore, developing sensors based on photoelectrochemical-electrochemical technologies to acquire multi-dimensional, cross-validated information for each biomarker, such as NT-proBNP and cTnI, or even developing sensor platforms capable of simultaneously detecting multiple biomarkers, is crucial for improving diagnostic accuracy and reliability, and is driving the development of sensing technology towards a truly "multi-dimensional" direction. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a photoelectrochemical-electrochemical sensor and detection method for the detection of NT-proBNP and cTnI. For any target substance, it can collect both photoelectrochemical and electrochemical signals, and the two signals can be cross-validated. At the same time, by integrating the two types of sensors into a single sensing platform, a sensor detection platform is constructed that can simultaneously output the multi-mode signals of the two analytes in a single detection, thereby improving the ability to obtain a comprehensive and high-fidelity information on the disease state in complex samples.

[0004] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a photoelectrochemical-electrochemical sensor for NT-proBNP detection, comprising an ITO glass substrate, mesoporous silica nanospheres attached to the ITO glass substrate, cadmium sulfide quantum dots coated with mercaptoacetic acid attached to the silica nanospheres, and an NT-proBNP detection probe connected to the cadmium sulfide quantum dots; the NT-proBNP detection probe is a double-stranded DNA1 (dsDNA1), the first strand of which is a 3'-amino-mediated NT-proBNP aptamer (3′NH2-GTC CCC CTG CCC AGC CCA ATG TCG ACT CAC ACC GG-5′), the sequence of which is shown in SEQ ID No. 1; the second strand is a 3'-Fc-modified complementary strand c-DNA1 (5′-CAG GGG GAC GGG TCG GGT TAC AGC TGA GTGTGG CC-Fc-3′), the sequence of which is shown in SEQ ID No. 2, wherein Fc is ferrocene.

[0005] Secondly, the present invention provides a photoelectrochemical-electrochemical sensor for cTnI detection, comprising an ITO glass substrate, mesoporous silica nanospheres attached to the ITO glass substrate, gold nanoparticles attached to the silica nanospheres, a cTnI detection probe coupled to the gold nanoparticles, and methylene blue attached to the cTnI detection probe; the cTnI detection probe is double-stranded DNA2 (dsDNA2), the first strand of which is a 3'-thiolized cTnI aptamer (3′SH-CGCATG CCA AAC GTT GCC TCA TAG TTC CCT CCC CGT GTC C-5′), the sequence of which is shown in SEQ ID No. 3; the second strand is a complementary c-DNA2 (5′-GCG TAC GGT TTG CAA CGG AGT ATC AAG GGA GGG GCA CAG G-3′), the sequence of which is shown in SEQ ID No. 4.

[0006] Thirdly, this invention provides a photoelectrochemical-electrochemical sensor platform for the detection of NT-proBNP and cTnI. This sensor platform includes both a photoelectrochemical-electrochemical sensor for NT-proBNP detection and a photoelectrochemical-electrochemical sensor for cTnI detection. Specifically, two independent detection functional regions are set on an ITO glass substrate: region I is used for NT-proBNP detection, and region II is used for cTnI detection. On the same ITO glass substrate, mesoporous silica nanospheres are attached to region I, and cadmium sulfide quantum dots coated with mercaptoacetic acid are attached to the silica nanospheres. An NT-proBNP detection probe is connected to the cadmium sulfide quantum dots. The NT-proBNP detection probe is a double-stranded DNA 1, where the first strand is a 3'-amino-mediated NT-proBNP aptamer (3′NH2-GTC CCC CTG CCC AGC CCAATG TCG ACT CAC ACC GG-5′), and the second strand is a 3'-Fc-modified complementary strand (5′-CAG). The structure consists of GGG GAC GGGTCG GGT TAC AGC TGA GTG TGG CC-Fc-3′; mesoporous silica nanospheres are attached to region II, gold nanoparticles are attached to the silica nanospheres, a cTnI detection probe is coupled to the gold nanoparticles, and methylene blue is attached to the cTnI detection probe; the cTnI detection probe is a double-stranded DNA2, the first strand of which is a 3′-thiolized cTnI aptamer (3′SH-CGC ATG CCA AAC GTT GCC TCA TAG TTC CCT CCC CGT GTC C-5′), and the second strand is a complementary strand (5′-GCG TAC GGT TTG CAA CGG AGT ATC AAG GGA GGG GCA CAG G-3′).

[0007] Furthermore, the preparation method of the mesoporous silica nanospheres (MSN) is as follows: using cationic surfactant CTAC as a template, TEOS as a silicon source, TEA as a catalyst, and 1-octane, decahydronaphthalene, and cyclohexane as emulsifiers, silica nanoparticles (SiO2 NPs) are first prepared by stepwise growth through a one-pot two-phase layering method; then the template is removed from the silica nanoparticles to obtain mesoporous silica nanospheres (MSNs).

[0008] Furthermore, the preparation method of the thioglycolic acid-coated cadmium sulfide quantum dots is as follows: thioglycolic acid is added to an aqueous solution of cadmium nitrate, and then the pH of the solution is adjusted to 11 with sodium hydroxide solution; then, freshly prepared sodium sulfide aqueous solution is rapidly added under vigorous stirring; the reaction system is stirred in the dark for 10 min, and anhydrous methanol is added until a precipitate is formed, and the water-soluble cadmium sulfide quantum dots coated with thioglycolic acid (denoted as COOH-CdS QDs) are obtained.

[0009] Furthermore, the preparation method of cadmium sulfide quantum dots (denoted as CdS@MSN) coated with mercaptoacetic acid on silica nanospheres is as follows: MSNs are dispersed in anhydrous ethanol solution containing 10 wt% 3-aminopropyltriethoxysilane (APTES), refluxed at 60 °C for 3 h under stirring, centrifuged and washed to obtain amino-functionalized MSNs (denoted as NH2-MSNs); then, NH2-MSNs are dispersed in deionized water under ultrasonic conditions to form a uniform suspension, and a pre-synthesized COOH-CdSQDs aqueous solution is added, stirred continuously at room temperature for 30 min, centrifuged and washed to obtain CdS QDs / NH2-MSNs composite nanomaterials for later use.

[0010] Furthermore, the preparation method of gold nanoparticles (denoted as Au@MSN) attached to silica nanospheres is as follows: MSNs are dissolved in deionized water and ultrasonically dispersed to ensure uniform dispersion; then chloroauric acid (HAuCl4) solution is added and stirred to mix uniformly; under continuous stirring, sodium citrate solution is added dropwise, and the reaction causes gold nanoparticles to be generated on the MSN surface. After centrifugation, washing, and drying, Au@MSN is obtained.

[0011] Furthermore, the NT-proBNP detection probe is prepared as follows: 100 μL of 1 μM 3'-amino-modified NT-proBNP aptamer (AptNT-proBNP) is mixed with 100 μL of 1 μM 3'-Fc-modified complementary DNA1 (c-DNA1), annealed at 95°C for 5 min, and then slowly cooled to room temperature to obtain double-stranded DNA1 (dsDNA1).

[0012] Furthermore, the cTnI detection probe is prepared as follows: 100 μL of 1 μM cTnI aptamer (AptcTnI) is reacted with 100 μL of 1 mM tris(2-carboxyethyl)phosphine (TCEP) for 1 h, then annealed at 95 °C for 5 min, and hybridized with 100 μL of complementary DNA2 (c-DNA2) to form double-stranded DNA2 (dsDNA2). The obtained dsDNA2 is mixed with an equal volume of 1 mg / mL Au@MSN aqueous solution and incubated overnight at 4 °C in the dark to achieve conjugation, obtaining the final dsDNA2-Au@MSN complex.

[0013] Fourthly, this invention provides a method for preparing a photoelectrochemical-electrochemical sensor for NT-proBNP detection: An ITO glass substrate is cleaned and dried, then 20 μL of a 4 mg / mL CdS@MSN aqueous solution is dropped onto it and allowed to dry; next, 20 μL of EDC / NHS solution is dropped onto it and incubated at room temperature for 1 h; subsequently, 20 μL of dsDNA1 is immobilized using a carbodiimide chemical method (37 °C, 1 h), where the carboxyl groups on the CdS surface bind to the amino groups of the NT-proBNP aptamer, thus achieving the immobilization of the capture probe.

[0014] Fifthly, the present invention provides a method for preparing a photoelectrochemical-electrochemical sensor for cTnI detection: an ITO glass substrate is cleaned and dried, then 20 μL of pre-assembled dsDNA2-Au@MSN is dropped onto it and dried; then 20 μL of 500 μM methylene blue (MB) solution is dropped onto it and incubated at room temperature for 1 h.

[0015] Sixthly, this invention provides a method for fabricating a photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection. An ITO glass substrate is cleaned and dried, and two working regions, region I and region II, are defined. A photoelectrochemical-electrochemical sensor for NT-proBNP detection is fabricated in region I, and a photoelectrochemical-electrochemical sensor for cTnI detection is fabricated in region II. The sensors in regions I and II are in parallel, and the fabrication order is not important.

[0016] In a seventh aspect, the present invention also provides an NT-proBNP detection kit, the kit comprising: (1) Multiple photoelectrochemical-electrochemical sensors for NT-proBNP detection prepared in the first aspect mentioned above; (2) NT-proBNP standard solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL; (3) PBS electrolyte with pH 7.4 and concentration of 0.01M.

[0017] Eighthly, the present invention also provides a cTnI detection kit, the kit comprising: (1) Multiple photoelectrochemical-electrochemical sensors for cTnI detection prepared in the second aspect above; (2) cTnI standard solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL; (3) PBS electrolyte with pH 7.4 and concentration of 0.01M.

[0018] In a ninth aspect, the present invention also provides an NT-proBNP and cTnI detection kit, the kit comprising: (1) A number of photoelectrochemical-electrochemical sensor platforms for NT-proBNP and cTn detection prepared in the third aspect mentioned above; (2) NT-proBNP standard solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL; (3) cTnI standard solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL; (4) PBS electrolyte with pH 7.4 and concentration of 0.01M.

[0019] All three kits should be stored at -20℃ for later use.

[0020] In a tenth aspect, the present invention also provides a method for detecting NT-proBNP and / or cTnI, employing a three-electrode system of an electrochemical workstation equipped with dual laser sources of 450 nm and 650 nm. Using the aforementioned kit, and with the photoelectrochemical-electrochemical sensor platform for NT-proBNP and / or cTnI detection as the working electrode, the working electrode is first incubated with NT-proBNP or cTnI standard samples of different concentrations according to different functional regions. A standard curve is plotted by measuring the photocurrent and / or electrochemical signal using chronoamperometry. Then, the current of the sample to be tested is measured, and the concentration of the sample to be tested is obtained according to the standard curve.

[0021] Beneficial Effects: This patent application constructs a multidimensional biosensor, sensing platform, and detection kit. For a single sensor, it enables multi-mode detection of a single analyte; for the sensor platform, it not only enables single-mode multi-analyte detection but also multi-mode multi-analyte detection. Specifically, for the detection of NT-proBNP and cTnI, based on functionalized mesoporous silica nanoparticles (MSN), the MSN substrate is designed into two different functional regions, modified respectively using Fc-dsDNA1 / CdS@MSN and MB-dsDNA2 / Au@MSN nanocomposites. Each region can operate independently in PEC-EC mode or simultaneously. When operating simultaneously, the two regions operate in dual PEC-EC mode, enabling parallel, non-interfering measurements. This achieves simultaneous and accurate measurement of NT-proBNP and cTnI, and each functional region synergistically combines photoelectrochemical PEC and electrochemical EC detection modes. This invention enables mutual calibration and complementary verification between signals, effectively improving the accuracy, reliability, and anti-interference capability of detection results, while also broadening the sensor's detection range and applicable scenarios. The above-mentioned and other advantages of this invention are further explained in the specific embodiments. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the sensing platform constructed by the present invention and its working principle.

[0023] Figure 2 These are electron microscope images of MSNs, where image B is SEM and image C is TEM.

[0024] Figure 3 These are electron microscope images of CdS@MSNs, where image C is SEM and image D is TEM.

[0025] Figure 4 These are electron microscope images of Au@MSNs, where image B is SEM and image C is TEM.

[0026] Figure 5 These are high-resolution TEM images and elemental mapping images: (A) TEM image of MSNs and (B) its corresponding elemental mapping image; (C) TEM image of CdS@MSNs and (D) its corresponding elemental distribution image; (E) TEM image of Au@MSNs and (F) elemental distribution image of Au@MSNs.

[0027] Figure 6 These are nitrogen adsorption-desorption isotherms for SiO2 NPs (A) and MSNs (B); and pore size distribution images for SiO2 NPs (C) and MSNs (D).

[0028] Figure 7 These are the XRD patterns of MSN, CdS@MSN, and Au@MSN.

[0029] Figure 8 These are the XPS spectra of MSN, CdS@MSN, and Au@MSN.

[0030] Figure 9 These are high-resolution XPS spectra: (A) Si 2p, (B) O 1s, (C) S 2p, (D) Cd 3d, (E) Au 4f.

[0031] Figure 10 (A) Schematic diagram of the construction process of the multidimensional sensing platform; (B) EIS response map of region I (a. blank ITO, b. MSN, c. CdS@MSN, d. Fc-dsDNA1 / CdS@MSN) and (C) EIS response map of region II (a. blank ITO, b. MSN, c. Au@MSN, d. dsDNA2 / Au@MSN, e. MB-dsDNA2 / Au@MSN).

[0032] Figure 11 (A) Photocurrent response of region I under 450 nm irradiation; (B) Photocurrent response of region II under 650 nm irradiation; (C) Current response of region I at +0.3 V and (D) Current response of region II at -0.2 V.

[0033] Figure 12 (A) UV-Vis spectra of MSN and Fc-dsDNA1 / CdS@MSN; (B) UV-Vis spectra of MSN, Au@MSN and MB-dsDNA2 / Au@MSN.

[0034] Figure 13 (A) Schematic diagram of dual-zone interference-free dual-signal output: a. Photocurrent response of the dual-zone sensor under alternating illumination conditions of 650 nm and 450 nm (three rounds of testing were conducted); b. Electrical signal response of the dual-zone sensor under continuous voltage scan from 0.5 V to -0.4 V.

[0035] Figure 14 The photocurrent responses of regions (A) I and (B) II under alternating illumination at 450 nm and 650 nm, respectively; the electrical signal responses of regions I and (D) II under continuous voltage scans from +0.5 V to -0.4 V; the photoelectrochemical signals under irradiation at 450 nm and 650 nm and the current signals from +0.6 V to -0.4 V.

[0036] Figure 15The following are linear scanning voltammetric curves of different modified electrodes under dark light, 450 nm and 650 nm monochromatic light excitation: (A) ITO bare electrode; (B) Fc-dsDNA1 / CdS@MSNs modified electrode; (C) MB-dsDNA2 / Au@MSNs modified electrode.

[0037] Figure 16 The sensing parameters of regions I and II were optimized; the photocurrent response (A) and current response (B) of different concentrations of CdS (a. 0 μM, b. 1 μM, c. 5 μM, d. 10 μM, e. 20 μM); the photocurrent response (C) and current response (D) of different concentrations of MB (a. 0 μM, b. 100 μM, c. 200 μM, d. 300 μM, e. 400 μM, f. 500 μM, g. 600 μM).

[0038] Figure 17 (A) Photocurrent response curves of region II under 650 nm excitation light irradiation with different concentrations of cTnI (ah: 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 ng / mL) and photocurrent response curves of region I under 450 nm excitation light irradiation with different concentrations of NT-pro BNP (ah: 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 ng / mL); (B) Linear fitting plots of region I (top) and region II (bottom).

[0039] Figure 18 (A) Electrochemical signal (DPV) curves of region I and region II at different concentrations of cTnI and NT-pro BNP, and (B) corresponding linear fitting plots of region I and region II, where ah represents concentrations of 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL.

[0040] Figure 19 Selectivity for NT-proBNP in (A) PEC mode and (B) EC mode; Selectivity for cTnI in (C) PEC mode and (D) EC mode.

[0041] Figure 20 Stability test of integrated dual-zone sensor under (A) 450 nm and (B) 650 nm irradiation in PEC mode.

[0042] Figure 21 Storage stability test of integrated dual-zone sensor in (A) PEC mode and (B) EC mode. Detailed Implementation

[0043] The present invention will now be described in a clear and complete detail with reference to the accompanying drawings.

[0044] 1. The main biochemical reagents used in this invention are shown in Table 1. Other reagents not listed can be purchased from the market.

[0045]

[0046] 2. The main instruments used in this patent are shown in Table 2.

[0047]

[0048] 3. Experimental Procedure 3.1 Synthesis of Mesoporous Silica Nanospheres (MSN) Three-dimensional mesoporous silica nanospheres were synthesized by a stepwise growth method. The cationic surfactant CTAC was used as a template, TEOS as a silicon source, TEA as a catalyst, and organic solvents such as 1-octane as emulsifiers. The mesoporous silica nanospheres were prepared by a one-pot two-phase layering method. The typical synthesis steps are as follows: First, in a 100 mL round-bottom flask, 24 mL of 25% CTAC solution and 0.18 g of TEA were added to 36 mL of deionized water, and the mixture was gently stirred at 60 °C for 1 h. Then, 20 mL of 20% TEOS 1-octane solution was slowly added to the above CTAC-TEA aqueous solution, and the mixture was magnetically stirred at 60 °C in an oil bath for 12 h to obtain the first-generation product. Next, the upper 1-octane oil phase was completely removed and replaced with a fresh 20% TEOS-decalin solution. The reaction was continued for 12 h under the same conditions to grow the second-generation product. For the third-generation synthesis, the upper oil phase was replaced with a 20% TEOS-cyclohexane solution, and the reaction was continued for 12 h under the same conditions to obtain silica nanoparticles (SiO2NPs). SiO2 NPs were placed in a 1M ethanol / water mixture and refluxed at 80 °C for 8 h to remove the template, resulting in mesoporous silica nanospheres (MSNs). Subsequently, the product was washed three times with alternating ethanol and deionized water. The entire process of template removal and mesoporous structure formation was repeated three times to ensure complete template removal and complete mesoporous structure formation.

[0049] 3.2 Synthesis of Cadmium Sulfide (CdS) Nanoparticles To 3.00×10⁻ 4A turbid white solution was formed by adding 150.00 μL of mercaptoacetic acid to an aqueous solution of cadmium nitrate tetrahydrate (270.00 mL). The pH of the solution was then adjusted to 11 with 0.01 M sodium hydroxide solution. (The last part, "1.50 × 10⁻", appears to be a typo and should be left as is.) 4 Dissolve mol of sodium sulfide in 10.0 mL of deionized water and add it rapidly to the above cadmium nitrate solution under vigorous stirring; stir the reaction system in the dark for 10 min, add anhydrous methanol until a precipitate is formed, and obtain water-soluble cadmium sulfide quantum dots (COOH-CdS QDs) coated with mercaptoacetic acid.

[0050] 3.3 Synthesis of CdS@MSN 0.1 g of MSNs were dispersed in 50 mL of anhydrous ethanol solution containing 10 wt% 3-aminopropyltriethoxysilane (APTES), and refluxed at 60 °C for 3 h with stirring to achieve amino functionalization of the MSN surface. After the reaction, the product was collected by centrifugation and washed three times to obtain amino-functionalized MSNs (denoted as NH2-MSNs). Subsequently, 0.01 g of NH2-MSNs was dispersed in 1 mL of deionized water under ultrasonic conditions to form a homogeneous suspension. 1 mL of a pre-synthesized 10 μM aqueous solution of COOH-CdS QDs was added, and the mixture was stirred continuously at room temperature for 30 min. CdS QDs were immobilized on the MSN surface through electrostatic interactions between amino and carboxyl groups and the formation of amide bonds. Finally, the CdS QDs / NH2-MSNs composite nanomaterials were collected by centrifugation and washed three times for later use.

[0051] 3.4 Synthesis of Au@MSN 0.05 g of MSN was dissolved in 3.8 mL of deionized water and sonicated for 10 min to ensure uniform dispersion. Then, 200 μL of 1% chloroauric acid (HAuCl4) solution was added, and the mixture was stirred for 5 min to ensure homogeneous mixing. Under continuous stirring, 1% sodium citrate solution was added dropwise, and the reaction was allowed to proceed for 1 h to allow gold nanoparticles to completely form on the MSN surface. Finally, the product was collected by centrifugation, thoroughly washed, and dried at 50°C for 6 h to obtain Au@MSN.

[0052] 3.5 Preparation of biomolecular recognition probes Probe preparation for NT-proBNP detection: 100 μL of 1 μM 3'-amino-modified NT-proBNP aptamer (AptNT-proBNP) was mixed with 100 μL of 1 μM 3'-Fc-modified complementary DNA (c-DNA1), annealed at 95℃ for 5 min, and then slowly cooled to room temperature to obtain double-stranded DNA1 (dsDNA1); wherein: AptNT-proBNP: 3′NH2-GTC CCC CTGCCC AGC CCA ATG TCG ACT CAC ACC GG-5′; c-DNA1: 5′CAG GGG GAC GGG TCG GGT TAC AGC TGA GTG TGG CC-Ferrocene-3′.

[0053] Probe preparation for cTnI detection: 100 μL of 1 μM cTnI aptamer (AptcTnI) was reacted with 100 μL of 1 mM tris(2-carboxyethyl)phosphine (TCEP) for 1 h to reduce the disulfide bonds generated by cohesion. Then, under the same annealing conditions (annealing at 95 °C for 5 min), it was hybridized with 100 μL of complementary DNA (c-DNA2) to form double-stranded DNA2 (dsDNA2). The obtained dsDNA2 was incubated overnight at 4 °C in the dark with an equal volume of 1 mg / mL Au@MSN aqueous solution to achieve coupling, obtaining the final dsDNA2-Au@MSN complex. This complex was stored at 4 °C in the dark for subsequent experiments.

[0054] Among them, AptcTnI: 3′SH-CGC ATG CCA AAC GTT GCC TCA TAG TTC CCT CCC CGT GTCC-5′; c-DNA2: 5′-GCG TAC GGT TTG CAA CGG AGT ATC AAG GGA GGG GCA CAG G-3′.

[0055] 3.6 Fabrication of the Sensor Platform The ITO glass substrate (0.7 cm × 3.3 cm) was ultrasonically cleaned sequentially in acetone, ethanol, and deionized water, and then dried. Two circular working areas (each 4 mm in diameter) were defined on the electrode surface, specifically by covering the ITO film with insulating tape featuring two circular cutouts. The specific construction steps of the sensor are as follows: Region I: 20 μL of CdS@MSN (4 mg / mL) was dropped onto an ITO electrode and air-dried. 20 μL of EDC / NHS solution (5 mM EDC and 10 mM NHS were mixed at a 1:1 ratio) was used to activate the carboxyl groups on the CdS surface and incubated at room temperature for 1 h to prepare for the subsequent amino condensation reaction with dsDNA1. Subsequently, 20 μL of dsDNA1 was immobilized by carbodiimide chemical method (37 °C, 1 h). The carboxyl groups on the CdS surface bound to the amino groups of the NT-proBNP aptamer, thereby immobilizing the capture probe and obtaining the NT-proBNP detection sensor.

[0056] Region II: First, drop 20 μL of pre-assembled dsDNA2-Au@MSN onto the substrate and let it air dry; then drop 20 μL of 500 μMMB solution onto the substrate and incubate at room temperature for 1 h to allow the MB load to be linked to the dsDNA2 backbone, thereby achieving the modification of electrochemical signal transduction function and obtaining the cTnI detection sensor.

[0057] The above-mentioned sensor platform, which integrates both NT-proBNP and cTnI detection sensors on the same ITO glass substrate, constitutes a sensor platform for NT-proBNP and cTnI detection (e.g., Figure 1 It should be understood that sensors targeting only one object, NT-proBNP or cTnI, can also be fabricated on an ITO glass substrate.

[0058] 3.7 Detection of NT-proBNP and cTnI A laser light source with wavelengths of 445±5 nm and 650 nm was installed on an electrochemical workstation. A traditional three-electrode system was used, with the prepared sensor or sensor platform as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The electrolyte was 0.01 M PBS (pH 7.4). At a bias voltage of 0 V, the photocurrent signal under different illuminations was measured by chronoamperometry, and the electrochemical signal was measured by differential voltammetry. When detecting different concentrations (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 ng / mL) of NT-proBNP, the sensor in region I was incubated with different concentrations of NT-proBNP for 30 min beforehand. Similarly, when detecting different concentrations (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 ng / mL) of cTnI, the sensor in region II was incubated for 30 min before detection. In addition, to verify the selectivity of the sensor, single solutions and mixed solutions containing 500 pg / mL interleukin (IL)-6, 500 pg / mL IL-17, 500 pg / mL IL-23, 10 mg / mL bovine serum albumin (BSA), and 1 μg / mL human immunoglobulin (IgG) were used for selectivity evaluation. Recovery was performed using normal human serum. All serum samples were diluted 10-fold with PBS buffer and incubated for 30 min before measuring the photocurrent signal.

[0059] 4 Results and Discussion 4.1 Morphological characterization of MSN, CdS@MSN and Au@MSN Figure 2 Electron microscopy images of MSNs grown continuously using a one-pot two-phase layering strategy are presented. Figure 2 Scanning electron microscopy (SEM) in section -B showed that the synthesized MSNs had a uniform size distribution (approximately 100 nm) and a homogeneous morphology. Further morphological analysis was performed using transmission electron microscopy (TEM), and low-magnification TEM images were obtained (…). Figure 2 -C) Preliminary findings show the mesoporous structure on the surface of the nanoparticles, indicating the successful preparation of MSNs.

[0060] CdS@MSNs were synthesized via a co-precipitation reaction between CdS NPs and MSNs. (SEM image) Figure 3 -C) shows that CdS@MSNs still maintain high dispersion and morphological uniformity; low-magnification TEM images ( Figure 3 -D) indicates that the particle size is slightly larger than that of pure MSNs, and a distinct core-shell structure is observed, indicating the successful preparation of CdS@MSNs.

[0061] Au@MSNs were synthesized by in-situ reconstruction of Au NPs on the MSN surface, resulting in SEM images ( Figure 4 -B) shows that its particle size is uniform and the surface exhibits a distinct granular morphology; low-magnification TEM image ( Figure 4 -C) As can be seen, the dark Au NPs are clearly distributed and anchored on the light gray MSN matrix.

[0062] To further characterize the morphology of MSN, CdS@MSN, and Au@MSN, high-resolution TEM characterization was performed, and the results are as follows: Figure 5 As shown, high-resolution TEM images of MSNs ( Figure 5 -A) clearly shows mesoporous channels radiating outward from the center. These channels have a large specific surface area, which is beneficial for the uniform deposition of subsequent materials and ensures good interfacial contact with MSNs. (Elemental mappings image of MSNs) Figure 5 -B) confirmed the presence of Si and O elements. Figure 6 A comparison of the specific surface area and pore size distribution of MSNs and SiO2NPs (non-mesoporous SiO2) was conducted, and BET data analysis revealed that MSNs exhibited superior pore structure performance compared to SiO2 NPs in all aspects: the BET specific surface area of ​​MSNs reached as high as 635.7 m². 2 / g, is SiO2 NPs (172.7m) 2 The concentration of MSNs was 3.7 times that of g; meanwhile, the mesopore volume of MSNs (0.66 cm³) was 3.7 times that of g. 3 The pore size ( / g) is significantly higher, with a mesoporous content exceeding 97%, indicating a richer and denser mesoporous structure. Pore size distribution results show that the mesopores of MSNs are concentrated in a small size range (average approximately 3.5–3.8 nm), exhibiting good structural uniformity, while SiO2 NPs have larger mesopore sizes (average 11–14 nm) and a relatively dispersed distribution. In summary, MSNs are more suitable for applications requiring high adsorption capacity and rapid mass transfer. Furthermore, TEM images of CdS@MSNs (…) are also provided. Figure 5 -C) analysis revealed a well-crystallized CdS NPs coating layer on the MSN surface. The inset shows a high-resolution lattice image of the surface CdS NPs, where clearly visible lattice fringes correspond to the (200) crystal plane of CdS. Element mappings image ( Figure 5 -D) clearly shows the distribution of the four elements Si, O, Cd and S, confirming that CdS NPs have been successfully coated on the MSN surface. Figure 5-E is a TEM image of Au@MSNs. Unlike the core-shell structure of CdS@MSNs, Au NPs are uniformly embedded on the MSN surface without altering its overall morphology. The inset is a high-resolution lattice image of a single Au NP, in which lattice fringes corresponding to the Au(111) crystal plane are clearly visible, with a plane spacing of 0.239 nm. Element mappings image ( Figure 5 In the -F) array, in addition to the characteristic signals of Si and O elements, a clear distribution of Au elements was also detected. All of the above characterization data together provide preliminary evidence for the successful preparation of MSNs, CdS@MSNs, and Au@MSNs.

[0063] 4.2 Characterization of crystal structure and surface chemical state of MSNs, CdS@MSNs and Au@MSNs Figure 7 X-ray diffraction (XRD) patterns of MSNs, CdS@MSNs, and Au@MSNs are presented. Pure MSNs exhibit low-intensity characteristic diffuse peaks of amorphous silica, which perfectly match the standard pattern of orthorhombic MSNs (2θ≈24°). Compared with the XRD patterns of pure MSNs and CdS QDs, the XRD pattern of CdS@MSNs composite material exhibits dual structural characteristics: on the one hand, it retains the broadened diffraction peaks near 2θ≈24°, consistent with pure MSN, which are characteristic peaks of amorphous SiO2, indicating that the basic amorphous structure of the MSNs support has not changed; on the other hand, clear diffraction peaks appear at 28.12°, 43.58°, and 51.69°, which match the diffraction peaks of pure CdS. These diffraction peaks correspond to the (101), (110), and (112) crystal planes of cubic CdS (JCPDS No. 89-0440), respectively. The above results confirm that CdS has been successfully loaded onto the surface of MSNs without disrupting the structural integrity of the silica framework. The amorphous morphology retained by the support and the clear crystalline characteristics of CdS provide a solid structural foundation for the functional stability of the composite material. For the Au@MSNs composite material, in addition to the characteristic peaks corresponding to pure MSNs, a series of sharp crystalline diffraction peaks are superimposed on the broadened amorphous diffuse peaks: a clear and sharp diffraction peak appears at 2θ≈38.2°, corresponding to the (111) crystal plane of face-centered cubic (fcc) gold; additional diffraction peaks appear at 44.4°, 64.6°, and 77.5°, corresponding to the (200), (220), and (311) crystal planes of fcc gold, respectively. These XRD results provide supplementary evidence for the successful synthesis of the Au@MSNs heterostructure.

[0064] To further analyze the elemental composition and chemical state of each material, X-ray photoelectron spectroscopy (XPS) was used for characterization. The full-spectrum XPS spectra are shown below. Figure 8As shown in the figure. Compared with the spectrum of pure MSN, the characteristic peak of Cd 3d orbital is clearly visible in the spectrum of CdS@MSN; in contrast, the signals of Au 4f and S 2p are not obvious because they are close to the peaks of Si 2p and Si 2s orbitals, respectively. Therefore, high-resolution spectra were acquired for more detailed analysis of these regions.

[0065] High-resolution XPS spectra of Si 2p, such as Figure 9 As shown in -A, the Si 2p peak in all samples is located at ~103.5 eV, corresponding to Si in SiO2. 4 ⁺ indicates that the MSN skeleton remained stable during the modification process. Figure 9 -B provides a high-resolution spectrum of the O 1s peak. For pure MSNs, the O 1s peak can be decomposed into two components: a main peak (Si–O–Si) at ~533.0 eV and a shoulder peak (surface silanol groups) at ~531.5 eV. For the modified sample, the spectral shape of the O 1s peak remains essentially unchanged, indicating that the loading process did not significantly alter the surface oxygen structure of the MSN. For CdS@MSNs, the Cd 3d spectrum ( Figure 9 -D) exhibits a typical spin-orbit bimodal structure, Cd 3d 5 / 2 and Cd 3d 3 / 2 The peaks are located at 405.2 eV and 412.0 eV, respectively, with a peak spacing of approximately 6.8 eV, similar to that of Cd in CdS. 2+ The chemical states were consistent, confirming the successful loading of CdS. The high-resolution S 2p spectrum ( Figure 9 After peak fitting using -C), the value is ~161.6 eV (S 2p). 3 / 2 ) and ~162.8 eV (S 2p 1 / 2 A pair of double peaks appeared at ( ), with a peak spacing of approximately 1.2 eV, attributed to S²⁻ in CdS; no obvious high binding energy sulfur oxide peaks (>164 eV) were detected, indicating that the loaded CdS did not undergo significant surface oxidation under the test conditions. For Au@MSN, the Au 4f high-resolution spectrum ( Figure 9 -E) exhibits a pair of sharp double peaks, with binding energies Au 4f and f respectively. 7 / 2 =84.0 eV and Au 4f5 / 2=87.7 eV, with a peak spacing of 3.7 eV, corresponding to metallic gold (Au). 0 The characteristics indicate that gold exists in the form of zero-valent nanoparticles.

[0066] 4.3 Assembly process of the sensing interface Figure 10 -A provides a schematic diagram illustrating the construction process of a multi-dimensional sensing platform; such as Figure 10As shown in Figure B, the electrochemical impedance spectroscopy (EIS) of the bare ITO electrode (curve a) exhibits a very small semicircle (minimum charge transfer resistance Rct) in the high-frequency region, indicating its excellent conductivity. When MSNs are modified onto the ITO surface (curve b), the semicircle diameter increases significantly, attributed to the insulating properties of MSNs hindering electron transfer. After modifying the CdS@MSNs composite layer (curve c), the EIS semicircle size and Rct decrease significantly, attributed to the reduced interfacial impedance due to CdS-mediated electron transfer. Subsequently, when Fc-dsDNA1 is assembled onto the CdS@MSN-modified electrode surface (curve d), the EIS semicircle and Rct increase again, due to the AptNT-proBNP biomolecular film increasing electron transfer resistance and inhibiting ion migration. For region II, the EIS characteristics of the bare ITO electrode (curve a) and the MSN-modified electrode (curve b) are shown in Figure B. Figure 10 -C) Consistent with Region I. After modifying Au@MSNs (curve c), the semicircle diameter and Rct significantly decreased, due to the enhanced conductivity of the composite layer; subsequently, incubation with dsDNA2 formed by AptcTnI and its complementary strand (curve d) resulted in an increase in the EIS semicircle and Rct, reflecting the increased electron transfer resistance and hindered ion migration caused by the AptcTnI biomolecular layer; further embedding MB into dsDNA2 resulted in a continued increase in the semicircle radius and Rct (curve e). The consistent changes in the EIS spectra confirmed the successful construction of Region II, and its trend was consistent with the modification pattern of Region I, further validating the effectiveness of Region II assembly.

[0067] To further investigate the PEC and EC signal variations in the two regions, the PEC and EC responses of regions I and II were systematically evaluated. For region I under 450 nm photoexcitation (… Figure 11 -A), the bare ITO electrode showed no PEC response, while the CdS@MSNs modified electrode generated approximately 340 nA of photocurrent due to the presence of CdS QDs; subsequently, after coupling with the aptamer, the photocurrent decreased significantly, because the biomolecular layer formed at the interface increased steric hindrance, hindering the migration of photogenerated carriers to the electrode surface. For region II ( under 650 nm light excitation) Figure 11 -B), the bare ITO, Au@MSNs, and dsDNA2 / Au@MSNs modified electrodes all showed almost no significant photocurrent; however, after MB embedding, a clear photocurrent of approximately 120 nA was generated. The changes in surface electrochemical behavior during the fabrication of region I and region II electrodes are as follows: Figure 11 -C and Figure 11As shown in Figure -D, no characteristic redox peak corresponding to ferrocene (Fc) was observed near +0.3 V on the unmodified ITO substrate or the CdS@MSNs modified electrode surface. However, after covalently immobilizing Fc-dsDNA1, the Fc-dsDNA1 / CdS@MSNs modified electrode showed a clear Fc characteristic current response peak at +0.3 V. Similarly, in the bare ITO electrode, Au@MSNs modified electrode, and dsDNA2 / Au@MSNs electrode system without MB embedding, no characteristic electrochemical signal of MB was detected at -0.2 V. Conversely, when MB is embedded in the dsDNA2 double strand to form an MB-dsDNA2 complex and immobilized on the Au@MSNs electrode surface, the MB-dsDNA2 / Au@MSN electrode exhibits a sharp and stable MB characteristic reduction peak at -0.2 V. The above results clearly demonstrate the evolution of PEC and electrochemical signals throughout the electrode preparation process, confirming the specificity and reliability of the dual-region dual-mode output signal.

[0068] 4.4 Verification of Dual-Signal Coordinated Output of the Multi-Dimensional Sensing Platform PEC-EC The light absorption characteristics of materials in two regions were studied using ultraviolet-visible spectrophotometry (UV-Vis). Figure 12 -A compares the UV-Vis diffuse reflectance spectra of MSNs and Fc-dsDNA1 / CdS@MSNs. In the wavelength range of 200–800 nm, MSNs only exhibit a characteristic absorption peak in the ultraviolet region (~200 nm), which is attributed to the absorption of Si-O bonds, and shows almost no absorption in the visible region (400–800 nm). In contrast, Fc-dsDNA1 / CdS@MSNs exhibits a significant absorption characteristic in the 200–450 nm range, while the absorption at 650 nm is negligible. This phenomenon is attributed to the inherent optical properties of the loaded CdS QDs. This indicates that Fc-dsDNA1 / CdS@MSNs can efficiently utilize light at a wavelength of 450 nm and is unaffected by light sources around 650 nm, providing a basis for achieving targeted photoelectric signal output in different spectral regions by controlling the light source. Figure 12 In the -B group, pure MSNs also exhibit absorption only in the ultraviolet region; while Au@MSNs show a characteristic absorption peak at 530 nm, which is attributed to the localized surface plasmon resonance of Au NPs; for MB-dsDNA2 / Au@MSNs, although the absorption peak at 530 nm is weakened, a distinct characteristic absorption band appears in the red region (600~700 nm), which is due to the incorporation of MB. These optical properties indicate that MB-dsDNA2 / Au@MSNs can serve as red-light-sensitive components and can generate a photoelectric response under 650 nm light excitation.

[0069] Figure 13 -A explains the working principle of the multi-dimensional sensing platform, which can output photoelectric and electrochemical signals through light modulation and voltage changes. Specifically, Figure 13 -Aa demonstrates the platform's photoelectrochemical response under alternating illumination of 450 nm and 650 nm, exhibiting two stable and alternating photoelectric signals; such as Figure 13 As shown in Figure -Ab, under continuous voltage stimulation, the differential voltammetry curve of the plateau shows two clear electrochemical peaks at 0.3 V and -0.2 V, corresponding to the redox processes of Fc and MB, respectively.

[0070] To verify that there is no cross-interference between the two signals, electrodes that are modified only in a single functional area were used to verify the signal output in each mode. Figure 14 -A shows the photoelectrochemical response of the electrode modified only in region I under alternating illumination at 450 nm and 650 nm: under 450 nm illumination, the photocurrent reaches 321.2 nA, consistent with the PEC signal obtained at the same wavelength for the dual-region modified electrode; while under 650 nm illumination, almost no photoelectrochemical response was observed. Conversely, Figure 14 -B shows the PEC response of the electrode modified only in region II under the same alternating illumination conditions: unlike region I, this electrode has almost no PEC response under 450 nm illumination, but produces a photocurrent of 122.3 nA under 650 nm illumination, which matches the PEC signal recorded by the dual-region electrode at this wavelength. Figure 14 -C shows the electrochemical signal scanned only at region I electrode in the range of 0.5 V to -0.4 V, with a clear Fc characteristic redox peak appearing at approximately 0.3 V, and its peak current is similar to... Figure 13 The current value at +0.3 V in Ab is highly consistent. Similarly, Figure 14 -D shows the electrochemical spectrum of only the Region II electrode within the same potential range, with a clear MB redox peak observed near -0.2 V, and its peak current is similar to... Figure 13 The corresponding current value at -0.2 V in -Ab is consistent. The above results together confirm that the photoelectrochemical and electrochemical signals generated by the two functional regions are independent and do not cross-interfere, thus verifying the reliability of the dual-signal output of the integrated sensing platform.

[0071] 4.5 Feasibility Verification of the Multi-Dimensional Sensing Platform To further investigate the signal response characteristics of the sensing system, linear sweep voltammetry (LSV) was used to systematically evaluate the electron transfer efficiency and interface stability of different modified electrodes under dark conditions and with monochromatic light excitation at 450 nm and 650 nm. The results are as follows: Figure 15As shown, the ITO electrode exhibited a near-zero current response under all test conditions, with no significant dark current or photocurrent signal. Figure 15 -A indicates that it possesses excellent electrochemical inertness and stability, making it suitable as a low-background substrate for functionalization. The Fc-dsDNA1 / CdS@MSNs electrode exhibits extremely low current density under dark conditions, reflecting its weak intrinsic electrochemical activity; under 450 nm light excitation, the system photocurrent increases significantly, while no significant response is observed under 650 nm light irradiation ( Figure 15 The response behavior is consistent with the bandgap absorption characteristics of CdS, confirming its selective photoelectrochemical response to short-wavelength visible light. For the MB-dsDNA2 / Au@MSNs electrode, a relatively high current signal was observed even under dark conditions, mainly attributed to the synergistic effect of the redox activity of MB and the electron conduction-promoting effect of Au nanoparticles; furthermore, the current increased significantly under 650 nm irradiation, while no significant change was observed under 450 nm irradiation. Figure 15 The photoresponse behavior is highly consistent with the characteristic absorption peak of MB, indicating that the composite system has efficient photoelectric conversion capability in the red light region. In summary, the two functionalized electrodes achieved specific photoelectrochemical responses to visible light of different wavelengths, while exhibiting significantly different intrinsic electrochemical activities. These results provide key experimental support for constructing a dual-mode high-sensitivity detection platform based on the synergistic response of electrical and photoelectrochemical signals.

[0072] Building upon the previous section's systematic photoelectrochemical and electrochemical characterization, which confirmed the stable dual-channel signal output capability of the fabricated sensor, this study further explores the core detection functions of Regions I and II, particularly their recognition and response performance to their respective target analytes. The aim is to clarify the changing patterns and feasibility of photoelectrochemical and electrochemical signals in the presence of the target analyte. Figure 14 As shown in Figure -E, the photocurrent responses of regions I and II were recorded under excitation at 450 nm and 650 nm, respectively. When region I was treated with 1 ng / mL of its specific target (NT-proBNP), the photocurrent signal significantly decreased compared to the background signal without the target. Similarly, when region II was treated with 1 ng / mL of its specific target (cTnI), the photocurrent output also showed a significant decrease. These phenomena indicate that the specific binding of the target to its corresponding aptamer can effectively regulate the interfacial charge transfer process. Furthermore, the changes in electrochemical signals before and after the addition of the target were evaluated, such as... Figure 14 As shown in -F, the electrochemical signal also decreased significantly compared to the background level after incubation with the specific target analyte. Both regions exhibited consistent and specific signal attenuation for their respective target analytes, confirming the successful construction of a multichannel sensor capable of simultaneously detecting multiple analytes.

[0073] 4.6 Optimization of Experimental Conditions To optimize the performance of the constructed sensor, key experimental parameters were systematically investigated. For region I, the amount of COOH-CdS QDs was optimized during the preparation of CdS@MSN. For example... Figure 16 -A and Figure 16 As shown in Figure -B, the photocurrent response gradually increases with increasing CdS QDs concentration; however, the electrochemical signal peaks at a concentration of 10 μM. Considering both photoelectrochemical (PEC) and electrochemical (EC) performance, 10 μM is optimally selected as the CdS QDs concentration for subsequent experiments. Of course, concentrations of 5-20 μM are also acceptable. These comprehensive optimization studies provide ideal experimental conditions for maximizing the performance of the biosensor platform. Similarly, for Region II, considering both PEC and EC signals, the optimal concentration of MB during sensor fabrication is determined. By monitoring the PEC and electrochemical responses at different MB concentrations, the concentration that produces the highest signal output is screened. Figure 16 -C and Figure 16 As shown in -D, both signals reach their maximum values ​​when the MB concentration is 500 μM. Therefore, 500 μM is chosen as the optimal concentration for MB. Of course, the MB concentration can be in the range of 500-600 μM.

[0074] 4.7 Multi-mode quantitative detection of specific targets using dual-region sensors Figure 17 -A shows the dynamic changes in photocurrent response in regions I and II under 450 nm and 650 nm photoexcitation with varying cTnI and NT-proBNP incubation concentrations. The results indicate that within the concentration range of 0.001 pg / mL to 10 ng / mL, the photocurrent signal in both regions significantly decreases with increasing NT-proBNP and cTnI concentrations, exhibiting a clear negative correlation. This signal attenuation is attributed to the specific binding of NT-proBNP to AptNT-proBNP in region I and cTnI to AptcTnI in region II. This binding induces conformational folding of the aptamers, forming dense biomolecular complexes. These structures significantly increase interfacial steric hindrance and charge transfer resistance, thereby reducing the migration efficiency of photogenerated carriers to the electrode surface. Figure 17 The figure above (-B) shows the linear fitting results of NT-proBNP concentration and photocurrent response. This detection system exhibits a wide linear range (0.001~100 ng / mL, fitting equation I = -31.99 lg c(NT-proBNP) + 257, R² = 0.9989), and a limit of detection (LOD) of 0.11 pg / mL (S / N = 3), verifying its high sensitivity for the quantitative detection of NT-proBNP. Similarly, Figure 17The figure below shows the linear correlation between cTnI concentration and photocurrent signal. This detection method has a wide dynamic range (0.0001~100 ng / mL) and good linearity (fitting equation: I = -14.85 lg c(cTnI) + 105.29, R² = 0.9945). Based on the signal-to-noise ratio S / N = 3, the limit of detection (LOD) is calculated to be 0.26 pg / mL, confirming its high sensitivity for quantitative detection of cTnI.

[0075] To further verify the quantitative detection performance of the sensor, differential pulse voltammetry (DPV) was used for supplementary electrochemical analysis. Figure 18 As shown in Figure -A, when the concentrations of NT-proBNP and cTnI increased from 0.001 pg / mL to 10 ng / mL, the DPV current response in both regions I and II exhibited a significant concentration-dependent decrease, showing a negative correlation consistent with the photocurrent. This signal attenuation is mainly attributed to the biomolecular layer formed by the specific binding of NT-proBNP and cTnI to their respective aptamers. This layer not only hinders the redox kinetics of Fc and MB labels but also inhibits the migration of electrolyte ions to the electrode surface. Therefore, the electrochemical current gradually decreases with increasing target concentration. Figure 18 -B shows the linear relationship between NT-proBNP concentration and its corresponding current response. The sensor has a wide dynamic range (0.001~100 ng / mL) and excellent linear correlation (fitting equation: I = -0.49 lg c(NT-proBNP) + 2.99, R² = 0.9994). Based on the signal-to-noise ratio S / N = 3, the limit of detection (LOD) is calculated to be 0.24 pg / mL, indicating its high sensitivity for quantitative detection of NT-proBNP. Similarly, the linear calibration curve of cTnI concentration and current signal also has a wide linear range (0.001~10 ng / mL, fitting equation: I = -1.01 lg c(cTnI) + 6.49, R² = 0.9981), and the limit of detection (LOD) of cTnI is 0.18 pg / mL (S / N = 3), confirming that the detection system has high sensitivity and detection consistency for both cardiac biomarkers.

[0076] The above results fully demonstrate that the proposed dual-region sensor exhibits excellent quantitative detection performance in both detection modes, including a wide linear range (0.0001 pg / mL to 100 ng / mL), high linear correlation coefficient, and low detection limit. The consistent results across different detection modes mutually validate each other, highlighting the enormous application potential of this multi-mode dual-region sensor in the high-sensitivity simultaneous quantitative detection of various targets.

[0077] 4.8 Selectivity and Stability To comprehensively evaluate the practical application value of the developed biosensor platform, its selectivity for detecting NT-proBNP was systematically investigated. Under the same experimental conditions, potential interfering substances, including interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), bovine serum albumin (BSA), human immunoglobulin (IgG), and cytochrome C (CytC), were tested. Figure 19 As shown in Figures A and B, the results indicate that the signal changes caused by each individual interfering agent are negligible (less than 4% of the target analyte response signal), and only the sample containing 10 ng / mL NT-proBNP produced a recognizable and obvious signal. The mixed solution containing 10 ng / mL NT-proBNP and each interfering agent at 100 ng / L (Mix) produced a signal (PEC: 161.33±4.77 nA, EC: 1.50±0.15 μA) almost identical to that of the single NT-proBNP sample. The signal deviation between single detection and mixed detection was within 3%, confirming the excellent selectivity of the Region I PEC / EC biosensor for NT-proBNP detection.

[0078] To evaluate the sensor's specificity for cTnI, a series of potential interfering agents (including IL-6, IL-17, IL-23, BSA, IgG, and CytC) were tested individually and then mixed with cTnI for testing. Figure 19 As shown in -C and D, the PEC and EC signals caused by the six single interfering agents are negligible (less than 3% of the target analyte response signal), with significant signal changes observed only after the addition of cTnI. Notably, the platform exhibits almost identical PEC and EC responses to single cTnI (10 ng / mL) and to a mixture of cTnI and interfering agents (10 ng / mL cTnI + 100 ng / mL of each interfering agent), with signal deviations less than 3% (PEC: 57.61 ± 2.50 nA, EC: 3.50 ± 0.15 μA). These results confirm the high selectivity of the Region II PEC / EC dual-mode platform for cTnI detection. In summary, the constructed dual-region dual-mode sensor demonstrates excellent selectivity, achieving specific detection of NT-proBNP and cTnI through unique photoelectrochemical and electrochemical signal changes.

[0079] Stability is a key factor in evaluating the feasibility of sensor applications. This study comprehensively investigated the stability of the developed sensor under different conditions. Figure 20As shown in -A and 20-B, under illumination of 450 nm and 650 nm, the photocurrent response of the sensor did not show significant attenuation during 400 seconds of continuous or alternating illumination, indicating that it has excellent operational photostability.

[0080] like Figure 21 -A and Figure 21 As shown in Figure -B, the storage stability of the sensor was evaluated over a 7-day period. The results showed that both the photoelectrochemical and electrochemical signals exhibited only negligible attenuation, confirming the sensor's good long-term stability. Furthermore, batch-to-batch repeatability is a key indicator for the large-scale application of the sensor. This study evaluated it using seven independently prepared sensor batches. The photocurrent and current signals of all batches remained highly consistent, indicating that the sensor possesses excellent repeatability and minimal batch-to-batch variability, highlighting the reliability of the fabrication process.

[0081] 4.9 Recovery Rate Test To evaluate the clinical feasibility of the dual-region, dual-mode biosensor, standard addition and recovery experiments were conducted using serum samples from healthy individuals. As shown in Table 3, the recovery rate of the PEC detection mode was 99.7%–103.48%, with a relative standard deviation (RSD) of 1.52%–6.82%. Similarly, the EC detection mode exhibited considerable precision, with a recovery rate of 98.46%–101.2% and an RSD of 0.32%–3.76%. These results confirm that the region I PEC / EC dual-mode sensor has high accuracy and reliability in detecting NT-proBNP in complex biological matrices, meeting the stringent requirements for clinical detection of NT-proBNP in human serum. As shown in Table 4, the recovery rate of the PEC detection mode was 99.2%–101.43%, with an RSD of 0.82%–4.28%; while the recovery rate of the EC detection mode was 99.6%–102.44%, with an RSD of 0.16%–6.82%. These results demonstrate that the Region II PEC / EC dual-mode platform can achieve accurate and reliable detection of cTnI in serum, further validating the clinical application potential of this sensor.

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[0083]

[0084] 4.10 Serum Sample Analysis To evaluate the analytical capabilities of the proposed multidimensional biosensor in detecting NT-proBNP and cTnI in clinical samples, this study analyzed serum samples from six patients with cardiovascular disease. The results (Tables 5 and 6) showed that the detection results of NT-proBNP and cTnI in all operating modes of the multidimensional sensor were highly consistent with the reference data provided by the hospital (NT-proBNP was quantified using a Roche e801 chemiluminescence analyzer, and cTnI was detected using an ELISA fluorescence kit). This consistency confirms the accuracy of the proposed multidimensional biosensor in identifying specific targets in complex clinical samples, highlighting its significant potential for simultaneous detection of cardiac biomarkers. This sensor is expected to provide valuable guidance for early diagnosis of myocardial infarction, cardiovascular risk assessment, clinical inflammation monitoring, and postoperative intervention. In conclusion, this multidimensional biosensor exhibits high accuracy and reliability, demonstrating promising prospects for practical clinical applications.

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[0086] .

[0087] 5 Summary: (1) This application designs a novel array dual-region sensor, which utilizes the high specific surface area, superior load capacity, wavelength-resolved excitation and continuous voltage change of MSNs, and can be used for simultaneous dual-target, dual-mode detection of key biomarkers of cardiovascular diseases NT-proBNP and cTnI; (2) The platform exhibits excellent sensitivity in both modes (PEC mode: NT-proBNP is 0.18 pg / mL, cTnI is 0.26 pg / mL; EC mode: NT-proBNP is 0.24 pg / mL, cTnI is 0.11 pg / mL), and has reliable quantitative analysis capabilities in the clinical setting; (3) The platform achieves synchronous dual electrical signal output and parallel PEC signal generation through optical modulation, which significantly simplifies the operation process, reduces detection costs, and increases detection throughput. This integrated multidimensional detection method not only provides complementary and self-verifying data streams, significantly improving the selectivity and accuracy of detection, but also establishes a new methodological framework for the application of PEC-EC joint technology in the precise and comprehensive diagnosis of complex diseases.

Claims

1. A photoelectrochemical-electrochemical sensor platform for the detection of NT-proBNP and cTnI, characterized in that, The sensor platform includes an ITO glass substrate, on which two independent detection functional regions, I and II, are set. Region I contains a photoelectrochemical-electrochemical sensor for NT-proBNP detection, and Region II contains a photoelectrochemical-electrochemical sensor for cTnI detection. The NT-proBNP sensor includes mesoporous silica nanospheres attached to the ITO glass substrate, with cadmium sulfide quantum dots coated with mercaptoacetic acid attached to the silica nanospheres, and an NT-proBNP detection probe connected to the cadmium sulfide quantum dots. The cTnI sensor includes mesoporous silica nanospheres attached to the ITO glass substrate, with gold nanoparticles attached to the silica nanospheres, a cTnI detection probe coupled to the gold nanoparticles, and methylene blue connected to the cTnI detection probe.

2. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 1, characterized in that, The NT-proBNP detection probe is a double-stranded DNA1. The first strand of the double-stranded DNA1 is a 3′-amino-mediated NT-proBNP aptamer with the sequence 3′NH2-GTC CCC CTG CCC AGC CCA ATG TCG ACT CAC ACC GG-5′. The second strand is a complementary c-DNA1 modified with 3′-Fc with the sequence 5′-CAG GGG GAC GGG TCG GGT TACAGC TGA GTG TGG CC-Fc-3′.

3. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 1, characterized in that, The cTnI detection probe is a double-stranded DNA2. The first strand of the double-stranded DNA2 is a 3′-thiolized cTnI aptamer with the sequence 3′SH-CGC ATG CCA AAC GTT GCC TCA TAG TTC CCT CCC CGT GTC C-5′, and the second strand is the complementary strand c-DNA2 with the sequence 5′-GCG TAC GGT TTG CAA CGG AGT ATC AAG GGA GGGGCA CAG G-3′.

4. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 1, characterized in that, The preparation method of the mesoporous silica nanospheres is as follows: using cationic surfactant CTAC as a template, TEOS as a silicon source, TEA as a catalyst, and 1-octane, decahydronaphthalene, and cyclohexane as emulsifiers, silica nanoparticles are first prepared by stepwise growth through a one-pot two-phase layering method; then the template is removed from the silica nanoparticles to obtain mesoporous silica nanospheres (MSNs).

5. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 1, characterized in that, The preparation method of the cadmium sulfide quantum dots coated with mercaptoacetic acid is as follows: Mercaptoacetic acid is added to an aqueous solution of cadmium nitrate, and then the pH of the solution is adjusted to 11 with sodium hydroxide solution; then an aqueous solution of sodium sulfide is rapidly added under vigorous stirring; the reaction system is stirred in the dark for 10 min, and anhydrous methanol is added until a precipitate is formed, and water-soluble cadmium sulfide quantum dots coated with mercaptoacetic acid are obtained, denoted as CdS QDs.

6. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 1, characterized in that, The preparation method of cadmium sulfide quantum dots coated with mercaptoacetic acid on silica nanospheres is as follows: Silica nanospheres are dispersed in anhydrous ethanol solution containing 10 wt% 3-aminopropyltriethoxysilane, refluxed at 60 °C for 3 h under stirring, centrifuged and washed to obtain amino-functionalized MSNs, denoted as NH2-MSNs; Subsequently, NH2-MSNs were dispersed in deionized water under ultrasonic conditions to form a uniform suspension. A cadmium sulfide quantum dot aqueous solution coated with mercaptoacetic acid was added, and the mixture was stirred continuously at room temperature for 30 min. After centrifugation and washing, CdS QDs / NH2-MSNs composite nanomaterials were obtained for later use.

7. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 1, characterized in that, The preparation method of gold nanoparticles attached to silica nanospheres is as follows: dissolve silica nanospheres in deionized water and sonicate to ensure uniform dispersion; then add chloroauric acid solution and stir to mix evenly; add sodium citrate solution dropwise under continuous stirring, and react to generate gold nanoparticles on the surface of MSNs. Centrifuge, wash and dry to obtain Au@MSN.

8. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 2, characterized in that, The NT-proBNP detection probe is prepared as follows: 100 μL of 1 μM 3'-amino-modified NT-proBNP aptamer is mixed with 100 μL of 1 μM 3'-Fc-modified c-DNA1, annealed at 95℃ for 5 min, and then slowly cooled to room temperature to obtain double-stranded DNA1, i.e., dsDNA1.

9. The photoelectrochemical-electrochemical sensor platform for NT-proBNP and cTnI detection according to claim 3, characterized in that, The cTnI detection probe is prepared as follows: 100 μL of 1 μM cTnI aptamer is reacted with 100 μL of 1 mM tris(2-carboxyethyl)phosphine for 1 h, then annealed at 95 °C for 5 min, and hybridized with 100 μL of c-DNA2 to form double-stranded DNA2, i.e., dsDNA2. The obtained dsDNA2 is mixed with an equal volume of 1 mg / mL aqueous solution of silica nanospheres with attached gold particles, and incubated overnight at 4 °C in the dark to achieve coupling between the two, thus obtaining the final dsDNA2-Au@MSN complex.

10. A method for preparing a photoelectrochemical-electrochemical sensor platform for the detection of NT-proBNP and cTnI, characterized in that, Includes the following steps: Clean and dry the ITO glass substrate, and set up two working areas, namely Area I and Area II; In Region I, a photoelectrochemical-electrochemical sensor for NT-proBNP detection was prepared: 20 μL of a 4 mg / mL CdS@MSN aqueous solution was dropped onto Region I and allowed to air dry; then 20 μL of EDC / NHS solution was dropped onto the sensor and incubated at room temperature for 1 h; subsequently, 20 μL of dsDNA1 was immobilized using a carbodiimide chemical method. In region II, a photoelectrochemical-electrochemical sensor for cTnI detection was prepared: 20 μL of pre-assembled dsDNA2-Au@MSN was dropped onto region II and allowed to dry; then 20 μL of 500 μM methylene blue solution was dropped onto the sensor and incubated at room temperature for 1 h.

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