Construction method of dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers

Abstract

The method for constructing a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers includes the following steps: A: preparation of MoS2; B: preparation of Cd-MoS 2； C: Preparation of cadmium sulfide quantum dots; D: Preparation of gold nanoparticles; E: Preparation of Au NPs@dsDNA; F: Construction of the sensing platform: Cd-MoS2 solution was dropped onto an ITO glass substrate, which was then immersed in a polydimethylammonium chloride solution and a CdS QD solution and activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Au NPs@dsDNA were dropped onto the ITO glass substrate and incubated for 1 hour, then immersed in a methylene blue solution and blocked with BSA. NT-pro BNP was dropped onto the ITO glass substrate and incubated for 1 hour. The photocurrent was measured at a wavelength of 450 nm. This invention has good reliability.

Description

Technical Field

[0001] The present invention relates to the detection field of amino-terminal pro-brain natriuretic peptide (NT-pro BNP), belonging to the field of chemical technology. Background Art

[0002] N-terminal pro-brain natriuretic peptide (NT-pro BNP) is widely considered an ideal marker for the diagnosis and prognosis of heart failure (HF) patients due to its high circulating concentrations and long-term stability. Current methods for detecting NT-pro BNP include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (Siemens, CLIA), immunochromatographic assay (ICA), and electrochemiluminescent immunoassay (ECLIA). Despite rapid development, these assays suffer from long analysis times, cross-reactivity, and, in some cases, inability to meet clinically required critical levels. Therefore, developing novel, sensitive, cost-effective, and robust NT-pro BNP detection strategies is highly attractive for the diagnosis and treatment of early cardiovascular disease (CVD). Compared to traditional single-signal output sensors, which are susceptible to human and equipment effects and have low accuracy, dual-signal output sensors can be cross-validated through two modes, further improving detection precision and target quantification, ensuring the accuracy and reliability of test results. Therefore, designing a dual-mode sensor for NT-pro BNP detection can achieve high sensitivity and a wide detection range, making it suitable for complex biological and chemical analyses. Photoelectrochemical (PEC) and electrochemical (EC) techniques have attracted considerable attention due to their excellent sensitivity, low background noise, and simple operation. Combining these two methods can significantly improve quantitative analysis and correct for the systematic errors and background signals of single-mode output sensing platforms. Three-dimensional MoS2, a representative transition metal material, possesses large surface area, excellent electronic properties, and a high density of electronic states, making it widely used as a material for electronic and optoelectronic devices. However, MoS2 exhibits strong charge carrier recombination, significantly reducing hydrogen production rates due to its low reduction potential. This limitation can be overcome through metal doping and heterojunction formation, such as La-MoS2 and MoS2 / ZnS. CdS has recently attracted considerable attention due to its relatively narrow band gap, low cost, and excellent performance. Therefore, the photoelectric performance of MoS2 can be enhanced by synthesizing Cd-MoS2 through elemental doping. Therefore, the development of a dual-mode photoelectrochemical / electrochemical sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers holds great promise in CVD research. Summary of the Invention

[0003] The present invention aims to provide a method for constructing a sensing platform for photoelectrochemical and electrochemical dual-mode detection of NT-pro BNP on the same electrode, which has the advantages of high sensitivity, low detection limit and wide detection range.

[0004] To achieve the purpose of the present invention, the following technical solution is adopted: a method for constructing a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers, comprising the following steps:

[0005] A: (NH4)6Mo7O 24 . 4H2O and CH3CSNH2 were dissolved in 15 mL of ultrapure water, ultrasonically treated for 10 min until completely dissolved, and then the two solutions were transferred to the polytetrafluoroethylene liner of a 50 mL high-pressure reactor and ultrasonically treated until the mixed solution turned light blue. The mixture was kept at 200 °C in an oven for 10 hours, cooled to room temperature, and centrifuged to remove the supernatant. The precipitate was washed alternately with ethanol and ultrapure water three times and freeze-dried to obtain MoS2.

[0006] B: (NH4)6Mo7O 24 . 4H2O, CH3CSNH2 and Cd(NO3)2 . 4H2O were dissolved in 10 mL of ultrapure water and ultrasonicated for 10 min until completely dissolved; then (NH4)6Mo7O 24 . 4H2O solution and Cd(NO3)2 . The 4H2O solution was transferred to the polytetrafluoroethylene liner of a 50 mL high-pressure reactor, ultrasonically treated, mixed evenly, and then CH3CSNH2 solution was added and ultrasonicated until the mixed solution turned light blue. It was kept in an oven at 200 °C for 10 hours; cooled to room temperature, centrifuged to remove the supernatant, and the precipitate was washed three times with ethanol and ultrapure water alternately; by changing the Cd(NO3)2 . 4H2O was added to synthesize products with a molar ratio of Cd to Mo of 0.25, 0.5, 1, 2, and 4; finally, the product was freeze-dried in a freeze dryer to obtain Cd-MoS 2；

[0007] C: Cd(NO3)2 . 4H2O, 3-mercaptopropionic acid, and β-mercaptoethanol were mixed evenly under vigorous shaking, and the pH was adjusted to 7-8 with NaOH. When heated to 110°C, Na2S was added and the mixture was allowed to react under condensation reflux for one hour to obtain cadmium sulfide quantum dots.

[0008] D: First, HAuCl4 was added to ultrapure water and heated to 148°C with stirring at 300 rpm. Then, a 1 wt% sodium citrate solution was slowly added dropwise. The speed was adjusted to 1000 rpm. After five minutes, the speed was adjusted back to 300 rpm. The reaction was continued for another 5 minutes before stopping heating to obtain gold nanoparticles.

[0009] E: The aptamer was treated with an equal volume of tris(2-carboxyethyl)phosphine hydrochloride for 1 hour to disrupt the SS bond and annealed at 95°C for 5 minutes. An equal concentration of complementary strands was also annealed at 95°C for 5 minutes and then bound via complementary base pairing. The synthesized DNA duplex was mixed with an equal volume of gold nanoparticles from step D and incubated at 4°C overnight to obtain Au NPs@dsDNA.

[0010] F: Construction of a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers: The ITO glass was ultrasonically cleaned with acetone, ethanol and water in sequence, and then dried; subsequently, the insulating glue was punched and the electrodes were fixed to form a circular working area with a diameter of 0.4 mm; 20 μL of Cd-MoS2 solution was dropped onto the ITO glass and dried, and then rinsed with ultrapure water; thereafter, the ITO glass was immersed in a polydimethylammonium chloride solution and a CdS QDs solution for five minutes, repeated twice, and rinsed with ultrapure water; then, the carboxyl groups on the surface of the CdS QDs were activated using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a 1:1 ratio; 20 μL of Au NPs@dsDNA was dropped onto the ITO glass and incubated at 37 °C for 1 hour. After rinsing with ultrapure water, the ITO glass was immersed in a methylene blue solution for 90 minutes, and then rinsed with 20 μL BSA was used for blocking. Finally, after rinsing, 20 μL of NT-pro BNP was dropped onto the ITO glass substrate and incubated for 1 hour, and then rinsed with ultrapure water. The photocurrent was measured under a UV flashlight with a wavelength of 450 nm and an electrochemical workstation. PBS solution with a pH of 7.4 was used as the electrolyte solution. Before performing the electrochemical test, nitrogen was introduced into the PBS solution to remove oxygen from the solution.

[0011] Further; Step A, Step B (NH4)6Mo7O 24 . The mass of 4H2O is 621 mg; the mass of CH3CSNH2 is 532 mg.

[0012] Further; in step C, Cd(NO3)2 . The concentration of 4H2O was 16 mM, the volume of 3-mercaptopropionic acid was 50 μL, the volume of β-mercaptoethanol was 10 μL, the concentration of NaOH was 10 M, and the concentration of Na2S was 32 mM.

[0013] Furthermore, in step D, the mass fraction of HAuCl4 in ultrapure water is 1 wt%.

[0014] Furthermore, in step E, the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 1 mM, and the concentrations of the aptamer and the complementary chain are both 1 μM.

[0015] Furthermore, in the electrode construction process of step F, the mass fraction of the polydimethylammonium chloride solution is 1 wt%, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 5 mM, the concentration of N-hydroxysuccinimide is 1 mM, the concentration of methylene blue is 500 μM, and the concentration of BSA is 1 wt%.

[0016] Compared with existing technologies, this invention has the following advantages: 1) Cd-MoS2 and CdS quantum dots, two materials with excellent photoelectric properties, were prepared. 2) A flashlight with a wavelength of 450 nm was used as the light source, potentially enabling portable detection. 3) The NT-pro BNP aptamer was paired with its complementary strand and then bound to gold nanoparticles to form Au NPs@dsDNA. Methylene blue (MB) was embedded in the double-stranded DNA as a bifunctional probe to amplify the photocurrent and generate an electrochemical signal. 4) A photoelectrochemical / electrochemical dual-mode biosensor platform for NT-pro BNP detection was constructed. The preparation method is simple and low-cost, and it can achieve high-sensitivity and wide-range detection of NT-pro BNP. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the incubation process of Au NPs@dsDNA.

[0018] Figure 2 is the particle size characterization diagram of Au NPs.

[0019] Figure 3 is a transmission electron microscopy (HRTEM) image of gold nanoparticles.

[0020] Figure 4 This is the energy dispersive spectroscopy (EDS) image of gold nanoparticles.

[0021] Figure 5 This is the UV-visible spectrum of gold nanoparticles.

[0022] Figure 6 UV-visible diffuse reflectance spectroscopy characterization of CdS, MoS2, and Cd-MoS2.

[0023] Figure 7 This is a feasibility verification diagram of the photoelectric signal of different concentrations of NT-pro BNP.

[0024] Figure 8 This is a feasibility verification diagram of the electrical signal of different concentrations of NT-pro BNP.

[0025] Figure 9 Schematic diagram of the photoelectrochemical (PEC) sensing mechanism.

[0026] Figure 10 This is the photocurrent diagram of the photoelectrochemical sensor corresponding to different concentrations of NT-pro BNP.

[0027] Figure 11 yes Figure 10 Linearization diagram of .

[0028] Figure 12 This is a graph showing the experimental results of the photocurrent of the photoelectrochemical sensor under different interferences.

[0029] Figure 13 The current diagram of the electrochemical sensor corresponding to different concentrations of NT-pro BNP.

[0030] Figure 14 yes Figure 13 Linearization diagram of .

[0031] Figure 15 This is a graph showing the current test results of the electrochemical sensor under different interferences.

[0032] Figure 16 is a reproducible schematic diagram of the photoelectrochemical sensor.

[0033] Figure 17 is a schematic diagram of the repeatability of the electrochemical sensor.

[0034] Figure 18 Schematic diagram of the stability of the photoelectrochemical sensor.

[0035] Figure 19 Schematic diagram of the stability of electrochemical sensors. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below by way of examples, but these examples should not be considered as limiting the present invention.

[0037] In this application, Cd-MoS2 refers to cadmium-doped molybdenum disulfide, CdS QDs refers to cadmium sulfide quantum dots; Au NPs refers to gold nanoparticles; Au NPs@dsDNA refers to gold nanoparticles bound to double-stranded DNA; MPA refers to 3-mercaptopropionic acid; β-ME refers to β-mercaptoethanol; EDC refers to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS refers to N-hydroxysuccinimide; MB refers to methylene blue; and PDDA refers to polydimethylammonium chloride solution.

[0038] The construction method of the photoelectrochemical / electrochemical dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers is as follows:

[0039] A: (NH4)6Mo7O 24 . 4H2O and CH3CSNH2 were dissolved in 15 mL of ultrapure water and sonicated for 10 minutes until completely dissolved. Both solutions were then transferred to a 50 mL autoclave lined with polytetrafluoroethylene and sonicated until the mixed solution turned light blue. The reactor was assembled and oven-dried at 200°C for 10 hours. After cooling to room temperature, the supernatant was removed by centrifugation, and the precipitate was washed alternately with ethanol and ultrapure water three times and freeze-dried to yield MoS2.

[0040] B: (NH4)6Mo7O 24 . 4H2O, CH3CSNH2 and a certain amount of Cd(NO3)2 . 4H2O were dissolved in 10 mL of ultrapure water and ultrasonicated for 10 min until completely dissolved. 24 . 4H2O solution and Cd(NO3)2 . The 4H2O solution was transferred to the polytetrafluoroethylene liner of a 50 mL autoclave, ultrasonicated, and mixed thoroughly. Then, CH3CSNH2 solution was added and ultrasonicated until the mixed solution turned light blue. The reactor was assembled and kept in an oven at 200°C for 10 hours. Cooled to room temperature, the supernatant was removed by centrifugation, and the precipitate was washed three times with ethanol and ultrapure water alternately. By changing the Cd(NO3)2 . 4H2O was added to synthesize products with molar ratios of Cd to Mo of 0.25, 0.5, 1, 2, and 4. Finally, the product was freeze-dried in a freeze dryer to obtain Cd-MoS2.

[0041] C: Cd(NO3)2 . 4H2O, 3-mercaptopropionic acid (MPA), and β-mercaptoethanol (β-ME) were mixed evenly under vigorous shaking, and the pH was adjusted to 7-8 with NaOH. When heated to 110°C, Na2S was added and the mixture was reacted under condensation reflux for one hour to obtain cadmium sulfide quantum dots.

[0042] D: First, add HAuCl4 to 49.25 mL of ultrapure water, stir and heat to 148 °C at 300 rpm, then slowly add 5.4 mL of 1 wt% sodium citrate solution dropwise, adjust the speed to 1000 rpm, and after five minutes, adjust it back to 300 rpm. Continue the reaction for 5 minutes and then stop heating to obtain gold nanoparticles.

[0043] E: The aptamer was treated with an equal volume of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 1 hour to disrupt the SS bond, followed by annealing at 95°C for 5 minutes. An equal concentration of complementary strand (C-DNA) was also annealed at 95°C for 5 minutes, allowing binding through complementary base pairing. The synthesized DNA duplex was mixed with an equal volume of gold nanoparticles and incubated at 4°C overnight to yield Au NPs@dsDNA.

[0044] Further; in steps A and B, (NH4)6Mo7O 24 . The mass of 4H2O is 621 mg; the mass of CH3CSNH2 is 532 mg.

[0045] Further; in step C, Cd(NO3)2 . The concentration of 4H2O was 16 mM, the volume of 3-mercaptopropionic acid (MPA) was 50 μL, the volume of β-mercaptoethanol (β-ME) was 10 μL, the concentration of NaOH was 10 M, and the concentration of Na2S was 32 mM.

[0046] Furthermore, in step D, the mass fractions of HAuCl4 and sodium citrate solution are both 1 wt%.

[0047] Furthermore, in step E, the concentration of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is 1 mM, and the concentrations of the aptamer (T-DNA) and the complementary chain (C-DNA) are both 1 μM.

[0048] Example 1

[0049] This embodiment is based on the construction of a photoelectrochemical / electrochemical dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers, and its construction method includes the following steps:

[0050] (1) (NH4)6Mo7O 24 .4H2O (621 mg, 0.033 M) and CH3CSNH2 (532 mg, 0.47 M) were each dissolved in 15 mL of ultrapure water and sonicated for 10 min until completely dissolved. Both solutions were then transferred to a 50 mL autoclave lined with polytetrafluoroethylene and sonicated until the mixed solution turned light blue. The reactor was assembled and oven-dried at 200°C for 10 hours. After cooling to room temperature, the supernatant was removed by centrifugation, and the precipitate was washed three times alternately with ethanol and ultrapure water and freeze-dried for later use.

[0051] (2) (NH4)6Mo7O 24 . 4H2O (621 mg, 0.033 M), CH3CSNH2 (532 mg, 0.47 M) and a certain amount of Cd(NO3)2 . 4H2O were dissolved in 10 mL of ultrapure water and sonicated for 10 min until they were completely dissolved. 24 . 4H2O solution and Cd(NO3)2 . The 4H2O solution was transferred to the polytetrafluoroethylene liner of a 50 mL autoclave, ultrasonicated, and mixed thoroughly. Then, CH3CSNH2 solution was added and ultrasonicated until the mixed solution turned light blue. The reactor was assembled and kept in an oven at 200°C for 10 hours. Cooled to room temperature, the supernatant was removed by centrifugation, and the precipitate was washed three times with ethanol and ultrapure water alternately. By changing the Cd(NO3)2 . 4H2O was added to synthesize products with Cd to Mo molar ratios of 0.25, 0.5, 1, 2, and 4. Finally, the obtained products were freeze-dried in a freeze dryer.

[0052] (3) Add Cd(NO3)2 . 4H2O (10 mL, 16 mM), 3-mercaptopropionic acid (50 μL), and β-mercaptoethanol (10 μL) were mixed thoroughly under vigorous shaking, and the pH was adjusted to 7-8 with NaOH (10 M). After heating to 110°C, Na2S (5 mL, 32 mM) was added and the reaction was allowed to proceed under condensation reflux for one hour.

[0053] (4) First, add HAuCl4 (1 mL, 1 wt%) to 49.25 mL of ultrapure water, stir and heat to 148 °C at 300 rpm, then slowly add 5.4 mL of 1 wt% sodium citrate solution, adjust the speed to 1000 rpm, and return to 300 rpm after five minutes. Continue the reaction for 5 minutes and then stop heating.

[0054] (5) The synthesis of Au NPs@dsDNA involves two steps. First, the aptamer was treated with an equal volume of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 1 hour to break the SS bond and annealed at 95 °C for 5 minutes. An equal concentration of complementary strand (C-DNA) was also annealed at 95 °C for 5 minutes and then bound through complementary base pairing. The synthesized DNA duplex was mixed with an equal volume of gold nanoparticles and incubated at 4 °C overnight to obtain Au NPs@dsDNA.

[0055] The synthesis process of Au NPs@dsDNA is as follows Figure 1 As shown in the figure, a thiol-terminated T-DNA (NT-pro BNP aptamer) and an amino-terminated complementary strand (C-DNA) were paired after treatment to form double-stranded DNA (dsDNA). Subsequently, the dsDNA and gold nanoparticles (Au NPs) were incubated overnight at 4°C to synthesize Au NPs@dsDNA with an amino group at one end. The synthesized gold nanoparticles were then characterized. Figure 2 The Au NPs have a particle size of about 13 nm and are evenly distributed. Figure 3 ) The calculated lattice spacing of the gold nanoparticles is 0.115 nm, corresponding to the (222) crystal plane. The corresponding energy dispersive spectroscopy (EDS) image is shown in Figure 4 As shown in Figure 2, these results together indicate the successful preparation of gold nanoparticles. To verify the successful binding of Au NPs to DNA, UV-visible spectroscopy was performed, and the results are shown in Figure 2. Figure 5 As shown. In the UV-visible spectrum, Au NPs exhibit their unique characteristic absorption peak at a wavelength of 520 nanometers, which is a typical performance of AuNPs in the visible light range. At the same time, at a wavelength of 260 nanometers, which usually represents the characteristic absorption of DNA, the absorption intensity of individual AuNPs is very weak and almost negligible. However, when observing the UV-visible spectrum of Au NPs@DNA, the situation is different. Compared with individual Au NPs, the absorption intensity of Au NPs@DNA at 260 nanometers is significantly increased, indicating that effective binding occurs between Au NPs and double-stranded DNA.

[0056] . Figure 6 UV-visible diffuse reflectance spectroscopy characterization of CdS, MoS2, and Cd-MoS2.

[0057] Example 2

[0058] (1) (NH4)6Mo7O 24 .4H2O (621 mg, 0.033 M) and CH3CSNH2 (532 mg, 0.47 M) were each dissolved in 15 mL of ultrapure water and sonicated for 10 min until completely dissolved. Both solutions were then transferred to a 50 mL autoclave lined with polytetrafluoroethylene and sonicated until the mixed solution turned light blue. The reactor was assembled and oven-dried at 200°C for 10 hours. After cooling to room temperature, the supernatant was removed by centrifugation, and the precipitate was washed three times alternately with ethanol and ultrapure water and freeze-dried for later use.

[0059] (2) (NH4)6Mo7O 24 . 4H2O (621 mg, 0.033 M), CH3CSNH2 (532 mg, 0.47 M) and a certain amount of Cd(NO3)2 . 4H2O were dissolved in 10 mL of ultrapure water and sonicated for 10 min until they were completely dissolved. 24 . 4H2O solution and Cd(NO3)2 . The 4H2O solution was transferred to the polytetrafluoroethylene liner of a 50 mL autoclave, ultrasonicated, and mixed thoroughly. Then, CH3CSNH2 solution was added and ultrasonicated until the mixed solution turned light blue. The reactor was assembled and kept in an oven at 200°C for 10 hours. Cooled to room temperature, the supernatant was removed by centrifugation, and the precipitate was washed three times with ethanol and ultrapure water alternately. By changing the Cd(NO3)2 . 4H2O was added to synthesize products with Cd to Mo molar ratios of 0.25, 0.5, 1, 2, and 4. Finally, the obtained products were freeze-dried in a freeze dryer.

[0060] (3) Add Cd(NO3)2 . 4H2O (10 mL, 16 mM), 3-mercaptopropionic acid (50 μL), and β-mercaptoethanol (10 μL) were mixed thoroughly under vigorous shaking, and the pH was adjusted to 7-8 with NaOH (10 M). After heating to 110°C, Na2S (5 mL, 32 mM) was added and the reaction was allowed to proceed under condensation reflux for one hour.

[0061] (4) First, add HAuCl4 (1 mL, 1 wt%) to 49.25 mL of ultrapure water, stir and heat to 148 °C at 300 rpm, then slowly add 5.4 mL of 1 wt% sodium citrate solution, adjust the speed to 1000 rpm, and return to 300 rpm after five minutes. Continue the reaction for 5 minutes and then stop heating.

[0062] (5) The synthesis of Au NPs@dsDNA involves two steps. First, the aptamer was treated with an equal volume of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 1 hour to break the SS bond and annealed at 95 °C for 5 minutes. An equal concentration of complementary strand (C-DNA) was also annealed at 95 °C for 5 minutes and then bound through complementary base pairing. The synthesized DNA duplex was mixed with an equal volume of gold nanoparticles and incubated at 4 °C overnight to obtain Au NPs@dsDNA.

[0063] Feasibility analysis verification: In the presence of NT-pro BNP, the double helix structure of Au NPs@dsDNA will unwind, causing the aptamer with gold nanoparticles (AuNPs) to fall off and disrupting the energy transfer between cadmium sulfide quantum dots (CdS QDs) and Au NPs@dsDNA. As a result, the photoelectric signal is restored, as shown in Figure 2. Figure 7 As shown in Figure 3, curves a, b, and c correspond to NT-pro BNP concentrations of 0, 10 pg / mL, and 100 pg / mL, respectively. It can be concluded that the designed NT-pro BNP photoelectric biosensor follows the "signal enhancement" mechanism and has been successfully constructed. At the same time, as the DNA double helix structure unwinds, the aptamer binds to the target, causing some methylene blue to fall off, resulting in a decrease in current, as shown in Figure 3. Figure 8 As shown in Figure 1, curves a, b, and c correspond to NT-pro BNP concentrations of 0, 10 pg / mL, and 100 pg / mL, respectively. As the concentration of the target increases, more and more DNA double strands unwind, causing the methylene blue signal to gradually weaken. Therefore, an electrochemical sensor that follows the "signal attenuation" mechanism has been successfully constructed. The photoelectrochemical (PEC) sensing mechanism is as follows: Figure 9 As shown in Figure 2, a heterojunction can be formed between Cd-MoS2 and cadmium sulfide (CdS). When Cd-MoS2 is illuminated by a light source, holes in the Cd-MoS2 migrate to the valence band of CdS, while electrons in CdS transfer to the conduction band of Cd-MoS2. These electrons then flow to the ITO electrode, generating an anodic current.

[0064] After optimizing the material ratio and concentration and confirming the feasibility, the analytical performance of the dual-mode sensor was tested. Figure 10As shown in the figure, the corresponding NT-pro BNP concentrations are 0, 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 10, 50, 100 and 200 ng / mL. In the photoelectrochemical sensor, as the NT-pro BNP concentration increases from 0 to 200 ng / mL, the photocurrent gradually increases and shows a satisfactory wide linear range in the range of 0.1-50 ng / mL. The linear regression equation of the logarithm of the concentration is I-I0= 0.28LogC + 1.14, and the regression coefficient (R²) is 0.999 ( Figure 11 The detection limit was 0.028 pg / mL, calculated as: LOD = 3 SD / k (SD: standard deviation of blank control, n = 3; k: slope of linear regression equation). Figure 13 As shown in Figure 2, in the electrochemical sensor, the curves correspond to NT-pro BNP concentrations of 0, 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 10, 50, 100, 200, and 500 ng / mL, respectively. As the NT-pro BNP concentration increases from 0 to 500 ng / mL, the current gradually decreases, and the linear range is 0.1 - 100 ng / mL. The linear regression equation for the logarithm of the concentration is I0 - I = 0.83LogC + 4.08, and the regression coefficient (R²) is 0.991 (e.g., Figure 14 ). The detection limit was 0.029 pg / mL, which was calculated using the same formula. The results showed that the constructed photoelectrochemical sensor and electrochemical sensor both had a wide detection range and a low detection limit. In order to evaluate the selectivity of the photoelectrochemical sensor and the electrochemical sensor, the changes in the response signals of cytochrome C (a), immunoglobulin G (b), myoglobin (c), interleukin-6 (d), interleukin-17 (e), interleukin-23 (f), NT-pro BNP (g) and the mixed solution (h) were studied under the same conditions. Figure 12 As shown in Figure 2, when NT-pro BNP was detected using the PEC sensor, the photocurrent was significantly higher than that of other interferents, and the photocurrents corresponding to NT-pro BNP and the mixed solution were similar. Figure 15 As shown in the electrochemical sensor, the current in the presence of interfering substances decreased significantly, and the currents corresponding to NT-pro BNP and the mixed solution were close, indicating that both the photoelectrochemical sensor and electrochemical sensor constructed in this study exhibited good specificity, were almost unaffected by other interfering substances, were able to distinguish NT-pro BNP from other proteins, and could achieve accurate detection even in the presence of complex interfering substances.

[0065] To verify the reproducibility of the proposed photoelectrochemical sensor and electrochemical sensor, five BSA / MB / Au NPs@dsDNA / CdS QDs / Cd-MoS2 / ITO electrodes were used to detect NT-pro BNP under the same conditions. Each electrode was prepared under the same conditions and tested with the same concentration (1 ng / mL) of NT-pro BNP. Figure 16 and Figure 17 As shown, the measurements of the five electrodes all showed similar response signals, with relative standard deviations of 1.43% and 4.1%, respectively. This demonstrates the good reproducibility of both the photoelectrochemical and electrochemical sensors. Furthermore, storage stability is an important factor to consider. Here, the stability of the sensors was tested for 18 days. Figure 18 It was shown that after 18 days, the photocurrent of the photoelectrochemical sensor decreased to 93.7% of its initial current; Figure 19 The results showed that after 18 days, the current of the electrochemical sensor decreased to 90.7% of its initial current. These results show that the biosensors based on both methods exhibit good storage stability and can maintain excellent photoelectrochemical and electrochemical performance for a relatively long time, indicating their potential for further commercialization.

[0066] After elaborating on the embodiments of the present invention in detail, professionals in the relevant technical field can clearly realize that the present invention allows for a variety of adjustments and improvements without departing from the scope of its patent protection and the core spirit. Any simple modification, equivalent transformation or optimization based on the technical essence of the present invention is considered to be part of the technical solution of the present invention. These adjustments are not limited to specific technical details, but also cover the broad scope of the entire technical solution. At the same time, it should be emphasized that the scope of application of the present invention is not limited to the examples listed in the specification, but has a wider applicability and flexibility, and can adapt to different application scenarios and needs.

Claims

1. Construction method of dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers, characterized by The following steps are involved: A: (NH4)6Mo7O 24 . 4H2O and CH3CSNH2 were dissolved in 15 mL of ultrapure water, ultrasonically treated for 10 min until completely dissolved, and then the two solutions were transferred to the polytetrafluoroethylene liner of a 50 mL high-pressure reactor and ultrasonically treated until the mixed solution turned light blue. The mixture was kept at 200 °C in an oven for 10 hours, cooled to room temperature, and centrifuged to remove the supernatant. The precipitate was washed alternately with ethanol and ultrapure water three times and freeze-dried to obtain MoS2. B: (NH4)6Mo7O 24 . 4H2O, CH3CSNH2 and Cd(NO3)2 . 4H2O were dissolved in 10 mL of ultrapure water and ultrasonicated for 10 min until completely dissolved; then (NH4)6Mo7O 24 . 4H2O solution and Cd(NO3)2 . The 4H2O solution was transferred to the polytetrafluoroethylene liner of a 50 mL high-pressure reactor, ultrasonically treated, mixed evenly, and then CH3CSNH2 solution was added and ultrasonicated until the mixed solution turned light blue. It was kept in an oven at 200 °C for 10 hours; cooled to room temperature, centrifuged to remove the supernatant, and the precipitate was washed three times with ethanol and ultrapure water alternately; by changing the Cd(NO3)2 . 4H2O was added to synthesize products with a molar ratio of Cd to Mo of 0.25, 0.5, 1, 2, and 4; finally, the product was freeze-dried in a freeze dryer to obtain Cd-MoS 2； C: Cd(NO3)2 . 4H2O, 3-mercaptopropionic acid, and β-mercaptoethanol were mixed evenly under vigorous shaking, and the pH was adjusted to 7-8 with NaOH. When heated to 110°C, Na2S was added and the mixture was allowed to react under condensation reflux for one hour to obtain cadmium sulfide quantum dots. D: First, add HAuCl4 to ultrapure water, stir and heat at 300 rpm to 148°C, then slowly add 1 wt% sodium citrate solution dropwise, adjust the speed to 1000 rpm, and after five minutes, adjust it back to 300 rpm. Continue the reaction for 5 minutes before stopping heating to obtain gold nanoparticles; E: The aptamer was treated with an equal volume of tris(2-carboxyethyl)phosphine hydrochloride for 1 hour to disrupt the SS bond and annealed at 95 °C for 5 minutes. An equal concentration of complementary strands was also annealed at 95 °C for 5 minutes and then bound through complementary base pairing. The synthesized DNA duplex was mixed with an equal volume of gold nanoparticles from step D and incubated at 4 °C overnight to obtain Au NPs@dsDNA. F: Construction of a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers: The ITO glass was ultrasonically cleaned with acetone, ethanol, and water in sequence, and then dried; subsequently, holes were punched in the insulating glue and the electrodes were fixed to form a circular working area with a diameter of 0.4 mm; 20 μL of Cd-MoS2 solution was dropped onto the ITO glass and dried, and then rinsed with ultrapure water; thereafter, the ITO glass was immersed in a polydimethylammonium chloride solution and then in a CdS QDs solution for five minutes, repeated twice, and rinsed with ultrapure water; then, the carboxyl groups on the surface of the CdS QDs were activated using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a 1:1 ratio; 20 μL of Au NPs@dsDNA was dropped onto the ITO glass and incubated at 37°C for 1 hour. After rinsing with ultrapure water, the ITO glass was immersed in a methylene blue solution for 90 minutes and then blocked with 20 μL of BSA. Finally, after rinsing, 20 μL of NT-pro BNP was dropped onto the ITO glass substrate and incubated for 1 hour, and then rinsed with ultrapure water. The photocurrent was measured under a UV flashlight with a wavelength of 450 nm and an electrochemical workstation. PBS solution with a pH of 7.4 was used as the electrolyte solution. Before performing the electrochemical test, nitrogen was passed into the PBS solution to remove oxygen from the solution.

2. The method for constructing a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers according to claim 1, characterized in that: Step A, Step B (NH4)6Mo7O 24 . The mass of 4H2O is 621 mg; the mass of CH3CSNH2 is 532 mg.

3. The method for constructing a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers according to claim 1, characterized in that: Cd(NO3)2 in step C . The concentration of 4H2O was 16 mM, the volume of 3-mercaptopropionic acid was 50 μL, the volume of β-mercaptoethanol was 10 μL, the concentration of NaOH was 10 M, and the concentration of Na2S was 32 mM.

4. The method for constructing a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers according to claim 1, characterized in that: The mass fraction of HAuCl4 in ultrapure water in step D is 1 wt%.

5. The method for constructing a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers according to claim 1, characterized in that: In step E, the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 1 mM, and the concentrations of the aptamer and complementary chain are both 1 μM.

6. The method for constructing a dual-mode sensing platform for NT-pro BNP detection based on Cd-MoS2 nanoflowers according to claim 1, characterized in that: During the electrode construction process in step F, the mass fraction of the polydimethylammonium chloride solution was 1 wt%, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was 5 mM, the concentration of N-hydroxysuccinimide was 1 mM, the concentration of methylene blue was 500 μM, and the concentration of BSA was 1 wt%.

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