AuSt@Cu 2-x Se nanoparticles and their preparation methods and applications, NIR-II PT-LFIA test paper, sST2 detection method, photothermal detection system

Abstract

This invention relates to the field of nanoprobe technology, and particularly to AuSt@Cu 2‑x Se nanoparticles, their preparation methods and applications, NIR-II PT-LFIA test paper, sST2 detection method, and photothermal detection system. The nanoparticles exhibit a core-shell structure, with the core being AuSt gold nanoparticles and the shell being Cu. 2‑x Se shell. This invention provides an AuSt@Cu shell. 2‑x Se nanoparticles were deposited on the AuSt surface, forming an amorphous Cu layer. 2‑x Se shell, through modulation of AuSt@Cu 2‑x The size and morphology of Se nanoparticles cause the absorption peak of their LSPR to redshift from the visible light region to the NIR-II region, improving the photothermal conversion performance of the photothermal probe under NIR-II laser irradiation. Furthermore, an sST2 detection method and photothermal detection system were constructed, filling the gap in the field of existing high-performance NIR-II photothermal lateral flow immunochromatography.

Description

Technical Field

[0001] This invention relates to the field of nanoprobe technology, and particularly to AuSt@Cu 2-x Se nanoparticles and their preparation methods and applications, NIR-II PT-LFIA test paper, sST2 detection method, photothermal detection system. Background Technology

[0002] Despite advancements in cardiovascular diagnostic and treatment technologies, heart failure (HF) remains a significant global socioeconomic burden, characterized by high mortality and frequent readmissions. Recent data shows that nearly 64 million people worldwide suffer from HF, representing 1-3% of the adult population, with particularly high prevalence in East Asia, North America, and Western Europe. As a progressive syndrome, early risk stratification and intervention are crucial for patient prognosis, often requiring intervention during the asymptomatic stage. However, current diagnostic methods struggle to achieve large-scale, decentralized screening. While echocardiography remains the gold standard for structural assessment, its bulky equipment and reliance on specialized operation limit its application in bedside settings. Furthermore, the serum biomarker NT-proBNP levels currently used in clinical testing are often affected by non-cardiac factors such as age, obesity, and renal insufficiency, leading to ambiguity in HF diagnosis.

[0003] sST2 protein, soluble growth-stimulating gene 2 protein, is a key biomarker for the diagnosis and prognostic assessment of heart failure, as confirmed in recent years. Compared with traditional NT-proBNP biomarkers, sST2 exhibits low biological variability and is largely unaffected by confounding factors such as age, renal function, and body mass index. Based on these advantages, sST2 has been included in the American College of Cardiology / American Heart Association guidelines for incremental risk stratification of acute and chronic heart failure. Currently, the clinical gold standard for sST2 quantification is enzyme-linked immunosorbent assay (ELISA), particularly the FDA-approved Presage® ST2 assay. However, ELISA methods suffer from drawbacks such as long turnaround times, complex procedures, and reliance on central laboratory equipment, fundamentally limiting their application in rapid point-of-care testing.

[0004] The core concept of point-of-care testing (POCT) technology lies in extending laboratory analytical capabilities to the sampling site to meet the urgent needs for rapid testing in scenarios such as public health emergencies, primary healthcare deployment, and family health management. Achieving clinically acceptable sensitivity and accuracy is the fundamental standard for qualified POCT equipment development. However, to achieve large-scale application, it is necessary to simplify the operating procedures and control equipment costs, enabling non-professionals to successfully complete tests and obtain reliable results at a low cost. Lateral flow immunochromatography (LFIA) is a membrane chromatography detection technology based on the capillary action of porous media. It uses capillary action to direct the flow of liquid samples between porous membranes to achieve rapid separation and detection of target analytes. It is a crucial cornerstone of POCT technology, favored for its speed, economy, ease of operation, and lack of complex equipment, and has become one of the most widely used POCT technologies in disease diagnosis, environmental monitoring, and food safety.

[0005] Photothermal signal-based lateral flow immunochromatography (PT-LFIA) is a novel detection modality developed in recent years. Its mechanism utilizes the photothermal conversion effect of nanoparticles. A nanoprobe with strong absorption is irradiated with a laser of a specific wavelength, causing the nanoprobe to exhibit a light-to-thermal (LSPR) effect. Subsequently, through a non-radiative relaxation process, the absorbed light energy is rapidly converted into lattice vibrational heat energy, leading to a local temperature increase in the particle, thus completing the light-to-thermal energy conversion process. The resulting thermal signal is monitored by an infrared thermal imager and converted into a visually intuitive temperature distribution image or temperature value. The near-infrared light window is divided into the near-infrared region I (700-900 nm) and the near-infrared region II (1000-1700 nm). In the NIR-I band, biological tissues and commonly used nitrocellulose membranes (NC membranes) have strong light absorption, generating background signals. In the NIR-II band, the light absorption of biological tissues and nitrocellulose membranes is extremely low, making it an ideal detection window with low background interference. Therefore, tuning the LSPR band to the NIR-II region becomes an effective strategy for suppressing thermal background and eliminating false positives. Despite its theoretical advantages, high-performance NIR-II photothermal lateral flow analysis is still rarely reported. Summary of the Invention

[0006] This invention provides an AuSt@Cu 2-x The invention relates to Se nanoparticles, their preparation methods and applications, NIR-II PT-LFIA test strips, sST2 detection methods, and photothermal detection systems. The aim is to fill the gap in existing high-performance NIR-II photothermal lateral flow analysis and provide a technical solution for biological detection of sST2 protein based on the NIR-II band, which can be applied to the diagnosis of heart failure.

[0007] To achieve the above objectives, the present invention provides an AuSt@Cu 2-xSe nanoparticles, wherein the nanoparticles have a core-shell structure, the core structure being AuSt gold nanoparticles, and the shell structure being Cu. 2-x Se shell, where the value of x is in the range of 0 < x < 1; The core structure of the nanoparticles has a particle size of 63.5 ± 7.6 nm, and the particle size of the nanoparticles is 65.2 ± 7.3-92.1 ± 7.2 nm.

[0008] Gold nanoparticles exhibit a strong localized surface plasmon resonance effect, with an optical extinction cross section 4-5 orders of magnitude higher than that of organic dyes and inorganic nanomaterials, enabling highly efficient photothermal conversion. Therefore, photothermal lateral flow analysis can achieve sensitivity far exceeding that of visual detection. Despite progress, current photothermal lateral flow analysis still faces a key bottleneck: high background thermal interference. Most systems employ first near-infrared (NIR-I, 700-900 nm) laser irradiation, where nitrocellulose membranes and biological matrices (such as hemoglobin) exhibit significant light absorption. This non-specific photothermal effect reduces the signal-to-noise ratio, hindering the detection of low-abundance analytes. In contrast, the second near-infrared (NIR-II, 1000-1700 nm) window is characterized by significantly reduced light absorption in nitrocellulose membranes and biological substances.

[0009] Therefore, this application modulates AuSt@Cu 2-x The size and morphology of Se nanoparticles cause the absorption peak of their LSPR to redshift from the visible light region to the NIR-II region, thereby improving the photothermal conversion performance of the photothermal probe under NIR-II laser irradiation.

[0010] Preferably, the AuSt@Cu 2-x The Au content in the Se nanoparticles is 34.9%, and the Cu content is... 2-x The Se content is 65.1%.

[0011] Under the same technical concept, the present invention also provides an AuSt@Cu 2-x The preparation method of Se nanoparticles includes the following steps: (1) Synthesis of AuSt gold nanoparticles: Prepare an aqueous solution mixture with HAuCl4·4H2O and C6H5Na3O7·2H2O as solutes, prepare a NaBH4 solution with C6H5Na3O7·2H2O as solvent, slowly add the NaBH4 solution to the mixed solution of HAuCl4·4H2O and C6H5Na3O7·2H2O, stir, and obtain an aqueous solution of gold nanoseeds; PVP was dissolved in DMF, and an aqueous solution of HAuCl4·4H2O was added. Then, an aqueous solution of gold nanoparticles was added, and the mixture was stirred at room temperature for 2-3 hours. After centrifugation and washing, AuSt gold nanoparticles were obtained. The obtained AuSt gold nanoparticles were dispersed in deionized water for storage. (2) Synthesis of AuSt@Cu 2-x Se nanoparticles: A dispersion of AuSt gold nanoparticles was mixed with a PVP aqueous solution and heated. An AA aqueous solution was then added, followed immediately by dropwise addition of a SeO2 aqueous solution, while maintaining a constant temperature and stirring. Next, a CuSO4·5H2O aqueous solution and the AA aqueous solution were added to initiate the reaction. After the reaction was complete, the mixture was centrifuged and washed to obtain AuSt@Cu. 2-x Se nanoparticles.

[0012] When the SeO2 solution concentration is 0.3 mM, the elemental content ratio of the nanoparticles reaches 0.97 (CuSe / Au), and the material exhibits both NIR-I and NIR-II window LSPR responses, but with relatively weak intensity. When the SeO2 solution concentration is 0.6 mM, the Au elemental content is 34.9%, and the Cu... 2-x With a Se content of 65.1%, the material exhibits extremely strong NIR-II window light absorption and significantly enhanced LSPR.

[0013] Preferably, the concentration of HAuCl4·4H2O in the aqueous solution mixture described in step (1) is 0.5-0.6 mM, and the ratio of the concentration of HAuCl4·4H2O to the concentration of C6H5Na3O7·2H2O is 1:1.5-1:1.7; the amount of NaBH4 solution added is 1.0-1.2 mL of 0.06-0.09 wt% NaBH4 solution added to every 100 mL of the mixed solution of HAuCl4·4H2O and C6H5Na3O7·2H2O; the concentration of the gold nanoseed aqueous solution is 0.4-0.7 nM; and the stirring time described in step (1) is 10-12 h. The concentration of PVP is 8-10 mM, and the amount of HAuCl4·4H2O aqueous solution added is 0.005-0.006 mL of 50-60 mM HAuCl4·4H2O aqueous solution added per 1 mL of PVP solution; the amount of gold nano-seed aqueous solution added is 0.001-0.008 mL of gold nano-seed aqueous solution added per 1 mL of PVP solution. The concentration of the AuSt gold nanoparticle dispersion in step (2) is 0.5-1.0 mg / mL; the mass ratio of AuSt gold nanoparticles to PVP in the AuSt gold nanoparticle dispersion and PVP aqueous solution is 1:40-1:50; the amount of AA aqueous solution added is 0.1-0.4 mL of 90-120 mM AA aqueous solution per 1 mL of AuSt gold nanoparticle solution; the amount of SeO2 aqueous solution added is 0.15-0.6 mL of 0.01-0.8 mM SeO2 aqueous solution per 1 mL of AuSt gold nanoparticle solution; the mixing and heating temperature in step (2) is 40℃; the constant temperature stirring time is 15-20 min; and the reaction time is 14-18 h.

[0014] Under the same technical concept, the present invention also provides an AuSt@Cu 2-x Application of Se nanoparticles, namely an AuSt@Cu 2-x Se nanoparticles were used to synthesize NIR-II photothermal probes, the LSPR peak of which is located in the NIR-II region at 1000-1100 nm.

[0015] Preferably, the method for synthesizing the NIR-II photothermal probe includes: S1. Prepare PBS buffer, add AuSt@Cu 2-x An aqueous suspension of Se nanoparticles was added to a PBS solution, mixed and stirred, and centrifuged to obtain AuSt@Cu. 2-x Se-3MPA, AuSt@Cu 2-x Se-3MPA was dispersed in PBS, and an aqueous solution of EDC / NHS was added. The activation reaction was carried out at room temperature for 0.5-1 h. After the reaction was completed, the mixture was centrifuged, washed, and redispersed to obtain the activated AuSt@Cu. 2-x Se-3MPA; S2. Dissolve the sST2 monoclonal antibody anti-ST2 mAb and mix it with the activated AuSt@Cu. 2-x Se-3MPA was mixed and incubated, then PBS solution containing BSA was added, and the mixture was shaken at low speed on a shaker at room temperature. After reaction, centrifugation and washing were performed to obtain the NIR-II photothermal probe.

[0016] Preferably, the pH of the PBS buffer in step S1 is 7.2-7.8, and the AuSt@Cu 2-x The concentration of the Se nanoparticle aqueous suspension is 0.2-1.0 mg / mL; the concentration of the 3-MPA aqueous solution is 40-100 mM; the AuSt@Cu 2-xThe volume ratio of the aqueous suspension of Se nanoparticles to the aqueous solution of 3-MPA is 50:1-250:1; the molar ratio of EDC to NHS in the EDC / NHS aqueous solution is 1:1.0-1:2.0, and the concentration of the EDC / NHS aqueous solution is 4.0-10.0 mg / mL; the aqueous solution of EDC / NHS and AuSt@Cu 2-x The volume ratio of the Se nanoparticle aqueous suspension is 1:5-1:25; The sST2 monoclonal antibody anti-ST2 mAb was dissolved in sterile PBS solution at a concentration of 0.2-2.0 mg / mL.

[0017] Under the same technical concept, the present invention also provides an NIR-II PT-LFIA test strip, wherein the NIR-II PT-LFIA test strip comprises a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; the conjugate pad and the sample pad are immersed and washed in a standard running buffer, and the conjugate pad is immersed in the NIR-II photothermal probe solution in the application; the NC membrane is coated with a detection line and a control line, and the sample is coated on the sample pad; The sample pad, conjugate pad, NC membrane, and absorbent pad are bonded to a PVC base plate, with a 1.5 mm overlap between each pad, and are cut to obtain the NIR-II PT-LFIA test paper. The standard running buffer is prepared from a 0.01 M PBS solution at pH 7.2-7.8, containing 0.1-1.0 wt% BSA, 1.0-10.0 wt% sucrose, 0.01-0.1 wt% PVP, and 0.1-1.0 wt% Tween-20.

[0018] Under the same technical concept, the present invention also provides a method for detecting sST2 in NIR-II PT-LFIA, comprising the following steps: (1) The sample was subjected to a chromatographic reaction using the NIR-II PT-LFIA test strip described above; (2) Irradiate the test strip detection line area after reaction with a near-infrared II laser with a wavelength of 1000-1700 nm; (3) Detect the temperature change in the detection line area due to the photothermal effect, and quantify the concentration of sST2 in the sample based on the temperature change value.

[0019] Under the same technical concept, the present invention also provides a NIR-II PT-LFIA photothermal detection system, the detection system comprising: Laser device; the laser device is used in conjunction with the AuSt@Cu. 2-x Se nanoparticles or AuSt@Cu prepared by the aforementioned preparation method2-x The laser irradiation source is a NIR-II laser that matches the absorption peak of the Se nanoparticles, or the NIR-II photothermal probe described in the application, or the NIR-II PT-LFIA test paper described. Laser head; Infrared thermal imager; Sample to be tested; The laser device is connected to the laser head and controls the laser head; the laser head is set to correspond with the sample to be tested and emits laser light onto the sample to be tested; the infrared thermal imager records the temperature curve and thermal image of the sample to be tested in real time.

[0020] The above-described solution of the present invention has the following beneficial effects: (1) This invention provides an AuSt@Cu 2-x Se nanoparticles were deposited on the AuSt surface, forming an amorphous Cu layer. 2-x By controlling the size and morphology of AuSt@Cu2-xSe nanoparticles, the absorption peak of their LSPR (Laser-Laser Resonance Probability) is redshifted from the visible light region to the NIR-II region, thus improving the photothermal conversion performance of the photothermal probe under NIR-II laser irradiation. When the incident light frequency matches the collective oscillation frequency of the free electrons on the surface of the metal nanoparticles, the LSPR effect is excited, resulting in light absorption and scattering. By controlling the morphology, size, and elemental composition of the nanoparticles, their LSPR peak position can be tuned to achieve enhanced absorption of light at specific wavelengths. Furthermore, the absorbed light energy is efficiently converted into lattice vibrational heat energy through a non-radiative relaxation process, exhibiting a photothermal conversion effect.

[0021] (2) This invention is based on AuSt@Cu 2-xSe nanoparticles were used to design the NIR-II photothermal probe and NIR-II PT-LFIA test strip for sST2 monoclonal antibodies. Compared with traditional detection methods, the photothermal probe and test strip have the following significant advantages: 1) Ultra-high signal-to-noise ratio: By detecting through the NIR-II window, the background thermal interference from the nitrocellulose membrane and biological matrix is ​​completely eliminated, allowing the specific signal to stand out. The signal-to-noise ratio is improved by orders of magnitude compared to NIR-I PT-LFIA; 2) Significantly improved detection sensitivity: The high signal-to-noise ratio creates conditions for detecting extremely low concentrations of sST2, achieving a sensitivity several orders of magnitude higher than traditional colorimetric methods, meeting the screening needs of the early asymptomatic stage of heart failure; 3) Accurate and reliable detection results: Low background interference and high sensitivity ensure the accuracy of quantitative results, avoid false positive results, and enable precise quantification of sST2, providing a reliable basis for clinical risk stratification and treatment monitoring. 4) It combines the advantages of speed and simplicity: It retains all the advantages of lateral flow analysis, such as simple operation, fast response (usually completed within 15-30 minutes), and no need for complicated equipment, truly realizing accurate detection at the bedside or in primary healthcare settings. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 AuSt@Cu in Embodiment 1 of the present invention 2-x Schematic diagram of the synthesis process of Se nanoparticles; Figure 2 These are TEM images of AuSt and AuSt@Se in Embodiment 1 of the present invention; where (a) is AuSt particles and (b) is AuSt@Se particles. Figure 3 This is a statistical diagram of the particle size distribution of AuSt and AuSt@Se in Example 1 of the present invention; where (a) is AuSt particles and (b) is AuSt@Se particles; Figure 4 AuSt@Cu synthesized from SeO2 solutions of different concentrations in Example 1 of this invention 2-x TEM images of Se nanoparticles; where (a) is a 0.05 mM SeO2 solution, (b) is a 0.1 mM SeO2 solution, (c) is a 0.2 mM SeO2 solution, (d) is a 0.3 mM SeO2 solution, (e) is a 0.4 mM SeO2 solution, and (f) is a 0.6 mM SeO2 solution. Figure 5 AuSt@Cu synthesized from 0.6 mL MSeO2 solution in Example 1 of this invention 2-x HAAD-STEM image and EDS elemental distribution map of Se nanoparticles; Figure 6 AuSt and Cu in Embodiment 1 of the present invention 2-x Se and AuSt@Cu 2-x XRD diffraction pattern of Se-0.6mMSeO2 nanoparticles; Figure 7 The images show the UV-Vis-NIR extinction spectra of the various nanomaterials in Example 1 of this invention and photographs of their suspensions in quartz cuvettes; where (a) shows AuSt, AuSt@Se NPs, and AuSt@Cu. 2-x Se NPs and AuSt@Cu 2-x (a) Se@mAB; (b) AuSt@Se nanoparticles synthesized from SeO2 solutions of different concentrations; (c) AuSt@Cu nanoparticles synthesized from SeO2 solutions of different concentrations. 2-x Se nanoparticles; Figure 8 This is a schematic diagram of the photothermal detection system in Embodiment 1 of the present invention; Figure 9 The photothermal detection system in Embodiment 1 of this invention is used to detect AuSt@Cu under 808 nm and 1064 nm laser irradiation. 2-x Temperature rise curve of Se NPs suspension; Figure 10 AuSt@Cu in Embodiment 1 of the present invention 2-x The trend of photothermal conversion efficiency η of Se NPs with SeO2 concentration conditions; Figure 11 Different concentrations of AuSt@Cu in Example 1 of this invention 2-x Heating curves of Se NPs suspension under 808 nm and 1064 nm laser irradiation; where (a) is under 808 nm laser and (b) is under 1064 nm laser. Figure 12 Different concentrations of AuSt@Cu in Example 1 of this invention 2-x Photothermal images of Se NPs suspension under 808 nm and 1064 nm laser irradiation; Figure 13 This is a schematic diagram of laser irradiation of the NC film area of ​​the NIR-I PT-LFIA test paper and thermal signal monitoring in Embodiment 2 of the present invention; Figure 14This is an analysis diagram of the results of laser irradiation of the NC film area of ​​the NIR-I PT-LFIA test strip and thermal signal monitoring in Example 2 of the present invention; wherein (a) represents the specified position of the NC film of the test strip irradiated by laser, (b) is a photograph of the test strip detection results of 0, 10, 20 and 200 ng / mL sST2 solution, and (c) and (d) are the photothermal detection results of irradiation by 808 nm and 1064 nm lasers, respectively; Figure 15 The following are the dual-modal quantitative analysis and evaluation results of the NIR-I PT-LFIA test strip under laser irradiation in Example 2 of this invention: (a) T-line SEM images of 0, 200, and 800 ng / mL sST2 samples; (b) optical photographs and photothermal images of 0-5000 ng / mL sST2 samples; and (c) detection signal-sST2 concentration calibration curves and linear fitting curves in colorimetric mode and (d) photothermal mode. Detailed Implementation

[0024] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0025] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0026] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a locking connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0027] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] Example 1 This embodiment provides an AuSt@Cu 2-x Se nanoparticles, exhibiting a core-shell structure, with the core being AuSt gold nanoparticles and the shell being Cu. 2-x Se shell; AuSt@Cu 2-x The Au content in the Se nanoparticles is 34.9%, and the Cu content is... 2-x The Se content is 65.1%; Cu 2-x The range of values ​​for x in Se is not fixed; it varies depending on the specific characteristics of the material. In this example, it does not affect the investigation of the material's properties, therefore, the specific value of x is not specified, and the range of x is 0 < x < 1; AuSt@Cu 2-x The preparation method of Se nanoparticles includes the following steps: 1. Synthesis of gold nanostars (AuSts) 1 mL of a 50 mM HAuCl4·4H2O aqueous solution was diluted with 89 mL of H2O, and then 2 mL of a 38.8 mM C6H5Na3O7·2H2O aqueous solution was added. Then, 1 mL of freshly prepared 0.075 wt% NaBH4 solution was slowly added, prepared using a 38.8 mM C6H5Na3O7·2H2O aqueous solution as the solvent. After stirring at room temperature for 12 hours, a 0.58 nM gold nanoseed aqueous solution was obtained. 1.5 g of PVP was dissolved in 15 mL of DMF, and 84 μL of a 50 mM HAuCl4·4H2O aqueous solution was added, followed by 20 μL of the synthesized gold nanoseed aqueous solution. The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was centrifuged at 8000 rpm, and the resulting AuSts were washed three times with ultrapure water. Finally, the AuSts were dispersed in H2O and stored at 4 °C.

[0029] 2. Synthesis of AuSt@Cu 2-x Se NPs photothermal materials First, 1 mL of an 800 μg / mL AuSts suspension was mixed with 3.5 mL of a 10 mg / mL PVP aqueous solution and heated to 40 °C. After the temperature stabilized, 0.2 mL of freshly prepared 100 mM AA aqueous solution was added, followed immediately by dropwise addition of 0.3 mL of SeO2 aqueous solution to achieve final concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.6 mM in the solution system. The mixture was stirred at 40 °C for 15 minutes, and then 60 μL of a 100 mM CuSO4·5H2O aqueous solution and 240 μL of a 100 mM AA aqueous solution were added, reacting for 16 hours. After the reaction was completed, the synthesized AuSt@Cu 2-x After centrifugation and washing with water, the Se nanoparticles were redispersed in water at a concentration of 0.4 mg / mL and stored at 4 °C for further use.

[0030] AuSt@Cu 2-x Characterization and LSPR property analysis of Se nanoparticles: (1) Microscopic morphology and structural characterization AuSts and AuSt@Cu were synthesized in the experiment. 2-x Se nanoparticles were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (STEM) for AuSts and AuSt@Cu. 2-x Microstructure of Se nanoparticles.

[0031] First, the morphology of AuSts was characterized, with a particle size of 63.5 ± 7.6 nm, as shown below. Figure 2 (a) and Figure 3 As shown in (a).

[0032] Secondly at AuSt@Cu 2-x During the synthesis and regulation of Se, samples were taken and analyzed for the intermediate product AuSt@Se and the reaction products AuSt@Cu. 2-x The appearance of Se. Figure 2 (b) TEM image of AuSt@Se and Figure 3 (b) The particle size distribution of AuSt@Se shows that Se atoms were successfully deposited on the surface of AuSts and formed an amorphous, non-uniform shell. The particle size of AuSt@Se is 67.1 ± 7.0 nm. Figure 4 AuSt@Cu synthesized from SeO2 solutions of different concentrations 2-xTEM images of Se nanoparticles; where (a) is a 0.05 mM SeO2 solution, (b) is a 0.1 mM SeO2 solution, (c) is a 0.2 mM SeO2 solution, (d) is a 0.3 mM SeO2 solution, (e) is a 0.4 mM SeO2 solution, and (f) is a 0.5 mM SeO2 solution; AuSt@Cu synthesized at SeO2 solution concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.6 mM. 2-x The Se particle sizes were 65.2 ± 7.3 nm, 67.5 ± 7.6 nm, 71.2 ± 7.0 nm, 77.1 ± 8.9 nm, 82.9 ± 6.8 nm and 92.1 ± 7.2 nm, respectively.

[0033] The above TEM results indicate that in Cu 2+ After the ions react with the Se layer, Cu is formed. 2-x Se shell, and as the concentration of SeO2 solution increases, Cu 2-x Se shell increases, AuSt@Cu 2-x The average particle size of Se also increased. TEM characterization results verified the synthesis principle of the nanomaterials as shown in the diagram. Figure 1 As shown.

[0034] AuSt@Cu was studied using scanning transmission electron microscopy. 2-x Further analysis was conducted on the microstructure and elemental composition of the Se nanoparticles. HAAD-STEM and EDS surface distribution analysis confirmed the presence of AuSt@Cu. 2-x The core-shell structure of Se nanoparticles, namely AuSt core surrounded by Cu 2-x The shell completely encloses the area, as... Figure 5 As shown.

[0035] In addition, AuSts and Cu were characterized by powder X-ray diffraction. 2-x Se and AuSt@Cu 2-x The crystal structure and composition of Se nanoparticles were analyzed. (AuSt@Cu) 2-x The XRD patterns of Se NPs show different diffraction peaks, such as Figure 6 As shown, all diffraction peaks correspond to cubic Au (JCPDS #: 01-1172) and cubic Cu. 2-x Se (JCPDS #: 06-0680) confirmed the synthesis of AuSt@Cu. 2-x The high crystallinity of Se nanoparticles. The above characterization results verify the AuSt@Cu 2-x Se NPs exhibit a core-shell structure with uniform morphology and particle size, good dispersibility, and a structure that simultaneously possesses cubic AuSt and Cu properties. 2-xCrystal system characteristics of Se.

[0036] (2) AuSt@Cu 2-x Elemental content analysis of Se nanoparticles With changes in the concentration of SeO2 solution in the synthesis conditions, AuSt@Cu was synthesized. 2-x The content of Au, Cu, and Se elements in Se nanoparticles also varies. The specific elemental composition was determined using an Agilent 5100 inductively coupled plasma optical emission spectrometer (ICP-OES), and the results are shown in Table 1 below: Table 1

[0037] Note: NP-c in the table above represents AuSt@Cu synthesized at SeO2 solution concentrations (c) of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.6 mM, respectively. 2-x Se nanoparticles.

[0038] According to the ICP-OES analysis results, when the SeO2 solution concentration is 0.6 mM, AuSt@Cu 2-x The Au content in Se NPs is 34.9%, and the Cu content is... 2-x The Se content is 65.1%.

[0039] (3) AuSt@Cu 2-x Optical property analysis of Se nanoparticles The experiment analyzed AuSt@Cu using ultraviolet-visible-near-infrared extinction spectroscopy. 2-x The localized surface plasmon effect of Se nanoparticles, compared with the experimentally prepared AuSts and the intermediate product AuSt@Se, yielded AuSt@Cu with the best near-infrared II photothermal conversion effect. 2-x Se@mAb photothermal probe. Extinction spectrum analysis results are as follows: Figure 7 As shown.

[0040] according to Figure 7 (a) Analysis shows that AuSts (#1) exhibits strong absorption at 808 nm. However, due to the higher refractive index of Se compared to water molecules, the LSPR peak of the AuSts nanoparticles deposited on the AuSts surface shows a significant red shift and weakening. Correspondingly, the color of the AuSts and AuSt@Se (#2) suspensions changes from dark blue to brown. With increasing SeO2 concentration in the solution, the LSPR peak of the generated AuSt@Se nanoparticles gradually red-shifts, such as... Figure 7 As shown in (b). The synthesized product AuSt@Cu 2-xThe light absorption of Se NPs gradually increases in the NIR-II region, and the LSPR peak redshifts from 808 nm in the NIR-I region to approximately 1010 nm in the NIR-II region. Figure 7 As shown in (c), the color of the product suspension changed from blue to green. The LSPR peak near 1010 nm originates from Cu. 2-x The light absorption of Se nanoparticles was investigated. Experimental results showed that by modulating Cu... 2-x The Se shell thickness was successfully achieved for AuSt@Cu. 2-x Precise regulation of the LSPR effect of Se nanoparticles, and the synthesis of AuSt@Cu from 0.6 mM SeO2. 2-x Se(#3) has the LSPR peak position closest to 1064 nm.

[0041] Then, select AuSt@Cu 2-x Se(#3) as a synthetic photothermal nanoprobe AuSt@Cu 2-x The Se@mAb (#4) conjugate was dispersed in water for testing after specific blocking was completed. No significant change was observed in the LSPR peak, indicating that the presence of the biological antibody does not affect the optical properties of the nanoparticles.

[0042] (4) AuSt@Cu 2-x Photothermal performance testing of Se nanoparticles First, AuSt@Cu with the best photothermal conversion effect was screened. 2-x Se nanoparticles: All suspensions were irradiated with 808 nm and 1064 nm lasers at a laser power density of 1.0 W / cm². 2 Irradiation was performed for 10 minutes, followed by cooling to ambient temperature. During the laser irradiation process, AuSt@Cu was recorded in real-time using a FLIR A35 infrared thermal imager. 2-x Temperature profile and thermal imaging of Se suspension.

[0043] Figure 8 This is a schematic diagram of the NIR-I PT-LFIA photothermal detection system in Embodiment 1 of the present invention; the system is constructed as follows: Figure 8 The photothermal detection system shown includes: Laser device; the laser device is used with AuSt@Cu 2-x The light source for the laser is a NIR-II laser whose absorption peak matches that of Se nanoparticles, NIR-II photothermal probes, or NIR-II PT-LFIA test paper. Laser head; Infrared thermal imager; Sample to be tested; The laser device is connected to the laser head and controls the laser head; the laser head is positioned correspondingly to the sample to be tested, and the laser is emitted onto the sample; the infrared thermal imager records the temperature curve and thermal image of the sample in real time. The test object is AuSt@Cu stored in a PE tube. 2-x Se suspension.

[0044] The experiment first examined the core-shell AuSt@Cu synthesized under different SeO2 concentrations. 2-x The photothermal conversion performance of Se NPs was evaluated. For example... Figure 9 As shown, AuSt@Cu synthesized under different SeO2 concentrations under 808 nm and 1064 nm laser irradiation (1.0 W / cm2) conditions. 2-x Se nanoparticles all exhibited significant concentration- and time-dependent photothermal conversion effects. With increasing nanoparticle concentration, the suspension heated up faster and reached a higher final saturation temperature.

[0045] To quantitatively evaluate the photothermal conversion capability of the material, the experiment monitored AuSt@Cu 2-x The heating and cooling temperatures of the SeNPs suspension were analyzed, and the photothermal conversion efficiency η was calculated based on the cooling curves and the final saturation temperature. The results are as follows: Figure 10 As shown. AuSt@Cu 2-x The concentration of Se NPs in the suspensions was quantified by ICP-OES analysis, and the Au concentration in each suspension was 0.03 mg / mL. With the increase of Cu... 2-x The increase in Se shell thickness, under 808 nm laser excitation, AuSt@Cu 2-x The η value of Se NPs increased from 21% to 37.2%, while under 1064 nm laser excitation, η increased from 29.1% to 52.8%. Photothermal performance test results indicate that thicker Cu... 2-x The Se shell can enhance AuSt@Cu 2-x Se's absorption and thermal conversion capabilities for near-infrared light, all AuSt@Cu 2-x Se NPs exhibited higher photothermal conversion efficiencies under 1064 nm laser excitation in the NIR-II window compared to those under 808 nm excitation in the NIR-I window. In contrast, pure AuSts showed photothermal conversion efficiencies of 15.4% and 28.4% under 1064 nm and 808 nm laser irradiation, respectively, significantly lower than AuSt@Cu. 2-x Se NPs. The reason is that the strong local electric field of AuSt excites Cu. 2-x The charge carriers in the Se shell oscillate strongly, and the hot electrons generated by Au are injected into Cu. 2-x The conduction band of Se promotes thermal radiation of charge carriers, thus enabling AuSt@Cu2- x The LSPR effect of Se exhibits a synergistic influence on photothermal conversion.

[0046] Experimental results show that AuSt@Cu synthesized from 0.06 mM SeO2 solution 2-x Se(#3) nanoparticles exhibit the highest photothermal conversion efficiency. Therefore, these nanoparticles were selected as the final photothermal material for the subsequent preparation of the photothermal probe.

[0047] To further explore AuSt@Cu 2-x The heating performance of Se NPs under NIR laser irradiation was investigated to screen for the optimal photothermal nanoprobe precursor. The synthesis of AuSt@Cu under 0.6 mM SeO2 conditions was experimentally tested. 2-x Photothermal heating curves of Se NPs ( Figure 11 ) and photothermal imaging ( Figure 12 As the laser irradiation time increased, the temperature of all suspensions gradually increased and reached a steady state within 10 minutes, and AuSt@Cu 2-x The heating temperature of Se NPs under 1064 nm laser irradiation is significantly higher than that under 808 nm laser irradiation.

[0048] Pure water (0 mg / mL) exhibited a steady-state temperature of only 25-28 °C after heating under 808 nm and 1064 nm laser irradiation. Under 808 nm laser irradiation, 1.0 mg / mL AuSt@Cu... 2-x The steady-state temperature of the Se suspension is around 60℃, while the temperature of both nanoparticles exceeds 70℃ under 1064 nm laser irradiation, exhibiting significant NIR-II photothermal heating characteristics.

[0049] In summary, this study developed a series of core-shell AuSt@Cu core-shell structures. 2-x Se nanomaterials exhibit excellent photothermal conversion effects, and AuSt@Cu synthesized under 0.6 mM SeO2 conditions... 2-x Se NPs exhibit stronger LSPR response characteristics in NIR-II than in NIR-I.

[0050] Example 2: Based on the synthesis of AuSt@Cu 2-x Se nanoparticles were used to prepare the NIR-II photothermal nanoprobe AuSt@Cu. 2-x Se@mAb includes the following steps: 1. Preparation of NIR-II photothermal probe AuSt@Cu 2-x Se@mAb Prepare a phosphate-buffered saline (PBS) buffer solution with a pH of 7.4 and a concentration of 0.01 M using NaH₂PO₄·2H₂O and NaH₂PO₄·12H₂O. Add 5 mL of 0.4 mg / mL AuSt@Cu... 2-x A suspension of Se nanoparticles was mixed with 20 μL of 60 mM 3-MPA aqueous solution in 4 mL of PBS pH 7.4 solution. After magnetic stirring for 2 hours, the solution was centrifuged to obtain 3-MPA-modified nanoparticles, namely AuSt@Cu. 2-x Se-3MPA was then redispersed in PBS pH 7.4. 0.2–1.0 mL of an aqueous solution of 4.0–10.0 mg / mL EDC / NHS (e.g., EDC to NHS molar ratio 1:1.0–1:2.0) was added, and the activation reaction was carried out at room temperature for 1 hour. After the reaction, the activated nanoparticles were centrifuged at 6000 rpm, washed with ultrapure water, and redispersed in 1 mL of PBS pH 7.4. The sST2 monoclonal antibody anti-ST2 mAb was thoroughly dissolved in sterile PBS (pH 7.4, 0.01 M), and then 160 μL of 0.25 mg / mL anti-ST2 mAb solution was added to the activated AuSt@Cu. 2-x Se-3MPA nanoparticles were mixed and incubated overnight at 4 ºC for coupling.

[0051] Subsequently, 1.0 mL of PBS solution containing 1.0 wt% BSA was added, and the mixture was shaken slowly on a shaker at room temperature for 1 hour. Finally, the photothermal nanoprobe AuSt@Cu 2-x Se@mAb was centrifuged at 3000 rpm, washed twice with ultrapure water, and redispersed in standard running buffer. It was then stored at 4 ºC and designated as the NIR-II photothermal probe.

[0052] The standard running buffer for the NIR-II photothermal probe is prepared from a 0.01 M PBS pH 7.4 solution containing 1.0 wt% BSA, 4.0 wt% sucrose, 0.1 wt% PVP, and 0.5 wt% Tween-20.

[0053] 2. The preparation of NIR-II PT-LFIA test strips based on NIR-II photothermal probes includes the following steps: Prepare sample pads, conjugate pads, NC membranes, and absorbent pads, and cut them into strips 30 cm long and 2.5 cm wide. Store them at room temperature, dry, and protected from light. Immerse the conjugate pads and sample pads in a mixture of 10 mL 0.01 M PBS, 1.0 wt% BSA, and 4 mL 0.5 wt% Tween-20 aqueous solution for 10 minutes. After discarding the solution, wash the pads twice with 10 mL ultrapure water and discard the water. Immerse the conjugate pads in 1.8 mL 0.8 mg / mL NIR-II photothermal probe solution for 10 minutes, then remove the conjugate pads. Subsequently, evenly spread a 1 mg / mL mouse anti-human sST2 polyclonal antibody (anti-ST2 pAb) solution and a goat anti-mouse IgG polyclonal antibody (anti-IgG Ab) solution in PBS onto the NC membrane using a multi-functional coating apparatus, serving as capture antibodies for the T and C lines, respectively. Dry all pads in a 37 °C oven for 3 hours.

[0054] The sample pad, conjugate pad, NC membrane, and absorbent pad are bonded to a PVC base plate, with a 1-2 mm overlap between each pad. The strips are then cut into 3 mm wide strips using a paper cutter, forming the NIR-II PT-LFIA detection platform. The test strips should be stored in a dry, sealed bag in a cool, dark place.

[0055] All nanomaterials used in the above experiments were synthesized in the laboratory. The sST2 protein and antibody were purchased from R&D Systems under the trade names: Recombinant human ST2 Fc, Human ST2 affinity purified polyclonal antibody (anti-ST2 pAb), and Human ST2 monoclonal antibody (anti-ST2mAb). These two antibodies recognize different epitopes of the sST2 protein, respectively. The goat anti-mouse IgG polyclonal antibody (anti-IgG Ab) was purchased from Sangon Biotech Co., Ltd.

[0056] 3. Construction of the NIR-II PT-LFIA Detection Platform Prepare sST2 sample solutions of different concentrations and use NIR-II PT-LFIA test strips to detect PBS solutions containing 0, 10, 20, and 200 ng / mL sST2. Detection is performed after the colors on the T and C lines have stabilized. The NIR-II PT-LFIA sST2 detection method generally includes the following steps: (1) Use NIR-II PT-LFIA test strips to perform chromatographic reactions on the samples; (2) Irradiate the test strip detection line area after reaction with a near-infrared II laser with a wavelength of 1000-1700 nm; (3) Detect the temperature change in the detection line area due to the photothermal effect, and quantify the concentration of sST2 in the sample based on the temperature change value.

[0057] A FLIR A35 infrared thermal imager was used to monitor thermal signals. Connected to a portable computer, a 1064 nm laser was used to irradiate the NC film of the test strip for thermal signal analysis. Figure 13 As shown. The actual device diagram and... Figure 8 Similar, the difference lies in that the photothermal detection object is the NIR-II PT-LFIA test paper.

[0058] To address the issue of thermal radiation affecting the thermal background signal analysis of the blank NC film via LFIA, the experiment investigated background thermal interference by testing the steady-state temperature at different locations on the LFIA detection film. Lasers were used to irradiate designated areas of the T-line, C-line, and NC film, as shown in the example below. Figure 14 As shown in (ab).

[0059] The results showed that as the concentration of the sST2 standard sample increased, the color intensity of the C line of the NIR-II PT-LFIA test strip remained almost unchanged, while the color intensity of the T line increased significantly. Meanwhile, based on the analysis of the photothermal heating test results... Figure 14 (cd) Under 1064 nm laser irradiation, the temperature of line C was significantly higher than that under 808 nm laser irradiation, indicating that the PT-LFIA test strip has better photothermal signal conversion performance in the NIR-II window. Simultaneously, the steady-state absolute temperature T and temperature difference ΔT after laser irradiation of the T line and NC film region were analyzed, and the average and standard deviation of three experiments were calculated. It was observed that compared to the absolute temperature TT-line value of the T line, the ΔT signal is more sensitive to the concentration response of the target molecule sST2. Therefore, the experiment was conducted using... T = TT-line - TNC, as the detection signal of the NIR-II PT-LFIA test strip, is more conducive to improving the detection sensitivity of the test strip.

[0060] To systematically evaluate the quantitative analysis performance of the NIR-II PT-LFIA detection platform, it is necessary to examine the accuracy and sensitivity of its colorimetric and photothermal modes. By detecting a series of standard sST2 sample solutions at different concentrations, the curves showing the change in T-line grayscale value in the colorimetric method and the ΔT signal value in the photothermal mode as a function of sST2 concentration are analyzed to form a dual-modal quantitative calibration analysis curve, providing a signal analysis model for the practical application of NIR-II PT-LFIA. Based on the sigmoidal logistic four-parameter equation, the curve of the detection signal changing with antigen concentration can be simulated as an S-shaped curve, and its calculation formula is as follows: (1) in, and These are the signal values ​​corresponding to the maximum asymptote and the minimum asymptote, respectively. The inflection point concentration represents the half-maximal effective concentration (EC50). Let be the Hill slope, and be the slope factor of the curve. For sST2 concentration, This represents the LFIA response signal value. The p-value reflects the sensitivity of the response signal value to changes in concentration; the larger the absolute value of the p-value, the steeper the curve.

[0061] In LFIA biosensors, the limit of detection (LOD) refers to the lowest target analyte concentration that can distinguish between background and response signals, typically with a confidence level of 99%. Geometrically, the LOD is the concentration value at the intersection of the LOD line and the response curve. The LOD can be defined by the following formula: (1) Where x and δ are the mean and standard deviation of the blank sample detection signal obtained from six independent tests (n=6), respectively.

[0062] Furthermore, by calculating the percentage of the measured average of three independent experiments to the theoretical value of sample addition, the recovery rate and relative standard deviation (RSD) of the standard sample detection can be analyzed to verify the accuracy and precision of the calibration curve analysis method. The calculation formula is as follows: (1) (2) Generally speaking, the recovery rate is used to measure how close the measured value is to the true value, and the optimal range is 90%-110%. The relative standard deviation (RSD) is used to measure the dispersion of repeated measurement results. LFIA biosensors with an RSD ≤ 10% exhibit better precision detection performance.

[0063] In this experiment, 200 μL of standard sST2 solutions of various concentrations were prepared using 0.01M pH 7.4 PBS buffer to achieve concentrations of 0, 0.1, 1, 20, 100, 250, 400, 600, 800, 1000, 2000, 3000, and 5000 ng / mL. NIR-II PT-LFIA test strips prepared in the same batch were used for testing. The test strips were irradiated with a 1064 nm laser for 2 minutes at a laser power density of 1.0 W / cm². 60 μL of the standard sample solution was added to the sample pad, followed by chromatography for 30 minutes to stabilize the signal. Each concentration point was tested three times independently. The T-line grayscale value of the NIR-II PT-LFIA detection bands was analyzed using ImageJ software. The T-line and NC membrane temperatures were monitored using a FLIR A35 thermal imager, and the ΔT signal curve during the temperature saturation phase was recorded. The trend of the detection signal with the change of sST2 concentration was analyzed, and calibration analysis curves for the full response range and linear analysis curves for the intermediate response range were fitted. Each concentration point was tested three times independently, and all results are expressed as Mean ± SD (n=3).

[0064] To verify the responsive detection capability of NIR-II PT-LFIA to sST2 antigen, the microstructure of the T-lines formed in 0, 200, and 800 ng / mL sST2 samples was analyzed using SEM, and the distribution of the nanoprobes within them was observed. The characterization results are as follows: Figure 15 As shown in (a), the test strip showed a negative result with a blank sample (0 ng / mL sST2), with the C line showing color but the T line remaining colorless. Furthermore, the T line region lacked fine nanoparticles, and the NC membrane exhibited a porous structure and a relatively smooth surface. The results at 200 and 800 ng / mL sST2 were positive, with both the T and C lines showing color, and the T line at 800 ng / mL sST2 showing a significantly deeper color. SEM characterization analysis revealed that the high-concentration sample resulted in a significantly higher enrichment of nanoparticles at the T line compared to the low-concentration sST2, and this nanoparticle enrichment caused a rougher surface to the nitrocellulose pore structure in the T line region. Therefore, SEM characterization effectively validated the analytical performance of the NIR-II PT-LFIA test strip for sST2 samples.

[0065] The experiment used a set of standard sST2 solutions with concentrations ranging from 0-5000 ng / mL for detection, and the detection results were obtained by... Figure 15 (b) indicates that, obviously, as the concentration of sST2 increases, the color of the T line and the ΔT signal gradually deepen, while the color and ΔT of the C line do not change significantly.

[0066] Figure 15(c) and (d) show the calibration curves and corresponding linear fitting curves under CL-LFIA and PT-LFIA detection modes, respectively (see inset). As shown in the figure, the extremely low concentration (0-10 ng / mL) and extremely high concentration (2000-5000 ng / mL) ranges constitute the plateau region for NIR-Ⅱ PT-LFIA dual-modal detection. Using Origin 2019b software, the signal response curves across the entire concentration range were fitted. Based on the Sigmoidal logistic four-parameter equation model, the Gray value-sST2 concentration calibration formula for CL-LFIA detection was obtained as follows: (R²=0.9868), the calibration formula for ΔT -sST2 concentration detected by PT-LFIA is as follows: (R² = 0.9904). Within the sST2 concentration range of 100-1000 ng / mL, the gray value detected by CL-LFIA showed a good linear relationship with the sST2 concentration, and the regression equation was as follows: (R² = 0.9913). PT-LFIA exhibits a wider linear range than CL-LFIA, within the ST2 concentration range of 20-1000 ng / mL, with the regression equation being... (R² = 0.9915). Simultaneously, the colorimetric and photothermal signals of six blank samples were analyzed. According to the calculation formula, the limit of detection (LOD) for LODCL-LFIA in colorimetric mode was 29.4 ng / mL, while the limit of detection (LOD) for LODPT-LFIA in photothermal mode was as low as 5.6 ng / mL. The results indicate that the sensitivity of PT-LFIA is significantly superior to that of the CL-LFIA method, with a detection limit approximately five times higher, better meeting the detection needs of low-abundance sST2 in clinical samples.

[0067] Table 2 Sample response rate and relative standard deviation of NIR-II PT-LFIA dual-modal detection

[0068] Based on the quantitative detection and curve fitting results of the NIR-II PT-LFIA method, this study evaluated the accuracy and sensitivity of the dual-modal detection method by calculating the response rate and relative standard deviation of the sST2 standard samples. The results are shown in Table 2. Four sST2 standard samples with signal response values ​​in the middle of the S-shaped calibration curve were selected for calculation: 250, 400, 800, and 1000 ng / mL sST2. Analysis of Table 2 shows that the 400 ng / mL sST2 solution had the highest response rate among the four samples, at 110.1% in the PT-LFIA (NIR-II) detection mode and 113.2% in the CL-LFIA mode. However, the response rates at other concentrations were all within the range of 90%-110%. Furthermore, the analysis results indicate that for the same sample detection, the response rate of PT-LFIA (NIR-II) is lower than that of the CL-LFIA detection mode, while the RSD values ​​of both detection methods are within the normal range.

[0069] In summary, this invention successfully constructed a lateral flow immunochromatographic detection platform based on the NIR-II photothermal effect for the sensitive quantitative detection of serum sST2.

[0070] First, AuSt@Cu was synthesized. 2-x Se core-shell nanoparticles, used as photothermal probes, exhibit strong absorption characteristics of the LSPR effect in the NIR-II window. Through 3-MPA modification, EDC / NHS activation, and antibody conjugation, an sST2 monoclonal antibody (anti-ST2 mAb) was successfully covalently linked to the nanoparticle surface. Non-specific sites were then blocked using BSA, thus constructing an NIR-II photothermal probe with good biocompatibility and stability.

[0071] Secondly, the pretreatment and assembly of each component of the LFIA test strip were completed, including optimization of the sample pad, conjugate pad, NC membrane, and absorbent pad. sST2 polyclonal antibody (anti-ST2 pAb) and goat anti-mouse IgG antibody were immobilized on the NC membrane as the detection line (T line) and control line (C line), respectively. By loading photothermal probes onto the conjugate pad, a complete NIR-II PT-LFA detection platform was constructed.

[0072] The platform achieves a colorimetric detection limit of 29.4 ng / mL for sST2 in serum, while the detection limit for photothermal quantitative detection based on 1064 nm laser excitation reaches 5.6 ng / mL, demonstrating a significant improvement in sensitivity. This detection limit matches the clinical risk stratification threshold for heart failure (low risk <35 ng / mL), meeting the needs of early screening. Furthermore, the platform exhibits excellent performance in terms of specificity, stability, and repeatability, providing reliable technical support for subsequent clinical sample validation.

Claims

1. An AuSt@Cu 2-x Se nanoparticles, characterized in that, The nanoparticles exhibit a core-shell structure, with the core being AuSt gold nanoparticles and the shell being Cu. 2-x Se shell, where the value of x is in the range of 0 < x < 1; The core structure of the nanoparticles has a particle size of 63.5 ± 7.6 nm, and the particle size of the nanoparticles is 65.2 ± 7.3-92.1 ± 7.2 nm.

2. The nanoparticles as described in claim 1, characterized in that, The AuSt@Cu 2-x The Au content in the Se nanoparticles is 34.9%, and the Cu content is... 2-x The Se content is 65.1%.

3. An AuSt@Cu 2-x The method for preparing Se nanoparticles is characterized by, Includes the following steps: (1) Synthesis of AuSt gold nanoparticles: Prepare an aqueous solution mixture with HAuCl4·4H2O and C6H5Na3O7·2H2O as solutes, prepare a NaBH4 solution with C6H5Na3O7·2H2O as solvent, slowly add the NaBH4 solution to the mixed solution of HAuCl4·4H2O and C6H5Na3O7·2H2O, stir, and obtain an aqueous solution of gold nanoseeds; PVP was dissolved in DMF, and an aqueous solution of HAuCl4·4H2O was added. Then, an aqueous solution of gold nanoparticles was added, and the mixture was stirred at room temperature for 2-3 hours. After centrifugation and washing, AuSt gold nanoparticles were obtained. The obtained AuSt gold nanoparticles were dispersed in deionized water for storage. (2) Synthesis of AuSt@Cu 2-x Se nanoparticles: A dispersion of AuSt gold nanoparticles was mixed with a PVP aqueous solution and heated. An AA aqueous solution was then added, followed immediately by dropwise addition of a SeO2 aqueous solution, while maintaining a constant temperature and stirring. Next, a CuSO4·5H2O aqueous solution and the AA aqueous solution were added to initiate the reaction. After the reaction was complete, the mixture was centrifuged and washed to obtain AuSt@Cu. 2-x Se nanoparticles.

4. The preparation method according to claim 3, characterized in that, The concentration of HAuCl4·4H2O in the aqueous solution mixture described in step (1) is 0.5-0.6 mM, and the ratio of the concentration of HAuCl4·4H2O to the concentration of C6H5Na3O7·2H2O is 1:1.5-1:1.7; the amount of NaBH4 solution added is 1.0-1.2 mL of 0.06-0.09 wt% NaBH4 solution added to every 100 mL of the mixed solution of HAuCl4·4H2O and C6H5Na3O7·2H2O; the concentration of the gold nanoseed aqueous solution is 0.4-0.7 nM; the stirring time described in step (1) is 10-12 h; The concentration of PVP is 8-10 mM, and the amount of HAuCl4·4H2O aqueous solution added is 0.005-0.006 mL of 50-60 mM HAuCl4·4H2O aqueous solution added per 1 mL of PVP solution; the amount of gold nano-seed aqueous solution added is 0.001-0.008 mL of gold nano-seed aqueous solution added per 1 mL of PVP solution. The concentration of the AuSt gold nanoparticle dispersion in step (2) is 0.5-1.0 mg / mL; the mass ratio of AuSt gold nanoparticles to PVP in the AuSt gold nanoparticle dispersion and PVP aqueous solution is 1:40-1:50; the amount of AA aqueous solution added is 0.1-0.4 mL of 90-120 mM AA aqueous solution per 1 mL of AuSt gold nanoparticle solution; the amount of SeO2 aqueous solution added is 0.15-0.6 mL of 0.01-0.8 mM SeO2 aqueous solution per 1 mL of AuSt gold nanoparticle solution; the mixing and heating temperature in step (2) is 40℃; the constant temperature stirring time is 15-20 min; and the reaction time is 14-18 h.

5. An AuSt@Cu 2-x The application of Se nanoparticles is characterized by... The AuSt@Cu 2-x Se nanoparticles were used to synthesize NIR-II photothermal probes, the LSPR peak of which is located in the NIR-II region at 1000-1100 nm.

6. The application as described in claim 5, characterized in that, The method for synthesizing the NIR-II photothermal probe includes: S1. Prepare PBS buffer, add AuSt@Cu 2-x An aqueous suspension of Se nanoparticles was added to a PBS solution, mixed and stirred, and centrifuged to obtain AuSt@Cu. 2-x Se-3MPA, AuSt@Cu 2-x Se-3MPA was dispersed in PBS, and an aqueous solution of EDC / NHS was added. The activation reaction was carried out at room temperature for 0.5-1 h. After the reaction was completed, the mixture was centrifuged, washed, and redispersed to obtain the activated AuSt@Cu. 2-x Se-3MPA; S2. Dissolve the sST2 monoclonal antibody anti-ST2 mAb and mix it with the activated AuSt@Cu. 2-x Se-3MPA was mixed and incubated, then PBS solution containing BSA was added, and the mixture was shaken at low speed on a shaker at room temperature. After reaction, centrifugation and washing were performed to obtain the NIR-II photothermal probe.

7. The application as described in claim 6, characterized in that, The pH of the PBS buffer in step S1 is 7.2-7.8, and the AuSt@Cu... 2-x The concentration of the Se nanoparticle aqueous suspension is 0.2-1.0 mg / mL; the concentration of the 3-MPA aqueous solution is 40-100 mM; the AuSt@Cu 2-x The volume ratio of the aqueous suspension of Se nanoparticles to the aqueous solution of 3-MPA is 50:1-250:1; the molar ratio of EDC to NHS in the EDC / NHS aqueous solution is 1:1.0-1:2.0, and the concentration of the EDC / NHS aqueous solution is 4.0-10.0 mg / mL; the aqueous solution of EDC / NHS and AuSt@Cu 2-x The volume ratio of the Se nanoparticle aqueous suspension is 1:5-1:25; The sST2 monoclonal antibody anti-ST2 mAb was dissolved in sterile PBS solution at a concentration of 0.2-2.0 mg / mL.

8. A NIR-II PT-LFIA test strip, characterized in that, The NIR-II PT-LFIA test strip comprises a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; the conjugate pad and the sample pad are immersed and washed in a standard running buffer, and the conjugate pad is immersed in the NIR-II photothermal probe solution in any of the applications described in claims 5-7; the NC membrane is coated with a detection line and a control line, and the sample is coated on the sample pad. The sample pad, conjugate pad, NC membrane and absorbent pad are bonded to the PVC base plate, with a 1-2 mm overlap area between each pad, and the NIR-II PT-LFIA test paper is cut to obtain the NIR-II PT-LFIA test paper. The standard running buffer is prepared from a 0.01 M PBS solution at pH 7.2-7.8, containing 0.1-1.0 wt% BSA, 1.0-10.0 wt% sucrose, 0.01-0.1 wt% PVP, and 0.1-1.0 wt% Tween-20.

9. A method for detecting sST2 in NIR-II PT-LFIA, characterized in that, Includes the following steps: (1) Perform a chromatographic reaction on the sample using the NIR-II PT-LFIA test strip as described in claim 8; (2) Irradiate the test strip detection line area after reaction with a near-infrared II laser with a wavelength of 1000-1700 nm; (3) Detect the temperature change in the detection line area due to the photothermal effect, and quantify the concentration of sST2 in the sample based on the temperature change value.

10. A photothermal detection system, characterized in that, The detection system includes: Laser device; the laser device uses the AuSt@Cu laser as described in any one of claims 1-2. 2-x Se nanoparticles or AuSt@Cu prepared by the preparation method according to any one of claims 3-4 2-x The laser irradiation source is a NIR-II laser that matches the absorption peak of the Se nanoparticles, or the NIR-II photothermal probe as described in any one of claims 5-7, or the NIR-II PT-LFIA test paper as described in claim 8. Laser head; Infrared thermal imager; Sample to be tested; The laser device is connected to the laser head and controls the laser head; the laser head is set to correspond to the sample to be tested and emits laser light onto the sample to be tested; the infrared thermal imager records the temperature curve and thermal image of the sample to be tested in real time.

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