Low-potential electrochemiluminescence immunosensor based on scandium-gold nanocluster / DIPEA as well as construction method and application of low-potential electrochemiluminescence immunosensor

Abstract

The invention relates to the technical field of electrochemiluminescence immunosensors, in particular to a low-potential electrochemiluminescence immunosensor based on scandium-gold nanocluster / DIPEA as well as a construction method and application of the low-potential electrochemiluminescence immunosensor. The construction method comprises the following steps: sequentially carrying out polishing, washing and activation pretreatment on a working electrode, dropwise adding an Ab1 solution to the surface of the electrode, and carrying out constant-temperature incubation; closing the Ab1 / AuE electrode by adopting BSA (Bovine Serum Albumin); dropwise adding an AD7c-NTP antigen solution into the BSA / Ab1 / AuE electrode, and carrying out constant-temperature incubation; an Ab2-Sc-coated AuNC solution is dropwise added to the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode, constant-temperature incubation is carried out, Ab2-Sc-coated AuNC / AD7c-NTP / BSA / Ab1 / AuE is formed, and the high-sensitivity electrochemical luminescence immunosensor is formed by taking Ab2-Sc-coated AuNC as a marker through a sandwich immunization method. The invention develops an electrochemical luminescence immunosensing platform with low excitation potential and high sensitivity, realizes noninvasive, rapid, low-cost, wide-detection-range and high-sensitivity detection of the urine marker (AD7c-NTP) of the Alzheimer's disease, and overcomes the problems of oxidative damage and background interference of high-potential ECL.

Description

Technical Field

[0001] This application relates to the field of electrochemiluminescence immunosensor technology, and in particular to a method for constructing and applying a low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA. Background Technology

[0002] With the continued rise in the proportion of the elderly population globally, the problem of age-related dementia is becoming increasingly prominent. The World Health Organization projects that 139 million people worldwide will have dementia by 2050, placing enormous pressure on individuals and socioeconomic systems. Among these, Alzheimer's disease (AD), with its irreversible chronic central nervous system degeneration, poses a significant challenge to global public health. Developing and implementing effective early AD screening strategies is urgently needed to improve people's quality of life and lifespan.

[0003] Currently, early screening for Alzheimer's disease (AD) mainly relies on positron emission tomography (PET), magnetic resonance imaging (MRI), and cerebrospinal fluid (CSF) testing. However, these methods have limitations: PET / MRI is expensive and difficult to popularize; CSF testing requires a CSF sample, is highly invasive, and has poor patient compliance. In contrast, the recently emerging blood tests for AD have been a research hotspot in the field of AD diagnosis due to their advantages of being less invasive, simpler to perform, and more economical. However, blood tests are still invasive and still suffer from poor patient compliance.

[0004] Furthermore, blood protein homeostasis mechanisms influence biomarker accumulation. Drastic changes in protein levels in cerebrospinal fluid and blood trigger homeostatic regulatory mechanisms in the body to maintain internal environmental stability, making it difficult for proteins to accumulate persistently in cerebrospinal fluid and blood. In contrast, urine, derived from blood, lacks this mechanism and can accumulate and tolerate considerable changes without harming the body. This characteristic makes urine a better source of biomarkers compared to other bodily fluids. Non-invasive urine testing for Alzheimer's disease (AD) is easy to perform, has higher patient compliance, and is more suitable for early clinical screening of the AD population. Alzheimer-associated neuronal thread protein (AD7c-NTP) is a transmembrane phosphoprotein expressed in the cell body of neurons. Numerous studies have shown that AD7c-NTP is an AD biomarker with specifically elevated expression levels in the brain tissue, cerebrospinal fluid, and urine of AD patients.

[0005] AD7c-NTP is a transmembrane protein that can easily cross the cell barrier. With a molecular weight of only 41 kDa and an isoelectric point of 9.89, it is a basic protein that binds tightly to most acidic plasma proteins in the blood. Upon reaching the kidneys, due to a layer of negatively charged viscous glycoproteins on the glomerular basement membrane, the positively charged AD7c-NTP is concentrated in the primary urine via ultrafiltration. The concentration of AD7c-NTP protein in urine is stable, making its detection relatively accurate and reliable. Since Monte et al. first reported AD7c-NTP in 1997 (The Journal of Clinical Investigation, 100.12: 3093-3104.), and after multi-center clinical validation (Journal of Clinical Laboratory Analysis, 12.5: 285-288; American Journal of Alzheimer's Disease and Other Dementias, 35;), the sensitivity and specificity of specifically assessing AD7c-NTP in the urine of AD patients are comparable to those of CSF. Therefore, the concentration of AD7c-NTP in urine has been recommended as a practical tool for diagnosing AD.

[0006] Traditional methods for detecting AD7c-NTP include in situ hybridization, immunoradiography, and enzyme-linked immunosorbent assay (ELISA). While these methods can detect AD7c-NTP protein in urine, they are complex, time-consuming, costly, and require specialized technicians. Electrochemiluminescence (ECL) has gained significant attention in clinical diagnostics, environmental monitoring, and food safety due to its advantages of low background, high sensitivity, and simple equipment. Electrochemiluminescence immunoassay (ECLIA) has become an important scientific tool for clinical diagnostics, food testing, and environmental pollution monitoring.

[0007] Traditional ECL systems typically consist of Ru(bpy)3 2+ The composition of tripropylamine (TPA) requires a relatively high positive ECL potential (typically around 1.2 V) to excite Ru(bpy)3. 2+ The tripropylamine (TPA) system produces a strong ECL. Recently, Liang et al. (Analytical Chemistry, 95.6: 3434-3441) reported a dry chemistry-based bipolar electrochemiluminescence detection method for AD7c-NTP, which exhibits high sensitivity. However, the chip fabrication steps used in this method are relatively complex, and it suffers from high signal background due to high driving voltage. Furthermore, when the excitation potential is too high, the antibody undergoes irreversible oxidative damage, affecting the specific binding of the antigen and antibody.

[0008] Among numerous luminescent organisms, gold nanoclusters (AuNCs) possess excellent stability, tunable luminescence properties, and superior biocompatibility, holding a prominent position among metal nanoclusters and representing a newly emerging ECL probe in recent years. In particular, high-performance AuNC probes employing different strategies for low-potential ECL mechanisms have attracted considerable attention from researchers. For example, Wei et al. (Sensors and Actuators B: Chemical, 367: 132034-132034.) designed an ion-doping strategy to synthesize Co-AuNCs, reducing the potential by 0.2 V and achieving ECL luminescence at 0.95 V, thus making a preliminary exploration of low-potential ECL. However, achieving low-potential AuNC ECLIA remains a significant challenge.

[0009] In summary, existing early AD screening programs have the following limitations: Although existing methods for detecting AD7c-NTP protein in urine (such as enzyme-linked immunosorbent assay) are publicly available, these methods are complex, time-consuming, and require improved sensitivity, and also require professional operation. Liang et al. (Anal. Chem. 2023, 95, 3434−3441) reported a dry chemistry-based bipolar electrochemiluminescence detection method for AD7c-NTP, which exhibits high sensitivity. However, the chip fabrication steps used in this method are relatively cumbersome, and it suffers from high signal background due to high driving voltage. Furthermore, when the excitation potential is too high, the antibody undergoes irreversible oxidative damage, affecting the specific binding of the antigen and antibody. Additionally, the use of traditional bovine serum albumin (BSA) gold clusters for detecting AD7c-NTP protein in urine suffers from low ECL efficiency, resulting in low sensitivity for detection methods using BSA gold clusters as ECL probes.

[0010] In addition, existing methods for detecting Alzheimer's disease-associated neurofilament protein (AD7c-NTP) in urine have the following shortcomings: Main drawbacks: Existing ECL technology has an excessively high excitation potential (≥1.2 V), which leads to irreversible oxidation of antibodies and affects the specificity of antigen-antibody binding; in addition, traditional bovine serum albumin gold nanoclusters ECL has low efficiency, resulting in low detection sensitivity.

[0011] Secondary drawbacks: Traditional AD7c-NTP detection methods involve complex high-potential ECL chip fabrication processes (such as the Liang et al. scheme in the literature) and high costs.

[0012] Therefore, how to develop a low-excitation-potential, high-sensitivity electrochemiluminescence immunosensing platform using simple methods to achieve non-invasive, rapid, and low-cost detection of Alzheimer's disease urine biomarkers (AD7c-NTPs) and overcome the oxidative damage and background interference problems of high-potential ECLs is precisely the problem that those skilled in the art are committed to solving. Summary of the Invention

[0013] To address the aforementioned technical problems in the prior art, this application develops a low-excitation-potential, high-sensitivity electrochemiluminescence immunosensing platform, enabling non-invasive, rapid, highly sensitive, and low-cost detection of Alzheimer's disease urinary biomarker (AD7c-NTP), overcoming the oxidative damage and background interference problems associated with high-potential ECL. The technical solution of this application is as follows: The method for constructing a low-potential electrochemiluminescence immunosensor based on scandium gold nanoclusters / DIPEA provided in this application includes the following steps: This application provides a method for constructing a low-potential electrochemiluminescence immunosensor based on scandium gold nanoclusters / DIPEA, which includes the following steps: S10. After polishing, washing, and activation pretreatment of the working electrode, add Ab1 solution to the electrode surface and incubate at a constant temperature to form an Ab1 / AuE electrode; block the Ab1 / AuE electrode with BSA to form a BSA / Ab1 / AuE electrode; add AD7c-NTP antigen solution to the BSA / Ab1 / AuE electrode and incubate at a constant temperature to form an AD7c-NTP / BSA / Ab1 / AuE electrode. S20. Add the Ab2-Sc@AuNC solution to the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubate at a constant temperature to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. Construct a highly sensitive electrochemiluminescent immunosensor using Ab2-Sc@AuNC as a signal probe via a sandwich immunoassay.

[0014] In some embodiments, the preparation process of Ab2-Sc@AuNC is as follows: Sc@AuNC is prepared using HAuCl4∙3H2O as the gold ion source, scandium trichloride as the scandium ion source, and sodium hydroxide as the reducing agent; then, phosphate buffer containing EDC and NHS is added to the Sc@AuNC and incubated at a constant temperature; then, Ab1 solution is added, incubated at a constant temperature, and separated; BSA is used for blocking to obtain the Ab2-Sc@AuNC bioconjugate; finally, the Ab2-Sc@AuNC bioconjugate is separated, washed, and dispersed to obtain the final product.

[0015] In some embodiments, the preparation process of Sc@AuNC is as follows: chloroauric acid solution, scandium trichloride solution, and NaOH solution are added to BSA solution and reacted at 35–39°C for 12 hours or more; the crude product is dialyzed and purified to obtain ultra-bright red fluorescent water-soluble gold nanoclusters Sc@AuNC; wherein the molar ratio of the chloroauric acid solution, scandium trichloride solution, NaOH solution, and BSA solution is (8–12):(4–6):(0.5–1.5):(8–12); the concentration of the NaOH solution is 0.5–1.5 M. Preferably, the reaction is carried out at 35–39°C for 12–24 hours. Preferably, the reaction is carried out at 35–39°C for 12 hours, the molar ratio of scandium trichloride solution to chloroauric acid solution is 0.5, and the concentration of NaOH solution is 1.0 M.

[0016] In some embodiments, the synthesis process of the Ab2-Sc@AuNC solution includes the following steps: 1) Adding a phosphate buffer containing 15-25 mg / mL EDC and NHS to Sc@AuNC and incubating at 4°C or 25°C for 0.5-4 hours; 2) Adding an Ab2 solution with a concentration of 50-500 μg / mL and incubating with shaking at 3.5-4.5°C for 10-14 hours; centrifuging the mixture again to remove unbound EDC, NHS, and Ab2; then dispersing the precipitate with 0.1% BSA solution and incubating for 30 minutes to block any non-specific binding sites; centrifuging the Ab2-Sc@AuNC bioconjugate, washing it multiple times with phosphate buffer solution, and dispersing it in a phosphate buffer solution for dispersion at pH 7.4; wherein the volume ratio of the phosphate buffer solution containing 15-25 mg / mL EDC and NHS, the Ab2 solution, and the phosphate buffer solution for dispersion is 0.1:1:1; In some embodiments, the preparation steps of Sc@AuNC are as follows: 8–12 mM chloroauric acid solution, 4–6 mM scandium trichloride solution, and 0.5–1.5 M NaOH solution are added to 8–12 mM BSA solution under vigorous stirring, and the reaction is carried out at 35–39 °C for 12 hours or more; the crude product is dialyzed and purified to obtain ultra-bright red fluorescent water-soluble gold nanoclusters Sc@AuNC; the volume ratio of the chloroauric acid solution, scandium trichloride solution, NaOH solution, and BSA solution is 1:1:0.5:1.

[0017] In some embodiments, the method includes the following steps: S10, preparing an AD7c-NTP / BSA / Ab1 / AuE electrode: S11, the working electrode is sequentially polished, washed, and activated pretreated, and then cleaned and dried; S12, Ab1 solution is dropped onto the electrode surface and incubated at 2-6°C for 6-12 hours to form an Ab1 / AuE electrode, and then cleaned and dried; wherein, the concentration of the Ab1 solution is 50-500 μg / mL; S13, after blocking the unbound active sites on the Ab1 / AuE electrode with BSA, it is incubated at 24-26°C to form a BSA / Ab1 / AuE electrode, and then cleaned and dried; S14, the BSA / AD7c-NTP antigen solution was added to the Ab1 / AuE electrode and incubated at 35-39°C for 45 minutes to 2 hours to form the AD7c-NTP / BSA / Ab1 / AuE electrode. The electrode was then washed and dried. S20: Preparation of electrochemiluminescence immunosensor: Ab2-Sc@AuNC solution was added to the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubated at 35-39°C to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. The electrode was then washed and dried. A highly sensitive electrochemiluminescence immunosensor was formed using Ab2-Sc@AuNC as a marker via a sandwich immunoassay.

[0018] In some embodiments, in step S11, the working electrode is thoroughly polished using alumina powder with a particle size of 0.3 μm and 0.05 μm in sequence; then, the surface of the working electrode is ultrasonically cleaned for 1–5 minutes using ultrapure water, anhydrous ethanol, and ultrapure water in sequence; then, the working electrode is activated using cyclic voltammetry with 0.5 M sulfuric acid solution as electrolyte until a stable redox peak appears; the working electrode is then rinsed clean with ultrapure water and finally dried with nitrogen gas; wherein, the working electrode is a gold electrode; the activation parameters are: a scan potential range of -0.2 V to 1.6 V and a scan rate of 0.1 V / s.

[0019] In some embodiments, in step S12, an Ab1 solution is dropped onto the electrode surface, and the electrode is incubated at a constant temperature of 2–6°C for 6–12 hours to form an Ab1 / AuE electrode. After thorough rinsing with pure water, the electrode is dried with nitrogen gas. The concentration of the Ab1 solution is 50–500 μg / mL, and its volume is 5–15 μl. Preferably, the concentration of the Ab1 solution is 200–500 μg / mL.

[0020] In some embodiments, in step S13, after blocking the unbound active sites on the Ab1 / AuE electrode with BSA, the electrode is incubated at a constant temperature of 23–27°C for 0.5–1 hour to form a BSA / Ab1 / AuE electrode. After thorough rinsing with ultrapure water, the electrode is dried with nitrogen gas. The concentration of the BSA solution is 0.4%–0.6%, and its volume is 5–15 μl.

[0021] In some embodiments, in step S14, AD7c-NTP antigen solution is added dropwise to the BSA / Ab1 / AuE electrode, and the electrode is incubated at a constant temperature of 35-39°C for 45 minutes to 2 hours to form an AD7c-NTP / BSA / Ab1 / AuE electrode. After thorough rinsing with ultrapure water, the electrode is dried with nitrogen gas. The concentration of the AD7c-NTP antigen solution is 1 pg / mL to 100 ng / mL, and its volume is 5-15 μl.

[0022] In some embodiments, in step S20, the Ab2-Sc@AuNC solution is dropped onto the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubated at a constant temperature of 35–39°C for 1–2 hours to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. After thorough rinsing with ultrapure water, the electrode is dried with nitrogen. The concentration of the Ab2-Sc@AuNC solution is 50–500 μg / mL, and its volume is 5–15 μl.

[0023] In some embodiments, the following steps are included: S10. Preparation of AD7c-NTP / BSA / Ab1 / AuE electrode: S11. The gold electrode is thoroughly polished using alumina powder with a particle size of 0.3 μm and 0.05 μm in sequence; then the surface of the working electrode is ultrasonically cleaned using ultrapure water, anhydrous ethanol, and ultrapure water in sequence; then, the working electrode is activated using cyclic voltammetry with 0.5 M sulfuric acid aqueous solution as electrolyte until a stable redox peak appears; the working electrode is then rinsed clean with ultrapure water and finally dried with nitrogen gas; wherein, the activation parameters are: scan potential range of -0.2 V to 1.6 V, scan rate of 0.1 V / s; S12. Add Ab1 solution to the electrode surface and incubate at 4℃ for 8 hours to form an Ab1 / AuE electrode. Rinse thoroughly with pure water and then dry with nitrogen. The concentration of Ab1 solution is 200 μg / mL and the volume is 7 μl. S13. After blocking the unbound active sites on the Ab1 / AuE electrode with BSA, incubate at 25°C for 1 hour to form a BSA / Ab1 / AuE electrode. Rinse thoroughly with ultrapure water and dry with nitrogen. The concentration of the BSA solution is 0.5% and its volume is 7 μl. S14. Add AD7c-NTP antigen solution to the BSA / Ab1 / AuE electrode, incubate at 37°C for 45 minutes to form AD7c-NTP / BSA / Ab1 / AuE electrode, rinse thoroughly with ultrapure water and dry with nitrogen. S20. Preparation of electrochemiluminescence immunosensor: The Ab2-Sc@AuNC solution was dropped onto the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubated at 37°C for 1 hour to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. After thorough rinsing with ultrapure water, the electrode was dried with nitrogen. The concentration of the Ab2-Sc@AuNC solution was 200 μg / mL and its volume was 7 μl.

[0024] 8. A low-potential electrochemiluminescence immunosensor based on scandium gold nanoclusters / DIPEA, characterized in that: it is prepared by adding Ab2-Sc@AuNC solution to the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubating at a constant temperature; wherein, the preparation process of Ab2-Sc@AuNC is as follows: Sc@AuNC is prepared by using HAuCl4∙3H2O as the gold ion source, scandium trichloride as the scandium ion source, and sodium hydroxide as the reducing agent; then, phosphate buffer containing EDC and NHS is added to the Sc@AuNC and incubated at a constant temperature; then, Ab1 solution is added, and the mixture is incubated at a constant temperature and separated; BSA is used for blocking to obtain the Ab2-Sc@AuNC bioconjugate; finally, the Ab2-Sc@AuNC bioconjugate is separated, washed, and dispersed to obtain the final product.

[0025] In some embodiments, the preparation process of Sc@AuNC is as follows: chloroauric acid solution, scandium trichloride solution, and NaOH solution are added to BSA solution and reacted at 35-39°C for 12 hours or more; the crude product is dialyzed and purified to obtain ultra-bright red fluorescent water-soluble gold nanoclusters Sc@AuNC; wherein the molar ratio of the chloroauric acid solution, scandium trichloride solution, NaOH solution, and BSA solution is (8-12):(4-6):(0.5-1.5):(8-12); wherein the concentration of the NaOH solution is 0.5-1.5M.

[0026] This application provides an application of a low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA in the non-diagnostic detection of the urinary biomarker AD7c-NTP. The key feature is that, using AuE as the working electrode, platinum as the auxiliary electrode, and Ag / AgCl as the reference electrode, the electrochemiluminescence immunosensor Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE is immersed in a PBS buffer containing 200-300 mM of the co-reactant DIPEA, and cyclic voltammetry is performed to measure the electrochemiluminescence signal. The low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA is constructed using the method described above. Preferably, the concentration of the co-reactant DIPEA is 200 mM.

[0027] Compared with the prior art, this application has the following beneficial effects: This application develops a low-excitation-potential, high-sensitivity electrochemiluminescence immunosensor platform by constructing a low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA. This platform enables non-invasive, rapid, low-cost, wide-range, and highly sensitive detection of Alzheimer's disease urine biomarker (AD7c-NTP), overcoming the oxidative damage and background interference problems of high-potential ECL.

[0028] Compared to the complex fabrication process of high-potential ECL chips used in existing traditional AD7c-NTP detection methods, the construction method in this application employs activation, isothermal incubation, and washing procedures, which are simple to operate, require simple equipment, use readily available raw materials, and have higher construction efficiency. Furthermore, its usage is simple and easy to learn, effectively saving costs and time.

[0029] Other features and beneficial effects of this application will be set forth in the following description, and some of the technical features and beneficial effects may be obvious from the description or learned by practicing this application. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a cluster characterization diagram of Sc@AuNC provided in Embodiment 1 of this application; Figure 2 This is an inductively coupled plasma mass spectrometry (ICP-MS) image of element Sc in Sc@AuNC provided in Embodiment 1 of this application; Figure 3 The luminescence properties and luminescence mechanism of AuNC and Sc@AuNC prepared in Example 1 are illustrated. Figure 4 A schematic diagram of the sandwich-type ECLIA platform for detecting AD7c-NTP and Nyquist plots and ECL curves for different electrodes; Figure 5 Different electrodes were used in a 5 mM solution containing 0.1 M KCl. 3- / 4- CV plot in solution; Figure 6 A graph showing normalized intensity data under different incubation or reaction conditions; Figure 7 ECL response curves and AD7c-NTP calibration curves for different concentrations of AD7c-NTP on the ECL immunosensing platform. Figure 8 The illustration shows the specificity data of the immune sensor for AD7c-NTP detection under different interference conditions; Figure 9 A graph showing the stability data of a biosensor used for AD7c-NTP detection; Figure 10 The electrochemiluminescence (ECL) curves of the Sc@AuNC / DIPEA system and the Sc@AuNC / DIPEA-OH system in phosphate buffer containing different co-reactants are shown. Figure 11 This is a comparison of luminescence intensity data of electrochemiluminescent immunosensors prepared under different NaOH concentrations during the synthesis of Sc@AuNC. Figure 12 This is a comparison of luminescence intensity data of electrochemiluminescence immunosensors prepared under different synthesis time conditions during the synthesis of Sc@AuNC; Figure 13 This is a comparison of luminescence intensity data of electrochemiluminescent immunosensors prepared under different molar ratios of Sc and Au during the synthesis of Sc@AuNC. Figure 14 The ECL curve is shown for the electrochemiluminescence immunosensor prepared in Comparative Example 18. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them; the technical features designed in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other; based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. To verify the effects of this application, the following embodiments are provided: Example 1: Preparation method of Sc@AuNC, comprising the following steps: Under vigorous stirring, chloroauric acid solution (1 mL, 10 mM), scandium trichloride solution (1 mL, 5 mM), and NaOH solution (500 μL, 1 M) were added to bovine serum albumin solution (BSA, 1 mL, 10 mM), and the reaction was carried out at 37°C for 12 h. The crude product was dialyzed and purified, and then stored at 4°C in the dark.

[0033] Example 2: Construction method of low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA, as detailed below: a. After polishing the gold electrode (AuE) with alumina powder with particle sizes of 0.3μm and 0.05μm, the electrode was then ultrasonically cleaned with ultrapure water, anhydrous ethanol, and ultrapure water in sequence. b. Then, using 0.5 M sulfuric acid as the electrolyte, the electrode was activated using cyclic voltammetry with the following parameters: scan potential range of -0.2 to 1.6 V, scan rate of 0.1 V / s, until a stable redox peak appeared. The electrode was then rinsed thoroughly with ultrapure water and dried with nitrogen.

[0034] c. Modify the electrode surface prepared in step b with 7 μl of 200 μg / mL Ab1 solution, incubate at 4°C for 8 hours to form Ab1 / AuE, rinse thoroughly with ultrapure water, and then dry with nitrogen.

[0035] d. Modify the electrode prepared in step c with 7 μl of 0.5% BSA solution to seal the unbound active sites on the electrode, incubate at 25°C for 1 hour to form BSA / Ab1 / AuE, rinse thoroughly with ultrapure water, and then dry with nitrogen.

[0036] e. Modify the electrode prepared in step d with AD7c-NTP antigen solution, incubate at 37°C for 45 minutes to form AD7c-NTP / BSA / Ab1 / AuE, rinse thoroughly with ultrapure water, and dry under nitrogen. Then, modify the electrode surface with 7 μl of 200 μg / mL Ab2-Sc@AuNC, incubate at 37°C for 1 hour, rinse thoroughly with ultrapure water, and dry under nitrogen to obtain the electrochemiluminescent immunosensor Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE.

[0037] The synthesis process of the Ab2-Sc@AuNC solution includes the following steps: 1) Add 100 μl of phosphate buffer containing 20 mg / mL EDC and NHS to the fully purified Sc@AuNC prepared in Example 1, and incubate at 25°C for 40 minutes; 2) Add 1 mL of 300 μg / mL Ab2 solution and incubate with shaking at 4 °C for 12 hours; centrifuge the mixture again to remove unbound EDC, NHS and Ab2; then disperse the precipitate with 0.1% BSA solution and incubate for 30 minutes to block any non-specific binding sites; centrifuge the Ab2-Sc@AuNC bioconjugate, wash it several times with phosphate buffer solution, disperse it in 1 mL of PBS at pH 7.4, and store it in the dark at 4 °C.

[0038] f. Electrochemiluminescence signal testing: Electrochemiluminescence analysis was performed using a computer-controlled electrochemiluminescence detector (MPI-E, Xi'an Ruimai), with AuE as the working electrode, platinum as the auxiliary electrode, and Ag / AgCl (saturated KCl) as the reference electrode. During the measurement, the prepared electrode (i.e., the electrochemiluminescence immunosensor Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE) was immersed in 10 mM PBS containing the co-reactant DIPEA (200 mM). The electrochemiluminescence signal was then measured using cyclic voltammetry. The initial voltage was 0 V, the termination voltage was 0.8 V, the scan rate was 100 mV / s, the photomultiplier tube was biased at 700 V, and all electrochemiluminescence measurements were performed at room temperature.

[0039] Examples 3-4: The only difference between Examples 3-4 and Example 2 is that 300 μg / mL and 400 μg / mL Ab1 solutions were used, respectively. For detailed comparison data of luminescence intensity in Examples 2-4, please refer to... Figure 6 .

[0040] Examples 5-6: The only difference between Examples 5-6 and Example 2 is that after adding Ab1 solution, incubation for 6 hours and 12 hours respectively forms Ab1 / AuE. For detailed comparison data of luminescence intensity in Examples 5-6, please refer to... Figure 6 .

[0041] Examples 7-8: The only difference between Examples 7-8 and Example 2 is that after adding the Ab2 solution, they were incubated for 45 min and 75 min respectively. For detailed comparison data of luminescence intensity in specific examples, please refer to... Figure 6 .

[0042] Examples 9-10: The only difference between Example 9 and Example 2 is that the co-reactant DIPEA is 250 mM and 300 mM, respectively. For detailed comparison data of luminescence intensity in specific examples, please refer to... Figure 6 .

[0043] Comparative Examples 1-4: The only difference between Comparative Examples 1 to 4 and Example 2 is that the concentration of NaOH is 0.25M, 0.5M, 1.5M and 2.5M respectively, while the other preparation processes and conditions remain unchanged.

[0044] Comparative Examples 8-13: The only difference between Comparative Examples 8 to 13 and Example 2 is that the synthesis time is 1h, 2h, 5h, 7h, and 24h, respectively, while other preparation processes and conditions remain unchanged.

[0045] Comparative Examples 14-17: The only difference between Comparative Examples 14 to 17 and Example 2 is that the molar ratios of Sc and Au are 0.2, 0.25, 1.0, and 2.0, respectively, while the other preparation processes and conditions remain unchanged.

[0046] Comparative Example 18: This comparative example differs from Example 2 only in that scandium is replaced with cerium. The specific preparation method of metallic Ce@AuNC includes the following steps: Under vigorous stirring, chloroauric acid solution (1 mL, 10 mM), cerium nitrate solution (1 mL, 5 mM), and NaOH solution (500 μL, 1 M) are added to bovine serum albumin solution (BSA, 1 mL, 10 mM), and the reaction is carried out at 37°C for 12 h. The crude product is dialyzed and purified, and then stored at 4°C in the dark.

[0047] The products obtained in the examples and comparative examples were subjected to corresponding performance tests, and the test results are as follows: Figure 1 This is a cluster characterization diagram of Sc@AuNC provided in Embodiment 1 of this application. Wherein, Figure 1 (A) is the TEM of Sc@AuNC; Figure 1 (B) is the EDS mapping for Sc@AuNC; Figure 1 (C) is the Sc(2p) photoelectron spectrum of Sc@AuNC; Figure 1 (D) is the Au(4f) photoelectron spectrum of Sc@AuNC; Figure 1 (E) shows the UV-Vis absorption spectrum and the photoluminescence spectrum of Sc@AuNC; Figure 1 In (F), curve a represents naked AuE, and curve b represents the ECL curve of Sc@AuNC / AuE in 10mM PBS containing 200mM DIPEA.

[0048] Figure 2 The image shows the inductively coupled plasma mass spectrometry (ICP-MS) spectra of element Sc in Sc@AuNC.

[0049] Figure 3 The illustrations show the luminescence properties and luminescence mechanism of AuNC and the Sc@AuNC prepared in Example 1; wherein, Figure 3 (A) is the ECL curve of AuNC and Sc@AuNC in 10 mM PBS containing 200 mM of co-reactant, with a potential range between 0 and 0.8 V; Figure 3 (B) ECL efficiency of AuNC and Sc@AuNC in 10 mM PBS containing 200 mM co-reactant, with a potential range of 0 to 0.8 V; Figure 3 (C) is the EPR diagram of the mixture of AuNC and Sc@AuNC with DIPEA, with DMPO as the trapping agent. Figure 3 (D) shows the CV curves of Sc@AuNC / AuE in DIPEA solution recorded at different scan rates of 0.05 V / s (a), 0.1 V / s (b), 0.15 V / s (c), 0.2 V / s (d), and 0.25 V / s (e). The inset shows the relationship between peak current and scan rate. Figure 3 (E) is a diagram of possible ECL mechanisms for the AuNC / TEA, AuNC / DIPEA, and Sc@AuNC / DIPEA systems.

[0050] Figure 4 (A) is a schematic diagram of the sandwich-type ECLIA platform for detecting AD7c-NTP. Figure 4 (B) and Figure 4 (C) Nyquist plots and ECL curves for different electrodes, respectively; Figure 4 (B) and Figure 4 In (C), a represents the bare gold electrode AuE, and b, c, d, and e represent Ab1 / AuE, BSA / Ab1 / AuE, AD7c-NTP / BSA / Ab1 / AuE, and Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE prepared in Example 2, respectively, all in 5 mM [Fe(CN)6] containing 0.1 M KCI. 3- / 4- Tested in solutions and 10 mM PBS containing 200 mM DIPEA.

[0051] Figure 5 Different electrodes were used in a 5 mM solution containing 0.1 M KCl. 3- / 4- CV diagram in solution; where a represents bare gold electrode AuE, and b, c, d, and e represent Ab1 / AuE, BSA / Ab1 / AuE, AD7c-NTP / BSA / Ab1 / AuE, and Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE prepared in Example 2, respectively.

[0052] Figure 6This is a graph showing normalized intensity data under different incubation or reaction conditions; among them, Figure 6 (A) The horizontal axis represents the concentration of Ab1. Figure 6 (B) The horizontal axis represents the incubation time of Ab1. Figure 6 (C) The horizontal axis represents the reaction time of Ab1 and AD7c-NTP. Figure 6 (D) The horizontal axis represents the concentration of DIPEA.

[0053] Figure 7 The graphs show the ECL response curves and AD7c-NTP calibration curves of the ECL immunosensing platform to different concentrations of AD7c-NTP. Figure 7 (A) is the ECL response curve of the ECL immunosensing platform to different concentrations of AD7c-NTP, where (af) represents: 0, 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL concentrations, respectively. Figure 7 (B) is the calibration curve of AD7c-NTP.

[0054] Figure 8 The illustration shows the specificity data of the immunosensor for AD7c-NTP detection under different interference conditions, namely Hb (10 ng / mL), PSA (10 ng / mL), ALB (10 ng / mL), RBP (10 ng / mL), NGAL (10 ng / mL), β2-MG (10 ng / mL) and AD7c-NTP (1 ng / mL) mixed with the above interfering proteins.

[0055] Figure 9 A graph showing the stability data (1 ng / mL) of the biosensor used for AD7c-NTP detection.

[0056] Figure 11 This is a comparison of the luminescence intensity data of electrochemiluminescent immunosensors prepared under different NaOH concentrations during the synthesis of Sc@AuNC. Figure 12 This is a comparison chart of luminescence intensity data of electrochemiluminescence immunosensors prepared under different synthesis time conditions during the synthesis of Sc@AuNC. Figure 13 This is a comparison of luminescence intensity data of electrochemiluminescent immunosensors prepared under different molar ratios of Sc and Au during the synthesis of Sc@AuNC. Figure 14 The ECL curve is shown for the electrochemiluminescence immunosensor prepared in Comparative Example 18.

[0057] The specific data analysis is as follows: Analysis Figure 1 TEM and EDS characterization confirmed that Sc was successfully doped into AuNC (see details). Figure 1 A and Figure 1 B). High-resolution XPS spectra of Sc(2p) Figure 1 C) shows peaks at 398.8 eV and 400.9 eV, attributed to Sc(2p3 / 2) and Sc(2p1 / 2), respectively. This implies that Sc(III) is reduced back to Sc(0), and Sc(III) and Sc(0) may coexist in Sc@AuNC. The binding energies of Au(4f5 / 2) and Au(4f7 / 2) are 87.6 and 83.8 eV, respectively, indicating that Au(0) and Au(I) coexist in Sc@AuNC, with a distribution of 43.5% Au(0) and 56.5% Au(I). Figure 1 D). UV-Vis absorption spectrum of Sc@AuNC ( Figure 1 E) exhibited only a significant protein absorption peak at 280 nm, attributed to the absorption rates of ligand heterocycles and aromatic residues. However, no absorption peak corresponding to the surface plasmon resonance (SPR) of gold at around 520 nm was observed in the spectrum, indicating the successful preparation of gold nanoclusters. No ECL signal was detected at the bare gold electrode (AuE) in the presence of 0.2 M DIPEA. Figure 1 F, curve a). However, after modifying AuE with Sc@AuNC, a significant ECL signal appeared ( Figure 1 F (curve b) indicates the creation of a new Sc@AuNC / DIPEA ECL system.

[0058] Analysis Figure 2 This proves that Sc was successfully doped into AuNC.

[0059] Analysis Figure 3 To achieve optimal ECL performance at low excitation potentials, three ECL systems were further compared under the same conditions in the 0-0.8 V range. Figure 3 As shown in Figure A, compared to the peak signal of the classic ECL system AuNC / TEA, the AuNC / DIPEA system achieved a significantly higher ECL intensity, an increase of 18.13 times. These results demonstrate that DIPEA, as a co-reactant, exhibits excellent efficiency in amplifying the ECL signal of AuNC-based systems. Furthermore, the Sc@AuNC / DIPEA system, when doped with Sc ions, shows a significant improvement in ECL performance, displaying a higher signal intensity than the AuNC / DIPEA system. Subsequently, the inventors further calculated the ECL intensity of the three ECL systems. Φ ECL, the AuNC / TEA system has low ECL strength ( Φ The ECL concentration (12.48%) highlights the limitations of traditional co-reactants. In contrast, the AuNC / DIPEA ECL system... Φ ECL was significantly enhanced. In particular, Sc@AuNC containing DIPEA exhibited significantly higher ECL strength and Φ ECL highlights the effectiveness of the Sc ion doping strategy in improving ECL performance. Finally, compared with the AuNC / TEA system, the ECL intensity and... Φ ECL increased by 40.89 times and 2.82 times, respectively. It is worth noting that the Sc@AuNC / DIPEA system... Φ The ECL value reached 35.19%, indicating the great potential of this method in ECL applications.

[0060] Subsequently, the inventors further investigated the mechanism of the Sc@AuNC / DIPEA ECL system. Sc 3+ The Sc ion is known for its strong Lewis acid properties, significantly enhancing electron transfer rates by acting as an electron acceptor. 3+ It may have played a crucial role in accelerating electron transfer with co-reactants. Furthermore, Figure 3 C shows the DIPEA captured by DMPO. •+ EPR spectrum. DIPEA captured by DMPO after mixing with Sc@AuNC. •+ The signal is stronger than AuNC / DIPEA. It can be inferred that Sc 3+ Ion doping can effectively promote the generation of greater amounts of DIPEA radicals. Notably, for the Sc@AuNC / DIPEA system, the current intensity gradually increases with increasing scan rate. The derived linear regression equation is current = 1.916 × scan rate + 0.1786, indicating that the electrolysis process of DIPEA on Sc@AuNC / AuE follows surface control. Figure 3 D). Therefore, due to DIPEA •+ Both Sc@AuNC and DIPEA substances, present at high concentrations, successfully generated highly efficient ECL reactions. The possible mechanisms of ECL in the Sc@AuNC / DIPEA system are as follows: Figure 3 As shown in E.

[0061] Analysis Figure 4 The basic mechanism of AD7c-NTP ECL detection relies on the increased concentration of Ab2-Sc@AuNC after the immune response, leading to enhanced ECL intensity. To verify the successful construction of the ECL immunosensor, the inventors observed and evaluated the interfacial properties of AuE using EIS. Figure 4As shown by curve a in B, the naked AuE exhibits the lowest resistance, while the subsequent assembly of Ab1, BSA, AD7c-NTP, and Ab2-Sc@AuNC leads to a further increase in Ret, because the protein layer hinders electron transfer. Furthermore, in the presence of AD7c-NTP, a significant increase in the ECL signal was observed in the Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE structure. Figure 4 C).

[0062] Analysis Figure 5 The naked AuE exhibited the largest current, while the subsequent assembly of Ab1, BSA, AD7c-NTP, and Ab2-Sc@AuNC resulted in a gradual decrease in current, as the protein layer hindered electron transfer. This result further confirms the successful construction of the ECL immunosensor.

[0063] Analysis Figure 6 It can be seen that the concentration of Ab1 reaches a plateau after 200 μg / mL, and the incubation time reaches a plateau after 6 hours. Therefore, the preferred concentration of the added Ab1 solution is greater than 200 μg / mL and less than 500 μg / mL, and the preferred incubation time is 6–12 hours. The reaction time between Ab1 and AD7c-NTP reaches a plateau after 45 minutes. Therefore, the optimal reaction time for adding the AD7c-NTP antigen solution is 45 minutes to 2 hours. The concentration of DIPEA reaches a plateau after 200 mM. When performing electrochemiluminescence signal testing, the preferred concentration of DIPEA as a co-reactant is 200 mM. It should be noted that: Figure 6 In the control experimental group other than the examples, the index data on the horizontal axis is used as the variable (as opposed to the single variable in Example 2).

[0064] Analysis Figure 7 The performance of the ECL immunosensor was evaluated by detecting the ECL intensity of different concentrations of AD7c-NTP. Figure 7 A, Figure 7 B). ECL strength ( I There is a strong linear relationship between the concentration of AD7c-NTP and the logarithm (lgC) (from 1 pg / mL to 100 ng / mL) (R0). 2 = 0.997), the linear equation is I = 4585.7 + 1359.4 × lg CThe detection limit is as low as 0.45 pg / mL, which is superior to previously reported AD7c-NTP detection methods. This highlights the superior sensitivity of the immunosensor constructed in this application for detecting AD7c-NTP, mainly due to the efficient and high-performance Sc@AuNC / DIPEA system based on the ECLIA platform. Therefore, this application not only introduces a novel ECL dual enhancement strategy based on a co-reactant-mediated ion doping system, but also creates an ECLIA platform for early AD screening.

[0065] Analysis Figure 8 Specificity is another important aspect of immune sensors. The inventors tested specificity by introducing common urinary interfering proteins, such as hemoglobin (Hb), prostate-specific antigen (PSA), albumin (ALB), retinol-binding protein (RBP), neutrophil gelatinase-associated lipotransferase (NGAL), and β2-microglobulin (β2-MG) (all at 10 ng / mL). Figure 8 As shown, the ECL signal of AD7c-NTP (1 ng / mL) and the mixture of the above-mentioned interfering proteins (both 10 ng / mL) with AD7c-NTP (1 ng / mL) showed no significant change, which strongly confirms the high specificity and anti-interference performance of the immunosensor.

[0066] Analysis Figure 9 The sensor maintained stable ECL reaction with 1 ng / mL AD7c-NTP after 4 weeks of storage. These findings confirm the excellent stability and reproducibility of the immunosensor. Specific storage conditions were 4°C, protected from light.

[0067] Analysis Figure 11-13 This application yields Sc@AuNC with optimal luminescent performance by optimizing three key conditions in the Sc@AuNC synthesis process. First, the concentration of NaOH was optimized, such as... Figure 11 As shown, the luminescence intensity was strongest when the NaOH concentration was 1 M (Example 2), making a 1 M sodium hydroxide concentration a preferred choice (Example 2). Then, as... Figure 12 As shown, the luminescence intensity gradually increases with the extension of synthesis time, reaching a plateau after 12 hours (Example 2). Therefore, 12 hours was determined to be the optimal synthesis time. Figure 13 As shown, the luminescence intensity of Sc@AuNC is the highest when the Sc / Au molar concentration ratio is 0.5. Therefore, the optimal choice for the molar concentration ratio of Sc and Au is determined to be 0.5.

[0068] Analysis Figure 14In Example 2, the scandium doping was replaced with cerium, i.e., the prepared Sc@AuNC was replaced with Ce@AuNC. Under the same conditions, the ECL intensity of Ce@AuNC was found to be much weaker than that of Sc@AuNC.

[0069] In summary, the effects of the solution provided in this application are as follows: 1) Non-invasive and convenient: This application method directly tests urine samples, resulting in high patient compliance; 2) High sensitivity: The doping of scandium ions increases the ECL efficiency of the scandium gold nanoclusters / DIPEA system from 12.48% to 35.19%, with a detection limit as low as 0.45 pg / mL, which is superior to the traditional method; the traditional method is J. Alzheimer's Dis. 74 (2020) 237-244.

[0070] 3) Wide linear range: 1 pg / mL to 100 ng / mL; 4) Rapid testing: The entire testing process takes only 1 hour and 45 minutes; 5) High specificity and anti-interference performance: The ECL signal of the mixture of AD7c-NTP and interfering protein with AD7c-NTP did not change significantly, and the immunosensor exhibited high specificity and anti-interference performance. 6) Excellent stability and repeatability: The ECL signal of the AD7c-NTP remained stable after 4 weeks of storage.

[0071] In summary, this application has developed an electrochemiluminescence immunosensing platform with a low excitation potential (≤0.75 V) to achieve non-invasive, rapid, low-cost, wide detection range and high sensitivity detection of Alzheimer's disease urine biomarker (AD7c-NTP), overcoming the problems of oxidative damage and background interference of high-potential ECL.

[0072] It should be noted that the technical terms used in this article are abbreviated as follows: EDC: N -(3-Dimethylaminopropyl)- N ′-Ethylcarbonyldiamine; NHS: N -Hydroxysuccinimide; PBS: Phosphate buffer; Ab1 solution: Primary antibody solution (containing specific antibody + buffer system), wherein the buffer system uses 10 mM PBS; Ab2 solution: Secondary antibody solution (with label, used to amplify the detection signal); BSA solution: Bovine serum albumin solution; DIPEA: N,N-diisopropylethylamine; AuE: Gold electrode.

[0073] In summary, the method for constructing a low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA provided in this application has at least the following technological innovations, key designs, and beneficial effects: Key design points of this application 1) Rare earth doping improves ECL efficiency: Sc³⁺ doping forms scandium gold nanoclusters (Sc@AuNC), which enhances luminescence intensity and increases ECL efficiency; 2) Low-potential co-reactant design: DIPEA was selected as the co-reactant to reduce the excitation potential to 0.7 V; 3) Anti-interference sensing platform: Low potential avoids antibody oxidative damage and ensures detection specificity; 4) Versatile design: By changing the capture / detection antibody, it can be adapted to the detection of other protein biomarkers.

[0074] Beneficial effects of this application 1) Non-invasive and convenient: This application method directly tests urine samples, resulting in high patient compliance; 2) High sensitivity: The doping of scandium ions increases the ECL efficiency of the scandium gold nanoclusters / DIPEA system from 12.48% to 35.19%, with a detection limit of 0.45 pg / mL; 3) Wide linear range: 1 pg / mL to 100 ng / mL; 4) Rapid testing: The entire testing process takes only 1 hour and 45 minutes; 5) High specificity and anti-interference performance: The ECL signal of the mixture of AD7c-NTP and interfering protein with AD7c-NTP did not change significantly, and the immunosensor exhibited high specificity and anti-interference performance. 6) Excellent stability and repeatability: The ECL signal of the AD7c-NTP remained stable after 4 weeks of storage.

[0075] This application develops a low-excitation-potential (≤0.75 V) and high-sensitivity electrochemiluminescence immunosensor platform by constructing a low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA. This platform enables non-invasive, rapid, low-cost, wide-range, and highly sensitive detection of Alzheimer's disease urine biomarker (AD7c-NTP), overcoming the challenges of oxidative damage and background interference from high-potential ECL.

[0076] Compared to the complex fabrication process of high-potential ECL chips used in previously reported AD7c-NTP detection methods, the construction method in this application employs a combination of activation, isothermal incubation, and washing / cleaning steps, which is simpler to operate, requires less equipment, uses readily available raw materials, and has higher construction efficiency. Furthermore, its ease of use effectively saves cost and time.

[0077] The novelty of this application is: 1. Existing Solution 1: In their research paper titled "Cobalt ion doping to improve electrochemiluminescence emission of gold nanoclusters for sensitive NIR biosensing," Hongying Jia et al. disclosed a scheme for improving the electrochemiluminescence emission of gold clusters by doping with cobalt ions.

[0078] Although the existing cobalt ion doping schemes for improving the electrochemiluminescence emission of gold clusters are similar in design to the present application, both employing metal-doped gold nanoclusters to enhance ECL performance by modulating the electronic structure. Furthermore, their immunosensing architecture and nanocluster functionalization strategies are similar: both use a sandwich method (Ab1 / antigen / Ab2-AuNCs) to construct the sensor, relying on antigen-antibody specific binding; both use EDC / NHS to activate carboxyl groups to couple antibodies to the AuNCs surface (Ab2-AuNCs as signal tags); and both aim to address the oxidation damage and background interference problems caused by high potentials (≥1.2V) in traditional ECL systems. However, there are significant technical differences between the two schemes in terms of implementation, technical effects, or applications, as follows: (1) Differences in technical implementation: Differences between doped elements and core materials: This application (scandium nanoclusters) utilizes the rare earth element scandium (Sc). 3+ Cobalt (Co) is used as a dopant to form Sc@AuNC with gold clusters, with bovine serum albumin (BSA) as a stabilizer. BSA coordinates with metal ions through amino groups to form a stable protein-metal cluster structure, which endows the material with excellent properties. Existing scheme 1 (cobalt-doped gold clusters) selects cobalt (Co) as a dopant. 2+ Cysteine ​​and N-acetyl-L-cysteine ​​are used as bis-stabilizers as doping ions. Small molecule thiol compounds react with Co via the thiol group. 2+ Au 3+ Coordinate bonds are formed, constituting Co²-AuNCs.

[0079] Co-reactant design: Existing scheme 1 uses conventional TEA, which has a high oxidation potential (1.3 V) and requires 0.95 V for excitation. This application innovatively designs DIPEA, reducing the excitation potential to 0.75 V.

[0080] Differences in Detection Target and Sample Type: This application targets the Alzheimer's disease biomarker AD7c-NTP, using urine as the sample. AD7c-NTP specifically exists in urine. A sandwich immunoassay is used, employing Ab1 and Ab2-Sc@AuNC to capture and detect AD7c-NTP. Urine samples offer the advantage of non-invasive collection, high patient compliance, and suitability for large-scale early screening. Existing scheme 1 targets the small cell lung cancer biomarker NSE, using serum as the sample. NSE is a protein biomarker in serum, specifically recognized by antibodies and bound to Co... 2+ -AuNCs enable highly sensitive detection; serum testing requires specialized sampling and is suitable for cancer diagnosis and monitoring.

[0081] Electrochemiluminescence mechanism and performance differences: For the ECL mechanism, this application uses DIPEA as a co-reactant, which can induce ECL signals at a low excitation potential of 0.7 V. DIPEA has a low oxidation potential, forming an efficient electron transport chain with Sc@AuNC. The low potential avoids oxidative damage to the antibody and reduces background interference. Existing scheme 1 uses TEA as a co-reactant with an excitation potential of 0.95 V. Cobalt doping lowers the electron transfer barrier by adjusting the electronic structure of the gold cluster surface. 2+ / Co 3+ The redox pair accelerates charge transfer and enhances ECL emission in the near-infrared region.

[0082] Sensor Structure Difference: The sensor in this application uses a gold electrode as a substrate, sequentially modified with Ab1, BSA, AD7c-NTP, and Ab2-Sc@AuNC. The good conductivity of the gold electrode is suitable for low-potential detection. Existing Solution 1 uses a glassy carbon electrode (GCE), with surface modification of ABA, Ab1, NSE, and Ab2-Co. 2+ -AuNC, the glassy carbon electrode surface is easy to modify in multiple steps.

[0083] Biocompatibility optimization: Existing scheme 1 does not clearly define the protective mechanism of low potential for antibodies. The 0.75 V operating potential in this application overcomes the oxidative damage of high-potential ECL, avoids irreversible antibody oxidation, and ensures detection specificity.

[0084] Application scenario expansion: Existing solution 1 requires blood collection for serum NSE detection. This application is adapted for non-invasive detection of urine AD7c-NTP, utilizing the characteristic of urine lacking protein homeostasis, making the target easier to detect and significantly improving patient compliance.

[0085] (2) Differences in technical effects Sensitivity and efficiency: Sc 3+ Doping increases ECL efficiency to 35.19% (higher than Co).2+ (33.8%), combined with the catalytic effect of DIPEA, the detection limit for urine samples is as low as 0.45 pg / mL.

[0086] Anti-interference capability: The low potential of 0.75 V reduces electrode side reactions and lowers background signal (the existing scheme 1 still has electrochemical interference at 0.95 V).

[0087] Clinical applicability: Non-invasive urine testing + rapid procedure in 1 hour and 45 minutes, overcoming the problems of high cost of traditional PET / MRI and high invasiveness of CSF testing.

[0088] Platform versatility: This application emphasizes that "other biomarkers can be detected by changing the antibody", which demonstrates good scalability.

[0089] (3) Core innovations and effects of this application This application achieves dual innovation through rare earth doping and co-reactant molecular engineering: employing Sc 3+ Doping: Utilizing the energy level characteristics of rare earth ions to overcome the ECL efficiency bottleneck; DIPEA design achieves a minimum excitation of 0.75 V, and the low potential avoids antibody oxidative damage, ensuring detection specificity; Non-invasive detection closed loop: Optimized for the characteristics of urine AD7c-NTP, forming a "high sensitivity-low damage-non-invasive sampling" solution.

[0090] Summarize: Existing Solution 1 uses cobalt ions (Co) 2+ ) doped gold nanoclusters (Co 2+ The luminescent material is cysteamine and N-acetyl-L-cysteine, the co-reactant is triethylamine (TEA), the working electrode is glassy carbon electrode (GCE), and the detection target is neuron-specific enolase (NSE) in serum.

[0091] In this application, the luminescent material is scandium ion (Sc). 3+ The study used gold nanoclusters (Sc@AuNC) doped with bovine serum albumin (BSA) as a stabilizer, diisopropylethylamine (DIPEA) as a co-reactant, and gold electrode (AuE) as the working electrode. The target for detection was AD7c-NTP, a biomarker for Alzheimer's disease, in urine.

[0092] Both have core doped ions (Co) 2+ vs Sc 3+Significant differences exist in aspects such as stabilizer type (cysteamine / N-acetyl-L-cysteine ​​vs BSA), co-reactant (TEA vs DIPEA), working electrode material (GCE vs AuE), and detection object and sample type (serum NSE vs urine AD7c-NTP), which are not disclosed in the existing scheme 1.

[0093] In addition, although the existing scheme 1 also adopts the idea of ​​metal ion doping of gold nanoclusters, the difference is that this application specifically selects Sc 3+ As a dopant ion rather than Co 2+ Furthermore, the addition of BSA as a stabilizer represents a novel design for the doped system—the synergistic effect of Sc³⁺ and gold, and the biocompatibility optimization of BSA, are not easily derived from the existing scheme 1. Regarding the co-reactant, the existing scheme 1 uses TEA, while this application selects DIPEA. The oxidation potential is reduced (from 1.15 V to 0.7 V) through optimization of the selected DIPEA; this structural optimization strategy is not disclosed or inspired in the existing scheme 1. In addition, the detection sample in this application is not serum, but specific urine. The detection system is designed specifically for the accumulation characteristics of the urine biomarker AD7c-NTP, making it suitable for non-invasive screening scenarios, which is also original.

[0094] The advantages of this proposed solution are: the excitation potential is reduced from 0.95 V to ≤0.75 V, avoiding irreversible antibody oxidation; the ECL efficiency is increased from 33.8% to 35.19%, resulting in higher detection sensitivity; the use of urine samples enables non-invasive testing, significantly improving patient compliance; and the entire testing process takes 1 hour and 45 minutes, making it more suitable for high-throughput clinical analysis.

[0095] 2. Existing Solution 2: The research materials published by Guolin Hong et al., entitled "Co-Reactant-Mediated Low-Potential Anodic Electrochemiluminescence Platform and Its Immunosensing Application," disclose a co-reactant-mediated low-potential anodic electrochemiluminescence platform and its immunosensing application scheme.

[0096] Although the existing scheme 2 is similar to the scheme in this application in terms of the low-potential excitation mechanism design point, both using isopropyl-substituted amine compounds (DIPEA-OH / DIPEA) as co-reactants to avoid antibody damage by utilizing their low oxidation potential characteristics, there are significant technical differences between the two in terms of technical implementation and technical effects or applications, as follows: (1) Differences in technical implementation: 1) Design of doping elements and core materials: This application uses the rare earth element scandium (Sc). 3+ Gold-doped nanoclusters (Sc@AuNC) were used with bovine serum albumin (BSA) as a stabilizer. 3+ Through Au 3+ Co-reduction and embedding of gold clusters is employed to enhance electrochemiluminescence (ECL) efficiency through the effect of scandium doping with rare-earth ions. Existing scheme 2 does not use metal ion doping; instead, it optimizes ECL performance solely through co-reactant molecular design. The core material is BSA-stabilized gold nanoclusters (BSA-AuNC), paired with 2-(diisopropylamino)ethanol (DIPEA-OH) as a co-reactant.

[0097] Technical principle comparison: This application relies on metal ion doping to change the electronic structure of the cluster and form the Sc@AuNC alloy luminescent center; the existing scheme 2 reduces the oxidation potential and promotes charge transfer through co-reactant chemical modification (hydroxyl catalysis).

[0098] Technical differences: This application is for "material doping modification", while the existing scheme 2 is for "co-reactant molecular engineering". The former changes the nature of the luminescent material, while the latter optimizes the reaction conditions.

[0099] 2) Differences in preparation process and detection targets: In the preparation method of this application, chloroauric acid, scandium trichloride and NaOH are reacted in BSA solution at 37°C for 12 hours, and Sc@AuNC is obtained by dialysis purification; the sensor is constructed by sandwich immunoassay, and the gold electrode is modified with Ab1, BSA, AD7c-NTP antigen and Ab2-Sc@AuNC in sequence.

[0100] In the existing scheme 2, BSA-AuNC is prepared by reduction of chloroauric acid without doping; DIPEA-OH is synthesized by isopropyl substitution and hydroxyl addition, and assembled with BSA-AuNC into an ECL system. The sensor structure consists of gold electrodes modified with Ab1, BSA, N protein and Ab2-BSA-AuNC.

[0101] This application targets the Alzheimer's disease urinary biomarker AD7c-NTP, leveraging the advantages of non-invasive urine sampling, making it suitable for large-scale early screening. Existing protocol 2 targets the SARS-CoV-2 viral N protein (serum sample), suitable for clinical diagnosis of COVID-19.

[0102] 3) Sensor structure and performance This application: Gold electrode → Ab1 → BSA → AD7c-NTP → Ab2-Sc@AuNC, relying on Sc@AuNC as a signal tag. Existing scheme 2: Gold electrode → Ab1 → BSA → N protein → Ab2-BSA-AuNC, directly labeling the antibody with BSA-AuNC.

[0103] Differences in applicable detection targets due to sensor construction: The wide linear range of this application is suitable for monitoring the progression of Alzheimer's disease; Existing solution 2 is suitable for early diagnosis of COVID-19.

[0104] (2) Differences in application scenarios and technological advantages This application is for: early screening of Alzheimer's disease (AD), utilizing non-invasive urine samples, with high patient compliance, suitable for preliminary screening of large populations, and promoting early intervention for AD. Existing Solution 2 is for: clinical diagnosis of COVID-19, targeting the detection of SARS-CoV-2 N protein in serum.

[0105] Summary of core technological advantages: This application addresses the non-invasiveness requirement of AD screening through "material innovation," while existing solution 2 overcomes the sensitivity bottleneck of virus detection through "reaction condition optimization."

[0106] (3) Core innovations of this application This application represents a breakthrough compared to the existing scheme 2: Dual-path synergy: enhancing intrinsic efficiency through rare-earth doping + optimizing reaction kinetics with DIPEA, rather than solely relying on co-reactant engineering. Scenario innovation: pioneering the use of low-potential ECL technology for Alzheimer's disease urinary biomarker detection, addressing the clinical pain point of "difficulty in popularizing non-invasive screening." Platform-based design: emphasizing "the ability to detect other biomarkers simply by changing the antibody," offering superior scalability compared to the single application of the existing scheme 2.

[0107] Summarize: Existing scheme 2 uses bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) without metal ions as the luminescent material, and hydroxyl-containing 2-(diisopropylamino)ethanol (DIPEA-OH) as the co-reactant. The target for detection is SARS-CoV-2 N protein in serum, and it does not explicitly mention standardized pretreatment steps for the working electrode. In this application, the luminescent material is scandium ions (Sc). 3+ The gold nanoclusters (Sc@AuNC) are doped with BSA as a stabilizer, but their performance is optimized through doping. The co-reactant is diisopropylethylamine (DIPEA) without hydroxyl groups. The target for detection is AD7c-NTP, a biomarker for Alzheimer's disease in urine. The working electrode (gold electrode) needs to undergo standardized pretreatment such as polishing and sulfuric acid activation.

[0108] Both have differences in core luminescent materials (undoped AuNCs vs Sc). 3+ Significant differences exist in aspects such as doping with AuNCs, co-reactant structure (hydroxyl-containing DIPEA-OH vs hydroxyl-free DIPEA), detection targets and sample types (serum N protein vs urine AD7c-NTP), and electrode pretreatment procedures, which are not disclosed in the existing scheme 2.

[0109] Existing scheme 2 achieves low-potential ECL by optimizing the co-reactant (DIPEA-OH), but does not involve a metal ion doping strategy. This application introduces Sc 3+ Doped gold nanoclusters, via Sc 3+ The synergistic effect with gold enhances electron transfer efficiency. This design cannot be easily derived from existing scheme 2—existing scheme 2 does not indicate the optimizing effect of metal ion doping on the ECL performance of AuNCs, while Sc 3+ The choice further demonstrates originality.

[0110] Regarding the co-reactant, the existing scheme 2 relies on the hydroxyl group enhancement efficiency of DIPEA-OH. This application selects DIPEA, which retains the advantage of low potential (0.7 V) and avoids the interference that hydroxyl groups may cause in the urine matrix. Figure 10 Electrochemiluminescence (ECL) signal data for two systems, Sc@AuNC / DIPEA and Sc@AuNC / DIPEA-OH, are shown in 100 mM phosphate-buffered saline (PBS) containing 200 mM co-reactants (DIPEA-OH or DIPEA), with potentials cycling between 0 and 0.8 V. Figure 10 As shown, a comparison between the Sc@AuNC / DIPEA system and the Sc@AuNC / DIPEA-OH system reveals that while the ECL signal of Sc@AuNC in DIPEA-OH is slightly enhanced compared to the Sc@AuNC / DIPEA system, the excitation potential is also increased. In previous research, the inventors' research group developed the AuNC / 2-(diisopropylamino)ethanol (DIPEA-OH) ECL system (Anal. Chem. 2022, 94, 12500-12506). Therefore, this application focuses on the low-potential AuNC / DIPEA system, proposing not only a novel co-reactant acceleration strategy to improve the ECL efficiency of the ECL system and constructing a new low-potential, high-performance ECL system, but also creating a highly efficient and sensitive detection method, bringing promise for large-scale early screening of AD. This targeted optimization design of the application is innovative.

[0111] Furthermore, the sample to be tested in this application is non-invasive urine, which is adapted to the characteristic of AD7c-NTP accumulation in urine, so as to achieve non-invasive screening. The scenario selection and system adaptation are not disclosed in the existing scheme 2, and are original.

[0112] It should be noted that the symbol "~" is used in this document to represent a numerical range, which includes two endpoints. Besides the specific choices shown in the above embodiments, the above formulation range can be used in any specific implementation of this application, including but not limited to the above-described embodiments. The specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of this application, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of this application. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them; although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for constructing a low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA, characterized in that, Includes the following steps: S10. After polishing, washing, and activation pretreatment of the working electrode, add Ab1 solution to the electrode surface and incubate at a constant temperature to form an Ab1 / AuE electrode; block the Ab1 / AuE electrode with BSA to form a BSA / Ab1 / AuE electrode; add AD7c-NTP antigen solution to the BSA / Ab1 / AuE electrode and incubate at a constant temperature to form an AD7c-NTP / BSA / Ab1 / AuE electrode. S20. Add the Ab2-Sc@AuNC solution to the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubate at a constant temperature to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. Construct a highly sensitive electrochemiluminescent immunosensor using Ab2-Sc@AuNC as a signal probe via a sandwich immunoassay.

2. The construction method according to claim 1, characterized in that, The preparation process of Ab2-Sc@AuNC is as follows: Sc@AuNC was prepared using HAuCl4∙3H2O as the gold ion source, scandium trichloride as the scandium ion source, and sodium hydroxide as the reducing agent. Then, phosphate buffer containing EDC and NHS was added to the Sc@AuNC and incubated at a constant temperature. Next, Ab1 solution was added, and the mixture was incubated at a constant temperature and then separated. BSA was used for blocking to obtain the Ab2-Sc@AuNC bioconjugate. Finally, the Ab2-Sc@AuNC bioconjugate was separated, washed, and dispersed to obtain the final product.

3. The construction method according to claim 2, characterized in that, The preparation process of Sc@AuNC is as follows: chloroauric acid solution, scandium trichloride solution and NaOH solution are added to BSA solution and reacted at 35-39℃ for 12 hours or more; the crude product is dialyzed and purified to obtain red fluorescent water-soluble gold nanoclusters Sc@AuNC; The molar concentration ratio of the chloroauric acid solution, scandium trichloride solution, NaOH solution, and BSA solution is (8-12):(4-6):(0.5-1.5):(8-12); the concentration of the NaOH solution is 0.5-1.5M.

4. The construction method according to claim 1, characterized in that, The synthesis process of the Ab2-Sc@AuNC solution includes the following steps: 1) Add phosphate buffer containing 15-25 mg / mL EDC and NHS to Sc@AuNC and incubate at 4°C or 25°C for 0.5-4 hours; 2) Add an Ab2 solution with a concentration of 50–500 μg / mL and incubate with shaking at 3.5–4.5 °C for 10–14 hours; centrifuge the mixture again to remove unbound EDC, NHS, and Ab2; then disperse the precipitate with 0.1% BSA solution and incubate for 30 minutes to block any non-specific binding sites; centrifuge the Ab2-Sc@AuNC bioconjugate, wash it several times with phosphate buffer solution, and disperse it in a phosphate buffer solution for dispersion at pH 7.4; the volume ratio of phosphate buffer solution containing 15–25 mg / mL EDC and NHS, Ab2 solution, and phosphate buffer solution for dispersion is 0.1:1:

1. The preparation steps of Sc@AuNC are as follows: Under vigorous stirring, 8-12 mM chloroauric acid solution, 4-6 mM scandium trichloride solution and 0.5-1.5 M NaOH solution are added to 8-12 mM BSA solution, and the reaction is carried out at 35-39℃ for 12 hours or more; the crude product is dialyzed and purified to obtain ultra-bright red fluorescent water-soluble gold nanoclusters Sc@AuNC; the volume ratio of chloroauric acid solution, scandium trichloride solution, NaOH solution and BSA solution is 1:1:0.5:

1.

5. The construction method according to claim 1, characterized in that, Includes the following steps: S10. Preparation of AD7c-NTP / BSA / Ab1 / AuE electrode: S11. The working electrode is polished, washed, and activated in sequence, and then cleaned and dried. S12. Add Ab1 solution to the electrode surface and incubate at 2-6℃ for 6-12 hours to form an Ab1 / AuE electrode, then clean and dry; wherein the concentration of Ab1 solution is 50-500 μg / mL. S13. After sealing the unbound active sites on the Ab1 / AuE electrode with BSA, incubate at a constant temperature of 24-26℃ to form a BSA / Ab1 / AuE electrode, and then clean and dry it. S14. Add AD7c-NTP antigen solution to the BSA / Ab1 / AuE electrode, incubate at 35-39°C for 45 minutes to 2 hours to form the AD7c-NTP / BSA / Ab1 / AuE electrode, and then clean and dry it. S20. Preparation of electrochemiluminescence immunosensor: The Ab2-Sc@AuNC solution was dropped onto the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubated at a constant temperature of 35-39°C to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. After washing and drying, a highly sensitive electrochemiluminescent immunosensor was formed by sandwich immunoassay using Ab2-Sc@AuNC as a marker.

6. The construction method according to claim 1, characterized in that: In step S11, the working electrode is thoroughly polished using alumina powder with a particle size of 0.3 μm and 0.05 μm in sequence; then, the surface of the working electrode is ultrasonically cleaned for 1–5 minutes using ultrapure water, anhydrous ethanol, and ultrapure water in sequence; then, the working electrode is activated using cyclic voltammetry with 0.5 M sulfuric acid solution as electrolyte until a stable redox peak appears; the working electrode is then rinsed clean with ultrapure water and finally dried with nitrogen gas; wherein, the working electrode is a gold electrode; the activation parameters are: scan potential range of -0.2 V to 1.6 V, scan rate of 0.1 V / s; In step S12, an Ab1 solution is dropped onto the electrode surface and incubated at a constant temperature of 2–6°C for 6–12 hours to form an Ab1 / AuE electrode. After thorough rinsing with pure water, the electrode is dried with nitrogen gas. The concentration of the Ab1 solution is 50–500 μg / mL, and the volume is 5–15 μl. In step S13, after blocking the unbound active sites on the Ab1 / AuE electrode with BSA, the electrode is incubated at a constant temperature of 23–27°C for 0.5–1 hour to form a BSA / Ab1 / AuE electrode. After thorough rinsing with ultrapure water, the electrode is dried with nitrogen. The concentration of the BSA solution is 0.4%–0.6%, and its volume is 5–15 μl. In step S14, AD7c-NTP antigen solution is added dropwise to the BSA / Ab1 / AuE electrode, and the electrode is incubated at a constant temperature of 35-39°C for 45 minutes to 2 hours to form an AD7c-NTP / BSA / Ab1 / AuE electrode. After thorough rinsing with ultrapure water, the electrode is dried with nitrogen gas. The concentration of the AD7c-NTP antigen solution is 1 pg / mL to 100 ng / mL, and its volume is 5-15 μl. In step S20, the Ab2-Sc@AuNC solution is dropped onto the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubated at a constant temperature of 35-39°C for 1-2 hours to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. After thorough rinsing with ultrapure water, the electrode is dried with nitrogen. The concentration of the Ab2-Sc@AuNC solution is 50-500 μg / mL, and its volume is 5-15 μl.

7. The construction method according to claim 1, characterized in that, Includes the following steps: S10. Preparation of AD7c-NTP / BSA / Ab1 / AuE electrode: S11. The gold electrode is thoroughly polished using alumina powder with a particle size of 0.3 μm and 0.05 μm in sequence; then the surface of the working electrode is ultrasonically cleaned using ultrapure water, anhydrous ethanol, and ultrapure water in sequence; then, the working electrode is activated using cyclic voltammetry with 0.5 M sulfuric acid aqueous solution as electrolyte until a stable redox peak appears; the working electrode is then rinsed clean with ultrapure water and finally dried with nitrogen gas; wherein, the activation parameters are: scan potential range of -0.2 V to 1.6 V, scan rate of 0.1 V / s; S12. Add Ab1 solution to the electrode surface and incubate at 4℃ for 8 hours to form an Ab1 / AuE electrode. Rinse thoroughly with pure water and then dry with nitrogen. The concentration of Ab1 solution is 200 μg / mL and the volume is 7 μl. S13. After blocking the unbound active sites on the Ab1 / AuE electrode with BSA, incubate at 25°C for 1 hour to form a BSA / Ab1 / AuE electrode. Rinse thoroughly with ultrapure water and dry with nitrogen. The concentration of the BSA solution is 0.5% and its volume is 7 μl. S14. Add AD7c-NTP antigen solution to the BSA / Ab1 / AuE electrode, incubate at 37°C for 45 minutes to form AD7c-NTP / BSA / Ab1 / AuE electrode, rinse thoroughly with ultrapure water and dry with nitrogen. S20. Preparation of electrochemiluminescence immunosensor: The Ab2-Sc@AuNC solution was dropped onto the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubated at 37°C for 1 hour to form Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE. After thorough rinsing with ultrapure water, the electrode was dried with nitrogen. The concentration of the Ab2-Sc@AuNC solution was 200 μg / mL and its volume was 7 μl.

8. A low-potential electrochemiluminescence immunosensor based on scandium gold nanoclusters / DIPEA, characterized in that: It was prepared by adding Ab2-Sc@AuNC solution to the surface of the AD7c-NTP / BSA / Ab1 / AuE electrode and incubating at a constant temperature. The preparation process of Ab2-Sc@AuNC is as follows: Sc@AuNC is prepared using HAuCl4∙3H2O as the gold ion source, scandium trichloride as the scandium ion source, and sodium hydroxide as the reducing agent; then, phosphate buffer containing EDC and NHS is added to the Sc@AuNC and incubated at a constant temperature; then, Ab1 solution is added, incubated at a constant temperature, and separated; BSA is used for blocking to obtain the Ab2-Sc@AuNC bioconjugate; finally, the Ab2-Sc@AuNC bioconjugate is separated, washed, and dispersed to obtain the final product.

9. The low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA according to claim 8, characterized in that, The preparation process of Sc@AuNC is as follows: chloroauric acid solution, scandium trichloride solution and NaOH solution are added to BSA solution and reacted at 35-39℃ for 12 hours or more; the crude product is dialyzed and purified to obtain ultra-bright red fluorescent water-soluble gold nanoclusters Sc@AuNC; The molar concentration ratio of the chloroauric acid solution, scandium trichloride solution, NaOH solution, and BSA solution is (8-12):(4-6):(0.5-1.5):(8-12); and the concentration of the NaOH solution is 0.5-1.5M.

10. The application of a low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA in the non-diagnostic detection of the urinary biomarker AD7c-NTP, characterized in that: Using AuE as the working electrode, platinum as the auxiliary electrode, and Ag / AgCl as the reference electrode, the electrochemiluminescent immunosensor Ab2-Sc@AuNC / AD7c-NTP / BSA / Ab1 / AuE was immersed in PBS buffer containing 200-300 mM co-reactant DIPEA, and cyclic voltammetry was performed to measure the electrochemiluminescence signal. The low-potential electrochemiluminescence immunosensor based on scandium nanoclusters / DIPEA is constructed using the construction method described in claims 1-7.

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