UiO-67 confined mononuclear iridium complex-based electrochemiluminescence sensor, construction method and application thereof

Abstract

This invention belongs to the field of biosensors and electrochemiluminescence (ECL) detection technology, and discloses an electrochemiluminescence sensor based on a UiO-67 confined single-site iridium complex, its construction method, and applications. The system uses zirconium-based metal-organic framework (MOF) material UiO-67 as a carrier, and iridium(III) bridging compounds are bonded through coordination to construct an in-situ Ir@UiO-67 nano-signal probe with high ECL activity. This probe is labeled with P-tau 181 secondary antibody to obtain an Ab2-Ir@UiO-67 complex. Simultaneously, a boron-doped diamond electrode modified with a gold film is used as the substrate electrode, and P-tau 181 primary antibody is immobilized to construct a specific recognition interface. A highly sensitive electrochemiluminescence sensing platform is then assembled via a sandwich immunoassay. This invention improves upon the bottleneck problems of poor water solubility and easy aggregation and quenching of traditional iridium complexes through MOF coordination anchoring and pore spatial confinement effects, achieving ultrasensitive quantitative detection of P-tau 181. It exhibits excellent specificity, reproducibility and stability in plasma samples.

Description

Technical Field

[0001] This invention relates to the fields of biosensors and electrochemiluminescence detection technology, specifically to an electrochemiluminescence sensor for ultrasensitive detection of Alzheimer's disease biomarkers, its construction method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and progressive worsening. It has become one of the leading causes of cognitive impairment, disability, and even death among the elderly worldwide, imposing a heavy medical and care burden on families and society. The core pathological changes in AD often begin more than a decade before the appearance of clinical symptoms, and currently there are still no effective means to reverse the disease course or cure it. Therefore, achieving accurate, early, and non-invasive diagnosis in the asymptomatic preclinical stage or the stage of mild cognitive impairment is of irreplaceable and crucial significance for initiating intervention as early as possible, delaying cognitive decline, and improving patient prognosis.

[0003] Traditional AD diagnosis relies on positron emission tomography (PET) imaging or cerebrospinal fluid analysis, but these methods are costly, involve radiation risks, or are highly invasive, making them unsuitable for large-scale population screening. Blood biomarkers have become a research hotspot for early AD diagnosis due to their non-invasiveness, accessibility, and cost-effectiveness. Among them, Tau protein phosphorylated at threonine 181 (P-tau 181) is a highly specific indicator distinguishing AD from other neurodegenerative diseases and is highly correlated with neurofibrillary tangles and β-amyloid plaque deposition in the brain.

[0004] However, the concentration of P-tau 181 in peripheral blood is extremely low and easily affected by complex matrices, which places extremely high demands on the sensitivity and anti-interference ability of detection technologies. Electrochemiluminescence (ECL) technology combines the controllability of electrochemistry with the low background advantage of chemiluminescence, and has the advantages of high signal-to-noise ratio, wide linear range, and no need for external light source, making it an ideal technology for the detection of trace biomarkers.

[0005] Traditional ECL luminescent agents such as luminol and tris(2,2-bipyridine)ruthenium(II) still have room for improvement in quantum efficiency and photochemical stability, while cyclometalated iridium(III) complexes possess tunable emission wavelengths, long luminescence lifetimes, and high quantum yields, making them a new generation of high-performance ECL luminescent materials. However, iridium(III) complexes are highly hydrophobic, almost insoluble in aqueous solutions, and prone to aggregation quenching due to intermolecular π-π stacking when modified onto electrode surfaces, greatly limiting their application in aqueous biosensing.

[0006] Zirconium-based metal-organic framework (MOF) material UiO-67 possesses excellent chemical stability, large specific surface area, and open pore structure. Its organic ligands contain bipyridine chelating groups, which can bind to iridium complexes through coordination reactions, making it an ideal carrier for confined single-site iridium complexes. Currently, there are no reports on using the coordination confinement effect of UiO-67 to in-situ convert non-electrochemiluminescent iridium bridging compounds into single-site iridium complex luminescent centers, and on constructing electrochemiluminescence sensors for ultrasensitive detection of P-tau 181. Summary of the Invention

[0007] Based on the shortcomings of the existing technology, one objective of this invention is to provide a water-soluble UiO-67-confined iridium complex (Ir@UiO-67) as a signal probe to construct an electrochemiluminescence (ECL) sensor and apply it to the detection of P-Tau181; the second objective is to provide a method for preparing the ECL sensor.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: using boron-doped diamond electrode (BDD) with gold electrodeposition as the sensing substrate and Ir@UiO-67 as the ECL signal probe, an ECL sensor that can be used for ultrasensitive detection of P-tau 181 is constructed.

[0009] The following technical solution is specifically adopted: (1) Construction of the substrate of BDD electrode: The clean BDD electrode was placed in tetrachloroauric acid (HAuCl4) electrolyte, and a gold film was loaded on the surface of the BDD electrode by constant potential electrochemical deposition.

[0010] (2) Antibody fixation and construction of sensing interface: P-tau 181 primary antibody (Ab1) solution was dropped onto the surface of BDD electrode modified with gold film, incubated, Ab1 was fixed, and finally the electrode was blocked with blocking agents such as bovine serum albumin to eliminate non-specific binding sites.

[0011] (3) Preparation of signal probe: Ir@UiO-67 was constructed in situ on the UiO-67 framework using a ligand engineering strategy. Specifically, the organic ligand 2,2'-bipyridine-5,5'-dicarboxylic acid of UiO-67 was used to coordinate with the dichloro-bridged iridium dimer to achieve molecular-level integration of the iridium(III) complex with UiO-67, resulting in a confined iridium complex nanocomposite. Then, the obtained Ir@UiO-67 was coupled with P-tau 181 secondary antibody (Ab2) to prepare the Ab2-Ir@UiO-67 signal probe, which served as an electrochemiluminescence signal source.

[0012] (4) Sensor assembly and detection: The detection substrate obtained in step (2) and the P-tau 181 antigen in the sample to be tested are incubated together at a suitable temperature to complete the identification and capture of the target by utilizing the specific interaction between antigen and antibody; then the Ir@UiO-67 signal probe modified with secondary antibody prepared in step (3) is added, and incubation is continued to form a sandwich immune complex.

[0013] The target analyte was quantitatively determined by linear scanning voltammetry using electrochemiluminescence, and the relationship between the change in peak intensity signal and the concentration of P-tau181 was used.

[0014] The luminescent material in the signal probe is a cyclic metallized iridium(III) complex, which is confined to the surface of a porous UiO-67 nanocarrier.

[0015] In the UiO-67 confined unit-point iridium complex Ir@UiO-67 material, the iridium(III) complex is anchored by forming a coordinate bond with the nitrogen atom of the 2,2'-bipyridine-5,5'-dicarboxylic acid (dcbpy) ligand in the UiO67 framework, and its structural formula is as follows:

[0016] The iridium (III) complex The structure is shown in Equation II:

[0017] The synthetic route for the compound of formula II is as follows:

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) In this invention, an iridium-based MOF composite material (Ir@UiO-67) with high ECL activity is generated in situ by co-bonding the iridium-bridged complex, which originally had no ECL activity, with the ligand C=N bond through the confinement effect of UiO-67. Relying on the spatial confinement effect of MOF coordination, atomic-level dispersion of iridium active centers and stable anchoring of single active sites are achieved, avoiding aggregation quenching and improving luminescence efficiency and sensing stability.

[0019] (2) This invention differs from the traditional strategy of directly loading active complexes. It achieves the controllable construction of ECL performance from scratch through an integrated process of coordination bonding, structural reconstruction, and activation. Furthermore, the UiO-67 porous structure used can effectively promote reactant diffusion and electron transfer, further improving the ECL reaction efficiency and providing an excellent luminescent material basis for constructing highly sensitive ECL sensors.

[0020] (3) The present invention selects BDD electrodes with low background signal and strong anti-pollution ability as the sensing substrate, and optimizes the electrode interface by gold film modification, which effectively improves the stability and reproducibility of ECL signal and provides a reliable sensing platform for the construction of sensor.

[0021] (4) This invention innovatively constructs a sandwich-type electrochemiluminescence analysis system, which combines the excellent luminescence performance and signal amplification capability of Ir@UiO-67 to achieve ultrasensitive detection of P-tau 181. This strategy has both high specificity and ease of operation, and significantly improves the sensitivity and accuracy of detection.

[0022] (5) This invention effectively improves the bottleneck problems of poor water solubility and easy aggregation and quenching of traditional iridium complexes by using MOF coordination anchoring and pore space confinement effect, and realizes ultrasensitive quantitative detection of P-tau 181. It exhibits excellent specificity, reproducibility and stability in plasma samples.

[0023] The ECL sensor constructed in this invention exhibits excellent detection performance for P-tau 181, with a detection limit as low as 0.12 fg·mL⁻¹. -1 The linear range is wide, from 0.0001 to 100 ng / mL. -1 The sensor exhibits superior detection performance compared to existing related technologies. It is simple to operate, low in cost, and can be directly used to detect P-tau 181 in plasma samples without complex sample pretreatment. It provides an efficient and reliable analytical tool for the early screening and clinical diagnosis of Alzheimer's disease and has broad application prospects in the preparation of early AD diagnostic reagents and kits. Attached Figure Description

[0024] Figure 1 This is a technical roadmap for the present invention.

[0025] Figure 2 Scanning electron microscope images and elemental distribution diagrams of the Ir@UiO-67 composite material.

[0026] Figure 3 The results of X-ray photoelectron spectroscopy analysis of Ir@UiO-67 are shown.

[0027] Figure 4 The image shows a comparison of the ECL emission spectrum and photoluminescence (PL) of Ir@UiO-67.

[0028] Figure 5 The ECL response curves and linear relationship diagrams of the sensor of the present invention to different concentrations of P-tau 181 are shown.

[0029] Figure 6To demonstrate the specificity of the sensor of this invention, bovine serum albumin (BSA), tumor necrosis factor-α (TNF-α), Alzheimer's disease-associated neurofilament protein (AD7c-NTP), immunoglobulin G (IgG), C-reactive protein (CRP), interleukin-6 (IL-6), and interferon-γ (IFN-γ) were used as interfering substances, and the selectivity of the sensor for each of these substances was determined. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the embodiments. The following embodiments are only used to illustrate the present invention, but do not limit the scope of the present invention in any way.

[0031] Example 1: Preparation of the Ir@UiO-67 complex (1) Weigh H2bpdc (biphenyl dicarboxylic acid) and H2dcbpy (2,2'-bipyridine-5,5'-dicarboxylic acid) as organic ligands in a molar ratio of 3:1, and add zirconium tetrachloride, benzoic acid, and place them in a solution of N,N-dimethylformamide (DMF) to prepare UiO-67 powder; (2) Take 19.16 mg Dissolve it in a mixed solution of 1 mL dichloromethane and 1 mL methanol and stir until completely dissolved to obtain an iridium dimer solution; (3) Add 35.53 mg of UiO-67 powder prepared in step (1) to the above iridium dimer solution, sonicate for 10 min to make UiO-67 uniformly dispersed, and then place the mixed solution in a 55 ℃ water bath for 24 h to obtain an orange precipitate; (4) The orange precipitate was rinsed repeatedly with dichloromethane and methanol in sequence, and then the precipitate was soaked in methanol for 3 days, with fresh methanol solution replaced every 24 hours to remove unbound iridium dimers. (5) The solid sample was collected by centrifugation and dried in a vacuum drying oven to constant weight to obtain the Ir@UiO-67 complex.

[0032] Example 2: Fabrication of Ab2-Ir@UiO-67 signal probe (1) Weigh an appropriate amount of the Ir@UiO-67 nanocomposite prepared in Example 1, disperse it in 0.1 mol / L PBS (pH 7.4), and prepare a 1 mg / mL Ir@UiO-67 solution; (2) Take 600 μL of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) / NHS (N-hydroxysuccinimide) mixed solution (10 mmol / L EDC, 40 mmol / L NHS) and add it to 1 mL of the above Ir@UiO-67 solution. Stir magnetically at 4 °C for 2 h to activate the carboxyl groups on the surface of Ir@UiO-67. (3) Add 200 μL of P-tau 181 secondary antibody with a concentration of 1 μg / mL to the activated Ir@UiO-67 solution and incubate slowly at 4℃ for 12 h to allow Ir@UiO-67 to undergo a coupling reaction with P-tau 181 secondary antibody; (4) Centrifuge the solution after reaction at 10000 r / min for 3 min, discard the supernatant and collect the precipitate; (5) Wash the precipitate three times with PBS at pH 7.4, then redisperse the precipitate in PBS and incubate on a shaker at 4 °C for 6 h to allow the unbound P-tau 181 secondary antibody to be fully desorbed. (6) Remove unbound P-tau 181 secondary antibody by high-speed centrifugation, and redisperse the final product in 1 mL of pH 7.4 PBS to obtain the Ab2-Ir@UiO-67 signal probe, which is stored at 4 ℃ for later use.

[0033] Example 3: Construction of an electrochemiluminescence sensor based on a UiO-67 confined unit-site iridium complex (1) Pretreatment of BDD electrode: The BDD electrode was ultrasonically cleaned with alumina powder and ultrapure water in sequence to remove physical adsorbed impurities on the surface, and then dried with nitrogen to obtain a clean BDD electrode. (2) Preparation of gold film modified BDD electrode: The clean BDD electrode was immersed in 10 mmol / L HAuCl4 solution. Using Ag / AgCl as the reference electrode, a thin gold film was deposited on the surface of the BDD electrode by constant potential electrochemical deposition for 60 s at a potential of -0.6 V. Then the electrode surface was rinsed with ultrapure water and dried with nitrogen to obtain the gold film modified BDD electrode. (3) Preparation of primary antibody modified detection substrate: 8 μL of P-tau 181 primary antibody was dropped onto the surface of the gold membrane modified BDD electrode. The electrode was placed in a constant temperature incubator at 37 ℃ and incubated for 1 h to allow the P-tau 181 primary antibody to be firmly fixed on the gold membrane surface to form an antibody membrane. Then, 3 μL of 0.1% BSA solution was dropped to block the non-specific active sites on the electrode surface. The electrode surface was washed three times with 0.10mol / L PBS at pH 7.4 and dried with nitrogen to obtain the primary antibody modified detection substrate. (4) Construction of sandwich immunosensor: The above-mentioned primary antibody modified detection substrate and the standard solution or test solution containing P-tau 181 were incubated at 37 °C for 40 min to allow P-tau 181 to specifically bind to the primary antibody. The electrode surface was washed with 0.10 mol / L PBS at pH 7.4 to remove unbound antigen. Subsequently, 8 μL of the Ab2-Ir@UiO-67 signal probe prepared in this invention was dropped onto the electrode surface and incubated at 37 °C for 1 h to allow the secondary antibody to specifically bind to the antigen, forming a sandwich immunosensor structure of "primary antibody-antigen-secondary antibody". The electrode surface was washed three times with 0.10 mol / L PBS at pH 7.4 and dried with nitrogen to obtain an electrochemiluminescence sensor based on the UiO-67 confined unit site iridium complex.

[0034] Application Example 1: Scanning electron microscope image and elemental distribution map of Ir@UiO-67 composite material The size and morphology of UiO-67 and Ir@UiO-67 were studied using scanning electron microscopy. Figure 2 As shown in Figure A, UiO-67 exhibits a regular octahedral shape. The elemental composition of Ir@UiO-67 was characterized using energy dispersive spectroscopy (EDS) and structural electron microscopy (SEM). Figure 2 As can be seen from Figure B, C, N, O, Zr and Ir elements are uniformly distributed in the synthesized Ir@UiO-67, providing solid evidence for the successful synthesis of coordinated confined iridium complexes.

[0035] Application Example 2: X-ray photoelectron spectroscopy analysis of Ir@UiO-67 XPS analysis was performed to further investigate the chemical states of Zr, O, C, N, and Ir elements in Ir@UiO-67. Figure 3 As shown, It is immobilized through coordination between Ir in Ir@UiO-67 and the N atom of dcbpy, rather than through physical adsorption.

[0036] Furthermore, to monitor the evolution and decay of ECL emission from Ir@UiO-67, three-dimensional (3D) ECL potential-wavelength images and corresponding two-dimensional (2D) contour plots were recorded under a potential scan range of 0 V to 1.7 V, as shown below. Figure 4 As shown in Figures A and B, the maximum electroluminescence emission wavelength of Ir@UiO-67 is 622 nm. For comparison, the PL spectrum of Ir@UiO-67 (…) Figure 4The maximum emission wavelength of the C spectrum is 654 nm, and the excitation wavelength is (Ex = 425 nm). Compared with the PL spectrum, the ECL emission spectrum shows a blue shift of approximately 32 nm, which may be due to self-absorption or instrumentation effects.

[0037] Application Example 3: Determination of AD7c-NTP using the bioelectrochemical sensor of the present invention. Potentiometric testing employed a three-electrode system, using the modified electrode of this invention as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. At 0.1 mol·L⁻¹ -1 PBS, 30 mmol / L -1 In a triethanolamine (pH 7.4) electrolyte, at 100 mV.s -1 The scan rate was measured using linear scanning voltammetry within the range of 0–1.7 V, and the ECL response signal of the sensor was recorded. For example... Figure 5 As shown in Figure A, by measuring different concentrations of P-tau 181 standard solutions, the ECL intensity was recorded when different concentrations of P-tau 181 standard solutions were detected, and a standard curve was plotted as the logarithm of ECL intensity versus concentration. Figure 5 As shown in Figure B, the concentration of P-tau 181 was 0.0001~100 ng·mL. -1 At that time, the linear regression equation was I = 2993.87lgC + 14679.45 (R² = 0.9985). Based on a signal-to-noise ratio (S / N) of 3, the detection limit of this sensor is 0.12 fg / mL. -1 This indicates that the electrochemiluminescence sensor has high sensitivity for the determination of P-tau 181 standard solution.

[0038] In actual plasma sample testing, based on the ECL signal intensity response and the above linear equation, the concentration of P-tau 181 in the human plasma can be determined, thereby achieving quantitative detection.

[0039] Application Example 4: Performance Evaluation of This Bio-ECL Sensor To demonstrate the specificity of the proposed detection method, the ECL response of P-tau 181 was monitored in the presence of other interfering substances (such as BSA, IgG, TNF-α, CRP, IL-6, IFN-γ, AD7c-NTP, and mixtures of interfering substances). Figure 6As shown in Figure A, only P-tau 181 showed a significant ECL signal, while the ECL signal intensities of the other interfering substances were extremely low. Furthermore, the response change was less than 1.01% when all interfering substances coexisted with P-tau 181, indicating that the designed sensor exhibits high selectivity for P-tau 181. This high selectivity can be attributed to the specific binding ability of the P-tau 181 antibody to the antigen. Therefore, this ECL sensing strategy can distinguish P-tau 181 from other interfering substances in plasma.

[0040] Due to the stability of the substrate electrode and the biocompatible microenvironment, this method exhibits good reproducibility. Repeatability tests were conducted using six independent sensors to detect P-tau 181, with a relative standard deviation of 1.60%. Figure 6 B. To investigate the stability of the sensor of this invention, the same sensor was used to test 1 pg·mL⁻¹ of samples. -1 100 pg / mL -1 and 1 ng.mL -1 The P-tau181 was measured 10 times repeatedly, such as Figure 6 As shown in Figure C, the relative standard deviations of their ECL signal responses are 1.34%, 0.43%, and 1.35%, respectively.

[0041] Application Example 5: Determination of AD7c-NTP in Plasma Samples To verify the practical application performance and reliability of the developed ECL biosensor in complex clinical matrices, this study performed five parallel assays on seven human plasma samples with known concentrations provided by the hospital to evaluate its clinical detection potential. Table 1 shows that the recovery rate of this method for actual plasma samples was 99.56%–103.4%, with a relative standard deviation (RSD) of 0.14%–1.99%. The recovery rate was close to 100%, and the RSD was far below 5%, indicating that the sensor has good accuracy and reproducibility. These results confirm that the constructed ECL biosensor can reliably detect P-tau 181, a core biomarker of Alzheimer's disease (AD) in blood, providing effective technical support for blood-based AD diagnosis and possessing great potential to meet the urgent needs of clinical applications.

[0042] Table 1. Plasma sample test results

Claims

1. A UiO-67 confined unit-point iridium complex signal probe, characterized in that, The Ir@UiO-67 complex with the following structure was co-incubated with P-tau 181 secondary antibody Ab2 and linked through a coupling reaction to prepare the Ab2-Ir@UiO-67 signal probe. The structural formula of the Ir@UiO-67 complex is as follows: Abbreviation: Ir@UiO-67; The luminescent material in the signal probe is an iridium(III) complex; The iridium(III) complex is anchored by forming a coordinate bond with the nitrogen atom of the 2,2'-bipyridine-5,5'-dicarboxylic acid ligand in the UiO-67 framework, and the iridium(III) complex is confined on the surface of the porous UiO-67 nanocarrier. The iridium(III) complex has the following structural formula: 。 2. An electrochemiluminescence sensor based on a UiO-67 confined unit-site iridium complex, characterized in that, It is prepared through the following steps: (1) The BDD electrode was polished to a mirror surface with alumina polishing powder and blown with nitrogen. A gold film was deposited in HAuCl4 solution at a constant potential to obtain the modified electrode. (2) Add the P-tau 181 primary antibody Ab1 solution to the surface of the modified electrode prepared in step (1), incubate together, and then seal the electrode surface with BSA solution; (3) The electrode obtained in step (2) was incubated with P-tau 181 containing different concentrations, and then the electrode was rinsed with pure water. (4) The activated Ir@UiO-67 was incubated with Ab2, and then centrifuged to discard the supernatant to obtain the Ab2-Ir@UiO-67 complex. The complex was dispersed in a buffer solution and stored for later use. (5) Take the Ab2-Ir@UiO-67 suspension and drop it onto the surface of the electrode obtained in step (3). After incubation, a sandwich immune structure "Ab1-P-tau181-Ab2-Ir@UiO-67" is formed. Then rinse the electrode with PBS.

3. The application of the electrochemiluminescence sensor based on the UiO-67 confined unit-point iridium complex as described in claim 2, characterized in that, Using it as the working electrode, a standard curve was prepared by electrochemical method, and it was used for the quantitative detection of P-tau 181 in blood for non-diagnostic treatment.

4. The application of the electrochemiluminescence sensor based on the UiO-67 confined unit-point iridium complex as described in claim 3, characterized in that, The potential test uses a three-electrode system, with the electrode described in claim 2 as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The intensity of the electrochemiluminescence signal generated by different concentrations of P-tau 181 is detected, and a standard working curve is plotted. Based on the standard working curve, P-tau 181 in the test sample is quantitatively detected.

5. The application of the UiO-67 confined unit-point iridium complex signal probe as described in claim 1, characterized in that, It was prepared into a diagnostic kit for detecting Alzheimer's disease.

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