Visual quantitative analysis method for alpha fetoprotein in saliva

Abstract

The invention belongs to the technical field of alpha fetoprotein visual quantitative analysis, and particularly relates to a visual quantitative analysis method for alpha fetoprotein in saliva, a double-recognition multiple-quenching molecular imprinting and electrochemical luminescence microscope is constructed, and MI-ECLM takes self-developed SECLM as a signal output device and comprises a molecular imprinting polymer and a double-mechanism quencher; preparing a saliva sample and performing pretreatment; the pretreated saliva sample is placed on an MI-ECLM sensor, and an optical image is obtained through electrochemical luminescence imaging; and analyzing the optical image to realize visual quantitative detection of alpha fetoprotein in saliva.

Description

Technical Field

[0001] This invention belongs to the field of visual quantitative analysis technology of alpha-fetoprotein, and particularly relates to a method for visual quantitative analysis of alpha-fetoprotein in saliva. Background Technology

[0002] Alpha-fetoprotein (AFP) is a key biomarker for the early diagnosis of malignant diseases such as liver cancer, and its quantitative detection is of great significance for disease screening, efficacy monitoring, and prognostic assessment. Traditional AFP detection methods mostly rely on blood samples, which require invasive collection methods, are cumbersome, and can easily cause discomfort to patients. Saliva samples, on the other hand, have unique advantages such as non-invasive collection, convenient acquisition, and real-time monitoring, and are gradually becoming the ideal sample type for biomarker detection. However, the concentration of AFP in saliva is extremely low and the matrix is ​​complex. Existing detection technologies, such as traditional electrochemiluminescence immunoassay, although possessing a certain sensitivity, rely on large equipment such as photomultiplier tubes, making it difficult to achieve on-site visual detection. Furthermore, the single recognition element and quenching mechanism lead to insufficient selectivity and a high risk of false positive results, failing to meet the needs of rapid point-of-care testing and early diagnosis.

[0003] Molecularly imprinted polymers (MIPs) have been widely used in biosensor construction due to their low preparation cost, good stability, and strong specific recognition capabilities. However, traditional MIP sensors often employ single recognition and single quenching mechanisms, resulting in drawbacks such as high detection limits and weak signal responses. Furthermore, existing visualization detection technologies often struggle to balance sensitivity and reliability, limiting their application in the detection of low-concentration biomarkers. Addressing the technical challenges of AFP detection in saliva, there is an urgent need to develop a quantitative analysis method that combines high sensitivity, high selectivity, visualization, and convenience. This would overcome the limitations of existing technologies in sample applicability, detection performance, and application scenarios, thereby facilitating early disease diagnosis and rapid on-site monitoring. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned technical problems by providing a method for the visual quantitative analysis of alpha-fetoprotein in saliva.

[0005] In view of this, the present invention provides a method for visual quantitative analysis of alpha-fetoprotein in saliva, comprising the following steps: Step 1: Construct a molecularly imprinted electrochemiluminescence microscope with dual recognition and multiple quenching. The MI-ECLM uses a self-developed SECLM as the signal output device and contains a molecularly imprinted polymer and a dual-mechanism quencher. Step 2: Prepare saliva samples and pretreat them; Step 3: Place the pretreated saliva sample on the MI-ECLM sensor and acquire optical images through electrochemiluminescence imaging; Step 4: Analyze the optical images to achieve visualized quantitative detection of alpha-fetoprotein in saliva.

[0006] Preferably, the dual-mechanism quencher in step one is an AuNRs@PEI@Fc-Ab complex formed by modifying the surface of gold nanorods, polyethyleneimine, and ferrocene complex with AFP monoclonal antibody. The AuNRs and Fc generate quenching effects through different mechanisms.

[0007] Preferably, the synthesis process of AuNRs@PEI@Fc includes the following steps: Seed solution preparation: Add 0.2 mL of 0.01 mol / L tetrachloroauric acid solution to 9.75 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution, then quickly inject 0.6 mL of freshly prepared 0.01 mol / L ice-cold sodium borohydride solution. Stir vigorously for 12 min. After the solution changes from yellow to yellowish-brown, incubate in a 27°C water bath for 1.5 h. Preparation of growth solution: 0.5 mL of 0.01 mol / L tetrachloroauric acid solution was added to 8 mL of 0.1 mol / L CTAB solution, followed by 0.05 mL of 0.01 mol / L silver nitrate solution, 0.2 mL of 1 mol / L hydrochloric acid and 80 μL of 0.1 mol / L ascorbic acid solution, and finally 2 mL of the seed solution prepared in step one. The solution was incubated in a water bath at 27°C for 18 h. The resulting purple-red solution was centrifuged at 12000 rpm for 20 min, washed three times with ultrapure water, dispersed in 1 mL of water, and stored at 4°C to obtain AuNRs solution. Preparation of AuNRs@PEI: Take 1 mL of AuNRs solution, add 5 μL of 1% Tween 20, mix well, centrifuge to remove the supernatant, disperse in 1 mL of ultrapure water, add 2 mL of 10 mg / mL polyethyleneimine solution, mix and react for 1 h, and then... After the bonds are fixed, centrifuge at 8000 rpm for 10 min to remove excess PEI, and then redisperse in 1 mL PBS; Preparation of AuNRs@PEI@Fc: 1 mL of 0.1 mmol / L 1,1'-ferrocene dicarboxylic acid (Fc(COOH)2) solution was reacted with 1 mL of 40 / 10 mmol / L EDC / NHS mixture for 15 min to activate the carboxyl groups. 1 mL of AuNRs@PEI solution was added and the mixture was stirred for 3 h. After centrifugation to remove excess solute, the mixture was dispersed in 1 mL of PBS.

[0008] Preferably, the preparation steps of AuNRs@PEI@Fc-Ab are as follows: add 500 μL of 50 μg / mL AFP monoclonal antibody to the AuNRs@PEI@Fc solution, stir at 37°C for 2 h, centrifuge to remove the supernatant, add 500 μL of 1% bovine serum albumin (BSA) solution and stir for 2 h to block non-specific active sites, centrifuge at 8000 rpm for 10 min to remove excess BSA, and disperse in 1 mL PBS.

[0009] Preferably, the method for preparing the MIP in step one is as follows: using catechol as a monomer and AFP as a template molecule, the MIP film is formed by electropolymerization on the surface of a glassy carbon electrode through cyclic voltammetry, specifically including electrode pretreatment and electropolymerization process.

[0010] Preferably, the specific parameters for the preparation of the MIP are as follows: Electrode pretreatment: The GCE electrode was polished to a mirror finish with 0.3μm and 0.05μm Al2O3 powders, then sonicated with ethanol / ultrapure water for 1 min, and dried with nitrogen for later use. Electropolymerization system: Prepare a 0.1 mol / L, pH 7.4 PBS solution containing 2.5 mmol / L catechol and 1 μg / mL AFP antigen; Electropolymerization conditions: The pretreated GCE electrode was placed in the above polymerization system, and cyclic voltammetry was used to... Scan 20 times within the potential range at a rate of 0.05 V / s; Template elution: The electropolymerized electrode was immersed in 50% ethanol aqueous solution and stirred for 1 h, then transferred to PBS solution and stirred for 10 min, rinsed with pure water and air-dried.

[0011] Preferably, the assembly steps of the MI-ECLM sensor in step one are as follows: 6 μL of AFP antigen solution of different concentrations is dropped onto the surface of the MIP-modified GCE electrode, incubated at 37°C for 50 min, rinsed with ultrapure water, and air-dried. Then, 8 μL of AuNRs@PEI@Fc-Ab solution is dropped onto the electrode, incubated at 37°C for 2 h, and rinsed with ultrapure water.

[0012] Preferably, the detection parameters for electrochemiluminescence imaging in step three are: Reaction solution: PBS solution containing 20 μmol / L ruthenium terpyridine chloride hexahydrate and 80 mmol / L tripropylamine; Excitation potential: 1.4V; Exposure time: 10s; ISO: 6400; Image analysis: Image J 1.53t software was used to process the images and obtain the average gray value for quantitative analysis.

[0013] Preferably, the pretreatment method for the saliva sample in step two is as follows: in the morning on an empty stomach, without rinsing the mouth or brushing the teeth and under no irritant conditions, collect 0.2 mL of naturally flowing clear saliva, dilute it tenfold with PBS and set it aside for later use.

[0014] The beneficial effects of this invention are: This invention significantly improves the core performance of AFP detection in saliva through an innovative dual recognition and multiple quenching mechanism. The dual specificity recognition of molecularly imprinted polymers and AFP monoclonal antibodies effectively avoids interference from the complex saliva matrix, drastically reducing false positive results and significantly improving detection reliability. Meanwhile, the hindering effect of the MIP, the internal filtration effect of gold nanorods, and the electron transfer quenching effect of ferrocene synergistically form a powerful quenching system, reducing the method's detection limit to as low as 4.5 fg / mL. It exhibits a good linear relationship within the concentration range, and its sensitivity and analytical performance are comparable to those of the electrochemiluminescence method using traditional photomultiplier tube detection. It has successfully solved the technical problem of extremely low AFP concentration in saliva and the difficulty in accurate quantification.

[0015] Meanwhile, this invention utilizes a self-developed SECLM as the signal output device, enabling intuitive visualization of the detection results. Combined with a non-invasive and convenient saliva sample collection and simple preprocessing procedure, it eliminates the need for large, precision equipment, meeting the practical needs of point-of-care and rapid on-site testing. This overcomes the limitations of traditional detection methods, which rely on invasive sampling, are cumbersome to operate, and cannot provide real-time monitoring. This method not only broadens the application scenarios of electrochemiluminescence imaging technology in biomarker detection but also provides an efficient tool for early disease diagnosis and real-time monitoring of treatment. Furthermore, it offers innovative ideas for optimizing the performance of molecularly imprinted sensors through dual recognition and multiple quenching, demonstrating broad application prospects in medical diagnostics and life science research. Attached Figure Description

[0016] Figure 1 (A) Flowchart of AuNRs@PEI@Fc-Ab fabrication, (B) Flowchart of MI-ECLM sensor construction, (C) Schematic diagram of MI-ECLM and (D) Schematic diagram of quenching mechanism.

[0017] Figure 2 (A) TEM image of AuNRs, (B) XRD patterns of AuNRs and AuNRs@PEI@Fc, (C) UV-Vis spectra of Fc, AuNRs and AuNRs@PEI@Fc and ECL spectrum of Ru-bpy, (D) TEM image of AuNRs@PEI@Fc and corresponding elemental distribution maps (D1-D5), (E) XPS full spectrum of AuNRs@PEI@Fc and (F) high-resolution spectrum of Au and (G) high-resolution spectrum of Fe.

[0018] Figure 3 (A) AFM image of MIP / without AFP, (B) Atomic force microscopy image of MIP / AFP.

[0019] Figure 4 Effects of different experimental conditions on sensing performance: (A) concentration of co-reactant TPA; (B) elution time; (C) incubation time. (Detection conditions: ECL: reaction solution: PBS (containing 20 μmol / L Ru(bpy)3Cl2 and 80 mmol / L TPA), excitation potential: 1.4 V, exposure time: 10 s, sensitivity (ISO): 6400, AFP: 100 pg / mL, error bar = standard deviation, n = 3).

[0020] Figure 5 (A) ECL spectrum, (B) CV curve and (C) EIS plot of different materials modified on GCE surface, (D) fluorescence lifetime plot of Ru-bpy and Ru-bpy+AuNRs mixture, (E) SEM image of MI-ECLM sensor on electrode surface, (F) DPV curves of TPA, Ru-bpy and Fc. (Detection conditions (A): ECL reaction solution containing PBS (20 μmol / L Ru(bpy)3Cl2 and 80 mmol / L LTPA), potential scan range: 0-1.4 V, potential scan rate: 0.1 V / s. Detection conditions (B, C): containing 0.1 mol / L KCl and (0.1 mol / L PBS (pH 7.4) solution).

[0021] Figure 6 (A) Electrochemiluminescence imaging at different AFP concentrations, (B) Linear relationship between AFP concentration and quenching efficiency.

[0022] (Detection conditions: reaction solution: PBS (containing 20 μmol / L Ru(bpy)3Cl2 and 80 mmol / L TPA), excitation potential: 1.4 V, exposure time: 10 s, sensitivity (ISO): 6400. error bar = standard deviation, n=3). Detailed Implementation

[0023] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0024] A dual-mechanism quencher was designed and introduced into a traditional MI-ECL sensor using a monoclonal antibody with specific recognition capabilities. This adds two quenching pathways to the existing MIP-inhibiting quenching effect, improving analytical performance while significantly enhancing selectivity and reliability due to the presence of two different types of specific recognition elements. Simultaneously, a molecularly imprinted electrochemiluminescence microscopy (MI-ECLM) with dual recognition and multiple quenching was constructed using a self-developed SECLM as the signal output device. This method offers advantages such as excellent analytical performance, high selectivity, and reliable visualization of results. We used catechol as a monomer to form a MIP that specifically recognizes AFP via electropolymerization on a glassy carbon electrode surface. Simultaneously, a gold nanorod-polyethyleneimine-ferrocene complex (AuNRs@PEI@Fc) was used as a quencher, and an AFP monoclonal antibody (Ab) was modified onto its surface to construct a dual-recognition, multiple-quenching MI-ECLM. When AFP is present, it can be specifically recognized and captured by the MIP. At this point, the electrode impedance increases, electron transport is restricted, which is unfavorable for electrochemical reactions, thus reducing the luminescence intensity. The carboxyl groups of AuNRs@PEI@Fc and the amino groups of Ab are bonded via amide bonds to form AuNRs@PEI@Fc-Ab. This is then incubated with AFP and adsorbed onto the electrode surface. Because both AuNRs and Fc have strong quenching effects but different quenching mechanisms, a strong quenching effect is generated, resulting in a significant decrease in the electrode ECL signal. A schematic diagram of the MI-ECLM sensor's construction and sensing principle is shown below. Figure 1 As shown. The dual recognition of MIP and Ab significantly improves the sensor's specificity and avoids false positives. Based on SECLM, rapid, sensitive, and visualized quantitative analysis of AFP was successfully achieved. A multi-mechanism synergistic quenching strategy improved the performance of the MI-ECLM method. This method has advantages such as low detection limit, high sensitivity, and wide linear range, and its performance is comparable to the traditional ECL method using photomultiplier tubes as detectors. This work proposes a novel approach to improve ECLM performance through a dual-recognition, multi-quenching design, broadening the application scope of electrochemiluminescence imaging and potentially enabling rapid and sensitive point-of-care and field detection of biomarkers, aiding in early disease diagnosis and real-time monitoring during treatment. Molecularly imprinted electrochemiluminescence microscopy provides a powerful tool for disease diagnosis, exploration of plant and animal physiological activities, pathogenesis research, and molecular biology research. We believe this method has promising applications in the medical and life sciences fields.

[0025] Field emission transmission electron microscope (JEM-2100, JEOL, Japan); field emission environmental scanning electron microscope (Vario Micro Cube, Elementar, Germany); ultraviolet-visible spectrophotometer (Cary-60, Agilent Technologies, USA); X-ray powder diffractometer (Rigaku D / max2500 / pc, Rigaku, Japan); high-speed centrifuge (H1650-W, Hunan Xiangyi Centrifuge Instrument Co., Ltd., China); electrochemical workstation (CHI660, Shanghai Chenhua Instrument Co., Ltd., China); multi-parameter chemical analysis and detection system (MPI-B, Xi'an Ruimai Analytical Instrument Co., Ltd., China); steady-state transient fluorescence spectrometer (QuantaMaster 8000, HORIBA, Japan); X-ray photoelectron spectrometer (Thermoescalab 250XI, Thermo Fisher Scientific, USA); smartphone (Mi10S, Xiaomi Technology Co., Ltd., China); optical microscope (PH100-2B41L-IPL, Phoenix Optics Co., Ltd., China).

[0026] Tripropylamine (TPA, AR) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Ruthenium terpyridine chloride hexahydrate (98%), catechol (99.0%), tetrachloroauric acid trihydrate (99.9%), 1,1'-ferrocenedicarboxylic acid (Fc(COOH)2, 98%), and hexadecyltrimethylammonium bromide (CTAB, 99%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Silver nitrate (AgNO3, 99.8%), ascorbic acid (99.7%), sodium borohydride (NaBH4, 98.0%), sodium dihydrogen phosphate dihydrate (99.0%), disodium hydrogen phosphate dodecahydrate (99.0%), and hydrochloric acid (38.0%) were purchased from Xilong Chemical Co., Ltd. (Guangdong, China). Bovine serum albumin (BSA) was purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). Alpha-fetoprotein and its corresponding antibody, neuron-specific enolase (NSE), procalcitonin (PCT), carcinoembryonic antigen (CEA), and β2-microglobulin (β2-MG) were purchased from Beijing Bio-Sens Biotechnology Co., Ltd. (Beijing, China). Ultrapure water was used in the experiments. All chemical reagents were used directly without further purification.

[0027] The synthesis of gold nanorods was based on references with some modifications, as follows: Seed solution: Add 0.2 mL of 0.01 mol / L tetrachloroauric acid solution to 9.75 mL of 0.1 mol / L CTAB solution, quickly inject 0.6 mL of freshly prepared ice-cold NaBH4 (0.01 mol / L), stir vigorously for 12 min, and after the solution changes from yellow to yellowish-brown, incubate in a 27°C water bath for 1.5 h.

[0028] Growth solution: 0.5 mL of 0.01 mol / L tetrachloroauric acid solution was added to 8 mL of 0.1 mol / L CTAB solution, followed by the addition of 0.05 mL AgNO3 (0.01 mol / L), 0.2 mL HCl (1 mol / L), and 80 μL ascorbic acid solution (0.1 mol / L). Finally, 2 mL of seed solution was added, and the mixture was incubated in a 27°C water bath for 18 h. A purple-red solution was obtained, centrifuged at 12000 rpm for 20 min, washed three times with ultrapure water, and finally dispersed in 1 mL of water to obtain the AuNRs solution, which was stored at 4°C.

[0029] Take 1 mL of AuNRs solution, add 5 μL of 1% Tween 20, mix well, centrifuge to remove the supernatant, and disperse in 1 mL of ultrapure water. Add 2 mL of PEI solution (10 mg / mL), mix well, and react for 1 h. AuNRs were immobilized in PEI by bonding. After the reaction was complete, the supernatant was removed by centrifugation at 8000 rpm for 10 min to wash away excess PEI. The AuNRs were then redispersed in 1 mL PBS to obtain the AuNRs@PEI solution.

[0030] Meanwhile, 1 mL of Fc(COOH)₂ (0.1 mmol / L) was mixed thoroughly with 1 mL of EDC / NHS (40 / 10 mmol / L) and reacted for 15 min to activate the carboxyl groups. After the reaction was complete, 1 mL of AuNRs@PEI solution was added, and the mixture was stirred for 3 h. Excess solute was removed by centrifugation, and the solution was dispersed in 1 mL of PBS to obtain the AuNRs@PEI@Fc solution.

[0031] Add 500 μL Ab (50 μg / mL) to the AuNRs@PEI@Fc solution, stir at 37°C for 2 h, centrifuge to remove the supernatant, add 500 μL 1% BSA and stir for 2 h to block non-specific active sites, finally centrifuge at 8000 rpm for 10 min, remove the supernatant to remove excess BSA, and disperse in 1 mL PBS to obtain the AuNRs@PEI@Fc-Ab solution.

[0032] First, the electrode was polished onto a polishing cloth with 0.3 μm and 0.05 μm Al2O3 powder to form a mirror surface. Then, it was sonicated with ethanol / ultrapure water for 1 min each time, and finally dried with nitrogen gas for later use.

[0033] The preparation of the MIP was based on references with some modifications. The detailed steps are as follows: Prepare a PBS solution containing 2.5 mmol / L catechol and 1 μg / mL AFP antigen (0.1 mol / L, pH 7.4; unless otherwise specified, the concentration and pH remain unchanged). Place a clean GCE electrode in the above PBS solution and perform cyclic voltammetry... The GCE was scanned 20 times at a rate of 0.05 V / s between 1 V and 1 V to obtain MIP-modified GCE. Finally, the GCE was immersed in 50% ethanol aqueous solution and stirred for 1 h to wash out the antigen template. Then it was transferred to PBS solution and stirred for 10 min to remove residual ethanol. After rinsing with pure water, it was air-dried for later use.

[0034] 6 μL of AFP antigen solutions of different concentrations were dropped onto the electrode surface, incubated at 37°C for 50 min, rinsed with ultrapure water, and air-dried. Then, 8 μL of AuNRs@PEI@Fc-Ab was dropped onto the electrode surface, incubated at 37°C for 2 h, rinsed with ultrapure water, and finally, optical images were obtained under the same conditions.

[0035] Electrochemiluminescence imaging was achieved using a self-developed electrochemiluminescence microscope. The method parameters were as follows: reaction solution: PBS (containing 20 μmol / L Ru(bpy)3Cl2 and 80 mmol / L TPA), excitation potential: 1.4 V, exposure time: 10 s, and sensitivity (ISO): 6400. The obtained images were processed using ImageJ software (version: 1.53t, National Institutes of Health, USA) to obtain the mean gray value for quantitative analysis, denoted as G.

[0036] In the morning, without rinsing your mouth or brushing your teeth, and on an empty stomach, without any irritants, allow saliva to flow naturally. Collect 0.2 mL of the clear saliva in a sterile centrifuge tube. After centrifuging the saliva sample at 3000 rpm for 10 min, aspirate the supernatant and dilute it tenfold with PBS before analysis.

[0037] To confirm the successful synthesis of AuNRs@PEI@Fc and its potential as a quencher, it was characterized using transmission electron microscopy (TEM), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and UV-Vis spectrophotometry. Figure 2As can be seen from A, AuNRs exhibit a typical rod-like structure with an average length of 31.9 nm ± 3.8 nm and an average diameter of 8.2 nm ± 0.7 nm (statistics of 120). The XRD pattern of AuNRs shows four diffraction peaks, corresponding to the (110), (200), (220), and (311) crystal planes. Figure 2 (B black line) confirms the face-centered cubic structure of gold. These results demonstrate the successful synthesis of AuNRs. The XRD pattern of AuNRs@PEI@Fc is basically consistent with the diffraction peak positions of AuNRs ( Figure 2 (B-line red) indicates that the PEI encapsulation and Fc modification did not alter the gold lattice structure. From Figure 2 As shown in Figure C, AuNRs exhibit broad absorption peaks at 520 nm and 700 nm (red lines). The synthesis of AuNRs@PEI@Fc did not alter the intrinsic absorption peaks of AuNRs (blue lines), indicating that the composite material has the potential to serve as a quenching material. The TEM image shows that AuNRs are encapsulated by PEI, forming irregular clusters with a size of approximately 400 nm (Figure C). Figure 2 D), as can be seen from the elemental distribution diagram, AuNRs@PEI@Fc is mainly composed of elements such as C, N, O, Au, and Fe, with Fe element evenly distributed on the surface of the PEI clusters (Figure). Figure 2 D1-D5). The XPS full spectrum shows that AuNRs@PEI@Fc is mainly composed of C, N, O, Au, and Fe elements, with contents of 58.83%, 8.68%, 27.67%, 3.41%, and 1.42%, respectively. Figure 2 E). After peak separation of the XPS high-resolution plot of Au 4f, double peaks of 83.74 eV and 87.41 eV were obtained, corresponding to the binding energies of Au 4f7 / 2 and Au4f5 / 2, respectively, consistent with the values ​​of the Au0 state. Figure 2 F). After peak separation of the XPS high-resolution image of Fe 2p, double peaks at 710.45 eV and 723.25 eV were obtained, corresponding to Fe 2p and Fe 2p, respectively. 2+ 2p3 / 2 and Fe 2+ The binding energy of 2p¹ / ² indicates that the iron element in ferrocene exists in a divalent form. Figure 2 G). The above results indicate the successful synthesis of AuNRs@PEI@Fc.

[0038] To verify the preparation of the electropolymerized molecularly imprinted film and its recognition of target analytes, the electrode surface was characterized using AFM. Figure 3 As can be seen from A, after electropolymerization, the electrode surface is covered with a thin film with a height of approximately 26 nm, exhibiting a rough, porous structure; from Figure 3As shown in Figure B, after MIP binds to the target, the thin film on the electrode surface thickens to about 40 nm, and the membrane surface is filled with protein to form an irregular blocky structure. These results indicate the successful synthesis of the MIP membrane, which also has the ability to specifically recognize the target.

[0039] Figure 2 (A) TEM image of AuNRs, (B) XRD patterns of AuNRs and AuNRs@PEI@Fc, (C) UV-Vis spectra of Fc, AuNRs and AuNRs@PEI@Fc and ECL spectrum of Ru-bpy, (D) TEM image of AuNRs@PEI@Fc and corresponding elemental distribution maps (D1-D5), (E) XPS full spectrum of AuNRs@PEI@Fc and (F) high-resolution spectrum of Au and (G) high-resolution spectrum of Fe.

[0040] To achieve optimal sensing performance, we optimized experimental conditions such as co-reactant concentration, elution time, and incubation time. The results are as follows: Figure 4 As shown, TPA, acting as a sacrificial co-reactant, is gradually consumed during the electrochemical reaction. Sufficient TPA concentration is crucial for ensuring the luminescent performance of the photoluminescent material. As the TPA concentration increases from 10 mmol / L to 80 mmol / L, the average gray value gradually increases, reaching an optimal value at 80 mmol / L. When the TPA concentration increases from 80 mmol / L to 100 mmol / L, the average gray value decreases. This may be due to the competitive oxidation reaction between excess TPA and ruthenium terpyridine on the electrode surface. Therefore, 80 mmol / L TPA was chosen as the co-reactant. Figure 4 A). Template elution is a crucial step in the construction of the MIP sensor. Elution time determines the adequacy of elution. As elution time increases, the average gray value first increases and then tends to plateau, reaching its maximum at 60 min, indicating that elution is complete at 60 min. Figure 4 B). Sufficient incubation time is beneficial to improving binding efficiency. As the incubation time increases, the average gray value first decreases and then tends to stabilize, reaching its optimal value at 50 min. Therefore, the incubation time is set to 50 min. Figure 4 C).

[0041] Figure 4 Effects of different experimental conditions on sensing performance: (A) concentration of co-reactant TPA; (B) elution time; (C) incubation time.

[0042] (Detection conditions: ECL: reaction solution: PBS (containing 20 μmol / L Ru(bpy)3Cl2 and 80 mmol / L TPA), excitation potential: 1.4 V, exposure time: 10 s, sensitivity (ISO): 6400, AFP: 100 pg / mL, error bar=standarddeviation, n=3) from Figure 5 As shown in Figure A, when the electrode surface is modified with MIP, the luminescence intensity is significantly weaker than that of the bare electrode (red line). This is because MIP is an organic membrane with high resistance, which is not conducive to electron transfer and inhibits the electrochemical reaction. When the template molecules on the MIP are eluted, the luminescence intensity is stronger than that of the uneluted electrode (blue line), indicating that the elution of the template molecules exposes the pores on the MIP with specific recognition activity, which improves the electrochemical reaction to some extent. When the template molecules rebind to the MIP, the luminescence intensity is lower than that of the eluted electrode (magenta line), indicating that the pores in the MIP are filled, the electrode surface resistance increases, and the occurrence of the electrochemical reaction is inhibited. When the Ab binds to the target molecule, the luminescence intensity is lower than that of the electrode incubated with an equal amount of target substance (green line). This is because antibodies, as biomolecules, have poor conductivity, which further increases the electrode surface resistance and is not conducive to the occurrence of the electrochemical reaction. When the electrode surface is modified with AuNRs-Ab, the luminescence intensity is significantly lower than that of the electrode bound with an equal amount of Ab (dark blue line), indicating that AuNRs have a quenching effect on the luminescence of Ru-bpy. When the electrode surface was modified with AuNRs@PEI@Fc-Ab, the luminescence intensity further decreased (purple line). Since PEI has no quenching effect on Ru-bpy (orange line), it indicates that Fc also has a quenching effect on the luminescence of Ru-bpy. The combination of AuNRs and Fc forms a double quenching effect, improving quenching efficiency and thus improving detection sensitivity. The above results show that the quenching effect is mainly generated by three parts: MIP, AuNRs, and Fc.

[0043] To verify the quenching mechanism of MIP, electrochemical characterization was performed on different materials. From Figure 5 As shown in Figure B, after MIP is formed on the electrode surface through electropolymerization, the current peak of the corresponding CV curve drops sharply to almost zero (red line), indicating that electrons have difficulty passing through the MIP. After template elution, the MIP exposes pores with specific recognition functions, which is conducive to electron transfer, thus increasing the current peak of the corresponding CV curve (blue line). Subsequently, after a certain amount of target material is recombined, the current peak of the CV curve decreases (green line). After the target material is combined with Ab, the current peak of the CV curve decreases (magenta line). Finally, after modification with AuNRs@PEI@Fc-Ab, due to the conductivity of AuNRs, the current peak of the corresponding CV curve is significantly increased (purple line). Figure 5As shown in Figure C, the bare electrode exhibits low impedance (black line), while the MIP shows high impedance (red line). After template elution, the impedance decreases significantly (blue line), increases further after incubation with a certain amount of antigen (green line), and increases again after Ab binds to the antigen (magenta line). Finally, modification with AuNRs@PEI@Fc-Ab significantly reduces the impedance (purple line). The CV curves are consistent with the EIS spectral results. All these results indicate that the target analyte forms a dense organic film with the MIP, which has poor electrochemical activity, hindering the electrochemical reaction between the luminescent material and the co-reactant on the electrode surface. This inhibits the occurrence of the electrochemical reaction and thus suppresses ECL. Therefore, the mechanism by which the binding of the MIP to the target analyte quenches ECL is due to the "impedance effect."

[0044] To investigate the quenching mechanism of AuNRs on Ru-bpy. From Figure 2 As shown in Figure C, AuNRs exhibit a large absorption peak (black line) between 580 nm and 800 nm, while the electrochemiluminescence spectrum of Ru-bpy peaks at 650 nm, indicating spectral overlap. Therefore, the quenching mechanism may be an internal filtering effect or resonant energy transfer. The fluorescence lifetime of Ru-bpy was determined using steady-state transient fluorescence spectroscopy. Without AuNRs, the fluorescence lifetime was 1.09 ns, while with the addition of AuNRs, it was 1.04 ns. Figure 5 D), the fluorescence lifetime did not change significantly, ruling out the possibility of resonance energy transfer; in addition, the SEM image of the MI-ECLM sensor shows that the material has a porous layered structure, indicating that the luminescent material and co-reactant can enter through the pores, reach the electrode surface to generate ECL, and be absorbed by AuNRs, providing the spatial conditions for generating an internal filtration effect. Figure 5 Therefore, the quenching mechanism of AuNRs on Ru-bpy is the internal filtration effect.

[0045] Fc, as a reducing agent, can participate in the ECL reaction of Ru-bpy. Figure 5 As can be seen from F, the oxidation potential of Fc is 0.52 V, the oxidation potential of TPA is 0.87 V, and the oxidation potential of Ru(bpy)3 is... 2+ The oxidation potential of Fc is 1.08 V, indicating that Fc has strong reducing properties and can react with Ru(bpy)3. 3+ and TPA + The reaction consumes Ru(bpy)3. 3+ and TPA + The amount of light-emitting body is reduced. The generation of [something] quenches the ECL. The specific process is as follows: ; ; ECL process: ; ; ; Quenching reaction: ; ; In summary, the sensing mechanism of the MI-ECLM sensor is the "obstruction effect" after the MIP identifies the target object, the internal filtering effect of AuNRs on Ru-bpy, and the consumption of Ru(bpy)3 by Fc. 3+ and TPA + The combination of electron transfer and multiple quenching processes endows this sensor with excellent sensing performance.

[0046] To investigate the analytical performance of the MI-ECLM sensor, we measured electrochemiluminescence imaging at different AFP concentrations. As the AFP concentration increased, the brightness of the MI-ECLM sensor gradually decreased. Figure 6 A). A linear relationship between AFP concentration and quenching efficiency was obtained by calculating the average gray value. There is a good linear relationship between them, and the linear equation is: Correlation coefficient R 2 = 0.9976, detection limit is 4.5 fg / mL ( Figure 6 B). Comparing this method with existing methods (Table 4-1), this method exhibits advantages such as low detection limit, high sensitivity, and intuitive, visual results, possessing analytical performance comparable to traditional ECL sensors. This indicates that this visual quantitative analysis method can be used for rapid, visual, and quantitative detection at the bedside, with a wide range of applications.

[0047] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for visual quantitative analysis of alpha-fetoprotein in saliva, characterized in that: Includes the following steps: Step 1: Construct a molecularly imprinted electrochemiluminescence microscope with dual recognition and multiple quenching. The MI-ECLM uses a self-developed SECLM as the signal output device and contains a molecularly imprinted polymer and a dual-mechanism quencher. Step 2: Prepare saliva samples and pretreat them; Step 3: Place the pretreated saliva sample on the MI-ECLM sensor and acquire optical images through electrochemiluminescence imaging; Step 4: Analyze the optical images to achieve visualized quantitative detection of alpha-fetoprotein in saliva.

2. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 1, characterized in that: The dual-mechanism quencher mentioned in step one is an AuNRs@PEI@Fc-Ab complex formed by modifying the surface of gold nanorods, polyethyleneimine, and ferrocene complex with AFP monoclonal antibody. The AuNRs and Fc quench the antibody through different mechanisms.

3. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 2, characterized in that: The synthesis process of AuNRs@PEI@Fc includes the following steps: Seed solution preparation: Add 0.2 mL of 0.01 mol / L tetrachloroauric acid solution to 9.75 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution, then quickly inject 0.6 mL of freshly prepared 0.01 mol / L ice-cold sodium borohydride solution. Stir vigorously for 12 min. After the solution changes from yellow to yellowish-brown, incubate in a 27°C water bath for 1.5 h. Preparation of growth solution: 0.5 mL of 0.01 mol / L tetrachloroauric acid solution was added to 8 mL of 0.1 mol / L CTAB solution, followed by 0.05 mL of 0.01 mol / L silver nitrate solution, 0.2 mL of 1 mol / L hydrochloric acid and 80 μL of 0.1 mol / L ascorbic acid solution, and finally 2 mL of the seed solution prepared in step one. The mixture was incubated in a water bath at 27°C for 18 h. The resulting purple-red solution was centrifuged at 12000 rpm for 20 min, washed three times with ultrapure water, dispersed in 1 mL of water, and stored at 4°C to obtain the AuNRs solution. Preparation of AuNRs@PEI: Take 1 mL of AuNRs solution, add 5 μL of 1% Tween 20, mix well, centrifuge to remove the supernatant, disperse in 1 mL of ultrapure water, add 2 mL of 10 mg / mL polyethyleneimine solution, mix and react for 1 h, and then... After the bonds were fixed, the mixture was centrifuged at 8000 rpm for 10 min to remove excess PEI and then redispersed in 1 mL PBS. Preparation of AuNRs@PEI@Fc: 1 mL of 0.1 mmol / L 1,1'-ferrocene dicarboxylic acid (Fc(COOH)2) solution was reacted with 1 mL of 40 / 10 mmol / L EDC / NHS mixture for 15 min to activate the carboxyl group. 1 mL of AuNRs@PEI solution was added and the mixture was stirred for 3 h. After centrifugation to remove excess solute, the mixture was dispersed in 1 mL of PBS.

4. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 3, characterized in that: The preparation steps of AuNRs@PEI@Fc-Ab are as follows: 500 μL of 50 μg / mL AFP monoclonal antibody is added to the AuNRs@PEI@Fc solution, stirred at 37°C for 2 h, centrifuged to remove the supernatant, 500 μL of 1% bovine serum albumin solution is added and stirred for 2 h to block non-specific active sites, centrifuged at 8000 rpm for 10 min to remove excess BSA, and dispersed in 1 mL PBS.

5. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 4, characterized in that: The preparation method of MIP in step one is as follows: using catechol as monomer and AFP as template molecule, MIP film is formed on the surface of glassy carbon electrode by electropolymerization through cyclic voltammetry, specifically including electrode pretreatment and electropolymerization process.

6. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 5, characterized in that: The specific parameters for the preparation of the MIP are as follows: Electrode pretreatment: The GCE electrode was polished to a mirror finish with 0.3μm and 0.05μm Al2O3 powders, then sonicated with ethanol / ultrapure water for 1 min, and dried with nitrogen for later use. Electropolymerization system: Prepare a 0.1 mol / L, pH 7.4 PBS solution containing 2.5 mmol / L catechol and 1 μg / mL AFP antigen; Electropolymerization conditions: The pretreated GCE electrode was placed in the above polymerization system, and cyclic voltammetry was used to... Scan 20 times within the potential range at a rate of 0.05V / s; Template elution: The electropolymerized electrode was immersed in 50% ethanol aqueous solution and stirred for 1 h, then transferred to PBS solution and stirred for 10 min, rinsed with pure water and air-dried.

7. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 6, characterized in that: The assembly steps of the MI-ECLM sensor described in step one are as follows: 6 μL of AFP antigen solution of different concentrations is dropped onto the surface of the MIP-modified GCE electrode, incubated at 37°C for 50 min, rinsed with ultrapure water, and air-dried. Then, 8 μL of AuNRs@PEI@Fc-Ab solution is dropped onto the electrode, incubated at 37°C for 2 h, and rinsed with ultrapure water.

8. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 7, characterized in that: The detection parameters for electrochemiluminescence imaging in step three are: Reaction solution: PBS solution containing 20 μmol / L ruthenium terpyridine chloride hexahydrate and 80 mmol / L tripropylamine; Excitation potential: 1.4V; Exposure time: 10s; ISO: 6400; Image analysis: Image J 1.53t software was used to process the images and obtain the average gray value for quantitative analysis.

9. The method for visual quantitative analysis of alpha-fetoprotein in saliva according to claim 8, characterized in that: The pretreatment method for the saliva sample described in step two is as follows: In the morning, on an empty stomach, without rinsing the mouth or brushing the teeth, and under conditions of no irritation, collect 0.2 mL of naturally flowing clear saliva, dilute it tenfold with PBS, and set it aside for later use.

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