A method of constructing an electrochemical immunosensor for detecting apolipoprotein a4
By modifying the electrode with NG-PEI-COF composite material and tracer probe, an electrochemical immunosensor was constructed, which solved the problem of interference in the detection of apolipoprotein A4 and achieved detection results with high sensitivity and high selectivity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- LANZHOU FOCI PHARM CO LTD
- Filing Date
- 2023-08-23
- Publication Date
- 2026-07-10
AI Technical Summary
Existing technologies for detecting apolipoprotein A4 are hampered by interfering substances such as arginine succinate synthase 1, brain-derived neurotrophic factor, heat shock protein 70, and cortisol, which affect the accuracy of detection and make it difficult to achieve selective, reproducible, and sensitive detection of apolipoprotein A4.
An electrochemical immunosensor was constructed by modifying the electrode with NG-PEI-COF composite material, gold nanoparticles, apolipoprotein A4 antibody, and toluidine blue using a method that combines NG-PEI-COF composite material and tracer probes. Detection was performed using square wave voltammetry.
Selective detection of apolipoprotein A4 was achieved, with good anti-interference ability, reproducibility and sensitivity, detection limit as low as 2.16 pg/mL, and linear response in the range of 0.01 ~ 300 ng/mL. It has good reproducibility and high stability.
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Figure CN117110398B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of apolipoprotein A4 detection and analysis, specifically relating to a method for constructing an electrochemical immunosensor for detecting apolipoprotein A4. Background Technology
[0002] Apolipoprotein A4 (Apo-A4), a biomarker closely related to the development of depression, has been revealed and discovered by numerous researchers in recent years. It plays a crucial role in regulating various physiological and emotional / behavioral responses to stressor exposure. Besides apolipoprotein A4, serum biomarkers for depression include arginine succinate synthase 1 (ASS1), brain-derived neurotrophic factor (BDNF), heat shock protein 70 (HSP70), and cortisol. These biomarkers can interfere with the detection of Apo-A4, affecting the accuracy of the test. Summary of the Invention
[0003] The purpose of this invention is to provide a method for constructing an electrochemical immunosensor for detecting apolipoprotein A4. This electrochemical immunosensor can achieve selective detection of apolipoprotein A4 and has good anti-interference ability, reproducibility, stability and sensitivity.
[0004] The technical solution adopted by the present invention to achieve the above objectives is as follows:
[0005] A method for constructing an electrochemical immunosensor for detecting apolipoprotein A4, comprising:
[0006] (1) Preparation of NG-PEI-COF composite material and tracer probe
[0007] Nitrogen-doped graphene and polyethyleneimine were thoroughly stirred and compounded in a solvent, and then bipyridine-functionalized covalent organic framework material was added and thoroughly stirred and compounded to obtain NG-PEI-COF composite material;
[0008] Using bipyridine-functionalized covalent organic framework materials as carriers, gold nanoparticles, apolipoprotein A4 antibody, and toluidine blue were sequentially loaded to obtain tracer probes.
[0009] (2) The NG-PEI-COF composite material, gold nanoparticles, apolipoprotein A4 and tracer probe were sequentially modified on the electrode to obtain the electrochemical immunosensor.
[0010] Preferably, when preparing the NG-PEI-COF composite material, the mass ratio of nitrogen-doped graphene, bipyridine-functionalized covalent organic framework material, and polyethyleneimine is 1~2:1~3:5.
[0011] Preferably, the preparation process of the tracer probe includes:
[0012] Bipyridine-functionalized covalent organic framework material and chloroauric acid were thoroughly mixed in a solvent, and then a reducing agent was added to react and obtain AuNPs / COF. After centrifugation and washing, the AuNPs / COF was resuspended in buffer solution.
[0013] Apolipoprotein A4 antibody and toluidine blue were added sequentially to the AuNPs / COF suspension. After thorough stirring, bovine serum albumin was added for blocking to obtain the tracer probe. After centrifugation and washing, the probe was resuspended in buffer.
[0014] More preferably, the mass ratio of bipyridine-functionalized covalent organic framework material to chloroauric acid is 1:0.3, the mass ratio of AuNPs / COF to toluidine blue is 1:1, and the volume ratio of AuNPs / COF to apolipoprotein A4 antibody solution is 1 mg:10 μL.
[0015] The apolipoprotein A4 antibody solution was obtained by diluting the apolipoprotein A4 antibody 100 times.
[0016] More preferably, the reducing agent is NaBH4, and the amount used is kept in excess relative to chloroauric acid.
[0017] More preferably, the buffer solution is a PBS solution with pH=7.4.
[0018] More preferably, the tracer probe is resuspended in the buffer at a ratio of AuNPs / COF to buffer of 1 mg: 1 mL.
[0019] Preferably, the modification process in step (2) includes:
[0020] First, the NG-PEI-COF composite material was modified onto the electrode using a drop-coating method;
[0021] Then the electrodes were placed in a chloroauric acid solution, and gold nanoparticles were electrodeposited by chronoamperometry.
[0022] Next, lipoprotein A4 solution was added dropwise to the electrode, and after overnight incubation, bovine serum albumin was added for blocking.
[0023] Finally, tracer probe suspension was added to the electrode for incubation to obtain the electrochemical immunosensor.
[0024] Preferably, the amount of NG-PEI-COF composite material used is 4~8 μg.
[0025] More preferably, the amount of NG-PEI-COF composite material used is 6~7 μg;
[0026] Preferably, the electrode is placed in a chloroauric acid solution with a concentration of 0.6~2 mg / mL.
[0027] More preferably, the electrode is placed in a chloroauric acid solution with a concentration of 0.8 to 1 mg / mL.
[0028] Preferably, the electrodeposition voltage is -0.3V and the time is 100~500 s.
[0029] More preferably, the electrodeposition time is 300~400 s.
[0030] Preferably, the volume of apolipoprotein A4 solution used is 10 μL, and the concentration is 0.01~300 ng / mL.
[0031] Preferably, after adding the tracer probe suspension, incubate for 2-3 hours.
[0032] Preferably, the bipyridine-functionalized covalent organic framework material has the following repeating structural units:
[0033] .
[0034] The above-mentioned electrochemical immunosensor is used in the detection of apolipoprotein A4.
[0035] Preferably, square wave voltammetry is used to detect apolipoprotein A4.
[0036] The specific procedures for testing include:
[0037] (I) Electrochemical immunosensors were constructed using a series of apolipoprotein A4 standard solutions according to the above method. The response peak current of each electrochemical immunosensor was measured by square wave voltammetry to obtain the linear relationship between apolipoprotein A4 concentration and response peak current.
[0038] (II) Construct an electrochemical immunosensor using the above method with the apolipoprotein A4 sample solution to be tested. Measure the response peak current of the electrochemical immunosensor using the square wave voltammetry method. Based on the linear relationship obtained in step (I), the concentration of the apolipoprotein A4 sample solution can be calculated. Attached Figure Description
[0039] Figure 1 A schematic diagram illustrating the process for constructing the electrochemical immunosensor of this invention.
[0040] Figure 2 XRD pattern of COF (A), N 1s XPS image of COF (B), infrared characterization of COF, NG, and NG-PEI-COF (C), SEM images of COF (D), NG-PEI-COF (E), and AuNPs / NG-PEI-COF (F).
[0041] Figure 3CV plot (A) and EIS plot (B) of GCE, NG-PEI-COF, AuNPs / NG-PEI-COF, and Apo-A4 / AuNPs / NG-PEI-COF in 0.1 MKCl and 5.0 mM K3[Fe(CN)6] / K4[Fe(CN)6].
[0042] Figure 4 The curves show the effects of the ratio of NG to COF (A), the amount of NG-PEI-COF modification (B), the deposition time of AuNPs (C), the deposition concentration of AuNPs (D), and the incubation time of the tracer label (E) on the current signal.
[0043] Figure 5 The image shows the SWV response (A) of the electrochemical immunosensor of the present invention after incubation with different concentrations of Apo-A4 in PBS solution, and the calibration curve (B) of the SWV response versus the concentration of Apo-A4.
[0044] Figure 6 The present invention relates to an electrochemical immunosensor for detecting the difference in response of Apo-A4 when it coexists with and is present alone with other markers of depression. Detailed Implementation
[0045] The technical solution of the present invention will be further described in detail below with reference to the embodiments.
[0046] The names and corresponding abbreviations of some substances in this invention are as follows:
[0047] Apolipoprotein A4, abbreviated as Apo-A4
[0048] Apolipoprotein A4 antibody, abbreviated as Ab
[0049] Covalent organic framework materials, abbreviated as COF, in most cases in this invention, COF represents bipyridine-functionalized covalent organic framework materials.
[0050] Polyethyleneimine, abbreviated as PEI
[0051] Nitrogen-doped graphene, abbreviated as NG
[0052] Gold nanoparticles, abbreviated as AuNPs
[0053] Toluidine blue, abbreviated as TB
[0054] Bovine serum albumin, abbreviated as BSA
[0055] Unless otherwise specified, the concentration units mM in this invention represent mmol / L and M represents mol / L.
[0056] This invention constructs an electrochemical immunosensor through cascaded amplification of signal transduction, target recognition, and signal conversion: TB-Ab-AuNPs-COF / Apo-A4 / AuNPs / NG-PEI-COF / electrode. It can be used in quantities ranging from 0.01 to 300 ng / mL. -1 Accurate determination of Apo-A4 (I(μA) = 42.70 lgC) within the linear range Apo-A4 (pg mL) -1 +165.5, R 2 =0.9902), with a detection limit as low as 2.16 pg mL. -1 (S / N=3). In addition, the biosensor also has good reproducibility (RSD=2.31%) and stability (98.82% after 7 days of storage at 4°C), and shows good anti-interference performance against a variety of other biomarkers of depression (such as heat shock 70, cortisol, etc.).
[0057] The NG-PEI-COF composite material possesses excellent specific surface area and electron transport capability, enabling first-stage amplification of sensor signals in electrical signal conduction. In this composite material, COF and NG form a nanocomposite through π-π bonding, promoting electron transport efficiency. PEI is used for functional modification to enhance the water solubility of the composite material.
[0058] AuNPs, which are functionalized with bipyridine and coordinate with COF, endow the sensor with good biocompatibility. They can effectively bind to -NH2 and -SH on the surface of antigens and antibodies through covalent bonds, achieving efficient fixation of antigens and antibodies. This provides abundant binding sites for the fixation of the antigen to be tested and realizes a second-stage signal amplification in target recognition and binding.
[0059] To address the issue of insufficient direct detection efficiency due to the lack of redox properties in the antigen itself, a tracer probe was formed by loading AuNPs, Apo-A4 antibody, and toluidine blue using the high loading efficiency of COF and the coordination properties of its internal bipyridine. The signal changes of TB indicate the concentration differences of Apo-A4, achieving a third-stage amplification in sensor signal conversion. Example
[0060] This embodiment details the specific process for constructing an electrochemical immunosensor for sensitive Apo-A4 detection. Figure 1 As shown. Specifically, firstly, bipyridine-functionalized COF materials are prepared ( Figure 1 -A), on the one hand, it is combined with NG, and then functionalized with polyethyleneimine (PEI) to improve its water solubility, to obtain a composite material with good electrical conductivity, NG-PEI-COF ( Figure 1-B) is used as an electrode modification material for electrochemical sensors. AuNPs are then modified by electrochemical deposition, utilizing Au-NH2 and Au-SH interactions to effectively immobilize the Apo-A4 antigen. On the other hand, taking advantage of the high loading efficiency of COF and the coordination properties of its internal bipyridine, AuNPs, Apo-A4 antibody, and TB are successively loaded to form a tracer probe (…). Figure 1 -C). Under the interaction of antigen and antibody, the tracer probe specifically binds to Apo-A4 immobilized on the electrode surface, and the resulting electrochemical signal of TB can provide quantitative evidence for the sensitive detection of Apo-A4. Figure 1 -D).
[0061] 1. Instruments and reagents:
[0062] The system includes an AUTOLAB PGSTAT302N electrochemical workstation (Metroën China Ltd.), with an electrochemical detection cell consisting of a three-electrode system: a glassy carbon electrode (GCE, d=3 mm) and its modified electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire electrode as the auxiliary electrode; a JSM-6701F cold field emission scanning electron microscope (SEM, Nippon Electron Optics Co., Ltd.); a Kratos AXIS Ultra DLD X-ray photoelectron spectrometer (XPS, Kratos Ltd., UK); an X-ray diffractometer (XRD, Rigaku Denki Instruments Co., Ltd., Japan); and an FTIR-650 Fourier transform infrared spectrometer (Tianjin Gangdong Technology Co., Ltd.).
[0063] Nitrogen-doped graphene (NG, Nanjing Xianfeng Nanomaterials Technology Co., Ltd.), 2,3,6,7,10,11-hexaaminotriphenylhexahydrochloride, 2,2'-bipyridine-5,5'-dicarboxylic acid, polyethyleneimine, N,N-dimethylformamide (Shanghai Maclean Biochemical Technology Co., Ltd.), Apo-A4 antigen and antibody (Wuhan Elabscience Biotechnology Co., Ltd.), chloroauric acid (Shanghai Co., Ltd.), 0.01 M pH=7.4 PBS buffer. All experimental water was ultrapure water. Nitrogen was purged into the electrolyte solutions used in each experiment to remove oxygen.
[0064] 2. Preparation of COF materials
[0065] 2,3,6,7,10,11-hexaaminotriphenylhexahydrochloride (0.04 mmol, 21.48 mg) and 2,2'-bipyridine-5,5'-dicarboxylic acid (0.06 mmol, 14.60 mg) were placed in a Pyrex tube, and 2–3 mL of polyphosphoric acid was added. The mixture was heated at 80 °C for 30 min to ensure thorough mixing. The tube was then evacuated for 15 min, followed by nitrogen purging for 5 min. This process was repeated three times. The Pyrex tube was sealed and incubated at 150 °C for 24 h, then at 180 °C for 24 h. After cooling to room temperature, the precipitate was washed successively with saturated sodium bicarbonate solution and deionized water. Finally, the product COF was obtained by vacuum drying at low temperature.
[0066] 3. Preparation of NG-PEI-COF composite materials
[0067] 1 and 2 mg of NG were ultrasonically dispersed in 1 mL of DMF, and 0.5 mL of 1% PEI solution was slowly added dropwise while stirring at room temperature for 4 h. Then, 1 mL of the COF suspension prepared above (dispersed in water, 1, 2, and 3 mg / mL) was added. -1 Stir at room temperature for 8 h. Finally, wash with DMF and water successively, and resuspend the precipitate in 1 mL of water for later use.
[0068] 4. Preparation of tracer probes
[0069] In 1 mL of 1 mg·mL -1 Add HAuCl4 solution (0.3 mL, 1 mg·mL⁻¹) to the COF suspension. -1 Stirring at room temperature for 4 h, followed by rapid addition of NaBH4 solution (0.2 mL, 2 mg·mL⁻¹). -1 Continue stirring for 2 h. Wash the resulting precipitate three times with ultrapure water, 3 minutes each time, at 10,000 rpm. Redisperse the centrifuged product with PBS to obtain an AuNPs / COF suspension (1 mg / mL). -1 ).
[0070] Take 1 mL of AuNPs / COF suspension and add 10 μL of apolipoprotein A4 antibody solution (Elabscience 100x, 10 μL). Stir at 4 °C for 3 h. Then add TB solution (1 mL, 1 mg·mL⁻¹). -1 Stir at 4℃ for 12 h. Finally, add 100 μL of BSA (1%) to block non-specific binding sites to obtain the tracer probe TB-Ab-AuNPs-COF. Centrifuge, wash, resuspend in 1 mL of PBS, and store at 4℃ for later use.
[0071] 5. Construction of electrochemical immunosensors
[0072] Before electrode modification, the GCE surface was polished with alumina powder, thoroughly rinsed with ultrapure water, nitric acid, and acetone, followed by ultrasonic cleaning to remove alumina residue and drying with nitrogen. The prepared NG-PEI-COF composite material (7 μL, 1 mg·mL⁻¹) was then collected. -1 Apply dropwise to the treated GCE and allow to air dry. Place the dried NG-PEI-COF / GCE in a 1 mg / mL solution. -1 AuNPs / NG-PEI-COF / GCE were prepared by deposition at -0.3 V for 400 s in a chloroauric acid (HAuCl4) solution using a chronoamperometry method. Then, 10 μL of Apo-A4 solution was added to the surface of the AuNPs / NG-PEI-COF / GCE, and the mixture was incubated overnight at 4 °C to firmly immobilize Apo-A4 on the electrode surface. Next, 5 μL of 1% BSA solution was added, and the mixture was incubated at 37 °C for 1 h to block non-specific binding sites, yielding Apo-A4 / AuNPs / NG-PEI-COF / GCE. Finally, 6 μL of the prepared tracer probe suspension was added to the modified electrode, and the mixture was incubated at 37 °C for 2 h. After each modification step, the free material was removed by rinsing with PBS solution.
[0073] 6. Electrochemical Measurement
[0074] All electrochemical measurements were performed on an AUTOLAB PGSTAT302N electrochemical workstation. Electrochemical impedance spectroscopy (EIS), CV, and DPV measurements were performed using 0.1 M KCl and 5.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] as electrolytes. The EIS frequency range was set to 0.1 Hz to 10 Hz. 5 The sine wave signal amplitude was 5 mV, and the voltage was 0.2 V. The applied voltage range for the CV method was -0.3 V to 0.5 V, with a scan rate of 20 mV / s. The DPV method was performed within the range of -0.3 V to 0.6 V, with a pulse amplitude of 25 mV, a pulse width of 50 ms, a pulse period of 0.5 s, and a potential increment of 5 mV. The immunoreaction process and Apo-A4 were detected using SWV, with 0.01 M pH=7.4 PBS buffer as the electrolyte. The voltage range was -0.6 V to 0 V, the pulse amplitude was 60 mV, the square wave frequency was 25 Hz, and the potential increment was 5 mV. All the above electrochemical detections were performed at room temperature.
[0075] 7. Results
[0076] 7.1 Characterization of COF materials
[0077] COF materials were obtained by a carboxyl-diamino condensation reaction of 2,3,6,7,10,11-hexaaminotriphenylhexahydrochloride and 2,2'-bipyridine-5,5'-dicarboxylic acid. XRD results of the COF materials showed ( Figure 2 -A), the material exhibits a characteristic diffraction peak at 22°, indicating the successful preparation of a COF material with a crystalline structure. Furthermore, in the N 1s XPS results ( Figure 2 -B), the peaks at 395.8 and 397.6 eV correspond to =N- and –NH-, respectively, indicating that the benzimidazole structure in the COF material was successfully prepared. The repeating structural unit is as follows:
[0078] .
[0079] 7.2 Characterization of NG-PEI-COF composite materials
[0080] COF, NG, and NG-PEI-COF were detected and analyzed using FTIR spectroscopy, and the results are as follows: Figure 2 -C is shown. The characteristic peak of COF is shown at 3425 cm⁻¹. -1 and 846 cm -1 These are caused by the stretching vibration of NH and the vibration of substituents in the benzene ring, respectively, at 1591 cm⁻¹. -1 This is due to the C=C vibration in the benzene ring and the C=N vibration in the benzimidazole ring. Simultaneously, the CN in the imidazole ring causes the vibration at 1307 cm⁻¹. -1 and 1382 cm -1 A corresponding peak appears at this location. Among the NG characteristic peaks, a peak at 1552 cm⁻¹ can be observed. -1 CN vibration caused by nitrogen doping and 3423 cm -1 The stretching vibration of NH is observed. Furthermore, after COF is functionalized to form the composite material NG-PEI-COF, the characteristic peaks of the COF material are still retained in the composite material, indicating that the COF structure is not destroyed by functionalization. Moreover, due to the π-π conjugation effect with NG, the infrared absorption of the compound undergoes a certain degree of redshift, indicating that the preparation of the NG-PEI-COF composite material is successful.
[0081] 7.3 Morphological Characterization of Modifying Materials
[0082] The surface morphology of different modified materials was observed using a scanning electron microscope (SEM). Figure 2 -D、 Figure 2 -E and Figure 2-F represent COF, NG-PEI-COF, and an electrode modified with electrochemically deposited AuNPs (AuNPs / NG-PEI-COF), respectively. It can be observed that the COF material surface exhibits a certain degree of stacked and interwoven wrinkled structure accompanied by the formation of some pores. The morphology of the NG-PEI-COF composite material, after NG functionalization, changes, exhibiting a layered structure characteristic of NG. Furthermore, the good conjugation effect between the two materials prevents excessive aggregation and stacking. Further deposition of AuNPs results in the surface wrinkled texture being covered by fine particles, making the electrode surface rougher. This indicates that AuNPs were successfully modified onto the NG-PEI-COF surface, significantly increasing the specific surface area and effectively enhancing the subsequent antigen immobilization and binding sites.
[0083] 7.4 Electrochemical Characterization of Basic Electrodes
[0084] The modification process of the base electrode was electrochemically characterized using CV and EIS methods. Figure 3 -A is a comparison of the CV response during the modification process of the Apo-A4 / AuNPs / NG-PEI-COF electrode. It can be seen that the peak current value obtained after modifying the NG-PEI-COF composite material is significantly larger than that of the bare glassy carbon electrode GCE. This is because the NG-PEI-COF composite material has a large specific surface area, which promotes electron transfer. The current decreases slightly after depositing AuNPs using the chronoamperometry method. This may be because when AuNPs are deposited on the electrode surface, electrons encounter more interface and surface defects in the transport path between AuNPs and the electrode, thus slightly increasing the resistance to electron transfer, but greatly increasing the number of antigen binding sites. When Apo-A4 is incubated on the electrode, a significant decrease in peak current is observed. This is because the antigen, as a biomacromolecule, binds to the electrode surface, creating a hindering effect that inhibits electron transfer and reduces conductivity. This also indicates that Apo-A4 was successfully immobilized on the electrode surface. This is consistent with... Figure 3 The EIS results for -B are consistent, which also proves that the electrochemical sensor has been successfully constructed.
[0085] 7.5 Condition Optimization
[0086] 7.5.1 Optimization of NG-PEI-COF Preparation Ratio and Conditions
[0087] Adjust the amount of NG used in the above section "3. Preparation of NG-PEI-COF composite material" to 1 or 2 mg, and the concentration of COF suspension to 1, 2, or 3 mg·mL. -1NG-PEI-COF composites with NG:COF ratios of 2:1, 1:1, 1:2, and 1:3 were prepared, and the DPV response of the prepared NG-PEI-COF composites in K3[Fe(CN)6] / K4[Fe(CN)6] solution was investigated. The results are as follows: Figure 4 As shown in Figure A, the conductivity is optimal when the NG:COF ratio is 1:1. This is because NG has superior conductivity compared to COF. Increasing the NG content can significantly improve the conductivity of the composite material. However, at higher concentrations, due to the structural characteristics of NG, it is prone to stacking and agglomeration, which hinders electron transfer.
[0088] 7.5.2 Optimization of NG-PEI-COF Modification Amount
[0089] 4, 5, 6, 7, and 8 μL of 1 mg·mL⁻¹ electrode solution were dropped onto the bare glassy carbon electrode (GCE). -1 The NG-PEI-COF suspension was investigated, and the results are as follows: Figure 4 As shown in Figure -B, the current response is maximized when the modification amount is 7 μL, which is the optimal amount of NG-PEI-COF.
[0090] 7.5.3 Optimization of AuNPs / NG-PEI-COF preparation conditions
[0091] With other experimental conditions fixed, the electrodeposition time and HAuCl4 solution concentration were varied for further investigation. Figure 4 -C indicates that the peak current response of the modified electrode increases with increasing deposition time. This is because AuNPs effectively increase the electrode surface area and improve the local electric field effect, promoting electron transport. The longer the deposition time, the more AuNPs are modified, and the more pronounced this promoting effect becomes. When the deposition time exceeds 400 s, the current value decreases instead. This is because excessively long deposition times lead to particle aggregation and uneven deposition, causing stacking of electron mass transfer pathways and resulting in performance degradation. Similarly, the concentration of the HAuCl4 solution regulates the gold ion supply rate and deposition rate, thereby further affecting the morphology, size, and distribution of AuNPs. When the concentration is too high, it leads to an increase in AuNP size and number, also resulting in aggregation and uneven deposition, inhibiting electron transport. Figure 4 The DPV response value of -D determines the concentration of HAuCl4 solution (1 mg / mL). -1 This is the optimal concentration.
[0092] 7.5.4 Optimization of tracer probe incubation time
[0093] The tracer label is modified with a specific antibody against the analyte Apo-A4, which is immobilized on the electrode surface through antigen-antibody binding. The redox probe in the tracer label is the main source of the detection signal. Therefore, the redox current signal of TB was investigated using the SWV method after different incubation times (0.5, 1.0, 1.5, 2.0, and 2.5 h) in PBS solution. Figure 4 As shown in Figure -E, with increasing incubation time, more tracer labels are immobilized on the electrode surface under the action of antigen and antibody, and more TB is detected. When the incubation time exceeds 2.0 h, the current signal tends to stabilize and only increases slightly, indicating that the amount of immobilized tracer probes has approached saturation. Considering ease of use, the relatively short 2.0 h is selected as the optimal incubation time.
[0094] 7.6 Analytical Performance of Electrochemical Immunosensors
[0095] 7.6.1 Sensitivity Test
[0096] The analytical performance of the electrochemical immunosensor for Apo-A4 was investigated using square wave voltammetry (SWV). After blocking non-specific binding sites with BSA solution, a limited amount of the analyte antigen Apo-A4 was immobilized on the electrode surface. The tracer label was then immobilized onto the immunosensor via antigen-antibody interactions. Under the same incubation time, the amount of tracer label bound was positively correlated with the immobilized Apo-A4 content. The amount of immobilized tracer label could be determined by detecting the electrochemical redox signal corresponding to TB on the tracer probe, thus allowing for further measurement of the Apo-A4 content modified on the electrode.
[0097] Using [amounts] containing 300, 100, 50, 10, 5, 1, 0.1, and 0.01 ng·mL respectively. -1 Following the method described in "5. Construction of Electrochemical Immunosensors" above, standard solutions of Apo-A4 were used to construct immunosensors TB-Ab-AuNPs-COF / Apo-A4 / AuNPs / NG-PEI-COF / GCE with different TB concentrations. Sensitive detection of Apo-A4 was achieved using SWV in PBS buffer solution. It can be seen that as the concentration of Apo-A4 in the standard solution decreases, the SWV response of the constructed sensor gradually decreases. Figure 5 -A), which also proves that the construction of the electrochemical immunosensor was successful. The calibration curve between the concentration of the Apo-A4 standard solution and its SWV response value is shown in Figure 1. Figure 5 -B is shown. The concentration ranges from 0.01 to 300 ng / mL. -1 Within the specified concentration range, the peak current of the constructed immunosensor exhibits a good linear relationship with the logarithm of the Apo-A4 concentration, with the linear equation: I(μA) = 42.70lgC. Apo-A4(pg·mL) -1 +165.5, linear correlation coefficient R 2 =0.9902, detection limit (LOD) = 2.16 pg·mL -1 (S / N=3).
[0098] 7.6.2 Selection, reproducibility, and stability tests
[0099] Arginine succinate synthase 1 (ASS1), brain-derived neurotrophic factor (BDNF), heat shock protein 70 (HSP70), and cortisol, all coexisting markers of depression in blood, were selected as interfering agents. As shown in Figure 6, the constructed immunosensor maintained high selectivity for Apo-A4 regardless of whether high concentrations of interfering agents coexisted with Apo-A4 or only the interfering agents were present. When Apo-A4 was mixed with interfering agents, the SWV response of each mixed solution was not significantly different from that of the Apo-A4 standard solution; when only interfering agents were present, the SWV response decreased to less than 40 μA. This indicates that the constructed TB-Ab-AuNPs-COF / Apo-A4 / AuNPs / NG-PEI-COF / GCE has good selectivity for the detection of Apo-A4.
[0100] The reproducibility of the electrochemical immunosensor was assessed by repeatedly measuring six solutions of the same concentration of Apo-A4. The titration was performed at 100 pg·mL⁻¹. -1 Six parallel replicates of TB-Ab-AuNPs-COF / Apo-A4 / AuNPs / NG-PEI-COF / GCE were prepared using Apo-A4 solution. The current response was measured by SWV, with a relative standard deviation of 2.31%, indicating good reproducibility of the constructed electrochemical immunosensor. Furthermore, to investigate the stability of the constructed electrochemical immunosensor, after storage at 4°C for 7 days, the sensor still retained 98.82% of the initial current response to the same concentration of Apo-A4 solution, demonstrating the good stability of the constructed immunosensor.
[0101] 7.6.3 Detection of Apo-A4 in actual samples
[0102] An electrochemical immunosensor was applied to detect apolipoprotein A4 in serum samples, and the results were compared with reference values obtained by ELISA. The analytical procedure was performed in triplicate using the standard addition method. The results are shown in Table 1. The RSD of the ELISA method varied from 2.13% to 3.75%, and the recovery rate varied from 102.15% to 105.40%. The constructed sensor achieved recoveries between 97.70% and 108.70%, with RSDs all less than 7.64%. There was no significant difference between the results obtained by the two methods. This indicates that our constructed sensor has good anti-interference capabilities and can be used for the detection of Apo-A4 in real samples.
[0103] Table 1. Electrochemical immunosensors used to determine Apo-A4 in real serum samples.
[0104]
[0105] a is the average of three consecutive measurements.
[0106] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for constructing an electrochemical immunosensor for detecting apolipoprotein A4, comprising: (1) Preparation of NG-PEI-COF composite material and tracer probe Nitrogen-doped graphene and polyethyleneimine were thoroughly stirred and compounded in a solvent, and then bipyridine-functionalized covalent organic framework material was added and thoroughly stirred and compounded to obtain NG-PEI-COF composite material; Using bipyridine-functionalized covalent organic framework materials as carriers, gold nanoparticles, apolipoprotein A4 antibody, toluidine blue and bovine serum albumin were sequentially loaded to obtain tracer probes. (2) NG-PEI-COF composite material, gold nanoparticles, apolipoprotein A4, bovine serum albumin and tracer probe were sequentially modified on the electrode to obtain the electrochemical immunosensor; Bipyridine-functionalized covalent organic framework materials have the following repeating structural units: 。 2. The method according to claim 1, characterized in that: When preparing NG-PEI-COF composite materials, the mass ratio of nitrogen-doped graphene, bipyridine-functionalized covalent organic framework material and polyethyleneimine is 1~2:1~3:
5.
3. The method according to claim 1, characterized in that: The preparation process of the tracer probe includes: Bipyridine-functionalized covalent organic framework material and chloroauric acid were thoroughly mixed in a solvent, and then a reducing agent was added to react and obtain AuNPs / COF. After centrifugation and washing, the AuNPs / COF was resuspended in buffer solution. Apolipoprotein A4 antibody and toluidine blue were added sequentially to the AuNPs / COF suspension. After thorough stirring, bovine serum albumin was added for blocking to obtain the tracer probe. After centrifugation and washing, the probe was resuspended in buffer.
4. The method according to claim 3, characterized in that: The mass ratio of bipyridine-functionalized covalent organic framework material to chloroauric acid was 1:0.3, the mass ratio of AuNPs / COF to toluidine blue was 1:1, and the volume ratio of AuNPs / COF to apolipoprotein A4 antibody solution was 1 mg:10 μL.
5. The method according to claim 3, characterized in that: The reducing agent is NaBH4.
6. The method according to claim 5, characterized in that: The buffer solution is a PBS solution with pH=7.
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7. The method according to claim 6, characterized in that: The tracer probe was resuspended in the buffer at a ratio of 1 mg AuNPs / COF to 1 mL buffer.
8. The method according to claim 1, characterized in that: The modification process in step (2) includes: First, the NG-PEI-COF composite material was modified onto the electrode using a drop-coating method; Then the electrodes were placed in a chloroauric acid solution, and gold nanoparticles were electrodeposited by chronoamperometry. Next, lipoprotein A4 solution was added dropwise to the electrode, and after overnight incubation, bovine serum albumin was added for blocking. Finally, tracer probe suspension was added to the electrode for incubation to obtain the electrochemical immunosensor.
9. The method according to claim 8, characterized in that: The amount of NG-PEI-COF composite material used is 4~8 μg; The concentration of chloroauric acid solution is 0.6~2 mg / mL; The electrodeposition voltage was -0.3V, and the time was 100~500 s; The volume of apolipoprotein A4 solution used was 10 μL, and the concentration was 0.01~300 ng / mL; After adding the tracer probe suspension, incubate for 2-3 hours.
10. The method according to claim 9, characterized in that: The amount of NG-PEI-COF composite material used is 6~7 μg.
11. The method according to claim 9, characterized in that: The concentration of chloroauric acid solution is 0.8~1 mg / mL.
12. The method according to claim 9, characterized in that: The electrodeposition time is 300~400 s.
13. The electrochemical immunosensor constructed according to any one of claims 1-12.