A method for constructing an AFP-L3% electrochemical biosensor and application thereof

Abstract

The application belongs to the field of biosensors, and provides a construction method and application of an electrochemical biosensor for quantifying AFP-L3% (AFP-L3 / AFP), and the specific process is as follows: Au@UiO-66 nanocomposites are obtained by using a hydrothermal method and an immersion reduction method, TCEP can open S-S in alpha-fetoprotein to obtain sulfydryl (-SH) and connect to Au@UiO-66, in-situ synthesis of AFP aptamer-copper clusters (Apt-CuNCs) is carried out by using DNA as a template, Apt-CuNCs are used to recognize total AFP, the content of total AFP is determined by using the generated Cu electrochemical signal, then, the Apt-CuNCs are subjected to enzymatic cleavage and stripping by using exonuclease I, alpha-fetoprotein heterogeneity (AFP-L3) is labeled by using lentil agglutinin (LCA) functionalized silver nanoparticles LCA@AgNPs, the content of AFP-L3 is determined by using the generated Ag electrochemical signal, and AFP-L3% can be calculated by combining the data of the two, and the electrochemical biosensor constructed by the method can be used for determining AFP-L3% in human serum.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a construction method and application of an electrochemical biosensor for detecting AFP-L3%. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal malignancies and one of the top three causes of cancer mortality worldwide. HCC develops insidiously and progresses rapidly, often reaching advanced or late-stage stages by the time it is clinically diagnosed, missing the optimal treatment window. Therefore, early screening for HCC is crucial for diagnosis and treatment.

[0003] Serum biomarker testing is an important tool in disease diagnosis, attracting significant attention due to its non-invasive or minimally invasive, low-cost, and ability to provide rich blood-based information. Alpha-fetoprotein (AFP) is a common serum tumor biomarker for HCC. Studies have shown that serum AFP levels in HCC patients are abnormally elevated compared to healthy subjects. However, due to limited specificity, AFP levels can also be elevated in the serum of patients with benign liver diseases. Recent studies have demonstrated that AFP can be classified into three isoforms based on their affinity for lentil agglutinin (LCA): AFP-L1, AFP-L2, and AFP-L3. AFP-L3 is specific for malignant cancer cells. Compared to traditional imaging techniques, AFP-L3 can be detected earlier in serum, enabling early intervention and improving survival rates for HCC patients. Existing studies have shown that an AFP-L3 percentage (AFP-L3%) exceeding 10% of total AFP predicts a liver cancer incidence exceeding 95%. Therefore, compared to serum AFP levels, AFP-L3% can be more accurately and reliably used to diagnose liver cancer. Studies have been conducted using fluorescence and Raman spectroscopy to detect AFP-L3%, but fluorescence detection is susceptible to interference from other substances in the solution and requires the elimination of numerous ionic and molecular interferences before testing. Raman spectroscopy, on the other hand, requires a high-energy laser beam to generate a high-intensity signal, making it a large instrument that is inconvenient to use and expensive. Summary of the Invention

[0004] To address the above technical issues, the present invention provides a method for constructing an electrochemical biosensor for detecting AFP-L3% and its application. When the target substance, total AFP (including three isomers), is present, it binds to the DNA probe Apt-CuNCs, and the working electrode detects the electrochemical signal of Cu. After the DNA probe is cleaved by EXO I, when the target substance, AFP-L3, is present, it specifically binds to LCA@AgNPs, and the working electrode detects the electrochemical signal of Ag. The combined electrical signals of these two signals are used to calculate specific detection of AFP-L3%.

[0005] A method for constructing an AFP-L3% electrochemical biosensor, comprising the following steps:

[0006] S1, preparing Au@UiO-66

[0007] Terephthalic acid and zirconium chloride were ultrasonically dissolved in N,N-dimethylformamide, glacial acetic acid was added, and the above mixed solution was reacted at 90°C for 48 h. Then, the white particle precipitate was obtained by centrifugal separation of the reacted mixture, the white particles were washed with DMF for several times, then the white particles were stirred with ultrapure water solvent for 3 days to obtain UiO-66 particles. The UiO-66 particles were ground into powder after vacuum drying at room temperature 25°C for 12 h and ultrasonically dispersed in pure water. The dispersion of the UiO-66 powder was stirred vigorously while adding chloroauric acid and continuously stirring in the dark for 8 h. Then, the precipitate was obtained by centrifugal separation of the reacted mixture, and the precipitate was redispersed in ultrapure water after washing with ultrapure water. Sodium borohydride solution was gradually added to the dispersion of the precipitate and continuously stirred in the dark for 4 h until the solution turned dark red. Finally, the precipitated Au@UiO-66 powder was obtained by centrifugal separation of the dark red mixture;

[0008] S2, preparing Apt-CuNCs

[0009] The DNA template solution was added to the 3-(N-morpholine) propanesulfonic acid (MOPS) solution, followed by the addition of copper sulfate solution (3-7 mM) and ascorbic acid. The Apt-CuNCs solution was obtained by reacting in the dark for 15 min;

[0010] S3, preparing LCA@AgNPs

[0011] A mixed solution of silver nitrate and trisodium citrate was prepared, followed by dropwise addition of NaBH4 solution under stirring conditions, and stirring in the dark at 0°C until yellow, and placing at 4°C for 12 h to obtain an AgNPs colloidal solution. The pH of the AgNPs colloidal solution was adjusted to 8.5 by taking a sodium phosphate buffer solution, followed by dropwise addition of LCA and stirring at 0°C for 40 min. Then, Tween 20 was added and stirred for 20 min. The precipitate LCA@AgNPs was obtained by centrifugal separation of the reacted mixture and washed with ultrapure water. The LCA@AgNPs dispersion was obtained by resuspension with PBS solution;

[0012] S4, constructing an AFP-L3% electrochemical biosensor

[0013] (a) The Au@UiO-66 obtained in S1 was dispersed in pure water to form a suspension, which was dropped on the surface of a pretreated glassy carbon (GC) electrode and dried;

[0014] (b) The AFP solution, which had reacted with the TCEP solution for 30 min, was dropped onto the electrode obtained in S4 (a) and reacted at 4 °C for 12 h. The unmodified AFP was then washed away with PBS solution.

[0015] (c) The electrode obtained in S4 (b) was immersed in the Apt-CuNCs solution obtained in S2 and incubated at 30–45 °C for 1.5–3 h. The unmodified Apt-CuNCs were then washed away with PBS solution to prepare Apt-CuNCs / AFP / Au@UiO-66 / GCE for LSV electrochemical testing.

[0016] (d) The electrode obtained in S4 (c) was immersed in EXO I enzyme solution and incubated at 35°C for 20 to 60 min. The digested DNA (Apt-CuNCs) was then washed away with PBS solution.

[0017] (e) The LCA@AgNPs dispersion obtained in S3 was dropped onto the electrode surface obtained in S4 (d) and incubated at a temperature of 30 to 45 °C for 30 to 75 min. The unmodified LCA@AgNPs were then washed away with PBS solution, and the LSV electrochemical test of LCA@AgNPs / AFP / Au@UiO-66 / GCE was performed.

[0018] Preferably, the DNA template sequence in step S2 is: 5'-GTG ACG CTC CTA ACG CTG ACT CAG GTGCAG TTC TCG ACT CGG TCT TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC TTT TTT TTTTTT TTT TTT TTT TTT TTT TTT T-3', SEQ ID NO.1.

[0019] Preferably, in step S2, the volume ratio of the DNA template solution, MOPS solution, copper sulfate solution and ascorbic acid solution is 6:49:2:3, wherein the concentration of the DNA template solution is 10 μM, the concentration of the MOPS solution is 10 mM, the pH of the MOPS solution is 7.5, the concentration of the copper sulfate solution is 5 mM, and the concentration of the ascorbic acid solution is 20 mM.

[0020] Preferably, in step S4 (a), the concentration of the Au@UiO-66 suspension is 1 mg / mL, the amount of the Au@UiO-66 suspension applied to the GC electrode is 10 μL, and the suspension is incubated in a 35°C constant temperature incubator for 30 min to dry.

[0021] Preferably, in step S4 (b), the volume ratio of the AFP solution to the TCEP solution is 1:1, wherein the concentration of the TCEP solution is 1 mM; the reaction conditions are incubation in a constant temperature incubator at 35°C for 30 min, and the drop coating volume is 15 μL.

[0022] Preferably, the concentration of the EXO I enzyme solution in step S4 (d) is 0.1 U / µL.

[0023] Preferably, the drop-coating amount of LCA@AgNPs in step S4 (e) is 5 μL.

[0024] An electrochemical biosensor obtained by the above construction method is used in the detection of AFP-L3%.

[0025] Preferably, a modified GC electrode is used as a working electrode, a platinum wire and an Ag / AgCl electrode are used as a counter electrode and a reference electrode, respectively, and immersed in a PBS solution to perform an LSV electrochemical test to obtain a linear relationship between the current response value and the AFP and AFP-L3 concentrations. When detecting solutions of unknown concentrations of AFP and AFP-L3, the corresponding concentration is found on the linear curve based on the measured current difference, thereby obtaining the specific concentrations of AFP and AFP-L3 in the solution. The AFP-L3% can be calculated by combining the concentration data of the two.

[0026] Preferably, the PBS solution in which the electrode is immersed has a concentration of 10 mM and a pH of 7.4.

[0027] The beneficial effects of the present invention are:

[0028] 1. This invention provides a method for constructing and applying an electrochemical sensor for detecting AFP-L3%. Two independent electrochemical signals are used to quantitatively detect the contents of AFP and AFP-L3, respectively. AFP-L3% can be calculated by combining the two data. This electrochemical biosensor for detecting AFP-L3% has a wide linear range, high stability, and high specificity, enabling the sequential detection of total AFP and subtyped AFP-L3. This electrochemical detection method is characterized by high sensitivity, good selectivity, ease of operation, and low detection cost.

[0029] 2. The linear range of the present invention in detecting total AFP is 10 ng mL -1 ~ 800 ng mL -1 , clinical lesion reference value 400 ng mL -1 The total AFP level in serum of healthy individuals ranges from 0 ng mL -1 ~25 ng mL -1The present invention can measure whether the content of total AFP in human serum exceeds the total AFP range of healthy human serum, that is, whether a pathological change occurs, which proves the application prospect of the present invention in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of a construction method of an electrochemical biosensor for detecting AFP-L3% proposed in the present invention;

[0031] Figure 2 Characterization diagram of the Au@UiO-66 material prepared in the present invention, where Figure a is the TEM image of Au@UiO-66, Figure b is the XRD pattern of Au@UiO-66 and UiO-66, and Figure c is the fluorescence characterization diagram of Apt-CuNCs;

[0032] Figure 3 Figures showing a feasibility study of the AFP-L3% electrochemical biosensor prepared according to the present invention, wherein Figure a is a CV graph of the sensor's gradual construction, Figure b is an impedance graph of the sensor's gradual construction, Figures c and d are graphs and statistical diagrams of the current change before and after the sensor captures AFP, and Figures e and f are graphs and statistical diagrams of the current change before and after the sensor captures AFP-L3 under different conditions;

[0033] Figure 4 Figures 1 and 2 show the optimization conditions and performance study for detecting total AFP in the AFP-L3% electrochemical biosensor prepared according to the present invention. Figure a shows the optimization curve for the Apt-CuNCs synthesis process, Figure b shows the optimization curve for the incubation time of AFP and Apt-CuNCs, Figure c shows the optimization curve for the incubation temperature of AFP and Apt-CuNCs, Figure d shows the current response curve for detecting total AFP, Figure e shows the linear relationship between total AFP concentration and current response value, and Figure f shows the specificity study for detecting total AFP.

[0034] Figure 5 Figure 3 is a graph showing the optimization of conditions and performance of the AFP-L3% electrochemical biosensor prepared by the present invention for detecting the typing AFP-L3 portion, wherein Figure a is a graph showing the optimization curve of the incubation time for EXO I enzyme digestion, Figure b is a graph showing the optimization curve of the incubation time for AFP-L3 and LCA@AgNPs, Figure c is a graph showing the optimization curve of the incubation temperature for AFP-L3 and LCA@AgNPs, Figure d is a graph showing the current response when detecting typing AFP-L3, Figure e is a graph showing the linear relationship between the typing AFP-L3 concentration and the current response value, and Figure f is a graph showing the specificity of detecting the AFP-L3 portion;

[0035] Figure 6Figure 3 is a stability study graph of the electrochemical biosensor for detecting AFP-L3% of the present invention, wherein Figure a is a stability study graph of the sensor for total AFP and isotype AFP-L3 on different electrodes, and Figure b is a stability study graph of the sensor for total AFP and isotype AFP-L3 stored at 4°C for 0 to 8 days. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments: Specific details are set forth in the following description to facilitate a full understanding of the present application, but the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application. Therefore, the present application is not limited to the specific implementation disclosed below.

[0037] Metal-organic frameworks (MOFs) offer advantages such as large surface area, diverse structures, tunable properties, and a wide range of synthetic methods. They are widely used in a variety of fields, including adsorption, catalysis, sensing, and drug delivery. In the development of sensing materials, MOFs have become a research hotspot due to their high designability. For example, functional groups such as carboxyl or amino groups can be introduced into MOF ligands to facilitate subsequent modification. For example, metal elements such as Ru and Ir can be introduced to enhance the photophysical properties of MOFs, making them more sensitive in electrochemiluminescence (ECL) or photoelectric sensing. Among them, the representative MOF, UiO-66, possesses outstanding hydrothermal and chemical stability, maintaining its structural stability in a variety of solutions. Modifications of UiO-66, such as loading other metal materials into its pore structure or attaching molecules via modifying groups, can broaden its applications in a variety of fields. AuNPs, as a classic nanomaterial, hold great promise for the construction of biosensors and the study of electrochemical catalysis, optoelectronics, and physicochemical properties. At the same time, since AuNPs can undergo a variety of chemical modifications, they can be given functionality and applied to chemical analysis, biomedicine and other fields. Based on the in situ generation of AuNPs from UiO-66, Au@UiO-66 is synthesized. It not only has the characteristics of large specific surface area of ​​MOF, but also takes into account the biocompatibility and easy attachment of functional groups of AuNPs, and can be applied to the field of biosensing.

[0038] The present invention provides a method for constructing and applying an electrochemical biosensor for quantifying AFP-L3% (AFP-L3 / AFP). The specific process is as follows: an Au@UiO-66 nanocomposite is prepared using a hydrothermal method and an impregnation reduction method. Tris(2-carboxyethyl)phosphine (TCEP) is used to convert the SS group in alpha-fetoprotein (AFP) into a thiol group (-SH), which is then attached to the Au@UiO-66. AFP aptamer-copper clusters (Apt-CuNCs) are synthesized in situ using DNA as a template. Total AFP is recognized by the Apt-CuNCs, and the resulting Cu electrochemical signal is used to determine the total AFP content. The Apt-CuNCs are then enzymatically exfoliated using exonuclease I (EXO I). Alpha-fetoprotein isoforms (AFP-L3) are then labeled with silver nanoparticles (LCA@AgNPs) functionalized with lentil lectin (LCA). The resulting Ag electrochemical signal is used to determine the AFP-L3 content. The AFP-L3% can be calculated by combining the two data.

[0039] The main drugs and experimental instruments used in this application are as follows:

[0040] Terephthalic acid (PTA, AR) and zirconium chloride (ZrCl4, AR) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Chloroauric acid (HAuCl4, AR) and tris(2-carboxyethyl)phosphine (TCEP, AR) were purchased from Sigma-Aldrich. N,N-dimethylformamide (DMF, AR), sodium borohydride (NaBH4, AR), silver nitrate (AgNO3, AR), acetic acid (AcOH, AR), copper sulfate pentahydrate (CuSO4·5H2O, AR), ascorbic acid (AA, AR), sodium phosphate (Na3PO4, AR), and trisodium citrate dihydrate (Na3C6H5O7·2H2O, AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. Tween 20 was purchased from Shanghai Beyotime Biotechnology (China). Exonuclease I (EXO I) and PBS (0.1 M, pH 7.4) were purchased from Shanghai Yuanye Biotechnology (China). Human AFP antigen was purchased from Sino Biological (USA). AFP-L3 was purchased from Shanghai Lianshuo Biotechnology Co., Ltd. (China). Lentil agglutinin (LCA) was purchased from Vectorlabs Co., Ltd. (Switzerland). Tumor marker quality control serum was purchased from Bio-Rad Life Sciences, Inc. (USA). 3-Morpholinepropanesulfonic acid (MOPS) and DNA (Aptamer-30T (used for synthesizing Apt-CuNCs): The DNA template sequence used in Example 1 was 5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAGTTC TCG ACT CGG TCT TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC T TT TTT TTT TTT TTT TTT TTT TTT TTT TTT T -3', SEQ ID NO. 1, where the Aptamer segment is used to identify total AFP, and the 30T segment (underlined) is used to synthesize copper nanoclusters (CuNCs). Both were purchased from Shanghai Sangon Biotechnology Co., Ltd. (China). All aqueous solutions were prepared using ultrapure water (18.2 MΩ·cm). A CHI104 glassy carbon (GC) electrode and a CHI660D electrochemical workstation were purchased from Shanghai Chenhua Instrument Co., Ltd., and an Autolab PGSTAT302N was purchased from Metrohm (China).

[0041] Example 1

[0042] like Figure 1 As shown, the construction method of the electrochemical biosensor for detecting AFP-L3% comprises the following steps:

[0043] (1) Preparation of Au@UiO-66

[0044] 62.3 mg of PTA and 106 mg of ZrCl₄ were dissolved in 50 mL of DMF. The mixture was sonicated for 10 minutes, then transferred to a Teflon autoclave, 2 mL of AcOH was added, and the mixture was shaken. The autoclave was then heated in a 90°C oven for 48 hours. The mixture was centrifuged (8000 rpm, 10 minutes) to obtain a white precipitate. This precipitate was washed three times with DMF to remove unreacted precursors. The precipitate was then exchanged with ultrapure water for three days with stirring (the ultrapure water was changed once per day), yielding UiO-66 particles. The resulting UiO-66 particles were dried in a vacuum at room temperature (25°C) for 12 hours, activated at 150°C for 12 hours, and ground into a powder using an agate mortar (the particle size was irrelevant to the experimental results). 10 mg of the resulting powder was dispersed in 2 mL of ultrapure water under ultrasonication. Next, under vigorous stirring, 100 µL of 25.4 mM HAuCl4 was dropped into the bottle of ultrasonically dispersed powder, the vial was sealed and vigorously stirred in the dark for 8 h. The precipitate in the vial was collected by centrifugation (8000 rpm, 10 min), the precipitate was washed once with ultrapure water and then redispersed in 2 mL of ultrapure water. Afterwards, under vigorous stirring, 102 µL of 0.1M freshly prepared NaBH4 ice water solution was gradually added to the dispersion of the precipitate. The dispersion turned deep red and was vigorously stirred in the dark for 4 h, and finally Au@UiO-66 powder was obtained. The prepared Au@UiO-66 powder was collected by centrifugation (8000 rpm, 10 min) and washed 3 times with ultrapure water. Its TEM characterization is as follows Figure 2 As shown in a: AuNPs are loaded on octahedral UiO-66, proving the successful preparation of Au@UiO-66; its XRD characterization is as follows Figure 2 As shown in (b): The XRD results of Au@UiO-66 match those of UiO-66 and the standard card of Au (04-0783), proving the successful preparation of Au@UiO-66.

[0045] (2) Preparation of Apt-CuNCs:

[0046] Apt-CuNCs were synthesized based on previously reported literature with slight modifications. The specific steps are as follows: 30 µL of 10 µM DNA template solution was added to 245 µL of MOPS (10 mM, pH 7.5) solution. Then, 10 µL of 5 mM CuSO4 and 15 µL of 20 mM ascorbic acid were added to the mixed solution in sequence and mixed well. The mixture was reacted at room temperature (25°C) in the dark for 15 minutes to obtain the Apt-CuNCs solution. Its fluorescence characterization is shown in Figure 2. Figure 2As shown in Fig. 3: under the condition of excitation wavelength of 340 nm, a fluorescence characteristic peak appeared at about 650 nm, which proved that CuNCs with DNA chains were successfully prepared.

[0047] (3) Preparation of LCA@AgNPs

[0048] A 100 mL mixture solution containing 0.25 mM AgN03 and 0.25 mM trisodium citrate was prepared at 25 °C, and the solution was colorless and transparent. Subsequently, 6 mL of 5 mM NaBH4 solution prepared with ice water was added dropwise into the above mixture solution, and the colorless solution was stirred at 0 °C in the dark until it turned yellow, and then it was placed at 4 °C for 12 h to obtain a stable AgNPs colloidal solution, and then the pH was adjusted to 8.5 using a 0.01 M sodium phosphate solution. To obtain LCA@AgNPs, 1 mL of the above AgNPs solution with pH of 8.5 was taken, and then 100 μΐ of 2 mg / mL LCA was added dropwise, and it was stirred at 0 °C for sufficient reaction for 40 min, and then 10 μΐ of 1% Tween 20 was added dropwise, and the stirring was continued for 20 min. After the mixture was centrifuged and washed with ultrapure water for three times, it was resuspended in 0.5 mL of PBS to obtain a LCA@AgNPs dispersion.

[0049] (4) Pretreatment of GC electrode:

[0050] Firstly, the GC electrode was polished with 0.3 μιη and 0.05 μιη particle size aqueous alumina slurry on a polishing cloth, and after ultrasonic cleaning in ethanol and water, the clean GC electrode surface was obtained by drying with nitrogen.

[0051] (5) Construction of AFP-L3% electrochemical biosensor:

[0052] Firstly, 10 μΐ of 1 mg / mL Au@UiO-66 suspension was dropped on the surface of glassy carbon electrode and dried at 35 °C to obtain Au@UiO-66 / GCE. 7.5 μΐ of AFP sample with different concentrations was mixed with 7.5 μΐ of 1 mM TCEP at 35 °C for 30 min, and then it was dropped on the surface of prepared Au@UiO-66 / GCE, and incubated at 4 °C for 12 h to obtain AFP / Au@UiO-66 / GCE. In order to test total AFP, the prepared AFP / Au@UiO-66 / GCE was washed with PBS to remove unbound total AFP, and then the electrode was immersed in 50 μΐ of synthesized Apt-CuNCs solution for 2 h at 35 °C incubation temperature. After that, the electrode was washed with PBS to remove unbound Apt-CuNCs to obtain Apt-CuNCs / AFP / Au@UiO-66 / GCE for subsequent electrochemical detection. In order to test AFP-L3, the electrode was immersed in 50 μΐ of 0.1 U / μΐ of EXO I enzyme solution for 30 min at 35 °C to remove the DNA (Apt-CuNCs) connected to the surface of AFP. The AFP / Au@UiO-66 / GCE surface treated with EXO I enzyme was washed with PBS, and 5 μΐ of prepared LCA@AgNPs dispersion was dropped on the electrode surface and incubated at 35 °C for 45 min. Finally, the electrode was washed with PBS to remove unbound LCA@AgNPs for subsequent linear sweep voltammetry (LSV) detection of LCA@AgNPs / AFP / Au@UiO-66 / GCE.

[0053] Figure 3 Figures a and b are CV and impedance diagrams of the electrochemical sensor obtained in step (3), respectively. The impedance gradually increases and the CV curve current peak gradually decreases due to the hindering of electron transfer by AFP and outer DNA, which proves the successful layer-by-layer modification of the sensor.

[0054] From Figure 3 Figures c and d show that the sensor can exhibit obvious Cu current signal after incubation with signal probe Apt-CuNCs after capturing total AFP, which indicates that Apt-CuNCs successfully recognize total AFP, further indicating the feasibility of the sensor for detecting total AFP. Figure 3Figures e and f show the Ag current signals obtained by incubating the sensor with LCA@AgNPs under different conditions. It can be seen that after completing the first step of total AFP detection, enzymatic cleavage of Apt-CuNCs with EXO I followed by incubation with LCA@AgNPs avoids surface steric hindrance and achieves the highest current response value, further demonstrating the feasibility of the sensor in detecting AFP-L3. These data demonstrate the feasibility of the sensor in detecting AFP-L3%.

[0055] Example 2

[0056] Analytical experiments for detecting AFP-L3% electrochemical biosensor:

[0057] Electrochemical LSV tests were performed on a CHI660D electrochemical workstation, using the Apt-CuNCs / AFP / Au@UiO-66 / GCE and LCA@AgNPs / AFP / Au@UiO-66 / GCE electrodes from Example 1 as working electrodes, and platinum wire and Ag / AgCl electrodes as counter and reference electrodes, respectively. The electrodes were immersed in PBS (10 mM, pH = 7.4). For total AFP detection, the Apt-CuNCs / AFP / Au@UiO-66 / GCE electrode was used as the working electrode. The scan range was -0.25 V to 0 V at a scan rate of 50 mV / s, and the current response was observed. The results are shown in Figure 2. Figure 4 As shown in Figures d and e, with the increase of total AFP concentration, the current response value gradually increased. -1 ~ 800 ng mL -1 In the concentration range of 100 μg / cm2, there is a good linear relationship between the total AFP concentration (C) and the current response value ΔI (ΔI = current response peak height), ΔI (µA) = 0.00104×C+0.14806, R 2 = 0.997, the detection limit can reach 0.18 ng mL -1 When detecting AFP-L3, LCA@AgNPs / AFP / Au@UiO-66 / GCE was used as the working electrode, with a scan range of 0.05V to 0.25V and a scan rate of 50 mV / s. The current response changes were observed, and the results were as follows: Figure 5 As shown in Figures d and e, with the increase of AFP-L3 concentration, the current response value gradually increased. -1 ~ 80 ng mL -1 In the concentration range of 100 μg / mL, there was a good linear relationship between the AFP-L3 concentration (C) and the current response value ΔI (ΔI = current response peak height), ΔI (µA) = 0.25985×C+2.02779, R 2= 0.992, the detection limit can reach 0.18 ng mL -1 When detecting AFP and AFP-L3 solutions of unknown concentration, the corresponding concentration is found on the linear curve based on the measured current difference, thereby obtaining the specific concentrations of AFP and AFP-L3 in the solution. By combining the concentration data of the two, AFP-L3% can be calculated.

[0058] Figure 4 Panels a, b, and c correspond to signal optimization for total AFP detection. The optimal current response signal was obtained when 5 mM CuSO₄ was used to synthesize Apt-CuNCs, the incubation time with total AFP was 2 hours, and the incubation temperature was 35°C. Excellent current response signals were also obtained under the conditions of 3 to 7 mM CuSO₄, 1.5 to 3 hours of incubation time with total AFP, and 30 to 45°C.

[0059] Figure 5 Panels a, b, and c correspond to signal optimization for AFP-L3 typing detection. The optimal current response signal was obtained when the EXO I digestion incubation time was 30 minutes, the LCA@AgNPs and AFP-L3 incubation time was 45 minutes, and the incubation temperature was 35°C. Excellent current response signals were also obtained under the conditions of an EXO I digestion incubation time of 20 to 60 minutes, a LCA@AgNPs and AFP-L3 incubation time of 30 to 75 minutes, and an incubation temperature of 30 to 45°C.

[0060] Figure 4 The f graph and Figure 5 Figure f corresponds to the specific analysis of total AFP and type AFP-L3, respectively. The target total AFP and type AFP-L3 have obvious current response signals compared with other tumor markers (PSA, CEA, cTnI), indicating that the sensor has excellent specificity.

[0061] Figure 6 Figure a corresponds to the stability analysis of total AFP and type AFP-L3 on different electrodes. It was found that the current response signals were almost the same when tested under the same conditions on different electrodes; Figure b corresponds to the stability analysis of total AFP and type AFP-L3 stored at 4°C for 0 to 8 days. It was found that the current response values ​​did not decrease significantly when tested under the same conditions under different storage days. Both stability tests show that the sensor has excellent stability.

[0062] Example 3

[0063] Electrochemical biosensor detection of AFP-L3% in serum samples:

[0064] Table 1: Analysis of AFP-L3% in serum samples by electrochemical biosensor

[0065]

[0066] The total AFP concentration was 20 ng mL using a 100-fold diluted serum sample. -1 , 100 ng mL -1 , 500ng mL -1 The concentration of AFP-L3 in the experimental samples was adjusted to 2 ng mL -1 , 10 ng mL -1 , 50 ng mL -1 Spiked recovery tests were performed using the same method as in Example 2. As shown in Table 1, the relative error of the measured AFP-L3% compared to the actual spiked amount ranged from 1.61% to 3.63%, less than 5% (in medical testing, a relative deviation of less than 5% ensures data accuracy and reliability). This demonstrates the high accuracy and reliability of the measurement results, indicating that this sensor can be used to measure AFP-L3% in serum samples.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing an electrochemical biosensor for detecting AFP-L3%, the method comprising the following steps: S1. Preparation of Au@UiO-66 Terephthalic acid and zirconium chloride were ultrasonically dissolved in N,N-dimethylformamide, glacial acetic acid was added, and the mixed solution was reacted at 90 °C for 48 h. The mixed solution after reaction was then centrifuged to obtain white granular precipitate. The white granules were washed with DMF multiple times, and then the white granules were stirred and exchanged with ultrapure water solvent for 3 days to obtain UiO-66 particles. The UiO-66 particles were vacuum dried at room temperature (25 °C) for 12 h, ground into powder, and ultrasonically dispersed in pure water. The dispersion of UiO-66 powder was vigorously stirred while adding chloroauric acid and stirring was continued in the dark for 8 h. The mixture after reaction was then centrifuged to obtain a precipitate. The precipitate was washed with ultrapure water and redispersed in ultrapure water. Sodium borohydride solution was gradually added to the dispersion of the precipitate and stirred in the dark for 4 h until the solution turned dark red. Finally, the dark red mixture was centrifuged to obtain precipitated Au@UiO-66 powder. S2. Preparation of Apt-CuNCs The DNA template solution was added to a 3-(N-morpholino)propanesulfonic acid (MOPS) solution, followed by addition of a copper sulfate solution and ascorbic acid. The concentration of the copper sulfate solution was 3 to 7 mM, and the reaction was carried out in the dark for 15 minutes to obtain an Apt-CuNCs solution. S3. Preparation of LCA@AgNPs A mixed solution of silver nitrate and trisodium citrate was prepared, and then NaBH4 solution was added dropwise under stirring. The mixture was stirred at 0°C in the dark until it turned yellow and then kept at 4°C for 12 h to obtain an AgNPs colloidal solution. Sodium phosphate buffer solution was used to adjust the pH of the AgNPs colloidal solution to 8.5, and then LCA was added dropwise and stirred at 0°C for 40 min. Tween 20 was then added dropwise and stirred for 20 min. The reaction mixture was centrifuged to obtain precipitated LCA@AgNPs, which was washed with ultrapure water and resuspended in PBS solution to obtain an LCA@AgNPs dispersion. S4. Construction of electrochemical biosensor for detecting AFP-L3% (a) The Au@UiO-66 obtained in S1 was dispersed in pure water to prepare a suspension, which was then dropped onto the surface of a pretreated glassy carbon (GC) electrode and dried. (b) The AFP solution, which had reacted with the TCEP solution for 30 min, was dropped onto the electrode obtained in S4 (a) and reacted at 4 °C for 12 h. The unmodified AFP was then washed away with PBS solution. (c) The electrode obtained in S4 (b) was immersed in the Apt-CuNCs solution obtained in S2 and incubated at 30–45 °C for 1.5–3 h. The unmodified Apt-CuNCs were then washed away with PBS solution to prepare Apt-CuNCs / AFP / Au@UiO-66 / GCE for LSV electrochemical testing. (d) The electrode obtained in S4 (c) was immersed in EXO I enzyme solution and incubated at 35°C for 20 to 60 min. The digested DNA (Apt-CuNCs) was then washed away with PBS solution. (e) The LCA@AgNPs dispersion obtained in S3 was dropped onto the electrode surface obtained in S4 (d) and incubated at a temperature of 30 to 45 °C for 30 to 75 min. The unmodified LCA@AgNPs were then washed away with PBS solution, and the LSV electrochemical test of LCA@AgNPs / AFP / Au@UiO-66 / GCE was performed.

2. The method for constructing an electrochemical biosensor for detecting AFP-L3% according to claim 1, characterized in that: The DNA template sequence in step S2 is: 5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCGACT CGG TCT TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC TTT TTT TTT TTT TTT TTT TTTTTT TTT TTT TTT T-3', SEQ ID NO.

1.

3. The method for constructing an electrochemical biosensor for detecting AFP-L3% according to claim 1, characterized in that: In step S2, the volume ratio of the DNA template solution, MOPS solution, copper sulfate solution, and ascorbic acid solution is 6:49:2:3, wherein the concentration of the DNA template solution is 10 μM, the concentration of the MOPS solution is 10 mM, the pH of the MOPS solution is 7.5, the concentration of the copper sulfate solution is 5 mM, and the concentration of the ascorbic acid solution is 20 mM.

4. The method for constructing an electrochemical biosensor for detecting AFP-L3% according to claim 1, characterized in that: In step S4 (a), the concentration of the Au@UiO-66 suspension was 1 mg / mL, and the amount of the drop coating on the GC electrode was 10 μL. The suspension was incubated in a 35 °C constant temperature incubator for 30 min to dry.

5. The method for constructing an electrochemical biosensor for detecting AFP-L3% according to claim 1, characterized in that: In step S4 (b), the volume ratio of AFP solution to TCEP solution was 1:1, and the concentration of TCEP solution was 1 mM. The reaction conditions were incubation in a constant temperature incubator at 35 °C for 30 min, and the drop coating volume was 15 μL.

6. The method for constructing an electrochemical biosensor for detecting AFP-L3% according to claim 1, characterized in that: The concentration of EXO I enzyme solution in step S4 (d) was 0.1 U / µL.

7. The method for constructing an electrochemical biosensor for detecting AFP-L3% according to claim 1, characterized in that: The drop-coating volume of LCA@AgNPs in step S4 (e) was 5 μL.

8. Use of the electrochemical biosensor obtained by the construction method according to any one of claims 1 to 7 in the detection of AFP-L3%.

9. The use according to claim 8, characterized in that The specific process is as follows: using a modified GC electrode as the working electrode, a platinum wire electrode and an Ag / AgCl electrode as the counter electrode and reference electrode, respectively, immersed in a PBS solution, LSV electrochemical testing is performed to obtain a linear relationship between the current response value and the AFP and AFP-L3 concentrations. When detecting AFP and AFP-L3 solutions of unknown concentrations, the corresponding concentration is searched on the linear curve based on the measured current difference, thereby obtaining the specific concentrations of AFP and AFP-L3 in the solution. By combining the concentration data of the two, the AFP-L3% can be calculated.

10. The use according to claim 9, characterized in that: The concentration of the PBS solution in which the electrode was immersed was 10 mM, pH = 7.4.

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