Construction method and application of electrochemical biosensor for detecting AFP-L3%

Abstract

The invention 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: an Au-coated UiO-66 nano composite material is obtained by using a hydrothermal method and an impregnation reduction method, TCEP can open S-S in alpha fetoprotein to obtain sulfydryl (-SH), the sulfydryl (-SH) is connected to the Au-coated UiO-66, and then the Au-coated UiO-66 nano composite material is obtained. The method comprises the following steps: by taking DNA as a template, carrying out in-situ synthesis of an AFP aptamer-copper cluster (Apt-CuNCs), identifying total type AFP by using the Apt-CuNCs, determining the content of the total type AFP by using a generated Cu electrochemical signal, then carrying out enzyme digestion stripping on the Apt-CuNCs by using exonuclease I, marking an alpha fetoprotein variant (AFP-L3) by using lentil lectin (LCA) functionalized silver nanoparticles LCA-AgNPs, and determining the content of subtype AFP-L3 by using a generated Ag electrochemical signal. The AFP-L3% can be calculated through the combination of the two data, and the electrochemical biosensor constructed by the method can be used for determining the AFP-L3% in human serum.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, 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 digestive tract malignancies and one of the top three cancers in terms of cancer mortality worldwide. HCC develops insidiously and progresses rapidly. When clinically diagnosed, it is usually in the middle or late stages, causing patients to miss the best time for treatment. Therefore, early screening of HCC is extremely important for the diagnosis and treatment of patients.

[0003] Serum biomarker detection is an important tool in disease diagnosis. It has attracted much attention because of its non-invasive or minimally invasive, low-cost and ability to provide rich blood information. Alpha-fetoprotein (AFP) is a common serum tumor biomarker for HCC. Studies have shown that the serum AFP level of HCC patients is abnormally elevated compared with healthy patients. However, due to limited specificity, AFP levels may also be elevated in the serum of some patients with benign liver diseases. Recent studies have shown that AFP can be divided into three different subtypes, AFP-L1, AFP-L2 and AFP-L3, based on the different affinities of AFP for lentil agglutinin (LCA), of which AFP-L3 is specific to malignant cancer cells. Compared with traditional imaging techniques, AFP-L3 can be detected earlier in serum, helping to achieve early intervention and thus improve the survival rate of HCC patients. Existing studies have shown that the proportion of AFP-L3 in total AFP (AFP-L3%) exceeding 10% indicates that the incidence of liver cancer exceeds 95%. Therefore, compared with the AFP level in serum, AFP-L3% can diagnose liver cancer more accurately and reliably. At present, there have been studies on the detection of AFP-L3% using fluorescence and Raman, but fluorescence detection is easily affected by other substances in the solution, and a large number of ions and molecules need to be eliminated before testing. Raman requires a high-energy laser beam to obtain a high-intensity signal. As a large instrument, it is inconvenient to use and has high costs. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a method for constructing an electrochemical biosensor for detecting AFP-L3% and its application. When the total type AFP (including three isomers) of the target to be detected is present, the target to be detected can bind to the DNA probe Apt-CuNCs, and the working electrode can detect the electrochemical signal of Cu. After the DNA probe is cleaved by EXO I, when the target AFP-L3 is present, the target to be detected can specifically bind to LCA@AgNPs, and the working electrode can detect the electrochemical signal of Ag. The specific detection of AFP-L3% is achieved by combining the electrical signals of the two.

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

[0006] S1. Preparation of Au@UiO-66

[0007] 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 hours, and then the mixed solution after the reaction was centrifuged to obtain a white particle precipitate, and the white particles were washed with DMF for multiple times, and then the white particles were stirred and exchanged with ultrapure water solvent for 3 days to obtain UiO-66 particles, and the UiO-66 particles were vacuum dried at room temperature 25°C for 12 hours and then ground into powder and ultrasonically dispersed in pure water, and the dispersion of UiO-66 powder was vigorously stirred and chloroauric acid was added and stirred continuously in the dark for 8 hours, and then the mixture after the reaction was centrifuged to obtain a precipitate, and the precipitate was washed with ultrapure water and redispersed in ultrapure water, and sodium borohydride solution was gradually added to the dispersion of the precipitate and stirred continuously in the dark for 4 hours until the solution turned dark red, and finally the dark red mixed solution was centrifuged to obtain precipitated Au@UiO-66 powder;

[0008] S2. Preparation of Apt-CuNCs

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

[0010] S3. Preparation of LCA@AgNPs

[0011] A mixed solution of silver nitrate and trisodium citrate was prepared, and then NaBH4 solution was added dropwise under stirring, and stirred at 0°C in the dark until it turned yellow, and then placed at 4°C for 12 hours 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 minutes; then Tween 20 was added dropwise and stirred for 20 minutes, and the mixture after reaction was centrifuged to obtain precipitated LCA@AgNPs, which was washed with ultrapure water and resuspended with PBS solution to obtain an LCA@AgNPs dispersion;

[0012] S4. Construction of electrochemical biosensor for detecting AFP-L3%

[0013] (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 the pretreated glassy carbon (GC) electrode and dried;

[0014] (b) The AFP solution after reacting with the TCEP solution for 30 min was dropped onto the electrode obtained in S4(a) and reacted at 4 °C for 12 h, and then the unmodified AFP was 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 for 20-60 min at 35°C, and then the digested DNA (Apt-CuNCs) was 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 30-45°C for 30-75 min. The unmodified LCA@AgNPs were then washed away with PBS solution. 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 (pH 7.5) is 10 mM, 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 suspension applied on 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 35°C constant temperature incubator 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 amount of LCA@AgNPs applied in step S4(e) is 5 μL.

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

[0025] Preferably, the modified GC electrode is used as the working electrode, the platinum wire and the Ag / AgCl electrode are used as the counter electrode and the reference electrode respectively, and are immersed in a PBS solution to perform an LSV electrochemical test to obtain a linear relationship between the current response value and the concentration of AFP and AFP-L3. When detecting AFP and AFP-L3 solutions of unknown concentrations, the corresponding concentration is found on the linear curve according to the measured current difference, thereby obtaining the specific concentrations of AFP and AFP-L3 in the solution. 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. The present invention provides a construction method and application of an electrochemical sensor for detecting AFP-L3%, using two independent electrochemical signals to quantitatively detect the contents of AFP and AFP-L3 respectively, and by combining the data of the two, AFP-L3% can be calculated, and an electrochemical biosensor for detecting AFP-L3% with a wide linear range, high stability, and high specificity is developed, which realizes the orderly detection of total AFP and subtype AFP-L3. The electrochemical detection method has the characteristics of high sensitivity, good selectivity, easy operation and low detection cost.

[0029] 2. The linear range of the present invention in detecting total AFP is 10 ng mL -1 ~800ng mL -1 , clinical lesion reference value 400ng mL -1 The total AFP range of healthy human serum is 0ng mL -1 ~25ng 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 A schematic diagram of a construction method of an electrochemical biosensor for detecting AFP-L3% proposed by the present invention;

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

[0032] Figure 3 A feasibility study diagram of the AFP-L3% electrochemical biosensor prepared by the present invention, wherein Figure a is a CV diagram of the sensor being gradually constructed, Figure b is an impedance diagram of the sensor being gradually constructed, Figures c and d are current change curves and statistical diagrams before and after the sensor captures AFP, and Figures e and f are current change curves and statistical diagrams before and after the sensor captures AFP-L3 under different conditions;

[0033] Figure 4 The figure is a condition optimization and performance research diagram for detecting the total AFP part in the AFP-L3% electrochemical biosensor prepared by the present invention, wherein figure a is an optimization curve diagram of the Apt-CuNCs synthesis process, figure b is an optimization curve diagram of the incubation time of AFP and Apt-CuNCs, figure c is an optimization curve diagram of the incubation temperature of AFP and Apt-CuNCs, figure d is a current response curve diagram when detecting total AFP, figure e is a linear relationship diagram between the total AFP concentration and the current value response value, and figure f is a specificity research diagram for detecting the total AFP part;

[0034] Figure 5 The figure is a condition optimization and performance research diagram of the AFP-L3% electrochemical biosensor prepared by the present invention for detecting the typing AFP-L3 part, wherein figure a is an optimization curve diagram of the incubation time of EXO I enzyme cleavage, figure b is an optimization curve diagram of the incubation time of AFP-L3 and LCA@AgNPs, figure c is an optimization curve diagram of the incubation temperature of AFP-L3 and LCA@AgNPs, figure d is a current response curve diagram when detecting the typing AFP-L3, figure e is a linear relationship diagram between the typing AFP-L3 concentration and the current value response value, and figure f is a specificity research diagram for detecting the AFP-L3 part;

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

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments: Specific details are elaborated 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) materials have the advantages of large specific surface area, diverse structures, adjustable properties, and diverse synthesis methods. They are widely used in many fields such as adsorption, catalysis, sensing, and drug delivery. In the development of sensing materials, metal-organic frameworks (MOFs) have become a research hotspot due to their high designability. For example, functional groups such as carboxyl or amino groups are introduced to the ligands of MOF to make them functionalized and easy to modify later; for example, metal elements such as Ru and Ir are introduced to enhance the photophysical properties of MOF to make it more sensitive in electrochemiluminescence (ECL) or photoelectric sensing. Among them, the typical MOF UiO-66 has outstanding hydrothermal stability and chemical stability, and can maintain structural stability in a variety of solutions. By modifying UiO-66, such as using the pore structure to load other metal materials or connecting molecules through modified groups, UiO-66 can be widely used in many fields. As a classic nanomaterial, AuNPs have broad application prospects in the construction of biosensors, the study of electrochemical catalysis, optoelectronics, and physicochemical properties. At the same time, since AuNPs can be subjected to a variety of chemical modifications, AuNPs are given functionality and can be applied to chemical analysis, biomedicine and other fields. Au@UiO-66, which is synthesized by in-situ generation of AuNPs based on UiO-66, not only has the characteristics of large specific surface area of ​​MOF, but also takes into account the biocompatibility of AuNPs and the characteristics of easy connection of functional groups, and can be applied to the field of biosensing.

[0038] The present invention provides a construction method and application of an electrochemical biosensor for quantitative AFP-L3% (AFP-L3 / AFP). The specific process is as follows: Au@UiO-66 nanocomposite material is obtained by hydrothermal method and impregnation reduction method, tri(2-carboxyethyl)phosphine (TCEP) can open SS in alpha-fetoprotein (AFP) to obtain sulfhydryl (-SH) and connect it to Au@UiO-66, AFP aptamer-copper cluster (Apt-CuNCs) is synthesized in situ using DNA as a template, Apt-CuNCs is used to identify total AFP, and the total AFP content is determined by the generated Cu electrochemical signal, then Apt-CuNCs is enzymatically cleaved and stripped by exonuclease I (EXO I), and alpha-fetoprotein heterogeneity (AFP-L3) is labeled by silver nanoparticles LCA@AgNPs functionalized with lentil lectin (LCA), and the typed AFP-L3 content is determined by the generated Ag electrochemical signal, and AFP-L3% can be calculated by combining the data of the two.

[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 tri(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 Bio-Tech (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 Products, Inc. (USA). 3-Morpholinepropanesulfonic acid (MOPS) and DNA (Aptamer-30T (used to synthesize Apt-CuNCs): The DNA template sequence used in Example 1 is 5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTCTCG ACT CGG TCT TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T -3', SEQ ID NO.1, where the Aptamer segment is used to identify the total AFP, and the 30T segment (underlined part) is used to synthesize copper nanoclusters (CuNCs), all of which were purchased from Shanghai Shenggong Biotechnology Co., Ltd. (China). Ultrapure water (18.2 MΩ·cm) was used to prepare all aqueous solutions. Glassy carbon (GC) electrode CHI104 and electrochemical workstation CHI660D were purchased from Shanghai Chenhua Instrument Co., Ltd., and Autolab PGSTAT302N was purchased from Swiss Metrohm China Co., Ltd.

[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 DMF (50 mL). The mixture was dissolved in ultrasound for 10 min, then transferred to a polytetrafluoroethylene autoclave, 2 mL of AcOH was added and shaken. The reactor was reacted in a 90 ° C oven for 48 h. The mixture in the reactor was centrifuged to obtain a white granular precipitate (8000 rpm, 10 min) and washed with DMF 3 times to remove the unreacted precursor. The white particles were then stirred and exchanged with ultrapure water solvent for 3 days (ultrapure water was replaced once a day), and finally UiO-66 particles were obtained. The obtained UiO-66 particles were vacuum dried at room temperature 25 ° C for 12 h, then vacuum activated at 150 ° C for 12 h, and ground into powder with an agate mortar (the particle size of the powder is irrelevant to the experimental results). 10 mg of the above powder was dispersed in 2 mL of ultrapure water under ultrasound. 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 hours. 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 0.1 M freshly prepared NaBH4 ice water solution was gradually added to the dispersion of the precipitate, the dispersion turned dark red and was vigorously stirred in the dark for 4 hours, 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 the 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 the XRD 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 was synthesized based on the 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 evenly. The mixture was reacted at room temperature of 25 °C in the dark for 15 min to obtain the Apt-CuNCs solution. Its fluorescence characterization is shown in Figure 2. Figure 2 As shown in Figure c: Under the condition of an excitation wavelength of 340 nm, a characteristic fluorescence peak appeared at around 650 nm, proving that CuNCs with DNA chains were successfully prepared.

[0047] (3) Preparation of LCA@AgNPs

[0048] A 100 mL mixed solution containing 0.25 mM AgNO3 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 to the above mixed solution, and stirred at 0 °C in the dark until the colorless solution turned yellow and 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 0.01 M sodium phosphate solution. To obtain LCA@AgNPs, 1 mL of the above AgNPs solution with a pH of 8.5 was taken, and then 100 μL of 2 mg / mL LCA was added dropwise, stirred at 0 °C for a full reaction for 40 min, and then 10 μL of 1% Tween20 was added dropwise and continued to stir for 20 min. The mixture was centrifuged and washed three times with ultrapure water and resuspended in 0.5 mL PBS to obtain an LCA@AgNPs dispersion.

[0049] (4) Pretreatment of GC electrode:

[0050] Firstly, the GC electrode was polished with 0.3 μm and 0.05 μm hydrated alumina slurry on a polishing cloth, respectively, and then ultrasonically cleaned in ethanol and water and dried with nitrogen to obtain a clean GC electrode surface.

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

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

[0053] Figure 3 Figures a and b are the CV graph and impedance graph of the electrochemical sensor obtained in step (3) that was gradually constructed. Since AFP and the outer layer of DNA hinder electron transfer, its impedance gradually increases and the current peak of the CV curve gradually decreases, proving the successful layer-by-layer modification of the sensor.

[0054] from Figure 3 As can be seen from Figures c and d, after the sensor captures total AFP and is incubated with the signal probe Apt-CuNCs, it can show an obvious Cu current signal, indicating that Apt-CuNCs successfully recognizes total AFP, further demonstrating the feasibility of the sensor in detecting total AFP. Figure 3Figures e and f are the current signals of Ag 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, Apt-CuNCs were cleaved with EXO I enzyme, and then LCA@AgNPs were incubated to avoid surface steric hindrance and obtain the highest current response value, further illustrating the feasibility of the sensor in detecting AFP-L3. The above data illustrate the feasibility of the sensor in detecting AFP-L3%.

[0055] Example 2

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

[0057] Electrochemical experiment LSV test was carried out on CHI660D electrochemical workstation, using Apt-CuNCs / AFP / Au@UiO-66 / GCE and LCA@AgNPs / AFP / Au@UiO-66 / GCE in Example 1 as working electrodes, platinum wire and Ag / AgCl electrode as counter electrode and reference electrode respectively. Soaked in PBS (10mM, pH=7.4) solution, Apt-CuNCs / AFP / Au@UiO-66 / GCE was used as the working electrode when detecting total AFP, the scanning range was -0.25V~0V, the scanning speed was 50mV / s, and the current response changes were observed. The results are shown in Figure 4 As shown in Figures d and e, with the increase of total AFP concentration, the current response value gradually increased. -1 ~800ng mL -1 In the concentration range of 1.377 W·m-1, the total AFP concentration (C) and the current response value ΔI (ΔI = current response peak height) showed a good linear relationship, ΔI (μA) = 0.00104 × C + 0.14806, R 2 =0.997, the detection limit can reach 0.18ng mL -1 ; When detecting the type AFP-L3, LCA@AgNPs / AFP / Au@UiO-66 / GCE was used as the working electrode, the scanning range was 0.05V~0.25V, and the scanning speed was 50mV / s. The changes in the current response 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 ~80ng mL -1 In the concentration range of 1.377 μm, 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 0.18ng mL-1 When detecting AFP and AFP-L3 solutions of unknown concentration, the corresponding concentration is found on the linear curve according to the measured current difference, so as to obtain the specific concentration of AFP and AFP-L3 in the solution. AFP-L3% can be calculated by combining the concentration data of the two.

[0058] Figure 4 Figures a, b, and c correspond to the signal optimization of total AFP detection. The best current response signal can be obtained when 5 mM CuSO4 is used to synthesize Apt-CuNCs, the incubation time of Apt-CuNCs and total AFP is 2 h, and the incubation temperature is 35 ° C. Among them, excellent current response signals can be obtained under the conditions of 3-7 mM CuSO4, 1.5-3 h Apt-CuNCs and total AFP incubation time, and 30-45 ° C incubation temperature.

[0059] Figure 5 Figures a, b, and c in the figure correspond to the signal optimization of the detection of typing AFP-L3. The optimal current response signal can be obtained when the incubation time of EXO I is 30 minutes, the incubation time of LCA@AgNPs and typing AFP-L3 is 45 minutes, and the incubation temperature is 35°C. Among them, excellent current response signals can be obtained under the conditions of 20-60 minutes EXO I incubation time, 30-75 minutes LCA@AgNPs and typing AFP-L3 incubation time, and 30-45°C incubation temperature.

[0060] Figure 4 The f graph and Figure 5 Figure f in the figure corresponds to the specific analysis of total AFP and the specific analysis of 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), which shows that the sensor has excellent specificity.

[0061] Figure 6 Figure a corresponds to the stability analysis of total AFP and subtype 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 subtype 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 serum samples were diluted 100 times to prepare the total AFP concentration of 20 ng mL -1 , 100ng mL -1 、500ngmL -1 The concentration of AFP-L3 in the experimental samples was adjusted to 2 ng mL -1 , 10ng mL -1 , 50ngmL -1 , the spike recovery test was performed in the same manner as in Example 2. As shown in Table 1: compared with the actual added amount, the relative error of the measured AFP-L3% was between 1.61% and 3.63%, which was less than 5% (in the field of medical testing, a relative deviation of less than 5% can ensure the accuracy and reliability of the data), indicating that the measurement results have high accuracy and reliability, that is, the sensor can be used for the determination of 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 protection scope 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 hours, and then the mixed solution after the reaction was centrifuged to obtain a white particle precipitate, and the white particles were washed with DMF for multiple times, and then the white particles were stirred and exchanged with ultrapure water solvent for 3 days to obtain UiO-66 particles, and the UiO-66 particles were vacuum dried at room temperature 25°C for 12 hours and then ground into powder and ultrasonically dispersed in pure water, and the dispersion of UiO-66 powder was vigorously stirred and chloroauric acid was added and stirred continuously in the dark for 8 hours, and then the mixture after the reaction was centrifuged to obtain a precipitate, and the precipitate was washed with ultrapure water and redispersed in ultrapure water, and sodium borohydride solution was gradually added to the dispersion of the precipitate and stirred continuously in the dark for 4 hours until the solution turned dark red, and finally the dark red mixed solution was centrifuged to obtain precipitated Au@UiO-66 powder; S2. Preparation of Apt-CuNCs The DNA template solution was added to 3-(N-morpholine) propanesulfonic acid (MOPS) solution, followed by copper sulfate solution (3-7 mM) and ascorbic acid, and the reaction was carried out in the dark for 15 min to obtain 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, and stirred at 0°C in the dark until it turned yellow, and then placed at 4°C for 12 hours 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 minutes; then Tween 20 was added dropwise and stirred for 20 minutes, and the mixture after reaction was centrifuged to obtain precipitated LCA@AgNPs, which was washed with ultrapure water and resuspended with 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 the pretreated glassy carbon (GC) electrode and dried; (b) The AFP solution after reacting with the TCEP solution for 30 min was dropped onto the electrode obtained in S4(a) and reacted at 4 °C for 12 h, and then the unmodified AFP was 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 for 20-60 min at 35°C, and then the digested DNA (Apt-CuNCs) was 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 30-45°C for 30-75 min. The unmodified LCA@AgNPs were then washed away with PBS solution. LCA@AgNPs / AFP / Au@UiO-66 / GCE was subjected to LSV electrochemical test.

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 TCGACTCGG TCT TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC TTT TTT TTT TTT TTT TTTTTTTT TTTTTTTT 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 (pH 7.5) is 10 mM, 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 Au@UiO-66 suspension was 1 mg / mL, the amount applied on the GC electrode was 10 μL, and 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 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 35°C constant temperature incubator for 30 min, and the drop coating volume is 15 μL.

6. The method for constructing an electrochemical biosensor for detecting AFP-L3% according to claim 1, characterized in that: The concentration of the 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 amount of LCA@AgNPs applied 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: the modified GC electrode is used as the working electrode, the platinum wire and the Ag / AgCl electrode are used as the counter electrode and the reference electrode respectively, and they are immersed in PBS solution to perform LSV electrochemical test to obtain the linear relationship between the current response value and the concentration of AFP and AFP-L3. When detecting AFP and AFP-L3 solutions of unknown concentration, the corresponding concentration is found on the linear curve according to the measured current difference, so as to obtain the specific concentration of AFP and AFP-L3 in the solution. AFP-L3% can be calculated by combining the concentration data of the two.

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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