An electrochemical aptamer sensor for detecting gp73 based on rGO-fc-pani nanocomposite

Abstract

An electrochemical aptamer sensor for detecting GP73 is constructed based on a reducing graphene oxide-ferrocene-polyaniline rGO-Fc-PANi nanocomposite. The rGO-Fc-PANi has a large specific surface area, high conductivity and good in-situ electrochemical activity, and is used as a carrier to combine with GP73 Apt The signal probe is formed by coupling, and the cDNA is supplemented by the aptamer to combine with GP73 Apt The cDNA-GP73 is formed by hybridization Apt The double-stranded GP73 protein specifically combines with GP73 Apt A competitive relationship is formed with the cDNA, and the change in the redox peak current of Fc in the rGO-Fc-PANi is recorded by a DPV method, so that the detection of GP73 is realized. The method has low cost, good selectivity and high sensitivity, and the minimum detection limit is 0.15 pg / mL.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and in particular relates to an electrochemical aptamer sensor for detecting GP73 based on a nanocomposite material. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Golgi transmembrane glycoprotein 73 (GP73), as an emerging tumor marker, offers advantages such as high specificity and sensitivity. Commonly used GP73 detection methods include radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, and flow cytometry. Patent publication number CN 114180202A relates to a GP73 assay kit and chemiluminescence assay method; patent publication number CN 113945713A relates to a novel biochip-based method for detecting tumor markers such as GP73, enabling large-scale sample testing. However, existing methods suffer from complex operation, high cost, and poor specificity. Therefore, a rapid, sensitive, and simple-to-use GP73 detection method is needed. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for detecting GP73 by constructing a sensor based on a nanocomposite material of reduced graphene oxide-ferrocene-polyaniline (rGO-Fc-PANi), and the minimum detection limit of GP73 is 0.15 pg / mL.

[0004] The detection principle of the present invention is as follows: rGO-Fc-PANi nanocomposite with large specific surface area, high conductivity and good in situ electrochemical activity is prepared; rGO-Fc-PANi is reacted with GP73 aptamer (GP73 Apt ) were coupled to prepare rGO-Fc-PANi-GP73 that can specifically bind to GP73 Apt Signal probe; rGO-Fc-PANi-GP73 was adsorbed by electrostatic adsorption Apt The signal probe was modified on the surface of the screen-printed electrode (AuNPs / SPE) deposited with gold nanoparticles; the aptamer complementary chain (cDNA) was combined with rGO-Fc-PANi-GP73 Apt GP73 in signaling probes Apt cDNA-GP73 Apt Double strands are arranged on the electrode surface to form an electrochemical biosensor interface. When GP73 is introduced into the biosensor interface, GP73 forms a competitive relationship with cDNA. Apt Specific binding, cDNA-GP73 AptDouble strand cleavage, cDNA shedding, forming protein-aptamer GP73-GP73 Apt The complex presents a stable spatial structure and is orderly arranged on the electrode surface. The redox peak current changes of Fc in rGO-Fc-PANi are recorded by differential pulse voltammetry (DPV) in electrochemical work, thereby realizing the detection of GP73.

[0005] The present invention is carried out according to the following steps:

[0006] Step 1: rGO-Fc-PANi-GP73 Apt Preparation of signaling probes

[0007] (1) Preparation of ferrocene-polyaniline (Fc-PANi): Fc and PANi were added to an acetic acid (CH3COOH) solution and mixed, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), stirred, centrifuged, and washed to obtain an Fc-PANi solution;

[0008] (2) Preparation of reduced graphene oxide-ferrocene-polyaniline (rGO-Fc-PANi): Graphene oxide (GO) was dissolved in pure water, ultrasonically crushed, ascorbic acid (AA) was added, and stirred to prepare rGO solution. Fc-PANi was then added, stirred, centrifuged, and washed to obtain rGO-Fc-PANi solution.

[0009] (3) Reduced graphene oxide-ferrocene-polyaniline-GP73 aptamer (rGO-Fc-PANi-GP73 Apt ) signal probe preparation: GP73 aptamer (GP73 Apt ) solution and rGO-Fc-PANi solution were evenly mixed, and then EDC and NHS solution were added, incubated, centrifuged and washed to obtain rGO-Fc-PANi-GP73 Apt Signal probe.

[0010] Step 2: Electrode modification and electrochemical aptasensor construction

[0011] (1) Preparation of AuNPs / SPE: The screen-printed electrode (SPE) was placed in a dilute sulfuric acid (H2SO4) solution and activated by cyclic voltammetry (CV). The activated SPE was immersed in a chloroauric acid (HAuCl4) solution for constant potential deposition, and then washed and dried to obtain AuNPs / SPE.

[0012] (2) rGO-Fc-PANi-GP73 AptPreparation of / AuNPs / SPE sensing interface: rGO-Fc-PANi-GP73 was added to AuNPs / SPE Apt Solution, incubation, washing, and drying to obtain rGO-Fc-PANi-GP73 Apt / Au NPs / SPE;

[0013] (3) cDNA / rGO-Fc-PANi-GP73 Apt Construction of / Au NPs / SPE electrochemical aptamer sensor: The aptamer complementary chain (cDNA) was dropped on rGO-Fc-PANi-GP73 Apt / Au NPs / SPE interface, incubated, washed, and dried, and then bovine serum albumin (BSA) solution was added dropwise to the sensing interface and naturally dried to obtain cDNA / rGO-Fc-PANi-GP73 Apt / AuNPs / SPE electrochemical aptasensor.

[0014] Step 3: Draw the working curve of GP73

[0015] (1) The standard GP73 solution was added dropwise to the electrochemical aptamer sensor obtained in step 2, incubated, washed, and dried to obtain GP73 / cDNA / rGO-Fc-PANi-GP73 Apt / AuNPs / SPE working electrode;

[0016] (2) The working electrode was placed in phosphate buffered saline (PBS) and differential pulse voltammetry (DPV) was used to scan the working electrode and record its peak current.

[0017] (3) Detect different concentrations of GP73 and record the peak current. Based on the relationship between the current response value of the sensor and the GP73 concentration, draw a working curve and calculate the minimum detection limit of the method.

[0018] Step 4: Detection of GP73 in actual serum samples

[0019] (1) Add the actual serum sample to be tested to the electrochemical aptamer sensor obtained in step 2, incubate, wash, and blow dry to obtain a working electrode.

[0020] (2) Place the working electrode in PBS solution and use DPV scanning of the electrochemical workstation to record its peak current.

[0021] (3) According to the working curve described in step 3, the concentration of GP73 in the actual sample to be tested is obtained.

[0022] Furthermore, in step 1, the molar ratio of EDC / NHS is 4:1.

[0023] Further, in the step 2, the H2SO4 solution is 0.5 mol / L, the CV scanning voltage is -0.8V-0.8V, and scanning is performed for 20 times.

[0024] Further, in the step 2, the mass fraction of the HAuCl4 solution used is 0.01%, the deposition potential is 0.4V, and the deposition time is 120s.

[0025] Further, in the step 2, the concentration of the rGO-Fc-PANi solution is 1.0mg / mL.

[0026] Further, in the step 2, the mass fraction of the BSA solution is 1%.

[0027] Preferably, in the step 1, the base sequence of the GP73 is 5'-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3'. Apt

[0028] Preferably, in the step 2, the base sequence of the cDNA is 5'-CCAGGGTATCCAAAGG ATCAACTGC-3'.

[0029] Preferably, in the step 3, the optimal incubation temperature of the GP73 is 25℃, and the optimal incubation time is 1h.

[0030] Preferably, in the steps 3 and 4, the concentration of the PBS solution is 0.2M, the pH value is 7.0, the DPV linear scanning range is -0.2V-0.4V, and the scanning rate is 0.01V / s.

[0031] In the step 1, an rGO-Fc-PANi nanocomposite with a large specific surface area, high conductivity and high electrochemical activity is provided; the rGO-Fc-PANi nanocomposite is coupled and combined with GP73 through an amide reaction to form an rGO-Fc-PANi-GP73 signal probe, and the rGO-Fc-PANi-GP73 signal probe provides a detection signal probe for steps 2, 3 and 4; the step 2 constitutes a biosensing interface for specifically recognizing the GP73, and is an essential and key step in the electrochemical detection of the GP73 in the steps 3 and 4; the GP73 working curve in the step 3 provides a basis for the actual serum sample detection in the step 4. Apt Apt Apt It can be seen that steps 1-4 support and act together to realize the detection of the GP73 by using the rGO-Fc-PANi, GP73, cDNA and the like to construct a sensor.

[0032] Compared with the prior art, the application has the following advantages:

[0033] ​​​The present invention makes full use of the ability of PANi to efficiently transfer electrons, the excellent electrochemical activity of Fc and the large loading capacity of rGO, and combines the high affinity of GP73 Apt , rGO-Fc-PANi-GP73 was successfully prepared Apt A signal probe specifically recognizes the GP73 protein, providing a new method for detecting GP73 in serum. The sensor exhibits excellent specificity, stability, and reproducibility, with a low detection limit of 0.15 pg / mL. When compared with a clinical enzyme-linked immunosorbent assay (ELISA), the relative error for specific detection of GP73 levels in serum ranged from 0.11% to 6.89%. This method offers simple and highly sensitive detection of GP73 in serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the electrochemical aptasensor for detecting GP73 based on rGO-Fc-PANi nanomaterials;

[0035] Figure 2 Transmission electron microscopy (TEM) images of nanocomposites, (A) PANi, (B) Fc-PANi, (C) rGO-Fc-PANi;

[0036] Figure 3 rGO-Fc-PANi-GP73 Apt UV spectrum of the signal probe;

[0037] Figure 4 Scanning electron microscopy (SEM) characterization images of different modification processes on the electrode surface;

[0038] Figure 5 DPV curves of the rGO-Fc-PANi-based electrochemical sensor detecting different GP73 concentrations. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] An electrochemical aptasensor for detecting GP73 based on rGO-Fc-PANi nanocomposite materials was constructed. The detection principle is shown in Figure 1 First, Au NPs were modified on the surface of activated screen-printed electrodes by electrodeposition technology; rGO-Fc-PANi-GP73 was adsorbed on the surface of the activated screen-printed electrodes by adsorption. Apt The signal probe is fixed on the electrode surface; the aptamer complementary chain (cDNA) is combined with rGO-Fc-PANi-GP73 Apt GP73 in signaling probes Apt Hybridization to form cDNA-GP73 AptDouble strands are arranged on the electrode surface to form an electrochemical biosensor interface. When GP73 is introduced into the biosensor interface, GP73 forms a competitive relationship with cDNA. Apt Specific binding, cDNA-GP73 Apt Double strand cleavage, cDNA shedding, forming protein-aptamer GP73-GP73 Apt The complex presents a stable spatial structure and is orderly arranged on the electrode surface. The redox peak current changes of Fc in rGO-Fc-PANi are recorded by DPV, thereby realizing the detection of GP73. The implementation steps are as follows:

[0041] 1.rGO-Fc-PANi-GP73 Apt Preparation of signal probe:

[0042] (1) 50.0 mg of PANi and 100.0 mg of Fc were dispersed in 100.0 mL of pure water, 0.5 mL of acetic acid solution was added, and the mixture was stirred evenly. Then, 10.0 mg of EDC and 2.5 mg of NHS catalyst were added, and the mixture was stirred for 12 h. The mixture was centrifuged at 8,000 rpm and washed with pure water to obtain an Fc-PANi solution.

[0043] (2) 50.0 mg of GO was dispersed in 100 mL of pure water and ultrasonically crushed for 1 h. 50.0 mg of AA was added and magnetically stirred for 3 h to prepare rGO solution.

[0044] (3) Then the rGO solution was added to 100.0 mL of Fc-PANi solution and stirred for 6 h to obtain rGO-Fc-PANi solution.

[0045] The nanocomposites were characterized using a JEM-1200EX transmission electron microscope (TEM). Figure 2 shown. Figure 2 A is the TEM image of PANi, showing a strip-like nanofiber structure; Figure 2 B is the TEM image of Fc-PANi, where the Fc attached to the outside of the strip structure makes the surface irregular; Figure 2 C is the TEM image of rGO-Fc-PANi, which shows a flake-like structure, proving that Fc-PANi has been successfully attached to the rGO surface, indicating that the rGO-Fc-PANi nanocomposite material was successfully constructed.

[0046] (4) 10.0 μL of 100.0 μM GP73Apt and 100.0 μL of 1.0 mg / mL rGO-Fc-PANi solution were mixed, and then 10.0 μg of EDC and 2.5 μg of NHS catalyst were added. The mixture was incubated at 4 °C for 12 h, centrifuged at 8,000 rpm, and washed with pure water to obtain rGO-Fc-PANi-GP73. Apt Signal probe.

[0047] The solution before and after centrifugation was scanned and analyzed using a Hitachi UH5300 UV-Vis spectrophotometer. Figure 3 GP73 Apt There is a more obvious UV absorption peak at 260nm (curve a), and rGO-Fc-PANi nanocomposite materials have absorption peaks at 198nm, 208nm and 262nm (curve b). Apt Afterwards, rGO-Fc-PANi-GP73 Apt The absorbance of the signal probe at 260 nm increased (curve c), and the free GP73 in the supernatant after centrifugation Apt The absorbance at 260 nm was weak (curve d), indicating that GP73 Apt It fully combines with rGO-Fc-PANi nanocomposites and forms a stable GP73 signaling probe.

[0048] 2. Electrode modification and construction of electrochemical aptamer sensor:

[0049] (1) Immerse the SPE electrode in a 0.5 mol / L H2SO4 solution for cyclic voltammetry scanning, scanning 20 segments within the voltage range of 0.4 V-1.0 V; after the scanning is completed, wash it with pure water and dry it to obtain an activated SPE electrode.

[0050] (2) The activated SPE electrode was immersed in HAuCl4 solution and stirred under a magnetic stirrer. The electrode was deposited at a constant potential of 0.4 V for 120 s. After the deposition was completed, it was washed with pure water three times and blown dry with an ear bulb to obtain Au NPs / SPE.

[0051] (3) Add 3.0 μL of rGO-Fc-PANi-GP73 Apt The signal probe was placed in an incubator and incubated for 1 h, washed with pure water 3 times, and blown dry to obtain rGO-Fc-PANi-GP73 Apt / AuNPs / SPE.

[0052] (4) Add 3.0 μL of cDNA solution dropwise to rGO-Fc-PANi-GP73 Apt / Au NPs / SPE, incubated in a 25°C incubator for 1 hour, and the complementary strands that failed to bind to the probe were washed. 2.5 μL of 1% bovine serum albumin solution BSA was added to block the active site and allowed to dry naturally to obtain cDNA / rGO-Fc-PANi-GP73 Apt / AuNPs / SPE sensing interface.

[0053] 3. Drawing of GP73 working curve:

[0054] (1) In cDNA / rGO-Fc-PANi-GP73 Apt 3.0 μL of GP73 standard solution was added to the / Au NPs / SPE sensing interface, incubated at 25°C for 1 h, washed with pure water, and blown dry to obtain a working electrode.

[0055] The electrode construction process was characterized using a SU8020 scanning electron microscope (SEM) produced by Hitachi, Japan. Figure 4 shown. Figure 4 A is the SEM image of SPE, and the electrode surface is rough and dark; Figure 4 B is Au NPs / SPE, the electrode surface is brightened and shiny particles are dispersed, indicating that Au NPs have been successfully modified onto the SPE surface; Figure 4 C is rGO-Fc-PANi-GP73 Apt / Au NPs / SPE, the surface shows a typical wrinkled structure, and contains many flaky nanoparticles, proving that rGO-Fc-PANi-GP73 Apt Successfully modified on the electrode surface; Figure 4 D is cDNA / rGO-Fc-PANi-GP73 Apt / AuNPs / SPE, a layer of membrane-like substance is attached to the electrode surface, which is formed by the hybridization of aptamer and cDNA; Figure 4 E is GP73 / cDNA / rGO-Fc-PANi-GP73 Apt / Au NPs / SPE, the membrane-like material on the electrode surface became thinner, which indicated that part of the cDNA fell off and GP73 attached to the electrode surface.

[0056] (2) The working electrode obtained above was placed in PBS (0.2M, pH=7.0) and scanned at a rate of 0.01 V / s in the range of -0.2 V to 0.4 V using the DPV method on a CHI660E electrochemical workstation, and the peak current was recorded. The DPV curves of different GP73 concentrations are shown in Figure 2. Figure 5As shown in Figure 2, as the concentration of GP73 increases, the DPV peak current value increases. Within the GP73 concentration range of 0.001ng / mL-100.0ng / mL, the sensor current response value (Y) and the GP73 concentration (X) show a logarithmic linear relationship. The working curve is Y=1.271*lgX+4.6719, and the correlation coefficient is 0.9912. According to formula C LOD =3S d / b(S d (a represents the standard deviation of six blank control groups, and b represents the slope of the working curve) The minimum detection limit was calculated to be 0.15 pg / mL.

[0057] 4. Detection of GP73 in actual serum samples:

[0058] (1) Nine serum samples of three types with known GP73 concentrations were collected (normal human serum: samples 1-3, serum from patients with liver disease: samples 4-6, and serum from patients with liver cancer: samples 7-9). The collection and processing of serum samples met the requirements of the Guangxi Key Laboratory of Metabolic Disease Research Ethics Committee. The concentration of GP73 in clinical serum was determined by ELISA.

[0059] (2) Take 3.0 μL of serum sample and add it dropwise to the cDNA / rGO-Fc-PANi-GP73 Apt / Au NPs / SPE electrode surface, incubated at 25°C for 1 h, washed with pure water, and blown dry to obtain a working electrode.

[0060] (3) The working electrode obtained above was placed in PBS (0.2 M, pH = 7.0), and the DPV method was used in a CHI660E electrochemical workstation to scan at a rate of 0.01 V / s within the scanning range of -0.2 V to 0.4 V, and the peak current was recorded.

[0061] (4) The GP73 concentration in each serum sample was calculated using the GP73 working curve Y = 1.271*lgX + 4.6719 obtained in step 3. Three experiments were performed for each serum sample, and the test results are shown in Table 1. As shown in Table 1, the relative error between the GP73 concentration measured by the constructed aptamer sensor and the GP73 concentration in the actual serum sample was between 0.11% and 6.89%, and the relative standard deviation was between 1.08% and 5.82%, indicating that the electrochemical aptamer sensor can be used to detect the GP73 concentration in actual serum samples.

[0062] Table 1 Detection results of GP73 in actual serum samples

[0063]

Claims

1. An electrochemical detection method for GP73 for non-diagnostic and / or therapeutic purposes based on reduced graphene oxide-ferrocene-polyaniline rGO-Fc-PANi nanocomposite material, characterized in that: Follow these steps: Step 1: Preparation of reduced graphene oxide-ferrocene-polyaniline-GP73 aptamer rGO-Fc-PANi-GP73Apt signal probe (1) Preparation of ferrocene-polyaniline Fc-PANi: Ferrocene Fc and polyaniline PANi were added to an acetic acid solution, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added. The mixture was stirred, centrifuged, and washed to obtain an Fc-PANi solution. (2) Preparation of reduced graphene oxide-ferrocene-polyaniline rGO-Fc-PANi: Graphene oxide (GO) was dissolved in pure water, ultrasonically crushed, ascorbic acid (AA) was added, and stirred; Fc-PANi was then added, stirred, centrifuged, and washed to obtain rGO-Fc-PANi; (3) Preparation of reduced graphene oxide-ferrocene-polyaniline-GP73 aptamer rGO-Fc-PANi-GP73Apt signal probe: GP73 aptamer GP73Apt solution and rGO-Fc-PANi solution were mixed, EDC and NHS were added, incubated, centrifuged and washed to obtain rGO-Fc-PANi-GP73Apt signal probe; Step 2: Electrode modification and biosensing interface construction (1) Preparation of gold nanoparticles / screen-printed electrodes AuNPs / SPE: The bare electrode SPE was activated in dilute sulfuric acid. The activated SPE was then placed in a 0.01% chloroauric acid (HAuCl4) solution for electrodeposition at a deposition potential of 0.4 V for 120 s to obtain AuNPs / SPE. (2) Preparation of reduced graphene oxide-ferrocene-polyaniline-GP73 aptamer / nano-gold / screen-printed electrode rGO-Fc-PANi-GP73Apt / AuNPs / SPE sensing interface: rGO-Fc-PANi-GP73Apt was added dropwise to AuNPs / SPE, incubated, washed, and dried to obtain rGO-Fc-PANi-GP73Apt / AuNPs / SPE; (3) Construction of aptamer complementary chain / reduced graphene oxide-ferrocene-polyaniline-GP73 aptamer / nano-gold / screen-printed electrode cDNA / rGO-Fc-PANi-GP73Apt / AuNPs / SPE electrochemical aptamer sensor: Aptamer complementary chain cDNA was dropped on / rGO-Fc-PANi-GP73Apt / AuNPs / SPE, incubated, washed, and blown dry. Then, 1% bovine serum albumin solution BSA was added to the interface and naturally dried to obtain cDNA / rGO-Fc-PANi-GP73Apt / AuNPs / SPE; Step 3: Draw the working curve of GP73 (1) adding the standard GP73 solution dropwise to the electrochemical aptamer sensor obtained in step 2, incubating, washing, and drying to obtain the GP73 / cDNA / rGO-Fc-PANi-GP73Apt / AuNPs / SPE working electrode; (2) Place the working electrode in phosphate buffered saline (PBS) and perform differential pulse voltammetry (DPV) scanning using an electrochemical workstation to record the peak current. (3) Detect different concentrations of GP73 and record the peak current. Based on the relationship between the current response value of the sensor and the GP73 concentration, draw a working curve and calculate the minimum detection limit of the method. Step 4: Detection of GP73 in actual serum samples (1) adding the actual serum sample to be tested to the electrochemical aptamer sensor obtained in step 2, incubating, washing, and drying to obtain a working electrode; (2) Place the working electrode in PBS solution and use DPV scanning on an electrochemical workstation to record its peak current; (3) According to the working curve described in step 3, the concentration of GP73 in the actual sample to be tested is obtained.

2. The method according to claim 1, characterized in that: In step 1, the molar ratio of EDC:NHS is 4:

1.

3. The method according to claim 1, characterized in that: The base sequence of GP73Apt is 5′-NH2-C6GCAGTTGATCCTTTGGATACCCTGG-3′.

4. The method according to claim 1, wherein: The concentration of the rGO-Fc-PANi solution in step 2 is 1.0 mg / mL.

5. The method according to claim 1, characterized in that: The base sequence of the cDNA in step 2 is 5′-CCAGGGTATCCAAAGGATCAACTGC-3′.

6. The method according to claim 1, characterized in that: The incubation temperature in steps 3 and 4 was 25° C., the incubation time was 1 h; the PBS concentration was 0.2 M, the pH value was 7.0; the DPV linear scan range was -0.2 V to 0.4 V, and the scan rate was 0.01 V / s.

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