CN111413385A - Method for detecting GPC3 based on RGO-CS-Fc/Pt-Pd NPs nano composite material - Google Patents
Method for detecting GPC3 based on RGO-CS-Fc/Pt-Pd NPs nano composite material Download PDFInfo
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Abstract
The invention relates to a novel aptamer sensor which takes a liver cancer marker phosphatidylinositol proteoglycan 3 (Glypican-3, GPC 3) as a research object, takes a GPC3 aptamer as a recognition probe, and is capable of specifically recognizing and combining GPC3 protein based on good electron transfer effect and excellent load capacity of a reduced graphene oxide-chitosan-ferrocene/nano platinum and palladium (Pt-Pd NPs/RGO-CS-Fc) composite material, so that the novel aptamer sensor capable of specifically recognizing and quantitatively analyzing the GPC3 protein is constructed, and is used for detecting the content of GPC3 in serum. The method has the advantages of simple operation, time saving, low cost and lower detection limit.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a method for detecting glypican-3 (GPC 3) based on a nanocomposite and a suitable ligand.
Background
Primary hepatocellular carcinoma (HCC) is a malignant tumor which is common in China and is easy to threaten the life of people. Common liver cancer markers include AFP, GPC3, GP73 and the like. GPC3 is present at a very low concentration in normal human tissues, but is present at a very high concentration in liver cancer patients, and therefore GPC3 is closely related to the occurrence and progression of liver cancer. The GPC3 detection method mainly includes radioimmunoassay, fluoroimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, flow immunoassay, electrochemical immunosensor, piezoelectric immunosensor, and the like. The invention patent publication No. CN 105717104B relates to a method for separating and obtaining CTC in peripheral blood of a liver cancer patient, from which a tissue specimen cannot be obtained, by using a membrane filter device, and detecting the expression condition of peripheral blood GPC3 of the hepatocellular carcinoma patient by using a cell wax block technology to make a thin layer slice. The invention discloses an invention patent with publication number CN 105759051B, and relates to a quantitative analysis kit for determining phosphatidylinositol proteoglycan-3 by GPC3 nanometer magnetic microsphere chemiluminescence immunoassay with acridine ester as a luminescent substance. The instruments used in the methods are expensive, complex to operate, time-consuming and high in technical requirement, and a rapid and simple GPC3 detection method needs to be established.
Disclosure of Invention
The invention aims to provide a method for detecting GPC3 by combining a nanocomposite material based on reduced graphene oxide-chitosan-ferrocene/nano platinum-palladium (RGO-CS-Fc/Pt-Pd NPs) with a suitable ligand, so that the sensitivity is improved and the specificity is enhanced.
In order to solve the technical problem, an electro-deposition technology and electrostatic adsorption are adopted to manufacture a GPC3 nanometer aptamer electrochemical biosensor based on RGO-CS-Fc/Pt-Pd NPs, Differential Pulse Voltammetry (DPV) of an electrochemical workstation is adopted to record peak current, the incubation temperature, the incubation time, the pH value of PBS, the use amount of RGO-CS-Fc composite material and the concentration of GPC3 aptamer of GPC3 are optimized, a standard curve is drawn, accurate GPC3 concentration is calculated by comparing with the standard working curve, and compared with the existing method, the method is relatively simple in operation, high in specificity, less in time and cost consumption and capable of reaching the detection limit of 3.67ng/m L.
The detection principle of the invention is that the electro-deposition technology and the electrostatic adsorption are adopted to modify RGO-CS-Fc/Pt-PdNPs on the surface of a screen printing electrode, GPC3 aptamer is loaded on the surface of RGO-CS-Fc/Pt-Pd NPs material through non-covalent binding action and intermolecular action, the aptamer is connected with the composite material in a single chain form due to the unstable spatial structure of the aptamer and can be modified on the electrode, after GPC3 is added on a biosensing interface, the GPC3 aptamer specifically binds with GPC3 protein to form a protein-aptamer complex with a stable spatial structure so as to be orderly arranged on the surface of the electrode, the electrochemical signal (the scanning voltage is-0.4V-1.0V and the scanning rate is 0.01V/s) in a PBS solution (0.2 mol/L and pH 7.4) before and after GPC3 is detected through a DPV method, and the relation curve of the current and the concentration of GPC3 is drawn, thereby realizing the detection of GPC 3.
The invention is carried out according to the following steps:
step 1: preparation of RGO-CS-Fc Material
(1) Preparation of Reduced Graphene Oxide (RGO): pouring Graphene Oxide (GO) into distilled water, and performing ultrasonic treatment by using an ultrasonic cell disruption instrument to fully and uniformly dissolve the Graphene Oxide (GO) to prepare a GO aqueous solution. Placing GO aqueous solution in a beaker, and adding Ascorbic Acid (AA) to reduce GO to obtain RGO.
(2) Preparation of Chitosan-ferrocene (CS-Fc): adding Chitosan (CS) into the acetic acid solution to obtain a chitosan solution. Mixing ferrocenecarboxylic acid (Fc) with the chitosan solution, activating by carbodiimide/N-hydroxysuccinimide (EDC/NHS), and stirring to obtain the CS-Fc complex.
(3) Preparing a reducing graphene oxide-chitosan-ferrocene (RGO-CS-Fc) composite material: adding the RGO suspension into the CS-Fc solution, activating by EDC/NHS, and centrifuging to obtain RGO-CS-Fc suspension.
Step 2: electrode modification and biosensing interface construction
(1) Placing a screen-printed electrode (SPE) in H2SO4And (3) in the solution, performing cyclic voltammetry scanning to obtain an activated screen printing electrode, and washing the screen printing electrode with water.
(2) And (3) placing the activated screen printing electrode into a chloroplatinic acid and palladium nitrate solution, carrying out constant potential deposition, and after the deposition is finished, washing the electrode with water to obtain the Pt-Pd NPs/SPE electrode.
(3) Soaking the Pt-Pd NPs/SPE electrode with glutaraldehyde, washing with PBS, drying, then dropwise adding RGO-CS-Fc suspension for incubation for a period of time, washing with PBS, and drying in the air to obtain the RGO-CS-Fc/Pt-Pd NPs/SPE electrode.
(4) Taking aminated GPC3 aptamer (GPC 3)apt) Adding dropwise into sensor interface, incubating for a period of time, washing GPC3 aptamer which is not fixed on the interface with PBS solution, adding Bovine Serum Albumin (BSA) solution, and blocking to obtain GPC3aptthe/RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface is dried for standby.
And step 3: GPC3 Standard Curve
(1) And (3) dropwise adding a standard GPC3 solution to the GPC3apt/RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface obtained in the step 2, incubating for a period of time, washing with a PBS solution to obtain a working electrode, and airing for later use.
(2) The working electrode was placed in PBS solution and its peak current was recorded using DPV scanning at the electrochemical workstation.
(3) GPC3 was detected at different concentrations, and a calibration curve was plotted to calculate the minimum detection limit of the method.
And 4, step 4: detection of GPC3 in actual samples
(1) And (3) dripping the actual sample to be detected on the GPC3apt/RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface obtained in the step (2), incubating for a period of time, washing with a PBS solution to obtain a working electrode, and airing for later use.
(2) The working electrode was placed in PBS solution and its peak current was recorded using DPV scanning at the electrochemical workstation.
(3) And (4) obtaining the concentration of GPC3 in the actual sample to be tested according to the standard curve in the step 3.
Further, the acetic acid solution in the step 1 was 100 m L1%.
Further, the EDC/NHS concentration in step 1 was 10 mmol/L.
Further, H in the step 22SO4Concentration of solutionIt was 0.5 mol/L.
Further, in the step 2, the scanning voltage is-0.4V-1.0V, and the number of scanning sections is 20.
Further, in the step 2, the concentrations of the chloroplatinic acid and the palladium nitrate are both 0.01%, the deposition potential is-0.2V, and the deposition time is 120 s.
Further, in the step 2, the concentration of glutaraldehyde is 2.5%.
Further, in the step 2, the concentration of the BSA solution is 0.5%, the concentration of PBS is 0.2 mol/L, and the pH value is 7.4.
Further, the GPC3 aptamer concentration in step 2 was 1. mu. mol/L.
Further, the GPC3 aptamer in step 2 was incubated at 37 ℃ for 3 hours at the electrode.
Preferably, the optimum incubation temperature of GPC3 in step 3 is 37 ℃ and the optimum incubation time is 20 min.
Preferably, the DPV linear scanning range in the steps 3 and 4 is-0.4V-1.0V, and the scanning speed is 0.01V/s.
Wherein, step 1 provides a high-conductivity nanocomposite material for step 2. Step 2 constitutes a biosensing interface that specifically recognizes GPC3 and facilitates the transfer of electrons. The construction of biosensing interface in step 2 is an essential key step in the electrochemical detection of GPC3 in step 3 and step 4. The working curve of GPC3 from step 3 provides a basis for the determination of GPC3 concentration in the actual sample from step 4. It can be seen that steps 1-4 support each other and act together to enable GPC3 detection using RGO-CS-Fc/Pt-Pd NPs composites and GPC3 aptamers as recognition probes.
Compared with the prior art, the invention has the following advantages:
1. nanometer platinum and palladium particles modified on the surface of an electrode have the effect of enhancing an electron transfer effect, are combined with a graphene material with excellent load capacity and capable of effectively amplifying a current signal and the specific recognition effect of a GPC3 aptamer on a GPC3 protein to obtain a nanometer platinum, palladium and reduced graphene oxide (RGO-CS-Fc/Pt-Pd NPs) composite material, and a nanometer aptamer electrochemical sensor capable of specifically detecting the GPC3 level in serum based on the composite nanometer material is constructed. Compared with the traditional sensor, the novel nano material sensor has the advantages of smaller volume, higher speed, higher precision and higher reliability.
2. The method for detecting GPC3 by using GPC3 aptamer as a recognition probe has the characteristic of small background interference and can reach the detection limit of 3.67ng/m L.
Drawings
FIG. 1 is a schematic diagram of the detection of GPC3 based on RGO-CS-Fc/Pt-Pd NPs nanocomposites in combination with aptamers;
FIG. 2 Transmission Electron micrographs of RGO (A) and RGO-CS-Fc (B);
FIG. 3 is a scanning electron microscope characterization of various modification processes on the electrode surface;
FIG. 4 working curves for a GPC3 nanoaptamer sensor based on RGO-CS-Fc/Pt-Pd NPs; fig. 4A is a DPV curve for different GPC3 concentrations and fig. 4B is a working curve for a GPC3 aptamer sensor.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
A method for detecting GPC3 based on RGO-CS-Fc/Pt-Pd NPs nanocomposite, the detection principle is shown in figure 1. Firstly, depositing platinum and palladium on the surface of the activated screen printing electrode. Then, the RGO-CS-Fc composite material is dripped on the surface of the electrode, and the composite material is fixed on the surface of the electrode through adsorption and chemical crosslinking. And then, by means of incubating the GPC3 aptamer, the aminated GPC3 aptamer can be covalently bound with RGO-CS-Fc on the surface of the electrode under the action of EDC/NHS, and is connected with the composite material in a single-chain form due to the unstable spatial structure of the RGO-CS-Fc, so that the modified GPC3 aptamer can be modified on the electrode. After GPC3 protein is dripped, GPC3 aptamer can be specifically combined with GPC3 protein to form a protein-aptamer complex to form a stable spatial structure, so that the protein-aptamer complex is orderly arranged on the surface of an electrode. The change of the electrochemical signal of the Fc in the sensor is detected by a DPV method, so that the GPC3 protein can be effectively quantitatively analyzed. The implementation steps are as follows:
1. preparation of RGO-CS-Fc composite nanomaterial:
weighing 5 mg of Graphene Oxide (GO), pouring GO into 50 m L distilled water, using an ultrasonic cell disruptor to carry out ultrasonic treatment for 2h to fully and uniformly dissolve GO to prepare 0.1 mg/m L aqueous solution of GO, putting 10m L aqueous solution of GO into a beaker, adding 10 mg of Ascorbic Acid (AA) to reduce GO, putting the beaker on a constant-temperature digital display magnetic heating stirrer to continuously stir for 12 h to obtain RGO, adding 2 mg of Chitosan (CS) into 100 m L% acetic acid solution, continuously stirring uniformly with a glass rod until no bubbles are observed in the solution to obtain a uniform and stable 2.0 mg/m L CS solution, weighing 2 mg of ferrocenecarboxylic acid (Fc) to mix with 10m L chitosan, activating with 10 mmol/L/NHS/EDC/NHS, stirring for 24 h to obtain a chitosan-ferrocene compound, adding 10m L RGO suspension into 10m L-chitosan (CS-Fc) solution, carrying out centrifugal separation on a plurality of RGO sheets which are shown in original RGCS-10 m L-Fc-chitosan (10 mmol/EDC) and a thin film which is combined with a plurality of RGO and a thin film which is shown in a drawing and a drawing which shows that RGO is subjected to be subjected to a more transparent RGO-RG-RGO-RG-RGO composite material which is combined by a more-Mg-10 m-10-.
2. Modification of the electrode and construction of a biosensing interface:
the screen-printed electrode (SPE) was first soaked in 0.5 mol/L H before use2SO4Scanning with Cyclic Voltammetry (CV) in solution, scanning at-0.4-1.0V voltage for 20 sections, washing with water, air drying to obtain activated SPE, putting the activated SPE electrode into 4 m L0.01.01% chloroplatinic acid and palladium nitrate solution, depositing at-0.2V constant potential for 120s, washing with pure water for 3 times, drying to obtain Pt-Pd NPs/SPE electrode, soaking the Pt-Pd NPs/SPE electrode in 2.5% glutaraldehydeWashing with PBS (pH 7.4) for 3 times for 15 min, drying, adding RGO-CS-Fc suspension 5 μ L dropwise, incubating for 30min, washing with PBS for 3 times, and air drying to obtain RGO-CS-Fc/Pt-Pd NPs/SPE, and subjecting to GPC3 aptamer (5' -NH) 2 μ L amination2-TAA CGC TGA CCT TAG CTG CAT GGC TTT ACA TGT TCC A-3') is added to RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface in a dropwise manner, the mixture is incubated for 3 hours, aptamers which can not be fixed to the interface are washed, 6 mu L0.5% BSA solution is added in a dropwise manner for blocking, and the mixture is naturally dried to obtain GPC3aptthe/RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface. As shown in fig. 3, the modification process of the electrode surface is characterized by using a Scanning Electron Microscope (SEM). The bare electrode surface in FIG. 3A is smooth; after the electrode in fig. 3B modifies the platinum and palladium metal materials, the surface of the electrode has obvious granular sensation, and shiny particles are dispersed, which indicates that the platinum and palladium metal materials are successfully modified on the surface of the electrode by electrodeposition; FIG. 3C clearly shows a lamellar structure, indicating that the RGO-CS-Fc material has been immobilized to the electrode; the electrode in FIG. 3D has some more filaments, indicating that the GPC3 aptamer has been modified to the electrode surface; the white sphere is clearly visible on fig. 3E, indicating that the GPC3 protein specifically binds to the GPC3 aptamer on the electrode.
3. Drawing of GPC3 Standard Curve:
at GPC3aptThe working electrode is placed in PBS supporting liquid (0.2 mol/L, pH 7.4) to record the peak current by DPV scanning of an electrochemical workstation, and the DPV curve diagram of different GPC3 concentrations is shown in figure 4A. CGP3 concentrations are from 0.001 μ g/m L to 10 μ g/m L, the current response value of the sensor is in linear relation with the concentration of GPC3, the linear equation is =88.7852-3.2873x = 5392, wherein the GPC response intensity is shown in the formula 685 2C 685: (685-685) where GPC3 is fixed on the biosensing interfaceLOD=3SbCalculated asThe detection limit of the sensor is 3.67ng/m L (S)bStandard deviation calculated for 6 replicates of blank samples, b is the slope of the standard curve).
4. Detection of GPC3 in actual serum samples:
mixing serum with standard solution of GPC3 protein at ratio of 1:1, respectively, 1 μ g/m L, 5 μ g/m L, and 10 μ g/m L, making into mixed solution, and adding dropwise 2 μ L to GPC3aptthe/RGO-CS-Fc/Pt-Pd NPs/SPE electrode surface. And (4) placing the working electrode in the PBS supporting solution for DPV scanning, and recording the current value. According to the standard curve y =88.7852-3.2873x of the step 4, the corresponding concentration of GPC3 in the actual serum sample can be calculated, and the detection result is shown in Table 1.
TABLE 1 results of GPC3 detection in actual serum samples
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
Claims (9)
1. A method for detecting GPC3 based on RGO-CS-Fc/Pt-Pd NPs nanocomposite, which comprises the following steps:
step 1: preparation of RGO-CS-Fc Material
(1) Preparing reducing graphene oxide: pouring the graphene oxide into distilled water, and performing ultrasonic treatment by using an ultrasonic cell disruption instrument to fully and uniformly dissolve the graphene oxide to prepare a GO aqueous solution; placing the GO aqueous solution in a beaker, and adding ascorbic acid to reduce GO to obtain RGO;
(2) preparation of chitosan-ferrocene: adding chitosan into an acetic acid solution to obtain a chitosan solution; mixing ferrocenecarboxylic acid with the chitosan solution, activating with EDC/NHS, and stirring to obtain a CS-Fc complex;
(3) preparing a reductive graphene oxide-chitosan-ferrocene composite material: adding the RGO suspension into the CS-Fc solution, activating EDC/NHS, and centrifuging to obtain RGO-CS-Fc suspension;
step 2: electrode modification and biosensing interface construction
(1) Placing the screen-printed electrode in H2SO4In the solution, cyclic voltammetry scanning is carried out to obtain an activated screen printing electrode, and the screen printing electrode is washed clean by water;
(2) placing the activated screen printing electrode into a chloroplatinic acid and palladium nitrate solution, carrying out constant potential deposition, and washing the electrode clean by water after the deposition is finished to obtain a Pt-Pd NPs/SPE electrode;
(3) soaking the Pt-Pd NPs/SPE electrode with glutaraldehyde, washing with PBS, drying, then dropwise adding RGO-CS-Fc suspension for incubation for a period of time, washing with PBS, and drying in the air to obtain an RGO-CS-Fc/Pt-Pd NPs/SPE electrode;
(4) dropping aminated GPC3 aptamer to sensor interface, incubating for a while, washing GPC3 aptamer not fixed to interface with PBS solution, dropping BSA solution for blocking to obtain GPC3aptthe/RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface is dried for standby;
and step 3: GPC3 working curve plotting
(1) Dropwise adding standard GPC3 solution to GPC3 obtained in step 2aptThe method comprises the following steps of (1) incubating an/RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface for a period of time, washing the sensing interface with a PBS solution to obtain a working electrode, and airing the working electrode for later use;
(2) putting the working electrode into a PBS solution, adopting DPV scanning of an electrochemical workstation, and recording the peak current of the working electrode;
(3) detecting GPC3 with different concentrations, drawing a standard curve, and calculating the lowest detection limit of the method;
and 4, step 4: detection of GPC3 in actual samples
(1) GPC3 obtained in step 2aptThe method comprises the following steps of (1) dropwise adding an actual sample to be detected into an/RGO-CS-Fc/Pt-Pd NPs/SPE sensing interface, incubating for a period of time, cleaning with a PBS solution to obtain a working electrode, and airing for later use;
(2) putting the working electrode into a PBS solution, adopting DPV scanning of an electrochemical workstation, and recording the peak current of the working electrode;
(3) and (4) obtaining the concentration of GPC3 in the actual sample to be tested according to the standard curve in the step 3.
2. A method of detecting GPC3 according to claim 1, wherein the acetic acid in step 1 is 100 m L1%.
3. The method of detecting GPC3 of claim 1, wherein the EDC/NHS concentration in step 1 is 10 mmol/L.
4. The method of detecting GPC3 of claim 1, wherein the electrode is placed at 0.5 mol/L H in step 22SO4The voltage range is-0.4V-1.2V.
5. A method of detecting GPC3 according to claim 1, characterized in that: the deposition solution for nano platinum and palladium in the step 2 is chloroplatinic acid with the concentration of 0.01 percent and palladium nitrate with the concentration of 0.01 percent, the deposition potential is-0.2V, and the deposition time is 120 s.
6. The method of detecting GPC3 according to claim 1, wherein the BSA solution in step 2 is 6. mu. L0.5.5% BSA solution.
7. A method of detecting GPC3 according to claim 1, characterized in that: the incubation temperature in steps 3 and 4 was 37 ℃ and the incubation time was 20 minutes.
8. The method of detecting GPC3 of claim 1, wherein the solution used in the DPV scanning in steps 3 and 4 is 0.2 mol/L in PBS at pH 7.4.
9. A method of detecting GPC3 according to claim 1, characterized in that: the scanning range in the step 3 and the step 4 is-0.4V-1.0V, and the scanning speed is 0.01V/s.
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