CN114965637A - Method for constructing sandwich type aptamer sensor based on nanocomposite to detect GPC3 - Google Patents
Method for constructing sandwich type aptamer sensor based on nanocomposite to detect GPC3 Download PDFInfo
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- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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Abstract
A method for detecting GPC3 by constructing a sandwich type aptamer sensor based on a nano composite material comprises the steps of modifying gold-reduced graphene oxide (Au NPs @ rGO) on the surface of a screen-printed electrode, and fixing a GPC3 aptamer (GPC 3) through physical adsorption Apt ) The H-rGO-Pt @ Pd NPs-GPC3 is prepared by taking heme-reduced graphene oxide-platinum @ palladium (H-rGO-Pt @ Pd NPs) as a carrier Apt And a signal probe is used for constructing the sandwich type electrochemical nano aptamer sensor. The method comprises the steps of catalyzing Ag deposition to carry out effective current amplification by using the peroxidase-like property of an H-rGO-Pt @ Pd NPs nano composite material, scanning by using a DPV (differential pulse velocimetry) method, recording the peak current of the Ag deposition, and realizing GPC3 detection, wherein the minimum detection limit is 0.4801 mu g/mL.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a method for detecting GPC3 by constructing a sandwich type aptamer sensor based on a nano composite material.
Background
Examples of the detection method of glypican-3 (GPC 3) include ELISA, radioimmunoassay, fluoroimmunoassay, chemiluminescence immunoassay, and electrochemical immunosensor. The invention patent with publication number CN 112014577A discloses an immunoassay kit, which adopts magnetic bead particles to couple a reagent M diluted by a mouse anti-human GPC3 monoclonal antibody and a reagent R diluted by an alkaline phosphatase marked rabbit anti-human GPC3 polyclonal antibody to realize the detection of GPC3 in serum. The invention patent with publication number CN 105717104A utilizes a membrane filtration device to separate and obtain CTC in peripheral blood of a liver cancer patient, and utilizes a cell wax block technology to manufacture a thin layer slice, and then detects GPC 3. Therefore, there is a need to establish a rapid, sensitive, portable method for detecting GPC 3.
Disclosure of Invention
The invention aims to solve the technical problem of providing a sandwich type aptamer sensor based on a heme-reduced graphene oxide-platinum @ palladium nanocomposite (H-rGO-Pt @ Pd NPs) to realize GPC3 detection, wherein the minimum detection limit is 0.4801 mu g/mL.
In order to solve the technical problem, the H-rGO-Pt @ Pd NPs nano composite material with high specific surface area, high conductivity and peroxidase-like property is synthesized by utilizing the adsorption effect of pi-pi bonds, and the nano composite material is combined with a GPC3 aptamer (GPC 3) in a pi-pi bond combination mode Apt ) Combining to prepare H-rGO-Pt @ Pd NPs-GPC3 capable of being specifically combined with GPC3 Apt A signal probe; modifying Au NPs @ rGO on the surface of the screen-printed electrode by adopting an electrodeposition technology; GPC3 aptamer is modified on the surface of Au NPs @ rGO electrode through electrostatic adsorption, and as GPC3 can be specifically combined with GPC3 aptamer to form a stable chemical structure, H-rGO-Pt @ Pd NPs-GPC3 is constructed Apt /GPC3/GPC3 Apt Sandwiched electrochemical adaptation of/Au NPs @ rGO/SPCEA body sensor. Dropping hydrogen peroxide (H) on the surface of the electrode 2 O 2 ) And silver nitrate (AgNO) 3 ) H-rGO-Pt @ Pd NPs can effectively catalyze H due to peroxidase-like property 2 O 2 With AgNO 3 Reaction of Ag + Electrons are obtained, the metal Ag is reduced to be deposited on the surface of the electrode, the current response of the deposited Ag sensor is detected by Differential Pulse Voltammetry (DPV), the current response of the Ag is in positive correlation with the concentration of GPC3, and the detection of GPC3 is realized.
The invention is carried out according to the following steps:
step 1: H-rGO-Pt @ Pd NPs-GPC3 Apt Preparation of Signaling probes
(1) Preparation of reduced graphene oxide (rGO): dissolving Graphene Oxide (GO) in pure water, and carrying out ultrasonic crushing; adding Ascorbic Acid (AA) and stirring to obtain rGO suspension;
(2) preparation of heme-reduced graphene oxide (H-rGO): adding ammonia water (NH) into heme (Hemin) 3 ·H 2 O), adding rGO solution and hydrazine hydrate (N) 2 H 4 ·H 2 O) solution, stirring in water bath, centrifuging, and cleaning to obtain H-rGO suspension;
as an improvement, the method also comprises the following steps:
(3) preparation of H-rGO-Pt @ Pd NPs: adding poly (diallyldimethylammonium chloride) (PDDA) and sodium chloride (NaCl) into the H-rGO suspension, stirring, and adding sodium tetrachloropalladate (Na) 2 PdCl 4 ) And sodium tetrachloroplatinate (Na) 2 PtCl 4 ) Then stirring is continued, and hydrazine hydrate (N) is added 2 H 4 ·H 2 O) solution is stirred, stored, centrifuged, cleaned and dried to obtain H-rGO-Pd @ Pd NPs solid;
（4）H-rGO-Pt@Pd NPs-GPC3 Apt preparation of a signal probe: coupling GPC3 aptamer (GPC 3) Apt ) The solution H-rGO-Pt @ Pd NPs solution is evenly mixed by ultrasound, incubated, centrifuged and cleaned to obtain H-rGO-Pt @ Pd NPs-GPC3 Apt A signaling probe.
Step 2: electrode modification and biosensing interface construction
（1）Placing a screen printing electrode (SPCE) in dilute sulfuric acid (H) 2 SO 4 ) Activating in a solution;
(2) placing the activated SPCE in a container containing chloroauric acid (HAuCl) 4 ) Carrying out electrodeposition in the mixed solution of the Au and the GO to obtain an Au NPs @ rGO/SPCE electrode;
(3) GPC3 Apt Dripping on the surface of Au NPs @ rGO/SPCE, incubating, washing and drying to obtain GPC3 Apt /Au NPs@rGO/SPCE。
And step 3: plotting of GPC3 working curves
(1) Dripping standard GPC3 solution with different concentrations on GPC3 electrochemical biosensing interface, incubating, cleaning, and blow-drying to obtain GPC3/GPC3 Apt /Au NPs@rGO/SPCE；
(2) At GPC3/GPC3 Apt H-rGO-Pt @ Pd NPs-GPC3 is dripped on Au NPs @ rGO/SPCE Apt Incubating, washing and drying the solution to obtain H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/GPC3 Apt / Au NPs@rGO/SPCE；
(3) H is to be 2 O 2 And AgNO 3 Dropwise adding the solution into H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/ GPC3 Apt Reacting on Au NPs @ rGO/SPCE in a dark place, and cleaning to obtain a working electrode Ag/H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/GPC3 Apt Au NPs @ rGO/SPCE for standby;
(4) immersing the working electrode in nitric acid (HNO) 3 ) And potassium nitrate (KNO) 3 ) In the glycine-sodium hydroxide (NaOH) buffer solution, DPV of an electrochemical workstation is adopted for scanning, and the response current value of the sensor is recorded;
(5) respectively detecting GPC3 working electrodes with different concentrations, recording the response current of the sensor, and drawing a standard working curve according to the relationship between the current response value of the sensor and the concentration of GPC 3; and calculating the lowest detection limit of the method.
And 4, step 4: detection of GPC3 in actual serum samples
(1) Immersing working electrode prepared by actual serum sample to be tested into the sample containing HNO 3 And KNO 3 In glycine-NaOH buffer solution, scanning is carried out by adopting DPV of an electrochemical workstationRecording the response current value of the sensor;
(2) and (4) calculating the concentration of GPC3 in the actual sample to be detected according to the working curve obtained in the step 3.
Preferably, the method comprises the following steps:
said GPC3 described in step 1 Apt The DNA sequence of (a) is 5' -TAA CGC TGA CCT TAG CTG CAT GGC TTT ACA TGT TCC A-NH 2 -3'；
and 3, in the step 4 and the step 3, the incubation temperature of the electrode is 25 ℃, the incubation time is 30 min, and when the pH value of the PBS buffer solution is 7.0, the response current of the sensor is maximum.
Wherein, the step 1 provides a H-rGO-Pt @ Pd NPs nano composite material with high specific surface area, high conductivity and peroxidase-like property, and GPC3 Apt H-rGO-Pt @ Pd NPs-GPC3 is formed through pi-pi bond combination Apt A nano signal probe for providing a detection signal for the step 2; and 2, forming a sandwich type biosensing interface for specifically identifying GPC3, and utilizing the specific combination of a GPC3 aptamer and GPC3 protein and the peroxidase-like property of the H-rGO-Pt @ Pd NPs nanocomposite, so that the electron transfer and catalysis are facilitated. The construction of the biosensing interface in step 2 provides feasibility conditions for the measurement of GPC3, an essential prerequisite for the electrochemical detection of GPC3 in steps 3 and 4. It can be seen that steps 1-4 are carried out layer by layer, and each step is indispensable, so that H-rGO-Pt @ Pd NPs nano composite material and GPC3 aptamer (GPC 3) can be utilized Apt ) And the detection of GPC3 by the sandwich type aptamer sensor is realized by the Au NPs @ rGO nano hybrid.
Compared with the prior art, the invention has the following advantages:
1. the H-rGO-Pt @ Pd NPs nanocomposite material constructed by the method has the characteristics of large specific surface area, excellent peroxidase-like catalytic activity, good biocompatibility and the like, and has a conductive substance Pt @ Pd NPs in the material, so that the electron transfer efficiency is enhanced. In addition to this, the present invention is,the high-efficiency peroxidase-like property of the H-rGO-Pt @ Pd NPs nano composite material can effectively catalyze H 2 O 2 And AgNO 3 Oxidation-reduction reaction is carried out to AgNO 3 Ag in (1) + Reducing the Ag into metal Ag, depositing the metal Ag on the surface of the electrode, amplifying effective current, detecting the current response of the deposited Ag sensor by a DPV method, and realizing the sensitive detection of GPC3 protein.
2. The electrode surface is modified by Au NPs @ rGO and H-rGO-Pt @ Pd NPs nano composite materials, so that GPC3 can be effectively fixed Apt And the conductive material has excellent conductivity, enhances electron transfer and effectively amplifies detection signals.
3. Hemin in H-rGO-Pt @ Pd NPs nano composite material is used as an electroactive substance to construct H-rGO-Pt @ Pd NPs-GPC3 Apt To identify the probe, GPC3 Apt A sandwich-type electrochemical sensor designed for capture probes detects GPC 3. The sandwich structure is more stable, the precision is high, the stability is good, the detection of the target molecules is facilitated, and the minimum detection limit of the method is 0.4801 mu g/mL.
Drawings
FIG. 1 is a schematic diagram of a sandwich type electrochemical aptamer sensor constructed based on H-rGO-Pt @ Pd NPs nano-materials for detecting GPC 3;
FIG. 2 SEM and XRD patterns of H-rGO-Pt @ Pd NPs nanocomposites;
FIG. 3 is a graph showing the validation of peroxidase-like properties of H-rGO-Pt @ Pd NPs nanocomposites;
FIG. 4H-rGO-Pt @ Pd NPs-GPC3 Apt A signal probe verification diagram;
FIG. 5 SEM image of electrode modification process;
FIG. 6 DPV profile of different GPC3 concentrations.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The principle of constructing a sandwich type electrochemical aptamer sensor based on H-rGO-Pt @ Pd NPs nano material for detecting GPC3 is shown in figure 1. Firstly, preparing H-rGO-Pt @ Pd NPs nano composite material, and then fixing GPC3 by using the material Apt Formation of H-rGO-Pt @ Pd NPs-GPC3 Apt A signal probe; then activating the SPCE electrode, and modifying Au NPs @ rGO on the surface of the activated electrode through an electrodeposition technology to enhance the conductivity of the electrode; GPC3 was synthesized specifically with H-rGO-Pt @ Pd NPs-GPC3 Apt And GPC3 Apt Combined to form a stable chemical structure to construct H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/GPC3 Apt a/Au NPs @ rGO/SPCE sandwich type electrochemical aptamer sensor; catalysis of H by peroxidase-like properties of H-rGO-Pt @ Pd NPs nanocomposite 2 O 2 And AgNO 3 Reaction of H 2 O 2 Can mix AgNO 3 Ag in (C) + Reducing the metal Ag to be deposited on the surface of the electrode, recording current signals before and after GPC3 detection by adopting an electrochemical workstation DPV method, and then drawing a working curve according to the relation between the concentration of GPC3 and response current to obtain the level of GPC3 in serum, thereby achieving the purpose of detecting GPC 3.
The implementation steps are as follows:
1、H-rGO-Pt@Pd NPs-GPC3 Apt preparation of Signaling probes
(1) Weighing 30.0 mg of GO, dispersing in 30.0 mL of pure water, crushing, adding 30.0 mg of Ascorbic Acid (AA), and magnetically stirring for 3 hours to prepare 1.0 mg/mL of rGO suspension; then adding the rGO suspension into 30.0 mL of Hemin solution, stirring for 30 min, adding 5.0 mu L of hydrazine hydrate solution, carrying out vortex oscillation, and carrying out water bath at 50 ℃ for 2 h; centrifuging and washing to obtain H-rGO suspension.
(2) Adding 2.0 mL of PDDA solution containing 2% and 2.0 mL of NaCl solution containing 2% into 30.0 mL of H-rGO suspension liquid containing 1.0 mg/mL, and stirring for 18H; 1.0 mL of Na 2 PdCl 4 And 1.0 mL of Na 2 PtCl 4 And adding the solution into the H-rGO mixed solution, adding 10.0 mu L of hydrazine hydrate solution, and stirring for 18H to obtain the H-rGO-Pt @ Pd NPs nano composite material.
Characterization of the H-rGO-Pt @ Pd NPs nanocomposites was performed using the Scanning Electron Microscope (SEM) SU8020 from Hitachi, Japan, as shown in FIG. 2A, where it can be seen that small particles attached to the surface of the pleated membrane, indicating that Pt and Pd particles were successfully attached to the H-rGO material. The H-rGO-Pt @ Pd NPs nanocomposite was characterized by using a D8 ADVANCE X-ray diffractometer manufactured by bruker, usa, and as shown in fig. 2B, the C (002) crystal plane of rGO corresponds to the diffraction peak of 2 θ =23.51 °, the (111), (200) and (220) of Pt correspond to the diffraction peaks of 2 θ =39.76 °, 46.24 ° and 67.45 °, and the (111), (200) and (220) of Pd correspond to the diffraction peaks of 2 θ =40.12 °, 46.66 ° and 68.12 °, which indicates that the H-rGO-Pt @ Pd NPs nanocomposite was successfully prepared.
The peroxidase-like properties of the H-rGO-Pt @ Pd NPs nanocomposite material prepared by Hitachi UH5300 ultraviolet-visible spectrophotometer of Shanghai, China and Chi660E electrochemical workstation manufactured by Chenghua instruments, Inc. of China were verified, as shown in FIG. 3. FIG. 3A is a demonstration of the peroxidase-like nature of H-rGO-Pt @ Pd NPs by color reaction, 3',5,5' -Tetramethylbenzidine (TMB) (a) alone and H-NPs alone 2 O 2 (b) Colorless and transparent, when the TMB and H are mixed 2 O 2 (c) Mixing, developing to light blue, adding H-rGO-Pt @ Pd NPs into TMB (d) and H 2 O 2 (e) The natural color is formed, and H-rGO-Pt @ Pd NPs, TMB and H are mixed 2 O 2 (f) When mixed, the dark blue, light blue c tubes were compared to the dark blue f tubes, indicating that H-rGO-Pt @ Pd NPs have peroxidase-like properties and catalyze the reaction of TMB and H2O 2. FIG. 3B is a ultraviolet test of peroxidase-like properties of H-rGO-Pt @ Pd NPs, wherein a, B, d and e have no absorption peaks, c has a tiny peak, and f has a strong absorption peak, which shows that TMB and H 2 O 2 The color development reaction occurs, and the nano composite material H-rGO-Pt @ Pd NPs has a catalytic action, so that the nano composite material has strong catalase-like property.
(3) 50.0. mu.L of GPC3 at a concentration of 5.0. mu. mol/L Apt Mixing with 100.0 μ L of H-rGO-Pt @ Pd NPs solution with concentration of 1.0 mg/mL, incubating at 4 deg.C for 12H, and cleaning to obtain H-rGO-Pt @ Pd NPs-GPC3 Apt A signaling probe.
H-rGO-Pt @ Pd NPs-GPC3 is subjected to ultraviolet-visible spectrophotometer by adopting Hitachi UH5300 in Shanghai of China Apt The signaling probe was verified as shown in FIG. 4, amino GPC3 aptamer (NH) 2 -GPC3 Apt ) The wavelength of the light has an obvious absorption peak at 260 nm, and the wavelengths of H-rGO-Pt @ Pd NPs at 260 nm and 397.5 nmAbsorption peaks exist at about 260 nm and 400 nm of the probe and the centrifuged probe supernatant, and the absorption peak intensity of the probe is greater than that of the probe supernatant, which indicates that H-rGO-Pt @ Pd NPs and NH 2 -GPC3 Apt And (4) successfully combining. The binding rate calculation formula is as follows:
K=(X 0 -X)/X 0 ×100%
wherein X 0 And X are each the same concentration and the same amount of signal probe (H-rGO-Pt @ Pd NPs-GPC 3) Apt ) And absorbance intensity of the signal probe supernatant, calculated to give a binding rate of K = 82.06%.
Electrode modification and biosensing interface construction
(1) Immersing the electrode (SPCE) in dilute H 2 SO 4 In the method, under the scanning voltage of 0.4-1.2V, the scanning is carried out at the speed of 0.5V/s and the CV scanning is carried out for 20 circles; the pretreated electrode was placed in 1 mL of HAuCl containing 0.01% 4 And depositing the solution and 1.0 mL of rGO solution with the concentration of 1.0 mg/mL for 120 s under the scanning voltage of 0-1.2V by adopting an i-t technology in an electrochemical workstation, cleaning and drying to obtain Au @ rGO/SPCE.
(2) 2.0. mu.L of 5.0. mu.M GPC3 Apt Dropwise adding the solution onto the surface of an Au NPs @ rGO/SPCE electrode, incubating for 30 min at 25 ℃, cleaning and drying; adding dropwise 2.0 μ L of 1% BSA solution on the electrode to block active site, incubating at 25 deg.C for 30 min, washing, and blow-drying to obtain GPC3 Apt /Au NPs@rGO/SPCE。
GPC3 working Curve
(1) Dripping 1.0 mu g/mL-70.0 mu g/mL GPC3 standard solution into the GPC3 electrochemical biosensing interface constructed in the step 2, and incubating for 1 h at 25 ℃ to obtain GPC3/GPC3 Apt /Au NPs@rGO /SPCE。
(2) 3.0 mu L of 0.63 mg/mL H-rGO-Pt @ Pd NPs-GPC3 is dropwise added on the sensing interface prepared in the step (1) Apt Incubating the solution at 25 ℃ for 1H, cleaning and drying to obtain H-rGO-Pt @ Pd NPs-GPC3 Apt / GPC3/GPC3 Apt /Au NPs@rGO/SPCE。
(3) Dropwise adding 2.0 muL of 100 mM H on the electrode 2 O 2 And a 1.0 μ L concentration of 50.0 mM AgNO 3 The solution is prepared by mixing a solvent and a solvent,reacting at 25 deg.C in dark for 30 min, cleaning, and blow-drying to obtain working electrode Ag/H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/GPC3 Apt /Au NPs@rGO/SPCE。
(4) Placing the electrode in HNO 3 And KNO 3 In the glycine-NaOH buffer solution, the concentration of GPC3 in the range of 1.0 mu g/mL-70.0 mu g/mL is detected by using DPV of an electrochemical workstation, the peak current is recorded, and a DPV curve graph of different GPC3 concentrations is shown in FIG. 6. The linear equation is Y =0.282183X +22.64276(Y is response current, X is GPC3 concentration), and the correlation coefficient R 2 =0.99564, standard deviation calculated by measuring blank sample multiple times 0.045, LOD =3S by formula b B, wherein S b The standard deviation was measured for the six blank groups and b is the slope of the standard curve, by which method the minimum detection limit of sensitivity was calculated to be 0.4801. mu.g/mL.
The electrode construction process was characterized using Scanning Electron Microscopy (SEM), as shown in fig. 5. FIG. 5A is a SEM image of SPCE showing the electrode surface as uniformly aligned particles due to its inherent carbon particles; FIG. 5B is an SEM image of Au NPs @ rGO/SPCE showing that the electrode is covered with a black film and a number of bright white spherical particles are uniformly distributed, indicating that Au NPs @ rGO is successfully deposited on the surface of the electrode; FIG. 5C is GPC3 Apt SEM image of/Au NPs @ rGO/SPCE, a thin film covering the surface, known as GPC3 Apt Successfully fixed on the surface of the electrode. The film surface is seen to be smoother in FIG. 5D due to GPC3 and GPC3 Apt The reaction between the components formed a stable structure, and it was found that GPC3 was successfully adsorbed on the electrode surface. FIG. 5E is H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/GPC3 Apt The surface of the/Au NPs @ rGO/SPCE shows a typical corrugated structure and is wrapped by a plurality of spherical nano-particles, and H-rGO-Pt @ Pd NPs-GPC3 is proved Apt The surface of the electrode is uniformly modified. FIG. 5F is Ag/H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/ GPC3 Apt the/Au NPs @ rGO/SPCE surface has bright particles, which indicates that Ag is effectively deposited on the surface.
Detection of GPC3 in actual serum sample
(1) GPC3 levels in human serum samples were determined by the addition of standards under optimal conditions. A normal human serum sample was mixed well with 10.0. mu.g/mL, 20.0. mu.g/mL, and 40.0. mu.g/mL of a GPC3 standard solution at a ratio of 1:1, respectively, to prepare a mixed solution.
(2) Dripping 2.0 μ L of mixed solution into the electrochemical biosensing interface of GPC3 constructed in step 2, and incubating for 30 min at 25 ℃, wherein GPC3/GPC3 Apt /Au NPs@rGO/SPCE。
(3) At GPC3/GPC3 Apt 3.0 mu L of 0.63 mg/mL H-rGO-Pt @ Pd NPs-GPC3 is dripped on/Au NPs @ rGO/SPCE Apt Incubating the solution at 25 ℃ for 1H, cleaning and drying to obtain H-rGO-Pt @ Pd NPs-GPC3 Apt / GPC3/GPC3 Apt /Au NPs@rGO/SPCE。
(4) Dropwise adding 2.0 muL of 100 mM H on the electrode 2 O 2 And a 1.0 μ L concentration of 50.0 mM AgNO 3 The solution reacts for 30 min in the dark at 25 ℃, and is cleaned and dried to obtain a working electrode Ag/H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/GPC3 Apt /Au NPs@rGO/SPCE。
(5) Placing the working electrode in HNO as described in step 3 3 And KNO 3 DPV scans were performed in glycine-NaOH buffer and the current values recorded. The GPC3 concentration of the human serum sample is calculated according to the GPC3 working curve obtained in the step 3, and the result is shown in Table 1, wherein the recovery rate is in the range of 100.96% -121.15%, and the RSD value is 0.05% -2.24%. The results show that the electrochemical aptamer sensor can be used for detecting the GPC3 concentration in an actual serum sample.
TABLE 1 results of GPC3 detection in actual serum samples
Sample 1: normal serum, AFP = 5.14 ng/mL
Sample 2: liver cancer serum, AFP = 88.27 ng/mL
Sample 3: liver cancer serum, AFP = 223.88 ng/mL
(Note: serum samples were obtained from Guangxi Metabolic disease research Key laboratory in hospital 924 (Guilin, China) of the Chinese people's liberation force and were in compliance with the ethical Committee of the Guangxi Metabolic disease research Key laboratory in hospital 924 of the Chinese people's liberation force.).
Claims (3)
1. A method for constructing a sandwich type aptamer sensor based on a nano composite material for detecting GPC3 for non-diagnostic purposes, comprising the following steps:
step 1: heme-reduced graphene oxide-platinum @ palladium nanocomposite H-rGO-Pt @ Pd NPs-GPC3 Apt Preparation of Signal Probe
Preparation of rGO: pouring graphene oxide GO into ultrapure water, uniformly crushing, and adding ascorbic acid AA for reduction to obtain a rGO suspension;
preparation of H-rGO: adding Hemin Hemin into ammonia water for dissolving, adding rGO suspension and hydrazine hydrate N 2 H 4 ·H 2 O, performing water bath reaction, and performing centrifugal cleaning to obtain an H-rGO suspension;
preparation of H-rGO-Pt @ Pd NPs: adding PDDA and NaCl into the H-rGO suspension, stirring, and adding Na sodium tetrachloropalladate 2 PdCl 4 Sodium tetrachloroplatinate Na 2 PtCl 4 And N 2 H 4 ·H 2 O, stirring to obtain an H-rGO-Pt @ Pd solution;
H-rGO-Pt@Pd NPs-GPC3 Apt preparation of a signal probe: adapting GPC3 to GPC3 Apt Mixing with H-rGO-Pt @ Pd solution, incubating, centrifuging, and removing supernatant to obtain H-rGO-Pt @ Pd NPs-GPC3 Apt A solution;
step 2: electrode modification and biosensing interface construction
Placing a screen printing electrode SPCE in a dilute sulfuric acid solution for activation;
(2) placing the activated SPCE into HAuCl containing chloroauric acid 4 Carrying out constant potential deposition in a mixed solution of the Au and the GO solution to obtain Au NPs @ rGO/SPCE;
(3) GPC3 Apt Dropwise adding the solution on the surface of Au NPs @ rGO/SPCE, incubating, washing and drying to obtain GPC3 Apt /Au NPs@rGO/SPCE；
(4) Dropping H on the electrode 2 O 2 And AgNO 3 The solution is reacted in the dark, washed,drying by blowing to obtain the working electrode Ag/H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3 /GPC3 Apt /Au NPs@rGO/SPCE；
And step 3: plotting of GPC3 working curves
(1) Dropwise adding GPC3 standard liquid to GPC3 Apt /Au NPs @ rGO/SPCE surface, incubating, cleaning and drying to obtain GPC3/GPC3 Apt /Au NPs@rGO/SPCE；
(2) At GPC3/GPC3 Apt H-rGO-Pt @ Pd NPs-GPC3 is dripped on Au NPs @ rGO/SPCE Apt Incubating, washing and drying the solution to obtain the electrode H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3/ GPC3 Apt /Au NPs@rGO/SPCE；
(3) H was added dropwise to the resulting electrode 2 O 2 And AgNO 3 The solution is reacted away from light, cleaned and dried to obtain the working electrode Ag/H-rGO-Pt @ Pd NPs-GPC3 Apt /GPC3 /GPC3 Apt /Au NPs@rGO/SPCE；
(4) Immersing the working electrode in a solution containing HNO 3 And KNO 3 The glycine-sodium hydroxide buffer solution is scanned by DPV, and the response current value of the sensor is recorded;
(5) detecting GPC3 with different concentrations respectively, and recording peak current; drawing a GPC3 working curve according to the relation between the current response value of the sensor and the concentration of GPC3, and calculating the lowest detection limit of the method;
and 4, step 4: detection of GPC3 in actual serum samples
(1) The working electrode prepared by the actual serum sample to be tested is immersed into the sample containing HNO 3 And KNO 3 The glycine-sodium hydroxide buffer solution is scanned by DPV of an electrochemical workstation, and the response current value of the sensor is recorded;
(2) and (4) calculating the concentration of GPC3 in the actual sample to be detected according to the working curve obtained in the step 3.
2. The method of claim 1, wherein: in the step 1, the concentration of the H-rGO-Pt @ Pd NPs nanoenzyme is 1.0 mg/mL, and the H-rGO-Pt @ Pd NPs-GPC3 Apt The concentration of the signal probe was 0.63 mg/mL.
3. The method of claim 1, wherein: HAuCl described in step 2 4 And rGO in a ratio of 1: 8; said H 2 O 2 And AgNO 3 Is 2: 1.
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