CN114252489A - Method for detecting GPC3 based on H-rGO-Pd NPs and Au NPs @ rGO nano-materials - Google Patents

Method for detecting GPC3 based on H-rGO-Pd NPs and Au NPs @ rGO nano-materials Download PDF

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CN114252489A
CN114252489A CN202111596298.3A CN202111596298A CN114252489A CN 114252489 A CN114252489 A CN 114252489A CN 202111596298 A CN202111596298 A CN 202111596298A CN 114252489 A CN114252489 A CN 114252489A
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gpc3
rgo
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李桂银
王博
刘卫兵
李羚
周治德
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Guangdong University of Petrochemical Technology
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Abstract

A non-diagnosis purpose method for detecting GPC3 based on H-rGO-Pd NPs and Au NPs @ rGO nano materials is characterized in that Au NPs @ rGO are modified on the surface of SPE through electrodeposition, and GPC3 is achieved through adsorptionAbLoading an Au NPs @ rGO/SPE surface; H-rGO-Pd NPs-GPC3 is prepared by taking H-rGO-Pd NPs nano material as carrierAptDetection probes by GPC3 and GPC3AbAnd GPC3AptSpecifically combining to construct a sandwich type electrochemical aptamer sensor; the detection signal is amplified based on the catalytic silver deposition effect of the H-rGO-Pd NPs nano-mimetic enzyme material, the DPV of an electrochemical workstation is adopted for scanning, and the peak current of the DPV is recorded, so that the detection of GPC3 is realized.

Description

Method for detecting GPC3 based on H-rGO-Pd NPs and Au NPs @ rGO nano-materials
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a method for detecting GPC3 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 alpha-fetoprotein (AFP), GPC3 (glypican-3), Golgi protein 73 (GP 73), and the like. 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 peripheral blood of a liver cancer patient, from which a tissue specimen cannot be obtained, by using a membrane filtration device, and further detecting the expression of peripheral blood GPC3 of the liver cancer patient. The invention patent of publication No. CN 105759051B relates to a quantitative analysis kit of nanometer magnetic microsphere chemiluminescence immunoassay GPC3, which takes acridinium as a luminescent marker. These methods are expensive, complicated, time-consuming and technically demanding, and therefore, it is desirable to establish a rapid and easy-to-operate GPC3 detection method.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a non-diagnosis purpose nano material based on heme-reduced graphene oxide-nano palladium (H-rGO-Pd NPs) and nano gold @ reduced graphene oxide (Au NPs @ rGO), construct a sandwich type aptamer electrochemical sensor, realize GPC3 detection, and achieve the lowest detection limit of 2.12 mug/mL.
In order to solve the technical problem, an H-rGO-Pd NPs nano material is used as a carrier and combined with a GPC3 aptamer (GPC 3)Apt) Preparing H-rGO-Pd NPs-GPC3AptA signal probe; au NPs @ rGO is modified on an activated screen-printed electrode (SPE) through an electrodeposition technology, and a GPC3 antibody (GPC 3) is adsorbedAb) Fixed on the surface of the electrode; because of GPC3 and GPC3AbAnd GPC3 aptamer can be specifically combined to form a stable structure, and H-rGO-Pd NPs-GPC3 is constructedApt/GPC3/GPC3AbAu NPs @ rGO/SPE sandwich type aptamer electrochemical sensors; high-efficiency peroxidase-like property catalysis of H by using H-rGO-Pd NPs nano material2O2With AgNO3Reacting to deposit nanometer silver particle (Ag) on the surface of sensor, recording its peak current by Differential Pulse Voltammetry (DPV) of electrochemical workstation, and collecting the peak currentThereby realizing the detection of GPC 3.
The invention is carried out according to the following steps:
step 1: H-rGO-Pd NPs-GPC3AptPreparation of Signal Probe
(1) Preparation of Graphene Oxide (GO) solution
Dispersing Graphene Oxide (GO) in pure water, performing ultrasonic centrifugation, and removing GO particles to obtain a GO solution;
(2) preparation of heme-reduced graphene oxide (H-rGO)
Uniformly mixing the GO solution with a heme (Hemin) aqueous solution, adding ammonia water and a hydrazine hydrate solution, oscillating, carrying out water bath, centrifuging, and cleaning to obtain an H-rGO solution;
as an improvement, the method also comprises the following steps:
(3) preparation of H-rGO-Pd NPs
PDDA (poly dimethyl diallyl ammonium chloride) and NaCl are added into the H-rGO solution to be stirred, the mixture is centrifugally cleaned, glycol is added, and the pH value of the mixed solution is adjusted by NaOH; centrifuging, cleaning and drying to obtain H-rGO-Pd NPs solid;
(4)H-rGO-Pd NPs-GPC3Aptpreparation of Signal Probe
Amino GPC3 aptamer (GPC 3)Apt) Uniformly mixing the solution and H-rGO-Pd NPs solution by ultrasound, incubating, centrifuging, cleaning, and removing free aptamer to obtain H-rGO-Pd NPs-GPC3AptA signaling probe.
Step 2: electrode modification and sensing interface construction
(1) Placing a screen-printed electrode (SPE) into H2SO4Activating;
(2) the activated SPE was placed in chloroauric acid (HAuCl)4) Carrying out electrodeposition in the mixed solution of the Au and the rGO to obtain an Au NPs @ rGO/SPE electrode;
(3) GPC3AbDropwise adding the mixture on the surface of an Au NPs @ rGO/SPE electrode, incubating and cleaning to obtain GPC3Ab/Au NPs@rGO/SPE；
Further, HAuCl for deposition described in step 24The solution is 0.05 percent, the concentration of the rGO solution is 1.0 mg/mL, the deposition potential is-0.5V, and the deposition time is 120 s;
and step 3: plotting of GPC3 working curves
(1) GPC3 Standard solution was added dropwise to the sensing interface obtained in step 2 (GPC 3)Ab/Au NPs @ rGO/SPE), incubating and washing to obtain GPC3/GPC3Ab/Au NPs@rGO/SPE；
(2) At GPC3/GPC3AbH-rGO-Pd NPs-GPC3 is dripped on Au NPs @ rGO/SPEAptSignal probe solution, incubation and cleaning to obtain H-rGO-Pd NPs-GPC3Apt/GPC3/GPC3Ab/Au NPs@rGO/SPE；
(3) In H-rGO-Pd NPs-GPC3Apt/GPC3/GPC3AbH is dripped on Au NPs @ rGO/SPE2O2And AgNO3The solution is reacted away from light and is cleaned to obtain a working electrode (Ag/H-rGO-Pd NPs-GPC 3)Apt/GPC3/GPC3AbAu NPs @ rGO/SPE) for standby;
(4) immersing the working electrode in KNO3-HNO3In the solution, DPV of an electrochemical workstation is adopted for scanning, and the response current value of the sensor is recorded;
(5) detecting GP73 standard solutions with different concentrations respectively, and recording peak current; drawing a working curve according to the relation 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) And (3) fully mixing the actual human serum sample with the GP73 standard solution in a ratio of 1:1 to prepare a mixed solution to be detected, dripping the mixed solution to be detected on the sensing interface prepared in the step (2), incubating and cleaning. Then dropwise adding H-rGO-Pd NPs-GPC3AptDetecting probe, incubating, washing, and dropping H again2O2And AgNO3Carrying out light-resistant reaction on the solution, washing to obtain a working electrode, and airing for later use;
(2) immersing working electrode prepared by mixed liquid to be tested into KNO3-HNO3In the solution, DPV of an electrochemical workstation is adopted for scanning, and a response current value is recorded;
(3) the GPC3 concentration in the actual serum sample was calculated from the GPC3 working curve obtained in step 3.
Preferably, the method comprises the following steps:
the incubation temperature of the electrode in the steps 3 and 4 is 25 ℃, and the incubation time is 1 h;
KNO described in step 3 and step 43-HNO3KNO in solution3Concentration of 0.6 mol/L, HNO3The concentration is 0.1 mol/L;
the scanning range in the step 3 and the step 4 is-0.2V-0.4V, and the scanning speed is 0.1V/s.
Wherein, step 1 is used for preparing H-rGO-Pd NPs-GPC3 with large specific surface area and high electron transfer efficiencyAptA signal probe; step 2 constitutes a biosensing interface that specifically recognizes GPC3 and is an essential key step in the electrochemical detection of GPC3 in steps 3 and 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 H-rGO-Pd NPs composites and GPC3 aptamers as recognition probes.
Compared with the prior art, the invention has the following advantages:
1. this patent has firstly prepared new H-rGO-Pd NPs nanocomposite, has then prepared a H-rGO-Pd NPs-GPC3 based on H-rGO-Pd NPs nanocompositeAptThe signal probe has the characteristics of large specific surface area, strong adsorbability and strong conductivity; the high-efficiency peroxidase-like property of the H-rGO-Pd NPs nano composite material is utilized to catalyze H2O2With AgNO3The reaction causes the nano silver particles to be deposited on the surface of the sensor, so that a stronger current signal is generated, and the high-sensitivity detection of GPC3 is realized.
2. Enhancement of electron transfer and signal amplification of rGO and excellent load capacity by using Pd NPs in H-rGO-Pd NPs nano composite material, and GPC3 to GPC3AbAnd GPC3AptThe sandwich type electrochemical aptamer sensor capable of specifically detecting the serum GPC3 level is constructed, and a sandwich type structure can more stably and firmly fix a target molecule; the sensor has good stability and high reliability, and the detection limit can reach 2.12 mu g/mL.
Drawings
FIG. 1 is a schematic diagram of GPC3 detection based on H-rGO-Pd NPs and Au NPs @ rGO nanomaterials;
FIG. 2 Transmission Electron Microscopy (TEM) of rGO (A) and H-rGO-Pd NPs (B);
FIG. 3 is a Scanning Electron Microscope (SEM) representation of various modification processes on the surface of an electrode;
FIG. 4 DPV graph (A) of different GPC3 concentrations; working curve (B) of GPC3 electrochemical sensor.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The principle of the method for detecting GPC3 based on H-rGO-Pd NPs and Au NPs @ rGO nano-materials is shown in FIG. 1. Firstly, preparing H-rGO-Pd NP nano composite material, and then fixing GPC3 by using the materialAptTo form H-rGO-Pd NP-GPC3AptA signal probe; modifying Au NPs @ rGO on an activated screen-printed electrode (SPE) by an electrodeposition technology, and adsorbing GPC3 by adsorptionAbFixed on the surface of the electrode; then GPC3 and H-rGO-Pd NPs-GPC3 are added dropwise in sequenceAptBecause of GPC3 and GPC3AbAnd GPC3 aptamer can be specifically combined to form stable secondary structure, and the H-rGO-Pd NPs-GPC3 of the patent is constructedApt/GPC3/GPC3Aba/Au NPs @ rGO/SPE sandwich type electrochemical aptamer sensor; high-efficiency peroxidase-like property catalysis of H by using H-rGO-Pd NPs nano composite material2O2With AgNO3And (3) reacting to deposit nano silver particles on the surface of the sensor, recording electrochemical signals before and after detecting GPC3 by adopting Differential Pulse Voltammetry (DPV) of an electrochemical workstation, and obtaining a standard curve of GPC3 concentration-sensor response current, thereby realizing the detection of GPC 3.
The specific implementation steps are as follows:
1、H-rGO-Pd NPs-GPC3Aptpreparation of Signal Probe
(1) Weighing 20.0 mg of GO to be dispersed in 20.0 mL of pure water, carrying out ultrasonic treatment for 2 h by using a cell disruptor to prepare 1.0 mg/mL of GO solution, uniformly mixing 10.0 mL of GO solution with 5.0 mL of Hemin aqueous solution, adding 30.0 mu L of ammonia aqueous solution and 100.0 mu L of hydrazine hydrate solution, carrying out vortex oscillation, and carrying out water bath for 4 h at 60 ℃; centrifuging and cleaning to obtain the H-rGO solution.
(2) Adding 2.0 mL of PDDA (poly dimethyl diallyl ammonium chloride) and 5.0 mL of NaCl into 10.0 mL of H-rGO solution, and stirring for 12H; 2.0 mL of Na2PdCl4Adding the solution into a PDDA modified H-rGO solution, and stirring overnight; adding 10.0 mL of glycol, adjusting the pH value to 12 by NaOH, and carrying out reflux reaction at 140 ℃ for 4 h; centrifuging, cleaning and drying to obtain the H-rGO-Pd NP nano composite material.
The nanomaterial was characterized using a JEM-1200EX Transmission Electron Microscope (TEM), as shown in FIG. 2. FIG. 2A is a TEM image of H-rGO in a pleated membranous structure; FIG. 2B is a TEM image of H-rGO-Pd NPs, and FIG. 2B shows that the surface of H-rGO-Pd NPs is increased by a plurality of dark particles with more uniform shapes, i.e. Pd particles, compared with the surface of H-rGO; the Pd NPs are proved to be successfully attached to the surface of the H-rGO, and the construction of the H-rGO-Pd NPs nano composite material is proved to be successful.
(3) mu.L of amino GPC3 aptamer (GPC 3)Apt，5'- NH2-TAACGCTGACCTTAG CTGCATGGCTTTACATGTTCCA-3') solution and 200.0 μ L H-rGO-Pd NPs solution, incubating, washing, and removing free aptamer to obtain H-rGO-Pd NPs-GPC3AptA signaling probe.
2. Electrode modification and biosensing interface construction
(1) The activated electrode (SPE) was immersed in 0.5 mol/L H2SO4Performing CV scanning for 20 circles under a scanning voltage of-0.4V-1.2V; the activated SPE electrode was placed in 5.0 mL of HAuCl containing 0.05%4And depositing at a constant potential of-0.5V in a mixed solution of the solution and a 1.0 mg/mL rGO solution, cleaning for 3 times by using pure water after deposition is finished, and drying by blowing to obtain Au NPs @ rGO/SPE.
(2) mu.L of a 100.0. mu.g/mL solution of GPC3 antibody (GPC 3)Ab) Dropwise adding the solution on the surface of Au NPs @ rGO/SPE, and incubating and washing; dripping 6.0 μ L BSA (1.0%) solution on the surface of Au NPs @ rGO/SPE, sealing for 30 min, cleaning, and air drying to obtain GPC3Ab/Au NPs@rGO/SPE。
Adopting SU8020 Scanning Electron Microscope (SEM) manufactured by Nippon Hitachi to repair the surface of the electrode differentlyThe decorating process was characterized as shown in fig. 3. FIG. 3A shows a bare electrode with a flat surface; FIG. 3B is an SEM image of Au NPs @ rGO/SPE with two uniform particles present, one darker and the other brighter, illustrating the successful deposition of Au and rGO onto the bare electrode; FIG. 3C is GPC3AbSEM image of/Au NPs @ rGO/SPE with some white film added to the surface, illustrating GPC3AbHave been successfully modified onto electrodes.
3. Plotting of GPC3 working curves
(1) GPC3 constructed in step (2)AbAdding 3.0 mu L of GPC3 standard solution (different concentrations) dropwise onto the/Au NPs @ rGO/SPE sensing interface, incubating, washing and airing to obtain GPC3/GPC3Ab/Au NPs@rGO/SPE。
(2) 4.0 mu L of H-rGO-Pd NPs-GPC3 is drippedAptDetecting a probe solution, and after incubating for 1h, dropwise adding 6.0 mu L H2O2And 3.0 μ L AgNO3Reacting the solution at room temperature in a dark place for 30 min, washing and drying to obtain a working electrode Ag/H-rGO-Pd NPs-GPC3Apt/GPC3/GPC3Ab/Au NPs@rGO/SPE。
Fig. 3D adds some white spheres to the electrode surface, indicating that GPC3 has been incubated onto the electrode. FIG. 3E shows the electrode surface is relatively flat and a wrinkled membrane is visible, illustrating H-rGO-Pd NPs-GPC3AptIncubation onto the electrodes has been successful. FIG. 3F is Ag/H-rGO-Pd NPs/GPC3Apt/GPC3/GPC3AbAu NPs @ rGO/SPE, the electrode surface presents a wrinkled film shape and is covered with silver white shiny particles, namely a large number of silver particles are deposited on the electrode surface. The results show that Au NPs @ rGO and GPC3Ab、GPC3、H-rGO-Pd NPs-GPC3AptAnd Ag is modified to the surface of the electrode one by one.
(3) Immersing the working electrode in 0.6 mol/L KNO3And 0.1 mol/L HNO3KNO of3-HNO3In the solution, peak currents were recorded using Differential Pulse Voltammetry (DPV) scans in the CHI660E electrochemical workstation, and DPV spectra for different concentrations of GPC3 are shown in fig. 4A, from which it can be seen that as the concentration of GPC3 increases, the corresponding current of the sensor increases. GPC3 concentrated when the GPC3 concentration was in the range of 10.0. mu.g/mL to 100.0. mu.g/mLDegree (X) is linearly related to the sensor response current (Y) (see fig. 4B), the working curve equation is Y =0.12775X +23.89707, and the correlation coefficient (R) is 0.98293. By the formula CLOD=3SbThe detection limit of the sensor is calculated to be 2.12 mu g/mL.
4. Detection of GPC3 in actual serum samples
(1) Three parts of normal human serum and three standard solutions of 50.0 mu g/mL, 80.0 mu g/mL and 100.0 mu g/mL of GPC3 with different concentrations are respectively taken to be uniformly mixed in equal proportion to prepare a mixed solution to be detected.
(2) Dripping 3.0 mu L of mixed liquid to be detected on the electrochemical biosensing interface constructed in the step 2, and then dripping 4.0 mu L of H-rGO-Pd NPs-GPC3AptDetecting a probe solution, and after incubating for 1h, dropwise adding 6.0 mu L H2O2And 3.0 μ L AgNO3And (5) reacting the solution at room temperature in a dark place for 30 min, cleaning to obtain a working electrode, and airing for later use.
(3) The working electrode was measured 3 times with DPV using an electrochemical workstation and the current values were recorded.
(4) According to the working curve equation Y =0.12775X +23.89707 of the step 3, the corresponding concentration of GPC3 in the actual serum sample can be calculated, and the detection result is shown in Table 1. The recovery rate is 103.78-106.52%, and the relative standard deviation is 1.89-8.81%. The results show that the developed GPC3 sandwich type electrochemical sensor has better application prospect and is expected to be used for GPC3 detection of actual clinical serum samples.
TABLE 1 concentration of GPC3 in actual serum samples
Figure 438285DEST_PATH_IMAGE002
(Note: actual human serum samples were provided by the ninth second and fourth hospitals of the United nations 'Council guarantee of the people's liberation force, China).

Claims (5)

1. A method for detecting GPC3 based on H-rGO-Pd NPs and Au NPs @ rGO nano-materials for non-diagnostic purposes comprises the following steps:
step 1: H-rGO-Pd NPs-GPC3AptPreparation of Signal Probe
Preparation of GO solution
Dispersing the GO solid in pure water, carrying out ultrasonic crushing, and carrying out centrifugal cleaning to obtain a GO solution;
preparation of H-rGO
Uniformly mixing the GO solution with a Hemin aqueous solution, adding ammonia water and a hydrazine hydrate solution, oscillating, carrying out water bath, and carrying out centrifugal cleaning to obtain an H-rGO solution;
the method is characterized in that: also comprises the following steps:
preparation of H-rGO-Pd NPs
Adding PDDA and NaCl into the H-rGO solution, stirring, and centrifugally cleaning; adding glycol and adjusting the pH value of the mixed solution; centrifuging, cleaning and drying to obtain H-rGO-Pd NPs;
H-rGO-Pd NPs-GPC3Aptpreparation of
Amino GPC3AptUltrasonically mixing the solution with H-rGO-Pd NPs solution, incubating, centrifuging and cleaning to obtain H-rGO-Pd NPs-GPC3AptA signal probe;
step 2: electrode modification and sensing interface construction
Placing the screen printing electrode in a dilute sulfuric acid solution, performing cyclic voltammetry scanning to obtain an activated screen printing electrode, and cleaning;
the activated screen-printed electrode is placed in HAuCl4Carrying out constant potential deposition of nano gold in a mixed solution of the nano gold and the rGO solution to obtain Au NPs @ rGO/SPE;
GPC3AbDropwise adding the solution on the surface of Au NPs @ rGO/SPE, incubating, cleaning and airing to obtain GPC3Ab/AuNPs@rGO/SPE；
And step 3: plotting of GPC3 working curves
Dropwise adding GPC3 standard liquid to GPC3AbThe surface of the/AuNPs @ rGO/SPE is incubated, cleaned and dried to obtain GPC3/GPC3Ab/AuNPs@rGO/SPE；
At GPC3/GPC3AbH-rGO-Pd NPs-GPC3 is dripped on/AuNPs @ rGO/SPEAptIncubating, washing and drying the solution to obtain H-rGO-Pd NPs-GPC3Apt/GPC3/GPC3Ab/Au NPs@rGO/SPE；
In H-rGO-Pd NPs/GPC3Apt/GPC3/GPC3AbH is dripped on/AuNPs @ rGO/SPE2O2And AgNO3The solution is reacted away from light, washed and dried to obtain the working electrode Ag/H-rGO-Pd NPs-GPC3Apt/GPC3/GPC3Ab/Au NPs@rGO/SPE；
Immersing the working electrode in KNO3-HNO3In the solution, DPV is adopted for scanning, and the response current of the sensor is measured;
detecting GPC3 with different concentrations, and recording peak current; drawing a working curve according to the relation between the current response value of the sensor and the concentration of GPC 3; calculating the lowest detection limit of the method;
and 4, step 4: detection of GPC3 in actual serum samples
Mixing an actual human serum sample with the GP73 standard solution to prepare a mixed solution to be detected, dripping the mixed solution on the sensing interface prepared in the step 2, and dripping H-rGO-Pd NPs-GPC3AptDetecting probe solution, incubating, cleaning, and dripping H2O2And AgNO3The solution is reacted in the dark at room temperature, washed to obtain a working electrode, and dried for standby;
immersing working electrode prepared by actual serum sample to be detected into KNO3-HNO3In the solution, DPV of an electrochemical workstation is adopted for scanning, and the response current of a sensor is measured;
and (4) calculating the GPC3 concentration in the actual sample to be detected according to the working curve obtained in the step 3.
2. A method of detecting GPC3 according to claim 1, characterized in that: the concentration of the H-rGO-Pd NPs solution in the step 1 is 0.5 mg/mL, and the H-rGO-Pd NPs-GPC3AptThe concentration of the solution was 0.5 mg/mL.
3. A method of detecting GPC3 according to claim 1, characterized in that: in step 2, the incubation temperature is 25 ℃, and the incubation time is 1 h.
4. A method of detecting GPC3 according to claim 1, characterized in that: and 3, scanning ranges of-0.2V to 0.4V in the steps 4 and 3.
5. A method of detecting GPC3 according to claim 1, characterized in that: the incubation temperature in step 3 and step 4 is 25 ℃, and the incubation time is 1 h.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114965637A (en) * 2022-04-24 2022-08-30 桂林电子科技大学 Method for constructing sandwich type aptamer sensor based on nanocomposite to detect GPC3

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114965637A (en) * 2022-04-24 2022-08-30 桂林电子科技大学 Method for constructing sandwich type aptamer sensor based on nanocomposite to detect GPC3
CN114965637B (en) * 2022-04-24 2023-08-18 桂林电子科技大学 Method for detecting GPC3 by constructing sandwich aptamer sensor based on nanocomposite

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