CN113203859B - Method for visually detecting GPC3 based on H-rGO-Pt @ Pd NPs nanoenzyme - Google Patents
Method for visually detecting GPC3 based on H-rGO-Pt @ Pd NPs nanoenzyme Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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Abstract
A method for visually detecting GPC3 based on H-rGO-Pt @ Pd NPs nanoenzyme is characterized in that H-rGO-Pt @ Pd NPs-Apt is used as a detection probe, GPC3-Ab is used as a capture probe, a H-rGO-Pt @ Pd NPs-Apt/GPC3/Ab sandwich type compound is formed, catalase-like catalytic activity based on H-rGO-Pt @ Pd NPs nanoenzyme is catalyzed by the compound, and the H-rGO-Pt @ Pd NPs nanoenzyme is catalyzed by the compound 2 O 2 The color development substrate TMB is oxidized, so that the solution of the system is changed from colorless to blue, and the blue color of the system is darker and darker along with the increase of the concentration of GPC3, thereby realizing the visual detection of GPC 3. The method is simple, convenient and quick to operate, consumes less materials and has a lower detection limit.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a method for visually detecting GPC3 based on a composite nano enzyme material.
Background
Glypican-3 (GPC 3) is a novel liver cancer marker discovered in recent years, and common detection methods include: enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay, fluorescence detection, immunohistochemistry, etc. The invention patent of publication No. CN 112014577A relates to a kit for detecting GPC3 by using a magnetic bead particle coupled mouse anti-human GPC3 monoclonal antibody and an alkaline phosphatase labeled rabbit anti-human GPC3 polyclonal antibody. The invention patent of publication No. CN 105759051B relates to a kit for measuring GPC3 by GPC3 nanometer magnetic microsphere chemiluminescence immunoassay using acridinium ester as luminescent substance, the instrument used in the method is expensive, and the method has higher requirements for operation skills. Therefore, it is required to establish a rapid and easy-to-operate method for detecting GPC 3.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for visually detecting GPC3 by adopting a spectrophotometric method based on a detection system constructed by heme-reduced graphene oxide-platinum @ palladium (H-rGO-Pt @ Pd NPs) nanoenzyme.
In order to solve the technical problem, a one-step reduction method is adopted to prepare H-rGO-Pt @ Pd NPs nanoenzyme, pi-pi conjugation of the nanoenzyme and GPC3 aptamer (GPC 3-Apt) is utilized to form an H-rGO-Pt @ Pd NPs-Apt detection probe, and GPC3 protein is captured by taking a GPC3 antibody (GPC 3-Ab) as a capture probe, so that an aptamer-protein-antibody sandwich type compound is formed. Hydrogen peroxide (H) is catalyzed by utilizing catalytic property of peroxidase-like H-rGO-Pt @ Pd NPs 2 O 2 ) Oxidation of chromogenic substrate 3,3',5,5' -Tetramethylbenzidine (TMB) to TMB OX So that the system solution changes from colorless to blue. With the increase of the concentration of GPC3, the blue color of the system becomes darker, so that the visual detection of GPC3 is realized.
The invention is carried out according to the following steps:
step 1: preparation of H-rGO-Pt @ Pd NPs nanoenzyme
(1) Preparation of reduced graphene oxide (rGO):
weighing Graphene Oxide (GO), placing in ultrapure water, and crushing. Adding Ascorbic Acid (AA) and stirring to obtain rGO solution.
(2) Preparation of heme-reduced graphene oxide (H-rGO):
weighing heme, dissolving with ammonia water and ultrapure water, adding a rGO solution, adding a hydrazine hydrate solution, uniformly stirring, carrying out constant-temperature water bath, and carrying out centrifugal washing to obtain the H-rGO nano material.
(2) Preparation of H-rGO-Pt @ Pd NPs nanoenzyme:
and adding a poly (diallyldimethylammonium chloride) (PDDA) solution and a sodium chloride (NaCl) solution into the H-rGO solution, uniformly stirring and mixing, and performing centrifugal washing to obtain the PDDA modified H-rGO solution. Mixing sodium chloropalladate (Na) 2 PtCl 6 ) And sodium chloroplatinate (Na) 2 PdCl 4 ) Adding the solution into a PDDA modified H-rGO solution, and stirring for reaction. Adding an ethylene glycol solution into the solution for reduction, adjusting the pH value by using a NaOH solution, carrying out constant-temperature water bath, and then carrying out centrifugal washing to obtain the H-rGO-Pt @ Pd NPs nanoenzyme.
Step 2: preparation of H-rGO-Pt @ Pd NPs-Apt detection probe
And (3) uniformly mixing GPC3-Apt and H-rGO-Pt @ Pd NPs solution, incubating, centrifuging and washing to obtain the H-rGO-Pt @ Pd NPs-Apt detection probe.
And step 3: construction of GPC3 visual detection system
Adding GPC3-Ab dropwise into a test tube, adding BSA solution to block nonspecific binding sites, adding GPC3 protein solutions with different concentrations to be detected into the sealed test tube, adding H-rGO-Pt @ Pd NPs-Apt into the test tube after a period of time, and adding a chromogenic substrate system TMB-H into the test tube 2 O 2 And reacting for a period of time to form a visual detection system of GPC 3. And (3) optimizing the system under the experimental conditions of H-rGO-Pt @ Pd NPs-Apt concentration, incubation temperature, incubation time, pH value and ionic strength of the detection system and the like to obtain the optimal GPC3 visual detection system.
And 4, step 4: GPC3 working curve plotting
And (3) in the optimal GPC3 visual detection system obtained in the step (3), acquiring an ultraviolet-visible absorption curve with the wavelength of 500-800 nm of a GPC3 protein solution to be detected in different concentrations, and recording the absorbance of the maximum absorption peak at 652 nm. According to the relation between the absorbance and the concentration of the GPC3 protein, a working curve is drawn, and the detection limit is calculated.
And 5: detection of GPC3 in actual serum samples
Known concentrations of GPC3 protein and actual human serum samples were mixed and added to the optimal GPC3 visual detection system obtained according to step 3 and absorbance values at 652nm were recorded. Wavelength scanning was performed using an ultraviolet-visible spectrophotometer, and its absorbance at 652nm was recorded. The GPC3 concentration that can be actually detected was calculated from the GPC3 working curve obtained in step 3.
Wherein, step 1 provides a nano enzyme material with high-efficiency catalase-like catalytic activity for the visual detection of GPC 3; step 2 provides a detection probe capable of specifically recognizing GPC3 protein for visual detection of GPC 3. Step 3 provides feasibility conditions for visual determination of GPC 3. The working curve of GPC3 from step 4 provides a basis for the determination of GPC3 concentration in the actual serum sample from step 5. Therefore, the steps 1-5 are mutually supported and act together, so that the visible detection of GPC3 can be realized by using H-rGO-Pt @ Pd NPs nanoenzyme.
Compared with the prior art, the invention has the following advantages:
1. the unique network structure and the large specific surface area of the H-rGO-Pt @ Pd NPs nanoenzyme formed by the method enhance the adsorption efficiency of GPC3-Apt, further enhance the specific binding with GPC3 and improve the detection efficiency of GPC 3. In addition, the high specific surface area of rGO, the property of Hemin peroxidase and the excellent catalytic performance of metal Pt @ Pd NPs improve the catalytic performance of H-rGO-Pt @ Pd NPs nanoenzyme by the synergistic effect of the rGO, the Hemin peroxidase and the metal Pt @ Pd NPs, so that the detection is more sensitive, and the detection limit can reach 1.801 ng/mL.
The system adopts H-rGO-Pt @ Pd NPs-GPC3-Apt as a detection probe and GPC3-Ab as a capture probe, and utilizes the strong affinity among GPC3-Apt, GPC3-Ab and a target GPC3 to construct an aptamer-target-antibody sandwich structure. The structure can more firmly fix the target molecules, so that the target molecules are not easy to fall off, and the detection of the target molecules is more facilitated. In addition, a detection system constructed by using the nano enzyme can detect GPC3 molecules, and can be used for detecting other target molecules such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and the like by changing the structures of antibodies and aptamers.
Drawings
FIG. 1 is a schematic diagram of GPC3 based on the visualization of H-rGO-Pt @ Pd NPs;
FIG. 2 Scanning Electron Microscopy (SEM) images of H-rGO (A), H-rGO-Pt @ Pd NPs (B);
FIG. 3 is a graph of the ultraviolet-visible absorption spectrum (UV-vis) of the H-rGO-Pt @ Pd NPs-Apt detection probe;
FIG. 4 is a working curve of GPC3 at various concentrations.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
A method for visually detecting GPC3 based on H-rGO-Pt @ Pd NPs nanoenzyme is shown in figure 1. Preparation of peroxidase-like enzyme by one-step reduction methodA catalytically active H-rGO-Pt @ Pd NPs nanoenzyme forms an H-rGO-Pt @ Pd NPs-Apt detection probe by utilizing pi-pi conjugation of the nanoenzyme and a GPC3 aptamer (GPC 3-Apt), and a GPC3-Ab is fixed in a test tube to serve as a capture probe, when a target GPC3 protein exists, the H-rGO-Pt @ Pd NPs-Apt/GPC3/GPC3-Ab sandwich type compound is formed by the specific recognition of GPC3-Apt and GPC3-Ab and GPC3 protein respectively, and the compound catalyzes H-rGO-Pt @ Pd NPs-Apt/GPC3/GPC3-Ab sandwich type compound 2 O 2 The chromogenic substrate TMB was oxidized, causing the system solution to change from colorless to blue. As the concentration of GPC3 increases, the system becomes darker and darker in blue, so that visual detection by GPC3 is realized.
The specific implementation steps are as follows:
(1) preparation of heme-reductive graphene oxide (H-rGO) nano material
Weighing 30mg of Graphene Oxide (GO) and placing the graphene oxide in a beaker, adding 30 mL of purified water, and carrying out ultrasonic crushing on GO for 60 min by using a BILON92-IID type ultrasonic cell crusher produced by Shanghai Bilang instruments, so that the GO is fully and uniformly dissolved to obtain a GO solution with the concentration of 1.0 mg/mL.
② adding 30mg of Ascorbic Acid (AA) into the GO solution dissolved uniformly, placing the mixture on a digital display constant temperature magnetic stirrer, and stirring the mixture for 12 hours at room temperature to obtain the reducing graphene oxide (rGO) solution.
③ weighing 30mg of heme (Hemin), dissolving in 10 mu L of ammonia water, adding 30 mL of distilled water to obtain Hemin solution, mixing with rGO solution according to the volume ratio of 1:1, adding 8.0 mu L of hydrazine hydrate solution into the mixed solution of rGO and Hemin, stirring for 10min by vortex, and placing the mixed solution in a digital display type electric heating constant temperature water bath kettle for 60 min o C, water bath for 4 h.
And fourthly, placing the solution in a TGL-20M type desk-top high-speed centrifuge produced by Hunan instrument laboratory instruments company, centrifuging for 10min at the rotating speed of 12000 r/min, removing supernatant, and washing for multiple times to obtain the heme-reducing graphene oxide (H-rGO) solution. Drying the obtained solution into solid for later use.
(2) Preparation of H-rGO-Pt @ Pd NPs nanoenzyme
Weighing 5.0 mg of H-rGO solid, adding distilled water to a constant volume of 10 mL to prepare 0.5 mg/mL of H-rGO solution, adding 2.0mL of PDDA solution and 5.0mL of 0.2mol/L of NaCl solution into the solution, and stirring for reaction for 12 hours. Centrifuging at 12000 r/min for 10min, removing supernatant, and washing for 3 times to remove impurities to obtain PDDA modified H-rGO solution. Drying the obtained solution into solid for later use.
② weighing 5.0 mg of PDDA modified H-rGO solid, adding distilled water to constant volume of 10 mL, preparing 0.5 mg/mL solution, adding 2.0mL of 20mmol/L Na into the solution 2 PtCl 6 Solution and 2.0mL of 20mmol/L Na 2 PdCl 4 The solution was stirred and reacted for 12 h.
③ adding 10 mL of glycol solution as a reducing agent into the solution, adjusting the pH of the solution to 12.0 by using 1.0 mol/L NaOH solution, placing the solution into a water bath kettle and heating the solution to 60 DEG o C, water bath for 4 h.
Placing the obtained solution in a TGL-20M desk type high-speed centrifuge produced by Hunan instruments laboratory instruments company to centrifuge for 10min at the rotating speed of 12000 r/min, removing supernatant, and washing for many times to remove impurities to obtain the H-rGO-Pt @ Pd NPs nanoenzyme with catalase-like catalytic activity.
Performing characterization analysis on the H-rGO-Pt @ Pd NPs nanoenzyme by using an SU8020 Scanning Electron Microscope (SEM) produced by Hitachi, Japan, as shown in FIG. 2, wherein FIG. 2 (A) is an SEM scanning image of an H-rGO compound, and the appearance of the H-rGO compound is like a layer of film gauze; FIG. 2 (B) is an SEM scanning of H-rGO-Pt @ Pd NPs nanoenzyme, from which the morphology of the H-rGO-Pt @ Pd NPs nanoenzyme can be clearly seen, and a film gauze is coated with a plurality of white fine particles, and the white particulate matters are Pt NPs and Pd NPs with the size of about 100 nm, so that the H-rGO-Pt @ Pd NPs nanoenzyme can be successfully prepared.
(3) Preparation of H-rGO-Pt @ Pd NPs-Apt detection probe
mu.mmol/L of GPC3-Apt (3 '-TATGAGTCGGGTAACCCTGGTGTTAACGAACGT TCACGGGACTCATAAAAAAAA-5' -NH) 2 ) And 1.0mg/mL H-rGO-Pt @ Pd NPs nanoenzyme are ultrasonically mixed according to the volume ratio of 1:1, and-NH on the aptamer 2 and-COOH on H-rGO-Pt @ Pd NPs to form an amido bond. In addition to this, the present invention is,the nanoenzyme and GPC3-Apt have pi-pi conjugation, so that GPC3-Apt is adsorbed to the surface of H-rGO-Pt @ Pd NPs nanoenzyme to form an H-rGO-Pt @ Pd NPs-Apt detection probe.
The H-rGO-Pt @ Pd NPs-Apt detection probe is characterized by utilizing a UH5300 type ultraviolet-visible spectrophotometer of Japan Instrument Co. As shown in FIG. 3, GPC3-Apt has an absorption maximum at 260 nm. The H-rGO-Pt @ Pd NPs-Apt detection probe has maximum absorption at 275.5 nm, which means that the H-rGO-Pt @ Pd NPs and GPC3-Apt have pi-pi conjugation effect and the absorption peak is red-shifted. These results indicate that GPC3-Apt has stably bound with H-rGO-Pt @ Pd NPs nanoenzymes to form H-rGO-Pt @ Pd NPs-Apt detection probes.
(4) Construction of GPC3 visual detection system
100 μ L of 10 μ M GPC3-Ab capture probe was added drop-wise to a blank tube, 37% o C, incubating for 2 h; 100 mu L of GPC3 protein solution (or serum) with different concentrations is respectively added into the test tube coated with the capture probe, 37 o C, incubation for 2 hours and washing; adding 100 μ L of 1% BSA blocking solution into the test tube for sample addition, 37% o C incubate for 2h and wash. Adding 100 mu L H-rGO-Pt @ Pd NPs-Apt into the sealed detection test tube, and washing after incubation. Finally, adding a 100 mu L chromogenic substrate system TMB-H into the test tube 2 O 2 And reacting for a period of time to form a visual detection system of GPC 3. Optimizing factors such as the concentration of H-rGO-Pt @ Pd NPs-Apt in the detection system, incubation time, incubation temperature, pH value and ionic strength of the detection system, and the like to obtain the optimal condition for detecting GPC3 by the system.
(5) Plotting of GPC3 working curves
In an optimal GPC3 visual detection system (the concentration of H-rGO-Pt @ Pd NPs-Apt is 5.0 mu g/mL, the incubation time is 2H, and the incubation temperature is 37 o And C, the ionic strength is 30 mmol/L, the pH value is 4.5), an ultraviolet-visible spectrophotometer is adopted to carry out full-wavelength scanning, an ultraviolet-visible absorption curve (figure 4) of GPC3 protein solutions with different concentrations and the wavelength of 500-800 nm is obtained, and the maximum absorption peak is at 652 nm. As the concentration of GPC3 protein increased, the absorbance at 652nm increased. Within the range of 10-300 ng/mL, the absorbance and the GPC3 concentration are positiveThe correlation is that the working curve equation is Y =0.46284+0.00127X (Y is the absorbance at 652nm, X is the GPC3 concentration), R 2 = 0.9922. By the formula C LOD =3S b The detection limit was calculated to be 1.801 ng/mL.
(6) Detection of GPC3 in actual serum samples
GPC3 solutions of known concentrations of 100 ng/mL, 150 ng/mL, and 200 ng/mL and normal human serum samples were mixed at a volume ratio of 1:1, and absorbance at 652nm was obtained in an optimal GPC3 visual detection system. The GPC3 working curve was compared to determine the actual detectable GPC3 concentration, and the results are shown in Table 1.
TABLE 1 results of GPC3 detection in actual serum samples
(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 (8)
1. A method for visually detecting GPC3 based on a heme-reduced graphene oxide-platinum @ palladium nanoenzyme-aptamer H-rGO-Pt @ Pd NPs-Apt detection probe for non-disease diagnosis is characterized by comprising the following steps:
(1) preparation of heme-reduced graphene oxide H-rGO
Mixing reduced graphene oxide (rGO) with heme (Hemin), carrying out water bath, centrifuging and washing to obtain a heme-reduced graphene oxide (H-rGO) nano composite material;
(2) preparation of heme-reduced graphene oxide-platinum @ palladium nanoenzyme H-rGO-Pt @ Pd NPs
Adding a PDDA solution and a NaCl solution of poly (diallyldimethylammonium chloride) into the H-rGO solution, and stirring for reaction; centrifuging and washing to obtain the PDDA modified H-rGO composite nano material;
② Na 2 PtCl 6 And Na 2 PdCl 4 Adding the PDDA modified H-rGO composite nano material into the solution, and stirring; adding ethylene glycol solutionAdjusting the pH value with NaOH solution, and carrying out constant-temperature water bath reaction; centrifuging and washing to obtain H-rGO-Pt @ Pd NPs nanoenzyme;
(3) preparation of H-rGO-Pt @ Pd NPs-Apt detection probe
Taking GPC3 aptamer GPC3-Apt and H-rGO-Pt @ Pd NPs nano enzyme solution, ultrasonically mixing, incubating the mixed solution at room temperature, centrifuging and washing to obtain an H-rGO-Pt @ Pd NPs-Apt detection probe;
(4) construction of GPC3 visual detection system
Dropwise adding a capture probe GPC3 antibody GPC3-Ab into a test tube, adding a bovine serum albumin BSA solution for sealing, and adding a GPC3 solution; adding an H-rGO-Pt @ Pd NPs-Apt detection probe; then adding a chromogenic substrate system TMB-H 2 O 2 Forming a GPC3 visual detection system;
(5) GPC3 working curve plotting
Dropwise adding the GPC3 solution with different concentrations to be detected into the optimal GPC3 visual detection system obtained in the step (4), performing wavelength scanning by adopting an ultraviolet-visible spectrophotometer, recording the absorbance of the ultraviolet-visible spectrophotometer at 652nm, drawing a working curve according to the relation between the absorbance and the concentration of GPC3, and calculating the detection limit;
(6) detection of GPC3 in actual serum samples
Mixing a GPC3 solution with a known concentration with an actual human serum sample, adding into the optimal GPC3 visual detection system obtained according to the step (4), performing wavelength scanning by using an ultraviolet-visible spectrophotometer, and recording the absorbance at 652 nm; using the GPC3 working curve obtained in step (5), the GPC3 concentration of the actual serum sample was calculated.
2. The method of claim 1, wherein: in the step (1), the mixing volume ratio of rGO to Hemin is 1: 1-4.
3. The method of claim 1, wherein: in the step (2), the volume of the PDDA solution is 2.0mL and 0.2%, and the volume of the NaCl solution is 5.0mL and 0.2 mol/L.
4. The method of claim 1, wherein: in the step (2), the pH value is 11-13.
5. The method of claim 1, wherein: step (2) Na 2 PtCl 6 And Na 2 PdCl 4 The solution was 2.0mL and 20 mmol/L.
6. The method of claim 1, wherein: in the step (3), the concentration of GPC3-Apt is 5.0 mu mol/L, the concentration of H-rGO-Pt @ Pd NPs is 1.0mg/mL, and the mixing volume ratio is 1: 1-5.
7. The method of claim 1, wherein: the capture probe GPC3-Ab in the step (4) is 100 mu L10 mu mol/L; the GPC3 solution is 100 mu L; the BSA solution is 100 mu L and 1 percent; the H-rGO-Pt @ Pd NPs-Apt is 100 mu L; the 100 mu L of TMB-H 2 O 2 Medium TMB is 12.84mmol/L, H 2 O 2 The concentration was 28.44 mmol/L.
8. The method of claim 1, wherein: in the step (4), the concentration of H-rGO-Pt @ Pd NPs-Apt is 1.0-7.0 mu g/mL, the incubation time is 0.3-3H, and the incubation temperature is 15-45 ℃.
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Application publication date: 20210803 Assignee: Guangxi Silizhao Biotechnology Co.,Ltd. Assignor: GUILIN University OF ELECTRONIC TECHNOLOGY Contract record no.: X2023980045668 Denomination of invention: A method based on H-rGO-Pt@Pd Method for visualizing the detection of GPC3 using NPs nanoenzymes Granted publication date: 20220826 License type: Common License Record date: 20231102 |
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