Research On Specific Determination Of GPC3 By Aptamer Nano-biosensor

Glypican-3（GPC3）is a membrane-bound heparin sulfate proteoglycan that is highly expressed in liver cancer tissues and negative in normal liver tissues or benign hepatocyte nodules.It is an important tumor marker for screening and diagnosis of liver cancer.Aptamers were repeatedly screened from a library of random oligonucleotide sequences synthesized in vitro using SELEX technology.They can bind to a target molecule with a high degree of affinity and specificity.It has the advantages of wide target molecules,strong specificity and stability.An aptamer biosensor is a type of analysis device that tightly combines an aptamer as a molecular biological recognition element and a signal conversion element to detect a target compound.Therefore,we use GPC3 as the target,GPC3 aptamer as the recognition element,and combined the use of enzyme-catalyzed signal amplification and nanomaterial signal amplification technology to construct two aptamer nanobiosensors to achieve specific detection of GPC3 in serum.The main research contents are as follows:（1）Research on aptamer nanosensor based on light addressable potential sensor（LAPS）to detect GPC3:LAPS chip,light source drive circuit,signal amplification circuit,and Lab VIEW platform constitute LAPS real-time test system.Gold nanoparticles/polyethyleneimine-graphene oxide（Au NPs/PEI-r GO）composite materials were designed and synthesized.Au NPs/PEI were measured using transmission electron microscope（TEM）,scanning electron microscope（SEM）,particle size analysis and Zeta potential.Au NPs/PEI-r GO has excellent conductivity and large specific surface area,increasing the loading of GPC3-Apt.Modified Au NPs/PEI-r GO and GPC3-Apt on the LAPS chip to form sensitive units.GPC3 standard solution was added dropwise on the LAPS sensitive unit to form the LAPS test substrate.Put the LAPS test substrate into the LAPS test system.Under the effect of the applied bias voltage,the specific combination of GPC3 and GPC3-Apt on the LAPS test substrate causes the potential of the sensitive cell surface to change.shift.The offset and GPC3 concentration showed a good linear relationship between 0.1-100μg/m L,the correlation coefficient was 0.98998,the minimum detection limit was 0.04μg/m L（[S/N]=3）,and the sensitivity was 1.33638 m V/μg/m L.At the same time,the LAPS detection system has good specificity and reproducibility.When used in actual serum detection,the recovery rate of LAPS sensor is between 99.03%-119.32%,and the relative standard deviation is between 1.05%-6.85%.（2）Research on the GPC3 electrochemical aptamer nanosensor based on heme/reduced graphene（HGNs）-like enzyme-catalyzed silver deposition:using hydrazine hydrate as a reducing agent,HGNs nanozymes were prepared by a one-step reduction method.The structure and morphological characteristics of HGNs were studied using Fourier transform infrared spectroscopy（FT-IR）,ultraviolet spectroscopy（UV-vis）,and transmission electron microscope（TEM）.The GPC3 electrochemical aptamer nanobiosensor was constructed using HGNs non-destructively labeled GPC3 aptamer as the signal probe（HGNs-Apt）and GPC3 aptamer as the capture probe.With the catalysis of HGNs-like peroxidases,H₂O₂reduces silver ions（Ag⁺）in solution to metallic Ag and deposits on the electrode surface.GPC3 is highly sensitively detected by differential pulse voltammetry（DPV）.Under the best conditions,the current response of Ag has a good positive correlation with the concentration of GPC3 in the range of 10.0-100.0μg/m L,with a correlation coefficient of 0.99576.The minimum detection limit is 3.16μg/m L（[S/N]=3）,and the sensitivity is 0.807μA·μM^-1·cm^-2.The applicability of this method was verified by analyzing spiked human serum samples（good recovery from 101.25%to 122.25%）.In addition,GPC3 electrochemical aptamer nanobiosensor has acceptable selectivity,stability and repeatability.