Dual-Mode Colorimetric-Electrochemical Aptamer Sensing For Low-Density Lipoprotein Detection

Low-density lipoprotein（LDL）is a valid biomarker involved in the risk assessment of cardiovascular disease（CVD）.Accurate and sensitive monitoring of LDL can provide a theoretical and practical basis for diagnosing,preventing,and treating CVD,reducing mortality,improving people’s quality of life,and reducing the global economic burden caused by CVD.In this thesis,a simple and intuitive colorimetric method and a fast-response electrochemical method are combined to detect LDL in human serum.The LDL aptamer（LDLapt）with specificity and reduced graphene oxide@molybdenum disulfide-ferrocenecarboxylic acid（rGO@MoS₂-Fc）nanozyme with excellent peroxidase-like properties and electroactive fast response were introduced to construct a colorimetric and electrochemical aptasensor for the accurate and sensitive analysis of serum LDL.The main studies are as follows:（1）Synthesis and properties of rGO@MoS₂-Fc nanozyme:The rGO@MoS₂-Fc nanozyme was synthesised,and a variety of methods were used to characterize the rGO@MoS₂-Fc nanozyme,such as scanning electron microscope,transmission electron microscope.The rGO@MoS₂-Fc nanozyme was verified to have the enzyme-like property of catalyzing the generation of hydroxyl radical（·OH）from hydrogen peroxidase（H₂O₂）,which oxidizes the colorless o-phenylenediamine（OPD）to yellow color 2,3-diamino phenothiazine（DAP）.The enzymatic kinetic constants of the rGO@MoS₂-Fc nanozyme were investigated and compared with those of horseradish peroxidase（HRP）,and furthermore,the peroxidase-like catalytic mechanism of the rGO@MoS₂-Fc nanozyme was investigated.In addition,the temperature and p H stability of rGO@MoS₂-Fc nanozyme were investigated.（2）Colorimetric aptasensor for LDL detection:The specificity of LDLapt binding to LDL was investigated.Then,rGO@MoS₂-Fc/LDLapt was formed to use as a signal probe based on LDLapt and rGO@MoS₂-Fc/LDLapt,and LDLapt was used as a capture probe to construct a sandwich-type colorimetric aptasensor.As the LDL concentration increases,the number of bound rGO@MoS₂-Fc/LDLapt signal probes increases and the color becomes darker,thus enabling visualization of LDL detection.Under optimal conditions,the absorbance（Y）showed a good linear relationship with the LDL concentration(C_LDL)in the range of 30.0-190.0μg/m L,and the linear equation was:Y=0.00375*C_LDL+0.3049,R²=0.9963 with a limit of detection（LOD）of 15.04μg/m L.The relative standard deviation（RSD）of the LDL assay was between 0.12%and 2.45%,with a relative error of 1.70%-4.71%when detecting actual serum samples using the colorimetric aptasensor.Furthermore,the colorimetric aptamer sensor has good specificity,reproducibility,and stability.（3）Electrochemical dual-signal aptasensor for LDL detection:A sandwich-type electrochemical dual-signal aptasensor was constructed using screen-printed electrode（SPCE）as the sensing substrate,electro-deposited gold-polyethyleneimine（Au-PEI）as the substrate modifier,then,immobilizing LDLapt on the Au-PEI/SPCE surface as a capture probe,and rGO@MoS₂-Fc/LDLapt as signal probe.The oxidation peak current of Fc in rGO@MoS₂-Fc/LDLapt and the electrochemical signals generated by decomposing H₂O₂that catalyzed by rGO@MoS₂-Fc/LDLapt were measured by differential pulse voltammetry（DPV）method and amperometry（i-t）method,respectively.Under optimal conditions,the DPV peak current（I）showed a good logarithmic relationship with C_LDL in the range of 0.001-100.0μg/m L with the linear equation:I=0.9175*lg C_LDL+6.6432,R²=0.9941 and LOD of 0.91 ng/m L.Meanwhile,the difference in i-t currents(current value of C_LDL detected minus the current value of the blank,ΔI)showed a good linear relationship with C_LDL in the range of 1.0-80.0μg/m L,with a linear equation ofΔI=2.7989E-9*C_LDL+6.7257E-9,R²=0.9896 and LOD of 0.8042μg/m L.In the actual serum samples assayed by the DPV method,the RSD was between 1.49%and 2.96%with a relative error of 1.02%-9.44%.For the actual serum samples by i-t method,the RSD was between 3.94%and 7.85%with a relative error of 2.76%-2.78%.Moreover,the electrochemical aptasensor has good specificity,reproducibility,and stability.（4）Comparative analysis between the colorimetric aptasensor and the electrochemical dual-signal aptasensor for the detection of LDL:The colorimetric aptasensor has the advantage of visual recognition and a wider detection range（30.0-190.0μg/m L）than the electrochemical dual-signal aptasensor,indicating that the colorimetric aptasensor is more suitable for relatively high LDL concentrations,however,the LOD of colorimetric aptasensor（15.04μg/m L）is higher than that of the electrochemical dual-signal aptasensor（0.91 ng/m L）,indicating the electrochemical dual-signal aptasensor has higher sensitivity.In a conclusion,each aptasensor has its own advantages and both have some research significance.