Construction Of Electrochemical Aptasensor Based On Graphene And Its Detection Of Disease Markers

Graphene shows potential application value in the fields of electrochemical biosensors,energy storage,battery catalysis and adsorption of environmental pollutants due to its large specific surface area,excellent electrical conductivity and catalytic performance.Noble metal nanomaterials,with good biocompatibility,ultra-high catalytic and electrical conductivity,have also become one of the ideal materials for the construction of electrochemical sensors.Because the composite of the two materials not only enhances the electrochemical signal response,but also greatly enriches the binding sites of the sensor,so as to fix a large number of aptamers to identify the target,and ultimately improve the selection performance and sensitivity of the electrochemical sensor.Therefore,in this paper,four kinds of porphyrin-functionalized grapheme/noble metal nanocomposites were synthesized,which were used to design different types of aptasensing strategies and realized the detection of disease markers.The main content has the following four aspects:(1)Electrochemical aptasensor for myoglobin-specific recognition based on porphyrin functionalized graphene-conjugated gold nanocomposites.Firstly,a novel electrochemical aptasensor was developed for sensitive and selective detection of myoglobin(Mb)based on meso-tetra(4-carboxyphenyl)porphyrin-functionalized graphene-conjugated gold nanoparticles(TCPP-Gr/Au NPs).Due to its good electric conductivity,large specific surface area,and excellent mechanical properties,TCPP-Gr/Au NPs can act as an enhanced material for the electrochemical detection of Mb.Meanwhile,it provides an effective matrix for immobilizing myoglobin-binding aptamer(Mb BA).The electrochemical aptasensor has a sensitive response to Mb in a linear range from 2.0×10^-11 M to 7.7×10^-7 M with a detection limit of 6.7×10^-12 M(S/N=3).Furthermore,the method has the merits of high sensitivity,low price,and high specificity.Our work will supply new horizons for the diagnostic applications of graphene-based materials in biomedicine and biosensors.(2)Electrochemical prostate specific antigen aptasensor based on hemin functionalized graphene-conjugated palladium nanocomposites.An electrochemical aptasensor is described for the detection of prostate specific antigen(PSA).The aptasensor is based on the use of hemin-functionalized graphene-conjugated palladium nanoparticles(H-Gr/Pd NPs)deposited on a glassy carbon electrode.The nanocomposites integrate the high electrical conductivity of graphene to take advantage of the easily functionalized surface chemistry of Pd NPs and their excellent electrical conductivity and catalytic property.The hemin placed on graphene acts as both protective agent and in-situ redox probe.The Pd NPs provide numerous binding sites for the immobilization of DNA-Biotin by the coordinative bond between Pd and amino groups.A sensitive and specific PSA assay was attained by immobilizing the PSA aptamer via biotin-streptavidin.The resulting aptasensor has a linear response that covers PSA concentration range of 0.025 to 205 ng/m L,with a 8.0 pg/m L lower detection limit(at-0.36 V,scan rate:0.10 m V/s,S/N=3).The method was applied to the quantitation of PSA in spiked serum samples giving recoveries range from 95.0%to 100.3%.(3)A novel dual signal and label-free electrochemical aptasensor for mucin 1 based on hemin/graphene@PdPtNPs.A dual signal and label-free electrochemical aptasensor for mucin 1(MUC1)was constructed based on hemin/graphene@PdPtNPs nanocomposite(H-Gr@PdPtNPs).Hemin attached on the graphene surface not only improves the solubility of graphene and acts as an in-situ electrochemical probe but also exhibits excellent peroxidase-like properties to electrocatalyze the reduction of H₂O₂.PdPtNPs also show outstanding catalytic capacity to the reduction of H₂O₂ and provide numerous binding sites for loading dsDNA(MUC1 aptamer and c DNA)to form the sensing interface.In the presence of MUC1,due to the specific affinity between aptamer and MUC1,double helix would be induced dissociation and the aptamer would be pulled off from the electrode.As a result,the electrochemical signals of hemin and H₂O₂ were recovered.Based on these properties,the label-free and sensitive dual signal electrochemical biosensor for MUC1 detection has been developed.The one is differential pulse voltammetry(DPV)signal of hemin and the other is chronoamperometry signal arisen from the catalytic reduction of H₂O₂.The linear ranges for MUC1 were 8.0 pg/m L-80 ng/m L and 0.80 pg/m L-80 ng/m L with the limit of detections 2.5 pg/m L and 0.25 pg/m L by DPV and chronoamperometry,respectively.The recovery of MUC1 in human blood serum samples was from 95.0%to 104.2%.The detection platform does not need signal labeling which greatly reduced the sophisticated and expensive procedures.The aptasensor provide a promising strategy for the determination of MUC1 in clinical diagnostics.(4)A triple-amplification ratiometric electrochemical aptasensor for CA 125 analysis based on H-Gr/SH-?-CD@Pd Pt NFs and Exo I assisted strategy.A triple-amplified and ratiometric electrochemical aptasensor was designed for CA125 determination on the basis of hemin-graphene/SH-?-cyclodextrin@Pd Pt NFsnanocomposites(H-Gr/SH-?-CD@Pd Pt NFs)and Exo I assisted strategy.Here,hemin still acts as a protective agent preventing graphene from sedimentation and it is also considered as the internal reference signal due to the reversible hemin_ox/hemin_red pair.Pd Pt NFs as an enhancer can promote electron transfer rate and amplify the reference signal from hemin.In the presence of CA125,due to the specific binding between aptamer and CA125,double helixd(dsDNA designed by CA125 aptamer and c DNA)conformational changes results in desorption of free quercetins(QUE).Subsequently,the released QUE and DNA fragments are enriched on H-Gr/SH-?-CD@Pd Pt NFs through host-guest recognition by SH-?-CD and non-specific adsorption by Gr-based materials.Thereby,the direct electron transfer of QUE exhibited a stronger electrical signal and that of hemin is partially blocked leading to a current reduction of hemin.In addition,recycling amplification of CA125 by Exo I effectively assisted quercetin increase and QUE signal amplification leading to background reduction.Based on these properties,the triple-amplified and ratio signal electrochemical biosensor for CA125 detection was proposed.Testing demonstrates the assay for sensitive detection with a wider linear range is from 6.0×10^-4 to 1007 ng/m L and a lower detection limit 0.14 pg/m L.The recovery of CA125 in human blood serum samples is from 99.2%to104.4%.This work provides new opportunities for the development of triple-amplification and ratiometric signal strategies for detecting other tumor markers in clinical diagnostics.(5)The research results are summarized,and the existing problems and improvement directions are discussed and prospected.