Construction Of Electrochemical Biosensors Based On Three-dimensional Porous Material And Tetrahedral DNA Nanomaterial

Three-dimensional（3D）porous nano-material is a kind of material which is composed of reticulated structure through closed or permeable pores.Covalent organic skeleton material（COFs）is a new porous crystalline material synthesized by light elements（B,C,N,O,Si）.Metal-organic skeleton material（MOFs）is also a kind of 3D porous material,which has the advantages of super-high porosity,large specific surface area,abundant pore structure,expandable structure,good thermal stability and chemical stability,etc.It has become a promising new material for the construction of new electrochemical sensors.In this paper,gold nanoparticles-cobalt sulfide/graphene oxide（Au NPs@CoS/GO）composite,Fe-MOF（Fe-MIL-88 NH₂）material,Au NPs@COF-LZU8 nanocomposites,tetrahedral DNA nanomaterials,with high specific surface area,high electron mobility and low cost,were synthesized and used to develop electrochemical biosensors for the detection of C-reactive protein and single nucleotide polymorphism（SNP）.（1）In the first chapter,an ultrasensitive electrochemical immunoassay based on porous covalent organic framework materials and Au NPs@CoS/graphene oxide（GO）nanocomposite was developed for the detection of C-reactive protein（CRP）.Au NPs@CoS/GO nanocomposite was synthesized,and employed as substrate because of their high conductivity to immobilize CRP antibody.A new porous covalent organic framework material（COF-LZU8）was functionalized by gold nanoparticles and doped with thionine（Thi）as the electron transfer mediator to obtain nanocomposite.The reduction peak signal of Thi/Au NPs@COF-LZU8 at-0.2V could be directly detected by highly sensitive differential pulse voltammetry（DPV）.Due to the high surface area of COF-LZU8,a great quantity of Thi could be adsorbed on the surface of COF,thus,the signal could be greatly amplified.Moreover,Thi/Au NPs@COF-LZU8 has strong adsorption capacity to the anti-CRP protein macromolecules due to the large specific surface area,good bio-affinity.The concentration of CRP antigen was proportional to the response current,so the quantitative detection of CRP was realized.Under the optimum conditions,the linear range of CRP was 0.05-150 ng/mL,with the detection limit of 0.016 ng/mL.This immunosensor provides a more effective and sensitive method for detection of CRP.（2）In the second chapter,a three-dimensional porous nanomaterial Fe-MIL-88NH₂ was synthesized by hydrothermal method,and a dual-signal proportional immunosensor was developed for the detection of C-reactive protein by using Fe-MOFs（Fe-MIL-88 NH₂）as matrix material.The octahedral MOFs not only provide a larger effective surface area,but also increase the amount of immobilized biomolecules and promote the transport of electrons and ions,and also exhibit good electrical conductivity.Modification of Au NPs onto MOFs can further increase specific surface area to capture large amounts of antibodies and increase electron transfer capacity.With the increase of CRP concentration,the redox peak current of K₃Fe（CN）₆/K₄Fe（CN）₆ decreases,while the reduction peak current of Fe³⁺in MOFs is relatively constant.The ratio of the response current of K₃Fe（CN）₆/K₄Fe（CN）₆ to the response current of MOFs was used as a response signal for quantitative determination of CRP.Fe-MOFs modified by Au NPs was used as internal reference probes（MOFs-rP）,K₃Fe（CN）₆/K₄Fe（CN）₆ was used as a signal probe（K₃Fe（CN）₆/K₄Fe（CN）₆-sP）.This ratio probe integrates sP and rP into a single structure,ensuring exactly the same modification conditions and the interdependence of sP and rP on a sensing interface.Therefore,this ratio probe has a stronger ability to eliminate environmental variation interference.The linear response of the immunosensor proposed in this paper ranges from 1 ng/mL to 200 ng/mL,and the detection limit is0.3 ng/mL（S/N=3）.The sensor has good selectivity and is expected to be used for early screening and diagnosis of cardiovascular disease.The method also lays the foundation for the preparation of other ratiometric signal amplification sensors for detecting biomarkers.（3）In this study,Au NPs@Fe-MIL-88 NH₂ was used as a marker to label CRP antibody,and silver ions were reduced by gold nanoparticles（Au NPs）using gold label silver stain technique（GLSS）.A sandwich paper-based immunosensor was constructed.A simple image-J software was used to detect the gray value of the system,and the quantitative detection of CRP was realized.The porous structure of Fe-MOFs material were used to adsorb more gold nanoparticles（Au NPs）.Using Au NPs as nucleating site,silver ions were automatically chemically reduced to silver metal in the presence of reductant,and the enhancement effect of silver was determined by the density of gold nanoparticles on the surface.The higher the concentration of CRP,the higher the density of gold nanoparticles on the marker and the more silver ions are reduced,the higher the gray value of the image.The CRP concentration has a linear relationship with the gray value.Under the optimal conditions,the linear range is from 0.05 ng/mL to 100 ng/mL,detection limit 0.017ng/mL.This method has the advantages of low cost,simple operation,quick and practical,and has practical significance.（4）Studies have shown that SNP is associated with a variety of diseases.Therefore,it is of great significance to detect SNP.In chapter 4,an electrochemical sensing platform based on DNA tetrahedral nanostructures is proposed for highly sensitive SNP detection.DNA tetrahedral nano-materials were synthesized by using four specially designed single-stranded DNA sequences.Gold nanoparticles/graphene oxide（Au NPs@GO）were modified on the glass-carbon（GC）electrode as the substrate of the electrode.DNA tetrahedral nano-materials were assembled onto the surface of the electrode through the thiol group（-SH）modified at the end of DNA.When the target DNA exists,the end of the target DNA hybridizes with the DNA terminal of the DNA tetrahedral nanomaterials.At this point,the signal probe labeled with Au NPs@Fe-MOF material can hybridize with the other end of the target DNA.Through the detection of Fe³⁺signal peaks in Fe-MOF materials,the detecting of SNP can be achieved.In the range of 0.5⁵00 nmol/L,the peak current value of the sensor has a good linear relationship with the logarithm of target DNA concentration.The linear correlation coefficient of R² is 0.9943 with detection limit of 0.166 nmol/L.Tetrahedral DNA nanomaterials synthesized by DNA self-assembled nanotechnology are the simplest and most firm three-dimensional pyramid structure model,which can improve the specificity of the system.