Studies On Electrochemical Biosensor Based On Metal Nanomaterials And Biological Amplification Strategies

As a new analytical model that combines the advantages of biosensors and electrochemical technology,electrochemical biosensors have been widely used in food analysis,environmental monitoring,drugs and clinical diagnosis.With the rapid development of scientific research,new challenges have been raised for the development of electrochemical biosensors with better stability and higher sensitivity.In this paper,a series of electrochemical biosensors for ultrasensitive detection of Pb²⁺,b2 microglobulin and melamine were constructed by combining a high-performance metal nanomaterials with DNA self-assembly,enzyme cascade amplification,target-transition recycling amplification and other techniques.The studies of this thesis are as follows:1.Dual triggers induced disassembly of DNA polymer decorated silver nanoparticle for ultrasensitive electrochemical Pb²⁺detectionThe decomposition of branched DNA polymer is typically accomplished by hybridization of a single initiator to DNA at the bottom of the electrode surface.However,due to the steric hindrance between dendritic DNA structure and electrodes,and the limited DNA initiator to decompose the dendritic DNA polymer at a slower rate,the decomposition efficiency of the DNA polymer is limited.In order to resolve this problem,a DNAzyme-assisted target recycling strategy was designed to generate a large number of dual triggers（T₁ and T₂）induced the efficient depolymerization of branched DNA polymer,which could improve the sensitivity of the biosensor.On the one hand,a dendritic DNA polymer was constructed to immobilize a large number of electrochemical signal tag silver nanoparticles（AgNPs）on the electrode by hybridization chain reaction（HCR）,which increased the electrochemical response signal.On the other hand,the target Pb²⁺cyclically cleaved the substrate in the Pb²⁺-DNAzyme,generating a large amount of free T₁ and T₂ and achieving target circular amplification and improving the sensitivity of the sensor.In addition,the resulted dual triggers induced efficient depolymerization of the branched DNA polymer by strand displacement reaction（SDR）,which was an isothermal process involved no enzymes and could selectively depolymerize the backbone and side chain of the branched DNA polymer,which enabled ultrasensitive detection of Pb²⁺with a detection range of 1 pmol/L to 100 nmol/L and a detection limit of 0.24 pmol/L.2.A novel electrochemiluminescence sensor based on functionalizedb-cyclodextrin-ferrocene host-guest complex with multiple signal amplificationHow to improve the luminous efficiency has become a research hotspot in the development of highly sensitive electrochemiluminescence sensors in recent years.Among them,the shortened distance between the luminophor and the coreactant can effectively improve the luminous efficiency in electrochemiluminescence（ECL）system.In addition,since natural peroxidases are easily inactivated and difficult to separate,metal nanomaterials with excellent stability and mimicking peroxidase properties have received extensive attention.In view of this,b-cyclodextrin（β-CD）functionalized N-（aminobutyl）-N（ethylisoluminol）（ABEI）was bound to the ferrocene（Fc）functionalized coreactant L-cysteine（L-Cys）by host-guest recognition,resulting in a greatly shortened distance between the ABEI and L-Cys and leading to a higher ECL signal.At the same time,a simple and sensitive ECL immunosensor was constructed based on the multi-catalytic signal amplification strategy of HAuNPs with mimic enzyme properties and Fc toward H₂O₂,and the detection ofβ2-microglobulin（b2M）was successfully realized.The developed strategy utilized functionalized metal nanocomposites with multiple signal amplification achieved efficient ECL luminescence efficiency and a new sensitive detection method for the detection of other proteins.3.Highly sensitive electrochemical detection of melamine based on side-by-side gold nanorods selfassembly-induced the regulation of enzyme cascade catalytic efficiencyThe enhancement of the electrochemical signal by the enzyme cascade amplification technique is more significant than the single-enzyme catalytic signal amplification method.The regulation of the interenzyme distance to the optimal distance is an important means of achieving a more efficient enzyme cascade.However,for the traditional supporter regulating interenzyme distance（metal organic framework,DNA nanostructure,etc.）,there are deficiencies in preparation complexity and structural instability,resulting in limited enzyme cascade efficiency.In order to solve the above problem,we synthesized anisotropic CTAB-capped gold nanorods（AuNRs）and used them as scaffolds to regulate the interenzyme distance,and the optimal interenzyme distance was achieved by the target melamine-induced AuNRs self-assembly in a homogeneous solution.And thus highly efficient enzyme cascade amplification was obtained.Specifically,the two terminal regions of AuNRs could be preferentially replaced by glucose oxidase（GOx）and horseradish peroxidase（HRP）,and a large number of regular side-by-side assembly modes were formed under the inducing of the target,which would lead to highly ordered,stable and optimal alignment of GOx and HRP,improving the enzyme cascade efficiency.At the same time,AuNRs could also be used as a mimetic enzyme to further catalyze the enzyme cascade to increase the efficiency of the enzyme cascade.In addition,our homogeneous electrochemical biosensors avoided the cumbersome and time-consuming modification process on the electrode surface.The recognition of the target analyte and the signal molecule were in the homogeneous solution,thereby avoiding the steric hindrance effect on the electrode surface and enhancing the recognition and response efficiency.Through the mentioned double amplification strategy,the sensing platform achieved sensitive detection of melamine.