The Construction And Application Of Electrochemical Immunosensor Based On Nanozymes

Objective:Alpha-fetoprotein（AFP）is a significant tumor maker,which could express in primary liver cancer.The quantitative detection of AFP play a vital role in screening and diagnosis of liver cancer.Recently,electrochemical immunosensors have garnered extensive attention due to their advantages of fast response,excellent selectivity,ease of handling and high sensitivity.Enzyme amplification technology has been regarded as useful signal amplification strategy due to its high electrocatalytic properties and biocompatible ability.However,natural enzymes are easily affected by environmental factors such as pH,temperature and humidity.Nanoenzyme,as a new type of artificial enzyme,has strong catalytic activity and stability,and can be a good substitute for biological enzymes for enzyme amplification technology.In this paper,we apply nanoenzymes with excellent properties to the construction of electrochemically active nanoprobes for signal amplification.Methods:1.A relatively stable and sensitive label-free electrochemical immunosensor based on hierarchically flower-like gold microstructures/polyaniline/reduced graphene oxide/prussion blue（HFG/PANI/rGO/PB）composite modified sensing interface was stepwise fabricated for determination ofα-fetoprotein（AFP）.The experimental conditions was optimized by method of differential pulse voltammetry（DPV）.Meanwhile,the analysis performance of the HFG/PANI/PB modified immunosensor and HFG/rGO/PB modified immunosensor were also studied.2.The core-shell Pd@Pt nanoparticles were synthesized and characterized by transmission electron microscopy.Gold nanoparticles（AuNPs）were electrodeposited on a gold electrode（AuE）surface,and then the primary anti-AFP（Ab₁）was anchored on the surface of AuNPs by the adsorption of AuNPs and Ab₁.A novel amperometric immunosensor was developed by using core-shell Pd@Pt nanoparticles to associate thionine（Thi）and Ab₂ as electrochemical signal probe for the sensitive detection of AFP.3.Gold nanoparticles（AuNPs）film,which formed by electrodepositing of AuNPs on the glod electrode,was used for the immobilization of the primary Ab₁.A novel electrochemical immunosensors was developed by using core-shell Pd@Pt nanoparticles/polyaniline-functionalizedcarbonnanotubenanocomposite（Pd@Pt/PANI@CNTs）to associate thionine（Thi）and Ab₂ as electrochemical signal probe for the sensitive detection of AFP.Results:1.Construction of immunosensing interface based on flower-like gold microstructure/polyaniline/reduced graphene oxide/Prussian blue complex.Compared with HFG/PANI/PB modified immunosensor,the HFG/PANI/rGO/PB modified immunosensor achieves high sensitivity.Compared to HFG/rGO/PB modified immunosensor,the HFG/PANI/rGO/PB modified immunosensor have a wider detection range.Under the optimal conditions,the DPV response decreased with the increase of AFP levels from 0.01 to 30 ng/mL,implying a good linear relationship between the DPV response and logarithm of AFP level.The linear equation was I（μA）=-6.5608lgC_AFP（ng/m L）+24.5159,where R² was 0.9289.The detection limit was0.003 ng/mL.2.Preparation of electrochemical immunosensor based on Pd@Pt core-shell nanoenzyme as signal amplification strategy.The amplified electrochemical signals derived from the Pd@Pt nanozyme toward the reduction of H₂O₂ using thionine as electron mediator.Under the optimal conditions,the DPV peak current was linearly dependent on the logarithm of AFP concentration in the range of 0.1-100 ng/m L with a detection limit of 0.035 pg/m L（S/N=3）。The linear equation wasΔI（μA）=0.9576lgC_AFP（ng/mL）+5.1557,where R² was 0.993.3.Preparation of electrochemical immunosensor based on Pd@Pt/PANI@CNTs nanoenzyme as signal amplification strategy.Under the optimal conditions,the DPV peak current was linearly dependent on the logarithm of AFP concentration in the range of 0.1-100 ng/m L with a detection limit of3.5 pg/m L（S/N=3）。The linear equation wasΔI（μA）=0.4415lgC_AFP（ng/mL）+4.0751,where R² was 0.9877.Conclusion:1.The effect of PANI and rGO on the proposed immunosensor was studied.In detail,PANI/rGO due to the unique electrochemical properties can effectively prevent PB leakage and form a stable sensing platform,which cause sensitive current responsiveness and thus the more satisfied detection limit.2.Anovel sandwich-type electrochemical immunosensor capable of detecting AFP was fabricated using core-shell Pd@Pt nanoparticles nanozyme as signal amplification strategy.Platinum（Pt）and palladium（Pd）,as two important precious metal-based nanoenzymes,have good biocompatibility and excellent enzyme-like catalytic performance,which can effectively improve detection sensitivity.Moreover,the bimetallic nanoenzyme with a core-shell structure has a large interconnection area and abundant active sites,resulting in immobilization of a large amount of antibodies.The immunosensor has good reproducibility,stability and selectivity,and can be used for the detection of AFP.3.In order to load more Ab₂,Pd@Pt were loaded onto the polyaniline functionalized carbon nanotubes（PANI@CNTs）which have high conductivity,adsorption capacity and large specific surface area.Thus,more Ab₂ and Thi were bonded to the tracer via the conjugates between PtNPs and thiols or alkylamines of Ab₂ and Thi.As a result,the immunosensor displayed obviously response toward target AFP due to the as-prepared nanozyme（Pd@Pt/PANI@CNTs）with high catalytic activity of Thi toward H₂O₂.