Gold Nanoparticles Mediated Electrochemiluminescence For Nucleic Acid Analysis

As a highly sensitive analytical detection technology,electrochemiluminescence is not only used in environmental detection,immunoassay,food detection,and drug analysis,but also surface analysis,kinetic research,electronic transfer research.Among them,tris（2,2′-bipyridine）ruthenium,[Ru（bpy）₃]²⁺,and its derivatives with co-reactant systems,such as tripropylamine（TPA）,are the most widely used electrochemiluminescence systems.The application of distinctive metal nanoparticles,such as gold nanoparticles,is beneficial to the improvement of electrochemiluminescence analysis characteristics.As a typical metal nanoparticle,the main role of gold nanoparticles in Ru（bpy）₃²⁺/TPA-based electrochemiluminescence biosensing system includes:good biocompatibility and large surface area of gold nanoparticles for carrying more biomolecules;good conductivity and electrocatalytic properties are modified on the surface of the working electrode to catalyze the electrochemical reaction,accelerate the electron transfer rate to enhance the luminescent signal;gold nanoparticles enhancing the luminescent signal by using its surface effect.Therefore,it is particularly important to have a systematically study of the role of gold nanoparticles in the electrochemiluminescence analysis of Ru（bpy）₃²⁺system,investigate the effects of various factors on the action of gold nanoparticles,and fully exploit the function of gold nanoparticle to construct a new type of Electrochemiluminescence biosensorsThis dissertation mainly includes two parts:The first part is the literature review,we introduced the electrochemiluminescence,especially the electrochemical luminescence biosensing analysis of Ru（bpy）₃²⁺/TPA system.On this basis,the function of gold nanoparticle in electrochemiluminescence sensing is further reviewed.The second part is the research report.It mainly includes two researches on the functional research of gold nanoparticles in the electroluminescence of Ru（bpy）₃²⁺system and the research work of biosensing applications:1.An electrochemiluminescence nucleic acid analysis based on the electrocatalytic effect of gold nanoparticle.In this work,we constructed gold nanoparticle modified ITO（ITO/AuNPs）electrodes by electrostatic adsorption on an indium tin oxide electrode（ITO）.The results showed that gold nanoparticles can enhance the electrochemica-luminescence signal of Ru（bpy）₃²⁺/TPA system,and the efficiency is up to 300 times.This enhancement is due to the electrochemical oxidation of the gold nanoparticle-catalyzed co-reactant TPA when the co-reactant is replaced by oxalate or compared to a plate gold electrode modified gold nanoparticle.In addition,the study also found that gold nanoparticles modified to the electrode can also annihilate luminescent radiation.Therefore,based on the above experimental results,we concluded that when gold nanoparticle is modified onto the electrode,the role of gold nanoparticle in the Ru（bpy）₃²⁺/TPA electrochemiluminescence reaction is the result of electrocatalysis and quenching co-competition:it can catalyze the electrochemical oxidation of the co-reactant TPA to enhance luminescence,and at the same time quench the luminescent radiation on the surface of the gold nanoparticle.On this basis,we designed an electrochemical luminescence nucleic acid analysis method based on gold nanoparticle electrocatalytic effect.First,a hairpin DNA sequence was introduced,one end of hairpin DNA was covalently immobilized on the surface of the electrode by the interaction of streptavidin and biotin,and the gold nanoparticle was labeled at the other end.When the hairpin DNA is in a closed state,gold nanoparticle is immobilized on the surface of the electrode,catalyzing the oxidation of TPA to enhance luminescence.When the hairpin DNA hybridizes with the complementary DNA,the gold nanoparticles at the end of the hairpin DNA are separated from the electrode surface,and the electrocatalytic effect disappears,resulting in a decrease of the luminescence signal.According to this principle,a method for detecting gold nanoparticle-labeled electrochemiluminescence nucleic acid with reduced signal was established.At the same time,electrochemical methods and scanning electron micrograph characterization confirmed our design.Under the optimized experimental conditions,the decrease of the electrochemiluminescence signal is linear with the logarithm of the DNA concentration in the range of 0.5pM-0.5 nM.The linear equation is I_ECL=-870.9logC+2466（C:pM,R²=0.9879）.The detection limit is 0.2 pM.At the same time,the method can effectively distinguish single-base mismatches from fully complementary DNA sequences,indicating that the method has better selectivity.2.A Competition between quenching and enhancing of electrochemiluminescence nucleic acid analysis by gold nanoparticle in“turn-on”mode.According to reports in the literature,the quenching and surface enhancement of gold nanoparticle is dependent on the competition of distance.However,in these reports,the electrocatalytic enhancement of gold nanoparticle is often overlooked.Therefore,in this research,we still introduce a hairpin DNA sequence,one end of which is covalently immobilized on the surface of the electrode by the interaction of streptavidin and biotin,and one end is labeled with gold.At the same time,Ru（bpy）₃²⁺derivatives were labeled on streptavidin surface to investigate the effect of gold nanoparticle on the luminescence behavior of Ru（bpy）₃²⁺in the close and open state of hairpin DNA.The results indicated that gold nanoparticle catalyzes the oxidation of TPA but does not enhance the luminescence signal in the close state of hairpin DNA.The main reason may be that the luminescent molecules are immobilized on the streptavidin on the electrode,and the distance from the gold nanoparticle is relatively close,and the quenching effect is dominant.When the hairpin DNA was opened by the hybridization reaction,the electrochemiluminescence signal was increased by 69 times compared with the close state.The reason is the quenching of gold nanoparticle in the closed state dominates the background signal,and when it is in the open state,the surface enhancement is dominant at this spatial distance,and the signal is tripled when there is no gold nanoparticle.At the same time,the length of the DNA sequence was changed,and the surface enhancement of the gold nanoparticle in the open state of the hairpin DNA was verified and correlated with the spatial distance.According to this principle,a method for detecting gold-labeled electrochemiluminescence nucleic acid with elevated signal was established.Under the optimal experimental conditions,the increase of the electrochemiluminescence signal is linear with the logarithm of the DNA concentration in the range of 0.05 pM-0.5 nM.The linear equation is I_ECL=536.9logC+879.1（C:pM,R²=0.9949）.The detection limit of the calculated method（DL=3σ/S）is 12 fM.In addition,the method can effectively distinguish single-base mismatches from fully complementary DNA sequences,indicating that the method has better selectivity.