Study On Single Particle Collision Electrochemistry And Its Quantitative Detection Of Biological Macromolecules

The emergence of single particle collision electrochemistry（SPCE）allows in situ detection of single analyte entity one at a time with simplicity,fast response and high throughput.SPCE method was originally designed to characterize physical and chemical properties of the corresponding single nanoparticles,and has been later extended into the field of bio-analysis,enabling better understanding of biological heterogeneity and providing new route for developing new diagnostic devices for quantifying biological analytes.So far,SPCE methods have been used to detect a variety of biological species such as DNA,RNA,enzymes,bacteria,vesicles,and cells.At present,the specificity,sensitivity and accuracy of disease marker detection still face huge challenges.The unique high sensitivity of the SPCE method can provide a new way for the analysis of disease markers.However,disease marker detection based on SPCE is still in its infancy,and most studies cannot achieve selective analysis.Based on this,this paper uses the SPCE method,using the electrocatalytic oxidation of platinum nanoparticles（Pt Nanoparticles,PtNPs）and the direct electrooxidation of silver nanoparticles（AgNanoparticles,AgNPs）as signal transduction means to conduct high-sensitivity detection of disease markers.Homogeneous detection with specificity provides an accurate,low-cost,fast and effective new solution for disease marker detection,and shows certain development prospects in point-of-care detection.The main work content of this paper includes the following four parts:1.Stochastic collision electrochemistry from single Pt nanoparticles:electrocatalytic amplification and miRNA sensing:single-particle collisions have made many achievements in basic research,but challenging is still existed due to their low collision frequency and selectivity in complex samples.In this work,we developed an"on-off-on"strategy based on Pt nanoparticles（PtNPs）that catalyze N₂H₄ collision signals on the surface of carbon ultramicroelectrodes（CUME）,and established a new method for the detection of miRNA-21 with high selectivity and sensitivity.PtNPs catalyze the reduction of N₂H₄ on the surface of the carbon ultramicroelectrodes to generate a stepped collision signal,which is in the"on"state.The single-stranded DNA paired with miRNA-21 is coupled with PtNPs to form a complex DNA/PtNPs.Because PtNPs are covered by DNA,the electrocatalytic collision of N₂H₄ oxidation is inhibited.At this time,the signal is in the"off"state.When miRNA-21 is added,the strong complementary pairing between miRNA-21 and DNA destroys the electrostatic adsorption of DNA/PtNPs conjugates and restores the electrocatalytic performance of PtNPs,and the signal is in the"on"state again.Based on this,a new method for detecting miRNA-21 was established.It provides a new way for small molecules sensing and has a wide range of applications in electroanalysis,electrocatalysis and biosensing.2.Stochastic collision electrochemistry of single silver nanoparticles and their biosensing:In this work a SPCE-based immunoassay was developed for the specific detection of IgG.AgNPs are conjugated with coated/labeled anti-IgG as capture/detection probes,which bind to IgG through specific molecular biological recognition to form AgNPs dimers or multimers.IgG-induced aggregation of AgNPs leads to a decrease in nanoparticle concentration and an increase in nanoparticle size.By examining the anodization of individual AgNPs in SPCE measurements,a self-verifying dual-mode analysis of shock frequency and oxidation charge was performed.Unlike most SPCE-based analyzes that use surface amplification strategies,where only surface atoms are available for electrochemical readout,the current principle fully utilizes all atoms of AgNPs to conduct signals,greatly improving detection sensitivity.This immunosensor shows great potential for point-of-care detection.3.Multiplex assays of miRNAs by using single particle collision electrochemistry in a single run:it is important to quantify multiple biomarkers in a single run due to the advantages of precious samples and diagnostic accuracy.Based on the distinguishability of two types of current signals from single particle electrochemical collision（SPCE）,step-type current transients produced by Pt nanoparticles（PtNPs）catalyzed hydrazine oxidation and peak-type current transients produced by Agnanoparticles（AgNPs）oxidation,a kind of multiplex immunoassay of target miRNAs（miRNA-21 and Let-7a）have been established during SPCE in a single run.When the single-stranded DNA（ssDNA1）that was perfectly complementary to miRNA-21 was coupled to the surface of PtNPs,the SPCE of PtNPs electrocatalysis was inhibited,and the step-type current transients disappeared,while the single-stranded DNA（ssDNA2）perfectly complementary to Let-7a was coupled to the surface of AgNPs,the SPCE of AgNPs oxidation was inhibited,and the peak-type current transients were disappeared,thus the signals were in the"off"state at this time.After that,miRNA-21 and Let-7a were added into solution,complementary base pairing disrupted the weak DNA-NP interaction and restored the electrocatalysis of PtNPs and the electrooxidation of AgNPs,and the step-type current signals and peak-type current signals were in the“on”state.Moreover,the frequencies from two different recovered signals（PtNPs catalysis and AgNPs oxidation）corresponded to the amount of added miRNA-21 and Let-7a,thus a multiplex immunoassay method for dual quantification of miRNA-21 and Let-7a in a single run was established.4.Single-particle collision electrochemical immunoassay for multiplex gastric cancer biomarker monitoring:In this work,a method for multiplex detection of gastric cancer biomarkers based on single-particle stochastic collision electrochemistry is proposed,which is combined with a nanoparticle labeling strategy,achieving ultra-sensitive detection of CA199 and CEA in complex samples.In this work,a mixed solution of PtNPs and AgNPs containing N₂H₄ can produce two different shapes of current transients,and the two types of current transients are distinguishable and do not interfere with each other.After PtNPs and AgNPs were labeled with the antibody CA199 and the antibody CEA respectively,the current changes of the two types of current transients were reduced because the continuous PEG layer and antibody on the surface of the nanoparticles partially hindered and shielded electron transfer and electrochemical signals.PtNPs and AgNPs are conjugated with coated/labeled CA199 and CEA antibodies as capture/detection probes respectively,and combine with CA199 and CEA through specific molecular biological recognition to form dimers or multimers of PtNPs and AgNPs.The aggregation of PtNPs and AgNPs induced by CA199 and CEA will lead to a decrease in nanoparticle concentration,an increase in nanoparticle size,and a decrease in collision frequency,thus enabling simultaneous detection of CA199 and CEA.