Single-Molecule Studies Of Enzymes By Nanopore Sensing Integrated With Nanoparticle Translocation

As a single-molecule level biomolecular detection and one of the hot spots in the field of DNA sequencing,nanopore sensors have developed and matured with the attention of more and more researchers in recent years.Nanopores have many outstanding characteristics,such as high throughput,low cost,no labeling,and high resolution,which make nanopores show great application prospects in DNA sequencing,mi RNA detection,small molecule and polymer detection,etc.Although there are also protein detection solutions based on nanopores,in fact,there are still huge challenges in using nanopores to analyze proteins in practical applications due to some physical structures and chemical properties of proteins.Nowadays,scholars have used gold nanomaterials in the fields of biosensing and biodetection and analysis order to its unique optical properties and excellent biocompatibility,overcoming many obstacles.In this paper,by combining gold nanoparticles with nanopore sensing platform,three gold nanoparticles with different morphologies are used,and the difference in ionic current generated due to the morphology or aggregation state when they pass through the nanopore is used to break through the limitation of protein detection in nanopore platform,realizing the purpose of ultra-sensitive detection of different proteins.1.There is a method for detecting telomerase activity that combines solid-state nanopore sensors and gold nanoparticles(Au NPs).As Telomerase is an important cancer target substance,how to achieve efficient and rapid detection and monitoring of telomerase has always been a research hotspot in early detection and treatment of cancer.This proposal can solve the low signal-to-noise ratio when detecting small molecules due to the process limitations of solid-state nanopores as well as can achieve rapid and efficient detection of telomerase activity.In this work,a poly A DNA-modified Au NPs assembly was designed for the detection.By adding telomerase primer sequence(TP)and using the principle of base complementary pairing,combined with a solid-state nanopore sensor,DNA extension and displacement reaction will occur in the presence of telomerase,causing a change in the aggregation state of Au NPs,thereby achieving the purpose of signal amplification.The change of the aggregation state of Au NPs will change the ionic current of the nanopore witch can be analyzed to detect the telomerase activity efficiently and sensitively.2.There is a solid-state nanopore-based enzymatic reaction-mediated gold nanorods(Au NRs)etching method for detecting hydrogen peroxide(H₂O₂).Horseradish peroxidase(HRP)catalyzes the oxidation of hydrogen peroxide to generate strong oxidizing hydroxyl radicals(·OH),which accelerates the etching of Au NRs greatly.In this process,the diameter of Au NRs remains almost unchanged while the length of Au NRs gradually decreases during the etching progresses.Au NRs will generate a specific signal when passing through the solid nanopore,the characteristic translocation signal would changed as the etching progresses happened.Combined with the characteristics of the single-molecule level of the solid-state nanopores,it can break through the limitations of existing methods such as colorimetry methods.These colorimetry methods are observed by the naked eye making them quite subjective What is more,the color change is a relatively macroscopic phenomenon.In fact,when the etching has started in the early period,the color of the solution has not changed yet.Therefore,the advantages of real-time,high sensitivity and low detection limit of nanopores can be used for monitoring the early period of etching,providing an ultra-sensitive detection platform for H₂O₂ detection.3.A proposal based on solid-state nanopore platform to study the morphology of enzymatically etched gold nanotriangles(Au NTs),and use horseradish peroxidase to etch the gold nanotriangles into nanodiscs(Au NDs).Further we studied the nanopore translocation process of Au NTs and Au NDs as two different morphologies of gold nanoparticles.Solid-state nanopores are imitated from nanochannels in biological membranes,and can be used to simulate the exchange and signal transduction of internalized substances in cells.From the experimental results in the previous section,the morphology of gold nanoparticles has a obvious influence on the frequency of nanopores translocation.Except the conventional spherical nanoparticles which are used as cell-targeted transport,there are more and more reports of tetrahedral and star-shaped particles which have higher fluxes when entering cells across membranes.The results of this work indicate that the gold nanotriangles have higher flux when transported across the membrane due to their cutting-edge advantages.At the same time,due to the anisotropy of the particles,they have more conformations and dynamics when passing through the nanochannel.The dynamic process is also more diverse than round particles,which has a certain guiding significance for the current geometric shapes of nanoparticles and viruses to infect cells-targeted therapies,leaving a large stage for the expansion and application of biological experiments in the future.