Polymer Nanochannel-based Nanodevices For Single-molecule Studies

Nanochannel single molecule sensing is an analytical technique with high throughput,sensitivity and without labeling or amplification.Solid nanochannel has been widely used in DNA sequencing,protein conformational analysis,biological macromolecules interaction,enzyme kinetics studies and single molecule detection of a variety of species,owing to its advantages such as good mechanical properties,adjustable in size,easy to modify and integrate.It is currently the most promising single molecule detection technology and is expected to become an important means of precision medical.However,this technology still faces a series of big challenges in the aspects of accurate preparation,signal-to-noise ratio(SNR),molecular translocation speed control,sensitivity and selectivity.Based on these problems,this paper systematically studied the main factors and the affecting laws of the properties and rectification effect of the nanochannel,for the purpose of realizing the accurate preparation and understanding the regulation mechanism of the properties of the nanochannel.Taking a polymer nanochannel as the sensor,we successfully realized the removal of heavy metal ions and highly sensitive sensing of the small size analyte such as lysozyme protein and the circulating tumor DNA,and systematically described the dynamic process of the analyte in the nanochannel as well as the generation and regulation mechanism of the corresponding pulse signal.The specific research content is as follows: 1.The preparation of polymer nanochannel and the regulation of its propertiesInspired by living organisms in the ion channels,this paper had prepared a conical nanochannel on the polyethylene terephthalate(PET)backing material by an asymmetric track etching method.The pore size of the nanochannel was fine-tuned by two-step etching method.The cross-membrane current was monitored by a picoammeter to realize the controllable and repeatable preparation of the nanochannel.The effects of the size,surface charge density and electrolyte solution concentration on the ion transport properties and the ion rectification characteristics of the nanochannels are systematically studied in this paper.In addition,based on the chelation between PEI and heavy metal ions,the polymer nanochannel modified by PEI was further prepared.Taking the ion rectification curve as the output signal,we constructed a nanochannel sensor in response to heavy metal ions to realize the removal of heavy metal ions at the nanoscale,which has a certain guiding significance toward effectively removing the excess heavy metal ions in the living body.2.Construction and Application of Nanopore Biphasic-pulse BiosensorWe report a simple and efficient method for small-sized protein detection by constructing biphasic-pulse nanopore biosensor.The generation,regulation and mechanism of biphasic pulse signals are systemically investigated in PET nanopore by using lysozyme as a translocating molecule.Experiments have demonstrated that the size of nanopore,the charges of translocating molecule and nanopore material are especially important for making biphasic-pulse nanopore.According to the instruction of typical PET-lysozyme nanopore model,other biphasic-pulse nanopores can be achieved by designing custom nanopore using diffident materials and molecules.Focusing on the problems of fast translocation of the analyte in the nanochannel and low resolution as well as signal-to-noise ratio,conical PET nanochannels of the size and surface charge properties of which well matched with the analyte was prepared.Based on the regulation of surface charge polarity and charge density of the nanochannel and the lysozyme,the translocation velocity of protein molecules and the characteristic signals of translocation events can be effectively adjusted.As expected,distinct and regular biphasic-pulses were observed on the recorded current-time trace.Unlike the traditional resistive pulse sensing,the biphasic-pulse event can provide unique and abundant fingerprint information.The biphasic-pulse nanopore as a detector possessed high sensitivity and selectivity duo to good signal-to-noise ratio,the resolution and signal reproducibility.And it is expected to be developed as a single molecular sensor,with potential application in the detection and diagnosis of protein-related diseases.3.Hybridization Chain Reaction(HCR)for Amplifying Nanopore SignalsWe have successfully constructed HCR-nanopore sensing system and applied in the sensitive detection of ct DNA.The HCR can be harnessed in solid nanopore sensing,overcoming the limitation that has precluded the short DNA or small target.Here,we combine hybridization chain reaction(HCR)with nanopore detection to translate the presence of a small DNA target to characteristic nanopore signals of a long nicked DNA polymer.The amplification of nanopore signals obtained by HCR not only overcomes the functional limitation of solid nanopore,but also significantly elevates both selectivity and signal-to-noise ratio.Moreover,this method exhibits an excellent selectivity between perfectly matched target sequences and single-base mismatched sequence.This method is expected to be developed as a general detection method for the detection and analysis of nucleic acids,proteins and small biological molecules,and has a broad application prospect in clinical medical diagnosis and analysis.4.Ultrasensitive Nanopore Sensing using Dielectrophoretic(DEP)TrappingBy combination the dielectrophoresis trapping with the nanopore technology,the enrichment and detection efficiency of DNA was significantly improved by introducing sucrose into KCl solution.In this experiment,since the DEP force can be controlled by simply tuning the frequency of the applied AC voltage,the effect of frequency on DNA trapping was investigated.The results showed that the event frequency of DNA translocation is raised 5 times than that without dielectrophoresis trapping.