Application Of Label-free Glass Nanopores For Protein Detection

As a powerful tool for single molecule analysis,nanopore has been widely used in the analysis of DNA,peptide nucleic acids,proteins and other molecules,and shows the good performance.However,protein owns complex conformation including different sizes,three-dimensional structures and uneven charge distribution.The speed of protein through the nanopore becomes faster with the decreasing protein size,resulting decreasing signal strength.Therefore,it is urgent to improve the signal-to-noise ratio(SNR)in the nanopore detection of protein.Herein,molecularly imprinted nanoparticle with low cost and high selectivity was used as a carrier to improve the selectivity and SNR in the nanopore detection.In addition,a thermally responsive hydrogel-filled nanopore is proposed to further improve the SNR of nanopore detection by reducing the protein translocation speed and the effective pore size.The main contents and results are as follows:(1)Combining glass nanopore technology and molecularly imprinted technology,a sensing platform for high selectivity of bovine serum albumin(BSA)was built.Experiments were carried out using silicon dioxide(Si O₂)nanoparticles as the carrier,BSA as the target to get molecularly imprinted polymer coated Si O₂nanoparticles(MIP/Si O₂).Based on the specific adsorption of MIP/Si O₂to BSA,the nanoparticles at different imprinting stages can be distinguished due to the difference in volume and surface charge when they pass through the nanopores under the effect of applied potential and electroosmotic flow in turn,achieving high selective and sensitive detection of BSA in complex samples.The linear range and the limit of detection of this method are 0.5?50 nmol L^-1and 0.3 nmol L^-1respectively.This sensor can also be used for the detection of BSA in real samples.The strategy presented in this work opens up new application fields for nanopore sensing.(2)To solve the problem of low SNR in protein detection,thermally responsive hydrogel-filled nanopores were prepared.The thermally responsive hydrogel effectively reduces the ion flux of the nanopores,and it interacts with BSA molecules,which can increase the translocation resistance of BSA,reduce the translocation speed of BSA,and enhance the signal-to-noise ratio of the translocation signal.By controlling the temperature to control the release of BSA,resistive-pulse method was used to record the real-time release of BSA.The translocation behavior of BSA was analyzed from the obtained biphasic-pulse signal,which provided a new idea for the use of conventional nanopore real-time monitoring and controlled protein molecule release.