Detecting and Monitoring the Formation of Biological Nanoassemblies with Resistive-Pulse Sensing.

Resistive-pulse Sensing;Nanopore;Immunoassay;Coulter Counting;Noise;Submicrometer Pore;Biomedical Engineering;Engineering;Biomedical Engineering

Uram, Jeffrey DanielWise, Kensall D. ;

University of Michigan

关键词: Resistive-pulse Sensing;    Nanopore;    Immunoassay;    Coulter Counting;    Noise;    Submicrometer Pore;    Biomedical Engineering;    Engineering;    Biomedical Engineering;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/57599/uramj_1.pdf?sequence=2&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

This thesis describes innovative applications for resistive-pulse sensing with submicrometer pores and discusses the noise and bandwidth characteristics of the experimental setup.In the initial study, resistive-pulse sensing was used to monitor the formation of antibody-antigen complexes (immune complexes or biological nanoassemblies).The developed technique was rapid (detection in ≤ 15 minutes), label-free, could be performed in small volumes (≤ 40 μL), and required no immobilization of the antibody or antigen.This assay was able to detect purified antigens at concentrations as low as 30 nM, and to detect antigens in complex media such as serum.It also enabled the characterization of the time course of immune complex formation and growth with a precision that made it possible to detect single complexes.In the second study, resistive-pulse sensing was used to characterize and quantify antibody-virus interactions.These experiments demonstrated that resistive-pulse sensing can be used to detect a specific virus or a virus-specific antibody in solution, probe the ability of an antibody to immunoprecipitate the virus, determine the average number of antibodies bound to virus particles, and monitor the time course of the assembly of antibodies onto viruses in situ.The third study developed theory for extracting thermodynamic parameters of antibody-antigen interactions from resistive-pulse data.A model system presented in the literature, antibodies binding to spherical nanoparticles that expose antigens, was used to validate the theory; the calculated solid phase affinity constant of the antibody (2.6x10^8 ± 0.8x10^8 M^-1) was in agreement with the specifications of the supplier of the antibody.The fourth and final study examined in detail the theoretical and experimental noise and bandwidth of current recordings from resistive-pulse sensing experiments. The theory presented in this study combined with its experimental validation enables the development of resistive-pulse sensing systems optimized for low-noise (high sensitivity) and high-bandwidth (high accuracy).The experiments presented here demonstrate that resistive-pulse sensing is a simple, yet powerful technique for examining the formation of biological nanoassemblies.Based on these findings, resistive-pulse sensing holds great promise as a tool for nanotechnology and for use in portable or high-throughput assays.