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Improved Detection of Native Conform...

Acharya, Shiv Jagat.

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Improved Detection of Native Conformation Proteins with Hydrogel Interfaced Nanopores.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Improved Detection of Native Conformation Proteins with Hydrogel Interfaced Nanopores./
Author:	Acharya, Shiv Jagat.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	152 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-04, Section: B.
Contained By:	Dissertations Abstracts International81-04B.
Subject:	Biomedical engineering. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=22582507
ISBN:	9781085692175

Improved Detection of Native Conformation Proteins with Hydrogel Interfaced Nanopores.
Acharya, Shiv Jagat.

Improved Detection of Native Conformation Proteins with Hydrogel Interfaced Nanopores. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 152 p.

Source: Dissertations Abstracts International, Volume: 81-04, Section: B.

Thesis (Ph.D.)--University of California, Los Angeles, 2019.

This item must not be sold to any third party vendors.

Nanopores are powerful sensors capable of directly measuring the physical characteristics of single molecules through electrical measurement. Native proteins are particularly interesting targets because of their relevance in medicine and biology. However, theoretical and experimental work has shown that proteins translocate through pores too rapidly to be resolved accurately, resulting in misleading attenuated blockade amplitudes, reduced event frequencies, and incomplete dwell time distributions. Here, it is shown that a hydrogel placed on the distal side of a nanopore can sufficiently increase the residence time of proteins to be resolved accurately by typical measurement apparatus. Measurements of proteins with hydrogel backed nanopores are shown to have event frequencies that exceeded theoretical predictions at concentrations multiple orders of magnitude below those used in typical protein studies, dwell time distributions that extend to the millisecond time scale, and event amplitudes that correctly predict the volume of proteins. It is demonstrated that IgG and BSA can be measured simultaneously in mixture and current blockades can inform on the shape of a protein as a result of the improved detection. Analytical and computational models are used in conjunction with experimental data to investigate the mechanism of capture, indicating that IgG and BSA do not penetrate the hydrogel, but instead escape by diffusion back into the cis chamber. The residence time is also shown to be much more sensitive to the diffusivity and electrophoretic mobility of the protein as a result of this capture mechanism. Lastly, a method to analyze nanopore measurements is presented using hidden Markov models and a modified Canny edge detection algorithm. The method addresses difficulty in analyzing data that shows significant flicker or capacitive noise or has long duration events, such as data produced by hydrogel facilitated detection of protein. The methods shown here serve to introduce interfacing hydrogels with solid state nanopores as an effective, amenable, and robust approach to addressing issues in protein detection related to short dwell times.

ISBN: 9781085692175Subjects--Topical Terms:

535387
Biomedical engineering.
Subjects--Index Terms:

Biosensors

Improved Detection of Native Conformation Proteins with Hydrogel Interfaced Nanopores.
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300 $a 152 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-04, Section: B.
500 $a Advisor: Schmidt, Jacob J.
502 $a Thesis (Ph.D.)--University of California, Los Angeles, 2019.
506 $a This item must not be sold to any third party vendors.
520 $a Nanopores are powerful sensors capable of directly measuring the physical characteristics of single molecules through electrical measurement. Native proteins are particularly interesting targets because of their relevance in medicine and biology. However, theoretical and experimental work has shown that proteins translocate through pores too rapidly to be resolved accurately, resulting in misleading attenuated blockade amplitudes, reduced event frequencies, and incomplete dwell time distributions. Here, it is shown that a hydrogel placed on the distal side of a nanopore can sufficiently increase the residence time of proteins to be resolved accurately by typical measurement apparatus. Measurements of proteins with hydrogel backed nanopores are shown to have event frequencies that exceeded theoretical predictions at concentrations multiple orders of magnitude below those used in typical protein studies, dwell time distributions that extend to the millisecond time scale, and event amplitudes that correctly predict the volume of proteins. It is demonstrated that IgG and BSA can be measured simultaneously in mixture and current blockades can inform on the shape of a protein as a result of the improved detection. Analytical and computational models are used in conjunction with experimental data to investigate the mechanism of capture, indicating that IgG and BSA do not penetrate the hydrogel, but instead escape by diffusion back into the cis chamber. The residence time is also shown to be much more sensitive to the diffusivity and electrophoretic mobility of the protein as a result of this capture mechanism. Lastly, a method to analyze nanopore measurements is presented using hidden Markov models and a modified Canny edge detection algorithm. The method addresses difficulty in analyzing data that shows significant flicker or capacitive noise or has long duration events, such as data produced by hydrogel facilitated detection of protein. The methods shown here serve to introduce interfacing hydrogels with solid state nanopores as an effective, amenable, and robust approach to addressing issues in protein detection related to short dwell times.
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