The study of single molecule protein biophysics using a solid-state nanopore

Kevin J. Freedman

doi:10.17918/etd-7020

The kinetics of protein folding and binding has been studied for several decades and continues to reveal links to overall cellular health,cell functionality, and responses to therapeutic agents. Despite numerous methods to purify and detect proteins, there is still a growing need to develop technology that can enhance sensing and reveal potentially hidden properties of proteins. This is important not only from a scientific perspective but also in practically achieving the goals of personalized healthcare. Next generation sensors should ideally have three main characteristics: (1) high resolution sensing, (2) high-throughput sensing, and (3) the potential to be automated and used by untrained personnel. Future devices will revolutionize the healthcare industry by decreasing both the time and cost to do basic scientific research, diagnostic testing, and drug development. A likely candidate for next-generation protein sensing is solid-state nanopores. The pores developed here are fabricated in a 50 nm thick silicon nitride membrane and a single nanopore is drilled using a focused ion beam or a focused electron beam. The detection method employed is largely based on resistive pulse sensing where analytes are electrokinetically transported through a pore and identified by their unique modulation of ionic current (i.e. an ionic blockade). Since the dimensions of the nanopore are on the same scale as the molecule being sensed, only a single molecule can enter the pore allowing individual protein kinetics to be probed. Traditionally proteins are detected by ensemble averaging which hides important kinetics and sub-populations of molecules that may be important to understanding the early stages of a disease or detect a disease early. In the first section of this study, the prominent issue of protein adsorption onto the sensing device (i.e. the nanopore) is addressed and resolved by using a modified voltage protocol. The rationale behind the new sensing scheme is explained in terms of the interplay of diffusive and entropic (barrier-dominated) forces on a protein. In the second section, we discovered that the voltage which drives the protein through the pore also has denaturing effects. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein. In the final section of this study, voltage-mediated unbinding of a single protein complex is studied. We argue that determining the unbinding forces between two proteins adds an additional level of specificity which is needed for eventual use as a diagnostic tool. In this study, nanopores are developed not only as a sensor but also a single molecule or protein complex manipulator that can locally unfold or unbind molecules.

The study of single molecule protein biophysics using a solid-state nanopore

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The study of single molecule protein biophysics using a solid-state nanopore

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