Monitoring Protein Adsorption with Solid-state Nanopores

Summary

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Abstract

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ^1-6, the unfolding of proteins ⁷, and binding affinity of different ligands ⁸. By coupling these nanopores to the resistive-pulse technique ^9-12, such measurements can be done under a wide variety of conditions and without the need for labeling ³. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions ¹³. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

Protocol

1. Manufacture of solid-state nanopores in silicon nitride membranes

1. Bring the FEI Tecnai F20 S/TEM to an acceleration voltage of 200 kV. If using a different S/TEM, the acceleration voltage should be greater than or equal to 200 kV ⁹
2. Load a 20 nm thick SPI silicon nitride window grid into the TEM sample holder and clean with Oxygen plasma for 30 seconds to remove any contaminants from the holder.
3. Load the sample into the S/TEM and allow for the vacuum to pump down. Once the S/TEM has pumped down to vacuum, find the nitride window in bright-field TEM mode by looking for a bright square on the Ronchigram. Make sure the TEM is ....

Discussion

Spontaneous adsorption of proteins onto solid-state surfaces ^27-29 is fundamentally important in a number of areas, such as biochip applications and design of a new class of functional hybrid biomaterials. Previous studies have shown that proteins adsorbed to solid-state surfaces do not show lateral mobility or significant desorption rates, and therefore protein adsorption is generally considered an irreversible and nonspecific process ^30-32. Protein adsorption onto solid state surfaces is thought t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tecnai F20 S/TEM	FEI		S/TEM requires acceleration voltage ≥200kV and field-emission source.
20 nm thick silicon nitride membrane window for TEM	SPI Supplies	4163SN-BA
Axon Axopatch 200B patch-clamp amplifier	Molecular Devices
Axon Digidata 1440A	Molecular Devices
pCLAMP 10 software	Molecular Devices		Electrophysiology Data Acquisition and Analysis Software
Sulfuric Acid	Fisher Scientific	A300
hydrogen peroxide	Fisher Scientific	H325
silicone O-rings	McMaster-Carr	003 S70	Alternatively use PDMS
silver wire	Sigma-Aldrich	348759	For electrodes
SPC Technology, D sub contact, pin	Newark Inc	9K4978	For electrodes
potassium chloride	Sigma-Aldrich	P9541
potassium phosphate dibasic	Sigma-Aldrich	P2222
potassium phosphate monobasic	Sigma-Aldrich	P5379
PDMS	Dow Corning		Sylgar 184 Elastomer set. For making chamber.
Kwik-Cast Sealant	World Precision Instruments, Inc.	KWIK-CAST	Fast acting silicone sealant
hot plate	Fisher Scientific
Faraday cage