Nanostructured separation and analysis devices for biological membranes

Abstract

The present invention provides a nanostructured device comprising a substrate including nanotroughs therein; and a lipid bilayer suspended on or supported in the substrate. A separation method is also provided comprising the steps of supporting or suspending a lipid bilayer on a substrate; wherein the substrate comprises nanostructures and wherein the lipid bilayer comprises at least one membrane associated biomolecule; and applying a driving force to the lipid bilayer to separate the membrane associated biomolecule from the lipid bilayer and to drive the membrane associated biomolecule into the nanostructures.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a Continuation-in-Part of and claims priority to U.S. patent application No. 10 / 073,935, entitled “Nanostructured Devices for Separation and Analysis,” filed on Feb. 14, 2002, now U.S Pat. No. 6,685,841 B2 issued on Feb. 3, 2004, which claims priority to U.S. Provisional Patent Application No. 60 / 268,365, entitled “Nanostructured Devices for Separation and Analysis,” filed Feb. 14, 2001. This application also claims priority to U.S. Provisional Patent Application No. 60 / 347,002, entitled “Nanostructured Devices,” filed on Jan. 11, 2002. The entire contents and disclosures of the above applications are hereby incorporated by reference.[0002]Notice: more than one reissue application has been filed for the reissue of U.S. Pat. No. 6,913,697 B2. The reissue applications include co-pending U.S. patent application Ser. Nos. 12 / 215,893(the present application), 12 / 217,113, and 12 / 217,114 all of which are Divisional Reissue Ap...

AI Technical Summary

Benefits of technology

[0015]It is therefore an object of the present invention to provide an efficient nanostructured matrix for separation and analysis of molecules.

[0016]It is a further object of the present invention to provide a matrix that enables gradient or non-uniform transport of molecules across a plane parallel to the surface of the matrix.

[0019]It is yet another object of the present invention is to provide a nanostructured matrix that may cater to different ranges of molecular separations, in terms of resolution and dynamics.

[0020]Another object of the present invention is to enable consistency in the composition of the nanostructures forming the separation matrix.

[0024]A further object of the present invention is to enable parallel production of separation matrices at relatively low cost.

[0025]In all of the above embodiments, it is an object to provide enhanced reproducibility and resolution in the separation of molecules.

Problems solved by technology

However, the set of separation strategies that rely on this technique are hampered by: (1) inconvenience of preparation of the variety of gels needed for the separations, (2) inherent inconsistencies in production conditions; and therefore, irreproducibility between different batches of gels, (3) susceptibility of the polymer to degradation under high electric fields, (4) lack of reusability, (5) difficulty in incorporation of these techniques into strategies for development of multi-dimensional (multi-technique) integrated separation systems, and (6) limited resolution and dynamic range of biomolecular separations.

However, their utility is greatly hampered by the need for cumbersome gel preparation protocols and lack of reproducibility.

These currently available systems, however, suffer from a number of drawbacks: (1) the matrices formed are generally composed of non-uniform structures, (2) even where a gradation in size of structures is required, they may be random or at best have to be serially and sequentially arrayed through a cumbersome process of lithography, (3) fabrications of separation devices pose problems in terms of batch-to-batch variations; and consequently, poor reproducibility of results therefrom, (4) lack of efficiency of separation, (5) loss of sample volume, and (6) biomolecules may not be amenable to separation by many of the available systems.

Although such prior work demonstrated that relatively simple 3-dimensional architectures could lead to effective separation, the developments have not gained ground with the biotechnological community.

The primary reasons for this lack of acceptance being the difficulty of preparation of the nanofluidic systems and the associated high-cost of fabrication.

Although these nanolithographic structures are useful in separation, the systems suffer from drawbacks: (1) resolution limitations, (2) flexibility limitations, and (3) difficulty in integrating the system with other, more complex, separation devices.

Thus, the need for an efficient, highly-resolving, flexible, cost-efficient, and reproducible molecular-separation matrix, is largely unmet.

The analysis and characterization of biomolecules is further limited by the difficulty in separating membrane-associated molecules.

Typically, detergents are used to remove transmembrane molecules, but even mild detergents may denature such molecules, rendering them inactive and / or disrupting necessary functional interactions with other membrane components including other proteins or lipid components.

Additionally, the study of biomolecules is limited by the difficulty in fabricating a cellular environment that allows for the interaction of molecules.

However, in these techniques, the close proximity or constraint of the lower leaflet to the supporting surface reduces their usefulness in analyzing transmembrane proteins or interactions between cytoplasmic and extracellular components of the membrane.

Although these types of suspended bilayers have been used for studying membrane permeability and transmembrane protein function, the use of such suspended lipid bilayers in the separation of transmembrane proteins has not been examined.

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