Assay Chip, and Uses of Said Assay Chip to Determine Molecular Structures and Functions

• US 20070275480A1
• Filed: 11/15/2004
• Published: 11/29/2007
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Leister Process Technologies, Paul Scherrer-Institut (Schweizerische Eidgenossenschaft)
• Original Assignee: Leister Process Technologies, Paul Scherrer-Institut (Schweizerische Eidgenossenschaft)
• Inventors: Tiefenauer, Louis, Brander, Karl
• Granted Patent: US 8,105,820 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

An assay chip for investigation the functionality of membrane proteins and their interactions with molecules includes a nanopore array having a plurality of nanopores in a suitable support layer deposited on the nanopore substrate and being a substantially planar support layer having a plurality of nanopores corresponding to the nanopores of the nanopore substrate. The chip includes further a biologically effective layer capable to host at least a non-lipid molecule or functional molecule, deposited on the support layer and covering the plurality of nanopores, resulting in accesible nanopores from both sides of the biologically effective layer for measurement or imaging. The structured support allows generating a biologically effective membrane, such as a lipid bilayer membrane, having high and reliable stability in a manner that its fluidity is sustained in order to keep integrated membrane proteins in the lipid bilayer in its full biological functionality.

Claims

1. An assay chip for investigation of a functionality of non-lipid molecules and their interactions with molecules, comprising:

a) a nanopore substrate having a plurality of nanopores;

b) a substantially planar support layer deposited on said nanopore substrate and having a plurality of nanopores corresponding to said nanopores of said nanopore substrate;

c) a biologically effective layer configured to host at least one of a non-lipid molecule and functional molecule, deposited on said support layer and covering the plurality of nanopores, resulting in accessible nanopores from both sides of the biologically effective layer for measurements.

2. The assay chip according to claim 1, wherein a surface of the support layer is chemically modified by at least one of activated hydrophobic and hydrophilic silanes resulting in a support promotion layer.

3. The assay chip according to claim 1, wherein the support layer is selected from the group consisting of silicon nitride (Si₃N₄) and silicon oxide substrate, and wherein the substrate is selected from the group consisting of silicon and carbon containing materials, polymers, metals, dielectrica, glass and ceramics.

4. The assay chip according to claim 1, wherein a thickness of the substrate and a diameter of the nanopores is chosen in order to have an aspect ratio in the range of 0.25 to 5.

5. The assay chip according to claim 4, wherein the diameter of the nanopores is in the range of 50 to 2000 nm.

6. The assay chip according to claim 1, wherein said nanopores are arranged in a plurality of nanopore array sections having an area in the range of 1×

10<sup>−

6</sup> mm² to 1 mm² on a total free standing silicon nitride membrane area of 1×

10<sup>−

6</sup> mm² to 10 mm².

7. The assay chip according to claim 1, wherein said nanopores have a distance from each other in the range of 0.5 to 5-times of their diameter.

8. The assay chip according to claim 1, wherein the biologically effective layer is a biomembrane isolated from one of prokaryotic and eukaryotic cells, and wherein a lipid bilayer is formed by preparation and later fusion of lipid vesicles or is a functional layer of supramolecular assembly.

9. The assay chip according claim 1, wherein the non-lipid molecules are from a natural source selected from the group consisting of eukaryotes and prokaryotes.

10. The assay chip according to claim 1, wherein the biologically effective layer hosts a non-lipid molecule, and wherein

the non-lipid molecule is a synthetic compound.

11. The assay chip according to claim 8, wherein the biomembrane and the lipid bilayer each comprise at least one of a non-lipid and functional molecule, whereby the functional molecule is produced using one of recombinant DNA and RNA technologies.

12. The assay chip according to claim 8, wherein the biologically effective layer is made from at least one intact living cell.

17. The assay chip according to claim 4, wherein the diameter of the nanopores is chosen in order to result with an aspect ratio in the range of 0.75 to 2.

18. The assay chip according to claim 5, wherein the diameter of the nanopores is in the range of 100 to 2000 nm.

19. The assay chip according to claim 7, wherein said nanopores have a distance from each other in the range of 0.8 to 2-times of their diameter.

13. A process for analyzing a functionality of at least one of a non-lipid molecule and functional molecule, being integrated in a biologically effective layer of an assay chip comprising a nanopore substrate having a plurality of nanopores, a substantially planar support layer deposited on said nanopore substrate and having a plurality of nanopores corresponding to said nanopores of said nanopore substrate, and a biologically effective layer configured, the process comprising:

a) applying a fluid containing a binding compound to one side of the biologically effective layer in order to allow the binding compound to interact with the non-lipid molecule;

b) monitoring the response of the non-lipid molecule induced by at least one of effector binding and an interaction of binding molecules in the biologically effective layer by measuring physical or chemical changes on cis- or trans-sides of the assay chip.

14. (canceled)

15. (canceled)

16. (canceled)