Method for Identifying and Quantifying Organic and Biochemical Substances

• US 20100184062A1
• Filed: 07/04/2008
• Published: 07/22/2010
• Est. Priority Date: 07/04/2007
• Status: Abandoned Application

• Current Assignee: Roswell Biotechnologies Inc.
• Original Assignee: Rho-Best Coating Hartstoffbeschichtigungs GmbH
• Inventors: Steinmüller, Detlef, Roppert, Kriemhilt, Köck, Anton, Steinmüller-Nethl, Doris
• Application Number:
  US12/667,583
• Publication Number:
  US 20100184062A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C08G 71/02 Polyureas
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2537/162 Helper probe
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

The invention relates to a method for identifying organic or biochemical substances and for determining their concentration in a fluid medium using a nanogap sensor that comprises at least two electrodes. The invention is characterized in that: a nanogap sensor) with electrodes of different materials is used, a respective probe molecule is bonded to each surface of the two electrodes of the sensor and the free remainder of the probe molecules have at least one bondable group with specificity for bonding to a sought substance or to an analyte molecule in the fluid medium. The analyte molecule has at least two binding sites and passes selectively out of the fluid medium in which it is contained, binds to the free ends of the probe molecules, forming a bridge with the probe molecule, and modifies the resulting impedance between the electrodes. The concentration of the substance in the fluid medium can be determined as a result of the modification.

Claims

1-10. -10. (canceled)

11. A method for identifying organic and biochemical substances, said substances being molecules, molecule sequences, molecule parts or similar molecular substances, and for determining the quantity or concentration of said substances in a fluid, said fluid being a liquid or gaseous medium, wherein a nanogap sensor having at least two electrodes is used for said identifying method, said method comprising:

a) providing a nanogap sensor, said sensor has at least two electrodes separated from one another by an electrically isolating layer or by a non-material gap, said electrodes being formed of materials being different from one another, said electrodes are electrically conductive and/or principally semi-conductive, wherein said conductivity is a relatively high conductivity with regard to conductivity of a semiconductor;

b) providing on a surface of a first electrode of said sensor, a first affinity or probe molecule, said first affinity molecule is oriented in at least partial longitudinal orientation in relation to said first electrode, said first affinity molecule has a sensor-binding area wherein said sensor-binding area is specifically or individually, respectively, sensor-bound to the material of said first electrode at one of its ends or in the proximity of one of its ends, and said sensor binding area is immobilized on said first electrode, wherein a free residue of said first affinity molecule has at least one free binding or bondable group, molecule sequences or similar structure providing suitable affinity binding sites which have at least one certain specificity for bonding to a sought substance, said sought substance being an analyte, analyte molecule or auxiliary molecule;

C) providing on the surface of said second electrode of said nanogap sensor, a second affinity or probe molecule said second affinity molecule is oriented in at least partial longitudinal orientation in relation to said second electrode wherein said second affinity molecule orientation differs in relation to said first affinity or probe molecule orientation, said second affinity molecule having a binding area that is specifically or individually sensor-bound to the material of said second electrode, the material of said second electrode differs from the material of said first electrode said binding area being at or in proximity to one end of said second electrode, and said binding area being immobilized on said second electrode, wherein said second probe molecule has a free residue and an affinity binding site having at least one free binding or bondable group, molecule sequences or similar structure having at least certain specificity for bonding to a sought substance said sought substance being an analyte, analyte molecule, or auxiliary molecule;

d) providing a fluid medium) to be checked by said method, said fluid medium having various molecules, molecule sections, molecule parts, molecule sequences, or combinations thereof flowing around each of said electrodes and said gap, there between, said fluid medium having an analyte molecule or auxiliary molecule to be detected in its quantity and/or concentration, said analyte molecule or auxiliary molecule is formed by a known molecule, known molecule section, or known molecule part, known molecule sequence having any shape, said electrodes having at least two binding sites at a distance from one another for said sensor-bound, immobilized probe molecules or for mobile free ends having free affinity binding sites;

e) selectively passing said fluid medium to be analyzed between said electrodes, wherein said electrodes have exposed binding sites arranged at exposed points, at said binding sites on ends or in proximity to ends of said electrodes;

f) forming a bond with specific binding or bondable groups, molecule sequences or combinations thereof with binding or bondable, groups, molecule sequences representing affinity binding sites respectively, at free mobile ends of said first and second affinity or probe molecules wherein each of said first and second probe molecules have specifically sensor-bound sites attached to said electrodes, each of said electrodes being formed of different materials,

g) forming a bridge molecule or bridge whereby said two electrodes of different materials are connected with one another, whereby said connection is by said probe molecules and said analyte molecule or auxiliary molecule respectively bridging said isolator layer or gap between said two electrodes of different materials;

h) alternatively, containing an auxiliary molecule in an existing bridge, binding said auxiliary molecule to said two probe molecules whereby said binding is by sensor-bound binding with sensor binding sites on said electrodes whereby said auxiliary molecule is separated from at least one of said probe molecules and bound to said electrodes by the interaction of an analyte molecule contained in said fluid medium said analyte molecule being capable of binding to at least one binding site of said auxiliary molecule with a resultant bond being stronger than at least one of said binding groups between said auxiliary molecule and at least one of said two probe molecules;

i) dissolving a previously existing bridge, and

j) detecting modification or change of impedance or of a frequency spectrum of an alternating current applied to said two electrodes, occurring during the bridge formation or the bridge dissolution process, the presence, the quantity or concentration of the sought molecule is determined, wherein in case of a bridge formation, the absence of the sought analyte molecule at the electrodes is present before bridge molecule formation and the presence of said sought analyte molecule at the electrodes present following the bridge formation;

or in bridge dissolution, a selective connection of said analyte molecule to at least one component of a bridge pre-manufactured using an auxiliary molecule resulting in said modification of said analyte molecule having a direct or indirect connection with said sensor surface or dissolving said connection following a reaction.

12. The method according to claim 11 further comprising dissolution of a bridge formed with said probe molecules bound to each of said two electrodes with said auxiliary molecule bilaterally bound via affinity binding sites with a DNA sequence strand, said dissolution is executed by supplying an analyte molecule, said analyte molecule being piece of complementary DNA sequence attachable to said auxiliary molecule and highly bondable to the same, using the fluid medium which analyte molecule binds to said auxiliary molecule to a respective DNA sequence strand, and forming a double-molecule DNA double-strand, which ultimately migrates into the fluid medium, said dissolution resulting in said two affinity bonds to said two probe molecules sensor-bound to said electrodes of different materials being dissolved.

13. The method according to claim 11 further comprising a dissolution of a bridge formed with said probe molecules and sensor-bound to each of said two electrodes with said auxiliary molecule bound via affinity binding sites on a DNA sequence strand and executing said dissolution using said analyte molecule present in the fluid medium having a molecule group attachable and bondable to only one of two exposed binding sites or groups of said auxiliary molecule initially bound to said affinity binding or binding groups of said two immobilized probe molecules sensor-bound to said two electrodes of different material, said method dissolving only one of said two affinity bonds with one of said two sensor-bound probe molecules and thus dissolving the bridge resulting in binding to one of said two exposed binding sites or binding groups of said auxiliary molecule.

14. The method according to claim 11 wherein said nanogap sensor has distance or thickness, between said two electrodes up to 500 nm.

15. The method according to claim 11 wherein said nanogap sensor is used, in which the distance between said two electrodes is formed by a layer of a solid or liquid dielectric material, said dielectric material being inorganic isolator materials from the fields of microelectronics or field-effect transistor applications being oxides, nitrides and/or chalcogenides, silicon oxide, silicon nitride, aluminum oxide, zirconium oxide, silicone nitride, tantalum pentoxide or thin-layer films of various origins, like in particular Langmuir-Blodgettfilms, polyelectrolyte multi-layers and self-organized monolayers of different materials and material combinations, as well as diverse polymers, which compared to the lower conductivity of the two measuring electrodes have a conductivity, which is lower by at least three powers of ten, like for example Kapton®

, Nafion®

or others, which are known to the person skilled in the art, particularly preferred isolation materials routinely used today already or in future, producible in reproducible layer thicknesses within the scope of micro-system technology, like in particular SiO₂ and Si-nitride. In case of completely undercut nano-belts, the isolating layer may likewise be identical with the electrolyte.

16. The method according to claim 11 wherein said nanogap sensor is used, said two electrodes formed of property-determining material respectively be formed of a combination of any two of the materials comprising metals, gold, platinum, silver, mercury;

doped semiconductors, silicon and germanium;

HI-V or M-VI semiconductors GaAs, CdS, CdSe, CdTe;

carbonaceous layers of graphite, fullerenes, nano-tubes, diamond-like carbon, diamond in various versions, e.g. as mono-crystal, micro-crystalline, nano- or ultra-nano-crystalline materials, as well as material combinations, alloys, including doping, or further electrode materials known per se, like in particular gallium nitride, SiC (silicon carbide), AlN (aluminum nitride), ATO or ITO, wherein a combination of two highly doped non-metals is particularly preferred, in this connection particularly preferred a combination of highly doped, almost metallically conductive silicon with highly doped diamond, in particular UNCD (ultra-nano-crystalline diamond).

17. The method according to claim 11 for the verification of a biochemical process, in which affinity bonds of said auxiliary molecule to said two probe molecules for bridging said two electrodes are dissolved, but said bridge-forming auxiliary molecule is destroyed at least one random point, for the detection of DNases or proteolytically effective enzymes.

18. The method according to claim 11 for the verification of a biochemical process, in which different biomolecules serve as at least one of said affinity or probe molecules said affinity molecule being antibodies, which via said or using said, analyte molecule cause the formation of a bridge, or nucleic acid sequences, which by means of hybridization cause bridging of said electrodes of different materials, wherein the possibility of linker sequences and spacers, which guarantee an increased mobility of said probe molecules compared to the surfaces of said electrodes of different materials, is included.

19. The use of the method according to claim 11 for the verification of a biochemical process, in which artificially generated analogues of biomolecules, like e.g. PNAs and LNAs, are used as said affinity or probe molecules.

20. The use of the method according to claim 11 for the verification of a biochemical process, in which by means of a continuous chemical reaction, by means of polymerase chain reaction (PCR), a band between said two electrodes of different materials or between said two probe molecules and unilaterally sensor-bound to these, respectively, is linked or destroyed, which is used for detection.

21. The method according to claim 14 wherein said nanogap sensor has distance or thickness, between said two electrodes up to 200 nm.

22. The method according to claim 14 wherein said nanogap sensor has distance or thickness, between said two electrodes in the range from 20 to 70 nm.