U.S. patent application number 12/813494 was filed with the patent office on 2010-09-30 for immobilizing molecules on a solid support.
This patent application is currently assigned to TACNIA PTY LTD. Invention is credited to Daniel Luke Johnson, Lisandra Lorraine Martin.
Application Number | 20100249375 12/813494 |
Document ID | / |
Family ID | 31983136 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100249375 |
Kind Code |
A1 |
Martin; Lisandra Lorraine ;
et al. |
September 30, 2010 |
IMMOBILIZING MOLECULES ON A SOLID SUPPORT
Abstract
A method for selectively orienting molecules on a surface of a
solid support. The method includes: (a) attaching a linker molecule
to the surface of the solid support, the linker molecule including
a head group that is capable of binding to the solid support, and a
tail group that is capable of chelating to a metal ion; (b)
subsequently treating the solid support with a solution containing
the metal ion; (c) attaching a metal ion chelating tag to the
molecules to form tagged molecules; and (d) capturing the tagged
molecules on the solid support by contacting it with the tagged
molecules to form a monolayer of molecules on the surface of the
solid support in which a majority of the molecules are held in the
same orientation with respect to the surface.
Inventors: |
Martin; Lisandra Lorraine;
(Toorak, AU) ; Johnson; Daniel Luke; (Unley,
AU) |
Correspondence
Address: |
Virtual Law Partners LLP
555 Bryant Street, Suite 820
Palo Alto
CA
94301
US
|
Assignee: |
TACNIA PTY LTD
Adelaide
AU
|
Family ID: |
31983136 |
Appl. No.: |
12/813494 |
Filed: |
June 10, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11327660 |
Jan 6, 2006 |
7759114 |
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12813494 |
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PCT/AU04/00923 |
Jul 8, 2004 |
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11327660 |
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Current U.S.
Class: |
530/345 ;
530/402 |
Current CPC
Class: |
B82Y 30/00 20130101;
Y10S 514/836 20130101; B05D 1/185 20130101; B01J 2219/00626
20130101; B82Y 40/00 20130101; C40B 40/10 20130101; G01N 33/54353
20130101; B01J 2219/00612 20130101; C40B 70/00 20130101; B05D 3/105
20130101; B01J 2219/00605 20130101; B01J 2219/00653 20130101; B01J
2219/0075 20130101; B01J 2219/00725 20130101; B01J 2219/00745
20130101; C40B 40/18 20130101; B05D 2202/40 20130101; B01J
2219/00572 20130101; B01J 2219/00637 20130101; B01J 2219/0061
20130101; B01J 2219/00738 20130101 |
Class at
Publication: |
530/345 ;
530/402 |
International
Class: |
C07K 1/04 20060101
C07K001/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2003 |
AU |
2003903504 |
Claims
1. A method for selectively orienting and immobilizing molecules on
a surface of a solid support, the method comprising: attaching a
linker molecule to the surface of the solid support, the linker
molecule including a head group that is capable of binding to the
solid support, and a tail group that is capable of chelating to a
metal ion; treating the solid support with a solution containing
the metal ion so that the metal ion is chelated to the tail group;
attaching a metal ion chelating tag to the molecules to form tagged
molecules; and capturing the tagged molecules on the solid support
by contacting it with the tagged molecules to form a monolayer of
molecules on the surface of the solid support in which a majority
of the molecules are held in the same orientation with respect to
the surface.
2. The method of claim 1 wherein the tail group of the linker
molecule is a cyclic ligand containing three or more atoms that are
capable of coordinating to the metal ion.
3. The method of claim 2 wherein the tail group of the linker
molecule is a macrocycle that is capable of chelating with the
metal ion.
4. The method of claim 3 wherein the macrocycle is a multidentate
macrocycle.
5. The method of claim 4 wherein the macrocycle is a derivative of
1,4,7-triazacyclononane.
6. The method of claim 5 wherein the macrocycle is
1-acetato-4-benzyl-1,4,7-triazacyclononane.
7. The method of claim 1 wherein the metal ion is selected from the
group consisting of zinc, copper, cobalt, nickel, sodium,
potassium, magnesium, calcium and lithium.
8. The method of claim 1 wherein the tag is a peptide chain
containing one or more chelate forming amino acids independently
selected from the group consisting of cysteine, lysine, histidine,
and arginine.
9. The method of claim 8 wherein the tag is a peptide containing
four to six histidine residues.
10. The method of claim 1 wherein the solid support onto which the
linker molecule is attached comprises a noble metal selected from
the group consisting of silver, gold, platinum and palladium.
11. The method of claim 1 wherein the solid support includes a gold
surface onto which the head group of the linker molecule binds.
12. The method of claim 1 wherein the linker molecule includes a
spacer between the head and tail groups.
13. The method of claim 12 wherein the spacer is a carbon-based
chain between the head and tail groups.
14. The method of claim 13 wherein the carbon-based chain is two to
fifteen atoms in length.
15. The method of claim 14 wherein the carbon-based chain is two to
six atoms in length.
16. The method of claim 1 wherein the head group is sulphur, oxygen
or selenium.
17. The method of claim 1 wherein the molecule is a peptide or a
protein.
18. The method of claim 17 wherein the tag in the tagged molecule
is covalently bound to the protein at a position on the protein
that is separated from the active site or recognition region of the
protein.
19. The method of claim 1 wherein the solid support is an
electrode.
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. A method for selectively orienting and immobilizing molecules
on a solid support having a gold surface, the method comprising:
treating the gold surface with mercaptopropionic acid to form a
mercaptopropionic acid self-assembled monolayer; treating the
mercaptopropionic acid self-assembled monolayer with a tetradentate
derivative of 1,4,7-triazacyclononane to form a macrocycle modified
mercaptopropionic self-assembled monolayer; treating the macrocycle
modified mercaptopropionic self-assembled monolayer with a metal
desirous of octahedral coordination to form a metallo macrocycle
modified mercaptopropionic self-assembled monolayer; and treating
the metallo macrocycle modified mercaptopropionic self-assembled
monolayer with a solution containing a 6*his tagged molecule to
form a highly orientated monolayer of immobilized molecules on the
solid support.
Description
PRIORITY CLAIM
[0001] This application is a divisional application of U.S.
application Ser. No. 11/327,660, field Jan. 6, 2006, which is a
continuation-in-part of International Application PCT/AU2004/000923
having an international filing date of Jul. 8, 2004, which itself
claims the benefit of priority to Australian application
2003903504, filed Jul. 8, 2003, all of which are incorporated
herein by reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to sensor chips of the type
that have a monolayer of molecules immobilised on a substrate
surface. The sensor chips may be used to investigate interactions
between the immobilised molecules and other molecules or to
investigate interactions between the immobilised molecules and the
substrate surface.
BACKGROUND
[0003] The investigation and/or analysis of ligand-molecule
interactions and/or the electrochemical behaviour of biomolecules
are fundamentally important in many fields, including biology,
immunology, chemistry and pharmacology.
[0004] A number of analytical techniques can be used to investigate
ligand-molecule interactions. For example, biological analytes can
be detected or quantified based on ligand-specific binding between
a ligand and a receptor. Common ligand/receptor binding pairs
include antigen-antibody, hormone-receptor, drug-receptor, cell
surface antigen-lectin, biotin-avidin, substrate-enzyme, and
complementary nucleic acid strands. The analyte to be detected may
be either member of the binding pair; alternatively, the analyte
may be a ligand analogue that competes with the ligand for binding
to the receptor.
[0005] Other analytical techniques utilise the oxidation or
reduction of a molecule on the surface of a solid support. For
example, glucose sensors may include an enzyme, such as glucose
oxidase, which converts glucose into reaction products including
hydrogen peroxide. A suitable electrode can then measure the
formation of hydrogen peroxide as an electrical signal. The signal
is produced following the transfer of electrons from the peroxide
to the electrode, and under suitable conditions the enzyme
catalysed flow of current is proportional to the glucose
concentration in a sample. Alternatively, an electrode surface may
be used in combination with current or impedance measuring elements
for detecting a change in current or impedance in response to the
presence of a ligand-receptor binding event.
[0006] Many of the aforementioned analytical techniques involve
immobilising a molecule on a solid support. Other than immobilising
the molecules, the solid support may play no role in subsequent
chemical or biological investigations of the immobilised
molecules.
[0007] Alternatively, the solid support may interact with the
immobilised molecule, such as when the solid support is an
electrode which is used to investigate the electrochemistry of the
molecule.
[0008] A common technique for the immobilization of molecules on
the surface of a solid support is to covalently attach molecules
onto a surface of a solid support that has previously been modified
with an alkanethiol. Covalent attachment of the molecules to the
surface prohibits diffusion of the molecules away from the solid
support and also typically results in the formation of a film of
molecules that is limited to a single monolayer, thereby limiting
the required sample volume. The formation of self-assembled
monolayers ("SAMs") of molecules in this way has enabled the design
of new interfaces for the study of ligand-molecule binding
interactions as well as specific redox-active analytes. For
example, monolayers have been formed via alkanethiol-gold linkage
and related linkages between carboxylates and phosphonates and
metal oxide surfaces. Monolayers formed on gold surfaces are
particularly suited for studying biomolecular recognition at
surfaces because the well-defined structures are amenable to
detailed characterization at a molecular level by using, for
example, scanning tunneling microscopy, atomic force microscopy as
well as other optical and electrochemical bioanalytical
techniques.
[0009] Solid supports having a monolayer of immobilised molecules
are commonly referred to as "chips" or "sensor chips". The sensor
chips are routinely used in biosensor instruments where one or more
properties of the immobilised molecules may be measured. A
representative class of biosensor instrumentation is sold by
Biacore AB (Uppsala, Sweden) under the trade name BIAcore.TM.
(hereinafter referred to as "the BIAcore instrument"). The BIAcore
instrument includes a light emitting diode, a sensor chip covered
with a thin gold film, an integrated fluid cartridge and a
photodetector. Molecules that are receptors of an analyte of
interest are immobilised on the surface of the sensor chip and the
chip is contacted with a flow of sample containing the analyte of
interest. Any change in the surface optical characteristics of the
sensor chip arising from the binding of the analyte of interest are
then measured by detecting any intensity loss or "dip" in light
that is reflected from the gold film on the surface of the sensor
chip.
[0010] Numerous devices for determination of analytes that are
based on the use of sensor chips are now available. However, many
of the available sensor chips have some limitation with respect to
sensitivity, test sample volume, reproducibility, speed of
response, number of effective uses, or the range of detection. In
the clinical setting, it is a goal to maximize the data obtainable
from relatively small test sample volumes during analysis of
fluids.
[0011] The discussion of the background to the invention herein is
included to explain the context of the invention. This is not to be
taken as an admission that any of the material referred to was
published, known or part of the common general knowledge in any
country. Further, throughout this specification reference may be
made to documents for the purpose of describing various aspects of
the invention. However, no admission is made that any document
cited in this specification forms part of the common general
knowledge in the art in any country.
SUMMARY
[0012] The present invention arises out of studies conducted by the
inventors that have shown that by uniformly orienting molecules on
the surface of a solid substrate so that the active site is
uniformly orientated on the surface it is possible to increase the
efficiency of any interaction of the immobilized molecule with a
solution phase or with the solid support. If the active sites or
recognition domains of every immobilized molecule are facing an
appropriate solution phase, then each molecule is capable of
interacting with an appropriate substrate and contributing to the
overall biosensor response. An optimized detection layer can be
expected to display increased sensitivity and reliability,
potentially enhancing the range of suitable applications and
reducing manufacturing costs.
[0013] The present invention provides a method for selectively
orienting and immobilizing molecules on a surface of a solid
support, the method comprising: [0014] attaching a linker molecule
to the surface of the solid support, the linker molecule including
a head group that is capable of binding to the solid support, and a
tail group that is capable of chelating to a metal ion; [0015]
treating the solid support with a solution containing the metal ion
so that the metal ion is chelated to the tail group; [0016]
attaching a metal ion chelating tag to the molecules to form tagged
molecules; and [0017] capturing the tagged molecules on the solid
support by contacting it with the tagged molecules to form a
monolayer of molecules on the surface of the solid support in which
a majority of the molecules are held in the same orientation with
respect to the surface.
[0018] The present invention also provides a sensor chip including
a monolayer of immobilised molecules captured on a surface of a
solid support, wherein a majority of the immobilised molecules are
in the same orientation with respect to the surface.
[0019] The present invention also provides a sensor chip
including:
[0020] a linker molecule attached to the surface of a solid
support, the linker molecule including a head group that is bound
to the solid support, and a tail group that is chelated to a metal
ion;
[0021] a monolayer of molecules on the surface of the solid
support, wherein each molecule contains a metal ion chelating tag
and is held on the surface of the solid support by co-ordination of
the tag with the metal ion such that a majority of the molecules
are held in the same orientation with respect to the surface.
[0022] To determine whether or not "a majority of the molecules are
held in the same orientation with respect to the surface" it is
possible to compare the redox response for molecules oriented on a
solid support using the method of the present invention and
measuring the electrochemical response over increasing scan rates.
The molecules are taken to be oriented if an improved
electrochemical reversibility is observed.
[0023] Preferably, the tail group of the linker molecule is a
cyclic ligand containing three or more atoms that are capable of
coordinating to the metal ion. More preferably, the tail group is a
tetradentate macrocycle that is capable of chelating with the metal
ion.
[0024] The present invention further provides a method for
selectively orienting and immobilizing molecules on a surface of a
solid support, the method including the steps of: [0025] attaching
a linker molecule to a gold surface on the solid support, the
linker molecule including a head group that is capable of binding
to the gold surface, a tetradentate macrocycle tail group that is
capable of chelating to a metal ion and a spacer extending between
the head group and the tail group; [0026] contacting the solid
support with a solution containing the metal ion so that the metal
ion is coordinated with the macrocycle; [0027] attaching a tag that
is capable of coordinating with the metal to the molecules to form
tagged molecules; and [0028] capturing the tagged molecules on the
solid support by contacting it with the tagged molecules such that
the tag is coordinated with the metal ion to form a monolayer of
molecules on the surface of the solid support in which a majority
of the molecules are held in the same orientation with respect to
the surface.
[0029] As a result of the macrocyclic effect, the use of a
macrocycle tail group may provide for tighter binding of the metal
ion to thereby reduce leakage of metal ions from the support. This
ultimately leads to a reduction in "metal bleeding" from the
biosensor. The term "macrocyclic effect" refers to the greater
thermodynamic stability of a complex with a cyclic polydentate
ligand when compared to the complex formed by a comparable
non-cyclic ligand.
[0030] The present invention also provides a biosensor device
including: [0031] a sensor chip of the present invention; and
[0032] a transducer for detecting a change in a parameter in the
immobilized molecules on the sensor chip.
[0033] The present invention also provides a method for increasing
the sensitivity of measurement of a parameter of a molecule that is
immobilized on a solid support, the method including the step of
orienting the molecule on the solid support using the method
described herein.
[0034] The present invention also provides a method for increasing
the reproducibility and/or sensitivity of measurement of an
electrochemical parameter of a redox active molecule that is
immobilized on a solid support electrode, the method including the
steps of: [0035] attaching a linker molecule to a surface on the
solid support electrode, the linker molecule including a head group
that is capable of binding to the solid support, a tail group that
is capable of chelating to a metal ion, and an alkyl chain of more
than two atoms in length between the head group and the tail group;
[0036] treating the solid support with a solution containing the
metal ion so that the metal ion is chelated to the tail group;
[0037] attaching a metal ion chelating tag to the redox active
molecules to form tagged molecules; and [0038] capturing the tagged
redox active molecules on the solid support by contacting it with
the tagged molecules to form an immobilised monolayer of redox
active molecules on the surface of the solid support in which a
majority of the molecules are held in the same orientation with
respect to the surface.
[0039] The present invention also provides a method of determining
the presence of an analyte in a sample, the method including the
steps of: [0040] providing a self-assembled monolayer on a surface
of a solid support, said monolayer comprising linker molecules
having head groups that are bound to the a surface of the solid
support, and tail groups that are chelated to a metal ion; and
analyte binding molecules having a tag that is bound to the metal
ion, said analyte binding molecules oriented uniformly with respect
to the surface of the solid support; [0041] contacting the
self-assembled monolayer having the analyte binding molecule
immobilised thereon with a sample suspected of containing a target
analyte; and [0042] detecting binding of the target analyte with
the analyte binding molecule to thereby determine the presence or
absence of the target analyte.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 is a schematic flow diagram showing a procedure for
producing SAMs according to the methods of the present
invention.
[0044] FIG. 2 shows a plot of wavenumber vs absorbance for a
Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy
study on the surface chemistry of SAMs that were formed according
to the methods of the present invention.
[0045] FIG. 3 shows typical cyclic voltammograms of histidine
tagged thioredoxin obtained at a AcBztacn modified gold electrode
(5, solid line) compared with those obtained when the protein is
covalently bound to the electrode via surface lysine residues at
stage 2 (dashed line). The one-electron reduction of the oxidised
thioredoxin to the radical anion is shown. Scan rates are (a) 0.2
mV/s (b) 1.0 mV/s and (c) 10 mV/s. Current responses are indicated
for each scan rate in 20 mM phosphate buffer (pH 7.4).
[0046] FIG. 4 shows a cyclic voltammogram for histidine tagged
plastocyanin (a) at AcBztacn modified gold/Zn electrode in 0.1 M
phosphate buffer, with a scan rate of 20 mV/s (vs NHE) and
histidine tagged bovine cytochrome P450c17 (b) at AcBztacn modified
gold/Zn electrode in 0.1 M phosphate buffer, and the surfactant
DDAB (didodecylammonium bromide), with a scan rate of 5 mV/s vs
NHE.
[0047] FIG. 5 shows binding of His-tagged thioredoxin, plastocyanin
and green fluorescent protein to Acbztacn- and NTA-modified
surfaces. Protein concentration is 30 .mu.gmL.sup.-1 in 10 mM
HEPES, 150 mM NaCl, pH 7.4.
DESCRIPTION
[0048] Before proceeding with a description of the present
invention, some of the terms that will be used throughout this
specification will now be defined.
[0049] The term "molecule" as used throughout the specification is
to be understood to mean any carbon-based molecule for which a
chemical and/or physical parameters or characteristics can be
measured or determined by capturing the molecule on a solid support
surface. The molecule could be any protein, peptide, nucleic acid,
carbohydrate, lipid or synthetic molecule.
[0050] The term "orientation" when used in the specification in
relation to the placement of a molecule with respect to the surface
of a solid support is to be understood to mean the position and
attitude of the molecule with respect to the solid support
surface.
[0051] The term "immobilized" when used in the specification in
relation to molecules on the surface of a solid support is to be
understood to mean that the molecules are bound to the solid
support under conditions in which the solid support is intended to
be used. Under such conditions, the molecules are not able to
migrate from the surface of the solid support. However, it is to be
understood that the molecules may be reversibly bound so that they
can be removed from the solid support under specific
conditions.
[0052] The term "biosensor" when used in the specification is to be
understood to mean a system, substrate or device that detects a
chemical or biological species with high selectivity on the basis
of molecular recognition. A biosensor uses a biological element,
such as an immobilized peptide, protein, poly- or oligonucleotide
or cell, as a sensor. A biosensor may use specific biochemical
reactions to detect molecules by electrical, thermal or optical
signals. Alternatively, a biosensor may be in the form of a solid
support having a layer of immobilised biological elements for use
in immunoassays, including, but not limited to, solid phase
enzyme-linked immunosorbent assays, radioimmunoassays,
fluoroimmunoassays etc.
[0053] As previously discussed, the present invention provides a
method for immobilizing and selectively orienting molecules on a
surface of a solid support. The method includes the steps of:
[0054] attaching a linker molecule to the surface of the solid
support, the linker molecule including a head group that is capable
of binding to the solid support, and a tail group that is capable
of chelating to a metal ion; [0055] treating the solid support with
a solution containing the metal ion so that the metal ion is
chelated to the tail group; [0056] attaching a metal ion chelating
tag to the molecules to form tagged molecules; and [0057] capturing
the tagged molecules on the solid support by contacting it with the
tagged molecules to form a monolayer of molecules on the surface of
the solid support in which a majority of the molecules are held in
the same orientation with respect to the surface.
[0058] By selectively orienting the molecules on the surface of the
solid support it may be possible to increase the sensitivity of an
analytical determination that is being made when compared to
existing methods because most of the molecules are oriented with an
active site in a position suitable for reaction. In the case of
proteins, using existing methods control over protein orientation
is limited, since any surface amines can provide a point of
attachment of the protein to the linker molecule. Consequently, two
adjacent protein molecules may well be up to 180.degree. with
respect to each other, since the monolayer forms by random coupling
events. In order to analyze for a specific substrate, specific
region(s) on the protein surface must be available to optimize
sensor performance. Under the random coupling scenario, as much as
half of the protein monolayer may be in an orientation unsuitable
for substrate conversion.
[0059] The tag in the tagged molecule is preferably covalently
bound to the molecule at a position on the molecule that is
separated from the active site (or recognition region) of the
molecule. It will be appreciated that the active site of the
molecule is that portion of the molecule that is involved in the
chemical and/or physical transformation that is being investigated
whist the molecule is captured on the solid support. For example,
in the case of a polypeptide or protein, the tag may be covalently
bound anywhere on the protein, as long as the protein activity is
not affected. In this way the protein is attached to the solid
support at a modified portion of the protein and not a native
portion of the protein. In the case of a small organic molecule the
tag may be attached to the small organic molecule via a flexible
linker chain that acts to separate the tag from the active site of
the molecule. The linker chain may be a functionalized alkyl chain,
for example.
[0060] The tag is preferably a small peptide chain containing
chelate forming amino acids. Suitable chelate forming amino acids
include cysteine, lysine, histidine, arginine. More preferably, the
tag is a peptide containing a minimum of two but preferably four to
six histidine residues. Preferably, the peptide chain contains a
histidine rich domain. The tag may contain three histidine residues
and three other amino acid residues that assist with removal of the
tag from the molecule. Most preferably the tag is a peptide
containing six consecutive histidine residues herein referred to as
a "6*his" tag. The 6*his tag has been found to be particularly
suitable for binding to a chelating metal ion.
[0061] The solid support onto which the linker molecule is attached
may be in any suitable form, such as foils, wires, wafers, chips,
micro- or nano-particles, semiconductor devices and coatings
deposited by any known deposition process. The solid support
material may be a metal, metal oxide, silica, glass, quartz,
plastic, or polymer surface (modified or unmodified). Suitable
polymer surfaces include dextran, polycarbonate etc.
[0062] In one preferred form of the invention the solid support
onto which the linker molecule is attached is a noble metal.
Suitable noble metals include silver, gold, platinum and palladium.
In addition, graphite-based materials, TiO.sub.2, IrO.sub.2,
SnO.sub.2, Si-based surfaces or clays may also be used as a solid
support. The solid support may be a chip having a surface that is
formed from one of the aforementioned materials. The chip itself
may be formed from any suitable material including but not limited
to glass, plastic or ceramic material. Most preferably, the solid
support includes a gold surface.
[0063] The solid support may also be a chromatography medium, such
as dextran, silica etc, and derivatives thereof. In this form, the
present invention may be used for binding and separation of
biomolecules, such as peptides and proteins. Thus, the present
invention may be used in immobilized metal affinity
chromatography.
[0064] The linker molecule may be attached to the solid support
either directly or indirectly. For example, the head of the linker
molecule may be bonded to a noble metal surface on the solid
support. Alternatively, the solid support may have a coating, such
as a dextran coating, onto which the linker molecule is
attached.
[0065] The linker molecule comprises the head group, the tail
group, and a spacer group between the head group and tail group.
The spacer group is covalently attached to the head and tail
groups, and one function of the spacer group is to locate the tail
group away from the surface of the solid support. Preferably the
spacer is a carbon-based chain between the head and tail groups.
Preferably, the carbon-based chain is an alkyl chain or an alkyl
chain containing heteroatoms such as O, N, Si, P, or S. For
example, the carbon based chain may be a --(CH.sub.2).sub.n--
group, where n is an integer from 1 to 50, more preferably 2 to 15,
and even more preferably 3 to 10. Alternatively, the carbon based
chain may include a heteroatom and, for example, may be a
--(CH.sub.2CH.sub.2O).sub.n--group, where n is an integer from 1 to
50, more preferably 3 to 10. The length of the carbon-based chain
can be chosen depending on the application envisaged, and may be
from 1 to 50 atoms in length. In one preferred form of the
invention the carbon based chain is two to six atoms in length and
most preferably three atoms in length between the tail group and
the head group. However, depending on the use of the sensor, the
linker may be up to 50 atoms in length.
[0066] Preferably, the head group is a heteroatom that has a
relatively high affinity for the noble metal surface or other
surface to which it is to be attached. Sulfur and selenium are
particularly preferred head groups for noble metal substrates,
whilst oxygen is preferred for silica solid support substrates.
However, any atom (e.g., Si) or group that can react with the
surfaces listed above may be used.
[0067] In a particularly preferred form of the invention the tail
group is a cyclic ligand containing three or more atoms that are
capable or coordinating to the metal ion. Multidentate macrocycles
containing three or more heterodonor atoms including O, S, Se, N,
P, As or any atom that can act as a Lewis base are particularly
preferred. Multidentate macrocycles may have greater than three
heterodonor atoms, and more preferably 3 to 15 heterodonor atoms.
In a preferred form of the invention, the multidentate macrocycle
is a tetradentate macrocycle. The macrocycle is preferably a
derivative of 1,4,7-triazacyclononane. More preferably, the
derivative of 1,4,7-triazacyclononane contains a heterodonor atom
that is pendant from the triazacyclononane ring. For example, the
heterodonor atom may be the oxygen atom of a carbonyl group (ester,
carboxylic acid, ketone, aldehyde, amide etc.). Most preferably the
macrocycle is 1-acetato-4-benzyl-1,4,7-triazacyclononane.
[0068] The derivative of 1,4,7-triazacyclononane may contain an R
group at the 4 position. The R group may be any suitable
substituted or non-substituted alkyl or aryl group. In one
preferred form of the invention, the R group is a benzyl group. The
R group could also be any one of the nitrogen protecting or
terminal groups that are known in the art. Examples of nitrogen
protecting or terminal groups can be found in Greene and Wuts,
Protective Groups in Organic Synthesis, 2nd ed, John Wiley &
Sons, New York, 1991, the disclosure of which is hereby
incorporated by reference in its entirety.
[0069] The metal ion can be any ion that is capable of chelating
with heteroatoms such as N, S, O and the like. Preferably, the
metal ion is capable of octahedral coordination. Suitable metal
ions include zinc, copper, cobalt and nickel. Other metal ions that
can be used include the alkali and alkaline earth metals,
particularly sodium, potassium, magnesium, calcium and lithium.
[0070] In one preferred form of the invention the solid support is
an electrode. In this way the solid support can form part of an
electrochemical array so that the redox activity of the molecules
immobilized on the surface can be determined. More specifically,
the method of the present invention may be particularly suitable
for direct electrochemical measurements of redox active proteins,
such as thioredoxin and other proteins having a redox active site,
including metalloproteins, or those containing organic cofactors.
In this way it is possible to measure directly the redox
`signature` of a protein of interest. In a preferred form of the
present invention the linker molecule is three atoms in length and
thus the bound protein is held relatively close to the electrode.
Close proximity to the electrode is associated with strong and
reproducible signals for electrode sensors.
[0071] Alternatively, the solid support may form part of a chip,
the surface of which is formed from a suitable material according
to the present invention, for example a noble metal surface. The
chip may be formed from glass, plastic, ceramic or the like. In
this case, the immobilized molecules may be used to capture a
specific analyte in a sample suspected of containing the
analyte.
[0072] In particularly preferred embodiments of the present
invention, the method involves: (i) the formation of a self
assembled monolayer of mercaptopropionic acid ("MPA") on gold; (ii)
the activation of the carboxylic acid groups to reaction with a
nitrogen nucleophile; (iii) treatment of the activated substrate
with a suitable derivative of 1,4,7-triazacyclononane; (iv)
treatment of the subsequent reaction product with a solution
containing the chelating metal ion Zn.sup.2+; and (v) formation of
a self assembled monolayer of protein (or other) molecules by
treating the product from (iv) with 6*his tagged molecules.
[0073] Advantageously, the sensor chip of the present invention can
be regenerated to bind the same or different molecules by washing
the surface of the solid support with imadazole, acidic pH or
addition of any ligand which is a Lewis base. In addition, the
metal ion can be changed using EDTA or another known metal
chelating ligands.
[0074] The present invention also provides a biosensor device
including a sensor chip as hereinbefore described. The biosensor
also includes a transducer for detecting a change in a parameter in
the immobilised molecules on the sensor chip. The transducer may
measure an electrical parameter, such as in a glucose biosensor, an
optical parameter, such as in a Biacore instrument, or any other
suitable parameter.
[0075] The skilled person will appreciate that the sensor chip of
the present invention may be used in existing apparatus for
measuring one or more parameters of the molecules immobilized on
the surface of the sensor chip and/or molecules that bind to the
molecules immobilized on the surface of the sensor chip. The
apparatus typically includes a reaction substrate and a separate
reader or detector device, such as a scintillation counter or
spectrophotometer. Assays based on surface plasmon resonance (SPR)
effects, such as the BIAcore.TM. instrument and methodology, may be
used. These assays exploit the shift in SPR surface reflection
angle that occurs with perturbations, e.g., binding events, at the
SPR gold-glass interface. Applications include direct
electrochemistry, including bioelectrochemistry, fluorescence or
luminescence studies between interacting species or functional
groups, and applications involving immunological interactions, such
as antibody-antigen interactions.
[0076] It will also be appreciated that included within the scope
of the present invention is the use of the layer of molecules held
in the same orientation with respect to the surface for
applications that do not involve measuring a parameter of molecules
immobilized on the surface, but rather use the uniform orientation
of the immobilized molecules as a scaffold. In this case, the
uniform orientation of the immobilized molecules may be used as a
scaffold, for example, for other molecules, molecular complexes, or
cells.
[0077] Accordingly, in another form the present invention provides
a solid support including a monolayer of immobilized molecules
captured on the surface of the solid support, wherein a majority of
the immobilized molecules are in the same orientation with respect
to the surface, and further including one or more molecules
attached or captured by the immobilized molecules on the surface of
the solid support.
[0078] In addition, the methods of selectively orienting and
immobilizing molecules on the surface of a solid support may
include a further step of attaching or capturing one or more
secondary molecules to the tagged molecules.
[0079] It will also be appreciated that included within the scope
of the present invention is the use of the layer of molecules held
in the same orientation with respect to the surface for
applications in which the properties of the immobilized molecules
are assessed without interaction with an analyte. For example,
direct electrochemistry provides the ability to measure properties
of a protein by electron exchange.
[0080] Accordingly, the present invention also provides a sensor
device for measuring a parameter of an immobilized molecule on the
surface of the sensor device.
[0081] Therefore, the present invention also provides a sensor
device including a sensor chip of the present invention and a
transducer for measuring a parameter in the immobilized molecules
on the sensor chip.
Representative Embodiments
[0082] Representative embodiments of the invention will now be
described by way of the following non-limiting examples.
Example 1
Preparation of Self Assembled Monolayers (SAMs)
[0083] Self assembled monolayers of molecules of interest were
formed on a gold surface. The immobilization protocol is outlined
in FIG. 1.
[0084] A clean gold surface was modified with a mercaptopropionic
acid (MPA) SAM, designated 1 and the monolayer was subsequently
modified with EDC/NHS to create the NHS intermediate 2.
Specifically, a gold sheet was placed in a 5 mM solution of MPA
(75:25 w/w Ethanol:H.sub.2O) overnight. The surface was then washed
with ethanol to remove unbound MPA, and dried under a stream of
N.sub.2. The dried gold sheet was placed into an aqueous solution
containing 75 mM EDC and 25 mM NHS for 30 minutes, washed with
water and used immediately.
[0085] The modified macrocyclic amine, 1-acetato-4 benzyl-1,4,7
triazacyclononane 3, was synthesized according to the method
described in Warden et al. (Org. Lett. (2001) 3(18) 2855-2858)
incorporated herein by reference. The gold sheet that had been
modified to 2 was then placed into an aqueous solution containing
20 nM 1-acetato-4-benzyl-1,4,7-triazacyclononane 3 for 3 days. The
pH of the solution was kept at .gtoreq.10. The gold was then washed
exhaustively with water and used immediately.
[0086] Addition of a metal desirous of octahedral coordination
(pictured is Zn.sup.2+) gives the coordination sites 5.
Specifically, the gold sheet that had been modified to 4 was placed
into a 20 mM solution of ZnCl.sub.2 for 20 minutes. The gold was
washed thoroughly with water, and then immersed in a dilute
solution containing 6*his tagged molecules to afford the highly
orientated protein monolayer 6. For example, the modified gold
surface may be placed in an aqueous solution containing 6*his
tagged thioredoxin (0.2 mg of protein/mL in 10 mM phosphate buffer,
pH 6.0) at 4.degree. C. overnight, washed in 10 mM phosphate
buffer, pH 6.0 and used immediately.
Example 2
Analysis of SAMs
[0087] The SAMs prepared according to Example 1 were examined using
electrochemical cell and surface characterization techniques that
are described in the literature (see, e.g., D. L. Johnson, J.
Thompson, S. M. Brinkmann, K. L. Schuller & L. L. Martin,
"Electrochemical Characterisation of purified Rhus vernicifera
laccase--voltametric evidence for a sequential 4-electron
transfer," Biochemistry (2003), 42:10229-10237 and references
therein).
[0088] DRIFT (FIG. 2) and XPS experiments (not shown) were
consistent with the proposed surface chemistry. DRIFT analysis was
performed on fine silver powder modified according to FIG. 1. DRIFT
analysis of the preparatory stages 1, 2, and 4 are shown in FIG. 2.
Successful surface modification can be clearly seen in the C--H and
C.dbd.O stretching regions. Structure 1 displays one C.dbd.O
stretching vibration resulting from the COOH functionality,
Structure 2 displays two distinct C.dbd.O stretching vibrations
resulting from the --COON-- moiety and from the carbonyl groups on
the succinimide, and Structure 4 displays two different C.dbd.O
stretching vibrations from the amide and from the COOH group on the
macrocyclic amine. XPS measurements concur, with significant
variation in the N.sub.1s, and O.sub.1s, binding regions.
Example 3
Electrochemical Behaviour of Immobilised Thioredoxin: Comparison of
Oriented and Non-Oriented Monolayers
[0089] The self assembled monolayer was studied using direct
electrochemistry on histidine-tagged E. coli thioredoxin.
Thioredoxin is the simplest member of a growing superfamily which
regulate redox operation through a disulfide bond(s). The highly
conserved active site contains two cysteine residues with the
sequence Cys-XXX--XXX-- Cys where X is any amino acid. The oxidized
form contains a disulfide bond, and the reduced form, generated by
the sequential addition of two electrons, contains two thiol
groups.
[0090] The thioredoxin used in this study was expressed in E. coli
using the Novagen expression plasmid vector pET-32a(+) containing
the coding region of E. coli thioredoxin gene and six codons for
additional histidine residues at the C terminus. The
histidine-tagged thioredoxin was then purified to homogeneity as
assessed by a single band on SDS-PAGE using a nickel affinity
(Ni/NTA) column (QIAGEN). The sample was concentrated to 5 mg/mL
and stored in potassium phosphate buffer (pH 7.4) at -80.degree. C.
before use.
[0091] Macrocycle modified electrodes were prepared by placing a
gold electrode in a 5 mM solution of mercaptopropionic acid (MPA)
(75:25 v/v ethanol/water) overnight. The electrode surface was then
washed with ethanol to remove unbound MPA, and dried under a stream
of nitrogen gas. The dried gold electrode was placed into an
aqueous solution containing 75 mM 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) and 25 mM N-hydroxysuccinimide (NHS) for 30
minutes, washed with water and used immediately. The SAM-modified
electrode was placed into an aqueous solution containing 20 mM
1-acetato-4-benzyl-1,4,7-triazacyclononane at pH 8 for .about.30 h.
The gold electrode was then washed with water and used immediately.
Metal ions were incorporated into the macrocycle by placing the
modified electrode into a 20 mM solution of ZnCl.sub.2 for 20
minutes. The electrode was washed thoroughly with water, placed
into an aqueous solution of thioredoxin (0.2 mg of histidine tagged
thioredoxin/mL in 10 mM phosphate buffer, pH 7.4) at 4.degree. C.
for between 30 mM and 3 h, then washed in the same buffer and used
immediately. Alternatively, His-tagged cytochrome P450c17 and
plastocyanin were immobilised overnight at 4.degree. C. using a 20
mM protein solution in 10 mM phosphate buffer, pH 7.4. Covalent
attachment of the thioredoxin directly to the electrode for
comparison was done following the same procedure but without
addition of the macrocyclic moiety, thereby resulting in its
attachment to a surface amine on the thioredoxin protein.
[0092] All electrochemical measurements were obtained using a BAS
100B electrochemical analyser (Bioanalytical Systems Inc.,
Lafayette, USA) at room temperature. A conventional electrochemical
set-up was employed, involving an Ag/AgCl (3 M KCl) reference
electrode, a platinum auxiliary electrode and a gold electrode
either in the form of a button electrode (from BAS) or a gold
sheet. Potentials were corrected to the normal hydrogen electrode
(NHE).
[0093] We compared the electrochemical behaviour of E. coli
thioredoxin specifically captured via a histidine-tag using the
methods of the present invention with thioredoxin covalently
immobilized on a SAM with random orientation. FIG. 3 illustrates
typical cyclic voltammograms obtained at a gold electrode. The
solid line represents an electrode modified using the procedure
described in Example 1, leading to an oriented monolayer; the
dashed line represents an electrode modified by coupling surface
amines on the protein to the electrode, leading to a randomly
oriented monolayer. The scan rates shown are a) 200 .mu.V/s, b) 1
mV/s and c) 10 mV/s. 20 mM phosphate buffer, pH 7.0. We observed a
superior electrochemical response using the oriented protein layer,
with a faster electron transfer rate constant (more reversible
redox signal) and an improved signal to background ratio.
Example 4
Surface Plasmon Resonance (SPR) Analysis of a Gold Chip Modified
According to the Present Invention Compared with a Commercial
BIAcore NTA Chip
[0094] Other histidine tagged proteins that were immobilized
included bovine cytochrome P450c17, Synechocystis plastocyanin and
Montipora efflorescens green fluorescent protein (GFP).
[0095] BIAcore spectra were obtained on a BIAcore X analytical
system using modified J1 (J1 is a plain gold surface) and NTA
sensor chips (BIAcore AB, Uppsala, Sweden). The J1 chip was
modified according to the methods described in Example 1, resulting
in a 1-acetato-4-benzyl-1,4,7-triazacyclononane ("AcBztacn") chip,
and the NTA chip was used as purchased. The running buffer was 10
mM HEPES, 150 mM NaCl, the activation buffer was 500 .mu.M
NiCl.sub.2, 10 mM HEPES, 150 mM NaCl, the sample buffer was 30
.mu.g/mL thioredoxin in 10 mM HEPES, 150 mM NaCl and the
regeneration buffer was 350 mM EDTA, 10 mM HEPES, 150 mM NaCl (all
at pH 7.4). All solutions were freshly prepared, degassed and
filtered through a 0.2 .mu.m Millipore filter. Oriented binding was
evaluated by charging the chip with activating buffer (30 .mu.L/min
for 1 min) followed by sample buffer (30 .mu.L/min for 1 min). The
stability of the protein--chip interaction was monitored by passing
running buffer (20 .mu.L/min) over the surface and monitoring the
sensorgram over time. The dissociation rate was determined as the
percentage of original binding lost per minute across a period of
between 2 and 14 h, assuming a linear decay. Non-specific protein
binding was evaluated by injecting sample buffer before Ni
activation, thus comparing the weakly attached protein material
prior to activation of the chips by metal ions.
[0096] Protein binding to the chip was assessed using SPR, as shown
in FIG. 5. We compared our AcBztacn electrode with the commercially
available NTA chip from BIAcore for two proteins histidine-tagged
thioredoxin and Montipora efflorescens green fluorescent protein
(GFP (Table 1)). In particular, we examined the relative binding
magnitude, stability (as assessed by protein dissociation) and the
degree of non-specific binding of histidine-tagged thioredoxin and
Montipora efflorescens green fluorescent protein (GFP). Despite a
lower surface coverage, the AcBztacn modified surface displayed
greater than five-fold increase in binding magnitude (Table 1) as
assessed by the amount of bound protein to Ni-treated surface. The
stability of the protein-Ni interaction was assessed by the rate of
decay of the initial binding strength, as determined by the amount
of protein dissociation after protein loading and expressed as the
percentage of dissociation per minute. The AcBztacn-Ni-protein
complex is eight times more stable than NTA-Ni-protein (Table 1).
The AcBztacn electrode therefore has potential applications for
long term biosensor use, with 50% of bound protein dissociated from
the NTA chip in less than 40 min, compared with a loss of only 40%
of total protein even after 15 h for the AcBztacn electrode (shown
by a comparison of the dissociation rates in Table 1). Binding of
protein prior to Ni ion activation was an order of magnitude
greater for NTA, indicating that a component of non-specific
binding occurs in the NTA chips. The increased binding of protein,
improved stability and a reduction in non-specific binding
indicates that the AcBztacn surface of the sensor chips of the
present invention has superior qualities to this
commercially-available system.
TABLE-US-00001 TABLE 1 Improvement Ni-NTA Ni-AcBztacn Factor
Thioredoxin Immobilized protein (RU) 828 4504 5.4 Dissociation rate
1.7 0.2 8.5 (% of initial RU lost per min) Non-specific protein
binding (%) 46.7 5.0 9.2 Green Fluorescent Protein Immobilized
protein (RU) 376 2325 6.2 Dissociation rate 7.6 0.9 8.4 (% of
initial RU lost per min) Non-specific protein binding (%) 38 4
9.5
[0097] Finally, it is to be understood that various other
modifications and/or alterations may be made without departing from
the spirit of the present invention as outlined herein and as
described in the claims appended hereto.
* * * * *