U.S. patent application number 14/974736 was filed with the patent office on 2016-07-14 for enzyme cascade methods for e-trace assay signal amplification.
This patent application is currently assigned to OHMX Corporation. The applicant listed for this patent is OHMX Corporation. Invention is credited to Michael J. Ahrens, Paul A. Bertin.
Application Number | 20160202203 14/974736 |
Document ID | / |
Family ID | 47559760 |
Filed Date | 2016-07-14 |
United States Patent
Application |
20160202203 |
Kind Code |
A1 |
Bertin; Paul A. ; et
al. |
July 14, 2016 |
ENZYME CASCADE METHODS FOR E-TRACE ASSAY SIGNAL AMPLIFICATION
Abstract
The disclosure also relates to novel methods for detection of
biological targets using amplification steps in conjunction with
conversion of functional groups attached to a transitional metal
complex, resulting in quantifiable electrochemical signal at two
unique potentials, E.sup.o.sub.1 and E.sup.o.sub.2.
Inventors: |
Bertin; Paul A.; (Western
Springs, IL) ; Ahrens; Michael J.; (West Lafayette,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OHMX Corporation |
Evanston |
IL |
US |
|
|
Assignee: |
OHMX Corporation
Evanston
IL
|
Family ID: |
47559760 |
Appl. No.: |
14/974736 |
Filed: |
December 18, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13737634 |
Jan 9, 2013 |
9250203 |
|
|
14974736 |
|
|
|
|
61584563 |
Jan 9, 2012 |
|
|
|
Current U.S.
Class: |
205/777.5 |
Current CPC
Class: |
G01N 33/581 20130101;
G01N 33/54306 20130101; G01N 27/4166 20130101; G01N 27/26 20130101;
G01N 27/3271 20130101; G01N 27/3277 20130101; G01N 27/3273
20130101; C12Q 1/005 20130101; G01N 33/5438 20130101; G01N 27/27
20130101 |
International
Class: |
G01N 27/327 20060101
G01N027/327; G01N 33/58 20060101 G01N033/58; G01N 27/416 20060101
G01N027/416; C12Q 1/00 20060101 C12Q001/00 |
Claims
1-24. (canceled)
25. A method for detecting a target analyte in a test sample, said
method comprising: a. contacting said test sample with a binding
ligand under conditions wherein said target analyte, if present,
binds said binding ligand forming a first complex; b. contacting
said first complex with a labeled target analyte, wherein: i. said
labeled target analyte binds any binding ligands not already bound
to said target analyte forming a second complex and the second
complex is contacted with an intermediary enzyme of a peroxide
generating system comprising a labeled linker to form a third
complex; ii. or optionally said labeled target analyte is bound to
an intermediary enzyme of a peroxide generating system attached via
a labeled linker and binds any binding ligands not already bound to
said target analyte forms said third complex; and iii. said labeled
target analyte is labeled with a first member of a specific binding
pair and said labeled linker is labeled with a second member of
said specific binding pair c. isolating said third complex; d.
contacting said third complex with a substrate for said
intermediary enzyme under conditions such that product(s) are
generated to form a first assay mixture; e. contacting a
peroxide-generating enzyme and optionally a recycling enzyme with
first assay mixture under conditions wherein peroxide is generated
to form a second assay mixture; f. contacting the second assay
mixture with a first solid support comprising an electrode
comprising a covalently attached electroactive moiety (EAM) having
a first E.sup.0, said EAM comprising a transition metal complex
comprising a self-immolative moiety (SIM) and a peroxide sensitive
moiety (PSM), wherein said peroxide reacts with said PSM to release
said SIM from said EAM and result in said EAM having a second
E.sup.0; g. measuring the electrochemical properties of said EAM at
the first E.sup.0 and at the second E.sup.0; and h. detecting for
the presence or the concentration of said target analyte from said
electrochemical properties.
26. A method for detecting a target analyte in a test sample, said
method comprising: a. contacting said test sample with a first
complex comprising a labeled target analyte pre-bound to a binding
ligand and an intermediary enzyme of a peroxide generating system
attached via a labeled linker to the labeled target analyte under
conditions wherein said target analyte, if present, displaces said
labeled target analyte, said labeled linker, and said intermediary
enzyme from said first complex forming a second complex, wherein
said labeled target analyte is labeled with a first member of a
specific binding pair and said labeled linker is labeled with a
second member of said specific binding pair; b. isolating said
second complex; c. contacting said second complex with a substrate
for said intermediary enzyme under conditions such that product(s)
are generated to form a first assay mixture; d. contacting a
peroxide-generating enzyme and optionally a recycling enzyme with
first assay mixture under conditions wherein peroxide is generated
to form a second assay mixture; e. contacting the second assay
mixture with a first solid support comprising an electrode
comprising a covalently attached electroactive moiety (EAM) having
a first E.sup.0, said EAM comprising a transition metal complex
comprising a self-immolative moiety (SIM) and a peroxide sensitive
moiety (PSM), wherein said peroxide reacts with said PSM to release
said SIM from said EAM and result in said EAM having a second
E.sup.0; f. measuring the electrochemical properties of said EAM at
the first E.sup.0 and at the second E.sup.0; and g. detecting for
the presence or the concentration of said target analyte from said
electrochemical properties.
27. A method for detecting a target analyte in a test sample, said
method comprising: a. contacting said test sample with a first
complex comprising a labeled target analyte pre-bound to a binding
ligand and an intermediary enzyme of a peroxide generating system
attached via a labeled linker to the labeled target analyte under
conditions wherein said target analyte, if present, displaces said
labeled target analyte, said labeled linker, and said intermediary
enzyme from said first complex forming a second complex on the
binding ligand and a first solution phase assay mixture containing
a third complex comprising the said displaced labeled target
analyte with said labeled linker, and said intermediary enzyme, and
wherein said labeled target analyte is labeled with a first member
of a specific binding pair and said labeled linker is labeled with
a second member of said specific binding pair; b. isolating said
third complex; c. contacting said third complex with a substrate
for said intermediary enzyme under conditions such that product(s)
are generated to form a second assay mixture; d. contacting a
peroxide-generating enzyme and optionally a recycling enzyme with
second assay mixture under conditions wherein peroxide is generated
to form a third assay mixture; e. contacting the third assay
mixture with a first solid support comprising an electrode
comprising a covalently attached electroactive moiety (EAM) having
a first E.sup.0, said EAM comprising a transition metal complex
comprising a self-immolative moiety (SIM) and a peroxide sensitive
moiety (PSM), wherein said peroxide reacts with said PSM to release
said SIM from said EAM and result in said EAM having a second
E.sup.0; f. measuring the electrochemical properties of said EAM at
the first E.sup.0 and at the second E.sup.0; and g. detecting for
the presence or the concentration of said target analyte from said
electrochemical properties.
28. The method according to claim 1, wherein said labeled target
analyte binds any binding ligands not already bound to said target
analyte forming a second complex and the second complex is
contacted with an intermediary enzyme of a peroxide generating
system comprising a labeled linker to form a third complex.
29. The method according to claim 1, wherein the target analyte is
a small molecule.
30. The method according to claim 5, wherein said target analyte is
testosterone.
31. The method according to claim 1, wherein said binding ligand is
attached to a second solid support.
32. The method according to claim 7, wherein said second solid
support is chosen from the group consisting of microparticles,
magnetic microparticles, beads, and microchannels.
33. The method according to claim 1, wherein said specific binding
pair is biotin/streptavidin.
34. The method according to claim 1, wherein said electrode further
comprises a self-assembled monolayer (SAM).
35. The method according to claim 1, wherein said substrate for
said intermediary enzyme of peroxide-generating system is FADP,
NADP.
36. The method according to claim 1, wherein said product(s) is a
substrate for said peroxide generating enzyme.
37. The method according to claim 1, further comprising the
presence of a substrate for said peroxide generating enzyme and
wherein said product(s) is a cofactor for said peroxide-generating
enzyme.
38. The method according to claim 1, wherein said recycling enzyme
is included to recycle said cofactor.
39. The method according to claim 1, wherein said intermediary
enzyme of a peroxide generating system is alkaline phosphatase (AP)
or another dephosphorylating enzyme.
40. The method according to claim 1, wherein said
peroxide-generating enzyme is D-amino acid oxidase (DAAO) or
another flavin dependent oxidoreductase enzyme.
41. The method according to claim 17, wherein said
peroxide-generating enzyme is NADH Oxidase and said recycling
enzyme is Alcohol Dehydrogenase (ADH).
42. A method according to claim 1, wherein said first binding
ligand and said second binding ligand are independently chosen from
the group consisting of monoclonal antibodies, fragments of
monoclonal antibodies, polyclonal antibodies, fragments of
polyclonal antibodies, proteins, and peptides.
43. A method according to claim 1, wherein said transition metal is
chosen from the group consisting of iron, ruthenium, and
osmium.
44. A method according to claim 1, wherein said EAM is chosen from
the group consisting of ferrocene and substituted ferrocene.
45. A method for detecting a target analyte in a test sample, said
method comprising: (a) contacting a target specific enzyme, a
recycling enzyme and a peroxide-generating enzyme with said target
sample under conditions wherein a peroxide is generated in the
presence of said target analyte to form an assay mixture; (b)
contacting the assay mixture with a first solid support comprising
an electrode comprising a covalently attached electroactive moiety
(EAM) having a first E.sup.0, said EAM comprising a transition
metal complex comprising a self-immolative moiety (SIM) and a
peroxide sensitive moiety (PSM), wherein said peroxide reacts with
said PSM to release said SIM from said EAM and result in said EAM
having a second E.sup.0; (c) measuring the electrochemical
properties of said EAM at the first E.sup.0 and at the second
E.sup.0; and (d) detecting for the presence or concentration of
said target analyte from said electrochemical properties.
46. A method for detecting a target analyte in a test sample, said
method comprising: (a) contacting said test sample with a first
complex or a first binding ligand, wherein: i. when said test
sample is contacted with the first complex, the first complex
comprises a labeled target analyte pre-bound to a binding ligand
said test sample under conditions wherein said target analyte from
said test sample, if present, displaces said labeled target from
said first complex forming a second complex, wherein said labeled
target analyte is labeled with a first member of a specific binding
pair; and the method further comprises: a. contacting said second
complex with an intermediary enzyme of a peroxide generating system
comprising a labeled linker forming a third complex, wherein said
labeled linker is labeled with a second member of said specific
binding pair; b. isolating said third complex; and c. contacting
said third complex with a substrate for said intermediary enzyme of
peroxide-generating system under conditions such that product(s)
are generated to form a first assay mixture; ii. when said test
sample is contacted with the first binding ligand, the first
binding ligand is contacted with said test sample under conditions
wherein said first binding ligand binds said target analyte, if
present, in said test sample to form a first complex, the method
further comprises: a. contacting said first complex with a second
binding ligand under conditions wherein said first complex and said
second binding ligand bind to form a second complex, wherein said
second binding ligand comprises an intermediary enzyme of a
peroxide-generating system and isolating said second complex; and
b. contacting said second complex with a substrate for said
intermediary enzyme of peroxide-generating system under conditions
such that products are generated to form a first assay mixture; (b)
contacting a peroxide-generating enzyme and optionally a recycling
enzyme with first assay mixture under conditions wherein peroxide
is generated to form a second assay mixture; (c) contacting the
second, assay mixture with a first solid support comprising an
electrode comprising a covalently attached electroactive moiety
(EAM) having a first E.sup.0, said EAM comprising a transition
metal complex comprising a self-immolative moiety (SIM) and a
peroxide sensitive moiety (PSM), wherein said peroxide reacts with
said PSM to release said SIM from said EAM and result in said EAM
having a second E.sup.0; (d) measuring the electrochemical
properties of said EAM at the first E.sup.0 and at the second
E.sup.0; and (e) detecting for the presence or the concentration of
said target analyte from said electrochemical properties.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application No. 61/584,563, filed Jan. 9, 2012,
the entire disclosures of which are hereby incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to novel compositions and methods for
the detection of biological targets using amplification steps in
conjunction with conversion of functional groups attached to a
transitional metal complex, resulting in quantifiable
electrochemical signal at two unique potentials, E.sup.o.sub.1 and
E.sup.o.sub.2. In particular, the methods of the invention utilize
enzyme-triggered redox altering chemical elimination (E-TRACE)
reaction.
BACKGROUND OF THE INVENTION
[0003] The electromotive force (EMF) is the maximum potential
difference between two electrodes of a galvanic or voltaic cell,
where the standard hydrogen electrode is on the left-hand side for
the following cell:
TABLE-US-00001 1 2 Pt Electrode H.sub.2 Aqueous Electrolyte
10.sup.-3M Fe(ClO.sub.4).sub.3 Pt Solution 10.sup.-3M
Fe(ClO.sub.4).sub.2
The EMF is called the electrode potential of the electrode placed
on the right-hand side in the graphical scheme of the cell, but
only when the liquid junction between the solutions can be
neglected or calculated, or if it does not exist at all.
[0004] The electrode potential of the electrode on the right-hand
side (often called the oxidation-reduction potential) is given by
the Nernst equation
E.sub.Fe.sub.3+.sub./Fe.sub.2+=E.sub.Fe.sub.3+.sub./Fe.sub.2+.sup.0+(RT/-
F)ln(a.sub.Fe.sub.3+/a.sub.Fe.sub.2+)
[0005] This relationship follows from equation (2.21) when
(.mu..sub.Fe.sub.3-.sup.0-.mu..sub.Fe.sub.2+.sup.0)/F is set equal
to E.sub.Fe.sub.3+.sup.0.sub./Fe.sub.2+ and the pH and ln
p.sub.H.sub.2 are equal to zero. In the subscript of the symbol for
the electrode potential the symbols for the oxidized and reduced
components of the oxidation-reduction system are indicated. With
more complex reactions it is particularly recommended to write the
whole reaction that takes place in the right-hand half of the cell
after symbol E (the `half-cell` reaction); thus, in the present
case
E.sub.Fe.sub.3+.sub./Fe.sub.2+.ident.E(Fe.sup.3++e=Fe.sup.2+)
[0006] Quantity E.sub.Fe.sub.3+.sub./Fe.sub.2+.sup.0 termed the
standard electrode potential. It characterizes the oxidizing or
reducing ability of the component of oxidation-reduction systems.
With more positive standard electrode potentials, the oxidized form
of the system is a stronger oxidant and the reduced form is a
weaker reductant. Similarly, with a more negative standard
potential, the reduced component of the oxidation-reduction system
is a stronger reductant and the oxidized form a weaker oxidant.
[0007] The standard electrode E.sup.0, in its standard usage in the
Nernst equation, equation (1-2) is described as:
E = E 0 + 2.3 RT nF log C 0 ( 0 , t ) C R ( 0 , t )
##EQU00001##
where E.sup.0 is the standard potential for the redox reaction, R
is the universal gas constant (8.314 JK.sup.-1mol.sup.-1), T is the
Kelvin temperature, n is the number of electrons transferred in the
reaction, and F is the Faraday constant (96,487 coulombs). On the
negative side of E.sup.0, the oxidized form thus tends to be
reduced, and the forward reaction (i.e., reduction) is more
favorable. The current resulting from a change in oxidation state
of the electroactive species is termed the faradaic.
[0008] Previous work describes using conversion of functional
groups attached to a transitional metal complex resulting in
quantifiable electrochemical signal at two unique potentials,
E.sup.o.sub.1 and E.sup.o.sub.2. See for example, U.S. Patent
Publication Nos. US 2011 0033869 and US 2012-0181186, all herein
incorporated by reference in their entirety. The methods generally
comprise binding an analyte within a sandwich of binding ligands
which may have a functional tag, on a solid support other than the
electrode. After target binding, a peroxide generating moiety or an
intermediary enzyme and substrate are added which generates
hydrogen peroxide. The redox active complex is bound to an
electrode and comprises a peroxide sensitive moiety (PSM). The
peroxide generated from the enzyme system reacts with the PSM,
removing a self-immolative moiety (SIM) and converting functional
groups attached to a transitional metal complex resulting in
quantifiable electrochemical signal at two unique potentials,
E.sup.o.sub.1 and E.sup.o.sub.2.
SUMMARY OF THE INVENTION
[0009] The present invention provides composition and methods for
the detection of target analytes using amplification methods to
enhance catalytically electrochemical signal from the conversion of
functional groups attached to a transitional metal complex
resulting in quantifiable electrochemical signal at two unique
potentials, E.sup.o.sub.1 and E.sup.o.sub.2. In particular, the
present invention discloses the advantages of combining the
amplification steps. In one aspect, the invention provides
compositions and methods for the detection of target analyte in a
test sample. Thus, the invention provides a first solid support
comprising an electrode comprising: a self-assembled monolayer
(SAM). (ii) a covalently attached electroactive active moiety (EAM)
comprising a transition metal complex comprising a self-immolative
moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said
EAM has a first E.sup.0 and a self-assembled monolayer (SAM). The
capture binding ligand that binds the analyte, is on a second solid
support other than the electrode.
[0010] In one embodiment a small molecule target, adenosine
triphopsphate (ATP) can be detected utilizing an enzymatic
amplification system. The ATP, along with glycerol, acts as a
substrate for Glycerol Kinase (GK), generating Adenosine
Diphosphate (ADP) and glycerol-3-phosphate. The ADP byproduct and
phosphocreatine act as substrates for Creatine Kinase (CK) which
transfers the phosphate group from phosphocreatine to ADP to
regenerate ATP and Creatine. The ATP is then utilized to generate
another glycerol-3-phosphate by Glycerol Kinase. Thus the ATP/ADP
cycling allows multiple glycerol-3phosphate molecules to be
generated for one starting molecule of ATP. The
glycerol-3-phosphate then is oxidized to form H.sub.2O.sub.2 and
glycerone phosphate by Glycerol-3-Phosphate Oxidase (G3PO).
Therefore H.sub.2O.sub.2 is generated in a manner dependent on the
starting ATP concentration but results in a concentration of
H.sub.2O.sub.2 orders of magnitude higher.
[0011] In one embodiment, the disclosure provides methods for
detecting a target analyte in a test sample, said method
comprising:
[0012] (a) contacting a target specific enzyme, a recycling enzyme
and a peroxide-generating enzyme with said target sample under
conditions wherein a peroxide is generated in the presence of said
target analyte to form an assay mixture;
[0013] (b) contacting the assay mixture with a first solid support
comprising an electrode comprising a covalently attached
electroactive moiety (EAM) having a first E.sup.0, said EAM
comprising a transition metal complex comprising a self-immolative
moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said
peroxide reacts with said PSM to release said SIM from said EAM and
result in said EAM having a second E.sup.0;
[0014] (c) measuring the electrochemical properties of said EAM at
the first E.sup.0 and at the second E.sup.0; and
[0015] (d) detecting for the presence or concentration of said
target analyte from said electrochemical properties.
[0016] In one embodiment of the disclosure any preceding embodiment
is where said electrode further comprises a self-assembled
monolayer (SAM). In another embodiment of the disclosure any
preceding embodiment is where the target analyte is a small
molecule.
[0017] In one embodiment of the disclosure said target analyte is
reduced Nicotinamide Adenine Dinucleotide (NADH). In another
embodiment, said recycling enzyme is Alcohol Dehydrogenase (ADH),
said target specific enzyme is NADH Oxidase, and said peroxide
generating enzyme is NADH oxidase.
[0018] In one embodiment of the disclosure said target analyte is
Adenosine Triphosphate (ATP). In another embodiment, said target
specific enzyme is Glycerol Kinase (GK), said recycling enzyme is
Creatine Kinase (CK) or Glycerol 3-Phosphate Dehydrogenase (GPDH),
and said peroxide generating enzyme is Glycerol-3-Phosphate Oxidase
(G3PO).
[0019] In one embodiment of the disclosure any preceding embodiment
is where the target analyte is testosterone.
[0020] In one embodiment of the disclosure any preceding embodiment
is where said EAM comprises a transition metal. In one embodiment,
said transition metal is chosen from the group consisting of iron,
ruthenium and osmium.
[0021] In one embodiment of the disclosure any preceding embodiment
is said EAM is chosen from the group consisting of ferrocene and
substituted ferrocene.
[0022] In another embodiment a small molecule target, adenosine
triphosphate (ATP) can be detected utilizing a different enzymatic
amplification system. The ATP, along with glycerol, acts as a
substrate for Glycerol Kinase (GK), generating Adenosine
Diphosphate (ADP) and glycerol-3-phosphate. The
glycerol-3-phosphate then is oxidized to form H.sub.2O.sub.2 and
glycerone phosphate by Glycerol-3-Phosphate Oxidase (G3PO). The
glycerone phosphate is then reduced by Glycerol-3-phosphate
Dehyrodgenase (GPDH) utilizing NADH, regenerating
glycerol-3-phosphate which in turn is re-oxidized by G3PO
generating another molecule of H.sub.2O.sub.2. Therefore
H.sub.2O.sub.2 is generated in a manner dependent on the starting
ATP concentration but results in a concentration of H.sub.2O.sub.2
orders of magnitude higher.
[0023] In one embodiment a small molecule target, reduced
Nicotinamide Adenine Dinucleotide (NADH) can be detected utilizing
an enzymatic amplification system. The NADH, acts as a substrate
for NADH oxidase (NAOX), generating NAD+ and H.sub.2O.sub.2. The
NAD+ byproduct and ethanol act as substrates for alcohol
dehydrogenase (ADH) which produces acetaldehyde and regenerates
NADH. The NADH is then re-oxidized by NADH oxidase generating
another H.sub.2O.sub.2. Thus the NADH/NAD+ cycling allows multiple
NADH molecules to be generated from one starting molecule of NADH.
Therefore H.sub.2O.sub.2 is generated in a manner dependent on the
starting NADH concentration but results in a concentration of
H.sub.2O.sub.2 orders of magnitude higher.
[0024] In one embodiment, the disclosure provides methods for
detecting a target analyte in a test sample, said method
comprising:
[0025] (a) contacting said test sample with a first complex
comprising a labeled target analyte pre-bound to a binding ligand,
with said test sample under conditions wherein said target analyte
from said test sample, if present, displaces said labeled target
from said first complex forming a second complex, wherein said
labeled target analyte is labeled with a first member of a specific
binding pair;
[0026] (b) contacting said second complex with an intermediary
enzyme of a peroxide generating system comprising a labeled linker
forming a third complex, wherein said labeled linker is labeled
with a second member of said specific binding pair;
[0027] (c) isolating said third complex;
[0028] (d) contacting said third complex with a substrate for said
intermediary enzyme of peroxide-generating system under conditions
such that product(s) are generated to form a first assay
mixture;
[0029] (e) contacting a peroxide-generating enzyme and optionally a
recycling enzyme with first assay mixture under conditions wherein
peroxide is generated to form a second assay mixture;
[0030] (f) contacting the second assay mixture with a first solid
support comprising an electrode comprising a covalently attached
electroactive moiety (EAM) having a first E.sup.0, said EAM
comprising a transition metal complex comprising a self-immolative
moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said
peroxide reacts with said PSM to release said SIM from said EAM and
result in said EAM having a second E.sup.0;
[0031] (g) measuring the electrochemical properties of said EAM at
the first E.sup.0 and at the second E.sup.0; and
[0032] (h) detecting for the presence or the concentration of said
target analyte from said electrochemical properties.
[0033] As used in this disclosure, the term "isolating" or
"isolate" means separating the particular components from others
through any suitable means including Physical separation or
purification.
[0034] In one embodiment of the disclosure, said specific binding
pair is biotin/streptavidin. In another embodiment of the
disclosure, said binding ligand is attached to a second solid
support. In yet another embodiment of the disclosure, said second
solid support is chosen from the group consisting of
microparticles, magnetic microparticles, beads, and
microchannels.
[0035] In one embodiment of the disclosure any preceding embodiment
is where said electrode further comprises a self-assembled
monolayer (SAM). In another embodiment of the disclosure any
preceding embodiment is where the target analyte is a small
molecule.
[0036] In one embodiment of the disclosure said product(s) is a
substrate for said peroxide generating enzyme. In another
embodiment of the disclosure method further comprises the presence
of a substrate for said peroxide generating enzyme and wherein said
product(s) is a cofactor for said peroxide-generating enzyme. In
yet another embodiment said recycling enzyme is included to recycle
said cofactor.
[0037] In one embodiment of the disclosure any preceding embodiment
is wherein said intermediary enzyme of a peroxide generating system
is alkaline phosphatase (AP) or any other dephosphorylating
enzyme.
[0038] In another embodiment of the disclosure any preceding
embodiment is where said peroxide-generating enzyme is selected
from the group consisting of D-amino acid oxidase (DAAO), or any
flavin dependent oxidoreductase enzyme.
[0039] In another embodiment of the disclosure any preceding
embodiment is where said peroxide-generating enzyme is NADH Oxidase
and said optional recycling enzyme is Alcohol Dehydrogenase
(ADH).
[0040] In one embodiment of the disclosure any preceding embodiment
is where the target analyte is testosterone.
[0041] In another embodiment of the disclosure any preceding
embodiment is where said first binding ligand and said second
binding ligand are independently chosen from the group consisting
of monoclonal antibodies, fragments of monoclonal antibodies,
polyclonal antibodies, fragments of polyclonal antibodies,
proteins, and peptides.
[0042] In one embodiment of the disclosure any preceding embodiment
is where said EAM comprises a transition metal. In one embodiment,
said transition metal is chosen from the group consisting of iron,
ruthenium and osmium.
[0043] In one embodiment of the disclosure any preceding embodiment
is said EAM is chosen from the group consisting of ferrocene and
substituted ferrocene.
[0044] In one embodiment a small molecule target, (e.g.,
testosterone) can be detected utilizing a competition based assay.
The sample is added to a preformed first complex comprising a
binding ligand and a bound biotinylated target molecule attached to
a solid support. Target molecule in sample displaces a proportion
of the prebound biotinylated target molecule in a concentration
dependent manner forming a second complex. Sample is then washed
away, retaining both first and second complexes attached to solid
support. A streptavidin linker tagged with an intermediary enzyme
of a peroxide generating system e.g., Alkaline phosphatase (AP) is
added binding only to the biotinylated targets of first complexes
forming a third complex. Substrate for intermediary enzyme (e.g.,
FADP, NADP), oxidase enzyme (e.g., D-amino acid oxidase, NADH
oxidase) and, optionally cycling enzymes (Alcohol Dehyrodgenase)
are added to the complexes and H.sub.2O.sub.2 is generated. The
signal output is only dependent on the number of third complexes
and is inversely proportional to the concentration of target in the
test sample. The higher the signal measured the lower the
concentration of target molecule in the test sample.
[0045] In another embodiment a small molecule target, (e.g.,
testosterone) can be detected utilizing a competition based assay.
The sample is added to a preformed first complex comprising a
binding ligand attached to a solid support, a bound biotinylated
target molecule, and an intermediary enzyme of a peroxide
generating system tag (e.g., Alkaline Phosphatase) attached via a
streptavidin linker to the biotinylated target. Target molecule in
sample displaces a proportion of the prebound biotinylated target
molecule in a concentration dependent manner forming a second
complex. Sample is then washed away, retaining complexed binding
ligands. Substrate for intermediary enzyme (e.g., FADP, NADP),
oxidase enzyme (e.g., D-amino acid oxidase, NADH oxidase) and,
optionally cycling enzymes (Alcohol Dehyrodgenase) are added to the
complexes and H.sub.2O.sub.2 is generated. The signal output is
only dependent on the number of first complexes and inversely
proportional to the concentration of target in the test sample. The
higher the signal measured the lower the concentration of target
molecule in the test sample.
[0046] In another embodiment a small molecule target, (e.g.,
testosterone) can be detected utilizing a competition based assay.
The sample is added to a preformed first complex comprising a
binding ligand attached to a solid support, a bound biotinylated
target molecule, and an intermediary enzyme of a peroxide
generating system tag (e.g., Alkaline Phosphatase) attached via a
streptavidin linker to the biotinylated target. Target molecule in
sample displaces a proportion of the prebound biotinylated target
molecule in a concentration dependent manner forming a second
complex. Supernatant, containing displaced biotinylated target
labeled with streptavidin and intermediary enzyme, is removed.
Substrate for intermediary enzyme (e.g., FADP, NADP), oxidase
enzyme (e.g., D-amino acid oxidase, NADH oxidase) and, optionally
cycling enzymes (Alcohol Dehyrodgenase) are added to the
supernatant and H.sub.2O.sub.2 is generated. The signal output is
only dependent on the amount of biotinylated target displaced in
the supernatant and is directly proportional to the concentration
of target in the test sample. The higher the signal measured the
higher the concentration of target molecule in the test sample.
[0047] In another embodiment a small molecule target, (e.g.,
testosterone) can be detected utilizing a competition based assay.
The sample is added to a binding ligand attached to a solid
support. Target molecule in sample binds capture ligand forming a
first complex. Sample is washed away. A bound biotinylated target
molecule is introduced and displaces target molecule forming a
second complex. The biotinylated target molecule may optionally
have and an intermediary enzyme of a peroxide generating system tag
(e.g., Alkaline Phosphatase) attached via a streptavidin linker.
Remaining unbound biotinylated target is washed away. Optionally,
an intermediary enzyme of a peroxide generating system tag (e.g.,
Alkaline Phosphatase) attached to a streptavidin linker is added if
not precomplexed with biotinylated target in previous step.
Optionally, wash away excess intermediary enzyme. Substrate for
intermediary enzyme (e.g., FADP, NADP), oxidase enzyme (e.g.,
D-amino acid oxidase, NADH oxidase) and optionally cycling enzymes
(Alcohol Dehyrodgenase) are added to the complexes and
H.sub.2O.sub.2 is generated. The signal output is only dependent on
the number of second complexes and inversely proportional to the
concentration of target in the test sample. The higher the signal
measured the lower the concentration of target molecule in the test
sample.
[0048] In one embodiment, the disclosure provides methods for
detecting a target analyte in a test sample, said method
comprising:
[0049] (a) contacting a first binding ligand with said test sample
under conditions wherein said first binding ligand binds said
target analyte, if present, in said test sample to form a first
complex;
[0050] (b) contacting said first complex with a second binding
ligand under conditions wherein said first complex and said second
binding ligand bind to form a second complex, wherein said second
binding ligand comprises an intermediary enzyme of a
peroxide-generating system;
[0051] (c) isolating said second complex;
[0052] (d) contacting said second complex with a substrate for said
intermediary enzyme of peroxide-generating system under conditions
such that products are generated to form a first assay mixture;
[0053] (e) contacting a peroxide-generating enzyme and optionally a
recycling enzyme with a first assay mixture under conditions
wherein peroxide is generated to form a second assay mixture;
[0054] (f) contacting the second assay mixture with a first solid
support comprising an electrode comprising a covalently attached
electroactive moiety (EAM) having a first E.sup.0, said EAM
comprising a transition metal complex comprising a self-immolative
moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said
peroxide reacts with said PSM to release said SIM from said EAM and
result in said EAM having a second E.sup.0;
[0055] (g) measuring the electrochemical properties of said EAM at
the first E.sup.0 and at the second E.sup.0; and
[0056] (h) detecting for the presence or the concentration of said
target analyte from said electrochemical properties.
[0057] In one embodiment of the disclosure, the target analyte is a
protein. In another embodiment of the disclosure, said binding
ligand is attached to a second solid support. In yet another
embodiment of the disclosure, said second solid support is chosen
from the group consisting of microparticles, magnetic
microparticles, beads, and microchannels.
[0058] In one embodiment of the disclosure any preceding embodiment
is where said electrode further comprises a self-assembled
monolayer (SAM). In another embodiment of the disclosure any
preceding embodiment is where the target analyte is a small
molecule.
[0059] In one embodiment of the disclosure said product(s) is a
substrate for said peroxide generating enzyme. In another
embodiment of the disclosure method further comprises the presence
of a substrate for said peroxide generating enzyme and wherein said
product(s) is a cofactor for said peroxide-generating enzyme. In
yet another embodiment said recycling enzyme is included to recycle
said cofactor.
[0060] In one embodiment of the disclosure any preceding embodiment
is wherein said intermediary enzyme of a peroxide generating system
is alkaline phosphatase (AP) or any other dephosphorylating
enzyme.
[0061] In another embodiment of the disclosure any preceding
embodiment is where said peroxide-generating enzyme is selected
from the group consisting of D-amino acid oxidase (DAAO), or any
flavin dependent oxidoreductase enzyme.
[0062] In another embodiment of the disclosure any preceding
embodiment is where said peroxide-generating enzyme is NADH Oxidase
and said optional recycling enzyme is Alcohol Dehydrogenase
(ADH).
[0063] In another embodiment of the disclosure any preceding
embodiment is where said first binding ligand and said second
binding ligand are independently chosen from the group consisting
of monoclonal antibodies, fragments of monoclonal antibodies,
polyclonal antibodies, fragments of polyclonal antibodies,
proteins, and peptides.
[0064] In one embodiment of the disclosure any preceding embodiment
is where said EAM comprises a transition metal. In one embodiment,
said transition metal is chosen from the group consisting of iron,
ruthenium and osmium.
[0065] In one embodiment of the disclosure any preceding embodiment
is said EAM is chosen from the group consisting of ferrocene and
substituted ferrocene.
[0066] In one embodiment a protein target can be detected utilizing
a sandwich based assay. The sample is added to a first binding
ligand attached to a solid support. Target molecule in sample binds
capture ligand forming a first complex. Test sample and excess
first binding ligand are washed away. A second capture ligand
comprising an intermediary enzyme of a peroxide generating system
tag is added to first complex. Second binding ligand binds target
on first complex forming a second complex. Excess second binding
ligand is washed away. Substrate for intermediary enzyme (e.g.,
FADP, NADP), oxidase enzyme (e.g., D-amino acid oxidase, NADH
oxidase) and optionally cycling enzymes (Alcohol Dehyrodgenase) are
added to the complexes and H.sub.2O.sub.2 is generated. The signal
output is directly proportional to the concentration of target in
the test sample. The higher the signal measured the higher the
concentration of target molecule in the test sample.
[0067] In another aspect, the invention provides compositions
comprising (a) a first solid support comprising an electrode
comprising: (i) a self-assembled monolayer (SAM); (ii) a covalently
attached electroactive active moiety (EAM) comprising a transition
metal complex comprising a self-immolative moiety and a peroxide
sensitive moiety (PSM), wherein said first EAM has a first E.sup.0;
and (b) a second solid support comprising of a peroxide-generating
enzyme that generates peroxide in the presence of a substrate for
said peroxide-generating enzyme.
[0068] In one embodiment the reaction mechanism for representative
ferrocene-based EAMs that undergo a peroxide-triggered change in
apparent formal potential (E-TRACE) follow the following steps:
[0069] a) starting ferrocenyl EAM that contains an
electron-withdrawing carbamate-linked boronate ester-substituted
ligand; and [0070] b) reaction with peroxide leads to an
electron-donating amino ligand on the ferrocene which results in a
distinct the redox potential from starting species.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIG. 1. illustrates structure of electroactive molecule
(EAM) 1 and mechanism of peroxide-induced ligand dissociation. The
change in ligand electronics is responsible for the shift in redox
potential.
[0072] FIG. 2. illustrates the synthetic scheme of one of the
embodiments of the invention as depicted in FIG. 1.
[0073] FIG. 3. shows solution CV data for EAM 1 and a control
compound following H.sub.2O.sub.2 induced cleavage of the POM
ligand. The change in E0 following the self-immolative process is
331 mV. Experiments were run in THF with TBAClO.sub.4 (150 mM)
supporting electrolyte using a carbon working electrode, Ag/AgCl
reference electrode, and a Pt wire counter electrode.
[0074] FIG. 4. shows overlaid cyclic voltammograms from a SAM of
EAM 1 before (dotted) and after solid incubation with 1 mM hydrogen
peroxide in NaHCO.sub.3 buffer (pH 8.5) for 10 min, followed by a
5-min wash in Na.sub.2CO.sub.3 buffer (pH 10.1; lower peaks).
Supporting electrolyte was 1M LiClO.sub.4, silver quasi reference
electrode, platinum wire counter electrode. Scan rate was 10000
mV/s.
[0075] FIG. 5. shows overlaid cyclic voltammograms from a SAM of
EAM 1 before (dotted) and after (solid) incubation with 1 mM
glucose and 100 uM glucose oxidase in NaHCO.sub.3 buffer (pH 8.5)
for 10 min, followed by a 5 min wash in Na.sub.2CO.sub.3 buffer (pH
10.1). Supporting electrolyte was 1M LiClO.sub.4, silver quasi
reference electrode, platinum wire counter electrode. Scan rate was
10000 mV/s.
[0076] FIG. 6. Illustrates a sample self-immolative spacer groups
based on substituted quinone methides.
[0077] FIG. 7. shows cyclic voltamogram for SAM of EAM 1 following
antibody sandwich formation with human cardiac troponin I (10
ng/mL) before (dotted) and after (solid) incubation with glucose
for 10 min. Inset shows the peak at -0.10V magnified.
[0078] FIG. 8. depicts a variety of self-immolative moieties which
find use in the present invention. "PSM" stands for "peroxide
sensitive moiety" and "EAM" stands for "electroactive moiety". As
is shown in the figures, a variety of monomeric self-immolative
moieties (sometimes referred to herein as "SIM") can be used;
Figure XA depicts a first type of self-immolative moiety, which
relies on the PSM contributing an --OH group upon contact with
peroxide, resulting a phenol-based linker that releases from the
EAM. n can be an integer of 1 or higher, with from 1 to 5 finding
particular use in the invention. m is an integer of at least one;
as will be appreciated by those in the art, m will depend on the
transitional metal complex used and the number of positions in the
EAM; for example, when a metallocene such as ferrocene is used,
there can be up to 5 PSM-SIMs per cyclopentadienyl ring, with at
least one of the positions on one of the rings being used for
attachment to the electrode. Figures B, C and D show multimers of
SIMs. X can be --NH-- or --O--.
[0079] FIG. 9. depicts additional peroxide sensitive moieties.
Figure A depicts the PSB ether (parasiletanylbenzyl) moiety and
Figure B depicts the pentafluorophenylsulfonate (PFPS) moiety. As
shown in Figure (next), there can be more than one self-immolative
moiety per EAM and/or more than one PSM-SIM per EAM. As for the
boron containing PSMs, there can be multiple PSB ethers or PFPS
moieties per EAM as well.
[0080] FIG. 10. depicts a ferrocene that has R groups. The moiety
shown has the attachment linker and the self-immolative moiety and
the peroxide sensitive moiety on different rings, although as
described herein, they can be on the same ring. In addition, any or
all of the R groups can be an additional -SIM-PSM substituent, as
well as traditional substituents (alkyl (including substituted
alkyl, heteroalkyl, cyclic alkyl, etc.), aryl (including
substituted aryl and heteroaryl), etc.).
[0081] FIG. 11 is similar to FIG. 1 in showing the reaction
mechanism for a representative ferrocene based EAM that undergoes a
peroxide-triggered change in the apparent formal potential. (A) is
the starting ferrocenyl EAM that contains an electron-withdrawing
carbamate-linked boronate ester-substituted ligand. (B) Shows that
the reaction with peroxide leads to an electron-donating amino
ligand on the ferrocene which changes the redox
potential)(E.sup.0).
[0082] FIG. 12 shows example Cyclic Voltammagrams of typical
response prior to and after reaction of hydrogen peroxide with
electro-active moiety (EAM) on a self-assembled monolayer.
[0083] FIG. 13. illustrates typical dose response to hydrogen
peroxide titration reaction on a E-TRACE modified self-assembled
monolayer (SAM).
[0084] FIG. 14. shows cascade signal amplification schematic for
NADH detection via E-TRACE.
[0085] FIG. 15. shows cascade signal amplification schematic for
ATP detection via E-TRACE.
[0086] FIG. 16. shows cascade signal amplification strategies for
AP-based cascade amplification for small molecules. A competitive
assay is described based on the displacement of biotinylated small
molecules. The peroxide generated is detected via E-TRACE.
[0087] FIG. 17. shows FADP-based cascade amplification for protein
targets. The peroxide generated is detected via E-TRACE.
[0088] FIG. 18. shows NADP-based cascade amplification for protein
targets. The peroxide generated is detected via E-TRACE.
[0089] FIG. 19. illustrates calibration curve for a serial dilution
of NADH in buffer following the signal amplification for NADH with
the E-TRACE assay. The limit of detection is improved one thousand
fold from 3 uM using only NADH oxidase to 1 nM using the NADH/NAD
cycling amplification approach using Alcohol Dehydrogenase.
[0090] FIG. 20. illustrates calibration curve for a serial dilution
of ATP in buffer following the signal amplification for ATP with
the E-TRACE assay. The limit of detection is improved nearly one
thousand fold from 1 uM using Glycerol Kinase/Glycerol3-Phosphate
oxidase to the 3 nM using the ATP/ADP cycling amplification
approach with Creatine Kinase.
[0091] FIG. 21. shows a typical calibration curve expected for a
serial dilution of a small molecule using a competitive assay that
utilizes the cascade signal amplification approach.
[0092] FIG. 22. A. illustrates calibration curve for a serial
dilution of cTnl in serum following the FADP-based cascade
amplification E-TRACE assay. The limit of detection is improved
many fold from a 100 ng/mL using the unamplified approach to 3
pg/mL using the amplified approach. B. Correlation study measuring
levels of cTnl in clinical samples using the E-TRACE assay approach
comparing it to the data provided from a clinical
immunoanalyzer.
[0093] FIG. 23. A. illustrates calibration curve for a serial
dilution of thyroid stimulating hormone in serum following the
FADP-based cascade amplification E-TRACE assay. B. Correlation
study measuring levels of TSH clinical samples using the E-TRACE
assay approach comparing it to the data provided from a clinical
immunoanalyzer.
[0094] FIG. 24. illustrates a calibration curve obtained using the
NADP-based cascade amplification for a serial dilution of Alkaline
Phosphatase.
DETAILED DESCRIPTION OF THE INVENTION
[0095] U.S. patent application Ser. Nos. 13/653,931, 12/253,828,
12/253,875, 12/853,204, 61/332,565, 61/347,121, 61/394,458,
61/366,013 and 61/407,828 are all incorporated by reference in
their entirety.
Overview
[0096] The sensitivities of E-TRACE assays for proteins, DNA, and
small molecules may be exponentially enhanced through signal
amplification strategies that rely on target-dependent enzyme
cascades for generating hydrogen peroxide. FIG. 1a shows a general
approach for cascade signal amplification based on an alkaline
phosphatase (AP)-tagged sandwich immunocomplex with a protein
target. A similar colorimetric assay has been reported previously.1
In this system AP catalyzes the dephosphorylation of FADP to yield
FAD, an enzyme cofactor that turns "on" a dormant apo-D-amino acid
oxidase (D-AAO). In turn, each active D-AAO generated oxidizes
D-proline and produces hydrogen peroxide which is detected using
the Ohmx E-TRACE technology, which is described in U.S. Patent
Publication No. US 20120181186, filed Jan. 19, 2012 which claims
the benefit of priority to U.S. provisional application nos.
61/434,122, filed Jan. 19, 2011 and 61/523,679, filed Aug. 15, 2011
and Ser. No. 12/853,204, filed Aug. 9, 2010, which claims the
benefit of priority to U.S. provisional application nos.
61/232,339, filed Aug. 7, 2009, and in U.S. patent application Ser.
No. 13/653,931, filed Oct. 17, 2012, all which are incorporated by
reference in their entirety. An example protein target detected in
this manner is shown in FIG. 1b, where a dilution series of cardiac
troponin-I (cTnl) in serum is analyzed. The data suggest a
detection limit in the low pg/mL regime is possible for cTnl in
serum using this E-TRACE amplification assay.
[0097] The assay relies on the use of an electroactive moiety
("EAM"), which is attached to the electrode and comprises a
self-immolative moiety, whose presence gives the EAM a first
E.sup.0, whose absence, upon irreversible cleavage, gives the EAM a
second E.sup.0. The electrode also contains capture binding ligands
that will bind the target analyte upon its introduction. A soluble
capture ligand is introduced, which also binds the target analyte
and comprises a peroxide generating moiety, such as a glucose
oxidase enzyme. Upon the addition of oxygen and a substrate for the
peroxidase generating moiety (e.g., an oxygen saturated buffer and
glucose, in the case of a glucose oxidase enzyme as the peroxidase
generating moiety) peroxide is generated, attacking the
self-immolative moiety and causing the removal of the
self-immolative moiety from the EAM, which results in a change in
the E.sup.0 of the EAM. This difference is detected, and if such a
change occurs, it is an indication of the presence of the target
analyte.
[0098] Thus, to determine whether a target analyte is present in
the sample, the sample is applied to the detection electrode
surface, optionally washed, and an oxidase enzyme-conjugated
secondary binding ligand (for example, an antibody) that binds an
alternative epitope of the target analyte is added, creating a
"sandwich assay" format with the target. The surface is optionally
washed, and treated with an oxygen-saturated buffer containing a
high concentration of glucose. The presence of the substrate
oxidase enzyme (sometimes referred to herein as "SOX") on the
surface results in the enzymatic creation of hydrogen peroxide in
solution which diffuses to the monolayer surface and triggers a
chemical elimination reaction ("self-immolative" reaction) in the
immobilized EAMs. This irreversible elimination reaction changes
the electronic environment of the EAM, for example by altering the
"R" groups (e.g., substituent groups) of the transition metal
complex, thus shifting the apparent formal potential of the EAM to
a second E.sup.0 to signal the presence of the target.
[0099] Accordingly, the present invention provides methods and
compositions for detecting target analytes in samples. The single
measurement approach for detecting directly the percentage of
glycated Hemoglobin is not affected by the amount of total
Hemoglobin present in the sample. Since total Hemoglobin, can vary
physiologically from 5-20 g/dL, a direct measurement of the same
percentage of glycated Hemoglobin across this range is feasible
with this approach.
[0100] Target Analytes
[0101] By "target analyte" or "analyte" or grammatical equivalents
herein is meant any molecule, compound or particle to be detected.
Target analytes bind to binding ligands (both capture and soluble
binding ligands), as is more fully described below.
[0102] Suitable analytes include organic and inorganic molecules,
including biomolecules. In a preferred embodiment, the analyte may
be an environmental pollutant (including pesticides, insecticides,
toxins, etc.); a chemical (including solvents, polymers, organic
materials, etc.); therapeutic molecules (including therapeutic and
abused drugs, antibiotics, etc.); biomolecules (including hormones,
cytokines, proteins, lipids, carbohydrates, cellular membrane
antigens and receptors (neural, hormonal, nutrient, and cell
surface receptors) or their ligands, etc); whole cells (including
procaryotic (such as pathogenic bacteria) and eukaryotic cells,
including mammalian tumor cells); viruses (including retroviruses,
herpesviruses, adenoviruses, lentiviruses, etc.); and spores;
etc.
[0103] In some embodiments, the target analyte is a protein. As
will be appreciated by those in the art, there are a large number
of possible proteinaceous target analytes that may be detected
using the present invention. By "proteins" or grammatical
equivalents herein is meant proteins, oligopeptides and peptides,
derivatives and analogs, including proteins containing
non-naturally occurring amino acids and amino acid analogs, and
peptidomimetic structures. The side chains may be in either the (R)
or the (S) configuration. In a preferred embodiment, the amino
acids are in the (S) or L configuration. As discussed below, when
the protein is used as a binding ligand, it may be desirable to
utilize protein analogs to retard degradation by sample
contaminants.
[0104] Suitable protein target analytes include, but are not
limited to, (1) immunoglobulins, particularly IgEs, IgGs and IgMs,
and particularly therapeutically or diagnostically relevant
antibodies, including but not limited to, for example, antibodies
to human albumin, apolipoproteins (including apolipoprotein E),
human chorionic gonadotropin, cortisol, a-fetoprotein, thyroxin,
thyroid stimulating hormone (TSH), antithrombin, antibodies to
pharmaceuticals (including antieptileptic drugs (phenytoin,
primidone, carbariezepin, ethosuximide, valproic acid, and
phenobarbitol), cardioactive drugs (digoxin, lidocaine,
procainamide, and disopyramide), bronchodilators (theophylline),
antibiotics (chloramphenicol, sulfonamides), antidepressants,
immunosuppresants, abused drugs (amphetamine, methamphetamine,
cannabinoids, cocaine and opiates) and antibodies to any number of
viruses (including orthomyxoviruses, (e.g. influenza virus),
paramyxoviruses (e.g., respiratory syncytial virus, mumps virus,
measles virus), adenoviruses, rhinoviruses, coronaviruses,
reoviruses, togaviruses (e.g. rubella virus), parvoviruses,
poxviruses (e.g. variola virus, vaccinia virus), enteroviruses
(e.g. poliovirus, coxsackievirus), hepatitis viruses (including A,
B and C), herpesviruses (e.g. Herpes simplex virus, varicella
zoster virus, cytomegalovirus, Epstein Barr virus), rotaviruses,
Norwalk viruses, hantavirus, arenavirus, rhabdovirus (e.g. rabies
virus), retroviruses (including HIV, HTLV I and II), papovaviruses
(e.g. papillomavirus), polyomaviruses, and picornaviruses, and the
like), and bacteria (including a wide variety of pathogenic and non
pathogenic prokaryotes of interest including Bacillus; Vibrio, e.g.
V. cholerae; Escherichia, e.g. Enterotoxigenic E. coli, Shigella,
e.g. S. dysenteriae; Salmonella, e.g. S. typhi; Mycobacterium e.g.
M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C.
tetani, C. difficile, C. perfringens; Cornyebacterium, e.g. C.
diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae;
Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;
Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G.
lambliaY. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;
Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;
Treponema, e.g. T. palladium; and the like); (2) enzymes (and other
proteins), including but not limited to, enzymes used as indicators
of or treatment for heart disease, including creatine kinase,
lactate dehydrogenase, aspartate amino transferase, troponin T,
myoglobin, fibrinogen, cholesterol, triglycerides, thrombin, tissue
plasminogen activator (tPA); pancreatic disease indicators
including amylase, lipase, chymotrypsin and trypsin; liver function
enzymes and proteins including cholinesterase, bilirubin, and
alkaline phosphotase; aldolase, prostatic acid phosphatase,
terminal deoxynucleotidyl transferase, and bacterial and viral
enzymes such as HIV protease; (3) hormones and cytokines (many of
which serve as ligands for cellular receptors) such as
erythropoietin (EPO), thrombopoietin (TPO), the interleukins
(including IL-1 through IL-17), insulin, insulin-like growth
factors (including IGF-1 and -2), epidermal growth factor (EGF),
transforming growth factors (including TGF-.alpha. and TGF-.beta.),
human growth hormone, transferrin, epidermal growth factor (EGF),
low density lipoprotein, high density lipoprotein, leptin, VEGF,
PDGF, ciliary neurotrophic factor, prolactin, adrenocorticotropic
hormone (ACTH), calcitonin, human chorionic gonadotropin, cotrisol,
estradiol, follicle stimulating hormone (FSH), thyroid-stimulating
hormone (TSH), leutinzing hormone (LH), progeterone, testosterone,
and (4) other proteins (including a-fetoprotein, carcinoembryonic
antigen CEA.
[0105] In addition, any of the biomolecules for which antibodies
may be detected may be detected directly as well; that is,
detection of virus or bacterial cells, therapeutic and abused
drugs, etc., may be done directly.
[0106] Suitable target analytes include carbohydrates, including
but not limited to, markers for breast cancer (CA15-3, CA 549, CA
27.29), mucin-like carcinoma associated antigen (MCA), ovarian
cancer (CAl25), pancreatic cancer (DE-PAN-2), and colorectal and
pancreatic cancer (CA 19, CA 50, CA242).
[0107] Targets include small molecules such as glucose or
cholesterol or ATP, FADP, NADH and other metabolites, or hormones
such as testosterones etc, or proteins such as thyroid stimulating
hormone, troponin I etc.
[0108] In one embodiment, a single measurement method for
determining the proportion of target analyte in a sample can be
performed according to the methods described herein by an
electrochemical measurement using the enzyme-triggered redox
altering chemical elimination (E-TRACE) reaction, or a standard
immunoassay optical test detecting H.sub.2O.sub.2 in solution and
is described in the following steps:
[0109] Step 1: Modification with Primary Antibody:
[0110] The surface of the electrochemical platform is modified to
include the sensing molecule (EAM) for the E-TRACE detection of
peroxide. Additionally a second solid support is modified with a
capture probe. This capture probe, e.g., antibody, binds
selectively and equivalently to all variant types of target (e.g.,
hemoglobin including hemoglobin and glycated hemoglobin). As
defined herein, the terms "binds selectively" means binding to a
predetermined target (e.g. total hemoglobin including hemoglobin
A1c) and "binds equivalently" mean non-preferentially to both the
protein (e.g., hemoglobin) and the glycated protein (e.g.,
hemoglobin A1c).
[0111] Step 2: Addition of Target:
[0112] Target can be small molecule or protein. In certain
embodiments, the primary binding occurs and is assumed to saturate
nearly all binding sites on the surface of the secondary support.
The importance of this is that samples with different total target
concentrations will still yield a representative proportion of the
target analyte bound to the surface.
[0113] Step 3: Addition of Detection Antibody:
[0114] In certain embodiments, the secondary antibody is introduced
to the surface and only binds to the immobilized target analyte.
This means the ELISA-like sandwich complex only forms on sites
occupied by target analyte and not on sites occupied by non-target
(e.g., non-glycated hemoglobin).
[0115] Step 4: Signal Transduction and Detection:
[0116] The anti-target antibody that selectively binds to target is
labeled with an intermediary enzyme of a peroxide generating
system, e.g., an oxidase enzyme (SOx). The intermediary enzyme
label, generates a product that is a cofactor or substrate for an
oxidase enzyme which produces hydrogen peroxide. The hydrogen
peroxide generated reacts with the electrochemical surface of the
electrochemical platform to provide an electrochemical signal.
[0117] The amount of signal is directly correlated to the number of
sandwich complexes, which in turn is dependent on how much Target
immobilized on the surface. Since the amount of immobilized Target
is directly dependent on the percentage of Target to total
hemoglobin in the original sample, the signal observed provides an
assessment of the ratio (percentage) of hemoglobin Target to total
hemoglobin.
[0118] For HbTarget, one of the binding ligands, either the capture
binding ligand or the soluble binding ligand has specificity for
the glycated form of hemoglobin. That is, in one embodiment, the
capture binding ligand can bind either form of hemoglobin; after
washing the surface, a soluble binding ligand that has specificity
only for the glycated form (i.e. HbTarget) with the
peroxide-generating moiety is added. Alternatively, the capture
binding ligand has specificity for Hb1Ac over other forms of
hemoglobin, and a soluble capture ligand without such specificity
can be used after appropriate washing of the surface. This approach
can be used for other target analytes where detection of either the
glycated or nonglycated form is desired. As will be appreciated by
those in the art, there are also target analytes for which
detection of both forms is desired, and in those embodiments, using
binding ligands that do not have specificity for one or the other
is used.
[0119] This single-measurement detection of Target coupled to an
electro-active SAM provides a less complex means for determining
the proportion of Target to total hemoglobin in a sample. It offers
the advantages of not needing to perform two measurements, as well
as eliminating optical measurements, multiple antibody pairs, or
percentage calculation algorithms, that introduces further error,
based on two separate measurements for Target and total
hemoglobin.
[0120] Target analytes of the disclosure may be labeled. Thus, by
"labeled target analyte" herein is meant a target analyte that is
labeled with a member of a specific binding pair.
[0121] Samples
[0122] The target analytes are generally present in samples. As
will be appreciated by those in the art, the sample solution may
comprise any number of things, including, but not limited to,
bodily fluids (including, but not limited to, blood, urine, serum,
lymph, saliva, anal and vaginal secretions, perspiration and semen,
of virtually any organism, with mammalian samples being preferred
and human samples being particularly preferred); environmental
samples (including, but not limited to, air, agricultural, water
and soil samples); plant materials; biological warfare agent
samples; research samples, purified samples, raw samples, etc.; as
will be appreciated by those in the art, virtually any experimental
manipulation may have been done on the sample. Some embodiments
utilize target samples from stored (e.g. frozen and/or archived) or
fresh tissues. Paraffin-embedded samples are of particular use in
many embodiments, as these samples can be very useful, due to the
presence of additional data associated with the samples, such as
diagnosis and prognosis. Fixed and paraffin-embedded tissue samples
as described herein refers to storable or archival tissue samples.
Most patient-derived pathological samples are routinely fixed and
paraffin-embedded to allow for histological analysis and subsequent
archival storage.
[0123] Solid Supports
[0124] The target analytes are detected using solid supports
comprising electrodes. By "substrate" or "solid support" or other
grammatical equivalents herein is meant any material that can be
modified to contain discrete individual sites appropriate of the
attachment or association of capture ligands. Suitable substrates
include metal surfaces such as gold, electrodes as defined below,
glass and modified or functionalized glass, fiberglass, teflon,
ceramics, mica, plastic (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polyimide, polycarbonate,
polyurethanes, Teflon.TM., and derivatives thereof, etc.), GETEK (a
blend of polypropylene oxide and fiberglass), etc, polysaccharides,
nylon or nitrocellulose, resins, silica or silica-based materials
including silicon and modified silicon, carbon, metals, inorganic
glasses and a variety of other polymers, with printed circuit board
(PCB) materials being particularly preferred.
[0125] The present system finds particular utility in array
formats, i.e. wherein there is a matrix of addressable detection
electrodes (herein generally referred to "pads", "addresses" or
"micro-locations"). By "array" herein is meant a plurality of
capture ligands in an array format; the size of the array will
depend on the composition and end use of the array. Arrays
containing from about 2 different capture substrates to many
thousands can be made.
[0126] In a preferred embodiment, the detection electrodes are
formed on a substrate. In addition, the discussion herein is
generally directed to the use of gold electrodes, but as will be
appreciated by those in the art, other electrodes can be used as
well. The substrate can comprise a wide variety of materials, as
outlined herein and in the cited references.
[0127] In general, preferred materials include printed circuit
board materials. Circuit board materials are those that comprise an
insulating substrate that is coated with a conducting layer and
processed using lithography techniques, particularly
photolithography techniques, to form the patterns of electrodes and
interconnects (sometimes referred to in the art as interconnections
or leads). The insulating substrate is generally, but not always, a
polymer. As is known in the art, one or a plurality of layers may
be used, to make either "two dimensional" (e.g. all electrodes and
interconnections in a plane) or "three dimensional" (wherein the
electrodes are on one surface and the interconnects may go through
the board to the other side or wherein electrodes are on a
plurality of surfaces) boards. Three dimensional systems frequently
rely on the use of drilling or etching, followed by electroplating
with a metal such as copper, such that the "through board"
interconnections are made. Circuit board materials are often
provided with a foil already attached to the substrate, such as a
copper foil, with additional copper added as needed (for example
for interconnections), for example by electroplating. The copper
surface may then need to be roughened, for example through etching,
to allow attachment of the adhesion layer.
[0128] Accordingly, in a preferred embodiment, the present
invention provides biochips (sometimes referred to herein "chips")
that comprise substrates comprising a plurality of electrodes,
preferably gold electrodes. The number of electrodes is as outlined
for arrays. Each electrode preferably comprises a self-assembled
monolayer as outlined herein. In a preferred embodiment, one of the
monolayer-forming species comprises a capture ligand as outlined
herein. In addition, each electrode has an interconnection, that is
attached to the electrode at one end and is ultimately attached to
a device that can control the electrode. That is, each electrode is
independently addressable.
[0129] Finally, the compositions of the invention can include a
wide variety of additional components, including microfluidic
components and robotic components (see for example U.S. Pat. Nos.
6,942,771 and 7,312,087 and related cases, both of which are hereby
incorporated by reference in its entirety), and detection systems
including computers utilizing signal processing techniques (see for
example U.S. Pat. No. 6,740,518, hereby incorporated by reference
in its entirety).
[0130] Electrodes
[0131] The solid supports of the invention comprise electrodes. By
"electrodes" herein is meant a composition, which, when connected
to an electronic device, is able to sense a current or charge and
convert it to a signal. Preferred electrodes are known in the art
and include, but are not limited to, certain metals and their
oxides, including gold; platinum; palladium; silicon; aluminum;
metal oxide electrodes including platinum oxide, titanium oxide,
tin oxide, indium tin oxide, palladium oxide, silicon oxide,
aluminum oxide, molybdenum oxide (Mo2O6), tungsten oxide (WO3) and
ruthenium oxides; and carbon (including glassy carbon electrodes,
graphite and carbon paste). Preferred electrodes include gold,
silicon, carbon and metal oxide electrodes, with gold being
particularly preferred.
[0132] The electrodes described herein are depicted as a flat
surface, which is only one of the possible conformations of the
electrode and is for schematic purposes only. The conformation of
the electrode will vary with the detection method used.
[0133] The electrodes of the invention are generally incorporated
into biochip cartridges and can take a wide variety of
configurations, and can include working and reference electrodes,
interconnects (including "through board" interconnects), and
microfluidic components. See for example U.S. Pat. No. 7,312,087,
incorporated herein by reference in its entirety. In addition, the
biochips generally include a working electrode with the components
described herein, a reference electrode, and a counter/auxiliary
electrode.
[0134] The biochip cartridges include substrates comprising the
arrays of biomolecules, and can be configured in a variety of ways.
For example, the chips can include reaction chambers with inlet and
outlet ports for the introduction and removal of reagents. In
addition, the cartridges can include caps or lids that have
microfluidic components, such that the sample can be introduced,
reagents added, reactions done, and then the sample is added to the
reaction chamber comprising the array for detection.
[0135] Self Assembled Monolayers
[0136] The electrodes comprise a self assembled monolayer ("SAM").
By "monolayer" or "self-assembled monolayer" or "SAM" herein is
meant a relatively ordered assembly of molecules spontaneously
chemisorbed on a surface, in which the molecules are oriented
approximately parallel to each other and roughly perpendicular to
the surface. Each of the molecules includes a functional group that
adheres to the surface, and a portion that interacts with
neighboring molecules in the monolayer to form the relatively
ordered array. A "mixed" monolayer comprises a heterogeneous
monolayer, that is, where at least two different molecules make up
the monolayer. As outlined herein, the use of a monolayer reduces
the amount of non-specific binding of biomolecules to the surface,
and, in the case of nucleic acids, increases the efficiency of
oligonucleotide hybridization as a result of the distance of the
oligonucleotide from the electrode. Thus, a monolayer facilitates
the maintenance of the target enzyme away from the electrode
surface. In addition, a monolayer serves to keep charge carriers
away from the surface of the electrode. Thus, this layer helps to
prevent electrical contact between the electrodes and the ReAMs, or
between the electrode and charged species within the solvent. Such
contact can result in a direct "short circuit" or an indirect short
circuit via charged species which may be present in the sample.
Accordingly, the monolayer is preferably tightly packed in a
uniform layer on the electrode surface, such that a minimum of
"holes" exist. The monolayer thus serves as a physical barrier to
block solvent accessibility to the electrode.
[0137] In some embodiments, the monolayer comprises conductive
oligomers, and in particular, conductive oligomers are generally
used to attach the EAM to the electrode surface, as described
below. By "conductive oligomer" herein is meant a substantially
conducting oligomer, preferably linear, some embodiments of which
are referred to in the literature as "molecular wires". By
"substantially conducting" herein is meant that the oligomer is
capable of transferring electrons at 100 Hz. Generally, the
conductive oligomer has substantially overlapping .pi.-orbitals,
i.e. conjugated .pi.-orbitals, as between the monomeric units of
the conductive oligomer, although the conductive oligomer may also
contain one or more sigma (.sigma.) bonds. Additionally, a
conductive oligomer may be defined functionally by its ability to
inject or receive electrons into or from an associated EAM.
Furthermore, the conductive oligomer is more conductive than the
insulators as defined herein. Additionally, the conductive
oligomers of the invention are to be distinguished from
electroactive polymers, that themselves may donate or accept
electrons.
[0138] A more detailed description of conductive oligomers is found
in WO/1999/57317, herein incorporated by reference in its entirety.
In particular, the conductive oligomers as shown in Structures 1 to
9 on page 14 to 21 of WO/1999/57317 find use in the present
invention. In some embodiments, the conductive oligomer has the
following structure:
##STR00001##
[0139] In addition, the terminus of at least some of the conductive
oligomers in the monolayer is electronically exposed. By
"electronically exposed" herein is meant that upon the placement of
an EAM in close proximity to the terminus, and after initiation
with the appropriate signal, a signal dependent on the presence of
the EAM may be detected. The conductive oligomers may or may not
have terminal groups. Thus, in a preferred embodiment, there is no
additional terminal group, and the conductive oligomer terminates
with a terminal group; for example, such as an acetylene bond.
Alternatively, in some embodiments, a terminal group is added,
sometimes depicted herein as "Q". A terminal group may be used for
several reasons; for example, to contribute to the electronic
availability of the conductive oligomer for detection of EAMs, or
to alter the surface of the SAM for other reasons, for example to
prevent non-specific binding. For example, there may be negatively
charged groups on the terminus to form a negatively charged surface
such that when the target analyte is nucleic acid such as DNA or
RNA, the nucleic acid is repelled or prevented from lying down on
the surface, to facilitate hybridization. Preferred terminal groups
include --NH, --OH, --COOH, and alkyl groups such as --CH.sub.3,
and (poly)alkyloxides such as (poly)ethylene glycol, with
--OCH.sub.2CH.sub.2OH, --(OCH.sub.2CH.sub.2O).sub.2H,
--(OCH.sub.2CH.sub.2O).sub.3H, and --(OCH.sub.2CH.sub.2O).sub.4H
being preferred.
[0140] In one embodiment, it is possible to use mixtures of
conductive oligomers with different types of terminal groups. Thus,
for example, some of the terminal groups may facilitate detection,
and some may prevent non-specific binding.
[0141] In some embodiments, the electrode further comprises a
passivation agent, preferably in the form of a monolayer on the
electrode surface. For some analytes the efficiency of analyte
binding (i.e. hybridization) may increase when the binding ligand
is at a distance from the electrode. In addition, the presence of a
monolayer can decrease non-specific binding to the surface (which
can be further facilitated by the use of a terminal group, outlined
herein. A passivation agent layer facilitates the maintenance of
the binding ligand and/or analyte away from the electrode surface.
In addition, a passivation agent serves to keep charge carriers
away from the surface of the electrode. Thus, this layer helps to
prevent electrical contact between the electrodes and the electron
transfer moieties, or between the electrode and charged species
within the solvent. Such contact can result in a direct "short
circuit" or an indirect short circuit via charged species which may
be present in the sample. Accordingly, the monolayer of passivation
agents is preferably tightly packed in a uniform layer on the
electrode surface, such that a minimum of "holes" exist.
Alternatively, the passivation agent may not be in the form of a
monolayer, but may be present to help the packing of the conductive
oligomers or other characteristics.
[0142] The passivation agents thus serve as a physical barrier to
block solvent accessibility to the electrode. As such, the
passivation agents themselves may in fact be either (1) conducting
or (2) nonconducting, i.e. insulating, molecules. Thus, in one
embodiment, the passivation agents are conductive oligomers, as
described herein, with or without a terminal group to block or
decrease the transfer of charge to the electrode. Other passivation
agents which may be conductive include oligomers of
--(CF.sub.2).sub.n--, --(CHF).sub.n-- and --(CFR).sub.n--. In a
preferred embodiment, the passivation agents are insulator
moieties.
[0143] In some embodiments, the monolayers comprise insulators. An
"insulator" is a substantially nonconducting oligomer, preferably
linear. By "substantially nonconducting" herein is meant that the
rate of electron transfer through the insulator is slower than the
rate of electron transfer through the conductive oligomer. Stated
differently, the electrical resistance of the insulator is higher
than the electrical resistance of the conductive oligomer. It
should be noted however that even oligomers generally considered to
be insulators, such as --(CH.sub.2).sub.16 molecules, still may
transfer electrons, albeit at a slow rate.
[0144] In some embodiments, the insulators have a conductivity, S,
of about 10.sup.-7 .OMEGA.-1 cm.sup.-1 or lower, with less than
about 10.sup.-8.OMEGA..sup.-1 cm.sup.-1 being preferred. Gardner et
al., Sensors and Actuators A 51 (1995) 57-66, incorporated herein
by reference.
[0145] Generally, insulators are alkyl or heteroalkyl oligomers or
moieties with sigma bonds, although any particular insulator
molecule may contain aromatic groups or one or more conjugated
bonds. By "heteroalkyl" herein is meant an alkyl group that has at
least one heteroatom, i.e. nitrogen, oxygen, sulfur, phosphorus,
silicon or boron included in the chain. Alternatively, the
insulator may be quite similar to a conductive oligomer with the
addition of one or more heteroatoms or bonds that serve to inhibit
or slow, preferably substantially, electron transfer. In some
embodiments the insulator comprises C6-C16 alkyl.
[0146] The passivation agents, including insulators, may be
substituted with R groups as defined herein to alter the packing of
the moieties or conductive oligomers on an electrode, the
hydrophilicity or hydrophobicity of the insulator, and the
flexibility, i.e. the rotational, torsional or longitudinal
flexibility of the insulator. For example, branched alkyl groups
may be used. In addition, the terminus of the passivation agent,
including insulators, may contain an additional group to influence
the exposed surface of the monolayer, sometimes referred to herein
as a terminal group ("TG"). For example, the addition of charged,
neutral or hydrophobic groups may be done to inhibit non-specific
binding from the sample, or to influence the kinetics of binding of
the analyte, etc. For example, there may be charged groups on the
terminus to form a charged surface to encourage or discourage
binding of certain target analytes or to repel or prevent from
lying down on the surface.
[0147] The length of the passivation agent will vary as needed.
Generally, the length of the passivation agents is similar to the
length of the conductive oligomers, as outlined above. In addition,
the conductive oligomers may be basically the same length as the
passivation agents or longer than them, resulting in the binding
ligands being more accessible to peroxide.
[0148] The monolayer may comprise a single type of passivation
agent, including insulators, or different types.
[0149] Suitable insulators are known in the art, and include, but
are not limited to, --(CH.sub.2).sub.n--, --(CRH).sub.n--, and
--(CR.sub.2).sub.n--, ethylene glycol or derivatives using other
heteroatoms in place of oxygen, i.e. nitrogen or sulfur (sulfur
derivatives are not preferred when the electrode is gold). In some
embodiments, the insulator comprises C6 to C16 alkyl.
[0150] In some embodiments, the electrode is a metal surface and
need not necessarily have interconnects or the ability to do
electrochemistry.
[0151] Electroactive Moieties
[0152] In addition to the SAMs, the electrodes comprise an EAM. By
"electroactive moiety (EAM)" or "transition metal complex" or
"redox active molecule" or "electron transfer moiety (ETM)" herein
is meant a metal-containing compound which is capable of reversibly
or semi-reversibly transferring one or more electrons. It is to be
understood that electron donor and acceptor capabilities are
relative; that is, a molecule which can lose an electron under
certain experimental conditions will be able to accept an electron
under different experimental conditions.
[0153] It is to be understood that the number of possible
transition metal complexes is very large, and that one skilled in
the art of electron transfer compounds will be able to utilize a
number of compounds in the present invention. By "transitional
metal" herein is meant metals whose atoms have a partial or
completed shell of electrons. Suitable transition metals for use in
the invention include, but are not limited to, cadmium (Cd), copper
(Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium
(Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt),
scandium (Sc), titanium (Ti), Vanadium (V), chromium (Cr),
manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc),
tungsten (W), and iridium (Ir). That is, the first series of
transition metals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt),
along with Fe, Re, W, Mo and Tc, find particular use in the present
invention. Metals that find use in the invention also are those
that do not change the number of coordination sites upon a change
in oxidation state, including ruthenium, osmium, iron, platinium
and palladium, with osmium, ruthenium and iron being especially
useful. Generally, transition metals are depicted herein (or in
incorporated references) as TM or M.
[0154] The transitional metal and the coordinating ligands form a
metal complex. By "ligand" or "coordinating ligand" (depicted
herein or in incorporated references in the figures as "L") herein
is meant an atom, ion, molecule, or functional group that generally
donates one or more of its electrons through a coordinate covalent
bond to, or shares its electrons through a covalent bond with, one
or more central atoms or ions.
[0155] In some embodiments, small polar ligands are used; suitable
small polar ligands, generally depicted herein as "L", fall into
two general categories, as is more fully described herein. In one
embodiment, the small polar ligands will be effectively
irreversibly bound to the metal ion, due to their characteristics
as generally poor leaving groups or as good sigma donors, and the
identity of the metal. These ligands may be referred to as
"substitutionally inert". Alternatively, as is more fully described
below, the small polar ligands may be reversibly bound to the metal
ion, such that upon binding of a target analyte, the analyte may
provide one or more coordination atoms for the metal, effectively
replacing the small polar ligands, due to their good leaving group
properties or poor sigma donor properties. These ligands may be
referred to as "substitutionally labile". The ligands preferably
form dipoles, since this will contribute to a high solvent
reorganization energy.
[0156] Some of the structures of transitional metal complexes are
shown below:
##STR00002##
[0157] L are the co-ligands, that provide the coordination atoms
for the binding of the metal ion. As will be appreciated by those
in the art, the number and nature of the co-ligands will depend on
the coordination number of the metal ion. Mono-, di- or polydentate
co-ligands may be used at any position. Thus, for example, when the
metal has a coordination number of six, the L from the terminus of
the conductive oligomer, the L contributed from the nucleic acid,
and r, add up to six. Thus, when the metal has a coordination
number of six, r may range from zero (when all coordination atoms
are provided by the other two ligands) to four, when all the
co-ligands are monodentate. Thus generally, r will be from 0 to 8,
depending on the coordination number of the metal ion and the
choice of the other ligands.
[0158] In one embodiment, the metal ion has a coordination number
of six and both the ligand attached to the conductive oligomer and
the ligand attached to the nucleic acid are at least bidentate;
that is, r is preferably zero, one (i.e. the remaining co-ligand is
bidentate) or two (two monodentate co-ligands are used).
[0159] As will be appreciated in the art, the co-ligands can be the
same or different. Suitable ligands fall into two categories:
ligands which use nitrogen, oxygen, sulfur, carbon or phosphorus
atoms (depending on the metal ion) as the coordination atoms
(generally referred to in the literature as sigma (.sigma.) donors)
and organometallic ligands such as metallocene ligands (generally
referred to in the literature as pi (.pi.) donors, and depicted
herein as Lm). Suitable nitrogen donating ligands are well known in
the art and include, but are not limited to, cyano (C.ident.N),
NH2; NHR; NRR'; pyridine; pyrazine; isonicotinamide; imidazole;
bipyridine and substituted derivatives of bipyridine; terpyridine
and substituted derivatives; phenanthrolines, particularly
1,10-phenanthroline (abbreviated phen) and substituted derivatives
of phenanthrolines such as 4,7-dimethylphenanthroline and
dipyridol[3,2-a:2',3'-c]phenazine (abbreviated dppz);
dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene (abbreviated
hat); 9,10-phenanthrenequinone diimine (abbreviated phi);
1,4,5,8-tetraazaphenanthrene (abbreviated tap);
1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and
isocyanide. Substituted derivatives, including fused derivatives,
may also be used. In some embodiments, porphyrins and substituted
derivatives of the porphyrin family may be used. See for example,
Comprehensive Coordination Chemistry, Ed. Wilkinson et al.,
Pergammon Press, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898)
and 21.3 (pp 915-957), all of which are hereby expressly
incorporated by reference.
[0160] As will be appreciated in the art, any ligand
donor(1)-bridge-donor(2) where donor (1) binds to the metal and
donor(2) is available for interaction with the surrounding medium
(solvent, protein, etc) can be used in the present invention,
especially if donor(1) and donor(2) are coupled through a pi
system, as in cyanos (C is donor(1), N is donor(2), pi system is
the CN triple bond). One example is bipyrimidine, which looks much
like bipyridine but has N donors on the "back side" for
interactions with the medium. Additional co-ligands include, but
are not limited to cyanates, isocyanates (--N.dbd.C.dbd.O),
thiocyanates, isonitrile, N.sub.2, O.sub.2, carbonyl, halides,
alkoxyide, thiolates, amides, phosphides, and sulfur containing
compound such as sulfino, sulfonyl, sulfoamino, and sulfamoyl.
[0161] In some embodiments, multiple cyanos are used as co-ligand
to complex with different metals. For example, seven cyanos bind
Re(III); eight bind Mo(IV) and W(IV). Thus at Re(III) with 6 or
less cyanos and one or more L, or Mo(IV) or W(IV) with 7 or less
cyanos and one or more L can be used in the present invention. The
EAM with W(IV) system has particular advantages over the others
because it is more inert, easier to prepare, more favorable
reduction potential. Generally that a larger CN/L ratio will give
larger shifts.
[0162] Suitable sigma donating ligands using carbon, oxygen, sulfur
and phosphorus are known in the art. For example, suitable sigma
carbon donors are found in Cotton and Wilkenson, Advanced Organic
Chemistry, 5th Edition, John Wiley & Sons, 1988, hereby
incorporated by reference; see page 38, for example. Similarly,
suitable oxygen ligands include crown ethers, water and others
known in the art. Phosphines and substituted phosphines are also
suitable; see page 38 of Cotton and Wilkenson.
[0163] The oxygen, sulfur, phosphorus and nitrogen-donating ligands
are attached in such a manner as to allow the heteroatoms to serve
as coordination atoms.
[0164] In some embodiments, organometallic ligands are used. In
addition to purely organic compounds for use as redox moieties, and
various transition metal coordination complexes with .delta.-bonded
organic ligand with donor atoms as heterocyclic or exocyclic
substituents, there is available a wide variety of transition metal
organometallic compounds with .pi.-bonded organic ligands (see
Advanced Inorganic Chemistry, 5th Ed., Cotton & Wilkinson, John
Wiley & Sons, 1988, chapter 26; Organometallics, A Concise
Introduction, Elschenbroich et al., 2nd Ed., 1992, VCH; and
Comprehensive Organometallic Chemistry II, A Review of the
Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10
& 11, Pergamon Press, hereby expressly incorporated by
reference). Such organometallic ligands include cyclic aromatic
compounds such as the cyclopentadienide ion [C5H5 (-1)] and various
ring substituted and ring fused derivatives, such as the indenylide
(-1) ion, that yield a class of bis(cyclopentadieyl)metal
compounds, (i.e. the metallocenes); see for example Robins et al.,
J. Am. Chem. Soc. 104:1882-1893 (1982); and Gassman et al., J. Am.
Chem. Soc. 108:4228-4229 (1986), incorporated by reference. Of
these, ferrocene [(C.sub.5H.sub.5).sub.2 Fe] and its derivatives
are prototypical examples which have been used in a wide variety of
chemical (Connelly et al., Chem. Rev. 96:877-910 (1996),
incorporated by reference) and electrochemical (Geiger et al.,
Advances in Organometallic Chemistry 23:1-93; and Geiger et al.,
Advances in Organometallic Chemistry 24:87, incorporated by
reference) electron transfer or "redox" reactions. Metallocene
derivatives of a variety of the first, second and third row
transition metals are potential candidates as redox moieties that
are covalently attached to either the ribose ring or the nucleoside
base of nucleic acid. Other potentially suitable organometallic
ligands include cyclic arenes such as benzene, to yield
bis(arene)metal compounds and their ring substituted and ring fused
derivatives, of which bis(benzene)chromium is a prototypical
example. Other acyclic .pi.-bonded ligands such as the allyl(-1)
ion, or butadiene yield potentially suitable organometallic
compounds, and all such ligands, in conduction with other
.pi.-bonded and .delta.-bonded ligands constitute the general class
of organometallic compounds in which there is a metal to carbon
bond. Electrochemical studies of various dimers and oligomers of
such compounds with bridging organic ligands, and additional
non-bridging ligands, as well as with and without metal-metal bonds
are potential candidate redox moieties in nucleic acid
analysis.
[0165] When one or more of the co-ligands is an organometallic
ligand, the ligand is generally attached via one of the carbon
atoms of the organometallic ligand, although attachment may be via
other atoms for heterocyclic ligands. Preferred organometallic
ligands include metallocene ligands, including substituted
derivatives and the metalloceneophanes (see page 1174 of Cotton and
Wilkenson, supra). For example, derivatives of metallocene ligands
such as methylcyclopentadienyl, with multiple methyl groups being
preferred, such as pentamethylcyclopentadienyl, can be used to
increase the stability of the metallocene. In a preferred
embodiment, only one of the two metallocene ligands of a
metallocene are derivatized.
[0166] As described herein, any combination of ligands may be used.
Preferred combinations include: a) all ligands are nitrogen
donating ligands; b) all ligands are organometallic ligands; and c)
the ligand at the terminus of the conductive oligomer is a
metallocene ligand and the ligand provided by the nucleic acid is a
nitrogen donating ligand, with the other ligands, if needed, are
either nitrogen donating ligands or metallocene ligands, or a
mixture.
[0167] As a general rule, EAM comprising non-macrocyclic chelators
are bound to metal ions to form non-macrocyclic chelate compounds,
since the presence of the metal allows for multiple proligands to
bind together to give multiple oxidation states.
[0168] In some embodiments, nitrogen donating proligands are used.
Suitable nitrogen donating proligands are well known in the art and
include, but are not limited to, NH2; NHR; NRR'; pyridine;
pyrazine; isonicotinamide; imidazole; bipyridine and substituted
derivatives of bipyridine; terpyridine and substituted derivatives;
phenanthrolines, particularly 1,10-phenanthroline (abbreviated
phen) and substituted derivatives of phenanthrolines such as
4,7-dimethylphenanthroline and dipyridol[3,2-a:2',3'-c]phenazine
(abbreviated dppz); dipyridophenazine;
1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);
9,10-phenanthrenequinone diimine (abbreviated phi);
1,4,5,8-tetraazaphenanthrene (abbreviated tap);
1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and
isocyanide. Substituted derivatives, including fused derivatives,
may also be used. It should be noted that macrocylic ligands that
do not coordinatively saturate the metal ion, and which require the
addition of another proligand, are considered non-macrocyclic for
this purpose. As will be appreciated by those in the art, it is
possible to covalent attach a number of "non-macrocyclic" ligands
to form a coordinatively saturated compound, but that is lacking a
cyclic skeleton.
[0169] In some embodiments, a mixture of monodentate (e.g., at
least one cyano ligand), bi-dentate, tri-dentate, and polydentate
ligands can be used in the construction of EAMs.
[0170] Of particular use in the present invention are EAMs that are
metallocenes, and in particular ferrocenes, which have at least a
first self-immolative moiety attached, although in some
embodiments, more than one self-immolative moiety is attached as is
described below (it should also be noted that other EAMs, as are
broadly described herein, with self-immolative moieties can also be
used). In some embodiments, when more than one self-immolative
moiety is attached to a ferrocene, they are all attached to one of
the cyclopentydienyl rings. In some embodiments, the
self-immolative moieties are attached to different rings. In some
embodiments, it is possible to saturate one or both of the
cyclopentydienyl rings with self-immolative moieties, as long as
one site is used for attachment to the electrode.
[0171] In some embodiments, the EAMs comprise substituted
1,1'-ferrocenes. Ferrocene is air-stable. It can be easily
substituted with both capture ligand and anchoring group. Upon
binding of the target protein to the capture ligand on the
ferrocene which will not only change the environment around the
ferrocene, but also prevent the cyclopentadienyl rings from
spinning, which will change the energy by approximately 4 kJ/mol.
WO/1998/57159; Heinze and Schlenker, Eur. J. Inorg. Chem. 2974-2988
(2004); Heinze and Schlenker, Eur. J. Inorg. Chem. 66-71 (2005);
and Holleman-Wiberg, Inorganic Chemistry, Academic Press 34th Ed,
at 1620, all incorporated by reference.
##STR00003##
[0172] In some other embodiments, the EAMs comprise
1,3-disubstituted ferrocenes. While 1,3-disubstituted ferrocenes
are known (see, Bickert et al., Organometallics 1984, 3, 654-657;
Farrington et al., Chem. Commun. 2002, 308-309; Pichon et al.,
Chem. Commun. 2004, 598-599; and Steurer et al., Organometallics
2007, 26, 3850-3859), electrochemical studies of this class of
molecules in SAMs have not been reported in the literature. In
contrast to 1,1'-disubstituted ferrocenes where cyclopentadienyl
(Cp) ring rotation can place both Cp substituents in an eclipsed
conformation, 1,3-disubstituted ferrocene regioisomers provide a
molecular architecture that enforces a rigid geometry between these
Cp groups. Thus, 1,3-disubstituted ferrocenes that possess an
anchoring group, such as an organosulfur group for gold anchoring,
and a capture ligand such as a receptor group, protein capture
ligands and/or enzyme-reactive moieties are suited for SAM-based
electrochemical biosensing applications where the receptor is
displayed at the solution/SAM interface with limited degrees of
freedom (see FIG. 1, where X is an anchoring group and Y can
comprise a capture ligand). Representative examples of
1,3-disubstituted ferrocenes are shown in FIG. 12, such as
compounds 1-5. An example of a 1,3-disubstituted ferrocene for
attaching both anchoring and capture ligands is shown below:
##STR00004##
[0173] A series of 1,3-disubstituted ferrocene derivatives (1-4)
were synthesized with different functional moieties and
organosulfur anchoring groups for SAM formation on gold, and are
shown below.
##STR00005##
[0174] Additional ferrocene EAMs suitable for use in methods of
disclosure are disclosed in U.S. patent application Ser. No.
13/667,713, filed Nov. 2, 2012, which claims the benefit of U.S.
Provisional Application No. 61/555,945, filed Nov. 4, 2011, all
which are expressly incorporated by reference in their
entirety.
[0175] In addition, EAMs generally have an attachment moiety for
attachment of the EAM to the conductive oligomer which is used to
attach the EAM to the electrode. In general, although not required,
in the case of metallocenes such as ferrocenes, the self-immolative
moiety(ies) are attached to one of the cyclopentydienyl rings, and
the attachment moiety is attached to the other ring, as is
generally depicted in FIG. 1, although attachment to the same ring
can also be done. As will be appreciated by those in the art, any
combination of self-immolative moieties and at least one attachment
linker can be used, and on either ring.
[0176] In addition to the self-immolative moiety(ies) and the
attachment moiety(ies), the ferrocene can comprise additional
substituent groups, which can be added for a variety of reasons,
including altering the E.sup.0 in the presence or absence of at
least the self-immolative group. Suitable substituent groups,
frequently depicted in associated and incorporated references as
"R" groups, are recited in U.S. patent application Ser. No.
12/253,828, filed Oct. 17, 2008; U.S. patent application Ser. No.
12/253,875, filed Oct. 17, 2008; U.S. Provisional Patent
Application No. 61/332,565, filed May 7, 2010; U.S. Provisional
Patent Application No. 61/347,121, filed May 21, 2010; and U.S..
Provisional Patent Application No. 61/366,013, filed Jul. 20, 2010,
hereby incorporated by reference.
[0177] In some embodiments, such as depicted below, the EAM does
not comprise a self-immolative moiety, in the case where the
peroxide-sensitive moiety is attached directly to the EAM and
provides a change in E.sup.0 when the peroxide-sensitive moiety is
exposed to peroxide. As shown below, one embodiment allows the
peroxide-sensitive moiety to be attached directly to the EAM (in
this case, a ferrocene), such that the ferrocene has a first
E.sup.0 when the pinacol boronate ester moiety is attached, and a
second E.sup.0 when removed, e.g., in the presence of the
peroxide.
##STR00006##
[0178] Self-Immolative Moieties
[0179] The EAMs of the invention include at least one
self-immolative moiety that is covalently attached to the EAM such
that the EAM has a first E.sup.0 when it is present and a second
E.sup.0 when it has been removed as described below.
[0180] The term "self-immolative spacer" refers to a bifunctional
chemical moiety that is capable of covalently linking two chemical
moieties into a normally stable tripartate molecule. The
self-immolative spacer is capable of spontaneously separating from
the second moiety if the bond to the first moiety is cleaved. In
the present invention, the self-immolative spacer links a peroxide
sensitive moiety, e.g., a boron moiety, to the EAM. Upon exposure
to peroxide, the boron moiety is removed and the spacer falls
apart, as generally depicted in FIG. 1. Generally speaking, any
spacer where irreversible repetitive bond rearrangement reactions
are initiated by an electron-donating alcohol functional group
(i.e. quinone methide motifs) can be designed with boron groups
serving as triggering moieties that generate alcohols under
oxidative conditions. Alternatively, the boron moiety can mask a
latent phenolic oxygen in a ligand that is a pro-chelator for a
transition metal. Upon oxidation, the ligand is transformed and
initiates EAM formation in the SAM. For example, a sample chelating
ligand is salicaldehyde isonicotinoyl hydrazone that binds
iron.
[0181] As will be appreciated by those in the art, a wide variety
of self-immolative moieties may be used with a wide variety of EAMs
and peroxide sensitive moieties. Self-immolative linkers have been
described in a number of references, including US Publication Nos.
20090041791; 20100145036 and U.S. Pat. Nos. 7,705,045 and
7,223,837, all of which are expressly incorporated by reference in
their entirety, particularly for the disclosure of self-immolative
spacers.
[0182] A few self-immolative linkers of particular use in the
present invention are shown in FIG. 6. The self-immolative spacer
can comprise a single monomeric unit or polymers, either of the
same monomers (homopolymers) or of different monomers
(heteropolymers). Alternatively, the self-immolative spacer can be
a neighboring group to an EAM in a SAM that changes the environment
of the EAM following cleavage analogous to the chemistry as recited
in previous application "Electrochemical Assay for the Detection of
Enzymes", U.S. Ser. No. 12/253,828, PCT/US2008/080363, hereby
incorporated by reference.
[0183] Peroxide Sensitive Moieties
[0184] The self-immolative spacers join the peroxide sensitive
moieties (PSMs, sometimes referred to herein as POMs) and the EAMs
of the invention. In general, a peroxide sensitive moiety is one
containing boron as depicted in FIG. 1.
[0185] For example, the figures herein depict the use of ferrocene
derivatives, where the peroxide triggers a change from a benzyl
carbamate with a p-substituted pinacol borate ester to an amine.
This self-eliminating group has been described previously for
generating amine-functionalized florophores in the presence of
hydrogen peroxide (Sella, E.; Shabat, D. Self-immolative dendritic
probe for the direct detection of triacetone triperoxide. Chem.
Commun. 2008, 5701-5703; and Lo, L.-Cl; Chu, C.-Y. Development of
highly selective and sensitive probes for hydrogen peroxide. Chem.
Commun. 2003, 2728-2729 both of which are incorporated by
reference. Other such groups (aryl borate esters and arylboronic
acids) are also described in Sella and Lo. In addition,
ferrocenylamines are known to exhibit redox behavior at lower
potentials (-150 mV) as compared to their corresponding carbamate
derviatives (see Sagi et al., Amperometric Assay for Aldolase
Activity; Antibody-Catalyzed Ferrocenylamine Formation. Anal. Chem.
2006, 78, 1459-1461, incorporated by reference herein).
[0186] Capture and Soluble Binding Ligands
[0187] In addition to SAMs and EAMs, in some embodiments, the
electrodes comprise capture binding ligands. By "binding ligand" or
"binding species" herein is meant a compound that is used to probe
for the presence of the target analyte and that will bind to the
target analyte. In general, for most of the embodiments described
herein, there are at least two binding ligands used per target
analyte molecule; a "capture" or "anchor" binding ligand that is
attached to the detection surface, and a soluble binding ligand,
that binds independently to the target analyte, and either directly
or indirectly comprises at least one label such as a SOX. By
"capture binding ligand" herein is meant a binding ligand that
binds the target analyte that is attached to the electrode surface
that binds the target analyte. By "soluble binding ligand" herein
is meant a binding ligand that is in solution that binds the target
analyte at a different site than the capture binding ligand.
[0188] As will be appreciated by those in the art, the composition
of the binding ligand will depend on the composition of the target
analyte. Binding ligands for a wide variety of analytes are known
or can be readily found using known techniques. For example, when
the analyte is a protein, the binding ligands include proteins
(particularly including antibodies or fragments thereof (FAbs,
etc.)) or small molecules.
[0189] In general, antibodies are useful as both capture and
soluble binding ligands.
[0190] The soluble binding ligand also comprises a peroxide
generating moiety such as an enzyme that generates peroxide. As
defined herein, the term "peroxide generating system" or
"peroxide-generating system" means one or more enzymes that
directly generates a peroxide from its substrate and/or an
intermediary enzyme that generates an intermediate, e.g., a
cofactor or pre-substrate, for another enzyme that in turn
generates a peroxide. In one example, a peroxide generating moiety
may be an enzyme that generates peroxide, e.g., "peroxide
generating enzyme". A wide variety of such enzymes are known,
including glucose oxidase, acyl CoA oxidases, alcohol oxidases,
aldehyde oxidases, etc. A wide variety of suitable oxidase enzymes
are known in the art (see any glucose oxidase enzyme classified as
EC 1.1.3.4, including, but not limited to, glucose oxidase, D-amino
acid oxidase (DAAO) and choline oxidase). Glucose oxidase enzymes
from a wide variety of organisms are well known, including
bacterial, fungal and animal (including mammalian), including, but
not limited to, Aspergillus species (e.g. A. niger), Penicillum
species, Streptomyces species, mouse, etc.). Also of use are acyl
CoA oxidases, classified as EC 1.3.3.6.
[0191] By the term "an intermediary enzyme" herein is meant an
enzyme that generates a product that is a substrate or a cofactor
for another enzyme such as another intermediary enzyme or a
peroxide-generating enzyme. For instance, the soluble binding
ligand may contain an enzyme, such as alkaline phosphatase (AP),
that catalyzes the generation of a necessary cofactor from a
phosphorylated precursor for a soluble apo-oxidase enzyme (i.e.,
FADP converted to FAD which binds to apo-DAAO) which in turn
generates peroxide by reaction with substrate. This strategy
enables cascade amplification of target binding events if the
concentrations of apo-enzyme, phosphorylated cofactor, and oxidase
enzyme substrate are high with respect to the surface immobilized
target.
[0192] As defined herein, the term "target specific enzyme" herein
is meant an enzyme, e.g., Glycerol Kinase, that is specific for a
target analyte, e.g. ATP.
[0193] As defined herein, the term "recycling enzyme" herein is
meant an enzyme that regenerates or recycles a necessary substrate
of another enzyme such as the generation of NADH from NAD+.
[0194] Generally, the capture binding ligand allows the attachment
of a target analyte to the detection surface, for the purposes of
detection. In one embodiment, the binding is specific, and the
binding ligand is part of a binding pair. By "specifically bind"
herein is meant that the ligand binds the analyte, with specificity
sufficient to differentiate between the analyte and other
components or contaminants of the test sample. By "specific binding
pair" herein is meant a complimentary pair of binding ligands such
as an antibody/antigen and receptor/ligand. The binding should be
sufficient to allow the analyte to remain bound under the
conditions of the assay, including wash steps to remove
non-specific binding. In some embodiments, for example in the
detection of certain biomolecules, the binding constants of the
analyte to the binding ligand will be at least about 10.sup.-4 to
10.sup.-9 M.sup.-1, with at least about 10.sup.-5 to 10.sup.-9
being preferred and at least about 10.sup.-7 to 10.sup.-9 M.sup.-1
being particularly preferred.
[0195] Binding ligands to a wide variety of analytes are known or
can be readily found using known techniques. For example, when the
analyte is a single-stranded nucleic acid, the binding ligand is
generally a substantially complementary nucleic acid.
Alternatively, as is generally described in U.S. Pat. Nos.
5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,
5,705,337, and related patents, hereby incorporated by reference,
nucleic acid "aptamers" can be developed for binding to virtually
any target analyte. Similarly the analyte may be a nucleic acid
binding protein and the capture binding ligand is either a
single-stranded or double-stranded nucleic acid; alternatively, the
binding ligand may be a nucleic acid binding protein when the
analyte is a single or double-stranded nucleic acid. When the
analyte is a protein, the binding ligands include proteins
(particularly including antibodies or fragments thereof (FAbs,
etc.)), small molecules, or aptamers, described above. Preferred
binding ligand proteins include antibodies and peptides. As will be
appreciated by those in the art, any two molecules that will
associate, preferably specifically, may be used, either as the
analyte or the binding ligand. Suitable analyte/binding ligand
pairs include, but are not limited to, antibodies/antigens,
receptors/ligand, proteins/nucleic acids; nucleic acids/nucleic
acids, enzymes/substrates and/or inhibitors, carbohydrates
(including glycoproteins and glycolipids)/lectins, carbohydrates
and other binding partners, proteins/proteins; and protein/small
molecules. These may be wild-type or derivative sequences.
[0196] The capture binding ligands (e.g. a capture antibody) can be
covalently coupled to the electrode (usually through an attachment
linker) or bound tightly but not covalently; for example, using
biotin/streptavidin reactions (e.g. biotin on the surface of the
SAM, streptavin-conjugated capture ligand such as an antibody, or
vice versa), bound via a nucleic acid reaction (for example, the
capture ligand can have a nucleic acid ("Watson") and the surface
can have a complementary nucleic acid ("Crick"), bound using
protein G binding to the Fc fragment of the antibody, etc.
[0197] It should also be noted that the invention described herein
can also be used as a sensor for the illicit explosive triacetone
triperoxide (TATP).
[0198] Anchor Groups
[0199] The present invention provides compounds including the EAM
(optionally attached to the electrode surface with a conductive
oligomer), the SAM, and the capture binding ligands on the
electrode surface. Generally, in some embodiments, these moieties
are attached to the electrode using anchor group. By "anchor" or
"anchor group" herein is meant a chemical group that attaches the
compounds of the invention to an electrode.
[0200] As will be appreciated by those in the art, the composition
of the anchor group will vary depending on the composition of the
surface to which it is attached. In the case of gold electrodes,
both pyridinyl anchor groups and thiol based anchor groups find
particular use.
[0201] The covalent attachment of the conductive oligomer may be
accomplished in a variety of ways, depending on the electrode and
the conductive oligomer used. Generally, some type of linker is
used, as depicted below as "A" in Structure 1, where X is the
conductive oligomer, and the hatched surface is the electrode:
##STR00007##
[0202] In this embodiment, A is a linker or atom. The choice of "A"
will depend in part on the characteristics of the electrode. Thus,
for example, A may be a sulfur moiety when a gold electrode is
used. Alternatively, when metal oxide electrodes are used, A may be
a silicon (silane) moiety attached to the oxygen of the oxide (see
for example Chen et al., Langmuir 10:3332-3337 (1994); Lenhard et
al., J. Electroanal. Chem. 78:195-201 (1977), both of which are
expressly incorporated by reference). When carbon based electrodes
are used, A may be an amino moiety (preferably a primary amine; see
for example Deinhammer et al., Langmuir 10:1306-1313 (1994)). Thus,
preferred A moieties include, but are not limited to, silane
moieties, sulfur moieties (including alkyl sulfur moieties), and
amino moieties.
[0203] In some embodiments, the electrode is a carbon electrode,
i.e. a glassy carbon electrode, and attachment is via a nitrogen of
an amine group. A representative structure is depicted in Structure
15 of US Patent Application Publication No. 20080248592, hereby
incorporated by reference in its entirety but particularly for
Structures as described therein and the description of different
anchor groups and the accompanying text. Again, additional atoms
may be present, i.e. linkers and/or terminal groups.
[0204] In Structure 16 of US Patent Application Publication No.
20080248592, hereby incorporated by reference as above, the oxygen
atom is from the oxide of the metal oxide electrode. The Si atom
may also contain other atoms, i.e. be a silicon moiety containing
substitution groups. Other attachments for SAMs to other electrodes
are known in the art; see for example Napier et al., Langmuir,
1997, for attachment to indium tin oxide electrodes, and also the
chemisorption of phosphates to an indium tin oxide electrode (talk
by H. Holden Thorpe, CHI conference, May 4-5, 1998).
[0205] In one preferred embodiment, indium-tin-oxide (ITO) is used
as the electrode, and the anchor groups are phosphonate-containing
species.
[0206] 1). Sulfur Anchor Groups
[0207] Although depicted in Structure 1 as a single moiety, the
conductive oligomer may be attached to the electrode with more than
one "A" moiety; the "A" moieties may be the same or different.
Thus, for example, when the electrode is a gold electrode, and "A"
is a sulfur atom or moiety, multiple sulfur atoms may be used to
attach the conductive oligomer to the electrode, such as is
generally depicted below in Structures 2, 3 and 4. As will be
appreciated by those in the art, other such structures can be made.
In Structures 2, 3 and 4 the A moiety is just a sulfur atom, but
substituted sulfur moieties may also be used.
[0208] Thus, for example, when the electrode is a gold electrode,
and "A" is a sulfur atom or moiety, such as generally depicted
below in Structure 6, multiple sulfur atoms may be used to attach
the conductive oligomer to the electrode, such as is generally
depicted below in Structures 2, 3 and 4. As will be appreciated by
those in the art, other such structures can be made. In Structures
2, 3 and 4, the A moiety is just a sulfur atom, but substituted
sulfur moieties may also be used.
##STR00008##
[0209] It should also be noted that similar to Structure 4, it may
be possible to have a conductive oligomer terminating in a single
carbon atom with three sulfur moieties attached to the
electrode.
[0210] In another aspect, the present invention provide anchor
comprise conjugated thiols. Some exemplary complexes with
conjugated thiol anchors are shown in FIG. 10. In some embodiments,
the anchor comprises an alkylthiol group. Some of the examples are
shown in FIGS. 10A and 4B. The two compounds depicts in FIG. 10B
are based on carbene and 4-pyridylalanine, respectively.
[0211] In another aspect, the present invention provides conjugated
multipodal thio-containing compounds that serve as anchoring groups
in the construction of electroactive moieties for analyte detection
on electrodes, such as gold electrodes. That is, spacer groups
(which can be attached to EAMs, ReAMCs, or an "empty" monolayer
forming species) are attached using two or more sulfur atoms. These
mulitpodal anchor groups can be linear or cyclic, as described
herein.
[0212] In some embodiments, the anchor groups are "bipodal",
containing two sulfur atoms that will attach to the gold surface,
and linear, although in some cases it can be possible to include
systems with other multipodalities (e.g. "tripodal"). Such a
multipodal anchoring group display increased stability and/or allow
a greater footprint for preparing SAMs from thiol-containing
anchors with sterically demanding headgroups.
[0213] In some embodiments, the anchor comprises cyclic disulfides
("bipod"). Although in some cases it can be possible to include
ring system anchor groups with other multipodalities (e.g.
"tripodal"). The number of the atoms of the ring can vary, for
example from 5 to 10, and also includes multicyclic anchor groups,
as discussed below
[0214] In some embodiments, the anchor groups comprise a
[1,2,5]-dithiazepane unit which is seven-membered ring with an apex
nitrogen atom and a intramolecular disulfide bond as shown
below:
##STR00009##
[0215] In Structure (111a), it should also be noted that the carbon
atoms of the ring can additionally be substituted. As will be
appreciated by those in the art, other membered rings are also
included. In addition, multicyclic ring structures can be used,
which can include cyclic heteroalkanes such as the
[1,2,5]-dithiazepane shown above substituted with other cyclic
alkanes (including cyclic heteroalkanes) or aromatic ring
structures. In some embodiments, the anchor group and part of the
spacer has the structure shown below
##STR00010##
[0216] The "R" group herein can be any substitution group,
including a conjugated oligophenylethynylene unit with terminal
coordinating ligand for the transition metal component of the
EAM.
[0217] The anchors are synthesized from a bipodal intermediate (I)
(the compound as formula III where R.dbd.I), which is described in
Li et al., Org. Lett. 4:3631-3634 (2002), herein incorporated by
reference. See also Wei et al, J. Org, Chem. 69:1461-1469 (2004),
herein incorporated by reference.
[0218] The number of sulfur atoms can vary as outlined herein, with
particular embodiments utilizing one, two, and three per
spacer.
[0219] As will be appreciated by those in the art, the compositions
of the invention can be made in a variety of ways, including those
outlined below and in U.S. patent application Ser. No. 12/253,828,
filed Oct. 17, 2008; U.S. patent application Ser. No. 12/253,875,
filed Oct. 17, 2008; U.S. Provisional Patent Application No.
61/332,565, filed May 7, 2010; U.S. Provisional Patent Application
No. 61/347,121, filed May 21, 2010; U.S. Provisional Patent
Application No. 61/366,013, filed Jul. 20, 2010. In some
embodiments, the composition are made according to methods
disclosed in of U.S. Pat. Nos. 6,013,459, 6,248,229, 7,018,523,
7,267,939, U.S. patent application Ser. No. 09/096,593 and
60/980,733, and U.S. Provisional Application No. 61/087,102, filed
on Aug. 7, 2008, all are herein incorporated in their entireties
for all purposes.
[0220] Applications
[0221] The systems of the invention find use in the detection of a
variety of target analytes, as outlined herein. In some
embodiments, "sandwich" type assays are used, as are generally
depicted in FIGS. 11-13. In other embodiments, for example for the
detection of particular metabolites such as ATP and NADH, other
formats are used.
[0222] In some embodiments, for example in "sandwich" type formats,
the target analyte, contained within a test sample, is added to the
electrode with the PSM-SIM-EAM mixture, a capture binding ligand,
and optionally a SAM. This addition is followed by an optional
washing step and the addition of the soluble binding ligand,
although as will be appreciated by those in the art, these
additions can be done simultaneously or the solution binding ligand
can be added to the sample containing the target analyte prior to
addition to the chip. The surface is again optionally washed, and
the substrate for the peroxide sensitive moiety, e.g. glucose, is
added under conditions that if present, peroxide is generated and
the SIM is cleaved.
[0223] In some embodiments, for example when there is a specific
peroxidase-generating enzyme that uses a metabolite of interest
(e.g. a target analyte or target metabolite), as depicted in FIGS.
12 and 13, the system takes on different configurations. For
example, in FIG. 12, the electrode, generally with a SAM, contains
at least two species: (1) an EAM with a self-immolative moiety,
depicted in FIG. 12 as a ferrocene-based derivative (although as
described herein and as will be appreciated by those in the art,
other EAMs can be used); and (2) an attached peroxidase-generating
enzyme, in this case, as depicted, NADH oxidase. As outlined
herein, the peroxidase-generating enzyme can be attached to the
electrode surface in a number of different ways. The attachment can
be "direct" when the enzyme is attached to the terminus of a
monolayer forming species, as is generally outlined in
PCT/US2008/080379, using a coupling chemistry. Alternatively, and
as depicted in FIG. 12, the enzyme can be attached using a number
of "sandwich" type attachments, for example using a monolayer
species with a biotin attached, a streptavidin and a biotin-labeled
enzyme.
[0224] In some embodiments, these two species can be attached as a
single moiety to the electrode surface, for example as generally
depicted in Structure 7 of U.S. Pat. No. 7,595,153, hereby
incorporated by reference in its entirety and specifically for the
schematics of attachment configurations.
[0225] These conditions are generally physiological conditions.
Generally a plurality of assay mixtures is run in parallel with
different concentrations to obtain a differential response to the
various concentrations. Typically, one of these concentrations
serves as a negative control, i.e., at zero concentration or below
the level of detection. In addition, any variety of other reagents
may be included in the screening assay. These include reagents like
salts, neutral proteins, e.g. albumin, detergents, etc. which may
be used to facilitate optimal binding and/or reduce non-specific or
background interactions. Also reagents that otherwise improve the
efficiency of the assay, such as protease inhibitors, nuclease
inhibitors, anti-microbial agents, etc., may be used. The mixture
of components may be added in any order that provides for the
requisite binding.
[0226] [The generation of peroxidase results in the loss of the
PGM-SIM component of the complex, resulting the a change in the
E.sup.0 of the EAM. In some embodiments, the E.sup.0 of the EAM
changes by at about 20 mV, 30 mV, 40 mV, 50 mV, 75 mV, 80 mV, 90 mV
to 100 mV, some embodiments resulting in changes of 200, 300 or 500
mV being achieved. In some embodiments, the changes in the E.sup.0
of the EAM is a decrease. In some embodiments, the changes in the
E.sup.0 of the EAM is an increase.
[0227] Electron transfer is generally initiated electronically,
with voltage being preferred. Precise control and variations in the
applied potential can be via a potentiostat and either a three
electrode system (one reference, one sample and one counter
electrode) or a two electrode system (one sample and one counter
electrode). This allows matching of applied potential to peak
electron transfer potential of the system which depends in part on
the choice of redox active molecules and in part on the conductive
oligomer used.
[0228] Detection
[0229] Electron transfer between the redox active molecule and the
electrode can be detected in a variety of ways, with electronic
detection, including, but not limited to, amperommetry,
voltammetry, capacitance and impedance being preferred. These
methods include time or frequency dependent methods based on AC or
DC currents, pulsed methods, lock in techniques, and filtering
(high pass, low pass, band pass). In some embodiments, all that is
required is electron transfer detection; in others, the rate of
electron transfer may be determined.
[0230] In some embodiments, electronic detection is used, including
amperommetry, voltammetry, capacitance, and impedance. Suitable
techniques include, but are not limited to, electrogravimetry;
coulometry (including controlled potential coulometry and constant
current coulometry); voltametry (cyclic voltametry, pulse
voltametry (normal pulse voltametry, square wave voltametry,
differential pulse voltametry, Osteryoung square wave voltametry,
and coulostatic pulse techniques); stripping analysis (aniodic
stripping analysis, cathiodic stripping analysis, square wave
stripping voltammetry); conductance measurements (electrolytic
conductance, direct analysis); time dependent electrochemical
analyses (chronoamperometry, chronopotentiometry, cyclic
chronopotentiometry and amperometry, AC polography,
chronogalvametry, and chronocoulometry); AC impedance measurement;
capacitance measurement; AC voltametry, and
photoelectrochemistry.
[0231] In some embodiments, monitoring electron transfer is via
amperometric detection. This method of detection involves applying
a potential (as compared to a separate reference electrode) between
the electrode containing the compositions of the invention and an
auxiliary (counter) electrode in the test sample. Electron transfer
of differing efficiencies is induced in samples in the presence or
absence of target analyte.
[0232] The device for measuring electron transfer amperometrically
involves sensitive current detection and includes a means of
controlling the voltage potential, usually a potentiostat. This
voltage is optimized with reference to the potential of the redox
active molecule.
[0233] In some embodiments, alternative electron detection modes
are utilized. For example, potentiometric (or voltammetric)
measurements involve non faradaic (no net current flow) processes
and are utilized traditionally in pH and other ion detectors.
Similar sensors are used to monitor electron transfer between the
redox active molecules and the electrode. In addition, other
properties of insulators (such as resistance) and of conductors
(such as conductivity, impedance and capicitance) could be used to
monitor electron transfer between the redox active molecules and
the electrode. Finally, any system that generates a current (such
as electron transfer) also generates a small magnetic field, which
may be monitored in some embodiments.
[0234] It should be understood that one benefit of the fast rates
of electron transfer observed in the compositions of the invention
is that time resolution can greatly enhance the signal to noise
results of monitors based on electronic current. The fast rates of
electron transfer of the present invention result both in high
signals and stereotyped delays between electron transfer initiation
and completion. By amplifying signals of particular delays, such as
through the use of pulsed initiation of electron transfer and "lock
in" amplifiers of detection, orders of magnitude improvements in
signal to noise may be achieved.
[0235] In some embodiments, electron transfer is initiated and
detected using direct current (DC) techniques. As noted above, the
first E.sup.0 of the redox active molecule before and the second
E.sup.0 of the reacted redox active molecule afterwards will allow
the detection of the analyte. As will be appreciated by those in
the art, a number of suitable methods may be used to detect the
electron transfer.
[0236] In some embodiments, electron transfer is initiated using
alternating current (AC) methods. A first input electrical signal
is applied to the system, preferably via at least the sample
electrode (containing the complexes of the invention) and the
counter electrode, to initiate electron transfer between the
electrode and the second electron transfer moiety. Three electrode
systems may also be used, with the voltage applied to the reference
and working electrodes. In this embodiment, the first input signal
comprises at least an AC component. The AC component may be of
variable amplitude and frequency. Generally, for use in the present
methods, the AC amplitude ranges from about 1 mV to about 1.1 V,
with from about 10 mV to about 800 mV being preferred, and from
about 10 mV to about 500 mV being especially preferred. The AC
frequency ranges from about 0.01 Hz to about 10 MHz, with from
about 1 Hz to about 1 MHz being preferred, and from about 1 Hz to
about 100 kHz being especially preferred
[0237] In some embodiments, the first input signal comprises a DC
component and an AC component. That is, a DC offset voltage between
the sample and counter electrodes is swept through the
electrochemical potential of the second electron transfer moiety.
The sweep is used to identify the DC voltage at which the maximum
response of the system is seen. This is generally at or about the
electrochemical potential of the redox active molecule. Once this
voltage is determined, either a sweep or one or more uniform DC
offset voltages may be used. DC offset voltages of from about 1 V
to about +1.1 V are preferred, with from about 500 mV to about +800
mV being especially preferred, and from about 300 mV to about 500
mV being particularly preferred. On top of the DC offset voltage,
an AC signal component of variable amplitude and frequency is
applied. If the redox active molecule has a low enough solvent
reorganization energy to respond to the AC perturbation, an AC
current will be produced due to electron transfer between the
electrode and the redox active molecule.
[0238] In some embodiments, the AC amplitude is varied. Without
being bound by theory, it appears that increasing the amplitude
increases the driving force. Thus, higher amplitudes, which result
in higher overpotentials give faster rates of electron transfer.
Thus, generally, the same system gives an improved response (i.e.
higher output signals) at any single frequency through the use of
higher overpotentials at that frequency. Thus, the amplitude may be
increased at high frequencies to increase the rate of electron
transfer through the system, resulting in greater sensitivity. In
addition, as noted above, it may be possible to the first and
second E.sup.0 of the redox active molecules, molecules on the
basis of the rate of electron transfer, which in turn can be used
either to distinguish the two on the basis of frequency or
overpotential.
[0239] In some embodiments, measurements of the system are taken at
least two separate amplitudes or overpotentials, with measurements
at a plurality of amplitudes being preferred. As noted above,
changes in response as a result of changes in amplitude may form
the basis of identification, calibration and quantification of the
system.
[0240] In some embodiments, the AC frequency is varied. At
different frequencies, different molecules respond in different
ways. As will be appreciated by those in the art, increasing the
frequency generally increases the output current. However, when the
frequency is greater than the rate at which electrons may travel
between the electrode and the redox active molecules, higher
frequencies result in a loss or decrease of output signal. At some
point, the frequency will be greater than the rate of electron
transfer through even solvent inhibited redox active molecules, and
then the output signal will also drop.
[0241] In addition, the use of AC techniques allows the significant
reduction of background signals at any single frequency due to
entities other than the covalently attached nucleic acids, i.e.
"locking out" or "filtering" unwanted signals. That is, the
frequency response of a charge carrier or redox active molecule in
solution will be limited by its diffusion coefficient. Accordingly,
at high frequencies, a charge carrier may not diffuse rapidly
enough to transfer its charge to the electrode, and/or the charge
transfer kinetics may not be fast enough. This is particularly
significant in embodiments that do not utilize a passavation layer
monolayer or have partial or insufficient monolayers, i.e. where
the solvent is accessible to the electrode. As outlined above, in
DC techniques, the presence of "holes" where the electrode is
accessible to the solvent can result in solvent charge carriers
"short circuiting" the system. However, using the present AC
techniques, one or more frequencies can be chosen that prevent a
frequency response of one or more charge carriers in solution,
whether or not a monolayer is present. This is particularly
significant since many biological fluids such as blood contain
significant amounts of redox active molecules which can interfere
with amperometric detection methods.
[0242] In some embodiments, measurements of the system are taken at
least two separate frequencies, with measurements at a plurality of
frequencies being preferred. A plurality of frequencies includes a
scan. In a preferred embodiment, the frequency response is
determined at least two, preferably at least about five, and more
preferably at least about ten frequencies.
[0243] Signal Processing
[0244] After transmitting the input signal to initiate electron
transfer, an output signal is received or detected. The presence
and magnitude of the output signal will depend on the
overpotential/amplitude of the input signal; the frequency of the
input AC signal; the composition of the intervening medium, i.e.
the impedance, between the electron transfer moieties; the DC
offset; the environment of the system; and the solvent. At a given
input signal, the presence and magnitude of the output signal will
depend in general on the solvent reorganization energy required to
bring about a change in the oxidation state of the metal ion. Thus,
upon transmitting the input signal, comprising an AC component and
a DC offset, electrons are transferred between the electrode and
the redox active molecule, when the solvent reorganization energy
is low enough, the frequency is in range, and the amplitude is
sufficient, resulting in an output signal.
[0245] In some embodiments, the output signal comprises an AC
current. As outlined above, the magnitude of the output current
will depend on a number of parameters. By varying these parameters,
the system may be optimized in a number of ways.
[0246] In general, AC currents generated in the present invention
range from about 1 femptoamp to about 1 milliamp, with currents
from about 50 femptoamps to about 100 microamps being preferred,
and from about 1 picoamp to about 1 microamp being especially
preferred.
[0247] Apparatus
[0248] The present invention further provides apparatus for the
detection of analytes using AC detection methods. The apparatus
includes a test chamber which has at least a first measuring or
sample electrode, and a second measuring or counter electrode.
Three electrode systems are also useful. The first and second
measuring electrodes are in contact with a test sample receiving
region, such that in the presence of a liquid test sample, the two
electrodes may be in electrical contact.
[0249] In yet another embodiment, the first measuring electrode
comprises a redox active complex, covalently attached via a spacer,
and preferably via a conductive oligomer, such as are described
herein. Alternatively, the first measuring electrode comprises
covalently attached redox active molecules and binding ligands.
[0250] The apparatus further comprises a voltage source
electrically connected to the test chamber; that is, to the
measuring electrodes. Preferably, the voltage source is capable of
delivering AC and DC voltages, if needed.
[0251] In an embodiment, the apparatus further comprises a
processor capable of comparing the input signal and the output
signal. The processor is coupled to the electrodes and configured
to receive an output signal, and thus detect the presence of the
target analyte.
EXAMPLES
Example 1
[0252] General Methods and Materials. Unless otherwise noted, all
synthetic manipulations were performed under a dry argon atmosphere
using standard Schlenk techniques. For reaction media, solvents
were dried over neutral alumina via the Dow-Grubbs solvent
system.sup.1 acquired from Glass Contours (Laguna Beach, Calif.).
These solvents were deoxygenated with argon prior to use. Reactions
were monitored by TLC using EMD precoated aluminum plates (silica
gel 60, F.sub.254, EMD Chemicals, Inc., Gibbstown, N.J.). Spots
were visualized by one of the following methods: iodine vapor,
exposure to UV light, or staining with phosphomolybdic acid
followed by heating. Flash chromatography was carried out on silica
(silica gel 60 particle size: 40-63 .mu.m; Sorbent Technologies,
Atlanta, Ga.) under a positive pressure of laboratory air. .sup.1H
NMR and proton-decoupled .sup.13C NMR spectra were recorded on a
Bruker Avance III spectrometer (499.37 MHz for .sup.1H, 125.58 MHz
for .sup.13C) and were processed with Bruker TOPSPIN 2.1 software.
High-resolution mass spectrometry (HRMS) was obtained using an
Agilent 6210 time-of-flight (TOF) LC/MS instrument using
electrospray ionization (ESI) or atmospheric pressure
photoionization (APPI) methods.
[0253] Chloroform-d.sub.1 was purchased from Cambridge Isotope
Laboratories. Compound 2 and p-pinacolborate benzyl alcohol were
synthesized as described previously (Bertin, P. A.; Meade, T. J.
Tetrahedron Lett. 2009, 50, 5409-5412; Sella, E.; Shabat, D. Chem.
Commun. 2008, 5701-5703, both of which are expressly incorporated
by reference). All other reagents were purchased from commercial
sources and used without further purification unless otherwise
noted.
[0254] Compound 3.
[0255] To a solution of compound 2 (0.500 g, 1.2 mmol) and
triethylamine (0.25 mL, 1.8 mmol) in tetrahydrofuran (15 mL) was
added diphenylphosphoryl azide (DPPA) (0.285 mL, 1.32 mmol). The
reaction was stirred at rt for 1.5 hours and concentrated under
reduced pressure. The crude residue was purified by column
chromatography (methanol:ethyl acetate:dichloromethane 0.5:1.5:8)
to yield the title compound as a red/orange solid (0.460 g, 1.04
mmol, 87%). .sup.1H NMR, .sup.13C{.sup.1H} NMR, and HRMS were
consistent with the title compound.
[0256] Compound 4.
[0257] A solution of compound 3 (0.460 g, 1.04 mmol) in toluene (20
mL) was vigorously degassed with Ar and heated to 100.degree. C.
for 1.5 hours, p-pinacolborate benzyl alcohol (0.268 g, 1.14 mmol)
and dibutyltin-dilaurate (DBTL) (0.018 mL, 0.03 mmol) were added
and the reaction maintained at 100.degree. C. for an additional 2
hours. The reaction was concentrated under reduced pressure and the
crude residue purified by column chromatography (diethyl
ether:ethyl acetate:dichloromethane, 1:2:2) to yield the title
compound as a pale orange solid (0.480 g, 0.741 mmol, 71%). .sup.1H
NMR, .sup.13C{.sup.1H} NMR, and HRMS were consistent with the title
compound.
[0258] Compound 5.
[0259] A solution of compound 4 (0.135 g, 0.209 mmol) in
dichloromethane (5 mL) was cooled in an ice bath. Trifluoroacetic
acid:dichloromethane (1:1 v/v, 5 mL) was added dropwise over 5 min.
After 15 min, the ice bath was removed and the reaction warmed to
room temperature. After 45 min, the volatiles were removed in vacuo
to yield the trifluoroacetate salt of the title compound as a
brown/orange solid (quantitative). .sup.1H NMR, .sup.13C{.sup.1H}
NMR, and HRMS were consistent with the title compound.
[0260] Compound 1.
[0261] To a solution of 11-mercaptoundecanoic acid (0.045 g, 0.206
mmol) and HATU (0.078 g, 0.206 mmol) in
dichloromethane:N,N-dimethylformamide (1:1 v/v, 5 mL) was added
compound 5 (0.105 g, 0.159 mmol) and diisopropylethylamine (0.083
mL, 0.477 mmol). The reaction was stirred at room temperature for 2
hours. The reaction mixture was diluted into ethyl acetate (150 mL)
and washed with brine (3.times.50 mL). The organic phase was dried
over Na.sub.2SO.sub.4, filtered, and concentrated to crude residue
that was purified by column chromatography (methanol:ethyl
acetate:dichloromethane, 0.5:1.5:8) to yield the title compound as
a yellow solid (0.035 g, 0.047 mmol, 30%). .sup.1H NMR,
.sup.13C{.sup.1H} NMR, and HRMS were consistent with the title
compound.
[0262] Electrochemistry.
[0263] Cyclic voltammetry was carried out with a CHI model 660A
electrochemical analyzer (CHI Instruments Inc.) in THF with 0.15 M
n-Bu.sub.4NClO.sub.4 supporting electrolyte (0.5 mL) using a three
electrode system of SAM-modified gold as the working electrode, a
Ag/AgCl wire reference electrode, and a platinum wire counter
electrode (Bioanalytical Systems). Model compound (green) was
prepared by treating compound 4 with hydrogen peroxide. The results
are shown in FIG. 3.
Example 2
Measurement of a quantifiable electrochemical signal at two unique
potentials, E.sup.o.sub.1 and E.sup.o.sub.2.as a result of direct
addition of H.sub.2O.sub.2
A. Purpose
[0264] The goal of this study was to test the effect of 5-minute
and 10-minute H.sub.2O.sub.2 incubation times on a diluted SAM of
EAM 1 (PB25_49) washed at pH 10.1 and incubated at pH 8.5.
H.sub.2O.sub.2 would decompose the Ferrocene on the EAM into a new
derivative which would show up at a new potential.
B. Materials
TABLE-US-00002 [0265] BATCH # / MATERIALS Name MW Final C
Stock/Solvent NOTES 1. EAM for SAM: PB25_49 MW = 747.57 0.1 mM 0.5
mg/0.5 mL Stock: 0.5 mg EtOH 2. Diluent solutions for (C6S).sub.2
MW = 234.47 0.5 mM -- Stock C = 9.13x SAM (4.56 mM stock)
(HO-C6S).sub.2 MW = 266.47 0.5 mM -- Stock C = 7.51x (3.75 mM
stock) 3. Electrode testing solution: 1M LiClO.sub.4 MW = 106.39 1M
10.6 g/1 L H.sub.2O Aqueous solution 1X 4. Hydrogen peroxide
Hydrogen MW = 34.01 1M 57 .mu.L/943 .mu.L Made fresh peroxide
H.sub.2O (50.4%) 5. Buffer Na.sub.2CO.sub.3 MW = 105.99 100 mM 0.53
g/50 mL pH 10.1 H.sub.2O 6. Washing buffers EtOH, -- -- -- --
nanopure water, Na.sub.2CO.sub.3, 1M LiClO.sub.4 7. Electrodes:
Reference Counter Working Wash and store -- electrode electrode
Electrode Quasi 1 Pt Wire Au Chip Rinse before 13 chips reference d
= 0.25 and after each (1M LiClO.sub.4) um use Note: All
calculations were based on the equation below; Nanopure water was
18 megaohm water from a Millipore purification system. with
Molecular Weight (MW or FW) found usually on the product bottles,
making sure units are correct. Concentration ( M : mol L ) = Weight
( g ) Volume ( L ) .times. MolecularWeight ( g mol )
##EQU00002##
EAM 1 structure:
##STR00011##
C. Procedure
[0266] Day 1:
[0267] Wash and Assemble Chips
[0268] 13 (12 for assay+1 for internal reference testing) chips
were washed as follows: the chips were placed in glass jar with
inserts, sonicated in 0.2% TWEEN 20 solution for 5 minutes, rinsed
with nanopure water and ethanol, and then dried with Argon. The
chips were then cleaned in a Plasma Cleaner for 10 minutes, and
rinsed with ethanol and dried with Argon. Metal bases, gaskets, and
PDMS stamps were hand washed with hand soap, rinsed with ethanol
and air dried. Chips were assembled by placing the chip on the
double sided tape on center of the metal base, PDMS stamp on the
chip, gasket on the PDMS stamp, all clamped together with binder
clips.
[0269] Prepare Experimental Stocks
[0270] EAM stock was prepared by combining the following into the
EAM aliquot:
TABLE-US-00003 # Amount Final Stocks EAM Aliquots (mg ea) MW EtOH
THF Conc. A PB25_49 1 0.5 747.57 500 .mu.L none 1.34 mM
[0271] Prepare SAM Solutions
[0272] SAM Solution was prepared by combining the following into
separate glass vials:
TABLE-US-00004 [EAM] AMT [D1] AMT [D2] AMT EtOH Tot Vol. mM .mu.L
D1 mM .mu.L D2 mM .mu.L .mu.L .mu.L 1.34 493 (C6S).sub.2 4.56 723
(HOC6S).sub.2 3.75 879 4505 6600
[0273] SAM Incubation
[0274] To chips 1-12, 500 .mu.L of above prepared SAM solution was
added, followed by overnight incubation. All chips were placed in
plastic containers containing ethanol, sealed with parafilm (to
avoid ethanol evaporation and drying of chips). The setup was
covered with Aluminum foil.
[0275] Day 2:
[0276] Internal Reference Measurements:
[0277] A 1 mM solution of 1 1' Ferrocene dimethanol was prepared in
1M LiClO.sub.4 solution. 1.3 mg of 1 1' Ferrocene dimethanol were
combined with 5 mL 1M LiClO.sub.4. MW 1 1' Ferrocene
dimethanol=246.09. 500 .mu.L of 1 mM 1 1' Ferrocene dimethanol
solution was added to a clean chip. Quasi 1 reference and platinum
counter electrodes were added to the system and CVs were
recorded.
[0278] Initial testing to check for proper SAM formation on
chips:
[0279] After overnight incubation, the chips were removed from the
incubation container. The SAM deposition solution was collected in
a vial and dried to obtain recycled EAM for future use. After
overnight incubation, chips 1 through 12 were washed by following
the steps shown below:
TABLE-US-00005 Ethanol 8 times Nanopure water 4 times Testing
buffer, 1M LiClO.sub.4 2 times
500 .mu.L of 1M LiClO.sub.4 was added to chips 1, 3, 5, 6, 7, 9,
11, and 12, and then chip was plugged in the switchbox. Reference
and counter electrodes were added to the EC system. The white
alligator clip from the CHI 650C was connected to the reference
electrode (Quasi 1), green clip to the working electrode and the
red clip to the counter electrode (Platinum wire, flamed in
advance, rinsed with EtOH and water).
[0280] The CHI 650C system was used to test all chips. For each
test, six files were used: 10000 mV/s, 100 mV/s, 10000 mV/s long,
multi CV (20 cycles) and ACV (forward and backward).
[0281] The multiplexer was used for testing all chips in this
experiment.
[0282] After initial testing, chips 1 through 5 and 7 through 11
were washed as follows:
TABLE-US-00006 Nanopure water 8 times 100 mM Na.sub.2CO.sub.3 (pH
10.1) 2 times
Chips 6 and 12 were washed as follows:
TABLE-US-00007 Nanopure water 8 times 100 mM NaHCO.sub.3 (pH 8.5) 2
times
[0283] Preparation of Different Concentrations of Hydrogen
Peroxide:
[0284] Different concentrations of H.sub.2O.sub.2 solution were
made in 100 mM Na.sub.2CO.sub.3 buffer (pH 10.1) immediately before
use. Original stock of H.sub.2O.sub.2 was at 1M which was made by
combining 57 .mu.L of 50% H.sub.2O.sub.2 with 943 .mu.L of nanopure
water. This stock was left in the 4.degree. C. fridge overnight to
allow for muta-rotation. From there on the dilutions were made as
shown below:
TABLE-US-00008 Final Amount concentration Ratio (previous of
previous Amount Total of to final concentration of of buffer volume
H.sub.2O.sub.2 (mM) concentration) H.sub.2O.sub.2 (.mu.L) (.mu.L)
(.mu.L) 1 1:1000 2 1998 2000 0.1 1:10 200 1800 2000 0.01 1:10 200
1800 2000 0.001 1:10 200 1800 2000 0 -- 0 2000 2000
[0285] Addition of Different Concentrations of H.sub.2O.sub.2 to
the Chips and Testing:
[0286] The hydrogen peroxide solutions made were vortexed well. 500
.mu.L of the respective hydrogen peroxide solutions was added to
each chip (1-12) and the solution was mixed thoroughly. The
incubation was carried out at room temperature for 5 minutes, while
mixing the solution in between with pipette tips at 4:30, 2:30, and
0:30 times, and for 10 minutes, while mixing the solution in
between with pipette tips at 7:30, 5:00, and 2:30 times. After the
respective H.sub.2O.sub.2 incubations, the chips were washed as
follows:
TABLE-US-00009 Nanopure water 8 times 100 mM Na.sub.2CO.sub.3 (pH
10.1) or 100 mM NaHCO.sub.3 (pH 8.5) 2 times
[0287] Each well was incubated with 500 .mu.L of their respective
buffers for 5 minutes. After the chips were incubated with buffer,
the chips were washed as follows:
TABLE-US-00010 Nanopure water 8 times 1M LiClO.sub.4 2 times
[0288] The switchbox was used for testing all chips as shown in
steps VI d, e and f. After testing, the chips were washed, cleaned
with ethanol and water and then disassembled.
TABLE-US-00011 Experiment Outline Chip Chip Name 1
#1_2_H2O2_0uM_5min_pH10pt1 2 #2_2_H2O2_1uM_5min_pH10pt1 3
#3_2_H2O2_10uM_5min_pH10pt1 4 #4_2_H2O2_100uM_5min_pH10pt1 5
#5_2_H2O2_1mM_5min_pH10pt1 6 #6_2_H2O2_0uM_5min_pH8pt5 7
#7_2_H2O2_0uM_10min_pH10pt1 8 #8_2_H2O2_1uM_10min_pH10pt1 9
#9_2_H2O2_10uM_10min_pH10pt1 10 #10_2_H2O2_100uM_10min_pH10pt1 11
#11_2_H2O2_1mM_10min_pH10pt1 12 #11_2_H2O2_0uM_10min_pH8pt5 13
#11_3_post-H2O2_FcMe2
Example 3
ATP Amplification Protocol
[0289] ATP Full Dose Response Curve in Complete Lysis Buffer
(0.5.times.HEPES)
[0290] Glycerol (2 mM), Glycerol kinase (GK, 1.33 U/mL), Glycerol
3-phosphate oxidase (G3PO-1.33 U/mL), Creatine kinase 18.times. (18
U/mL), Phosphocreatine (PC, 30 mM), ATP 0 nM-10 .mu.M were
used.
[0291] The following buffers were used: 0.5.times.HEPES (pH 7.4, 29
mM Maltoside), 1:200 benzonase, 1:2000 protease inhibitor, and
1:100 phosphatase inhibitor cocktail (0.5.times.), which was
modified as follows: 2.5 mM NaF, 3 .mu.M sodium orthovanadate, 500
.mu.M sodium pyrophosphate decahydrate, 500 .mu.M
beta-Glycerolphosphate, and 2.5 mM NaN.sub.3 (added to the
phosphatase inhibitor cocktail as a catalase inhibitor for
H.sub.2O.sub.2).
[0292] ATP 0 nM-10 .mu.M was prepared as follows: ATP serial
dilutions were made 10-fold higher in nanopure water containing 10
mM MgCl.sub.2, such that the [MgCl.sub.2] in the final assay is 1
mM. Nanopure water containing 10 mM MgCl2 was used to reconstitute
enzymes such as GK and G3PO. The following were the desired final
assay concentration of each component:
[0293] GK 10U/aliquot was prepared as follows: reconstituted in 250
.mu.L of water containing 10 mM MgCl.sub.2-0.04 U/.mu.L. 5 .mu.L of
0.04 U/.mu.L stock was taken and added to each well to a final
assay concentration of 1.33 U/mL. (For 140 .mu.L assay volume, 5
.mu.L of GK was added for each data point)
[0294] G3PO 5U/aliquot was prepared as follows: reconstituted in
125 .mu.L buffer to 0.04 U/uL. 5 .mu.L of 0.04 U/uL stock was taken
and added to each well to a final assay concentration of 1.33 U/mL.
5 .mu.L of G3PO was taken for total of 140 .mu.L assay volume.
[0295] Creatine kinase at 350 U/mg was prepared as follows: 0.5 mg
of CK was dissolved in 300 .mu.L of 20% glycerol in water to give
0.58 U/.mu.L. Then 5 .mu.L of 0.58 U/.mu.L stock was added to data
point for 140 .mu.L total assay volume.
[0296] Phosphocreatine (30 mM) was obtained by dissolving 574 mg of
solid in 5 mL water, and taking 5 .mu.L of that stock to each tube
to make 140 .mu.L total assay volume.
[0297] Glycerol (2 mM) was prepared by taking 22 .mu.L of Glycerol
stock and adding it to 4978 .mu.L buffer to make a 5 mL total
stock, and 5 .mu.L of this 5 mL stock was added to each well to
make 14 .mu.L total volume.
[0298] 5 .mu.L of Benzonase stock was added to 1 mL of
0.5.times.HEPES, pH 7.4 buffer to give Benzonase: 1:200 in
0.5.times.HEPES buffer, pH 7.4.
[0299] Phosphatase inhibitor cocktail (modified, 1:100) was
prepared as follows. 250 mM NaN.sub.3 was added to 5 mL (50.times.)
phosphatase inhibitor cocktail solution such as that final
concentration in 0.5.times. buffer was 2.5 mM NaN.sub.3. 1 .mu.M of
1 M stock of Zn acetate was added to 100 .mu.L of 50.times.
Phosphatase inhibitor cocktail aliquot such as that the final
concentration of Zn acetate in 0.5.times. lysis buffer is 100
.mu.M. 10 .mu.L of phosphatase inhibitor cocktail added to
0.5.times.HEPES, lysis buffer.
[0300] 0.5 .mu.L Protease inhibitor was added to 1 mL
0.5.times.HEPES, lysis buffer to give Protease inhibitor (1:2000
dilution) in 0.5.times.HEPES, pH 7.4.
[0301] Assay conditions constituted of 140 .mu.L of each sample
incubated for 2.5 hrs to be run in triplicates. The following
components were added to the tube for 2.5 hrs incubation before
putting them on chips for target testing: [0302] Upon incubation in
0.5.times.HEPES, pH 7.4 for 2.5 hrs, pH was adjusted to 10 by
adding 46.7 .mu.L Na.sub.2CO.sub.3 (100 mM); [0303] Total of 186.7
.mu.L sample (per concentration) and 25% sample dilution occurred
in the process of adjusting to pH 10 [0304] 30 .mu.L sample was
added to each chip in triplicates all at once and incubate (for 10
mins) and washed at two different washing stations
simultaneously.
[0305] The following volumes of each component were added: buffer
100 .mu.L; GK 5 .mu.L; G3PO 5 .mu.L; CK 5 .mu.L; phosphocreatine
(30 mM) 5 .mu.L; glycerol (2 mM) 5 .mu.L; ATP 15 .mu.L; ADHP 5
.mu.L; and HRP 5 .mu.L
[0306] E-Chip Experiment
[0307] Average Peak current ratio and Area ratio were analyzed for
all ten concentrations to obtain standard error on the full dose
response curve. No initial testing was done and the chips were
washed eight times with Ethanol, four times with water and
incubated with target solution of 10 mins right away. Upon 10 min
of incubation, chips were washed four times with water and two
times with LiClO.sub.4 and tested with 1M LiClO.sub.4 testing
solution.
Example 4
NADH Amplification Protocol
[0308] The following buffers were used: 0.5.times.HEPES, pH 7.4 (29
mM Maltoside), 1:200 benzonase, 1:2000 phosphatase inhibitor
cocktail (0.5.times.), which was modified as follows: 2.5 mM NaF, 3
.mu.M sodium orthovanadate, 500 .mu.M sodium pyrophosphate
decahydrate, 500 .mu.M beta-Glycerolphosphate, 2.5 mM NaN.sub.3,
and 100 .mu.M zinc acetate.
[0309] Desired concentrations in well/tube were as follows:
TABLE-US-00012 Solution Concentration NADH oxidase 5 ng/.mu.L
Ethanol 4% ADH 80 ng/.mu.L FAD 10 .mu.M NADH 3 .mu.M, 1 .mu.M, 300
nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM
[0310] NADH oxidase was prepared by adding 1.45 .mu.L of stock per
100 .mu.L of total NADH reaction solution. Ethanol was prepared by
adding 4 .mu.l 100% ethanol per 100 .mu.L NADH reaction solution.
ADH (4 .mu.g/.mu.L) was prepared by adding 2.4 mg/600 .mu.L in
water and 20% glycerol, and 2 .mu.L of this solution was added per
100 .mu.L of reaction sample.
[0311] FAD was prepared by dissolving 5.8 mg in 1000 .mu.L nanopure
water to give 7.5 mM stock. 7.5 mM stock was used to make 1 mM
stock by adding 13.3 .mu.L of 7.5 mM stock and diluting up to 100
.mu.L. Finally, 1 .mu.L of 1 mM stock was added per100 .mu.L
reaction.
[0312] NADH was prepared to a final concentration of 1 .mu.M, 300
nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, and 0.03
nM.
[0313] Each reaction had the following concentration per well:
TABLE-US-00013 buffer with NADH NADH inhibitor sample Alcohol FAD
ADH oxidase cocktail and Buffer (3 .mu.M-0.03 nM) 100% 1 mM 4
.mu.g/.mu.L 0.344 .mu.g/.mu.L sample benzonase (.mu.L) (.mu.L)
(.mu.L) (.mu.L) (.mu.L) (.mu.L) 1 HEPES 100 1 4 1 2 1.45 2 HEPES
100 1 4 1 2 1.45 3 HEPES 100 1 4 1 2 1.45 4 HEPES 100 1 4 1 2 1.45
5 HEPES 100 1 4 1 2 1.45 6 HEPES 100 1 4 1 2 1.45 7 HEPES 100 1 4 1
2 1.45 8 HEPES 100 1 4 1 2 1.45 9 HEPES 100 1 4 1 2 1.45 10 HEPES
100 1 4 1 2 1.45 11 HEPES 100 0 4 1 2 1.45
[0314] Each component was added to a 96 well plate and incubated
for 2 hours. After 2 hrs 90 .mu.L from each reaction sample was
taken and pH adjusted to pH 10 by adding 30 .mu.L 400 mM carbonate
solution. 30 .mu.L was then incubated on e-chips in
triplicates.
[0315] E-chip materials consist of [0316] EAM 2 (1 mg/l [0317] mL
EtOH):
[0317] ##STR00012## [0318] Target Incubation Buffer: 1.times.PBS,
100 mM NaHCO.sub.3 pH 10 [0319] Testing Buffer: 1M LiClO.sub.4.
3H.sub.2O [0320] Washing Buffer: Nanopure water [0321] Electrodes:
External Quasi and platinum wire
##STR00013## ##STR00014##
[0322] To a 0.degree. C. solution of compound 6 (0.215 g, 0.75
mmol) in DCM (10 mL) was added sodium borohydride (0.114 g, 3.00
mmol). MeOH (6 mL) was added slowly over 15 min. The reaction was
stirred and warmed to room temperature for 1 h. The reaction was
concentrated under reduced pressure and the crude residue was
dissolved in EtOAc (100 mL), washed with brine (3.times.100 mL),
dried over Na.sub.2SO.sub.4, filtered, and concentrated under
reduced pressure to a crude solid. Purification by column
chromatography yielded 7 as an orange solid (197 mg, 0.68 mmol,
91%).
[0323] A solution of compound 7 (0.535 g, 1.86 mmol) and sodium
azide (0.726 g, 11.2 mmol) in AcOH (35 mL) was stirred at
60.degree. C. for 20 h. The reaction was diluted with EtOAc (200
mL), washed with NaHCO.sub.3 (aq) (3.times.100 mL), dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure
to a crude brown oil. Purification by column chromatography yielded
8 as an orange oil (0.441 g, 1.41 mmol, 76%).
[0324] A solution of compound 8 (0.430 g, 1.37 mmol) and
triphenylphosphine (0.431 g, 1.64 mmol) in THF (25 mL) was stirred
at 60.degree. C. for 20 h. The reaction was concentrated under
reduce pressure to a crude oil which was purified by column
chromatography to yield 9 as a dark orange oil (0.371 g, 1.29 mmol,
94%).
[0325] A solution of 11-mercaptoundecanoic acid (2.70 g, 12.4
mmol), trityl chloride (4.14 g, 14.8 mmol), and DIPEA (5.17 mmol,
28.7 mmol) in toluene (40 mL) was stirred at room temperature for
20 h. The reaction was concentrated under reduced pressure and the
crude residue was dissolved in DCM (100 mL), washed with H.sub.2O
(3.times.100 mL), dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure to a crude yellow oil.
Purification by column chromatography yielded 10 as a white solid
(4.14 g, 73%).
[0326] To a 0.degree. C. solution of compound 9 (0.355 g, 1.24
mmol) and 10 (0.571 g, 1.24 mmol) in DCM (30 mL) was added
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide HCl (0.249 g, 1.30
mmol). After stirring for 5 h the reaction was concentrated under
reduced pressure to a crude brown oil. Purification by column
chromatography yielded 11 as a viscous orange oil (0.724 g, 0.99
mmol, 80%).
[0327] To a solution of compound 11 (0.720 g, 0.99 mmol) in EtOH
(30 mL) was added a solution of potassium hydroxide (0.333 g, 5.93
mmol) in H.sub.2O (3 mL). The reaction was heated to 70.degree. C.
After stirring for 24 h, the reaction was concentrated under
reduced pressure to crude residue. The crude residue was dissolved
in H.sub.2O (100 mL), acidified to pH=4.0, extracted with DCM
(4.times.100 mL), dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure to an orange oil. The orange
oil was purified by column chromatography to yield 12 as a golden
yellow solid (0.589 g, 0.84 mmol, 85%).
[0328] To a solution of compound 12 (0.456 g, 0.65 mmol) in THF (30
mL) was added diphenylphosphoryl azide (168 .mu.L, 0.78 mmol)
followed by triethylamine (136 .mu.L, 0.98 mmol). The reaction was
stirred for 20 h and concentrated under reduced pressure to a crude
red oil. Purification by column chromatography yielded 13 as a
red/orange solid (0.400 g, 0.55 mmol, 85%).
[0329] To a solution of compound 13 (0.298 g, 0.41 mmol) and 14
(0.106 g, 0.45 mmol) in toluene (30 mL) was added di-n-butyltin
dilaurate (12 .mu.L, 0.002 mmol). The reaction was stirred at
100.degree. C. for 4 h and concentrated under reduced pressure to a
crude brown oil. Purification by column chromatography yielded 15
as a golden yellow oil (0.305 g, 0.33 mmol, 80%).
##STR00015##
[0330] To a solution of compound 15 (0.134 g, 0.14 mmol) in DCM (2
mL) was added a solution of trifluoroacetic acid (200 .mu.L),
triethylsilane (115 .mu.L, 0.72 mmol) in DCM (2 mL). The reaction
was stirred at room temperature for 3 h and concentrated under
reduced pressure to a crude brown oil. Purification by column
chromatography yielded 2 as a yellow solid (0.072 g, 0.10 mmol,
72%).
[0331] For all chips, the following procedure was performed to
deposit the SAM. Chips were rinsed again with EtOH and dried with
argon. Accessory parts (gasket, tubs) were cleaned by washing
machine, rinsed with EtOH and air-dried. Bases were cleaned by
hand. 200 .mu.L of the deposition solution prepared above was added
to each chip, and chips were incubated overnight in EtOH incubation
chamber. Following overnight incubation, chips removed from
containers. The chips were then washed as follows: 8.times.EtOH,
4.times. nanopure water.
[0332] Each chip was incubated as designated in table below. Buffer
used for the samples was complete lysis buffer with modified
Phosphatase inhibitor with zinc acetate and sodium azide. The
samples were pH adjusted to pH 10 with 400 mM of carbonate in 1:4
ratio. Each concentration was run in triplicates on chips. After
target incubation of 10 mins the chips were washed 4.times. water,
2.times. LiClO.sub.4, and tested.
TABLE-US-00014 Macro Chip No. Solution NADH 1 1 .mu.M 2 300 nM 3
100 nM 4 30 nM 5 10 nM 6 3 nM 7 1 nM 8 0.3 nM 9 0.1 nM 10 0.03 nM
11 0 nM
Example 5
TSH Protein Measurement
[0333] The reagents used were as follows: anti-TSH loaded magnetic
beads (capture, MP), 10 .mu.g/.mu.L MP; TSH antigen, 1 mU/mL;
biotin-anti-TSH conjugate (Secondary), 0.85 mg/mL; Alkaline
Phosphatase-Streptavidin conjugate (SA-AP), 1.5 mg/mL; FADP, 10 mM;
Apo-DAAO enzyme (DAAO), 3.9 mg/mL-100 .mu.M; D-Proline, 1 M; TBS,
20.times. (1 M Tris); PBS, 20.times. (1 M PO.sub.4); BSA, 10%
filtered solution; Tween20, 10% filtered solution; and Sodium
Carbonate, 400 mM.
[0334] E-chip materials consist of [0335] EAM (1 mg/1 mL EtOH):
[0335] ##STR00016## [0336] Target Incubation Buffer:1.times.PBS,
100 mM NaHCO.sub.3 pH 10 [0337] Testing Buffer: 1M LiClO.sub.4.
3H.sub.2O [0338] Washing Buffer: Nanopure water [0339] Electrodes:
External Quasi and platinum wire
[0340] Clinical serum samples with known TSH content were obtained
from the NorthShore core facility and diluted as shown below:
TABLE-US-00015 TSH (.mu.lU/mL) Sample ID Dilution (%) after
dilution 142 1.25 0.13473 144 1.25 0.47359 145 12.50 0.74813 146
1.25 0.05525 147 12.50 0.54663
[0341] Magnetic bead binding assay similar in structure to previous
magnetic bead assays. Targets consisted of serial dilution of
recombinant TSH antigen, and serial dilution concentrations were:
30, 3, 0.3, 0.03, and 0 .mu.lU/mL, in binding buffer (1.times.PBS,
1% BSA, 0.3% tween). Magnetic bead stocks with capture antibody
were pre-washed and pre-blocked at 10 .mu.g/.mu.L. Stock in PBS was
diluted 50-fold; and added 100 .mu.L to each tube (20 .mu.g/tube).
Pellet beads and remove buffer before adding target. TSH antigen
was serial diluted to 30, 3, 0.3, 0.03, and 0 by making 3.16-fold
serial dilutions. After removing buffer from beads, 100 .mu.L of
each target dilution was added to individual tubes, vortexed
quickly to mix after each target dilution is added, and incubated
for 30 minutes on shaker (200-300 rpm).
[0342] Antibody solution (4 ng/.mu.L) was prepared in binding
buffer with enough for 100 .mu.L per tube. Beads were washed by
pelleting against magnetic bar, removing supernatant and adding 500
.mu.L of wash buffer, then vortexed briefly and spined down to
avoid droplet formation on the walls of the tube. Beads were
pelleted against magnetic bar and remove wash buffer, and 100 .mu.L
of antibody solution was added to each tube. The solution was
vortexed quickly to mix and incubated for 15 minutes at room
temperature on shaker (200-300 rpm).
[0343] SA-AP solution of 1 mL at 22.5 ng/.mu.L was prepared in
binding buffer (BB). Beads were washed by pelleting against magnet,
and wash buffer was removed. The solution was briefly vortexed to
mix, then it was spun down. Beads were pelleted against the bar
magnet and wash buffer was removed. 100 .mu.L of SA-AP solution was
added to each tube, and vortexed quickly to mix and incubated for
10 minutes at room temperature on shaker (200-300 rpm).
[0344] Beads were washed 4 times as described above. Following
washes in wash buffer, 100 .mu.L TBS pH 8.5 was added, and the
beads in the buffer were carefully transferred to clean tubes. FADP
solution of 20 .mu.M was prepared by adding 2 .mu.L of 10 mM FADP
stock to 1 mL of TBS pH 8.5. Beads were pelleted against bar magnet
and removed for final incubation. 95 .mu.L of substrate solution
was added to each tube, and vortexed quickly to mix and incubated
for 30 minutes on shaker (200-300 rpm). During the incubation,
E-chips were prepared by washing 8.times. with ethanol and 4.times.
with nanopure water.
[0345] After incubation is complete, beads were pelleted on magnet
and 90 .mu.L of substrate solution was moved to a new tube to stop
FADP conversion. 10.times.DAAO solution was prepared by making 2
.mu.M DAAO enzyme and 350 mM D-proline in TBS pH 8.5 buffer.
10.times.DAAO solution was added to each tube to start reaction,
and start of each reaction was staggered by 15 seconds, vortexed
briefly each addition and incubated 5 minutes in the tubes. pH was
adjusted to 10. 14.28 .mu.L of 400 .mu.M sodium carbonate was added
to 100 .mu.L, and added in the same order as the DAAO and again
waited 15 seconds between tubes to maintain constant incubation
time across tubes; and vortexed briefly after each addition.
[0346] The water was aspirated from E-chips. 36 .mu.L of pH
adjusted solution was added to each of three replicate chips.
Volume was added quickly to replicates and each target dilution was
stagger to maintain constant E-Chip incubation time across samples.
Samples were incubated for 10 minutes. After incubation, solution
from E-Chips was aspirated and washed 4.times. with water and
2.times. with 1 M LiClO.sub.4. The chips were tested on CHI
multiplexer with a 0.5 V/s scan rate.
[0347] Data Analysis
[0348] Data Analysis was performed in Matlab using the EDAS
executable version. The peak current ratio (i.sub.2/i.sub.1-2) was
determined from the bottom curve of each voltammogram.
[0349] Results
[0350] The standard curved generated in this experiment had a
higher signal than in previous experiments, where a shortened
protocol with reduced incubation times was used to check
consistency in signal between the old and new bead lots. The
standard curve had a small error at each TSH dose that increased
slightly at higher doses of TSH. The signals from the clinical
samples (142, 144, 145, 147, 146) all fell within the range of the
standard curve and are shown in FIG. 23. The signal increases with
increasing TSH value as determined by Northshore.
[0351] It is to be understood that the examples and embodiments
described herein are for illustrative purposes only. Unless clearly
excluded by the context, all embodiments disclosed for one aspect
of the invention can be combined with embodiments disclosed for
other aspects of the invention, in any suitable combination. It
will be apparent to those skilled in the art that various
modifications and variations can be made to the present invention
without departing from the scope of the invention. Thus, it is
intended that the present invention cover the modifications and
variations of this invention provided they come within the scope of
the appended claims and their equivalents.
* * * * *