U.S. patent application number 11/431247 was filed with the patent office on 2006-11-30 for water relaxation-based sensors.
Invention is credited to Lee Josephson, Yi Sun, Ralph Weissleder.
Application Number | 20060269965 11/431247 |
Document ID | / |
Family ID | 37397220 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060269965 |
Kind Code |
A1 |
Josephson; Lee ; et
al. |
November 30, 2006 |
Water relaxation-based sensors
Abstract
This invention relates to magnetic resonance-based sensors and
related methods.
Inventors: |
Josephson; Lee; (Reading,
MA) ; Sun; Yi; (Malden, MA) ; Weissleder;
Ralph; (Peabody, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
37397220 |
Appl. No.: |
11/431247 |
Filed: |
May 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60679437 |
May 9, 2005 |
|
|
|
Current U.S.
Class: |
435/7.1 ; 435/14;
435/287.2; 977/900 |
Current CPC
Class: |
A61B 5/14503 20130101;
G01R 33/50 20130101; G01N 33/54326 20130101; G01N 27/745 20130101;
G01N 33/54366 20130101; G01R 33/465 20130101 |
Class at
Publication: |
435/007.1 ;
435/014; 435/287.2; 977/900 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12Q 1/54 20060101 C12Q001/54; C12M 1/34 20060101
C12M001/34 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The work described herein was carried out, at least in part,
using funds from National Institutes of Health (NIH) Grants R01
EB004626 and EB00662. The government therefore has certain rights
in the invention.
Claims
1. A water relaxation-based sensor for detecting the presence of an
analyte in a sample, the sensor comprising: (i) a walled enclosure
enveloping a chamber, wherein the wall comprises an opening for
passage of the analyte into and out of the chamber; (ii) a
plurality of magnetic nanoparticles located within the chamber,
each nanoparticle having at least one moiety that is covalently or
noncovalently linked to the nanoparticle; and optionally, (iii) at
least one binding agent located within the chamber; wherein the
opening is smaller in size than the nanoparticles, and is larger in
size than the analyte; and wherein the moiety and the analyte each
bind reversibly to the binding agent, when present; or the analyte
binds reversibly to the moiety.
2. The sensor of claim 1, wherein the opening is smaller in size
than the binding agent.
3. The sensor of claim 1, wherein the wall comprises a plurality of
openings for passage of the analyte into and out of the chamber,
wherein each of the openings is smaller in size than the
nanoparticles and the binding agent, and each of the openings is
larger in size than the analyte.
4. The sensor of claim 1, wherein the moiety comprises a
carbohydrate, an antibody, an amino acid, a nucleic acid, an
oligonucleotide, a therapeutic agent or a metabolite thereof, a
peptide, or a protein.
5. The sensor of claim 1, wherein the moiety comprises a molecular
fragment of the analyte being detected or a molecular fragment of a
derivative, isostere, or mimic of the analyte being detected.
6. The sensor of claim 1, wherein the moiety is linked to the
nanoparticle by a functional group comprising --NH--, --NHC(O)--,
--(O)CNH--, --NHC(O)(CH.sub.2).sub.nC(O),
--(O)C(CH.sub.2).sub.nC(O)NH--, --NHC(O)(CH.sub.2).sub.nC(O)NH--,
--C(O)O--, --OC(O)--, or --SS--, and wherein n is 0 to 20.
7. The sensor of claim 6, wherein the functional group is
--NHC(O)(CH.sub.2).sub.nC(O)NH--.
8. The sensor of claim 7, wherein n is 2.
9. The sensor of claim 1, wherein the binding agent is absent.
10. The sensor of claim 9, wherein the moiety comprises a
protein.
11. The sensor of claim 9, wherein: (a) when the analyte is absent,
the chamber comprises substantially disaggregated nanoparticles;
and (b) when the analyte is present, the chamber comprises a
nanoparticle aggregate, wherein the nanoparticle aggregate
comprises nanoparticles bound to the exogenous analyte through the
moiety.
12. The sensor of claim 1, wherein the binding agent is
present.
13. The sensor of claim 12, wherein the binding agent comprises a
protein or a monoclonal antibody.
14. The sensor of claim 12, wherein the moiety comprises a
molecular fragment of the analyte being detected or a molecular
fragment of a derivative, isostere, or mimic of the analyte being
detected.
15. The sensor of claim 12, wherein: (a) when the analyte is
absent, the chamber comprises a nanoparticle aggregate, wherein the
nanoparticle aggregate comprises nanoparticles bound to the binding
agent through the moiety; and (b) when the analyte is present, the
nanoparticles are displaced from the binding agent by the analyte,
and the chamber comprises substantially disaggregated
nanoparticles.
16. The sensor of claim 1, wherein the opening has a size of from
about 1 kDa to about 3 kDa.
17. The sensor of claim 1, wherein each of the nanoparticles has an
overall size of from about 30 nm to about 60 nm.
18. The sensor of claim 11 or 15, wherein the nanoparticle
aggregate has a an overall size of at least about 100 nm.
19. The sensor of claim 11 or 15, wherein the change in
nanoparticle aggregation between (a) and (b) alters the proton
relaxation of water inside of the chamber, but does not
substantially alter the proton relaxation of water outside of the
chamber.
20. The sensor of claim 19, wherein the change in nanoparticle
aggregation between (a) and (b) produces a measurable change in the
T2 relaxation times of water inside the chamber.
21. The sensor of claim 1, wherein the moiety comprises a chiral
compound.
22. The sensor of claim 1, wherein the moiety comprises a
carbohydrate.
23. The sensor of claim 1, wherein the moiety comprises the
structure: ##STR3##
24. The sensor of claim 1, wherein the binding agent is a protein
that comprises at least two binding sites.
25. The sensor of claim 1, wherein the binding agent is a protein
that comprises at least four binding sites.
26. The sensor of claim 1, wherein the binding agent is a protein
that binds to a carbohydrate.
27. The sensor of claim 26, wherein the carbohydrate is
glucose.
28. The sensor of claim 27, wherein the protein is conconavalin
A.
29. The sensor of claim 1, wherein the binding agent comprises a
monoclonal antibody, a polyclonal antibody, or a
oligonucleotide.
30. The sensor of claim 1, wherein the magnetic nanoparticles each
comprise a magnetic metal oxide.
31. The sensor of claim 30, wherein the magnetic metal oxide
comprises a superparamagnetic metal oxide.
32. The sensor of claim 30, wherein the metal oxide comprises iron
oxide.
33. The sensor of claim 32, wherein each of the magnetic
nanoparticles is an amino-derivatized cross-linked iron oxide
nanoparticle.
34. The sensor of claim 1, wherein the nanoparticles are
substantially aggregated.
35. A method of detecting an analyte in an aqueous sample, the
method comprising: (i) providing the sensor of claim 1; (ii)
measuring relaxation times of the water inside of the chamber of
the sensor in the absence of the analyte or under conditions that
mimic the absence of the analyte; (iii) contacting the sensor with
the sample; (iv) measuring relaxation times of the water inside of
the chamber of the sensor; and (v) comparing the T2 relaxation
times measured in step (ii) and step (iv); wherein a change in T2
relaxation times measured in step (iv) relative to the T2
relaxation times measured in step (ii) indicates the presence of
the analyte.
36. The method of claim 35, wherein the analyte is a monovalent
analyte.
37. The method of claim 35, wherein the analyte is a multivalent
analyte.
38. The method of claim 35, wherein the change in the T2 relaxation
times is measured using a magnetic resonance imaging method.
39. The method of claim 35, wherein the change in the T2 relaxation
times is measured using a magnetic resonance non-imaging
method.
40. The method of claim 35, wherein the analyte is a
carbohydrate.
41. The method of claim 35, wherein the analyte is glucose.
42. The method of claim 35, wherein the analyte is chiral.
43. The method of claim 42, wherein the chiral analyte is present
together with one or more optically active moieties in the
sample.
44. The method of claim 43, wherein the chiral analyte is present
together with a stereoisomer of the chiral analyte in the
sample.
45. The method of claim 44, wherein the chiral analyte is present
together with an enantiomer of the chiral analyte in the
sample.
46. The method of claim 42, wherein the chiral analyte is an amino
acid.
47. The method of claim 35, wherein the analyte is a nucleic acid
or an oligonucleotide.
48. The method of claim 35, wherein the analyte is a therapeutic
agent or a metabolite of a therapeutic agent.
49. The method of claim 35, wherein the analyte is peptide or a
protein.
50. The method of claim 35, wherein steps (ii) and (iv) comprise
measuring T2 relaxation times.
51. The method of claim 50, wherein an increase in T2 relaxation
times measured in step (iv) relative to the T2 relaxation times
measured in step (ii) indicates the presence of the analyte.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Application 60/679,437, filed on May 9, 2005, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] This invention relates to magnetic resonance-based sensors
and related methods.
BACKGROUND
[0004] Magnetic resonance (MR)-based reporting methods, such as
magnetic resonance imaging (MRI), offer certain known advantages as
non-invasive methods. For example, MRI can be used at tissue depths
where optical reporting methods can sometimes be complicated by
light scattering and absorption by the tissue, e.g., tissue depths
greater than about 250 .mu.m.
[0005] One application of nanotechnology in medicine is the
development of biocompatible nanomaterials as environmentally
sensitive sensors and molecular imaging agents. Preparations of
magnetic particles designed for separation and extraction use
particles that are amenable to easy manipulation by weak applied
magnetic fields. These materials are typically micron sized and
have a high magnetic moment per particle. However, nanoparticles do
not respond to the weak, magnetic fields of hand held magnets.
SUMMARY
[0006] This invention relates generally to magnetic resonance-based
sensors (e.g., water relaxation and equilibrium-based sensors) and
related methods, and is based, in part, on the discovery that
sensors having magnetic nanoparticles encapsulated within a
semipermeable enclosure can be used as remote sensors for detecting
various analytes in an aqueous, e.g., a water-containing, sample
and can be used for the continuous monitoring of changing levels of
the analytes.
[0007] In its broadest aspects, the invention provides a water
relaxation-based sensor for detecting the presence of an analyte in
a sample. The sensor includes an enclosure defining an opening for
entry of the analyte, e.g., a semipermeable membrane, and confined
within said enclosure, a plurality of nanoparticles. The
nanoparticles are suspended or suspendable in an aqueous liquid
phase, have a magnetic moment, e.g., comprise crystalline iron
oxide or other magnetic material, and are covalently or non
covalently linked to, or otherwise have immobilized thereon, one or
more moieties selected to alter the state of aggregation of the
nanoparticles as a function of the presence or concentration of the
analyte in the enclosure.
[0008] In one aspect, this invention features water
relaxation-based sensors for detecting the presence of an analyte
(e.g., an exogenous analyte) in a sample. The sensors include: (i)
a walled enclosure enveloping a chamber, wherein the wall includes
one or more openings (e.g., a single opening or a plurality of
openings) for passage of the analyte into and out of the chamber;
(ii) a plurality of magnetic nanoparticles located within the
chamber, each nanoparticle having at least one moiety that is
covalently or noncovalently linked to (immobilized on) the
nanoparticle; and optionally, (iii) at least one binding agent
located within the chamber, in which the opening can be smaller in
size than the nanoparticles and the binding agent and larger in
size than the analyte; and the moiety and the analyte can each bind
reversibly to the binding agent, when present; or the analyte can
bind reversibly to the moiety. In some embodiments, the openings
can be larger than the binding agent, as long as the binding agents
remain within the chamber when bound to the nanoparticles.
[0009] Embodiments can include one or more of the following
features.
[0010] The nanoparticles can be suspended or be suspendable in an
aqueous liquid phase. The nanoparticles can have a magnetic moment
generally, or under certain conditions.
[0011] The wall can include more than one opening. When the wall
contains more than one opening (e.g., a plurality of openings),
then at least one of the openings is smaller in size than the
nanoparticles and optionally the binding agent, and larger in size
than the analyte. In certain embodiments, the wall includes a
plurality of openings, in which each of the openings is smaller in
size than the nanoparticles and the binding agent, and each of the
openings is larger in size than the analyte.
[0012] The moiety can be selected to alter the state of aggregation
of the nanoparticles as a function of the presence or concentration
of the analyte in the enclosure.
[0013] In its several alternative embodiments, the sensor may
exploit different detection formats. For example, the moiety may be
selected to bind to the analyte to produce an aggregate of plural
linked nanoparticles as a function of the presence or concentration
of the analyte in the enclosure. The sensor may include an
aggregate of plural linked nanoparticles, which is disaggregated as
a function of the presence or concentration of the analyte in the
enclosure. The moiety may be a fragment of an authentic sample of
the analyte or a structural mimic thereof, in which case the sensor
further includes a multivalent binding agent, which binds to the
analyte and the mimic (if used) to produce an aggregate of plural
linked nanoparticles. The sensor can further include a multivalent
binding agent which binds to the moiety to produce an aggregate.
The sensor also can include a binding agent, which binds to the
moiety in the presence of the analyte to disassociate an aggregate.
In yet another form, the sensor can include a binding agent that
binds to the moiety in the presence of the analyte to produce an
aggregate. In one embodiment, the sensor further includes a
plurality of aggregates confined within the enclosure. In another
embodiment, the sensor includes a sample flow path in communication
with the interior of the enclosure. Thus, the sample can flow into,
or into and out of, the enclosure to permit periodic sampling of
the analyte.
[0014] In some embodiments, the sensors include features (i), (ii),
and (iii) above; the moiety can be, or can include as part of its
chemical structure, a molecular fragment of the analyte being
detected or a molecular fragment of a derivative, isostere, or
mimic of the analyte being detected; and the binding agent can be a
protein. When the analyte is absent, the chamber can include one or
more nanoparticle aggregates. Each of the nanoparticle aggregates
can include one or more nanoparticles and the binding agent.
Formation of the nanoparticle aggregate can occur through binding
of a moiety on the nanoparticle to the binding agent (e.g., the
binding agent can include one or more binding sites that are
recognized for binding by the moiety).
[0015] When the analyte is present, the chamber can include
substantially disaggregated nanoparticles. The analyte, when
present, can displace the nanoparticles from the nanoparticle
conjugates, thereby providing substantially disaggregated
nanoparticles (e.g., the analyte and the moiety can be selected
such that the analyte and the nanoparticles can compete for binding
with the binding agent, and the analyte, when present, can displace
the nanoparticles from the binding agent in the aggregate to
provide disaggregated nanoparticles).
[0016] In certain embodiments, (a) when the analyte is absent, the
chamber can include a nanoparticle aggregate, wherein the
nanoparticle aggregate can include nanoparticles bound to the
binding agent through the moiety; and (b) when the analyte is
present, the nanoparticles are displaced from the binding agent by
the analyte, and the chamber comprises substantially disaggregated
nanoparticles. In the presence of a water-containing liquid media,
the change in nanoparticle aggregation (from nanoparticle
aggregates to substantially disaggregated nanoparticles and vice
versa, e.g., the difference between (a) and (b) above) alters the
proton relaxation of water inside of the chamber, but does not
substantially alter the proton relaxation of water outside of the
chamber.
[0017] In some embodiments, the sensors include features (i) and
(ii) above, feature (iii) is absent; and the moiety can be, or can
include as part of its chemical structure, a protein. When the
analyte is absent, the chamber can include substantially
disaggregated nanoparticles. When the analyte is present, the
chamber can include one or more nanoparticle aggregates. Each of
the nanoparticle aggregates can include one or more nanoparticles
and the analyte. Formation of the nanoparticle aggregate can occur
through binding of the analyte to the moiety on the nanoparticle
(e.g., the moiety can include one or more binding sites that are
recognized for binding by the analyte).
[0018] In certain embodiments, (a) when the analyte is absent, the
chamber comprises substantially disaggregated nanoparticles; and
(b) when the analyte is present, the chamber comprises a
nanoparticle aggregate, wherein the nanoparticle aggregate
comprises nanoparticles bound to the analyte through the moiety. In
the presence of a water-containing liquid media, the change in
nanoparticle aggregation (from nanoparticle aggregates to
substantially disaggregated nanoparticles and vice versa e.g., the
difference between (a) and (b) above) alters the proton relaxation
of water inside of the chamber, but does not substantially alter
the proton relaxation of water outside of the chamber.
[0019] In one aspect, this invention features methods of detecting
an analyte in an aqueous sample (e.g., monitoring the presence or
concentration of an analyte in a sample stream), the methods
include: (i) providing a sensor as described herein; (ii) measuring
relaxation times (e.g., T2 or T1 relaxation times) of the water
inside of the chamber of the sensor in the absence of the analyte
or under conditions that mimic the absence of the analyte; (iii)
contacting the sensor with the sample (e.g., the nanoparticles can
be suspended, or suspendable, in an aqueous liquid phase and can
also have a magnetic moment); (iv) measuring relaxation times
(e.g., T2 or T1 relaxation times) of the water inside of the
chamber of the sensor; and (v) comparing the T2 relaxation times
measured in step (ii) and step (iv). A change (e.g., an increase or
decrease) in T2 relaxation times measured in step (iv) relative to
the T2 relaxation times measured in step (ii) indicates the
presence of the analyte.
[0020] For example, one can flow a sample stream into the
enclosure, allow analyte in the sample to alter the state of
aggregation of nanoparticles (e.g., suspended nanoparticles), and
measures perturbation of the magnetic resonance relaxivity of water
protons disposed adjacent the nanoparticles. These steps can be
repeated to obtain a temporal profile of the concentration of the
analyte in the stream.
[0021] Embodiments can include one or more of the following
features.
[0022] The nanoparticles can be suspended or be suspendable in an
aqueous liquid phase. The nanoparticles can have a magnetic
moment.
[0023] The wall can include more than one opening. When the wall
contains more than one openings (e.g., a plurality of openings),
then at least one of the opening is smaller in size than the
nanoparticles and the binding agent, and larger in size than the
analyte. In certain embodiments, the wall includes a plurality of
openings, in which each of the openings is smaller in size than the
nanoparticles and the binding agent, and each of the openings is
larger in size than the analyte.
[0024] The moiety can be selected to alter the state of aggregation
of the nanoparticles as a function of the presence or concentration
of the analyte in the enclosure. The moiety and the analyte can
each bind reversibly to the binding agent, when present; or the
analyte can bind reversibly to the moiety. The analyte can be a
monovalent or multivalent analyte.
[0025] The moiety can be, or can include as part of its structure,
a carbohydrate, an antibody, an amino acid, a nucleic acid, an
oligonucleotide, a therapeutic agent or a metabolite thereof, a
peptide, or a protein. The moiety can be a covalently or
noncovalently linked analyte (e.g., the analyte that is being
detected, sometimes referred to as a bound analyte or a bound
binding protein), a covalently or noncovalently linked analyte
derivative, or a covalently or noncovalently linked analyte
isostere or mimic (e.g., a derivative, isostere, or mimic of the
analyte that is being detected).
[0026] In some embodiments, the moiety can be, or can include as
part of its structure, a molecular fragment of the analyte being
detected or a molecular fragment of a derivative, isostere, or
mimic of the analyte being detected.
[0027] As used herein and throughout, a moiety that includes "a
molecular fragment of the analyte being detected" (or that includes
a molecular fragment of a derivative, isostere, or mimic of the
analyte being detected) is one in which a portion (e.g., a
substantial portion) of the chemical structure of the analyte being
detected (or a derivative, isostere, or mimic thereof) is
incorporated into the chemical structure of the moiety. In these
embodiments, the nanoparticle can have the general formula (A):
(A).sub.z-NP.sup.c; in which "A" is a molecular fragment of an
analyte, A-X, in which X is a hydrogen atom or a functional group
that is present in the analyte, but not incorporated into the
nanoparticle of formula (A); "NP.sup.c" is the nanoparticle core,
"-" is a covalent linkage (e.g., a chemical bond or linking
functional group) that connects any atom of the fragment to the
nanoparticle; and z is 1-50 (e.g., 1-40, 1-30, 1-25, 1-20, 2-20).
"A" in the above formula can also be the molecular fragment of a
derivative, isostere, or mimic of the analyte being detected.
[0028] The moiety can be a protein or a nucleic acid.
[0029] The binding agent can be absent. The moiety can be, or
include as part of its structure, a protein. In some embodiments,
(a) when the analyte is absent, the chamber includes substantially
disaggregated nanoparticles; and (b) when the analyte is present,
the chamber can include one or more nanoparticle aggregates. Each
of the nanoparticle aggregates can include one or more
nanoparticles and the analyte. Formation of the nanoparticle
aggregate can occur through binding of the analyte to the moiety on
the nanoparticle (e.g., the moiety can include one or more binding
sites that are recognized for binding by the analyte).
[0030] In some embodiments, (a) when the analyte is absent, the
chamber comprises substantially disaggregated nanoparticles; and
(b) when the analyte is present, the chamber comprises a
nanoparticle aggregate, wherein the nanoparticle aggregate
comprises nanoparticles bound to the analyte through the
moiety.
[0031] The binding agent can be present, it can be, for example, a
protein or a monoclonal antibody. The moiety can be, or can include
as part of its structure, a molecular fragment of the analyte being
detected or a molecular fragment of a derivative, isostere, or
mimic of the analyte being detected.
[0032] In some embodiments, (a) when the analyte is absent, the
chamber can include one or more nanoparticle aggregates; and (b)
when the analyte is present, the chamber can include substantially
disaggregated nanoparticles. Each of the nanoparticle aggregates
can include one or more nanoparticles and the binding agent.
Formation of the nanoparticle aggregate can occur through binding
of a moiety on the nanoparticle to the binding agent (e.g., the
binding agent can include one or more binding sites that are
recognized for binding by a chemical group that is present as all
or part of the chemical structure of the moiety). The analyte, when
present, can displace the nanoparticles from the nanoparticle
conjugates, thereby providing substantially disaggregated
nanoparticles (e.g., the analyte and the nanoparticle substituent
can be selected such that the analyte and the nanoparticle moiety
can compete for binding with the binding agent, and the analyte,
when present, can displace the nanoparticles from the binding agent
in the aggregate to provide disaggregated nanoparticles).
[0033] In some embodiments, (a) when the analyte is absent, the
chamber can include a nanoparticle aggregate, wherein the
nanoparticle aggregate can include nanoparticles bound to the
binding agent through the moiety; and (b) when the analyte is
present, the nanoparticles are displaced from the binding agent by
the analyte, and the chamber comprises substantially disaggregated
nanoparticles. The change in nanoparticle aggregation between (a)
and (b) can alter the proton relaxation of water inside of the
chamber, but does not substantially alter the proton relaxation of
water outside of the chamber.
[0034] The change in nanoparticle aggregation between (a) and (b)
can produce a measurable change in the T2 relaxation times of water
inside the chamber, and the change in the T2 relaxation times can
be measurable using a magnetic resonance imaging or non-imaging
method.
[0035] The moiety can be linked to the nanoparticle by a functional
group such as --NH--, --NHC(O)--, --(O)CNH--,
--NHC(O)(CH.sub.2).sub.nC(O), --(O)C(CH.sub.2).sub.nC(O)NH--,
--NHC(O)(CH.sub.2).sub.nC(O)NH--, --C(O)O--, --OC(O)--, or --SS--,
in which n can be 0-20, e.g., 2, 5, 10, or 15. Each of the openings
can have a size (pore size) of from about 1 kDa to about 1 .mu.m
(e.g., about 1 kDa to about 300,000 kDa; about 1 kDa to about
100,000 kDa; about 1 kDa to about 5 kDa; 1 kDa to about 3 kDa; 1
kDA to about 1 .mu.m).
[0036] Each of the nanoparticles can have a particle size or
overall size of from about 10 nm to about 500 nm (e.g., about 10 nm
to about 60 nm, about 30 nm to about 60 nm). The overall size is
the largest dimension of a particle. The nanoparticle aggregate can
have an overall size (particle size) of at least about 100 nm. The
nanoparticles can be substantially aggregated (e.g., include on or
more nanoparticle aggregates) or substantially disaggregated. The
analyte can be a carbohydrate (e.g., glucose).
[0037] The analyte can be chiral. The chiral analyte can be present
together with one or more optically active moieties in the sample.
The chiral analyte can be present together with a stereoisomer of
the chiral analyte in the sample. The chiral analyte can be present
together with the enantiomer of the chiral analyte in the sample.
The chiral exogenous analyte can be an amino acid.
[0038] The analyte can be a nucleic acid or an oligonucleotide.
[0039] The analyte can be a therapeutic agent, which as used herein
refers to a bioactive moiety, which when administered to a subject
(e.g., a human or animal subject) confers a therapeutic,
biological, or pharmacological effect (e.g., treats, controls,
ameliorates, prevents, delays the onset of, or reduces the risk of
developing one or more diseases, disorders, or conditions or
symptoms thereof) on the subject, or a metabolite thereof. The
analyte can be, e.g., folic acid.
[0040] The analyte can be a peptide or a protein (e.g., influenza
hemagglutinin peptide).
[0041] The moiety can be, or can include as part of its structure,
a chiral moiety. The moiety can be, or can include as part of its
structure, an amino acid, a nucleic acid, an oligonucleotide, a
therapeutic agent, a metabolite of a therapeutic agent, a peptide,
or a protein. The moiety can be, or can include as part of its
structure, a carbohydrate (e.g., having the structure: ##STR1##
[0042] The binding agent can be a protein that includes at least
two binding sites or at least four binding sites. The binding agent
can be a recombinant protein or be a complex of proteins each with
binding sites. The complex of proteins may be assembled by
crosslinking. The binding agent can be a protein that binds to a
carbohydrate (e.g., glucose). The protein can be conconavalin A.
The binding agent can be a monoclonal antibody, a polyclonal
antibody, or a oligonucleotide. The binding agent can be, e.g., an
antibody to folic acid or an antibody to influenza hemagglutinin
peptide. The analyte and the nanoparticle can bind reversibly to
the binding agent.
[0043] The magnetic nanoparticles each can include a magnetic metal
oxide (e.g., a superparamagnetic metal oxide). The metal oxide can
be iron oxide. Each of the magnetic nanoparticles can be an
amino-derivatized cross-linked iron oxide nanoparticle.
[0044] The sensor can be configured to be an implantable sensor.
For example, the sensor can be implanted subcutaneously. In certain
embodiments, the sensor can be implanted in an extremity of a
subject (e.g., a human or animal).
[0045] Steps (ii) and (iv) can include measuring T2 relaxation
times or T1 relaxation times. An increase in T2 relaxation times
measured in step (iv) relative to the T2 relaxation times measured
in step (ii) can indicate the presence of the analyte. A decrease
in T2 relaxation times measured in step (iv) relative to the T2
relaxation times measured in step (ii) can indicate the presence of
the analyte.
[0046] The term "analyte" or "exogenous analyte" refers to a
substance or chemical constituent (e.g., glucose, folic acid, or
influenza hemagglutinin peptide) in a sample (e.g., a biological or
industrial fluid) that can be analyzed (e.g., detected and
quantified) and monitored using the sensors described herein.
[0047] The term "subject" includes mice, rats, cows, sheep, pigs,
rabbits, goats, horses, primates, dogs, cats, and humans.
[0048] A nanoparticle having at least one moiety described herein
that is covalently or noncovalently linked to the nanoparticle and
that can switch from being in an aggregated and disaggregated state
is sometimes referred to herein as a "magnetic nanoswitch" or
"nanoswitch."
[0049] Embodiments can have one or more of the following
advantages.
[0050] While not wishing to be bound by theory, it is believed that
nanoparticle aggregation (formation of nanoparticle aggregates,
e.g., microaggregates) and disaggregation (formation of
disaggregated or dispersed nanoparticles from microaggregates or
nanoparticle agregates) is an equilibrium controlled process, and
that the position of this equilibrium is dependent upon (and
therefore maintained by) analyte concentration. When analyte
concentration changes, the position of the equilibrium changes, at
least in a range of sensitivity depending on several factors. This
change in the position of this equilibrium is manifested by changes
in proton relaxation of the water inside of the sensor chamber,
which is measurable. As such, the sensors have the advantage of
being useful for the continuous monitoring of changing levels of
analytes because there is generally no need to re-condition or
replace the sensors during the course of most ongoing (e.g., long
term) measurements because essentially nothing is created or
produced during detection; the equilibrium between aggregated and
disaggregated nanoparticles is shifted by the analyte. As long as
one can continuously monitor changes in the aforementioned
equilibrium that occur inside the sensor chamber (e.g., by
periodically or continuously monitoring T2 relaxation times of the
water inside of the chamber), then one can continuously monitor
changing levels of analytes as those changes occur.
[0051] The sensors can be used to detect a chemically diverse array
of analytes, which include without limitation, carbohydrates (e.g.,
glucose), peptides (e.g., influenza hemagglutinin peptide), and
therapeutic agents (e.g., folic acid).
[0052] The sensors are relatively simple devices lacking moving
parts, electronics and any connection to an outside recording
device such as a sampling tube or wire. Instead, the sensor
operates by absorbing and emitting radiation at the Larmour
precession frequency of water protons, which is interpretable as T2
and exogenous analyte (e.g., glucose) concentration. The radiation
employed (e.g., 60 MHz for the 1.5 T MRI) penetrates biological
systems, e.g., at depths where optical reporting methods can
sometimes be complicated by light scattering and absorption by the
tissue, e.g., tissue depths greater than about 250 .mu.m. The
sensor can therefore be essentially a remote sensor, reporting on
its local environment through water relaxation measurements while
unconnected to an outside recording device or power source.
[0053] Analyte detection can take place in solution rather than on
a surface, so as to avoid the need for developing and optimizing
sensor surface chemistry. This enables the features of an assay
(sensitivity, specificity, kinetics) to be determined in a tube
format, independently from the semi-permeable device or
instrumentation needed to distinguish sensor water from bulk water.
Binding agents, e.g., proteins and antibodies, and nanoparticles
can readily be tested as reagents for new water relaxation assays,
leading a panel of relaxation-based sensors for different
analytes.
[0054] By sensing the position of the reversible equilibrium of
nanoparticle aggregation/dispersion, the production or consumption
of molecules is avoided as compared with an assay that includes
irreversible reactions (e.g., single use assays). Thus, again there
is no need to "recharge" the sensor with a substrate to continue
operation. The sensors can be prepared for reuse, for example, by
equilibrating in the absence of the analyte or under conditions
chosen to mimic the absence of the analyte (e.g., relatively low
concentrations of the analyte).
[0055] The radiofrequency radiation used with the water relaxation
sensor interacts with water protons, rather than nanoparticles or
biological molecules, thereby minimizing the likelihood of
radiation induced damage.
[0056] The sensors are amenable for use in the detection of two or
more analytes (e.g., a panel of different sensors each having,
e.g., a different binding agent and moiety, can be used in the same
screening or testing environment to detect multiple analytes).
[0057] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present application, including definitions will
control. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0058] Although methods and materials similar or equivalent to
those described herein can be used in the practice of the present
invention, preferred methods and materials are described below. The
materials, methods, and examples are illustrative only and not
intended to be limiting. Other features and advantages of the
invention will be apparent from the detailed description and from
the claims.
DESCRIPTION OF DRAWINGS
[0059] FIG. 1A is a cross-sectional view of an embodiment of a
water relaxation-based sensor for detecting a monovalent analyte.
Also shown are the equilibrium controlled processes that occur in
the sensor chamber in the absence and presence of the analyte and a
summary of the water relaxation properties of the aggregated and
dispersed nanoparticles.
[0060] FIG. 1B is a cross-sectional view of an embodiment of a
water relaxation-based sensor for detecting a monovalent analyte.
Also shown are the equilibrium controlled processes that occur in
the sensor chamber in the absence and presence of the analyte and a
summary of the water relaxation properties of the aggregated and
dispersed nanoparticles.
[0061] FIG. 1C is a schematic representation of a sensor
configuration embodiment in which the nanoparticles can bind
directly to each other, and the analyte can mediate
aggregation/disaggregation. The surface of this nanoparticle (left
side of equation) is capable of binding to an analyte and binding
of the analyte can alter the physical properties of the surface,
e.g., charge or hydrophobicity, resulting in
aggregation/disaggregation (right side of equation). Here,
aggregation/disaggregation is mediated by changes in pH (H+).
[0062] FIG. 1D is a schematic representation of a sensor
configuration embodiment in which the detection of a sequence of
bases on a nucleic acid fragment can mediate self-assembly of the
nanoparticles. When the two types of nanoparticles are mixed, they
can self-assemble via the hybridization that occurs between bases
of the oligonucleotides (left side of equation). An analyte that
can bind to one of the types of particles can induce the
dissociation of aggregates (right side of equation).
[0063] FIG. 2 is a reaction scheme showing the synthesis of glucose
linked cross-linked iron oxide nanoparticle (Glu-CLIO).
[0064] FIG. 3A is a graphical representation of changes in T2
relaxation times obtained in a tube based water relaxation assay
for glucose using the Glu-CLIO/ConA configuration. Amino-CLIO does
not react with ConA binding protein, as indicated by a stable T2
relaxation time after ConA addition. Attachment of 2-amino-glucose
(G) results in a functionalized nanoparticle, Glu-CLIO, which shows
a T2 drop upon addition of a glucose-binding protein (ConA). The T2
drop is reversed by the addition of glucose. The data is indicative
of nanoparticle aggregation and disaggregation. For T2
measurements, 0.5 mL of Glu-CLIO (10 ug Fe/mL), 800 ug/mL ConA in
PBS with 1 mM CaCl.sub.2 and 1 mM MgCl.sub.2 were used.
[0065] FIG. 3B is a photograph of the assay apparatus. Iron
concentration in the photograph was increased to 100 ug Fe/mL for
better contrast.
[0066] FIG. 4A is a graphical representation of changes in T2
relaxation times obtained in a tube based assay for glucose using
the Glu-CLIO nanoswitch/ConA configuration. Addition of ConA to
Glu-CLIO caused a initial T2 drop, which was reversed by the
addition of increasing concentrations of glucose. FIG. 4B is a
graphical representation of changes in T2 values (at the plateau)
obtained with different glucose concentrations. FIGS. 4C, 4D, and
4E are graphical representations of particle size distribution as
obtained by light scattering experiments. FIG. 4C shows the
particle size distribution for dispersed Glu-CLIO nanoparticles.
FIG. 4D shows the switch from dispersed nanoparticles (dark bars)
to the microaggregate state (light bars) upon ConA addition. FIG.
4E shows the switch of the Glu-CLIO nanoparticles back to the
dispersed state upon glucose addition.
[0067] FIG. 5A is a graphical representation of changes in T2
relaxation times obtained by contacting a water relaxation sensor
with solutions of varying external glucose concentrations. Sensor
was first placed in PBS with 0.1 mg/mL glucose, then in buffer with
1.0 mg/mL glucose and returned to a solution of 0.1 mg/mL glucose.
Conditions were essentially the same as described with respect to
FIGS. 3A and 3B.
[0068] FIG. 5B is a photograph of the sensor and the apparatus for
containing the glucose-containing sample media.
[0069] FIGS. 6A, 6B, and 6C are images corresponding to the
reaction of a water relaxation sensor to glucose visualized by MRI.
FIG. 5A shows sensors with ConA and Glu-CLIO placed in 50 mL tubes.
FIG. 5B shows an MR image of a 50 mL tube with external glucose of
0.5 mg/mL FIG. 5C shows an MR image of with an external glucose
concentration of 1.4 mg/mL. Conditions were essentially the same as
described with respect to FIGS. 3A and 3B.
[0070] FIG. 7 is a graphical representation of time dependent
changes in T2 with increasing and decreasing glucose
concentrations. The Glu-CLIO nanoswitch/ConA system used in the
experiments described in FIGS. 4A-4E was placed in a semipermeable
device so that glucose could be cycled between low and high
concentrations, causing nanoparticles in the sensor to shift back
and forth between a low T2 state and high T2 state.
[0071] FIG. 8A is a graphical representation of changes in T2
relaxation times obtained in a tube based assay for influenza
hemagglutinin peptide (HA) using the HA-CLIO nanoswitch/antibody to
HA (anti-HA) configuration. Addition of anti-HA to HA-CLIO caused
an initial T2 drop, which was reversed by the addition of
increasing concentrations of HA. FIG. 8B is a graphical
representation of changes in T2 values obtained with different HA
concentrations. FIGS. 8C, 8D, and 8E are graphical representations
of particle size distribution as obtained by light scattering
experiments. FIG. 8C shows the particle size distribution for
dispersed HA-CLIO nanoparticles. FIG. 8D shows the switch from
dispersed nanoparticles (dark bars) to the microaggregate state
(light bars) upon anti-HA addition. FIG. 8E shown the switch of the
HA-CLIO nanoparticles back to the dispersed state upon HA
addition.
[0072] FIG. 9A is a graphical representation of changes in T2
relaxation times obtained in a tube based assay for folic acid (FA)
using the FA-CLIO nanoswitch/antibody to FA (anti-FA)
configuration. Addition of anti-FA to FA-CLIO caused a initial T2
drop, which was reversed by the addition of increasing
concentrations of FA. FIG. 9B is a graphical representation of
changes in T2 values obtained with different FA concentrations.
FIGS. 9C, 9D, and 9E are graphical representations of particle size
distribution as obtained by light scattering experiments. FIG. 9C
shows the particle size distribution for dispersed FA-CLIO
nanoparticles. FIG. 9D shows the switch from dispersed
nanoparticles (dark bars) to the microaggregate state (light bars)
upon anti-FA addition. FIG. 9E shows the return of the FA-CLIO
nanoparticles back to the dispersed state upon FA addition.
[0073] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0074] This invention relates generally to magnetic resonance-based
sensors (e.g., water relaxation-based sensors) and methods for
detecting various analytes (e.g., exogenous analytes) in
water-containing media (e.g., in vitro or in vivo media).
[0075] Sensors
[0076] In general, the sensors described herein include magnetic
nanoparticles, or nanoparticles with a magnetic moment under
certain conditions, encapsulated within a semipermeable walled
enclosure, e.g., an enclosure that retains the nanoparticles, but
allows for passage of the analyte into and out of the confines of
the sensor chamber. The walled enclosure can have one or more
openings sized to enable the passage of the analyte, but not the
nanoparticles (and binding agent, when present). Each of the
nanoparticles has at least one moiety (e.g., a molecular fragment
of the analyte being detected or a molecular fragment of a
derivative, isostere, or mimic of the analyte being detected; or a
protein) that is covalently or noncovalently linked to the
nanoparticle. The sensor can further include a binding agent (e.g.,
a protein or a monoclonal antibody) also encapsulated within a
semipermeable enclosure. The binding agent, when present, is
capable of binding to the analyte and the moiety; and the moiety is
capable of binding to the analyte or the binding agent. In general,
the moiety and the analyte can each bind reversibly to the binding
agent, when present; or the analyte can bind reversibly to the
moiety. Currently preferred chemistries for use in the practice of
the invention are described herein. Generally, the chemistry of the
analyte, binding moiety, and binding agent, per se, unless
indicated otherwise herein, may be and typically is conventional,
and may be adapted from other arts for use in the novel sensors and
methods disclosed herein.
[0077] Referring to FIG. 1A, in some embodiments, a water
relaxation-based sensor 10 for detecting a monovalent analyte
(A.sup.ex in FIG. 1A) includes a walled enclosure 12, a plurality
of nanoparticles 20, and at least one binding agent (e.g., a
protein) 18. The walled enclosure encapsulates a chamber 16 and is
perforated with a plurality of openings 14. Both the nanoparticles
20 and the binding agent 18 are located within the confines of the
chamber 16. Each of the nanoparticles 20 has at least one moiety
(A.sup.b in FIG. 1A) that is covalently or noncovalently linked to
the nanoparticle and that includes a molecular fragment of the
analyte being detected. The binding agent 18 is capable of binding
(e.g., reversibly binding) to the analyte and the moiety A.sup.b.
In all embodiments, the analyte is smaller in size than either the
nanoparticles 20 or the binding agent 18. In all embodiments, the
openings 14 are (i) larger in size than the analyte so as to allow
the analyte to pass freely into and out of the chamber 16 (arrows
17) and (ii) smaller in size than either the nanoparticles 20 or
the binding agent 18 so as to retain the nanoparticles 20 and the
binding agent 18 within the chamber 16.
[0078] When the analyte is absent, the nanoparticles 20 bind to the
binding agent 18 to form a nanoparticle aggregate 22 within the
sensor chamber 16. It is believed that binding of the nanoparticles
20 to the binding agent 18 occurs through the moiety A.sup.b (see
FIG. 1A). In general, formation of the nanoparticle aggregate 22 is
an equilibrium controlled process (arrows 23).
[0079] When the exogenous analyte is present and enters the chamber
16 through opening 14, the binding agent-bound (e.g., binding
protein-bound) nanoparticles of aggregate 22 are displaced from the
binding agent by the analyte (A.sup.ex in FIG. 1A), thereby
altering the nanoparticle-binding agent (binding protein)
equilibrium (arrows 23). As a result, a second equilibrium is
established (arrows 25) in the chamber 16 among the analyte, s
analyte-binding agent (binding protein complex 24, and
(regenerated) nanoparticles 20. The regenerated nanoparticles
produced in the second equilibrium controlled process (arrows 25)
are substantially disaggregated relative to the bound nanoparticles
of aggregate 22 formed in the first equilibrium controlled process
(arrows 23).
[0080] Referring to FIG. 1B, in some embodiments, a water
relaxation-based sensor 10 for detecting a multivalent analyte
(.ident.A.sup.ex in FIG. 1B) includes a walled enclosure 12 as
described elsewhere and a plurality of nanoparticles 26. The
nanoparticles are located within the confines of the chamber, and
each of the nanoparticles has at least one moiety (e.g., at least
one protein; at least 2, at least 3, at least 4) that is linked to
the nanoparticle (hollow wedges in FIG. 1B). In all embodiments,
the exogenous analyte is smaller in size than the nanoparticles 26.
In all embodiments, the openings 14 are (i) larger in size than the
exogenous analyte so as to allow the analyte to pass freely into
and out of the chamber 16 (arrows 17) and (ii) smaller in size than
the nanoparticles 26 so as to retain the nanoparticles 26 within
the chamber 16.
[0081] When the analyte is absent, the nanoparticles 26 are
substantially disaggregated within the sensor chamber 16.
[0082] When the analyte is present and enters the chamber 16
through opening 14, the nanoparticles 26 bind to the multivalent
analyte (.ident.A.sup.ex in FIG. 1B) to form a nanoparticle
aggregate 28 within the chamber (arrows 27). The nanoparticles that
form part of aggregate 28 are substantially aggregated relative to
nanoparticles 26. It is believed that binding of the nanoparticles
26 to the analyte occurs through the moiety (e.g., a protein). In
general, formation of the nanoparticle aggregate 28 is an
equilibrium controlled process (arrows 27).
[0083] Referring to FIG. 1C, in some embodiments, the sensors can
be configured such that an analyte (e.g., a proton, H.sup.+) can
directly mediate self-assembly (aggregation and disaggregation of
the nanoparticles). Sensors having the configuration shown in FIG.
1C can have one or more of the following properties: (i) the
nanoparticles can bind directly to each other (i.e., there are no
molecules serving a "bridges" between nanoparticles, as shown in
FIGS. 1A and 1B); (ii) a single type of nanoparticle can be
employed, and (iii) an analyte can control the self-assembly by
binding to the surface of the nanoparticle. In these embodiments,
the surface of the nanoparticle is capable of binding to an
analyte. Binding of the analyte can alter the physical properties
of the surface, e.g. charge or hydrophobicity. While not wishing to
be bound by theory, it is believed that the change in surface
properties can alter the attraction between the nanoparticles, and
self-assembly (or disassembly) of nanoparticles can occur. In
certain embodiments, the surface of the nanoparticle can be
designed to have a surface that can be charged or uncharged, as the
pH is varied over some range of interest. For example, a peptide
such as Ac-LLLLLL-KHHHE-G-K(FITC)-C--NH.sub.2, pI=6.48, can be
attached to nanoparticles using bifunctional crosslinking agents
such as SPDP or SIA, (see, e.g., Koch et al. "Uptake and metabolism
of a dual fluorochrome Tat-nanoparticle in HeLa cells." Bioconjug
Chem. 2003; 14(6): 1115). At a pH of about 6.48 or above, the
histidine is substantially unprotonated, and aggregation can occur
through self-association among the hydrophobic leucine side chains.
At a pH below about 6.48, the histidine is substantially
protonated, the nanoparticles carry a positive charge and are
dispersed.
[0084] Referring to FIG. 1D, in some embodiments, the sensors can
be configured such that detection of a sequence of bases on a
nucleic acid fragment can mediate self-assembly of the
nanoparticles. Sensors having the configuration shown in FIG. 1D
can have one or more of the following properties: (i) two types
nanoparticles can be prepared which have an affinity for each
other, and (ii) one of the two types of nanoparticles is capable of
binding the analyte. In these embodiments, two types of
nanoparticles can be synthesized, each having a specific sequence
of synthetic oligonucleotide attached. When the two types of
nanoparticles are mixed, they can self-assemble via the
hybridization that occurs between bases of the oligonucleotides. An
example of such double-stranded, oligonucleotide-mediated,
nanoparticle aggregate is given in Perez et. al., "DNA-based
magnetic nanoparticle assembly acts as a magnetic relaxation
nanoswitch allowing screening of DNA-cleaving agents." J Am Chem
Soc. 2002; 124:2856. An analyte (e.g. a sequence of bases present
on a nucleic acid fragment) can then enter the sensor and by
binding to one of the types of particles can induce the
dissociation of aggregates.
[0085] Since the concentration dependent reaction of the analyte
with the nanoparticle aggregate alters the nanoparticle aggregation
state, the presence and quantity of the exogenous analyte can be
sensed, for example, as a change in the T2 relaxation times of
water inside of the sensor chamber. It is known, for example, that
water T2 relaxation times shorten upon aggregation or clustering of
previously dispersed (e.g., monodispersed, polydispersed) magnetic
nanoparticles. While not wishing to be bound by theory, it is
believed that during nanoparticle self-assembly into higher order
nanoassemblies, the superparamagnetic iron oxide core of individual
nanoparticles becomes more efficient at dephasing the spins of the
surrounding water protons (i.e., enhancing spin-spin relaxation
times, e.g., T2 relaxation times).
[0086] Thus, in some embodiments, the analyte can be detected and
quantified in the sampling media by monitoring the relaxation
properties of the water that is present within the sensor chamber
16 (e.g., measuring changes, e.g., increases and decreases, in T2
relaxation times of water that is present within the sensor
chamber). For example, referring to FIG. 1A, the T2 relaxation
times of the water inside of the sensor chamber 16 are expected to
decrease in the absence of analyte (due to formation of the
nanoparticle aggregate 22) and then increase relative to these
depressed values in the presence of analytes (due to displacement
and subsequent disaggregation of the bound nanoparticles of
aggregate 22). Alternatively, referring to FIG. 1B, the T2
relaxation times of the water inside of the sensor chamber 16 are
expected to increase in the absence of multivalent analyte and then
decrease relative to these values in the presence of the
multivalent analytes. Since the binding agent and/or the
nanoparticles are confined within the chamber 16, the changes in
nanoparticle aggregation occurring within the sensor chamber 16 in
general do not substantially alter the proton relaxation of water
outside of the chamber (i.e., bulk water).
[0087] Although measuring T2 can be a desirable method for
determining nanoparticle aggregation, any water relaxation
phenomena associated with nanoparticles or with their change in
aggregation state can be used. T2 can generally be determined in a
relatively fast and facile manner. However, measurements of
nanoparticle aggregation can use T2 in conjunction with other
relaxation processes such as T1. Measurements of T1 and T2 can be
used to correct for small changes in nanoparticle aggregation state
within the sensor, due to a small expansion of contraction of the
chamber. Accordingly, as used herein, references to measurement of
relaxation phenomenon or magnetic relaxivity is intended to embrace
all such relaxation related processes, including measurement of
TI.
[0088] Sensor Components and Specifications
[0089] In general, the size and shape of the sensor 10 can be
selected as desired.
[0090] In some embodiments, the sensors can be, for example,
tubular, spherical, cylindrical, or oval shaped. The sensors
described herein can have other shapes as well.
[0091] In some embodiments, the size and shape of the sensor can be
selected to accommodate a desired or convenient sample holder size
and/or sample volume (e.g., in in vitro sensing applications). In
general, the volume of sensor can be selected to enable the sensor
to distinguish between the relaxation properties of water inside of
the chamber and the water outside of the chamber. For example, the
sensor size can be selected so as to accommodate a sample volume of
from about 0.1 microliters (.mu.L) to about 1000 milliliters (mL)
(e.g., about 1 .mu.L (e.g., with animal imagers), 10 .mu.L (e.g.,
with clinical MRI instruments) or 0.5 mL. In certain embodiments,
the sensor can have a tubular shape in which the open end of the
tube has a diameter of from about 1 millimeter (mm) to about 10 mm
(e.g., 5 mm 7.5 mm).
[0092] In some embodiments, the sensor size and shape can be
selected on the basis of the spatial resolution capabilities of
conventional magnetic resonance technology (e.g., in in vitro
sensing applications). In certain embodiments, the longest
dimension of the sensor can be from about 0.01 mm to about 2 mm
(e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1). In
certain embodiments, the applied magnetic field can be, for
example, about 0.47 Tesla (T), 1.5 T, 3 T, or 9.4 T (animal assays
generally).
[0093] The walled enclosure 12 separates the chamber 16 from the
bulk sample media and provides one or more conduits (e.g., openings
14) for entry of the exogenous analyte (if present) from the bulk
sample media. In general, the walled enclosure 12 can be any
semipermeable material (e.g., a biocompatible semipermeable
material) that is permeable to the exogenous analyte and water and
substantially impermeable to the nanoparticles and the binding
agent. In some embodiments, the semipermeable material can be an
ultrafiltration or dialysis membrane. In some embodiments, the
semipermeable material can be a polymeric substance (e.g.,
polymeric substances used for encapsulating transplanted cells,
see, e.g., M. S. Lesney, Modern Drug Discovery 2001, 4, 45). In
some embodiments, the semipermeable material can be a material used
in small implantable, sustained release devices (e.g., those used
in implantable, sustained release birth control devices, e.g.,
Depo-Provera, Norplant, Progestasert; or those described in C. I.
Thompson et al., Can J Physiol Pharmacol 80, 180-92 (Mar, 2002) or
D. C. Stoller, S. R. Thornton, F. L. Smith, Pharmacology 66, 11-8
(Sep, 2002)).
[0094] In some embodiments, the walled enclosure is relatively
resistant to fouling or coating under the sampling conditions,
thereby increasingly the likelihood that the walled enclosure can
maintain the specified pore size of the openings 14 (e.g.,
increasing the likelihood that openings 14 will remain
substantially unblocked during sensing). Fouling is the closure of
pores (e.g., openings 14) due to the adsorption of protein that
blocks pore. Fouling can be ascertained by placing materials in
biological fluids (e.g., blood) and evaluating their performance
using biocompatibility testing methods known in the art.
[0095] In some embodiments, the walled enclosure 12 can be
essentially nonimmunogenic, thereby minimizing the likelihood of
causing unwanted immune or toxic side effects in a subject (e.g., a
human).
[0096] Examples of biocompatible, semipermeable materials include
without limitation polysaccharide based materials (cellulose),
modified carbohydrate (cellulose ester), or polyvinyl
pyrolidine.
[0097] In some embodiments, the walled enclosure 12 can be made of
a relatively inflexible semipermeable material, meaning that the
encapsulated chamber 16 is a true space or void that does not
substantially change in volume when contacted with the fluid sample
media. In other embodiments, the walled enclosure can be a
relatively flexible semipermeable material, meaning, for example,
that the encapsulated chamber can expand in volume when contacted
with the fluid sample media (e.g., by intake of the fluid sample
media).
[0098] In general, the walls of the enclosure 12 are sufficiently
thin to allow rapid sensor equilibration to changes in exogenous
analyte levels. In some embodiments, the membrane that forms the
wall can have a thickness of from about 1 and about 500 hundred
microns.
[0099] In general, the pore size of the openings 14 can be selected
so as to meet the molecular exclusion criteria described herein
(i.e., permeable to the exogenous analyte and water and
substantially impermeable to the nanoparticles and the binding
agent).
[0100] In some embodiments, molecular exclusion can be exclusion by
molecular weight. In certain embodiments, each of the openings can
have a pore size of from about 1 kDa to about 500,000 kDa (e.g., a
pore size that allows passage of molecules that have a certain
molecular weight Each of the openings can have a size (pore size)
of from about 1 kDa to about 1 .mu.m (e.g., about 1 kDa to about
300,000 kDa; about 1 kDa to about 100,000 kDa; about 1 kDa to about
5 kDa; 1 kDa to about 3 kDa; 1 kDA to about 1 .mu.m). In certain
embodiments, the openings can have a pore size of about 1 kDa or
about 3 kDa.
[0101] In certain embodiments, the semipermeable material can be
Spectra/Por.RTM. tubing, Slide-A-Lyzer.RTM. microcassetes or
dialysis fibers. Such materials are generally preferred for
applications not involving implantation. In general, the
semipermeable material has a pore size that is larger in size than
the analyte to permit passage of the analyte into and out of the
chamber, but sufficiently small to retain magnetic nanoparticles
and other reagents such as binding agents (e.g., a binding protein)
within the confines of the chamber. The semipermeable material can
be selected for the stability (long term function) in the fluid,
which contains the analyte to be measured (e.g., blood plasma,
interstitial fluid, cerebral spinal fluid of a human or animal
subject). The semipermeable material can be further selected on the
basis of whether the sensor is implanted or whether the fluid to be
assayed is contained within a vessel that is outside of the subject
(e.g., a bioreactor, tube or pipe).
[0102] The magnetic particles can be nanoparticles (e.g., having a
particle size of from about 10 nanometers (nm) to about 200 nm) or
particles (e.g., having a particle size of from about 200 nm to
about 5000 nm) provided that the particles remain essentially
suspended (i.e., the particles do not settle). As used herein, the
term "magnetic nanoparticles" refers to any particle that is always
magnetic and any particle that has a magnetic moment under certain
conditions (e.g., in an applied electromagnetic field). Particle
settling can generally be avoided by using relatively small
particles (e.g., nanoparticles) or relatively large particles whose
density is comparable to that of water. The density of particles
can be altered by using polymers of different densities in their
synthesis. In all embodiments, the nanoparticles or particles have
a surface that permits the attachment of biological molecules. In
some embodiments, the magnetic particles can be nanoparticles
having a particle size of from about 10 nm to about 500 nm (e.g.,
about 15 to about 200 nm, about 20 to about 100 nm, about 10 nm to
about 60 nm, about 20 nm to about 40 nm, about 30 nm to about 60
nm, about 40 to 60 nm; or about 50 nm). The unfunctionalized metal
oxides are generally crystals of about 1-25 nm, e.g., about 3-10
nm, or about 5 nm in diameter.
[0103] Magnetic materials larger than nanoparticles (particles) can
be used. In general, such particles can have one or more of the
following properties: (i) it is desirable that the particles have a
relatively high R2, i.e., alter water relaxation, (ii) it is
desirable that the particles not to have a high susceptibility to
settle significantly by gravity during the time course of the
assay, (iii) it is desirable that the particles have a surface for
the attachment of biomolecules, preferably amino or carboxyl
groups. Examples include microspheres of from about 1-5 micron in
diameter. Such particles can be obtained, e.g., from commercial
suppliers, which include Dynbead magnetic microspheres from
Invitrogen (Carlsbad, Calif.), microspheres from Bangs Laboratories
(Fishers, Ind.), and Estapor.RTM. Microspheres from Merck or EMD
Life Sciences (Naperville, Ill.).
[0104] In some embodiments, the particles (e.g., nanoparticles 20
or 26) can be unfunctionalized magnetic metal oxides, such as
superparamagnetic iron oxide. The magnetic metal oxide can also
include cobalt, magnesium, zinc, or mixtures of these metals with
iron. The term "magnetic" as used herein means materials of high
positive magnetic susceptibility such as paramagnetic or
superparamagnetic compounds and magnetite, gamma ferric oxide, or
metallic iron. In some embodiments, the nanoparticles 20 include
those having a relatively high relaxivity, i.e., strong effect on
water relaxation.
[0105] In general, the particles can have a relatively high
relaxivity owing to the superparamagnetism of their iron or metal
oxide. In some embodiments, the nanoparticles (e.g., 20 or 26) have
an R1 relaxivity between about 5 and 30 mM.sup.-1 sec.sup.-1, e.g.,
10, 15, 20, or 25 mM.sup.-1 sec.sup.-1. In some embodiments, the
nanoparticles (e.g., 20 or 26) have an R2 relaxivity between about
15 and 100 mM.sup.-1 sec.sup.-1, e.g., 25, 50, 75, or 90 mM.sup.-1
sec.sup.-1. In some embodiments, nanoparticles (e.g., 20 or 26)
have a ratio of R2 to R1 of between 1.5 and 4, e.g., 2, 2.5, or 3.
In some embodiments, the nanoparticles (e.g., 20 or 26) have an
iron oxide content that is greater than about 10% of the total mass
of the particle, e.g., greater than 15, 20, 25 or 30 percent.
[0106] In some embodiments, when the magnetic nanoparticle is an
iron oxide-based nanoparticle, concentrations of iron (Fe) can be
from about 2 micrograms (.mu.g)/mL to about 50 .mu.g/mL Fe. In
general, the iron concentration is selected so as to be
sufficiently high to alter the relaxation properties of water. For
particles with relatively high relaxivities, lower iron
concentrations can be used. For particles with relatively low
relaxivities, higher iron concentrations can be used.
[0107] Each of the nanoparticles (e.g., 20 or 26) includes at least
one moiety (e.g., at least 2, at least 3, at least 4) that is
covalently or noncovalently linked to the nanoparticle.
[0108] In some embodiments, the moiety can be linked to the
nanoparticle via a functional group. The functional group can be
chosen or designed primarily on factors such as convenience of
synthesis, lack of steric hindrance, and biodegradation properties.
Suitable functional groups can include --O--, --S--, --SS--,
--NH--, --NHC(O)--, --(O)CNH--, --NHC(O)(CH.sub.2).sub.nC(O)--,
--(O)C(CH.sub.2).sub.nC(O)NH--, --NHC(O)(CH.sub.2).sub.nC(O)NH--,
--C(O)O--, --OC(O)--, --NHNH--, --C(O)S--, --SC(O)--,
--OC(O)(CH.sub.2).sub.n(O)--, --O(CH.sub.2).sub.nC(O)O--,
--OC(O)(CH.sub.2).sub.nC(O)--, --C(O)(CH.sub.2).sub.nC(O)O--,
--C(O)(CH.sub.2).sub.nC(O)--, --NH(CH.sub.2).sub.nC(O)--,
--C(O)(CH.sub.2).sub.nNH--, --O(CH.sub.2).sub.nC(O)--,
--C(O)(CH.sub.2).sub.nO--, --S(CH.sub.2).sub.nC(O)--,
--C(O)(CH.sub.2).sub.nS--, --NH(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH--, --O(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nO--, --S(CH.sub.2).sub.n--, or
--(CH.sub.2).sub.nS--, in which each n can be 1-100 (e.g., n can be
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96,
97, 98, 99). Functional groups having cyclic, unsaturated, or
cyclic unsaturated groups in place of the linear and fully
saturated alkylene linker portion, (CH.sub.2).sub.n, can also be
used to attach the moiety to the nanoparticle. In certain
embodiments, the functional group can be selected so as to render
the nanoparticle larger in size than the opening(s) in the wall of
the chamber so as to retain the nanoparticle within the confines of
the chamber (e.g., where a relatively large analyte is being
detected such as a lipoprotein).
[0109] In certain embodiments, the functional group can be
--NHC(O)(CH.sub.2).sub.nC(O)NH--, in which n can be 0-20. In
certain embodiments, n can be 2, 3, 4, 5, or 6 (preferably, 2).
[0110] The functional group can be present on a starting material
or synthetic intermediate that is associated with either the
nanoparticle portion or the moiety portion of the nanoparticles
(e.g., 20 or 26).
[0111] In some embodiments, a nanoparticle-based starting material
can contain one or more functional groups for attachment of one or
more moieties, (e.g., 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45,
or 50 functional groups). In certain embodiments, the nanoparticle
can be an amino-derivatized cross-linked iron oxide nanoparticle
(e.g., NH.sub.2-CLIO). The number of moieties (e.g., a molecular
fragment of the analyte being detected or a molecular fragment of a
derivative, isostere, or mimic of the analyte being detected; or a
protein) that are ultimately linked to the nanoparticle can be
equal to or less than the number of functional groups that are
available for attachment of the moiety(ies) to the nanoparticle. In
any event, it is permissible for the number of moieties per
nanoparticle to vary within a given population of nanoparticles
(e.g., a population of 20 and/or 26).
[0112] In general, the number of moieties per nanoparticle can be
selected as desired (e.g., depending on the number and location of
binding sites on the binding agent (e.g., a protein) and/or if it
is desired to have the nanoparticle cross link binding agents
(e.g., proteins) when more than one binding agent is present). In
some embodiments, the magnetic particles can be multivalent
particles in which multiple copies of a monovalent material are
attached to the same particle. In general, the valency can be from
about 2.5 to about 20 copies of bound moiety per nanoparticle
(i.e., average numbers of copies per nanoparticle, thus some
particles can be monovalent within a given population of generally
multivalent particles). Higher levels are not necessarily believed
to be needed for function. Multivalent nanoparticles can be
prepared by attaching two or more functional groups per
nanoparticle. Multivalent nanoparticles can also be prepared by
attaching either multivalent or monovalent binding agents (e.g.,
proteins).
[0113] In general, moieties can include, without limitation,
carbohydrates (e.g., glucose, polysaccharides), antibodies (e.g.,
monoclonal antibodies, biotinylated anti-GFP polyclonal antibody),
amino acids as well as derivatives and stereoisomers thereof (e.g.
D-phenylalanine), chiral moieties, lipids, sterols,
lipopolysaccharides, lipoproteins, nucleic acids, oligonucleotides,
therapeutic agents (e.g., folic acid), metabolites of therapeutic
agents, peptides (e.g., influenza hemagglutinin peptide), or
proteins.
[0114] In some embodiments, the moiety can be, or include as part
of its chemical structure, a molecular fragment of the analyte
being detected or a molecular fragment of a derivative, isostere,
or mimic of the analyte being detected.
[0115] In general, such a moiety is one that is (i) recognized by
the binding agent (e.g., protein, e.g., a binding protein) and (ii)
displaceable from the binding agent by the analyte (i.e., the
analyte can compete with the moiety for binding to the binding
agent (e.g., a protein)). Thus, in some embodiments, the moiety and
the analyte being detected can be substantially similar in
structure to one another and have substantially similar binding
affinities towards the binding agent. In other embodiments (such as
when the moiety is, or includes as part of its chemical structure,
a molecular fragment of a derivative, isostere, or mimic of the
analyte being detected), the moiety and the analyte being detected
may not necessarily be substantially similar in structure, but may
have substantially similar binding affinities towards the binding
agent.
[0116] Nanoparticles having at least one moiety that is a molecular
fragment of the analyte being detected or a molecular fragment of a
derivative, isostere, or mimic of the analyte being detected can
have the general formula (A): (A).sub.z-NP.sup.c (A)
[0117] in which:
[0118] moiety "A" is a molecular fragment of an analyte (or a
derivative, isostere, or mimic thereof), A-X, wherein X is a
hydrogen atom or a functional group that is present in the free
analyte (or a derivative, isostere, or mimic thereof), but not
incorporated into the nanoparticle of formula (A);
[0119] "NP.sup.c" is the nanoparticle core;
[0120] "-" is a covalent linkage (e.g., a chemical bond or any
linking functional group described herein) that connects any atom
of the fragment to the nanoparticle; and
[0121] z is 1-50 (e.g., 1-40, 1-30, 1-25, 1-20, 2-20, 2, 4, 6, 8,
10, and 15).
[0122] In certain embodiments, X can be a hydrogen atom that forms
part of an amino or hydroxy group that is present in the analyte;
or X can be functional group, such as an amino group or a hydroxy
group. By way of example, for a given analyte A-OH (X=OH=hydroxy
group), the corresponding nanoparticle can have, for example and
without limitation, the structure (A-O).sub.z-NP.sup.c or
(A-NH).sub.z-NP.sup.c.
[0123] In some embodiments, the moiety can include a carbohydrate
as part of its chemical structure (e.g., glucosyl). In certain
embodiments, the moiety can include a molecular fragment of a
carbohydrate analyte being detected or a molecular fragment of a
derivative, isostere, or mimic of a carbohydrate analyte being
detected. For example, the moiety can have formula (I): ##STR2## in
which the wavy line indicates the point of connection of the moiety
to the nanoparticle. The moiety of formula (I) can be used in
conjunction with a sensor for detecting and quantifying
glucose.
[0124] The moiety can be a monovalent or multivalent protein (e.g.,
having at least two (e.g., three, four, five, or six)) binding
sites.
[0125] In certain embodiments, the nanoparticle can further include
a substituent that serves to render the nanoparticle larger in size
than the opening(s) in the wall of the chamber so as to retain the
nanoparticle within the confines of the chamber (e.g., where a
relatively large analyte is being detected such as a
lipoprotein).
[0126] In some embodiments, the binding agent can be absent (e.g.,
when detecting multivalent analytes with nanoparticles in which the
covalently or noncovalently linked moiety is, e.g., a protein; see,
e.g., assay configuration delineated in FIG. 1B). Thus the assay
can measure multivalent analyte proteins, using a sensor with a
pore size that is large enough to allow analyte to enter and leave
the chamber, while retaining nanoparticles, i.e. the pore size in
FIG. 1A and FIG. 1B. can be adjusted. Various assay configurations
are described herein.
[0127] In some embodiments, the binding agent can be present (e.g.,
when detecting monovalent analytes with nanoparticles, which
include a molecular fragment of the analyte being detected or a
molecular fragment of a derivative, isostere, or mimic of the
analyte being detected; see, e.g., assay configuration delineated
in FIG. 1A). The binding agent can be, for example, a protein, an
antibody, a lectin, a receptor binding protein, a binding domain of
a protein, a synthetic material, or a non-protein material.
[0128] In some embodiments, the binding agent can be a protein. In
certain embodiments, the binding protein can be a multi-valent
binding protein having at least two binding sites (e.g., three,
four, five, or six).
[0129] In general, binding between the binding agent (e.g., a
protein or antibody) and the nanoparticle (via the moiety) and the
analyte is reversible; and binding between a moiety and an analyte
is reversible. As such, the analyte can be detected and quantified
in a non-consumptive manner (i.e., the binding agent or the moiety
reversibly binds, but does not consume, the analyte). This
reversibility provides a steady state condition for bound and
unbound analyte that can be quantitated. Analyte concentration can
then be mathematically calculated using conventional methods. A
person of ordinary skill in the art would recognize that, for
example, the reaction kinetics associated with binding and release
of the analyte can be different for each protein selected as a
binding agent.
[0130] In certain embodiments, the protein binding agent can bind a
therapeutic agent or metabolite thereof; a carbohydrate (e.g.,
glucose; e.g., the protein can be conconavalin A); or an amino acid
(e.g., the binding protein can selectively bind one enantiomer over
another, e.g., D-alanine versus L-alanine). In certain embodiments,
the protein can be green fluorescent protein (GFP).
[0131] In certain embodiments, the protein binding agent can be a
monomer. In other embodiments, the protein binding agent can be
multimeric binding agent (e.g., prepared by making a fusion protein
that includes several copies of one protein or cross-linking
monomers to create a multivalent binding moiety). In still other
embodiments, the binding agent can be an enzyme, which is modified
so that it can bind to a substrate, but does not catalyze a
reaction.
[0132] In certain embodiments, the binding agent can bind a lipid,
a sterol, a lipopolysaccharide, or a lipoprotein. For example, the
binding agent can be a protein that binds a sterol (e.g., apoSAAp
for binding cholesterol, see, e.g., Liang and Sipe, 1995,
"Recombinant human serum amyloid A (apoSAAp) binds cholesterol and
modulates cholesterol flux", J. Lipid Res, 36(1): 37). As another
example, the binding agent can be a receptor that binds a
lipoprotein (e.g., a soluble low density lipoprotein and/or mutants
thereof, see, e.g., Bajari et al, 2005, "LDL receptor family:
isolation, production, and ligand binding analysis", Methods,
36:109-116; or Yamamoto et al., 2005, "Characterization of low
density lipoproteinreceptor ligand interactions by fluorescence
resonance energy transfer", J. Lipid Research, 47:1091. As a
further example, the binding agent can be a protein that binds a
fatty acid (e.g., human serum albumin, see, e.g., Fang et al, 2006,
"Structural changes accompanying human serum albumin's binding of
fatty acids are concerted", 1764(2):285-91. Epub Dec. 27,
2005).
[0133] In other embodiments, the binding agent can be a monoclonal
antibody, a polyclonal antibody, or a oligonucleotide. The binding
agent can be, e.g., an antibody to folic acid or an antibody to
influenza hemagglutinin peptide.
[0134] In some embodiments, the analyte can be chiral. The chiral
analyte can be present together with one or more optically active
moieties in the sample (e.g., a stereoisomer of the chiral analyte
in the sample, e.g., the enantiomer of the chiral analyte in the
sample). In certain embodiments, the chiral analyte can be an amino
acid.
[0135] The sensors described herein can be used to detect, monitor,
and quantify a of analytes that can include, without limitation,
ions, small molecules, proteins, viruses and lipoproteins (see
Table 1). TABLE-US-00001 TABLE 1 Analytes that can be measured by
water relaxation sensors Type of analyte Analyte (size in kDa
unless otherwise noted) T4, T3, cortisol Small molecule hormone
(<1) Thyroid stimulating hormone (TSH), Protein hormones
(10-100) human chorionic gonadotropin (hCG), leutinizing hormone
(LH), follicle stimulating hormone (FSH) Troponin, C-reactive
protein (CRP), Proteins for inflammation or Creatine phosphokinase
(CPK-MB, CPK- heart attack (10-100) BB), myoglobin Prostatic
specific antigen (PSA), Proteins for cancer detection (10-100)
Carcinoma embryonic antigen (CEA), alphafetoprotein (AFP) Low
density lipoprotein (LDL), high Lipoproteins for lipid status
density lipoprotine (HDL) (500-2000) Ferritin Protein for iron
anemia (400-600) Paclitaxel Small molecule cancer chemotherapeutic
(<1) B12/Folate Small molecule nutrients and cofactors
Theophyline, gentamycin, tobramycin, Therapeutic drug (<1)
valproate Digoxin, digitoxin Therapeutic drug (<1) Glucose
Glucose (<1) Hydrogen ions Metabolite (<1) Calcium ion
Metabolite (<1) Herpes simplex virus Virus (1 .mu.m) Human
immunodeficiency virus Virus (1 .mu.m) (HIV) Hepatitis A, B or C
Virus (1 .mu.m)
[0136] In certain embodiments, the analyte can be a carbohydrate
(e.g., glucose); a lipid, a sterol, a lipopolysaccharide, a
lipoprotein, a nucleic acid or an oligonucleotide; therapeutic
agents (e.g., folic acid), metabolites of therapeutic agents,
peptides (e.g., influenza hemagglutinin peptide), or a protein.
[0137] Sensor Manufacture and Use
[0138] In some embodiments, nanoparticles having reactive
functional groups, (e.g., electrophilic functional groups such as
carboxy groups or nucleophilic groups such as amino groups) can be
employed as starting materials for the nanoparticles used in
conjunction with the sensors.
[0139] Carboxy functionalized nanoparticles can be made, for
example, according to the method of Gorman (see WO 00/61191). In
this method, reduced carboxymethyl (CM) dextran is synthesized from
commercial dextran. The CM-dextran and iron salts are mixed
together and are then neutralized with ammonium hydroxide. The
resulting carboxy functionalized nanoparticles can be used for
coupling amino functionalized groups, (e.g., a further segment of
the functional group or the substrate moiety).
[0140] Carboxy-functionalized nanoparticles can also be made from
polysaccharide coated nanoparticles by reaction with bromo or
chloroacetic acid in strong base to attach carboxyl groups. In
addition, carboxy-functionalized particles can be made from
amino-functionalized nanoparticles by converting amino to carboxy
groups by the use of reagents such as succinic anhydride or maleic
anhydride.
[0141] Nanoparticle size can be controlled by adjusting reaction
conditions, for example, by using low temperature during the
neutralization of iron salts with a base as described in U.S. Pat.
No. 5,262,176. Uniform particle size materials can also be made by
fractionating the particles using centrifugation, ultrafiltration,
or gel filtration, as described, for example in U.S. Pat. No.
5,492,814.
[0142] Nanoparticles can also be synthesized according to the
method of Molday (Molday, R. S. and D. MacKenzie, "Immunospecific
ferromagnetic iron-dextran reagents for the labeling and magnetic
separation of cells," J. Immunol. Methods, 1982, 52(3):353-67, and
treated with periodate to form aldehyde groups. The
aldehyde-containing nanoparticles can then be reacted with a
diamine (e.g., ethylene diamine or hexanediamine), which will form
a Schiff base, followed by reduction with sodium borohydride or
sodium cyanoborohydride.
[0143] Dextran-coated nanoparticles can be made and cross-linked
with epichlorohydrin. The addition of ammonia will react with epoxy
groups to generate amine groups, see, e.g., Josephson et al.,
Angewandte Chemie, International Edition 40, 3204-3206 (2001);
Hogemann et al., Bioconjug. Chem., 2000, 11(6):941-6; and Josephson
et al., "High-efficiency intracellular magnetic labeling with novel
superparamagnetic-Tat peptide conjugates," Bioconjug. Chem., 1999,
10(2):186-91. This material is known as cross-linked iron oxide or
"CLIO" and when functionalized with amine is referred to as
amine-CLIO or NH.sub.2-CLIO.
[0144] Carboxy-functionalized nanoparticles can be converted to
amino-functionalized magnetic particles by the use of water-soluble
carbodiimides and diamines such as ethylene diamine or hexane
diamine.
[0145] Nanoparticles 20 having a moiety corresponding to formula
(I) can be prepared by contacting amino-CLIO (NH.sub.2-CLIO) with
succinic anhydride (pH 8.5) followed by 2-aminoglucose in the
presence of a carbodiimide (e.g. a water soluble carbodiimide,
e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC)/N-hydroxysuccinimide (NHS), pH 6.0 (see FIG. 2). Such
nanoparticles are referred to herein as "G-CLIO," "Glu-CLIO," or
"Glu-CLIO nanoswitches."
[0146] Folic acid (FA) can be conjugated to NH.sub.2-CLIO using a
water soluble carbodiimide (e.g. EDC/NHS, pH 6.0) to provide
nanoparticles 20 having a folic acid-containing moiety linked to
the nanoparticle. Such nanoparticles are referred to herein as
"FA-CLIO" or "FA-CLIO nanoswitches."
[0147] Influenza hemagglutinin peptide (HA) can be conjugated to
NH.sub.2-CLIO with, e.g., N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP) (PBS buffer, pH 7.4 to provide
nanoparticles 20 having an HA-containing moiety linked to the
nanoparticle. Such nanoparticles are referred to herein as
"HA-CLIO" or "HA-CLIO nanoswitches."
[0148] The nanoparticles used in the sensors described herein can
also be prepared using the conjugation chemistry described in,
e.g., Sun, E. Y., Josephson, L., Kelly, K., Weissleder, R.
Bioconjugate Chemistry 2006, 17, 109-113, which is incorporated by
reference herein. The nanoparticles used in the sensors described
herein can also be prepared using "click chemistry" methodology
described in, e.g., Kolb et al, Angew Chem Int Ed Engl., 2001,
40:2004-2021.
[0149] The combination of nanoparticles 20, analyte, and protein
binding agent 18 used in the sensors described herein can be
selected as desired.
[0150] In some embodiments, the combination of nanoparticles,
analyte, and binding agent used in the sensors can be based on the
assay configurations described in, e.g., Josephson, et al.,
Angewandte Chemie, International Edition 40, 3204-3206 (2001);
Perez et al., Nat Biotechnol 20, 816-20 (2002); J. M. Perez et al.
J Am Chem Soc 124, 2856-7 (2002); and Tsourkas et al. Angew Chem
Int Ed Engl 43, 2395-9 (2004) (a water relaxation assay using
antibodies and surface functionalized nanoparticles detecting
enantiomeric impurities indicating the ability of the MR-based
assay to measure the levels of drugs or metabolites rather than
glucose), each of which is incorporated by reference herein.
[0151] By way of example, Table 2 illustrates representative assay
configurations. TABLE-US-00002 TABLE 2 Relaxation Sensor Assay Type
or Example and Configuration Exogenous Analyte Binding Agent
Comment See, e.g., FIG. 1A glucose conA See Examples See, e.g.,
FIG. 1A Chiral Moieties (e.g., Monoclonal See, e.g., All Figures
Small molecule antibodies of Tsourkas, A., Enantiomers) Hofstetter,
O., Hofstetter, H., Weissleder, R., and Josephson, L. (2004).
Magnetic relaxation switch immunosensors detect enantiomeric
impurities. Angew Chem Int Ed Engl 43, 2395-2399. See, e.g., FIG.
1A Monovalent analytes Multivalent binding Multivalent (e.g.,
therapeutic agent (e.g., able to Functionalized agents, peptides,
bind two or more nanoparticle must be nucleic acids, etc.)
nanoparticles (able to two binding simultaneously; e.g., proteins
Lectin, polyclonal or simultaneously) monoclonal antibody) See,
e.g., FIG. 1B Nucleic acid oligonucleotide See e.g., all figures of
Josephson, L., Perez, J. M., and Weissleder, R. (2001). Magnetic
nanosensors for the detection of oligonucleotide sequences.
Angewandte Chemie, International Edition 40, 3204-3206. See, e.g.,
FIG. 1B mRNA oligonucleotide E.g., FIG. 4 of Perez, J. M.,
Josephson, L., O'Loughlin, T., Hogemann, D., and Weissleder, R.
(2002). Magnetic relaxation switches capable of sensing molecular
interactions. Nat Biotechnol 20, 816-820 See, e.g., FIG. 1B GFP
Polyclonal antibodies E.g., FIG. 5A, Perez, J. M., Josephson, L.,
O'Loughlin, T., Hogemann, D., and Weissleder, R. (2002). Magnetic
relaxation switches capable of sensing molecular interactions. Nat
Biotechnol 20, 816-820. See, e.g., FIG. 1B Proteins, Proteins,
Multivalent polysaccharide Exogenous Analyte (able to
simultaneously bind two or more nanoparticles). Multivalent
functionalized nanoparticle (able to bind to two or more analytes
simultaneously.)
[0152] In some embodiments, the sensors described herein can be
used to monitor physiological concentrations of glucose (see
Examples section).
[0153] In general, any MR-based method that is capable of
discriminating water T2 relaxation times in the sensor chamber from
those outside of the sensor chamber can be used to monitor the
sensors. Such methods can be MR imaging or MR non-imaging
methods.
[0154] For example, in applications where a single water relaxation
sensor is employed, and the T2 of non-sensor, bulk water is
uniform, determining the relaxation properties of sensor water may
not necessarily require an MR imager or a two-dimensional matrix of
water relaxation data. Any non-imaging method capable of
distinguishing the properties of the MR signal emanating from
inside the sensor from those of the bulk water could be used. Thus,
far simpler and less costly types of instrumentation than a
clinical MRI instrument can distinguish the relaxation properties
sensor water from bulk water. First, the sensor can be implanted in
a tube of flowing tube of fluid, minimizing the volume of a
homogeneous magnetic field needed but still using the spatial
encoding methods of MRI instrumentation. Second, the applied
magnetic field need not be homogeneous, a requirement of magnets
used to generate MR images. Selective excitation magnets were
considered in early MR imager designs (see, e.g., Z. Abe, K.
Tanaka, Y. Yamada, Radiat Med 2, 1-23). A variable field strength
hand-held magnet and excitation/receiver coil are used for
analyzing the relaxation properties of samples within several
millimeters of the magnet in commercial devices, see, e.g.,
http://www.minispec.com/products/ProFiler.htm. These devices can be
used with the new sensors.
[0155] Solvent, (e.g., water), spin-spin relaxation times (T2) can
be determined by relaxation measurements using a nuclear magnetic
resonance benchtop relaxometer. In general, T2 relaxation time
measurements can be carried out at 0.47 T and 40.degree. C. (Bruker
NMR Minispec, Billerica, Mass.) using solutions with a total iron
content of 10 .mu.g Fe/mL.
[0156] Alternatively, T2 relaxation times can be determined by
magnetic resonance imaging of 384-well plates (50 .mu.L sample
volume), allowing parallel measurements at higher throughput. In
general, magnetic resonance imaging can be carried out using a 1.5
T superconducting magnet (Sigma 5.0; GE medical Systems, Milwaukee,
Wis.) using T2-weighted spin echo sequences with variable echo
times (TE=25-1000 ms) and repetition times (TR) of 3,000 ms to
cover the spectrum of the anticipated T2 values. This technique is
described in, for example, Perez, J. M., et al. Nat Biotechnol
2002, 20, 816-820; and Hogemann, D., et al. Bioconjug Chem 2002,
13, 116-121.
[0157] While not wishing to be bound by theory, it is believed that
nanoparticle aggregation is associated with an increase in R2
relaxivity of nanoparticles, but not necessarily with R1
relaxivity. See, e.g., Table 1 Josephson, L., Perez, J. M., and
Weissleder, R. (2001). "Magnetic nanosensors for the detection of
oligonucleotide sequences." Angewandte Chemie International
Edition: 3204-3206. Relaxivity, R=change in relaxation rate (1/T)
per change in concentration by 1 mM. Since there is essentially no
change or relatively little change in R1 associated with
nanoparticle aggregation, the measurement of T1 can be used to
determine nanoparticle concentration, while measurements of T2 can
be used to determine nanoparticle aggregation. This method can use
by way of example equations (1)-(3) below, where NP=nanoparticle,
and T1.sub.o and T2.sub.o are the relaxation times of sensor water
in the absence of nanoparticles. [A], the concentration of analyte,
can be a simple or complex function of R2, which reflects the
aggregation state of the particles.
[NP]=(1/T1.sub.o-1/T1.sub.NP)/R1 (1)
(1/T2.sub.o-1/T2.sub.NP)/[NP]=R2 (2) [A]=kR2 or [A]=f(R2) (3)
[0158] Nanoparticle aggregation can also be determined without
measurement of T2 as the examples below indicate.
[0159] 1. Measurement of the T2*, or free induction decay, rather
than T2.
[0160] 2. Measurement of amount of relaxation properties of
specific class of water protons in the sample using an off
resonance radiation, that is radiation that is not precisely at the
Larmour precession frequency. In this measure a frequency of
incident radiation not precisely at the Larmour precession
frequency is employed.
[0161] 3. Measurement of the height of a single echo obtained with
a T2 measuring pulse sequence rather than a complete echo train.
Normal T2 measurements utilize the declining height of a number of
echoes to determine T2.
[0162] 4. Shifting the frequency or strength of the applied
magnetic field, measuring the broadness of the proton absorption
peak. Broader the peaks or energy absorption are correlated with
higher values of T2.
[0163] In some embodiments, with instrumentation designed solely
for distinguishing the relaxation of sensor water protons from bulk
water protons, relaxation based sensors can be used to monitor
exogenous analytes in any enclosed, aqueous system including
bioreactors or fluids in a variety of industrial applications.
[0164] In some embodiments, the sensor can be an implantable sensor
implanted subcutaneously (e.g., the sensor can be implanted in an
extremity of a subject so as to avoid having the entire body of the
subject surrounded by the magnetic field).
EXAMPLES
[0165] The invention is further illustrated by the following
Examples. The Examples are provided for illustrative purposes only,
and are not to be construed as limiting the scope or content of the
invention in any way.
[0166] General
[0167] Synthesis of surface functionalized nanoparticles: EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride),
sulfo-NHS (sulfosuccinimidyl ester of N-hydroxysuccinimide) were
purchased from Pierce. SPDP (N-succinimidyl 3-(2-pyridyldithio)
propionate) was purchased from Molecular Biosciences. All other
chemicals were purchased from Sigma Aldrich and used as received.
Amino-CLIO nanoparticles were synthesized by crosslinking the
dextran coating with epichlorohydrin and reacting it with ammonia,
to provide primary amine groups (see, e.g., Josephson, L.; Tung, C.
H.; Moore, A.; Weissleder, R. Bioconjug Chem. 1999, 10, (2),
186-91.; and Josephson, L.; Perez, J. M.; Weissleder, R. Angewandte
Chemie, International Edition 2001, 40, (17), 3204-3206). The
number of amines was determined by reaction with SPDP and treatment
with dithiothreitol that releases pyridine-2-thione (P2T) (see,
e.g., Zhao, M.; Kircher, M. F.; Josephson, L.; Weissleder, R.
Bioconjug Chem 2002, 13, (4), 840-4). Protein binding agents ConA,
anti-folate acid antibody (anti-FA) and anti-HA antibody (anti-HA)
were purchased from Sigma.
[0168] For T2 measurements, a 0.47T relaxometer (Bruker) was used.
Measurements were made in 0.5 mL of PBS at 40.degree. C. The
concentration of surface functionalized nanoparticles was between 8
and 15 .mu.g/ml Fe, adjusted to give a starting T2 of about 150
msec. The concentrations of binding proteins were 1 mg/mL (Con A),
0.1 mg/mL (anti-HA) and 0.1 mg/mL (anti-FA). After addition of each
amount of analyte, T2 was recorded several times until it reached a
stable value.
[0169] The size of nanoparticles was measured by a laser light
scattering (Zetasizer, Malvern Instruments) in 1 mL PBS at
23.degree. C. and is the volume-based size. The concentration of
surface functionalized nanoparticles was between 25 and 35 .mu.g/ml
Fe, which is believed to be optimal for obtaining nanoswitch size
distribution on this instrument. The concentrations of binding
proteins were 1 mg/mL (Con A), 0.5 mg/mL (anti-HA) and 0.5 mg/mL
(anti-FA). Repeated size measurements were made at 40-80 minutes
post addition of binding protein or analyte and were essentially
constant over than period. Results shown are typical size
distributions. For reversion to the dispersed states (FIGS. 4E, 8E
and 9E), 600 mg/dl glucose, 500 nM HA and 30 nM FA were
employed.
[0170] For some of the experiments enclosing the nanoswitches in a
semipermeable device, 0.25 ml of Glu-CLIO (10 .mu.g/mL Fe) and ConA
(1 mg/mL) were placed in membrane with a 10 kDa cutoff
(Spectra/Por, Fischer). The device was transferred back and forth
between solutions with glucose concentrations of 20 mg/dl and 200
mg/dl. It was removed at different times and placed in a NMR tube.
T2 values were obtained in less than 30 seconds and the device
placed in the original glucose solution of a glucose solution with
a different concentration.
[0171] Data processing: The line drawn for cyclical changes in T2
was obtained by use of the following equation:
T2=A*sin(B*time+C)+Y, A=15.58, B=0.0253836, C=50.283 and
Y=83.8099.
Example 1
Glu-CLIO Nanoswitches
[0172] Summary
[0173] To demonstrate the water relaxation sensor we designed a
prototype for monitoring physiological concentrations of glucose.
We employed conconavalin A as a binding protein and synthesized a
glucose functionalized magnetic nanoparticle (Glu-CLIO).
Conconavalin A (ConA) is a tetravalent lectin that is known to
react with glucose. Some of the sensors were prepared having a
walled enclosure with a pore size of 3 kDa. In general, the walled
enclosure of the sensors retained the Glu-CLIO nanoparticle and
ConA, while permitting glucose to freely enter or leave the
sensor.
Example 1A
Preparation of Glu-CLIO
[0174] MION47 and amino-CLIO (25-35 nm) were prepared as described
elsewhere. D-Glucose, D-(+)-Glucosamine hydrochloride, succinic
anhydride, Concanavalin A (ConA) and Sephadex G-25 were from Sigma
Aldrich Co. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) and sulfo-N-hydroxysuccinimide (sulfo-NHS) were
from Pierce (Rockford, Ill.). To synthesize a
glucose-functionalized nanoparticle (Glu-CLIO), NH.sub.2-CLIO was
first converted to a carboxylic group functionalized nanoparticle,
followed by coupling of 2-amino-glucose using a water-soluble
carbodiimide. To obtain a carboxylic functionalized CLIO, 2.0 mg
succinic anhydride was added into 200 uL NH.sub.2-CLIO (10 mg
Fe/mL, 42 NH.sub.2 per 2064 Fe) with 300 uL (0.1 M) NaHCO.sub.3
buffer, pH 8.5. The mixture was incubated at room temperature for
two hours and succinic acid removed using a Sephadex G-25 column
eluted with MES buffer (0.5 M NaCl, 0.05 M MES), pH 6.0. To
conjugate 2-amino-glucose to carboxy functionalized CLIO
(CLIO-COOH), 2 mg EDC and 2 mg sulfo-NHS were added into 500 uL
CLIO-COOH (mg Fe/mL) in MES buffer, pH 6.0. The mixture was allowed
to react for two hours at room temperature and purified by Sephadex
G-25 column eluted with PBS buffer at pH 7.4. Subsequently 2 mg
Glucosamine was added into above solution and the mixture reacted
for one hour at room temperature. Unreacted glucosamine was removed
with Sephadex G-25 in PBS.
Example 1B
Glu-CLIO-ConA-Glucose Tube Assay
[0175] Relaxation times were obtained at 0.47T, 40.degree. C. using
a Minispec relaxometer (Bruker).
[0176] To demonstrate the interaction between Glu-CLIO, ConA and
glucose, experiments were performed directly in NMR tube (no
semi-permeable walled enclosure), 10 ug Fe/mL, 800 ug/mL ConA. All
experiments were performed in PBS with 1 mM CaCl.sub.2 and 1 mM
MgCl.sub.2. Transverse relaxation times (T2's) were measured in the
relaxometer, Bruker Minispec.RTM. NMS 120 at 0.47 T and 40.degree.
C. Size was determined with a Zetasizer HS1000.RTM. (Malvern
Instruments, Marlboro, Mass.) in the buffer above with Glu-CLIO at
20 ug Fe/mL, followed by the addition of ConA to 1 mg/mL and 1.5
mg/mL glucose. All experiments used these concentration of ions,
buffer, ConA and Glu-CLIO.
[0177] As shown in FIG. 3A, the addition of ConA to Glu-CLIO
resulted in a drop in T2 that reached a plateau after about 50
minutes, while no change was obtained with amino-CLIO. Associated
with the ConA induced T2 change was an increase in the size of the
Glu-CLIO nanoparticle from 30 nm to 301 nm by laser light
scattering, indicating nanoparticle clustering was associated with
ConA addition and the T2 increase, and that the system was behaving
like a magnetic relaxation switch. Addition of glucose then caused
a partial reversibility of the T2 drop, with T2 values again
reaching a plateau after about 100 minutes, 150 minutes, and 200
minutes. This type of response was seen with additions that
produced concentrations of 0.4, 0.8 and 1.8 mg/mL glucose. The
sensor responded to 0.2 to 1.8 mg/mL glucose, which is
approximately the physiological range of plasma glucose in humans.
The apparatus is shown in FIG. 3B.
Example 1C
Glu-CLIO-ConA-Glucose Tube Assay
[0178] Glu-CLIO nanoswitches were diluted into tubes to obtain a T2
of 153 msec. Upon addition of ConA, T2 decreased and reached a
plateau value of 65 msec (see FIG. 4A).
[0179] Addition of increasing concentrations of glucose reversed
this effect, with a constant T2 value observed at each glucose
concentration (see FIG. 4B). The change in plateau T2 values
occurred in a linear fashion over the physiological range of
glucose concentrations (see FIG. 4B).
[0180] To further investigate the interconversion (switch) between
the initial dispersed nanoparticle state and the microaggregated
state, light scattering measurements were obtained (see FIGS. 4C,
4D, and 4E). Dispersed nanoswitches had a mean diameter of 26 nm
(see FIG. 4C), which increased to a mean diameter of 230 nm upon
addition of ConA (see FIG. 4D), and which returned to its original
size distribution with the addition of glucose (400 mg/dl) (see
FIG. 4E). The initial T2 value of 153 msec (see FIG. 4A) was not
achieved upon addition of the glucose. The discrepancy between the
initial and final T2 value is believed to be due to a slight
dilution of iron that accompanied the addition of concentrated
solutions of glucose. Based on the return of nanoswitches to their
original size distribution (see FIG. 4E), a complete
interconversion between dispersed and microaggregated nanoswitch
states was achieved by glucose addition.
Example 1D
Glu-CLIO-ConA-Glucose Sensor Assay
[0181] We next placed the components of the tube-based relaxation
assay (ConA and Glu-CLIO as used in FIGS. 3A and 3B) in the
semi-permeable device shown in FIG. 5B, to obtain a water
relaxation based sensor (volume .about.0.5 mL, 3 kDa pores). We
allowed the sensor to equilibrate with 0.1 mg/mL glucose overnight
and obtained a stable T2 of 98 msec (see FIG. 5A), obtained by
placing it in a glass tube used with the MR relaxometer. The sensor
was then placed in a second tube (1 mg/mL glucose) and T2 values
monitored (see FIG. 5A). A new plateau of 70 msec was attached
after about 100 minutes, indicating glucose-induced dispersion of
the Glu-CLIO nanoparticles (see FIG. 5A). The sensor was then
placed in a third tube with 0.1 mg/mL glucose. T2 increased and
returned to a plateau of T2 again at 98 msec (see FIG. 5A). Thus,
the water relaxation based glucose sensor uses a T 2 dependent
equilibrium between ConA and Glu-CLIO to sense external glucose in
a reversible fashion.
[0182] Similar results were obtained when Glu-CLIO and ConA were
enclosed in a semi-permeable sensor to interact with glucose in the
external sensor environment (FIG. 5B), using 500 uL of G-ConA and
ConA as above were placed in a membrane of cellulose ester with a 1
kDa cutoff and diameter of 7.5 mm (Spectra/Por.RTM., Fisher
Scientific). The sensor was placed in 50 mL tube with a magnetic
stir at the bottom of tube for mixing. At various times the
external concentration of glucose was varied, and the sensor
removed, placed in an NMR tube, and T2 determined as above (results
not shown).
Example 1E
Glu-CLIO-ConA-Glucose Sensor Assay with MRI Imaging
[0183] We placed Glu-CLIO-ConA sensors in two test tubes, one with
2 mg/mL glucose and without glucose and imaged the tubes using a
clinical MR imager (see FIG. 6A). To demonstrate the ability of MRI
to detect the interaction between Glu-CLIO and ConA the
semi-permeable membrane, Glu-CLIO (10 ug Fe/mL) and Con A (800
ug/mL) were placed in a 1 kDa cutoff, 5 mm diameter semi-permeable
tube (Spectra/Por Irradiated Dispodialyzer, Fisher) and the sensor
placed in a 50 mL tube as above. After 2 hours, the stir bar was
removed and images obtained on a clinical GE Signa 1.5 T unit.
(Image size 256.times.192, field of view 7.times.14 Cm, slice
thickness 1.5 mm using a turbo spin echo pulse sequence, TR 2500,
TE65).
[0184] As shown in FIGS. 6B and 6C, the sensor in the high glucose
environment had higher signal intensity (brighter image),
reflecting nanoparticle dissociation and a higher (longer) T2.
Thus, the concentration of external glucose altered the signal
intensity of water within the sensor that was evident on an MR
image.
Example 1F
Glu-CLIO-ConA-Glucose Sensor Assay with MRI Imaging
[0185] We further examined whether the nanoswitch/binding protein
equilibrium would be maintained if the nanoswitches and binding
proteins were enclosed in a semipermeable device, with pores that
would allow analyte to enter but which would retain nanoparticles
and binding protein. This would allow analyte concentrations to be
raised or lowered, depending on the sensor environment (dialysate).
A number of different units were investigated as possible including
Spectra/Por tubing, Slide-A-Lyzer microcassettes and dialysis
fibers. Spectra/Por tubing permitted the rapid and repeated
transfer of the sensor from a 100 mL beaker, where glucose
concentrations were cycled between 20 mg/dl and 400 mg/dl to an MR
relaxometer, where T2 measurements were made in less than a minute.
The T2 of sensor water changed in cycled between about 68 and 100
msec as it responded to changing concentrations of glucose (see
FIG. 7). Both increases and decreases in glucose concentration
resulted in alteration of the nanoswitch microaggregation state,
evident by changes in T2, further demonstrating the equilibrium
nature of nanoswitches.
Example 2
HA-CLIO Nanoswitches
Example 2A
Preparation of HA-CLIO
[0186] To synthesize hemagglutinin peptide-CLIO, a thiolated
influenza hemagglutinin (HA) peptide with a C-terminal cysteine
(YPYDVPDVAGGC) was synthesized by using Fmoc chemistry on Rink
amide resin (Calbiochem, NovaBiochem) and was purified by reverse
phase HPLC. The molecular weight of HA was confirmed by MALDI-TOF.
To attach HA to nanoparticle, amino-CLIO was first reacted with
SPDP. After purification, 200 .mu.L SPDP modified CLIO (5.0 mg/mL
Fe) in PBS buffer, pH 7.4 was mixed with 100 .mu.L of HA (50 mM) in
DMSO. Reaction proceeded for 2 hours at room temperature. The CLIO
conjugate was separated by Sephadex G-25 column and eluted with PBS
buffer, pH 7.4. The number of peptides per nanoparticle was
determined by the SPDP method. Nanoparticles had 25 HA per 2000 Fe
and the size distribution shown in FIG. 3.
Example 2B
HA-CLIO-anti-HA-HA Tube Assay
[0187] Relaxation times were obtained at 0.47T, 40.degree. C. using
a Minispec relaxometer (Bruker).
[0188] Nanoswitches had similar properties when HA-CLIO replaced
Glu-CLIO and antibody to HA (anti-HA) replaced ConA as shown in
FIGS. 8A-8E. As shown in FIG. 8A, T2 dropped from 162 to 141 msec
with the addition of anti-HA. Plateau values of T2 changed over a
range of HA concentrations between 50 and 400 nM (FIG. 8B), which
was about 80 fold lower than the concentrations of glucose needed
to change T2 (2.5 .mu.M-20 .mu.M, FIG. 4B). Again light scattering
data indicated that the analyte (HA) was capable of essentially
completely reversing microaggregate formation (see FIGS. 8C, 8D,
and 8E).
Example 3
FA-CLIO Nanoswitches
Example 3A
Preparation of FA-CLIO
[0189] To synthesize FA-CLIO, amino-CLIO in PBS buffer, pH 7.4 was
first exchanged with MES buffer (50 mM MES hydrate, 0.1 M NaCl), pH
6.0 and the solution was concentrated to 5.0 mg/mL. Then 100 .mu.L
(50 mM) folic acid in DMSO was added to 200 .mu.L amino-CLIO (5.0
mg/mL Fe) in MES solution, pH 6.0. This was followed by the
addition of excess EDC (0.96 mg, 5 .mu.mol) and sulfo-NHS (1.1 mg,
5 .mu.mol) in 100 .mu.L DMSO. Reaction proceeded at room
temperature for 2 hours and the product was purified by Sephadex
G-25 column and eluted with PBS buffer, pH 7.4. Attachment of FA
was quantified by the loss of amine groups using SPDP, see above,
and was 33 FA per 2000 Fe with the size distribution shown in FIG.
9C.
Example 3B
FA-CLIO-anti-FA-FA Tube Assay
[0190] Relaxation times were obtained at 0.47T, 40.degree. C. using
a Minispec relaxometer (Bruker).
[0191] The properties of the nanoswitch system were examined with
FA-CLIO nanoparticles and anti-FA as the binding protein. As shown
in FIG. 9A, T2 dropped from 155 msec to 113 msec with anti-FA
addition. The range or concentrations of FA associated with
changing T2 values are shown in FIG. 9B (5-20 nM) and was about
1000 fold lower than the concentrations of glucose measured (2.5
.mu.M-20 .mu.M, FIG. 4B). Again, light scattering data indicated
that the analyte (HA) was capable of essentially completely
reversing microaggregate formation (see FIGS. 9C, 9D, and 9E).
[0192] We conclude that surface functionalized nanoparticles and
binding proteins maintained an equilibrium, continuously switching
between a dispersed (disaggregated), low T2 state (20-40 nm) and
microaggregated high T2 state (200-250 nm), depending the
concentration of exogenous analyte. Based on light scattering data,
high concentrations of exogenous analytes completely reversed
microaggregate formation, returning the system to its original
dispersed state expected for an equilibrium process. As indicated
by the use of nanoswitches and binding proteins for glucose, FA,
and HA, nanoswitches were able to detect chemically diverse
analytes over a relatively wide range of concentration.
Other Embodiments
[0193] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. For example, two or more chelating moieties
can be incorporated into a single monomeric substrate molecule.
Accordingly, other embodiments are within the scope of the
following claims.
* * * * *
References