U.S. patent application number 17/583548 was filed with the patent office on 2022-05-12 for polymeric organic nanoparticles with enhanced emission.
This patent application is currently assigned to Sony Group Corporation. The applicant listed for this patent is Sony Group Corporation. Invention is credited to Vitor DEICHMANN, Michaela MAI, Tzenka MITEVA, Gabriele NELLES, Markus OBERMAIER, Anthony ROBERTS, Jan ROTHER, Vladimir YAKUTKIN.
Application Number | 20220145172 17/583548 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-12 |
United States Patent
Application |
20220145172 |
Kind Code |
A1 |
MITEVA; Tzenka ; et
al. |
May 12, 2022 |
POLYMERIC ORGANIC NANOPARTICLES WITH ENHANCED EMISSION
Abstract
The present disclosure relates to luminescent including photon
up-conversion nanoparticles. These nanoparticles include a
polymeric organic matrix, at least one light emitter distributed
within this matrix, a stabilizing agent, and at least one metal
particle enclosed within the matrix, wherein the metal particles
are plasmonic nanoparticles. The present disclosure further relates
to methods of manufacture and to uses of such nanoparticles.
Inventors: |
MITEVA; Tzenka; (Stuttgart,
DE) ; ROBERTS; Anthony; (Stuttgart, DE) ; MAI;
Michaela; (Stuttgart, DE) ; OBERMAIER; Markus;
(Stuttgart, DE) ; NELLES; Gabriele; (Stuttgart,
DE) ; DEICHMANN; Vitor; (Stuttgart, DE) ;
YAKUTKIN; Vladimir; (Stuttgart, DE) ; ROTHER;
Jan; (Stuttgart, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sony Group Corporation |
Tokyo |
|
JP |
|
|
Assignee: |
Sony Group Corporation
Tokyo
JP
|
Appl. No.: |
17/583548 |
Filed: |
January 25, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16085741 |
Sep 17, 2018 |
11230663 |
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PCT/EP2017/056905 |
Mar 23, 2017 |
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17583548 |
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International
Class: |
C09K 11/02 20060101
C09K011/02; C09K 11/06 20060101 C09K011/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2016 |
EP |
16163430.8 |
Claims
1: A nanoparticle, comprising: a polymeric organic matrix; at least
one light emitter distributed within said matrix; a stabilizing
agent; and at least one metal nanoparticle enclosed within said
matrix.
2: The nanoparticle according to claim 1, wherein said polymeric
organic matrix comprises at least one material selected from the
group consisting of a polyacrylonitrile, a polystyrene, an
oligostyrene, a styrene copolymer, a styrene-butadiene copolymer, a
polystyrene-based elastomer, a polyethylene, an oligoethylene, a
polyphenylene, a polyphenylene dendrimer, a polypropylene, a
polytetrafluoroethylene, an extended polytetrafluoroethylene, a
polyacrylate, a polymethylmethacrylate, an ethylene-co-vinyl
acetate, a polysiloxane, a polysiloxane copolymer, a substituted
polysiloxane, a modified polysiloxanes, a polyether, a
polyurethane, a polyether-urethane, a polyethylene terephthalate, a
polysulphone, and a copolymer thereof.
3: The nanoparticle according to claim 1, wherein said light
emitter that emits light by luminescence.
4: The nanoparticle according to claim 1, wherein said nanoparticle
is a photon up-conversion nanoparticle.
5: The nanoparticle according to claim 1, further comprising: at
least one sensitizer, that absorbs light at a first wavelength
region w.ltoreq..lamda..sub.1.ltoreq.x, and said at least one light
emitter emits light at a second wavelength region
y.ltoreq..lamda..sub.2.ltoreq.z, where
.lamda..sub.2.ltoreq..lamda..sub.1, upon absorption of light by
said at least one sensitizer at said first wavelength region
w.ltoreq..lamda..sub.1.ltoreq.x, said at least one light emitter
emits light at said second wavelength region
y.ltoreq..lamda..sub.2.ltoreq.z.
6: The nanoparticle according to claim 1, wherein said at least one
light emitter is a molecule selected from the group consisting of
an anthracene, a perylene, a perylene derivative, a coumarin and a
BODIPY dye, said at least one light emitter has a structure of
Formula (I) or (II) or includes a molecule having the structure of
Formula (I) or (II), ##STR00076## wherein R.sub.1 and R.sub.2 are
independently selected from the group consisting of hydrogen and a
moiety with a structure of Formula (III), at least one of R.sub.1
and R.sub.2 is a moiety with the structure of Formula (III),
##STR00077## wherein R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7
are independently selected from the group consisting of H, F, and
tri-fluoro-methyl (--CF.sub.3), and wherein at least one of
R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is F or
tri-fluoro-methyl (--CF.sub.3); or wherein said at least one light
emitter has a structure of Formula (X) or includes a molecule
having the structure of Formula (X), ##STR00078## wherein R.sub.8
and R.sub.9 are independently selected from the group consisting of
hydrogen and a moiety with a structure of Formula (XI), wherein at
least one of R.sub.8 and R.sub.9 is a moiety with the structure of
Formula (XI), ##STR00079## wherein R.sub.10, R.sub.11, R.sub.12,
R.sub.13 and R.sub.14 are independently selected from the group
consisting of H, F, and tri-fluoro-methyl (--CF.sub.3), and wherein
at least one of R.sub.10, R.sub.11, R.sub.12, R.sub.13 and R.sub.14
is F or tri-fluoro-methyl (--CF.sub.3); or wherein said at least
one light emitter has one of structures or includes a molecule
having one of the structures, ##STR00080## ##STR00081## or wherein
said light emitter has a structure of Formula (XXIII), (XXIV) or
(XXV) or includes a molecule having the structure of Formula
(XXIII), (XXIV) or (XXV), ##STR00082## wherein W in Formulas
(XXIII-XXV) is one selected from groups, ##STR00083## wherein Y as
used in formula W is selected from the group consisting of
CH.sub.2, S, O, Se and N--R.sub.2, and wherein R.sub.2 is selected
from the group consisting of H, a linear alkyl group, a branched
alkyl group, a cycloalkyl group, a halogenated alkyl group, a
halogen atom, an alkyl sulfanyl group, an aryl sulfanyl group, an
amino alkyl group, an amino aryl group, an aryl group, a heteroaryl
group, a fluorenyl group, an amino group, a nitro group, an OH
group, an SH group, and a group --O--R.sub.3, wherein R.sub.3 is
selected from the group consisting of a linear alkyl group, a
branched alkyl group, a cycloalkyl group, a halogenated alkyl
group, an alkyl sulfanyl group, an aryl sulfanyl group, an amino
alkyl group, an amino aryl group, an aryl group, a heteroaryl group
and a fluorenyl group, wherein X and Y in Formulas (XXIII-XXV) are
independently selected from groups, ##STR00084## ##STR00085##
wherein R is selected from the group consisting of H, a linear
alkyl group, a branched alkyl group, a cycloalkyl group, a
halogenated alkyl group, a halogen atom, an alkyl sulfanyl group,
an aryl sulfanyl group, an amino alkyl group, an amino aryl group,
an aryl group, a heteroaryl group, a fluorenyl group, an amino
group, a nitro group, an OH group, an SH group, and a group
--O--R.sub.3, wherein R.sub.3 is selected from the group consisting
of a linear alkyl group, a branched alkyl group, a cycloalkyl
group, a halogenated alkyl group, an alkyl sulfanyl group, an aryl
sulfanyl group, an amino alkyl group, an amino aryl group, an aryl
group, a heteroaryl group and a fluorenyl group, wherein Z in
Formulas (XXIII-XXV) is selected from groups, ##STR00086## wherein
R.sub.2 is selected from the group consisting of H, a linear alkyl
group, a branched alkyl group, a cycloalkyl group, a halogenated
alkyl group, a halogen atom, an alkyl sulfanyl group, an aryl
sulfanyl group, an amino alkyl group, an amino aryl group, an aryl
group, a heteroaryl group, a fluorenyl group, an amino group, a
nitro group, an OH group, an SH group, and a group --O--R.sub.3,
wherein R.sub.3 is selected from the group consisting of a linear
alkyl group, a branched alkyl group, a cycloalkyl group, a
halogenated alkyl group, an alkyl sulfanyl group, an aryl sulfanyl
group, an amino alkyl group, an amino aryl group, an aryl group, a
heteroaryl group and a fluorenyl group, and wherein Ri is selected
from groups, ##STR00087## ##STR00088## or wherein said at least one
light emitter has one of structures, ##STR00089## wherein R is a
linear or branched alkyl group; or wherein said light emitter has a
structure of Formula (XXVI) or includes a molecule having the
structure of Formula (XXVI) ##STR00090## wherein R is selected from
groups, ##STR00091## wherein Y is selected from the group
consisting of CH.sub.2, S, O, Se and N--R.sub.2, and wherein
R.sub.2 is selected from the group consisting of H, a linear alkyl
group, a branched alkyl group, a cycloalkyl group, a halogenated
alkyl group, a halogen atom, an alkyl sulfanyl group, an aryl
sulfanyl group, an amino alkyl group, an amino aryl group, an aryl
group, heteroaryl group, a fluorenyl group, an OH group, an SH
group, and a group --O--R.sub.3, and wherein R.sub.3 is selected
from the group consisting of a linear alkyl group, a branched alkyl
group, a cycloalkyl group, a halogenated alkyl group, an alkyl
sulfanyl group, an aryl sulfanyl group, an amino alkyl group, an
amino aryl group, an aryl group, a heteroaryl group and a fluorenyl
group, or wherein R is selected from groups, ##STR00092## wherein
R.sub.1 is a linear or branched alkyl group; or wherein said at
least one light emitter has one of structures or includes a
molecule having one of the structures, ##STR00093## wherein R is a
linear or branched alkyl group.
7: The nanoparticle according to claim 5, wherein said at least one
sensitizer has a structure of Formula (XXVII), Formula (XXVIII),
Formula (IXXX), Formula (XXX) or Formula (XXXI) or includes a
molecule having the structure of Formula (XXVIII), Formula (IXXX),
Formula (XXX) or Formula (XXXI), ##STR00094## wherein R.sub.1 is
hydrogen, a linear or branched alkyl group, in particular with up
to 6 carbon atoms, or a benzene ring, and wherein R is a linear or
branched alkyl group, with up to 6 carbon atoms, ##STR00095##
##STR00096##
8: The nanoparticle according to claim 1, wherein said nanoparticle
includes 1 to 4 metal nanoparticles and/or does not include metal
nanoparticles that are in contact with each other.
9: The nanoparticle according to claim 1, wherein said at least one
metal nanoparticle has a diameter in a range of from 1 to 100 nm
and/or consists of a material selected from the group consisting of
Ag, Au and Co and/or plasmonic and/or magnetic.
10: The nanoparticle according to claim 1, further comprising: at
least one antioxidant selected from the group consisting of lipoic
acid, vitamin E, a carotenoid, an ascorbic acid derivative soluble
in an organic solvent.
11: The nanoparticle according to claim 1, further comprising: at
least one functional group positioned at a surface of said matrix
and selected from the group consisting of --COOH, --NH.sub.2, --SH
(thiol), --NHS, alkynyl, --N.sub.3, aldehyde, ketone and biotin
group.
12: The nanoparticle according to claim 1, further comprising:
antibody molecules that are attached to a surface of said matrix
and binds to a biomolecule.
13: A sensing layer, comprising: nanoparticles each comprising the
nanoparticle of claim 1.
14: A method of producing nanoparticles, comprising: providing a
polymer or a combination of polymers or a combination of polymers
with small molecules from which the polymeric organic matrix is to
be formed, a stabilizing agent, at least one light emitter, and
metal nanoparticles; preparing a dispersion of said metal
nanoparticles in an organic water-miscible solvent; preparing a
mixture of said polymer or combination of polymers or combination
of polymers with small molecules from which the polymeric organic
matrix is to be formed, said stabilizing agent, and said at least
one light emitter in an organic water-miscible solvent; adding said
mixture of said polymer or combination of polymers or combination
of polymers with small molecules, said stabilizing agent, said at
least one light emitter to said dispersion of metal nanoparticles
or vice versa, thus forming a mixture including said metal
nanoparticles; and inducing said mixture including said metal
nanoparticles to form nanoparticles, thus forming a dispersion of
nanoparticles, wherein said nanoparticles include a polymeric
organic matrix with said at least one light emitter distributed
therein, and said metal nanoparticles are enclosed in said
polymeric organic matrix.
15. (canceled)
16: The nanoparticle according to claim 1, further comprising: at
least one antioxidant distributed in the said matrix.
17: The nanoparticle according to claim 1, wherein said polymeric
organic matrix comprises one of a combination of a polystyrene and
an oligostyrene, a combination of a polyethylene and an
oligoethylene, and a combination of a polyphenylene and a
polyphenylene dendrimer.
18: The nanoparticle according to claim 1, wherein said at least
one light emitter is a molecule selected from the group consisting
of an anthracene, a perylene, a perylene derivative, a coumarin and
a BODIPY dye.
19: The nanoparticle according to claim 1, wherein said at least
one light emitter has a structure of Formula (VII), Formula (VIII)
or Formula (IX), or includes a molecule having the structure of
Formula (VII), Formula (VIII) or Formula (IX): ##STR00097## or
wherein said at least one light emitter has a structure of Formula
(XVII), Formula (XVIII), Formula (XIX), Formula (XX), Formula (XXI)
or Formula (XXII), or includes a molecule having the structure of
Formula (XVII), Formula (XVIII), Formula (XIX), Formula (XX),
Formula (XXI) or Formula (XXII), ##STR00098## ##STR00099## or
wherein said at least one light emitter has one of structures or
includes a molecule having one of the structures, ##STR00100##
##STR00101## or wherein said light emitter has a structure of
Formula (XXIII), (XXIV) or (XXV) or includes a molecule having the
structure of Formula (XXIII), (XXIV) or (XXV), ##STR00102## wherein
X and Y in Formulas (XXIII-XXV) are independently selected from
groups, ##STR00103## ##STR00104## wherein R is selected from the
group consisting of H, a linear alkyl group, a branched alkyl
group, a cycloalkyl group, a halogenated alkyl group, a halogen
atom, an alkyl sulfanyl group, an aryl sulfanyl group, an amino
alkyl group, an amino aryl group, an aryl group, a heteroaryl
group, a fluorenyl group, an amino group, a nitro group, an OH
group, an SH group, and a group --O--R.sub.3, wherein R.sub.3 is
selected from the group consisting of a linear alkyl group, a
branched alkyl group, a cycloalkyl group, a halogenated alkyl
group, an alkyl sulfanyl group, an aryl sulfanyl group, an amino
alkyl group, an amino aryl group, an aryl group, a heteroaryl group
and a fluorenyl group, wherein Z in Formulas (XXIII-XXV) is
selected from groups, ##STR00105## wherein R.sub.2 is selected from
the group consisting of H, a linear alkyl group, a branched alkyl
group, a cycloalkyl group, a halogenated alkyl group, a halogen
atom, an alkyl sulfanyl group, an aryl sulfanyl group, an amino
alkyl group, an amino aryl group, an aryl group, a heteroaryl
group, a fluorenyl group, an amino group, a nitro group, an OH
group, an SH group, and a group --O--R.sub.3, wherein R.sub.3 is
selected from the group consisting of a linear alkyl group, a
branched alkyl group, a cycloalkyl group, a halogenated alkyl
group, an alkyl sulfanyl group, an aryl sulfanyl group, an amino
alkyl group, an amino aryl group, an aryl group, a heteroaryl group
and a fluorenyl group, wherein, in particular, R.sub.2 has not more
than 6 carbon atoms, wherein Ri is selected from groups,
##STR00106## ##STR00107## wherein W in Formulas (XXIII-XXV) is
selected from groups, ##STR00108## ##STR00109## ##STR00110##
wherein R is selected from the group consisting of H, a linear
alkyl group, a branched alkyl group, a cycloalkyl group, a
halogenated alkyl group, a halogen atom, an alkyl sulfanyl group,
an aryl sulfanyl group, an amino alkyl group, an amino aryl group,
an aryl group, a heteroaryl group, a fluorenyl group, an amino
group, a nitro group, an OH group, an SH group, and a group
--O--R.sub.3, wherein Ri is selected from groups, ##STR00111##
##STR00112## and wherein R.sub.2 is selected from the group
consisting of H, a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, a halogen atom, an
alkyl sulfanyl group, an aryl sulfanyl group, an amino alkyl group,
an amino aryl group, an aryl group, a heteroaryl group, a fluorenyl
group, an amino group, a nitro group, an OH group, an SH group, and
a group --O--R.sub.3, or wherein said at least one light emitter
has one of structures, ##STR00113## wherein R is a linear or
branched alkyl group; or wherein said light emitter has a structure
of Formula (XXVI) or includes a molecule having the structure of
Formula (XXVI), ##STR00114## wherein R is selected from groups,
##STR00115## wherein Y is selected from the group consisting of
CH.sub.2, S, O, Se and N--R.sub.2, and wherein R.sub.2 is selected
from the group consisting of H, a linear alkyl group, a branched
alkyl group, a cycloalkyl group, a halogenated alkyl group, a
halogen atom, an alkyl sulfanyl group, an aryl sulfanyl group, an
amino alkyl group, an amino aryl group, an aryl group, a heteroaryl
group, a fluorenyl group, an OH group, an SH group, and a group
--O--R.sub.3, and wherein R.sub.3 is selected from the group
consisting of a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, an alkyl sulfanyl
group, an aryl sulfanyl group, an amino alkyl group, an amino aryl
group, an aryl group, a heteroaryl group and a fluorenyl group; or
wherein R is selected from groups, ##STR00116## wherein R.sub.1 is
a linear or branched alkyl group; or wherein said at least one
light emitter has one of structures or includes a molecule having
one of the structures, ##STR00117## wherein R is a linear or
branched alkyl group.
20: The nanoparticle according to claim 1, wherein said at least
one light emitter has a structure of Formula (VII), Formula (VIII)
or Formula (IX), or includes a molecule having the structure of
Formula (VII), Formula (VIII) or Formula (IX): ##STR00118## or
wherein said at least one light emitter has a structure of Formula
(XVII), Formula (XVIII), Formula (XIX), Formula (XX), Formula (XXI)
or Formula (XXII), or includes a molecule having the structure of
Formula (XVII), Formula (XVIII), Formula (XIX), Formula (XX),
Formula (XXI) or Formula (XXII), ##STR00119## ##STR00120## or
wherein said at least one light emitter has one of structures or
includes a molecule having one of the structures, ##STR00121##
##STR00122## or wherein said at least one light emitter has one of
structures, ##STR00123## wherein R is a linear or branched alkyl
group: or wherein said at least one light emitter has one of
structures or includes a molecule having one of the structures,
##STR00124## wherein R is a linear or branched alkyl group.
21: The nanoparticle according to claim 5, wherein said at least
one sensitizer has a structure of Formula (XXVIII), ##STR00125##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of and claims the
benefit of priority to U.S. application Ser. No. 16/085,741, filed
Sep. 17, 2018, which is the National Stage of the International
Patent Application No. PCT/EP2017/056905, filed Mar. 23, 2017,
which claims priority to European Patent Application No.
16163430.8, filed Mar. 31, 2016. The entire contents of these
applications are incorporated herein by reference.
BACKGROUND
[0002] The field of the DISCLOSURE is the field of luminescent,
fluorescent, phosphorescent and photon up-conversion polymeric
organic nanoparticles.
[0003] The present disclosure relates to luminescent including
photon up-conversion nanoparticles. These nanoparticles include a
polymeric organic matrix, at least one light emitter distributed
within this matrix, a stabilizing agent, and at least one metal
particle enclosed within the matrix, wherein the metal particles
are plasmonic nanoparticles. The present disclosure further relates
to methods of manufacture and to uses of such nanoparticles.
DESCRIPTION OF THE RELATED ART
[0004] The "background" description provided herein is for the
purpose of generally presenting the context of the disclosure. Work
of the presently named inventors, to the extent it is described in
this background section, as well as aspects of the description
which may not otherwise qualify as prior art at the time of filing,
as well as aspects described in this background section in relation
to nanoparticles, sensors and sensing layers are neither expressly
nor implicitly admitted as prior art against the present
disclosure.
[0005] In recent years, nanoparticles have emerged as important
materials with numerous applications ranging from display devices
to the use as optical reporters and bioprobes in the life
sciences.
[0006] Among the organic nanoparticles capable of emitting light,
different types of nanoparticles have been developed, such as
organic nanoparticles capable of fluorescence emission or capable
of phosphorescent emission. Both types are commercially available,
especially the fluorescent types. But they mostly have low emission
quantum yield and low photostability. They are used for multiple
applications so enhancement of the emissive properties is urgently
needed. In contrast to the fluorescent and phosphorescent
nanoparticles, the existing photon upconversion nanoparticles other
than those disclosed in EP 2298849 A1 or US 2010/0330026 A1 are
inorganic.
SUMMARY
[0007] The present disclosure provides a nanoparticle including
[0008] a polymeric organic matrix, [0009] at least one light
emitter distributed within said matrix, [0010] a stabilizing agent,
and [0011] at least one metal nanoparticle enclosed within said
matrix, wherein said at least one metal nanoparticle is a plasmonic
nanoparticle.
[0012] The present disclosure provides a sensing layer including
nanoparticles according to the present disclosure.
[0013] The present disclosure provides a method of producing
nanoparticles as defined in any of the embodiments above, said
nanoparticles including [0014] a polymeric organic matrix, [0015]
at least one light emitter distributed within said matrix, [0016]
optionally at least one sensitizer and/or at least one antioxidant
distributed within said matrix, [0017] a surface stabilizing agent,
and [0018] at least one metal nanoparticle enclosed within said
matrix, wherein said at least one metal nanoparticle is a plasmonic
nanoparticle and/or is capable of increasing the photostability of
the organic nanoparticle, said method including the steps of:
[0019] providing a polymer or combination of polymers or
combination of polymers with small molecules from which the
polymeric organic matrix is to be formed, a stabilizing agent, at
least one light emitter, plasmonic metal nanoparticles and
optionally at least one sensitizer and/or at least one antioxidant,
[0020] preparing a dispersion of said plasmonic metal nanoparticles
in an organic water-miscible solvent, optionally allowing ligand
exchange of said plasmonic metal nanoparticles, [0021] preparing a
mixture of said polymer or said combination of polymers or said
combination of polymers and small molecules from which the
polymeric organic matrix is to be formed, said surface stabilizing
agent, said light emitter(s) and optionally said sensitizer(s)
and/or said antioxidant(s) in an organic water-miscible solvent,
[0022] adding said mixture of said polymer or said combination of
polymers or said combination of polymers and small molecules, said
surface stabilizing agent, said light emitter(s) and optionally
said sensitizer(s) and/or said antioxidant(s) to said dispersion of
plasmonic metal nanoparticles or vice versa, thus forming a mixture
including said plasmonic metal nanoparticles, [0023] inducing said
mixture including said organic matrix components and surface
stabilizer, emitters and plasmonic metal nanoparticles to form
nanoparticles, thus forming a dispersion of nanoparticles wherein
said nanoparticles include a polymeric organic matrix with said
light emitter(s) and, optionally, said sensitizer(s) and/or said
antioxidant(s), distributed therein, and wherein said metal
nanoparticles are enclosed in said polymeric organic matrix.
[0024] The present disclosure also provides the use of
nanoparticles according to the present disclosure in a biological
application selected from the group consisting of labeling and/or
detection of cells, biological (macro-)molecules or other analytes,
fluorescence microscopy, (flow) cytometry, fluorescence-activated
cell sorting (FACS), fluorescence resonance energy transfer (FRET),
immunohistochemistry, clinical immunoassays,
fluorescence-quenching-based enzyme-activity assays,
high-throughput screening, molecular diagnostics, sensing of
temperature, sensing of pressure and sensing of oxygen.
[0025] In the present disclosure the detection/sensing of
neurotransmitters and other singlet oxygen scavengers are provided.
Examples are given of preparation of sensing layers containing the
organic nanoparticles for detection (sensing) of neurotransmitters
and other singlet oxygen scavengers. The targeted application is
using the layers for sensing/imaging of neurotransmitters as
released from live cells--functional live cell imaging.
[0026] The foregoing paragraphs have been provided by way of
general introduction, and are not intended to limit the scope of
the following claims. The described embodiments, together with
further advantages, will be best understood by reference to the
following detailed description taken in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] A more complete appreciation of the disclosure and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0028] FIG. 1 shows examples of nanoparticles according to the
present disclosure. (A) Luminescent organic nanoparticles. Emitters
could be singlet or triplet emitters, or a combination thereof. The
circles shown in different shades of grey illustrate different
types of emitters as also shown in the text. The different shades
of grey mean that the emitters can have variable
absorption/emission wavelengths as described in the
embodiments/claims. (B) Luminescent organic nanoparticles for
photon upconversion. An emitter and a sensitizer are included. The
darkest grey circles are the sensitizer molecules. Also these
sensitizers can have variable absorption wavelengths as described
in the embodiments/claims. Again, for the emitters which are shown
in differently shaded lighter grey circles, the different shades of
grey mean that the emitters can have variable absorption/emission
wavelengths as described in the embodiments/claims. Both in A and B
in the center of the nanoparticle, there is the enhancing plasmonic
nanoparticle; additionally, the matrix can contain an
antioxidant.
[0029] FIG. 2 shows a characterization of nanoparticles (NPs) as
prepared in Example 1. (A) Z-average diameter. (B) Absorption. (C)
Emission when excited with 405 nm. (D) Emission when excited with
488 nm. In each case, the zeta potential was -55 mV The chosen
batches of NPs (81) illustrate 4 different variations; in two
batches, the size of the Ag nanoparticles was varied (batches 81.2
and 81.3), and in two batches, the concentration of the antioxidant
lipoic acid used was varied (batches 81.1 and 81.4) The
concentration of lipoic acid is higher in batch 81.4, and the same
lipoic acid concentration is used in batches 81.2 and 81.3.
[0030] FIG. 3 shows the effect of plasmon enhancement by
incorporation of 4 nm silver nanoparticles in the photon
up-conversion nanoparticle matrix of nanoparticles (NP), with the
chromophore in (A) being
3,10-Bis((4-tert-butylphenyl)ethynyl)perylene. These are also the
same NPs as shown in FIG. 2 as batch 81.4 (without incorporation of
4 nm Ag NPs) and batch 81.2 (with incorporation of 4 nm Ag NPs). In
(B) the standard chromophore coumarin was used as light emitter. As
can be seen, the emission decay time of the Coumarin 153 is
shortened due to the Ag NP plasmon. The fluorescence signal (not
shown) was correspondingly also enhanced by >2x.
[0031] FIG. 4 shows examples for light emitters suitable for use in
nanoparticles according to the present disclosure.
[0032] FIG. 5 shows examples of emission spectra and maximum
wavelengths in nanoparticles with different light
emitter/sensitizer/metal nanoparticle combinations. The graphs on
the left side are the spectra as measured by standard fluorometer
(with a big spot of ca few mm.sup.2 area, low excitation
intensity). The graphs on the right side are the spectra as
measured in flow cytometry relevant (FCM) conditions (i.e. a small
spot, ca. 130 .mu.m diameter, 30 mW or 200/W/cm2 excitation
intensity).
[0033] FIG. 6 shows confocal microscopy data (A) and spectroscopic
data (B-D) obtained with nanoparticles according to the present
disclosure conjugated to microparticles as cell models. For details
on the conjugation reaction of the nanoparticles to microparticles,
see also FIGS. 16 and 17. For (B) Absorbance: 488 nm, Emission: 514
nm and 555 nm, HWHM (half-width at half-maximum) at 514 nm: 15 nm,
HWHM at 555 nm: 18 nm; (C) Absorbance: 488 nm, Emission: 535 nm,
HWHM: 14 nm; (D) Absorbance: 488 nm, Emission: 590 nm, HWHM: 21 nm;
Reference FITC HWHM: 30 nm.
[0034] FIG. 7 shows a schematic diagram of the band filter cube(s)
for photon up conversion imaging and microscopic images obtained by
live cell imaging of HepG2 cells after uptake of nanoparticles
according to the present disclosure. (A) shows band filter cubes
used for photon up-conversion imaging: Excitation with band filter
centered at 640 nm with 14 nm transmission band. The upconversion
emission is detected through band filter center at 520 nm with 84
nm transmission band. (B) Standard fluorescence mode with
excitation at 405 nm. (C) Standard fluorescence mode with
excitation at 488 nm. (D) Unique up-conversion imaging with
excitation at ca. 630 nm and emission at 450-520 nm. No
autofluorescence.
[0035] FIG. 8 shows: [0036] (A) Jablonski Diagram of the
up-conversion mechanism in presence and absence of molecular
oxygen. The sensitizer molecule is excited by absorption a photon
with h.nu.1. Via intersystem crossing, the triplet state of the
sensitizer is predominantly occupied within ns. In the absence of
oxygen, triplet-triplet energy transfer takes place between the
sensitizer and the emitter molecule. Two emitters in triplet state
can now undergo triplet-triplet annihilation leading to one emitter
back in ground state and one emitter with an occupied excited
singlet state, which can emit anti-Stokes shifted light with energy
h.nu..sub.2. In the presence of oxygen, the energy is transferred
to molecular oxygen in triplet state producing singlet oxygen.
[0037] (B) Schematic drawing of a PUC (photon upconversion) NP
(nanoparticle) composition. [0038] (C) Characterization of PUC
(photon upconversion) NPs (nanoparticles) by QY (quantum yield): QY
of PUC in absence of molecular oxygen (anaerobic conditions) as a
function of illumination intensity. The QY remains constant for
intensity variation between 1 and 10 W/cm2. [0039] (D) The
intensity dependence of the PUC emission integral amount for
different excitation intensities (anaerobic conditions). In the
range 0.4 to 10 W/cm2 excitation a linear dependence is
confirmed.
[0040] FIG. 9 shows: emission spectra of PUC-NP in aqueous
dispersions at different serotonin and dopamine concentrations
under 633 nm. [0041] (A) and (B) Emission spectra of PUC-NPs at
dopamine concentrations ranging from 1 mM to 20 mM (A) or at
serotonin concentrations ranging from 0.1 mM to 1 mM (B),
respectively. [0042] (C) PUC emission and phosphorescence as a
function of the dopamine (C) or serotonin (D) concentration.
DA=dopamine; ST=serotonin
[0043] FIG. 10 shows: An embodiment of a sensor including an
emissive layer (also sometimes referred to as "sensing layer" or
"emissive sensing layer"=ESL) from organic nanoparticles with metal
nanoparticles and antioxidant inside, an attachment (link) layer
for the cell adhesion layer, a cell adhesion layer (in this case
collagen). The same sensors have been used further in the examples.
PAA=polyacrylamide; PUC NPs=photon upconversion nanoparticles; Ag
NPs=silver nanoparticles; GDA=glutardialdehyde;
[0044] FIG. 11 shows: [0045] (A) normalized PUC intensity in
dependence of the illumination time with non-coherent light at 638
nm with an intensity of 2.3 W/cm.sup.2 at different dopamine
concentrations. [0046] (B) normalized PUC intensity as a function
of the dopamine concentration at different illumination times.
[0047] (C) Images of PUC emissive sensing layer at different
concentrations of dopamine after 7.2 s of illumination with
non-coherent light at 638 nm with an intensity of 2.3 W/cm2. All
images were taken at the same area. [0048] (D) normalized PUC
intensity in dependence of the illumination time with non-coherent
light at 638 nm with an intensity of 2.3 W/cm.sup.2 at different
serotonin concentrations. [0049] (E) normalized PUC intensity as a
function of the ST concentration at different illumination times.
[0050] (F) Images of PUC emissive sensing layer at different
concentrations of serotonin after 2.4 s of illumination with
non-coherent light at 638 nm with an intensity of 2.3 W/cm.sup.2.
All images were taken at the same area.
[0051] FIG. 12 shows: A comparison between sensing capabilities of
emissive sensing layer (ESL) between ascorbic acid and dopamine at
concentrations from 0.0 mM to 0.5 mM. Brightness of images was
increased by 40%.
[0052] FIG. 13 shows: [0053] (A) Visualization of local dopamine
release from a micropipette positioned close to the layer surface
using ESL under illumination with non-coherent light at 638 nm with
an intensity of 2.3 W/cm.sup.2. Dopamine was released from the
pipette shortly before t.sub.5 10 s after start of the respective
time series and then every 10 s. At t.sub.12=23.6 s the maximum of
PUC signal after the second release shortly before t.sub.11=20s.
Release pressures were varied between 1000 hPa and 31 hPa. Images
are represented at a gamma value of 0.45 (scale bar: 20 .mu.m)
[0054] (B) normalized PUC intensity (n=3, mean.+-.std) as a
function of time after dopamine release from a micropipette.
Dopamine solution droplet was first released at 10 s by the
micropipette and then every 10 s. [0055] (C) maximum normalized PUC
intensity after first and second release of 2 mM dopamine droplet
from a micropipette (mean.+-.std) as a function of the release
pressure. Data were fitted linearly without weights (dotted
line).
[0056] FIG. 14 shows data obtained with a sensor according to the
present disclosure for a polymer matrix. [0057] (A) Control image
of sensor in the presence of complete growth medium without
neurotransmitter (sometimes abbreviated herein also as "NT") imaged
in photon up-conversion mode. The medium is on top of the
sensor/sensing layer. The control image is completely black. [0058]
(B)+(C) Sensor in presence of complete growth medium including 0.5
mM (B) and 1 mM (C) of the neurotransmitter (NT) dopamine, imaged
in photon up-conversion mode. The images obtained are clearly
brighter than the control image.
[0059] The images shown in (A), (B) and (C) are photon
up-conversion (PUC) images taken with an excitation of 640 nm (band
filter centered at 640 nm with 14 nm transmission band). The
up-conversion emission is detected through a band filter centered
at 520 nm with 84 nm transmission band (for the microscope cube see
FIG. 7A). Standard imaging duration was 2 s. After each PUC image,
a fluorescence image with 488 nm excitation for 100 ms (control or
image for normalization) was taken.
[0060] FIGS. 14 (B) and (C) shows data obtained with a sensor
according to the present disclosure adapted to the detection of
dopamine in an experiment (C) in the presence of 0.5 mM dopamine
(B), and in the presence of 1 mM dopamine (C). The medium is on top
of the sensor/sensing layer. Then, 0.5 mM and 1 mM (final
concentration) of the neurotransmitter dopamine was pipetted on the
layer into the complete growth medium. All images are Photon
up-conversion (PUC) images (excitation 638 nm). Standard imaging
duration was 2 s. After each PUC image, a fluorescence image with
488 nm excitation for 100 ms (control or image for normalization)
was taken. The control image obtained in the absence of dopamine is
completely black (data shown in 14A), the images obtained in the
presence of 0.5 mM dopamine are clearly brighter than the control
image obtained in the absence of dopamine, whereas the images
obtained in the presence of 1 mM dopamine are again much brighter
than the image obtained in the presence of 0.5 mM dopamine. [0061]
(D) shows the normalization of a photon up-conversion image (633
nm) to a fluorescence image (488 nm) leading to improvement of
sensing quality for both, 0.5 mM and 1 mM dopamine. The normalized
images confirmed a resolution higher than 10 .mu.m density. The
normalized image obtained in the presence of 1 mM dopamine had the
expected increase in brightness compared to the normalized image
obtained in the presence of 0.5 mM dopamine. [0062] (E) shows the
successful imaging of dopamine at a concentration as low as 0.1 mM
with a sensor according to the present disclosure (same procedure
as described above). The image obtained in the presence of 0.1 mM
dopamine is clearly brighter than the image obtained in the absence
of dopamine.
[0063] FIG. 15 shows data obtained from an experiment to examine
layer stability of sensing layers as included in the sensor
according to the present disclosure. For the figures shown in A and
B the ESL as shown in C with NPs with higher sensitizer
concentration were used. FIG. 15A: Left: Photon upconversion signal
of an ESL directly after preparation. The dark image is taken with
HBSS without dopamine (DA) and below it is the image with 0.1 mM
dopamine (DA) in the HBSS incubated with 0.0 mM dopamine (DA) and
0.1 mM (DA) in HBSS. Right: corresponding fluorescence imaged at
488 nm illumination. FIG. 15B: Left: Photon upconversion signal of
an ESL after 15 days at 37.degree. C. and 5% CO.sub.2 with CDI
iCell DopaNeuron cells growing on the layers. The dark image is
HBSS without dopamine (DA) and below it with 0.1 mM dopamine (DA)
in the HBSS. Right: corresponding fluorescence imaged at 488 nm
illumination. FIG. 15C: Changes of the normalized PUC signal (under
nitrogen 95% and CO2 5% atmosphere at 37.degree. C.) over 24 h. The
PUC NPs batches 105.1 and 105:3 contain different amount of
sensitizer PdTBP.
[0064] FIG. 16 shows the mechanism of crosslinking of carboxylic
acid groups to a primary amine, forming an amide bond, by using the
crosslinking agents 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
Hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This reaction
can be used for the attachment of the NPs when they have COOH on
their surface to any NH2 (amino) group containing surface or
molecule. The same reaction can be used for the formation of
cross-linked gels of e.g. collagen with NPs (chemically crosslinked
gels).
[0065] FIG. 17 shows components of the (TTA-UC)-NP (triplet-triplet
annihilation supported upconversion). (A) Glycolic acid ethoxylate
4-nonylphenyl ether (CoPEG) can be used as surface stabilizing
agent at the surface. As component of the matrix (the inner side of
the NP) phenylheptadecane, Polymethylmetacrylate (PMMA) as polymer
and Ascorbic acid palmitate as singlet oxygen scavenger may be
used. (B) Also in the inner part of the nanoparticle, PdTBP may be
used as sensitizer, DPhP-C.sub.4 as emitter, and 4 nm Ag particles
can be used as plasmonic nanoparticles. (C) Illustrates the process
of attachment of the organic nanoparticles on the surface of an
object (e.g. protein, a microparticle, a cell or any other surface
with present suitable functional groups, e.g. aminogroups). This
shows the possibility of labelling using the organic NPs in
accordance with the present disclosure. In FIG. 6 the labeled
microparticles (size 4.5 m) are shown in fluorescence confocal
microscopy images.
[0066] FIG. 18 shows the characterization of the crosslinked
nanoparticle-microparticle conjugate purified by filtration.
Confocal microscopy pictures. (a) area 70 .mu.m.times.70 .mu.m; (b)
area 20 .mu.m.times.20 .mu.m; (c) area 10 .mu.m.times.10 .mu.m; (d)
area 10 .mu.m.times.10 .mu.m.
[0067] FIG. 19 shows an example of a nanoparticle fabrication
chamber with valves.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0068] The following embodiments can, wherever this does not lead
to logical contradictions, be combined with each other without
restrictions. Thus, the present disclosure shall encompass, even
where not explicitly spelled out in the following, any feasible
combination of the embodiments described below.
[0069] As discussed above, the present disclosure provides a
nanoparticle (NP) including [0070] a polymeric organic matrix,
[0071] at least one light emitter distributed within said matrix,
[0072] a surface stabilizing agent, and [0073] at least one metal
nanoparticle enclosed within said matrix, [0074] an antioxidant, in
particular a singlet oxygen scavenger, a reactive oxygen scavenger
(ROS) scavenger or a radical scavenger. It has been found that said
at least one metal nanoparticle is capable of enhancing the
intensity of the light emitted by said light emitter(s) by way of
plasmon enhancement. It has also been found that the shape and the
reactivity of the NPs when used in crosslinking/attachment to
aminogroup containing surfaces or molecules is stabilized. It has
furthermore been found that the presence of said metal nanoparticle
in said nanoparticle (NP) enhances the photostability of such
NP.
[0075] In one embodiment, said polymeric organic matrix is a solid
matrix. As the skilled person will appreciate, said polymeric
organic matrix is chemically inert with respect to the other
components of the nanoparticle or the surroundings/milieu in which
said nanoparticle is used. As the skilled person will appreciate,
the polymeric organic matrix is optically inert (i.e. it does not
affect the light used to excite the light emitter(s) or the light
that is emitted by the light emitter(s)). In one embodiment, said
polymeric organic matrix is transparent in the range of from 300 to
1600 nm.
[0076] In one embodiment, said polymeric organic matrix is composed
of a material selected from the group consisting of
polyacrylonitriles, polystyrenes, styrene copolymers,
styrene-butadiene copolymers, polystyrene-based elastomers,
polyethylenes and oligoethylenes, polypropylenes,
polytetrafluoroethylenes, extended polytetrafluoroethylenes,
polyacrylates, polymethylmethacrylates, ethylene-co-vinyl acetates,
polysiloxanes, such as polymethylsiloxanes and
polyphenylmethylsiloxanes, e.g. polydimethylsiloxane or
polyphenylmethylsiloxane, their copolymers as well as substituted
and modified polysiloxanes, polyethers, polyurethanes,
polyether-urethanes, polyethylene terephthalates and polysulphones,
particularly a material selected from the group consisting of
polystyrenes, polyacrylonitriles and polymethylmethacrylates, more
particularly polymethylmethacrylate.
[0077] In one embodiment, said nanoparticle (NP) has a diameter in
the range of from 10 to 1000 nm, particularly in the range of from
10 to 500 nm, more particularly in the range of from 10 to 200 nm,
even more particularly in the range of from 10 to 100 nm.
[0078] In one embodiment, said at least one light emitter is
distributed homogeneously within said polymeric organic matrix.
[0079] In one embodiment, said at least one light emitter is an
organic molecule. In one embodiment, said at least one light
emitter consists of one molecule (i.e. only one molecular species).
In one embodiment, said at least one light emitter consists of a
combination of more than one, in particular two, molecules.
[0080] In one embodiment, the light emitted by said at least one
light emitter has a wavelength in the range of from 360 to 800 nm,
particularly in the range of from 400 to 700 nm.
[0081] In one embodiment, said at least one light emitter is
capable of emitting light by luminescence, in particular by
fluorescence or by phosphorescence, more particularly by
fluorescence. In particular said fluorescence is with high quantum
yield when the emitter is in the nanoparticle. In one embodiment,
said at least one light emitter is a fluorescent or phosphorescent
emitter, in particular a fluorescent emitter. In nanoparticles that
include both light emitter(s) and a sensitizer(s), the sensitizer
molecule(s) are phosphorescent emitters. can have highly populated
excited triplet state (via strong intersystem crossing
singlet-triplet). This triplet state can be emissive
(phosphorescence) or not. This depends on the molecular
structure.
[0082] In one embodiment, said at least one light emitter has a
high quantum yield (in the NP matrix) of fluorescence when the
light emitter is present within the nanoparticle. In one
embodiment, said at least one light emitter is present in said
nanoparticle at a high concentration. A relatively high emitter
concentration in the confined nanoparticle has the advantage that a
high photon outcome with high quantum yield is achieved. This is
achieved via optimization of the molecular structure of the emitter
and/or via adjustment of the matrix. As used herein, a "high
quantum yield" refers to a quantum yield >60%.
[0083] In one embodiment, said at least one light emitter is a
fluorescence chromophore (in particular having an emissive singlet
state). In one embodiment, said at least one light emitter is a
phosphorescent chromophore. In one embodiment, said at least one
light emitter has a highly populated excited triplet state.
[0084] In one embodiment, the light emitted by said at least one
light emitter has a wavelength in the range of from 360 to 850 nm,
particularly in the range of from 420 to 700 nm. In one embodiment
the excitation wavelength of said at least one light emitter is in
the range of from 350 to 840 nm, particularly in the range of from
390 to 810 nm.
[0085] In one embodiment, said nanoparticle further includes at
least one sensitizer. As the skilled person will appreciate, the
presence of a sensitizer is obligatory for photon up-conversion
(PUC) nanoparticles.
[0086] In one embodiment, said at least one sensitizer and said at
least one light emitter are separate entities (i.e. the at least
one sensitizer and the at least one light emitter are not
covalently linked to each other and do not form part of the same
molecule).
[0087] In one embodiment, said at least one sensitizer is
distributed, in particular homogeneously, within said polymeric
organic matrix.
[0088] In one embodiment, said at least one sensitizer is an
organic molecule. In one embodiment, said at least one sensitizer
consists of one molecule (i.e. only one molecular species). In one
embodiment, said at least one sensitizer consists of a combination
of more than one, particularly two, molecules.
[0089] In one embodiment, the light emitted by said at least one
light emitter has a wavelength in the range of from 360 to 750 nm,
particularly in the range of from 420 to 640 nm.
[0090] In one embodiment, said at least one sensitizer absorbs
light at a wavelength in the range of from 450 to 1600 nm, in
particular in the range of from 530 to 860 nm, more particularly in
the range of from 620 to 750 nm.
[0091] In one embodiment, said at least one sensitizer is capable
of absorbing light at a first wavelength region
w.ltoreq..lamda..sub.1.ltoreq.x, and said at least one light
emitter is capable of emitting light at a second wavelength region
y.ltoreq..lamda..sub.2.ltoreq.z, wherein
.lamda..sub.2.ltoreq..lamda..sub.1, wherein, upon absorption of
light by said at least one sensitizer at said first wavelength
region w.ltoreq..lamda..sub.1.ltoreq.x, said at least one light
emitter emits light at said second wavelength region
y.ltoreq..lamda..sub.2.ltoreq.z.
[0092] In one embodiment, said light emitted by said light emitter
at said second wavelength region .lamda..sub.2 is due to an
up-conversion process based on triplet-triplet transfer (sensitizer
triplet to emitter triplet) followed by triplet-triplet
annihilation of the emitter triplets. Particularly, the
up-conversion process occurs upon absorption of light by said
sensitizer at said first wavelength region .lamda..sub.1, or said
light emitted by said light emitter at said second wavelength
region .lamda..sub.2 is due to an up-conversion process based on a
triplet-triplet transfer process between photoexcited molecules (in
triplet state) of said sensitizer molecules to the triplet state of
the emitter molecules followed by triplet-triplet annihilation
process between photo-excited molecules of said emitter.
[0093] In a preferred embodiment, said second wavelength region
.lamda..sub.2 is in the range 360-750 nm and said first wavelength
region .lamda..sub.1 is in the range 450-1600 nm.
[0094] In one embodiment said light emitter is an organic dye
molecule.
[0095] In one embodiment, said sensitizer is an organic dye or
molecule having a populated triplet or mixed triplet-singlet state,
a two-photon-absorbing (TPA)-dye, an optical limiting compound,
another molecule with a populated triplet state or an optical
limiting compound--e.g. a fullerene, or carbon nanotubes.
[0096] In one embodiment, said nanoparticle is capable of photon
up-conversion.
In one embodiment, said light emitter in combination with said
sensitizer is capable of, upon irradiation with light of
appropriate wavelength, photon up-conversion emission (i.e.
emission of light generated by photon up-conversion).
[0097] In one embodiment, said light emitter is capable of emitting
light by luminescence, particularly by fluorescence. Particularly
the fluorescence is with high quantum yield and/or high photon
outcome when the emitter is in the nanoparticle.
[0098] In one embodiment, said at least one light emitter is a
fluorescent chromophore.
[0099] In one embodiment, said at least one light emitter in
combination with said at least one sensitizer is capable of, upon
irradiation with light of appropriate wavelength (the excitation
wavelength for photon up-conversion), photon up-conversion emission
(i.e. emission of light generated by photon up-conversion).
[0100] In one embodiment, the energy levels of the triplet state of
said at least one light emitter and of the triplet state of said at
least one sensitizer are such that they allow for efficient
triplet-triplet excitation transfer from the light sensitizer to
the light emitter.
[0101] In one embodiment, said at least one light emitter is a
molecule selected from the group consisting of anthracenes,
perylenes, perylene derivatives such as perylene monoimides or
perylene diimides, coumarins and BODIPY dyes. In particular, said
at least one light emitter is a perylene, a substituted perylene or
a perylene derivative, such as perylene monoimide or perylene
diimide. A "substituted perylene" or a "perylene derivative" as
used herein, refers to a structure having a perylene core. A
"perylene derivative" may be a perylene that is substituted with
appropriate substituents.
[0102] In one embodiment, said at least one light emitter has the
structure represented by Formula (I) or (II) or includes a molecule
having the structure represented by Formula (I) or (II):
##STR00001##
wherein R.sub.1 and R.sub.2 are independently selected from the
group consisting of hydrogen and a moiety with the structure
represented by Formula (III), wherein at least one of R.sub.1 and
R.sub.2 is a moiety with the structure represented by Formula
(III):
##STR00002##
wherein R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are
independently selected from the group consisting of H, F, and
tri-fluoro-methyl (--CF.sub.3), wherein at least one of R.sub.3,
R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is F or tri-fluoro-methyl
(--CF.sub.3).
[0103] In one embodiment, R.sub.1 and/or R.sub.2 is the moiety
represented by Formula (IV):
##STR00003##
[0104] In one embodiment, R.sub.1 and/or R.sub.2 is the moiety
represented by Formula (V):
##STR00004##
[0105] In one embodiment, R.sub.1 and/or R.sub.2 is the moiety
represented by Formula (VI):
##STR00005##
[0106] In one embodiment, said at least one light emitter has the
structure represented by Formula (VII) or includes a molecule
having the structure represented by Formula (VII):
##STR00006##
[0107] In another embodiment, said at least one light emitter has
the structure represented by Formula (VIII) or includes a molecule
having the structure represented by Formula (VIII):
##STR00007##
[0108] In another embodiment, said at least one light emitter has
the structure represented by Formula (IX) or includes a molecule
having the structure represented by Formula (IX):
##STR00008##
[0109] In one embodiment, said at least one light emitter has the
structure represented by Formula (X) or includes a molecule having
the structure represented by Formula (X):
##STR00009##
wherein R.sub.8 an R.sub.9 independently selected from the group
consisting of hydrogen and a moiety with the structure represented
by Formula (XI), wherein at least one of R.sub.8 and R.sub.9 is a
moiety with the structure represented by Formula (XI):
##STR00010##
wherein R.sub.10, R.sub.11, R.sub.12, R.sub.13 and R.sub.14 are
independently selected from the group consisting of H, F, and
tri-fluoro-methyl (--CF.sub.3), wherein at least one of R.sub.10,
R.sub.11, R.sub.12, R.sub.13 and R.sub.14 is F or tri-fluoro-methyl
(--CF.sub.3).
[0110] In one embodiment, R.sub.8 and/or R.sub.9 is the moiety
represented by Formula (XII):
##STR00011##
[0111] In one embodiment, R.sub.8 and/or R.sub.9 is the moiety
represented by Formula (XIII):
##STR00012##
[0112] In one embodiment, R.sub.8 and/or R.sub.9 is the moiety
represented by Formula (XIV):
##STR00013##
[0113] In one embodiment, R.sub.8 and/or R.sub.9 is the moiety
represented by Formula (XV):
##STR00014##
[0114] In one embodiment, R.sub.8 and/or R.sub.9 is the moiety
represented by Formula (XVI):
##STR00015##
[0115] In one embodiment, said at least one light emitter has the
structure represented by Formula (XVII) or includes a molecule
having the structure represented by Formula (XVII):
##STR00016##
[0116] In another embodiment, said at least one light emitter has
the structure represented by Formula (XVIII) or includes a molecule
having the structure represented by Formula (XVIII):
##STR00017##
[0117] In another embodiment, said at least one light emitter has
the structure represented by Formula (XIX) or includes a molecule
having the structure represented by Formula (XIX):
##STR00018##
[0118] In another embodiment, said at least one light emitter has
the structure represented by Formula (XX) or includes a molecule
having the structure represented by Formula (XX):
##STR00019##
[0119] In preferred embodiment, said at least one light emitter has
the structure represented by Formula (XXI) or includes a molecule
having the structure represented by Formula (XXI):
##STR00020##
[0120] In another embodiment, said at least one light emitter has
the structure represented by Formula (XXII) or includes a molecule
having the structure represented by Formula (XXII):
##STR00021##
[0121] In one embodiment, said at least one light emitter has a
structure represented by one of the following structures or
includes a molecule having a structure represented by one of the
following structures:
##STR00022##
[0122] In one embodiment, said at least one light emitter has the
structure represented by Formula (XXIII), (XXIV) or (XXV) or
includes a molecule having the structure represented by Formula
(XXIII), (XXIV) or (XXV):
##STR00023##
wherein W in formulae XXIII-XXV is selected from one of the
following groups:
##STR00024##
wherein Y, as used in the formulae of W, is selected from the group
consisting of CH.sub.2, S, O, Se and N--R.sub.2, and wherein
R.sub.2 is selected from the group consisting of H, a linear alkyl
group, a branched alkyl group, a cycloalkyl group, a halogenated
alkyl group, a halogen atom, an alkyl sulfanyl group, an aryl
sulfanyl group, an amino alkyl group, an amino aryl group, an aryl
group, a halogenated alkyl group, a heteroaryl group, a fluorenyl
group, an amino group, a nitro group, an OH group, an SH group, and
a group --O--R.sub.3, [0123] wherein R.sub.3 is selected from the
group consisting of a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, an alkyl sulfanyl
group, an aryl sulfanyl group, an amino alkyl group, an amino aryl
group, an aryl group, a halogenated alkyl group, a heteroaryl group
and a fluorenyl group, wherein, in particular, R.sub.2 has not more
than 6 carbon atoms, wherein X and Y in formulae XXIII-XXV are
independently selected from the following
##STR00025## ##STR00026##
[0123] groups: and wherein R is selected from the group consisting
of H, a linear alkyl group, a branched alkyl group, a cycloalkyl
group, a halogenated alkyl group, a halogen atom, an alkyl sulfanyl
group, an aryl sulfanyl group, an amino alkyl group, an amino aryl
group, an aryl group, a halogenated alkyl group, a heteroaryl
group, a fluorenyl group, an amino group, a nitro group, an OH
group, an SH group, and a group --O--R.sub.3, [0124] wherein
R.sub.3 is selected from the group consisting of a linear alkyl
group, a branched alkyl group, a cycloalkyl group, a halogenated
alkyl group, an alkyl sulfanyl group, an aryl sulfanyl group, an
amino alkyl group, an amino aryl group, an aryl group, a
halogenated alkyl group, a heteroaryl group and a fluorenyl group,
wherein, particularly, R has not more than 6 carbon atoms, wherein
Z in formulae XXIII-XXV is selected from the following groups:
##STR00027##
[0124] and wherein R.sub.2 is selected from the group consisting of
H, a linear alkyl group, a branched alkyl group, a cycloalkyl
group, a halogenated alkyl group, a halogen atom, an alkyl sulfanyl
group, an aryl sulfanyl group, an amino alkyl group, an amino aryl
group, an aryl group, a halogenated alkyl group, a heteroaryl
group, a fluorenyl group, an amino group, a nitro group, an OH
group, an SH group, and a group --O--R.sub.3, [0125] wherein
R.sub.3 is selected from the group consisting of a linear alkyl
group, a branched alkyl group, a cycloalkyl group, a halogenated
alkyl group, an alkyl sulfanyl group, an aryl sulfanyl group, an
amino alkyl group, an amino aryl group, an aryl group, a
halogenated alkyl group, a heteroaryl group and a fluorenyl group,
wherein, in particular, R.sub.2 has not more than 6 carbon atoms,
wherein Ri in formulae XXIII-XXV is selected from the following
groups:
##STR00028## ##STR00029##
[0126] In one embodiment, W is selected from the following
groups:
##STR00030## ##STR00031## ##STR00032##
[0127] In one embodiment, said at least one light emitter has a
structure selected from one of the following:
##STR00033##
wherein R is a linear or branched alkyl group, particularly with
not more than 6 carbon atoms.
[0128] In one embodiment, said at least one light emitter has the
structure represented by Formula (XXVI) or includes a molecule
having the structure represented by Formula (XXVI):
##STR00034##
wherein R is selected from the following groups:
##STR00035##
wherein Y is selected from the group consisting of CH.sub.2, S, O,
Se and N--R.sub.2, and wherein R.sub.2 is selected from the group
consisting of H, a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, a halogen atom, an
alkyl sulfanyl group, an aryl sulfanyl group, an amino alkyl group,
an amino aryl group, an aryl group, a halogenated alkyl group, a
heteroaryl group, a fluorenyl group, an OH group, an SH group, and
a group --O--R.sub.3, [0129] wherein R.sub.3 is selected from the
group consisting of a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, an alkyl sulfanyl
group, an aryl sulfanyl group, an amino alkyl group, an amino aryl
group, an aryl group, a halogenated alkyl group, a heteroaryl group
and a fluorenyl group, wherein, particularly, R.sub.2 has not more
than 6 carbon atoms, or wherein R is selected from the following
groups:
##STR00036##
[0129] wherein R.sub.1 is a linear or branched alkyl group,
particularly with not more than 6 carbon atoms.
[0130] In one embodiment, said at least one light emitter has a
structure selected from one of the following or includes a molecule
having a structure selected from one of the following:
##STR00037##
wherein R is a linear or branched alkyl group, particularly with up
to 6 carbon atoms.
[0131] Said at least one sensitizer may be any dye with high
intersystem crossing resulting in highly populated triplet
state,
[0132] In one embodiment, said at least one sensitizer is or
includes a porphyrin, particularly a benzo porphyrin or naphto
porphyrins, or a phthalocyanine.
[0133] In one embodiment, said at least one sensitizer has the
structure represented by Formula (XXVII) or includes a molecule
having the structure represented by Formula (XXVI):
##STR00038##
wherein R.sub.1 is hydrogen, a linear or branched alkyl group, in
particular with up to 6 carbon atoms, or a benzene ring,
[0134] and wherein R is a linear or branched alkyl group, in
particular with up to 6 carbon atoms.
[0135] In one embodiment, said at least one sensitizer has a
structure represented by Formula (XXVIII), Formula (IXXX), Formula
(XXX) or Formula (XXXI) or includes a molecule having a structure
represented by Formula (XXVI), Formula (IXXX), Formula (XXX) or
Formula (XXXI):
##STR00039## ##STR00040##
[0136] In one embodiment, said sensitizer has the structure
represented by Formula (XXVIII):
##STR00041##
[0137] In one embodiment, said nanoparticle (NP) includes 1 to 4
metal nanoparticles (i.e. at least one but not more than 4 metal
nanoparticles).
[0138] In one embodiment, the nanoparticle (NP) includes more than
one metal nanoparticle.
[0139] In one embodiment, said nanoparticle (NP) does not include
metal nanoparticles that are in contact with each other.
[0140] In one embodiment, said metal (plasmonic) nanoparticles are
magnetic.
[0141] In one embodiment, said metal particles are plasmonic in
that they are metal nanoparticles.
[0142] In one embodiment, said metal nanoparticles include or are
composed of cobalt.
[0143] In one embodiment, said at least one metal nanoparticle has
a diameter in the range of from 1 to 100 nm, particularly in the
range of from 10 to 100 nm, more particularly in the range of from
10 to 50 nm.
[0144] As the skilled person will appreciate, for the plasmonic
nanoparticles any metal particles with plasmon may be used, e.g.
plasmonic metal nanoparticles. Typically, such metal nanoparticles
have a plasmon and are herein also referred to as "plasmonic" metal
nanoparticles. Without wishing to be bound by any theory, the
present inventors believe that the electron density of such
plasmonic metal nanoparticles can couple with radiation. i.e.
plasmonic nanoparticles are used as particles whose electron
density can couple with electromagnetic radiation of wavelengths
that are far larger than the particle itself. In such a way
plasmonic nanoparticles are capable of enhancing the emission of
the emitters distributed in the nanoparticles.
[0145] In one embodiment, said metal (plasmonic) nanoparticles have
a diameter in the range of from 1 to 100 nm, particularly in the
range of from 4 to 80 nm, more particularly in the range of from 10
to 60 nm, more particularly in the range of from 10 to 50 nm. In
one embodiment, such metal nanoparticle has an average diameter in
the range of from 1 nm to 50 nm, particularly 1 nm to 30 nm, more
particularly 1 nm to 20 nm. In one embodiment, it has an average
diameter of from 10 nm to 50 nm, particularly 10 nm to 30 nm. In
another embodiment, such metal nanoparticle has an average diameter
of from 1 nm to 40 nm, particularly 5 nm to 30 nm. Typically, such
metal nanoparticle having an average diameter of from 1 nm to 100
nm or an average diameter of any subrange therein is a plasmonic
nanoparticle. If the present application indicates that a diameter,
e.g. of metal nanoparticles, is in a certain range, this means that
the average diameter of said metal nanoparticles falls in that
certain range. In one embodiment, the diameter of individual metal
nanoparticles varies by not more than 30%, particularly by not more
than 20%, more particularly by not more than 10%.
[0146] In one embodiment, said nanoparticle (NP) includes a single
metal nanoparticle. In one embodiment, said single metal
nanoparticle forms the core of said nanoparticle (NP), wherein said
polymeric organic matrix forms a shell around said core.
[0147] The metal particles may be composed of any metal or
materials with plasmonic properties--and in this way are plasmonic
nanoparticles (e.g. nanospheres, nanoshells, nanocubes, nanorods
and nanoplates). In one embodiment, said at least one plasmonic
nanoparticle consists of a material selected from the group
consisting of Ag, Au and Co, Al, Cu, metal alloys/layered
structures like Ag/Au or of nitrides of transition metals (TiN,
ZrN). In one embodiment, said at least one plasmonic (metal)
nanoparticle is composed of Ag.
[0148] In one embodiment, said at least one metal nanoparticle
consists of only one element (i.e. it is composed of a pure
chemical substance consisting of a single type of atom).
[0149] In one embodiment, said at least one metal nanoparticle does
not include or consist of a rare-earth metal, rare-earth metal ions
or compounds/material formed from a rare-earth element.
[0150] In one embodiment, said nanoparticle (NP) does not include
rare-earth metal, rare-earth metal ions or compounds/material
formed from a rare-earth element.
[0151] In one embodiment, said metal nanoparticles are massive
particles (i.e. the interior of each metal nanoparticle is
completely filled by the metal which said metal particle is made
of, and the metal particle does not enclose any other material than
said metal nor does it enclose any void space).
[0152] In one embodiment, the nanoparticle further (NP) includes at
least one antioxidant. Inclusion of an antioxidant
improves/optimizes the chromophore distribution and increases
emission yield and photostability (emission enhancement and
photostability enhancement). In one embodiment, said at least one
antioxidant is an organic antioxidant. In one embodiment, said at
least one antioxidant is soluble in an organic, water-miscible
solvent, more particularly in THF or DHF. In one embodiment In one
embodiment, said at least one antioxidant is distributed
homogeneously within said polymeric organic matrix. In one
embodiment In one embodiment, said at least one antioxidant is
selected from the group consisting of lipoic acid, vitamin E, a
carotenoid and a ascorbic acid ester.
[0153] Different antioxidants--singlet oxygen scavengers or other
reactive oxygen species scavengers (ROS scavengers) can be used.
Generally, any antioxidant, especially singlet oxygen
scavenger/quencher, can be used as far as it is soluble in an
organic phase. The antioxidant does not need to be soluble in water
(or at best only needs to have very limited solubility in water),
but it should be well soluble in THF, DMF or ethanol (or other
water miscible organic solvents which are also used as solvents for
the other NP components). The following exemplary antioxidants can
be used but the application is not limited to these examples:
ascorbic acid palmitate (6-O-Palmitoyl-L-ascorbic acid), ascorbic
acid esters, caffeic acid esters, lipoic acid (all racemic forms),
lauryl gallate and other galic acid esters--octyl, butyl, ethyl
esters; Vitamin E (.alpha.-Tocopherol, .delta.- or
.gamma.-Tocopherol and tocopherol acetates)--all racemic forms;
Tocotrienol--all racemic forms, resveratrol; Pyrocatechol;
3-ethylbenzophenone; Magnolol, carnosol; Vitamin A--retinol
(retinoic acid), retinol palmitate, retinol acetate, retinol
esters, vitamin A aldehyde (retinal), carotene s--e.g.
beta-carotene I and II, carotenal, mixtures of beta-carotenes, also
lycopene; Ubiquinone (Coenyme Q-10), bromadiolon, vitamin K2,
vitamin K3, flavones/flavonols (catehins, etc.), eugenol and
others.
Designed combinations thereof--e.g. ascorbic acid palmitate with
lipoic acid--can also be used.
[0154] In one embodiment, the nanoparticle (NP) includes functional
groups at its surface that allow to covalently couple a molecule to
the nanoparticle. In one embodiment, said functional groups are
selected from the group consisting of --COOH (carboxylate),
--NH.sub.2, --SH (thiol), --NHS, alkynyl, --N.sub.3, aldehyde,
ketone and biotin group, more particularly said functional groups
are --COOH or --NH.sub.2.
[0155] In one embodiment, the nanoparticle (NP) includes molecules
or chemical groups attached to its surface that are capable of
specifically binding to an analyte molecule or that have an
enzymatic activity that allows to detect an analyte molecule. In
one embodiment, said molecules attached to the surface of the
nanoparticle are protein molecules. In one embodiment, said
molecules attached to the surface of the nanoparticle are antibody
molecules. In one embodiment, said analyte molecule is a
biomolecule. In one embodiment, said analyte molecule is selected
from the group consisting of a nucleic acid/(poly-)nucleotide, such
as DNA or RNA, (poly-)peptide/protein, carbohydrate, lipid,
glycoprotein, lipoprotein, viral and/or bacterial antigen, and
pharmaceutical.
[0156] In a situation where said molecules attached to the surface
of the nanoparticle are antibody molecules, the skilled person will
appreciate that a requirement for the detection of an antigen as
analyte is the stable binding (covalent or non-covalent) of the
antibody on the surface of the nanoparticles without losing the
selectivity of the antibody (i.e. a free active site is needed). If
the antigen is attached to the fluorescent label, detection can be
achieved by a competition assay.
[0157] In one embodiment, said nanoparticle (NP) has a modified
surface such that their uptake by cells is increased. In one
embodiment, the surface of said nanoparticles is modified by
attachment of proteins. Such a surface modification that allows for
increased uptake by cells facilitates is advantageous if the
nanoparticles are to be used for cell imaging after uptake or for
flow cytometry after uptake.
[0158] In one embodiment, the surface of said nanoparticles is
modified by attachment of specific antibodies or with functional
moieties. Such a surface modification allows for attachment of the
nanoparticles to the surface of different cells, when the
nanoparticles are used for flow cytometry or fluorescence assisted
cell sorting.
[0159] An example for detection of an analyte upon binding to the
surface of a nanoparticle (NP) according to the present disclosure
may, for example, be a protein like bovine serum albumin (BSA),
which may be detected via luminescence resonance energy transfer
(LRET) in up-conversion regime. The nanoparticle (NP) has
analyte-specific surface modifications like carboxy- or amino
groups. Specific binding of the analyte can be supported also e.g.
by antibody, affibody or aptamers. In case of luminescence
resonance energy transfer (LRET) the size of the analyte is small,
particularly 1-10 nm. The conjugation of an analyte onto the photon
up-conversion nanoparticle surface allows a LRET in presence of a
fluorescent label. In the absence of the analyte, no photon
up-conversion signal is observed.
[0160] The nanoparticles according to the disclosure may detect the
presence of analytes by effects on the photon up-conversion of the
particles upon binding of the analyte to the surface of the
nanoparticles.
[0161] As discussed above, the present disclosure also provides a
sensing layer including nanoparticles according to the present
disclosure as defined in any of the embodiments above.
[0162] The nanoparticles (NP) according to the present disclosure
may be included in a layer prepared from a biodegradable material
that allows to detect the presence of analyte molecules, i.e. in a
sensing layer. Such sensing layer or "emissive layer" or "emissive
sensing layer" (ESL) may form part of a sensor. A "sensor", as used
herein, refers to an arrangement of an "emissive sensing layer" on
a substrate. Such sensor may include one or several additional
layers, as necessary, adding functionality e.g. selected from cell
adhesion layers, coating layers, enhancement layers for enhancing
the intensity of the light emitted from the emissive layer,
attachment layers etc.
[0163] The polymeric organic matrix of said nanoparticles may be
prepared from a biodegradable material. In one embodiment, said
biodegradable material is a material that is degraded by cells
growing on said layer or nanoparticles or in proximity to said
layer or nanoparticles.
[0164] By detecting the disappearance over time of the
fluorescence/phosphorescence/up-conversion emission of the
layer/nanoparticles, the presence of such cells can be
detected.
[0165] As discussed above, the present disclosure also provides a
method of producing nanoparticles (NP) as defined in any of the
embodiments above, said nanoparticles (NP) including [0166] a
polymeric organic matrix, [0167] at least one light emitter
distributed within said matrix, [0168] optionally at least one
sensitizer and/or at least one antioxidant distributed within said
matrix, [0169] a stabilizing agent, and [0170] at least one
plasmonic metal nanoparticle enclosed within said matrix, wherein
said at least one metal nanoparticle is capable of enhancing the
intensity of the light emitted by said light emitter(s) by way of
plasmon enhancement, said method including the steps of: [0171]
providing a polymer or combination of polymers or combination of
polymers with small molecules from which the polymeric organic
matrix is to be formed, a stabilizing agent, at least one light
emitter, plasmonic metal nanoparticles, and optionally at least one
sensitizer and/or at least one antioxidant, [0172] preparing a
dispersion of said plasmonic metal nanoparticles in an organic
water-miscible solvent, particularly upon ligand exchange of said
plasmonic metal nanoparticles, [0173] preparing a mixture of said
polymer or combination of polymers or combination of polymers with
small molecules from which the polymeric organic matrix is to be
formed, said stabilizing agent, said light emitter(s) and
optionally said sensitizer(s) and/or said antioxidant in an organic
water-miscible solvent, [0174] adding said mixture of said polymer
or combination of polymers or combination of polymers with small
molecules, said stabilizing agent, said light emitter(s) and
optionally said sensitizer(s) and/or said antioxidant(s) to said
dispersion of metal nanoparticles or vice versa, thus forming a
mixture including said metal nanoparticles, [0175] inducing said
mixture including said plasmonic metal nanoparticles to form
nanoparticles (NP), thus forming a dispersion of nanoparticles (NP)
wherein said nanoparticles (NP) include a polymeric organic matrix
with said light emitter(s) and, optionally, said sensitizer(s)
and/or said antioxidant(s), distributed therein, and wherein said
metal nanoparticles are enclosed in said polymeric organic
matrix.
[0176] In this method of producing nanoparticles (NP), said
nanoparticles (NP), said polymeric organic matrix, said at least
one light emitter, said at least one sensitizer, said at least one
antioxidant, said metal nanoparticles and said stabilizing agent
are as defined in any of the embodiments above.
[0177] Said mixture including said plasmonic metal nanoparticles
may be induced to form nanoparticles (NP) by adding cold water.
Upon formation of said dispersion of nanoparticles (NP), said
organic, water-miscible solvent(s) may be removed again from said
dispersion of nanoparticles by evaporation from the dispersion with
a rotary evaporator at low pressure, e.g. subatmospheric pressure,
and elevated temperature.
[0178] Subsequently, said dispersion may be subjected to
centrifugation using a filter with a molecular weight exclusion
limit of <7 000 Da (such that the nanoparticles (NP) are
recovered, whereas water and components with a molecular weight
below the exclusion limit of the filter (e.g. not used surfactant
molecules, some emitter aggregates or matrix components not
incorporated in the nanoparticles) are removed).
[0179] Upon obtaining said nanoparticles (NP) from the
centrifugation step, said nanoparticles can be re-distributed in
pure distilled water. Optionally, the nanoparticles may be purified
by dialysis or with HiTrap filters (desalting).
[0180] In one embodiment, in the step of providing said polymer or
combination of polymers or combination of polymers with small
molecules from which the polymeric organic matrix is to be formed,
said stabilizing agent, said at least one light emitter, said
plasmonic metal nanoparticles, and optionally said at least one
sensitizer and/or said at least one antioxidant, all these
components are provided dissolved/suspended in an organic,
water-miscible solvent, wherein, more particularly, said organic,
water-miscible solvent is THF or DMF.
[0181] In one embodiment, prior to inducing said mixture including
said metal (plasmonic) nanoparticles to form nanoparticles (NP)
said mixture including said plasmonic metal nanoparticles is
brought to a temperature of 0.degree. C.
[0182] In one embodiment, said step of inducing said mixture
including said plasmonic metal nanoparticles to form nanoparticles
(NP) is achieved by adding water, particularly cold water, more
particularly water having a temperature of 4.degree. C.
[0183] In one embodiment, upon formation of said dispersion of
nanoparticles (NP), said organic, water-miscible solvent(s) is
removed again from said dispersion of nanoparticles (NP), e.g. by
evaporation.
[0184] As discussed above, the present disclosure provides the use
of nanoparticles (NP) as defined in any of the embodiments
described above in a biological application selected from the group
consisting of labeling and/or detection of cells, biological
(macro-)molecules or other analytes, fluorescence microscopy,
(flow) cytometry, fluorescence-activated cell sorting (FACS),
fluorescence resonance energy transfer (FRET),
immunohistochemistry, clinical immunoassays,
fluorescence-quenching-based enzyme-activity assays,
high-throughput screening, molecular diagnostics, sensing of
temperature, sensing of pressure and sensing of oxygen.
[0185] In this use, said nanoparticles (NP) are as defined in any
of the embodiments above.
[0186] The nanoparticles (NP) according to the present disclosure
have various advantages that allow to use them in diverse
applications. Thus, nanoparticles according to the present
disclosure show for example fluorescent and/or up-conversion
emisson with increased brightness, increased emission stability and
increased signal-to-noise ratio compared to known nanoparticles in
the art. Such advantages are achieved, to a differing extent, by
the features of the different embodiments described above as well
as by combinations thereof. The light emitters according to the
present disclosure show a higher brightness/higher fluorescence
quantum yield and a higher stability compared to known light
emitters in the art. The inclusion of a radical scavenger, such as
an antioxidant into the nanoparticles has the further advantage
that protection of all chromophores (emitter(s) and sensitizer(s)
(when used)) against reactive radicals and reactive oxygen species
is provided. Additionally, the radical scavenger, e.g. the
antioxidant hinders the transfer of any excited triplet state to
oxygen molecules, which stops the singlet oxygen formation as
well.
[0187] Different antioxidants--singlet oxygen scavengers or other
reactive oxygen species scavengers (ROS scavengers) can be used.
Generally, any antioxidant, especially singlet oxygen
scavenger/quencher, can be used as far as soluble in the organic
phase. They need to be not soluble, or have very limited
solubility, in water but be well soluble in THF, DMF or ethanol (or
other water miscible organic solvents which are also solvents for
the other NP components). The following antioxidants can be used
but the application is not limited to these examples: ascorbic acid
palmitate (6-O-Palmitoyl-L-ascorbic acid), ascorbic acid esters,
caffeic acid esters, lipoic acid (all racemic forms), lauryl
gallate and other galic acid esters--octyl, butyl, ethyl esters;
Vitamin E (.alpha.-Tocopherol, .delta.- or .gamma.-Tocopherol and
tocopherol acetates)--all racemic forms; Tocotrienol--all racemic
forms, resveratrol; Pyrocatechol; 3-ethylbenzophenone; Magnolol,
carnosol; Vitamin A--retinol (retinoic acid), retinol palmitate,
retinol acetate, retinol esters, vitamin A aldehyde (retinal),
carotene s--e.g. beta-carotene I and II, carotenal, mixtures of
beta-carotenes, also lycopene; Ubiquinone (Coenyme Q-10),
bromadiolon, vitamin K2, vitamin K3, flavones/flavonols (catehins,
etc.), eugenol and others Designed combinations thereof--e.g.
ascorbic acid palmitate with lipoic acid--can also be used.
[0188] Furthermore, the nanoparticles according to the present
disclosure can have a functional surface and/or be biocompatible or
biodegradable. Such nanoparticles can be used for e.g. flow
cytometry (or any cytometry in general, including imaging).
Moreover, they can be used for live cell imaging (e.g. after uptake
of the nanoparticles into the cells). In addition, the
nanoparticles can be optimized specifically via attachment of
proteins, antibodies, enzymes and other specific groups on their
surface and used for sensing/imaging of oxygen (photon
up-conversion nanoparticles), temperature (typically by using an
emitter that is a phosphorescent chromophore--i.e. the sensitizer
type of molecules as emitter), biologically active molecules, e.g.
in neurotransmitters sensing (photon up-conversion nanoparticles or
fluorescence intensity change), or the detection of singlet oxygen.
The sensing/imaging/detection can be carried out in/around live
cells or in any other biotechnological context. Moreover, the
nanoparticles according to the present disclosure can be
incorporated into biocompatible/cell compatible sensing layers.
Alternatively, the nanoparticles can be made cell-permeable for
imaging/sensing inside live cells.
[0189] The term "polymeric organic matrix", as used herein, is
meant to refer to a matrix that includes a polymer or is made up of
a polymer which includes carbon-carbon bonds For example it may
include or be composed of a polysiloxane. In one embodiment, the
term refers to a polymer that has a carbon chain backbone. A
"polymer" is a substance composed of molecules characterized by the
multiple repetition of one or more species of monomers. In this
context, a "multiple repetition of monomers" is meant to refer to
10 or more, particularly 50 or more, more particularly 100 or more
monomers linked to each other.
[0190] If the present application states that a component A is
"chemically inert" with respect to a component B, this means that
component A does not chemically react with component B.
[0191] The term "light emitter", as used herein, refers to a
molecule or combination of molecules that, upon irradiation with
light of a certain excitation wavelength, is capable of emitting
light of a certain emission wavelength. The emitted light may be
generated by luminescence, particularly fluorescence.
[0192] A "metal nanoparticle", as used herein, is a metal particle
having an average diameter <1 um. As the skilled person will
appreciate, for the metal nanoparticles any metal particles with
plasmon may be used, e.g. metal nanoparticles. Typically, such
metal nanoparticles have a plasmon and are herein also sometimes
referred to as "plasmonic" metal nanoparticles. Without wishing to
be bound by any theory, the present inventors believe that the
electron density of such plasmonic metal nanoparticles can couple
with electromagnetic radiation of wavelengths that are far larger
than the particle itself.
[0193] In one embodiment, said metal nanoparticles have a diameter
in the range of from 1 to 100 nm, particularly in the range of from
4 to 80 nm, more particularly in the range of from 10 to 60 nm,
more particularly in the range of from 10 to 50 nm. In one
embodiment, such metal nanoparticle has an average diameter in the
range of from 1 nm to 50 nm, particularly 1 nm to 30 nm, more
particularly 1 nm to 20 nm. In one embodiment, it has an average
diameter of from 10 nm to 50 nm, particularly 10 nm to 30 nm. In
another embodiment, such metal nanoparticle has an average diameter
of from 1 nm to 40 nm, particularly 5 nm to 30 nm. Typically, such
metal nanoparticle having an average diameter of from 1 nm to 100
nm or an average diameter of any subrange therein is a plasmonic
nanoparticle. If the present application indicates that a diameter,
e.g. of metal nanoparticles, is in a certain range, this means that
the average diameter of said metal nanoparticles falls in that
certain range. In one embodiment, the diameter of individual metal
nanoparticles when prepared in different runs from the same
composition of the organic phase and the same mixing conditions
varies by not more than 30%, particularly by not more than 20%,
more particularly by not more than 10%.
[0194] If the present disclosure refers to a metal particle being
"enclosed within" a matrix, this designates a situation where said
metal particle is surrounded at all sides by said matrix, such that
the surface of said metal particle is completely covered by said
matrix. If more than one metal particle is enclosed within the
organic nanoparticle--they are enclosed as single particles with no
contact to each other which is essential for keeping their plasmon
intact to ensure plasmon enhancement.
[0195] If the present disclosure refers to a metal nanoparticle
being capable of "enhancing the intensity" of light emitted by a
light emitter, this designates a situation where said metal
nanoparticle is capable of enhancing the number of photons emitted
per number of photons absorbed and/or the photostability. If the
present disclosure refers to a metal nanoparticle being capable of
"enhancing the luminescence or photon up-conversion emission" of
the nanoparticles (NP) by plasmon enhancement, this designates a
situation where said metal nanoparticle is capable of enhancing the
intensity (number of photons emitted per number of photons
absorbed) and/or the photostability of said nanoparticle (NP). With
the nanoparticles (NP) according to the disclosure, such effects
are even more pronounced at higher excitation intensities, i.e.
where the photostability is more difficult to keep otherwise.
[0196] A "perylene", as used herein, is a molecule having the
following structure:
##STR00042##
wherein W, X, Y and Z are suitable substituents.
[0197] A "perylene monoimide", as used herein, is a molecule having
the following structure:
##STR00043##
wherein W, X, Y, Z and Ri are suitable substituents.
[0198] A "perylene diimide", as used herein, is a molecule having
the following structure:
##STR00044##
wherein X, Y, Z and Ri are suitable substituents. "Suitable"
substituents" are manifold and can be determined by a person
skilled in the art.
[0199] A "sensitizer" is a chromophore molecule which is able to
absorb light, particularly with high populated triplet states, and
capable to transfer the excited triplet state to a suitable light
emitter. The combination of sensitizer and emitter molecules with
triplet-triplet transfer can achieve photon up-conversion--also as
described in EP 2298849 or US 2010/0330026 A1. The sensitizer may
be a metal-organic complex. Upon irradiation, the sensitizer
absorbs light at the excitation wavelength. By an up-conversion
process e.g. based on direct or sequential two-photon excitation or
on direct or sequential multi-photon excitation or on excitation of
molecules populating high vibrational state(s) ("hot-band
absorption"), or an up-conversion process based on a
triplet-triplet annihilation process between photoexcited molecules
of said light emitter and/or based on a triplet-triplet
annihilation process between photo-excited molecules of said
sensitizer, a higher energy state is generated, leading to emission
of light at the emission wavelength by the light emitter.
[0200] Examples of sensitizers suitable for photon up-conversion
are the compounds shown in Formulas IXXX-XXXI above. Examples of
light emitters suitable for photon up-conversion are the compounds
shown in Formulas VII-IX and XVII-XXII.
[0201] A "stabilizing agent", as used herein, is a non-ionic or
ionic surfactant (surface active molecule or polymer) and ensures
the nanoparticle formation/dispersion and the functional groups on
the surface (COOH or NH.sub.2, or other) of the nanoparticles. The
stabilizing agent occupies the surface of the organic nanoparticles
(NP). Through its hydrophobic part, the stabilizing agent interacts
with the organic core of the nanoparticle (NP) (where the
chromophores, which are not water-soluble, are present). Through
its hydrophilic part, the stabilizing gent is exposed to the
aqueous/polar environment surrounding the nanoparticles (NP). Thus,
the stabilizing agent allows for formation of nanoparticles and
stabilizes the nanoparticles in the dispersion.
[0202] A "metal nanoparticle", as used herein, is used synonymously
with "plasmonic" nanoparticle. Plasmon enhancement of emission by
plasmonic nanoparticle is the target. Without wishing to be bound
by any theory, the term "plasmonic nanoparticles" refers to
nanoparticles whose electron density can couple with
electromagnetic radiation of wavelengths that are far larger than
the particle. It refers to a plasmonic nanoparticle consisting of a
metal, a combination of different metals or a metal alloy or a
nitride of transition metal. In one embodiment, a "metal
nanoparticle" consists of one metal (i.e. of atoms of only one
chemical element)--Ag.
[0203] If the present disclosure refers to a metal nanoparticle
being "enclosed within" a matrix, this designates a situation where
said metal particle is surrounded at all sides by said matrix, such
that the surface of said metal particle is completely covered by
said matrix.
[0204] At some instances, the present application may refer to
metal particles or metal nanoparticles "that are in contact with
each other". If a metal nanoparticle A "is in contact with" a metal
nanoparticle B, this designates a situation where metal
nanoparticle A and metal nanoparticle B directly touch each other,
i.e. there is no intervening layer of a material that is neither
part of metal nanoparticle A nor part of metal nanoparticle B, nor
a gap, between said metal nanoparticle A and said metal
nanoparticle B.
[0205] If the present disclosure indicates that a molecule is
capable of "specifically binding" to a certain analyte, this refers
to a situation where the dissociation constant for the interaction
of said molecule and said analyte is <1 .mu.M, particularly
<100 nM, more particularly <10 nM.
[0206] A molecule with an "enzymatic activity that allows to detect
an analyte molecule" is a molecule that catalyzes an enzymatic
reaction that is dependent on or strongly influenced by the
presence/absence of the analyte molecule to be detected (e.g.
because it is a substrate of the catalyzed reaction or because the
catalytic activity of the molecule with enzymatic activity is
strongly influenced by the presence/absence of the analyte molecule
to be detected), such that by monitoring the reaction catalyzed by
said molecule with enzymatic activity of said molecule changes
regarding the presence/absence of the analyte molecule with an
enzymatic activity that allows to detect said analyte molecule,
conclusions about the presence/absence of said analyte molecule or
about changes in the concentration of said analyte molecule can be
detected.
[0207] As the skilled person will appreciate, the detection of an
analyte via enzyme activity requires the stable binding (covalent
or non-covalent) of the enzyme to the surface of the nanoparticle
without losing the activity of the enzyme (i.e. a free active site
is needed). The molecule with an enzymatic activity that allows to
detect the analyte molecule may for example be an oxidase that is
covalently attached to the nanoparticle surface, and the analyte to
be detected the substrate of said oxidase. In the presence of the
analyte, the oxidase reaction generates light that excites the
light emitter(s) (or sensitizer(s)) in the nanoparticle, wherein
the light eventually emitted by the nanoparticle is further
enhanced by the metal particles present in the nanoparticle
[0208] The term "biomolecule" and "biological molecule" are used
interchangeably herein and refer to any molecule produced by a
living cell or a living organism (including viruses). This may
include, but is not limited to macromolecules such as proteins,
polysaccharides, lipids, and nucleic acids (including DNA and RNA),
as well as small molecules such as primary metabolites, secondary
metabolites, and natural products. In particular, it refers to
neurotransmitters, oxygen and reactive oxygen species, hormones,
antioxidants and vitamins,
[0209] The term "biologically active molecule" refers to a molecule
that is capable of facilitating or inducing a specific cellular or
tissue response.
[0210] Note that the present technology can also be configured as
described below in the following embodiments:
Embodiments
[0211] 1. A nanoparticle (NP) including [0212] a polymeric organic
matrix, [0213] at least one light emitter distributed within said
matrix, [0214] a stabilizing agent, and [0215] at least one metal
nanoparticle enclosed within said matrix, wherein said at least one
metal nanoparticle is a plasmonic nanoparticle. 2. The nanoparticle
according to embodiment 1, wherein said polymeric organic matrix is
composed of a material or a combination of materials selected from
the group consisting of polyacrylonitriles, polystyrenes and
oligostyrenes, styrene copolymers, styrene-butadiene copolymers,
polystyrene-based elastomers, polyethylenes and oligoethylenes,
polyphenylenes and polyphenylene dendrimers, polypropylenes,
polytetrafluoroethylenes, extended polytetrafluoroethylenes,
polyacrylates, polymethylmethacrylates, ethylene-co-vinyl acetates,
polysiloxanes, such as polymethylsiloxanes and
polyphenylmethylsiloxanes, e.g. polydimethylsiloxane or
polyphenylmethylsiloxane, their copolymers as well as substituted
and modified polysiloxanes, polyethers, polyurethanes,
polyether-urethanes, polyethylene terephthalates and polysulphones,
or any copolymers of the listed polymers, particularly a material
selected from the group consisting of polystyrenes,
polyacrylonitriles and polymethylmethacrylates, more particularly
polymethylmethacrylate. 3. The nanoparticle according to any of the
foregoing embodiments, wherein said light emitter is capable of
emitting light by luminescence, in particular by fluorescence or by
phosphorescence, more particularly by fluorescence. 4. The
nanoparticle according to any of the foregoing embodiments, wherein
said nanoparticle is capable of photon up-conversion. 5. The
nanoparticle according to any of the foregoing embodiments, wherein
said nanoparticle further includes at least one sensitizer,
wherein, in particular, said at least one sensitizer is capable of
absorbing light at a first wavelength region
w.ltoreq..lamda..sub.1.ltoreq.x, and said at least one light
emitter is capable of emitting light at a second wavelength region
y.ltoreq..lamda..sub.2.ltoreq.z, wherein
.lamda..sub.2.ltoreq..lamda..sub.1, wherein, upon absorption of
light by said at least one sensitizer at said first wavelength
region w.ltoreq..lamda..sub.1.ltoreq.x, said at least one light
emitter emits light at said second wavelength region
y.ltoreq..lamda..sub.2.ltoreq.z. 6. The nanoparticle according to
any of the foregoing embodiments, wherein said at least one light
emitter is a molecule selected from the group consisting of
anthracenes, perylenes, perylene derivatives such as perylene
monoimides or perylene diimides, coumarins and BODIPY dyes,
wherein, in particular, said at least one light emitter has a
structure represented by Formula (I) or (II) or includes a molecule
having the structure represented by Formula (I) or (II):
##STR00045##
[0215] wherein R.sub.1 and R.sub.2 are independently selected from
the group consisting of hydrogen and a moiety with the structure
represented by Formula (III), wherein at least one of R.sub.1 and
R.sub.2 is a moiety with the structure represented by Formula
(III):
##STR00046##
wherein R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are
independently selected from the group consisting of H, F, and
tri-fluoro-methyl (--CF.sub.3), wherein at least one of R.sub.3,
R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is F or tri-fluoro-methyl
(--CF.sub.3), wherein, in particular, R.sub.1 and/or R.sub.2 is a
moiety represented by Formula (IV), Formula (V) or Formula
(VI):
##STR00047##
wherein, more particularly, said at least one light emitter has a
structure represented by Formula (VII), Formula (VIII) or Formula
(IX), or includes a molecule having the structure represented by
Formula (VII), Formula (VIII) or Formula (IX):
##STR00048## ##STR00049##
or wherein said at least one light emitter has the structure
represented by Formula (X) or includes a molecule having the
structure represented by Formula (X):
##STR00050##
wherein R.sub.8 and R.sub.9 are independently selected from the
group consisting of hydrogen and a moiety with the structure
represented by Formula (XI), wherein at least one of R.sub.8 and
R.sub.9 is a moiety with the structure represented by Formula
(XI):
##STR00051##
wherein R.sub.10, R.sub.11, R.sub.12, R.sub.13 and R.sub.14 are
independently selected from the group consisting of H, F, and
tri-fluoro-methyl (--CF.sub.3), wherein at least one of R.sub.10,
R.sub.11, R.sub.12, R.sub.13 and R.sub.14 is F or tri-fluoro-methyl
(--CF.sub.3), wherein, more particularly, R.sub.8 and/or R.sub.9 is
the moiety represented by Formula (XII), Formula (XIII), Formula
(XIV), Formula (XV) or Formula (XVI):
##STR00052##
or wherein, more particularly, said at least one light emitter has
the structure represented by Formula (XVII), Formula (XVIII),
Formula (XIX), Formula (XX), Formula (XXI) or Formula (XXII), or
includes a molecule having the structure represented by Formula
(XVII), Formula (XVIII), Formula (XIX), Formula (XX), Formula (XXI)
or Formula (XXII):
##STR00053## ##STR00054##
or wherein said at least one light emitter has a structure
represented by one of the following structures or includes a
molecule having a structure represented by one of the following
structures:
##STR00055## ##STR00056##
or wherein said light emitter has the structure represented by
Formula (XXIII), (XXIV) or (XXV) or includes a molecule having the
structure represented by Formula (XXIII), (XXIV) or (XXV):
##STR00057##
wherein W in formulae XXII-XXV is selected from one of the
following groups.
##STR00058##
wherein Y as used in formula W is selected from the group
consisting of CH.sub.2, S, O, Se and N--R.sub.2, and wherein
R.sub.2 is selected from the group consisting of H, a linear alkyl
group, a branched alkyl group, a cycloalkyl group, a halogenated
alkyl group, a halogen atom, an alkyl sulfanyl group, an aryl
sulfanyl group, an amino alkyl group, an amino aryl group, an aryl
group, a halogenated alkyl group, a heteroaryl group, a fluorenyl
group, an amino group, a nitro group, an OH group, an SH group, and
a group --O--R.sub.3, wherein R.sub.3 is selected from the group
consisting of a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, an alkyl sulfanyl
group, an aryl sulfanyl group, an amino alkyl group, an amino aryl
group, an aryl group, a halogenated alkyl group, a heteroaryl group
and a fluorenyl group wherein, particularly, R.sub.2 has not more
than 6 carbon atoms; wherein X and Y in formulae XXIII-XXV are
independently selected from the following groups:
##STR00059## ##STR00060##
and wherein R is selected from the group consisting of H, a linear
alkyl group, a branched alkyl group, a cycloalkyl group, a
halogenated alkyl group, a halogen atom, an alkyl sulfanyl group,
an aryl sulfanyl group, an amino alkyl group, an amino aryl group,
an aryl group, a halogenated alkyl group, a heteroaryl group, a
fluorenyl group, an amino group, a nitro group, an OH group, an SH
group, and a group --O--R.sub.3, wherein R.sub.3 is selected from
the group consisting of a linear alkyl group, a branched alkyl
group, a cycloalkyl group, a halogenated alkyl group, an alkyl
sulfanyl group, an aryl sulfanyl group, an amino alkyl group, an
amino aryl group, an aryl group, a halogenated alkyl group, a
heteroaryl group and a fluorenyl group, wherein, particularly, R
has not more than 6 carbon atoms, wherein Z in formulae XXIII-XXV
is selected from the following groups:
##STR00061##
and wherein R.sub.2 is selected from the group consisting of H, a
linear alkyl group, a branched alkyl group, a cycloalkyl group, a
halogenated alkyl group, a halogen atom, an alkyl sulfanyl group,
an aryl sulfanyl group, an amino alkyl group, an amino aryl group,
an aryl group, a halogenated alkyl group, a heteroaryl group, a
fluorenyl group, an amino group, a nitro group, an OH group, an SH
group, and a group --O--R.sub.3, wherein R.sub.3 is selected from
the group consisting of a linear alkyl group, a branched alkyl
group, a cycloalkyl group, a halogenated alkyl group, an alkyl
sulfanyl group, an aryl sulfanyl group, an amino alkyl group, an
amino aryl group, an aryl group, a halogenated alkyl group, a
heteroaryl group and a fluorenyl group, wherein, in particular,
R.sub.2 has not more than 6 carbon atoms, wherein Ri is selected
from the following groups.
##STR00062## ##STR00063##
wherein, particularly, W is selected from the following groups:
##STR00064## ##STR00065## ##STR00066##
With R, R.sub.1, R.sub.2 being as defined above; or wherein said at
least one light emitter has a structure selected from one of the
following:
##STR00067##
wherein R is a linear or branched alkyl group, particularly with
not more than 6 carbon atoms; or wherein said light emitter has the
structure represented by Formula (XXVI) or includes a molecule
having the structure represented by Formula (XXVI):
##STR00068##
wherein R is selected from the following groups:
##STR00069##
wherein Y is selected from the group consisting of CH.sub.2, S, O,
Se and N--R.sub.2, and wherein R.sub.2 is selected from the group
consisting of H, a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, a halogen atom, an
alkyl sulfanyl group, an aryl sulfanyl group, an amino alkyl group,
an amino aryl group, an aryl group, a halogenated alkyl group, a
heteroaryl group, a fluorenyl group, an OH group, an SH group, and
a group --O--R.sub.3, wherein R.sub.3 is selected from the group
consisting of a linear alkyl group, a branched alkyl group, a
cycloalkyl group, a halogenated alkyl group, an alkyl sulfanyl
group, an aryl sulfanyl group, an amino alkyl group, an amino aryl
group, an aryl group, a halogenated alkyl group, a heteroaryl group
and a fluorenyl group, wherein, particularly, R.sub.2 has not more
than 6 carbon atoms; or wherein R is selected from the following
groups:
##STR00070##
wherein R.sub.1 is a linear or branched alkyl group, particularly
with not more than 6 carbon atoms; or wherein said at least one
light emitter has a structure selected from one of the following or
includes a molecule having a structure selected from one of the
following.
##STR00071##
wherein R is a linear or branched alkyl group, particularly with up
to 6 carbon atoms. 7. The nanoparticle according to any of
embodiments 5 and 6, wherein said at least one sensitizer is or
includes a porphyrin or a phthalocyanine, wherein, in particular,
said at least one sensitizer has a structure represented by Formula
(XXVII), Formula (XXVII), Formula (IXXX), Formula (XXX) or Formula
(XXXI) or includes a molecule having a structure represented by
Formula (XXVIII), Formula (IXXX), Formula (XXX) or Formula
(XXXI).
##STR00072##
wherein R.sub.1 is hydrogen a linear or branched alkyl group, in
particular with up to 6 carbon atoms, or a benzene ring, and
wherein R is a linear or branched alkyl group, in particular with
up to 6 carbon atoms;
##STR00073## ##STR00074##
wherein, in particular, said sensitizer has the structure
represented by Formula (XXVIII):
##STR00075##
8. The nanoparticle according to any of the foregoing embodiments,
wherein said nanoparticle (NP) includes 1 to 4 metal nanoparticles,
and/or wherein said nanoparticle (NP) does not include metal
nanoparticles that are in contact with each other. 9. The
nanoparticle according to any of the foregoing embodiments, wherein
said at least one metal nanoparticle has a diameter in the range of
from 1 to 100 nm, in particular in the range of from 10 to 100 nm,
more particularly in the range of from 10 to 50 nm, and/or wherein
said at least one metal nanoparticle consists of a material
selected from the group consisting of Ag, Au and Co, and/or wherein
said metal nanoparticle(s) is(are) plasmonic and/or magnetic. 10.
The nanoparticle according to any of the foregoing embodiments,
wherein said nanoparticle further includes at least one
antioxidant, in particular a singlet oxygen scavenger or reactive
oxygen scavenger (ROS), particularly a singlet oxygen
scavenger/quencher, wherein, in particular, said at least one
antioxidant is selected from the group consisting of lipoic acid,
vitamin E, a carotenoid, ascorbic acid derivatives soluble in
organic solvents, particularly an ascorbic acid palmitate. 11. The
nanoparticle according to any of the foregoing embodiments, wherein
said nanoparticle includes functional groups at its surface that
allow to covalently couple a molecule to the nanoparticle, wherein,
in particular, said functional groups are selected from the group
consisting of --COOH (carboxylate), --NH.sub.2, --SH (thiol),
--NHS, alkynyl, --N.sub.3, aldehyde, ketone and biotin group,
wherein, more particularly, said functional groups are --COOH
and/or --NH.sub.2. 12. The nanoparticle according to any of the
foregoing embodiments, wherein said nanoparticle includes molecules
or chemical groups attached to its surface that are capable of
specifically binding to an analyte molecule or that have an
enzymatic activity that allows to detect an analyte molecule,
wherein, in particular, said molecules attached to the surface of
the nanoparticle are antibody molecules, and wherein, in
particular, said analyte molecule is a biomolecule. 13. A sensing
layer including nanoparticles as defined in any of the foregoing
embodiments. 14. A method of producing nanoparticles (NP) as
defined in any of embodiments 1-13, said nanoparticles (NP)
including [0216] a polymeric organic matrix, [0217] at least one
light emitter distributed within said matrix, [0218] optionally at
least one sensitizer and/or at least one antioxidant distributed
within said matrix, [0219] a stabilizing agent, and [0220] at least
one metal nanoparticle enclosed within said matrix, wherein said at
least one metal nanoparticle is a plasmonic nanoparticle, said
method including the steps of: [0221] providing a polymer or
combination of polymers or combination of polymers with small
molecules from which the polymeric organic matrix is to be formed,
a stabilizing agent, at least one light emitter, plasmonic metal
nanoparticles, and optionally at least one sensitizer and/or at
least one antioxidant, [0222] preparing a dispersion of said
plasmonic metal nanoparticles in an organic water-miscible solvent,
particularly upon ligand exchange of said plasmonic metal
nanoparticles, [0223] preparing a mixture of said polymer or
combination of polymers or combination of polymers with small
molecules from which the polymeric organic matrix is to be formed,
said stabilizing agent, said light emitter(s) and optionally said
sensitizer(s) and/or said antioxidant in an organic water-miscible
solvent, [0224] adding said mixture of said polymer or combination
of polymers or combination of polymers with small molecules, said
stabilizing agent, said light emitter(s) and optionally said
sensitizer(s) and/or said antioxidant(s) to said dispersion of
metal nanoparticles or vice versa, thus forming a mixture including
said metal nanoparticles, [0225] inducing said mixture including
said plasmonic metal nanoparticles to form nanoparticles (NP), thus
forming a dispersion of nanoparticles (NP) wherein said
nanoparticles (NP) include a polymeric organic matrix with said
light emitter(s) and, optionally, said sensitizer(s) and/or said
antioxidant(s), distributed therein, and wherein said metal
nanoparticles are enclosed in said polymeric organic matrix. 15.
Use of nanoparticles according to any of embodiments 1-13 in a
biological application selected from the group consisting of
labeling and/or detection of cells, biological (macro-)molecules or
other analytes, fluorescence microscopy, (flow) cytometry,
fluorescence-activated cell sorting (FACS), fluorescence resonance
energy transfer (FRET), immunohistochemistry, clinical
immunoassays, fluorescence-quenching-based enzyme-activity assays,
high-throughput screening, molecular diagnostics, sensing of
temperature, sensing of pressure and sensing of oxygen.
EXAMPLES
Example 1
Preparation of Nanoparticles
[0226] This example describes the optimised production of examples
of multicomponent nanoparticles in accordance with the present
disclosure. These special, original designed nanoparticles are
prepared by rapidly mixing water into a cooled, stirring, solution
of an optimised organic matrix, optimised surface stabiliser or
dispersant, dyes--including specially developed hydrophobic dyes
with even more efficient incorporation) an emitter and a sensitizer
in dry THF under an inert atmosphere. In addition to the organic
components, silver nanoparticles can be added to the organic phase
before mixing. The procedure and the components of the
nanoparticles were optimized for sensitivity, increase in
upconversion signal and size, which also effects transparency of
the solution.
[0227] The size and size distribution of the nanoparticles produced
in this method is very good if the aqueous phase and organic phase
are chilled and mixed very rapidly. To accomplish this two
electronically controlled valves, that quickly move from fully
closed to fully open in under 0.2 s are used. A partial vacuum of
30 mbar in the mixing chamber also facilitates the very fast
addition of the aqueous phase to the organic phase. This, along
with rapid stirring of the solution, allows a colloidal solution to
be formed upon the water addition. The dispersion was then stirred
under inert atmosphere for over an hour and the organic solvent
evaporated under reduced pressure. Following cooling overnight the
dispersion was filtered to remove large masses, and centrifuged
within a concentrator tube remove small molecules and aggregates
and collect the organic particles. Nanoparticles were collected in
water (2 ml or 0.5 ml) and stored at 4.degree. C.
[0228] What is described in this example is a representative
procedure.
[0229] Purpose/Aim:
[0230] This example details a method for the controlled,
repeatable, formation of nanoparticles with consistent size and
polydispersity index (PDI). This procedure was developed to yield
methods, which allows for the inclusion of fluorescent dye(s), or
upconversion systems (sensitizers & emitters) into the
nanoparticles--especially as aqueous dispersions.
[0231] The applications of such dye-loaded nanoparticles are
diverse (including fluorescence, or PUC, or both in combination),
for example:
[0232] To manufacture bright, stable, with controllable size
emissive nanoparticles in aqueous dispersions--for application as
labels for e.g. proteins and cells. In a similar manner the water
soluble dyes are used. [0233] For direct uptake by cells--imaging
of live cells or their flow cytomteric investigations (following
the uptake of the NPs by the cells) [0234] For attachment to cells
surface--flow cytometry/sorting--after the NPs are attached on the
cell surface (here polystyrene (PS) microparticles are used in the
examples as cell model) [0235] The formation of sensing/imaging
layers, e.g. in the development of a sensing layer which can be
(is) used in combination with neuronal cell cultures as
non-invasive neuroimaging system for the visualization of neuronal
activities. To manufacture PUC-NPs to sense the NT dopamine or
other NTs like serotonin, as well as other biomolecules which can
scavange/quench singlet oxygen--as added to the NPs dispersion--as
released by neuronal cells or from any other source. [0236] To
control the PUC outcome from the NPs as well as their incorporation
in ESL--for control of sensitivity and dynamic range of ESL for
dopamine sensing
[0237] Solution/Procedure (Experimental):
[0238] The main optimisation pathways of the NPs core: [0239]
Variation of the components: [0240] Variation of the ratio/amount
of the matrix components e.g. PMMA (polymethylmethacrylate) and PHD
(heptadecyl benzene) [0241] Variation of the surface stabilizer and
its concentration [0242] Variation also combinations of the emitter
molecules and there concentration [0243] Variation also combination
of the sensitizer molecules also their concentration [0244]
Addition/variation also combinations of antioxidant(s) (singlet
oxygen scavenger/quencher or ROS scavenger) and their
concentration, [0245] Variation of the mixing conditions [0246] Air
vs. inert atmosphere [0247] Temperature of the organic solution and
the water and as follows influence also the size and the surface
potential of the nanoparticles as well as on their emissive/sensing
properties and their further attachment/functionalisation
properties.
[0248] The following procedure details the production of
nanoparticles. Typically 4 to 6 variations (nanoparticles comprised
of, for example, differing Ag nanoparticles and antioxidant
concentration as shown in FIGS. 2 and 3) are typically prepared in
one day.
[0249] This method of preparing the nanoparticles is to inject cold
water into an organic phase containing the, matrix components, the
stabiliser, the antioxidants (if any), the metal nanoparticles and
the dye molecules. Then by adding water, quickly and with a large
amount of agitation, to this phase nanoparticles of consistent size
and polydispersity index (PDI) are produced in a controlled,
repeatable manner for the corresponding combination of
components.
Typical Materials:
[0250] Surface stabilizer: CoPEG (Glycolic acid ethoxylate
4-nonylphenyl ether) [0251] Matrix: PHD(Heptadecylbenzene), and
PMMA [0252] Emitters: e.g.
3,10-Bis((4-tert-butylphenyl)ethynyl)perylene or
3,9-diphenyl-perylene Or 3,9-Bis-(4-butyl-phenyl)-perylene or
further see FIG. 4 or all molecular structures Sensitizer: PdTBP
[0253] Organic Solvent: THF [0254] Additional Nanoparticles 4 nm
AgNP in 1 mg/ml THF [0255] Antioxidant: 6-O-Palmitoyl-L-ascorbic
acid or lipoic acid, or other (see the description above),
[0256] The general components of exemplary nanoparticles in
accordance with the present disclosure are depicted in FIG. 1. See
FIG. 1A--for luminescent, e.g. fluorescent NPs, and FIG. 1B for
photon upconversion nanoparticles (PUC NPs).
[0257] Results on Size and Polydispersity (Briefly):
[0258] Typical results are shown in the table below.
TABLE-US-00001 NP1 NP2 Silver Nanoparticles added No Yes Average
Size/nm 126 .+-. 1 144 .+-. 1 PDI 0.20 .+-. 0.22 .+-. Zeta
Potential/mv (neutral -51.5 .+-. 0.7 -54.8 .+-. 0.8 Conductivity
(.mu.S/cm) 11.3 .+-. 2.4 8.7 .+-. 0.04 Mobility (.mu.mcm/Vs) -4.0
.+-. -0.1 -4.3 .+-. 0.1
[0259] The polydispersity index (PDI) is a measure of the size
consistency of the nanoparticles. For nanoparticle batches prepared
via this method, the PDI is around 0.2, which is good.
[0260] The Zeta potential is a measure of colloidal stability. The
Zeta potential is calculated from the measured conductivity of the
solution, and the mobility of the particles. In general Zeta
potential values of a magnitude greater than 40 mV are indicative
of a stable colloid, furthermore, values above 60 mV are indicative
of excellent stability. Typically, for nanoparticles prepared via
this method the Zeta potential is .about.-50 mV this indicated that
these colloidal solutions are not likely to aggregate. Upon further
optimisation of the NPs components and their ration up to -65 mV
were achieved.
[0261] As an example of stability, the nanoparticles were measured
for size distribution following storage for 5 days to check their
stability and degree of aggregation--there was no change beyond
that within the region of the error of the measurement, indicating
stable nanoparticles.
TABLE-US-00002 Average size difference/ nm PDI difference NP1 4.68
.+-. 3.00 0.0288 .+-. 0.0212 NP2 1.82 .+-. 2.90 -0.0077 .+-.
0.0076
Exemplary Nanoparticle Preparation Method Overview Summary:
[0262] Nanoparticles are prepared in this method by firing water
[e.g. MilliQ, with controlled temperature and high speed (through
electronic valves) into a temperature controlled, stirring solution
of an organic phase [e.g. glycolic acid, heptadecylbenzene,
polymethylmethacrylate, and Pd tetrabenzoporphyrin (PdTBP)
sensitizer in dry THF under an inert N.sub.2 atmosphere. Silver
nanoparticles, [e.g. 4 nm Ag-Dodecanethiole NP or 50 nm SiO2 capped
Ag nanoparticles are compared in FIG. 9] and antioxidant (e.g.
lipoic acid in 2 different concentrations is shown in FIG. 9) can
be added to the organic phase before mixing. The dispersion was
then stirred under inert atmosphere for over an hour and the
organic solvent (THF) evaporated under reduced pressure. The
dispersion was filtered (Whatman 1 filter paper) to remove and
large masses, and then centrifuged (100 K MWCO Corning Spin-X UF
Concentrator 20 ml, to remove small molecules and particles.
Nanoparticles of interest were collected in MilliQ water and stored
cool (4.degree. C.). This yields stable nanoparticles with a good
polydispersity index (e.g. 0.2) and good zeta potential (e.g. -50
mV or higher).
[0263] Conclusion:
[0264] Organic nanoparticles with reproducible, controllable,
repeatable size and optimised size distribution are successfully
prepared via this method. Dyes and smaller nanoparticles have been
successfully incorporated into the nanoparticles.
[0265] A protocol for NP synthesis was developed and optimized step
by step. The set-up for the optimised procedure incorporates
pressure and temperature control as well as fast speed mixing
valves. Nanoprobes with completely reproducible and controlled
variable properties (e.g. size and surface potential) were
synthesized with the given set-up.
[0266] The nanoparticle dispersions developed by this method are
highly emissive, biocompatible and can be used in a variety of
biotechnological applications, especially for flow cytometry, live
cell imaging, or--live cell functions imaging--e.g.
neurotransmitters (or antioxidants) visualisation/imaging in
neuronal or any other tissue or cell culture and/or as released by
neuronal cells
Example 2
Preparation of an Example of a Sensor Comprising an Emissive
Sensing Layer
[0267] Here an exemplary method is presented to produce transparent
emissive sensing layers (ESLs) composed of emissive nanoparticles
as prepared in accordance with the present disclosure, i.e. photon
upconversion nanoparticles (PUC NPs) embedded into a polymer
matrix. The ESLs aim for the quantitative detection of different
biomolecules secreted from living cells in cell culture with high
spatiotemporal resolution. To achieve these goals the layers
necessitate to exhibit excellent homogeneity, good stability under
cell culture conditions as well as a high sensitivity and
selectivity towards the target molecules.
[0268] This example describes an optimization of the sensing layer.
Especially the homogeneity and the upconversion (UC) signal
achieved by the layers in this example are very good. Additionally,
stiffness of the layers can be tuned easily. The optimization
includes change from a biopolymer matrix towards an organic polymer
matrix. FIG. 10 shows the general structure and composition of an
embodiment of an ESL, in particular a schematic drawing showing the
general composition and structure of an emissive sensing layer
(ESL). The ESL is prepared on a modified glass support and is
afterwards functionalized with extracellular matrix proteins (ECM
matrix proteins), such as collagen which facilitate
biocompatibility of the layers.
[0269] Purpose/Aim:
[0270] The overall target is to develop a non-invasive tool to
image cellular functions such as neurotransmitter release from
neuronal cells (also in response to stimulation in real-time) using
a standard epifluorescence microscopic setup. The layers are
optimized for the use with neuronal cell models like PC-12 cells or
human induced pluripotent stem cells. To achieve detection of
target molecules from living cells the layers need to be permeable
for the target molecules and in close proximity to the side of
release. Furthermore the layers need to be stable for the duration
of the cell culture.
[0271] Solution/Procedure (Experimental):
[0272] 1. Procedure:
[0273] For optimization of ESLs an organic polymer matrix of
polyacrylamide was used. This polymer forms an elastic hydrogel and
can be varied in stiffness, which could also be of interest for
cell culture applications. Polyacrylamide is used for cell culture
applications like traction force microscopy. A detailed structure
of the ESL and its components is shown in FIG. 10 which shows a
schematic drawing showing the composition and structure of an
emissive sensing layer (ESL).
[0274] 1.1 Preparation of Activated Aminosilane-Coated Glass
Slides:
[0275] To achieve attachment of the ESL to the glass support, the
glass support is chemically modified. Aminofunctionalized glass
slides (e.g NEXTERION A+, Schott GmbH, Jena) are used here as a
starting point. Further activation is done by glutaraldehyde.
[0276] Preparation of Hydrophobic Coverslips:
[0277] Hydrophobic coverslips are needed to cover the polymerizing
gel solution on the activated aminosilane-coated coverslips to
prevent oxygen diffusion into the solution, which prevents
polymerization and to achieve a flat surface of the final gel.
Making the coverslips hydrophobic makes it easier to remove the
glass slips after polymerization and helps to avoid damage to the
gel. Different standard procedures for hydrophobisation can be
used, e.g. using RainX.
[0278] 1.2 Preparation of Gel:
[0279] To prepare the ESL solution one needs the PUC NPs, the
enhancer (Plasmonic) nanoparticles (Ag or Au nanoparticles) and
polyacrylamide (PAA) gel stock solution. The stock solution can be
prepared in different acrylamide/bisacrylamide solutions to adapt
gel stiffness and density. The mixtures, which have been used, are
summarized in the below table. Further mixtures can be found in
Plotnikov et al. (Plotnikov et al., Methods in Cell Biology, 2014;
"High-resolution Traction Force Microscopy" in Methods in Cell
Biology, Volume 123, 2014, ISSN 0091-679X)
TABLE-US-00003 TABLE Mixtures of PAA gel stock solutions Component
4 kPa stock solution 30 kPa stock solution Acrylamide 40% 3.75 ml 3
ml Bisacrylamid 2% 0.75 ml 1.4 ml MilliQ 0.50 ml 0.60 ml Total
volume 5.00 ml 5.00 ml
[0280] The PAA gel stock solution can be stored at 4.degree. C. for
at least a year.
[0281] In a first step, Ag NPs (40 nm, plasmonic nanoparticles) are
added to PUC NP solution in an 1.5 ml Eppendorf cup under sterile
conditions. Then, PAA gel stock solution are added to the NP
mixture and the complete solution is degassed either under argon or
nitrogen atmosphere for 1 h. Furthermore, a 4% (w/w)
ammonium-peroxosulfate (APS) solution is prepared in MilliQ. When
everything is ready prepared, polymerization is induced by addition
of TEMED and APS solution. The solution is quickly mixed using the
100 .mu.l pipette and the gel solution is added per well as a small
droplet on the surface of an 8-well sticky slide on an activated
aminosilane-coated glass slide. The droplet is quickly covered by a
hydrophobic coverslip. The rest of the solution is used as a
polymerization control. After 30 minutes of incubation at room
temperature, polymerization is complete and the hydrophobic
coverslips are removed carefully using a tweezer. The gels are
washed 3 times with MilliQ before they are stored at 4.degree.
C.
[0282] Further modification of the ESL can be achieved by various
measures, e.g. coating with e.g. polydopamine and ECM proteins.
Details about functionalization procedures can be found in the
parallel application concurrently filed with and copending with the
present application under applicant's reference number
S32783EP.
[0283] 2. Results of ESLs (Briefly):
[0284] 2.1 Emissive Sensing Layers: Phase Contrast, Fluorescence
and Upconversion
[0285] The emissive sensing layers prepared according to the
aforementioned protocol were tested for fluorescence intensity at
488 nm and also upconversion intensity at 638 nm illumination
wavelength under standardized conditions. Tests were conducted at
the MSL imaging platform (Zeiss Axiovert inverted microscope, HXP
lamp, 10.times. (tiled images) or 40.times. objective). To acquire
the upconversion of the ESL, the layers were incubated for 2 h
under N.sub.2-atmosphere at 37.degree. C. to remove molecular
oxygen.
[0286] 2.2 Emissive Sensing Layers: Dopamine Sensing
[0287] The layers were also tested for their sensing capabilities
towards the targeted neurotransmitter dopamine. The results are
shown in the figures, in particular FIG. 11-14. Dopamine
hydrochloride solution was dissolved either in PBS or HBSS.
Conclusion:
[0288] To improve homogeneity and sensitivity of the ESL
polyacrylamide was used as matrix component. Two different
acrylamide/bisacrylamide ratios were tested and both resulted in
ESL with improved homogeneity, upconversion signal and
transparency. Also the stability of gels under standard cell
culture conditions could be validated. The ESL preparation is
highly reproducible. ESLs are also stable over 3 weeks of
incubation under cell culture conditions (see FIG. 15 for 24 h and
2 weeks stability results). Dopamine could be detected at relevant
concentrations in the .mu.M range. To facilitate growth of cell
cultures or iPS cells, layers are further functionalized.
[0289] Thus, the foregoing discussion discloses and describes
merely exemplary embodiments of the present disclosure. As will be
understood by those skilled in the art, the present disclosure may
be embodied in other specific forms without departing from the
spirit or essential characteristics thereof. Accordingly, the
disclosure of the present disclosure is intended to be
illustrative, but not limiting of the scope of the disclosure, as
well as other claims. The disclosure, including any readily
discernible variants of the teachings herein, define, in part, the
scope of the foregoing claim terminology such that no inventive
subject matter is dedicated to the public.
* * * * *