of 101
Carbon
Nanomaterial
Fluorescent
Probes
and Their
Biological
Applications
Published
as part of Chemical
Reviews
virtual
special
issue “Fluorescent
Probes
in Biology”.
Andrew
T. Krasley,
+
Eugene
Li,
+
Jesus M. Galeana,
+
Chandima
Bulumulla,
+
Abraham
G. Beyene,
*
and Gozde S. Demirer
*
Cite This:
Chem.
Rev.
2024,
124,
3085−3185
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ABSTRACT:
Fluorescent
carbon
nanomaterials
have
broadly
useful
chemical
and
photophysical
attributes
that are conducive
to applications
in biology.
In this review,
we
focus
on materials
whose
photophysics
allow
for the use of these
materials
in biomedical
and
environmental
applications,
with
emphasis
on imaging,
biosensing,
and cargo
delivery.
The
review
focuses
primarily
on graphitic
carbon
nanomaterials
including
graphene
and its
derivatives,
carbon
nanotubes,
as well
as carbon
dots
and
carbon
nanohoops.
Recent
advances
in and
future
prospects
of these
fields
are discussed
at depth,
and
where
appropriate,
references
to reviews
pertaining
to older
literature
are provided.
CONTENTS
1. Introduction
3086
2. Carbon
Nanomaterial
Synthesis
and
Character-
ization
3088
2.1.
Carbon
Nanotubes
(CNTs)
3088
2.2.
Carbon
Dots
(CDs)
3088
2.3.
Carbon
Nanocones
(CNCs)
and
Carbon
Nanohoops
(CNHs)
3088
2.4.
Graphene,
Graphene
Oxide
(GO),
Reduced
Graphene
Oxide
(RGO),
and
Graphene
Nanoribbons
(GNRs)
3090
2.5.
CNM
Characterization
Methods
3091
2.5.1.
Chemical
Identification
3091
2.5.2.
Morphological
and
Structural
Charac-
terization
3091
2.5.3.
Optical
Characterization
3093
2.5.4.
Surface
Charge
and
Size
Character-
ization
3093
3. Material
Properties
of CNMs
3093
3.1.
Photophysical
Properties
3093
3.1.1.
Single-Walled
Carbon
Nanotubes
(SWCNTs)
3093
3.1.2.
Carbon
Dots
(CDs)
3094
3.1.3.
Carbon
Nanocones
(CNCs)
3098
3.1.4.
Carbon
Nanohoops
(CNHs)
3098
3.1.5.
Graphene,
Graphene
Oxide
(GO),
and
Reduced
Graphene
Oxide
(RGO)
3098
3.2.
Mechanical
Properties
3099
3.2.1.
Single-Walled
Carbon
Nanotubes
(SWCNTs)
3099
3.2.2.
Carbon
Dots
(CDs)
3099
3.2.3.
Carbon
Nanocones
(CNCs)
3099
3.2.4.
Carbon
Nanohoops
(CNHs)
3099
3.2.5.
Graphene,
Graphene
Oxide
(GO),
and
Reduced
Graphene
Oxide
(RGO)
3099
3.3.
Electronic
Properties
3100
3.3.1.
Single-Walled
Carbon
Nanotubes
(SWCNTs)
3100
3.3.2.
Carbon
Dots
(CDs)
3100
3.3.3.
Carbon
Nanocones
(CNCs)
3100
3.3.4.
Carbon
Nanohoops
(CNHs)
3101
3.3.5.
Graphene,
Graphene
Oxide
(GO),
and
Reduced
Graphene
Oxide
(RGO)
3101
4. Surface
Functionalization
Chemistry
3104
4.1.
Non-covalent
Functionalization
Chemistries
3104
4.1.1.
Single-Walled
Carbon
Nanotubes
(SWCNTs)
3104
4.1.2.
Carbon
Dots
(CDs)
3104
4.1.3.
Carbon
Nanocones
(CNCs)
3104
4.1.4.
Carbon
Nanohoops
(CNHs)
3104
4.1.5.
Graphene,
Graphene
Oxide
(GO),
and
Reduced
Graphene
Oxide
(RGO)
3104
4.2.
Covalent
Functionalization
Chemistries
3105
4.2.1.
Single-Walled
Carbon
Nanotubes
(SWCNTs)
3105
Received:
August
16, 2023
Revised:
February
1, 2024
Accepted:
February
9, 2024
Published:
March
13,
2024
Review
pubs.acs.org/CR
© 2024
The
Authors.
Published
by
American
Chemical
Society
3085
https://doi.org/10.1021/acs.chemrev.3c00581
Chem.
Rev.
2024,
124,
3085
3185
This article is licensed under CC-BY 4.0
4.2.2.
Carbon
Dots
(CDs)
3105
4.2.3.
Carbon
Nanocones
(CNCs)
3106
4.2.4.
Carbon
Nanohoops
(CNHs)
3106
4.2.5.
Graphene,
Graphene
Oxide
(GO),
and
Reduced
Graphene
Oxide
(RGO)
3106
5. CNM
Fluorescent
Probe
Applications
3107
5.1.
Fluorescence
Imaging
in Biomedical
Appli-
cations
3107
5.1.1.
In Vivo
Vasculature
Imaging
3107
5.1.2.
In Vivo
Whole
Organ
Imaging
3109
5.1.3.
In
Vitro
Imaging
in
Reduced
Prepara-
tions
3112
5.2.
Environmental
and
Food
Sample
Imaging
with
CNMs
3115
5.2.1.
Bacteria
Imaging
3115
5.2.2.
Plant
Imaging
3119
5.3.
Biosensing
Applications
of
Fluorescent
CNMs
3121
5.3.1.
Biomedically
Relevant
Sensing
3121
5.3.2.
Environmentally
Relevant
Sensing
3130
5.4.
Tracking
of Delivery
with
CNM
Fluorescent
Probes
3141
5.4.1.
Delivery
of Drugs
in Nanomedicine
3141
5.4.2.
Gene
and
Protein
Delivery
3141
6. Biomedical
and
Environmental
Translation
of
CNMs
3143
6.1.
Cytotoxicity
of CNMs
3143
6.1.1.
In Vitro
Cytotoxicity
3143
6.1.2.
In Vivo
Cytotoxicity
3145
6.1.3.
In Planta
Cytotoxicity
3146
6.2.
Environmental
Accumulation
and
Fate
of
CNMs
3146
6.3.
Scale-Up,
Economical,
and
Regulatory
Con-
siderations
of CNMs
3149
6.3.1.
Scalability
of CNM
Synthesis
3149
6.3.2.
Economic
Feasibility
of CNM
Technolo-
gies
3150
6.3.3.
Regulation
of CNM
Usage
3150
7. Perspectives
and
Outlook
3151
Author
Information
3152
Corresponding
Authors
3152
Authors
3152
Author
Contributions
3152
Notes
3152
Biographies
3152
Acknowledgments
3153
References
3153
1. INTRODUCTION
Fluorescent
carbon
nanomaterials
(CNMs)
probes
have
garnered
significant
attention
in the fields
of biomedicine
and environmental
science
due to a desirable
array
of optical,
electrical,
chemical,
and
material
properties.
These
CNMs
encompass
several
classes
of nanoparticles
whose
primary
constituent
is elemental
carbon.
The first in the family
of these
materials
is graphene,
with
a characteristic
planar
structure
made
from
sp
2
hybridized
carbon
atoms
arranged
in an
extended
honeycomb
network,
and
its derivatives
such
as
graphene
oxide
(GO)
and
graphene
nanoribbons
(GNR).
1
While
graphene
is an allotrope
of elemental
carbon
and has
dimensions
on the scale
of
10
μ
m, GO and related
family
of
graphene
derivatives
constitute
a mix of sp
2
and sp
3
hybridized
carbon
atoms,
typically
contain
epoxide,
carbonyl,
or
carboxylic
acid
functional
groups,
and have
dimensions
that
are on the order
of
10
nm.
Graphene
is a zero-bandgap
nanomaterial
with
metallic
character
and,
despite
a wealth
of
fascinating
material
properties,
is nonfluorescent
and hence
not
a focus
of this
review.
However,
it forms
the basis
for
understanding
GO and GNR
family
of nanomaterials,
which
can be synthesized
from
graphene,
and can be fluorescent
with
demonstrated
use for biological
and environmental
applica-
tions.
We will therefore
introduce
graphene
and discuss
its
properties
as it enables
us to explain
the synthesis
and material
properties
of GO and related
derivatives.
Later
sections
of this
review,
which
focus
on applications
and use cases
of CNMs,
will primarily
focus
on GO and other
fluorescent
CNMs,
and
not graphene.
Carbon
nanotubes
(CNTs)
constitute
another
important
class
of fluorescent
CNMs
included
in this review.
Carbon
nanotubes
are cylindrical
nanocrystals
of sp
2
hybridized
carbon
atoms
that can be conceptualized
as rolled
sheets
of graphene.
While
the diameter
of CNTs
is typically
on the order
of single
nanometers,
their
length
could
extend
for up to
1
μ
m. Within
CNTs,
one
can
distinguish
between
single-walled
CNTs
(SWCNTs),
and double
and multiwalled
CNTs
(DWCNTs
and MWCNTs,
respectively).
SWCNTs
are single
rolled
sheets
of graphene,
whereas
DWCNTs
and MWCNTs
can have
two
or multiple
coaxial
rolled
sheets
of graphene
that are nested
within
each
other.
Quantum
confinement
effects
give rise to a
set of unique
photophysical
properties
in semiconducting
SWCNTs,
including
a nonphotobleaching
fluorescence
in the
near-infrared
and shortwave
infrared
(NIR/SWIR)
regions
of
the electromagnetic
spectrum
(850
1400
nm).
Despite
the
reported
low
quantum
yield
(QY)
of SWCNTs
(typically
1%),
their
stable
photoemission
spectra,
sharp
optical
transitions
(full
width
at half-maximum
180
200
cm
1
, or
20
nm),
and large
absorption
cross
section
(10
15
10
17
cm
2
/C atom)
can be advantageous
for biological
imaging
applications.
2,3
Moreover,
the
fact
that
SWCNT
photo-
emission
emanates
from
surface
bound
(and
hence
environ-
mentally
sensitive)
excitons
make
SWCNTs
excellent
scaffolds
for biosensing
with
single
molecule
sensitivity.
4,5
Other
members
of the
CNT
family,
including
DWCNTs
and
MWCNTs,
are nonfluorescent
because
the coaxial
geometry
of nested
nanotubes
facilitates
efficient
nonradiative
relaxation
from
otherwise
fluorescent
single
tubes.
6,7
Therefore,
DWCNTs
and
MWCNTs
are not a focus
of this
review.
Carbon
nanocones
(CNCs,
also known
as carbon
nanohorns)
encompass
another
class
of sp
2
hybridized
rolled
graphene
sheets
with
conical,
as opposed
to cylindrical,
geometry.
Although
they
are easier
to synthesize
than
CNTs,
and have
been
used
as nanohybrids
in conjunction
with
other
fluorescent
nanomaterials
and
dyes,
CNCs
do not
have
intrinsic
fluorescence
of their
own
and are therefore
not a
focus
of the later
sections
of this review.
8
Carbon
dots (CDs)
refer
to a major
class
of CNMs
that also
includes
carbon
quantum
dots
(CQDs)
and
carbonized
polymer
dots
(CPDs)
and
are an important
focus
of this
review.
9
CDs
are quasi
zero-dimensional,
spherical
CNMs,
with
diameters
that are in the range
of
1
10
nm. They
can
be synthesized
from
a wide
range
of precursor
materials,
are
intrinsically
fluorescent,
and exhibit
diverse
photophysical
and
material
properties
that are functions
of the carbon
source
and
the synthetic
strategy
used
to produce
them.
Indeed,
the latest
synthetic
strategies
can now
furnish
bright
CDs
with
quantum
Chemical
Reviews
pubs.acs.org/CR
Review
https://doi.org/10.1021/acs.chemrev.3c00581
Chem.
Rev.
2024,
124,
3085
3185
3086
yields
up to 80%,
and highly
tunable
and stable
photoemission
ranging
from
blue to NIR,
from
a wide
range
of abundant
low-
cost
source
materials.
Relative
to other
fluorescent
CNMs,
CDs
permit
a better
degree
of control
and ease
over
their
synthesis
and purification,
which
has enabled
the generation
of
CDs
exhibiting
a wide
range
of photophysical
and chemical
properties.
This
diversity
has also led to the use of CDs
in a
wide
range
of applications,
including
bioimaging,
which
we
extensively
explore
in this review.
9
Carbon
nanohoops
(CNHs)
constitute
the smallest
and
newest
class
of fluorescent
CNMs
discussed
in this review.
CNHs
are composed
of aromatic
rings
that
are fused
to
generate
a macrocyclic
structure
that
resembles
the smallest
slice
of a SWCNT.
CNHs
are unique
among
fluorescent
CNMs
in that
they
are synthesized
bottom
up from
small
molecule
precursors
using
strategies
that benefit
from
advances
in modern
synthetic
organic
chemistry,
including
precise
control
over
molecular
structure,
excellent
characterization,
and purification
to produce
monodisperse
products
with
well-
behaved
photophysical
and chemical
properties.
As the newest
member
of fluorescent
CNMs,
applications
of CNHs
for
bioimaging
are still in their
infancy,
but early
results
have
been
highly
encouraging,
and
weexplore
these
advances
in the
review.
Small
molecule
organic
fluorophores
and
fluorescent
proteins
(FPs)
still constitute
the primary
reagents
of choice
in scientific
research
where
imaging
or sensing
is employed.
There
are several
reasons
for this.
These
reagents
are better
characterized,
are
monodisperse
(pure),
and
therefore
generally
well behaved
compared
to fluorescent
CNMs.
They
are also optically
compatible
with
most
commercially
available
microscopes.
FPs
are typically
expressed
through
common
genetic
strategies
widely
available
to experimental
biologists,
which
facilitates
their
ease
of use.
Similarly,
some
organic
fluorescent
dye
reagents
are
straightforward
in their
application.
However,
as we highlight
in this review,
there
are some
unique
advantages
that
fluorescent
CNMs
provide
that make
them
a rational
or only
choice
for certain
biological
applications.
First,
the optical
properties
of fluorescent
CNMs
can be
quite
advantageous
for applications
in biology.
A commonly
encountered
theme
in the emissive
properties
of all CNMs
is a
remarkable
photostability,
with
some
fluorescent
CNMs
exhibiting
nonphotobleaching
fluorescence.
Additionally,
Figure 1.
Carbon
nanomaterial
(CNM)
types
and their
structures.
(A) Pristine
carbon
nanotubes
are cylindrical
nanocrystals
of sp
2
hybridized
carbon
atoms.
(B) Carbon
dots are quasi-spherical
nanoparticles
with
a mix of sp
2
and sp
3
carbon
atoms
and contain
a variety
of functional
handles.
(C) Carbon
nanocones
represent
sp
2
carbon
atoms
rolled
into a conical
geometry.
(D) Carbon
nanohoops
can be conceptualized
as a single
slice of
a carbon
nanotube.
(E, F) Pristine
graphene
is a 2-dimensional
material
made
of sp
2
carbon
atoms
in a honeycomb-like
arrangement,
whereas
graphene
oxide
contains
a mix of sp
2
and sp
3
carbon
atoms
and features
various
functional
moieties.
Chemical
Reviews
pubs.acs.org/CR
Review
https://doi.org/10.1021/acs.chemrev.3c00581
Chem.
Rev.
2024,
124,
3085
3185
3087
emission
is highly
tunable,
broadly
encompassing
the visible,
NIR,
and
SWIR
regions
of the spectrum.
A dearth
of
fluorophores
that emit
in the NIR/SWIR
means
that CNMs
could
be compelling
reagents
of choice
for imaging
and
biosensing
in that region
of the spectrum.
Second,
thanks
to a
unique
combination
of their
small
size,
and
surface
and
mechanical
properties,
some
CNMs
are able to reach
and enter
cell
and
tissue
types
that
otherwise
are inaccessible
to
traditional
probes,
enabling
applications
in neural
tissues
and
plant
organelles
for instance.
Third,
preparation
of most
fluorescent
CNMs
does
not require
sophisticated
synthesis
and
purification
skills,
and these
materials
can be produced
at scale
and low cost compared
to other
laboratory
reagents.
Lastly
and
importantly,
fluorescent
CNMs
facilitate
multiplexed
use cases,
in which
the nanomaterials
can be functionalized
with
contrast
agents
that
allow
orthogonal
imaging
modalities,
or can be
loaded
with
therapeutics,
drugs,
or biomolecules,
such
as genes
and proteins,
for delivery
into cells.
Compared
to fluorescent
nanomaterials
synthesized
from
heavy
metals,
CNMs
are
biocompatible,
and their
abundant
functional
handles
can be
ligated
to fine-tune
their
biointerfacial
properties.
Although
not
in the scope
of this review,
CNMs
have
also found
applications
in a diverse
range
of the scientific
enterprise,
including
electrochemical
sensing,
optoelectronics,
catalysis,
and energy
storage.
In this
paper,
we provide
a review
of the synthesis,
functionalization,
characterization,
and material
properties
of
the CNMs
that
we introduced
in the preceding
paragraphs.
Subsequently,
we explore
the applications
of fluorescent
CNMs
in biological
imaging,
molecular
sensing,
and
cargo
delivery
both
in biomedical
and environmental
science
and
engineering.
We
conclude
the review
by discussing
the
important
topics
of CNM
cytotoxicity,
environmental
accumulation,
and fate,
and their
scale-up,
economical,
and
regulatory
considerations,
all of which
are critical
factors
for
the successful
translation
of CNMs
from
the lab to clinical
and
field
applications.
This
review
mostly
covers
advancements
made
in the last five
years,
with
relevant
comprehensive
reviews
suggested
for earlier
studies
for interested
readers.
However,
older
literature
are discussed
in cases
where
new
literature
is unavailable,
or the earlier
literature
still represent
the most
significant
advancements
for the topic
at hand.
2. CARBON
NANOMATERIAL
SYNTHESIS
AND
CHARACTERIZATION
In this
section,
we discuss
synthesis
and
characterization
methods
for CNMs
briefly
introduced
in the previous
section.
We
discuss
material
properties
in Section
3, chemical
modifications
in Section
4, and biological
and environmental
applications
in Sections
5 and 6.
2.1. Carbon
Nanotubes
(CNTs)
Carbon
nanotubes
(CNTs)
are nanocrystalline
materials
that
are composed
of a hexagonal
sp
2
hybridized
network
of carbon
atoms
that are rolled
into a cylindrical
form
(Figure
1A).
They
can contain
single,
double,
or multiple
coaxial
layers
resulting
in either
single-walled
(SWCNTs),
double-walled
(DWCNTs),
or multiwalled
carbon
nanotubes
(MWCNTs).
CNTs
can be synthesized
through
various
methods,
with
the
three
primary
modes
of synthesis
being
chemical
vapor
deposition
(CVD),
10
12
arc discharge,
13
and laser
ablation.
14
In contrast
to DWCNTs
and MWCNTs,
SWCNTs
possess
intrinsic
and unique
photophysical
properties,
and have
been
extensively
employed
for biosensing
and imaging
applications
and will therefore
be one of the primary
CNMs
discussed
in
this review.
Since
their
first discovery
in the late 20th
century,
SWCNTs
have
drawn
interest
from
a wide
range
of scientific
fields
due to
their
mechanical,
chemical,
electrical,
and
optical
proper-
ties.
15
17
While
SWCNTs
typically
have
a diameter
of 1
3
nm, DWCNTS
and MWCNTs
can have
a broader
diameter
distribution
ranging
from
2 to 100 nm.
18
Depending
on the
structure
of the graphitic
lattice,
SWCNTs
can be categorized
into
three
groups:
armchair,
zigzag,
or chiral.
19
SWCNT
electronic
band
gap
structure
is critical
for setting
their
electronic
and
optical
properties.
For
instance,
certain
SWCNTs
can serve
as field
effect
transistors,
20
and tracking
change
in electrical
or optical
properties
across
the nanotube
in
the presence
of adsorbed
molecules
can provide
a means
for
molecular
sensing.
Also
a consequence
of their
electronic
bandgap
structure,
certain
SWCNT
chiralities
exhibit
photo-
luminescence
by absorbing
light
in the NIR-I
and emitting
in
the NIR-II
region.
This
makes
them
excellent
reagents
for
biological
imaging
21,22
and
scaffolds
for biosensing
applica-
tions.
23,24
SWCNTs
have
also been
employed
as photoinduced
drug
delivery
vessels,
25,26
gene
and
protein
delivery
vehicles,
27,28
nanopores,
29,30
adjuvant
vaccines,
31
and
are
used
in tissue
engineering
applications.
32
34
2.2. Carbon
Dots
(CDs)
Carbon
dots
(CD),
quasi-spherical
in nature,
are typically
smaller
than
10 nm in diameter
and encompass
a collection
of
nanoparticles,
such
as graphene
quantum
dots
(GQDs),
carbon
quantum
dots
(CQDs),
and carbonized
polymer
dots
(Figure
1B).
35
CDs
have
been
extensively
used
in various
fields
due to their
tunable
photoluminescent
(PL)
properties,
35
38
chemical
diversity,
39,40
and biocompatibility.
41,42
Synthesis
routes
consist
of top-down
and
bottom-up
approaches.
For
bottom-up
synthesis,
polymers,
43,44
glu-
cose,
45,46
glycerol,
47,48
biowaste,
49,50
amino
acids,
51,52
among
others
have
been
used
to create
surface-functionalized
CDs
via
hydrothermal
methods
or microwave
pyrolysis.
Top-down
methods
for CD synthesis
require
cleavage
of larger
carbon
allotropes
like graphite,
53
57
GO,
58
carbon
fibers,
59
and other
carbon
materials.
60,61
Recently,
there
has also been
a growing
literature
on the “green
synthesis”
of CDs
from
biological
organisms.
62,63
The popularity
of CDs
for bioimaging
have
been
attributed
to their
unique
photoluminescence
properties
and
high
quantum
yields.
64,65
Their
π
-conjugated
system
absorbs
UV
light
and provides
emission
of visible
light
facilitated
by both
n
π
*
and
π
π
*
transitions
of C=N
and C=O,
and C=C
bonds,
respectively.
66,67
Many
studies
suggest
that N doping
(adding
nitrogen)
of CDs
leads
to higher
quantum
yields.
67
These
inherent
photoluminescence
properties
and biocompat-
ibility
of CDs
make
them
ideal
fluorescent
probes,
where
they
have
been
utilized
to image
cells,
68,69
biomolecules,
70
and
various
other
biological
systems.
71
73
2.3. Carbon
Nanocones
(CNCs)
and Carbon
Nanohoops
(CNHs)
Beyond
the aforementioned
CNMs,
carbon
nanocones
and
nanohoops
have
gained
increasing
attention
for their
distinctive
physicochemical
properties
(Figure
1C,D).
Although
their
applications
in biological
research
are less
developed,
these
materials
hold
a promising
potential
for
biosensing,
bioimaging,
and therapeutics.
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Table 1. Comparison
of CNM Characterization
Methods
Method
Typical
Information
Provided
Considerations
for Use
Ref
FTIR
Chemical
functional
groups
pre- and post-modification
for all
CNMs
Non-destructive,
real-time,
simple,
and fast
1, 39, 187
Availability
of extensive
reference
spectra
Requires
relatively
large
amount
of sample
Does
not provide
quantitative
information
Peaks
can be hard
to distinguish
from
background
in some
CNMs
(e.g.,
pristine
C
60
)
Raman
CNTs:
chirality,
diameter,
defects
Diverse
information,
simple,
non-destructive
138,
142,
188
190
CDs,
CNHs,
CNCs:
band
gap
The ratio
of D- and G-bands
can give quantitative
measure
of
defect
density
Graphene:
layers
and defects
Spectra
can be hard
to deconvolute
given
limited
availability
of
reference
spectra
GO:
chemical
structure,
doping,
band
gap
Higher
spatial
resolution,
wider
field
of view,
and faster
scan
rates
are needed
XPS
Surface
elemental
composition
and chemical
environment
of
surface
species
(e.g.,
CF vs CF
2
)
Higher
sensitivity
compared
to FTIR
191
195
Provides
quantitative
composition
information
Requires
relatively
large
sample
amount
and ultrahigh
vacuum
conditions
Deconvolution
of peaks
can be ambiguous
and requires
prior
insight
on functional
groups
present
NMR
CNTs,
CNHs,
CNCs:
chemical
structure
Non-destructive
analysis
of both
solid
and liquid
samples
80, 149,
196
200
CDs:
chemical
modification
and purity
Quantitative
chemical
composition
results
High
resolution
at the atomic
level
Difficult
to determine
structure
in large
and complex
CNMs
due to
many
peaks
Low
isotopic
abundance
of
13
C limits
sensitivity
SAXS
SWCNTs:
morphology,
diameter
Small
sample
amounts
needed
and fast
153,
154,
201
203
MWCNTs:
nanotube
alignment
Quantitative
characterization
of metastable
systems
with
multiple
conformations
Graphene,
GO:
molecular
mass
and physical
properties
Cannot
reconstruct
3D structure
from
1D data
and only
offers
surface-level
insights
Lower
resolution
compared
to electron
microscopy
Often
requires
use of a synchrotron
facility
SANS
Structure,
morphology,
porosity,
total
internal
surface
of CNMs
Higher
penetration
compared
to SAXS,
better
suited
for
multilayered
CNMs
(e.g.,
MWCNTs)
15, 51, 58,
204
Preserves
sample
integrity
Higher
contrast
between
CNM
and solution
Often
requires
use of neutron
facilities
that are sparsely
available
Measurement
time
can be long
AFM
Surface
morphology,
size and height
of CNMs
pre- and post-
modification,
determination
of cargo
loading
Analysis
of both
solid
and liquid
samples
159,
205
209
Higher
resolution
than
SEM,
providing
3D surface
topography
at
nm lateral
and sub-Å
vertical
resolution
No need
for vacuum,
non-destructive
Lower
scanning
areas
(
μ
m
2
) than
electron
microscopy
Slow
scan
speeds
SEM
Surface
morphology
of all CNMs
Can scan larger
area (mm
2
) than
AFM
and has large
depth
of field,
suitable
for imaging
rough
samples
206,
207,
210
213
Can
be combined
with
other
approaches
to provide
elemental
composition
analysis
Has lower
resolution
than
AFM
and cannot
provide
3D
information
CNMs
typically
have
low contrast
in electron
microscopy
compared
to other
nanoparticles
Requires
vacuum
conditions
STM
Surface
morphology
of conductive
and semiconductive
CNMs
Provides
sub-angstrom
resolution
in all three
dimensions
167,
214
216
Requires
conductive
or semiconductive
samples,
problems
when
π
-
conjugation
is disrupted
(e.g.,
GO)
Requires
vacuum
conditions
TEM
CNTs:
inner
and outer
tube
morphology
Can
be combined
with
other
approaches
to provide
elemental
composition
analysis
217
220
CDs:
size,
graphene
lattice
spacing
Can
give crystal
structure
information
CNCs:
surface
morphology
High
spatial
resolution
of 0.05
nm
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Carbon
nanocones
(CNCs),
also known
as nanohorns,
are
comprised
of carbon
atoms
arranged
within
a highly
conjugated
C
C
π
-system
akin to CNTs
and graphene
sheets.
CNCs
have
a diameter
of 2
5
nm and length
of 40
50
nm.
74
Diverging
from
CNTs
and graphene,
CNCs
have
one
end
enclosed
and the other
end open,
embodying
the shape
of an
ice cream
cone
(Figure
1C).
They
can be synthesized
by
various
processes
depending
on the desired
size,
including
cascade
annulation,
75
and
laser
and
solar
radiation
abla-
tion.
76,77
The
potential
applications
of CNCs
include
biosensing,
bioimaging,
therapeutics,
and cargo
delivery.
Carbon
nanohoops
(CNHs)
belong
to a new class
of CNMs
and can be thought
of as singular
cross
sections
of CNTs
(Figure
1D).
CNHs
are composed
of several
aromatic
rings
fused
together
to form
a closed
conjugated
π
-system
that
resembles
a macro-ring
structure.
Although
nanohoops
emerged
theoretically
in 1954,
their
synthesis
was not feasible
until
2008,
when
Jasti,
Bertozzi,
and colleagues
synthesized
[9],
[12],
and [18]-cycloparaphenylenes
([
n
]-CPPs).
78
This
groundbreaking
CNHs
synthesis
has been
followed
up by
various
innovative
approaches
leveraging
transition
metals
to
execute
reductive
eliminations
for formation
of highly
strained
CPP
macrocycles.
79
86
Unlike
many
other
π
-conjugated
CNMs,
nanohoops
have
radially
oriented
π
-systems
yielding
unique
optical,
electronic,
and charge
transport
properties,
87
making
them
attractive
for select
bioimaging
applications.
88,89
In terms
of optical
properties,
smaller
CNHs
demonstrate
red-
shifted
fluorescence
due to the narrowing
of the HOMO
LUMO
(highest
occupied
molecular
orbital
lowest
unoccu-
pied
molecular
orbital)
gap.
90
In addition
to size,
electron
donating
and accepting
rings
also change
fluorescent
emission
properties
via solvent-molecule
interactions
and
improve
quantum
yields.
91
2.4. Graphene,
Graphene
Oxide
(GO),
Reduced
Graphene
Oxide
(RGO),
and Graphene
Nanoribbons
(GNRs)
Graphene
made
its debut
in 2004
92
and the pioneering
work
of
Geim
and Novoselov
in graphene
physics
was recognized
with
the 2010
Nobel
Prize.
Graphene
has
unique
electronic,
magnetic,
optical,
and thermal
properties
that make
it suitable
for a wide
range
of applications.
93
97
Composed
of a single
layer
of hexagonal
sp
2
hybridized
carbon
atoms
arranged
in a 2-
dimensional
(2D)
sheet,
graphene’s
highly
conjugated
π
-
system
is responsible
for its electronic
properties
(Figure
1E).
Its zero-bandgap
enables
effective
electron
conduction
at
relativistic
speeds,
98,99
making
graphene
excellent
for electro-
chemical
processes.
100
102
Graphene
is the thinnest
and
strongest
nanomaterial
to date
with
atomic
thickness
and
mechanical
stiffness
of 1060
GPa.
103,104
Graphene
is synthesized
via two main
synthetic
routes.
Top-
down
approaches
include
mechanical
and
chemical
exfolia-
tion,
92,105
unzipping
of carbon
nanotubes,
106
108
and chemical
synthesis,
109,110
which
are typically
used
to synthesize
smaller
graphene
lattices
(nm
up to cm length).
For
bottom-up
approaches,
CVD
111
113
and
epitaxial
growth
114
116
are
preferred
to synthesize
larger
graphene
lattices
(up to several
cm in length).
GO
is comprised
of a graphene
parent
structure,
and
additionally
contains
hydroxyl
(
OH)
and epoxide
functional
groups
(C
O
C)
on the longitudinal
plane,
and carbonyl
oxygens
(=O),
ethers
(
O
),
and carboxylic
acids
(O=C
OH)
at the
edges
117,118
(Figure
1F).
These
chemical
modifications
contribute
to GO’s
solubility
in polar
protic
and
polar
aprotic
solvents,
119
and
give
rise
to photo-
luminescence
properties
that
broaden
its applications.
GO’s
decoration
with
oxygen-rich
moieties
also results
in a p-doping
effect
and lowers
its Fermi
level,
which
facilitates
development
of artificial
optoelectronic
systems
that
mimic
naturally
occurring
biological
phenomena.
120,121
Other
common
appli-
cations
of GO
include
drug
delivery,
122,123
antimicro-
bials,
124,125
fluorescent
probes
for biological
sensing,
126
and
cancer
biomarker
detection.
127
Until
recently,
synthesis
and homogeneous
functionalization
of highly
crystalline
GO
was
not feasible,
where
synthesis
mostly
relied
on the direct
oxidation
of graphite
to produce
graphite
oxide
followed
by an exfoliation
process.
128
High
degrees
of crystallinity
translate
to decreased
amounts
of defect
sites
and thus
improved
electrical
conductivity
and resistance
to oxidation.
Toward
this goal,
a route
for highly
crystalline
GO synthesis
has recently
been
developed,
achieving
a >99%
monolayer
ratio
with
uniform
epoxy
modification
and minimal
lattice
defects,
117
advancing
the robust
use of GO in many
applications.
Even
though
this approach
currently
only
works
for epoxy
modification,
its translation
to other
surface
modifications
with
high
crystallinity
will be highly
enabling.
Another
recent
CNM
of interest
is graphene
nanoribbons
(GNRs).
These
small
strips
of graphene
typically
have
width
to
Table 1. continued
Method
Typical
Information
Provided
Considerations
for Use
Ref
Graphene,
GO:
lattice
spacing,
surface
morphology
2D image
can offer
insights
on size and lattice
spacing
but cannot
give 3D structure
CNMs
typically
have
low contrast
in electron
microscopy
compared
to other
nanoparticles
Requires
vacuum
conditions
UV
vis-
IR
Absorption
and emission
spectra,
quantum
yield,
photophysical
properties
for optically
active
CNMs,
and purity
Non-destructive,
real-time,
simple,
and fast
221
226
Equipment
readily
available
for UV
vis
region
NIR
region
requires
expensive
equipment
for characterization
(e.g.,
SWCNTs)
DLS
and
ZETA
Size and surface
zeta potential
of CNMs,
dispersity
and colloidal
stability
of CNMs
Real-time,
simple,
and fast
227
232
Nondestructive
for hydrodynamic
size,
but destructive
for zeta
potential
measurement
Colored
or fluorescent
samples
may skew
the results,
though
there
are newer
equipment
available
to overcome
this
Can
only
be used
for spherical
CNMs
for accurate
measurement,
but algorithms
could
be adjusted
for other
shapes
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length
ratios
of 1:10,
and with
recent
advancements
in GNR
synthesis,
widths
less than
10 nm have
been
achieved.
129
The
GNR
band
gap is governed
by its size,
making
narrow
GNRs
desirable.
Similar
to other
CNMs,
top-down
synthesis
methods
involve
the fragmentation
of larger
carbon
allotropes,
such
as
CNTs
and graphene.
Specifically,
CNTs
can undergo
plasma
etching,
where
a localized
and controlled
exposure
to plasma
induces
their
unzipping
and facilitates
the formation
of smaller
GNRs.
130
Other
synthesis
approaches
include
lithographic
131
and sonochemical
132,133
methods
for the formation
of GNRs
from
graphene,
which
also requires
etching
for the controlled
removal
of atoms
from
higher
ordered
graphene.
Bottom-up
synthesis
of GNRs
has been
reported.
Halogenated
aromatic
substrates
are some
of the first chemical
precursors
to be used
for bottom
up GNR
synthesis.
134
These
chemical
moieties
provide
avenues
for the selective
polymerization
of smaller
aromatic
building
blocks
via radical
addition
reactions
at high
temperatures.
More
recently,
novel
methods
for the synthesis
of GNRs
exploited
the use of transition
metals
for the selective
polymerization
of smaller
building
blocks,
135,136
offering
tighter
control
for the formation
of thinner
GNRs.
2.5. CNM
Characterization
Methods
In this
section,
we describe
the
most
prevalent
CNM
characterization
techniques
including
methods
used
for
chemical
identification,
morphological,
structural,
optical,
size,
and surface
charge
characterization.
Table
1 provides
a
comparative
overview
of these
techniques
and their
limitations
to guide
readers
to choose
the most
suitable
characterization
method
for a given
CNM
and property
type.
2.5.1.
Chemical
Identification.
Methods
for characteriz-
ing CNM
chemical
identity
include
spectroscopic
approaches
such
as Fourier-Transform
Infrared
(FTIR)
and Raman.
FTIR
measures
the vibrations
of atoms
and bonds
when
they
absorb
infrared
light
(IR)
at various
wavelengths
providing
information
about
the chemical
functional
groups
present
within
a material
(Figure
2A).
Even
though
it is more
common
for the characterization
of CDs,
137
CNTs,
138
and GO,
139
it can
also be used
with
other
CNMs
as a standard
characterization
tool.
140,141
Another
technique
that relies
on IR and vibrational
frequencies
to provide
a molecular
fingerprint
is Raman
spectroscopy.
Raman
utilizes
scattering
of light
(instead
of
absorbance
in FTIR)
to measure
intrinsic
chemical
properties
of CNMs,
and can provide
information
on the mass
density,
optical
energy
gap, elastic
constants,
doping
levels,
presence
of
defects,
and
other
forms
of crystal
disorder.
It also
offers
insights
into
the edge
structure,
strain,
number
of graphene
layers,
nanotube
diameter,
chirality,
and curvature
of CNMs
(Figure
2B).
142
FTIR
and Raman
distinguish
different
bond
types,
each
with
unique
limitations
and strengths.
Therefore,
they
provide
a comprehensive
chemical
identification
of
CNMs
when
used
in combination.
Other
common
methods
for chemical
identity
character-
ization
of CNMs
include
X-ray
photoelectron
spectroscopy
(XPS)
and nuclear
magnetic
resonance
(NMR).
XPS
enables
quantitative
analysis
of elemental
composition
of most
CNMs.
It uses high
energy
X-ray
photons
to ionize
electrons
within
an
atom
to provide
data
on electron
binding
energies
associated
with
specific
atoms
within
the specimen.
For this reason,
XPS
is one of the standard
methods
of characterization
of most
CNMs
and their
surface
modifications
(Figure
3A).
27,143,144
In
addition
to XPS,
NMR
spectroscopy
allows
for the character-
ization
of the molecular
structure,
specifically
of carbon
and
hydrogen
atoms
via
13
C NMR
and
1
H NMR,
and can offer
information
on certain
surface-modified
CNMs
(CDs,
CNTs)
145
147
and
their
relative
purity
(Figure
3B).
148
However,
one major
limitation
is the difficulty
of interpreting
properties
of the whole
CNM
being
analyzed
given
their
large
and
complex
carbonaceous
structures.
Therefore,
relatively
small
CNMs,
such
as nanohoops,
are more
frequently
characterized
via NMR.
80,149
2.5.2.
Morphological
and Structural
Characterization.
Small-angle
neutron
scattering
(SANS)
and small-angle
X-ray
scattering
(SAXS)
are powerful
techniques
that
utilize
the
Figure 2.
FTIR
and Raman
characterization
of different
preparations
of SWCNTs.
(A) FTIR
spectra
for raw SWCNT
soot
(a), dry oxidized
(b),
H
2
O
2
refluxed
(c), purified
material
via HCl
and high-temperature
vacuum
anneal
(HTVA)
treatment
at 1100
°
C (d), and purified
material
via
HNO
3
and HTVA
treatment
at 1100
°
C (e). The top two spectra
on purified
SWCNTs
represent
the cleanest
material.
(B) Raman
spectra
for the
same
SWCNT
types
from
(A) showing
R-band
(100
300
cm
1
) region
(left panel)
and D- and G-band
region
(1230
1750
cm
1
) (right
panel).
Reproduced
from
ref 138.
Copyright
2005
American
Chemical
Society.
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3091
diffraction
of high
energy
particles
to investigate
the atomic
and magnetic
structures
of CNMs.
Both
techniques
leverage
the wave
characteristics
inherent
to particles
to analyze
the
diffraction
patterns
according
to Bragg’s
law, thereby
obtaining
information
about
the arrangement
and organization
of atoms
in the material’s
inner
and outer
layers.
SAXS
is particularly
well-suited
for studying
the external
surfaces
of CNMs,
making
it ideal
for analyzing
single-layered
materials,
such
as graphene
and SWCNTs
(Figure
4A).
153,154
On the other
hand,
SANS,
by using
neutrons,
possesses
greater
penetration
capabilities
and
is less
destructive,
allowing
the investigation
of bulk
materials
and
determination
of internal
structures
without
causing
significant
decomposition
(Figure
4B).
Consequently,
SANS
is especially
useful
for exploring
the chemical
structures
deeply
embedded
within
nanomaterials
like
MWCNTs.
155
While
SAXS
and SANS
provide
distinct
benefits,
they
also
complement
each
other
by offering
insights
on chemical
structures
in different
regions
of CNMs.
Therefore,
researchers
often
employ
both
techniques
in tandem
to gain
a
comprehensive
structural
understanding
of CNMs.
However,
it must
be noted
that SANS
does
have
an advantage
over SAXS
as it provides
higher
contrast
between
the sample
and
its
solvent
through
contrast
matching.
In SAXS,
contrast
matching
is also possible
but requires
the chemical
modification
of the
nanoparticles
making
it more
challenging
to perform.
156
Other
techniques
utilized
for morphological
characterization
of CNMs
are atomic
force
microscopy
(AFM),
scanning
electron
microscopy
(SEM),
scanning
tunneling
microscopy
(STM),
and transmission
electron
microscopy
(TEM).
AFM
measures
the intermolecular
forces
between
a sharp
probe
and
a sample
to afford
an image
of the surface
of CNMs.
AFM
is
routinely
used
for CNTs,
159
CDs,
160
graphene,
161
and GO
162
to provide
high
resolution
images
of CNM
surface
at the
nanometer
and even
atomic
scale
and is commonly
used
to
verify
the loading
of macromolecules
(Figure
5A).
SEM
focuses
a high
energy
electron
beam
to a sample
to measure
the secondary
electron
emissions.
It is commonly
employed
for
the characterization
of CNTs,
163
CDs,
164
graphene,
161
and
GO
165
and provides
a detailed
analysis
of surface
features,
such
as roughness,
texture,
and the presence
of defects
(Figure
5B).
STM
also visualizes
surface
characteristics
of CNMs
by using
a
sharp
conductive
probe
positioned
closely
to a sample,
which
tunnels
electrons
via quantum
tunneling
when
a bias voltage
is
applied.
Consequently,
small
aberrations
in the tunneling
Figure 3.
XPS
and NMR
characterization
of CNMs.
(A) XPS
spectra
of (a) unmodified
SWCNT
C 1s, (b) carboxylated
C 1s, (c) amide-
functionalized
C 1s, (d) amine-functionalized
C 1s, (e) amide-functionalized
N 1s, (f) amine-functionalized
N 1s. Reproduced
from
ref 150.
Copyright
2005
American
Chemical
Society.
(B) (Top
panel)
1
H NMR
spectrum
of Methylene-Bridged
[6]CPP.
Reproduced
from
ref 151.
Copyright
2020
American
Chemical
Society.
(Bottom
panel)
Solid-state
NMR
spectra
of polyethylenimine
(PEI)-functionalized
SWCNTs
via
13
C
MAS
NMR
spectrum
with
a 12 kHz
spinning
speed.
Reproduced
from
ref 152.
Copyright
2008
American
Chemical
Society.
Chemical
Reviews
pubs.acs.org/CR
Review
https://doi.org/10.1021/acs.chemrev.3c00581
Chem.
Rev.
2024,
124,
3085
3185
3092