An evaporation induced self-assembly approach to prepare polymorphic carbon dot fluorescent nanoprobes for protein labelling

Article abstract of DOI:10.1039/c8cc05860a

Herein, we report a novel route to prepare polymorphic carbon dot fluorescent probes via the evaporation-induced self-assembly of glutaraldehyde and carbon dots, which first usually form carbon nanoclusters which then could self-assemble to form carbon nanocrystals, nanospheres or nanofibers in different ionic strength solutions at room temperature. The aldehyde functionalized polymorphic C-dot fluorescent probe can easily covalently bond with amino groups on proteins.

Full text of DOI:10.1039/c8cc05860a

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An evaporation induced self-assembly approach
to prepare polymorphic carbon dot fluorescent
nanoprobes for protein labelling†
Cite this: Chem. Commun., 2018,
54, 13123
Received 18th July 2018,
Accepted 29th October 2018
Lei Li, Zhongyu Lian, Xi Yan, Meng Xia and Mingcui Zhang
*
DOI: 10.1039/c8cc05860a
rsc.li/chemcomm
Herein, we report a novel route to prepare polymorphic carbon dot Meanwhile, toxicity has greatly limited the application of these
fluorescent probes via the evaporation-induced self-assembly of glu- optical probes in the biomedical field.
taraldehyde and carbon dots, which first usually form carbon nanoclus-
Fluorescent carbon dots (C-dots) as a new class of fluorescent
ters which then could self-assemble to form carbon nanocrystals, materials have demonstrated great potential for applications in
nanospheres or nanofibers in different ionic strength solutions at room direct imaging and biosensing,10 disease diagnosis,11 print ink,12
temperature. The aldehyde functionalized polymorphic C-dot fluores- photocatalysis,13 and photovoltaic devices.14 Compared to traditional
cent probe can easily covalently bond with amino groups on proteins. fluorescent probes, the C-dots have their unique properties including
significantly improved photostability, biological compatibility, drug
The exploration and development of highly sensitive analytical delivery and green economy.15–18 In particular, C-dots as a class of
methods for the detection of proteins, peptides, and other important green labels have been used in bioanalysis, including immuno-
biological molecules in vivo and in life is a hot and difficult topic in assays, western blots and immunohistochemistry, as they interact
biomedical research. Therefore, biomarker technology is an indis- with specific biological molecules such as antibodies, peptides and
pensable research method in this field. The importance of efficient adapters, coupled to increase the sensitivity of the assay.19,20 How-
and sensitive fluorescent nanoprobes in biomedical research ever, the coupling reaction usually causes fluorescence quenching
and practice has been rapidly increasing.1,2 Many optical probes due to the aggregation of C-dots,21–23 and thereby the sensitivity
including organic dyes, semiconductor quantum dots, and upcon- reduces. To overcome this shortcoming, researchers performed
version nanoparticles have been widely used for biological labelling various functionalization reactions of C-dots. Min-Jung Kang
and imaging.3–7 Most of these materials have unsatisfactory aspects. designed silica nanospheres encapsulating a quantum dot-layer
Organic dyes offer the advantages of high fluorescence quantum (SQS) to increase the quantum dot concentration and make them
yields (QYs) and wide commercial availability. Unfortunately, organic highly photoluminescent.24 Aharon Gedanken used the C-dots
dyes have poor photostability and solubility, which limit their further embedded in a polyvinyl alcohol (PVA) polymer to prepare bright
practical application in the field of chemical and biological analysis. C-dots and improve the stability of the C-dots.25 Dongxu Zhao
To solve these problems, semiconductor quantum dots such as CdSe prepared C-dot–NaCl hybrid crystals through a simple process to
and CdTe have received increasing attention in recent decades owing extend their light life.26 Wei-Lung Tseng described a bottom-up
to their highly tunable fluorescence properties and increased photo- assembly route for monodisperse C-dots into different sizes of C-dot
stability. However, the flashing phenomenon of these traditional aggregates through the control of the concentration of fatty acids
quantum dots still exists. Recently, rare earth dopant upconversion provided in bright C-dots.27 Nonetheless, the induce C-dot self-
nanomaterials (UCNPs) that offer high photostability and thermal assembly method based on coupling agents to amplify marker
stability have received considerable attention for applications in signals for protein labelling is yet reported.
sensing and bioimaging. Regrettably, synthesis of UCNPs is
Recently, a new self-assembly method evaporation induced self-
cumbersome.8,9 Photobleaching and photoflashing may result assembly (EISA) has attracted much attention. In this method, the
in the degradation of the reliability of the measured results. molecules are introduced to the surface from solution, thus their
conformation in the aqueous medium, which is mediated by
Key Laboratory of Functional Molecular Solids, Ministry of Education, Key
intramolecular forces and surrounding intermolecular hydrogen
bonds, can be significantly altered upon adsorption.28 This self-
Laboratory of Chemo Biosensing, College of Chemistry and Materials Science, Anhui
Normal University, Wuhu 241000, Anhui Province, China.
assembly behaviour needs to be studied at the hydrophobic inter-
E-mail: zhangmc@mail.ahnu.edu.cn
face, however, most of them are based on silicon wafers.29,30 When
† Electronic supplementary information (ESI) available: Experimental details and
supplementary figures. See DOI: 10.1039/c8cc05860a
silicon is used as the carrier medium, edge enrichment is observed
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as a result of the interplay of contact line pinning, solvent evapora- self-assembly of C-dots into polymorphic nanostructures under dif-
tion and capillary flow.31 Evidently, the preconcentration of this ferent conditions and drying through solvent evaporation. Basically,
constituent cannot provide a decentralized reaction environment. glutaraldehyde-mediated C-dot reactions generate C-dot nano-
However, the hydrophobic carbon film of this phenomenon has yet clusters at room temperature (Fig. S2, ESI†). Next, different concen-
report. Inspired by this phenomenon, as a reference, we studied the trations of a sodium chloride or phosphate buffer solution were
self-assembly behavior of C-dots on hydrophobic carbon films based added to the carbon nanocluster solution, respectively, and then
on glutaraldehyde-mediated C-dots. Compared to self-assembly in mixed uniformly. All samples were diluted 1000-fold on a copper grid
solution, these C-dots’ self-assembly method does not need addition and evaporated at room temperature. The carbon film on the copper
of a surfactant. Here we report a facile, safe and green synthesis of network not only provides a good hydrophobic environment for
C-dot nanoclusters using glutaraldehyde coupled C-dots at room evaporation-induced C-dot self-assembly, but more importantly, the
temperature. We chose glutaraldehyde for the following reasons. morphology of the self-assembled C-dots formed on the carbon film
Firstly, hydroxylamine is formed directly from the aldehyde groups can be observed using a transmission electron microscope (TEM)
and amino groups in alkaline solutions without stringent conditions. any time. Scheme 1 represents the carbon nanostructures formed
Secondly, upon evaporation of the solvent, the products formed by under different conditions. Scheme 1a: the C-dot nanoclusters
the aldehyde groups and amino groups are greatly dehydrated and exhibit a monodispersed spherical structure in a pH = 7.4 (sodium
tend to be stable, which is beneficial for the self-assembly of C-dots. hydroxide-adjusted PB solution) aqueous solution. Scheme 1b: main-
Most importantly, the C-dots self-assemble into a uniform mono- taining pH = 7.4, the carbon nanoclusters exhibit a uniform linear
disperse spherical structure in pH = 7.4 phosphate buffer and the structure when 0.1 M NaCl is added. Scheme 1c: maintaining pH =
aldehyde groups on the surface can further bind to the amino 7.4 and keeping 0.1 M NaCl constant, the carbon nanoclusters
groups.32 As a result, the evaporation-induced C-dot self-assembly exhibit a nanobelt-shaped structure when 0.01 PBS is added.
gives rise to different morphologies under different ionic strength Scheme 1d: maintaining pH = 7.4, and keeping 0.1 M NaCl and
conditions. Crucially, the complex can further bind to the amino 0.01 M PBS constant, the carbon nanoclusters exhibit a nanofiber
groups on the protein under approximately neutral conditions. This morphology when 0.1 M PBS is added. The formation mechanism of
goes especially well with antibody labelling and bioanalytical condi- glutaraldehyde, which mediates the self-assembly of single C-dot
tions. This makes C-dots a powerful fluorescent labelling tool in the nanoparticles before solvent evaporation, is not clear yet. We propose
biomedical field.
the possible growth pathways: firstly, the aldehyde-functionalized
In this study, we first demonstrate the formation of nano- single C-dots and the glutaraldehyde-mediated C-dot nanoclusters
structures with different morphologies on a carbon film, which may exist simultaneously in the solution. Secondly, different ionic
was induced by evaporation, and then the C-dot nanoclusters were strengths and pHs have an important role in the nucleation process
used for protein labelling. The C-dots were obtained through solvent- as the solvent evaporates.
thermal treatment. After the hydrothermal reaction, a light yellow
The carbon nanoclusters aggregate to form a uniform sphere
solution with blue emission under UV light was obtained, which under approximately neutral conditions and show a clear
indicates that the C-dots have been successfully prepared (Fig. S1, morphology, which can be clearly seen from the embedded
ESI†). Scheme 1 schematically depicts the entire process of the graph (Fig. 1a). The high-crystallinity carbon spheres are formed
by evaporation-induced C-dot self-assembly under approximately
neutral conditions. It is worth noting that there is a layer of an
organic protective layer (red arrow in Fig. 1a) around these
carbon spheres (blue arrow in Fig. 1a). This just confirms that
the self-assembly of the C-dots into spherical structures induces
self-assembly through solvent evaporation. This means the
hydrogen bond is the most stable when the pH of the solution
is under approximately neutral conditions.
When the solvent is left, the distance between the C-dots is pulled
closer by the force of the hydrogen bond and they finally assembled
into a highly crystalline carbon nanosphere. When the pH was
changed from neutral to weakly acidic (pH = 6.0), the C-dot self-
assembly morphology was still spherical but gradually blurred.
Because the hydrogen bond is very unstable under acidic conditions,
the final formation is loose self-assembled C-dots; and the other
reason for the loose C-dots might be due to the pH responsiveness of
the coupled CQN bonds33 (Fig. S3, ESI†). The appearance of this
phenomenon drove us to study the effect of ion intensity. We found
that when 0.1 M NaCl was added to the reaction system and stirred
overnight after mixing at 4 1C, the C-dots exhibited a carbon fiber
Scheme 1 Proposed mechanism for the self-assembly of C-dots nano-
structure drying through solvent evaporation. a–d represent the C-dot
morphology which could be seen by TEM imaging. As shown in
Fig. 1b, the structure of the nanowire grows toward both ends
nanostructures formed under conditions of pH = 7.4, 0.1 M NaCl, 0.01 M
PBS and 0.1 M PBS, respectively.
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sp3-hybridized carbon) and G (ordered sp2-hybridized carbon) bands,
respectively, of the carbon materials. Raman spectra of the self-
assembly of the C-dots under conditions of pH (b), NaCl (c), 0.01 M
PBS (d) and 0.1 M PBS (e) are included in Fig. 2 for comparison. It
was observed that all of these self-assembly of the C-dots under
conditions of pH, NaCl, 0.01 M PBS and 0.1 M PBS contain D and G
bands, respectively. The energy dispersive X-ray (EDX) spectrum
also shows different self-assembly conditions with composition of
elements (Fig. S5, ESI†). On the other hand, the C-dots only show the
signals of C, N and O (Fig. S5, ESI†). The size distribution of the
C-dots and the multi-morphological self-assembled C-dots are
shown in Fig. S6, ESI.† The multi-morphological self-assembled
C-dots were further confirmed by X-ray photoelectron spectroscopy
(XPS) (Fig. S7, ESI†). All means of characterization proved that the
observed nanostructure was actually a C-dot-assembly instead of a
residual salt in the C-dot solution after evaporation.
Of particular interest to us is when proteins are added to the pre-
prepared C-dot nanoclusters, the C-dot protein complexes are further
formed, meanwhile the proteins are labelled. This provides a rapid
labelling method for proteins at room temperature. In this experi-
ment, we chose to use antibodies (antibody titer is 1 : 64 000, Fig. S8,
ESI†) as the research object, because we can prove whether or not it is
labelled by the specific reaction of antigen antibodies. Before valida-
tion, we performed FTIR spectroscopy (Fig. S9, ESI†) and TEM
Fig. 1 TEM images obtained for the C-dot nanostructures dried from
water solutions under different conditions: (a) pH = 7.4, (b) 0.1 M NaCl, (c)
0.01 M PBS, and (d) 0.1 M PBS, respectively.
centered on the nanoclusters in the evaporated area. The possible imaging of the C-dot antibody complexes, respectively. The TEM
reason is the electrostatic interaction between the sodium ions and images of the C-dots, carbon nanoclusters, and C-dot antibody
the carbon nanoclusters, which increased the density of the carbon complexes can be clearly seen in Fig. 3. Fig. 3a indicates good
atoms. Finally, this increases the assembling efficiency and dispersibility of the C-dots with slight random agglomerations. Upon
stability.34 Fortunately, as the solvent is reduced, the C-dot nanoclus- adding glutaraldehyde under stirring at room temperature, the
ters cluster into nanowires at high concentrations of sodium chloride C-dots assemble to form larger C-dot nanoclusters (Fig. 3b). The
solution. The traces left by the solvent around the nanowires can be C-dot nanocluster labelled antibody form composite was also demon-
clearly seen in the TEM image. At the same time, we continued to strated by TEM images. As shown in Fig. 3c, the C-dot nanoclusters
explore the impact of phosphate buffer under the same processing and antibodies are coupled by glutaraldehyde to form uniformly
conditions. The results correspond to previous ideas. As shown in dispersed nanospheres. Finally, C-dot antibody complexes formed
Fig. 1c, the C-dot nanoclusters self-assemble into obvious nanorib- when the antibodies were added. More importantly, from the inset of
bons in 0.01 M phosphate buffer. From the two ends of the the TEM image shown in Fig. 3c, we can clearly see the formation of a
nanoribbon, it can be clearly seen that this nanoribbon is formed layer of protein corona on the surface of a C-dot nanocluster. This is a
by the accumulation of many nanofibers. Next, we studied the better demonstration of the successful labelling of the antibody with
influence of increase of the concentration of phosphate buffer, for
which we treated the C-dot nanoclusters with 0.1 M phosphate
buffer. It can be seen from Fig. 1d that the C-dot nanofibers became
bigger in size and both ends were in a divergent state when the
concentration of the phosphate buffer solution was increased.
Compared to the sodium chloride buffer solution, the C-dot nano-
fibers formed in the phosphate buffer with no obvious solvent
around it. Therefore, phosphate buffer plays a major role in the
formation of C-dot nanofibers compared to sodium chloride solu-
tions. Because phosphate is a cross-shaped needle crystal, it can be
attached as a C-dot nanocluster to form large-sized C-dot nanofibers
(Fig. S4, ESI†).
To confirm the components of the multi-morphological self-
assembled C-dots, they were characterized by Raman spectroscopy,
energy dispersive X-ray (EDX) spectroscopy, dynamic light scattering
(DLS) and X-ray photoelectron spectroscopy (XPS). Raman spectra
Fig. 2 Raman spectra of the original C-dots (a), the self-assembly of
of the original C-dots are shown in Fig. 2a. The peaks centered
C-dots under conditions of pH = 7.4 (b), 0.1 M NaCl (c), 0.01 M PBS (d) and
0.1 M PBS (e), respectively.
at ca. 1343 and 1540 cmÀ1 are attributed to the D (disordered
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antibody is 2.5 mg mLÀ1 (Fig. S10, ESI†). Further, we performed a
direct fluorescent immunoassay to demonstrate the utility of the
fluorescent probe, where the experiment group fluorescence intensity
was noticeably raised about 44 025 as shown in Fig. 3d (green
column), which is about 10 times that of the blank control group.
By contrast, when only C-dots were examined, no significant fluores-
cence signal was observed (Fig. 3d, blue column), indicating the good
specificity and selectivity of the proposed fluorescent immunoassay.
Thus, these comparison results indicate adequately that the prepared
probes via C-dots labelled anti-nano-drug deliver antibody could be
used to sensitively detect nano-drug deliver through the amplification
of signals. At the same time, the ultraviolet absorption spectrum also
provides a good proof (Fig. S11, ESI†).
In summary, combined with evaporation-induced self-assembly
techniques, green non-toxic C-dots and glutaraldehyde coupling agents,
we first studied the morphology of C-dot nanostructures by solvent
evaporation on carbon films. Then, the C-dot nanoclusters further
formed complexes with the protein at room temperature. We further
verified the fluorescence intensity of the C-dot antibody complex by a
fluorescence immunoassay, which is about 10 times that of the blank
control group. As can be observed from these preliminary results,
glutaraldehyde-mediated carbon nanoclusters are not only intermedi-
ates for the formation of multi-modal nanomaterials, but more impor-
tantly, they can also form complexes with proteins. This will have a very
beneficial impact on the field of biomarkers.
The authors gratefully acknowledge the financial support
from the National Natural Science Foundation of China (No.
21175004).
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13126 | Chem. Commun., 2018, 54, 13123--13126
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