Pyrene-imidazolium complexed graphene for the selective fluorescent detection of G-quadruplex forming DNA

Article abstract of DOI:10.1039/c3cc46744a

A unique system has been developed for quantifying G-quadruplex forming DNA down to picomolar levels for future applications in telomeric assessment. Pyrene-imidazolium captured in high amounts on the surface of reduced graphene oxide shells allows specific DNA sequence detection by complex formation resulting in release and fluorescence enhancement of pyrene-imidazolium.

Full text of DOI:10.1039/c3cc46744a

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Pyrene-imidazolium complexed graphene for the
selective fluorescent detection of G-quadruplex
forming DNA†
Seung Jin Moon,a So Hyeon Park,a Justyn Jaworski*bc and Jong Hwa Jung*a
Cite this: Chem. Commun., 2013,
49, 11698
Received 4th September 2013,
Accepted 22nd October 2013
DOI: 10.1039/c3cc46744a
www.rsc.org/chemcomm
A unique system has been developed for quantifying G-quadruplex length of the telomeric DNA. Such a system could provide
forming DNA down to picomolar levels for future applications in critical information on the replicative history of the cells,
telomeric assessment. Pyrene-imidazolium captured in high amounts thereby offering an indication of their fate.11
on the surface of reduced graphene oxide shells allows specific DNA
In the following work, we reveal a sensitive and selective strategy
sequence detection by complex formation resulting in release and for identifying DNA capable of forming a G-quadruplex. By speci-
fluorescence enhancement of pyrene-imidazolium.
fically making use of graphene encapsulation of silica nanoparticle
molds to provide a hollow graphene shell, we produced a high
It is widely accepted that the DNA–protein complexes, known as surface area platform for capture and fluorescence quenching
telomeres, have a key influence on the state and lifetime of normal of pyrene-appended imidazolium (referred to as SiO2@rGO-PI).
cells as well as cancers. Located at the end of the chromosome as a Importantly, liberation of pyrene-appended imidazolium (PI) from
capping sequence, the length and interactions between telomeric the graphene shell (SiO2@rGO) can occur in the presence of even
DNA may ultimately dictate cell growth and death.1 The guanine trace concentrations of G-quadruplex forming DNA. As a result, the
rich sequences of human telomeric DNA, for example (TTAGGG)n, PI bound complex with DNA emits a high fluorescence signal in
are of particular interest as these terminal sequences are capable of the solution, which we confirmed by spectral observation to be
forming G-quadruplex structures that may inhibit telomerase proportional to the concentration of G-quadruplex forming DNA.
extension of the repeating telomeric DNA, thereby allowing cell PI has previously shown several important properties thereby
death to occur.2 Stabilization of the G-quadruplex structure may inspiring its use for our detection scheme. For instance, PI will
prevent the unwanted characteristic immortality of cancer cells; selectively destabilize G-quadruplex forming dsDNA sequences
hence, G-quadruplex stabilizers are actively under research as resulting in complex formation with one of the ssDNA strands
anti-tumor therapeutics.3–5
to create a G-quartet structure (hydrogen bonding among four
While G-quadruplex DNA structures have been extensively guanine bases), where the formation of this unique structure
utilized as sensors due to their affinity for metal ions,6 very little induces fluorescence emission from the excited state complex.12
literature exists on the detection of G-quadruplex forming DNA In our system, this complex formation allows the PI to be released
itself.7,8 Estimation of telomeric DNA length typically requires from the graphene shell allowing fluorescence-enhanced detection
laborious digest methods to determine the mean terminal restric- that is both sensitive and selective to G-quadruplex forming DNA.
tion fragment length,9 or alternatively fluorescence in situ hybridiza- The confirmation resulting from G-quartet formation, as induced
tion has been used to obtain the relative number of DNA repeats but by PI, results in excimer enhancement of the pyrene moiety of PI as
only for a predetermined sequence.10 Therefore, a need still exists observed by the fluorescence peak at 550 nm.
for the development of a detection system with selectivity for the
Prior studies making use of graphene encapsulation of nano-
presence of quadruplex forming DNA, or more importantly the particles have provided functional, high surface area structures
with applicability in enantioselective separation systems,13 hybrid
biomaterials for bone growth,14 multifunctional probes15 and
a Department of Chemistry and Research Institute of Natural Sciences,
sensor development.16 The significant enhancement in the surface
Gyeongsang National University, Jinju, South Korea. E-mail: jonghwa@gnu.ac.kr;
Fax: +82-055-758-6027; Tel: +82-055-772-1488
area-to-volume ratio seen in graphene encapsulated silica nano-
particles has proven particularly useful for FET-type protein
sensors resulting in significantly improved detection limits.17
From a DNA sensor perspective, graphene has previously demon-
strated the capability to bind with ssDNA via non-covalent
interactions with aromatic nucleobases;18 however, quadruplex
b Department of Chemical Engineering, Hanyang University, Seoul, South Korea.
E-mail: justynj@hanyang.ac.kr; Fax: +82-02-2220-1935; Tel: +82-02-2220-2339
c Institute of Nano Science and Technology, 222 Wangsimni-ro, Seoul, South Korea
† Electronic supplementary information (ESI) available: Detailed experimental
and synthesis procedures as well as sequence details and XPS. See DOI: 10.1039/
c3cc46744a
c
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Fig. 1 (A) Schematic of the preparation method for SiO2@rGO-PI; (a) SiO2 particles, (b) amino group functionalized SiO2 particles, (c) rGO wrapped SiO2 particles,
(d) PI adsorbed onto SiO2@rGO and (e) PI released from SiO2@rGO. (B) Chemical structure of PI and the various DNA used in this study. (C) Fluorescence spectra
of PI, SiO2@rGO-PI, and also SiO2@rGO-PI in the presence of various DNA.
DNA, in contrast, was shown to exhibit dramatically decreased formation of SiO2@rGO allowing the creation of a high surface area
binding.19
probe for heterogeneous phase detection. Following encapsulation
In our system, p–p stacking interactions between the aro- of the silica nanoparticles by simple stirring, the GO was reduced to
matic surfaces of graphene and pyrene allow the capture of PI rGO through addition of hydrazine and the silica particle template
on the surface of the hollow graphene shell (Fig. 1). This close was removed by incubation under highly alkaline conditions. The
proximity to graphene results in a fluorescently quenched state resulting hollow structures were observed by TEM as shown in Fig. 2.
for the PI moiety. This ability of graphene to be a good energy In addition, GO wrapped on the surface of SiO2 particles was
acceptor has encouraged its use in a number of sensing platforms confirmed by TEM using an elemental mapping technique
where high fluorescence quenching efficiency is required.18 (Fig. S2, ESI†). Exposure of 600 pM of PI to the high surface
Disassociation of PI motifs from the surface was found to occur area rGO structures allowed capture of PI as facilitated by p–p
selectively after exposure to dsDNA possessing the characteristic stacking between the pyrene moieties and the rGO surface.
G-quadruplex forming sequence (XG3). As a result, significant A significant amount of PI was found to be captured on the
fluorescent enhancement was observed with the release of the surface by the presence of F1s and N1s peaks through XPS
PI–DNA complex. In brief, our results demonstrate a selective (Fig. S3, ESI†). When captured, there is essentially no observable
detection limit of quadruplex forming dsDNA as low as 20 pM fluorescence from PI in the rGO-bound quenched state (Fig. 1C). In
for a 10-mer of dsDNA. Interestingly, an increase in the fluores- contrast, a high fluorescence signal was observed, with the peak
cence signal was found when using longer stretches of this residing at B550 nm, upon exposure to the same concentration
repeat sequence. In addition, non-specific sequences (i.e., XG2 of 10-mer (XG3) DNA. This repeat sequence has the ability to form
and X2G2) hardly affected the quenching of PI by graphene, since a G-quadruplex upon complex formation with PI resulting in
a selective G-quartet complex formation would be required to enhanced excimer fluorescence. Interestingly, X2G2 and XG2 dsDNA,
release the moiety from the graphene surface.
which are sequences incapable of G-quadruplex formation, did not
To begin our experiments, pyrene-appended imidazolium (PI) elicit any release or complex formation with PI, as no significant
was synthesized, precipitated in KPF6 (aq.) solution, and washed to variation in fluorescence intensity was found for these samples
provide a white solid as previously reported.12 Graphene oxide (GO) when compared to the quenched off-state of SiO2@rGO-PI.
was prepared by a modified Hummers’ method for oxidative
The dynamic range of our system for detecting G-quadruplex
exfoliation (details are provided in ESI† along with AFM images in forming dsDNA was investigated using 10-mer (XG3) dsDNA in
Fig. S1)13 and was subsequently mixed with SiO2 particles produced 10 mM sodium phosphate buffer (pH = 7.0) (Fig. 3). By observing the
to have an average diameter near 500 nm as described previously. fluorescence spectra as a function of target DNA (XG3) concentration,
The SiO2 particles serve an important role as the template for we found the fluorescence intensity to increase very linearly upon
exposure of 20 pM to 140 pM G-quadruplex forming DNA (20 pM
being the lowest concentration detected). The maximum signal at
140 pM is attributed to the maximal loading conditions of PI onto
rGO, as above 140 pM DNA exposure there will be complete complex
formation (saturation condition) of target DNA with all avail-
able PI hence resulting in release and maximal observation of
excimer fluorescence. On the basis of these results, our system
offers a very significant improvement in terms of sensitivity to
G-quadruplex forming DNA.7,8,20
In order to gain more information with respect to (XG3) DNA
complex formation with PI, we examined different lengths of
Fig. 2 (a) TEM image of SiO2@rGO. (b) TEM image of SiO2@rGO after washing
with basic solution (0.1 M NaOH).
G-quadruplex forming dsDNA with variation in the number of
c
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mode sensor. Extensive capture of PI onto high surface area rGO
shells provided the means for selective release of PI by complex
formation with specific DNA sequences resulting in fluorescence
enhancement. This unique strategy has succeeded in offering
unprecedented sensitivity, in the picomolar range, to G-quadruplex
forming DNA, representing a significant advancement in rela-
tion to existing techniques. We anticipate that the results of this
proof of concept may bring us one step closer to a robust sensor
for assessing telomeric DNA in the future by providing a system
for quantifying the level of G-quadruplex capable sequence
content in a DNA sample. Finally, we expect that the adaptability
of this strategy, with respect to new and existing fluorescent
probe technologies, may offer the means for implementation of
a diverse range of rGO based sensing systems.
Fig. 3 Fluorescence spectra originating from PI and SiO2@rGO-PI upon incuba-
tion with XG3 DNA (0–150 pM). Inset: 550 nm fluorescence intensity as a function
of picomolar XG3 concentration (pM) upon addition to SiO2@rGO-PI.
This work was supported by
a
grant from NRF
(2012R1A4A1027750 and 2012-002547). In addition, this work
was partially supported by a grant from the Next-Generation
BioGreen 21 Program (SSAC, grant#: PJ009041022012), Rural
Development Administration, Korea.
repeat units. Specifically, we examined 30-mer and 50-mer dsDNA
having 6 and 10 repeats of guanosine trimer (G3) motifs present
per DNA duplex (Fig. S4, ESI†), respectively (see ESI† for full DNA
sequence details). To provide a frame of reference, the 10-mer
(XG3) DNA mentioned above has only two repeats of (G3)-motifs
present per DNA duplex. Using these dsDNA as a comparison, we
found no significant variation in the fluorescence intensity in
regard to the number of repeats present per stretch of target DNA.
This indicates that an individual DNA molecule will complex with
only one molecule of PI to form an excimer even if the DNA
contains 2–10 repeat units of the (G3)-motif. Based on prior work
examining the specific interactions between PI and G-quadruplex
forming dsDNA, the interaction of a single molecule of PI is
expected to require at least two repeating units of the guanosine
trimer in the target DNA for effective formation of the G-quartet
and induction of excimer fluorescence enhancement,12 which
we also corroborate here (Fig. 1C).
The SiO2@rGO capture platform offers potential for adaptation
with other fluorophore based sensing probes. Rhodamine fluoro-
phore conjugates with pyrene, for instance, have already been
utilized for selective detection of metal ion species, including
Cu2+.21 Capture of such pyrene conjugates onto SiO2@rGO may
provide similar benefits in high surface area capture as demon-
strated in this work. Given the diversity of existing fluorescent
signalling probes,22 integration of such components in the form of
pyrene derivatives with our heterogeneous phase capture platform
may offer comparable advantages in terms of sensitivity as well as
quenching of background fluorescence for enhanced detection.
Detection schemes utilizing this type of approach may find higher
success in the detection of larger targets, as in our case of
G-quadruplex forming DNA, since a sizeable target interaction
may be necessary to release the probe from the rGO surface.
Because the electronic performance of graphene is sensitive to
fluctuations from the surrounding environment,23 future work
on the detection of the controlled release of PI by telomeric
DNA may make obvious use of a combined electrical/optical
readout sensor for enhancing the application diversity.
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c
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