Luminescent lanthanide graphene for detection of bacterial spores and cysteine

Article abstract of DOI:10.1039/c5cc02889b

Here, we describe a new approach for preparation of luminescent lanthanide graphene in the presence of dipicolinic acid (DPA). Hg²⁺ can competitively bind with DPA which greatly quenches the fluorescence and the resultant complex is able to selectively and sensitively detect cysteine with a detection limit of 5 nM.

Full text of DOI:10.1039/c5cc02889b

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DOI: 10.1039/C5CC02889B
Chemical Communications
COMMUNICATION
Luminescent lanthanide graphene for detection of bacterial
spores and cysteine
Yuzhen Wang,bc Ying Li,bc Wenjin Qibcd and Yujun Songabc
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Here, we describe
a
new approach for preparation of
luminescent lanthanide graphene in the presence of dipicolinic
acid (DPA). Hg2+ can competitively bind with DPA which greatly
quenches the fluorescence and the resultant complex is able to
selectively and sensitively detect cysteine with the detection
limit of 5 nM.
As a rising star in material sciences, two-dimensional graphene
has attracted tremendous attention in the past few years.1-4
Potential applications ranging from novel drug delivery,
biosensing and clinical diagnosis have been actively explored.5-8 To
meet this end, water-soluble, visible fluorescent graphene is a
suitable candidate because they can be easily tracked in situ
diagnosis or drug delivery path way in vivo. Due to the good water
solubility and easy functionalization, graphene oxide (GO) was
9
widely used in biological application.3,
However, GO only
exhibits a weak infrared fluorescence, which limits its use in vitro
and in vivo.10 Much effort has been devoted for developing
fluorescent graphene. Previous studies have demonstrated that
most organic compounds have short fluorescence lifetime, and
more importantly, their fluorescence are strongly quenched due to
photoinduced electron transfer or energy transfer to graphene
when organic fluorophores are bound to the graphene sheets.11 It
has been reported that rare-earth compounds have been widely
used as laser materials, optoelectronic devices and as fluorescence
probes in immunoassays.12, 13 This is attributed to the 4f orbitals of
rare-earth elements are shielded by 5s5p6s orbitals, rare-earth
compounds exhibit unique spectroscopic characteristics, such as
long luminescence lifetime, large Stoke’s shift and sharp line-like
atomic emission.13-15 As these properties can overcome
Scheme 1. Schematic illustration of GE-Tb-DPA based sensor
for detection of DPA and cysteine.
autofluorescence and light scattering, rare-earth compounds are
commonly used as fluorophores in chemical biology.13
In this work, we design and synthesize europium (Eu3+) or
terbium (Tb3+) complex covalently modified graphene (GE-Eu /GE-
Tb) (Fig. S1, Scheme 1). In the presence of dipicolinic acid (DPA), a
biomarker and major constituent of B. anthracis spores, the
complex exhibits strong red or green luminescence under UV
excitation which attributes to the formation of the GE-Eu-DPA or
GE-Tb-DPA complex. Due to the threats in biological attack, B.
anthracis spores have aroused particular concern throughout the
world in the past decades.16-20 Development of methods for rapid
and ultrasensitive detection of B. anthracis spores is greatly
important for prevention and control of anthrax disease. Both of
the luminescence complexes provide a rapid and simple method for
DPA detection. The GE-Tb-DPA complex shows higher sensitivity
than GE-Eu-DPA for detection of DPA. We found that mercury ions
can competitively bind to DPA and thereby decrease the
fluorescence intensity of GE-Eu-DPA, while the intensity was
recovered in the presence of cysteine. As an essential amino acid in
proteins and biologically activity, cysteine plays an important role in
the human body, such as protein synthesis, detoxification, and
a.Department of Biomedical Engineering, College of Engineering and Applied
Sciences, Nanjing University, Nanjing, Jiangsu, 210093, China.
b.Department of nanomedicine, Houston Methodist Hospital Research Institute,
6670 Bertner ave, Houston, Texas, USA, 77030.
c. Weill Medical College of Cornell University, New York, New York 10065
d.Department of Obstetrics & Gynecology, The First Affiliated Hospital of Kunming
Medical University, Kunming, China 650032
Email: ysong@houstonmethodist.org, wenjincookie@163.com
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C5CC02889B
Figure 1. (a, b) Photographs of GO, GA, GE-Eu and GE-Eu-DPA
(from left to right) in ddH2O taken under daylight lamp (a)
and 365 nm UV light (b). (c) AFM image of GE-Eu-DPA; (d)
Height profile taken across the white line in (c).
metabolism.21-25 The concentration of cysteine is highly correlated
to the physiological functions and useful in diagnosing the
underlying disease.26 Therefore, a graphene-based biosensor is
also developed to detect cysteine.
Figure 2. (a) Fluorescence response of 4 µgml-1 GE-Tb upon
addition of different concentrations of DPA. (b) The
fluorescence intensity of GE-Tb (545 nm, black) or GE-Eu (616
Syntheses of GE-Eu and GE-Tb are illustrated in Fig. S1 and
detailed in the Experimental section. We firstly obtained amino
functionalized graphene (GA) by covalently grafting 1,2-Bis(2-
aminoethoxy)ethane onto graphene through the reaction of epoxy
groups on graphene oxides and amino groups on 1,2-Bis(2-
aminoethoxy)ethane in the presence of KOH. Subsequently, NaBH4
was added to reduce the product. Then ethylenediamine tetraacetic
acid dianhydride (EDTAD) was covalently grafted onto the surface
of graphene by the reaction of the amino group presenting on the
graphene surface with the anhydride group of EDTAD molecules.27
The EDTA ligand on graphene is then readily converted into a
complex coordinating with Eu3+ or Tb3+(GE-Eu or GE-Tb). Upon
exposure of GE-Eu or GE-Tb to DPA, DPA will coordinate with EDTA-
Eu or EDTA-Tb group to form luminescent GE-Eu-DPA or GE-Tb-DPA.
The covalently grafted 1,2-bis(2-aminoethoxy)ethane onto
graphene was confirmed by FTIR spectra of GA (Fig. S2). The strong
CH2 (2925 cm-1, 2852 cm-1) vibrations and a characteristic C–N
stretch mode (1320 cm-1, ν C–N binding with an aromatic ring)
confirm that the 1,2-bis(2-aminoethoxy)ethane has been covalently
grafted to the graphene sheet successfully.28 Due to the reduction
of NaBH4, GA shows a maximum absorption at 269 nm. Conjugation
of EDTA ligands to GA (GE) was confirmed by TGA and FTIR
measurements. The new peaks at 1726 cm-1 and 1650 cm-1 in the
FTIR spectrum indicate the presence of -COOH and CO–NH groups
in the GE. The amount of EDTA grafted on GA has been evaluated
by thermogravimetric analysis (TGA) under N2 atmosphere and the
content of EDTA in GE was 21.8 % (Fig. S3a). After the graft of EDTA
on GA, the solution was redispersed, which attribute to the
enhancement of electrostatic repulsion in the presence of EDTA.
The formation of GE-Eu-DPA complex on graphene was evaluated
by Energy-dispersive X-ray (EDX) spectra (Fig. S3b). The signals in
the EDX spectrum indicate that Eu3+/Tb3+ are introduced onto
graphene surface. Furthermore, the XPS images showed that
Eu3+/Tb3+ are averagely distributed on the graphene sheets (Fig. S4).
After DPA was introduced in EDTA-Eu, an intense narrow band in
UV-Vis spectra at 274 nm was observed (Fig. S3c). The solution of
GE-Eu-DPA exposed in UV light exhibits strong red florescence, also
nm, red) as
a function of DPA concentration. Inset:
Photographs of GE-Tb-DPA (green) or GE-Eu-DPA (red) taken
under 365 nm UV light.
suggesting that EDTA-Eu-DPA complex was successfully grafted on
the graphene surface (Fig. 1a, b). Our atomic force microscopy
(AFM) images show that the height of EDTA-Eu-DPA is 3.8 nm,
which is higher than GO (~1 nm) (Fig. 1c, d).
In our graphene based system, we found Tb3+ is more sensitive
than Eu3+ for the detection of B. anthracis spore. In the absence of
DPA, the solution of GE-Tb was essentially nonemissive upon
excitation at 270 nm (Fig. 2). Due to nonradiative deactivation
from vibronic coupling of the OH oscillators with the excited
lanthanide, coordinated water molecules can quench Tb3+
luminescence.17 However, addition of DPA will make the solution
exhibit sharp emission bands at 492, 546, 586 and 621 nm,
respectively, corresponding to the deactivation of the TbIII excited
5
states D4-7Fn (n = 6, 5, 4 and 3). The DPA detection limit for this
system was estimated to be 30 nM. The result is comparable to that
previous reported by Lin et al, suggesting that graphene almost has
no impact on the sensitivity. The luminescent Tb3+-complex tag
29
was linked by
a diamine linker, which not only covalently
immobilized Tb3+-complex on the graphene sheets but also
efficiently prevented graphene luminescence quenching effect on
Tb3+ complex. We compared both of the complexes for detection of
DPA, GE-Tb shows higher sensitivity than GE-Eu for detection of
DPA.
With the addition of Hg2+, the fluorescent intensity of GE-Tb-DPA
at 546 nm decreased gradually. Due to the π→π* transitions of
bound DPA, the UV absorption spectrum of EDTA-Tb-DPA exhibited
two well observed peaks at 271 and 278 nm (Fig. S5). However, in
the presence Hg2+, the peak at 271 nm was shifted to 273 nm and
the peak at 278 nm was diminished. This resulted spectrum is very
similar to DPA-Hg complex, suggesting that Hg2+ was competitively
bound to DPA which inhibited EDTA-Tb binding to DPA. Therefore,
the formation of DPA-Hg complex leads to the decrease of
fluorescent intensity of GE-Tb-DPA.
It has been reported that cysteine can react with Hg2+ to form a
2 | J. Name., 2012, 00, 1-3
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Science Foundation of China (Projects 81160076) and the Fostering
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Talents Project for Young Academic andDTOeI:c1h0n.1o0lo39g/yC5leCaCd0e2r8s89iBn
Yunnan Province (Project 2012HB029).
Notes and references
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In conclusion, this work describes a new approach for preparation
of luminescent lanthanide graphene. Eu3+ or Tb3+ was covalently
and averagely grafted on the graphene sheets. In the presence of
DPA, the graphene complex exhibits strong red or green
luminescence under UV excitation. The GE-Tb complex was used to
sensitively detect DPA with a detection limit of 30 nM. Hg2+ can
competitively bind to DPA and thereby decrease the fluorescence
intensity of GE-Tb-DPA, while the intensity was recovered in the
presence of cysteine. Therefore, a biosensor was also developed for
selectively detection of cysteine with a detection limit as low as 5
nM. Our work will facilitate the utilization of unique luminescence
properties of rare-earth complex functionalized graphene in
medical diagnostics, bioimaging and environmental monitoring.
This work was financially supported by the National Natural
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This journal is © The Royal Society of Chemistry 20xx
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