Nanomolar pyrophosphate detection in water and in a self-assembled hydrogel of a simple terpyridine-Zn2+ complex

Article abstract of DOI:10.1021/ja4128949

A simple terpyridine-Zn(II) complex is shown to act as an efficient and highly selective fluorescent sensor for pyrophosphate in water at physiological pH. The sensor complex showed an unprecedented fluorescence response (～500 fold increase) and a record nanomolar sensitivity (detectable fluorescent response at 20 nM and LOD ～ 0.8 nM). It has successfully been used to stain and record confocal fluorescence microscopy images of HeLa cells. Moreover, the complex was found to self-assemble into a hydrogel which was subsequently used to coat disposable paper strips for easy, low-cost detection of pyrophosphate.

Full text of DOI:10.1021/ja4128949

Communication
pubs.acs.org/JACS
Nanomolar Pyrophosphate Detection in Water and in a Self-
Assembled Hydrogel of a Simple Terpyridine-Zn2+ Complex
Sandip Bhowmik,†,‡ Biswa Nath Ghosh,†,‡ Varpu Marjomaki,§ and Kari Rissanen*,†
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†Department of Chemistry, Nanoscience Center, University of Jyvaskyla, P.O. Box 35, 40014 Jyvaskyla, Finland
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§Department of Biological and Environmental Science, Nanoscience Center, University of Jyvaskyla, Survontie 9, 40500 Jyvaskyla,
Finland
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* Supporting Information
Herein, we report nanomolar level detection of PPi in water at
physiological pH utilizing a simple fluorescent terpyridine-Zn2+
receptor. The receptor exhibited excellent PPi binding selectivity
over other anions and structurally analogues NPPs.
The ligand 4′-(4-N,N′-dimethylaminophenyl)-2,2′:6′,2″-ter-
pyridine L was synthesized according to the literature method.34
Stirring of the dichloromethane solution of L with ZnCl2 in
methanol in 1:1 mixture resulted in the formation of metal
complex ZnCl2L. The crystal structure confirmed the formation
of the 1:1 metal complex in which Zn2+ adopts a distorted
trigonal bipyramidal N3Cl2 coordination (Figure 1).
ABSTRACT: A simple terpyridine-Zn(II) complex is
shown to act as an efficient and highly selective fluorescent
sensor for pyrophosphate in water at physiological pH.
The sensor complex showed an unprecedented fluores-
cence response (∼500 fold increase) and a record
nanomolar sensitivity (detectable fluorescent response at
20 nM and LOD ∼ 0.8 nM). It has successfully been used
to stain and record confocal fluorescence microscopy
images of HeLa cells. Moreover, the complex was found to
self-assemble into a hydrogel which was subsequently used
to coat disposable paper strips for easy, low-cost detection
of pyrophosphate.
n the past couple of decades, considerable effort had been
Iinvested in developing receptor probes to sense biologically
significant ions at physiologically relevant concentrations.1−4
Anions such as pyrophosphate (PPi) and ATP play a key role in
energy transduction and several major metabolic processes.5−7
The PPi concentrations can provide critical information on
important processes such as DNA replication and hence can be
used in cancer diagnosis by monitoring telomerase elongation
process.8 Detection and quantification of PPi can also help
identifying diseases such as chondrocalcinosis or calcium
pyrophosphate dihydrate (CPPD) crystal deposition disease.9,10
Considering its importance, several techniques have been
developed for the detection of PPi and other related NPPs
(nucleotide polyphosphates).11−14 Among these the fluores-
cence-based detection systems being the most popular mainly
due to its high sensitivity, fast response, and the possibility of in
vivo and in vitro imaging.15−18 Although several fluorescent
chemosensors have been developed for this purpose, certain key
issues still remain to be addressed. Due to strong hydration of
anions and low solubility of organic fluorescent probes, a
majority of the studies are limited to organic or mixed aqueous
media.19−24 Although a few recent studies have addressed this
issue, the sensitivity in water (or in organic or mixed media) was
invariably limited to micromolar levels.25−29 This meant that the
rapidly growing fields, such as cancer diagnosis based on PPi
detection, had to be solely dependent on fluorescent protein-
based techniques, as the existing chemosensors lack the required
sensitivity.30−33 Hence, there is a growing demand for highly
selective and sensitive probes for PPi detection for diagnostics
and medical applications.
Figure 1. ORTEP plot of the molecular structure of ZnCl2L. Thermal
ellipsoids are shown at 50% probability level. Selected bond distances
(Å) and angles (°): Zn1−N1 2.200(2), Zn1−N2 2.089(3), Zn1−N1i
2.200(2), Zn1−Cl1 2.2604(6), Zn1−Cl1i 2.2604(6), Cl1−Zn1−Cl1i
110.15(4), N1−Zn1−N2 74.62(5), N2−Zn1−N1i 74.62(5). Symmetry
element: i = −x + 2, y, −z + 1/2.
The changes in the UV−Vis spectra of ZnCl2L were
monitored in the presence of different anions (as sodium salts)
in water buffered with 0.01 M HEPES, pH 7.4, at 298 K. The
buffered complex solution was yellow in color and its UV−Vis
spectrum exhibited three major bands at 285, 320, and 410 nm.
No significant changes in the spectra were observed for anions
such as F−, Br−, I−, SO4 , AcO−, NO3 , CO32−, HPO42−, and
2‑
−
3−
PO4
.
However, the addition of PPi and other nucleotide
triphosphates resulted in a sharp bathochromic shift (∼ 30
nm) and a decrease in the peak intensity of 410 nm band (Figure
2a). The red shift was visibly evident in the slight change in color
of the resulting solution and clearly indicated PPi-to-receptor
binding. More drastic optical changes were observed in the
fluorescence spectra of ZnCl2L. Although ZnCl2L was found to
Received: December 26, 2013
Published: February 4, 2014
© 2014 American Chemical Society
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Communication
(60 nM) (Figure 4a). Competitive binding studies were also
carried out in the presence of these anions. As shown in Figure
Figure 4. (a) Emission of ZnCl2L (50 μM) in 10 mM HEPES buffer
(pH 7.4) in presence of different anions. (b) Competitive binding
affinity of ZnCl2L (50 μM) toward PPi (50 nM, 0.001 equiv) in the
presence of 1 equiv of different anions in water (10 mM HEPES buffer,
pH 7.4).
Figure 2. (a) UV−Vis spectra of ZnCl2L (50 μM) in 10 mM HEPES
buffer (pH 7.4) with different anions (50 μM). (b) Emission spectra of
ZnCl2L (50 μM) in 10 mM HEPES buffer (pH 7.4) upon addition of
PPi (2.6−22 μM) (inset: change in emission @ 591 nm; λ ext @ 440
nm). (c) Emission of ZnCl2L (50 μM) under 365 nm UV lamp with
different anions (15 μM).
4b, the fluorescence intensities of ZnCl2L did not change upon
addition of 1 equiv (50 μM) of any of these anions. However
addition of only 0.001 equiv (50 nM) of PPi resulted in a
remarkable enhancement in the fluorescence intensity further
asserting the excellent selectivity and sensitivity of the PPi
binding receptor. However it should be noted that a substantial
decrease in the fluorescence response from PPi was observed in
the presence of HPO42− and PO43− anions. Yet, considering the
competitive inhibition from a large excess of other phosphate
anions, the quenching effect is quite expected.
We further investigated the applicability of ZnCl2L in cell
imaging. HeLa cells were grown with DMEM (containing 10%
fetal bovine serum and antibiotics) for 16 h and used at
subconfluency. The cells were first washed with 10 mM HEPES
buffer, pH 7.4, containing 137 mM NaCl, then treated with 10
μM ZnCl2L for 30 min in the same buffer at 37 °C, and washed
extensively with buffer before imaging. Images were recorded
after laser excitation at 488 nm and using emission wavelengths
between 555 and 655 nm for orange-red fluorescence. As
depicted in Figure 5, ZnCl2L was successfully employed in the
be fluorescent in the solid state, its fluorescence was effectively
quenched in water.
An unprecedented ∼500-fold enhancement in fluorescence
intensity of ZnCl2L was observed at 591 nm upon addition of 50
μM of PPi (Figure 2b,c). The titration plot depicting the change
in fluorescence intensities (I/I0 − 1) as a function of PPi
concentrations showed the typical saturation characteristics.
However, it revealed an unusual binding stoichiometry of 1:3
(PPi:ZnCl2L) which was confirmed unambiguously by a Job plot
analysis conducted between PPi and ZnCl2L (see SI). The
excellent fluorescence response and the binding stoichiometry
prompted us to investigate the lower detection limit of PPi in
water. We were pleasantly surprised to find that probe was able to
detect PPi at nanomolar levels (∼20 nM; lowest limit of
detection (LOD) was calculated to be ∼0.8 nM) (Figure 3a).
Figure 3. (a) Emission spectra of ZnCl2L (50 μM) in 10 mM HEPES
buffer (pH 7.4) upon addition of PPi (20−180 nM) (λex = 440 nm). (b)
Emission spectra of ZnCl2L (50 μM) in 10 mM HEPES buffer (pH 7.4)
upon addition of 200 nM PPi, ATP, ADP, CTP, and GTP) (λex = 440
nm).
Figure 5. Confocal fluorescence microscopy images of (a) HeLa cells
incubated with probe ZnCl2L (50 μM), (c) single HeLa cell, and (e)
single HeLa cell without staining (control). Differential interference
contrast images of (b) single HeLa cell incubated with probe ZnCl2L
(50 μM) and (d) single HeLa cell without staining (control).
Only negligible changes in the fluorescence intensities of
ZnCl2L in presence of other nucleotide phosphates were
observed compared to PPi (Figure 3b). The stronger binding
affinity toward PPi can probably be attributed to higher negative
charge density. On the other hand, almost no change in the
fluorescence was induced by other anions such as F−, Br−, I−,
PPi staining of HeLa cells which exhibited a bright orange-yellow
emission. Due to the excellent sensitivity of the receptor almost
the entire cell could be mapped, even regions with minimal PPi
concentrations. The maximum emission intensities were
recorded at the nuclei as well as emissions were also observed
from the membranous cytoplasmic structures.
−
3−
SO42−, AcO−, NO3 , CO32−, HPO42−, and PO4 even when
present at much higher concentrations (10 μM) compared to PPi
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As a promising device development, ligand L was found to
form a self-assembled hydrogel (Figure 6c) in the presence of
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details, UV−Vis and fluorescence study, gelation
procedure, X-ray single crystal structure analysis (CCDC
977466). This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
kari.t.rissanen@jyu.fi
■
Author Contributions
‡These authors contributed equally
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Mr. Petri Papponen and Mr. Hannu Salo are kindly acknowl-
edged for their help in TEM and SEM measurements,
respectively. The Academy of Finland (KR, grant no. 256259,
265328, 263377 and 263256; VM, 257125) is kindly acknowl-
edged for financial support.
Figure 6. (a) SEM and (b) TEM images of hydrogels of L (6 mM) in
presence of ZnCl2 (6 mM) in 0.1N HCl. (c) Photograph of the
hydrogel. (d) Gel-coated paper strips under 365 nm UV lamp.
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