Rational design of a fluorescence-turn-on sensor array for phosphates in blood serum

Article abstract of DOI:10.1002/anie.200702611

Getting turned on: An order-of-magnitude fluorescence amplification of simple tripodal sensors has been observed in the presence of phosphate ions. An X-ray structure analysis of one of the complexes (see picture) shows the binding of three phosphate ions, which closely resembles the anionic part of ATP, by the sensor. The sensors were used to generate cross-reactive arrays that can detect anions in human serum. (Figure Presented).

Full text of DOI:10.1002/anie.200702611

Angewandte
Chemie
DOI: 10.1002/anie.200702611
Analytical Methods
Rational Design of a Fluorescence-Turn-On Sensor Array for
Phosphates in Blood Serum**
Grigory V. Zyryanov, Manuel A. Palacios, and Pavel Anzenbacher, Jr.*
In memory of Dmitry M. Rudkevich
Phosphate anions, both inorganic and organic, hold a unique
position in nature, as they take part in almost all metabolic
processes. Nucleotide phosphates such as AMP or ATP are
important for their role in bioenergetics, metabolism, and
sensitivity of the fluorescence-turn-on signaling could allow
for the sensing of phosphate ions in aqueous environments,
[
12]
including biological milieu such as blood serum. Here, we
[
13]
present tripodal sensor molecules S1–S8 (Scheme 1) based
[
1]
[14]
transfer of genetic information. Phosphates in biological
liquids are often present in high concentrations and may be
utilized in the diagnosis of certain dis-
on 1,3,5-triaminomethyl-2,4,6-triethylbenzene
form arrays of six hydrogen-bond donors.
which can
[
2]
eases. Human serum contains 0.80–
[
2b]
1
.45 mm phosphate; higher phosphate
levels are directly connected to cardio-
vascular disease and acute renal fail-
[
2,3]
ure. Similarly, ATP is present in resting
muscle at a concentration of 4 mm, and in
erythrocytes at a concentration of just
[
4]
under 2 mm.
Numerous materials have been devel-
oped to sense phosphate ions in water at
[
5]
biological pH values. The sensors based
on anion-induced enhancement of intrin-
sic fluorescence (turn-on) are attractive
as they offer the potential for high
[
6,7]
sensitivity
and, unlike the displace-
[
8]
ment assays, are instantly reversible.
Alas, few fluorescence-turn-on sensors
exist for phosphate that function in elec- Scheme 1. The sensor molecules S1–S8 utilize six hydrogen-bond donors to form the sensor–
[
9]
trolyte solutions. To the best of our anion complex.
knowledge, fluorescence-turn-on sensors
capable of sensing phosphate ions in complex biological
milieu, such as blood serum, have not yet been developed.
Recently, we demonstrated that simple anion sensors
based on hydrogen-bonding interactions can bind and sense
anions in hydrophilic polymer matrices, despite their inability
The binding of anions by S1–S8 was studied in solution by
1
H NMR and fluorescence titrations, as well as in the solid
1
state. The H NMR titrations at 228C and À808C showed a
concerted downfield shift of the NH resonances, which
suggests the formation of a 1:1 complex with C symmetry,
3
[
10,11]
to bind anions in water.
This method combined with the
in which all three receptor arms are equally involved in the
formation of the complex. These results confirm that S1–S8
were not deprotonated by the anions and that the observed
increase in fluorescence is due to anion–receptor association.
The fluorescence amplification suggests that the binding of
the anions to the thiourea moieties results in restriction of
their conformational freedom, thus improving the electronic
coupling between the chromophore and the hydrogen donors
while limiting the rotational and vibrational modes, which
would otherwise result in nonradiative decay without strong
[*] G. V. Zyryanov, M. A. Palacios, Prof. Dr. P. Anzenbacher, Jr.
Department of Chemistry and
Center for Photochemical Sciences
Bowling Green State University
Bowling Green, OH 43403 (USA)
Fax: (+1)419-372-9809
E-mail: pavel@bgsu.edu
[**] P.A. acknowledges support from the Alfred P. Sloan Foundation,
BGSU (Technology Innovations Enhancement grant), and the NSF
[6]
fluorescence output. These structural features result in an
(
SENSOR no. 0330267). M.A.P. acknowledges support from the
up to 200-fold increase in the fluorescence intensity for
phosphate, pyrophosphate, and acetate ions (Figure 1). The
anion-binding affinities of S2–S4 and S6–S8 follow the
McMaster Endowment. We would like to thank Prof. Franz P.
Schmidtchen for helpful discussions during the preparation of this
manuscript.
À
3À
À
À
À
general order: H P > HPP > AcO @ Cl > Br , which
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
2
also corresponds to the relative degree of amplification of
Angew. Chem. Int. Ed. 2007, 46, 7849 –7852
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7849

Communications
Figure 2. Top and side view of S5·3TBA·3H PO determined by X-ray
2
4
crystallography. S5 is shown as thermal ellipsoids at the 50%
probability level, while dihydrogen phosphate anions are in a space-
filling form. The TBA cations have been omitted. The complex shows
C3 symmetry.
Figure 1. Top: Response of sensor molecules S6 and S8 to anions in
solution (DMSO/H O 95:5). Bottom: Fluorescence amplification of S6
2
(
0.4 mm) in the presence of tetrabutylammonium hydrogen pyrophos-
+
À
phate (TBA H PO ).
2
4
solid state may be due to entropic effects which prevent
effective desolvation and organization of the multiple anions
inside the shallow cavity of the receptor. Conversely, in the
solid phase, where the receptors may be more preorganized
and enthalpic contributions to binding are generally stronger,
the fluorescence signal (I/I ). Anion affinities described by
o
À1
6
[16]
association constants (m ) range between 3.8–5.0 ” 10 for
the binding of anion clusters can be observed.
À
À
6
À
6
F , 10–100 for Cl , 1.0–5.0 ” 10 for AcO , 2.3–5 ” 10 for
We realized that we could detect biological phosphate
ions in an arrangement capable of emulating the solid state.
We decided to construct arrays utilizing S2–S4 and S6–S8
embedded in hydrophilic polyurethane matrices to circum-
vent the fact that S2–S4 and S6–S8 are not soluble in water.
The polymer/sensor solutions were cast onto a microtiter
À
6
dihydrogen phosphate (H P ), and 1.0–5.0 ” 10 for hydrogen
pyrophosphate (HP O7 , HPP ) in DMSO.
2
3
À
3À
2
The strong fluorescence-turn-on signal combined with the
relatively low selectivity of S2–S4 and S6–S8 for any specific
phosphate anion suggest that these materials may be excellent
candidates for differential sensing in arrays where the
individual sensors are not highly selective toward specific
analytes, and where the specificity comes from recognition of
[
17]
plate (well radius: 500 Æ 25 mm, depth: 230 Æ 10 mm). The
anions used for this purpose were fluoride, chloride, acetate,
À
benzoate (BzO ), phosphate, pyrophosphate, AMP, and ATP.
[
15]
the response pattern unique for the specific analyte.
The tests were performed in two ways: First, with the anions
We presume that the binding preference for phosphates is
due to the binding of three phosphate oxygen atoms by the
dissolved in water, and second with 1.0 mm anions in 10 mm 2-
[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid
C -symmetrical receptor. To investigate the S1–S8 structure
and anion association we performed several X-ray analyses of
the receptors. Interestingly, crystals of S5 grown from a
(HEPES) at pH 7.4. The first arrangement allows for playing
out the intrinsic pH value of the salts, while in the second
experiment the effect of the pH values is mitigated by the
buffer. In both experiments, the films doped with S2–S4 and
S6–S8 responded to the presence of anions by a dramatic
increase in the fluorescence intensity, the degree of which is
specific to a particular analyte (Figure 3). The changes in
fluorescence are unique to each anion, thus enabling the
identification of these anions.
3
concentrated phosphate solution revealed a C -symmetrical
3
complex comprising an all-up receptor conformation together
with three tightly bound phosphate anions (Figure 2). This
finding suggests that these simple tripodal receptors have the
unique capability to bind up to three phosphate anions, and
opens up the possibility of using these sensor molecules for
biologically important phosphates, including ATP.
The integration of the nonzero pixels in the blue and
green channels (Figure 3, bottom) is compared to that before
application of the sample. Multivariate analysis of the data
The differences in the propensity of S1–S8 to bind one
phosphate anion in solution as opposed to more anions in the
7850
www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 7849 –7852

Angewandte
Chemie
Figure 4. Top: Qualitative changes in the fluorescence of the sensor–
polyurethane films after addition of human blood serum and serum
with added anions (to increase the concentration to 5 mm). Bottom:
[
18]
PCA score plot: Proximity in space on the plot directly correlates to
similarities in the array responses induced by the anions added to the
blood serum.
In summary, the effective sensing of anions, including
biologically important phosphates, has been successfully
achieved by using simple sensors embedded in polyurethane.
We have shown that even simple receptors utilizing six
hydrogen bonds provide turn-on sensors capable of sensing
anions in aqueous media. These simple tripodal sensors are
capable of accommodating various phosphate ions including
AMP and ATP. Preliminary experiments suggest that sensors
in polyurethane films may be used for the fabrication of turn-
on arrays that will allow analysis of phosphate-type anions
even in complex biological milieu, such as blood serum. The
successful recognition of various biologically important
phosphate ions in human blood serum suggests the potential
for the development of a point-of-care method for monitoring
phosphate homeostasis.
Figure 3. Top: Changes in the fluorescence intensity of the sensor–
polyurethane films upon addition of aqueous solutions of anions. The
anions (400 nL, 1 mm, except ATP and AMP, which were 5 mm) were
added in water (left) and in 10 mm HEPES pH 7.4 (right). Bottom: The
gray pixels in the blue (380–500 nm) and green (480–600 nm) chan-
nels were integrated to quantify the changes in the fluorescence in the
array.
then allows for identification of the anions (Supporting
[
18]
information).
Blood serum, with its protein and electrolyte content, will
behave as a unique buffer which also contains phosphate and
[
2]
various carboxylate ions, and is therefore likely to give a
unique response. In the next array (Figure 4) we applied
water (control), serum, and serum samples with added anions
Received: June 14, 2007
Published online: August 15, 2007
(
phosphate, pyrophosphate, AMP, or ATP). As expected,
Keywords: analytical methods · anions · host–guest systems ·
sensors · supramolecular chemistry
because of its intrinsic anion content, the serum itself turned-
on the fluorescence of the array, thereby creating a unique
fluorescence response, which was further modulated by the
added anions. Principal component analysis (PCA) shows
that films of S2–S4 and S6–S8 allow phosphate, pyrophos-
phate, AMP, and ATP to be distinguished. Interestingly, in
human serum analysis, the nucleotide (adenine) in AMP and
ATP plays an important role in generating a unique response
for these anions over inorganic phosphates.
.
[
18]
[
1] J. M. Berg, L. Stryer, J. L. Tymoczko, Biochemistry, 5th ed.,
W. H. Freeman, New York, 2002.
[
2] a) W. J. Marshall, S. K. Bangert, Clinical Chemistry, 5th ed.,
Elsevier, Edinburg, 2004; b) R. F. Schmidt, G. Thews, Human
Physiology, 2nd ed., Springer, Berlin, 1989.
Angew. Chem. Int. Ed. 2007, 46, 7849 –7852
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7851

Communications
[
3] R. Dhingra, L. M. Sullivan, C. S. Fox, T. J. Wang, R. B. DꢀAg-
ostino, Sr. , J. M. Gaziano, R. S. Vasan, Arch. Intern. Med. 2007,
67, 879 – 885.
[11] M. A. Palacios, R. Nishiyabu, M. Marquez, P. Anzenbacher, Jr.,
J. Am. Chem. Soc. 2007, 129, 7538 – 7544.
[12] Unlike the previous efforts using colorimetric sensors, the
fluorescence-based signaling does not require stripping the
blood plasma or serum off colored proteins: S. L. Tobey, E. V.
Anslyn, Org. Lett. 2003, 5, 2029 – 2031.
[13] B. Kuswandi, Nuriman, W. Verboom, D. N. Reinhoudt, Sensors
2006, 6, 978 – 1017, and references therein.
[14] T. D. P. Stack, Z. Hou, K. N. Raymond, J. Am. Chem. Soc. 1993,
115, 6466 – 6467.
1
[
[
[
[
4] A. Ghosh, P. Ronner, E. Cheong, P. Khalid, F. M. Matschinsky, J.
Biol. Chem. 1991, 266, 22887 – 22892.
5] J. L. Sessler, P. A. Gale, W.-S. Cho, Anion Receptor Chemistry,
RSC Publishing, Cambridge-UK, 2006.
6] Principles of Fluorescence Spectroscopy, 2nd ed. (Ed.: J. R.
Lakowicz), Kluwer, New York, 1999.
7] a) R. Narayanaswamy, O. S. Wolfbeis Optical Sensors: Indus-
trial, Environmental and Diagnostic Applications. Springer,
Berlin, 2004; b) R. Martínez-Mµæez, F. Sancenón, J. Fluoresc.
[15] J. J. Lavigne, E. V. Anslyn, Angew. Chem. 2001, 113, 3212 – 3225;
Angew. Chem. Int. Ed. 2001, 40, 3118 – 3130.
2
005, 15, 267 – 285; c) D. Curiel, E. J. Hayes, P. D. Beer, Top.
[16] a) J. L. Sessler, D.-G. Cho, V. Lynch, J. Am. Chem. Soc. 2006, 128,
16518 – 16519; b) V. Amendola, M. Boiocchi, D. Esteban-
Gómez, L. Fabbrizzi, E. Monzani, Org. Biomol. Chem. 2005, 3,
2632 – 2639.
Fluoresc. Spectrosc. 2005, 9(Part A), 59 – 118; d) R. Martínez-
Mµæez, F. Sancenón, Chem. Rev. 2003, 103, 4419 – 4476.
[
8] a) K. Niikura, A. Metzger, E. V. Anslyn, J. Am. Chem. Soc. 1998,
1
20, 8533 – 8534; b) A. Metzger, E. V. Anslyn, Angew. Chem.
[17] T. Mayr, C. Igel, G. Liebsch, I. Klimant, O. S. Wolfbeis, Anal.
Chem. 2003, 75, 4389 – 4396.
1998, 110, 682 – 684; Angew. Chem. Int. Ed. 1998, 37, 649 – 652;
c) L. Fabbrizzi, N. Marcotte, F. Stomeo, A. Tagletti, Angew.
Chem. 2002, 114, 3965 – 3968; Angew. Chem. Int. Ed. 2002, 41,
[18] a) K. R. Beebe, R. J. Pell, M. B. Seasholtz, Chemometrics: a
practical guide, Wiley, New York, 1998; b) P. C. Jurs, G. A.
Bakken, H. E. McClelland, Chem. Rev. 2000, 100, 2649 – 2678;
c) principal component analysis (PCA) was used to reduce the
dimensionality of the data set. To identify patterns in the data, a
principal component (PC) axis is calculated to lie along the line
of maximum variance in the original data. The PC axes lie along
lines describing diminishing levels of variance. The coordinates
of the sample relative to the PC axes, termed scores, can be used
as an indicator of correlation between analytes. Proximity in
space on a score plot directly correlates to similarities in changes
of fluorescence (compared to the control) emitted by the sensors
in the array. For a detailed description of the technique see also
reference [11].
3
811 – 3814.
9] a) M. W. Hosseini, A. J. Blacker, J.-M. Lehn, J. Am. Chem. Soc.
990, 112, 3896 – 3904; b) M. E. Huston, E. U. Akkaya, A. W.
[
1
Czarnik, J. Am. Chem. Soc. 1989, 111, 8735 – 8737; c) S. E.
Schneider, S. N. OꢀNeil, E. V. Anslyn, J. Am. Chem. Soc. 2000,
122, 542 – 543; d) J. L. Sessler, J. M. Davis, V. Krµl, T. Kim-
brough, V. Lynch, Org. Biomol. Chem. 2003, 1, 4113 – 4123; e) A.
Ojida, S. Park, Y. Mito-oka, I. Hamachi, Tetrahedron Lett. 2002,
4
3, 6193 – 6195; f) A. Ojida, Y. Mito-oka, K. Sada, I. Hamachi, J.
Am. Chem. Soc. 2004, 126, 2454 – 2463.
10] R. Nishiyabu, P. Anzenbacher, Jr., J. Am. Chem. Soc. 2005, 127,
270 – 8271.
[
8
7852
www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 7849 –7852