Zinc(II)-dipicolylamine-functionalized polydiacetylene-liposome microarray: A selective and sensitive sensing platform for pyrophosphate ions

Article abstract of DOI:10.1002/asia.201000621

A microarray-chip assay system for the fluorescence detection of phosphate-containing analytes in aqueous media has been constructed from stimuli-responsive polymerized poly(diacetylene)-liposomes for the first time. Proper combination of the liposome components (Zn^II-dipicolylamine for phosphate binding and an amine-terminated component for anchoring the liposome onto an aldehyde-derivatized glass plate), has led to a microarray chip that selectively detects pyrophosphate, an important biomarker, over competing anions, such as phosphate and adenosine triphosphate, with nanomolar sensitivity. The chip-based assay shows advantages, such as high specificity and sensitivity, over solution-based assays that use the same liposomes, and over known homogeneous molecular sensing systems. Microchip 'N Dale: A microarray-chip system based on a poly(diacetylene) liposome functionalized with a zinc(II)-dipicolylamine ligand detects pyrophosphate ions with unparalleled sensitivity and selectivity. These chips show advantages over solution-based assays that use the same liposomes. Copyright

Full text of DOI:10.1002/asia.201000621

FULL PAPERS
DOI: 10.1002/asia.201000621
Zinc(II)-Dipicolylamine-Functionalized Polydiacetylene–Liposome
Microarray: A Selective and Sensitive Sensing Platform for Pyrophosphate
Ions
Kyung Mi Kim, Dong Ju Oh, and Kyo Han Ahn*[a]
Abstract:
A
microarray-chip assay
amine-terminated component for an-
choring the liposome onto an alde-
hyde-derivatized glass plate), has led to
a microarray chip that selectively de-
tects pyrophosphate, an important bio-
marker, over competing anions, such as
phosphate and adenosine triphosphate,
with nanomolar sensitivity. The chip-
based assay shows advantages, such as
high specificity and sensitivity, over so-
lution-based assays that use the same
liposomes, and over known homogene-
ous molecular sensing systems.
system for the fluorescence detection
of phosphate-containing analytes in
aqueous media has been constructed
from stimuli-responsive polymerized
poly(diacetylene)-liposomes for the
first time. Proper combination of the
liposome components (ZnII-dipicolyla-
mine for phosphate binding and an
Keywords: liposomes · microreac-
tors · pyrophosphate · sensors · zinc
Introduction
over competing derivatives such as phosphate and ATP. Fur-
thermore, existing sensing systems are small-molecule-based
probes, and thus operate only in a homogenous state. Heter-
ogeneous sensing systems that contain multiple ZnACTHNUGRTNEUNG(DPA) li-
gands remain to be explored even though they have poten-
tial for application in a chip-based assay. We became inter-
ested in such an “integrated” sensing system of ZnACTHNUGRTNEUNG(DPA) li-
gands, which would facilitate a chip-based assay for phos-
phate-containing molecules. An interesting question that
The development of a selective and sensitive detection
method for pyrophosphate ions has been a subject of intense
research effort because pyrophosphate ions are involved in
a number of key biological processes, such as calcium–phos-
phate metabolism, adenosine triphosphate (ATP) hydrolysis,
and DNA polymerization.[1] Among various approaches,
fluorescence-sensing methods have received considerable at-
tention owing to their versatile and sensitive features. In
particular, fluorescent molecular probes based on zinc(II)-
follows is whether the integrated ZnACHTNUTRGNEUNG(DPA) system on a
solid substrate would show sensing behavior similar to, or
different from, that in solution.
dipicolylamine (ZnACHTUNGTRENNUNG(DPA)) ligands have received much at-
tention[2] because the metal complexes show high affinity
toward phosphate derivatives even in aqueous media, owing
to the strong metal–phosphate coordination bonding. Al-
though several notable fluorescence-sensing systems for py-
rophosphate ions have been developed recently,[3] they are
deficient either in the detection limit (usually at micromolar
concentrations) or in their selectivity for pyrophosphate
The poly(diacetylene) (PDA) liposome system[4] would to
be an ideal platform for introducing ZnACTHNUTRGNE(NUG DPA) ligands onto
a solid substrate with a signaling function. PDA liposomes
are well known, as sensing platforms, for their stimuli-sensi-
tive chromogenic and fluorogenic behavior. In solution,
PDA liposomes show a color change from blue to red in re-
sponse to various stimuli, such as heat and pH, ligand–re-
ceptor interactions, as the conjugation length of the polydia-
cetylene backbone is perturbed by the stimuli.[5] Important-
ly, the “red-phase” PDA liposomes show turn-on-type red
fluorescence. The groups of Ahn and Kim fabricated PDA
liposomes on solid substrates,[6] and demonstrated the poten-
tial of the liposome assembly on solid substrates as sensing
platforms for the detection of cyclodextrins,[7a] protein–pro-
tein interactions,[7b] and ammonia.[7c] PDA–liposome-micro-
array systems with structurally more-elaborate ligands have
appeared very recently that have demonstrated usefulness in
[a] K. M. Kim, D. J. Oh, Prof. K. H. Ahn
Department of Chemistry and
Center for Electro-Photo Behaviors in Advanced Molecular Systems
Pohang University of Science and Technology (POSTECH)
San 31 Hyoja-dong, Pohang 790-784 (Korea)
Fax : (+82)54-279-3399
E-mail: ahn@postech.ac.kr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000621.
122
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Chem. Asian J. 2011, 6, 122 – 127

the detection of potassium ions,[8a] mercury ions,[8b] and
anionic surfactants.[8c]
In our efforts to develop efficient molecular-sensing sys-
tems, particularly turn-on-type fluorescence-sensing systems
for anions and metal cations,[9] we investigated microarray-
chip systems based on PDA liposomes that allow use of
fluorescence turn-on signaling for phosphate-containing
molecules. Our goal is to develop chip-based-assay systems
for analytes of biological interest, which might be developed
into molecular-diagnosis tools for clinical purposes. Herein,
we report the first chip-based fluorescence-sensing system
for phosphate-containing molecules—specifically pyrophos-
phate ions—which shows unparalleled sensitivity and selec-
tivity toward this important biomarker.
Results and Discussion
To realize a liposome-based assay system for phosphate-con-
taining molecules, the liposomes must be constructed to
function in solution and on a solid substrate. We have de-
signed Zn
G
2
(Scheme 1) from diacetylenic amine 1, which has two amide
groups, and can thus provide two strands of intermolecular
hydrogen bonds, and stabilize the liposome structure during
fabrication. The synthesis of component 2 can be carried out
using standard organic transformations. Details of these syn-
theses can be found in the Supporting Information.
Initially, we prepared a liposome solution solely from the
ZnACHTUNGTRENNUNG(DPA) component (2); however, the liposome system
was unstable and readily underwent precipitation upon in-
teraction with analytes. To solve the precipitation problem,
we incorporated a second component as a dummy ligand for
the solution assay, and as a linker for loading the liposomes
onto a solid substrate. After several attempts, we found that
component 1, which contains an amine, fulfils all the re-
quirements. We also found that introduction of the second
component (2) was required for the detection of phosphate
and pyrophosphate ions (see below), which indicates that
the proper choice of liposome components can be a critical
factor in realizing a desired functional liposome system.
Scheme 1. Synthesis of the polymerized dipicolylamine-containing lipo-
some 3 from the liposome prepared from a 1:1 mixture of diacetylenic
amine 1 and diacetylenic dipicolylamine 2, and preparation of the corre-
sponding polymerized ZnACTHNUTRGNEUG(N DPA) liposome (4). Similarly, a liposome com-
posed of a hydroxy-terminated component (5) has been synthesized.
Only part of the chemical composition is shown for 3, 4, and 5.
Thus, the formation of the mixed liposome has been opti-
mized by changing the ratio of liposome components 1 and
2; the optimal ratio for our purpose is 1:1. The mixed lipo-
some thus prepared in solution was irradiated with UV light
to obtain the corresponding polymerized liposome (3). 3
was treated with zinc nitrate to obtain the corresponding
polymerized Zn
used for the solution study. The polymerized ZnACHTUNGTRENNUNG
ACHTUNGTRENNUNG
Abstract in Korean:
some solution was stable for months at 48C. The liposomes
so prepared have size distributions in the range of 40–
80 nm, as measured by scanning electron microscopy (SEM;
see Figure 1) and dynamic light scattering analyses (see Fig-
ure S1 in the Supporting Information).
Sensing Studies in Solution
First, we evaluated the sensing behavior of 4 toward various
anions such as chloride, bromide, nitrate, sulfate, perchlo-
rate, azide, acetate, carbonate, phosphate, and pyrophos-
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K. H. Ahn et al.
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Figure 3. UV/Vis spectra of 4 (0.25 mm solution) upon addition of various
anions (P2O74À, HPO42À, control (4 only), N3À, CH3CO2À, CO32À, BrÀ, ClÀ,
NO3À, and ClO4À) in a buffer solution (HEPES, pH 7).
Figure 1. SEM images of the polymerized Zn
90,000).
ACHTUNGTREN(NUNG DPA) liposome (4) (ꢁ
and phosphate or pyrophosphate anions distort the poly-
enyne conjugation in the liposome.[4] The molecular interac-
phate (as their sodium salts). The sensing experiment was
carried out by observing color and UV/Vis spectral changes
in the liposome solution (1.0 mL, 0.25 mm, pH 7.0 HEPES
buffer; HEPES=4-(2-hydroxyethyl)-1-piperazineethanesul-
fonic acid) upon addition of each anion solution (25 mL,
10 mm). The blue liposome solution became red–purple
upon interaction with only phosphate or pyrophosphate;
whereas no color change was observed with the other
anions (Figure 2). The molecular interaction was complete
tions involving the ZnACHTUNGETRN(UNG DPA) ligands are further supported
by the observation that the liposome prepared solely from 1
does not show any binding behavior (no color change)
toward the phosphate anions; merely precipitation occurred.
A quantitative value for the extent of the blue to red
color change is given by the colorimetric response,[7] which
shows the high selectivity of the ZnACHTNUTRGNEUNG(DPA) liposome 4
toward phosphate and pyrophosphate ions in solution
(Figure 4).
Figure 2. Photographs showing color changes of the ZnACTHNUTRGNE(UNG DPA) liposome
(4) solution (0.25 mm) upon addition of each anion as its sodium salt
(0.25 mm, pH 7 HEPES buffer) after 30 min at 258C: a) buffer only, b)
À
2À
À
2À
À
N3
, , , h) SO4 , i) ClO4 ,
c) AcOÀ, d) CO3 e) BrÀ, f) ClÀ, g) NO3
j) HPO42À, and k) P2O74À (pyrophosphate).
within 30 minutes, after which there was little change in ab-
sorbance or color. We also evaluated ATP and adenosine
monophosphate (AMP), but these phosphate derivatives
caused precipitation (even though color changes were ob-
served), probably owing to the poor solubility of the lipo-
somes bound with these anions. The color changes corre-
spond to the spectral changes (Figure 3): the absorbance
maxima at 602 and 612 nm, observed for the polymerized
liposome solution, decreased drastically, whereas the absorb-
ance maximum at 547 nm increased, to some extent, with a
blueshift (541 nm). The observed analyte selectivity can be
Figure 4. Colorimetric response (CR) of the ZnACTHNUTRGNE(NUG DPA) liposome 4 solu-
tion toward various anions. CR%=[(PB0ÀPBv)/PB0]ꢁ100, in which
PB=Ablue/(Ablue+Ared), A is the absorbance at either the “blue” compo-
nent in the UV/Vis spectrum (approx. 640 nm) or the “red” component
(approx. 540 nm), PB0 is the red/blue ratio of the control sample (before
color change), and PBv is the value obtained for the vesicle solution after
the color change (Ablue: 540 nm, Ared: 612 nm).
Although several polydiacetylenic liposomes functional-
ized with different chemical ligands are known, this is the
explained by evoking the strong affinity of the ZnACTHNUGRTNEUNG(DPA) li-
gands toward phosphate and pyrophosphate anions. The
color and thus absorbance changes observed suggest that
the molecular interactions between the ZnACTHNURTGNEU(GN DPA) ligands
first example in which phosphate-binding ZnACTHNGUTER(NNUG DPA) ligands
are incorporated into the polydiacetylenic liposome system
to recognize and sense phosphate derivatives in solution.[10]
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Zinc(II)-Dipicolylamine-Functionalized Polydiacetylene–Liposome Microarray
Sensing Studies on Microarray Chips
On the basis of the promising results from the solution
study, we fabricated a microarray chip based on the Zn-
ACHTUNGTRENNUNG(DPA) liposome. The preparation sequence is a little differ-
ent from that followed for the preparation of the liposome
solution. In this case, the 1:1 mixed liposome (from 1 and 2)
prepared in solution was spotted onto a glass substrate with
terminal aldehyde groups, and then zinc-complex formation
and polymerization between the diacetylene backbones
were carried out. As has been previously observed,[6a] a blue
to red color change was observed when the polymerized lip-
osome (3), instead of the nonpolymerized liposome, was
loaded onto the glass substrate through the aldehyde–amine
reaction (imine formation). This means that chemical liga-
tion of the polymerized liposome onto the glass substrate
also causes distortion of the liposome backbone prior to in-
teraction with analytes, which renders the chip system use-
less. Thus, we followed the previously reported sequence[6]
to prepare the microarray chip as follows.
The liposome-microarray chip was prepared by noncon-
tacted spotting of the liposome solution prepared from a 1:1
mixture of component 1 and 2 onto the aldehyde-modified
glass slide (spot size ~200 nm in diameter). The spotted
slide was stored at 80% humidity for 6 hours, and was then
rinsed with deionized water and dried under a stream of ni-
trogen at ambient temperature. The incubation time was op-
timized by checking the fluorescence intensity for the final
liposome chip prepared at various incubation times: if the
incubation time was shorter than 6 hours, the fluorescence
intensity was weaker; if it was longer (12, 24 h), there was
little intensity change. The dried slide was dipped into a zinc
nitrate solution (10 mm) for 30 minutes at room tempera-
ture, and was then rinsed with deionized water to remove
uncomplexed zinc nitrates before being dried under a
stream of nitrogen at ambient temperature. Finally, the slide
was exposed to UV light (254 nm, 1 mWcmÀ2) for 2 minutes
to polymerize the diacetylene groups. This provided the de-
sired blue-phase liposome-microarray chip (Scheme 2). The
liposome-microarray chip shows the blue to red phase
change upon addition of pyrophosphate ions (see below),
which indirectly indicates that the liposomes on the glass
substrate maintain their structural integrity during the chip-
preparation steps. As mentioned above, the terminal amino
group of 1 is necessary to covalently attach the mixed lipo-
somes onto the aldehyde-terminated glass surface. In addi-
tion to this role, the amino group, in the ammonium form,
seems to act as an auxiliary ligand in recognizing phosphate
Scheme 2. Fabrication of the liposome-microarray chip and molecular in-
teraction with pyrophosphate ion.
solution (pyrophosphate P2O74À, azide, acetate, carbonate,
bromide, chloride, nitrate, sulfate, perchlorate, and phos-
phate; all as their sodium salts). We also evaluated ATP and
AMP, which, in the above solution study, showed blue to
red color changes, but also caused precipitation. The micro-
array chip was immersed into each anion solution (10 nm,
pH 7 HEPES buffer) and incubated at room temperature
for 6 hours. The fluorescence images were then analyzed
with appropriate filters for excitation and emission lights.
The data clearly show red fluorescence only in the case of
pyrophosphate ions among the anions examined (Figure 5).
This is in contrast to the solution assay, in which the Zn-
AHCTUNGTREG(NNUN DPA) liposome responds to phosphate, pyrophosphate,
ATP, and AMP (even though precipitation occurs in the
latter two cases). The reason for the specific response of the
microarray chip toward pyrophosphate anions over other
phosphate derivatives is not clear; however, the results indi-
cate that the liposomes on the chip seem to provide confor-
mationally more rigid binding sites relative to the liposomes
in solution. In case of such rigid binding sites, the pyrophos-
phate ion seems to fit the space between the two zinc sites,
whereas other phosphate derivatives are too small or too
large to be accommodated in it. The binding of pyrophos-
ions, along with the ZnACHTUNRGTNE(NUG DPA) ligand, or to stabilize the lipo-
some structure through hydrogen bonding: when we used a
hydroxyl-terminated pentacosadiynoic acid derivative for
the formation of mixed liposome 5 instead of the amine-ter-
minated 1, there was little color change upon addition of
phosphate or pyrophosphate anions to the liposome solu-
tion.
phate ions between two nearby ZnACHTUNTRGNEUNG(DPA) groups distorts
the conjugated enyne backbone; hence, the conjugation
length becomes shorter, which results in the red fluorescent
phase. Note that known homogeneous sensing systems for
We evaluated the fluorescence-sensing behavior of the mi-
croarray system toward the anions examined in the buffer
pyrophosphate ions[2] use 1,3-bis[Zn
ACTHNUTRGNEUNG(DPA)]phenyl deriva-
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K. H. Ahn et al.
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Figure 6. Fluorescence images of the liposome chip to pyrophosphate
4À
(P2O7
) ions at various concentrations: a) buffer only, b) 100mm,
c) 10mm, d) 1mm, e) 100nm, f) 10nm, g) 1nm, h) 100pm, i) 10pm, and
j) 1pm. The images were taken after dipping the chip into each analyte
solution (10 mm, pH 7 HEPES buffer) and incubating for 6 h at room
temperature. Filters were used for the excitation (535Æ50 nm) and emis-
sion (610Æ75 nm). The diameter of each spot is about 200 nm.
Conclusions
We have fabricated the first microarray-chip system that de-
tects pyrophosphate ions with unparalleled selectivity and
sensitivity in aqueous media over competing phosphate de-
rivatives and other anions. The chip system is constructed
on a stimuli-responsive sensing platform of a PDA-based
polymerized liposome that is structurally optimized from
two components: a zinc(II)-dipicolylamine-functionalized
component for binding phosphate ions; and an amine-termi-
nated component, which acts as an auxiliary ligand, and also
facilitates chemical ligation of the liposome onto a solid sub-
strate. The microarray-chip system selectively responds to
pyrophosphate ions among examined analytes; it shows ap-
parent red fluorescence below 1nm, in contrast to the solu-
tion assay with the liposome, which shows blue to red color
changes toward phosphate, AMP, ATP, and pyrophosphate
ions. The chip-based-assay system is promising for the detec-
tion of pyrophosphate ions, which play important roles in
many biological processes; their in situ detection is currently
under investigation.
Figure 5. Fluorescence images of the liposome chip on interaction with
4À
À
various analytes: a) buffer only, b) P2O7
(pyrophosphate), c) N3
,
,
2À
d) OAcÀ, e) CO32À, f) BrÀ, g) ClÀ, h) NO3À, i) SO42À, j) ClO4À, k) HPO4
l) ATP, and m) AMP. The images were taken after dipping the chip into
each analyte solution (10 mm, pH 7 HEPES buffer) and incubating for
6 h at room temperature. Filters were used for excitation at 535Æ50 nm,
and for emission at 610Æ75 nm. The diameter of each spot is about
200 nm.
tives as ligands to obtain selectivity over phosphate ions. In
the present case, mono ZnACTHNUTRGENU(GN DPA) ligand is used; however, in
its integrated form on the surface of the liposomes, specifici-
ty for pyrophosphate is achieved. Also, in our chip-based
assay we can include analytes such as ATP and AMP, which
precipitate in the solution assay, because such a solubility
problem becomes irrelevant in the heterogeneous phase.
This is an advantage of the chip-based assay over the solu-
tion-based assay.
Next, we evaluated the sensitivity of the microarray-chip
system toward pyrophosphate ions by reducing the pyro-
phosphate concentration to 1pm and analyzing the resultant
fluorescence images. Red fluorescence spot images are clear-
ly observable in the presence of pyrophosphate ions from
the mm level to 1nm range, and boundary spot images are
observable even below that concentration (Figure 6). Even
better sensitivity may be achieved by optimizing the spotting
process; for example, simply by increasing the number of
spotting. Such high sensitivity is not easily achieved with
most known homogeneous sensing systems. A more signifi-
cant advantage of the chip assay is its excellent selectivity
toward pyrophosphate over other phosphate-containing
molecules, which is rarely achieved with most known homo-
geneous sensing systems. The correlation curve between
fluorescence intensity and pyrophosphate concentration is
linear in the low nanomolar range (see the Supporting Infor-
mation, Figure S2).
Experimental Section
The synthesis of liposome components 1 and 2 is described in the Sup-
porting Information.
Preparation of the ZnACTHNUGTRNEUNG(DPA) Liposomes
A solution of 1 (2.1 mg, 5 mmol) and 2 (3.4 mg, 5 mmol) in chloroform
(1 mL) was prepared in a vial from stock solutions of each component.
The solvent was removed by a stream of Ar before the addition of deion-
ized water (10 mL). The resulting suspension was sonicated for 20 min at
808C (with a Fisher Scientific sonic dismembrator, model 500) and then
filtered through a cellulose acetate filter (0.8 mm) to remove dispersed
lipid aggregates. The filtered solution was kept at 48C overnight to give a
solution of the mixed liposome. An aqueous solution of ZnACHTUNGTRENNUNG(NO3)2
(0.1 mL, 100mm) was added to the liposome solution, and the resultant
solution was exposed to 254 nm UV (1 mWcmÀ2) for 2 min to give a so-
lution of the polymerized Zn
solution assays below.
ACHTUNGTRENN(UNG DPA) liposome 4, which was used for the
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Chem. Asian J. 2011, 6, 122 – 127

Zinc(II)-Dipicolylamine-Functionalized Polydiacetylene–Liposome Microarray
Detection of Anions Using the Liposome Solution
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Solutions of azide, acetate, carbonate, bromide, chloride, nitrate, sulfate,
perchlorate, phosphate, and pyrophosphate anions (as their sodium salts;
25 mL, 10 mm) were added at room temperature to a solution of 4 (1 mL,
250 mm), prepared as above. Photos showing the color change of each so-
lution and UV/Vis spectra were taken within 30 min.
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Preparation of the ZnACHTUNGTRENNUNG(DPA) Liposome Microarray
The mixed-liposome solution prepared from a 1:1 mixture of components
1 and 2 (Scheme 1) was spotted onto an aldehyde-derivatized glass plate
by using a manual microarrayer (Piezorray Perkin–Elmer LAS) with a
pitch of 0.5 nm between spots. The printed slides were stored at room
temperature for 6 h at 80% humidity. After being rinsed with deionized
water and dried under a stream of N2, the slides were dipped into an
aqueous solution of ZnACHTUNGTRENNUNG(NO3)2 (10 mm, 20 mL) for 30 min at room tem-
perature. The slides were rinsed with deionized water and dried under a
stream of N2 before being exposed to 254 nm UV (1 mWcmÀ2) for 2 min
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[10] In the course of our study, a PDA–liposome system functionalized
A ZnACHTUNGTRENNUNG(DPA) microarray chip prepared as described above was dipped
into each analyte solution (pyrophosphate, azide, acetate, carbonate, bro-
mide, chloride, nitrate, sulfate, perchlorate, phosphate, ATP, and AMP, as
their sodium salts; 20 mL, 10 nm) and incubated for 6 h at room tempera-
ture. After being rinsed with deionized water, fluorescence images of the
solutions were obtained by using a fluorescence microscope (Zeiss Axio-
plan 2). Similarly, the ZnACTHNUGRTNEUNG(DPA)-microarray chip was incubated with a so-
lution that contained varying concentrations of pyrophosphate ions
(1pm–100mm: 2.0ꢁ10À11–2.0ꢁ10À3 mol in 20 mL) for 6 h; images were ob-
tained by using the fluorescence microscope. Quantitative fluorescence
intensities from the dots were analyzed by using HCImage 1.0 software
(Hamamatsu Corporation), and were repeated in four independent ex-
periments.
Acknowledgements
This work was supported by grants from the Center for Electro-Photo
Behaviors in Advanced Molecular Systems (R11-2008-052-01001).
with ZnACTHNURTGNEU(GN cyclen)ligands that responds to ATP as well as pyrophos-
phate ions at mmolar concentration in solution was reported: D. A.
Jose, S. Stadlbauer, B. Kçnig, Chem. Eur. J. 2009, 15, 7404.
[1] a) S. E. Mansuruva, Biochim. Biophys. Acta 1989, 977, 237; b) D. L.
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Received: August 30, 2010
Published online: December 9, 2010
Chem. Asian J. 2011, 6, 122 – 127
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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