Polydiacetylene-based colorimetric self-assembled vesicular receptors for biological phosphate ion recognition

Article abstract of DOI:10.1002/chem.200900339

Self-assembled vesicular polydiacetylene (PDA) particles with embedded metal complex receptor sites have been prepared. The particles respond to the presence of ATP and PPi (pyrophosphate) in buffered aqueous solution by visible changes of their color and

Full text of DOI:10.1002/chem.200900339

DOI: 10.1002/chem.200900339
Polydiacetylene-Based Colorimetric Self-Assembled Vesicular Receptors for
Biological Phosphate Ion ACTHNUTRGNERNUG ecognition
D. Amilan Jose, Stefan Stadlbauer, and Burkhard Kçnig*[a]
Abstract: Self-assembled vesicular
polydiacetylene (PDA) particles with
copper [CuII–IDA] modified diacety-
lenes, embedded in amphiphilic diacet-
tensity. Other anions such as ADP,
AMP, H2PO4 , CH3COOꢀ, Fꢀ, Clꢀ, Brꢀ
ꢀ
G
R
embedded metal complex receptor
sites have been prepared. The particles
respond to the presence of ATP and
PPi (pyrophosphate) in buffered aque-
ous solution by visible changes of their
color and emission properties. Blue
PDA vesicles of uniform size of about
200 nm were obtained upon UV irradi-
ation from mono- and dinuclear
zinc(II)–cyclen and iminodiacetato
A
and Iꢀ, failed to induce any spectral
changes. The zinc(II)–cyclen nanoparti-
cles are useful for the facile detection
of PPi and ATP in millimolar concen-
trations in neutral aqueous solutions,
while CuII–IDA modified vesicular
PDA receptors are able to selectively
discriminate between ATP and PPi.
PPi to the PDA vesicle solution induces
a color change from blue to red observ-
able by the naked eye. The binding of
ATP and PPi changes the emission in-
Keywords: anion
recognition
·
phosphates · nanosensors · vesicles ·
zinc
Introduction
merized to generate PDA in situ.[4–6] The polymers conjugat-
ed backbone provides absorbance and fluorescence proper-
ties which are useful for the transduction of the analytical
signal.[7] Functionalized polydiacetylene films and vesicles
have been used as chemosensors to monitor metal ions,[7a]
glucose,[8] proteins[9] and for the colorimetric detection of
the influenza virus.[10]
We have recently reported the use of amphiphilic metal
complex binding sites for molecular recognition on ordered
surface bilayers.[11] Lewis-acidic metal complexes, such as
The development of chemical sensors continues to be of
great interest in modern analytical chemistry.[1] Demands
arise from clinical diagnostics, environmental and food anal-
ysis or in the detection of illicit drugs, explosives and chemi-
cal warfare agents. A molecular chemical sensor typically
consists of the analyte binding site and a signaling unit,
which transposes the binding event into a macroscopically
observable output, for example, a color change or a light
emission. For the development of devices it is often useful
to immobilize the sensor molecule on surfaces, in mem-
branes, gels or on nanoparticles. Immobilization can be
zinc(II)–cyclen
(cyclen=1,4,7,10-tetraazacyclododecane)
complexes, can reversibly coordinate oxoanions and other
Lewis basic guest molecules with high affinity and selectivi-
ty.[12,13] The binding is even possible in the presence of com-
peting solvent molecules, such as water. This renders these
metal complexes particular suitable as binding sites for
anionic analytes[14] of biological origin under physiological
conditions.
ACHTUNGTRENNUNG
achieved, among other methods,[2] by the incorporation of
receptors into self-assembled monolayers or bilayer mem-
branes.[3] Polydiacetylene (PDA) is a particular interesting
material in this respect, as diacetylene surfactants will self-
assemble in water to form vesicles that can be photopoly-
We herein describe the preparation of self-assembled
PDA vesicles with embedded amphiphilic metal zinc(II)–
cyclen and CuII–IDA (IDA=iminodiacetato) complexes.
The PDA polymer nanoparticles act as solid support for the
receptor sites and as the chromophore of the signaling unit.
[a] Dr. D. A. Jose, Dipl.-Chem. S. Stadlbauer, Prof. Dr. B. Kçnig
Institut fꢀr Organische Chemie, Universitꢁt Regensburg
93040 Regensburg (Germany)
Fax : (+49)941-943-1717
E-mail: burkhard.koenig@chemie.uni-regensburg.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900339: General methods
and materials, details of binding studies, experimental procedures and
analytical data for all new compounds.
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FULL PAPER
at room temperature by irradiating the solutions with light
of 254 nm wavelength for 5 to 10 min, whereby the colorless
vesicle solution turned blue (Scheme 4). The average size of
the liposomes was 160–180 nm as determined by dynamic
light scattering (DLS). Liposomes containing 4-Zn are
named LP-4-Zn; liposomes containing 8-Zn are named LP-
8-Zn; and liposomes containing 11-Cu are named LP-11-Cu,
respectively.
Result and Discussion
Synthesis of amphiphilic diacetylene zinc(II)–cyclen and
copper(II)–IDA complexes: The cyclen, bisACTHNUGRTENUNG(cyclen) and imi-
nodiacetato (IDA)-modified diacetylene monomers 4, 8 and
11 were prepared by amide formation as shown in
Scheme 1. A methanolic solution of 4 and 8 was treated
with an aqueous solution of ZnACHTUNTRGNEUNG(ClO4)2 to yield the amphi-
philic receptor molecule 4-Zn (Scheme 1) and 8-Zn
(Scheme 2), respectively, in good yields. Receptor molecule
11-Cu (Scheme 3) was prepared by treating a methanolic so-
lution of 11 with an aqueous solution of CuCl2 at room tem-
perature and subsequently refluxed at 708C. All newly pre-
pared monomeric diacetylene complexes 4-Zn, 8-Zn and 11-
Cu were characterized by standard analytical and spectro-
scopic techniques and are very stable when stored under ni-
trogen in the dark. Detailed experimental procedures and
analytical data of the compounds are provided in the Sup-
porting Information.
Particle size measurement: Dynamic light scattering (DLS)
particle sizing measurements were preformed by a Zetasizer
3000 from Malvern instruments Ltd. Malvern, UK.
The vesicle solutions were diluted 5- to 6-fold and mea-
sured at room temperature by keeping the typical count rate
at 30–50 kcps. Each diameter value was an average result of
continuous measurements in 5 min. At least three measure-
ments were preformed for each solution. Table 1 summariz-
es the results. The obtained values are in good agreement
with comparable liposomes that have been reported earlier
in literature.[16] The two-compo-
nent vesicles have an average
size of 230 nm. Upon UV irra-
diation, the vesicle contract to
160–180 nm, likely due to the
covalent cross linking that re-
duces the distance between the
lipid molecules (Figure 1 and
Scheme 4).
Binding studies of LP-4-Zn,
LP-8-Zn and LP-11-Cu with
different anions: Zinc (II) com-
plexes of macrocyclic poly-
amines, such as ZnII–cyclen
show affinity to phosphate
anions and nucleotides.[17,18] We
therefore expected a response
of the ZnII-cyclen modified
Scheme 1. Synthesis of ZnII–cyclen complex modified 10,12-tricosodiynoic acid (TCDA, 6) 4-Zn.
PDA vesicles to phosphorylated
Table 1. Results of DLS particle size measurement for LP-4-Zn, LP-8-
Zn and LP-11-Cu vesicles.
Synthesis of vesicular PDA receptors: The liposomes were
prepared according to literature methods.[15] A mixture con-
taining the modified diacetylene monomer 4-Zn, 8-Zn or
11-Cu and the unmodified diacetylene monomer 10,12-trico-
sadiynoic acid (TCDA) or 10,12-pentacosadiynoic acid
(PCDA) in a molar ratio of 10:90, respectively, was dis-
solved in dichloromethane in a 25 mL round bottom flask.
The solvent was evaporated by a stream of N2 gas and an
appropriate amount of buffered aqueous solution (HEPES
10 mmol, pH 7.2) was added to the round-bottom flask to
give the desired concentration of the lipid (1ꢃ10ꢀ3 molLꢀ1).
This solution was sonicated at 808C for 40 min. The re-
sulting milky solution was filtered through a syringe filter
while hot and the filtrate was cooled and stored at 08C over-
night. The self-assembled bilayer vesicles were polymerized
Before irradiation [nm]
After irradiation [nm]
LP-4-Zn
LP-8-Zn
LP-11-Cu
230ꢁ20
220ꢁ25
205ꢁ25
180ꢁ15
160ꢁ10
145ꢁ10
anions. Figure 2 depicts schematically the assembly and ana-
lyte response of the vesicular metal complex sensor system.
Although there is still no clear evidence for the molecular
origin of the color change of PDAs upon analyte binding, it
has widely been accepted that the color change is associated
with a conformational change of the polydiacetylene back-
bone.[19] Accordingly, in the blue colored polymerized vesic-
ular receptor an extended conjugation of the p orbitals in
the main chain of the polydiacetylene polymers is present.
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B. Kçnig et al.
Initially, electronic absorption
spectra for a 5ꢃ10ꢀ5 molLꢀ1 so-
lution of the blue colored LP-4-
Zn vesicles were recorded in
the absence and presence of
various anions (Figure 3). The
absorption spectra of LP-4-Zn
in buffered aqueous solution
(10 mmol HEPES, pH 7.2) at
room temperature shows before
the addition of analytes distinct
absorption bands at lmax =640,
589 and 543 nm. Upon addition
of an excess amount of ATP,
PPi or CNꢀ ions the absorption
band at 640 nm disappeared
completely and an intense ab-
sorption band at 489 and
543 nm is observed. The color
of the solution turns red. No
changes in the absorption spec-
tra or color is observed upon
addition of other anions such as
ꢀ
Fꢀ, Clꢀ, Brꢀ, Iꢀ, H2PO4 ,
Scheme 2. Synthesis of bis(ZnII–cyclen) complex modified 10,12-tricosodiynoic acid (TCDA, 6) 8-Zn.
CH3COOꢀ, AMP or ADP
under
similar
conditions
(Figure 3b).
Scheme 3. Synthesis of copper(II)–IDA modified 10,12-pentacosadiynoic
acid (PCDA) 11-Cu.
Figure 1. Particle size distribution curves of a) LP-4-Zn and b) LP-8-Zn.
Upon analyte binding to the embedded receptor sites the
conjugated p orbitals undergo distortion, leading to a partial
twist of the p-orbitals. Thus, the dark blue color of the poly-
mers gradually shifts to a red color depending on the
amount of the stress induced.
The color change of the liposomes from blue to red was
quantified by calculating the colorimetric response (CR)
using Equation (1). The CR value is derived from the
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Self-Assembled Vesicular Receptors
FULL PAPER
absorption spectrum upon addi-
tion of ATP and PPi.
Recent studies[20] from our
group demonstrate that dinu-
clear ZnII–cyclen complexes
show an even higher affinity to
ATP and PPi ions than mono-
nuclear ZnII–cyclen complexes.
The vesicular receptors contain-
ing 8-Zn were therefore expect-
ed to have an increased affinity
to ATP and PPi ions. Titration
experiments revealed that ap-
proximately 20 equivalents of
ATP or PPi were sufficient to
achieve complete saturation
(see Figure 6). This is consider-
ably less as compared to LP-4-
Zn. Other anions such as Fꢀ,
ꢀ
Clꢀ,
Brꢀ,
Iꢀ,
H2PO4 ,
CH3COOꢀ, AMP and ADP did
not induce any spectral changes
of the liposome solution.
Table 2 and Figure 5 summarize
the results.
Liposome containing a mix-
ture of both metal complexes 4-
Zn and 8-Zn behave in their re-
sponse towards anions such as
Scheme 4. Representative mixed diacetylene liposome system of TCDA and 4-Zn after mixing and upon poly-
merization induced by irradiation with light of 254 nm wavelength; counter ions are not shown.
Figure 2. Schematic representation of the preparation and the analyte response of self-assembled polydiacetylene vesicles with embedded metal complex
binding sites for anions.
change in the ratio of absorbance at 640 and 550 nm in the
absence (A0) and presence (AX) of different analytes. The
absorption ratio before analyte addition is calculated as
A0 =I620/(I620 + I490) and the absorption ratio after analyte
addition follows from Ax =I620/(I620+I490), respectively.
the liposomes containing only 8-Zn. This shows that only
the presence of the dinuclear ZnII–cyclen metal complex
binding sites evokes an increased affinity of the vesicular re-
ceptors to the anion.
While the binding of ATP and PPi as highly charged oxo-
AHCTUNGTRENNUNG
anions to the bis(ZnII–cyclen) binding sites was expected
from previous studies, the response to the presence of cya-
nide ions is surprising (Figure 6). We suspected a pH effect
or a dissociation of the zinc ion from the metal complex. To
% CR ¼ ½ðA0ꢀAxÞ=A0ꢂ ꢃ 100
ð1Þ
Figure 4a and b show the induced changes in the LP-4-Zn
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B. Kçnig et al.
Figure 3. a) UV-visible spectra of LP-4-Zn (5ꢃ10ꢀ5 molLꢀ1) in the presence of different anions (aqueous solution, HEPES 10 mmol, pH 7.2, 100 equiv of
the anion salt added). b) Color change of the blank LP-4-Zn with different anions.
Figure 5. Colorimetric response (% CR) to different anions with lipo-
&
&
somes LP-4-Zn ( ), LP-8-Zn ( ) and LP-11-Cu (&).
vesicular receptor can therefore not be attributed as the
origin of the colorimetric change. Carboxy-terminated poly-
diacetylene vesicles are known to undergo a blue to red
chromic transition in the pH range 9.0–10.1.[21] The pH of
the aqueous buffered vesicles solution (HEPES 10 mm,
pH 7.2) was determined at the end of each titration. In the
case of added basic cyanide ions the pH of the solution con-
siderably increased to 10.0–10.5, in all other cases the pH
value remained between 7.2–8.0. This confirms that the pH
change of the solution causes the color change upon addi-
tion of a large excess of cyanide ions.
Figure 4. UV-visible absorption titration of LP-4-Zn (5ꢃ10ꢀ5 molLꢀ1).
a) Upon addition of ATP (5ꢃ10ꢀ6–3.5ꢃ10ꢀ3 molLꢀ1). b) Upon addition
of PPi (5ꢃ10ꢀ6–3.5ꢃ10ꢀ3 molLꢀ1).
Apparent binding constants of the liposome–anion com-
plex were derived from the change in UV-visible absorbance
at 490 nm with respect to the concentration of specific
anions using non-linear regression analysis (Figure 7). The
calculated binding constant (lgKapp) values are given in
Table 3. CR values and lgKapp values reveal that ATP and
PPi ions strongly interact with the interface of the vesicle re-
ceptor, while other phosphates and halide ions do not
induce a significant color and spectral change.
Table 2. Calculated colorimetric response (%CR) values of LP-4-Zn,
LP-8-Zn and LP-11-Cu upon addition of various anions.
ꢀ
None ATP PPi ADP AMP H2PO4
Fꢀ
CNꢀ
LP-4-Zn
LP-8-Zn
LP-11-Cu
–
–
–
54.0
73.7
9.7
67.2 21.7
81.8 20.8
–
–
–
0.9
2.4
–
12.6 46.1
2.5 79.8
86.4
6.5
–
51.6
investigate if the colorimetric response towards cyanide ions
is induced by decomplexation of the cyclen complexes, the
liposome solution was treated with the strong complexing
agent EDTA. The addition of EDTA did not lead to a color
change on the solution. Displacement of zinc ions from the
After we established the binding properties of ZnII–cyclen
based receptor LP-4-Zn and LP-8-Zn, we investigated the
binding of CuII–IDA based receptor LP-11-Cu to different
anions in more detail. Upon addition of an excess amount
of PPi (100 equivalent) the absorption band at 640 nm dis-
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Self-Assembled Vesicular Receptors
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ꢀ
Clꢀ,
Brꢀ,
Iꢀ,
H2PO4 ,
CH3COOꢀ, AMP, ADP and
ATP, under similar conditions.
It is interesting to note that
CuII–IDA complex modified
vesicles behave differently as
compared to ZnII–cyclen modi-
fied vesicles with phosphates.
Among different phosphates
LP-11-Cu responded only to
PPi with an apparent binding
constant lgKapp =2.7 and did
not show any spectral and color
changes with ATP as LP-4-Zn
and LP-8-Zn do (Figure 8).
Thus LP-11-Cu is suitable for
the selective detection of PPi
over ATP in aqueous media.
Coordinatively unsaturated CuII
complexes exhibit strong bind-
ing tendencies towards anionic
substrates due to the d9 elec-
tronic configuration of the
metal center, which ensures
Figure 6. UV-visible absorption titration of LP-8-Zn (8.2ꢃ10ꢀ5 mol Lꢀ1). a) Upon addition of ATP (5ꢃ10ꢀ6
–
3.5ꢃ10ꢀ3 molLꢀ1). b) Upon addition of PPi (5ꢃ10ꢀ6–3.5ꢃ10ꢀ3 molLꢀ1). c) Color change of LP-8-Zn with
100 equivalents of different anions.
Table 3. Calculated apparent binding constant (lgKapp) of LP-4-Zn, LP-8-
Zn and LP-11-Cu with different analytes.
Receptor
Anion affinity [lg Kapp]
ꢀ
liposomes ATP PPi ADP AMP H2PO4
Fꢀ
<1.0
–
CH3COOꢀ
–
–
–
LP-4-Zn
LP-8-Zn
LP-11-Cu
2.2
2.7
–
2.5
3.2
2.6
<1.0
<1.0
–
–
–
–
–
–
–
–
high ligand field stabilization effects. As a result, the anionic
substrate PPi is effectively bound even in the strongly sol-
vating aqueous conditions. Very few synthetic receptors
have been reported so far that allow a discrimination of PPi
and ATP in aqueous media.[22]
Emission studies with different anions: The room tempera-
ture emission spectra (Figure 9) of LP-4-Zn (5ꢃ
10ꢀ5 molLꢀ1), LP-8-Zn (8.2ꢃ10ꢀ4 molLꢀ1) and LP-11-Cu
(6ꢃ10ꢀ5 molLꢀ1) recorded in aqueous buffered solution
(HEPES 10 mmol, pH 7.2) show a very weak emission band
centered at 625 nm upon excitation at 510 nm. The intensity
of LP-4-Zn and LP-8-Zn emission at 570 nm and 640 nm in-
creases significantly upon addition of an excess of ATP and
PPi (Figure 9bnd Figure 10a)
Figure 7. UV-visible titration profile of a) LP-4-Zn (5ꢃ10ꢀ5 molLꢀ1) and
b) LP-8-Zn (8.2ꢃ10ꢀ5 molLꢀ1) with different anion in aqueous buffered
solution (HEPES 10 mmol, pH 7.2) at RT.
ꢀ
Other anions, such as ADP, AMP, CNꢀ, Brꢀ, H2PO4 or
Fꢀ, induce only very little or no change in the emission in-
tensity (Figure 9a). Although CNꢀ ions induce changes in
the absorption spectrum of the liposomes due to pH in-
crease, the effect on the emission spectrum is small
(Figure 10b). In the case of LP-11-Cu among different phos-
phorylated ions only PPi induced an emission enhancement.
Other phosphates, such as ATP, ADP, AMP and H2PO4, did
appeared completely and an intense absorption band at 489
and 543 nm is observed (Figure 8). The color of the solution
turned red. However, no changes in the absorption spectra
or color could be observed with other anions, such as Fꢀ,
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B. Kçnig et al.
Figure 8. UV-visible absorption titration of LP-11-Cu (6.0ꢃ10ꢀ5 molLꢀ1).
a) Upon addition of different anions (6.0ꢃ10ꢀ3 molLꢀ1). b) UV-visible ti-
tration profile with different anion in aqueous buffered solution (HEPES
10 mmol, pH 7.2) at RT.
Figure 10. Emission titration spectra of LP-8-Zn (8.2ꢃ10ꢀ5 molLꢀ1) a) ti-
tration with varying concentrations of ATP (1ꢃ10ꢀ5–3.2ꢃ10ꢀ3 molLꢀ1
)
b) titration with varying concentrations of CNꢀ (1ꢃ10ꢀ5–3.2ꢃ
10ꢀ3 molLꢀ1).
Figure 11. Relative changes in emission intensity (lex =510 nm) upon
treatment with different anions in aqueous buffered solution (HEPES
&
&
10 mmol, pH 7.2) of LP-4-Zn ( ), LP-8-Zn ( ) and LP-11-Cu (&).
not show any significant change in the emission spectra
(Figure 11).
Test paper analysis: The response in a color change of LP-4-
Zn, LP-8-Zn and LP-11-Cu in the presence of ATP and PPi
was used to prepare a test paper stripe, which allows the
simple detection of ATP and PPi at millimolar concentra-
tions in water.
Figure 9. Emission titration of LP-4-Zn (5ꢃ10ꢀ5 molLꢀ1) a) with different
anions (100 equivalents) b) titration with increasing amounts of PPi (5ꢃ
10ꢀ6–3.5 ꢃ10ꢀ3 molLꢀ1).
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Self-Assembled Vesicular Receptors
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Arrays of unpolymerized LP-4-Zn, LP-8-Zn and LP-11-
Cu vesicles containing test papers were prepared by soaking
filter papers in HEPES buffered aqueous solution of the
vesicles (pH 7.2) and drying them in air. These colorless test
papers were stored in the dark and irradiated with light of
254 nm before further experiments. For detecting the ana-
lyte, the blue colored test paper was immersed in the aque-
ous analyte solution for several seconds and then air-dried.
As shown in Figure 12a, in the case of LP-4-Zn and LP-8-
Zn a color change of the test papers is observed for aqueous
solutions containing ATP and PPi ions. However, in the case
of LP-11-Cu only with PPi a color change was obtained.
Other anions did not induce any color change at equal con-
centrations. Based on this color change of the test paper
ATP or PPi concentration levels in aqueous media may be
estimated (Figure 12b).
Next, the liposomes particles morphology of LP-8-Zn was
investigated in the presence of PPi, ATP or H2PO4 . Blank
ꢀ
blue color LP-8-Zn vesicles were exposed to the PPi, ATP
ꢀ
or H2PO4 for a few minutes and then viewed under the
light microscope with identical magnification. The images of
the PPi and ATP exposed liposomes show that the binding
of these anions induces aggregation of the liposomes[23]
ꢀ
(Figure 14a). However, in the case of H2PO4 this was not
observed due to the weaker binding of this ion (Figure 14b).
In ongoing more detailed investigations of the morphology
changes using high resolution electron microcopy we will try
to elucidate the mechanism of the surface anion binding in-
duced rapid liposome aggregation.
Figure 14. Light microscopy images (20 X, objective lens) of a) LP-8-Zn
ꢀ
with PPi, b) LP-8-Zn with H2PO4
.
Conclusions
In summary, we have shown that nanometer sized polymer-
ized vesicular receptors LP-4-Zn, LP-8-Zn and LP-11-Cu
can be prepared from amphiphilic diacetylene metal com-
plexes. The vesicular receptors obtained from ZnII–cyclen
functionalized polydiacetylene monomers respond to the
presence of ATP and PPi at neutral pH at millimolar con-
centrations with visible changes of their color and emission.
Other anions, such as halides, do not induce an analytic re-
sponse. The CuII–IDA modified vesicular PDA receptors
show a selective response to PPi, which allows a simple col-
orimetric and visible discrimination between ATP and PPi.
The induced aggregation of the liposomes by the specific
binding of ATP or PPi analytes to the surface receptors is
the likely origin of the intensive changes in their optical
properties. Polymerized vesicular receptors with metal com-
plex binding sites may find applications for simple analytic
tasks, as shown with anion test papers. However, the devel-
opment of more complex vesicular receptors with increased
affinity and specificity may also be envisaged.
Figure 12. a) Color changes of LP-4-Zn, LP-8-Zn and LP-11-Cu
(0.35 mmol) test papers treated with different anions (15 mmol) at room
temp. b) LP-4-Zn test paper dipped with two different concentration of
aqueous PPi solution.
Light microscopy images of the vesicles: To monitor the
changes of the morphology of the vesicles upon analyte
binding they were investigated under a light microscope
(Leica, Wetzlar, Germany). Freshly prepared vesicles solu-
tions LP-4-Zn, LP-8-Zn and LP-11-Cu were dropped on the
glass slide and viewed using normal light microscopy.
ACHTUNGTRENNUNGFigure 13a, b and c shows the appearance of the PDA vesi-
cles LP-4-Zn, LP-8-Zn and LP-11-Cu without any anionic
analyte. The images reveal that they were well separated
and spherical in shape.
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft for support of this work.
D.A.J. thanks the Alexander von Humboldt foundation for a fellowship.
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Received: February 7, 2009
Published online: June 23, 2009
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