Zn(II)-cyclam based chromogenic sensors for recognition of atp in aqueous solution under physiological conditions and their application as viable staining agents for microorganism

Article abstract of DOI:10.1021/ic200223g

Two chromogenic complexes, L.Zn (where L is (E)-4-((4-(1,4,8,11- tetraazacyclotetradecan-1-ylsulfonyl)phenyl)diazenyl)-N,N-dimethylaniline) and its [2]pseudorotaxane form (α-CD.L.Zn), were found to bind preferentially to adenosine triphosphate (ATP), amon

Full text of DOI:10.1021/ic200223g

ARTICLE
pubs.acs.org/IC
Zn(II)ꢀCyclam Based Chromogenic Sensors for Recognition of ATP in
Aqueous Solution Under Physiological Conditions and Their
Application as Viable Staining Agents for Microorganism
Prasenjit Mahato, Amrita Ghosh, Sanjiv K. Mishra, Anupama Shrivastav, Sandhya Mishra,* and Amitava Das*
Central Salt & Marine Chemicals Research Institute (CSIR), Bhavnagar, 364002 Gujarat, India
S Supporting Information
b
ABSTRACT:
Two chromogenic complexes, L.Zn (where L is (E)-4-((4-(1,4,8,11-tetraazacyclotetradecan-1-ylsulfonyl)phenyl)diazenyl)-N,
N-dimethylaniline) and its [2]pseudorotaxane form (r-CD.L.Zn), were found to bind preferentially to adenosine triphosphate
(ATP), among all other common anions and biologically important phosphate (AMP, ADP, pyrophosphate, and phosphate) ions in
aqueous HEPES buffer medium of pH 7.2. Studies with live cell cultures of prokaryotic microbes revealed that binding of these two
reagents to intercellular ATP, produced in situ, could be used in delineating the Gram-positive and the Gram-negative bacteria. More
importantly, these dyes were found to be nontoxic to living microbes (eukaryotes and prokaryotes) and could be used for studying
the cell growth dynamics. Binding to these two viable staining agents to intercellular ATP was also confirmed by spectroscopic
studies on cell growth in the presence of different respiratory inhibitors that influence the intercellular ATP generation.
’ INTRODUCTION
medium6 (pH ∼ 7.2) and, in some cases, a limited pH range
existence.7 Recognition of phosphate ions in an aqueous medium
is adversely affected for such reasons. However, recognition of
inorganic phosphates (PO43ꢀ and its different protonated form,
pyrophosphate (P2O74ꢀ), etc.) and phosphates that are the part
of respective metabolites like adenosine monophosphate
(AMP), adenosine diphosphate (ADP), and adenosine tripho-
sphate (ATP) is imperative due to their involvement in numer-
ous crucial biological processes ranging from intercellular energy
conversion and storage and metabolism to biosynthesis, signal
transduction, ion-channel regulation, muscle contraction, gene
regulation, etc.8 ATP also serves as a phosphate donor in kinase
catalyzed protein phosphorylation. ATP is present in all meta-
bolically active cells, and its concentration gets reduced when a
cell undergoes necrosis or apoptosis.9 Deficiency in ATP results
in ischemia, Parkinson’s disease, and hypoglycemia.10 Thus,
monitoring the ATP concentration level is crucial for the study
The realization that the tailor-made small molecule could be
used as a selective and efficient probe for recognition of certain
ionic and neutral species, having detrimental effect(s) on human
health and environment, has led to a recent exponential growth
in the area of molecular sensors.1,2 Among different types of
sensor molecules, research on the fluorescence-based chemosen-
sors has received more attention, as these generally offer a lower
detection limit for the desired analyte, as well as for their
application potential in imaging processes.3 This led to adverse
growth for small-molecule colorimetric chemosensors; however,
colorimetric or spectroscopic detection of biologically important
ions and, thus, the evaluation of various biological phenomena
have received considerable attention in recent years due to the
obvious ease in the detection procedure.4 Further, a colorimetric
sensor offers the possibility of the semiquantitative visual detec-
tion of the targeted analyte(s).5 Among different ions that have
biological significance, recognition of anionic analytes is gener-
ally more challenging as compared to their cationic counterparts
due to the high solvation energy of these anions in aqueous
Received: February 2, 2011
Published: March 30, 2011
r
2011 American Chemical Society
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of different cellular mechanisms, enzymatic processes, and cell
apoptosis, as well as for assessing cytocidal, cytostatic, and
proliferative effects of drugs and biological response modifiers.11
All these have necessitated the development of an appropriate
reagent for rapid and convenient determination system for ATP;
these include a luciferinꢀluciferase bioluminescence assay12,13
and electrochemical,14 fluorometric,6d,15 and colori-
metric16,17 sensors. Among these, for the reasons mentioned
above, research efforts for the development of chromogenic
sensors that allow visual detection of ATP in physiological
condition have been intensified in recent times. Despite wide-
spread interest and recent advances, there are only a few
examples of colorimetric sensors for ATP with desired sensitivity
and rapid response time, which promise real-time detection of
ATP under various biological conditions without special skills or
instruments.17 In our earlier communications,17b,c we have
shown that a Zn(II)ꢀdipicolylamine based coordination com-
plex with a pendant azo-functionality as the reporter group could
be used for staining yeast cells (Saccharomyces cerevisiae, an
eukaryotic microbe) or in prokaryotic microbes like Bacillus
megaterium (Gram-positive) and Pseudomonas fluorescence
(Gram-negative) through preferential binding to the ATP pro-
duced in situ, while time duration for the staining process was
about 5 min. More importantly, this reagent was found to be
nontoxic to the living cells, an ideal criterion for practical
applications. However, this Zn(II)-based reagent had a limited
solubility in pure aqueous or HEPES buffer medium. More recently,
we have synthesized a new Zn(II)ꢀcyclam complex and reported
our preliminary observations on ATP-recognition and cell viability
studies using live cells of Saccharomyces cerevisiae (patent application
details: 0093NF2009 [1242DEL2010], 24.05.2010).17a
In eukaryotes, most ATP is produced in chloroplasts (for
plants), or in mitochondria (for both plants and animals). In
prokaryotes, ATP is produced in the cell wall, as well as in the
cytosol by glycolysis.18 Among the prokaryotes, Gram-positive
and Gram-negative bacteria have different cell wall structures and
chemical composition,19 and the classification is made based on
the positive or negative results from Gram’s staining method,
which is generally a multistep process.20 Therefore, it would be
even more important for developing an appropriate staining
agent that could effectively delineate the membrane of the
smaller bacteria following a single step procedure.
Culture method is the most popular method for monitoring
bacteria in various sources of fresh water, which is a time-
consuming (g24 h) process for obtaining results, and more
importantly, some viable bacteria are difficult to culture. Further,
quantification and yeast viability are critical for fermentation,21
medical examination,22 wastewater treatment,23 dental biofilm,24
and bioengineering systems.25 This necessitates the development
of a more rapid and reliable staining method using reagents that
are nontoxic to the wide varieties of live cells. In many instances,
cells stained with certain fluorescent markers (e.g., 4,6-diamidi-
no-2-phenylindole dihydrochloride (DAPI)) are nonviable.26
Moreover, one of the major aspects that has received relatively
less attention is the time profile action of energy-dependent
processes in microbes, which would enable us to monitor the
bioprocess in a rapid, easy, and accurate manner through
ATP binding with the growing microbes, i.e., their correla-
tion to growth kinetics response. In this present Article, we
have addressed this specific issue using two viable staining
agents, L.Zn and its [2]pseudorotaxane form (r-CD-L.Zn)
(R-CD = R-cyclodextrin) (Scheme 1), for different eukaryotic
Scheme 1. Methodology Adopted for the Synthesis of L and
L.Zn
and prokaryotic cells. These reagents could even distinguish
the Gram-positive (Bacillus megaterium) and Gram-negative
(Pseudomonas fluorescence) bacteria following a simple one step
staining procedure. Cell growth dynamics was probed by mon-
itoring the spectral changes associated with the binding of the
ATP, produced during metabolic processes of the growing
microbes, to the Zn(II)-center of L.Zn or r-CD-L.Zn, and this
correlated well with actual cell growth kinetics response. Pre-
liminary results on synthesis and studies on staining live cells of
Saccharomyces cerevisiae were reported in our earlier work.17a
’ RESULTS AND DISCUSSION
Synthesis and Sensing Properties. The intermediate ligand
(E)-4-((4-(1,4,8,11-tetraazacyclotetradecan-1-ylsulfonyl)phenyl)
diazenyl)-N,N-dimethylaniline (L) was synthesized by reacting
1,4,8,11-tetraazacyclotetradecane with (E)-4-((4-(dimethylamino)
phenyl)diazenyl)benzene-1-sulfonyl chloride (Scheme 1 and
Supporting Information, SI).17a Absorption maxima for L in
CH3CN/CH2Cl2 (1:1, v/v) appeared at 425 nm. This charge
transfer (CT) band was attributed predominantly to the
πNMe [HOMO]- and π[NdN]*[LUMO]-based transition (SI
2
Figure 1). Systematic spectroscopic studies revealed a distinct
affinity for L toward Zn2þ, and 1:1 complex formation took place
in CH3CN/CH2Cl2 (1:1, v/v) medium with formation constant
of 3.45 ( 0.97 ꢁ 104 Mꢀ1 17a
,
while the color of the solution
changed from pale yellow to pale orange. This led us to the
synthesis of L.Zn by reacting L with Zn(NO3)2 6H2O in
3
methanol medium (SI).17a Both L and L.Zn were characterized
by standard analytical and spectroscopic techniques, and related
spectra are provided in the Supporting Information (SI Figures
1ꢀ5). UVꢀvis spectra recorded for L and L.Zn (SI Figure 1)
showed a broad absorption band with λmax at 422 and at 453 nm,
respectively, in CH3OHꢀCH3CN medium (3:7, v/v). This red-
shifted band maximum at 453 nm for L.Zn was attributed to a
more favored intercomponent (πꢀπ*) charge transfer (CT)
transition. This charge transfer nature of L.Zn is supported by the
bathochromic shift in absorption maxima with the gradual
increase in solvent polarity (SI Figure 6). This CT band for L.
Zn in 10 mM HEPES buffer medium appeared at 463 nm. In
order to examine the binding behavior of the reagent, L.Zn,
toward various anionic analytes (e.g., ATP, CTP (cytidine
2
ꢀ
triphosphate), ADP, AMP, PPi, H2PO4ꢀ, SO4 ꢀ, CH3CO2
,
Iꢀ, Brꢀ, Clꢀ, Fꢀ, CNꢀ, SCNꢀ, NO3ꢀ, and NO2ꢀ), UVꢀvis
spectra for L.Zn were recorded in the absence and presence of
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Scheme 2. Schematic Representation of the Formation of
[2]Pseudorotaxane, r-CD-L.Zn, and the Binding of ATP to
the Zn(II)-Center of L.Zn or r-CD-L.Zn
and O[R-PO ]ꢀ compared to the O[β-PO ]ꢀ unit accounts for the
4
4
smaller Δδ shift in 31P NMR spectra. This observation also
confirms the formation of a heptacoordinated Zn(II)-center in L.
Zn in the presence of ATP (Scheme 2). Examples of heptacoor-
dinated Zn(II)-centers are available in the literature.29 A very
insignificant shift in 31P signals was observed when similar
experiments were repeated for CTP, while no shift was observed
for ADP for similar experiments.17a The 31P NMR spectral data
also confirms the affinity that was established through spectral
studies (ATP . CTP > ADP . AMP). Presumably, the higher
electrostatic interaction between the Zn2þ-center and tripho-
sphates and the formation of a higher number of chelate rings
between ATP or CTP and L.Zn are crucial for efficient L.
ZnꢀOPhosphate binding, as compared to those for ADP and
AMP. The lack of any interaction between PPi, despite having
the similar “4ꢀ“ charge as that of CTP/ATP, and L.Zn perhaps is
better explained on the basis of its very high solvation energy.30
The 1:1 complex formation (L.ZnꢀATP) was further confirmed
by ESI-MS spectral studies (SI Figure 10). Signals at 1103.9 and
1082 correspond to m/z for [L.ZnꢀATP] ꢀ 2(NO3ꢀ)] (A) and
[(A ꢀ Naþ) þ Hþ], respectively, with anticipated isotope
distribution.
large excess (2000 mol equiv) of respective anions in 10 mM
HEPES buffer medium (pH 7.2). A further red-shifted CT
absorption band for L.Zn with a maximum at 503 nm was
observed on addition of excess sodium salt of ATP (SI Figure 7).
Thus, a red shift of 40 nm was achieved on binding to ATP, while
an insignificant shift of 9 nm was observed when CTP of
comparable concentration was added and the change for ADP
was even less significant (SI Figure 7A).17a Other anionic
analytes failed to induce any detectable change in electronic
spectra (SI Figure 7A). A visually detectable change in solution
color was also observed on addition of ATP to L.Zn solution
(10 mM HEPES buffer medium), whereas this change was barely
detectable for CTP and ADP. For all other anionic analytes used
in this study, solution color remained unchanged. No change in
spectra on addition of these anions suggests either no or a very
weak binding of these anions to the Zn(II)-center of L.Zn.
Systematic spectrophotometric titrations were carried out in
10 mM HEPES buffer medium in order to evaluate the relative
binding affinities of ATP, CTP, and ADP toward L.Zn (SI
Reversible binding of L.Zn to ATP could be demonstrated by
adding an aqueous solution of sodium citrate (5.0 ꢁ 10ꢀ4 M) to
L.ZnꢀATP. The original spectrum of L.Zn (with λmax
=
463 nm) was restored when an aqueous solution of excess
sodium citrate was added to the solution of L.ZnꢀATP (SI
Figure 11).
More interestingly, the solubility of the L.Zn (0.045 g Lꢀ1) in
water was found to be substantially enhanced (0.34 g Lꢀ1) in the
presence of excess R-CD (4.5 g Lꢀ1) (SI Figure 12). Favored
nonbonded interactions between the (dimethylamino)phe-
nyl)diazenyl)benzene fragment of L.Zn and the hydrophobic
interior of the R-CD on inclusion into the R-CD cavity could
account for the observed enhanced solubility of L.Zn in
water; such inclusion of analogous (E)-1,2-di(pyridin-4-yl)eth-
ene and (E)-1,2-di(pyridin-4-yl)diazene in R-CD was reported
earlier.31,32 The absorption maximum for L.Zn (463 nm) was
found to shift to shorter wavelength (458 nm) in the presence of
R-CD (SI Figure 13). The kinetics of such complex formation is
generally ultrafast, and thus, any spectral change associated with
the formation of an inclusion complex is expected to evade the
normal optical spectroscopic measurements.31j Weak perturba-
tion of the HOMOꢀLUMO gap absorption spectral bands31,32
was anticipated for such inclusion complex formation and
resulted in the 5 nm blue shift of the intercomponent (πꢀπ*)-
based CT band for L.Zn with λmax at 458 nm (SI Figure 13).
Thus, the blue shift of 5 nm could be attributed to the formation
of a [2]pseudorotaxane complex, r-CD.L.Zn. Presumably, for-
mation of the more polar excited state for r-CD.L.Zn is less
favored in the apolar interior of R-CD than in the surrounding
solvent for a nonincluded reagent L.Zn.33 Analogous studies
were repeated with β-CD (β-cyclodextrin). Evaluation of the
respective association constants from systematic spectrophoto-
metric titration revealed a higher value for r-CD.L.Zn formation
than that of β-CD.L.Zn (KfAssc(R-CD) = 255 ( 15 Mꢀ1 and
KfAssc(β-CD) = 221 ( 13 Mꢀ1 at 25 °C) (SI Figure 13 and
SI eq 3). However, these association constant values are close to
those reported earlier for related systems.31 As R-CD forms a
stronger inclusion complex with L.Zn, we have used r-CD.L.Zn
for further studies. In the presence of excess R-CD (10-fold),
the [2]pseudorotaxane form, i.e., r-CD.L.Zn (Scheme 2), is
Figures 7ꢀ9). Affinity constants for ATP (Kf[L.Zn-ATP]
=
(1.9 ( 0.15) ꢁ 103 Mꢀ1), CTP (Kf[L.Zn-CTP] = (9.43 ( 0.14) ꢁ
102 Mꢀ1), and ADP (Kf[L.Zn-ADP] = (4.38 ( 0.12) ꢁ 102 Mꢀ1
)
were evaluated under identical experimental conditions at 25 °C
in aq HEPES buffer (pH of 7.2) medium using a Benesiꢀ
Hildebrand plot (SI Figure 7C and SI Figures 8 and 9). The
respective binding constant for ATP, CTP, and ADP clearly
reveals a highest affinity of L.Zn toward ATP. Binding constants
evaluated in each case were an average of at least three indepen-
dent spectrophotometric titrations. Presumably, the presence of
the electron withdrawing pyrimidone functionality in CTP, as
compared to the purine base in ATP, has made it a weaker
electron donor, which accounts for the weaker binding of L.Zn to
CTP, than that of ATP. Binding of ATP with L.Zn was also
confirmed by 31P NMR spectral studies. Upfield shifts were
observed for the 31P signals for the R- (0.07 ppm), β- (0.09 ppm),
and γ- (0.05 ppm) phosphorus atoms of the ATP, when bound
to L.Zn.17a The shift in 31P NMR signals for R-, β-, and γ-P
atoms signify the binding to the Zn(II)-center of L.Zn through
the oxygen atom, bearing the negative charge of the respectivꢀe
phosphate unit. Relatively weaker interaction of the O[γ-PO ]
4
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Figure 1. (A) Partial 1H NMR spectra of L.Zn (1.9 mM) with varying [R-CD] (0ꢀ76 mM) (in D2O). (B) 1H NMR spectra of R-CD (4.1 mM) in
presence of L.Zn (1equiv) (in CD3OD/D2O = 1:1) for δ = 3.45ꢀ5.04 ppm, a region where L.Zn does not have a resonance signal. (D1 and D2)
Schematic representation of pyranose structure and gauche conformation of R-CD with θ as the dihedral angle involving two vicinal hydrogen atoms, H1
and H2, of the pyranose ring.
expected to be the major component (g96%) either in the
aqueous solution or in HEPES buffer. Interestingly, formation of
the inclusion complex (r-CD.L.Zn) allowed for dissolving a
higher amount of L.Zn in water.
We also studied the inclusion complex (r-CD.L.Zn) forma-
1
tion by H NMR spectroscopy. A gradual downfield shift for
three aromatic hydrogen atoms (Hb, Hd, and He) of L.Zn (SI
Scheme 1) were observed on the addition of increasing amounts
of R-CD, while a subsequent upfield shift for Hc of L.Zn was
observed (Figure 1A). These shifts were more prominent for He
(Δδ = 197.5 Hz) and Hd (Δδ = 90.5 Hz), respectively
(Figure 1A), whereas the extent of downfield shift for Hb
Figure 2. Change in absorption spectra of (A) L.Zn (9.46 μM) in the
(Δδ = 55 Hz) and upfield shift for Hc (Δδ = 33 Hz) were
presence of varying [ATP] (0ꢀ17.28 mM). (B) r-CD.L.Zn ([L.Zn] =
relatively small. Such upfield and downfield shifts of the host
2.66 μM; [R-CD] = 26.6 μM) with varying [ATP] (0ꢀ0.032 M) in aq
HEPES buffer (pH = 7.2). Inset: BenesiꢀHildebrand plot of 1/(A ꢀ A0)
vs 1/[ATP] for the change in absorbance at 536 nm, upon addition of
ATP to L.Zn/r-CD.L.Zn.
protons on inclusion complex formation with R-CD are not very
uncommon.31n
The extent and direction of such shifts indicate a difference in
the extent of shielding and deshielding interactions of the
respective proton (Hb, Hc, Hd, and He) of L.Zn (SI Scheme 1
and Figure 1) on forming the inclusion complex with r-CD.L.Zn
through inclusion of L.Zn in the R-CD cavity. The downfield
shift indicates the proximity of a hydrogen atom to an electro-
negative oxygen atom, while the local polarity variation due to
van der Waals' forces between the aromatic guest and cyclodex-
trin host could have accounted for the observed upfield shift of
certain protons. Presumably, both influences are operational on
each hydrogen atom of the bis(azo phenyl)-part of the guest
molecule (L.Zn), and the relative extent, as well as direction of
shift, is the result of the overall influence of these two opposing
effects.31nꢀr Upfield shifts of 25.5ꢀ54.8 Hz were also observed
for the hydrogens (H1ꢀH6) of the R-CD moiety (Figure 1B).
Similar upfield shifts were also reported for an analogous
[2]pseudorotaxane formation with 4,40-bipyridine derivatives
and R-CD.31,32 Presence of the shielding effect due to diamag-
netic anisotropy of the benzenoid moiety of the included azo
compound could account for such upfield shifts. In the present
situation, after initial inclusion of L.Zn in the R-CD cavity,
possible polar interaction between the ꢀSO2ꢀ moiety of the
sulphonamide fragment and ꢀCH2OH functionality of the
R-CD larger rim could induce conformational changes of the
host cavity. Further support for the conformational change(s) of
structure suggested a definite change in the coupling constant,
3J12, for the R-CD moiety (Figure 1D1,D2 and SI Table 1). This
change in coupling constant value (Δ3J12) can be used to
calculate the change in dihedral angle (Δθ) involving two vicinal
H1 and H2 atoms of the pyranose ring of the host R-CD moiety
on forming an inclusion complex (SI).31l Value for Δθ = 10.6 was
thus evaluated (SI Table 1). The broadness in the observed
spectra (Figure 1B) might have contributed to some inaccuracy
in evaluating this calculated dihedral angle (θ); however, such
change in θ is reported earlier for related inclusion complexes
with R-CD as host.31k,l
All these results clearly indicate the formation of a hostꢀguest
inclusion complex between L.Zn and R-CD. Further, the
[2]pseudorotaxane formation was also confirmed by ESI-MS
studies; a molecular ion peak for r-CD.L.Zn was observed at
m/z value of 1648.6 (SI Figure 14). More importantly, the
formation of such inclusion complex helped to dissolve a higher
amount of L.Zn (0.5 mM) in aqueous solution and thus resulted
in a more intense color change on binding to ATP (5 mM) (vide
infra). The binding constant for ATP to L.Zn or r-CD.L.Zn was
evaluated from spectroscopic titration data (Figure 2).
Systematic changes in the spectral pattern of L.Zn or [r-CD.L.
Zn] were recorded with increasing [ATP] in aq HEPES buffer
medium (pH 7.2) and are shown in Figure 2. The absorption
maximum around 457 nm was found to decrease with a
concomitant increase in absorbance at 503 nm for L.Zn
(isosbestic points at 420 and 466 nm) and at 499 nm for
r-CD.L.Zn (isosbestic point at 468 nm). Thus a red shift of 46
and 42 nm was observed on binding of ATP to the Zn(II)-center of
1
the host R-CD cavity was available in the detailed H NMR
studies. 1H NMR spectra of R-CD (4.1 mM) and R-CD in the
presence of L.Zn (1 mol equiv) were recorded in CD3OD/D2O
(1:1, v/v). A critical examination, comparison of these two 1H
NMR. spectra, and evaluation of the coupling constants for the
interaction between vicinal H1 and H2 hydrogen of the pyranose
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Figure 4. SEM images of (A) blank yeast cells, (B) yeast cells treated
with L.Zn, (C) blank Gram-positive bacterial cells, (D) Gram-negative
bacterial cells treated with L.Zn, (E) Gram-negative bacterial cells, and
(F) Gram-negative bacterial cells treated with L.Zn ([L.Zn] = 0.66 ꢁ
10ꢀ4 M).
Figure 3. Light microscope images of Gram-positive bacteria (A)
unstained and stained with (B) L.Zn (0.66 ꢁ 10ꢀ4 M) and (C)
r-CD.L.Zn {[L.Zn] = (5 ꢁ 10ꢀ4 M) and [R-CD] = (5 ꢁ 10ꢀ3 M)}
at 25 °C. Light microscope images of Gram-positive bacteria (D)
unstained and stained with (E) L.Zn (0.66 ꢁ 10ꢀ4 M) and (F)
r-CD.L.Zn {[L.Zn] = (5 ꢁ 10ꢀ4 M) and [R-CD] = (5 ꢁ 10ꢀ3 M)}
at 25 °C in 10 mM HEPES buffer solution (pH = 7.2).
[R-CD] = 5 ꢁ 10ꢀ3 M). Figure 3BꢀE, taken with identical
magnification, revealed that the both dyes (L.Zn and r-CD.L.
Zn) could distinguish Gram-positive and Gram-negative bacteria
through distinctly different color, color intensities, and shape of
the stained cells. As anticipated, stained cells for Gram-positive
bacteria appeared longer in the images (Figure 3B,C), while
more intense staining was observed Gram-negative bacteria
(Figure 3E,F). Again after staining with L.Zn/r-CD.L.Zn, the
color of the Gram-positive bacterial cells changed from colorless
to pink, whereas in the case of Gram-negative bacteria the color
change occurs from colorless to violet. The difference in the
staining intensity for two different bacteria could be better
understood if one considers the difference in cell structure and
cell wall composition of the respective bacteria. Further, the
amount of extracellular ATP being released by Gram-negative
bacteria is reported to be more than that of the Gram-positive
bacteria, and this could contribute to the more intense staining of
the Gram-negative bacteria.35 The thinner, hydrophilic, and
more porous cell walls of the Gram-negative bacteria are
expected to allow higher excretion of ATP to the cell surface,
where it gets bound to the present dye L.Zn or r-CD.L.Zn and
thereby contributes also to the more efficient staining. Existing
reports on the recognition of ATP are mostly based on the
changes in fluorescence or electrochemical properties,14,15 and
examples for the colorimetric detection of ATP in aqueous
solution are rather limited.16,17
Scanning Electron Microscopy. SEM images (Figure 4) of
blank and stained eukaryote (yeast) and prokaryote (Gram-
positive and Gram-negative) cells revealed the change(s) in the
morphology of the outer surface of the cells on staining. SEM
images of yeast cells without dye were found to be smooth in
contrast to the images of the yeast cells with dye, where stained
cell surfaces were found to be rough. This signifies the presence
of the extraneous material, i.e., L.Zn, on the cell surface. Similar
changes on the cell surfaces for Gram-negative and Gram-
positive bacteria in the absence and presence of staining agent,
L.Zn, were also evident from the SEM images recorded
(Figure 4CꢀF). The difference in the contrasts in the SEM
images of the sample surfaces also signifies the nonhomogeneous
chemical nature of the sample surface. Relatively brighter cell
exterior for Gram-negative bacteria, as compared to the Gram-
positive bacteria, indicates the accumulation of higher negative
L.Zn and r-CD.L.Zn, respectively. The association constants for
the formation of the complex L.Zn-ATP and r-CD.L.Zn-ATP
were evaluated using BenesiꢀHildebrand plot (Figure 2) and
thus found to be Kf[L.Zn-ATP] = (1.9 ( 0.15) ꢁ 103 Mꢀ1 and Kf[R-
CD.L.Zn-ATP] = (1.52 ( 0.09) ꢁ 103 Mꢀ1. This shows a slightly
lower value for r-CD.L.Zn-ATP, as compared to that for L.Zn-
ATP. A visually detectable color change was noticed for
r-CD.L.Zn on binding of ATP, and this change was little more
prominent than that observed for L.Zn under a comparable
condition (SI Figure 12). Such a prominent color change was not
observed when ADP or CTP was used instead of ATP for binding
to r-CD.L.Zn. This visual change in color for L.Zn and r-CD.L.
Zn on binding to ATP led us to explore the feasibility of using
these two reagents for staining of the live eukaryote (yeast) and
prokaryote (Gram-positive and Gram-negative bacteria) cells, as
these microbes produce ATP in their cell surface during meta-
bolic processes.35 Preferential binding of L.Zn toward ATP was
also established by spectroscopic studies carried out in the
presence of 5 mol equiv excess of all other anions as compared
to that for ATP (SI Figure 15).
Staining Studies of the Prokaryotes (Gram-Positive and
Gram-Negative Bacteria). Light Microscopy Image. In our ear-
lier studies we could demonstrate that L.Zn or r-CD.L.Zn could
be used as a colorimetric staining agent for yeast cells and the
staining nature could be viewed under a simple light micro-
scope.17a Binding of the ATP ions, produced in situ, on the cell
surface to L.Zn or r-CD.L.Zn accounted for the plausible
reason for the observed staining. We have also checked the
possibility of using these reagents for staining cells of Bacillus sp.
(Gram-positive) and Pseudomonas sp. (Gram-negative) bacteria.
Figure 3AꢀF revealed the images of cells of Gram-positive and
Gram-negative bacteria in the absence and presence of these two
reagents, when viewed under light microscope.
Figure 3B,E revealed images of the same bacteria when viewed
in the presence of L.Zn (66 μM). These images clearly showed a
distinct change in color of the bacterial strain in the presence of L.
Zn. Figure 3C,F revealed images of the same bacteria when
viewed in the presence of r-CD.L.Zn ([L.Zn] = 5 ꢁ 10ꢀ4 M and
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useful for checking the cell growth dynamics for the above
referenced microbes. Results of such experiments are expected
to confirm the viability of these two colorimetric staining
reagents.
Growth Curve Study. The growth profiles of eukaryotic
(Saccharomyces cerevisiae) and prokaryotic (Gram-negative
(Pseudomonas fluorescence) and Gram-positive (Bacillus mega-
terium)) cells in an aqueous culture medium in the absence and
presence of L.Zn and r-CD.L.Zn are shown in Figure 5. Change
in absorbance (at λmax = 600 nm) versus time (t) was plotted up
to 20 h, as no appreciable change in growth was observed
thereafter. Figure 5aꢀf revealed a nice correlation between the
cell growth and the ATP generation with progressive growth of
the respective cells during the metabolic processes.
These kinetic studies were carried out to monitor the different
phases of growth, wherein the increase in the ATP concentration
as the batch culture grows from lag phase to log phase and then
stationary phase was observed, and ultimately a steady decline in
the growth curve was observed. Figure 5a,c,e shows the actual
growth curve of live cells of Saccharomyces cerevisiae, Pseudomonas
fluorescence, and Bacillus megaterium, respectively, with time,
while Figure 5b,d,f shows a relative buildup of [ATP] in
cytoplasm, measured with time for S. cerevisiae, Pseudomonas
fluorescence, and Bacillus megaterium, respectively, at 27 °C in
10 mM aq HEPES buffer (pH = 7.2) medium. A close review of
Figure 5 reveals that the actual cell growth and the relative
buildup in ATP concentration, measured spectrophotometrically
using reagents L.Zn and r-CD.L.Zn, in the cultures under
identical experimental conditions matched nicely. The relative
decrease, observed in actual cell growth and ATP concentration
(Figure 5), can be explained if one considers that the catabolic
processes, which consume ATP, become more important after a
certain time. Neither ATP nor ADP/AMP/PPi can be stored at
high concentration within the cells of the living system. Their
relative concentrations in cytoplasm are generally regulated over
a narrow concentration limit by various biological processes. The
concentration of intercellular ATP is typically around (1.0ꢀ
10) ꢁ 10ꢀ3 M,37 and spectrophotometric measurements re-
vealed that the lower detection limit of ATP for both these
colorimetric staining agents are much lower (lower detection
limits of ATP for L.Zn and r-CD.L.Zn are 18 and 23 μM,
respectively) than this. Thus, kinetic studies revealed that cell
growth and release of ATP during the metabolic process could be
correlated using either L.Zn or r-CD.L.Zn as the staining agent.
Extent of absorbance changes and the higher slope observed in
the case of Gram-negative bacteria revealed a higher binding of L.
Zn or r-CD.L.Zn to ATP released during the cell growth, as
compared to that of Gram-positive.
To ascertain that L.Zn or r-CD.L.Zn binds to ATP produced
during metabolic processes, studies with the aqueous culture
medium of the yeast cells were carried out in the absence and
presence of three different respiratory inhibitors: namely, CCCP
(carbonylcyanide-m-chlorophenylhydrazone, 100 μL of 300 μg/
mL),38a rotenone (100 μL of 300 μg/mL),38b and cycloheximide
(1 μL of 2 μg/mL).38c CCCP is generally thought to increase
proton permeability across the cell membrane, with consequent
dissipation of the membrane potential and inhibition of ATP
synthesis.39 Rotenone interferes with the electron transport
chain in mitochondria and thus interferes with NADH during
the creation of usable cellular energy (ATP).40 Thus, CCCP and
rotenone are expected to inhibit the proton pump of yeast cells,
and this causes the inhibition of the plasma membrane ATPase
Figure 5. Growth curve of (a) Saccharomyces cerevisiae, (c) Pseudomo-
nas fluorescence, and (e) Bacillus megaterium obtained from change in cell
count with time. Relative change in absorbance at 600 nm for aqueous
culture medium of (b) Saccharomyces cerevisiae, (d) Pseudomonas
fluorescence, and (f) Bacillus megaterium in presence of 100 μL L.Zn
(0.106 mM)/r-CD.L.Zn (L.Zn = 0.164 mM; r-CD = 1.64 mM) in
10 mM HEPES buffer (pH = 7.2).
charge on the cell surface. Gram-negative bacteria possess a lipid-
rich outer membrane (as well as a plasma membrane) and a thin
peptidoglycan layer, along with a pore forming protein like porins
spanning the outer surface.36 All these favor a higher accumula-
tion of the ATP at its cell surface and thereby the higher
accumulation of L.Zn-ATP, which could account for the brighter
exterior in the SEM image for the Gram-negative bacteria
exposed to L.Zn. This also supports the more intense staining
of the Gram-negative bacteria on staining with L.Zn or r-CD.L.
Zn and viewing through optical microscope (Figure 3).
Viability Assay of the Cells after Staining with L.Zn Com-
plex. The cell division of the live yeast cells was checked before
and after staining with the L.Zn or r-CD.L.Zn under the light
microscope over a period of 2 h after the stained cell suspension
was taken as a hanging drop prepared on a concavity slide (SI
Figure 16). It is worth mentioning that unstained yeast cells
could not be viewed through optical microscope as these were
colorless and cells appeared as yellow on staining with L.Zn and
as yellow-orange on staining with r-CD-L.Zn (SI Figure 16).17a
In our earlier work we have reported that cell division of
Saccharomyces cerevisiae cells remains unaffected in absence and
presence of L.Zn and r-CD.L.Zn.17a To develop a better
understanding about the viability of these reagents, cell growth
and motility for cells were checked for eukaryotic (Saccharomyces
cerevisiae) and prokaryotic (Bacillus megaterium (Gram-positive)
and Pseudomonas fluorescence (Gram-negative)) microbes. Un-
affected cell proliferation confirmed that the staining agent
(either L.Zn or r-CD.L.Zn) was nontoxic and kept cells viable
even after staining. Thus, L.Zn and r-CD.L.Zn could be used as
chemosensors for bioactive molecules like ATP produced in live
cells through different metabolic processes and thereby can be
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CCCP/rotenone, a slight increase in ATP generation was
observed until 8 h. Then, beyond 10 h, where the stationary
phase was achieved, a gradual decrease in the ATP generation
curve (Figure 6b) was registered. Due to an appreciable increase
in the cell numbers up to ∼10 h (Figure 6a), concentration of
CCCP was not sufficient to completely inhibit the plasma
membrane ATPase activity, and a nominal increase in ATP
concentration was registered until ∼8 h, after which stationary
phase was attained. After stationary phase the cell numbers
remain unaltered, but the ATP generation gets inhibited. For
rotenone the decrease in ATP was little more prominent
(Figure 6c,d). Cell growth studies on Saccharomyces cerevisiae
in the presence of cycloheximide clearly revealed a complete
inhibition of the growth of the yeast cell, as well as the ATP
production (Figure 6e,f).38c Thus, these results obtained in the
presence of a different respiratory inhibitor tend to confirm that
the binding of ATP to the Zn(II)-center of L.Zn/r-CD.L.Zn
was solely responsible for the increase in absorbance and the
detectable color change of the stained yeast cells. Thus, these two
reagents could be used as viable and efficient colorimetric
staining agents for prokaryotic and eukaryotic microbes for
viewing through optical microscope. Further, these reagents also
could be used for studies on cell growth dynamics, which has
relevance in terms of industrial application.
Figure 6. Growth curve of Saccharomyces cerevisiae and relative change
in [ATP] with time. Change in absorbance at 600 nm for aqueous
culture medium of Saccharomyces cerevisiae in the presence of (a) 100 μL
of 300 μg/mL CCCP in 200 mL of culture, (c) 100 μL of 300 μg/mL
rotenone in 200 mL of culture, (e) 1 μL of 2 μg/mL cycloheximide in
200 mL of culture. Relative changes in ATP concentration in the
presence (control) of (b) 100 μL L.Zn/r-CD.L.Zn þ 100 μL of
300 μg/mL CCCP, (d) 100 μL L.Zn/r-CD.L.Zn þ 100 μL of 300 μg/
mL (rotenone), (f) 100 μL L.Zn/r-CD.L.Zn þ 1 μL of 2 μg/mL Cyh
{[L.Zn] = 0.106 mM, [r-CD.L.Zn] = ([L.Zn], 0.164 mM; [R-CD],
0.938 mM) in 10 mM HEPES buffer (pH = 7.2)}.
’ CONCLUSION
Two colorimetric reagents, L.Zn and its [2]pseudorotaxane
form (r-CD.L.Zn), were used successfully for recognition and
detection of ATP produced in situ during different metabolic
processes in living organisms in pure aq HEPES buffer (pH =7.2)
medium. Lower detection limits, evaluated for these two re-
agents, were found to be much lower than the typical concentra-
tion of ATP in live cells of eukaryotic and prokaryotic microbes.
Studies further revealed that both colorimetric reagents were
nontoxic to the living microbes and could be used for staining live
cells of respective microbes for viewing through light microscope.
Experimental results confirmed that viability of the live cells of
Saccharomyces cerevisiae, Pseudomonas fluorescence, and Bacillus
megaterium was maintained in the presence of these two reagents
and could be used for studying the cell growth dynamics of each
of these individual microbes.
activity only and does not interfere with the growth of the cells.
For control experiment when these were not added to the culture
media of the yeast cells, growth curves remain almost unaffected
(Figure 5). Cycloheximide blocks the peptidyl transferase of 80S
eukaryotic ribosomes and, thus, interferes with the translocation
step in protein synthesis, blocking translational elongation and
inhibiting the cell growth.
Figure 6aꢀf clearly reveals the cell growth and ATP genera-
tion with time for yeast cells, where ATP intracellular buildup was
evaluated spectrophotometrically using either L.Zn or r-CD.L.
Zn as the colorimetric reagent. Comparison of Figure 6a,b
suggests that cell growth for Saccharomyces cerevisiae remained
unaffected in absence or presence of CCCP, whereas comparison
of Figure 6c,d suggests that cell growth for Saccharomyces
cerevisiae remained unaffected in absence or presence of rote-
none. Additionally, comparison of Figure 6aꢀd suggests that
there was no increase in intracellular [ATP] during the lag phase
of the cell growth; on the contrary a slight decrease in [ATP] was
registered with time in Figure 6b,d for experiments carried out in
presence of inhibitors like CCCP and rotenone, respectively.
Thus, the initial ATP generation with progressive growth of the
respective cells during the metabolic processes was found to be
appreciably affected on addition of CCCP/rotenone to the yeast
cell culture stained with L.Zn/r-CD.L.Zn. This observation
agrees well with the previous reports which state that CCCP/
rotenone inhibits the generation of ATP.38a,b In the case of
’ ASSOCIATED CONTENT
S
Supporting Information. Details regarding synthesis,
b
characterization, solubility experiment, 31P NMR study, spectro-
photometric titrations, reversibility study, and interference study.
Additional information as noted in text. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: amitava@csmcri.org (A.D.).
’ ACKNOWLEDGMENT
AD thanks DST (New Delhi) and CSIR (India) for financial
support. PM, AG, SKM and AS acknowledge CSIR for their Sr.
Research Fellowship.
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ARTICLE
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