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Self-Assembled Tb3+ Complex Probe for Quantitative Analysis of ATP during Its Enzymatic Hydrolysis via Time-Resolved Luminescence in Vitro and in Vivo Sung Ho Jung,†,⊥,# Ka Young Kim,†,⊥ Ji Ha Lee,† Cheol Joo Moon,† Noh Soo Han,‡ Su-Jin Park,§ Dongmin Kang,*,§ Jae Kyu Song,*,‡ Shim Sung Lee,† Myong Yong Choi,*,† Justyn Jaworski,*,∥ and Jong Hwa Jung*,† †

Department Department § Department ∥ Department ‡

of of of of

Chemistry and Research Institute of Natural Sciences, Gyeongsang National University, Jinju, Korea Chemistry, Kyung Hee University, Seoul 130-701, Korea Life Science, Ewha Womans University, Seoul 120-750, Korea Chemical Engineering and Institute of Nanoscience and Technology, Hanyang University, Seoul, Korea

S Supporting Information *

ABSTRACT: To more accurately assess the pathways of biological systems, a probe is needed that may respond selectively to adenosine triphosphate (ATP) for both in vitro and in vivo detection modes. We have developed a luminescence probe that can provide real-time information on the extent of ATP, ADP, and AMP by virtue of the luminescence and luminescence lifetime observed from a supramolecular polymer based on a C3 symmetrical terpyridine complex with Tb3+ (S1-Tb). The probe shows remarkable selective luminescence enhancement in the presence of ATP compared to other phosphate-displaying nucleotides including adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), H2PO4− (Pi), and pyrophosphate (PPi). In addition, the time-resolved luminescence lifetime and luminescence spectrum of S1-Tb could facilitate the quantitative measurement of the exact amount of ATP and similarly ADP and AMP within living cells. The time-resolved luminescence lifetime of S1-Tb could also be used to quantitatively monitor the amount of ATP, ADP, and AMP in vitro following the enzymatic hydrolysis of ATP. The long luminescence lifetime, which was observed into the millisecond range, makes this S1-Tb-based probe particularly attractive for monitoring biological ATP levels in vivo, because any short lifetime background fluorescence arising from the complex molecular environment may be easily eliminated. KEYWORDS: lifetime, adenosine triphosphate (ATP), chemoprobe, supramolecular polymer, lanthanide

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INTRODUCTION Adenosine triphosphate (ATP) present in living organisms serves a number of crucial roles, perhaps the most well-known being the energy “currency” of cellular metabolism.1 Hydrolysis of a phosphate group from ATP to yield adenosine diphosphate (ADP) provides the means for energetic coupling to otherwise unfavorable enzymatic reactions. Hydrolysis of ATP also occurs upon polymerization into nucleic acids, as it is one of the purine monomers.2 Loss of pyrophosphate, PPi, by adenylate cyclase to yield cyclic adenosine monophosphate (cAMP) is similarly important, as this small molecule plays a key part in signal transduction.3 Probes for assessing ATP as well as its hydrolytic breakdown products are particularly important for understanding intracellular kinetics of enzymes and also for high-throughput screening assays. Specifically, determining intracellular signaling networks including the activity and presence of certain kinases, which are the key subset of enzyme that perform such ATP hydrolysis, remains a critical area of © XXXX American Chemical Society

research for both basic biochemical and cellular studies as well as applied work, such as drug screening and controlling stem cell differentiation. In the past, a number of sensing approaches for the detection of ATP4−9 or other phosphate-containing analytes10−12 have proven to be effective. These have often incorporated a spectroscopic approach including UV−visible absorption,13 fluorescence,14 circular dichroism,15 or nuclear magnetic resonance spectroscopy.5,15 While these work well for in vitro measurements, the use of such systems in vivo for selective measurement of ATP in living cells has, in contrast, resulted in relatively few reports.1,6,16,17 The implementation of fluorescent/luminescent-based probes for ATP detection has shown exciting developments recently. In a particular example wherein Received: October 11, 2016 Accepted: December 9, 2016 Published: December 9, 2016 A

DOI: 10.1021/acsami.6b12857 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Preparation of Supramolecular Nanostructure S1-Tb. Stock solution of 1 (2.0 mM) with Tb(NO3)3·5H2O (2.0 mM) in methanol was stirred for 60 min at RT. The solution mixture was added to deionized water and then heated at 50 °C for 10 min. The mixture was allowed to cool to RT (−0.5 K/min). This stock solution was diluted to desired concentration in water (fractions of water = 99 vol %) with 50.0 mM HEPES buffer at pH 7.4. Sample Preparation for Luminescence Measurement. Stock solutions of S1-Tb and nucleotide in water and 50.0 mM HEPES buffer (10 mM NaCl, 1 mM MgCl2, pH 7.4) were prepared. The titration experiments with the nucleotide were performed at 293 K with a solution (3.0 mL) of S1-Tb (20.0 μM), HEPES buffer, and various concentrations of the nucleotide. The luminescence emission spectral change (excitation wavelength λex = 325 nm, slit widths = 3) was monitored upon addition of a freshly prepared aqueous solution of the analyte with a microsyringe. Luminescence titration curves at 545 nm were analyzed with the nonlinear least-squares curve-fitting method to evaluate the apparent binding constant (Kapp, M−1). Abbreviations of the analytes listed in Table S1 are as follows: ATP = adenosine-5′-triphosphate, ADP = adenosine-5′-diphosphate, GTP = guanosine-5′-triphosphate, TTP = thymidine 5′-triphosphate. Hydration Number Obtained by Luminescence Lifetimes. The luminescence decay of an aqueous solution of S1-Tb (20.0 μM) was measured. For measurements in D2O, the samples were lyophilized and redissolved in D2O three times prior to analysis. All decay measurements monitored the emission at 545 nm. The luminescence lifetimes (τ) were determined by fitting the data to an exponential decay. The hydration number was calculated according to the following equation developed by Horrocks:27 q = 4.2[(1/τH2O) − (1/τD2O)]. Time-Resolved Luminescence Lifetime Measurements. By using a conventional laser system, the emission lifetime were measured upon generation with an excitation source (420 nm output of a Spectra-Physics Qunta- Ray Q-switched GCR-150-10 pulsed Nd/YAG laser). The signals for the luminescence decay were obtained using a Hamamatsu R928 PMT, and the data were recorded on a Tektronix model TDS-620A (500 MHz, 2 GS/s) digital oscilloscope from which it was exponentially fit for analysis. Lifetime (τ) was measured upon excitation of the S1-Tb at 325 nm generated by a frequency-doubled (using a BBO crystal) output of a pulsed nanosecond Nd:YAG (Continuum, Powerlite 8060)-pumped dye laser (Continuum, ND-6000, DCM as a dye) operating at 10 Hz with a 7 ns pulse width. The emitted lights were collected using an ARC SpectraPro-300i monochromator and detected with a Hamamatsu R2368 photomultiplier tube (PMT). The entrance and exit slits of the monochromator were opened to 0.5 mm, and the PMT voltage was 700 V (delay time: 0, gate time: 40 ms, time resolution: 1.0 μs). The signal was digitized and stored by a digital storage oscilloscope (LeCroy, LT322, 500 MHz, 200 MS/s) and subsequently processed with a homemade autodata collecting program using LabVIEW. ATP Hydrolysis Experiment. All samples for spectroscopic measurements were prepared by injecting the 2 × 10−3 M stock solution of S1-Tb into the required volume of aqueous HEPES buffer. The required amounts of phosphates were injected into the buffer, and the solution was mixed by manual stirring before measurement. Commercially available CIAP (calf intestinal alkaline phosphatase) was 2.8 U mg−1, and CIAP stock solution was prepared by dissolving 2 mg of CIAP in 80 mL of aqueous HEPES buffer. Each enzyme kinetic measurement was performed by adding an appropriate amount of CIAP stock solution into the phosphate-bound stacks, and all measurements were initiated immediately. All CIAP-based hydrolysis kinetics of ATP were studied with 1 equiv of ATP. Confirmation of Replacement of the Water Molecules by ATP. S1-Tb (10 mM) for 1H NMR measurements was prepared in CD3OD that was dried with molecular sieves and in CD3OD containing H2O (30 mM), without and with ATP (10 mM). The required amounts of ATP (10 mM) were injected into stock solutions of S1-Tb, and the solution was mixed by manual stirring before measurement.

the detection principle relied on excimer formation with aromatic groups, selectivity toward phosphate groups was achievable by metal coordination and hydrogen bonding.12 With such fluorescent-based sensing techniques, the probes have short emission wavelengths on the order of 350−500 nm and, as is often the case, they have particularly short (nanoseconds) lifetimes. This can be a drawback for in vivo assays because nontarget cellular features can also exhibit levels of fluorescence in this short wavelength range. Ongoing improvements in this area have shown lanthanidebased reporter as promising front-runners to overcome the problems of short luminescence lifetime.18 In particular, terbium- and europium-based probes have proven luminescence lifetimes of milliseconds.19−21 In a recent breakthrough report, ATP detection by making use of such lanthanide-based chemoprobes has been demonstrated in vitro using a “turn-off” mode of detection from a terbium-derived reporter.22−25 Being able to monitor not only ATP but also its hydrolytic breakdown products in living cells remains a key objective. With this goal in place, we pursued the use of a unique terbium-based reporter that could function in a practical “turn-on” mode for timeresolved luminescence detection and quantification of ATP and the hydrolytic products ADP and AMP, not only in vitro enzymatic systems but also for intracellular measurements. This represents the first results of detecting ATP by time-resolved luminescence lifetime within living cells.

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METHODS

General. Unless otherwise noted, chemical reagents and solvents were purchased from commercial suppliers (Tokyo Chemical Industry (TCI), Aldrich) and used without further purification. The UV−Vis absorption spectra of the samples were recorded on a Thermo Evolution 600 spectrophotometer equipped with a Peltier cell holder for temperature control. Using a Shimadzu Fourier transform infrared 8400S instrument, the IR spectra of samples from KBr pellets were observed over the range of 400−4000 cm−1. In addition, the 1H and 13 C NMR spectra were taken on a Bruker DRX 300, and mass spectroscopy samples were analyzed on a JEOL JMS-700 mass spectrometer. The elemental analysis was performed with a PerkinElmer 2400 series II instrument. Atomic force microscope (AFM) imaging was performed using a PPP-NCHR 10 M cantilever (Park Systems). The samples were prepared by spin-coating (1500 rpm) onto freshly cleaved muscovite mica, and images were recorded with the AFM operating in tapping mode in air at RT with resolution of 1024 × 1024 pixels, using moderate scan rates (0.3 Hz). All luminescence spectra were recorded with a RF-5301PC spectrophotometer. Synthesis of Ligand 1. Compound 1 was synthesized according to a literature procedure.26 A solution of compound 2 (0.014 g, 0.053 mmol) in dry dichloromethane was added dropwise to a solution of compound 3 (0.05 g, 0.164 mmol) in dry dichloromethane at 0 °C under nitrogen atmosphere. The mixture was stirred at room temperature for 24 h, and then the solid was obtained as a white precipitate and washed with dichloromethane. (0.051 g, 90%). mp 230 °C; 1H NMR (300 MHz, CD3OD) 8.74, 8.72 (d, 2H, CHtpy), 8.64 (s, 1H, CHbenzene), 8.25 (s, 2H, CHtpy), 8.03, 8.01, 7.99 (t, 2H, CHtpy), 7.61, 7.60, 7.57, 7.51 (4H, CHtpy), 3.69, 3.67, 3.64, 3.62, 3.60 (m, 4H, CH2), 2.15, 2.13, 2.11, 2.09, 2.06 (m, 2H, CH2); 13C NMR (125 MHz, CD3OD) 167.22, 160.36, 160.04, 149.99, 146.83, 145.90, 138.54, 138.25, 135.17, 131.91,131.67, 131.17, 129.31, 126.85, 121.73, 121.73,110.65, 106.05, 100.79, 41.30, 37.43, 28.21; IR (KBr, cm−1): 3245, 3094, 3058, 2942, 2871,1637, 1589, 1527, 1464, 1443, 1352, 1297, 1270, 1238, 1159, 1124, 1090, 1034, 993, 908, 867, 785, 736; ESI-MS: m/z 358.25 [M + 3H]3+/3, 536.92 [M + 2H]2+/2, 1072.58 [M + H]+; calculated for C63H59N15O3 1073.4925, found 1073.4930. B


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Scheme 1. “Turn-On” Luminescence Sensing Mechanism of Supramolecular Assembly 1-Tb (S1-Tb) for ATP: (a) No Morphology (turn-on), (b) Helical Structure (turn-off), and (c) Spherical Structure (turn-on)

Figure 1. (A) The luminescence spectra of S1-Tb (20.0 μM) upon addition of ATP (0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40.0 μM) in 50.0 mM HEPES buffer (10 mM NaCl, 1 mM MgCl2, pH 7.4) using a 10 mm width cell at 289 K (λex = 325 nm). Inset: curve-fitting analysis of luminescence change at 545 nm. Cell width = 10 mm. (B) Luminescence responses of S1-Tb (20.0 μM) to various anions (20.0 μM) and nucleotides (20.0 μM) at 545 nm. (C) Photograph was obtained under a UV lamp at 365 nm.

Figure 2. (A) Time-resolved luminescence decay curves of S1-Tb (20.0 μM) with ATP (0−20.0 μM) in 50.0 mM HEPES buffer (10 mM NaCl, 1 mM MgCl2, pH 7.4) at 289 K (λex = 325 nm). (b) Lifetime values as a function of concentration of ATP (0−20.0 μM). Determination of Amount of ATP, ADP, and AMP. The equation y = a + [n1 × b1e−b1t] + [n2 × b2e−b2t] + [n3 × b3e−b3t] was used for curve fitting where the number of components could be

changed from one to three (with the equation above specifically used for a three-component fit).28,29 A Levenberg−Marquardt method was used for fitting. The parameters used were as follows: (a: intercept of C


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ACS Applied Materials & Interfaces calculated linear line by curve fitting, b: slope of calculated linear line by curve fitting, n: signal magnitude, t: decay time). In Vivo Determination of ATP, ADP, and AMP in Cells. HeLa cells were cultured in low glucose DMEM (Lonza OEM/Pan biotech) supplemented with 10% FBS (Gibco) and 1% penicillin−streptomycin (Hyclone). To measure in vivo ATP, ADP, and AMP concentrations using luminescence, HeLa cells (4 × 105 cell/well) in 12-well dishes containing coverslips (diameter, 12 mm) coated with poly-L-lysine were loaded with 0.1 mM S1-Tb probe for 1 h. The cells with S1-Tb were incubated in media with or without 10 mM 2-deoxy-D-glucose (2-DG), (Sigma), an inhibitor of glycolysis, or alternatively with media containing extracelluar ATP, ADP, or AMP for 1 h. The luminescence was measured from cells collected in 100 μL of media.

(S1-Tb) that affords real-time measurement of ATP and its derivatives by its selective luminescence lifetime in the presence of ATP compared with ADP and AMP. In addition, this approach also afforded intracellular measurements of ATP and the ratio of ATP to AMP. Because of the long lifetimes and its fast, differential response to the presence of ATP and its breakdown products, we reveal the capabilities of this real-time probe as a unique “turn on” sensor for measuring the enzymatic hydrolysis of ATP for either in vitro or in vivo use. In recent works, researchers have used Zn or Tb ion chelating moieties for fluorescence- or luminescence-based sensors of ATP in vitro.22,31,32 To achieve a system with high sensitivity for ATP and a long luminescence lifetime, we synthesized 1, capable of forming a complex with the lanthanide Tb3+. The spectroscopic properties of 1 (1.0 × 10−5 M) in methanol upon titration of 0−9 equiv of Tb(NO3)3 revealed a substantial increase in the absorption spectra at 330 nm with a concomitant decrease at 280 nm. In looking closer at the absorption spectrum of 1, the absorption at 280 nm for the π → π* transition of the terpyridine moiety seems to shift to a shoulder at 330 nm when exposed to Tb3+ (Figure S1a). The luminescence spectra exhibited increasing emission peaks at 491, 544, 584, and 622 nm following the amount of Tb(NO3)3 (Figure S1). These spectral peaks are in accordance with the corresponding transition from the 5D4 excited state to the 7F6, 7 F5, 7F4, and 7F3 ground state of Tb3+. The observed plateau of the absorption and luminescence spectra at 1 equiv suggested that a complex of 1 with the Tb3+ ions occurs with 1:1 stoichiometry (Figure S2), which was also established by the corresponding m/z peak signals obtained by ESI-MS for [1(Tb)(NO3)2] and [1-(Tb)(NO3)3+H+] (Figure S3). Importantly, the above-mentioned luminescence was gradually quenched upon the addition of water to the 1-Tb3+ complex in methanol (Figure S4). As the amount of water was increased, almost complete quenching of the luminescence signal was observed at 1:99 v/v of MeOH:water. While the strong luminescence of 1-Tb3+ in methanol is attributed to the terpyridine moieties serving as sites of energy transfer to the Tb3+, the quenching effect is attributed to water serving as an additional ligand in coordination with sites on the Tb ion, resulting in nonradiative energy transfer via O−H vibrations.33 To assess the extent of water coordinating at the inner-sphere with the lanthanide ion complexed with 1, we assessed the luminescence lifetimes for the Tb-centered emission for both the case of H2O and of D2O based on Horrocks’s method.27 The emission lifetime (τ) for H2O was calculated to be 0.16 ms while that of D2O was 0.21 ms. From these calculations, a single Tb center, which has nine coordination sites available, would bind to three water molecules on open coordination sites and three nitrate anions and have three coordinate bonds with a single terpyridine moiety of 1. This suggests the weak emission of 1-Tb3+ in water

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RESULTS AND DISCUSSION In our approach, we synthesized terpyridine-appended ligand 1, which was capable of transforming into self-assembled

Figure 3. Luminescence decay curves of S1-Tb (20 μM) with ATP (20 μM) in the presence of CIAP (1 U/mL) over time (0−120 min) (excitation wavelength λex = 325 nm).

nanostructures owing to π−π stacking, hydrogen bonding, and coordination between the terpyridine moieties with Tb3+.30 In water, the supramolecular nanostructure with terbium ions (referred to as S1-Tb) resulted in strong intermolecular interactions between the stacked terpyridine moieties, thereby preventing photoelectron transfer from the Tb-complexed terpyridine and keeping the chromophore “off”. Conversely, upon addition of purine nucleotides, specifically ATP, a “turnon” response could be observed by intercalation and relaxation of the supramolecular structure. In addition, electrostatic interaction between the four minus charges of ATP favored the interaction with the positively charge terbium complex, thereby affording a preferential “turn-on” response to ATP over ADP or AMP (Scheme 1). We find that a time-resolved luminescence approach could facilitate accurate quantification of the amount of ATP, ADP, and AMP in the course of a hydrolyzing enzyme reaction. We present here our findings of this supramolecular 1-Tb probe

Table 1. Concentrations and Lifetimes of ATP, ADP, and AMP Calculated by the Lifetime Measurement of S1-Tb (20 μM) with ATP (20 μM) in the Presence of CIAP (1 U/mL) over Time (0−120 min) τtotala τATPb τADPb τAMPb a

0 min

20 min

40 min

80 min

100 min

120 min

347 347 (20 μM) (0 μM) (0 μM)

292 315 (16.8 μM) 106 (3.2 μM) (0 μM)

278 234 (10.6 μM) 109 (4.2 μM) 96 (5.2 μM)

167 123 (2.2 μM) 105 (2.6 μM) 109 (15.2 μM)

135 97 (0.2 μM) (0 μM) 114 (19.8 μM)

115 (0 μM) (0 μM) 115 (20 μM)

Experimental total lifetime. bTheoretical lifetime for phosphate species obtained from the fitting method. D


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Figure 4. (A) Time-resolved luminescence spectra of S1-Tb (0.1 mM) after incubation with ATP (10−60 μM) using 0.1 mm of cell width in living cells. (B) Luminescence decay curves of S1-Tb (0.1 μM) after incubation with ATP (10−60 μM) in the living cells.

Table 2. Lifetime Values of Phosphate Species Existing in the Living Cell by Using the S1-Tb Probe (0.1 mM) and Concentrations of ATP, ADP, and AMP Obtained by the Curve Fitting Method entry a

1 2a 3 4 5 6 7

injected concentration 0 0 ATP 20 μM ATP 40 μM ATP 60 μM ADP 60 μM AMP 60 μM

τtotalb 249.3 336.2 340.4 272.1 199.7 287.1 272.4

± ± ± ± ± ± ±

6.7 6.4 5.8 7.6 7.6 8.0 7.1

τATPc

τADPc

τAMPc

ATPd

ADPd

AMPd

ATP:AMP ratio

± ± ± ± ± ± ±

99 ± 2.1 100 ± 3.1 99 ± 2.5 102 ± 3.0 102 ± 3.1 103 ± 2.8 101 ± 2.7

97 ± 2.3 104 ± 3.1 95 ± 2.4 96 ± 1.8 97 ± 2.5 96 ± 2.3 112 ± 3.0

30.1 ± 3.0 46 ± 3.7 49.9 ± 4.0 73.6 ± 5.1 88.7 ± 4.2 26.5 ± 3.5 29.4 ± 3.4

2.8 ± 0.5 3.7 ± 0.4 7.9 ± 0.2 7.4 ± 0.8 2.3 ± 0.3 65.4 ± 2.0 4.9 ± 0.6

15.7 ± 1.2 44.3 ± 3.8 17.6 ± 1.0 14.3 ± 1.1 9.8 ± 1.1 12.6 ± 1.2 78.5 ± 3.4

2:1 1:1

233 354 311 244 178 186 188

6.4 6.7 7.5 9.2 6.6 9.1 9.7

a Entry 1: before treatment of the 2DG (inhibitor), entry 2: after treatment of the 2DG (inhibitor). bTotal lifetime (μs). cLifetime (μs) obtained by fitting from τtotal. dThe amounts (μM) of phosphate species calculated by the lifetime in the living cell.

was due to a decrease in the extent of coordination in the complex. Moreover, we have found that the addition of water causes the self-assembly of 1-Tb3+ into nanofibers according to AFM and SEM observations (Figure S5A and S5B) due in part to π−π stacking driven by the poor solubility of 1 in water. In looking to such a structure as a possible probe, the ability to limit the coordination of water to the cationic Tb3+ could serve as a means for enhancing luminescence, thereby providing a “turn-on” mode of sensing. Because this could have potential as a chemoprobe for detecting anionic biological components, we investigated the binding ability of S1-Tb (20 μM) upon addition of H2PO4− (Pi), pyrophosphate (PPi), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), and thymidine triphosphate (TTP) (Figure S6), as well as inorganic anions (SO42−, NO3−, HCO3−, ClO4−, and OAc− as sodium salts) (Figure S7) in an aqueous solution containing 1% methanol (v/v) (aqueous conditions; 50 mM HEPES, pH 7.4). To our surprise, we found there to be significant enhancement in the luminescence spectra upon addition of purines, specifically ATP, ADP, GTP, and GDP, with a pronounced selectivity for ATP among these. In looking to confirm this, we examined exposure of ATP (0−40 μM) to an aqueous solution of S1-Tb (20 μM) which revealed a remarkable increase in luminescence intensity for increasing ATP concentrations as shown in Figure 1A. The luminescence intensity showed a good linearity upon addition of ATP (2.0− 20.0 μM). These results indicate that ATP molecules were bound to S1-Tb quantitatively. Specifically, in looking at the luminescence intensity at 545 nm, we found there to be a 5-fold increase after addition of 40 μM ATP. In contrasting this result with a related prior report, the Pierre group has been successful at using a terbium complex in which the luminescence “turnsoff” upon addition of ATP, because the donor atoms of the

macrocyclic ligand were completely coordinated to the terbium ion without solvent molecules.18,34 In our study, we clearly observed a “turn-on” system in S1-Tb for ATP, due to a lack of a coordination site in one terpyridine moiety. Having identified the potential for S1-Tb to serve as a “turnon” sensor for ATP, we examined the selectivity in more detail. As seen in Figure 1b, specificity for signal enhancement in the presence of ATP and to a lesser extent ADP and GTP was observed for the S1-Tb probe. In a related work, it was found that a rationally designed terbium complex could elicit a luminescent signal for ATP and GTP over other nucleotide phosphates.31 To obtain evidence of π−π stacking between the adenine moiety and the terpyridine moiety, 1H NMR spectra of ATP were measured in the absence and presence of S1-Tb (Figure S8A). The proton peaks of the adenine group in the presence of S1-Tb were shifted to high field, which was attributed to π−π stacking interaction. 1H NMR spectra of S1Tb (10 mM) were also observed in the presence of H2O (30 mM) without and with ATP (10 mM) indicating replacement of the water molecules (Figure S8B). The broad proton peak of H2O in S1-Tb (10 mM) solution without ATP resulted in sharp peak upon addition of ATP. This broad proton peak of water without ATP was due to suppression of the rotation of water molecules by a coordination bond to the Tb3+ ion. The broad proton peak of H2O without ATP changed into the sharp peak with ATP. The result indicates that water molecules coordinated to Tb3+ were replaced by oxygen atoms of ATP. In addition, the proton of H2O in S1-Tb (10 mM) solution upon addition of ATP (10 mM) was shifted to high field, which was attributed to replacement of water molecules by ATP. Thus, the coordination of the oxygen atoms of the phosphate moiety of ATP resulted in increased luminescence. Disruption of the selfassembled nanowire complex may thereby facilitate excited terpyridine groups to again participate in energy transfer to the E


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ACS Applied Materials & Interfaces excited 5D state of terbium to allow phosphorescence of the Tb center to the 5F ground state. Indeed we find that the oxygen atoms of ATP interact with terbium as indicated by the 31P NMR results (Figure S9) that shows the alpha and gamma phosphate sites to have extensive deshielding upon exposure to S1-Tb, indicating their interactions with the probe. Moreover, the three phosphorus peaks experience signal broadening upon addition of S1-Tb to the ATP solution. Interestingly, this possible role of the anionic oxygens involved in interaction with the cationic Tb center can be further justified by the apparent increase in selectivity for ADP over AMP and similarly ATP over ADP. It may be expected that energy transfer from the adenosine to the terpyridine could enhance the terbium luminescence in addition to the phosphate groups of ATP replacing the coordinated water molecules upon complex formation with 1-Tb3+. In contrast, ADP and AMP did not participate in π−π stacking with the terpyridine moiety of 1, which could be attributed to a steric hindrance from the short binding lengths between the adenine moiety and the phosphate moiety. Furthermore, the weak electrostatic attraction favors binding to the four negatively charged groups on ATP over the three groups on ADP and two on AMP.34 This would in turn increase selectivity for ATP over ADP or AMP including other phosphate species. In looking at the binding isotherm of ATP titration in a solution of S1-Tb, we see a sigmoidal curve with an apparent binding constant (Kapp, M−1) of 2.24 × 1014 M−1 for ATP. This was found to be significantly higher affinity binding than the Kapp of ADP or TTP. Table S1 summarizes the apparent binding constants of S1-Tb wherein the binding affinities for GTP were also found to be considerably higher than that of ADP or TTP. This would suggest that the high selectivity arises from the purine nucleotide base as well as the three anionic phosphate groups. Interestingly, the calculated Hill coefficient for ATP and GTP interactions with S1-Tb suggests high cooperativity in binding, while ADP and TTP binding was independent. The possibility for purine intercalation and loosening of the self-assembled nanofiber-like structure could find a means for such cooperativity to expose other sites on the S1-Tb supramolecular assembly. According to AFM and SEM observations, the self-assembled nanofiber structure of S1-Tb was changed into the spherical structure after exposure to ATP (Figure S5C and S5D). This morphology change is in interesting contrast to a recent report of ATP-induced assembly of tetraphenylethene-linked guanidinium which was found to result in fluorescence signals.35 Furthermore, we observed luminescence changes of S1-Tb with ATP at different pH values (Figure S10). No significant luminescence response changes of S1-Tb with ATP were observed from 6 to 9 pH values. Thus, the self-assembled S1-Tb that has high sensing ability toward ATP works effectively under physiological pH conditions. In addition, the remarkable luminescence changes were not observed with an excess of NaCl (10 Mm) and MgCl2 (1 mM). These results indicate that the ionic strength did not affect the luminescence property in the S1-Tb system (Figure S11). To further assess the interaction of S1-Tb with ATP, the time-resolved luminescence was used to examine the luminescence lifetime of the S1-Tb probe in various concentrations of ATP (Figure 2). As compared to conventional organic dyes, the emission lifetime of S1-Tb was very long, beginning from 0.09 ms and extending to 0.35 ms after addition of 20.0 μM ATP. Interestingly, the luminescence

lifetime of the S1-Tb increased linearly as a function of ATP concentration (Figure 2b), and a similar response with shorter lifetimes was observed for ADP as well as AMP (Figures S12 and S13). This differentiation in fluorescence lifetime with respect to concentration could provide a means for distinguishing ATP as compared to ADP or AMP. The luminescence intensity of the ATP response was interestingly not impacted by the presence of other potentially competing species, such as ADP, AMP, Pi, PPi, GTP, and TTP, even at 10 equiv relative to ATP, presumably due to the high binding affinity of S1-Tb for ATP over the other analytes (Figure S14). A calibration curve of the intensity at 545 nm as a function of ATP concentration performed well at higher concentration even when in the presence of 10 equiv of GTP (Figure S15), confirming the ability of S1-Tb to sense ATP with high selectivity and low background noise despite the presence of other nucleoside triphosphates. For testing the probe capabilities of S1-Tb to detect ATP with in vitro enzymatic systems, we monitored the changes in luminescence lifetime upon hydrolysis of ATP by a direct enzymatic reaction with calf intestinal alkaline phosphatase (CIAP).4 This enzyme could provide hydrolytic cleavage of ATP to yield the derivative ADP as well as AMP, which we were able to monitor in real time by observing the S1-Tb signal by the time-resolved luminescence technique (Figure 3). To assess the lifetimes of S1-Tb over the course of the cleavage of ATP into ADP and AMP, the decay profiles were fitted by global analysis, which led to determination of the fractional contribution averaged lifetimes.36 From this, we quantified the amount of ATP converted to ADP and/or AMP by using three equations for the calibration curves of ATP, ADP, and AMP, respectively (Figure S16). As expected, the amount of ATP was found to decrease continuously over time, while AMP increased over time. The amount of ADP however reached a maximum as it was first formed by hydrolysis of one phosphate unit from ATP but then subsequently hydrolyzed again to AMP by CIAP. The exact values for the time-dependent concentrations of ATP, ADP, and AMP throughout the course of the reaction are provided in Table 1. ATP and ADP were completely hydrolyzed into AMP after 120 min. Importantly, we compared our time-resolved luminescence results for ATP hydrolysis with that of a reliable but labor-intensive method using NMR. As shown in Figure S17, our results were consistent with the actual concentrations determined by time-course NMR. Because the results from the S1-Tb-based probe provided a highly correlated signal for quantification of ATP and its hydrolytic breakdown products, we found our approach useful as a realtime “turn-on” sensor for measuring the in vitro enzymatic rates of ATP hydrolysis. Finally, we examined the in vivo capability of the S1-Tbbased probe to quantify ATP levels in living cells. From the sample of cell culture, time-resolved luminescence lifetime and luminescence spectrum measurement were performed to measure the amounts of ATP, ADP, and AMP. The luminescence spectrum and luminescence lifetime of S1-Tb provide clear ATP signals despite being obtained from within living cells (Figure 4). These results were consistent with our in vitro system. With our initial measurements, the ratio of ATP:AMP was found to be 2:1 prior to incubation with an inhibitor of glycolysis, 2-deoxy-D-glucose (2DG), while after exposure to the inhibitor we identified a significantly reduced amount of ATP but with increased levels of AMP to give an ATP:AMP ratio of 1:1 (Table 2). For the cell sample before F


Research Article

ACS Applied Materials & Interfaces 2DG, the concentration of ATP (30.01 μM, entry 1 in Table 2) obtained by time-resolved luminescence lifetime in the living cell is reasonable considering that the concentration of ATP in cells is expected to be nearly 3 mM and the cell volume represented 1/100 of the total sample volume in solution for our luminescence measurements.28,29,37 Importantly, we also observed that when the cells were provided a known amount of 60 μM ATP, ADP, or AMP supplemented to the culture media, the corresponding luminescence lifetime signals reflected an ∼60 μM respective increase in the given metabolite from our quantification. Furthermore, the observed lifetime of ATP after injection of 20, 40, and 60 μM in culture media with living cells showed good linearity (Figure S18). These indicate that extracellular metabolites will also impart a signal; however, more importantly we see that we can achieve accurate quantification within the cell environment by this approach. This method possesses several advantages over existing technologies; specifically, it does not require a modified substrate, and it enables continuous monitoring of the reaction kinetics such as ATP hydrolysis.

Myong Yong Choi: 0000-0001-5729-5418 Jong Hwa Jung: 0000-0002-8936-2272

CONCLUSION In summary, we synthesized S1-Tb which itself is quenched in aqueous solutions due to coordination of H2O and the π−π stacking-driven nanofiber assembly but interestingly exhibited restored luminescence in the presence of ATP and to a much lesser extent ADP, GTP, GDP, and TTP, thereby operating in a “turn-on” mode of detection. The intense selectivity for ATP led us to assess the capability of the S1-Tb to serve as an in vitro probe. We confirmed the quantification of the extent of ATP by luminescence intensity and even identified the amount of ATP, ADP, and AMP by virtue of the calibrated luminescence lifetime, which was distinct for the metabolites. Furthermore, we established the fact that the S1-Tb probe could be effective in monitoring ATP levels for enzymatic in vitro reactions as well as in vivo cell culture systems. In this work, we have assessed the selective binding affinities of the probe to ATP and have examined the binding mechanisms. Perhaps most importantly, we feel the long luminescence lifetime of the S1-Tb probe bodes well for the practicality of this probe in complex culture systems, as it may provide a unique signal in contrast to the short lifetime fluorescence signals that often suffer from interference due to the high background noise intrinsic to cell systems.

(1) Imamura, H.; Nhat, K. P. H.; Togawa, H.; Saito, K.; Iino, R.; Kato-Yamada, Y.; Nagai, T.; Noji, H. Visualization of ATP Levels inside Single Living Cells with Fluorescence Resonance Energy Transfer-Based Genetically Encoded Indicators. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 15651−15656. (2) Egli, M.; Saenger, W. Principles of Nucleic Acid Structure; Springer Science & Business Media, Germany: Berlin, 2013; Chapter 1, pp 1− 19. (3) Neary, J. T.; Zhu, Q. Signaling by ATP Receptors in Astrocytes. NeuroReport 1994, 5, 1617−1620. (4) Kumar, M.; Brocorens, P.; Tonnelé, C.; Beljonne, D.; Surin, M.; George, S. J. A Dynamic Supramolecular Polymer with StimuliResponsive Handedness for in Situ Probing of Enzymatic ATP Hydrolysis. Nat. Commun. 2014, 5, 5793. (5) Yan, Q.; Zhao, Y. ATP-Triggered Biomimetic Deformations of Bioinspired Receptor-Containing Polymer Assemblies. Chem. Sci. 2015, 6, 4343−4349. (6) Tantama, M.; Martínez-François, J. R.; Mongeon, R.; Yellen, G. Imaging Energy Status in Live Cells with a Fluorescent Biosensor of the Intracellular ATP-to-ADP Ratio. Nat. Commun. 2013, 4, 2550. (7) Westman, G.; Lidehall, A.-K.; Magnusson, P.; Ingelsson, M.; Kilander, L.; Lannfelt, L.; Eriksson, B.-M. Decreased Proportion of Cytomegalovirus Specific CD8 T-Cells but No Signs of General Immunosenescence in Alzheimer’s Disease. PLoS One 2013, 8, e77921. (8) He, H. Z.; Ma, V. P. Y.; Leung, K. H.; Chan, D. S. H.; Yang, H.; Cheng, Z.; Leung, C. H.; Ma, D. L. Label-Free G-Quadruplex-Based Switch-On Fluorescence Assay for the Selective Detection of ATP. Analyst 2012, 137, 1538−1540. (9) Xu, M.; Gao, Z.; Zhou, Q.; Lin, Y.; Lu, M.; Tang, D. Terbium Ion-Coordinated Carbon Dots for Fluorescent Aptasensing of Adenosine 5′-Triphosphate with Unmodified Gold Nanoparticles. Biosens. Bioelectron. 2016, 86, 978−984. (10) Lee, D. H.; Kim, S. Y.; Hong, J. I. A Fluorescent Pyrophosphate Sensor with High Selectivity over ATP in Water. Angew. Chem., Int. Ed. 2004, 43, 4777−4780. (11) Kurishita, Y.; Kohira, T.; Ojida, A.; Hamachi, I. OrganelleLocalizable Fluorescent Chemosensors for Site-Specific Multicolor Imaging of Nucleoside Polyphosphate Dynamics in Living Cells. J. Am. Chem. Soc. 2012, 134, 18779−18789. (12) Lee, H. N.; Xu, Z.; Kim, S. K.; Swamy, K.; Kim, Y.; Kim, S.-J.; Yoon, J. Pyrophosphate-Selective Fluorescent Chemosensor at Physiological pH: Formation of a Unique Excimer upon Addition of Pyrophosphate. J. Am. Chem. Soc. 2007, 129, 3828−3829. (13) Morikawa, M.-A.; Yoshihara, M.; Endo, T.; Kimizuka, N. ATP as Building Blocks for the Self-Assembly of Excitonic Nanowires. J. Am. Chem. Soc. 2005, 127, 1358−1359.

Present Address #

National Institute for Materials Science (NIMS), 1-2-1 Sengen, Tsukuba-city, Ibaraki, Japan.

Author Contributions ⊥

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from NRF (2012R1A4A1027750, 2014M2B2A9030338, 2015R1A2A1A10053576, and 2015R1A2A2A05001400). In addition, this work was partially supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant no. PJ011177022016), Rural Development Administration, Korea.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b12857. 1 H NMR and 13C NMR spectrum of the final product, AFM and SEM images, UV−vis data, fluorescence data, and time−resolved luminescence decay (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: *E-mail: *E-mail: *E-mail: *E-mail:

S.H.J. and K.Y.K. contributed equally to the work.

Notes

[email protected]. [email protected]. [email protected]. [email protected]. [email protected].

ORCID

Shim Sung Lee: 0000-0002-4638-5466 G

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Research Article

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