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Article Cite This: ACS Omega 2017, 2, 6782-6785
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Probing Charge-Transfer and Short-Lived Triplet States of a Biosensitive Molecule, 2,6-ANS: Transient Absorption and TimeResolved Spectroscopy Mohan Singh Mehata,*,† Yang Yang,‡ and Keli Han*,‡ †
Laser-Spectroscopy Laboratory, Department of Applied Physics, Delhi Technological University, Bawana Road, Delhi 110042, India State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences, Dalian 116023, China
‡
S Supporting Information *
ABSTRACT: We report the existence of a short-lived triplet electronic state of 2,6-ANS (2-anilinonaphthalene-6-sulfonic acid), which, together with nonplanar (NP) and planar [charge-transfer (CT)] states, is produced following photoexcitation; these results are based on nanosecond transient absorption and time-resolved decays. The short-lived triplet state has a lifetime of ∼126 ns and is observed via triplet−triplet (T−T) transitions after exciting 2,6-ANS with a pump laser pulse of 355 nm (probe wavelength range of 360−500 nm). Moreover, the CT state, which is very close to the NP state produced from the locally excited state/NP state, emits active fluorescence with a lifetime of ∼11 ns. The solvent plays a major role in the rotation of the phenylamino group during the conversion of the NP state to the CT state, and vice versa. Intersystem crossing occurs from the CT state. Thus, investigating the triplet state together with the CT/NP states of 2,6-ANS, a commonly used probe for sensing proteins and other biomolecules, is highly relevant and helps reveal its photoexcitation dynamics.
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environment in proteins and lipid nanotubes.6,7 These photoinduced electron-transfer-based probes are also sensitive to the pH and the presence of transition-metal ions in the environment.8 The mechanism of ANS involves the intramolecular rotation of the phenylamino group and the subsequent conversion of its nonplanar (NP) state to the stable charge-transfer (CT) state immediately after photoexcitation. However, different explanations for the massive solvent effects on the excited-state lifetime have been given.1−11 Here, we applied nanosecond transient absorption (TA) and time-resolved FL techniques to 2,6-ANS dissolved in degassed methanol and measured the nanosecond TA and FL decays. On the basis of the TA and FL decay curves, along with the steadystate spectra, the S−S (singlet−singlet) and T−T (triplet− triplet) transitions that occur following the photoexcitation
INTRODUCTION
Probes containing the anilinonaphthalene sulfonate (ANS) fluorophore, which is characterized by the dependence of its absorption, fluorescence (FL), FL quantum yield (QY), and lifetime on its environment, have been used for studying the structures of biological macromolecules for the past several years.1−4 ANS and the related molecules 1-anilinonaphthalene8-sulfonate and 2,6-ANS (2-anilinonaphthalene-6-sulfonic acid) are the most widely used molecules featuring this fluorophore. A dramatic change in the FL QY with different solvents, a change in FL intensity with the local environment, and a drastic blue shift of the FL wavelength maximum in low-polarity solvents relative to that in higher polarity solvents such as water have been observed.1−3 An increase in FL intensity in a hydrophobic environment was also reported in 1954.4 In biological fields, ANS has been used as a hydrophobicity probe for protein folding and for cell surface studies.5 The characteristic features of the ANS fluorophore have been used successfully to evaluate the micropolarity of the local © 2017 American Chemical Society
Received: July 4, 2017 Accepted: October 2, 2017 Published: October 17, 2017 6782
DOI: 10.1021/acsomega.7b00921 ACS Omega 2017, 2, 6782−6785
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is due to the slowdown of the nonradiative deactivation. When the temperature increases above room temperature, the FL intensity decreases without a considerable shift in band position, indicating that the FL originates from the CT state only, that is, from the planar molecule. However, the NP and CT states are very close to each other in alcoholic solvents and water, and both of them may exist.9,11 When the temperature increases further above 40 °C, the FL intensity again starts increasing without any significant shift, which may affect the rate of the ISC process. In addition, FL decay analysis was performed with a 336 nm excitation wavelength and was monitored using a 420 nm emission wavelength under degassed conditions and atmospheric conditions, both of which are shown in Figure 2. Both
have been confirmed, and the nonradiative transition through intersystem crossing (ISC) was found to be responsible for the decrease in FL QY.
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RESULTS AND DISCUSSION Figure 1a shows the absorption, FL, and FL excitation spectra of 2,6-ANS in methanol. The absorption spectrum shows the
Figure 2. FL decay curves of 2,6-ANS in methanol (solid black line) obtained at 298 K under degassed conditions and simulated curve (dotted red line). The excitation and emission wavelengths were 336 and 420 nm, respectively. The FL decay curve (solid green line) and simulated curve (dotted red line) obtained without degassing. The instrumental response function is shown with the solid blue line.
decay profiles could be well-simulated by assuming a singleexponential decay function, that is, A1 exp(−t/τ1), and the lifetime τ1 is determined. The fitted lifetime is 10.89 ± 0.02 ns with χ2 = 1.29. Under atmospheric conditions, the lifetime becomes 7.43 ± 0.02 ns (χ2 = 1.17). Thus, the observed lifetime is a single exponential and is quenched reasonably in the presence of oxygen. The observed FL lifetime originates from the relaxed state which is designated the CT state. Furthermore, the single lifetime of 2,6-ANS in methanol is ascribed to the fast interconversion between the CT and NP states. The reported lifetime of 2,6-ANS varies with different solvents and mixtures of solvents and is typically 0.3 ns of the CT state FL and approximately 1−5 ns of the NP state in water, which is debatable.3 In addition to the solvent, oxygen is an important factor that can alter the lifetime of such biomolecules, as observed in this study. Thus, the lifetime of ANS strongly depends on the solvent system. Figures 3 and 4 show the TA spectrum and dynamical curves, respectively, of 2,6-ANS in methanol under degassed conditions. The measurements were carried out at 5 mJ@355 nm excitation and every 10 nm interval in the 360−500 nm region. The corresponding instrumental response function of the dynamical curve is provided in Figure S1 in the Supporting Information. The pulse energy was maintained as small as possible to still obtain the transient signal while avoiding
Figure 1. (a) Absorption, normalized FL, and corrected FL excitation spectra of 2,6-ANS dissolved in methanol. The inset shows the chemical structure of 2,6-ANS. (b) FL spectra of 2,6-ANS in methanol as a function of temperature. The excitation and emission wavelengths were 350 nm (a,b) and 420 nm (a), respectively.
lowest energy band at approximately 350 nm as well as strong bands at approximately 319 and 273 nm, whereas the FL spectrum shows a band maximum at 420 nm. The FL excitation spectrum is nearly the same as the absorption spectrum, as shown in Figure 1a, which indicates that the QY does not change with excitation. Thus, the steady-state absorption represents the locally excited singlet state transitions. The FL may arise from the NP state and/or from a CT state produced following the rotation of the phenylamino group in polar solvents. FL from the NP state is observed in less polar solvents.3 Furthermore, the FL spectra were also measured at temperatures varying from −10 to 50 °C and are shown in Figure 1b. Cooling from room temperature to −10 °C was expected to double the solvent viscosity relative to that at room temperature, but the intensity of the FL band increases without any significant shift in wavelength. The increase in FL intensity 6783
DOI: 10.1021/acsomega.7b00921 ACS Omega 2017, 2, 6782−6785
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Figure 3. TA spectrum of 2,6-ANS in methanol as a function of probe wavelength under degassed conditions. The pumped wavelength was 355 nm with a pulse energy of 5 mJ.
Figure 4. TA dynamical curves of 2,6-ANS in methanol at different probe wavelengths under degassed conditions. The pumped wavelength was 355 nm with a pulse energy of 5 mJ. Figure 5. TA dynamical curves/absorbance (ΔAbs) of 2,6-ANS in methanol at probe wavelengths of 400 nm (a) and 425 nm (b) pumped by nanosecond laser pulses of 355 nm at 5 mJ under degassed conditions. The black and red curves represent the original data and the single-exponential fit, respectively.
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additional reactions. It can be seen from Figure 3 that a series of absorption/emission peaks last for several sub-microseconds. The positive (red color) and negative (blue) signals are attributed to the T−T and S−S transitions, respectively. Strong S−S transitions appear at approximately 360−380 nm, which may be due to the ground-state bleaching effect. Above this point, both positive and negative lobes were obtained at each probe wavelength, indicating that both T−T absorption and S− S emission occur (Figures 4 and 5). Different dynamical curves were selected because of their high signal-to-noise ratios for fitting. However, only the dynamical curves obtained at 400 and 425 nm are shown in Figure 5. The positive part of the TA signal is well-fitted with the single-exponential fit component: ΔAbs(t) = A2 exp(−t/τ2), where τ2 and A2 are the lifetime and the pre-exponential factor, respectively. The fitted lifetime is 0.126 ± 0.004 μs with an adjustment R2 of >0.9. Similarly, the negative part of the dynamical curves is fitted with the singleexponential fit. However, the fitted lifetime could not be considered accurate, as the lifetime of the FL is approximately 10.89 ns, which is beyond the resolution of the TA system. The long lifetime corresponds to the positive amplitude of the TA signal designated to the T−T transitions (absorption), that is, assigned to the short-lived triplet state. Thus, the TA signal of 2,6-ANS demonstrates the existence of a short-lived triplet
state. However, the emission measurement, which was conducted between −10 and 50 °C, did not show any emission from the triplet state, even at −10 °C. Following photoexcitation, an NP state is initially produced and subsequently forms a CT state that emits FL. However, the NP and CT states of 2,6-ANS lay very close to each other in alcoholic solvents9 and are not distinguishable in water.11 We did not observe any significant shift in the FL spectrum with decreasing temperature, that is, upon increasing the viscosity of the solvent, and vice versa, which indicates that the FL may originate from the CT state and that the solvent methanol allows the rotation of the phenylamino group. Importantly, both the NP and CT states can yield triplets.9,10 The CT state that emits active FL undergoes a nonradiative transition through ISC. The TA, together with FL decay analysis and steady-state spectra, indicates the existence of NP/CT (due to fast interconversion) and triplet states of 2,6-ANS; among these species, the CT state emits strong FL with a lifetime of ∼11 ns, as demonstrated in Figure 6. 6784
DOI: 10.1021/acsomega.7b00921 ACS Omega 2017, 2, 6782−6785
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ACKNOWLEDGMENTS M.S.M. thanks the Chinese Academy of Science (CAS), China, for supporting his stay at DICP, CAS, Dalian, China, under the CAS visiting scholar program and Dr. H.C. Joshi, IPR, India, for valuable comments. This work was financially supported by the National Basic Research Program of China (2013CB834604), the Natural Science Foundation of China (NSFC, grant no. 21403226), the Science and Engineering Research Board (SERB), DST (grant no. EMR/2016/001110), and the DSTFIST project (SR/FST/PSI-164/2011(C)).
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Figure 6. State diagram of the photoexcitation dynamics of 2,6-ANS.
The molecule 2,6-ANS has been studied based on the TA spectra/dynamical curves and time-resolved FL decay curves. The fitted TA and FL decay curves show the existence of NP/ CT and triplet states with lifetimes of approximately 11 and 126 ns, respectively. Thus, the present investigation of 2,6-ANS, a molecule commonly used to probe biomolecules, is highly important and unusual, as it opens up new avenues to understand the existence of the CT and triplet states of ANS and the variable QY.
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METHODS AND MATERIALS 2,6-ANS was obtained from Molecular Probes, USA, and was used without further purification. Steady-state absorption and FL spectra were recorded with a LAMBDA-750 UV/vis/NIR spectrometer (PerkinElmer) and a Fluorolog-3 spectrofluorometer (HORIBA Jobin Yvon), respectively. The details about the equipment were described in our previous report.13 A sample heater/cooler Peltier thermocouple device (FI-3004) equipped with a Fluorolog-3 was used for recording the temperature from −10 to 50 °C. Time-resolved FL decay curves were collected on a time-correlated single-photon counting system (DeltaFlex, HORIBA Jobin Yvon) with a PPD-850 detector using a delta diode as an excitation source. Nanosecond TA measurements were performed with pump− probe techniques, as described in our previous reports.12,14,15 All samples were thoroughly degassed (N2) before obtaining the spectra, FL decays, TA spectra, and dynamical curves. The concentration of the samples was 20 μM, and the path length of the sample quartz cuvette was 10 mm.
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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/acsomega.7b00921. Instrumental response function of the TA system recorded with a pulse energy of 5 mJ at 355 nm with a neat solvent (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (M.S.M.). *E-mail:
[email protected] (K.H.). ORCID
Mohan Singh Mehata: 0000-0003-4564-5243 Keli Han: 0000-0001-9239-1827 Notes
The authors declare no competing financial interest. 6785
DOI: 10.1021/acsomega.7b00921 ACS Omega 2017, 2, 6782−6785
