Luminescent Carbazole-Based Eu^III and Yb^III Complexes with High Two-Photon Absorption Cross-Section Enable Viscosity-Sensing in the Visible and Near IR with One- and Two-Photon Excitation

Abstract

The newly synthesized Eu^III and Yb^III complexes with the new carbazole-based ligands CPAD²⁻ and CPAP⁴⁻ display the characteristic long-lived metal-centered emission upon one- and two-photon excitation. The Eu^III complexes show the expected narrow emission bands in the red region, with emission lifetimes between 0.382 and 1.464 ms, and quantum yields between 2.7 and 35.8%, while the Yb^III complexes show the expected emission in the NIR region, with emission lifetimes between 0.52 and 37.86 μs, and quantum yields between 0.028 and 1.12 %. Two-photon absorption cross sections (σ_2PA) as high as 857 GM were measured for the two ligands. The complexes showed a strong dependence of the one- and two-photon sensitized emission intensity on solvent viscosity in the range 0.5 – 200 cP in the visible and NIR region.

Graphical Abstract

Lanthanide complexes with carbazole-based antennas and their one- and two-photon luminescence and viscosity-based emission intensity.

INTRODUCTION

Lanthanide (Ln^III) ions are interesting as luminescent labels and sensors for a variety of applications.^1-15 The luminescence is color pure, due to the core nature of the f orbitals involved in the emission, and is long-lived, due to the forbidden nature of the f-f transitions, which enables time-delayed emission spectroscopy with increased signal-to-noise ratio.^16-18 The forbidden transitions render direct excitation inefficient, and thus the emission is sensitized through an appended ligand chromophore (Figure 1). This is referred to as the antenna effect and leads to a large Stokes shift of sensitized emission, which is advantageous due to separation of excitation and emission wavelengths. In addition, ligands are easily derivatized, which enables tuning the chemical and spectroscopic properties of the complexes. Typically chromophores absorb in the high energy region of the spectrum,^{19, 20} and these wavelengths have low tissue penetration and result in radiation damage, thus making them undesirable for bioimaging applications.^21-24 Excitation at lower non-damaging energies is achieved using ligands with extended aromatic systems that absorb in the visible; however, the low energy singlet (S) and triplet (T) states cannot sensitize visible emitting Ln^III ions.^{16, 25} An alternative is to use two-photon absorption (2PA),^{21, 22, 26-29} a nonlinear process where two photons with half the energy required by the one-photon excitation (1PA) are absorbed simultaneously (Figure 1).^{30, 31} Organic dyes³² and transition metal complexes³³ can be efficient photoluminescent labels for 2PA imaging, but they are often affected by photobleaching, and have short emission lifetimes and narrow Stokes shifts, which limit their usefulness. Nanoparticles also have been frequently studied for this purpose, although their synthesis and purification are not always straightforward and might suffer from solubility issues.³⁴ LnIII ion complexes don’t suffer from these limitations, as indicated above, and are thus great candidates for 2PA imaging.

Figure 1.

A) Energy level diagram illustrating the antenna effect for Ln^III. 2PA and 1PA are the two- and one-photon absorption, F fluorescence, P phosphorescence, ISC intersystem crossing, ET energy transfer, BT back-transfer, L luminescence, NR non-radiative pathways, S states with singlet and T states with triplet multiplicity. B) Molecules studied in this work.

Lakowicz and co-workers pioneered the sensitization of Eu^III emission using 2PA.^{35, 36} Since then, the use of luminescent Ln^III complexes as 2PA labels and sensors has since flourished and examples of both visible and NIR emitters are known.^37-49 Andraud, Maury and co-workers studied 2PA-sensitization of Eu^III complexes with dipicolinato- and cyclononane-based ligands and concluded that ligands with charge-transfer (CT) states have improved 2PA cross-sections (σ_2PA).^49-51

Physicochemical properties of biological systems provide important information regarding physiological processes. For example, viscosity plays an important role in the intra- and extracellular mass transport^{52, 53} and abnormalities in blood and plasma viscosity are indicative of diabetes and hypertension.^{52, 54, 55} Viscosity of macroscopic systems is easily determined, yet the same methods cannot be applied at cellular level.⁵² Organic dyes, such as molecular rotors, have been used as luminescent viscosity sensors.^56-58 To date, only one Ln^III-based viscosity sensor has been reported by Zhang and co-workers. Their Yb^III porphyrinato complexes with a Kläui ligand displayed emission lifetimes τ that varied by a factor of ~2.8 in the viscosity range 0.52-1200 cP.⁵⁹ Since viscosity alters the population of intramolecular charge transfer states, molecules with such states should provide a unique opportunity to sense it.⁶⁰

De Bettencourt-Dias and co-workers reported a carbazole-substituted dipicolinato as sensitizer of Eu^III and Tb^III emission.^{61, 62} Carbazole is known for its 2PA properties.^63-65 Thus, with the goal of increasing our knowledge of 2PA-sensitized Ln^III emission and expand the availability of Ln^III-based viscosity sensors, we isolated two new carbazole-based ligands with potential CT states,^{50, 62} CPAD²⁻ (4-((4-(9H-carbazol-9-yl)phenyl)ethynyl)pyridine-2,6,dicarboxylate), and CPAP⁴⁻ (2,2’-(4-((4-(9H-carbazol-9-yl)phenyl)(pyridine-2,6-diyl)bis(methylene)dimalonate), and their Ln^III complexes (Ln^III = Eu^III, Gd^III and Yb^III).

RESULTS AND DISCUSSION

Synthesis and characterization of the H₂CPAD and H₄CPAP ligands and the Ln^III complexes

H₂CPAD and H₄CPAP (Figure 2A) were synthesized through modified literature procedures (Scheme S1) and characterized using standard techniques (Figures S2, S4, S6, S8, S10 and S12). The Ln^III (Ln^III = Eu^III, Gd^III, Yb^III) complexes were obtained as yellow solids in ~70% yield by reacting LnCl₃ with K₂CPAD in a 1:3 metal-to-ligand ratio and with K₄CPAD in a 1:1 metal-to-ligand ratio in water:DMF (1:20). They were characterized by mass spectrometry (Figures S13-S18) and shown to be photostable (Figure S19).

Figure 2.

A) 1PA excitation (black lines, left) and emission spectra (red lines, right) of Eu^III complexes (λ_exc = 380 nm). B) 2PA emission spectra of Eu^III complexes. C) Plot of the log of the emission intensity I at 615 nm upon 2PA excitation as a function of the log of the laser power P. From top to bottom in each figure (a) K₃[Eu(CPAD)₃] in DMSO, (b) K[Eu(CPAP)(DMSO)₂] in DMSO, and (c) K[Eu(CPAP)(H₂O)₂] in TRIS/HCl buffered aqueous system (pH ~7.4, 10% DMSO); λ_exc = 720 nm; [complex] = 1x10⁻⁴ M.

One photon spectroscopy studies of the K₃[Ln(CPAD)₃] and K[Ln(CPAP)(L)₂] (Ln = Eu^III or Yb^III and L = DMSO or H₂O) complexes

K₃[Ln(CPAD)₃] and K[Ln(CPAP)(L)₂] (Ln = Eu^III or Yb^III and L = DMSO or H₂O) in solution display the characteristic Eu^III-centered ⁵D₀→⁷F_J (J = 0 – 4) (Figure 2A right) and Yb^III-centered ²F_5/2→²F_7/2 transitions in the emission spectra (Figure 3 right). Different splitting patterns in the emission spectra are a result of different coordination environments around the metal ion, due to the different ligand binding.¹⁷ The excitation spectra (Figures 2B and 3 left) closely resemble the absorption spectra of the ligands (Figure S22), indicating that the ligands sensitize the metal-centered emission.

Figure 3.

Excitation (black lines, left) and emission spectra (pink lines, right) of Yb^III complexes. a) K₃[Yb(CPAD)₃] in DMSO (λ_exc = 370 nm), b) K[Yb(CPAP)(DMSO)₂] in DMSO (λ_exc = 350 nm), and c) K[Yb(CPAP)(H₂O)₂] in TRIS/HCl buffered aqueous system (pH ~7.4, 10% DMSO) (λ_exc = 350 nm) in TRIS/HCl buffered aqueous system (pH ~7.4, 4% DMSO);. [complex] = 1x10⁻⁴ M.

The excited state lifetimes (τ) and emission efficiencies (${\Phi }_L^{Eu}$) of the complexes are summarized in Table 1. τ of 1.464 ms for [Eu(CPAD)₃]³⁻ and 1.702 ms for [Eu(CPAP)(DMSO)₂]⁻ were measured in DMSO. In water:DMSO (9:1), the latter complex shows an emission lifetime of 382 μs, consistent with exchange of the coordinated DMSO molecules for water (vide infra). The long τ are accompanied by high intrinsic quantum yields (${\Phi }_{Eu}^{Eu}$) in DMSO and a lower value for the complex in water. ${\Phi }_L^{Eu}$ of 31.7%, 35.8% and 2.7% were observed for [Eu(CPAD)₃]³⁻, [Eu(CPAP)(DMSO)₂]⁻ and [Eu(CPAP)(H₂O)₂]⁻, respectively. Compared with Cs₃[Eu(dpa)₃] (dpa = dipicolinato) in DMSO (Table S1), the lower emission efficiencies are consistent with an increase in the donor-acceptor distances R_L (Table 1),⁶¹ and quenching caused by O-H vibrational coupling in the case of [Eu(CPAP)(H₂O)₂]⁻ (vide infra). The favorable energies of S and T states of the ligands result in high η_sens for the Eu^III complexes (Table 1). ${\Phi }_L^{Yb}$ of 0.75% and 0.028% were observed for [Yb(CPAP)(DMSO)₂]⁻ and [Yb(CPAP)(H₂O)₂]⁻, respectively (Table 1). These values are similar to related dipicolinato-based complexes and compare favorably with known compounds.^{45, 47, 48, 67, 68} The emission lifetimes for the [Yb(CPAP)(L)₂]⁻ complex (L = H₂O or DMSO) show a similar behavior. τ is longer for L = DMSO at 8.91 μs than for L = H₂O at 0.52 μs, as is to be expected due to quenching through the O-H oscillators.

Table 1.

Singlet S and triplet T state energies of the ligands, lifetime τ, intrinsic emission efficiency ${\Phi }_{Ln}^{Ln}$, quantum yield ${\Phi }_L^{Ln}$ of sensitized efficiency, sensitization efficiency η_sens, and number of coordinated water molecules q for the complexes. λ_exc = 360 nm.

Complex	Solvent	S[a][cm−1]	T[a][cm−1]	τ	${\Phi }_{Ln}^{Ln}[\%]$	${\Phi }_L^{Ln}$ [%]	ηsens [%]	RL[Å]	q
K3[Eu(CPAD)3]	DMSO	23,520±460	19,690±300	1.464±0.003[c]	69	31.7±4.0	46	8.8883	---
K3[Yb(CPAD)3]	DMSO			37.86±0.06[d]	-	1.12±0.12	-		---
K[Eu(CPAP)(DMSO)2]	DMSO	25,570±350	20,490±270	1.702±0.037[c]	63	35.8±3.7	57	9.2466	---
K[Yb(CPAP)(DMSO)2]	DMSO			8.91±0.16[d]	-	0.75±0.02	-		---
K[Eu(CPAP)(H2O)2]	Water: DMSO[e]			0.382±0.011[c]	11	2.7±0.4	25		1.9
K[Yb(CPAP)(H2O)2]	Water: DMSO[e]			0.52±0.01[d]	-	0.028±0.002	-		---

^[a]Obtained using the Gd^III complex at 77 K.⁶⁶

^[b]TRIS/HCl buffered aqueous solution (pH ~7.4, 4% DMSO).

^[c]In ms.

^[d]In μs.

^[e]TRIS/HCl buffered aqueous solution (pH ~7.4, 10% DMSO).

In the case of the Ln^III complexes with the ligand CPAP⁴⁻, a decrease in the emission efficiency and lower τ in TRIS/HCl buffered aqueous system (pH ~7.4, 10% DMSO), compared with the DMSO solution, is observed. This decrease is a result of the non-radiative deactivation of the Ln^III excited state by the O-H vibrations of coordinated water molecules. The presence of two coordinated water molecules q was confirmed for the Eu^III complex by comparing τ in water (τ_H2O) and D₂O (τ_D2O) (Equation S5 and Table 1).

Two photon spectroscopy studies of the K₃[Ln(CPAD)₃] and K[Ln(CPAP)(L)₂] (Ln = Eu^III or Yb^III and L = DMSO or H₂O) complexes

All Eu^III complexes can be excited with a wide range of low energy photons in a 2PA process (Figure S32). By exciting at 720 or 750 nm, the latter chosen for comparison with the 2PA standard rhodamine B,⁶⁹ the characteristic metal-centered emission pattern is observed (Figures 2B, S29a-S31a). The quadratic dependence of the emission intensity I on the laser power P (Figures 2C, S29b-S31b, Table 2, Table S2) confirmed the 2PA process. The emission spectra obtained by one- or two-photon excitation are the same (Figures 2, S29-S31), indicating that the same excited states are involved in the process.

Table 2.

Two-photon absorption cross-sections (σ_2PA) of the Eu^III complexes.

Complex	Solvent	σ2PA[a] [GM][b]
K3[Eu(CPAD)3]	DMSO	857±24
K[Eu(CPAP)(DMSO)2]	DMSO	266±34
K[Eu(CPAP)(H2O)2]	Water: DMSO[c]	228±36

^[a]λ_exc = 750 nm.

^[b]1 GM = 10⁻⁵⁰ cm⁴ s photon⁻¹ molecule⁻¹.

^[c]TRIS/HCl buffered aqueous solution (pH ~7.4, 10% DMSO).

The two-photon absorption cross-sections (σ_2PA) are summarized in Table 2. A value of 857 GM was obtained for K₃[Eu(CPAD)₃]. This is among the highest reported for Eu^III complexes in solution, such as 775 GM for [NBu4]₃[Eu(dpa5)₃] described by Maury, Andraud and co-workers.⁴⁵

Sensitization of Eu^III using 2PA is not commonly reported for complexes with coordinated solvent molecules, because the latter often quench the emission. Parker and co-workers found a σ_2PA of 1.7 and 0.4 GM for aqueous solutions of [Eu(do2ax1)(H₂O)](OTf)₃ and [Eu(do2ax2)(H₂O)](OTf)₃, respectively (do2ax1 and do2ax2 are dota derivatives with appended 5H-thiocromeno[2,3-b]pyridine-5-one and methyl 5-oxo-5H-thiocromeno[2,3-b]pyridine-7-carboxylate, respectively).⁴³ We were pleasantly surprised to determine a σ_2PA of 228 GM for the K[Eu(CPAP)(H₂O)₂] in TRIS/HCl buffered aqueous system (pH ~7.4, 10% DMSO), similar to the value in DMSO, consistent with the fact that σ_2PA is intrinsic to the ligand and not affected by the solvent.⁷⁰ This value also indicates that CPAP⁴⁻, which yields complexes soluble in water:DMSO, is viable for 2PA bioimaging, although this application is not shown here.

Viscosity sensing studies using the K₃[Ln(CPAD)₃] (Ln = Eu^III or Yb^III) complexes

The temperature-dependent phosphorescence spectra of K₃[Gd(CPAD)₃], with maxima at 510 nm at 298 K and 435 nm at 77 K (Figure S33), indicate that a twisted intramolecular charge-transfer (TICT) state is present in CPAD²⁻, involving the carbazole and phenyl rings.^{62, 71} As mentioned above, changes in the viscosity often alter the population CT states.⁷¹ Thus, we prepared 0.2 mM solutions of K₃[Ln(CPAD)₃] (Ln = Eu^III or Yb^III) with varying amounts of methanol and glycerol to obtain a wide range of viscosities. This allowed us to observe changes in the emission intensity as a function of the viscosity using one- and two-photon excitation for the K₃[Eu(CPAD)₃] complex, the first example of viscosity sensing using Eu^III-centered emission in the visible region of the spectrum using two-photon excitation. Upon excitation through the ligand at 400 nm or 800 nm the emission intensities increase by about 2-fold or 5.8-fold, respectively, as the viscosity increases (Figures 4(A) and 4(B)) in the range 0-200 cP. This is in the same order of magnitude as the increase observed for the Yb^III complex described by Ning and co-workers for the same viscosity range.⁵⁹

Figure 4.

A) Emission spectra of K₃[Eu(CPAD)₃] (λ_exc = 400 nm) upon one-photon excitation with varying viscosities. B) Emission spectra of K₃[Eu(CPAD)₃] (λ_exc = 720 nm) upon two-photon excitation with varying viscosities. The insets show the plot of the emission intensity at 615.5 nm upon one-photon or two-photon excitation as a function of viscosity. [complex] = 1x10⁻⁴ M.

Viscosity-dependent emission intensity is also observed for K₃[Yb(CPAD)₃] complex, as shown in Figure 5. An approximate 2-fold increase over the 0–200 cP range is seen, on the same order of magnitude of the only other example of Yb^III-centered emission used for viscosity sensing.⁵⁹

Figure 5.

Emission spectra of K₃[Yb(CPAD)₃] (λ_exc = 400 nm) upon one-photon excitation with varying viscosities. The inset shows the plot of the emission intensity ratios at 976 and 1020 nm upon one-photon excitation as a function of viscosity. MeOH:glycerol with various ratios were used as solvents with variable viscosity. [complex] = 1x10⁻⁴ M.

In addition to viscosity, the emission intensity can be influenced by the solvent oxygen content, as the interaction between oxygen and the triplet level of the ligand adds a non-radiative deactivation pathway that decreases the EuIII emission lifetime. ⁷² Methanol and glycerol have different amounts of dissolved oxygen; the mole fractions at 298 K and 101.3 kPa are 4.15×10⁻⁴ for methanol and 4.8×10⁻⁶-5.5×10⁻⁶ for glycerol.^{73, 74} To ensure that emission behavior is due to solvent viscosity, and not quenching through oxygen,^{72, 75} the emission lifetimes of the complexes were determined. At 1.318±0.052 ms in methanol, and 1.298±0.014 ms in 1:9 methanol:glycerol (Figures S26 and S27), these are equivalent. The emission intensity of both Eu^III and Yb^III complexes is independent of the amount of dissolved oxygen in solution as well (Figure S34). This confirms that there is no quenching and thus the intensity changes are due to the viscosity of the medium. Another solvent property, namely its polarity, can influence the emission intensity of the Ln^III as well, as high polarity solvents can stabilize ligand CT states,⁴⁹ and thus the excited state energy of the ligand, which results in changes to the ligand → Ln^III energy transfer rates. To ensure that the energy of the ligand excited state is not affected by solvent polarity, the phosphorescence spectra of the [Gd(CPAD)₃] complex obtained in 100% methanol and mixture of methanol:glycerol (1:9) (Figure S35). The lack of significant change of the phosphorescence emission bands confirms that the polarity does not affect the energy levels of the ligand and thus emission intensity changes are solely due to the viscosity of the medium.

CONCLUSIONS

A series of new Ln^III complexes containing new carbazole-based ligands were synthesized and their photophysical properties were measured. Eu^III emission efficiencies up to 35.8% were observed using one-photon excitation. Eu^III emission using 2PA was observed for all the complexes. The σ_2PA of 857 GM for K₃[Eu(CPAD)₃] is, to date, the highest determined for Eu^III complexes in solution. A substantial change in emission intensity as a function of the viscosity was observed for K₃[Eu(CPAD)₃] upon excitation with one-photon and, for the first time, using two-photon excitation. Viscosity dependent emission intensity is observed as well for the analogous Yb^III complex, only the second example of NIR Ln^III-based emission viscosity sensing. These complexes are thus effective sensors of the viscosity of the medium, which can be monitored through the intensity of the emitted light in the visible and NIR excited by either one- or two-photons. These results establish this carbazole-based family of ligands as a foundation for improved 2PA dyes and as viscosity sensors. This will contribute to the fields of imaging, sensing of physicochemical properties and diagnosis in microscale systems.

EXPERIMENTAL SECTION

All commercially obtained reagents were of analytical grade and were used as received. Solvents were dried and purified by standard methods unless otherwise noted. All synthetic steps were completed under N₂ unless otherwise specified. The detailed synthetic procedures of the ligands and their Ln^III complexes are provided in the Supporting Information. Details of the one- and two-photon photophysical characterization and viscosity measurements are provided in the Supporting Information as well.

NMR Spectroscopy.

All NMR spectra were recorded on Varian 400 and 500 MHz spectrometers with chemical shifts reported (δ, ppm) in deuterated chloroform (CDCl₃) against tetramethylsilane (TMS, 0.00 ppm) at 25.0 ± 0.1 °C.

Mass Spectrometry.

Electrospray ionization mass spectra (ESI-MS) were collected in positive ion mode on a Waters Micromass ZQ quadrupole in the low-resolution mode for the ligands and on an Agilent model G6230A with a QTOF analyzer in the high-resolution mode for the metal complexes. The samples were prepared by diluting acetonitrile solutions to a concentration of ~1 mg/mL and passing through a 0.2 mm microfilter.