Ratiometric detection of amyloid-β aggregation by a dual-emissive tris-heteroleptic ruthenium complex

Jiang-Yang Shao ab, Si-Hai Wu *a, Junjie Ma a, Zhong-Liang Gong b, Tian-Ge Sun b, Yulong Jin b, Rong Yang a, Bin Sun c and Yu-Wu Zhong *bd
aSchool of Medicine, Huaqiao University, Quanzhou, Fujian 362021, China. E-mail: wusihai@hqu.edu.cn
bBeijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Photochemistry, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. E-mail: zhongyuwu@iccas.ac.cn
cInstitute of BioPharmaceutical Research, Liaocheng University, Liaocheng 252000, China
dSchool of Chemical Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

Received 14th November 2019 , Accepted 14th January 2020

First published on 14th January 2020

A dual-emissive tris-heteroleptic ruthenium complex is designed, synthesized and applied for the ratiometric photoluminescent detection of amyloid-β (Aβ) aggregation in both steady and transient states. The Aβ aggregation is supported by transmission electron microscopy and confocal laser scanning microscopy analysis. In addition, molecular docking calculations have been performed to gain insights into the interaction mode between the ruthenium complex and Aβ fibrils.

Alzheimer's disease (AD) has grown into an increasingly serious health problem for mankind today, which is characterized by the loss of memory, cognitive decline, and behavioral and physical disability.1,2 The exact molecular pathogenesis is not clear yet; however, the aggregation of amyloid-β (Aβ) protein into fibrils in the brain is believed to play an essential role in the progression of AD.3,4 The detection of Aβ aggregation has thus become a critical method for the early diagnosis and pathology studies of AD. A range of techniques have been developed for the detection of Aβ fibril formation, including magnetic resonance imaging, immunoassay, and photoluminescent detection.5–8 Among them, photoluminescent detection is advantageous in terms of operation simplicity, high sensitivity, and the ability for real-time imaging.

In addition to commercial probes, e.g. Thioflavin T (Fig. 1a),9 a number of photoluminescent materials based on organic small molecules,10–13 nanostructures,14 and transition-metal complexes (TMCs)15–20 with different emission colors and excited-state lifetimes have been developed and applied for the detection of Aβ fibrils. In particular, TMCs are characterized by large Stokes shift, long lifetime, and good photostability, making them appealing for this application.15–20 For instance, ruthenium complex [Ru(bpy)2(dppz)]2+ (1, bpy = 2,2′-bipyridine; dppz = dipyrido[3,2-a:2′,3′-c]phenazine, Fig. 1b) was used by Martí to sense Aβ aggregation in a time-gated fashion.15

image file: c9cc08909h-f1.tif
Fig. 1 (a and b) Commercial probe Thioflavin T and the known ruthenium complex 1 (ref. 15) used for the detection of Aβ aggregation with an emission turn-ON response. (c) Complex 2 reported in this work for the ratiometric emissive detection of Aβ aggregation.

The above-discussed luminescent probes, either organic molecules or TMCs, are largely based on the intensity switching of a single emission band, with an emission turn-ON response in most cases. The accuracy and reliability of this method were interfered with by the fluctuation of excitation energy, environment effects, and the variation of the probe concentration. One strategy to improve the sensing accuracy and reliability is to use a ratiometric luminescent probe with two distinct emission bands. The sensing accuracy could be improved by setting one emission band as an internal standard and probing the changes of the other band. Such a ratiometric luminescent method has been well-established in the detection of cations, anions, and biomolecules.21–23 However, ratiometric detection of Aβ fibrils has been only reported in a few studies with organic probes.13b,13c,14b The main difficulty is the invention of a suitable dual-emitting chromophore24–26 that can bind efficiently to Aβ fibrils with distinct spectral changes. We present herein an example of a ratiometric TMC probe 2 (Fig. 1c) for this purpose.

Complex 2, [Ru(phen)(dpma)(dppz)](PF6)2 (phen = 1,10-phenanthroline, dpma27 = N,N-di(pyrid-2-yl)-methylamine), is a tris-heteroleptic ruthenium complex with three different bidentate ligands. Ruthenium complexes with dppz are well-known for DNA detections.28 In this study, dppz is used to interact with Aβ fibrils.15,29 The phen ligand with a rigid and planar structure is used to improve the emission property of the complex and further strengthen its interaction with Aβ fibrils. Ligand dpma is electron-rich and has a large bite angle ligand, which is used to induce energy-separated excited states with dual-emitting properties (see further discussion below).26,30

The synthesis and characterization of 2 are provided in the ESI. The high purity of the complex was confirmed by the elemental analysis, NMR, and HPLC analysis (Fig. S1–S3, ESI), which is crucial for the dual-emitting studies of a single molecular material. Complex 2 displays intense absorptions between 250 and 300 nm, assigned to the intraligand (IL) π → π* transitions (Fig. 2a). It also shows wide absorptions in the range of 320–550 nm. Interestingly, two well-separated emission bands at 400 and 650 nm, respectively, are observed for complex 2 in CH3CN solution (Fig. 2b). The excitation spectrum shows that the absorptions from 300 to 380 nm are responsible for the higher-energy emission band at 400 nm; while the lower-energy emission at 650 nm is mainly associated with the absorptions in the visible region. The blue emission at 400 nm has an excited-state lifetime τ of 2.0 ns at rt (N2-saturated) and 4.0 ns at 77 K, respectively (Fig. 2c). In contrast, the red emission band at 650 nm has a τ of 261 ns at rt and 2.16 μs at 77 K, respectively (Fig. 2d and Table S1, ESI). These data suggest that the blue and red emissions are of singlet and triplet excited-state character, respectively. The total emission quantum yield (Φ) of 2 in N2-saturated CH3CN is about 7.3%. In comparison, the ligand dpma exhibits a structureless emission band at 385 nm with a Φ of 0.8% (Fig. S4 and Table S1, ESI).

image file: c9cc08909h-f2.tif
Fig. 2 (a) Absorption and excitation spectra of 2 in CH3CN. (b) Emission spectrum of 2 in N2-saturated CH3CN (excited at 330 nm; 1 × 10−5 M). (c and d) Decay profile of the emission at (c) 400 nm and (d) 650 nm of 2 in CH3CN at rt and 77 K, respectively.

DFT calculations indicate that the HOMO and LUMO of the ligand dpma have major contributions from the amine unit and the pyridine rings, respectively, suggesting that the emission of dpma has a singlet charge transfer (1CT) character (Fig. S5, ESI). In contrast, both the Ru ion and the dpma ligand contribute to the HOMO of 2 (Fig. S6, ESI). The lower occupied orbitals HOMO−1 and HOMO−2 of 2 are dominated by the Ru ion and HOMO−3 is dominated by the dpma ligand. The LUMO of 2 has a major contribution from the dppz ligand. The phen ligand contributes to the higher unoccupied orbitals such as LUMO+1 and LUMO+2. TDDFT predicts that the vertical S1 excitation of 2 is associated with the HOMO → LUMO transition (λ = 485 nm; f = 0.0427; see Fig. S7 and Table S2, ESI). This state should be responsible for the observed low-energy emission at 650 nm. Considering that this emission has a triplet character, it is safe to assign it to the dppz-targeted triplet metal-to-ligand charge transfer (3MLCT) emission. The nature of the emission band at 400 nm of 2 is not clear yet. We tentatively attribute this emission to the dpma-localized 1CT character, because it has similar wavelength region and shape as the emission of dpma itself. However, these two emissions have different properties. For instance, the emission of the ligand dpma is only slightly dependent on the O2 content of the solution (Fig. S8a, ESI). In contrast, the higher-energy emission of 2 could be significantly quenched by O2 (Fig. S8b, ESI). The latter phenomenon could be caused by the O2-induced intersystem crossing in complex 2, followed by the energy transfer from the 3MLCT state to triplet O2.26

In aqueous buffer solution (30 mM NaCl/10 mM Tris, pH = 7.5), complex 2 only displays the higher-energy emission band at 415 nm with a Φ of 0.05% (Fig. S9, ESI). The lower-energy 3MLCT emission is completely quenched by the hydrogen bond formation of dppz with water.28,31,32 We then turned to monitor the Aβ40 aggregation process by adding complex 2 to the Aβ sample after different incubation duration and recording the emission spectral changes of the resulting mixture. When the incubation time was prolonged from 0 to 80 min, the emission intensity at 415 nm shows some fluctuations; whereas the 3MLCT emission at 665 nm was remarkably enhanced (Fig. S10, ESI). Fig. 3a displays the emission spectral changes normalized to the emission intensity at 415 nm. These data indicate that complex 2 could sense the formation of Aβ fibrils as a result of the interaction of the dppz ligand with the hydrophobic microenvironments of Aβ fibrils and thus re-activate the luminescent 3MLCT state.15,28,31,32 The higher-energy emission at 415 nm serves as an inherent internal standard to improve the detection accuracy. The changes of the emission intensity ratio between 665 and 415 nm (I665/I415), as a function of the incubation time, appear as a sigmoidal curve (Fig. 3b). This agrees with a typical fibril formation sequence of a lag phase followed by a fast aggregation and fibril elongation period and a steady aggregate state.33 The I665/I415 ratio is around 1.6 at the steady state versus 0.2 in the lag phase. A comparison study shows that, under the same measurement conditions, the monomeric emission at around 490 nm of Thioflavin T is enhanced twice in response to the aggregation of Aβ (Fig. S11, ESI).

image file: c9cc08909h-f3.tif
Fig. 3 (a) Emission spectral changes (normalized to the emission at 415 nm) monitored by complex 2 (14 μM) of Aβ40 aggregation (14 μM) after different incubation times (10, 20, 30, 40, 45, 50, 65, 70, 80 min) in aqueous buffer solution (excited at 330 nm). (b) Corresponding changes of the emission intensity ratio between 665 and 415 nm (I665/I415) of panel (a) versus incubation time. (c) TRPL spectra of the mixture of 2 with Aβ40 fibrils (excited by a 375 nm pulsed laser; the instrument response is around 70 ns). *Second-order bands of the excitation wavelength. (d) Time-dependent emission intensity ratio between 640 and 460 nm (I640/I460) of panel (c).

The excited lifetime of the emission at 415 and 665 nm of the mixture of 2 with Aβ fibrils is 7.4 and 178 ns, respectively (Fig. S12, ESI). Fig. 3c shows the nanosecond time-resolved photoluminescence (TRPL) spectra of the mixture of 2 with Aβ fibrils (the instrument response time is around 70 ns). Possibly due to the use of a different instrument setup and excitation wavelength, the emission wavelengths of the steady and transient emission spectra are slightly different (Fig. 3avs.Fig. 3c). In the transient emission spectra, the decays of two emission bands at 460 and 640 nm have been recorded. In the beginning 20 ns, the intensity at 460 nm decreased significantly. The emission intensity ratio between 640 and 460 increased linearly from 0.41 to 6.89 in the first 60 ns. In the following hundreds of ns, this ratio decreased linearly from 6.89 to around 3.0 with a shallower slope. Such a time-dependent ratiometric response makes this method potentially useful for time-gated sensing and imaging, which could allow us to detect the Aβ fibril formation in the presence of strong fluorescence background emission.15,34 In contrast, only the decay of the emission at 460 nm was observed in the TRPL of the mixture of 2 with free Aβ peptide (Fig. S13, ESI).

The formation of Aβ fibrils was confirmed by transmission electron microscope (TEM) analysis (Fig. 4). The original Aβ peptides appear as small dot images. After incubation for 80 min, they appear as entangled fibrils with a diameter of around 10 nm and length in the order of μm. The Aβ aggregation could also be imaged by confocal laser scanning microscopy (CLSM) in the presence of complex 2 (Fig. 5). The red emission was switched ON when the peptide was incubated for a certain period of time. The pictures in Fig. 5 show that short fibrils appear in 30 min, which grow into large plagues in 90 min. Due to the low emission quantum yield of complex 2, it is not possible at this stage to image smaller aggregated species using this complex. Because the CLSM instrument at hand has no ultraviolet excitation source, the higher-energy emission of complex 2 could not be imaged either.

image file: c9cc08909h-f4.tif
Fig. 4 TEM images of (a) original Aβ40 peptides and (b) Aβ40 fibrils after incubation for 80 min.

image file: c9cc08909h-f5.tif
Fig. 5 CLSM images of Aβ40 peptides after incubation for (a) 30 min, (b) 55 min, and (c) 90 min as monitored by complex 2. The samples were prepared by treating pre-formed Aβ40 aggregates with complex 2. Excited by a 488 nm laser.

Molecular docking calculations have been performed to gain insight into the binding mode of complex 2 with Aβ40 aggregates (Fig. 6). Similar to the previously reported interaction between [Ru(bpy)2(dppz)]2+ (complex 1) and Aβ fibrils,29 the hydrophobic cleft formed by Val18 and Phe20 provides a potential docking site for complex 2, as a result of the π/π interactions between Phe20 with dppz and phen rings and the C–H/π interactions between Val18 and dppz, respectively. In addition, the pyridyl ring of the dpma ligand was found to interact with Glu22 through hydrogen-bond interaction to further stabilize the complexation while exposing the dpma ligand to the solvent environment. Modeling of the complexation of 2 with a control peptide with mutated Val18, Phe20, and Glu22 shows that these interactions are important in accommodating the ruthenium complex (Fig. S14, ESI). This docking mode is consistent with the re-activation of the lower-energy 3MLCT phosphorescence and the essentially unchanged intensity of the dpma-localized higher-energy emission when complex 2 interacts with Aβ aggregates.

image file: c9cc08909h-f6.tif
Fig. 6 (a) Molecular modeling of complex 2 with Aβ fibrils. (b) Enlarged representation of the interaction of 2 with the hydrophobic cleft of Aβ fibrils.

The above results were all carried out with Aβ40. In comparison, Aβ42 is another primary yet less abundant and more toxic isoform of Aβ. The studies on the Aβ42 aggregates have attracted much attention.35 Preliminary studies indicated that a ratiometric emission response could also be observed upon mixing complex 2 with Aβ42 after incubation for around 1 h (Fig. S15, ESI). However, the degree of lower-energy emission enhancement is much less smaller with respect to that with Aβ40 fibrils, though further evidence is needed to clarify the difference in sensing the aggregation of Aβ40 and Aβ42 using complex 2.

In conclusion, a tris-heteroleptic monoruthenium complex [Ru(phen)(dpma)(dppz)](PF6)2 has been prepared. This complex shows dual emissions with fluorescence and phosphorescence properties, respectively. We have demonstrated that this complex could act as a photoluminescent and time-gated ratiometric probe for the real-time detection of Aβ aggregation without adding an additional reference. In the presence of Aβ fibrils, the lower-energy phosphorescence emission of the probe is dramatically enhanced by mutual interactions, while leaving the higher-energy fluorescence emission almost unchanged as an intrinsic internal reference. In this manner, the detection accuracy could be improved with respect to the commonly employed monomeric emission response. This work shows the appealing advantages of dual-emissive metal complexes for the luminescent detection of Aβ aggregation. We are currently working on the molecular optimization to obtain metal complexes with lower-energy dual emissions that could be both accessed by CLSM and lifetime imaging microscopy.

We thank the National Natural Science Foundation of China (81602970, 21601194, 21872154, 21975264, and 21925112), the Promotion Program for Young and Middle-aged Teachers in Science and Technology Research of Huaqiao University (ZQN-PY519), and Quanzhou City Science & Technology Program of China (2019C064R) for funding support. We thank Dr Nan Zhang and Prof. Dihua Shangguan of ICCAS for their help with the measurements of CLSM.

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Electronic supplementary information (ESI) available: Synthesis and characterization, spectroscopic and physical measurements, and calculation results (pdf). See DOI: 10.1039/c9cc08909h

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