Article Cite This: ACS Appl. Bio Mater. 2019, 2, 3120−3127
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Ratiometric Detection of Mitochondrial Thiol with a Two-Photon Active AIEgen Yuan Gu,†,‡ Zheng Zhao,†,‡ Guangle Niu,†,‡ Ruoyao Zhang,†,‡,§ Han Zhang,⊥ Guo-Gang Shan,†,‡ Hai-Tao Feng,†,‡ Ryan T. K. Kwok,†,‡ Jacky W. Y. Lam,†,‡ Xiaoqiang Yu,§ and Ben Zhong Tang*,†,‡,⊥
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Department of Chemical and Biological Engineering, Department of Chemistry, the Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction and Institute for Advanced Study, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077, China ‡ HKUST- Shenzhen Research Institute, No. 9 Yuexing first RD, South Area Hi-tech Park, Nanshan, Shenzhen 518057 China § Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, China ⊥ Center for Aggregation-Induced Emission, SCUT-HKUST Joint Research Institute, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China S Supporting Information *
ABSTRACT: In clinical studies, thiol measurement in the whole blood is of diagnostic and prognostic significance. In addition, the detection of mitochondrial thiol is very important for investigating cellular functions or dysfunctions. Here, a ratiometric aggregation-induced emission luminogen (AIEgen) called TPE-PBP for thiol detection was developed by introducing a para-dinitrophenoxy benzylpyridinium moiety to tetraphenylethylene. TPE-PBP exhibits excellent biocompatibility, high photostability, and large two-photon absorption cross-section. TPE-PBP emits red fluorescence in PBS buffer without thiol. Upon addition of thiol, there is a gradual blue shift of TPE-PBP’s emission with ratiometric fluorescent response because of cleavage of the dinitrophenyl ether bond by thiol followed by the self-immolation of the para-hydroxybenzyl moiety to produce a less conjugated AIEgen. The in vitro quantification of thiol is enabled by the linear ratiometric fluorescence response with a very low limit of detection. Considering the high sensitivity and good selectivity toward thiol detection, TPE-PBP was also utilized to detect thiol in the blood. Furthermore, mitochondrial thiols in vitro, ex vivo, and in vivo were quantitatively mapped by TPE-PBP using fluorescence microscopy under one- and two-photon conditions. KEYWORDS: aggregation-induced emission, ratiometric probe, two-photon imaging, thiol-specific mitochondria-selective imaging, in vivo imaging
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INTRODUCTION Disease diagnosis at the early time and monitoring the intracellular biomolecules’ changes are of critical importance to reduce mortality and medical costs.1−5 Thanks to the great contributions of scientists, a lot of sensitive and selective bioprobes for specific disease diagnosis have been well developed.6−9 Intracellular thiol, like cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), participates in crucial biological processes.10−14 Particularly, the mitochondrial pool of thiol is crucial for the protection against oxidative stress and contributes to removing ROS to keep ROS at a suitable level to satisfy the physiological demand. 15−20 A decrease in mitochondrial thiol level reduces the antioxidant defense system and may reflect several diseases, such as diabetes, mellitus, renal failure, malignancy, and neurodegenerative diseases.19,20 Therefore, valuable information can be provided by monitoring thiol status in mitochondria to study thiol’s © 2019 American Chemical Society
roles in mitochondria’s functions and dysfunctions, which is of clinical significance.19,20 Traditional analytical methods for the detection of thiol in mitochondria include HPLC, capillary electrophoresis, LC/ MS, enzyme assay, and electrochemical methods.10−14,19,20 Despite the advantages of high selectivity and sensitivity of these methods, they usually bring about the concerns of high equipment costs, complexity, complicated sample processing, and long runtime; as a result, their practical applications are actually limited.11 Also, these methods usually need the isolation and homogenization of mitochondria.20 Due to the inherited merits of simplicity, high sensitivity, and low background noise, visualizing the analytes in situ and Received: May 26, 2019 Accepted: May 31, 2019 Published: May 31, 2019 3120
DOI: 10.1021/acsabm.9b00447 ACS Appl. Bio Mater. 2019, 2, 3120−3127
Article
ACS Applied Bio Materials uncovering the information that is unlikely to be obtained by homogenates of cell can be completed by fluorescent probes.1,19−24 Great research endeavors have been devoted to the creation of new thiol-specific bioprobes for monitoring intracellular mitochondrial thiol’s level and location. These fluorescent probes typically take advantages of the nucleophilicity of thiol.15−17 For example, a turn-on thiol assay was realized by Hu et al. by utilizing BODIPY as fluorescent reporter and 2,4-dinitrophenyl sulfonyl (DNBS) moiety as the substrate and quencher.25 However, these probes are troubled by the aggregation-caused quenching (ACQ) effect. In concentrated solution or in the aggregated state, these probes’ emissions are weakened or even quenched. As a result, researchers have to dilute solutions at the expense of low sensitivity and poor photostability for biological applications.26−28 In 2001, a class of molecules with propeller shape, such as tetraphenylethylene (TPE) and siloles, were found by Tang and co-workers to be nonemissive in dilute solutions but turn on their emission in the aggregate or solid states.29−31 Accordingly, this unusual phenomenon was termed as “aggregation-induced emission (AIE)”.32,33 The theory of restriction of intramolecular motions (RIMs), including restriction of intramolecular vibrations (RIVs) and restriction of intramolecular rotations (RIR), can explain the mechanism of AIE.34−37 Guided by the principle of RIM, a lot of AIEbased bioprobes with merits of low cytotoxicity, long-term in situ retention ability, good photostability, and high brightness are successfully developed for various practical applications.38−43 Although several thiol-specific AIE probes for in vitro turn-on detection have been reported,44−49 most of them are single-wavelength indicators, whose turn-on signals can be affected by the experimental parameters like laser power, probe concentrations, and/or optical path length, making them unsuitable for quantitative measurements.50,51 Furthermore, most of them have scarcely been applied for detecting mitochondrial thiol. In consideration of this, ratiometric methods detecting the fluorescent intensities of the probe at two distinct wavelengths can serve as a build-in correction for the environmental effects and facilitate analyte quantification.52 On the other hand, compared with the traditional fluorescent imaging using one-photon excitation, two-photon-excited fluorescent imaging holds advantages of less interference from background autofluorescence, deeper penetration to tissues, and low phototoxicity to living biosamples.53−56 Therefore, it is highly desirable to develop a thiol-specific ratiometric AIEgen with two-photon characteristics for mitochondrial thiol detection. In this contribution, the design and facile synthesis of a ratiometric thiol-specific AIEgen (named TPE-PBP) with large two-photon imaging property is reported. Mitochondrial thiol in vitro, ex vivo, and in vivo can be specifically targeted and quantified in one- and two-photon microscopy. Moreover, this probe was successfully utilized for thiol detection in the blood, which shows potential applications in clinical diseases diagnosis.
Scheme 1. Speculated Mechanism for Selective Reaction of TPE-PBP toward Biothiol
HRMS with high purity (see Figures S1−S3 in the SI). The photoluminescnece (PL) property is investigated by adding TPE-PBP stock solution to the DMSO and water mixed solvents. As shown in Figures 1 and S4, with the water fraction
Figure 1. (A) Emission spectra of TPE-PBP in DMSO and DMSO/ H2O mixed solvents with varied water fractions. (B) Plots of the I/I0 value as well as maximum emission wavelength of TPE-PBP versus the water fraction in DMSO/H2O mixed solvents. I0 was the PL intensity at f w = 0%. Excitation wavelength: 405 nm.
of DMSO/water mixture increased from 0 to 50%, the emission maximum of TPE-PBP decreased first. When the water fraction further increased from 50% to 80%, there was a sudden increment in the emission intensity (∼11-fold) observed accompanied by the PL maximum blue-shifted from 638 to 586 nm. The initial PL intensity decrease should be caused by the twisted intramolecular charge transfer (TICT) effect,38−43 which is characterized by a PL intensity decrease with solvent polarity increase, while the following PL intensity enhancement is ascribed to the AIE property because the intramolecular motions of TPE-PBP are restricted by aggregate formation at high water fractions. The emission wavelength shift with the water fractions is also consistent with the TICT effect. At high water fractions from 80% to 99%, the PL intensity slightly decreased with the wavelength red-shifted a little, suggesting the amorphous particle possibly formed.55,56 Nanoparticles with smaller size are formed at high water fractions, as indicated by dynamic light-scattering (DLS) results, which is similar to the previous reports (Figure S5).55,56 Spectra Response toward Biothiol. In order to construct a thiol-specific ratiometric probe, the paradinitrophenoxy benzylpyridinium moiety was chosen as the
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RESULTS AND DISCUSSION Synthesis and Photophysical Properties of AIE Luminogens. The tetraphenylethylene containing a paradinitrophenoxy benzylpyridinium moiety (TPE-PBP) (Scheme 1) was synthesized by following the synthetic route shown in Scheme S1 in the Supporting Information (ESI). The product’s chemical structure was confirmed by NMR and 3121
DOI: 10.1021/acsabm.9b00447 ACS Appl. Bio Mater. 2019, 2, 3120−3127
Article
ACS Applied Bio Materials
and the results showed that the reaction speed decreased gradually after 120 min (Figure S8B). Noteworthy, TPE-PBP showed pH-dependent reaction with GSH (Figure S8C). In the pH range of 3−9.5, TPE-PBP itself was quite stable, while the mixture of GSH and TPE-PBP reacted efficiently when pH value is increased over 6, possibly due to the higher concentration of thiolate species at a relatively alkaline environment.15 Finally, pH of 7.4 was selected as the sensing conditions because it is close to the physiological conditions.16 GSH titration was implemented by increasing the GSH’s concentration from 0 to 300 μM to react with 10 μM TPEPBP. The fluorescent spectra were measured (Figure 3A), and
thiol recognition group. The dinitrophenyl ether will be cleaved by biothiol through SNAr attack. Through an intramolecular 1,4-elimination reaction, the produced parahydroxybenzyl moiety can undergo self-immolation to release the fluorophore.15 To test the validity of the design principle, glutathione (GSH) was used as a model compound of biothiol to react with TPE-PBP. The reaction between glutathione and TPE-PBP produced TPE-Py, as monitored by emission and absorption spectra (Figure 2). There was a gradual increase at
Figure 2. Absorption (A) and PL spectra (B) of TPE-PBP (10 μM, red) before and after GSH treatment (1 mM, blue) and reacted for 120 min in DMSO/PBS buffer (1:1, v/v) at 25 °C. Inset: photographs of TPE-PBP solutions prior to (right) and after (left) addition of GSH taken under visible light (A) and UV light (λex = 365 nm) (B). Excitation wavelength: 365 nm.
Figure 3. (A) PL spectra of TPE-PBP (10 μM) in the presence of increasing concentration of GSH (final concentration: 0, 25, 50, 75, 100, 125, 150, 175, 200, and 300 μM) DMSO/PBS buffer (1:1, v/v) for 120 min at 25 °C. (B) A linear calibration curve between the PL intensity of TPE-PBP and the GSH’s concentration between 0 and 125 μM after 120 min incubation. Excitation wavelength: 365 nm. Error bars are ± relative standard deviations, n = 3.
500 nm as well as a concomitant decrease at 631 nm in the emission spectra of TPE-PBP (10 μM) treated with 1 mM GSH in DMSO/PBS mixed solvents. The change of absorption and emission spectra of TPE-PBP before and after reaction with GSH could also be observed by the naked eye. The resulting products were identified by the ESI-MS measurements (Figure S7), which indicated that the resulted fluorophore’s chemical structure was the same as TPE-Py. Therefore, the speculated recognition and self-immolative cleavage indeed occurred. The proposed reaction mechanism was depicted in Scheme 1. Different from the 2,4dinitrobenzenesulfonyl moiety which was widely used as a fluorescence quencher through a photoinduced electron transfer (PET) process,10−14 the para-dinitrophenoxy benzylpyridinium moiety served as an electron-accepting unit to red shift the emission spectra of TPE-PBP through the intramolecular charge transfer (ICT) process as well as a mitochondrial targeting group. After reaction with biothiol, a para-dinitrophenoxy benzyl moiety will be cleaved to generate the tetraphenylethylene pyridinine (TPE-Py) with a blueshifted emission. The emission blueshift was ascribed to the weaker ICT effect of TPE-Py than TPE-PBP. Density functional theory (DFT) calculation confirms weaker charge separation of TPE-Py than TPE-PBP (Figure S6). The dual emission of TPE-PBP upon treatment with biothiol enables it to work as a ratiometric AIEgen for biothiol detection. To build a sensitive bioprobe, the test conditions need further optimization. First, the buffer’s influence on the reaction between GSH and TPE-PBP was evaluated. As shown in Figure S8A, when the f DMSO in DMSO/PBS buffer was 50% and 60%, the ratio of PL intensity between TPE-Py and TPE-PBP (I500/I631) was the largest, indicating a better reaction efficiency. The mixture of DMSO/PBS with volume ratio of 1:1 thus was chosen as the optimal reaction buffer. Next, we tested the reaction efficiency of GSH and TPE-PBP,
the fluorescent intensity ratio at 500 and 631 nm (I500/I631) was plotted against concentration (Figure 3B). As shown in Figure 3B, I500/I631 was linearly proportional to the GSH concentration (0−125 μM), suggesting the detection limit is 0.61 μM. To evaluate the selectivity of TPE-PBP for nonbiothiol analytes, the responses of TPE-PBP to glycine (Gly), phenylalanine (Phe), methionine (Met), histidine (His), proline (Pro), arginine (Arg), and aspartic acid (Asp) in addition to common metal ions (K+, Ca2+, Na+, Mg2+, Fe3+, Cu2+, Zn2+, Mn2+), O22− and ONOO−, were investigated. As shown in Figures 4 and S9, TPE-PBP exhibits a strong response toward thiol, including GSH, Cys, and Hcy, but slight response toward NaSH and negligible response toward other nonthiol-containing amino acids, metal ions, O22−, and ONOO−. It is also worthy to note that TPE-PBP has a better response to GSH than Cys and Hcy. The longer chain length of GSH than Cys and Hcy possibly enables the intermolecular electrostatic interaction between the pyridinium of TPE-PBP and the carboxyl of GSH to promote the SNAr attack according to the proposed reaction mechanism (Scheme 1). A blood test is widely used for clinical diagnosis.17 In the whole body, the conentration of GSH is around millimolar (1−3 mM) in most cells and around micromolar (2−20 μM) in the blood plasma.10−14,17 So, fresh rabbit blood was collected and used as a sample for thiol detection. As shown in Figure S10, TPEPBP could detect thiol in rabbit blood samples in a ratiometric manner which has the potential to be utilized in clinical disease diagnosis. These results indicate that TPE-PBP can work as a fluorescent sensor for selective ratiometric detection of thiol. 3122
DOI: 10.1021/acsabm.9b00447 ACS Appl. Bio Mater. 2019, 2, 3120−3127
Article
ACS Applied Bio Materials
Figure 5. Intracellular biothiol detection in HeLa cell lines using TPE-PBP. (A and D) Fluorescent images with emission range around 500 nm, (B and E) fluorescent images with emission range around 600 nm, and (C and F) merged images of green and red channels. (A, B, C) Incubating HeLa cells with 10 μM of TPE-PBP for 60 min and (D, E, F) pretreating HeLa cells with 500 μM of NMM for 20 min before incubation with 10 μM of TPE-PBP for 60 min. (G) Relative PL intensity of NMM and TPE-PBP treated HeLa cells. Scale bar = 20 μm. Error bars are ± relative standard deviations, n = 3.
Figure 4. Relative PL intensity of TPE-PBP (10 μM) incubated with 1 mM of various kinds of analytes in DMSO/PBS buffer (1:1, v/v) at 25 °C. Data were recorded after 120 min reaction. Excitation wavelength: 365 nm. Error bars are ± relative standard deviations, n = 3.
TPE-PBP and TPE-Py was retained even after 12 min, while more than 60% of the fluorescence of MitoTracker deep red was lost under the same conditions. Therefore, TPE-PBP and the derived TPE-Py showed a much higher photostability than MitoTracker Deep Red. Two-Photon Ratiometric Thiol Detection in Vitro, ex Vivo, and in Vivo. Thanks to its high spatial resolution as well as high signal-to-noise ratio, strong antiphotobleaching capacity, and high penetration depth enabled by near-infrared (NIR) excitation, two-photon-excited fluorescent imaging has been widely used in life science research.59,60 Predicting whether a luminogen is suitable for 2PM was typically realized by measuring the two-photon absorption (2PA) cross section (δ2PA).59,60 With strong electron-donating and -withdrawing moieties, TPE-PBP possessing a conjugated structure is speculated to show strong δ2PA. Studying TPE-PBP’s 2PA was realized by utilizing a femtosecond pulsed two-photonexcited fluorescence (TPEF) technique, and the δ2PA was measured using fluorescein as the standard.61 The measured wavelength varies from 800 to 900 nm with an interval of 20 nm, and the δ2PA values were calculated. The results showed that in the DCM/hexane mixture (f DCM = 30%) the maximum δ2PA value (1790 GM) of TPE-PBP was obtained at 860 nm (Figure S14). This value was much higher than most fluorescent proteins (normally
