Selective and rapid detection of ascorbic acid by a cobalt oxyhydroxide-based two-photon fluorescent nano-platform

Qingxin Hana, Huan Yanga, Shuting Wenb, Huie Jiangac, Li Wanga and Weisheng Liu*a
aState Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China. E-mail:; Fax: +86/931/8912582; Tel: +86/931/8915151
bLanzhou University Second Hospital, Lanzhou 730000, China
cCollege of Bioresources Chemistry and Materials Engineering, Shaanxi University of Science and Technology, Xi'an 710021, China

Received 3rd January 2018 , Accepted 9th February 2018

First published on 9th February 2018

The development of an efficient and easy fabricated approach to detect ascorbic acid (AA) is of physiological and pathological significance. In this work, a two-photon sensor platform which is constituted with a 1,8-naphthalimide-based fluorophore and CoOOH nanosheets was designed in which the blue two-photon fluorescence of the fluorophore was suppressed to a remarkable extent via a FRET process between CoOOH nanosheets and the fluorophore. The fluorescence inhibition could be removed through the specific reaction of CoOOH and AA. Based on this feature, we have demonstrated the prominent sensing performance of the sensor platform, including excellent two-photon induced fluorescence properties, a convenient fabrication pathway, a specific response to AA, a wider linear range and a high stability. This fluorescence assay is capable of detecting AA in living cells and has potential for further application such as AA associated disease diagnosis.


Recently, more comprehensive attention has been paid to ascorbic acid (AA) because it is one of the most crucial micronutrients in the human body. AA also acts as an effective antioxidant and is involved in many biochemical processes, for example, modulating the dopamine and glutamate-mediated neurotransmission and scavenging intracellular reactive species, as well as preventing oxidative damage of biological molecules (e.g., proteins, lipids and DNA).1 Many illnesses including cataracts, urinary stones, atherosclerosis, diarrhea, stomach convulsion and cancers are associated with the levels of AA.2 Moreover, the consumption of AA supplements is always tied up with some chronic disease incidence and deaths.3 In this regard, it is of physiological and pathological significance to detect AA effectively.4

Until now, some assays of capillary electrophoresis,5 spectrophotometry,6 chromatography,7 electrochemistry,8 and fluorometry9 have been developed for the detection of AA. Thereinto, the spectrophotometric methods always suffer from the interferences from many biological substances and cannot offer an ideal output signal. Other available methods usually show shortcomings such as being time-consuming and insensitive and require special instruments. Alternatively, fluorometric methods can convert the levels of the target species into an optical signal change which could be easily visualized by using a fluorescence spectrometer or a fluorescence microscope or even a simple light lamp. Fluorescent probes have consistently demonstrated their potential in detecting and imaging the molecules of interest because they can monitor extracellular and intracellular events with high selectivity and sensitivity, and they are feasible for AA determination.10 However, fluorescent probes of AA based on the interaction between AA and gold nanoclusters9a or MnO2 nanosheets9b lacked selectivity and failed to discriminate AA from other antioxidants efficiently. In addition, ascorbate oxidase was also needed for the case of MnO2 nanosheets to catalyze the reaction by which the sensing mechanism was based. The cerium metal–organic framework (ZJU-136-Ce) may suffer from UV excitation and a long reaction time, and this bulk material was against highly sensitive detection and applications in biological systems.9c In this regard, selective and sensitive AA probes are urgently needed.

Cobalt oxyhydroxide (CoOOH) was reported to specifically react with AA and several AA-selective probes have been developed based on this reaction: Tang's group has described a novel nanoprobe, i.e. persistent luminescence nanoparticles modified with CoOOH.11 Lin's group has designed a fluorescent nanoprobe using carbon dot (CD)-modified hexagonal cobalt.12 Besides this, a CoOOH modified upconversion nanosystem, silica nanoparticles and graphene quantum dots were also reported for sensing AA activity in the human plasma.13 Our group also developed a ratiometric nanoprobe for AA detection and imaging.14 Nevertheless, some limitations have restricted the practical application of these probes: (1) complicated and harsh fabrication processes are needed to prepare these probes. (2) The UV excitation for carbon dots could easily be affected by the background and the NIR excitation for upconversion systems requires expensive lasers with high power. Apart from that, the in vitro excitation for persistent luminescence nanoparticles also suffers from dramatic attenuation. (3) The sizes of these nanoprobes are relatively large, which limited their stability, biocompatibility and accuracy as a sensor. Therefore, there are still demand to fabricate simple, efficient and biocompatible fluorescent probes for ascorbic acid determination.

A molecular two-photon (TP) fluorophore can convert two near-infrared photons to one photon of higher energy and thus emit fluorescence efficiently. It provides potential to design well-defined fluorescent probes with improved three-dimensional spatial localization, deeper tissue penetration, low photo-damage to biosamples, and deeper tissue penetration.15 Taking the high selectivity and the large molar extinction coefficient of CoOOH into consideration, we present here the design and preparation of a highly selective TP fluorescent probe for AA sensing, in which the fluorescence of 4-methoxy-N-butyl-1,8-naphthalimide (MBNI) decayed by CoOOH nanosheets and the fluorescence could be released in the presence of AA.

Experimental section

Materials and instruments

NMR spectra were recorded on a JNM-ECS-400 spectrometer and referenced to the signals of TMS. Mass spectra (ESI) were recorded on a Bruker Esquire 6000 Ion Trap System. The two-photon spectra, absolute quantum yield Φ and lifetime were measured using an Edinburgh Instrument FLS920, with an integrating sphere (150 mm diameter, BaSO4 coating) for absolute quantum yield measurements and a 100 W μF 920H lamp as the excitation source for lifetime measurements. The steady-state fluorescence spectra measurements were performed on a Hitachi F-7000 fluorescence spectrometer with a Xe lamp as the excitation source. FT-IR spectra were recorded on KBr pellets in the region 4000–400 cm−1. XPS spectra were recorded by using an ESCALAB210 X-ray photoelectron spectrometer. TEM images were taken on a Tecnai-G2-F30 (300 kV) instrument. UV-vis spectra were recorded on an Agilent Cary 5000 UV-vis-NIR spectrophotometer. X-ray diffraction (XRD) data were recorded by using Cu Kα radiation from an X'Pert PRO MPD diffractometer. Fluorescence imaging studies were performed by using a LSM 510 META laser confocal fluorescence microscope with a mode-locked titanium–sapphire laser source set at a wavelength of 750 nm. All measurements were carried out at room temperature except where noted. Aqueous solutions were prepared with Milli-Q water (18.4 MΩ cm−1). The reagents and solvents were of analytical grade and obtained commercially. In the absence of a special note, they were used without further purification.

Synthesis of 4-methoxy-N-butyl-1,8-naphthalimide (MBNI)

The TP fluorophore, 4-methoxy-N-butyl-1,8-naphthalimide, was prepared by the same synthesis procedure reported in our previous reports.16 The product was obtained as a white solid. M. p. 105.1–105.8 °C. 1H NMR (400 MHz, CDCl3, δ ppm): δ 9.03–8.39 (m, 1H), 7.87–7.59 (m, 1H), 7.04 (d, J = 8.3 Hz, 1H), 4.28–4.15 (m, 1H), 4.13 (s, 1H), 1.72 (t, J = 7.0 Hz, 1H), 1.45 (dd, J = 15.0, 7.4 Hz, 1H), 0.98 (t, J = 7.3 Hz, 1H). 13C NMR (101 MHz, CDCl3, δ ppm): δ 164.42, 163.83, 160.64, 133.27, 131.36, 129.16, 128.44, 125.82, 123.32, 122.33, 115.04, 105.10, 56.17, 40.10, 30.29, 20.46, 13.93. ESI mass spectrum m/z: calcd for C17H17NO3 283.1, found: 284.1 [M + H]+.

Preparation of dye-CoOOH nanosheets

CoOOH nanosheets were prepared according to the literature17 with some modifications. To a 20 mL reaction vessel containing 5 mL of CoCl2 solution (10 mM), 5 mL of NaOH solution (0.8 M) was first added following an ultrasonic treatment (5 min). Then, 5 mL of NaClO (0.2 M) solution was added dropwise to the above solution with a continuous ultrasonic treatment. Subsequently, dark brown CoOOH nanosheets were collected by centrifugation and then washed with deionized water three times before re-dispersion in 10 mL of Milli-Q water for use.

The TP fluorescent nano-platform was fabricated by adding 0.5 mL of DMSO solution of MBNI (1.0 mM) to the above dispersion system. Then the mixture was subjected to 10 min ultrasound before diluting with 95 mL HEPES buffer solution (10 mM, pH 7.40, containing 0.5% DMSO) for the spectroscopic study. The aqueous solutions with different pH values were used instead of HEPES buffer solution to study the pH effects.

Spectroscopy studies

To study the TP fluorescence suppression of MBNI, increasing concentrations (0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.60, 0.70, 0.80, 1.00, 1.20, and 1.50 mM) of CoOOH nanosheets were added to the MBNI solution. And then the solution was stabilized for 10 min before being subjected to measure the TP fluorescence.

The two-photon absorption (TPA) cross-section values (δ) of MBNI were determined according to the reported method,18 with fluorescein solution (5 μM, pH = 13.0) as the reference molecule. In selectivity measurements, the aqueous solutions of various biologically relevant important cations (Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Cu2+, Co2+, and Fe3+), anions (Cl, Br, I, CO32−, HSO32−, NO3, OAc, and S2−), amino acids and reactive species (H2O2, t-BuOOH, GSH, L-Cys, L-Thr, L-Arg, Hcy and AA) were used to give a final concentration of 0.2 mM for each case. In these experiments, a system containing 5 μM of the TP fluorophore and 0.2 mM of CoOOH nanosheets was used for the sensing study. All fluorescence spectra were recorded with an excitation wavelength of 375 nm for one-photon measurements and 750 nm for two-photon measurements.

Fluorescence microscopy imaging

HepG2 cells were incubated with growth medium at 37 °C for 24 h, and then divided into three groups. For the first group, HepG2 cells were only exposed to MBNI (5 μM) for 30 min before washing with PBS three times, and then were subjected to imaging by confocal fluorescence microscopy. For the second group, HepG2 cells were incubated with MBNI (5 μM) for 30 min and CoOOH (0.5 mM) for another 30 min. The subsequent procedures were the same as those of the first group. For the last group, HepG2 cells were incubated with MBNI (5 μM) for 30 min, CoOOH (0.5 mM) for 30 min and AA (0.5 mM) for 30 min, successively. Fluorescent imaging of HeLa cells and AGS cells was almost the same as that of HepG2 cells.

Results and discussion

Design principle and characterization of MBNI-CoOOH

The sensing mechanism in the nano-platform is based on a fluorescence suppression, which experienced the fluorescence resonance energy transfer (FRET) process from 4-methoxy-N-butyl-1,8-naphthalimide (MBNI) to CoOOH nanosheets. Nevertheless, the presence of AA could restore the fluorescence as a result of the specific reaction between CoOOH and AA (Scheme 1). 1,8-Naphthalimide derived fluorophores were commonly employed for developing fluorescent chemosensors due to their excellent fluorescence properties, such as long wavelengths of absorption and emission, favourable TPA cross-section, high photostability and fluorescence quantum yield (Φ = 0.42), weak pH dependence, etc.16a,18b,19 Thus, MBNI was selected as the TP fluorophore in our sensing system. As shown in Fig. 1, CoOOH nanosheets were first prepared and characterized comprehensively. The XPS results indicated that the cobalt existed in the oxidation state of Co(III) (780 eV) and no other impurity oxidation state of cobalt existed (Fig. 1B & Fig. S1). The TEM images and XRD results clearly revealed that the morphology of the as-prepared CoOOH nanosheets was mainly amorphous (Fig. S2). The mesoporous CoOOH nanosheets were well-dispersed according to the graph. TEM-mapping analysis indicated that Co and O evenly distributed in the whole CoOOH nanosheets. The EDXA spectrum indicates the presence of the cobalt element in CoOOH nanosheets. The formation of cobalt oxyhydroxide was further proved by Fourier transform infrared (FT-IR) spectroscopy (Fig. S3). The IR absorption peak centered at 3437 cm−1 was assigned to the bond stretching of the hydroxyl group (–OH). The characteristic peaks at 1625 cm−1 and 571 cm−1 were attributed to the vibrations of the Co–O double and single bonds, respectively. The TG-DSC curves revealed that the CoOOH nanosheets were thermally stable. As shown in Fig. S2A, MBNI-CoOOH nanosheets basically maintained the original morphology and size of the CoOOH nanosheets.
image file: c8qi00003d-s1.tif
Scheme 1 Strategic design and the proposed mechanism for AA detection.

image file: c8qi00003d-f1.tif
Fig. 1 (A) TEM image of CoOOH nanosheets. (B) XPS spectrum of CoOOH nanosheets. (C) High-magnification HAADF-STEM image of CoOOH nanosheets. (D) Co TEM-mapping and (E) O TEM-mapping of CoOOH nanosheets corresponded to (C).

Under both one-photon (OP) and TP excitation, MBNI exhibits excellent blue fluorescence emission centered at 470 nm, which is largely overlapped with the broad absorption band (∼350–600 nm) of CoOOH nanosheets (Fig. 2A). Thus, the emission of the fluorophore could be quenched by CoOOH nanosheets effectively. When an increasing amount of CoOOH was added into the aqueous solution of MBNI, the fluorescence intensity was gradually decreased accordingly (Fig. 2B). Ultimately, 0.5 mM CoOOH nanosheets were used in our nano-platform to suppress the fluorophore fluorescence after a trade-off between the quenching effect and sensing performance. By comparing the fluorescence decay curves (Fig. 2C) of the MBNI to MBNI–CoOOH system, a decrease of fluorescence lifetime (470 nm) was observed in the presence of CoOOH nanosheets, suggesting the FRET process between MBNI and CoOOH. As shown in Fig. 2D, UV-vis absorption spectra indicated that CoOOH nanosheets displayed a broad absorption band centered at 405 nm, which was consistent with the previous reports.11,12 The presence of MBNI in the nano-system has no prominent impact on the UV-vis spectrum of CoOOH because the molar ratio of the fluorophore to CoOOH and the molar extinction coefficient of MBNI were too small to change the absorbance intensity in the nano-system.

image file: c8qi00003d-f2.tif
Fig. 2 (A) The UV-vis spectrum (left coordinate, blue line) of CoOOH nanosheets and the fluorescence spectra (right coordinate) of MBNI under OP excitation (λex = 365 nm, red line) and TP excitation (λex = 750 nm, black line). (B) The fluorescence spectra of MBNI (5 μM) in the presence of an increasing amount of CoOOH nanosheets (0.0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.60, 0.70, 0.80, and 1.0 mM). λex = 750 nm. (C) Fluorescence decays of MBNI in the absence (black) and presence (red) of CoOOH nanosheets, λem = 470 nm, λex = 365 nm. (D) UV-vis spectra of MBNI (5 μM, black line), CoOOH nanosheets (0.5 mM, red line), and the CoOOH-based nano-platform (blue line).

Spectra study and AA determination

The response of the nano-platform for AA was investigated. When the concentration of AA was increased in the nano-system, the absorbance peak assigned to the CoOOH nanosheets was decreased accordingly (Fig. 3A). Meanwhile, the color of the solution changed from yellowish-brown to colorless upon the addition of AA, which could be readily observed with the naked eye. The TPA cross-section value of MBNI in HEPES buffer solution (10 mM, pH 7.40, containing 0.5% DMSO) was around 110 GM at 750 nm (Fig. S4), implying that MBNI was an efficient TP fluorophore for fabricating fluorescent probes. When the nano-platform was treated with AA, the fluorescence intensity at 470 nm increased rapidly within the first 5 min, and reached a plateau afterwards (Fig. S5). Such a quick response is comparable to the reported AA fluorescent chemosensor. These absorption and fluorescence spectra changes could be described as the AA-mediated reduction of CoOOH into Co2+, the decomposition of the CoOOH nanosheets, and the elimination of the fluorescence suppression originating from the FRET process. In order to evaluate the fluorometric sensing performance of this CoOOH-based nano-platform, the fluorescence titration experiment was performed with varied concentrations of AA (Fig. 3B). When the AA concentration was increased from 0 to 0.1 mM, the fluorescence intensity of the nano-platform was linearly increased under TP excitation (750 nm), showing a typical “turn-on” fluorescence response (Fig. S6). Furthermore, the detection limit for AA was around 0.26 μM, which is well under the vast majority of CoOOH-based AA probes.
image file: c8qi00003d-f3.tif
Fig. 3 (A) UV-vis spectra of the CoOOH-based nano-platform with the titration of AA (0, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, and 240 μM) in HEPES buffer solution. Inset: color change of the CoOOH-based nano-platform upon the addition of AA. (B) TP fluorescence spectra of the CoOOH-based nano-platform with the titration of AA (0, 4, 8, 12, 16, 20, 24, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 200, 240 and 300 μM) in HEPES buffer solution. Inset: fluorescence change of the CoOOH-based nano-platform upon the addition of AA. λex = 750 nm.

Then, the stability of this kind of CoOOH-based nano-platform was studied. As shown in Fig. S7, the fluorescence intensity at 470 nm showed no obvious variation as the pH changed from 4.0 to 9.0, suggesting that the current dye–CoOOH nano-system was not affected by the pH alteration under physiological conditions. The stability of the nano-platform was also tested in HEPES buffer solution. Fig. S8 indicates that the fluorescence intensity at 470 nm was unchanged within 60 min (note that the intensities were recorded only for 60 min, which was long enough for AA sensing) in the absence of AA, proving that the identified system was stable. These results suggested that this dye–CoOOH system was ready for AA detection.

Since selectivity is crucial for a probe, it is necessary for the dye–CoOOH system to possess a highly selective response towards AA over other potentially interfering substances. Therefore, the selectivity of the CoOOH-based nano-platform was assessed with a variety of related analyte species: important cations (Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Cu2+, Co2+, and Fe3+), anions (Cl, Br, I, CO32−, HSO32−, NO3, OAc, S2O32−, and S2−), amino acids and reactive species (H2O2, t-BuOOH, GSH, L-Cys, L-Thr, L-Arg, and Hcy). As shown in Fig. 4, all these interfering species’ existence would not trigger remarkable fluorescence enhancement compared to AA, suggesting that this dye–CoOOH system possesses a specialized response to AA. This high selectivity could be described as the specific reaction of CoOOH toward AA, which was indicated in our previous work and other literature reports.11–14 As shown in Fig. S9, CoOOH showed a higher reduction activity among various interfering substances to convert CoOOH into Co2+. Therefore, other reductive species such as Fe2+, S2O32−, H2S, Cys, Hcy, and GSH caused negligibly small interferences in the determination of AA. Moreover, red bars in Fig. 4 consolidate the conclusion that the CoOOH-based nano-platform could hardly be affected by the coexistence of other potentially interfering substances.

image file: c8qi00003d-f4.tif
Fig. 4 Selectivity of the CoOOH-based nano-platform for AA (0.2 mM) in the presence of 0.2 mM various biologically relevant important cations (Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Cu2+, Co2+, and Fe3+), anions (Cl, Br, I, CO32−, HSO32−, NO3, OAc, and S2−), amino acids and reactive species (H2O2, t-BuOOH, GSH, L-Cys, L-Thr, L-Arg, and Hcy) in HEPES buffer solution. λex = 750 nm. Black bars represent the addition of an appropriate species to the solution of dye–CoOOH nanosheets. Red bars represent the subsequent addition of AA to the solution.

Detection of AA in diluted serum

Having demonstrated the AA-triggered fluorescence enhancement of the CoOOH-based nano-platform in HEPES buffer solution, we wondered whether this system could be used to detect AA in biological samples (e.g. serum). Keeping the superiority of the TP probe in mind, we have first studied the background fluorescence in diluted (25%) fetal bovine serum. As shown in Fig. 5A, the fluorescence background of serum solution was hardly observed under TP excitation. When AA was added into the diluted serum solution of the CoOOH-based nano-platform, a fluorescence enhancement similar to that of the buffer solution was observed, which implied that the probe had potential to sensing AA in diluted serum. Next, we have spiked the diluted serum with a standard solution that contains different amounts of AA. Interestingly, the fluorescence intensity at 475 nm increased linearly as the concentration of AA was added up to 100 μM (Fig. 5B). Since the concentration of AA in the human blood was documented in the range of 20–100 μM,20 the probe is practically useful for AA detection upon further bio-relevant study.
image file: c8qi00003d-f5.tif
Fig. 5 (A) TP fluorescence spectra of the CoOOH-based nano-platform in diluted (25%) fetal bovine serum (red line) and the solution was subsequently incubated with AA (blue line). The black line represents the background fluorescence. λex = 750 nm. (B) TP fluorescence intensity (470 nm) changes of the CoOOH-based nano-platform in diluted (25%) fetal bovine serum vs. AA concentration. λex = 750 nm.

TP fluorescent imaging

The excellent sensing performances of the CoOOH-based nano-platform under physiological conditions inspired us to evaluate whether our probe could work in living cells. The HepG2 cell line was applied to assess the feasibility of the probe for AA imaging. As shown in Fig. 6, the HepG2 cells of the control/blank group displayed no fluorescence under TP excitation (λex = 750 nm). The HepG2 cells incubated with 5 μM of MBNI displayed a strong blue TP fluorescence. And the blue fluorescence could be quenched to a great extent when the dye loaded cells were treated with CoOOH nanosheets. Meanwhile, when additional AA was added to the HepG2 cells which had been treated with CoOOH nanosheets previously, a significant fluorescence enhancement has been observed. The result evidenced the recovery of fluorescence, which is because the FRET process between MBNI and CoOOH nanosheets was destroyed when the added AA decomposed the CoOOH. Then, HeLa cells and AGS cells were employed to investigate whether the CoOOH-based nano-platform could respond to the changes of AA concentration in different kinds of living cells. As shown in Fig. S10 & S11, the same imaging results of HepG2 cells were achieved in both HeLa cells and AGS cells, implying that our probe could respond to varied levels of AA in the cell environment. Fig. S12 also implies that the CoOOH-based nano-platform had potential to determine intracellular AA concentrations. Moreover, as shown in Fig. S13, the image with TP excitation (λex = 750) has a higher signal to noise ratio than the image with OP excitation (λex = 405), indicating that TP cellular imaging is superior to that of OP. These results further confirmed the applicability of the CoOOH-based nano-platform to detect AA in living cells.
image file: c8qi00003d-f6.tif
Fig. 6 TP fluorescence images in living HepG2 cells. (A, E, and I) Control group of blank cells. (B, F, and J) HepG2 cells were incubated with MBNI (5 μM) for 15 min at 37 °C. (C, J, and K) HepG2 cells were incubated with MBNI (5 μM) for 15 min and further incubated with CoOOH nanosheets (0.1 mM) for another 15 min. (D, H, and L) HepG2 cells were successively incubated with MBNI (5 μM), CoOOH nanosheets (0.5 mM), and AA (250 μM) for 15 min. (E), (F), (G), and (H) are the bright-field images corresponding to (A), (B), (C), and (D), respectively. (I), (J), (K), and (L) are the overlay images.


In conclusion, we have successfully realized a fluorescent CoOOH-based nano-platform for the highly selective and sensitive sensing of AA. The FRET process between MBNI and CoOOH nanosheets makes it particularly useful for developing a novel and facilitated TP approach to detect AA in biological samples. Apart from the favorable TP fluorescence properties, this dye–CoOOH system displayed several advantages, such as being easy to fabricate, stable under normal conditions, with a wide linear response range, excellent specificity toward AA, etc. Moreover, the AA-mediated color and fluorescence changes of this nano-platform could be recognized with the naked eye. The probe is not only applicable for sensing AA under physiological conditions but also proved to potentially find application in intracellular AA detection. Furthermore, this study has proposed a new reasonable strategy to develop efficient TP sensor platforms for other species.

Conflicts of interest

There are no conflicts to declare.


This study was supported by the NSFC (Grant 21431002).

Notes and references

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Electronic supplementary information (ESI) available: Additional characterization and supporting figures. See DOI: 10.1039/c8qi00003d

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