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A Highly Sensitive Squaraine-Based Water-Soluble FarRed/Near-Infrared Chromofluorogenic Thiophenol Probe Li Xiong, Jun Ma, Yan Huang, Zihe Wang, and Zhiyun Lu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00151 • Publication Date (Web): 22 Mar 2017 Downloaded from http://pubs.acs.org on March 24, 2017

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A Highly Sensitive Squaraine-Based Water-Soluble Far-Red/NearInfrared Chromofluorogenic Thiophenol Probe Li Xiong, Jun Ma, Yan Huang, Zihe Wang, and Zhiyun Lu* Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, China. KEYWORDS: squaraine; thiophenol; colorimetric; fluorometric; water-soluble; high sensitivity.

ABSTRACT: A squaraine-based far-red/near-infrared fluorescent probe (SQ-DNBS) was exploited for thiophenol detection. SQ-DNBS is a colorimetric and “off‒on” fluorometric dual-channel “naked-eye” chemosensor showing high selectivity, high sensitivity (detection limit: 9.9 nM) and rapid response to thiophenol in aqueous solution. SQ-DNBS also can be used in practical applications for the detection of thiophenol in water samples. Photophysical and spectral characterization results revealed that the probing mechanism of SQ-DNBS toward thiophenol lies in the thiolatemediated cleavage reaction. Our discovery demonstrates the potential of arylmethylene-squaraine skeleton as promising fluorophore unit to construct high-performance far-red/near-infrared chemosensors.

Thiophenols, a class of important pharmaceutical raw materials1 and intermediates2, are also considered as a kind of widespread pollutant due to their high toxicity. In detail, their median lethal concentration (LC50) for fish3 and dose (LD50) for mouse4 are only 0.01−0.4 mM and 2.15-46.2 mg kg-1, respectively; and excessive ingestion of them will lead to serious systemic injuries like central nervous system damage, muscular weakness and even death.5 Therefore, it is highly demanded to develop simple, rapid, sensitive and selective analytical methods for the detection of thiophenols. Among the current detection techniques for thiophenols, optical probe is considered to be quite promising for its handy operation, noninvasiveness and real-time characters.6 To act as an ideal thiophenol fluorescent probe, it should not only show high selectivity, high sensitivity and prompt response to the analyte, but also possess good water solubility.7 Moreover, for biosensor applications, it should show intense fluorescence signals in far-red (FR) to near-infrared (NIR) region (λem ≥ 630 nm), so that the contamination signals from the endogenous fluorophores in biological systems could be effectively avoided.8 Yet generally, thiophenol fluorescent probes will suffer from severe interference from thiol species due to the quite similar chemical properties of thiophenols and thiols. In fact, it was not until 2007 that Wang et al.9 reported the first example of highly selective thiophenol fluorescent probe by taking advantage of the more nucleophilic property of thiophenols than thiols in neutral environments (pH = 7.3) due to their more efficient dissociation into thiolate species (Scheme 1). Based on this differentiation mechanism, many optical probes showing high selectivity toward thiophenols have been developed subsequently.4,10

Currently, for visible fluorescent thiophenol probes, the detection limit (LOD) could be as low as 1.8 nM,11 the response time could be less than 5 s,12 and the fluorescence quantum efficiency (ΦF) of the optical responding species could be up to 0.66.13 Nevertheless, in the case of FR/NIR fluorescent thiophenol probes, so far there are only five relevant reports,14 and their performance lags far behind that of their visible counterparts. For example, the lowest LOD is only 34 nM,14f and most of their signaling species show relatively low ΦF.14a-c Moreover, none of them could be applied to true aqueous media without organic cosolvent to realize the determination of thiophenol concentration.14 Therefore, it is highly desirable to develop FR/NIR fluorescent thiophenol-responding probes with integrated high selectivity, high sensitivity, good water solubility, prompt response and relatively high ΦF of the signaling species. Owing to their strong absorption and fluorescence in FR/NIR region and good photostability, squaraine derivatives15 have attracted considerable attention as the fluorophore core to construct chemosensors in ions,16 small molecules,17 proteins,18 and environmental polarity19 probing, and biosensors for cellular imaging applications as well.18b,20 Nevertheless, as far as squaraine- based thiophenol probes are concerned, no report could be found Scheme 1. Schematic illustration of the better nucleophilicity of thiophenols than thiols in neutral conditions.

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Scheme 2. Schematic illustration of: (a) the reaction of arene-squaraine derivative SQ-Arene with thiols; and (b) the design concept of the probe SQ-DNBS for the detection of thiophenols.

up to date. This might be ascribed to the poor chemical stability of their strongly electron-deficient central cyclobutene rings against nucleophiles like thiols and thiophenols, which will finally result in disrupted π-conjugation system of the fluorophore (e.g. SQ-Arene29, vide Scheme 2a). In fact, by using their cyclobutene ring as the recognition site, a number of squaraine-based thiol optical probes have been explored successfully.21 Yet in all cases, they show an “on-off” rather than “off-on” fluorescent response to thiols in FR/NIR region, which is detrimental to the in vivo detection applications. In addition, in view of the fact that in neutral environments (pH = 7.5), such probes could show prompt response toward thiols (in some cases, even < 6 s22), it should be difficult for them to differentiate thiophenol from thiol species. Therefore, the exploitation of squaraine-based high-performance optical thiophenol probe remains a challenging research work.

group was employed as the D building block of SQ-DNBS to endow it with not only good chemical stability on its cyclobutene ring (A unit) against nucleophiles,23 but also relatively long-wavelength fluorescence toward FR/NIR region. On the other hand, an electron-rich 2,4,6trihydroxyphenyl group capable of forming two intramolecular hydrogen bonds with the oxygen atoms of the central A unit24 was chosen as the D' unit to endow SQDNBS with a more extended conjugation system, further steric protection on its cyclobutene ring from nucleophilic attack,25 and increased molecular rigidity and planarity hence enhanced ΦF of the signaling species as well.24b,26 To gain optical response toward thiophenols, a 2,4-dinitrobenzenesulfonyl (DNBS)10a-c,27 group as the fluorescence quenching and thiophenol recognition site was grafted to the 4-position of the D' unit of the compound via an ester bond. And to enhance the water solubility of the probe, a mannose subunit was introduced to the benzoindole segment using a triethylene glycol ether chain as the bridging unit.28 As expected, SQ-DNBS can act as a high-performance water-soluble colorimetric and fluoro

Very recently, Karpenko et al.23 found that a compound bearing an arylidene-squaraine rather than arenesquaraine molecular framework could display good chemical stability against the nucleophilic attack from thiols. Enlightened by this discovery, we conjectured that arylidene-squaraine derivatives might be appropriate luminogen candidates to construct thiophenol probes whose central cyclobutene rings could be chemically stable against both thiols and thiophenols, so that their FR/NIR fluorescence properties could be maintained. To validate this hypothesis, herein, we designed and synthesized an unsymmetrical D-A-D'-structured 1,3-squaraine derivative SQ-DNBS as a novel thiophenol probe (molecular structure shown in Figure 1, design concept shown in Scheme 2b). On one hand, a π-extended benzindolylmethylene

Figure 1. Molecular structures of SQ-DNBS and SQ. 2


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Figure 2. (a) The absorption spectra in the presence of different concentration of thiophenol in PBS (0.01 M, pH =7.4); and (b) the correlation between absorbance of 5 μM SQ-DNBS at 626 nm and concentration of thiophenol in PBS (0.01 M, pH = 7.4). Insets: photographs of color changes of 5 μM SQ-DNBS upon addition of thiophenol (25 μM).

Figure 3. (a) The fluorescence spectra (λex = 538 nm) of 5 μM SQ-DNBS in the presence of different concentrations of thiophenol in PBS (0.01 M, pH =7.4); and (b) the correlation between fluorescence intensity of 5 μM SQ-DNBS at 645 nm (I645 nm) and concentration of thiophenol in PBS (0.01 M, pH = 7.4). Insets: photographs of fluorescence changes of 5 μM SQ-DNBS upon addition of thiophenol (25 μM).

metric (λem = 645 nm) dual-channel naked-eye FR/NIR thiophenol probe with integrated high selectivity, high sensitivity (LOD: 9.9 nM), high fluorescence efficiency (ΦF = 0.35), and rapid response (≤ 10 min). To the best of our knowledge, this is one of the best thiophenol fluorescent chemosensors developed so far.

tween the concentration and absorbance value of SQDNBS at 553 nm (A553 nm), indicating that SQ-DNBS shows good water solubility4 in the concentration range of 0.1-10 μM. Hence SQ-DNBS is a promising candidate to realize the detection of thiophenols in aqueous systems without the need of organic cosolvent. Spectral Response of SQ-DNBS toward Thiophenol. In dilute aqueous PBS solution (5 μM), the absorption maximum (λabs) of SQ-DNBS is located at 553 nm. Upon addition of thiophenol, the λabs is red-shifted to 626 nm and the absorbance is intensified gradually. As a consequence, obvious color change from pink to blue is discernable, indicating that SQ-DNBS could serve as a sensitive “naked-eye” colorimetric indicator for thiophenol (vide Figure 2a). Moreover, according to the absorption spectral titration experiment results (vide Figure 2b), for the 5 μM SQ-DNBS solution sample, its values of A626 nm shows good linear correlation with the concentration of thiophenol in the range of 0-5 μM and the slope k was calculated to be 5.05E4 M-1 (R2 = 0.9899). Through eleven times measurements on the value of A626 nm of 5 μM SQ-

Experimental section The details of experimental section, including reagents and apparatus, synthesis, determination of quantum yield, kinetics constant and half-time calculations and the determination of thiophenol in water samples, were all described in the supporting information. Results and discussion Water Solubility of SQ-DNBS. To verify whether SQDNBS possesses good water solubility, the UV-Vis absorption spectra of SQ-DNBS with concentration of 0.1-10 μM in 0.01 M phosphate buffer solutions (PBS, pH = 7.4) were recorded. As depicted in Figure S1 and S2, a good linear correlation (R2 = 0.9937) could be obtained be3


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Figure 4. (a) The absorption spectra of 5 μM SQ-DNBS in the presence of 25 μM thiophenol at different reaction time in PBS (0.01 M, pH = 7.4); (b) Kinetics of absorbance enhancement profile of SQ-DNBS (5 μM in 0.01 M PBS, pH = 7.4) at A626 nm in the presence of thiophenol (5 equiv.) or thiol species (20 equiv.); (c) Fluorescence spectra of 5 μM SQ-DNBS in the presence of 25 μM thiophenol at different reaction time in PBS (0.01 M, pH = 7.4); (d) Fluorescence enhancement profile at I645 nm of SQ-DNBS (5 μM in 0.01 M PBS, pH = 7.4) in the presence of thiophenol (5 equiv.) or thiol species (20 equiv.).

DNBS solution in the absence of thiophenol, the standard deviation (S. D.) of our UV–Vis spectrophotometer was calculated to be 6.3 × 10-4. According to the equation LOD = 3 × S. D./k, the LOD of SQ-DNBS toward thiophenol by absorption spectral titration was determined to be 37.4 nM.

be 9.9 nM. Therefore, SQ-DNBS could serve as a quite sensitive “naked-eye” FR/NIR chromofluorogenic dualchannel optical indicator for thiophenol. According to kinetic studies on the enhancement profiles of both A626 nm and I654 nm of SQ-DNBS in the presence of thiophenol (Figure 4), the reaction equilibrium could be attained within 10min with a relatively large kinetics constant (kʹ = 0.336 min-1) and short half- time (t1/2 = 2.06 min, vide Figure S5), confirming the rapid response of the probe SQ-DNBS to thiophenol.

As illustrated in Figure 3 and Figure S3, although SQDNBS is nearly non-emissive in aqueous PBS solution with different ionic activity (ΦF = 0.0005 in 0.01M PBS, pH = 7.4), the addition of thiophenol into it will trigger significantly enhanced FR/NIR photoluminescence (PL) with emission maximum (λPL) of 645 nm (under excitation at 538 nm), indicative of the fluorescence “turn-on” character of SQ-DNBS toward thiphenol. For the 5 μM and 10 μM SQ-DNBS samples, their fluorescence intensity (FI) values at 645 nm (I645 nm) show good linear correlation with the concentration of thiophenol in the range of 0-5 μM (Figure 3b) and 1-11μM (Figure S4), respectively. The slope k of the fluorescence spectral titration results of the 5 μM SQ-DNBS sample with thiophenol was calculated to be 4.16E7 M-1 (R2 = 0.9999). The standard deviation (S. D.) of our fluorescence spectrophotometer was calculated to be 0.14 through eleven times measurements on the FI value of I645 nm of 5 μM SQ-DNBS solution in the absence of thiophenol, hence the LOD of SQ-DNBS to thiophenol through fluorescence spectral titration was determined to

As the pH value is a key factor influencing interactions between thiophenol and probe molecule, the effect of pH value on the I645 nm of SQ-DNBS in the absence and presence of thiophenol was investigated. As shown in Figure S6, the PL property of SQ-DNBS is insensitive to pH environment variations within pH range of 1.0–12.0. In acidic environments (pH < 4.0), the coexisted thiophenol could not trigger significant variations on the I645 nm of SQDNBS, which should be attributed to the relatively weak nucleophilic capacity of neutral thiophenol molecule; but with increasing pH values from 4.0 to 11.0, the presence of thiophenol will induce significant enhanced fluorescence intensity at 645 nm, which should result from the dissociation of thiophenol into thiolate ion with much stronger nucleophilicity. In addition, the fluorescence intensity of 4


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Figure 5. A626 nm (a) and I645 nm (λex = 538 nm) (b) of 5 μM SQ-DNBS in the presence of 100 μM various thiol and other nucleophiles or 4 μM thiophenols in PBS (0.01 M, pH = 7.4). Insets: photographs of optical changes of 5 μM SQ-DNBS upon addition of various analytes. 1, free; 2. Cys; 3, Hcy; 4, GSH; 5, 3-mercaptopropionic acid; 6, aniline; 7, phenol; 8, KI; 9, Na2S; 10, Na2SO3; 11, 25 μM p-nitrothiophenol; 12, 25 μM p-methylthiophenol; 13, 25 μM p-methoxythiophenol; 14, 25 μM thiophenol.

with SNAr,10a-c,27 as illustrated in Figure 6. To elucidate the probing mechanism of SQ-DNBS toward thiophenol, spectral characterizations were carried on the proposed reaction product SQ (molecular structure shown in Figure 6). Photophysical characterization results revealed that both the absorption and fluorescence profiles of SQ resemble those of the mixture of SQ-DNBS and thiophenol (Figure S8). It is noteworthy that the ΦF of SQ was determined to be as high as 0.35. This could account for the high sensitivity of the probe SQ-DNBS. Moreover, after being stored for one week, the solution of SQ-DNBS in the presence of excessive thiophenol (5 equiv.) displays unchanged spectral properties, implying that the πconjugation skeleton of SQ is chemically stable toward thiophenol under these experimental conditions.

SQ-DNBS remains stable over 1 h under constant photoexcitation (Figure S7), indicative of its good photostability. Consequently, SQ-DNBS can serve as a photostable thiophenol probe in neutral and weak alkaline environments. Selectivity of SQ-DNBS toward thiophenols. As high selectivity is an essential character for thiophenol probes, various interference analyte species including different types of aliphatic thiols (i.e., 3-mercaptopropionic acid, Cys, Hcy and GSH) and other nucleophiles (i.e., aniline, phenol, KI, Na2S and Na2SO3) were examined in parallel to thiophenols under similar conditions. As depicted in Figure 5, for p-methylthiophenol and pmethoxythiophenol bearing an electron-donating group, they could induce distinct spectroscopic and visual signal changes on SQ-DNBS; but for p-nitrothiophenol bearing a strongly electron-deficient substituent, it could trigger much less significant optical changes on SQ-DNBS than thiophenol, which should be ascribed to the much weakened nucleophilicity of its mercapto functional group.4 As far as thiols and other nucleophiles are concerned, they all display quite weak interfering effects on the spectral properties of SQ-DNBS. Actually, as depicted in Figure 4, the presence of much excess of thiol species (20 equiv.) still could not induce competitive optical responses of SQ-DNBS, implying that SQ-DNBS is insusceptible to the interference from thiols and other nucleophiles. Upon further addition of 0.8 equiv. of thiophenol to the mixture of SQ-DNBS and the above-mentioned interfering species, significantly enhanced FI could be discerned. These competition experiment results indicated that SQ-DNBS shows high specificity toward thiophenols, hence could be used to detect thiophenol species in relatively complicated environments.

In line with the photophysical measurements, 1H NMR characterization results (Figure 6) indicated that upon addition of 5 equiv. of thiophenol into SQ-DNBS, the proton signals corresponding to the DNBS moiety of SQDNBS (8.98 ppm for proton 1, 8.76 ppm for proton 2, and 8.43 ppm for proton 3)27a all disappear completely; concurrently, the characteristic proton signals of SQ at 10.51 ppm (corresponding to proton a) and (2,4dinitrophenyl)(phenyl)sulfane (DN) at 8.91, 8.36, and 7.02 ppm (corresponding to protons I, II, and III in sequence30) emerge out, confirming the formation of SQ through the reaction of SQ-DNBS with thiophenol. Note that no characteristic proton signals corresponding to the addition product of thiophenol with the central cyclobutene ring of SQ or SQ-DNBS were discernable,31 indicating that the central cyclobutene rings of both SQ-DNBS and SQ are chemically stable against nucleophilic attack of thiophenols. These deductions were further validated by highresolution mass spectroscopy (HR-MS) characterization results, since upon addition of thiophenol, the m/z signal of 960.2122 corresponding to the [SQ-DNBS + Na]+ speci-

Probing Mechanism of SQ-DNBS toward Thiophenols. For thiophenol probes bearing a 2,4dinitrobenzenesulfonyl (DNBS) recognition site, they will generally undergo a thiolate-mediated cleavage process 5


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1

Figure 6. Partial H NMR spectra of SQ-DNBS, SQ, SQ-DNBS + thiophenol (5 equiv.) in DMSO-d6 and DN in CDCl3.

es disappears, but an m/z signal of 730.2481 that could be assigned to the [SQ + Na]+ species is discernable (Figures S13, S14, and S15 in the ESI†). In addition, no m/z signals that could be attributed to the addition product of thiophenol with the central four-membered ring of the probe could be observed.21a,32 Therefore, the optical response of SQ-DNBS toward thiophenols should be attributed to the thiolate-mediated cleavage mechanism, and the central four-membered rings of both SQ and SQ-DNBS are indeed chemically stable against the nucleophilic attack of thiophenols.

Real Water Samples Application. Considering the toxicity of thiophenols and their potential as pollutants, we applied SQ-DNBS to quantify thiophenol concentrations in water samples with the standard addition method4,10d,14b to validate its practical utility in environmental science. The crude water samples were obtained from Jinjiang River, East Lake of Chengdu and the tap water in Chengdu city. As depicted in Table 1, for all the pristine water samples, they could not induce significant optical response of SQ-DNBS. After spiking different concentration of thiophenol (1 μM, 2 μM or 4 μM) into the water samples, obvious fluorescence enhancement was observed, and the thiophenol concentration in these water samples could be accurately measured by the probe SQDNBS with good recovery (97%-104%). These results suggested that SQ-DNBS is quite promising in the quantitative detection of thiophenols in water samples.

Table 1. Determination of thiophenol concentration in water samples. sample

Jinjiang River water

East Lake water

Tap water

thiophenol spiked (μM)

thiophenol recovered (μM)

recovery (%)

0

Not detected

--

1

0.97 ± 0.013

97

2

2.05 ± 0.020

102

4

3.98 ± 0.049

100

0

Not detected

--

1

1.04 ± 0.10

104

2

1.93 ± 0.13

97

4

4.02 ± 0.45

100

0

Not detected

--

1

1.02 ± 0.065

102

2

1.97 ± 0.30

99

4

4.00 ± 0.31

100

Conclusions In summary, we demonstrated for the first time that squaraine dyes could serve as the fluorophore unit to construct thiophenol optical chemosensor. The resultant “naked-eye” far-red/near-infrared probe SQ-DNBS shows high selectivity, good sensitivity and rapid response time through colorimetric and “off‒on” fluorometric dual optical channels in aqueous solution without organic cosolvent, and the signaling mechanism lies in the thiolatemediated cleavage reaction on SQ-DNBS. Taking advantage of the chemical stability of the cyclobutene ring of SQ-DNBS against nucleophiles, the highly efficient farred/near-infrared fluorescence properties of the squaraine fluorophore of SQ-DNBS could be maintained successfully, and the fluorescence properties of SQ-DNBS are free 6


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of interference from thiol species in neutral environments. In fact, to our knowledge, SQ-DNBS is one of the best chemosensors for the detection of thiophenols. Our discovery could not only broaden the application area of squaraine fluorophores, but also shed light on the molecular design strategy for high-performance thiophenol probing materials.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. 1 13 The synthetic details, the H NMR, C NMR and HR-MS spectra, the concentration-dependent absorption spectra, kinetic, summary of fluorescent probes for thiophenols.

AUTHOR INFORMATION Corresponding Author *E-mail for Z.-Y.L.: [email protected]; Tel: 86-2885410059; Fax: 86-28-85410059.

Author Contributions All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors acknowledge the financial support for this work by the National Natural Science Foundation of China (Grant No. 21372168 and 51573108). We are grateful to the Analytical & Testing Center of Sichuan University for providing NMR data for the intermediates and objective molecules.

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