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Aggregation-switching strategy for promoting fluorescent sensing of biologically relevant species. A simple nearinfrared cyanine dye highly sensitive and selective for ATP Peng Zhang, Meng-Si Zhu, Hao Luo, Qian Zhang, Lin-E Guo, Zhao Li, and Yun-Bao Jiang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 8, 2017
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Aggregation-switching strategy for promoting fluorescent sensing of biologically relevant species. A simple near-infrared cyanine dye highly sensitive and selective for ATP †
†
Peng Zhang, Meng-Si Zhu, Hao Luo, Qian Zhang, Lin-E Guo, Zhao Li, and Yun-Bao Jiang* Department of Chemistry, College of Chemistry and Chemical Engineering, The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, and iChEM, Xiamen University, Xiamen 361005, China. * E-mail:
[email protected]; Fax: +86 592 218 6400 ABSTRACT: We report a strategy for enhanced performance of fluorescent sensing of biologically relevant species that often binds with natural receptors via multiple interactions. We propose to make the fluorescent sensory molecule to form H-aggregates that its emission is quenched leaving a low background, and upon binding to a biologically relevant species the aggregates are switched to another form in which the fluorescent species is better protected to afford a stronger emission signal. Meanwhile, the aggregated fluorescent dyes afford multiple interactions with the sensing species that requires multiple binding sites. The lower background, stronger binding and stronger signal would therefore lead to a much higher sensing performance, as improved selectivity would also result in along the signal amplification. We thus designed a near-IR cyanine dye bearing two boronic acid groups (Cy-BA) for fluorescent sensing of ATP, that the boronic acid groups in the dye molecule bind to the cis-diol moiety in ATP. Introduction of a cationic surfactant dodecyltrimethylammonium bromide (DTAB) below its critical aggregation concentration is a key, since Cy-BA molecules were made into H-aggregates being practically non-fluorescent. Upon mixing with ATP, a dramatic enhancement in the fluorescence occurred, because of the formation of ATP/Cy-BA/DTAB vesicles in which the fluorescent dye is well dispersed and protected. This sensing scheme, despite the dynamic nature of the boronic acid/cis-diol interaction and the weakness of the electrostatic interactions among ATP/Cy-BA/DTAB and the poor selectivity of these interactions, allows a highly sensitive and selective detection of ATP in aqueous solution.
INTRODUCTION A sensing system features a strong binding while low background and strong signal output would afford a high sensitivity.1-3 We proposed to establish such a highly sensitive fluorescent sensing scheme by making the fluorescent sensory dyes into H-aggregates in which the highly efficient internal conversion substantially quenches the fluorescence of the sensory dye.4-6 This would also allow a stronger binding to the target species in particular when the target species bears multiple groups to be bound, that an improved selectivity may result in because of the tunable orientations of the binding sites in the aggregates.7 If upon binding to the target species the aggregates could switch to other forms, in which the fluorescent component is well dispersed and/or protected, the fluorescence signal would be enhanced to a much higher extent, leading to stronger outputs.8,9 This scheme could well apply to biologically relevant targets, since in nature the strong yet highly selective binding of them is realized by flexible multiple binding receptors, such as saccharides by lectins. Reported here is our proof-of-the-concept attempt for a highly sensitive and selective fluorescent sensing for ATP, by designing a structurally simple cyanine that bears two boronic acid groups (Cy-BA, Figure 1a). Cyanine dye framework is chosen because it is able to form either H- or J-aggregates.5,10 Incorporating two boronic acid groups into the dye structure enables its binding to two ATP molecules at the cis-diol moie-
ty in the latter,11-13 which results in the formation of two negatively charged boronates at high pH.14,15 Cy-BA molecules were made into H-aggregates in the presence of a cationic surfactant dodecyltrimethylammonium bromide (DTAB), which upon binding to ATP were switched to supramolecular vesicles in which the bound fluorescent dye molecules were well dispersed and protected by the vesicles (Figure 1).16 Formation of the vesicles benefits from multiple interactions of ATP, with Cy-BA and DTAB via boronic acid/cis-diol motif and electrostatic interactions of the formed boronate and the phosphate anions in ATP. We succeeded in establishing a near-IR fluorescent sensing scheme for ATP in aqueous solutions with both high sensitivity and selectivity, despite the structurally simple structure of Cy-BA and poorly selective and relatively weak interactions of it with ATP and DTAB.
EXPERIMENTAL SECTION All reagents and solvents used in synthesis were commercially available at analytical grade or higher and were used as received. Solvents for spectral titrations were deionized water and redistilled MeOH. Details of the synthesis of Cy-BA and Cy-Ph are given in the supporting information (SI), together with full characterization by1H NMR, 13C NMR and HRMS. Copies of the 1H NMR and 13C NMR spectra are also supplied in the SI. Thinlayer chromatography (TLC) was performed on silica gel plates. Column chromatography was performed using silica
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Cy-BA in MeOH shows a maximum absorption at 785 nm and a near-IR fluorescence emission at 816 nm. Aggregation of Cy-BA was studied in MeOH-H2O binary solutions (aqueous phase, 50 mM NH3-NH4Cl buffer of pH 10.0). Figure 3a shows that the monomer absorption of Cy-BA in MeOH at 785 nm undergoes a prompt decrease when the buffer volume percentage is over 70% (Figure 3b), together with a blue shift in the absorption, suggesting the H-aggregation of Cy-BA in the water-rich solutions.17,18 Fluorescence titrations support the H-aggregation of the cyanine dye, by a dramatic quenching of the fluorescence (Figure 3c, d), because of the highly efficient internal conversion in the H-aggregates.19 In NH3-NH4Cl buffer, the absorption spectrum of the H-aggregated Cy-BA is dramatically broadened over the range of 560-900 nm while the emission is almost completely quenched (Figure 3c, d). 20
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Figure 1. Chemical structures of (a) Cy-BA and Cy-Ph and (b) ATP, ADP, and AMP. (c) Schematic diagram of enhanced fluorescent sensing of ATP by near-IR fluorescent cyanine dye Cy-BA.
RESULTS AND DISCUSSION Near-IR cyanine dyes Cy-BA and its control compound bearing no boronic acid group, Cy-Ph (Figure 1a) were synthesized by the reaction of quaternary salts and bisaldehyde in 7:3 (v/v) mixture of 1-butanol and benzene. They were purified by chromatography on a silica gel column (CH2Cl2-MeOH) and characterized by 1H NMR, 13C NMR, HRMS (Supporting Information) and crystal structure (Figure 2). Crystals of Cy-BA and Cy-Ph suitable for X-ray crystallography were grown by slow evaporation of their solutions in CH2Cl2. Crystal structure of Cy-BA(Figure 2a) shows that the two boronic acid groups position in the two sides of the cyanine plane, whereas the two phenyl groups in Cy-Ph sit in the same side of the conjugation plane (Figure 2b).
Figure 2. Crystal structures of Cy-BA (a) and Cy-Ph (b). All hydrogen atoms and bromide atoms were omitted for clarity.
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Figure 3. Absorption (a) and fluorescence (c) spectra of CyBA in MeOH-H2O binary solvents of varying composition and plots of A850nm/A785nm (b) and fluorescence intensity at 820 nm (d) versus water volume percentage. Aqueous solution was 50 mM NH3-NH4Cl buffer of pH 10.0. [Cy-BA] = 2 µM, λex= 700 nm. In order to enable aggregate switching upon analyte binding to the Cy-BA dye, we proposed to introduce a cationic surfactant into the solution that would interact with the anionic Cy-BA dye electrostatically. This was expected to enhance the H-aggregation of the dye molecules, since the long hydrophobic tails of attached surfactant molecules would further facilitate the aggregation.17 While upon interacting with the biologically relevant species, which in many cases contains multiple binding sites, for example ATP here, would afford negatively charged boronate at high pH, together with the three anionic phosphates in ATP framework. This ATP-boronate interacts much more strongly with the cationic surfactant to form conjugates that bear multiple hydrophobic tails. These conjugates, differing very much from the dye-surfactant conjugates, would aggregate in a different manner, for example to form vesicles. The length of the hydrophobic tail in the cationic surfactant is therefore a key factor to influence those interactions and the aggregations. We therefore examined the effect of cationic surfactant of varying length of alkyl tail, cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), dodecylltrimethylammonium bromide (DTAB), and decyltrimethylammonium bromide (DeTAB), on the aggregation of Cy-BA in the absence and presence of ATP, re-
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spectively. Figure 4 shows the absorption and fluorescence spectra of Cy-BA as a function of DTAB concentration in 50 mM NH3-NH4Cl buffer of pH 10.0 (see Figure S5 in SI for pH optimization). In the absence of ATP, the absorption of CyBA did not change with increasing DTAB concentration up to 10 mM, after which the broad absorption of the aggregates at 680 nm and 840 nm weakened, while a significant increase in the absorption at 795 nm occurred (Figure 4a). Meanwhile, the already very weak fluorescence at 790 nm experienced a further quenching at low DTAB concentration, and turned to grow at 820 nm at DTAB concentration beyond 10 mM (Figure 4b). Both phenomena suggested the enhanced Haggregation of Cy-BA by DTAB at low concentration, i.e.< 10 mM. Beyond this concentration DTAB micelles formed so that the Cy-BA molecules were dispersed in the micelles, displaying characteristic absorption and emission of Cy-BA monomer as shown in Figure 3 for the respective spectrum in MeOH.20 In the presence of 250 µM ATP, the absorption and fluorescence of Cy-BA began to grow at the concentration of DTAB of 6 mM (Figures S6 and S7), suggesting that the critical aggregation concentration (CAC) of DTAB drops from 10 mM to 6 mM (Figures 5c and S8).21
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Figure 4. Absorption (a) and fluorescence (b) spectra of CyBA in 50 mM NH3-NH4Cl buffer of pH 10.0 in the presence of DTAB of increasing concentration from 0 to 16 mM. Inset in (b) shows the further quenching of fluorescence of Cy-BA upon introduction of DTAB of low concentration. [Cy-BA] = 10 µM, λex= 700 nm. With CTAB, TTAB, and DeTAB, the absorption and fluorescence spectral titration profiles are in general similar to those of DTAB. Plots of fluorescence intensity (Figure 5) and absorbance (Figure S9) against concentration of the surfactant show that DTAB at 10 mM gives the maximum changes in the fluorescence or absorbance. The spectral changes led by the surfactant were attributed to the interaction of the cis-diol moiety in ribose of ATP with boronic acid group in Cy-BA and the electrostatic interactions of surfactant, e.g. DTAB, with the formed negatively charged boronates at pH 10 and the phosphate anions in ATP that switched the H-aggregates of Cy-BA to the supramolecular vesicles. In order to support this interaction pattern, control compound Cy-Ph bearing no boronic acid group (Figure 1a)
H NMR titrations offer further support to this interaction pattern.22 DTAB (10 mM) in D2O exhibits six resonances at 3.23, 3.02, 1.70, 1.28, 1.21, and 0.79 ppm, respectively. Addition of ATP (250 µM) or Cy-BA (10 µM) resulted in practically no change, whereas significant changes occurred when the same amount of ATP and Cy-BA were added together (Figure 6a). In the latter case, the resonance at 3.23 ppm shifted upfield to 3.16 ppm, 3.02 ppm to 2.99 ppm, 1.70 ppm to 1.62 ppm, and 1.28 ppm to 1.21 ppm, while downfield resonances were observed from 6.8 ppm to 8.0 ppm which refers to the signals of aromatic protons of Cy-BA (Cy-BA itself is insoluble in D2O, Figure S11). This indicates the occurrence of the multiple interactions among Cy-BA/ATP/DTAB. This conclusion was supported by the splitting of the signal of H2O in D2O, which only took place when DTAB and ATP were both present in the D2O solution of Cy-BA (Figure S12). The formation of supramolecular vesicles was characterized by dynamic light scattering (DLS, Figure 6b) and SEM (Figure 6c). Cy-BA (10 µM) in buffer solution formed aggregates of mean diameters of 880±176 nm. Little change took place when 250 µM ATP (780±115 nm) or 10 mM DTAB (615±105 nm) was added, while a significant increase in the size of existing species occurred when DTAB and ATP were both added together, with Dh amounting to 3,130±396 nm. Significantly larger DTAB-Cy-BA-ATP aggregates (ca.3,000 nm), compared to DTAB micelles (190±25nm) and aggregates of DTAB with ATP (256±18 nm) (Figures S13,S14), supported the evolution of the ATP/Cy-BA conjugates, upon interacting with DTAB, into supramolecular vesicles. The supramolecular vesicles were confirmed by the scanning electron microscope (SEM) images which showed their size of 2-3 µm (Figures6cand S15). The color of Cy-BA solution changes from bright green to yellow green only when DTAB and ATP were both added together (Figure 6d). This color response toward ATP, visible to the naked eyes, estab-
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On the basis of the above findings, we concluded that the Cy-BA dyes are dispersed and protected by the vesicles in which it interacts with both ATP and DTAB, with thereby substantially enhanced emission at 820 nm, whereas in the presence of low concentration DTAB only Cy-BA existed in H-aggregates with dramatically quenched emission at 790 nm (Figures 1c and 4). The spectral response toward ATP was then examined. Addition of ATP to the solution of Cy-BA containing 10 mM DTAB in pH 10.0 NH3-NH4Cl buffer resulted in a decrease in the broad absorption over 560-900 nm while an increase in the absorption at 795 nm (Figure 7a). This again probes the evolution of the aggregation of Cy-BA dye from H-aggregates with DTAB to vesicles with both DTAB
As the partially dephosphorylated products of ATP, adenosine 5’-diphosphate (ADP) and adenosine 5’-monophosphate (AMP, Figure 1b), structurally highly similar to ATP and are always present to some extent in all living cells together with ATP. Spectral responses of Cy-BA-DTAB toward ADP and AMP were also examined. Similar but to much less extent changes were observed upon addition of ADP or AMP, increasing of the absorbance at 795 nm and emission at 820 nm (Figure 8aand Figures S18-S21). High selectivity for ATP over ADP and AMP was shown (Figures8a and S22).
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Figure 6. (a) 1H NMR spectra of (i) DTAB (10 mM), (ii) DTAB with 250 µM ATP, (iii) DTAB with 10 µM Cy-BA, and (iv) DTAB with 250 µM ATP and 10 µM Cy-BA in NH3NH4Cl buffer in D2O of pD 10.0. (b) DLS analyses of Cy-BA (10 µM) itself and in the presence of ATP, DTAB and ATP+DTAB in NH3-NH4Cl buffer. (c) SEM image of Cy-BA (10 µM) with 10 mM DTAB and 250 µM ATP. Scale bar is 5µm. (d) Colors of solutions of Cy-BA (10 µM), Cy-BA with 250 µM ATP, Cy-BA with 10 mM DTAB and Cy-BA with 250 µM ATP and 10 mM DTAB in NH3-NH4Cl buffer.
and ATP, in which dye molecules are well dispersed in monomer form that are protected by the hydrophobic microenvironment. The aggregated organic dye Cy-BA is practically non-fluorescent when excited at 700 nm, but became highly fluorescent at 820 nm upon addition of ATP (Figure 7b). The fluorescence intensity increases linearly with ATP concentration over 0 to 20 µM, establishing the quantitative detection of ATP in aqueous solutions (Figure 8a).Job plots showed that Cy-BA to ATP stoichiometry is 1:2 (Figure S16), indicating that each boronic acid group in Cy-BA binds with an ATP molecule. The limit of detection (LOD) for ATP was determined to be 90 nM at 3σ/k, in which σ is the standard deviation of 20 blank measurements and k is the slope obtained from the linear dependence of fluorescence intensity of Cy-BA versus ATP concentration (Figure S17).23,24 This LOD for ATP in aqueous solutions is much lower than those of the previously reported ATP sensing using conventional organic dyes.25-28
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Figure 7. Absorption (a) and fluorescence (b) spectra of CyBA in NH3-NH4Cl buffer containing 10 mM DTAB in the presence of ATP from 0 to 300 µM. Arrows indicate the direction of increase in the concentration of added ATP. [Cy-BA] = 10 µM, λex= 700 nm.
Figure 8. Plots of fluorescence intensity at 820 nm of Cy-BA versus concentration of ATP, ADP, and AMP in the presence (a) or absence (b) of 10 mM DTAB in 50 mM NH3-NH4Cl buffer of pH 10.0. Inset in (a) is the expanded portion at low concentration of adenosine 5’-phosphates. [Cy-BA] = 10 µM, λex= 700 nm. In order to confirm the role of DTAB in the sensing, we investigated the response without DTAB. Addition of ATP to the buffered solution of Cy-BA led to a decrease in the absorption of the H-aggregates and further quenching of the emission at 790 nm (Figures S23, S24). Addition of ADP or AMP resulted in similar weak response (Figures S25-S28), as shown in Figures 8b and S29. It was therefore concluded that the presence of cationic surfactant DTAB is critical for the substantially enhanced response sensitivity and selectivity.
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The high selectivity of Cy-BA/DTAB toward ATP was also shown in extended assays in the presence of other nucleotides such as thymine monophosphate (TMP), cytidine monophosphate (CMP), guanine monophosphate (GMP) and uridine monophosphate (UMP), and small phosphate anions such as ppi, HPO42- and H2PO4-, and cis-diol containing saccharides such as sialic acid, ribose, glucose, fructose, mannose, xylose and galactose. It was found that only ATP influences the emission and absorption of Cy-BA/DTAB (Figure 9a and Figure S30). Excess amount (10 equivalents) of those foreign compounds did not change the fluorescence at 820 nm (Figure 9a) and the absorbance at 795 nm (Figure S30), ruling out the interference of the cis-diol moiety and/or the phosphate anions in those foreign compounds that may have interacted with CyBA and/or DTAB in their aggregates. Competitive assays showed that the coexistence of those compounds did not change the absorbance and emission of ATP/Cy-BA/DTAB system either (Figures 9b and S31). 250
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(Pi).29 During the apyrase-catalyzed ATP hydrolysis, the emission of Cy-BA at 820 nm decreases gradually during 0 to 30 minutes (Figure 10a), while that of the control solution containing no apyrase remains unchanged. Plots of the emission at 820 nm show that the hydrolysis finished within 30 minutes at the concentration of apyrase above 20 mU (Figure 10b). Thanks to the higher sensitivity and selectivity, the present fluorescence assay is facile for measuring the activity of apyrase, requiring a lower enzyme concentration and operating in a shorter time.30 180
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Figure 9. (a) Fluorescence spectra of Cy-BA and DTAB in the presence of 10 equivalents of ATP, ADP, AMP, TMP, CMP, GMP, UMP, ppi, HPO42-, H2PO4-, sialic acid, ribose, glucose, fructose, mannose, xylose or galactose and (b) intensity of CyBA/DTAB at 820 nm in pH 10.0 ammonia bufferin the presence of 100 µM ATP upon addition of 10 equivalents of competition species 1-17: none, ADP, AMP, TMP, CMP, GMP, UMP, ppi, HPO42-, H2PO4-, sialic acid, ribose, glucose, fructose, mannose, xylose, andgalactose. [Cy-BA] =10 µM, [DTAB] = 10 mM, λex= 700 nm. With the ability of Cy-BA-DTAB to recognize ATP from PPi containing anions, even ADP and AMP, the mixture of Cy-BA and DTAB was applied to the fluorescence assay of ATP-relevant enzyme activity. Apyrase is a hydrolytic enzyme that converts ATP or ADP into AMP and inorganic phosphate
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CONCLUSIONS In conclusion, we approve that our proposed aggregationswitching scheme for promoting fluorescent sensing operates successfully. We showed that with a structurally simple cyanine dye bearing two boronic acid groups that intrinsically holds low selectivity and affinity in its interaction with ATP turns to exhibit a high sensitivity and selectivity in its NIR fluorescent sensing toward ATP at an LOD of 90 nM in aqueous solutions. Using a cationic surfactant of appropriate alkyl tail at the critical aggregation concentration is a key to this success, since the surfactant promotes the H-aggregation of the anionic dyes that quenches the fluorescence of the dye, leaving low background. Upon interacting with the sensing target ATP, it promotes the formation of supramolecular vesicles in which the fluorescent species is well dispersed and protected thereby emitting much stronger NIR fluorescence and giving higher output signal. The biologically relevant target, such as ATP here, that bears multiple interacting groups affords the possibility of accommodating multiple interactions with the cyanine dye via its boronic acid groups and with DTAB electrostatically, forming a multi-component nDTAB@(ATP/Cy-BA/ATP) conjugate containing more than one DTAB molecules that tends to form vesicles. The Haggregates of the cyanine dyes as the interacting species would afford enhanced interactions with ATP, while upon these interactions the H-aggregates were switched into vesicles that again involved signal amplification, resulting in a
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highly sensitive fluorescent sensing for ATP, together with a high selectivity because of the signal amplification.31 The present scheme represents a sophisticated mimic of the biological receptor which consists of an array of binding sites that are in general not well-defined in a fixed configuration. It is believed to be of general use for fluorescent sensing of biologically relevant species.
ASSOCIATED CONTENT Supporting Information The Supporting Information (SI) is available free of charge on the ACS Publications website at DOI:xxx. Syntheses and full characterization of Cy-BA and Cy-Ph, 1H NMR and 13C NMR spectra of them (Figures S1-S4), and fluorescence, absorption and NMR spectral titration profiles, DLS data, and SEM images (Figures S5-S31).
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected].
ORCID Yun-Bao Jiang, 0000-0001-6912-8721.
Author Contributions †These authors contributed equally to this work.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT We thank the support of NSFC (21435003, 91427304, 21521004, and J1310024) and Program for Changjiang Scholars and Innovative Research Team in University, MOE of China (IRT13036).
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