A reversible fluorescent probe for directly monitoring protein-small molecules interaction utilizing vibration-induced emission

Dyes and Pigments 163 (2019) 425–432

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A reversible fluorescent probe for directly monitoring protein-small molecules interaction utilizing vibration-induced emission

T

Na Wanga, Chenqi Xina, Zheng Lia, Gaobin Zhanga, Lei Baia, Qiuyu Gonga, Chenchen Xua, Xu Hanb, Changmin Yua,∗∗, Lin Lia,∗, Wei Huanga,b a

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing, 211800, PR China b Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), 127 West Youyi Road, Xi'an, 710072, PR China

ARTICLE INFO

ABSTRACT

Keywords: Vibration-induced emission (VIE) Biotin-avidin system Bio-logic gate Reversible

The interactions between proteins and small molecules play an important role in the regulation of various cellular processes and modern drug discovery. Herein, we have developed a novel strategy for real-time monitoring the protein-ligand interaction based on vibration-induced emission (VIE) mechanism. These results demonstrated that the probe DPAC-DB could directly visualize the binding process in biotin-avidin system, which was undisturbed to other environmental stimuli, such as pH, interference species and temperature. Furthermore, the unique VIE effect caused by dramatic and reversible intramolecular vibrations enabled our strategy possible to build up the bio-logic gate. This method to efficiently control the emission of VIE-based probe through the reversible noncovalent protein-small molecules interactions opens new avenues for the development of potent protein pharmaceuticals and small molecule drugs.

1. Introduction High affinity protein interactions are ubiquitous that has played a key role in many spatiotemporal cellular structures, such as regulatory multiprotein complexes, immunological recognition processes, and the quaternary architecture of ligand-gated ion channels [1,2]. Amongst them, noncovalent reversible binding between small molecules and proteins, in particular, is of central importance in the regulation of various cellular processes, including transcription regulations, enzymatic activity, protein synthesis and degradation [3,4]. Furthermore, such interactions are also important in modern drug discovery as small molecule drugs are designed as potent protein pharmaceuticals to alter protein functions upon binding [5–7]. Thus, it is of substantial interest to accurately monitor the binding affinity between biomolecules and target proteins. Biophysical techniques such as mass spectrometry [8], microbiological method [9], atomic force microscopy [10], electrochemical [11], nuclear magnetic resonance (NMR) [12], isothermal titration calorimetry (ITC) [13] and surface plasmon resonance (SPR) [14] have been developed to determine protein-ligand binding affinities. The major shortcoming, however, is that these methods typically require multistep sample processing, large amounts of sample and/or very ∗

specialized equipment. Another problem is that they are unable to realize real-time visualization during the protein-ligand interaction process. Fluorescence technology has been proven to be a promising tool because of its unique advantages, such as excellent sensitivity, simplicity and real-time visual detection of analytes both in vitro and in vivo [15–21]. So far, fluorescence assays based on conventional spectroscopic manners such as fluorescence anisotropy (FA) and fluorescence resonance energy transfer (FRET) have also been designed to screen interactions between proteins and ligands [22–25]. Recently, a class of specific fluorophores with unique VIE effect have been reported by Tian's group [26–30], which intrinsically exhibit orange-red fluorescence in the free state, but abnormally display blue fluorescence in the constrained state. This fantastic phenomenon that undergoes an extremely distinct fluorescence color change has been demonstrated to be attributed to the limitation of intramolecular vibrations in disubstituted hydrophenazine derivatives [28]. Obviously, this novel VIE mechanism differs from conventional photophysical or photochemical processes in several aspects [31,32], such as distinctive fluorescence change caused by the intrinsic change in intramolecular planarity, large Stokes' shift up to over 250 nm, and instantaneously fluorescence response within femtosecond scale that could capture the dynamic process, etc. Furthermore, such a dramatic fluorescent

Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Yu), [email protected] (L. Li).

∗∗

https://doi.org/10.1016/j.dyepig.2018.12.027 Received 8 November 2018; Received in revised form 15 December 2018; Accepted 15 December 2018 Available online 18 December 2018 0143-7208/ © 2018 Elsevier Ltd. All rights reserved.

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Scheme 1. Schematic illustration of the ratiometric visualization of the binding process between avidin and VIE-based probe DPAC-DB.

response to environmental change is ratiometric and visible by naked eyes, which combines with the above features enable VIE with the capacity of real-time monitoring the changed stimuli, such as environmental response sensors (including viscosity and soluble cryogenic thermometer) [29], ion detection [30], hole transport materials [33], low-molecular-weight gelator [34] and mechanosensitive membrane probes [35]. Herein, as a proof of concept, we developed a novel probe to detect the binding interaction between protein and small molecule based on the VIE-active fluorogen N, N′-diphenyl-dihydrodibenzo[a,c]phenazine (DPAC). In this study, we chose the biotin-avidin system (BAS) as the model to evaluate the protein-ligand interaction. The capture of biotin with avidin is a powerful tool in biology, as well as an ideal model system for the study on interactions of high-affinity protein-ligand, based on the strong specific binding (Ka ≈ 1015 M−1) between avidin and biotin [36]. As shown in Scheme 1, the biotin moieties were coupled to the DPAC core through a nonconjugated spacer [37]. In this rationally designed layout, configuration change and the sequential VIE of DPAC could be modulated when the biotin moieties bind with the coordination site of avidin. In the absence of avidin, the free intramolecular vibrations bring the hydrophenazine unit to a stable planar state, resulting in the orange-red emission. However, upon binding of the biotin molecules with avidin, the restriction of the intramolecular vibrations in hydrophenazine unit will be achieved caused by the steric hindrance between the N, N′-disubstituents, just like the blocking of the butterflies wings. Thus, the fluorescence color changes from orange-red to blue emission due to the reduced electronic conjugation. Based on the VIE principle, the fluorescence response of this probe is from the intrinsic change in planarity of the molecular excited state caused by the binding interaction between biotin and avidin, which is essentially irrelevant to the physical formation and intermolecular relationships of the probes, thus enabling our strategy quite suitable to detect the interaction process between small molecule and its targeted protein. It is noted that such a dramatic and reversible fluorescent ratiometric response to the interaction between protein and small molecule could make our probe to be VIE-based bio-logic gate with different “input”.

purchased from Sigma. Petroleum ether (PE, 60–90 °C), DCM, ethyl acetate (EA) and methanol (MeOH) were used as eluents for flash column chromatography with Merck silica gel (0.040–0.063). Reaction progress was monitored by TLC on pre-coated silica plates (250 μM thickness) and spots were visualized by UV light or iodine. Distilled water was used throughout the experiments. Absorption spectra were recorded using Synergy HTX microplate reader or a Shimadzu UV-3600 UV–Vis–NIR spectrophotometer. Photoluminescent spectra were recorded using BioTek Cytatio5 Cell Imaging Multi-Mode Reader. All the measurements were performed at room temperature. 1H and 13C NMR spectra were collected in CDCl3 or DMSO‑d6 using an Avance AV-500 spectrometer and Avance AV-300 spectrometer. 1H NMR coupling constants (J) were reported in Hertz (Hz) and multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet). Mass spectra were recorded on Finnigan LCQ mass spectrometer and Shimadzu LC-IT-TOF spectrometer. 2.2. Synthesis and characterization 2.2.1. Synthesis of the N9,N10-diphenylphenanthrene-9,10-diamine (1) TiCl4 (4 mL) was dropwise added through an injector to a cooled solution (0 °C) of phenanthroquinone (5.0 g, 24.0 mmol) and aniline (8 mL, 84.3 mmol) in toluene (150 mL). The solution was stirred at room temperature for 12 h. The product was isolated with solvent under reduced pressure and dissolved in THF/EtOH (1/1, v/v) (100 mL). NaBH4 (1.0 g, 26.4 mmol) was added in portion at room temperature and refluxed for 2 h. Then water was added, and the product was extracted with DCM. After drying with MgSO4 and removal of DCM, the resulting precipitate was refluxed in ethanol, filtrated, washed with ethanol and dried under vacuum to give the compound 1, a pale yellow solid (Yield: 4.1 g, 47%). 1H NMR (300 MHz, DMSO‑d6) δ: 8.87 (d, J = 3.0 Hz, 2H), 7.94 (d, J = 6.0 Hz, 2H), 7.66 (t, J = 9.0 Hz, 2H), 7.61 (s, 2H), 7.56 (t, J = 9.0 Hz, 2H), 7.01 (t, J = 9 Hz, 4H), 6.62 (t, J = 9.0 Hz, 2H), 6.52 (d, J = 3 Hz, 4H). 2.2.2. Synthesis of the 9,14-diphenyl-9,14-dihydrodibenzo[a,c]phenazine (DPAC) To a solution of compound 1 (500 mg, 1.4 mmol) and iodo-aryl compound (428 mg, 2.1 mmol) in trichlorobenzene (10 mL), K2CO3 (387 mg, 2.8 mmol) and Cu(OTf)2 (127 mg, 2.8 mmol) were added. The mixture was stirred at 210 °C for 6 h. Most trichlorobenzene was removed by vacuum distillation, and black residue was obtained. After cooling to room temperature, the residue was washed with water and extracted with dichloromethane. The residue was purified by column chromatography (eluent: 5% dichloromethane in petroleum ether) to

2. Experimental section 2.1. Materials and instrumentation All reagents and solvents were purchased from commercial suppliers and used without further purification, unless otherwise noted. Biotin (67896B) was purchased from Adamas and avidin (A9275) was 426

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give pure product N, N′-diaryl-dihydrodibenzo[a, c]phenazines (DPAC) (Yield:160 mg, 27%). 1H NMR (300 MHz, CDCl3) δ: 8.76 (d, J = 9 Hz, 2H), 8.16 (d, J = 6 Hz, 2H), 7.76 (s, 2H), 7.65 (d, J = 6 Hz, 2H), 7.56 (d, J = 6 Hz, 2H), 7.35 (s, 2H), 7.01 (s, 8H), 6.80 (s, 2H).

2.2.6. Synthesis of the (dibenzo[a,c]phenazine-9,14-diylbis(4,1phenylene))bis(methylene)bis((4(2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)butyl)carbamate) (DPAC-DB) DPAC-OH (250 mg, 0.5 mmol), biotin (489 mg, 2.0 mmol), DPPA (413 mg, 2.0 mmol), and Et3N (141 mg, 2.0 mmol) were dissolved with anhydrous CH3CN (10 mL), and the solution was continuously stirred overnight in nitrogen at 55 °C. The reaction mixture was cooled before vacuum dry. The resulting residue was reconstituted in DCM (50 mL) and sequentially washed with 5% aq citric acid (2 × 50 mL), water (50 mL), saturated aq NaHCO3 (50 mL) and brine (50 mL). The organic phase was isolated and then dried with Na2SO4. Purification by flash chromatography (eluent: 10% CH3OH in DCM) gave DPAC-DB as a white solid (Yield: 200 mg, 41%). 1H NMR (500 MHz, DMSO‑d6) δ: 8.95 (d, J = 5 Hz, 2H), 7.92-7.90 (m, 4H), 7.72 (t, J = 15 Hz, 2H), 7.62 (t, J = 15 Hz, 2H), 7.44-7.42 (m, 2H), 7.14 (t, J = 10 Hz, 2H), 7.09 (d, J = 10 Hz, 4H), 7.00 (d, J = 10 Hz, 4H), 6.43 (s, 2H), 6.38 (s, 2H), 4.80 (s, 4H), 4.26-4.25 (m, 2H), 4.09-4.07 (m, 2H), 3.06-3.02 (m, 2H), 2.932.92 (m, 4H), 2.78-2.76 (m, 2H), 2.56 (d, J = 10 Hz, 2H), 1.59-1.54 (m, 2H), 1.45-1.41 (m, 6H), 1.36-1.33 (m, 4H); 13C NMR (125 MHz, DMSO‑d6) δ: 162.73, 156.10, 143.67, 136.86, 129.55, 129.29, 128.19, 127.03, 125.81, 123.76, 116.26, 64.89, 60.91, 59.20, 55.48, 29.20, 24.50, 22.10. HRMS (m/z): calcd [M+Na]+ for C54H54N8O6S2Na: 999.3764; found, 999.3721.

2.2.3. Synthesis of the 4,4'-(dibenzo[a,c]phenazine-9,14-diyl) dibenzaldehyde (DPAC-CHO) Phosphorus oxychloride (3.5 g, 22.8 mmol) was injected into N, N′dimethylformamide (5.0 mL) under a nitrogen atmosphere at 0 °C, and the mixture was vigorously stirred for 1 h to obtain the Vilsmeier reagent. The solution of DPAC (1.0 g, 2.3 mmol) in N, N′-dimethylformamide (10 mL) was then added by dropwise. The mixture was heated to 85 °C and stirred for 12 h. After cooling to room temperature, the reaction mixture was poured into ice water, followed by the adjustment of pH to neutral. The deep yellow precipitate was then filtered and purified by column chromatography eluting with 20% petroleum ether in dichloromethane. A light yellow product 4, 4'-(dibenzo[a,c]phenazine-9, 14-diyl)dibenzaldehyde (DPAC-CHO) was obtained (Yield: 0.9 g, 80%). 1H NMR (300 MHz, CDCl3) δ: 9.69 (s, 2H), 8.81 (d, J = 6 Hz, 2H), 8.07 (d, J = 9.0 Hz, 2H), 7.85 (dd, J1 = 3 Hz, J2 = 6 Hz, 2H), 7.76 (t, J = 15 Hz, 2H), 7.63 (t, J = 15 Hz, 2H), 7.54 (d, J = 9 Hz, 4H), 7.49 (dd, J1 = 3 Hz, J2 = 6 Hz, 2H), 7.01 (d, J = 9 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6) δ: 190.72, 150.78, 142.89, 137.06, 131.04, 129.70, 129.18, 127.91, 127.72, 127.69, 127.56, 126.77, 123.95, 123.72, 114.72. HRMS (m/z): [M+H]+ calcd for C34H23N2O2, 491.1754; found, 491.1754.

2.3. Preparation of probes and analytes Concentrated liquor of probe (1.0 mM) was prepared in DMSO. Test solution of probe (2 μM) in DMSO/H2O was obtained by diluting concentrated liquor. All solutions was stored in 1.5 mL centrifuge tube, and then shaken well and incubated at room temperature. Avidin stock solution was prepared by diluting commercial avidin solid with purified water with the concentration of 10 μg μL−1.

2.2.4. Synthesis of the (dibenzo[a,c]phenazine-9,14-diylbis(4,1phenylene))dimethanol (DPAC-OH) DPAC-CHO (1.0 g, 2.0 mmol) was dissolved in 30 mL methanol, and sodium borohydride (0.8 g, 21 mmol) was then added in an ice bath. The mixture was stirred overnight at room temperature. The reaction mixture was poured into ice water, followed by the filtration of beige precipitate to give the DPAC-OH (Yield: 0.9 g, 89%). 1H NMR (300 MHz, DMSO‑d6) δ: 8.92 (d, J = 6.0 Hz, 2H), 7.96 (d, J = 6.0 Hz, 2H), 7.88 (s, 2H), 7.69 (s, 2H), 7.59 (s, 2H), 7.40 (s, 2H), 7.03 (m, 8H), 4.95 (s, 2H), 4.30 (d, J = 3.0 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6) δ: 145.95, 143.88, 136.76, 135.57, 129.41, 128.82, 128.24, 128.12, 127.37, 127.12, 126.71, 125.46, 123.74, 123.55, 116.91, 62.36. HRMS (m/z): [M+H]+ calcd for C34H27N2O2, 495.2067; found, 495.2063.

2.4. The spectroscopic testing procedures The equilibrium and kinetics of the reaction between avidin and biotin were investigated at different feed avidin concentration with DPAC-SB/DPAC-DB (Final concentration: 2 μM) and the experiments were carried out at 298 K and incubation time of 1 min. For the effect of avidin concentration on avidin-biotin interaction, 1.5 mL centrifuge tube containing a uniform DPAC-SB/DPAC-DB concentration (2 μM) and a variable avidin concentration were incubated at 298 K for 1 min, which was then measured at 355 nm excitation and 450 nm emission wavelength (10 nm slit for standard experiments). The effect of pH on avidin-biotin interaction was investigated at different pH values (from 4.0 to 8.0). The fluorescence emission spectrum of the probe DPAC-DB (2 μM) and the DPAC-DB (2 μM) with avidin (1.03 μg μL−1) were measured after incubating for 1 min in various pH solutions In all the measurements, the effect of dilution was corrected being repeated each measurements by triplicate and the mean and standard deviation were calculated.

2.2.5. Synthesis of the 4-(14-(4-(hydroxymethyl)phenyl)dibenzo[a,c] phenazin-9(14H)- yl)benzyl(4-(2-oxohexahydro-1H-thieno[3,4-d]imidazol4-yl)butyl)carbamate (DPAC-SB) DPAC-OH (250 mg, 0.5 mmol), biotin (244 mg, 1.0 mmol), DPPA (206 mg, 1.0 mmol), and Et3N (71 mg, 1.0 mmol) were dissolved with anhydrous CH3CN (10 mL), and the solution was continuously stirred overnight in nitrogen at 55 °C. The reaction mixture was cooled before vacuum dry. The resulting residue was reconstituted in DCM (50 mL) and sequentially washed with 5% aq citric acid (2 × 50 mL), water (50 mL), saturated aq NaHCO3 (50 mL) and brine (50 mL). The organic phase was isolated and then dried with Na2SO4. Purification by flash chromatography (eluent: 5% CH3OH in DCM) gave DPAC-SB as a white solid (Yield: 140 mg, 38%). 1H NMR (500 MHz, DMSO‑d6) δ: 8.94 (d, J = 10 Hz, 2H), 7.94-7.90 (m, 4H), 7.72-7.68 (m, 2H), 7.64-7.57 (m, 2H), 7.42-7.40 (m, 2H), 7.13 (t, J = 15 Hz, 1H), 7.07-6.98 (m, 8H), 6.42 (s, 1H), 6.36 (s, 1H), 4.81 (s, 2H), 4.31 (s, 2H), 4.28-4.25 (m, 1H), 4.10-4.06 (m, 1H), 3.05-3.03 (m, 1H), 2.95-2.90 (m, 2H), 2.79-2.75 (m, 1H), 2.59-2.53 (m, 1H), 1.60-1.54 (m, 1H), 1.45-1.41 (m, 3H), 1.391.34 (m, 2H); 13C NMR (125 MHz, DMSO‑d6) δ: 162.74, 156.11, 146.74, 145.75, 137.12, 135.68, 129.28, 128.21, 127.49, 126.91, 123.89, 123.74, 116.24, 64.89, 62.40, 60.90, 59.19, 55.49, 29.83, 27.96, 25.77. HRMS (m/z): calcd [M+Na]+ for C44H40N5O4SNa: 758.2801; found, 758.2792.

2.5. The method for determining the limit of detection (DPAC-DB) First the calibration curve was obtained from the plot of fluorescence intensity ratio (I450/I600), as a function of the avidin concentration. The regression curve equation was then obtained for the lower concentration part. The detection limit = 3 × S.D. / k Where k is the slope of the curve equation, and S.D. represents the standard deviation for the probe DPAC-DB solution's fluorescence intensity ratio (I450/I600) in the absence of avidin [38]. I450/I600 = 0.65442 + 14.1773[avidin] (R2 = 0.9928) 427

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LOD = 3 × 0.0250 / 14.1773 = 5.3 ng μL−1

subscript s stands for the sample, Φ is the fluorescence quantum yield, A is the absorbance of the solution, n is the refractive index of the solvent, and F is fluorescence intensity of the solution at the excitation wavelength. The absorption maximum was kept during 0.02–0.05 [40]. Thus, the fluorescence quantum yields of DPAC-DB in the absence and presence of avidin at 298 K in DMSO/H2O mixtures (5/5, v/v, 2.0 μM, λex = 355 nm) were calculated to be 1.4% and 21.6%, respectively. The fluorescence quantum yields of DPAC-SB in the absence and presence of avidin at 298 K in DMSO/H2O mixtures (4/6, v/v, 2.0 μM, λex = 355 nm) were calculated to be 1.8% and 7.8%, respectively.

2.6. The method for determining the reversibility (DPAC-DB) Fluorescence intensity of DPAC-DB (2 μM) was investigated upon the alternate addition of avidin/biotin (Cycle 0: 0 μM, 0.5: avidin 0.26 μg μL−1, 1: biotin 45 μM, 1.5: avidin 0.61 μg μL−1, 2: biotin 80 μM, 2.5: 0.65 μg μL−1, 3: 150 μM) in DMSO/water mixture (5/5, v/v). 2.7. The SEM/TEM of DPAC-DB

3. Results and discussion

Prior to the TEM, SEM, the samples of DPAC-DB (2.0 μM), avidin (1.03 μg μL−1), and DPAC-DB (2.0 μM) incubated with avidin (1.03 μg μL−1) in the DMSO/H2O (5/5, v/v) were dropping onto the carbon-coated carbon support copper grids, Si, respectively, and then dried at 40 °C. TEM was operated at an acceleration voltage of 120 kV (JEM-1400). SEM images were obtained by using JSM-7800.

3.1. Design and synthesis In this study, two biotin moieties were respectively coupled to the “wings” of DPAC core through nonconjugated linker, namely DPACDB. As a comparison, another probe named DPAC-SB is further designed by incorporating one biotin moiety to the DPAC core as shown in Fig. 1. Their characterizations were confirmed by NMR and LCMS.

2.8. Determination of the affinity constant (Ka) of avidin-DPAC-DB binding

3.2. Photophysical properties

The affinity constant (Ka) of DPAC-DB with avidin in DMSO/H2O solution (5/5, v/v) was determined [39]. The fluorescence intensity ratio (I450/I600) was obtained by changing the concentration of DPACDB from 0.1 nM to 50 μM in the presence of avidin (0.13 μg μL−1). When the ratio was plotted against the concentration of DPAC-DB, a curve was obtained. The EC50 was calculated to be 48.05 nM, thus, Ka = 1/EC50 = 0.21 × 108 M−1.

First, the photophysical properties of DPAC-SB and DPAC-DB were studied in organic solvents with various polarities. As depicted in Fig. 2, both of DPAC-SB and DPAC-DB exhibited a maximum absorption at around 350 nm, which is virtually insensitive to the solvent polarity. Notably, the fluorescence spectra of DPAC-SB displayed a main orangered emission around 600 nm and a negligible blue emission in various solvents with extremely large Stokes’ shift (∼250 nm). However, the main emission peaks of DPAC-DB appeared in blue band around 450–470 nm in less polar solvents, while with the polarity of the solvents increasing, it gradually showed red shifted (∼600 nm), which is due to the reduced solubility of DPAC-DB in less polar solvents after the modification of two biotin moieties (see Fig. 2D). We could control the vibration of the phenazine unit by simply changing the portions between the good solvent (e.g. DMSO) and poor solvent (e.g. H2O) to achieve the obvious emission color-tunability. According to VIE mechanism, the phenazine unit vibration would be restricted as probe molecules aggregate in the high water fraction,

2.9. Quantum yield measurement The fluorescence quantum yields were measured in DMSO by using quinine sulfate at 365 nm (ФF = 56%). as a standard. The fluorescence quantum yield of DPAC-DB was calculated in terms of the following equation (eq (1)): S

=

r

Ar ( r ) As ( s )

ns2 nr2

Fs Fr

(1)

Where the subscript r stands for the reference molecule and

Fig. 1. Synthetic routes for DPAC-SB and DPAC-DB. Reagents and conditions. a) TiCl4, toluene 12 h; NaBH4, THF/EtOH (1/1, v/v) 2 h; 47%; b) Cu(OTf)2, K2CO3, trichlorobenzene, 6 h, 210 °C, 27%; c) POCl3, DMF, 12 h, 85 °C, 80%; d) NaBH4, MeOH, overnight, 89%; e) biotin (1.0 mmol), DPPA (1.0 mmol), Et3N (1.0 mmol) DPPA, Et3N, 55 °C, overnight, 38%; f) biotin (2.0 mmol), DPPA (2.0 mmol), Et3N (2.0 mmol) DPPA, Et3N, 55 °C, overnight, 41%. 428

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Fig. 2. The absorption spectra of DPAC-SB (A) and DPAC-DB (B) in various solvents. The fluorescent spectra of DPAC-SB (C) and DPAC-DB (D) in various solvents (10 μM, λex = 355 nm).

subsequently resulting in the blue-shift of fluorescence emission. As shown in Fig. S1, the fluorescence spectrum of DPAC-SB and DPAC-DB were investigated with increasing proportion of water in DMSO at the excitation of 355 nm. The fluorescence intensities in both colors showed weakly changes when the water content was increased from 0 to 70 vol %. However, an obvious blue emission band around 450 nm was observed with a further increase of the water fraction (fw). Thus, to completely dissolve the probes and ensure a strong ratiometric color change, we next detect the biotin-avidin interaction in the H2O/DMSO mixture below fw = 70% (60% for DPAC-SB and 50% for DPAC-DB).

avidin and reached a plateau within seconds due to the high binding affinity between avidin and biotin moiety (Fig. S3). To further demonstrate the VIE response, we have performed the negative control experiments with the DPAC-OH (no biotin moieties). As shown in Fig. S4, due to the lack of binding with avidin protein, DPAC-OH showed negligible fluorescence changes in comparison with DPAC-DB. Generally, the interaction between protein and ligands was affected by different pH values. We next measured the fluorescence signal changes of DPAC-DB response to avidin over a wide pH range of 4.0–8.0. Due to the emission arises intrinsically from the intramolecular conformational transitions, the fluorescence intensity of DPAC-DB itself was completely irrelevant to the changes of pH values, which is shown in Fig. S5. Meanwhile, significant fluorescence intensity changes were still observed after binding with avidin. In addition, slightly enhanced fluorescence ratio I450/I600 of the probe was observed with the increase of pH values, indicating the binding capacity between avidin and biotin was increased from pH 4.0 to 8.0, which is consistent with previous studies [41]. Furthermore, the excellent anti-interference performance of DPAC-DB that interacted with/without avidin was also observed in other potentially biological interference species, including ions, amino acids and other nontargeted proteins (Fig. S6). Notably, the thermal stability in the biotin-avidin system could be easily performed by detecting the fluorescence intensity of DPAC-DB upon addition of avidin in different temperature. As shown in Fig. S7, the remarkable fluorescent intensity remained from 0 to 65 °C, while reduced vastly up to 75 °C, which indicated the interaction between biotin and avidin was disrupted at such high temperature [42]. These above results clearly demonstrated that this VIE-based ratiometric system could be suitable to conveniently and accurately monitor the binding process between biotin and avidin.

3.3. The principle for spectral response of DPAC-DB with avidin The principle for spectral response of DPAC-DB with different concentrations of avidin was first carried out. As shown in Fig. 3A, the UV–vis absorption peak around 350 nm remained unchanged upon addition of avidin. However, a significant turn on fluorescence intensity at 450 nm appeared and enhanced gradually accompanied by further additions of avidin (Fig. 3B). Correspondingly, the changed ratio I450/ I600 of the probe showed a good linear relationship with the concentration of avidin ranged from 0 to 1.03 μg μL−1 and the response limit of avidin was calculated to be 5.3 ng μL−1 (Fig. 3C). In addition, the binding process between biotin and avidin was also directly visualized by fluorescence color changes. As shown in Fig. 3D, the color of probe DPAC-DB could pass from orange-red to blue through the white-light emission region with corresponding chromaticity coordinates (CIE) coordinates changing from (0.4, 0.38) to (0.2, 0.15) as the avidin concentration increased. Furthermore, the same fluorescence color changes could be observed by naked eyes with a handheld UV lamp (365 nm) (Fig. 3E). And, more remarkable, another probe DPACSB exhibited the same performance in the response to avidin (Fig. S2), which demonstrated the diversity in our designed probes based on VIE mechanism. However, due to only one binding site for the DPAC-SB with avidin, the probe still has weak vibrational freedom, and hence exhibiting relatively less fluorescence change upon avidin binding. It is noted that both of DPAC-DB and DPAC-SB showed a rapid response for

3.4. The response mechanism of DPAC-DB with avidin Herein, in order to directly visualize the interaction between avidin and biotin, we introduced the VIE-based fluorophore as the indicator. The proposed mechanism of DPAC-DB response to avidin is that the 429

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Fig. 3. (A) The absorption spectra of DPACDB (2.0 μM) in DMSO/H2O solution without/ with avidin (1.03 μg μL−1); (B) The fluorescence spectra of DPAC-DB (2.0 μM) with 0–1.29 μg μL−1 of avidin in DMSO/H2O mixture (λex = 355 nm); (C) Linear fit of fluorescence intensity ratio (I450/I600) of DPAC-DB (2.0 μM) versus avidin concentration (0–1.29 μg μL−1); (D) The corresponding chromaticity coordinates (CIE) of DPAC-DB (2.0 μM) in DMSO/H2O solution (λex = 355 nm) with avidin (0–1.29 μg μL−1); (E) The corresponding fluorescence photographs of DPAC-DB (2.0 μM) in DMSO/H2O mixture with avidin (0–1.29 μg μL−1, from left to right) under 365 nm UV light illumination.

biotin moieties coupled on the “wings” of DPAC core specifically bind with avidin, subsequently resulting in the restriction of the intramolecular vibrations in hydrophenazine unit. It is noted that there are four identical subunits for each avidin, which could crosslink with DPAC-DB and thus tends to further aggregate as depicted in Scheme 1. We next performed the scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis to explore the detail mechanism. As shown in Fig. S8, both of the SEM and TEM images revealed that nanoscopic aggregates were formed in the solution of DPAC-DB containing avidin (8.0 equiv.), while no aggregation was observed in DPAC-DB solution and avidin solution, respectively. Moreover, the above hypothesis could be further proved by the timeresolved experiments as shown in Figure S9 and Table S1. After adding the avidin into the DPAC-DB solution, the emission monitored at 450 nm exhibited a prolonged decay from 2.36 to 9.47 ns, manifesting the suppression of the vibrational motions of DPAC core by the interaction between biotin moieties and avidin. The prolonged decay time of the red emission at 600 nm correlated well with the observation from the blue region. Thus, the fluorescence changes could be observed by the suppression of the vibration along NeN axis in DPAC-DB even if there were only the biotin-avidin interactions without aggregation of DPAC-DB molecules [40].

anomalous photophysical phenomenon in VIE-based fluorophores is caused by the dramatic and reversible intramolecular vibrations, which makes our probe possible to build the bio-logic gate. The affinity constant between DPAC-DB and avidin obtained by measuring fluorescence intensity ratio (I450/I600) of DPAC-DB response to avidin was calculated to be 0.21 × 108 M−1 (Fig. S10). The equilibrium constant value was very small in comparison with the affinity constant of the avidin-biotin binding (Ka ≈ 1015 M−1) [36], which is possibly due to the labeling with DPAC and introduction of DMSO solvent [43]. Thus, a biotin elution assay can be achieved by the competition reaction of free biotin and DPAC-DB with the limited binding sites of avidin [44]. As shown in Fig. 4A, upon addition of avidin, the fluorescence emission was changed from orange-red to blue with a remarkable increase in the fluorescence intensity around 450 nm. Notably, as excess free biotin molecules was added to the solution, the fluorescence spectra of the solution recovered to that of initial DPAC-DB. Such reversible change could be attributed to competition between the free biotin and DPACDB with avidin. Several cycles were conducted, and interestingly, this probe exhibited cycling stability without obvious changes in fluorescence ratio I450/I600 during the state transition (Fig. 4B). Thus, depending on the two biological inputs (avidin and biotin), this probe DPAC-DB could switch between two different fluorescence emission states, orange-red and blue, which could display “Write-Read-EraseRead” behavior in binary logic. As shown in Table S2, in this system, the sequential logic operations are represented by two inputs: InA (avidin input) and InB (biotin input) as a function of a memory element. On the other hand, the orange-red emission in initial DPAC-DB solution

3.5. The reversible response and molecular logic gates Recently, a significant development has been achieved in the biological system which behaves as molecular logic gates. In principle, the 430

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Fig. 4. Fluorescence spectra of DPAC-DB (black line), DPAC-DB with of avidin (red line) and re-adding biotin (blue line) in DMSO/ water mixture (2.0 μM, λex = 355 nm). Repetitive write-erase cycles based on fluorescence emission of DPAC-DB obtained in the presence of avidin (Off state) and biotin (On state). (B) Fluorescence intensity of DPAC-DB (2.0 μM) upon the alternate addition of avdin/ biotin (Cycle 0: 0 μM, 0.5: avidin 0.26 μg μL−1, 1: biotin 45 μM, 1.5: avidin 0.61 μg μL−1, 2: biotin 80 μM, 2.5: 0.65 μg μL−1, 3: 150 μM) in DMSO/water mixture. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

was considered as “write” and “logic state 0“. Upon addition of avidin (InA = 1), the blue-shift emission was “read” out and saved as “logic state 1“. Finally, the blue fluorescence emission was “erased” by adding biotin (InB = 1).

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4. Conclusion In summary, we have developed a novel strategy for monitoring the protein-ligand interaction based on VIE mechanism. This ratiometric probe DPAC-DB, constructed by simply coupling the ligand (biotin) with VIE-based fluorophore, could directly visualize the binding process in BAS. Moreover, upon addition of avidin or not, this probe was proved to be undisturbed to environmental stimuli, such as pH, interference species and temperature. These results demonstrate the VIEbased probe is capable of responding to intramolecular dynamic changes in biological detection. In addition, the unique VIE effect caused by dramatic and reversible intramolecular vibrations makes our strategy possible to build the bio-logic gate. However, there are still several obstacles to be suffered in this study, such as the poor solubility of the probe in the water, the addition of organic solvent and chemical modification of small molecule ligand. Future research will be focused on these issues. Notwithstanding, we believe our strategy will provide a promising way to investigate the protein-ligand interaction for the development of potent protein pharmaceuticals and small molecule drugs. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (81672508, 61505076), Jiangsu Provincial Foundation for Distinguished Young Scholars (BK20170041), ChinaSweden Joint Mobility Project (51811530018), Postgraduate Research & Practice Innovation Program of Jiangsu Province and Natural Science Foundation of Guangdong Province (2017A030313299). We also warmly thank Prof. He Tian from East China University of Science and Technology for donating sample and experimental support, and Prof. Yao Shao Q. from National University of Singapore for insightful comments. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2018.12.027. References [1] Wolberger C. Multiprotein-dna complexes in transcriptional regulation. Annu Rev Biophys Biomol Struct 1999;28:29–56. [2] Mayer ML. Structure and mechanism of glutamate receptor ion channel assembly. Activation and modulation. Curr Opin Neurobiol 2011;21:283–90.

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