Article pubs.acs.org/IECR
Confined-Space Mechanism Inspired by the Ingenious Fabrication of a Fö rster Resonance Energy Transfer System as a Ratiometric Probe for Ag+ Recognition Jianjun Du,* Tao Zhu, Weijun Ma, Wenbing Cao, Quanyong Gu, Jiangli Fan, and Xiaojun Peng State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, P. R. China S Supporting Information *
ABSTRACT: In this work, a facile and interesting fabrication of the Förster resonance energy transfer (FRET) system is presented for the first time by introducing an independent donor and acceptor into a confined space, which is different from the typical “donor−linker−acceptor” FRET system. Human serum albumin (HSA), as a proof-of-concept, is selected to load a pair of spectra-matchable but independent fluorescent dyes (ES1 and R1) in its different binding sites. Furthermore, this space-confined FRET system could act as a ratiometric sensor for the quantitative recognition of Ag+ in the water samples with good selectivity and high sensitivity.
1. INTRODUCTION Fluorescent dyes play important roles in the field of bioengineering and gene engineering, as well as chemical and environmental research, such as in the cell/organism imaging and sensing/recognizing various targets.1−6 Some undesirable properties, however, constrain their further applications in practice; for example, the small Stokes shift could result in excitation backscattering effects. Besides, the changes of fluorescence intensity at a single wavelength could not supply the quantitative information because of the influences of surrounding environmental factors, concentration, and excitation. Nevertheless, the emergence of a novel technology and/or mechanism, for example, the Förster resonance energy transfer (FRET) mechanism which is a nonradiative energy transfer process, resolves the above problems by its inherent advantages.7−10 The FRET acceptor would emit fluorescence when the coupled donor is irradiated, so the Stokes shift is much larger than both donor and acceptor. Furthermore, the FRET system would provide a ratiometric signal by recording fluorescence changes at both donor and acceptor wavelengths simultaneously,11−14 which does well in the quantitative recognition and ratiometric imaging.15−18 The FRET efficiency heavily depends on the distance between the donor and the acceptor (less than 10 nm),19 which is usually controlled by a linker moiety between the donor and acceptor. In the classical FRET system, this linker is important and indispensable, but it needs an elaborate design and complicated synthesis. Therefore, how to develop a novel, free, and efficient FRET system in a facile mode is one of biggest challenges at present. In the FRET system, energy transfers from a donor to the corresponding acceptor in a dipole−dipole coupling mode, meaning the chemical bond-based linker is a main method, but not the only way to control the distance of donor−acceptor. © XXXX American Chemical Society
Therefore, there could be some different and interesting ways to satisfy the distance requirement and realize the energy transfer. Therefore, inspired by the concept of “confined space” which is often used in the field of catalysis,20 we hypothesize that the linker is needed no more if the donor and acceptor were locked into a proper defined geometry. To the best of our knowledge, this is the first time that a facile fabrication of the FRET system is presented using a pair of spectra-matchable but independent fluorophores based on a space-confined mechanism. This confined geometry needs to be less than 10 nm in size with different binding sites for loading a donor and an acceptor, respectively. Human serum albumin (HSA), as a proof-of-concept, is selected as a best candidate, because it has a suitable size (less than 10 nm) and several cavities for packing the donor and acceptor separately (Scheme 1). In our former works, the twisted intramolecular charge transfer (TICT)-based fluorescent dyes were proven very sensitive toward microenvironment changes and were used for “lighting up” the albumin.21 Therefore, the TICT-based dyes could be useful in this work as a visible signal to prove the loading of fluorophores inside HSA. Besides, the donor and acceptor should enter different cavities of the HSA respectively to avoid the dyes’ aggregationinduced quenching. However, almost all fluorescent dyes in the literature and our former works have been reported to locate in the normal subdomains IIA and IIIA, namely, site I and site II in HSA.22−24 The binding affinity offered by site I is mainly through hydrophobic interactions while in site II it involves a Received: July 14, 2017 Revised: August 25, 2017 Accepted: September 5, 2017
A
DOI: 10.1021/acs.iecr.7b02902 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research y = Bmax *x /(Kd + x)
Scheme 1. Evolution of the Novel and Convenient FRET System Based on the Confined-Space Mechanism
wherein Bmax is the top asymptote; x is the concentration of HSA, and y is the fluorescence intensity obtained from ES1 (1.0 μM) incubated with different concentrations of HSA. 2.4. Determination of the Detection Limit. The fluorescence emission spectra of the R1−HSA−ES1 complex were measured 10 times, and the standard deviation of the blank measurement was obtained. Then the solution with R1− HSA−ES1 was treated with Ag+ at different concentrations. A linear regression curve was then achieved according to fluorescence intensity ratio (I 483 nm /I 620 nm ) versus Ag + concentrations. The detection limit was calculated with the following equation: Detection limit = 3σ/k, where σ is the standard deviation of blank measurements and k is the slope between the fluorescence intensity ratio (I483 nm/I620 nm) versus Ag+ concentrations. 2.5. Determination of Quantum Yields. Rhodamine B in ethanol was used as the reference to determine the quantum yields,25 and we used the following equation to calculate the quantum yields
combination of hydrophobic interactions, hydrogen bonding, and electrostatic effect. Herein, in this work, Dye ES1 was designed and synthesized as an acceptor with several S and N atoms, and could adjust its binding capability in the abnormal FA1 site (hemin site). Together with the R1 in our former work which was used as a donor and located in site I, a facile and interesting FRET system (R1−HSA−ES1) was fabricated in the confined geometry of HSA. Furthermore, the acceptor ES1 could be quenched by Ag+ because of its thiophilic nature. Therefore, this FRET system could also act as a fluorescent probe for the ratiometric determination of Ag+ in practical water samples.
Φu =
As Funu 2 2 A uFn s s
× Φs
wherein Φ is the quantum yield; A is the optical density; F is the measured integrated emission intensity; and n is the refractive index. The subscript “u” refers to the unknown sample, and subscript “s” refers to the standard reference of known quantum yield. 2.6. Calculation of r in FRET. The FRET efficiency, EFRET, and the FRET distance (r) between R1 and ES1 were obtained by the following equations. First, the efficiency of energy transfer, EFRET is given by
2. MATERIAL AND METHODS 2.1. Materials. Chemicals and reagents used in this work, such as HSA, bovine serum albumin (BSA), chymotrypsinogen A, chymotrypsin, protease K, lysozyme, hemoglobin, histone, glutamic acid, glycine, histidine, trptophan, arginine, tyrosine, aspartate, cysteine, asparagines, lysine, NaCl, Na2SO4, NaNO3, NaClO4·H2O, NaCO3, Na2SO3, NaF, NaBr, NaI, CH3COONa, Na2Cr2O7·2H2O, KCl, CaCl2, BaCl2, FeSO4·7H2O, FeCl3· 6H2O, CuSO4·5H2O, MgCl2·6H2O, MnCl2, CrCl3·6H2O, CoCl2·6H2O, and Pb(NO3)2 were purchased from Energy Chemical and Aladdin Industrial Corporation and were used without further purification. All solvents used are of analytical grade without further purification. Solutions of different cations and anions were prepared by dissolving inorganic salts in distilled water at 5.0 mM, respectively. All proteins and small biomolecules were dissolved in distilled water to prepare stock solutions with concentrations of 5.0 mg/mL. The solutions of ES1 were prepared in DMSO (5.0 mM) and stored in the fridge before use. 2.2. Characterization. 1H and 13C NMR spectra were recorded on Varian INOVA-400 with chemical shifts (δ) reported in ppm relative to the solvent residual signals of CDCl3 (7.24 ppm) and coupling constants reported in Hz. UV−visible spectra were collected on a PerkinElmer Lambda 35 UV−vis spectrophotometer. Fluorescence measurements were performed on a VAEIAN CARY Eclipse fluorescence spectrophotometer. High resolution mass spectra (HRMS) were recorded on Agilent 6224 (TOF−LC/MS). The fluorescence lifetime was tested on FLS 920 of Edinburgh Analytical Instruments, and pH was adjusted using OHAUS ST2100. 2.3. Determine the Dissociation Constant Kd. To determine the dissociation constant, we used the following equation:
E=
R 06 R 06 + r 6
(1)
wherein, R0 is the critical distance at which the energy transfer efficiency is 50% and r is the distance between the donor and the acceptor. R0 can be obtained by the following equation: R 06 = 8.8 × 10−25k 2n−4 ΦJ
(2)
wherein, k2 is the spatial orientation factor related to orientation in space of the transition dipoles of the donor and acceptor, n is the average refractive index of the medium, φ is the fluorescence quantum yield of the donor in the absence of acceptor, and J is the spectral overlap integral between the emission spectra of the donor and the absorption spectra of the acceptor. The spectral overlap integral J is evaluated by the equation: ∞
J=
∫0 FD(λ)ε(λ)λ 4 dλ ∞
∫0 FD(λ) dλ
(3)
wherein, F(λ) is the fluorescence intensity of the donor at wavelength, λ and ε(λ) is the molar absorption coefficient of the acceptor at that wavelength. 2.7. Calculation of FRET Efficiency. The transfer efficiencies were calculated using steady state data with the following equation: B
DOI: 10.1021/acs.iecr.7b02902 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research E=1−
F F0
Table 1. Optical Properties of R1 and ES1 in PBS (10 mM, pH 7.4)
(4)
where F is the net integral of the emission of the donor in the presence of the acceptor and F0 is the integral in the absence of the acceptor. 2 . 8 . S y n t h e s i s o f th e C o m p o u n d E S 1 . 4(Dimethylamino)cinnamaldehyde (0.17 g, 1 mmol) and Rhodanine (0.26 g, 2.0 mmol) were dissolved in toluene and NH4OAc (0.20 g, 2.6 mmol) dissolved in AcOH (0.5 mL, 9.0 mmol) was added. The flask was equipped with a Dean−Stark apparatus, and the reaction mixture was heated to reflux and stirred for 5 h. After the mixture was cooled to room temperature, it was diluted with dichloromethane (20 mL), washed with water (2 × 30 mL) and brine (30 mL), and dried with MgSO4. Evaporation of the solvents gave the crude product, which was purified by silica gel column with ethyl acetate/petroleum ether in the ratio 1:20 as eluent to afford the pure ES1. Yield: 65%. 1H NMR (400 MHz, CDCl3), δ (ppm): 7.51 (d, 2H, J = 8 Hz), 7.21 (q, 2H, J = 36 Hz), 6.72 (m, 3H, J = 4 Hz), 3.07 (m, 1H, J = 4 Hz), 2.99 (s, 6H). 13C NMR (100 MHz, CDCl3): δ151.45, 145.41, 130.97, 129.73, 129.41, 118.48, 112.10, 111.89, 45.75, 8.64. HPLC−MS (API-ES): [MH]−289.0469; found, 289.0475.
R1 R1−HSA ES1 ES1−HSA
λab/nm
λem/nm
Stokes shift/nm
Φf
τs/ns
434 435 461 469
489 483 661 630
55 48 200 161
0.002 0.018 0.002 0.021
0.79 0.84 0.70 0.95
water in different ratios were prepared respectively to mimic the different viscosities and polarities in/out of HSA cavities as shown in Figure 1b,c. ES1 showed stronger fluorescent intensity in the microenvironment with a high viscosity and low polarity. Further, the fluorescence lifetimes of ES1 in the absence and the presence of HSA were tested and were 0.70 and 0.95 ns, respectively (Figure S2). The longer lifetime of ES1 after binding with HSA occurs because the free rotation of ES1 is hampered by the steric hindrance within the HSA cavity. The dissociation constant (Kd) for ES1−HSA complexes was also calculated, which reached 2.16 ± 0.23 μM, meaning ES1 had a strong affinity with HSA. Then, amino acids (glutamic acid, glycine, histidine, trptophan, arginine, tyrosine, aspartate, cysteine, asparagine, and lysine), common environment-related anions (F−, Cl−, Br−, NO3−, CH3COO−, CO32−, ClO4−, Cr2O72−, SO32−, and SO42−) and metal ions (K+, Ca2+, Cu2+, Cr3+, Mg2+, Ni2+, Mn2+, Ba2+, Fe2+, and Fe3+), and various proteins (chymotrypsinogen A, chymotrypsin, protease K, lysozyme, hemoglobin, and histone) were tested, which resulted in a negligible increase of fluorescent intensity except HSA (Figures S3−S6). A Job’s plot analysis was performed to confirm that the stoichiometry of the ES1−HSA complex is 1:1 proportion (Figure S7). To understand the binding site of ES1 in HSA, the drug displacement experiment proceeded using known site-selective drugs, that is, warfarin, propofolum, and ibuprofen.26,27 Unfortunately, none of them exhibited obvious fluorescence quenching, meaning ES1 did not locate in any of those normal binding sites. Then inspired by the first example of the HSA probe in the FA1 site (hemin) by Vendrell and Chang et al.,28 the positive response was obtained by introducing hemin in our site-screening experiment. As illustrated in Figure 1d, the displacement with hemin induced a significant decrease (72.9%) in the fluorescence response of ES1, indicating that ES1 specifically located in the site FA1 of HSA. This makes fabrication of FRET in a single HSA molecule possible by stuffing ES1 together with a spectroscopymatchable donor in site I (Figure 2a). Then R1, which is used as a reference in our former work,21 was selected as a donor in our FRET system, because it could
3. RESULTS AND DISCUSSION In PBS (10 mM, pH 7.4), ES1 exhibited an extremely weak fluorescence as expected with a maximum emission at 661 nm (Figure 1a), while in the presence of HSA, the emission
Figure 1. (a) Fluorescence emission spectra of ES1 in the absence and presence of HSA, excited at 507 nm in PBS (10 mM, pH 7.4). (b) Changes of fluorescence spectra for ES1 (10 μM) in aqueous media with different 1,4-dioxane contents. (c) Changes of fluorescence spectra for ES1 (10 μM) in ethanol solution with different glycerol contents. (d) Changes of F/F0 for ES1−HSA in the presence of drugs in different binding sites.
intensity of ES1 blue-shifted to 630 nm and increased dramatically (20-fold enhancements). This obvious fluorescent shift and enhancement proved its binding in a HSA site (Figure S1 in the Supporting Information and Table 1). The emission increase of ES1 occurs mainly because of the different microenvironments (e.g., viscosity and polarity) inside and outside of the protein cavities, similar to what was seen in our former works. Solutions of glycerol/ethanol and 1,4−dioxanes/
Figure 2. (a) Spectral overlap (marked by horizontal lines) between the donor R1−HSA emission (dashed blue line) and acceptor ES1− HSA absorption (solid red line). (b) Fluorescence emission spectra of R1−HSA with the addition of different contents of ES1. C
DOI: 10.1021/acs.iecr.7b02902 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research enter the site I of HSA and its spectra were matchable with ES1 (Table 1), which satisfied the FRET principle perfectly. There was a very large overlap of R1’s emission with ES1’s absorption, while ES1’s emission was negligible when it was excited at the excitation wavelength of R1 (Figure 2a).7 Along with increasing addition of ES1 into R1−HSA solution from 0 μM to 6 μM, the fluorescent emission of R1−HSA decreased gradually, while the fluorescent emission for ES1−HSA increased obviously (Figure 2b). This naked-eye observed ratiometric fluorescence change indicated that energy transfer from R1 to ES1 was quite satisfied (Figure 2b, inset). The FRET efficiency (EFRET) and distance (r) between the energy donor and energy acceptor was thus calculated by the formulas as shown in the experimental section. For the R1− HSA−ES1 complex, the value of EFRET was 0.84, wherein the concentration of R1, HSA, and ES1 were 5 μM, 2.5 μM, and 5 μM, respectively (In this case, J = 9.69 × 1014 M−1 cm3, k2 = 2 /3, N = 1.33 for water and φ = 0.018 for R1−HSA complex). The R0 was calculated as 2.63 nm and r was given as 2.13 nm, respectively. The FRET distance (r) between donor and acceptor satisfied the required distance (
