Microenvironment-Sensitive Fluorescent Dyes for Recognition of

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Microenvironment-Sensitive Fluorescent Dyes for Recognition of Serum Albumin in Urine and Imaging in Living Cells Tao Zhu, Jianjun Du, Wenbing Cao, Jiangli Fan, and Xiaojun Peng Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.5b04214 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on January 3, 2016

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Industrial & Engineering Chemistry Research

Microenvironment-Sensitive Fluorescent Dyes for Recognition of Serum Albumin in Urine and Imaging in Living Cells Tao Zhu, Jianjun Du,* Wenbing Cao, Jiangli Fan, and Xiaojun Peng

State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P.R. China, [email protected]

ABSTRACT: A series of microenvironment-sensitive fluorescent dyes, SA1−4, have been presented, which can light up human serum albumin (HSA) in aqueous media and solid state with colorful emissions as well as dramatic fluorescence enhancements respectively, based on twisted intramolecular charge transfer and molecular rotor strategy. These microenvironmentsensitive SA1−4 exhibited excellent fluorescent capabilities in the fast, convenient, selective, and sensitive recognition of HSA, especially in the quantitative albumin assay in human urine for assessment of kidney function and diagnosis of renal disease.

Moreover, SA1−4/HSA

complexes could be applied in fluorescence imaging in living cells.

KEYWORDS:

Fluorescent dye; Albumin; Twisted intramolecular charge transfer (TICT);

Microenvironment-sensitive; Fluorescence imaging

ACS Paragon Plus Environment

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1. Introduction

Albumin is one of the most abundant proteins responsible for various important physiological processes, such as maintaining the osmotic pressure of blood compartment, balancing nutrition, and especially transporting a wide variety of drugs and metabolites.1-3 The normal concentration, for human serum albumin (HSA) in serum, is around 35−50 g/L, while in urine it is usually less than 30 mg/L for healthy people because of filtering capability of kidneys.4 Therefore, serum albumin content could be considered as a reliable indicator for liver and kidney biological function and related disease. For example, the presence of an excess amount of HSA in urine, i.e. albuminuria, is an early warning for the impairment of kidney function as well as a diagnostic marker for related renal disease.5, 6 In contrast, a low level of HSA in the blood plasma, named hypoproteinemia, would be a sign of liver cirrhosis and chronic hepatitis.7 In the face of escalating concern over its indispensable factor on human health, therefore, it is necessary to present rapid, specific, and cost-effective tools for monitoring albumin level in body fluids.

Numerous methods, such as surface enhanced Raman scattering,8 electrochemistry,9 high performance liquid chromatography,10 and chemiluminescence,11 have been developed successfully for the albumin determination qualitatively or/and quantitatively in different matrixes.

However, the main limitations of these methodologies are related to their cost,

response time, sensitivity, interference, and need of professional operator, all of which made


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them ineffectual for the practical application, especially ill suited for on-site analysis and rapid in situ screening. Alternatively, fluorescence spectroscopy becomes one of the most widely applied techniques in studying DNA, enzyme activity, protein, and biomolecule. It exhibits capabilities in probing target in complicated microenvironment on the platform of fluorescent dye combined with strategies of coordination chemistry, host-guest interaction, and chemical reaction.12-17 As for HSA, several fluorescent probes for serum detection in aqueous media have been reported recently, for example, squaraine dye,18-22 BODIPY derivative,23 staining agent based on 3amino-N-alkyl-carbazole scaffold,24 polarity and viscosity sensitive fluorescent dye,25-27 aggregation induced emission (AIE) dye,28,

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and green fluorescent protein (GFP)-inspired

chromophore.30 Besides, our group developed a new dicyanomethylene-4H-chromene based probe for HSA sensing in urine with excellent sensitivity in recent.27 However, there are still big challenges in monitoring serum albumin in practical, such as the avoidance of complicated chemical synthesis, improvement of sensing efficiency (e.g. excitation and emission wavelengths, fluorescence quantum yield) to avoid the interference from biological autofluorescence, and quantifying low concentration of albumin (less than 30 mg/L) for the practical routine analysis. That motivates us to further explore novel, simple, and portable fluorescent probes for sensing serum albumins, while also possessing strong anti-jamming capability, high sensitivity, good repeatability, and especially near-infrared emission.


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In this context, with the above motivations, intense endeavor has been dedicated to recognizing HSA depending on the supramolecular interaction of microenvironment-sensitive fluorescent dyes with HSA. HSA, synthesized and secreted by the liver, exhibits a threedimensional structure consisting of several binding pockets because as much as 67% of the secondary structure is formed by α-helix of six turns, besides two main binding sites located in subdomain IIA and IIIA, namely, site I and site II.31

It exhibits quite a different

microenvironment (e.g. polarity, viscosity, and steric hindrance) inside these hydrophobic binding pockets from those in the bulk solution. Our hypothesis, herein, is designing and synthesizing an environment-sensitive fluorescent dye which could bind HSA and then exhibit fluorescence changes in both intensity and wavelength, because of 1) the fabrication of polarity sensitive donor-π-acceptor (D-π-A) structure undergoing intramolecular charge transfer (ICT); and 2) the introduction of polarity sensitive para-substituted benzene structure undergoing twisted ICT (TICT).15, 32-34 Therefore, the fluorescent dye would show weak fluorescence in aqueous media (high polarity environment) while exhibit dramatic fluorescence enhancement with wavelength shift once enter the binding site in HSA (low polarity microenvironment); 3) inspiration from the fluorescent molecular-rotor R1 (Scheme 1), which is very sensitive to viscosity through the 2-methoxyacetonitrile group resulting in lower background fluorescence by nonradiative decay.35

Therefore, both the TICT process and the motor rotation would be

restricted by steric effect in HSA cavities, resulting in a dramatic fluorescence increase.


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As a proof-of-concept, a series of fluorescent dyes, SA1−4, were designed and synthesized on the platform of classical D-π-A structure simply by just one or two steps of synthesis.36 As shown in Scheme 1, SA1 and SA3 exhibit typical structures with TICT property, and then malononitrile moiety was introduced as a molecular motor in SA2 and SA4 on the basis of SA1 and SA3, respectively. Compared with SA1 and SA2, SA3 and SA4 possess an extended conjugation system by introducing a C=C double bond, leading to red-shifts in absorption and emission. SA4, especially, shows a near-infrared fluorescence emission at around 685 nm after binding HSA with a maximum 428-fold fluorescence enhancement. Furthermore, SA1−4/HSA complexes show excellent fluorescence properties, which could be further applied in fluorescence imaging in living cells.

2. Experimental Section

2.1 Chemicals.

Chemicals and reagents used in this work, such as HSA, BSA,

chymotrypsinogen A, chymotrypsin, protease K, lysozyme, haemoglobin, 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 used without further purification. Solutions of different metal ions and anions were prepared by dissolving inorganic salts in DI water at 5 mM, respectively. All


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proteins and small biomolecules were dissolved in DI water to prepare stock solutions with concentration of 5.0 mg/mL. The solutions of SA1−4 were prepared in DMSO (5 mM) and stored in fridge before use.

2.2 Instruments.

1

H and

13

C NMR spectra were recorded on Varian INOVA-400 with

chemical shifts (δ) reported as ppm relative to the solvent residual signals of CDCl3 (7.24 ppm) and coupling constants reported in Hz.

UV-vis spectra were collected on a Perkin-Elmer

Lambda 35 UV−vis Spectrophotometer.

Fluorescence measurements were performed on a

Agilent Technologies CARY Eclipse Fluorescence Spectrophotometer. The quantum yields of SA1−4 in solid state in the absence and presence of HSA was measured by absolute quantum yield spectrometer (C11347-11, Hamamatsu).

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 Synthetic Procedures. The synthesis of R1 followed the literature,35 and the synthesis of SA1−4 followed the general route shown in Scheme 1.

2.3.1. Synthesis of SA1. 1 (1.00 g, 6.70 mmol), 2 (0.80 g, 6.70 mmol), and NaOH (300 mg) were dissolved in methanol (5 mL) and stirred at room temperature until heavy precipitates formed (24 h). The precipitate was isolated by filtration, washed with cold methanol, and further purified by silica gel column (dichloromethane/hexane, 1/1, v/v) to give SA1, as a bright yellow solid. Yield: 92.6%. 1H NMR (400 MHz, CDCl3), δ (ppm): 8.03 (d, 2H, J = 8 Hz), 7.83 (d, 1H,


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J = 16 Hz), 7.59 (m, 5H, J = 40 Hz), 7.38 (d, 1H, J = 16 Hz), 6.75 (d, 1H, J = 8 Hz), 3.07 (s, 6H);

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C NMR (100 MHz, CDCl3) δ: 190.887, 182.031, 145.722, 139.214, 132.322, 130.582,

128.477, 117.267, 112.188, 40.188, 29.876. TOF-LC/MS: m/z calcd for [M + H+]+, 252.1388; found, 252.1385.

2.3.2. Synthesis of SA2. SA1 (0.25 g, 1.00 mmol) and malononitrile 3 (0.13 g, 2.00 mmol) were dissolved in toluene, NH4OAc (0.20 g, 2.60 mmol) dissolved in AcOH (0.5 mL, 9.00 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 cooling to room temperature, the mixture was diluted with dichloromethane (20 mL), washed with water (2 × 30 mL), brine (30 mL) and dried with MgSO4. Evaporation of the solvents gave the crude product and purified by silica gel column (ethyl acetate/petroleum ether, 1/20, v/v) to afford SA2. Yield: 56.1%.

1

H NMR (400

MHz, CDCl3), δ (ppm): 7.53 (d, 3H, J = 4Hz), 7.43 (m, 5H, J = 32Hz), 6.81 (d, 1H, J = 24Hz), 6.68 (d, 2H, J = 8Hz), 3.07 (s, 6H); 13C NMR (100 MHz, CDCl3) δ: 171.610, 152.388, 149.378, 134.032, 130.431, 128.805, 124.399, 122.370, 120.717, 119.175, 110.515, 114.033, 113.299, 111.919, 86.080, 40.141, 39.209, 31.939, 29.711, 22.707, 14.140. TOF-LC/MS: m/z calcd for [M + H+]+, 300.1501; found, 300.1498.

2.3.3. Synthesis of SA3. 4 (0.53 g, 3.00 mmol) was gradually added into a mixture solution of NaOH solution (0.40 g in 1 mL H2O) and 2 (0.60 g, 4.50 mmol) in 7 mL ethanol at 0 °C. The mixture was then allowed to warm to room temperature and stirred for 5 h, after which a


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precipitate of product formed. The product was collected by filtration and washed repeated to remove all NaOH. Recrystallization from ethanol was applied to afford the product SA3. Yield: 85.3%. 1H NMR (400 MHz, CDCl3), δ (ppm): 7.97 (s, 2H), 7.62 (t, 1H, J = 20 Hz), 7.47 (m, 5H, J = 80Hz), 7.00 (t, 2H, J = 20 Hz), 6.88 (t, 1H, J = 20 Hz), 6.70(s, 2H), 3.02 (s, 6H); 13C NMR (100 MHz, CDCl3) δ: 190.594, 148.365, 143.034, 138.784, 132.251, 131.090, 128.471, 122.777, 112.159, 40.285, 31.882, 29.528, 22.819, 14.048.

TOF-LC/MS: m/z calcd for [M + H+]+,

278.1545; found, 278.1540.

2.3.4. Synthesis of SA4. To a solution of 5 (0.19 g, 1.10 mmol) and 4 (0.18 g, 1.00 mmol) in dichloromethane was gradually added several drops of piperidine. The mixture was warmed to room temperature and stirred for 1.5 h. After evaporation of the solvent, the residue was purified on a silica gel column (ethyl acetate/petroleum ether, 1/20, v/v) to afford the SA4. Yield: 76.3%. 1

H NMR (400 MHz, CDCl3), δ (ppm): 7.53 (d, 4H, J = 8 Hz), 7.38 (q, 4H, J = 20 Hz), 7.06 (d,

1H, J = 12 Hz), 6.88 (d, 1H, J = 8 Hz), 6.80 (d, 3H, J = 12 Hz), 3.05 (s, 6H);

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C NMR (100

MHz, CDCl3) δ: 171.030, 150.896, 145.583, 133.737, 130.635, 129.790, 129.442, 128.824, 128.541, 125.286, 114.356, 113.732, 112.176, 40.243. TOF-LC/MS: m/z calcd for [M + H+]+, 326.1657; found, 326.1650.

2.4 Limit of Detection (LOD).

The fluorescence emission spectral of SA1 and SA4 was

measured and the standard deviation of a blank measurement was achieved. The fluorescence intensity of SA1−4/HSA complexes was plotted with HSA concentration. The detection limit


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was calculated by using equation of 3σ/k, wherein “σ” is the standard deviation of the blank measurement and “k” is the slope of linear relationship of fluorescence intensity of SA1−4/HSA complexes with HSA concentration.

2.5 Solubility of SA1− −4 in Aqueous Media. To study the solubility of SA1−4 in aqueous media, different concentrations of SA1−4 were added to aqueous media. The absorbance at the maximum absorbance wavelength was monitored by UV−vis spectrophotometer, which was further plotted with concentrations of SA1−4.

2.6 Dissociation Constant Kd of SA1− −4/HSA Complexes. To determine the dissociation constant, we used the equation: y = Bmax * x / ( K d + x ) , wherein “Bmax” is the top asymptote; “x” is the concentration of HSA and “y” is the fluorescence intensity obtained from SA1−4 (1 µM) incubated with different concentrations of HSA.

2.7 Quantum Yields of SA1− −4. Rhodamine B in ethanol was used as the reference to determine the quantum yields37 in solution by using the equation: Φ u =

As Fu nu 2 ×Φ s , wherein Au Fs ns 2

“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 with a known quantum yield.

The solid quantum yields of SA1−4 in the absence and presence of HSA were measured by absolute quantum yield spectrometer (C11347-11, Hamamatsu).


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2.8 HSA Recognition in Human Urine. Urine was obtained from a healthy male donor. The HSA content in urine was assayed in local hospital. Then urine samples were prepared by adding different spiked HSA contents in urine samples, and the fluorescence changes were monitored by the fluorescence spectrophotometer, and each sample was performed for six parallel experiments.

2.9 Fluorescence Imaging in Living Cells. For confocal fluorescence imaging, MCF-7 cells were cultured under recommended conditions.

SA1−4/HSA complexes were added at a

concentration of 2.5 µM and cultured for 2 h before the experiment. Cells then were washing with PBS (pH 7.4) for three times and then imaged by a laser scanning confocal fluorescence microscope.

3. Results and Discussion

3.1 Spectroscopic Properties of SA1− −4 and Response to HSA. At first, we measured the solubility of SA1−4 in aqueous media. As shown in Figure S1 (Supporting Information), along with the increasing content of SA1−4, the intensities at their maximum absorption peaks increased linearly over ranges of 0−30 µM, 0−90 µM, 0−100 µM, and 0−80 µM respectively, meaning all of them exhibited very good solubility in water media. Then the spectroscopic properties of SA1−4 were measured in PBS (10 mM, pH 7.4).


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The UV−vis absorption of the SA1−4 was firstly studied in PBS (10 mM, pH 7.4). As shown in Figure 1a and Table 1, the maximum absorption peaks of SA1 and SA3 locate at 428 nm and 431 nm respectively, meaning the extending conjugated C=C moiety doesn’t lead to a obvious red-shift because of TICT mechanism, both of which are similar as absorption of R1 (434 nm). In contrast, for SA2 (484 nm) and SA4 (519 nm), the introduction of malononitrile group, which increases the electron-withdrawing capability of acceptor, results in large red-shifts compared with SA1 (428 nm) and SA3 (431 nm) respectively.

In PBS (10 mM, pH 7.4), R1 and SA1−4 exhibited extremely weak fluorescence as expected with maximum emissions at 489 nm, 541 nm, 647 nm, 620 nm, and 770 nm, respectively (Figure 1a and Table 1). Because of the additional rotor consumption, quantum yields (Qf) of SA2 (0.9%) and SA4 (0.3%) are lower than those of SA1 (2.1%) and SA3 (0.5%), respectively. In contrast, in the presence of HSA, the emission intensity and quantum yields of SA1−4 increased dramatically with large blue-shifts to 535 nm, 590 nm, 585 nm, and 685 nm, and the quantum yields of SA1−4 reached 10.2%, 7.1%, 9.3%, and 14.8% respectively (Figure 1b and 1c). Furthermore, obvious larger Stokes shifts were obtained for SA1−4 (96 nm, 102 nm, 153 nm, and 149 nm) than R1 (48 nm) after binding HSA by TICT mechanism. SA1−4 also exhibited solid-state fluorescence with maximum emissions at 535 nm, 688 nm, 660 nm, and 787 nm and quantum yields of 8.2%, 1.5%, 17.5%, 0.5% respectively, compared with HSA (Figure 1e and


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S2). After binding HSA, the quantum yields of SA1−4/HSA complexes in solid increased to 12.5%, 13.4%, 18.7% and 11.9%, respectively (Table 1).

Furthermore, to evaluate the sensitivity and maximum F/F0 value for HSA, quantitative analysis of HSA titration was performed. As shown in Figure 1d and Figure S3 (Supporting Information), with increasing amount of HSA, for example, 90-fold and 57-fold fluorescence enhancements for SA1 and SA4 were obtained respectively, when HSA concentration reached 615 mg/L. The detection limits (3σ/k) were as low as 125.2 ng/L and 76.3 ng/L for SA1 and SA4 respectively, which were enough for HSA assay in urine. To exhibit the fluorescence enhancement of SA1−4 in solution with different HSA contents, we calculated the F/F0 values of SA1−4 in the absence and presence of different HSA contents. As shown in Figure 2a, the fluorescence increases of SA1 and SA3 reached saturated when HSA concentration reached 500 mg/L; in contrast, for SA2 and SA4, obvious increasing tendency could be still observed even after addition of excess 2000 mg/L HSA.

We believe this

phenomenon arises from the

introduction of the malononitrile moiety, which can affect the interaction of dyes with HSA. For instance, the dissociation constants (Kd) of SA1/HSA and SA3/HSA are 0.77 ± 0.12 µM and 0.20 ± 0.05 µM, which are smaller than those of SA2/HSA (1.50 ± 0.36 µM) and SA4/HSA (5.08 ± 0.88 µM) respectively, meaning it needs more contents to reach maxmium fluorescence enhancement for SA2 and SA4. Furthermore, as much as 428-fold fluorescence enhancement


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was finally obtained for SA4/HSA because of its lower background, which is consistent with the changes of quantum yields (Table 1).

3.2 Selectivity of SA1− −4. To test the selectivity of SA1−4, firstly, the fluorescent responses were detected in the presence of various proteins, including chymotrypsinogen A, chymotrypsin, protease K, lysozyme, haemoglobin, histone, BSA (bovine serum albumin), and HSA (Figure 2b). All proteins exhibited little fluorescence changes except HSA and BSA. The structure and functions of HSA and BSA are similar, but the microstructure and microenvironment inside their hydrophobic binding pockets exhibit some difference, which can affect the selectivity of dyes with proteins. Compared with SA1-3, SA4 is maybe more fit in cavity of HSA than that of BSA because of its unique structure. Secondly, amino acids, such as glutamic acid, glycine, histidine, trptophan, arginine, tyrosine, aspartate, cysteine, asparagines, and lysine were used for the selective test. For examples, SA1 and SA4 showed negligible increase of fluorescence intensity in the presence of different amino acids (Figure S4− −S5, Supporting Information). Thirdly, common environment-related anions (F−, Cl−, Br−, I−, NO3−, CH3COO−, CO32−, ClO4−, Cr2O72−, SO32−, and SO42−) and metal ions (K+, Ca2+, Cu2+, Co2+, Cr3+, Mn2+, Mg2+, Ba2+, Pb2+, Fe2+, and Fe3+) were also studied, resulting in no fluorescence response at all (Figure S6−S9, Supporting Information). The excellent selectivity of SA1−4 to HSA clears the hurdle for the subsequent cell imaging and HSA recognition in real body fluid sample.


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3.3 Binding Site. A Job’s plot analysis was performed to confirm the stoichiometry of the SA1−4/HSA complexes, in which the fluorescence intensity of SA1 and SA2 (for instance) peaked at 1:1 proportion (Figure 2c and Figure S10, Supporting Information).

Generally, serum albumin possesses two major binding sites, binding affinity offered by site I is mainly through hydrophobic interactions, whereas site II involves a combination of hydrophobic, hydrogen bonding, and electrostatic effect.38 For understanding the binding site of SA1−4 in HSA, a ligand displacement strategy proceeded using known site-selective drug, i.e. warfarin for site I and ibuprofen for site II.25 As illustrated in Figure 2d, the displacement with warfarin induced significant decreases of about 61% and 55% in the fluorescence response of R1 and SA1 respectively, while ibuprofen almost had no effect. However, both addition of warfarin and ibuprofen resulted in gradual decrease of the fluorescence intensity of SA4, and the displacement proportions were about 49% and 45%, respectively. The results clearly indicates that the turn-on response of SA1 is because of the specific binding with site I, while both binding to site I and site II contributes to the fluorescence increase of SA4.

3.4 TICT Mechanism of SA1−4. Two excited states, e.g. locally excited and TICT states, are accessible upon photoexcitation. The locally excited state is proposed to be emissive, but the TICT state is usually not. The emission of the probes are sensitive to the polarity and viscosity of the solvent, that is, the intensity and wavelength of emission will change significantly by variation in the polarity and viscosity of the solvents. The microenvironment (polarity, viscosity,


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steric effect etc.) changed a lot for SA1−4 inside and outside the HSA cavities. The PBS is polar solution with light viscosity; while in the cavity of albumins, it provides a microenvironment with low polarity and strong steric hindrance. The increase of steric hindrance within the HSA will hinder rotation of the C−C single bond and C−N bond in donor moiety, thus suppress the transition from locally excited state to the TICT state. Therefore, the population of the locally excited state will increase, and correspondingly the fluorescence emission intensity will be enhanced. Therefore, to examine the contribution of solvent polarity in this dependency, we first analyzed the fluorescence intensity of SA1 in 1,4-dioxanes/water mixture, and observed the fluorescence intensity increased with large blue-shifts when the solvent polarity decreased (Figure 3a).

Then the emission spectra of SA1 in different viscosity were measured by

controlling the ratio of glycerol/water. As seen from Figure 3b, increased viscosity restrained free rotation of molecular rotors, which might minimize non-radiative energy loss and have fluorescence turned on. Besides the strong “pull–push” system in SA1−4, the introduction of molecular rotor in SA2 and SA4 also made them lower quantum yields (0.9% and 0.3%) than SA2 and SA4 (2.1% and 0.5%) in PBS. Furthermore, fluorescent molecular rotors usually show longer fluorescence lifetime in a restricted environment. Therefore, the lifetime of SA1 and SA4 before and after binding with HSA were measured. In the absence of HSA, SA1 and SA4 exhibits fluorescence lifetime of τSA1 = 0.96 ns and τSA4 = 0.61 ns, respectively. While after binding HSA, the complexes displayed fluorescence lifetimes of τ1 = 0.82 ns and τ2 = 2.68 ns for SA1/HSA, as well as τ1 = 1.95 ns and τ2 = 4.07 ns for SA4/HSA (Figure S11−S12, Supporting


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Information). We therefore proposed that SA1−4 could interact with the protein’s binding sites via noncovalent bonding and the polarity changes, and then caused great changes in the fluorescence spectrum.

3.5 Quantitative Recognition of HSA. Predominantly, pH value is one of the most important factors that may influence HSA sensing. As shown in Figure S13 (Supporting Information), the fluorescence intensity of SA1 kept stable over pH ranges of 4.0−7.0, while obvious increase was observed in alkaline solutions (pH 7.0−10.0). Obvious fluorescence change could be observed immediately after addition of HSA and become saturated within 10 sec, and the system showed good stability at least 60 min before and after binding SA1 and SA4 (Figure S14, Supporting Information).

The direct quantitative detection of HSA in biofluids shows a great clinical importance while it is usually hampered by the interference of other biological sub-stances and high background fluorescence of urine. The favorable fluorescence properties of SA1−4 for HSA recognition prompted us to further study their utility for the determination of HSA in biosystems, such as the protein content in the urine, which is helpful for the early diagnosis of renal diseases. The HSA content in healthy urine is normally less than 30 mg/L, which is hard to measure for most of fluorescent probes. In our experiment, human urine containing HSA at different concentrations was prepared by artificially mixing HSA in a pure urine sample (from normal healthy male donor and the HSA content of urine sample was detected as 3.73 mg/L using urinary albumin


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assay in local hospital). As seen from Figure 4a, the fluorescence response of SA4 showed an excellent linear correlation with the amount of HSA over a large range of 3.73−853.73 mg/L in real urine samples. Our proposed method is easy to operate and the detection limit is sufficient for HSA detection in human urine in lower range of 3.73−173.73 mg/L (Figure 4b), compared with radioimmunoassay, the commonly used method in clinical labs. For testing HSA in real urine samples quantitatively, three urine sample were prepared by spiking HSA artificially in healthy urine samples from volunteer.

Good recoveries were obtained over the range of

87.4−113.3% with RSD% from 5.9−10.5% as shown in Table 2 for HSA assay by using SA4− based linearity in Figure 4b, which revealed its acceptable veracity and reproducibility for HSA assay in real urine samples.

3.6 Fluorescence Imaging of SA1− −4/HSA Complexes in Living Cells. As shown in Table 1, the fluorescence intensities of SA1−4 increased obviously after binding HSA. Therefore, SA1− 4/HSA are good candidates for fluorescence imaging in living cells, because 1) HSA, as a kind of three-dimensional structure, could provide hydrophobic, non-polar, and restricted cavities for SA1−4; 2) HSA possesses excellent cell permeability that can bring SA1−4 into cells. Therefore, we incubated MCF-7 cells with SA1−4/HSA complexes.

As expected, all SA1−4/HSA

complexes were confirmed to be membrane-permeable, and stained cells with different fluorescence emissions mainly in cytoplasm areas as shown in Figure 5.

4. Conclusions


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In summary, we have reported a series of fluorescent dyes, SA1− −4, which showed great sensing properties for serum albumin with fluorescent emissions from visible region to near infrared region. SA4 exhibited a remarkable fluorescence enhancement with 428-fold and low detection limit as 76.3 ng/L toward HSA without interference from interfering ion, biomolecule, and protein in biosystem. Furthermore, SA4 could be used to detect the albumin level in real human urine and the fluorescence response showed a good linear correlation with HSA concentration. Finally, SA1− −4/HSA complexes were applied in fluorescence imaging in living cells, which are also believed good prospects in study of protein/enzyme labeling and their interactions.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website:

1

H NMR, 13C NMR and TOF-MS spectra of SA1−4, solubility test of SA1−4, photos of HSA

under white and UV irradiation in PBS and solid state, fluorescent titration of SA4 with HSA, selectivity experiments of SA1 and SA4 with different amino acid, metals, and anion respectively, Job’ plot of SA2, fluorescence lifetime test of SA1 and SA4 in the absence and presence of HSA respectively, fluorescence changes of SA1 in different pH values, response time and stability experiment of SA1 and SA4 respectively


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ACKNOWLEGEMENT This work was financially supported by NSFC (21421005, 21406028, and 21136002), Doctoral Scientific Fund (20130041120014), General Project of Liaoning Province Department of Education (L2013024), and the Fundamental Research Funds for the Central Universities (DUT14ZD214).

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Scheme 1. Chemical structure of R1 and synthesis of SA1−4

Figure 1. (a) Absorption (dots) and fluorescence emission (lines) spectra of SA1−4 (10 µM) in PBS (10 mM, pH 7.4); (b) Fluorescence emission of SA1−4 (10 µM) in the presence of HSA (300 mg/L) in PBS (10 mM, pH 7.4); (c) Photos of SA1−4 in the absence (up) and presence (down) of HSA in PBS (10 mM, pH 7.4) under UV irradiation (365 nm); (d) Fluorescence of SA1 (10 µM) in the absence and presence of different contents of HSA (0, 5, 10, 20, 30, 45, 75, 105, 135, 165, 215, 315, 415, and 615 mg/L), excitation wavelength is 428 nm in PBS (10 mM, pH 7.4); e) Photos of solid SA1−4 in the absence (up) and presence (down) of HSA under UV irradiation (365 nm).

Figure 2. (a) Increase of F/F0 for SA1−4 (10 µM) in the presence of HSA contents over 50−2000 mg/L; (b) Fluorescence increase of SA1−4 (10 µM) in the presence of different proteins (5 µM); (c) Job’s plot of SA1 for HSA, [SA1] + [HSA] = 10 µM; (d) Changes of F/F0 for R1/HSA, SA1/HSA, and SA4/HSA in the presence of Ibuprofen or Warfarin.


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Figure 3. (a) Changes of fluorescence for SA1 (10 µM) in the presence of different aqueous media with different 1,4-dioxane contents; (b) Changes of fluorescence for SA1 (10 µM) in the presence of different aqueous media with different glycerol contents.

Figure 4. Linear response of F690 nm for SA4 along with different HSA contents in urine over range of (a) 3.73−853.73 mg/L and (b) 3.73−173.73 mg/L.

Figure 5. Confocal fluorescence images of MCF-7 cells in the absence (a1−d1) and presence (a3−d3) of SA1/HSA, SA2/HSA, SA3/HSA, and SA4/HSA; and overlap photos (a4−d4) of bright field images (a2−d2) with fluorescence images (a3−d3).

Table 1. Photophysical parameters of R1 and SA1−4

Table 2. The determination of HSA content in urine samples based on SA4


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Scheme 1. Chemical structure of R1 and synthesis of SA1-4 183x97mm (200 x 200 DPI)


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Figure 1. (a) Absorption (dots) and fluorescence emission (lines) spectra of SA1-4 (10 µM) in PBS (10 mM, pH 7.4); (b) Fluorescence emission of SA1-4 (10 µM) in the presence of HSA (300 mg/L) in PBS (10 mM, pH 7.4); (c) Photos of SA1-4 in the absence (up) and presence (down) of HSA in PBS (10 mM, pH 7.4) under UV irradiation (365 nm); (d) Fluorescence of SA1 (10 µM) in the absence and presence of different contents of HSA (0, 5, 10, 20, 30, 45, 75, 105, 135, 165, 215, 315, 415, and 615 mg/L), excitation wavelength is 428 nm in PBS (10 mM, pH 7.4); e) Photos of solid SA1-4 in the absence (up) and presence (down) of HSA under UV irradiation (365 nm). 291x307mm (200 x 200 DPI)



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Figure 2. (a) Increase of F/F0 for SA1-4 (10 µM) in the presence of HSA contents over 50-2000 mg/L; (b) Fluorescence increase of SA1-4 (10 µM) in the presence of different proteins (5 µM); (c) Job’s plot of SA1 for HSA, [SA1] + [HSA] = 10 µM; (d) Changes of F/F0 for R1/HSA, SA1/HSA, and SA4/HSA in the presence of Ibuprofen or Warfarin. 277x219mm (200 x 200 DPI)


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Figure 3. (a) Changes of fluorescence for SA1 (10 µM) in the presence of different aqueous media with different 1,4-dioxane contents; (b) Changes of fluorescence for SA1 (10 µM) in the presence of different aqueous media with different glycerol contents. 275x103mm (200 x 200 DPI)



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Figure 4. Linear response of F690 nm for SA4 along with different HSA contents in urine over range of (a) 3.73-853.73 mg/L and (b) 3.73-173.73 mg/L. 276x103mm (200 x 200 DPI)


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Figure 5. Confocal fluorescence images of MCF-7 cells in the absence (a1-d1) and presence (a3-d3) of SA1/HSA, SA2/HSA, SA3/HSA, SA4/HSA; as well as overlap photos (a4-d4) of bright field images (a2-d2) with fluorescence images (a3-d3). 213x221mm (200 x 200 DPI)



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Table 1. Photophysical parameters of R1 and SA1-4 175x81mm (200 x 200 DPI)


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Table 2. The determination of HSA content in urine samples based on SA4 150x66mm (200 x 200 DPI)



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TOC 154x68mm (200 x 200 DPI)


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Microenvironment-Sensitive Fluorescent Dyes for Recognition of

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