English

• 1.   Introduction

  As one of the most essential elements in biological systems of human body, the Fe³⁺ ions perform crucial role in oxygen carrying capacity and act as a cofactor in many enzymatic electron transfer as well as oxidation reactions^{[1, 2]}. One hand, deficiency in Fe³⁺ ions could cause degradation of oxygen transportation in cells, especially in anemia, liver damage, diabetes, and cancers.^[3~5] The other hand, excessive accumulation of Fe³⁺ ions in cells causes a Fenton reaction, resulting in generation of reactive oxygen species (ROS) which is the major aetiological factors for Alzheimer's disease or Parkinson's disease.^[6~8] Thus, the qualitative and quantitative analysis of Fe³⁺ ions is of great significance in clinical medical applications.^[9]

  At present, conventional detection methods of Fe³⁺ ions include voltammetry, atomic absorption spectrometry and inductively coupled plasma mass spectrometry.^[10~13] How- ever, these methods suffer from such problems as destruction of testing samples and interference by other ions during testing. Recently, fluorescent probe technology has been considered as one of the most effective detection methods of microelements in human body due to good selectivity, high sensitivity, low detection limits, capability of both in vitro and in vivo detections and facile instrument operability.^[14~17] Especially, the rapid development in confocal fluorescence microscopy and optical imaging push forward the application process of fluorescent probe technology in biomedical field.

  Till now, many kinds of fluorescent probes for Fe³⁺ ions have been reported. For example, the rhodamine derivate fluorescent probes, benzimidazoquinoline derivate fluorescent probes, benzimidazole derivate fluorescent probes.^[18~20] However, current fluorescent probes for Fe³⁺ions are limited by several disadvantages, e.g. small Stokes shift, poor performance for detections in pure water, and worse interference immunity of other ions. For example, the cross-sensitivity of Cu²⁺ and Al³⁺ ions during the Fe³⁺ ions detection is very difficult to eliminate, which is owing to analogous ion size of Fe³⁺ ions.^{[6, 21]} In addition, the working medium of current fluorescent probes for Fe³⁺ions is usually mainly composed of organic solvents, which can damage microorganisms, destroy cellular structure functional integrity, inhibit cell growth and even lead to cell death.^[22] Hence, it is necessary for designing and preparing hydrophilic fluorescent probe for Fe³⁺ ions with high selectivity. Furthermore, the reversibility ability of the fluorescent probes is one of the important properties for Fe³⁺ ions detection applications. Hence, it is necessary for designing and preparing hydrophilic and reversible fluorescent probe with high selectivity.

  Herein, we report a novel reversible fluorescent probe for detection of Fe³⁺ ions, which is composed of nanoparticles derived from fluorescence material of TA-DF-PDM. It exhibits high selectivity for Fe³⁺ ions in pure water, high sensitivity with detection limit of 0.9833 μmol/L, excellent biocompatibility and low cytotoxicity.

  2.   Results and discussion

  2.1   Morphology of TA-DF-BDM/PSMA NPs

  The diameter distributions of TA-DF-BDM/PSMA NPs were characterized by dynamic light scattering (DLS). As shown in Figure 1a, the average diameter of TA-DF- BDM/PSMA NPs is estimated to be 64 nm. And, the morphology of the TA-DF-BDM/PSMA NPs in aqueous solution was observed by transmission electron microscope (TEM). As expressed in Figures 1c and 1d, the grain shape of TA-DF-BDM/PSMA NPs is nearly sphere with average diameter of about 50 nm, which is slightly smaller than that obtained using DLS method. It is because that hydrodynamic diameter of the nanoparticles measured by DLS is usually larger than their size obtained by TEM.^[23] Furthermore, the DPI data (0.136) exhibits good hydrophilicity and dispersibility, which retain absence of nanoparticles aggregation in water even after being stored for months. In addition, the zeta potential of the TA-DF-BDM/ PSMA NPs was -23.3 mV (Figure 1b). For comparison, the PSMA-free TA-DF-BDM NPs was also prepared, which exhibited the zeta potential of -0.716 mV. It is suggested that the PSMA successfully incorporated into TA-DF- BDM and formed stable nanoparticles of TA-DF-BDM/ PSMA NPs.

  Figure 1

  

  Figure 1.  (a) DLS of TA-DF-BDM/PSMA NPs in aqueous solution, (b) comparison of zeta potentials of TA-DF-BDM NPs and TA-DF-BDM/PSMA NPs in aqueous solution, (c) TEM image of TA-DF-BDM NPs, and (d) TEM image of TA-DF-BDM/PSMA NPs

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  2.2   Photophysical properties of TA-DF-BDM/PSMA NPs

  The molecule of TA-DF-BDM is constructed with twi- sting D-π-A configuration, in which triphenylamine (TA), 9, 9-dioctylfluorene (DF) and benzylidenemalononitrile (BDM) serve as electron donor (D) unit, π conjugated unit, and electron acceptor (A) unit, respectively. TA-DF-BDM realizes blue-light emission by charger transfer from D unit of TA to A unit of BDM. As shown in Figure 2a, the UV-vis absorption spectrum of TA-DF-BDM exhibits absorption band at 350~420 nm, which attributes to charge transfer from TA to BDM. It induces in blue-light emission peak (λ_PL) located at 450 nm in PL spectrum of TA-DF- BDM. But, it can be found that the UV-vis absorption spectrum and PL spectrum of TA-DF-BDM show rather bigger overlap, which lead to self-absorption phenomenon. Additional, due to smaller Stokes shifts (ca. 26 nm), the blue-light emission of TA-DF-BDM will easily be interfered with background noise from excitation source during fluorescence imaging.^[23~25]

  Figure 2

  

  Figure 2.  UV-vis absorption spectra and PL spectra (a) TA-DF-BDM in THF; (b) TA-DF-BDM/PSMA NPs in aqueous solution

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  To solve above problem, the TA-DF-BDM/PSMA NPs were fabricated by reprecipitation method. It was known that the nanoparticles of fluorescence molecules can lead to bigger red-shift of emission peak relative to fluorescence molecules, owing to molecular accumulation. So, it can be seen from Figure 2b that the λ_PL of TA-DF-BDM/ PSMA NPs shows bathochromic shift from 450 nm to 608 nm, which separates with its UV-vis absorption spectrum totally. It expresses rather larger Stokes shifts (ca. 210 nm).^[24] Furthermore, the TA-DF-BDM/PSMA NPs were fabricated by reprecipitation method, which will also improve hydrophilicity and dispersibility for satisfying requirement for biological measurement environment.

  2.3   Photophysical stability of TA-DF-BDM/PSMA NPs

  The pH values of different human organisms are quite different, which range from weak acidic in the lysosome and endosome (pH=4.7~6.5) to weak basic in active mitochondria (pH=8.0).^[25] Hence, the photophysical properties TA-DF-BDM/PSMA NPs need to be stable in measurement scenes with different pH value. For testifying it, the PL spectra of TA-DF-BDM/PSMA NPs in solutions with different pH value were recorded. As shown in Figure 3, the PL spectra of TA-DF-BDM/PSMA NPs show no variation with increasing pH values from 4 to 11, for example, the λ_PL and emission intensity. It is indicated excellent photophysical stability of TA-DF-BDM/PSMA NPs in different pH value ^[26].

  Figure 3

  

  Figure 3.  PL spectra of TA-DF-BDM/PSMA NPs in solutions with different pH value

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  2.4   Fluorescence response to metal ions

  Selectivity is an essential prerequisite to evaluate a fluorescent probe before its practical application. To evaluate selectivity of TA-DF-BDM/PSMA NPs for Fe³⁺ ions, the PL spectra of TA-DF-BDM/PSMA NPs in aqueous solutions with presence of common environmental metal ions (i.e. Fe³⁺, Zn²⁺, Ag²⁺, Fe²⁺, Co²⁺, Na⁺, Pb²⁺, Hg²⁺, Ni²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Cu²⁺, Mg²⁺, Ca²⁺, K⁺, Al³⁺) were recorded, respectively.^{[22, 29]}

  The UV absorbance spectra of TA-DF-BDM/PSMA NPs (10 μmol/L) in the presence of different metal ions (1 equiv.) in aqueous solution were shown in Figure 4a. It is obvious that, in the absorption peaks, there was no evident change except for Fe³⁺ ions. As shown in Figure 4b, the λ_PL located at around 610 nm corresponding to red emission of TA-DF-BDM/PSMA NPs is nearly vanished upon addition of Fe³⁺ ions, which distinguishes with other ions. Hence, it is can be drawn that only Fe³⁺ ions can quench red-emission of TA-DF-BDM/PSMA NPs, however, other metal ions including Cu²⁺ ions exhibit no quenching phenomenon. It is suggested that TA-DF-BDM/PSMA NPs possess excellent selectivity for detecting Fe³⁺ ions in potential.

  Figure 4

  

  Figure 4.  (a) UV absorbance and (b) PL spectra and photographs under UV lamp (365 nm) of TA-DF-BDM/PSMA NPs (10 μmol/L) in the presence of different metal ions (1 equiv.) in aqueous solution; From left to right are Fe³⁺, Zn²⁺, Ag²⁺, Fe²⁺, Co²⁺, Na⁺, Pb²⁺, Hg²⁺, Ni²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Cu²⁺, Mg²⁺, Ca²⁺, K⁺ in the illustration; (c) PL spectra of TA-DF-BDM/PSMA NPs (25 μmol/L) exposed to various concentration of Fe³⁺ ions in aqueous solution; (d) fluorescence titration curve of TA-DF-BDM/PSMA NPs (25 μmol/L) with Fe³⁺ in aqueous solution, inset: the relationship between emission peak intensity and Fe³⁺ ions concentration; (e) PL intensity at 610 nm of TA-DF-BDM/PSMA NPs (10 μmol/L) exposed to 1.00 equiv. of various metal ions and mixtures of 1.00 equiv. of Fe³⁺ with 1.00 equiv. of other metal ions in aqueous solution

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  Furthermore, the fluorescence quenching behavior of Fe³⁺ ions was studied using fluorescence titration at fixed concentration of TA-DF-BDM/PSMA NPs with varying concentrations of Fe³⁺ ions. As expressed in Figure 4c, the PL intensity of red emission peak of TA-DF-BDM/PSMA NPs degrades with increasing Fe³⁺ ions concentration, which is almost fully quenched when the concentration of Fe³⁺ ions rises above 40 μmol/L. Under low concentrations of Fe³⁺ ions (6~22 μmol/L), the PL intensity of emission peak decreases rapidly and exhibits good linear (R=0.9945) relationship with concentrations of Fe³⁺ ions (Figure 4d). Hence, it is deduced that TA-DF-BDM/ PSMA NPs can act as fluorescent probe for quantitative detection of microscale Fe³⁺ ions. And, the detection limit was calculated from the equation DL=3δ/k, where δ is the standard deviation of the blank measurement and k is the slope between emission peak intensity versus Fe³⁺ ions concentration. The detection limit of TA-DF-BDM/PSMA NPs for Fe³⁺ ions is 0.9833 μmol/L.

  To investigate the interference of other metal ions, Fe³⁺ions were detected by TA-DF-BDM/PSMA NPs in presence of different ions. The PL spectra of TA-DF-BDM/ PSMA NPs were further measured in complicated systems in the presence of Fe³⁺ ions mixed with 1.00 equiv. of Fe³⁺, Zn²⁺, Ag²⁺, Fe²⁺, Co²⁺, Na⁺, Pb²⁺, Hg²⁺, Ni²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Cu²⁺, Mg²⁺, Ca²⁺, K⁺, Al³⁺ ions, respectively. As shown in Figure 4e, when other ions were added, the PL intensity of emission peak at 610 nm did not change much. The PL intensity decreased obviously after dropping of Fe³⁺ ions. To investigate the effect of pH on Fe³⁺detection, furthermore, we test the change of the solution fluorescence intensity adding the Fe³⁺ under variety of the pH. All these results confirm that the detection of TA-DF-BDM/PSMA NPs to Fe³⁺ions is exclusive, which possesses a potential to be applied in practice.

  2.5   Binding mode of TA-DF-BDM with Fe³⁺ ions

  The Fe³⁺ ion is a type of paramagnetic metal ion, and its complex with TA-DF-BDM can affect the proton signals close to the Fe³⁺ binding site. As shown in Figure 5, the data revealed that proton signal of H_a at δ 8.589 shifted upfield to δ 8.550 and became board after the addition of 1.00 equiv. of Fe³⁺ ions. This might be attributed to coordination reaction between Fe³⁺ions and malononitrile of BDM unit in TA-DF-BDM, which can frustrate twisting D-π-A configuration and restrain emission originating from charge transfer from TA to BDM.^[27]

  Figure 5

  

  Figure 5.  ¹H NMR spectra in DMSO-d₆ of (a) TA-DF-BDM, (b) TA-DF-BDM+0.50 equiv. of Fe³⁺, and (c) TA-DF-BDM+1.00 equiv. of Fe³⁺

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  To determine the stoichiometry of TA-DF-BDM and Fe³⁺ ion, Job's method was employed. A series of mixture solutions of TA-DF-BDM and Fe³⁺ ion with various molar fractions were prepared by keeping the total TA-DF- BDM and Fe³⁺ ion concentration constant of 10 μmol/ L.^{[28, 29]} The PL in intensity of emission peak at 450 nm was recorded for each solution. As expressed in Figure 6, it was found that the maximum PL intensity was observed when the molar fraction of Fe³⁺ ions reached 0.48, indicating that 1:1 stoichiometry for the complex of TA-DF-BDM to Fe³⁺ ions. It is owning to coordination reaction between Fe³⁺ ions with malononitrile of BDM unit.

  Figure 6

  

  Figure 6.  Job's plot of TA-DF-BDM for Fe³⁺ in aqueous solution

  [TA-DF-BDM]+[Fe³⁺]=10 μmol/L. X is the molar fraction of TA-DF-BDM, I₀ and I indicate the emission intensity at 450 nm before and after addition of Fe³⁺ ions, respectively

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  2.6   Fluorescence reversibility of TA-DF-BDM/PSMA NPs

  The 200 μL of aqueous solution of Fe³⁺ ions with concentration of 10 μmol/L was injected into aqueous solution of TA-DF-BDM/PSMA NPs (names as Solution S-NP) to acquire solution S-NP-Fe. By comparing with PL spectra of solutions of S-NP and S-NP-Fe in Figure 7a, it can be found that the emission peak located at 610 nm corresponding to TA-DF-BDM/PSMA NPs vanished after injection of Fe³⁺ ions, which had been identified as above work. Then, 200 μL of L-ascorbic acid aqueous solution with concentration of 0.28 μmol/L was injected into solution S-NP-Fe to acquire solution S-NP-Fe-LAA. Then, it was observed that the emission peak located at 610 nm appeared again after injection of L-ascorbic acid. So, it is suggested the TA-DF-BDM/PSMA NPs have good reversibility of fluorescence probes for detection of Fe³⁺ ions. According to the reversibility of the fluorescent probe, it can be expected that composite probe of solution S-NP-Fe can also be used to detect L-ascorbic acid qualitatively and quantitatively as expressed in Figure 7b.

  Figure 7

  

  Figure 7.  (a) Fluorescence reversibility of TA-DF-BDM/PSMA NPs and photographs of TA-DF-BDM/PSMA NPs under UV excitation; (b) fluorescence titration curve of TA-DF-BDM/ PSMA NPs+Fe³⁺ (10 μmol/L) with LAA in aqueous solution

  Inset: the relationship between emission peak intensity and LAA concentration

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  For understanding the fluorescence reversibility, electron paramagnetic resonance (EPR) tests were carried on TA-DF-BDM/PSMA NPs.^[30] As shown in Figure 8a, the EPR spectrum of solution S-NP-Fe exhibits a strong paramagnetic signal in the area of 2500~3500 GS, due to paramagnetic effect Fe³⁺ ions. However, solution S-NP- Fe-LAA shows weaker paramagnetic signal. Upon addition of L-ascorbic acid, the paramagnetic signal of Fe³⁺ ions is weakened. It is because that L-ascorbic acid is a strong reducing agent that leads to transformation from Fe³⁺ ions to Fe²⁺ ions (as shown in Figure 8b).

  Figure 8

  

  Figure 8.  (a) EPR spectra of solution S-NP-Fe and solution S-NP-Fe-LAA, and (b) EPR spectra of solution S-Fe and solution S-Fe-LAA

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  2.7   Detection of Fe³⁺ ions in human serum samples

  For evaluating the feasibility of TA-DF-BDM/PSMA NPs for detection Fe³⁺ ions in complex biological environment, the chemosensory based on TA-DF-BDM/PSMA NPs was challenged by human serum.^[31] After human serum samples were centrifuged and removed the protein precipitate, the supernatant was diluted 100-fold with Tris- HCI buffer (10 mmol/L, pH=7.19). Then, 150 μL of aqueous solution containing TA-DF-BDM/PSMA NPs (20 μmol/L) and different concentration of Fe³⁺ ions dropped in 150 μL of diluted human serum. The PL spectra of testing sample were measured and the fluorescence titration curve was fitted. As shown in inset of Figure 9b, the unknown concentration of Fe³⁺ ions was obtained according to the relationship between the PL intensity of emission peak at 610 nm and concentration of Fe³⁺ ion. It can be seen from Table 1 that the recovery is in the range of 94.9%~108.2% and the relative standard deviation (RSD, n=5) is less than 3.5%, indicating high analytical precision of TA-DF-BDM/PSMA NPs.

  Figure 9

  

  Figure 9.  (a) PL spectra of TA-DF-BDM/PSMA NPs (10 μmol/ L) exposed to various concentration of Fe³⁺ ions in human serum; (b) fluorescence titration curve of TA-DF-BDM/PSMA NPs (10 μmol/L) with Fe³⁺ in human serum, inset: the relationship between PL intensity of emission peak at 610 nm and concentration of Fe³⁺ ions

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  Table 1

  

  Table 1.  Testing results for the determination of Fe³⁺ ions in human serum samples

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  No.	Added/(μmol•L-1)	Found/(μmol•L-1)	Recovery/%	RSD/%
  1	10	10.83	108.2	2.9
  2	20	19.35	96.7	3.5
  3	26	24.68	94.9	1.8
  4	50	50.19	100.4	1.2

  2.8   Cellular imaging

  The toxicity of TA-DF-BDM/PSMA NPs is a natural concern because of its potential for bioimaging. The cytotoxicity of TA-DF-BDM/PSMA NPs was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays using HeLa cells at various concentration for 24 h.^[27] In Figure 10, the results show that TA-DF-BDM/PSMA NPs is nontoxic to the cultured cells under the experimental conditions. We also used HeLa cells as model cells to study the subcellular distributions of TA-DF-BDM/PSMA NPs by confocal fluorescence microscopy. Herein, cultured HeLa cells were incubated with TA-DF-BDM/PSMA NPs (10 μmol/L) for 30 min under 37 ℃. As predicted, confocal fluorescence microscopy images showed that TA-DF-BDM/PSMA NPs with fluorescence were cell membrane permeable as shown in Figure 11. In a control experiment, the cells were pretreated with TA-DF-BDM/PSMA NPs for 30 min, and further incubated with Fe³⁺ (10 μmol/L) for 30 min, eliciting an obvious fluorescence decrease in the range of 550~650 nm, in agreement with the Fe³⁺ ions induced fluorescence response. The L-ascorbic acid was added to the cells incubate for 30 min, and the fluorescence reversibility is at 550~650 nm.

  Figure 10

  

  Figure 10.  Cytotoxicity test of HeLa cells treated with various concentrations of TA-DF-BDM NPs after 24 h

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  Figure 11

  

  Figure 11.  Fluorescence imaging of HeLa cells incubated with TA-DF-BDM/PSMA NPs (10 μmol/L) before (a) or after (b) addition of Fe³⁺ ions (10 μmol/L) and (c) addition of L-ascorbic acid

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  3.   Conclusions

  The reversible fluorescent probe for Fe³⁺ ions is pre- pared, i.e. TA-DF-BDM/PSMA NPs which is composed of nanoparticles based on fluorescence material of TA-DF- BDM. In this work, the fluorescence material of TA-DF- BDM was synthesized by Suzuki coupling reaction firstly, which was made into nanoparticles by reprecipitation method to acquire TA-DF-BDM/PSMA NPs. The spectral properties of TA-DF-BDM/PSMA NPs were discussed in detail. This fluorescence probe based on TA-DF-BDM/ PSMA NPs exhibited such properties as red emission (λ_PL=610 nm), larger Stokes shift (210 nm), excellent photophysical stability in different pH value, good selectivity, low detection of Fe³⁺ ions (0.9833 μmol/L) in pure water. In PL spectra, the PL intensity of red emission peak of TA-DF-BDM/PSMA NPs degrades with increasing Fe³⁺ ions concentration. It is attributed to coordination reaction between Fe³⁺ ions and malononitrile of BDM unit in TA-DF-BDM, which can frustrate the twisting D-π-A configuration and restrain red-emission originating from charge transfer from TA to BDM. Additionally, TA-DF- BDM/PSMA NPs have good reversibility of fluorescence probes for detection of Fe³⁺ ions. By human serum measurements and cellular imaging, it can be found that TA- DF-BDM/PSMA NPs shows high analytical precision, good biocompatibility and low cytotoxicity. Above all, TA-DF-BDM/PSMA NPs exhibits a huge potential to be applied in biomedical practice.

  4.   Experimental

  4.1   Synthetic section

  All reagents and organic solvents were purchased from commercial chemical reagent company and used without further purification. The synthetic route of TA-DF-BDM is shown in Figure 12.

  Figure 12

  

  Figure 12.  Synthetic route of TA-DF-BDM

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  4.1.1   Preparation of 4-(7-bromo-9, 9-dioctylfluoren-2- yl)-N, N-diphenylaniline (M1)

  The triphenylamine 4-borate (0.371 g, 1 mmol) and 9, 9- dioctyl-2, 7-dibromofluorene (1.192 g, 2 mmol) were dissolved in 15 mL of toluene, and K₂CO₃ aqueous solution (2 mol/L, 10 mL) was dropped into it. Then, tetrakistriphenylphosphine palladium (Pd(PPh₃)₄) (0.057 g, 0.05 mmol) was added into above reaction mixture. After refluxing for 4 h under nitrogen protection, the reaction mixture was poured into water and extracted with dichloromethane. The collected organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. Finally, the resultant residue was purified by silica gel column chromatography using mixture eluents of ethyl acetate and hexane (V:V=1:20) to afford M1 as intermediate product, 0.3123 g, 74.2% yield. ¹H NMR (600 MHz, DMSO-d₆)δ: 7.86 (d, J=7.9 Hz, 1H), 7.77 (d, J=8.1 Hz, 1H), 7.72 (d, J=1.3 Hz, 1H), 7.64 (ddd, J=16.5, 10.7, 8.7 Hz, 4H), 7.51 (dd, J=8.0, 1.8 Hz, 1H), 7.36~7.29 (m, 4H), 7.10~7.02 (m, 8H), 2.04 (tt, J=13.3, 8.3 Hz, 4H), 1.25~0.95 (m, 20H), 0.75 (t, J=7.2 Hz, 6H), 0.49 (s, 4H). Anal. cald for C₄₇H₅₄BrN: C 79.22, H 7.59, Br 11.22, N 1.97; found C 79.17, H 7.72, Br 11.22, N 1.97.

  4.1.2   Preparation of 4-(7-(4-(diphenylamino)phenyl)- 9, 9-dioctylfluoren-2-yl)benzaldehyde (M2)

  The intermediate product of M1 (0.381 g, 0.5 mmol) and 4-acylphenylboronic acid (0.234 g, 1 mmol) were dissolved in 15 mL of toluene, which mixed with K₂CO₃ aqueous solution (2 mol/L, 10 mL). Then, Pd(PPh₃)₄ (0.057 g, 0.05 mmol) was added into reaction mixture, which was been refluxing overnight under nitrogen protection. Then, the resulting mixture was poured into water and extracted with dichloromethane. The collected organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. Finally, the resultant residue was purified by silica gel column chromatography using ethyl acetate and hexane (V:V=1:15) as eluents to afford M2 as intermediate product, 0.2308 g, 60.6% yield. ¹H NMR (600 MHz, CDCl₃) δ: 10.08 (s, 1H), 8.00~7.96 (m, 2H), 7.85~7.82 (m, 2H), 7.81~7.75 (m, 2H), 7.64 (dd, J=7.9, 1.7 Hz, 1H), 7.61~7.54 (m, 5H), 7.30~7.27 (m, 3H), 7.26 (d, J=4.3 Hz, 1H), 7.20~7.13 (m, 6H), 7.04 (tt, J=7.5, 1.1 Hz, 2H), 2.08~1.98 (m, 4H), 1.21~1.00 (m, 20H), 0.78 (t, J=7.2 Hz, 6H), 0.70 (t, J=10.6 Hz, 4H). Anal. cald for C₅₄H₅₉NO: C 87.92, H 8.01, N 1.90, O 2.17; found C 87.75, H 8.12, N 1.90, O 2.17.

  4.1.3   Preparation of TA-DF-BDM

  The intermediate product of M2 (0.246 g, 0.33 mmol) and malononitrile (0.066 g, 1 mmol) were dissolved into mixture solution of absolute ethanol (15 mL) and tetrahydrofuran (THF) (5 mL). The reaction mixture was keeping refluxing for 3 h. Then, the solvent was concentrated under reduced pressure. Finally, The resultant residue was purified by silica gel column chromatography using ethyl acetate and hexane (V:V=1:15) as eluents to afford TA- DF-BDM as yellow solid powder 0.2206 g, 69.7% yield. m.p. 166.2~168.6 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 8.02 (d, J=8.4 Hz, 2H), 7.85 (t, J=8.3 Hz, 2H), 7.81 (t, J=3.9 Hz, 2H), 7.78 (d, J=7.9 Hz, 1H), 7.65 (dd, J=7.9, 1.7 Hz, 1H), 7.62~7.54 (m, 5H), 7.31~7.26 (m, 4H), 7.17 (ddd, J=9.7, 7.6, 1.5 Hz, 6H), 7.07~7.03 (m, 2H), 2.04 (dd, J=9.7, 6.8 Hz, 4H), 1.19~1.02 (m, 20H), 0.78 (t, J=7.2 Hz, 6H), 0.72~0.64 (m, 4H). Anal. cald for C₅₇H₅₉N₃: C 87.13, H 7.51, N 5.35; found C 87.23, H 7.53, N 5.27.

  4.2   Preparation of nanoparticles of TA-DF-BDM/ PSMA

  As shown in Figure 13, the TA-DF-BDM/PSMA nanoparticles (named as TA-DF-BDM/PSMA NPs) were prepared by reprecipitation method. First, TA-DF-BDM and PMSA were dissolved into THF to obtain a solution with concentration of 10^-3 mol/L. Then, 100 μL of THF solution of TA-DF-BDM and PSMA (5:1, m/m) was added quickly to deionized water (10 mL) during ultrasonication.The THF was removed by heating under nitrogen protection, followed by filtration through a 0.22 μm aqueous phase needle filter. And, the resulting TA-DF-BDM/ PSMA NPs solution was acquired.

  Figure 13

  

  Figure 13.  Illustration of preparation process of TA-DF-BDM/ PSMA NPs

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  4.3   Measurement section

  The aqueous solutions of FeCl₃, Zn(NO₃)₂, AgNO₃, FeCl₂•7H₂O, CoCl₂•6H₂O, NaCl, Pb(NO₃)₂, HgCl₂, Ni(NO₃)₂•6H₂O, CdCl₂•2.5H₂O, MnSO₄, BaCl₂, CuSO₄• 5H₂O, MgCl₂, CaCl₂, KCI, and Al(NO₃)₃•9H₂O with a concentration of 10^-3 mol/L were respectively prepared. Before spectral measurement, the high-concentration stock solution was diluted to the concentration required.

  The ¹H NMR spectra were recorded by an AVANCE 600 MHZ spectrometer. The infrared (IR) absorption spectra were measured using a Bruker TENSOR 27. The average diameter of TA-DF-BDM/PSMA NPs was determined by the dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90). The morphology of TA-DF-BDM/PSMA NPs was investigated by transmission electron microscope (TEM) (JEOL JEM-2010, 200 KV). The ultraviolet-visible (UV-vis) absorption spectra were recorded on a Hitachi U-3900 scanning spectrophotometer. Fluorescence spectra were measured on a HORIBA FluoroMax-4 fluorescence spectrometer, and the fluorescence spectra were all excited by 365 nm.

  4.4   MTT assay

  Cytotoxicity studies on TA-DF-BDM/PSMA NPs were performed using MTT assay. The HeLa Cells (10⁶ cell per mL) were dispersed within replicate 96-well microtiter plates to a total volume of 200 mL/well. The plates were maintained at 37 ℃ in incubator with mixture gas of CO₂ and air (V:V=5:95) for 4 h. The TA-DF-BDM/PSMA NPs were diluted to different concentrations with medium and added to each well after removing original medium. HeLa cells were incubated with probe concentrations for 24 h. The concentrations of the TA-DF-BDM/PSMA NPs were 2.5~30 μmol/L, and 200 mL of MTT solution (5.0 mg•mL^-1, HEPES) was added to each well. After 4 h, the remaining MTT solution was removed, and 150 mL of DMSO was added into each well to dissolve the formazan crystals. Absorbance was measured at 610 nm in a TRITURUS (Bio-Rad, Model 550) microplate reader.

  4.5   Cell research

  The HeLa cells were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum) at 37 ℃ in a humidified environment of 5% CO₂. The cells were plated on 6-well plates at 5×10⁴ cells per well and allowed to adhere for 12 h. TA-DF-BDM/PSMA NPs (0.5 μL, 10 μmol/L) were added to the cell medium (500 μL) at 10 mmol/L final concentration. After incubating for 60 min, excess TA-DF-BDM/PSMA NPs were removed by gentle rinsing with cold phosphate buffer saline (PBS, pH=7.4) three times. Then, the cells pretreated with TA- DF-BDM/PSMA NPs were treated with Fe³⁺ (50 μmol/L) in DMEM medium, incubated for further 30 min at 37 ℃ and washed with PBS buffer. Fluorescence images were collected by sequential line scanning using the Olympus FV1000 confocal laser-scanning microscope. Fluorescence images were recorded at red channels (550~650 nm).

  Supporting Information  ¹H NMR spectrum of M1, M2 and TA-DF-BDM, ¹³C NMR spectrum of TA-DF-BDM in CDCl₃, IR and ESI-MS spectra of TA-DF-BDM. Elements composites of M1 and M2, the high resolution ESI-mass spectrum of TA-DF-BDM, the change of the solution fluorescence intensity adding the Fe³⁺ ion under variety of the pH and the reducibility of glutathione at different concentrations.

  
  
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• 

  Figure 1  (a) DLS of TA-DF-BDM/PSMA NPs in aqueous solution, (b) comparison of zeta potentials of TA-DF-BDM NPs and TA-DF-BDM/PSMA NPs in aqueous solution, (c) TEM image of TA-DF-BDM NPs, and (d) TEM image of TA-DF-BDM/PSMA NPs

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  Figure 2  UV-vis absorption spectra and PL spectra (a) TA-DF-BDM in THF; (b) TA-DF-BDM/PSMA NPs in aqueous solution

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  Figure 3  PL spectra of TA-DF-BDM/PSMA NPs in solutions with different pH value

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  Figure 4  (a) UV absorbance and (b) PL spectra and photographs under UV lamp (365 nm) of TA-DF-BDM/PSMA NPs (10 μmol/L) in the presence of different metal ions (1 equiv.) in aqueous solution; From left to right are Fe³⁺, Zn²⁺, Ag²⁺, Fe²⁺, Co²⁺, Na⁺, Pb²⁺, Hg²⁺, Ni²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Cu²⁺, Mg²⁺, Ca²⁺, K⁺ in the illustration; (c) PL spectra of TA-DF-BDM/PSMA NPs (25 μmol/L) exposed to various concentration of Fe³⁺ ions in aqueous solution; (d) fluorescence titration curve of TA-DF-BDM/PSMA NPs (25 μmol/L) with Fe³⁺ in aqueous solution, inset: the relationship between emission peak intensity and Fe³⁺ ions concentration; (e) PL intensity at 610 nm of TA-DF-BDM/PSMA NPs (10 μmol/L) exposed to 1.00 equiv. of various metal ions and mixtures of 1.00 equiv. of Fe³⁺ with 1.00 equiv. of other metal ions in aqueous solution

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  Figure 5  ¹H NMR spectra in DMSO-d₆ of (a) TA-DF-BDM, (b) TA-DF-BDM+0.50 equiv. of Fe³⁺, and (c) TA-DF-BDM+1.00 equiv. of Fe³⁺

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  Figure 6  Job's plot of TA-DF-BDM for Fe³⁺ in aqueous solution

  [TA-DF-BDM]+[Fe³⁺]=10 μmol/L. X is the molar fraction of TA-DF-BDM, I₀ and I indicate the emission intensity at 450 nm before and after addition of Fe³⁺ ions, respectively

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  Figure 7  (a) Fluorescence reversibility of TA-DF-BDM/PSMA NPs and photographs of TA-DF-BDM/PSMA NPs under UV excitation; (b) fluorescence titration curve of TA-DF-BDM/ PSMA NPs+Fe³⁺ (10 μmol/L) with LAA in aqueous solution

  Inset: the relationship between emission peak intensity and LAA concentration

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  Figure 8  (a) EPR spectra of solution S-NP-Fe and solution S-NP-Fe-LAA, and (b) EPR spectra of solution S-Fe and solution S-Fe-LAA

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  Figure 9  (a) PL spectra of TA-DF-BDM/PSMA NPs (10 μmol/ L) exposed to various concentration of Fe³⁺ ions in human serum; (b) fluorescence titration curve of TA-DF-BDM/PSMA NPs (10 μmol/L) with Fe³⁺ in human serum, inset: the relationship between PL intensity of emission peak at 610 nm and concentration of Fe³⁺ ions

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  Figure 10  Cytotoxicity test of HeLa cells treated with various concentrations of TA-DF-BDM NPs after 24 h

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  Figure 11  Fluorescence imaging of HeLa cells incubated with TA-DF-BDM/PSMA NPs (10 μmol/L) before (a) or after (b) addition of Fe³⁺ ions (10 μmol/L) and (c) addition of L-ascorbic acid

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  Figure 12  Synthetic route of TA-DF-BDM

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  Figure 13  Illustration of preparation process of TA-DF-BDM/ PSMA NPs

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  Table 1.  Testing results for the determination of Fe³⁺ ions in human serum samples

  No.	Added/(μmol•L-1)	Found/(μmol•L-1)	Recovery/%	RSD/%
  1	10	10.83	108.2	2.9
  2	20	19.35	96.7	3.5
  3	26	24.68	94.9	1.8
  4	50	50.19	100.4	1.2

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