Polydopamine Dots-Based Fluorescent Nanoswitch Assay for

26 Sep 2018 - We developed a facile and feasible fluorescent nanoswitch assay for reversible recognition of glutamate (Glu) and Al3+ in human serum an...
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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

Polydopamine Dots-Based Fluorescent Nanoswitch Assay for Reversible Recognition of Glutamic Acid and Al3+ in Human Serum and Living Cell Qiaoqiao Ci,† Jinhua Liu,*,†,‡ Xiaofei Qin,† Linqi Han,† Hai Li,† Haidong Yu,† Kah-Leong Lim,§ Cheng-Wu Zhang,*,† Lin Li,*,† and Wei Huang†,∥

ACS Appl. Mater. Interfaces 2018.10:35760-35769. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 10/31/18. For personal use only.

†

Key Laboratory of Flexible Electronics (KLOFE), Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China ‡ State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China § Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593 ∥ Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), 127 West Youyi Road, Xi’an 710072, China S Supporting Information *

ABSTRACT: We developed a facile and feasible fluorescent nanoswitch assay for reversible recognition of glutamate (Glu) and Al3+ in human serum and living cell. The proposed nanoswitch assay is based on our recently developed method for controlled synthesis of fluorescent polydopamine dots (PDADs) at room temperature with dopamine as the sole precursor. The fluorescence of nanoswitch assay could be quickly and efficiently quenched by Glu (turn-Off), and the addition of Al3+ could recover the fluorescence of the PDADs−Glu system (turn-On). Meanwhile, the reversible recognition of Glu and Al3+ in this nanoswitch system was stable after three cycles. Additionally, the system displayed excellent performance for Glu and Al3+ determination with a low detection limit of 0.12 and 0.2 μM, respectively. Moreover, PDADs are successfully applied to determine Glu and monitor Al3+ in human serum. Noteworthy, the nanoswitch assay is transported into HepG2 cells and realized “Off” detection of Glu and “On” sensing Al3+ in the living cells. Therefore, this PDADs-based nanoswitch assay provides a strategy to develop reversible recognition biosensors for intracellular and external molecular analysis. KEYWORDS: polydopamine dots, nanoswitch assay, reversible recognition, glutamic acid, Al3+

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INTRODUCTION The use of polymer fluorescent nanoparticles (PFNPs) in chemical sensors, fluorescence (FL) imaging, drug delivery, and bioanalysis application has aroused great interest because of their low cytotoxicity, easy surface functionalization, and low cost.1−7 PFNPs are mainly composed of conjugated polymers, resulting in strong fluorescence emission.8−13 Although some conjugated polymers exhibit good water solubility,14,15 most conjugated polymers are facing the challenge of poor water solubility, and thus functional modifications are inevitable for further analysis and biological applications. Therefore, a progressive strategy of promoting water solubility is to generate nonconjugated polymer nanoparticles (NCPNs). In recent years, the applications of numerous NCPNs in biosensors, nucleic acid, and drug delivery have been widely investigated.16−18 However, some defects are still remain including sophisticated synthetic procedure, environmentally harmful organic solvents, and low fluorescence quantum yields (QYs) © 2018 American Chemical Society

in aqueous solution. Hence, developing autofluorescent polymer materials with ideal water solubility, simple preparation, and high fluorescence QYs still remains very challenging. Dopamine (3,4-dihydroxyphenylethylamine, DA), a member of the catecholamines family, is an important chemical neurotransmitter.19−21 DA can be oxidized to dopaminequinone in an alkaline and aerobic condition, and the latter can be further polymerized to form polydopamine (PDA) by deprotonation and intermolecular Michael addition reaction (eq S1).22,23 To date, PDA as a quencher has been broadly used for RNA,24 DNA,25 ATP,26 cancer cells,27 and cerebral antioxidants detection in biological studies,28 particularly by coupling with DNA probes based on π−π stacking and hydrogen bonding interactions.29 In these PDA assays, the size of PDA is usually Received: July 19, 2018 Accepted: September 26, 2018 Published: September 26, 2018 35760

DOI: 10.1021/acsami.8b12087 ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

Research Article

ACS Applied Materials & Interfaces

Preparation of PDADs. Series of concentration of DA (0.01, 0.05, 0.10, 0.2, 0.5, and 1 M) were prepared and kept in the dark at room temperature. The absorption and fluorescence intensity of the PDADs were checked every 20 days. After reaction for 3 months, the DA solution (0.05 M) was transferred into dialysis bag with distilled water for 2 days to remove the unreacted dopamine, then the solution in the dialysis bag was frozen and dried. We prepared the PDADs stock solution by dissolving 23.7 mg of the PDADs powders in 5.0 mL highly pure water; the concentration of obtained PDADs solution was 4.7 mg/ mL. Quantum Yield (QY) Measurement of PDADs. QY was measured by comparing the fluorescence intensity of quinine sulfate in 0.1 M H2SO4 according to the standard protocol. The quantum yield of PDADs was obtained through the following equation35

about 100−200 nm. Compared to quantum dots, polymer dot (PD) has stronger fluorescence and the size of PD is tunable. Moreover, PD with a small size is desirable so that they will not disrupt the native behavior of the biomolecules labeled and can better penetrate and distribute in the intercellular spaces.30,31 Considering these strengths, the study of fluorescent polydopamine dots (PDADs) with small size is of great significance to the development of PD. As we know, only a few studies have addressed the preparation of PDADs with small size. Zhang et al. reported a top-down approach to produce fluorescent PD by the reaction between large polyamine nanoparticles and H2O2.32 Bayindir et al. used NaOH and HCl to initiate and stop the polymerization reaction, generating fluorescent polydopamine.33 Tseng et al. developed an approach to obtain fluorescent PDADs by hydroxyl radical to induce the degradation of polydopamine nanoparticles.34 However, these approaches often suffer from low fluorescence QY and complex process of synthesis. Therefore, developing a facile and controllable method to synthesize highly fluorescent PDADs remains a huge challenge. Herein, we reported a facile and controllable method to synthesize PDADs by using one-step oxidation of the sole precursor dopamine (DA) at room temperature and then further verified its application for high sensitivity and selectivity recognition of Glu and Al3+ based on an “Off−On” strategy. Our optimized strategy for PDADs synthesis was proven to be a simple and efficient method without needing complicated instrumentation or harsh reaction conditions. The prepared PDADs-based fluorescent probe displayed excellent performance for Glu and Al3+ detection in a broad range. Moreover, to demonstrate the practical application of PDADs, we further applied it to detect Glu and Al3+ in fluid samples and living cell.

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ΦP =

A FP × D × ΦD AP FD

(1)

where Φ refers to the fluorescence QY, F refers to the integrated area of emitted fluorescence spectra, and A refers to the absorbance at the excitation wavelength. The subscript P and D represent the PDADs and quinine sulfate, respectively. Fluorescence Response of PDADs to Glu and Al3+. In this sensing system, Glu was detected in Tris−HCl buffer solution (50 mM) of pH 7.0. In a typical experiment, PDADs (10 μL, 4.7 mg/mL) dispersion was added to the Tris−HCl buffer (490 μL) and then a fixed amount of Glu was added. The solution was mixed on a vortex mixer and incubated. The fluorescence spectra were obtained (Ex = 310 nm, Em = 425 nm). Determination of Al3+ with the PDADs−Glu sensor system was conducted in the Tris−HCl buffer. To obtain the assay, 10 μL of PDADs (4.7 mg/mL) dispersion and 10 μL of Glu solution (100 mM) were incubated with 480 μL of the Tris−HCl buffer as described previously. Then, solutions with various concentrations of Al3+ were prepared and their fluorescence was measured with wavelength of 310 nm excitation after 10 min of incubation. Determination of Glu and Al3+ in Human Serum. Human blood sample was obtained from the healthy adult at ZhongDa Hospital of Nanjing and then immediately stored frozen until assayed. To obtain the spiked samples for the standard addition recovery experiment, the blood was centrifuged (8000 rpm, 20 min) to separate the clear supernatant layer serum, followed by filtration using a 0.45 μm Millipore filter for further sample analysis. The obtained serum was mixed with different concentrations of Glu and Al3+ for further analysis. Cell Cytotoxicity Activity Assay. Cytotoxicity of PDADs was determined by using the Cell Counting XTT (XTT, a commercially available cell viability dye) in HepG2 cells following the protocol reported by Martha et al.36 Briefly, HepG2 cells were seeded in a 96well plate and cultured in the humidified incubator ventilated with 5% CO2 at 37 °C for 24 h. After that, the cells were incubated with different concentrations of PDADs (2, 5, 10, 15, 20, and 30 μg/mL) for 24 h. Then, the cells were washed and incubated in serum-free Dulbecco’s modified Eagle’s medium with 2% XTT for 2 h. The absorbance was obtained at 450 nm with a microplate reader and cell viability was obtained according to the equation

EXPERIMENTAL SECTION

Materials. Dopamine hydrochloride (DA) were obtained from BBI Life Sciences. The amino acids valine (Val), glycine (Gly), phenylalanine (Phe), leucine (Leu), alanine (Ala), serine (Ser), isoleucine (Ile), proline (Pro), threonine (Thr), methionine (Met), glutamic acid (Glu), tyrosine (Tyr), histidine (His), aspartic acid (Asp), glutamate (Glu), lysine (Lys), tryptophan (Trp), arginine (Arg), cysteine (Cys), asparagine (Asn), glutamine (Gln), homocysteine (Hcys), and the metal salts Zn(NO3)2·6H2O, Cr(NO3)3·9H2O, NaNO3, Ba(NO3)2, Mg(NO3)·6H2O, KNO3, NaCl, Al(NO3)3·9H2O, Ca(NO3)2·4H2O, Fe(NO3)3·9H2O, Mn(NO3)2·4H2O, Co(NO3)2·6H2O, Cd(NO3)2· 4H2O, Ni(NO3)2·6H2O, AgNO3, FeCl2·4H2O, Cu(NO3)·3H2O, and Pd(NO3)2 were obtained from Shanghai Titan Scientific Co. Ltd. (Shanghai, China). Trihydroxymethyl aminomethane (C4H11O3) was obtained from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, China). Hydrochloric acid (HCl) was ordered from Shanghai Lingfeng Chemical Reagent Co. Ltd. The filtration of human serum samples used 0.45 μm × 13 mm syringe filter PES (FILTER-BIO, Nantong, China). Apparatus. Transmission electron microscope (TEM) images were acquired by using a JEM-2010 (UHR, JEOE, Japan) with a 200 kV accelerating voltage and atomic force microscope (AFM, XE-70, Park, Korea) in the Scan Asyst mode was used to obtain the size and height of the PDADs. X-ray photoelectron spectroscopy (XPS) was carried on a PHI Quantum 5000 XPS system (Physical Electronics). XPS was used to characterize the elemental composition and bonding configuration. Fourier transform infrared (FTIR) were performed on a VECTOR 22 spectrophotometer (Bruker, Germany). Thermogravimetric analysis was used on a TGA2 analyzer (Mettler Toledo, Swit). Mass spectrography was performed on a 4000 Q-TRAP (AB Sciex). Fluorescence spectra were recorded in a microplate reader (Cytation5, BIOTEK). To get the emission spectra, excitation wavelength at 310 nm was adopted. UV−vis absorption spectra of PDADs was recorded in a microplate reader (Cytation5, BIOTEK).

cell viability rate =

A − A0 × 100% AS − A 0

(2)

Live Cells Fluorescence Imaging.37−39 HepG2 cells were cultured on a 20 mm diameter glass-bottom dish following the same conditions as mentioned above. After 48 h, PDADs (20 μg/mL) were added into the medium and incubated for 2 h. After that, the culture dish was washed three times with phosphate-buffered saline and mounted on the microscope stage for imaging. Fluorescence images were captured by a Zeiss LSM880 NLO confocal microscope system. To reveal the ability of PDADs to detect Glu and Al3+ in living HepG2 cells, the cells were treated with Glu (160 μM) about 30 min, followed by treatment with Al3+ (200 μM) for 30 min. After that, those cells were subjected to imaging. 35761

DOI: 10.1021/acsami.8b12087 ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

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Figure 1. TEM images of PDADs (A) and the HRTEM of PDADs (B). Insets of the fast Fourier transform (FFT), AFM images (C), and the height distribution of PDADs (D) and profile analyses (E) of PDADs.

Figure 2. (A) FTIR and (B) Raman of PDADs. (C) UV−vis absorption spectra (abs) of PDADs. (D) Fluorescence spectra of PDADs under different excitation wavelengths.

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RESULTS AND DISCUSSION

concentrations of PDADs were monitored every 20 days. As shown in Figures S1−S3, the color, absorption, and fluorescence changed with the extension of reaction time. The as-prepared PDADs exhibited strong blue fluorescence (QYs: 9.1%) in water. The TEM image displayed that PDADs had a relative

Synthesis and Characterization of PDADs. The PDADs were synthesized by natural oxidation of DA solution at room temperature for 3 months without using an additional oxidizing agent. The absorption and fluorescence intensity of different 35762

DOI: 10.1021/acsami.8b12087 ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

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Figure 3. (A) Time-dependent fluorescence responses of the PDADs upon the addition of Glu (0.8 mM). (B) Fluorescence emission spectra of PDADs in the presence of different concentrations of Glu in Tris−HCl buffer (50 mM, pH = 7.0). Inset: a linear relationship of (F1/F0)−1 versus the concentration of Glu over the range from 0.2 to 7 μM (λem = 425 nm). (C) Relatively fluorescence signal response to Glu and other amino acids in the PDADs system: 1, none; 2, Pro; 3, Tyr; 4, Ala; 5, Leu; 6, Val; 7, Cys; 8, Ser; 9, Glu; 10, Asp; 11, Ile; 12, Thr; 13, Arg; 14, Phe; 15, His; 16, Gly; 17, Lys; 18, Hcy; 19, Asn; 20, Gln; and 21, Met; the concentrations of all the amino acids is 800 μM. (D) Fluorescence intensity of PDADs−Glu system in the presence of different concentrations of NaCl in Tris−HCl buffer (50 mM, pH = 7.0).

Figure 4. (A) Time-dependent fluorescence responses of the PDADs−Glu system upon the addition of Al3+ (1 mM). (B) Fluorescence emission spectra of the PDADs−Glu system in the presence of different concentrations of Al3+ in Tris−HCl buffer (50 mM, pH 7.0). Inset: a linear relationship of F1′/F0′ versus the concentration of Al3+ over the range from 0.6 to 2.1 μM (λem = 425 nm). (C) Relatively fluorescence signal response to Al3+ and other metal ions in the PDADs−Glu system: 1, Fe3+; 2, Fe2+; 3, Ca2+; 4, Cu2+; 5, Co2+; 6, Ni2+; 7, Zn2+; 8, Ag+; 9, Cr+; 10, Na+; 11, none; 12, K+; 13, Mg2+; 14, Na+; 15, Hg2+; 16, Pd+; 17, Mn2+; and 18, Al3+; the concentrations of all the metal ions is 1 mM. (D) Fluorescence emission spectra of PDADs−Glu−Al3+ system in the presence of different concentrations of NaP2O7 in Tris−HCl buffer.

narrow size distribution ranging from 4 to 8 nm (Figure 1A), different from the PDA nanoparticles reported previously.40

High-resolution TEM (HRTEM) images disclosed that PDADs had clear lattice structures with fringe spacing of 0.21 nm, which 35763

DOI: 10.1021/acsami.8b12087 ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

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ACS Applied Materials & Interfaces

Figure 5. (A) Fluorescence emission spectra of PDADs under different experimental conditions: (a) PDADs; (b) PDADs + Glu; (c) PDADs + Glu + Al3+; (d) PDADs + Glu + Al3+ + EDTA; (e) PDADs + Glu + Al3+ + EDTA + Mg2+. (B) Fluorescence change of cycle detection of Glu and Al3+ in this Off−On fluorescence nanoswitch system.

Table 1. Determination of Glu and Al3+ in Serum Samples by PDADs samples

original

added (μM)

measured (μM)

recovery (%)

RSD (%) (n = 3)

l(Glu) 2(Glu) 3(Glu) 4(A13+) 5(A13+) 6(A13+)

10 10 10 1 1 1

0 10 20 0 1.5 3

10.77 20.06 28.67 1.043 2.410 3.901

107.7 100.3 95.57 104.3 96.40 97.52

1.38 3.36 3.48 4.62 5.12 3.87

PDADs have a positive charge (+11.49 mV). Raman spectroscopy is a commonly adopted technology to characterize the structural and electronic properties of carbon materials. The Raman spectrum of PDADs is shown in Figure 2B. The peak at 1352 cm−1 correlated to the D band, which was obtained from the A1g breathing mode of the sp3 carbon. The peak at 1578 cm−1 correlated to the G band, which correlated to the in-plane bondstretching of the C sp2 atoms. Typical feature of the mixture of amorphous carbon and nanocrystalline graphite are shown in the Raman spectrum, which was consistent with our HRTEM results. The UV−vis absorption is presented in Figure 2C. The synthetic PDADs solution exhibited absorption bands around 310 and 360 nm, the peak (310 nm) corresponded to the π−π* transition of CC/CN and the absorption at 322 nm was ascribed to the n−π* transition of the CO/C−NH 2 bond.41,42 To explore the optical properties of the as-synthesized PDADs, a detailed emission spectral assay with excitation wavelengths from 310 to 530 nm was performed and the data are summarized in Figure 2D. The fluorescence emission remained constant at 450 nm (Ex: 320−370 nm). But, the emission shifted from 450 to 610 nm, corresponding to Ex wavelengths ranging from 370 to 530 nm. The red shift of the emission wavelength was well known as the excitation-dependent-emission phenomenon of carbon dots, which could be underlaid by the different size distributions and “surface states” formed by diverse functional groups. These results indicated that the PDADs showed excitation-dependent fluorescence behaviors.43 Additionally, the PDADs showed a pH-independent fluorescence behavior (Figure S6). The fluorescence of PDADs increased with change in pH from 3 to 7 and decreased from 7 to 10, with the maximum fluorescence intensity appearing at pH = 7, with a slight red shift (30 nm) from pH 3−5 to 6−10. This pHindependent fluorescence behavior of the PDADs could be attributed to the presence of free zigzag sites, which shared comparable structure with carbon quantum dots reported previously. Therefore, the result suggested that the excitationdependent and pH-independent fluorescence behaviors might be ascribed to the surface states and the size effect of PDADs. We also try to investigate the reaction mechanism based on NMR, mass spectrometry, IR, and UV−vis results. First, the UV−vis spectra were recorded to study the effect of reaction time on the mechanism of PDADs formation (Figure S7A). The broad band (310−400 nm) was correlated with the formation of a 1,4 Michael addition between quinones and amines, which were subsequently oxidized and polymerized into PDADs.44

was the typical distance between graphite layers. The corresponding fast Fourier transform (FFT) pattern exhibited a hexagonal lattice, indicating that the PDADs had crystalline hexagonal structures (Figure 1B). The AFM imaging and typical section analysis of PDADs were also performed (Figure 1C−E). All the above results indicated that PDADs had spherical shapes with a height distribution from 6 to 8 nm. The elemental composition and valence state of the PDADs were identified by XPS spectra. As shown in Figure S4A, the PDADs mainly contain C, O, and N elements, with the composition of C (72.29 atom %), O (21.48 atom %), and N (6.24 atom %). The XPS spectrum of C 1s showed three peaks (284.6, 286.1, and 287.8 eV), which belonged to C−C/CC, C−N, and CO/CN, respectively, indicating the existence of heteroatoms in PDADs (Figure S4B). The XPS N 1s spectrum was divided into three peaks (398.4, 399.2, and 400.5 eV), contributing to pyridinic N, amino N, and pyrrolic N, respectively (Figure S4C). Moreover, the XPS O 1s spectrum contains two peaks at 531.7 and 532.5 eV, which can be assigned to CO and C−OH/C−O−C groups, respectively (Figure S4D). This was consistent with the results of CO in the C 1s spectrum. The contents and ratios of the surface groups in PDADs on the basis of the XPS results are given in Table S1. The Fourier transform infrared (FTIR) was acquired to determine the surface functional groups of the PDADs. As shown in Figure 2A, the broad band (3343−3226 cm−1) could belong to the N− H and −OH stretching vibration modes and the band at 2960 cm−1 was the result of the C−H stretching mode. The band at 1608 cm−1 could be attributed to −NH2. The peaks (814−1483 cm−1) could belong to the in-plane and the out of-plane bending. Much information was obtained from the nitrogen heteroaromatic skeleton. These results suggested that the surface of PDADs contained a great deal of oxygen and nitrogen by the natural oxidation. The ζ-potential of PDADs is investigated in Figure S5. The result indicated that the 35764

DOI: 10.1021/acsami.8b12087 ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

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ACS Applied Materials & Interfaces

Figure 6. Fluorescent imaging of HepG2 cells: fluorescence image of HepG2 cells and incubated with PDADs, PDADs/Glu, and PDADs/Glu/Al3+, respectively (a, d, g, j). Corresponding bright-field image of (b, e, h, k). Corresponding merge image of (c, f, i, l). Scale bars = 50 μm.

Scheme 1. Schematic Illustration of the Formation of PDADs and the Detection of Glu and Al3+ Based on the Off−On Strategy

formation, the DA solutions with different reaction times were lyophilized and investigated by FTIR spectrum (Figure S7B). In the FTIR spectrum, the intensity of peaks at 3347 and 3237 cm−1 of PDADs reduced, resulting from the primary amine

Figure S7A shows the relationship between the absorption intensity of PDADs and the reaction time, indicating the formation of more PDADs over time. To elucidate how the reaction time of DA polymerization affects the PDADs 35765

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equilibrium is described in eq S2. The hydrogen ion released from acidic amino acids could change the pH of the solution when the concentration reached a certain value. As pH decreased, the fluorescence intensity of PDADs decreased gradually, which was in agreement with our results that pH effect of PDADs. A control experiment for detecting Glu in aqueous solution using PDADs was implemented (Figure S9). The results indicated that the FL of PDADs was effectively quenched by Glu in the aqueous solution, which was in coordination with the mechanism of the release of more hydrogen ions from acidic amino acids.52 Moreover, the salt tolerance of the PDADs−Glu system was evaluated by adding different concentrations (0−1 M) NaCl (Figure 3D); there was little effect on the fluorescence of PDADs−Glu system. Thus, the fluorescence of PDADs quenched by Glu was not due to electrostatic interactions. To further reveal the quenching mechanism between PDADs and Glu, time-correlated single photon counting was used to determine the photoluminescence (PL) lifetimes for 310 nm excitation. The average PL lifetime of PDADs without and with Glu was 4.30 ns (tau 1 = 4.30 ns, 100%) and 5.48 ns, respectively (tau 1 = 0.74 ns, 23.44%; tau 2 = 6.93 ns, 76.56%) (Figure S10). Introduction of Glu reduced the nonradiative rate of PDADs, which led to an increasing in transition time from excited state to ground state and eventually an increase in PL lifetime of PDADs. The results indicated that the quenching mechanism of PDADs in the presence of Glu was static quenching due to small differences of 1.18 ns in the PL lifetime. Fluorescence Response to Al3+ in PDADs−Glu System. Glu was one of the most metabolic protein amino acids and a potential chelator for Al3+. Binding Glu ions to trace amounts of Al3+ might interfere with many metabolic processes in the organisms.53 Therefore, the “Off” fluorescence of the PDADs− Glu system was sensitive to the presence of Al3+ due to the complexation between Al3+ and Glu, causing the elimination of Glu and fluorescence recovery of PDADs. To determine the response of the fluorescence of PDADs to Al3+, 1 mM Al3+ was added to the PDADs−Glu system. A rapid fluorescence recovery of PDADs was observed within 2 min. After that, the fluorescence recovery slowed down and reached a steady state within 5 min (Figure 4A). To appreciate the sensitivity of the sensing system, Al3+ was added in different concentrations. The fluorescence signal enhanced accordingly with increase in Al3+ (Figure 4B). The linear relationship of the fluorescence intensity versus the Al3+ concentration is shown in Figure S11. The enhancement of fluorescence efficiency was defined as F1′/F0′, where F1′ and F0′ referred to the fluorescence intensities with and without Al3+ in the PDADs−Glu system, respectively. The enhancement of fluorescence correlated perfectly with the concentration of Al3+ (0−4 μM) (R2 = 0.9927), with a detection limit of 0.2 μM. The sensitivity of our method was superior to that reported by others (Table S3).54−59 To assess the selectivity of Al3+, the effects of other metal ions (Fe3+, Co2+, Ca2+, Cu+, Fe2+,Ni2+, Ag+, Cr+, Zn2+, Na+, Mg2+, K+, Na+, Hg2+, Pd+, and Mn2+) on the system were investigated. It could be clearly seen from Figure 4C that only the addition of Al3+ could induce significant FL enhancement, whereas addition of other metal ions did not change the fluorescence signal of the PDADs−Glu system. This was because Al3+ binds to Glu to form Glu−Al3+ complex, which caused separation of Glu from the surface of the PDADs and subsequent recovery of the fluorescence of PDADs. The results suggested that the current sensing system possess excellent selectivity for Al3+.

being transformed into secondary or tertiary amines. Noteworthy, in the spectra, we could not find appreciable feature peaks for secondary or tertiary amines. However, the FTIR spectrum of the polymerization with different reaction times were comparable with that of the raw DA powder in the 2400− 3400, 500, and 700 cm−1 regions. This might be attributed to the precipitation of residual DA in the PDADs during the drying process. Figure S7C,D show the 13C NMR spectra of the PDADs obtained at 6 and 3 months, respectively, suggesting that the PDADs consisted of indoline species. The characteristic peaks at 144 ppm were attributed to diphenolic phenoxy C atoms. The bands (115−135 ppm) correlated with the C atoms of arene and indole units. Moreover, the peaks (33−42 ppm) were derived from aliphatic species.45 Matrix-assisted laser desorption ionization time-of-flight mass spectra of PDADs obtained at 3 and 6 months also confirm the existence of mixed oligomers (Figure S7E,F).46 The spectrum of the PDADs obtained at 6 months showed a larger polymer than that obtained at 3 months, indicating a slow polymerization process with increase in time. PDADs-Based Fluorescence Determination of Glu. On the basis of enriched oxygen and nitrogen on the surface of PDADs, we developed an optical sensor for Glu by quenching the fluorescence of PDADs. To determine how Glu affects the fluorescence signal of the PDADs, the change in fluorescence was measured upon Glu addition. In the first 2 min, the fluorescence of the PDADs was quenched rapidly. After that, the quenching rate slowed down and reached a steady stage within 2 more min (Figure 3A). To determine the sensitivity of the PDADs toward Glu, fluorescence titration assay was carried out. The as-prepared PDADs showed a featured fluorescence emission peak at 425 nm (λex = 310 nm) (Figure 3B). Addition of Glu rendered prominent fluorescence quenching and the decrease in fluorescence intensity of PDADs linearly correlated with the increase of Glu, which might be due to the binding of Glu to the surface of PDADs. Figure S8 demonstrates this phenomenon. The Stern−Volmer equation described the mechanism of fluorescence quenching: F0/F1 = 1 + Ksv[Q], of which F0 and F1 represented the fluorescence intensity of PDADs without and with Glu, respectively. [Q] and Ksv were the concentrations of Glu and the Stern−Volmer constant, respectively. The calibration curve displayed a linear relationship (R2 = 0.9954) of (F1/F0)−1 versus the Glu concentration (0−100 μM) with a detection limit of 0.12 μM, which was comparable to other reported methods (Table S2).47−51 The results demonstrated that PDADs could serve as probes for Glu detection with high sensitivity. Besides sensitivity, specificity is another prerequisite for ideal probes. To confirm the specificity of PDADs, we evaluated the fluorescence of PDADs with 20 kinds of amino acids (Pro, Tyr, Ala, Thr, Leu, Ile, Cys, His, Ser, Arg, Glu, Phe, Asp, Asn, Val, Gln, Gly, Lys, Hcy, and Met) under the same conditions. As shown in Figure 3C, some amino acids (Pro, Tyr, Ala) only caused light fluorescence change, less than 20%, whereas some (Leu, Ile, Cys, Arg, Ser, Glu, Asp, Val, Thr, Asn, Phe, Gly, Lys, Hcy, His, Gln, and Met) induced some fluorescence quenching. Only Asp and Glu can lead to significant FL quenching of 78 and 55%, respectively. The results indicated that the as-synthesized PDADs were highly selective toward Glu over other amino acids. This was because Glu contained more hydrogen ions. As is known, Glu are acidic amino acids and their pI is 3.22. Compared with other amino acids, Glu gave out one more hydrogen ion in water, and the detailed electrolytic dissociation 35766

DOI: 10.1021/acsami.8b12087 ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

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ACS Applied Materials & Interfaces

concentrations (2, 5, 10, 15, 20, and 30 μg/mL) for 12 h. As shown in Figure S17, the PDADs showed no obvious cytotoxicity to the HepG2 cell than that of control groups, and the cell viability was still more than 85% even after incubation with 20 μg/mL PDADs. Meantime, there was no obvious difference in cell morphology with and without the PDADs, as shown in the bright-field images (Figure 6). These results demonstrated that the PDADs had low cytotoxicity and excellent biocompatibility. Next, the PDADs were used to image Glu and Al3+ in HepG2 cell using a confocal microscope. HepG2 cells incubated with PDADs at 37 °C for 2 h showed intracellular fluorescence in the blue channel (Ex = 405 nm), and the control group without PDADs had no fluorescence (Figure 6). When HepG2 cells were treated with PDADs and Glu for 2 h, no intracellular blue fluorescence was observed. However, the outstanding blue fluorescence was detected after HepG2 cells were incubated with the PDADs−Glu−Al3+ system for 2 h. In addition, bright-field images also showed that all cells maintained a good morphology. These results indicated that the obtained PDADs can be applied in detecting Glu and Al3+ in living cells (Scheme 1).

To further investigate the competitive mechanism between Al3+ and PDADs−Glu system, pyrophosphate was added into the PDADs−Glu−Al3+ system in different concentrations. As we know, the pyrophosphate had strong binding ability with metal ions. The fluorescence of PDADs−Glu−Al3+ decreased gradually with different concentrations (0−1000 μM) of pyrophosphate in the Tris−HCl buffer (Figures 4D and S12), which promoted the Glu release from Glu−Al3+ complex and caused the interaction between Glu and PDADs again, leading to the fluorescence quenching of PDADs. These results demonstrated that pyrophosphate ions and Al3+ had a stronger binding ability than Glu and Al3+. To further understand the main mechanism of the PL recovery progress of PDADs, some relevant coordination experiments were implemented (Figures 5A and S13). Due to the stronger coordination ability between ethylenediaminetetraacetic acid (EDTA) and Al3+, the coordination of Glu and Al3+ in the system is destroyed, resulting in the FL quenching of PDADs by Glu. Moreover, the further coordination ability was investigated by using the stronger interaction of Mg2+ and EDTA. The addition of Mg2+ disrupted the coordination of EDTA and Al3+, causing the FL recovery of PDADs due to the released Al3+ coordinating with the Glu in the Glu−PDADs system. These relevant coordination experiments confirmed the coordination mechanism between Glu and Al3+ in the PL recovery progress of PDADs. More importantly, the switch system showed good stability for Off−On detection of Glu and Al3+ after three cycles in this Off−On PL switch system (Figure 5B). Further study of absorption spectrum for Glu and Al3+ detection confirms the results of fluorescent detection (Figure S14). Moreover, we used the conventional method to synthesize polydopamine nanoparticle.60 The absorbance of polydopamine nanoparticle for conventional method and our method is at 430 and 350 nm (Figure S15A), respectively. The results indicated that the size of polydopamine nanoparticle for our method was smaller than that of conventional method, and the obtained polydopamine nanoparticle using conventional method showed low fluorescence and cannot realize the Off−On detection of Glu and Al3+ (Figure S15B−D). Although the preparation of PDADs was a relatively time-consuming process, the obtained PDADs could be freeze-dried or stored in a fridge, which did not affect the spectrum properties of the PDADs and their application in detection of Glu and Al3+ (Figure S16). Determination of Glu and Al3+ in Human Serum. To evaluate the feasibility of the proposed approach, we applied PDADs to detect Glu and Al3+ in serum samples. Glu in different concentrations (10, 20, and 30 μM) was added to the serum and then detected by the proposed strategy. And, in the presence of certain concentration of Glu, different amounts of Al3+ (1, 2, and 3 μM) were added to the serum samples and detected. Table 1 summarizes the results according to the standard addition method. Meantime, the accuracy of our sensing system for the detection of Glu and Al3+ in human serum was assessed via the recovery rate. Finally, the recovery rates of both Glu and Al3+ were in the range of 95.6 to 107.7%, and the relative standard deviations (RSDs) were not over 5.2%. The results indicated that our proposed method was accurate and reliable and could be applied to detect Glu and Al3+ in serum. Image of Glu and Al3+ in Living Cells. To further explore the application of the assay in biological system, the cytotoxicity experiments in HepG2 cell were studied by using a standard XTT cell-proliferation assay. The viability of HepG2 cell was estimated by incubating with the PDADs at different

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CONCLUSIONS In summary, we developed a facile, one-step, and natural oxidation method to fabricate highly fluorescent PDADs for the Off−On detection of Glu and Al3+. The PDADs were successfully prepared at room temperature by using DA as the sole precursor, and the present approach is simple without strict reaction conditions and sophisticated synthesis process. Moreover, the as-obtained PDADs exhibited a highly selective fluorescence quenching response to Glu and recovery response to Al3+ with a low detection limit of 0.12 and 0.2 μM, respectively. Noteworthy, the Off−On strategy could be applied for determining Glu and Al3+ in the human serum based on this fluorescence quenching−recovery behavior. Moreover, PDADs successfully realized Off detection of Glu and “On” sensing Al3+ in HepG2 cells. It is believed that this efficient Off−On sensing strategy based on PDADs provides a feasible method for developing facile, low-cost, and highly sensitive and selective sensors for biological sensing.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b12087.

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UV−vis spectrum, fluorescence spectrum, and XPS of PDADs; prepared PDADs at different reaction times; fluorescence decay profiles of PDADs; cell viability in the different concentration of PDADs; XPS functional group percentages of obtained PDADs; comparison of some sensor platform for Glu and Al3+ detection (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.L.). Tel/Fax: +00862583587982. *E-mail: [email protected] (C.-W.Z.). *E-mail: [email protected] (L.L.). ORCID

Jinhua Liu: 0000-0002-4031-3031 Hai Li: 0000-0002-9659-1153 35767

DOI: 10.1021/acsami.8b12087 ACS Appl. Mater. Interfaces 2018, 10, 35760−35769

Research Article

ACS Applied Materials & Interfaces

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Haidong Yu: 0000-0001-7124-3630 Lin Li: 0000-0003-0426-6546 Author Contributions

Q.C., J.L., X.Q., L.H., H.L., H.Y., K.-L.L., C.-W.Z., L.L., and W.H. all contributed to conducting the experiments and data analysis. J.L. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Science Foundation of China (Grant Nos. 21505072, 81672508, 61505067, and 21675085), Six Talent Peaks Project of Jiangsu Province (2016-SWYY-033), China-Sweden Joint Mobility Project (51811530018), Jiangsu Key Research and Development Program (BE2015699), and the Opening Project of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (Grant No. 2016004).

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