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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, Hai-Dong Yu, Kah-Leong Lim, Cheng-wu Zhang, Lin Li, and Wei Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12087 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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ACS Applied Materials & Interfaces

Polydopamine Dots-Based Fluorescent Nanoswitch Assay for Reversible Recognition of Glutamic Acid and Al3+ in Human Serum and Living Cell Qiaoqiao Ci a, Jinhua Liu

a, b*

, Xiaofei Qin a, Linqi Han a, Hai Li a, Haidong Yu a,

Kah-Leong Lim c, Cheng-Wu Zhang a*, Lin Li a*and Wei Huang a, d

a

Key Laboratory of Flexible Electronics (KLOFE) and 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

b

State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410082, China

c

Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593

d

Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), 127 West Youyi Road, Xi'an 710072, China.

Keywords Polydopamine dots; nanoswitch assay; reversible recognition; glutamic acid; Al3+

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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 of controlled synthesis of fluorescent polydopamine dots (PDADs) at room temperature with dopamine (DA) 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). Meantime, 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 µM 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 living cells. Therefore, this PDADs-based nanoswitch assay provides a strategy to develop reversible recognition biosensors for intracellular and external molecules analysis.

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INTRODUCTION The use of polymer fluorescent nanoparticles (PFNPs) in chemical sensors, fluorescence 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,

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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 there including sophisticated synthetic procedure, environmentally harmful organic solvents, and low fluorescence quantum yields (QYs) in aqueous solution. Hence, developing auto-fluorescent polymer materials with ideal water solubility, simple preparation and high fluorescence QYs is very challenging and still in pursuit. Dopamine

(3,4-dihydroxyphenylethylamine,

DA),

a

member

of

the

catecholamines family, is an important chemical neurotransmitter.19-21 DA can be oxidized to dopaminequinone with an alkaline and aerobic condition,, and the later 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 cerebral antioxidants

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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 about 100-200 nm. Compared to quantum dots (QDs), polymer dot (PD) has stronger fluorescence and the size of PD is tunable. Moreover, PD with small size is desirable that they will not disrupt the native behavior of the biomolecules-labelled and can better penetrate and distribute in 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 FPD 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 (PDNPs).34 However, these approaches often suffer from the low fluorescence quantum yield 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 synthetic method for PDADs by using one-step oxidation the sole precursor dopamine (DA) at room temperature, and then further verified its application for highly 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

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complicated instrumentation or harsh reaction conditions. The prepared PDADs-based fluorescent probe displayed excellent performance for Glu and Al3+ detection with 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.

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•6H20, 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, Pd(NO3)2, were obtained from Shanghai Titan Scientific Co. Ltd. (Shanghai, China). Trihydroxymethyl aminomethane (C4H11O3) were 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 (FIL TER-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

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Atomic Force Microscope (AFM, XE-70, Park , Korea) in the Scan Asyst mode were used to obtain the size and height of the PDADs. X-ray photoelectron spectroscopy (XPS) were carried on a PHI Quantum 5000 XPS system (Physical Electronics, USA). XPS was used to characterize the elemental composition and bonding configuration. Fourier

transform

infrared

(FT-IR)

were performed

on a

VECTOR

22

spectrophotometer (Bruker, Germany). Thermogravimetric analysis (TGA) was used on a TGA2 analyzer (Mettler Toledo, Swit). Mass spectrography was performed on a 4000 Q-TRAP (AB Sciex, USA). Fluorescence spectra was recorded in a microplate reader (Cytation5, BIOTEK, USA). 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, USA). 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 three months, the DA solution (0.05 M) was transferred into dialysis bag with distilled water for two days to remove the unreacted dopamine, then the solution in 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 PDADs solution of 4.7 mg/mL was received. 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 with following

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equation: 35 ΦP =

FP AD × ×ΦD AP FD

(1)

of which 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 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 adding a fixed amount of Glu. 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 tris-HCl buffer. To do 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 concentration of Al3+ were prepared, the fluorescence of which was measured with wavelength of 310 nm excitation after 10 min 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 standard addition recovery experimental, the blood was centrifuged (8000 rpm, 20 min) to separate the clear supernatant layer serum, followed filter using a 0.45 µm Millipore filter for further sample analysis. The obtained serum was mixed with different 7



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concentration 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 Zhuo et al.36 Briefly, HepG2 cells were seeded in a 96-well plate and cultured in the humidified incubator ventilated with 5% CO2 at 37 °C for 24 h. After that, cells were incubated with different concentrations of PDADs (2, 5, 10, 15, 20 and 30 µg/mL) for 24 h. Then cells were washed and incubated in serum-free DMEM with 2% XTT for 2 h. The absorbance was obtained at 450 nm with microplate reader and cell viability was got following the equation:

Cellviabilityrate =

A - A0 × 100% AS - A0

(2)

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

RESULTS AND DISCUSSION Synthesis and characterization of PDADs. The PDADs were synthesized by 8


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natural oxidation of DA solution at room temperature for 3 months without using additional oxidizing agent. The absorption and fluorescence intensity of different concentration PDADs were monitored every 20 days. As shown in Figure S1, S2, and 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 narrow size distribution ranging from 4-8 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 was the typical distance between graphite layers. The corresponding fast Fourier transform (FFT) pattern exhibited hexagonal lattice, indicating that the PDADs were crystalline hexagonal structures (Figure 1B). AFM image and typical section analysis of PDADs were also performed (Figure 1C, 1D, and 1E). All the above results indicated that PDADs had spherical shapes with height distribution from 6 to 8 nm. The element composition and valence state of the PDADs were followed identified by XPS spectra. As shown in Figure S4A, the PDADs mainly contain C, O, and N elements with composition to be C (72.29 at%), O (21.48 at%) and N (6.24 at%). 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 heteroatoms existence 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

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two peaks at 531.7, 532.5 eV that can be assigned to C=O and C–OH/C–O–C groups , respectively (Figure S4D). This was in consistent with C=O in the C 1s spectrum. The contents and ratios of surface groups in PDADs had been given in table S1 based on XPS results. 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, 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 in-plane and out of-plane bending. Much information resulted from the nitrogen heteroaromatic skeleton. These results suggested that a great deal of oxygen and nitrogen-containing were produced on the surface of PDADs during the process of natural oxidation, which endowed as-obtained PDADs excellent water solubility and good complexation ability. The zeta potential of PDADs was investigated in Figure S5. The result indicated that the PDADs have positive charges (+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 was showed in Figure 2B. The peak appeared at 1352 cm−1 correlated to D band which got from A1g breathing mode of sp3 carbon. The Peak at 1578 cm−1 correlated to G band which correlated to in plane bond-stretching of C sp2 atoms. Typical feature of the mixture of amorphous carbon and nanocrystalline graphite were showed in the Raman spectrum, which was consistent with our HRTEM results.

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The UV-visible absorption was 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–NH2 bond.

41, 42

To explore the optical

properties of the as-synthesized PDADs, a detailed emission spectral assay with excitation wavelengths from 310 nm to 530 nm was performed and the data were 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 range 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 underlay by the different size distributions and "surface states" formed by diverse functional groups. These results indicated that the PDADs held excitation-dependent fluorescence behaviors.43 Additionally, the PDADs showed pH-independent fluorescence behavior (Figure S6). The fluorescence of PDADs increased with change pH from 3 to 7, and decreased from 7 to 10, the maximum fluorescence intensity appeared at pH = 7, with a slight red shift (30 nm) from pH(3-5) to pH(6-10). This pH-independent fluorescence behavior of PDADs could attribute to presence of free zigzag sites, which shared comparable structure with carbon quantum dots reported previously. Therefore, the result suggested that the excitation-dependent and pH-independent fluorescence behavior might ascribe to surface states and the size effect of PDADs.

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We also try to investigate the reaction mechanism based on NMR, MS, IR and UV-vis results. Firstly, UV-Vis spectra were adopted to study the effect of reaction time on the mechanism of PDADs formation (Figure S7A). The broad band (310-400 nm) was correlated with formation of a 1,4 Michael addition between quinones and amines, subsequently oxidized and polymerized into PDADs.44 Figure S7A showed the relationship between the absorption intensity of PDADs and reaction time, indicating the formation of more PDADs over time. To elucidate how reaction time of DA polymerization affect PDADs formation, DA solutions with different reaction time were lyophilized and investigated by FTIR spectrum (Figure S7B). In FTIR spectrum, DA polymerized into PDADs would made one of 3347 and 3237 cm-1 reduced, resulting from the primary amine 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 time were comparable with that of the raw DA powder in the 2400-3400 and 500, 700 cm-1 regions. This might be attributed to the precipitation of residual DA in the PDADs during drying process. Figure S7C and S7D showed the 13CNMR spectra of PDADs obtained from three and six months, respectively, suggesting that 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 arene and indole units’ C atoms. Moreover, the peaks (33 -42 ppm) were derived from aliphatic species.45 MALDI-TOF mass spectrums of PDADs obtained from three and six months also confirm the existence of mixed

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oligomers (Figure S7E and S7F).46 The spectrum of the PDADs obtained from six months showed a larger polymer than that of three months, indicating a slow polymerization process with the increase of time. PDADs-based fluorescence determination of Glu. Based on 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 affect the fluorescence signal of PDADs, the change of fluorescence was measured upon Glu addition. In the first 2 min, the fluorescence of PDADs was quenched rapidly. After that, the quenching rate slowed down and reached to steady stage within 2 more min. (Figure 3A). To determine the sensitivity of the PDADs towards 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 of fluorescence intensity of PDADs linearly correlated with the increase of Glu, which might be owed the binding of Glu to the surface of PDADs. Figure S8 demonstrated 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 was the concentration 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 to 100 µM) with a detection limit of 0.12 µM, which was comparable to other reported methods

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(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 %, while some amino (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 the significant FL quenching 78% and 55%. The results indicated that the as-synthesized PDADs were highly selective toward Glu over the other amino acids. This might be underlay by more hydrogen ion containing of Glu. As known, Glu were acidic amino acids, the pI of which was 3.22. Compared with other amino acids, Glu gave out one more hydrogen ion in water, and the detailed electrolytic dissociation equilibrium was described as follow (eq S2). The hydrogen ion was released from acidic amino acids could change the pH of solution when the concentration reached a certain amount. As the pH decreases, the fluorescence intensity was decreased gradually, which was in agreement with our results that pH effect of PDADs. A control experiment for detecting Glu in aqueous using PDADs was implemented (Figure S9). The results indicated that the FL of PDADs was effectively be quenched by Glu in

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aqueous, 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 concentration (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 reaction of electrostatic interactions. To further reveal the quenching mechanism between PDADs and Glu, time-correlated single photon counting was used to determine the PL lifetimes for 310 nm excitation. The average PL lifetime of PDADs without and with Glu was 4.30 (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 of transition time from excited state to ground state and eventually

increase of PL lifetime of

PDADs. The results indicated that the quenching mechanism of PDADs with the present of Glu was static quenching due to the few differences of PL lifetime by 1.18 ns. Fluorescence response to Al3+ in PDADs-Glu system. Glu was one of the most metabolically among protein amino acids, and potential chelators for Al3+. By binding to Glu ions trace amounts of Al3+ might interfere many metabolic processes in organisms.53 Therefore, the “off” fluorescence of 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 response of the fluorescence of PDADs to Al3+, 1 mM Al3+ was added to the PDADs-Glu system.

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A rapid fluorescence recovering of PDADs was observed within 2 min. After that, the fluorescence recovery slowed down and reached to a steady state within 5 min (Figure 4A). To appreciate the sensitivity of the sensing system, different Al3+ concentrations were added in. The fluorescence signal enhanced accordingly with the increase of Al3+ (Figure 4B). The linear relationship of the fluorescence intensity versus the Al3+ concentration was showed 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 was 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 of reported by others (Table S 3).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, only the addition of Al3+ could induce significant FL enhancement, while adding other metal ions had no change of fluorescence signal in the PDADs-Glu system. This was because Al3+ binding to Glu to form Glu-Al3+ complex, which caused Glu separating from the surface of PDADs and subsequent recovery the fluorescence of PDADs. The results suggested that current sensing system possess excellent selectivity for Al3+. In order to further investigate the competitive mechanism between Al3+ and PDADs-Glu system, pyrophosphate was added into the PDADs-Glu-Al3+ system with different concentrations. It was well known that pyrophosphate ions had a strong

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binding ability with metal ions as a complexing agent, thus there was a competitive complexation reaction between Glu and pyrophosphate ions, pyrophosphate ions prone to coordinate with Al3+, and the released Glu would quench the fluorescence of PDADs. The fluorescence of PDADs-Glu-Al3+ decreased gradually with different concentrations (0-1000 µM) of pyrophosphate in tris-HCl buffer (Figure 4D and S12), which promoted 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 stronger binding ability than that of Glu and Al3+. To further understand the main mechanism of the PL recovery progress of PDADs, some relevant coordination experiments were implemented (Figure 5A and S13). Due to the stronger coordination ability between EDTA and Al3+, the coordination of Glu and Al3+ in 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 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 confirm the results of fluorescent detection (Figure S14). Moreover, we used the conventional

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method to synthesize polydopamine nanoparticle.60 The absorbance of polydopamine nanoparticle for conventional method and our method is at 430 nm 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, C and D). Although the preparation of PDADs was a relatively time-consuming process, the obtained PDADs could be freeze-dried or stored in fridge, which did not affect the spectrum properties of 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 with different concentrations (10, 20 and 30 µM) were added to the serum and then detected by proposed strategy. And in the presence of certain concentration of Glu, different amount of Al3+ (1, 2 and 3 µM) were added to the serum samples and detected. Table 1 summarized the results according to the standard addition method. Meantime, the accuracy of our sensing system for detection Glu and Al3+ in human serum was assessed via the recovery rate. Finally, both the recovery rate for 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 accuracy and reliability, 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

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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 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 incubating with 20 µg/mL PDADs. Meantime, there was no obvious difference in the cell morphology with and without PDADs from the bright-field images (Figure 6). These results demonstrated that the PDADs had lowcytotoxicity and excellent biocompatibility. Next the PDADs were used to image of Glu and Al3+ in HepG2 cell using a confocal microscope. HepG2 cells incubated with PDADs at 37 oC 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 treated with PDADs and Glu for 2 h, no intracellular blue fluorescence was observed. However, the outstanding blue fluorescence was detected after HepG2 cells incubated with PDADs-Glu-Al3+ system for 2 h. In addition, bright-field images also showed that all cells maintained a good morphology. These results suggested that the PDADs had potential application of relay detecting Glu and Al3+ in living cells.

CONCLUSIONS In summary, we developed a facile, one-step and natural oxidation method to fabricate highly fluorescent PDADs for “Off-On” detection of Glu and Al3+. The

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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 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 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, highly sensitive and selective sensors in biological sensing.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the internet at http://pubs.acs.org. Uv-vis spectrum, fluorescence spectrum and XPS of PDADs; Photograph of prepared PDADs at different reaction time; 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 detection; Comparison of some sensor platform for Al3+ detection.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]; [email protected]. Fax/Tel: +00862583587982. Author Contributions QQC, JHL, XQ, LH, HL, HY, KLL, CWZ, LL, WH all contributed to conducting experiments and data analysis. JHL wrote the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors acknowledge the ﬁnancial support from the National Science Foundation of China (Grant nos. 21505072, 81672508, 61505067, 21675085), Six Talent Peaks Project of Jiangsu Province (2016-SWYY-033), China-Sweden Joint Mobility Project (51661145021), Jiangsu Key Research and Development Program (BE2015699), and the Opening Project of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (Grant nos. 2016004).

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Legend to Figures Scheme 1. Schematic illustration of the formation of PDADs and detection of Glu and Al3+ based on the “off-on” strategy. 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) FT-IR and (B) Raman of PDADs; (C) UV-vis absorption spectra (abs) of PDADs; (D) Fluorescence spectra of PDADs under different excitation wavelengths. Figure 3. (A) Time-dependent flourescence 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; all the concentrations of amino acids are 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 flourescence responses of the PDADs-Glu system upon the addition of Al3+ (1 mM); (B) Fluorescence emission spectra of PDADs-Glu system in the presence of different concentrations of Al3+ in tris-HCl buffer (50 mM,

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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+; all the concentrations of metal ions are 1 mM; (D) Fluorescence emission spectra of PDADs-Glu- Al3+ system in the presence of different concentrations of NaP2O7 in tris-HCl buffer. 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. Figure 6. Fluorescent imaging of HepG2 cells: fluorescence image HepG2 cells and incubated with PDADs, PDADs/Glu, 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. Table 1. Determination of Glu and Al3+ in serum samples by PDADs.

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Scheme 1.

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Figure 4.

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1000

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A 800

FI (a.u)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 41

600 400 200 1

3+

l +A

2

lu +G

Figure 5.

38


3

3+

l +A

4

5

+

3 lu l +G +A

6

7

3+ lu l +G +A

Page 39 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


l

Figure 6.

39



Table 1

40


Page 40 of 41

Page 41 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents Graphic (Only for TOC)

41


Polydopamine Dots-Based Fluorescent ... - ACS Publications

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