Dual Colorimetric and Fluorescent Sensor Based On

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Dual Colorimetric and Fluorescent Sensor Based On Semiconducting Polymer Dots for Ratiometric Detection of Lead Ions in Living Cells Shih-Yu Kuo,† Hsiang-Hau Li,† Pei-Jing Wu, Chuan-Pin Chen, Ya-Chi Huang, and Yang-Hsiang Chan* Department of Chemistry, National Sun Yat-sen University, 70 Lien Hai Road, Kaohsiung, Taiwan 80424 S Supporting Information *

ABSTRACT: Recently, semiconducting polymer dots (Pdots) have become a novel type of ultrabright fluorescent probes which hold great promise in biological imaging and analytical detection. Here we developed a visual sensor based on Pdots for Pb2+ detection. We first embedded near-infrared (NIR) dyes into the matrix of poly[(9,9-dioctylfluorene)-co2,1,3-benzothiadiazole-co-4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole] (PFBT-DBT) polymer and then capped the Pdots with polydiacetylenes (PDAs), in which parts of the PDAs were prefunctionalized with 15-crown-5 moieties to form Pdots. The high selectivity of these Pdots for lead ions is attributed to the formation of 2:1 15-crown-5-Pb2+-carboxylate sandwich complex on the Pdot surface. After Pb2+ chelation, the conjugation system of the PDA was perturbed and strained, causing a chromatic change of the PDA from blue to red. At the same time, the encapsulated NIR dyes were liable to leach out that resulted in an emission variation of the Pdots. Accordingly, lead ions can be recognized by either color change or emission variation of the Pdots. We also loaded these nanoprobes into live HeLa cells through endocytosis, and then monitored changes in Pb2+ levels within cells, demonstrating their utility for use in cellular and bioimaging applications. In addition, we fabricated easyto-prepare test strips impregnated with Pdot-poly(vinyl alcohol) films to identify Pb2+ in real samples, which proved their applicability for in situ on-site detection. Our results suggest that this Pdot-based visual sensor shows promising potential for advanced environmental and biological applications.

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of lead ions due to their change of localized surface plasmon resonance absorption upon aggregation and/or interaction with Pb2+.8−10 However, the broad absorption spectra of metal nanoparticles might not be able to precisely reflect subtle changes of Pb2+, and thus in many circumstances the color response of the nanoparticle solution cannot be distinctly visualized. Recently, there have been several groups developing fluorescence-based Pb2+-responsive probes by use of DNAzymes,11−13 proteins,14 polymers,15 small molecules,16,17 and quantum dots.18,19 Unfortunately, most of the Pb2+-sensitive small organic molecules can only be employed in organicaqueous mixtures due to their poor water solubility, which might limit their use in biologically relevant systems. Yet DNAzyme or protein-based reporters usually involve complicated instrumentation and delicate sample storage processes. To date, there has been a paucity of studies that can be successfully used for tracking lead ions in living cells.20,21 Therefore, the exploration of new probes that can be applied for selective and sensitive evaluation of Pb2+ in both biological and environmental applications is highly demanded.

ead is one of the three most abundant and hazardous heavy metal ions (including cadmium and mercury) in the environment. Lead pollution can cause serious problems, particularly in children, such as brain damage, neurological/ cardiovascular disorders, and mental retardation.1 In the human body, lead ions can readily bind to the thiol groups of enzymes or proteins and thus affect their activities in biological processes.2 As a result, the maximum concentration values of lead tolerated in electrical and electronic equipment are strictly regulated by the European Union’s Restriction on Hazardous Substances (RoHS) directive.3 The Centers for Disease Control and Prevention (CDC) also set a reference level for lead at 0.1 mg/L (0.5 μM or 100 ppb) to evaluate whether the children have blood lead levels higher than most children’s levels.4 Although several analytical methods, such as atomic absorption spectrometry (AAS),5 inductively coupled plasma atomic emission spectrometry (ICP-AES),6 and inductively coupled plasma mass spectrometry (ICP-MS),7 have been widely used to determine lead content, these techniques usually require sophisticated instrumentation and complicated sample pretreatment processes. There have been ongoing efforts to develop a rapid, reliable, and economical technique for on-site determination of lead ions. Among these detection techniques, several research groups have demonstrated the exploitation of metal nanoparticles (e.g., gold nanoparticles) for prompt visual detection © XXXX American Chemical Society

Received: December 27, 2014 Accepted: March 30, 2015

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Analytical Chemistry Scheme 1. Schematic Showing the Preparation of 15-Crown-5-Functionalized PFBT-DBT Pdots for Pb2+ Detectiona

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(A) First, semiconducting polymer PFBT-DBT and NIR695 dyes were mixed well in THF and then coprecipitated in water under vigorous sonication to form dye-encapsulated PFBT-DBT Pdots. A mixture of carboxyl- and 15-crown-5-functionalized polydiacetyelenes (PDAs) were then coated onto the surface of Pdots for subsequent Pb2+ sensing. (B) Hydrodynamic diameters of PDA-encloased dye-doped Pdots measured by DLS. The inset shows their corresponding TEM image. The scale bar represents 100 nm. (C) Gel electrophoresis of bare PFBT-DBT Pdots, PDA-coated PFBT-DBT Pdots, and PDA-enclosed PFBT-DBT Pdots after the addition of Pb2+.

In recent years, semiconducting polymer dots (Pdots) have attracted much attention owing to their unique photophysical properties, including high fluorescence brightness, good photostability, fast radiative rate, and minimal cytotoxicity.22−28 Additionally, the recent development of the surface functionalization of Pdots have endowed them with burgeoning applications both in vitro and in vivo.29−32 More recently, Chiu’s and our groups have created a variety of sensing platforms by taking advantage of the amplified energy transfer from the excited Pdot matrix to the fluorescent sensing molecules.33−37 Here we report a novel strategy for the ratiometric determination of Pb2+ in environmental and cellular studies. Ratiometric measurements hold several advantages over other typical nonratiometric (e.g., single-wavelength) approaches. For example, the fluctuation of the excitation source, concentration of probe, and any drifts in the environment/instrument can be effectively eliminated. Specifically, we first blended NIR695 dyes with poly[(9,9dioctylfluorene)-co-2,1,3-benzothiadiazole-co-4,7-di(thiophen2-yl)-2,1,3-benzothiadiazole] (PFBT-DBT) in THF, and then the mixtures were injected into H2O under intense sonication (nanoprecipitation) to form Pdots (Scheme 1). The NIR695 dye-encapsulated Pdots were subsequently capped by both 15crown-5- and carboxyl-functionalized diacetylene (10,12pentacosadiynoic acid) as two neighboring carboxyl acid groups and crown ether moieties can selectivity sandwich Pb2+,8,38 and at the time the diacetylene (DA) derivatives can be further polymerized to form polydiacetylenes (PDAs) via topochemical 1,4-addition39−46 to seal the doped NIR695 chromophores.47

This new type of nanomaterial possesses a dual spectral (fluorescent and colorimetric) response toward Pb2+ in that the formation of sandwich complexes can induce a shortening or partial distortion of the PDA conjugation, accompanied by the subsequent leaching of the embedded NIR695 dyes. The disturbance of PDAs produces a blue-to-red phase transition that can be observed by the naked eye, while the resulting emission change can be utilized for the quantitative determination of Pb2+. We have also demonstrated the applicability of these Pdots for Pb2+ detection in environmental samples and in living cells, and thus their potential for use in a wide range of bioimaging studies and diagnostic assays.

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EXPERIMENTAL SECTION Chemicals. All reagents were purchased from Sigma-Aldrich or Alfa Asear and used as received unless indicated elsewhere. The following salts were used: lead(II) nitrate, nickel(II) chloride, iron(II) chloride, calcium chloride, manganese(II) chloride, cobalt(II) chloride, zinc chloride, copper(II) chloride, and arsenic trioxide. All biorelated agents such as streptavidin, anitibody, or medium are purchased form Invitrogen (Life Technologies). High purity water (18.2 MΩ·cm) was used throughout the experiment. All 1HNMR and 13CNMR spectra were recorded on a Bruker AV300 spectrometer (400 MHz). PFBT-DBT was synthesized according to our reported literature.47 Preparation of NIR695-Doped PFBT-DBT Pdots. First, 200 μL of PFBT-DBT (1 mg/mL in THF), 20 μL of PS−PEGCOOH (1 mg/mL in THF), and 1.2 μL of NIR695 dyes (0.5 B

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prior to experiments until ∼80% confluence was reached. To prepare cell suspensions, the adherent cancer cells were quickly rinsed with media and then incubated in 0.8 mL of trypsinethylenediaminetetraacetic (EDTA) solution (0.25 w/v % trypsin, 0.25 g/L EDTA) at 37 °C for 3 min. The cell suspension solution was then centrifuged at 1000 rpm for 5 min to precipitate the cells to the bottom of the tube. After taking out the upper media, the cells were rinsed and resuspended in 5 mL of culture media. Approximately tens of thousands cells were split onto a glass-bottomed culture dish and allowed to grow for 12 h before Pdot tagging. For endocytosis, 100 μL of Pdots was added into the flask containing cells and then incubated for 2 h. Prior to fluorescence imaging, the cells were rinsed with PBS buffer to remove any nonspecifically bound Pdots on the cell surface. Cell Imaging. The fluorescence spectra of Pdot-tagged cells were acquired with a fluorescence confocal microscope (Nikon D-Eclipse C1) under ambient conditions (24 ± 2 °C). The confocal fluorescence images were collected using a diode laser at 488 nm (∼15 mW) as the excitation source and an integration time of 1.6 μs/pixel. A CF1 Plan Fluor 100× (N. A. 1.30, W.D. 0.16 mm) oil objective was utilized for imaging and spectral data acquisition; the laser was focused to a spot size of ∼7 μm2. The blue fluorescence was collected by filtering through a 450/35 band-pass (λex = 408 nm), while the red fluorescence was collected by filtering through a 605/37 bandpass (λex = 488 nm). MTT Assay. The cellular cytotoxicity of the Pdots was examined on HeLa cells. The number of viable cells was determined using the MTT assay with 3-(4,5-dimethylthiazole2-yl)-2,5-phenyltetrazolium bromide. HeLa cells were first seeded in each well of a 24-well culture plate and then incubated with various concentrations of Pdots (100, 200, and 400 pM) for 6, 12, and 24 h. After that, 20 μL (5 mg/mL) of MTT aqueous solution was added to each well, and the cells were further incubated for 4 h at 37 °C to deoxidize MTT. The medium was then washed out, and 300 μL of DMSO was added into each well to dissolve formazam crystals. Absorbance was measured by a BioTek ELx800 microplate reader at 570 nm, while the cells cultured with the pure medium (e.g., without Pdots) served as controls. Preparation of Pb2+-Responsive Test Strips Impregnated with Pdot-PVA Films. 1 g of poly(vinyl alcohol) (PVA, Mw: 89 000−98 000, purchased from Sigma-Aldrich) powder was first added into 9 mL of water with stirring for 10 min at room temperature and then heated to 70 °C inside a water bath for 2−3 h until PVA was completed dissolved. To a 5 mL glass vial was added 2 mL of PDA-capped Pdots, 2 mL of PVA solution, 40 μL of 1 M HEPES buffer, 80 μL of 5% PEG solution, and 100 μL of glycerol. The mixture was gently mixed to homogeneous and then added onto the top of the filter paper inside a glass dish. The filter paper was left to dry naturally and then cut into small species to make test strips.

mg/mL in THF) were mixed together in 5 mL of THF. Polystyrene graft ethylene oxide functionalized with carboxylic end group (PS−PEG-COOH, Mn = 6500 Da of PS moiety; 4600 Da of PEG-COOH; polydispersity, 1.3) was purchased from Polymer Source, Inc. (Dorval PQ, Canada) and used as received. The mixture was sonicated for 15 s and then injected into 10 mL of H2O under violent sonication. After that, THF was removed by purging with dry N2 on a 60 °C hot plate for 30 min. During Pdot formation, the hydrophobic polystyrene parts of PS−PEG-COOH tended to embedded inside the Pdot matrix while the hydrophilic PEG portions together with COOH groups extended outside into the aqueous environment. PEG units have been known to minimize nonspecific protein adsorption during biological labeling, and a small amount of COOH groups could reduce the size of the resulting Pdots. The resulting Pdot solution was filtered through a 0.2 mm poly(ether sulfone) membrane filter to remove large aggregations formed during preparation. Fabrication of 15-Crown-5-Functionalized PDAs on the Surface of Pdots. 9.03 mg of 10,12-pentacosadiynoic acid, 6.0 mg of 2-aminomethyl-15-crown-5, and 6.0 mg of 1ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was mixed in a 20 mL glass vial, and then 3.0 mL of THF was added. The reaction mixture was stirred at room temperature for 12 h and then stored at 4 °C as the stock solution. To prepare DA-enclosed Pdots, 70 μL of carboxylfunctionalized DA (i.e., 10,12-pentacosadiynoic acid, 3 mg/mL in THF) and 84 μL of 15-crown-5 functionalized DA from the stock solution was mixed together, and then the mixture solution was injected into 2 mL of Pdot solution under sonication. THF was then removed by heating the solution to 60 °C for 12 min with stirring. After that, the solution was cooled to 4 °C for 4 h. The solution was purged with N2 for 25 min, and then the coated DAs were polymerized upon exposure to a 254 nm UV light for 20 s, in which the color of the solution turned from orange to blue-green. The PDA could be further modified with more 15-crown-5 if needed. The solution was centrifuged at 7000 rpm for 5 min to discard the aggregates in the bottom. The filtrate was further purified by use of sizeexclusion chromatography (Sephacryl HR-300 gel medium, molecular mass cutoff 1.5 × 106 Da) to remove the empty PDA nanoparticles, free DAs, EDC, and unreacted 15-crown-5. Characterization of PDA-Enclosed NIR695-Embedded Pdots. The average particle size was determined by dynamic light scattering and transmission electron microscopy (TEM). TEM images of the synthesized Pdots were acquired using a JEOL 2100 transmission electron microscope at an acceleration voltage of 200 kV. For TEM, a drop of Pdot aqueous solution was placed onto a carbon-coated grid and allowed to evaporate at room temperature. The absorption spectra of Pdots were measured using UV−visible spectroscopy (Spectra System UV2000 HR, Thermo Separation Products). The fluorescence spectra were collected using a Hitachi F-7000 fluorometer (Hitachi, Tokyo, Japan) under 450 nm excitation. Cell Culture and Labeling. The cervical cancer cell line HeLa was ordered from Food Industry Research and Development Institute (Taiwan). Primary cultured HeLa cells were grown in Dulbecco’s Modified Eagle Medium (cat. no. 11885, Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (5000 units/mL penicillin G, 50 μg/mL streptomycin sulfate in 0.85% NaCl) at 37 °C with 5% CO2 humidified atmosphere. The cells were precultured in a T-25 flask and allowed to grow for 5−7 days

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RESULTS AND DISCUSSION Design and Surface Functionalization of NIR DyeEncapsulated Pdots for Selective Pb2+ Detection. In our recent work, we have successfully developed a strategy to obtain NIR-emitting Pdots by embedding near-infrared (NIR) dyes into the Pdot matrix where an efficient intraparticle energy was transferred from the excited polymer matrix to the doped NIR dyes (Scheme 1).47 The dye-encapsulated Pdots were then capped with PDAs to prevent any potential leakage of the C

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blue to red. The color change of the sample was attributed to the interfacial perturbation of PDA assemblies upon Pb2+ chelation and thus could be used for qualitative determination of Pb 2+ . However, sometimes slight changes in Pb 2+ concentration could not be reliably presented from the color transition. Therefore, we used the emission ratio of Pdots to plot the calibration curve of Pb2+. Figure 2A shows the

entrapped dyes. The average hydrodynamic size of the resulting PDA-enclosed NIR695-doped Pdots was determined by DLS to be 27 nm, which is consistent with the TEM measurements (Scheme 1B). Here we separately functionalized DAs with 15crown-5 derivatives and then blended with unmodified DAs at an optimized ratio to enclose dye-doped Pdots. In this scenario, these Pdots become Pb2+ responsive because each carboxylate group and its proximal crown ether moiety can selectively chelate a Pb2+ ion to form a 2:1 sandwich complexation.8 We expected that the formation of the multivalent and strong coordinations of 15-crown-5 and carboxylate to Pb2+ could cause the perturbation of the well-ordered PDA conjugation and thereby shorten the PDA conjugation network. This structural disturbance or strain of PDA backbones could result in a shift of their absorption band from ∼650 to ∼550 nm (i.e., blue-to-red chromatic transition), accompanied by the leaching of the doped NIR dyes from the defective PDAs. As displayed in Scheme 1C, gel electrophoresis showed that bare PFBTDBT Pdots moved faster toward the positive electrode than PDA-enclosed PFBT-DBT Pdots and PDA-enclosed PFBTDBT Pdots after the addition of Pb2+. This result indicated that the PDA encapsulation increased the total mass of Pdots, while the Pb2+ ions lowered the surface charges of these Pdots. Here we doped 0.3% (w/w) NIR695 dyes into the Pdot matrix and then capped the Pdots with a mixture of carboxyl-functionalized and 15-crown-5-modified DAs for further polymerization. The molecular weight of PFBT-DBT was determined to be ∼12 300 by gel-permeation chromatography, and the average nanoparticle size of Pdots was determined by DSL to be 27 nm. Based on the information, the average number of polymer chains and NIR dyes were estimated to be ∼504 and ∼25 per Pdot, respectively. Figure 1 shows the absorption and emission

Figure 2. (A) Effect of different concentrations of Pb2+ on the emission intensity of Pdot solutions. Concentrations of Pb2+ ranged from 0 to 100 μM (λex = 450 nm). Concentration of Pdots used was 20 nM at pH 7.4. (B) A plot of the ratio of the 650 nm peak (from PFBT-DBT Pdots) over the 715 nm peak (from NIR695 dyes) as a function of Pb2+ concentration. The red line is a linear fit to the data. The inset in (B) magnifies the regions of lower concentrations of Pb2+ (0−10 μM). (C) Effect of different ions (50 μM) on the emission intensity of Pdot solutions. Photographs on the top show each sample under ambient light (upper row) and 365 nm light (bottom row).

fluorescence spectra of these Pdots in the presence of different concentrations of lead ions. It is evident that the fluorescence intensity of NIR695 (715 nm) decreased gradually while the emission intensity of PFBT-DBT (650 nm) increased with increasing Pb2+ concentrations. Accordingly, we observed an apparent increase in red emission after the addition of Pb2+ (photographs on the top of Figure 2C). It should be noted that the emission wavelengths longer than 700 nm can barely be seen by naked eyes so that the fluorescence appeared very dim for the Pdot samples before and after the addition of other cations. We further employed a ratiometric method for the determination of lead ions by plotting the ratio of the emission intensities of PFBT-DBT (λem = 650 nm) to NIR695 (λem = 715 nm) as a function of Pb2+ concentration. As shown in Figure 2B, a linear correlation between the ratio of I650 nm/ I715 nm and Pb2+ concentration could be obtained, which ranged from 0 to 10 μM (R2 = 0.9964). For this set of experiments, the relative standard deviation of the blank signal from 10 replicates was 0.6%. An increased emission intensity of 7% from PFBTDBT with a concomitant decrease of 3% in NIR695 emission could be detected at 0.5 μM of lead ions. It is worth mentioning

Figure 1. UV−visible and fluorescence spectra of bare PFBT-DBT Pdots in water (black line) and PDA-crown-enclosed NIR695embedded Pdots (red line). Dashed lines show absorption spectra, while solid lines represent corresponding emission spectra.

spectra of bare PFBT-DBT and PDA-enclosed NIR695embedded Pdots. The emission of PFBT-DBT was greatly quenched along with the emergence of a new NIR emission peak at 715 nm after dye doping (solid red line), indicating that energy was transferred from Pdot to NIR dyes. Besides, the appearance of a new absorption at ∼670 nm (dashed red line) demonstrated the formation of highly ordered PDAs on the surface of Pdots. Colorimetric and Fluorescent Detection of Pb2+. Upon the addition of Pb2+, the color of the Pdot solution turned from D

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lifetime of 0.3% NIR965-encapsulated Pdots was shortened to 2.02 ns and then recovered back to 3.27 ns after the addition of 50 μM Pb2+. The change in Pdot lifetime (4.83 to 2.02 ns) reveals an energy transfer efficiency of 58%, which is fairly close to the energy transfer efficiency of 64% from steady-state quenching as shown in Figure 1. Upon the addition of lead ions, the energy transfer efficiency decreased to 32%, which is consistent with the results from the fluorescence recovery at 650 nm (Figure 2B). Pb2+ Measurements in Real Water Samples and within Live HeLa Cells. To demonstrate the feasibility of the ratiometric Pdot Pb2+ sensors, we introduced them into live HeLa cells via endocytosis, and then lead ions were added into the cell-culture medium. After incubation for 2 h, the cells were washed with PBS buffer to remove free Pdots in solution and any nonspecific adsorption on the cell surface. The confocal fluorescence microscopy images of HeLa cells before and after the addition of lead ions are shown in Figure 4A,B, respectively.

that we have tried various NIR695 doping concentrations and found that the best sensitivity for Pb2+ detection could be achieved when 0.3% NIR695 was doped into the Pdot matrix. We also optimized the ratio of 15-crown-5-modified DAs to carboxyl-functionalized DAs in an effort to obtain the most visual color change of PDAs from blue to red for Pb2+ sensing. Besides, we have evaluated the selectivity of these Pdots in solutions containing different cations (Figure 2C). We found that these Pdots exhibited excellent selectivity for Pb2+, and their emission ratio as well as solution color was unaffected or minimally influenced by other cations, as compared to that of the negative control sample (ion-free sample or blank). Selectivity of PDA-Pdot Nanocomposites for Detection of Pb2+. We investigated the interference effect of mixed cations by measuring the absorption spectra of Pdots in the mixture of cations containing Cu2+, Fe2+, Ca2+, As3+, Zn2+, Mn2+, Co2+, and Ni2+. As shown in Figure 3A, we found that

Figure 3. (A) Interference effect of mixed ions on the absorption spectra of Pdot solutions. UV−vis spectra of Pdots in pure water (black line) and in solution containing 10 μM of various ions before (blue line) and after (red line) the addition of 10 μM of lead ions. The response time was 20 min. (B) Time-resolved fluorescence decay of bare PFBT-DBT Pdots (experimental data, black circles; fitting, blue solid line), PDA-capped NIR695-doped PFBT-DBT Pdots (experimental data, blue circles; fitting, blue solid line), and PDA-capped NIR695-doped PFBT-DBT Pdots after the treatment of lead ions (experimental data, red circles; fitting, red solid line).

the absorption spectrum decreased slightly probably due to some inevitable Pdot aggregations in the presence of elevated ion concentrations.31 Besides, negligible shift in the absorption peak of 670 nm was observed, suggesting that the PDA capping layer remained intact on the surface of dye-embedded Pdots. Upon the addition of 10 μM Pb2+ into the mixture, the Pdot solution turned red gradually in which the relative absorption peaks of 540 and 670 nm changed with time (solid red line in Figure 3A). These results further suggested that the excellent selectivity of these PDA-Pdot nanocomposites toward Pb2+ should be stemmed from the formation of 2:1 15-crown-5Pb2+-carboxylate sandwich complex. Assuming that all of the diacetylenes were successfully capped onto the surfaces of Pdots, the average numbers of carboxyl-functionalized and 15crown-5-modified diacetylenes are calculated to be 104 340 and 86 950, respectively. Because each carboxylate group and its proximal 15-crown-5 ether can chelate a lead ion to form a 2:1 sandwich complexation, there are 86 950 Pb2+-chelating sites on average on each Pdot. Additionally, we measured the fluorescence lifetime (τ) of bare PFBT-DBT Pdots to be 4.83 ns using a time-correlated single-photon counting module system (Figure 3B). The

Figure 4. (A) Confocal microscopy images HeLa cells labeled by PDA-enclosed NIR695-emdedded Pdots through endocytosis. Blue fluorescence is from nuclear counterstain Hoechst 34580, and red fluorescence is from Pdots. The right panel represents fluorescence overlaid with the bright-field image. (B) Images of HeLa cells which were incubated with 20 μM Pb2+ for 2 h. The scale bars are 30 μm. (C) Fluorescence intensity ratios (I650 nm/I715 nm) of Pdots at different pH, ranging from 3 to 8. (D) The impact of Pdots on cell viability determined by use of MTT assay.

It clearly shows that the fluorescence intensity of PFBT-DBT (red fluorescence, 605/37 band-pass filter) increased after the treatment of Pb2+, demonstrating that these Pdots can be applied to track changes in intracellular Pb2+ levels in mammalian cells. To further confirm that the increase in the emission of PFBT-DBT was not due to the pH response when Pdots were introduced into the acidic organells such as endosomes or lysosomes via endocytosis, we evaluated the emission response of these Pdots to pH changes. As shown in Figure 4C, minimal pH influence on the fluorescence ratio (I650 nm/I715 nm) was found. In addition, we conducted in vitro E

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utilized the test strips for visual detection of lead ions by immersing into the target solutions containing Pb2+. As shown in Figure 5B,C, the color of the test paper changed from blue to red under ambient light as the concentration of Pb2+ increased while obvious red fluorescence could also be seen under 365 nm UV light, consistent with the spectral response (Figure 2B). We noted that red emission on the test strips might be partially attributed to the red-state PDA although its fluorescence quantum yield is very low (∼1%). The result demonstrated that these Pdot-based sensors have promising potential for on-site visual detection of lead residues in water.

cytotoxicity experiments and found no significant cytotoxic effects of these Pdot-based nanoprobes (Figure 4D). We also assessed the applicability of this ratiometric sensing system in environmental conditions and simulated physiological media. For environmental samples, we spiked known amounts of Pb2+ into tap water and seawater (see the Supporting Information, SI, Tables S1 and S2 for their certified trace metal concentrations). For simulated biological fluids, we added Pb2+ into RPMI-1640 cell culture solutions (SI, Table S3). As summarized in Table 1, the results exhibited a good

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Table 1. Application of Pdots for the Determination of Lead Ion in Real Samples and in Simulated Physiological Media sample tap water

a

seawaterb

commercial RPMI-1640c

[Pb2+] nominal (μM) 0.50 2.00 5.00 0.50 2.00 5.00 0.50 2.00 5.00

CONCLUSIONS In summary, we have developed a sensitive, selective, portable, and ratiometric approach for Pb2+ detection based on this new type of PDA-functionalized Pdot sensors. The detection results can be readily observed by naked eyes. The linear detection range for Pb2+ falls within the ecotoxicologically (environmentally) relevant concentration range (e.g., EPA limits of Pb2+ poisoning). We have also successfully applied the sensors in tracking changes of lead ions in live HeLa cells. Moreover, we have created an easy-to-read test strip for further on-site sensing. This facile, reliable, and economical method takes advantage of the high optical brightness, good biocompatibility, and easy surface functionalization of Pdots. We anticipate the Pb2+-sensitive Pdots to find broad utility both in basic analytical detection and in studies of lead biology.

[Pb2+] found ± SD (μM) 0.51 2.02 5.26 0.46 1.94 5.29 0.48 1.94 5.06

± ± ± ± ± ± ± ± ±

0.07 0.06 0.28 0.16 0.12 0.42 0.06 0.07 0.18

a

http://www.water.gov.tw/eng/04water/wat_e_water_detail.asp?id= 12 (see the SI). bhttp://www.nrc-cnrc.gc.ca/obj/doc/solutionssolutions/advisory-consultatifs/crm-mrc/nass_5_e.pdf (see the SI). c Purchased from Life Technologies (see the SI).

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ASSOCIATED CONTENT

S Supporting Information *

agreement between the experimentally determined and spiked values for all of the samples. This experiment indicates the feasibility of using this Pdot-based sensing system for lead ion detection in both environmental and physiological samples. Preparation of Lead Test strips Based on PdotBlended PVA Films. To further apply this platform for in situ on-site detection, we prepared a test paper following the procedures as illustrated in Figure 5A. Briefly, we first prepared a mixture solution containing PVA, HEPES, PEG, and glycerol, and then the Pdot solution was added and mixed well. The mixed solution was then dripped onto a test paper inside a glass dish, and we waited until it was naturally dry. After that, we

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

Both authors (S.-Y.K. and H.-H.L.) contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Ministry of Science (103-2113-M-110-004-MY2), NSYSU-KMU Joint Research Project (103-I 004), and National Sun Yat-sen University. We also gratefully acknowledge support from Prof. Chao-Ming Chiang, Prof. Wei-Lung Tseng, Prof. Chin-Hsing Chou, and Dr. Jiun-Yi Shen from National Taiwan University.

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REFERENCES

(1) Department of Health and Human Services and Prevention, Center for Disease Control; Surveillance for Elevated Blood Lead Levels Among Childrens: United States, 1997−2001. Morbidity and Mortality Weekly Report; Centers for Disease Control and Prevention: Atlanta, GA, 2003; Vol. 52, p 1. (2) Jedrychowski, W.; Perera, F.; Jankowski, J.; Rauh, V.; Flak, E.; Caldwell, K. L.; Jones, R. L.; Pac, A.; Lisowska-Miszczyk, I. Int. J. Hyg. Environ. Health. 2008, 211, 345. (3) The European Parliament and the Council of the European Union; Directive on the Restriction of the Use of Certain Hazardous Substances in Electrical and Electronic Equipment. 2011/65/EU.

Figure 5. (A) Schematic diagram showing the processes of fabrication of Pdot-based test strips for lead ion sensing. (B) Color changes of test strips impregnated with Pdot-PVA films, after immersion into solutions containing 0, 200, and 500 μM Pb2+ for 5 min, respectively. (C) Their corresponding fluorescence under 365 nm UV light. F

DOI: 10.1021/ac504845t Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ac504845t Anal. Chem. XXXX, XXX, XXX−XXX