Research Article pubs.acs.org/journal/ascecg
Equipment-Free, Single-Step, Rapid, “On-Site” Kit for Visual Detection of Lead Ions in Soil, Water, Bacteria, Live Cells, and Solid Fruits Using Fluorescent Cube-Shaped Nitrogen-Doped Carbon Dots Raksha Choudhary,† Santanu Patra,† Rashmi Madhuri,*,† and Prashant K. Sharma‡ †
Department of Applied Chemistry and ‡Functional Nanomaterials Research Laboratory, Department of Applied Physics, Indian School of Mines, Dhanbad, Jharkhand 826 004, India S Supporting Information *
ABSTRACT: In this work, we have designed an equipment free, single-step, rapid, cost-effective, “in-house”, and “outdoor” kit for visual detection of lead ions (Pb2+) in various real samples viz., soil, water, bacteria, live cells, and solid fruits based on cube-shaped fluorescent carbon dots. The cube-shaped nitrogen-doped carbon dots (CCDs) were prepared using calcein dye as precursor and have potential to be used as better and stable replacement of commercially available dyes. For the visual detection of Pb2+, the color of the CCDs changes from yellowish to brown in solution and a limit of detection (LOD) of 10.0 μg L−1 was obtained for the naked eye. In addition, the same CCD solution was also coated on a filter paper strip to fabricate an easy-toprepare paper sensor. The paper sensor was used to identify the Pb2+ in real samples which proves their applicability toward in-situ on-site detection. In addition, the prepared strip sensor was successfully implemented for analysis of lead ions inside the solid fruit also. For quantitative detection, a photoluminescence (PL) study was carried out for trace level determination of Pb2+ with LOD of 2.21 ng L−1. Both the visual as well as PL study also suggested that the results obtained from the CCD sensing probe is free from any interference. We also incubated the CCDs into live cells (E. coli and MCF-7) through endocytosis and monitored the changes in Pb2+ levels within cells. The study demonstrates the role of prepared fluorescent probe for live cell bioimaging and intracellular detection of metal ions. KEYWORDS: Cube-shaped nitrogen-doped carbon dots, On-site visual detection, Paper sensor, Cell bioimaging, Intracellular detection, Solid-fruit analysis
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INTRODUCTION Our anthropological activities and tremendous increase in the use of metal ions as a part of our life have started contaminating the air, water, food, and/or soil; these have turned out to be the root cause of ecological damage and environmental pollution.1 Among the various metal ions, lead has a special place in different applications and is classified as one of the three most abundant and hazardous heavy metal ions for the environment.2 Lead has a high tendency of accumulation inside the body via bonding with thiol groups of enzymes or proteins, and therefore, after accumulation, it is very difficult to remove, either via chemical or biological treatment processes.3 Accumulation of lead can generate high blood pressure and kidney disorders and have a severe impact on children between the ages of 1 and 5 years.4,5 Therefore, in several countries the tolerance limit for lead has been decided and is strictly followed in electrical or electronic equipment.6 The blood level of lead has also been decided and fixed at the value of 0.1 mg L−1 (or 100 ppb) by the Center for Disease Control and Prevention (CDC).7 On the basis of the necessity of lead monitoring in electronic gadgets, food, blood, and environmental components, various analytical methods such as inductively coupled © XXXX American Chemical Society
plasma with mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) have been developed and used for accurate identification of lead, but these techniques require sophisticated handling, high instrumentation, and complex sample preparation steps and are very costly with high sample analysis time.8 Therefore, in addition to the big instruments, several electrochemical, optical (fluorometry and colorimetry) methods have also been designed to detect toxic metal ions in the parts per billion range, but they require skilled operators and sophisticated instruments, which are not possible in remote areas, at the particular time.9−11 On the basis of the need of lead monitoring the visual sensors can be a solution for this problem in a simple, costeffective, and eco-friendly way. There is no need of heavy instruments or toxic chemicals for analysis, and the result can be observed by the naked eye. For example, Jacobi et al. have reported a label-free, DNA-based sensor immobilized within monolithic hydrogel for visual detection of lead(II).12 The Received: June 28, 2016 Revised: July 28, 2016
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DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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applied for detection of lead ion inside the solid fruit. In the quantitative manner, the sensor is able to detect the ultratrace level of lead ions in real samples, by direct interaction of CCDs and metal ions. This is the first sensor of its kind for detection of lead ions in three different platforms with good sensitivity and selectivity in very small time span (few second) with very lesser cost.
sensor showed the detection limit of nanomolar range, but DNA handling is not an easy task. Recently, Nath et al. have reported a gold nanosensor for easy sensing of lead and copper ions in solution using paper strips.13 The sensor was proposed to be handy but shows a poor detection range at the parts per million level only. Some of the researchers have also reported the fluorescence based color change analysis system, which have high sensitivity but get limited by the problem of dyebleaching.14,15 For example, Karak et al. have reported a xanthone based Pb2+ selective turn on fluorescent probe for living cell staining.14 In the past few years, the new member of carbon family, i.e. carbon dots, have covered a major area of research in the field of florescence sensor; however, their role in visual sensing is still in under developed condition. Carbon dots (CDs) possess the small size, unique optical properties, photostability, mutable photoluminescent property, nonblinking, high aqueous solubility, low photobleaching, outstanding biocompatibility, low toxicity, and low cost characteristics,16 which make them a superior fluorescence material for the preparation of fluorescence probes in comparison to the commercially available dyes. Recently, Xiong et al. have reported the in situ electro-polymerization of nitrogen doped CDs for detection of intracellular lead ion.17 Similarly, CDs prepared by acid hydrolysis of bovine serum albumin18 and semiconducting polymer dots based dual colorimetric and fluorescent sensor19 were also prepared for detection of lead ions. Researchers have been discovering the reliable, economical, and rapid technique, which could be operated by unskilled operators for monitoring of trace level of lead ions in real samples, like groundwater, food samples, soil, etc. Therefore, using the advantages and considering the drawbacks of existing visual sensors, we have developed here a smart, portable, sensitive, selective, and easy-to-use (can be handle by any unskilled people also) visual sensor for direct estimation of milligram to nanogram level concentration of lead ions in real samples. The sensor was fabricated using the aqueous solution of CDs synthesized from the commercially available calcein dye as precursor. For the first time, we have reported the synthesis of nonspherical nitrogen-doped carbon dots, i.e., cube-shaped, in a single and simple synthesis step. Calcein is a fluorescent dye used for complexometric titration and cell labeling in biological applications;20 however it suffers from problem of photobleaching, low stability, and weak fluorescence intensity.21 In contrast to the precursor, the nitrogen-doped CDs synthesized from calcein dye possess more stability, high photoluminescence property, almost negligible photobleaching with good biocompatibility. In order to reduce the use of harsh chemicals, the synthesis of cube-shaped nitrogen-doped CDs (CCDs) was accomplished in green-way using water as solvent via combination of microwave and hydrothermal approach. The combination of these two methods, not only reduces the reaction time but requires very few chemicals and results in formation of 3D carbon dots (cube-shaped), unlike to others i.e. zero-dimensional CDs. The as prepared CCDs have high quantum yield and shows better performance as fluorescent ink for calligraphy and staining of objects in comparison to the calcein dyes. The CCDs were also used for qualitative (paper-based), semiquantitative (solutionphase) and quantitative analysis (fluorescence) of lead ions. The CCDs-modified filter paper strips were used for qualitative analysis of lead ions, which shows a sharp change in the color of strip in the presence of lead ion. The strip sensor was also
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EXPERIMENTAL SECTION
Chemicals and Apparatus. All the chemicals were of analytical grade and used as received. Lead nitrate and silvernitrate were purchased from Alfa Aesar (USA). Calcein dye, interferents like iron sulfate, auric chloride, zinc chloride, arsenic trioxide, cadmium(II) chloride, copper(II) chloride, mercury(II) chloride, chromium(VI) oxide and solvents like ethanol and methanol were purchased from Spectrochem Pvt. Ltd. (India) and Loba Chemie Pvt. Ltd. (India). For the preparation of stock solutions, double distilled water was used. The wastewater samples were collected from the coal mines and industrial area of Dhanbad, India. Human urine and blood samples were collected from local pathological laboratory (Indian School of Mines, Dhanbad). The fluorescence spectra were recorded in a PerkinElmer LS55 fluorescence spectrometer. The UV−visible spectroscopic analysis was done by a UV−visible spectrophotometer (Shimadzu, UV-1800). The transmission electron microscopy (TEM) was performed on a Tecnai T-30(300 Kv FEG TEM) and FT-IR analysis was carried out on Varian Fourier Transform Infrared [FT-IR (USA)] spectrometer. The powder X-ray diffraction (XRD) study of the nanoparticles was carried by Bruker D8 Focus X-ray diffractometer using a Cu source. All the experiment was performed at room temperature (25 ± 2 °C). The camera pictures were taken by Nikon Coolpix camera (16 mega pixel). Synthesis of Cube-Shaped Nitrogen-Doped CDs from Calcein Dye (CCDs). For the synthesis of CCDs a combination of microwave and hydrothermal synthesis was implemented. For this, 0.5 g of the calcein dye was dissolved in distilled water (20.0 mL) and kept in domestic microwave oven (power intensity = 600 W) for 30 min. After that the solution was placed for hydrothermal reaction in Teflon lined stainless steel autoclave at 150 °C for 2 h. After cooling to room temperature, the solution was collected and centrifuged at 12 000 rpm for 10 min to separate the less fluorescent and larger size CDs. The obtained CCDs solution was further dialyzed against ultrapure water using tubular dialysis membrane for 48 h. The reaction parameters like temperature, time (microwave and hydrothermal treatments) were optimized and discussed in the Supporting Information (section S1, Figure S1, and Table S1). Measurement of Fluorescence Quantum Yield (QY). The fluorescence QY was calculated by using aqueous solution of fluorescein dye dispersed in 0.1 M H2SO4 as a standard reference. The QY of prepared CCDs was calculated using following equation:
QYX = QY stdIxA std nx 2 /IstdA x nstd 2
(1)
Here, “I” is the measured integrated emission intensity, “n” is the refractive index of the solvent, and “A” is the optical density. The subscript “std” refers to the standard sample with known QY and “x” for the unknown samples. Patterning and Staining from Calcein Dye and CCD Solution. In order to study the fluorescent behavior of prepared CDs and their use as a replacement of commercial dye, their use in patterning or calligraphy and staining of the 3D crafted papers was explored. For the study, the CCD solution was filled in sketch pen and the sketch was used to draw some structures or write some letters on a black paper. For staining purposes, the same CCD solution was used and 3D paper artwork was been dipped in that solution. Immediately, the papers were removed out from the solvent and placed at open air for drying. The photographs of different patterns drawn on black sheets and stained paper arts have been taken via digital camera (Nikon Coolpix, 16 MP) in both visible daylight and UV light B
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 1. (A) UV−vis spectra of CCDs and calcein dye. (inset) Photograph of CCDs in day and UV light. (B) PL spectra of CCDs. (C) TEM image of CCDs. (D) XRD of CCDs. (inset) Particle size distribution curve derived from TEM analysis. (E) FT-IR of CCDs. (inset) SAED pattern of CCDs. (F) Zeta potential plot of CCDs. conditions. For comparison calcein dye solution (5.0 mg mL−1) was also prepared, and the same study was performed with dye also. Qualitative and Semiquantitative Analysis of Lead Ion in Standard Solutions. For the qualitative (i.e., presence or absence of lead) and semiquantitative (a range of concentration) analysis of lead ions present in aqueous medium, two different kind of sensing protocols were employed. First, the fixed concentration of CCDs solution (0.5 mL) was taken and mixed with different concentration of Pb2+solution (200.0 μL). Through naked eye, a color change of solution from yellowish to brown was observed which indicates the presence or absence of lead ion in the sample. The change in the color of solution with respect to concentration of lead ions was captured by digital camera in day light and used as standard reference color. Herein, the change in the color of the CCD solution is proportional to the concentration of lead ions present in the solution. In order to improve the visual detection, the CCDs coated stripsensor was also fabricated. For this, the whatman filter paper (grade: 41) was cut in a strip of: length = 5.0 cm, width = 1.0 cm, and dipped in the CCDs solution. After that, the strips were dried in air and again immerged in the different concentrations of Pb2+ ion. The change in color was observed immediately under normal day light and their images were recorded as standards. The limit of detection (LOD) for visual detection can be defined as the least amount of analyte able to produce a different color change that can be confirmed by the independent observers. Herein, the least concentration of lead ion responsible for change in the color of solution or strip was considered as the LOD of visual detection. Quantitative and Ultratrace Level Determination of Pb2+ in Standard Solutions. For quantitative and trace level detection of lead ions, the photoluminescence (PL) study was performed at the optimized parameters like contact time and pH. For the PL study, the
standard stock solution of Pb2+ of varying concentration was mixed to the fixed concentration of CCDs and corresponding change in the PL spectra were recorded. The change in fluorescence intensity of solution was fitted in Stern−Volmer equation: (I0/I ) − 1 = KSVC
(2)
Where, “I0” is the initial fluorescence intensity of CCDs, in the absence of Pb2+, “I” is the fluorescence intensity after adding definite concentration of Pb2+ solution, “Ksv” is the Stem−Volmer constant, “C” is the concentration of Pb2+ solution. The LOD was calculated as three times the standard deviation for the blank measurement in the absence of CCDs, divided by the slope of calibration plot between PL intensity and concentration of Pb2+ solution. Cytotoxicity Study of Calcein Carbon Dots and Their Role in Cell Imaging. To detect cell cytocompability of prepared CCDs, standard methyl thiazoltetrazolium bromide (MTT) assay and live-cell imaging was done using MCF-7 breast cancer cell lines.22 MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) having 10% (v/v) bovine serum, 1% (v/v) antibiotics (penicillin and streptomycin) at 37 °C in 5% CO2 humidified atmosphere. After 24 h, the cell were incubated with different concentration of CCDs (0− 1500.0 mg L−1) and again cultured for next 24 and 48 h. Each (single) concentration added to three wells was taken as parallel control and wells containing cell but not treated with CCDs was taken as negative control. Some wells were also taken without cells and treated as blank control. After incubation for next 24 h and/or 48 h, 20.0 μL of MTT was added to each well and optical density was evaluated (at 570 nm). Cell viability was calculated using the formula given below: cell viability(%) = (A sample − A blank ) × 100%/(Acontrol − A blank ) (3) C
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 2. (a) XPS spectrum of CCDs. Deconvoluted (b) C 1s, (c) N 1s, and (d) O 1s XPS spectra of CCDs.
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Where “Asample” is absorbance of a well with cells, MTT solution, and CCDs; “Ablank” is absorbance of a well with medium and MTT solution, without cells; “Acontrol” is absorbance of a well with MTT solution and cell, without CCDs. The data was articulated as the percentages of viable cells compared to the survival of control (untreated cells as controls having 100% viability). Three parallel wells were run for each concentration and each experiment was repeated three times. Real Sample Analysis. In order to explore the real-world applicability of the proposed sensor, various water samples viz., drinking water (from well, tap and hand pump), wastewater (from coal mine and industrial areas), and human sera samples (blood, plasma, urine) have been collected and analyzed via visual and PL methods. The human sera samples were collected from the pathological laboratory, Indian School of Mines (ISM), Dhanbad. For semiquantitative detection, human sera samples were spiked with Pb (II) (120.0 μg L−1). Both solution phase (in vial) and solid phase (by paper strip) detection method shows a change in color depending on the concentration of Pb (II). The appeared color (in solution or strip) was matched with the standard colors and approximate concentration of Pb (II) was calculated. In another experiment, the wastewater samples and drinking water samples (collected from different area) were used for monitoring of lead ions. For this, the collected samples were taken in the vial (200.0 μL) and 0.5 mL of CCDs solution or the CCDs coated strip was added to them. The corresponding color change in the vial and paper strip will again match with the standard and approximate concentration of Pb (II) was calculated. For measurement of of Pb (II) in solid fruit, first a potato was taken and injected with (120.0 μg L−1) of Pb (II) and stored for overnight for homogeneous distribution of lead in the fruit. After that the contaminated potato was sliced and a CCDs coated strip was inserted in the dissected portion. The change in the color of strip will provide an idea about presence or absence of metal ion in the fruit. Intracellular Imaging of Lead Ions. To demonstrate the feasibility of the prepared CCDs for sensing of lead ions in complex matrices, we have performed an intracellular detection study. For this, to the CCDs incubated MCF-7 cells, different concentrations of lead ions (0, 1.0, 2.0, 5.0 μg L−1) were introduced. After 10 min of incubation, the cells were washed with buffer solution to remove free CCDs or any nonspecific adsorption on the cell surface. After this, the confocal fluorescence microscopy image of MCF-7 cells was recorded. The change in the PL intensity of MCF-7 cells in the presence of lead ions was also recorded for quantitative determination.
RESULTS AND DISCUSSION Optical Properties of Prepared Cube-Shaped CDs. The optical properties of prepared CCDs were done through UV− visible and photoluminescence spectroscopic techniques (Figure 1). The UV−visible spectrum of calcein dye shows a distinct and characteristic peak at 500 nm, which corresponds to the extended system of conjugated π-bonds (causes the molecule to absorb light in the visible range). However, the CCDs show two peaks in two different UV-regions. The first broad peak appeared in the range of 400−500 nm, which may correspond to the presence of calcein skeleton (Figure 1A). The presence of a sharp and intense peak at 280 nm could be ascribed to the π−π* transition of the aromatic sp2 domains (CC bonds).23 The fluorescence property of prepared CCDs was monitored in the wide range of wavelengths (Figure 1B). It was observed that with change in the excitation wavelength, the intensity of emission spectra starts increasing, without showing any shift in their position. The maximum intensity emission peak was observed with the excitation of 420 nm. Without any modification and post-treatments, the CCDs show a very intense yellow color in UV light (inset, Figure 1A). The quantum yield of CCDs was also calculated and found to be 86.9% (fluorescein as the reference), which is much higher than majority of the CDs synthesized from different chemical precursors. The high quantum yield is possibly due to the existence of electron rich nitrogen and oxygen-containing functional groups, which are generally known as excellent auxochromes as well as shape-specificty.24 Morphology and Composition of Cube-Shaped CDs. The morphology and size distribution of CCDs are characterized by TEM (Figure 1C and inset and D). The TEM image and particle size distribution curve indicated that the as-prepared CCDs were well dispersed, cubical in shape and showed a narrow size distribution between 2.0 and 3.0 nm with the average size of ±2.2 nm. In addition, the selected area electron diffraction (SAED) pattern of CCDs was also recorded, which shows the amorphous nature of dots (inset, Figure 1E). The XRD pattern of CCDs shows a broader (002) D
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 3. Different calligraphy patterns drawn by sketch pen filled with CCD solution under UV light (λ = 360 nm).
2B).26 The N 1s spectrum reveals three distinct peaks at 398.4, 399.1, and 400.0 eV, which can be attributed to the presence of pyridinic, 1°/2° amino and pyrrolic nitrogen, respectively, in the CCDs (Figure 2C). The O 1s spectrum shows two distinct peaks at 531.7 and 532.5 eV, which are attributed to CO and C−OH/C−O−C functional groups, respectively (Figure 2D), revealing the formation of oxygen functional groups on the surfaces of CCDs. Fluorescence Stability of CCDs. To evaluate the stability of as synthesized CCDs under harsh conditions, various experiments were performed. The relative activity of fluorescence at different pH is shown in Figure S2. The effect of ionic strength (in terms of the concentration of NaCl) on the fluorescence stability of CCDs was also investigated. The stability results toward extreme pH and ionic strengths showed that the fluorescence of synthesized CCDs possessed fantastic stability in the wide range of different pH conditions (2.0− 12.0), and the fluorescence intensities remained constant with the increasing NaCl concentration (Figure S2), which would be beneficial for use in the presence of various salt concentrations in practical applications. The stability of CCDs was tested at different temperature range and fluorescence intensity was found almost stable in temperature range (10−90 °C) as depicted in Figure S2. Furthermore, the fluorescence stability of CCDs was also tested in the presence of UV light illuminations and different storage conditions. First, the CCDs solution was illuminated with UV light (UV lamp of 366 nm) for 2 h and
peak centered at about 21.5°, attributed to highly disordered carbon atoms and confirms the graphene core-structure of the as-prepared CCDs.25 The broad nature of XRD pattern also suggests the amorphous nature of CCDs (Figure 1D). The functional groups present on the surface of CCDs were investigated by FT-IR (Figure 1E). The peaks at ∼3400 cm−1 (−O−H and N−H stretch), 2820 cm−1 (C−H stretch), 1641 cm−1 (CC stretch), 1350 cm−1 (C−C stretch), 1235 cm−1 (C−O stretch), and 1090 cm−1 (C−N stretch) in the FT-IR spectra support the presence of −OH and −NH2 groups on the surface of CDs. Moreover, the elemental analysis was carried out to verify the composition of CCDs. The results reveal that the CCDs are composed of C = 52.10%, H = 2.62%, N = 6.38%, and O = 38.90% (calculated). The above results suggest that the obtained particles have nitrogen and oxygen heteroatom with many functional groups including −COOH, −OH, and −NH2. In order to study the total charge on the surface of prepared CDs, their zeta potential was also estimated and found as −22.26 mV, owing to the existence of electron rich −OH/−NH2 groups on the surface of CCDs (Figure 1F). Furthermore, the XPS spectra were also performed to analyze the surface state and composition of the CCDs (Figure 2). Three peaks at 285, 398, and 534 eV exist in the XPS spectra, indicating the presence of C, N and O elements in CCDs, respectively. The C 1s spectrum displays four distinct peaks at 285.4 eV (C−C/−CH), 286.0 eV (C−N bond), 286.2 eV (−C−O bond), and 286.8 eV (−CO bond) (Figure E
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 4. Three-dimensional crafted artwork stained with CCD solution, in daylight (A, D, E, G, J) and UV-light (B, C, F, H, I).
Figure 5. (A) Photograph of change in color intensity of CCDs upon addition of varying concentration of standard Pb2+ ion (10.0 to 250.0 μg L−1). (B) Change in color intensity CCDs coated paper strip for standard solutions of Pb2+ ion. (C) PL emission spectra of CCDs upon addition of various concentrations of Pb2+: (1) 0 to (20) 162.19 ng L−1. (D) UV−visible spectra of CCDs upon addition of various concentration of Pb2+: (1) 0, (2) 6.62, (3) 16.39, (4) 85.23, (5) 162.19 ng L−1. (E) UV−visible spectra of calcein dye upon addition of various concentrations of Pb2+: (1) 0, (2) 6.62, (3) 16.39, (4) 85.23, (5) 162.19 ng L−1.
change in fluorescence intensity was recorded every 15 min interval. In the duration of 2 h, no observable changes were recorded in the fluorescence intensity of prepared CCDs (Figure S2). Similarly, the CCDs solution was stored for 90 days in two different conditions: (1) in common refrigerator (5 °C) and (2) in room temperature (30 °C). After 90 days of storage, no changes were observed in both the CCDs solutions, which represent good stability (Figure S2). This makes the CCDs as best candidate for room temperature sensing applications. Comparison of CCDs and Calcein Dye by Patterning and Staining Study. The recent advances in the development and designing of fluorescent nanomaterials, basically carbon dots have taken the topmost position of the competitor of
commercially available dyes. In general the commercially popular dyes (e.g., calcein) suffer from problem of unstable fluorescence intensity and fast photobleaching tendency.27 However, owing to the quantum size effect of nanoparticles, the tiny carbon dots are free-from such problems and have much higher suitability for commercial applications.28 Herein, the comparison between PL intensity of calcein dye and CCDs was performed in UV light, and then the PL spectra were also recorded. First, at the same concentration the PL intensity of calcein dye and CDs was compared in UV light, their camera pictures were recorded and thereafter the PL spectra were taken (Figure S3). As shown in the figure, in the camera pictures, initially the PL intensity of calcein dye is slightly less in comparison to the CCDs, which starts decreasing immediately F
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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ACS Sustainable Chemistry & Engineering Scheme 1. Probable Binding between Prepared CCDs and Lead Ions
and get faded in 2 min, while, the CCDs fluorescence intensity and color remains stable for months. In the PL study it was found that dye solution have ∼10 times lower intensity than CCDs (Figure S3). Furthermore, the CCDs solutions filled sketch pens were used to draw some sketches and write some words on a black sheet. The written or drawn structures are completely invisible in the day light, however, shows a very bright and intense yellow color in the UV light (Figure 3). Similarly, the camera pictures of CCDs solution stained 3D crafted paper arts were also recorded and shown in the Figure 4. All the crafts are white in color in daylight; however, they shows bright yellow color in the UV light. We have also tried to use the CCDs solution for making some decorative piece. For this, a small oval shaped structure was filled with CCDs solution, which is light yellow in color. But in UV-light, it shows an illuminating, beautiful, and bright yellow color. A similar study was also performed with calcein dye solution and found very poor and unstable yellow color, which diminishes with time and becomes colorless in only a few seconds. The study can conclude the real-time application of prepared CCDs as an alternative to commercially available dyes, due to their “vis−invisible” properties; it can also be used in optoelectronic devices as anticounterfeit measures. Equipment-Free and Rapid Detection of Pb2+ by the Naked Eye. Other than their use as replacement of commercial dyes, the prepared CCDs show a very bright visual color change with different concentrations of lead ions. Interestingly, the calcein dye does not show any such color change, after addition of lead ions (Figure S2F). Owing to the sharp and stable change in the color of CCDs in the presence of lead ions, the CCDs solution and CCDs-coated strips (filterpaper) were used as a visual-sensor for presence or absence/ semiquantitative detection of Pb2+ ions in aqueous medium. First, the different concentrations of standard solutions of Pb2+ (10.0 to 250.0 μg L−1) were prepared and added to the fixed concentration of CCDs solution (Figure 5A). A fast and random change of color from light yellow, yellow to brown was observed with increase in concentration of Pb2+. The images of each vial were captured by digital camera and stored as standards. It was also observed that, when the Pb 2+ concentration was less than 10.0 μg L−1, no observable change in color was observed (Figure 5A) and beyond 250.0 μg L−1 concentration of Pb2+, the color remain constant. Therefore, the sensing concentration range in the solution-phase has been limited from 10.0 to 250.0 μg L−1, with LOD of 10.0 μg L−1 (the minimum observable concentration). The same solutions
were placed under UV-light also but quenching in the UV-light is not very distinguishable with change in the concentration of Pb2+ (Figure S4). Therefore, the visual color change under normal day light has been used for sensing of Pb2+ in the solution. In the field of water pollutant sensing, the major requirement is for outdoor or at-home kits can able to detect pollutants in a rapid, easy, economic, and accurate manner. Now-a-days, the point-of-care (POC) devices have become very popular in these fields, owing to their easy mode of operation, cost-effectiveness, and on-field detection or portability. In order to improve the portability and easy-handling operation of Pb2+ sensing in the water samples, the CCDscoated paper strips were prepared. In the simple, two step method of analysis, the CCDs-coated strips were dipped in the various concentrations of Pb2+ standard samples and the change in color of the strips were observed through bare eyes (Figure 5B). Their camera pictures were recorded in the UV-light also, which was unable to show the distinct variation in color of the strip with change in the concentration of Pb2+ (Figure S5). Like litmus paper test, where each pH gives a separate distinct color, the colors of strips were also standardized for particular concentration of analytes. As shown in the figure, the paper-strip based sensor can able to detect the Pb2+ ions in the concentration range of 10.0 to 200.0 μg L−1, with LOD of 10.0 μg L−1. Lower or higher concentration than the given range is unable to produce any observable color change in the CCDscoated strips. Quantitative Detection of Pb2+ by PL Study. For the quantitative and ultratrace detection of Pb2+ ions, the photoluminescence (PL) study was performed. As shown in the Figure 5C, the fluorescence intensity of the CCDs decreased gradually with the increase of the concentration of Pb2+. Figure S6 indicated the linear relationship between quenching efficiency (I0/I −1) and Pb2+ concentrations in the range of 6.62 to 162.19 ng L−1. Therefore, the concentration of Pb2+ could be calculated using the following calibration equation: [(I0/I ) − 1] = (0.0176 ± 0.0007)C(ng L−1) − (0.1701 ± 0.0627),
n = 9,
R2 = 0.99,
K sv = 0.0176 L ng −1
Under the same experimental conditions, the limit of detection of Pb2+ ions was estimated to be 2.21 ng L−1 based on 3SD/k (where SD is the standard deviation of the corrected blank signal of the CCDs and k is the slope of the calibration G
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 6. CCD solution, after addition of different metal ions in (A) daylight and (B) UV light showing cross-reactivity study. (C) PL intensity of CCDs recorded in the presence of single (interfering ions only) and binary mixture of metal ions with Pb2+. (D) Cytotoxicity assay of different concentrations of CCDs against MCF-7 cancer cell, after 24 and 48 h incubation time. Visual detection of Pb2+ in solution phase and via strip-sensor in various water samples. (E and H) Drinking water collected from our institute (tap water), local village (well and hand pumps), and a well located adjacent to the industrial area. (F and I) Human sera samples (serum, plasma, urine). (G and J) Wastewater (from coal mine and industrial areas).
curve).29 The calculated LOD is found to be very low in comparison to the earlier reported values using alternative methods (Table S2). The results demonstrated that CCDs could serve as sensitive and selective probe for the determination of Pb2+. Binding Mechanism between CDs and Pb2+. To determine the binding mechanism between Pb2+ and CDs, UV−visible analysis was performed. The spectrum was recorded for CCDs with varying concentration of lead ions and shown in the Figure 5D. As shown in the spectra, with the addition of Pb2+ to the CCDs solution, a distinguishable change in the spectra of CCDs was observed. First, the intensity of main peak (480 nm) get decreased and shifted toward longer wavelength i.e. red-shift, which suggests the bonding interaction between CCDs and Pb2+ ions. The red-shift in the UVspectrum also supports the appearance of orange−red color, after mixing of Pb2+ and CCDs. In addition, to this peak, a new peak also appeared at 323 nm, when Pb2+ was added to the CCDs and get enhanced with increase in concentration of metal ion. The new peak could be attributed to the red color complex formed due to mixing of Pb 2+ and CCDs. Interestingly, when the same experiment was performed with
calcein dye, initially a single peak (478 nm) with two shoulders (on both sides) was observed. Among these, the shoulder peak appeared at 500 nm was diminished with increase in concentration of spiked Pb2+, owing to the dilution effect; however, the other peak remained constant in their original position and no extra peak appeared in the spectra, contrary to the CCDs (Figure 5E and Figure S7). On the basis of the UV− visible study, the possible bonding between Pb2+ with CCDs is shown in the Scheme 1. It can be concluded that the lead ions form a type of coordination complex with the surface groups of CCDs; however, the complex formation is not possible with calcein dye. The strong bonding interaction between CCDs and metal ions is responsible for a sharp change in the color of CCDs in the presence of Pb2+; whereas calcein dye failed to show any such color change. Interference Study. For sensor fabrication, selectivity is the key phenomena. Therefore, the effect of metal ions on the luminescence of CCDs was inspected by measuring the fluorescence intensities in the presence and absence of various metal ions including Ag+, Au3+, Cd2+, Hg2+, Cu2+, As3+, Fe2+, Cr3+, Zn2+, Fe3+, Ca2+, and Mg2+. First, their effect was visually observed by recording the camera picture of CCDs solution, H
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Table 1. Detection of Pb2+ in Human Sera, Waste Water, Soil, Leaf, and Vegetable Samples Using CCDs as Fluorescence Probes
after addition of different metal ions in day light and UV light (Figure 6A and B). As shown in the figure, no change in color of CCDs solution was observed with any of the interfering metal ions, either on day or UV light. The CCDs shows a bright orange color (in day light) or black color (due to quenching in UV light) only in the presence of Pb2+, which shows the high selectivity of the prepared visual sensor. The corresponding change in PL intensity was also recorded in the single (interfering ions only) and binary mixtures also (interfering ions and Pb2+) (Figure 6C). As expected, no change in the PL intensity was observed in the presence of interfering ions; however, appropriate quenching was observed in the binary mixture of lead and other interfering ions. Therefore, it can be concluded that, other metal ions do not have any interference effect in the quantitative detection of Pb2+ ion. Real Sample Analysis. To verify the applicability of proposed method, the sensor was used for detection of Pb2+ in some real samples also. In the study the samples used are basically drinking water (from well, tap, and hand pump), wastewater (from coal mine and industrial areas), and human sera samples (serum, plasma, urine). The drinking water was collected from our institute (tap water), local village (well and hand pumps), and a well located adjacent to the industrial area. The human sera samples did not contain Pb2+, in general; therefore, a sample spiked with known concentration of lead ions is used for analysis. First, the visual analysis was performed. For this, 200.0 μL of samples were directly (without any pretreatment) was added in the glass vial containing 0.5 mL of CCDs and corresponding change in the color was observed. The color that appeared was matched with the standard color, and a semiquantitative analysis was performed. As shown in the Figure 6E, F, and G, except the drinking water collected from our institute tap; all the other water samples contain some amount of lead ions, and the results were tabulated as Table S3. The maximum concentration of lead ions was found in the drinking water collected from the well adjacent to the industrial area. The water collected from wells also shows the presence of lead ions; however, the concentration is found less than tolerance limit. However, the wastewater collected from industries or coal mine areas has very high concentrations of lead ions (Table S3). Similarly, CCD coated paper strips were also used to check the quality of water samples in terms of concentration of lead ions. For this, the samples were taken in a glass vial and the strips were immersed in it. Immediately, the strip shows a change in color, which is matched with the standard color and the corresponding concentration was determined. As shown in the Figure 6H, I, and J, almost similar results were obtained with strip-sensors also, where the concentration of Pb2+ found in the different samples is portrayed in Table S3. It was also observed that the results obtained from visual sensing (either via solution-phase analysis or strip-sensing) are very similar to the results obtained via PL study. The PL data were also recorded for real samples and the results were given in Table 1. In each sample, approximately 99.1−100.5% recovery was obtained with 1−1.3% RSD values. Furthermore, for the validity of the proposed sensor, the real samples were also analyzed via atomic absorption spectrometry (AAS), which shows 100% agreement with the obtained results. Therefore, from the real sample analysis, it can be easily concluded that the proposed sensor have potential to analyze Pb2+ in any type of matrices using the visual (equipment-free) or instrumental based methods.
sample serum 1 serum 2 urine 1 urine 2 wastewater
spike (ng L−1) 16.39 32.71 16.39 32.71 16.39
industrial water
16.39
lake water 16.39 lake water 16.39 tap water 16.39 tap water 16.39 soil 16.39 soil 16.39 potato extract
16.39
Brinjal extract
16.39
tomato juice 16.39 mango juice 16.39 potato leaf 16.39 a
determined ± SDa (ng L−1)
recovery (%)
RSDb (%)
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
99.8 100.0 99.6 99.8
1.1 1.1 1.3 1.2 1.0 1.1 1.1 1.2 1.3 1.1 1.2 1.0 1.2 1.1 1.0 1.2 1.1 1.1 1.2 1.3 1.0 1.1 1.0 1.0 1.1 1.2 1.0 1.0 1.2 1.2
16.37 32.72 16.32 32.66 18.11 16.43 14.11 16.30 6.12 16.29 5.98 16.39 13.11 16.47 14.43 16.45 16.22 16.25 15.31 16.35 15.96 16.35 12.22 16.30 12.96 16.44 12.33 16.36 14.35 16.39
0.18 0.36 0.21 0.39 0.18 0.17 0.16 0.19 0.08 0.18 0.07 0.17 0.16 0.18 0.15 0.19 0.18 0.18 0.19 0.22 0.16 0.18 0.12 0.16 0.14 0.20 0.12 0.16 0.17 0.19
100.2 99.4 99.3 100.0 100.5 100.4 99.1 99.7 99.8 99.4 100.3 99.8 100.0
Standard deviation of three replicate values. deviation. cAtomic absorption spectroscopy.
b
AASc
18.12 14.10 6.13 5.96 13.12 14.44 16.24 15.30 15.96 12.22 12.95 12.31 14.33
Relative standard
In a very interesting analysis, the CCDs coated strips were used for detection of Pb2+ in solid fruit. The potato (already contaminated with Pb2+) was used for analysis, and as shown in the supporting video (SI video), the potato was sliced and the strip was inserted in the fruit. Within seconds, an orange color appeared on the strip shows the presence of lead ion in the solid fruit. Therefore, the strip sensor could also be used for quality check or purity and contamination checking of fruit/ vegetables etc., before their intake by common people. Toxicity of the CCDs and Their Application in Bioimaging. For effective bioimaging, it is essential that the selected fluorescent probe must have excellent photoluminiscent properties, high stability, and bright luminescence with low cytotoxicity. To evaluate the cytotoxicity of CCDs, the viability of MCF-7 cells treated with CCDs was measured by the methyl thiazolyltetrazolium (MTT) method. As shown in Figure 6D, the cell viabilities were assessed to be greater than 95% upon addition of the CCDs over a wide concentration range of 0− 1500.0 mg L−1 with incubation times of 24 and 48 h. The results indicate the low toxicity and excellent biocompatibility of prepared CCDs, which made them a suitable and safe probe for in vivo applications. To verify the ability of the CDs for cell imaging, in vitro cellular uptake experiment was carried out with MCF-7 cells I
DOI: 10.1021/acssuschemeng.6b01463 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Figure 7. Confocal microscopic images of MCF-7 and E. coli cells, before and after incubation with fixed Pb2+, captured at different time intervals: (A and E) 0, (B and F) 10, (C and G) 20, (D and H) 30 s, respectively. Confocal microscopic images of MCF-7 cells, after incubation with different concentrations of Pb2+: (I) 0, (J) 1.0, (K) 2.0, and (L) 5.0 μg L−1.
and E. coli cells. The cells were treated with CCDs and observed under a fluorescence microscope. Figure 7 showed the photographs of the MCF-7 and E. coli cells, captured by a laser scanning confocal microscope. As expected, the CCDs incubated E. coli and MCF-7 cells became quite bright and yellow colored owing to the strong fluorescence from the carbon dots, indicating a large amount of CCDs had been internalized into the cells. The yellow color emission is an important property of CCDs and has advantage over commercial dye. According to the literature, the nonfluorescent acetomethoxy (AM) derivate of calcein known as calcein-AM is very popular in the field of cell imaging, owing to their low cell toxicity. After transport into the cells, intracellular esterase removes the acetomethoxy group and converts the calcein-AM to calcein; the calcein molecule gets trapped inside and gives out strong green fluorescence. The green color emitted by the dye is some time difficult to observe in the presence of tissues having autofluorescence of internal fluorescence properties.30 In addition, since bacterial cells have a cell wall, calcein-AM will not pass through the bacteria cell wall and, therefore, cannot used for bacterial cell staining/imaging. Herein, the bright yellow color emitted from CCDs, is free from the problem of autofluorescence and could be used as replacement of calecin dye with more suitability and high fluorescence. The prepared
CCDs can able to stain and visualize both types of cells, i.e., animal cell as well as bacterial cells. Intracellular Imaging of Pb2+ Ion. Owing to the effective monitoring of Pb2+ ions in different complex environment and samples, the CCDs were also used to monitor the presence of Pb2+ inside the MCF-7 and E. coli cells. Figure 7 (A−D and E− H) shows the images of the MCF-7/E. coli cells, respectively, initially incubated with CCDs at 37 °C for 1 h and then exposed to Pb2+ ions (5.0 μg L−1) for different time intervals i.e. 10, 20, and 30 s. As the time passes, the yellow fluorescence intensity of CCDs starts fade away. In a few seconds (
