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Paper Based Photoluminescent Sensing Platform with Recognition Sites for Tributhyltin Esma Sari, Recep Uzek, and Arben Merkoçi ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01396 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019
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Paper Based Photoluminescent Sensing Platform with Recognition Sites for Tributhyltin
Esma Saria,b, Recep Üzeka,b, Arben Merkoçi*,a,c a Catalan
Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, Barcelona 08193, Spain
b Hacettepe
University, Faculty of Science, Department of Chemistry, 06800, Ankara, Turkey c ICREA,
Pg. Lluís Companys 23, 08010 Barcelona, Spain
* Correspondence Author Prof. Dr. Arben Merkoçi Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and The Barcelona Institute of Science and Technology, Campus UAB, Bellaterra, Barcelona 08193, Spain Tel: +34937374604 E-mail:
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ABSTRACT : In this study, a novel photoluminescence material for the detection of tributyltin (TBT) was developed by using a paper-based nanocomposite system. For this purpose, molecularly imprinted polymeric nanoparticles (MIN) were synthesized with mini-emulsion polymerization technique. Graphene quantum dots obtained by the hydrothermal pyrolysis were immobilized to the nanoparticle surface via EDC-NHS coupling. The fabrication of sensing platform for TBT can be divided into two steps that are the preparation of nanocomposite and the applying the nanocomposite onto nitrocellulose membrane. The selectivity constant and association kinetics were calculated to analyse the interaction of TBT with immobilized MINs. The results proved that the developed nanosensor is promising for the determination of TBT with high selectivity and sensitivity reaching a detection limit of 0.23 ppt in seawater. This novel photoluminescent nanosensor has the potential to pave the way for further studies and applications. KEYWORDS : Molecular imprinting, graphene quantum dots, organotin, nanocomposite, optical sensor
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In recent decades, polymer nanocomposites have attracted great attention in sensor applications because of their unusual multifunctional properties with unique design possibilities 1-3. In addition, graphene is the most popular precursor for the preparation of polymer nanocomposites due to its excellent physical and chemical properties such as biocompatibility, low toxicity, etc. 4, 5. Graphenebased polymer nanocomposites have been used to enhance the optical, mechanical, thermal and electrical properties of polymer nanocomposites 6-8. Paper-based systems are good alternatives to applications of polymer nanocomposites in the sensor field. Paper-based sensors have reached wide popularity because of their low cost, easy to operate, portable and simplicity of fabrication 9-12. Rapid and selective detection of trace analytes has become essential in many fields 13, 14. Especially, detecting organometals pollutants is a high priority over public safety and environmental protection 15-18. The detection of organometallic compounds present in groundwater is crucial not only for their toxicological effect but also bioaccumulation properties of these compounds 19. Tributyltin (TBT) is an organotin compound which is widely used for antifouling paints of-boats and ship hull to overcome bioinstruction problem. These paints lead to the leakage of TBT into the aquatic environments and so the TBT level in lakes, rivers and seawaters range from ppm to ppt 20, 21. Moreover, it has been reported to have toxic effects even below 1.0 ng.L-1 21. Since TBT is well known to have a highly toxic effect on biological systems and environment such as DNA damage, imposex problem, endocrine disruption, there is a serious concern about the detection of the trace amount of TBT 22-24. The methods involving the main steps of extraction or preconcentration, derivatization, separation and detection such as solid-phase microextraction combined with liquid chromatography-mass spectrometry (SPME-LC-MS), liquid-phase extraction combined with gas chromatography-mass spectrometry (LPE-GC-MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography combined with inductively coupled plasma–mass spectrometry (GC-ICP-MS), gas chromatographytandem mass spectrometry (GC-MS-MS), etc. were widely used to provide sufficient sensitivity and selectivity for the detection of TBT 20. As there are disadvantages such as time-consuming, the requirement of an expert, expensive and use high volumes of toxic, the novel methods like biosensors are necessary to determine TBT. Biosensors are analytical devices consisting of a biorecognition ligand such as an aptamer, DNA, enzyme, etc. and convert the biological signal into a detectable signal for the detection of an analyte. Furthermore, they present some advantages such as high sensitivity and selectivity, low cost, easy-to-use, fast and relatively simple instrumentation 25. Considering the instability and cost of biorecognition ligands, the molecular imprinting is a good alternative to biorecognition. Molecularly imprinting is a method used to tailor the analyte recognition center by copolymerization of a monomer mixture with the analyte as the template in the presence of cross-linkers. Molecular imprinting technique has attracted attention from the early 1990s until now for recognition due to their physical shape selectivity and chemical recognition sites which makes this technique highly selective and simple when it is compared with conventional methods such as antibodies and enzymes as a biomolecular recognition material 26-29. The approach of detection with the molecularly imprinted polymer combined with fluorescent materials recently has been widely used to meet the increasing demand for efficient, reliable, simple, and versatile nanodimensional sensor 30-33. The development of new type of fluorescent nanomaterials attracted wide attention to satisfy the increasing request of low toxicity, photo-stability, and biocompatibility. Considering this framework, Graphene Quantum Dots (GQDs) are excellent materials-owing to their marvelous properties related to band gap caused by quantum confinement and strong photoluminescence (PL) inflicted by edge effects 34, 35. In this study, the novel sensor platform was designed to detect TBT as a model analyte by using the nanocomposite with TBT recognition sites and photoluminescence on nitrocellulose. As mentioned above, the paper-based molecular imprinted nanocomposite system was developed for the first time for the TBT detection as an alternative to expensive systems such as GCMS, GC-MS-MS used in TBT determination and the detection limit at ppt level is comparable to these systems was achieved. The nanocomposite composed of the molecularly imprinted nanoparticles coupled with GQDs (GMIN) which photoluminescence was affected by TBT to be detected either in PBS buffer or in seawater was applied on the nitrocellulose to obtain the lower limit of detection. This novel photoluminescent sensor established in this research, has the potential to pave the way for further studies and applications. ACS Paragon Plus 3 Environment
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EXPERIMENTAL Materials Monobutyltin (MBT), dibuyltin (DBT), tributyltin hydride (TBT), L-glutamic acid (Glu), NHydroxysuccinimide (NHS), quinine sulfate, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), L-aspartic acid (Asp), ethylene glycol dimethacrylate (EDMA), methacrylic acid (MAA), ammonium persulfate (APS), sodium bisulfite, sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA, MW 30000-70000) and dialysis tubing cellulose membrane (MW cut-off, 14000) were purchased from Sigma (Madrid, Spain). Nitrocellulose membrane (SHF1800425) was obtained from Millipore (Billerica, MA). Phosphate-buffered saline (PBS) tablets, dialysis membranes (Spectra/Pore 5) were acquired from Sigma-Aldrich (Madrid, Spain). All chemicals were of analytical grade and used as received unless otherwise noted. The other reagents were in analytical grade and were from Sigma (Madrid, Spain). All aqueous solutions were freshly prepared in Milli-Q water produced using a Milli-Q system (>18.2 MW cm−1) purchased from Millipore. The stock solution of TBT was prepared in methanol and stored in the fridge at 4°C by covering with aluminum foil to prevent light degradation when not in use. Apparatus Particle sizes and distributions were estimated using Nano Zetasizer (NanoS, Malvern Instruments, London, UK). FTIR spectra of the nanoparticles were recorded with a Fourier transform infrared spectrometer (Bruker PMA 50 with Tensor 27, Coventry, UK). Scanning transmission electron microscopy (S-TEM) images were taken with a FEI Tecnai F20 S/TEM (Hillsboro, OR). UV-vis absorbance and photoluminescence measurements were carried out using a SpectraMax M2e spectrophotometer (Molecular Devices, Sunnyvale, CA). Sigma centrifuge (Sigma® 1-15, Fisher Bioblock Scientific, DE) was used. Q-Sonica Sonicator Q125 (Newtown, USA) was used to the homogenization of the samples. Synthesis and purification of carboxyl-terminated NPs The procedure for the fabrication of molecularly imprinted nanoparticles (MIN) is based on twophase mini-emulsion polymerization methods 36, 37. The amount of polar monomer in the organic phase is very important to the nanoparticle formation and polymerization in mini-emulsion polymerization. The use of excess amounts of polar monomers or target molecules may result in bulk polymer formation or inhibition. Therefore, the amount of MAA in the organic phase was examined, and the nanoparticles were prepared by using the optimal amount of MAA to obtain the higher sensor response. In a typical procedure, two different aqueous phases and oil phase were prepared, separately. Prior to polymerization, PVA (93.75 mg), SDS (14.425 mg) and sodium bicarbonate (11.725 mg) were dissolved in 5 mL Milli-Q ultrapure water to form the first aqueous phase. The second aqueous phase was prepared by dissolving of PVA (50 mg) and SDS (50 mg) in 100 mL of Milli-Q ultrapure water. In order to prepare an oil phase, TBT (80.4 µL, ~ 0.3 mmol) was mixed with MAA (200 µL, ~ 2.4 mmol) functional monomer under vigorous stirring with a molar ratio of 1:8 to obtain the pre-complexing solution. Crosslinking monomer EDMA (1.05 mL) was mixed to oil phase, being stirred for an additional 30 min. Thereafter, the oil phase was mixed with the first aqueous phase. The mixture was sonicated at 90% magnitude for 15 min in an ice bath (Sonicator, QSonica Q125, 125 Watt) to get a mini-emulsion. Afterwards, homogeneous solution was added into the second aqueous phase, and the mixture was transferred to the three-necked bottle and was stirred magnetically at 500 rpm (Radleys Carousel 6, UK). When the polymerization mixture was reached the polymerization temperature (i.e. 40°C), the initiator pairs, sodium bisulfite (58.1 mg) and ammonium persulfate (63.0 mg) were injected to the system and polymerization process was allowed to proceed for the next 24 hours under 500 rpm stirring. The solution consisting of nanoparticles was centrifuged at 5000 rpm for 30 min to remove any larger or agglomerated particles. Upon completion, in order to remove unreacted monomers and surfactant and initiators, nanoparticle solution was first dialyzed using dialysis membrane against Milli-Q ultrapure water overnight. After removing of the impurities, TBT in MIN was then extracted by dialysis against ethanol-Milli-Q ultrapure water (v/v, 50:50) for 4 hours and finally dialyzed against Milli-Q ultrapure water for 4 hours. Finally, the nanoparticle solution covered with aluminum foil was stored in the refrigerator. Non-imprinted ACS Paragon Plus 4 Environment
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nanoparticles (NIN) were also prepared in a similar manner as above, except without the addition of the template, TBT. Synthesis of amine-terminated GQDs Graphene quantum dots (GQDs) were prepared via the hydrothermal pyrolysis 17, 38, 39. Two precursors (glutamic acid and aspartic acid) mixture was used for the first time to obtain N-terminated GQDs. A 10 mL beaker with 2 g glutamic acid and 100 mg aspartic acid was heated to 185°C by using a silicone oil bath. After heating for 15 min, a clear transparent liquid of GQDs was obtained. The liquefied solution was poured into Milli-Q ultrapure water (25 mL). Following this step, GQDs solution was exposed under the UV lamp (365 nm) for 30 min to enhance PL intensity of GQDs. The quantum yield (φ) was calculated by comparing PL intensities and absorbance values of the GQDs with the standard quinine sulfate in H2SO4 (50 mM) solution (φ=54%). Preparation of GQDs conjugated NPs EDC/NHS coupling was applied for the immobilization of GQDs onto the NPs. 5 mL NPs were mixed with 62.5 mM 2 mL EDC and 12.5 mM 2 mL NHS and stirred for 2 hours at room temperature. Activated NPs were collected by centrifuging at 14000 rpm for 30 min and washed with twice with Milli-Q ultrapure water. Then 7 mL of GQDs was added to the solution and kept under gentle string for overnight. Afterward, the above solution was centrifuged at 14000 rpm for 1 hour to remove free QDs and washed with twice with Milli-Q ultrapure water. After washing step, the final precipitate called the nanocomposite (GMIN or GNIN) composed of NPs (MIN or NIN) and GQDs was dispersed in PBS buffer. Then, the nanocomposites in PBS buffer at the concentration of 30 mg/mL were kept in the refrigerator at 4°C with an aluminum foil cover. The stored solution was used until the end of the solution about 6 months without subsequent filtration, aggregation, microbial contamination, etc. Schematic illustration for the sensing principle of GMIN was given in Figure 1.
Figure 1. Schematic representation of the developed material (GMIN) and its sensing principle. Characterization of nanoparticles X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR) were used for the structural characterization of nanoparticles. The optical properties were investigated by UV-spectrophotometer (SpectraMax M2e spectrophotometer, Sunnyvale, CA). The size distribution and surface morphology of nanoparticles were characterized by using Nano Zetasizer (NanoS, Malvern Instruments, London, UK), scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HR-TEM). ACS Paragon Plus 5 Environment
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Preparation of paper-based sensing system During the experiments, all the fluorescence intensity detections were performed under the same conditions, i.e.; excitation wavelength was set at 350 nm with a recording emission range of 400-500 nm with 5 nm slit widths. Nitrocellulose (NC) membrane was cut into pieces of size ~ 6 mm by using a standard paper puncher. The NC disk was placed into a black microwell plate. Then, 2.5 µL of GMIN solution (diluted in 8× in PBS buffer pH 7.4) was manually pipetted into each disk. The NC disk was dried at 40°C for 5 min. The PL intensities of the test zones were readout as an initial value (peaking at 440 nm). Afterward 2.5 µL of the solutions, including different concentrations of TBT (from 0.2 ppb to 0.2 ppt) was added dropwise onto the test disk and dried at 40°C for 5 min. The application process was shown in supporting information (Video S1). The PL intensities of corresponding disks were measured as a final value. It should be noted that the signal of detection disk was recorded as a blank by treating with only PBS buffer in the above process. In addition, the detection zone was incubated only with GMIN as a control group. The design and application of paper-based system were illustrated in Figure S1 (Supporting Information). The response of the paper-based PL nanosensor was also tested for MBT and DBT with the same procedure to reveal the specificity and the selectivity of the nanosensor. For equalizing of the signal coming from an NC disk background, statistically analyze and overlapping criteria were applied to the quantitative data obtained from each NC test zone, as follows. The NC disks were selected to reduce the background noise for the application according to their PL intensities before the application of nanocomposites on the NC disk. The standard deviations of initial PL intensities of NC disks must be below 5% to choose the NC disks which have similar initial PL intensities before the application. The response of the nanosensor was described as below; 𝐼 𝐼 𝐼𝑜 ― 𝐼𝑜 𝑡 𝑐 Relative response = 𝐼 𝐼𝑜
[
[ ] [ ] [ ] 𝑐
]
where, I and Io are equilibrium and initial PL intensities of the sample (maximum emission intensity at 430 nm), respectively. c is the control experiment and t is the experiment with different analyte concentrations. The results with standard deviations were reported as an average of least ten measurement. Analysis of the seawater sample Seawater samples were collected from Poblenou Beach (Barcelona, Spain). Seawater samples were filtered through Whatman no. 1 filter paper to remove insoluble impurities. TBT, MBT and DBT standard solutions were spiked into the seawater to investigate the performance of the nanosensor system for real samples. For the paper-based sensing application, the PL intensities were scanned with the aforementioned procedure. RESULT AND DISCUSSION Preparation and characterization of GMIN The structural analysis before and after immobilization of GQDs on NPs and the inclusion of functional monomer into the structure were investigated by using X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR) (Figure 2). Baseline correction and smoothing were applied to FTIR spectra given in Figure 2a. The common bands in both spectra O-H stretching bands at around 3440 cm−1 the aliphatic C-H bending at 2950cm-1, C=O stretching at around 1700 cm-1, and C-O stretching bands at around 1145 cm-1 were due to EDMA and MAA. The characteristic bands for GQDs 39-41 were used to identify the structure of GQDs (Figure S2). The stretching vibrations of amine N–H (ca. 3360 cm-1) in GMIN nanoparticles spectra indicated the presence of amine groups in GQDs. As a conclusion, these results prove that the inclusion of the
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functional monomer into the structure and covalently immobilization of GQDs on the surface were carried out successfully. Further functional group analysis of GQDs, MIN and GMIN was performed by using XPS technique (Figure 2b). As seen in Figure 2b, C 1s, O 1s and N 1s signals were observed at 285 eV, 533 eV and 400 eV for GQDs, respectively. C 1s and O 1s signals come from EDMA and MAA were obtained at 286 eV and 533 eV, respectively for MIN. As seen in GMIN spectra, there are similar signals at the same energy with MIN but there is also some difference in signal intensity and GMIN has N 1s signal at 402 eV and Sn 3d signal at about 500 eV. The GMIN spectra was taken after incubation TBT and Sn 3d signal comes from TBT. After baseline correlation of the N 1s signal of nanoparticles, it can be clearly seen that GQDs and GMIN have an amine group of a surface. However, there is no N 1s signal in MIN spectra and the N 1s signal in GMIN spectra resulted from immobilization of GQDs. Moreover, the N 1s signal intensity must be decreased after immobilization as expected because the amine groups were used for immobilization of GQDs to MIN surfaces. These results clearly prove that the immobilization of GQDs on MIN surface and also corresponding to FTIR.
Figure 2. Spectroscopic characterization of NPs. (a) XPS spectra of GQDs, MIN and GMIN; (b) FTIR spectra of GQDs, MIN and GMIN, respectively. The size and surface properties of GQDs were investigated by TEM images. The TEM images and size histogram of GQDs were given in Figure S3. The average size of GQDs is around 3 nm according to the histogram. Furthermore, GQDs were spherical with narrow size distribution and have crystallinity with lattices of 0.20 nm which are sp2 clusters in GQDs (Figure 3a and 3b). The size distribution and surface morphology study of nanoparticles were performed by zetasizer, TEM and STEM. The size of MIN and NIN was changing from 30 nm to 90 nm and average size of nanoparticles around 40 nm according to zetasizer and STEM micrographs (Figure S4 and Figure S5A). After immobilization of GQDs on MIN and NIN, the average size of nanoparticles increased to 50 nm expected due to the fact that nanoparticles were covered with GQDs (Figure S4 and Figure S5). As seen in Figure 3c-3f, the core-shell structure of nanocomposites (GMIN and GNIN) was formed 42. These results indicate that the nanoparticles were successfully synthesized with spherical and narrow size distribution, and the nanoparticles surface was also covalently covered with GQDs. SEM technique was used to control the immobilization of the GMIN on nitrocellulose membrane (Figure S6). It is clearly seen that GMIN is immobilized after drying on the nitrocellulose membrane.
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Figure 3. Surface morphology of NPs: STEM and TEM micrographs of GQDs (a,b), GNIN (c) and GMIN (d-f). To confirm the successful preparation of the GMIN, UV−vis and PL spectra of NPs, GQDs and GMIN were recorded. As seen in Figure 4, GMIN and GNIN with fluorescence properties were prepared successfully. In addition, the aqueous solution of NPs, GNIN, and GMIN photographs were taken under visible light and 365 nm UV light. ACS Paragon Plus 8 Environment
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Figure 4. Spectrofluorophotometric analysis and optical images of NPs. Optical images of GQDs and nanoparticles (under sunlight (a) and under 365 nm UV light (b)), absorbance (c) and fluorescence spectra (d). Assay optimization and performance of the nanosensor for TBT detection The immobilization concentrations (EDC/NHS, GQDs and MIN) and GMIN concentration were investigated to find the optimal response of the nanosensor in assay optimization (Supporting information). After assay optimization results (Figure S7), optimal EDC/NHS ratio and GMIN concentration were determined as 62.5 mM/12.5 mM and 1/16 respectively. The aim of the nanosensor was to detect TBT using a paper based system employing TBT-induced photoluminescence (PL) signal change. The mechanism of TBT sensing based on electron-transferinduced fluorescence quenching and the quenching of the PL signal was carried out when TBT bound to the cavities that specific for TBT. According to the emission spectrum of PL intensity values at 440 nm of GMIN excited by a 350 nm UV light was evaluated for TBT concentrations in the range from 0.2 ppt to 0.2 ppb in PBS (Figure 5). As plotted in Figure 5, the relative response of GMIN increased by increasing TBT concentrations from 0.2 ppt to 0.2 ppb for GMIN in PBS and seawater. As seen in Figure 5, the nanosensor also has higher response to TBT in lower concentration than in higher concentration because of filling the cavities. Furthermore, the resulting calibration curves are fitted well in exponential growth in PBS and seawater (Figure S7). Limit of detections of the GMIN (LOD, calculated as the concentration corresponding to the signal equal to mean signal of the blank plus three times its standard deviation) were achieved as 0.16 ppt and 0.23 ppt by using the calibration curve in PBS and seawater, respectively. As seen in Figure 5, the nanosensor response was partially reduced because of the interferences by binding of some pollutants in seawater which contains many pollutants and salt with high concentration. However, the GMIN performance for the detection of ACS Paragon Plus 9 Environment
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TBT in seawater was too close to the performance in PBS. Furthermore, there was good agreement between PBS experimental measurements and seawater experimental measurements (Figure S8). It can be also seen that no significant change was observed in the PL intensities of GNIN with the increasing concentration of TBT. It should be noted that the experiments were repeated more than ten times and according to NC disk background signal at least coherent, six results were chosen to have the calibration curve.
Figure 5. Performance of the nanosensor for TBT detection at different concentration levels by GMIN (a) and GNIN (b) in PBS (left) and seawater (right). The adsorption isotherms are used for investigation of the relationship between the equilibrium of bound and free guest concentration over a certain concentration range and give information about surface homogeneity and binding properties 43-45. As it is seen in Table S1, Langmuir-Freundlich model is most suitable to explain the binding behavior of TBT on MIP surface according to the correlation coefficient. Therefore, the GMIN has heterogeneous binding sites (1/n, (0.5457)
