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A Colorimetric Probe for Dopamine Based on Gold Nanoparticles-electrospun Nanofibre Composite
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 2 (2015) 4060 – 4069
7th International Symposium On Macro- and Supramolecular Architectures and Materials
A colorimetric probe for dopamine based on gold nanoparticleselectrospun nanofibre composite Nokuthula Ngomanea, Nelson Tortob, Rui Krausea* and Sibulelo Vilakazic a
Department of Chemistry Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa Botswana Institute for Technology Research and Innovation, Private Bag 0082, Plot 50654, Machel Drive, Gaborone, Botswana c Advanced Materials Division, Nanoscience and Nanotechnology, 200 Malibongwe Drive 2125, Randburg, South Africa
b
Abstract
An easily prepared solid state colorimetric probe for detecting the neurotransmitter dopamine (DA) was developed. The probe, in the form of an electrospun Nylon−6 (N6) nanofibre with embedded un−functionalized gold nanoparticles (UF−AuNPs) produces a clear colour change in the presence of a DA that is detectable by the naked eye. Characterisation of the nanofibre using UV/vis spectroscopy and electron microscopy (TEM) confirmed the formation of the AuNPs in the polymer solution, and that the AuNPs were completely encapsulated within the composite nanofibres before exposure to the analytes. The probe exhibited very high sensitivity towards DA resulting in colour change of the composite fibres from purple to navy blue/black even under low concentrations of DA. The probe was also selective to DA since the colour remained unchanged in the presence of commonly encountered interfering species such as ascorbic acid, uric acid, catechol, epinephrine and norepinephrine. Moreover, the colour change was observed rapid, occurring either immediately on contact with higher concentrations (5 x10−4 M) or within about 3−5 min for the lower concentrations (e.g. 5 x10−7 M). Since this probe does not require the use of any instruments, and is both rapid and stable over time, it can be applied in the field by an inexperienced person. * Corresponding author. Tel.:+27-46-603-7030; fax:+27-46-622-5109.
E-mail address: [email protected] © 2015 2014Published Elsevier by Ltd. All rights © Elsevier Ltd. reserved. Selectionand andpeer-review peer-review under responsibility the Conference Committee Members of 7th International Symposium on Selection under responsibility of theofConference Committee Members of 7th International Symposium on MacroArchitectures and Materials. Macro-and andSupramolecular Supramolecular Architectures and Materials. Keywords: Dopamine; gold nanoparticles; electrospun nanofibres; colorimetric probe; neurotransmitters
2214-7853 © 2015 Published by Elsevier Ltd. Selection and peer-review under responsibility of the Conference Committee Members of 7th International Symposium on Macro- and Supramolecular Architectures and Materials. doi:10.1016/j.matpr.2015.08.036
Nokuthula Ngomane et al. / Materials Today: Proceedings 2 (2015) 4060 – 4069
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1.
Introduction Dopamine (DA) is a biogenic catecholamine neurotransmitter [1] that is intimately involved in the functions of the central nervous, hormonal and cardiovascular systems. In addition, various neurological disorders and their treatment such as the Parkinson’s disease and Schizophrenia are connected to DA concentrations [2-4]. For easy and fast detection of DA, many researchers have focused on developing colorimetric probes [5-7]. The advantage of using the colorimetric technique over other methods (e.g. electrochemical and spectroscopic) is that it excludes the use of expensive instruments that may require technically competent personnel to operate. To date, however, the reported colorimetric probes for DA are mostly solution based, thus requiring the measurement and handling of liquids. This makes it difficult to transport and use in the field. Moreover, the synthesis of most of the colorimetric probes involve following of complicated and long procedures and the use of complex functional groups [8-10].
As such, this paper reports for the first time a solid state colorimetric probe for DA in the form of an electrospun nanofibre containing embedded un−functionalized gold nanoparticles (UF−AuNPs). AuNPs are extremely sensitive owing to their higher extinction coeffients compared with traditional organic chromophores [11], hence they were chosen for colorimetric response. The nanofibres were prepared using a technique known as electrospinning, which is a simple technique that uses high voltage power to induce the formation of ultrafine fibres with diameters that range from few nanometres to several microns depending on conditions [12]. Nylon-6 (N6) was used as the solid support polymer for the UF−AuNPs owing to its good mechanical strength and chemical stability [13]. This allows us to cut the electrospun fibre mats into small strips for ease of use in matrices such as urine. The performance and mechanism of action of the probe was studied. The results showed that the composite N6 and UF-AuNPs probe could be a potential candidate in the detection of DA without the worry of interferences. 2.
Experimental
2.1. Reagents and materials Gold(III)chloride hydrate (99.999%) (HAuCl4•xH2O), sodium borohydride (98.0%) (NaBH 4), dopamine hydrochloride, uric acid (99.0%), ascorbic acid, (−)−epinephrine (EPI), (−)−norepinephrine (NORE), 3,4-dihydroxyL-phenylalanine (CAT), trisodium citrate dehydrate, 3−mercaptopropionic acid and mercaptosuccinic acid were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Hydrochloric acid (32.0%) and nitric acid (55.0%) were from B & M Scientific CC (Western Cape, RSA). Formic acid (85.0%) was purchased from Minema (Gauteng, RSA). Sodium dihydrogen phosphate was purchased from May & Baker (Dagenham, England) and sodium hydroxide was from Merck (Gauteng, RSA). All chemicals were of analytical grade and used as received. All the glassware and magnetic stirrer bar used were washed with freshly prepared aqua regia (1:3 HNO3: HCl) and rinsed thoroughly with water before use. 2.2. Instrumentation Absorbance measurements were performed using a Lambda 25 Perkin−Elmer UV spectrophotometer in 1 cm cuvettes (Santa Clara, CA, USA). Transmission electron microscopy (TEM) images were acquired using a Zeiss Libra® 120 Plus energy filter transmission electron microscope (Germany) with a megaview iTEM camera. High resolution scanning electron microscopy (HRSEM) analysis was conducted using JOEL JSM−700IF. The HRSEM, samples were carbon coated prior analysis. Ultrapure water obtained from a Millipore RiOS™ 16 and Milli−Q Academic® A10 system (Milford, MA, USA) was used throughout the study. 2.3. Preparation of the AuNPs−electrospun nylon−6 nanofibre composite 1.08 g of nylon−6 (N6) was dissolved in a mixture of acetic acid and formic acid (1:1) to make a 16 wt% solution. The mixture was stirred over night for complete dissolution of the polymer. About 0.115 mmol of HAuCl 4 • xH2O was dissolved in the polymer solution and vigorously stirred for 15 min at room temperature. The resulting
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solution was yellow (from whitish) in colour. On addition of 0.00793 mmol of NaBH4 (reducing agent), the solution turned black for a few seconds then wine red. After stirring for 2 min the solution became purple.
2.4. Electrospinning The electrospinning of the purple solution was conducted at an optimal voltage of 25 kV and a flow rate of 0.5 ml/h was controlled using a syringe pump. The continuous N6 fibres were collected on a stationary collector covered with an aluminium foil. The distance from the tip of the spinneret to the collector was 10 cm. 2.5. Colorimetric detection of DA employing the AuNPs + N6 nanofibres at pH 7.4 Portions of the UF−AuNPs +N6 nanofibre mat were cut and introduced to 2 mL of the interfering species (AA, UA, EPI, NORE and catechol, 50 μM) and 5 μM DA. Photo graphs of the fibres were taken within 5 min. Other portions of the nanofibre mat were introduced to various concentrations of DA to determine the visual LOD at pH 7.4. 3.
Results and discussion
3.1. UV/vis characterization of the polymer solution and TEM of the UF–AuNPs + N6 composite nanofibre The polymer composite solution was characterized using UV/vis spectroscopy before electrospinning to determine if nanoparticles had formed. Abroad absorption peak at 572 nm (Fig. 1), which is within the known range for AuNPs (500–600 nm) [14] confirmed the success of their formation in situ. Broad absorption peaks are usually associated with a wide range of particle sizes or aggregation of nanoparticles [6,15]. However, in this case the TEM image of the nanofibres obtained after electrospinning the polymer solution showed that before introduction of DA the AuNPs were spherically shaped and well dispersed within the nanofibre matrix and the average diameter of the AuNPs was estimated to be 8 nm (Fig. 2). Since in our situation we do not have a wide range of particle sizes nor aggregation before introduction of the analyte, we suggest that the broad peak is an indicator of the blending process of the UF–AuNPs and the N6. A similar process was observed in the synthesis of a composite of polycarbonate film with thiol AuNPs [16]. The dispersion of the UF–AuNPs suggested that the AuNPs were stabilized by the functional groups of N6.The nanofibre mat, which was purple in colour, was stored in a dark cupboard at room temperature until needed.
Fig. 1. The UV/vis spectrum of the N6 + AuNPs solution prior to electrospinning.
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Fig. 2. TEM images of the N6 + AuNPs composite nanofibres before DA.
3.2. Colorimetric detection of dopamine The purple fibres were evaluated as probes by introducing strips of the nanofibre mat to various analytes. In aqueous solutions of DA (5 μM) a colour change from the purple to navy blue/black was observed (Fig. 3). However, when the same fibres was dipped in solutions (50 μM each) of other compounds that are known to interfere with DA detection such as uric acid (UA), ascorbic acid (AA), catechol (CAT), norepinephrine (NORE) and epinephrine (EPI) the nanofibre mats remained almost unchanged. No matter how high the concentrations of AA and UA were made, colour change was not observed. The concentration of the catecholamines in urine and blood samples of healthy subjects is normally lower than those for DA for example, in urine NORE is ~0.088–0.47 μmol/24 h and EPI is ~0.002–0.11 μM/24 h [17]. On the other hand, the concentrations of AA and UA are normally found to be higher than those of DA (1.48–4.43 mmol/24 h for UA [18] and AA is ~100–1000 times the concentration of DA [19]).
Fig. 3. The UF−AuNPs + N6 composite nanofibres in DA (5 μM) and other interfering substances (50 μM) at pH 7.4.
3.3. Characterization using high resolution scanning electron microscopy (HRSEM) We have previously investigated the aggregation of nanoparticles in solution to explain colour change in nanoparticles, but this case presents gold nanoparticles in a solid composite. In order therefore to understand what could be happening to the AuNPs to result in such colour change, the fibres were subjected to high resolution scanning electron microscopy analysis was conducted (HRSEM) (Fig. 4). In
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the presence of DA, more AuNPs were observed on the surface of the nanofibres (Fig. 4C&D).There was no significant difference between the fibres that were in the other analytes and that of the control (Fig. 4A&B). The fibres in AA were used as a representative for the interfering substances.
Fig. 4. HRSEM image of (A) the UF−AuNPs +N6 composite nanofibre, (B) the fibres in AA and (C&D) in DA. C and D are the same just different magnifications.
3.4. Evaluation of the detection limit of the N6−AuNPs nanofibre composite probe The fibres were next placed in contact with solutions containing various concentrations of DA to investigate the detection limit for this process (Fig. 5). The colour change was evident already from 5 x 10 -7 M (the normal content of DA in healthy people was in the range of 1.3–2.6 μM [20]) displaying high sensitivity towards DA. The intensity of the colour increased with increasing concentrations of DA, and the development of the final colour was most rapid in DA concentrations over 5 x 10-7 M.
Fig. 5. The UF−AuNPs + N6 composite in various concentrations of DA.
For quantification of DA using the solid state probe (nanofiber mat), open–source software imageJ was used to assist in the analysis of the fibre colours (Fig. 6). It was observed that the intensity of the colour increased with the increase in concentration of DA in a linear fashion. The use of imageJ can be a great alternative where the colour changes are not so clear or for visually impaired people.
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Relative intensity
30 25 20 15 10 5 0 0M
5E-6 M 5E-5 M 5E-4 M 5E-3 M 5E-2 M
Fig. 6. Bar graph showing the relationship between concentration of DA and relative intensities of the colour of the fibres in the various concentration of DA.
In order to understand the mechanism of detection by the probe, further characterization of the materials was necessary.
3.5. Fourier transforms infrared characterization of the composite fibres and TEM image of the fibres in the presence of DA FTIR studies were conducted to investigate any possible interactions between the UF−AuNPs and the functional groups of N6 (Fig. 7). The FTIR spectrum of the N6+UF−AuNPs (Fig. 7a) showed an increase in % transmittance while the intensity of all the peaks remained similar to that of the clean N6 nanofibres (Fig. 7b). The absorption bands at 3299 cm−1 for the clean N6 nanofibres has shifted to 3301 cm−1, while the band at 1639 cm-1 shifted to 1642 cm-1 and the band at 690 cm−1 shifted to 698 cm−1 in the case of the N6 + UF−AuNPs nanofibres. When the N6+UF–AuNPs composite nanofibres were introduced to DA, a decrease in percentage transmittance was observed and the intensity of all the peaks increased significantly (Fig. 5c). In addition to these changes, the peak at 3301 cm−1 of the N6+UF–AuNPs fibres shifted to 3299 cm−1, whereas the peak at 1642 cm−1 moved to 1638 cm−1 and the peak at 698 cm−1 shifted to 687 cm−1 . The shifting of wave numbers from lower to higher numbers has been associated with the presence of interactions between functional groups of polymers and nanoparticles [21-23].
Fig. 7. FTIR spectra of (A) N6 and AuNPs, (B) clean N6 and (C) N6 and AuNPs fibres in DA.
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In this case, since the major shifts are observed at 1639 cm–1 and 690 cm–1 the results implies the existence of molecular interaction between the amide (I) and amide (V) groups of N6 [24] respectively and the AuNPs in the N6 + UF−AuNPs nanofibre composite. These data showed that the AuNPs were stabilized by the functional groups of N6. The shoulder around 3483 cm–1 was assigned to the free NH [25]. Since the bond formed by the interaction between the amide (I) and amide (V) of N6 and the AuNPs are not strong, they can be broken easily. Owing to the strong affinity of DA for AuNPs, putting the nanofibres in a solution with DA result in the breaking of those bonds and the AuNPs become free to interact with DA to form aggregates that can no longer be embedded by the nanofibres. Hence, the shifting of bands from higher to lower wave numbers was assumed to be indicative of broken interactions between the AuNPs and the N6. The TEM image in Fig. 8C confirmed the formation of aggregates which was also in agreement with the HRSEM results. N6 nanofibres have pores that are interconnected, this morphology enables DA to penetrate the nanofibre and interact with the embedded AuNPs. The interactions causes change in the shape of the nanoparticles from the spherical to hexagonal and star shapes, growth and formation of aggregates that can no longer be impregnated by the nanofibres hence they diffuse to the surface (Fig. 8A & B). The change is especially evident from Fig. 8B and D, which are images of the same nanofibre taken at difference magnifications.
Fig. 8. TEM images of the N6 + AuNPs composite nanofibres after DA.
3.6. X-ray photoelectron spectroscopy (XPS) characterization The Au4f XPS spectrum of the probe before introduction to DA (Fig. 9A) showed two peaks at 82.7 eV and 86.3 eV. The two peaks shifted slightly to 81.7 eV and 85.4 eV in the presence of DA (Fig. 9B). The latter peak around 86.3 eV has been assigned to Au3+ species, while the peak at 82.7 eV has been attributed to Au 0. Since there was an excess of gold salt used, a plausible mechanism for the formation of nanoparticles on the surface of the fibres that would be responsible for the colour change is further reduction of gold cations. This is possible since DA is a reducing agent, but unlikely in this case since we would have expected a third peak around 86.3 eV or even a small shoulder after introducing the probe to DA. The third peak together with a significant change to the peak at 85.4 eV would have meant that in the presence of DA excess Au3+ ions was reduced to Au0 or Au1+.
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Fig. 9. Au 4f XPS spectra of the UF–AuNPs + N6 composite fibres before (A) and after (B) DA.
These results implied that the mechanism of detection is based on a direct interaction of the UF−AuNPs and DA, resulting in an unexpected aggregation of nanoparticles near the surface of the nanofibers. In this case it should result in a change in the interaction of functional groups with the surface of the nanoparticles, which we sought to investigate by XPS analysis of nitrogen and oxygen. From the XPS analysis for the binding energy of nitrogen (Fig. 10) it is evident that the relative contributions of all the binding modes that make up the main peak are approximately the same. It is feasible that there is only a very slight decrease of the contribution from the 400 eV peak after exposure to DA. The observation suggests that there was no change in the binding mode with respect to nitrogen. Since the binding energy of different compounds vary and overlap quite a bit, one should expect that any significant variation would be reflected in a large change in the nitrogen binding energy. In our case, this is not seen which fits well with other data and our suggestion that the N6 nanofibre backbone stabilises the nanoparticles.
Fig. 10. XPS high resolution nitrogen 1s spectra of the UF–AuNPs + N6 composite fibres before (A) and after (B) DA.
The oxygen binding energy, on the other hand, showed quite a significant shift to favour the 531.0 eV contribution over the others (Fig. 11). The binding energy at 529.4 eV shifted to 529.0, 532.7 to 533.0, 534.6 to 535.3 and 536.7 to 538.0 eV after DA. This signifies that there is one specific binding mode of oxygen that changes significantly after exposure to DA. According to Beamson and Briggs, the 531.0 eV peak is likely to be that of a carbonyl. Since DA does not have a carbonyl this binding mode could be that of the ortho diphenol moiety (or its easily oxidised dicarbonyl or ortho-quinone form), or could reflect a different binding mode with the polymer backbone. This aspect is being investigated further.
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Fig. 11. XPS high resolution oxygen 1s spectra of the UF–AuNPs + N6 composite fibres before (A) and after (B) DA.
The interaction of the AuNPs with quinone carbonyl justifies the reason there could be no colour change in the other compounds more especially in epinephrine and norepinephrine. The two catecholamines form unstable protonated species that easily oxidize to form quinone derivatives at acidic pH [26,27]. Since the solutions were alkaline (pH 7.4), we do not expect epinephrine and norepinephrine to induce any colour change. 4.
Conclusion
In summary, we have successfully developed a solid state colorimetric probe for DA. The probe is based on un−functionalized AuNPs hosted in electropun N6 nanofibres. The probe does not need any additional functionality to be selective to DA as it displayed very high specificity and sensitivity for DA only and was not responsive in other interfering compounds. The simplicity at which the probe works makes it to have great potential to find application in clinical diagnosis of DA related neurological disorders and diseases. Any individual can be able to use it and take the necessary precautions. Acknowledgement Financial support was provided by the National Research Foundation (NRF) of South Africa, DST/Mintek Nanotechnology Innovation Centre and the Henderson postgraduate scholarship. References [1] N. Civjan, Chemical Biology: Approaches to Drug Discovery and Development to Targeting Disease, John Wiley & Sons, 2012. [2] Z. Guo, M.-L. Seol, M.-S. Kim, J.-H. Ahn, Y.-K. Choi, J.-H. Liu, X.-J. Huang, Analyst (Cambridge, U. K.) 138 (2013) 2683-2690. [3] Y.-R. Kim, S. Bong, Y.-J. Kang, Y. Yang, R.K. Mahajan, J.S. Kim, H. Kim, Biosens. Bioelectron. 25 (2010) 2366-2369. [4] T. Peik-See, A. Pandikumar, H. Nay-Ming, L. Hong-Ngee, Y. Sulaiman, Sensors 14 (2014) 15227-15243. [5] Y. Lin, C. Chen, C. Wang, F. Pu, J. Ren, X. Qu, Chem. Commun. (Cambridge, U. K.) 47 (2011) 1181-1183. [6] L. Liu, S. Li, L. Liu, D. Deng, N. Xia, Analyst (Cambridge, U. K.) 137 (2012) 3794-3799 [7] B. Kong, A. Zhu, Y. Luo, Y. Tian, Y. Yu, G. Shi, Angewandte Chemie 123 (2011) 1877-1880. [8] Y. Zheng, Y. Wang, X. Yang, Sensors and Actuators B: Chemical 156 (2011) 95-99. [9] J.-J. Feng, H. Guo, Y.-F. Li, Y.-H. Wang, W.-Y. Chen, A.-J. Wang, ACS Applied Materials & Interfaces 5 (2013) 1226 -1233. [10] H.-C. Lee, T.-H. Chen, W.-L. Tseng, C.-H. Lin, Analyst 137 (2012) 5352-5357. [11] Y. Zhang, B. Li, X. Chen, Microchimica Acta 168 (2010) 107-113. [12] Y. Qin, Micromanufacturing engineering and technology, William Andrew, 2010. [13] H.R. Pant, C.S. Kim, Polym. Int. 62 (2013) 1008-1013. [14] K. Patel, S. Kapoor, D.P. Dave, T. Mukherjee, Research on Chemical Intermediates 32 (2006) 103-108. [15] J. Polte, R. Erler, A.F. Thuenemann, S. Sokolov, T.T. Ahner, K. Rademann, F. Emmerling, R. Kraehnert, ACS Nano 4 (2010) 1076-1082. [16] C. Larosa, E. Stura, R. Eggenhoffner, C. Nicolini, Materials 2 (2009) 1193-1204.
Nokuthula Ngomane et al. / Materials Today: Proceedings 2 (2015) 4060 – 4069 [17] MedlinePlus, Catecholamines - urine, 2015. [18] WebMD, Uric acid in urine, 2012. [19] Z. Zhuang, J. Li, R. Xu, D. Xiao, Int. J. Electrochem. Sci. 6 (2011) 2149-2161. [20] Y. Dong, X. Chen, C. Li, X. Chen, Journal of Lanzhou Universiy (Natural Science) 45 (2009) 77- 88. [21] J. Bai, Y. Li, S. Yang, J. Du, S. Wang, J. Zheng, Y. Wang, Q. Yang, X. Chen, X. Jing, Solid State Commun. 141 (2007) 292-295. [22] A.E. Deniz, H.A. Vural, B. Ortac, T. Uyar, Mater. Lett. 65 (2011) 2941-43. [23] J. Bai, Q. Yang, M. Li, S. Wang, C. Zhang, Y. Li, Mater. Chem. Phys. 111 (2008) 200-205. [24] H.R. Pant, M.P. Bajgai, C. Yi, R. Nirmala, K.T. Nam, W.-i. Baek, H.Y. Kim, Colloids and Surfaces A: Physicochemical and Engineering Aspects 370 (2010) 87-89. [25] S. Oprea, V.O. Potolinca, Polymer-Plastics Technology and Engineering 52 (2013) 1550-1556. [26] A. Babaei, S. Mirzakhani, B. Khalilzadeh, Journal of the Brazilian Chemical Society 20 (2009) 1862-1869. [27] L.L. Iversen, The uptake and Storage of Noradrenaline in Sympathetic Nerves, Cambridge Univ. Press, 1967.
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ScienceDirect Materials Today: Proceedings 2 (2015) 4060 – 4069
7th International Symposium On Macro- and Supramolecular Architectures and Materials
A colorimetric probe for dopamine based on gold nanoparticleselectrospun nanofibre composite Nokuthula Ngomanea, Nelson Tortob, Rui Krausea* and Sibulelo Vilakazic a
Department of Chemistry Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa Botswana Institute for Technology Research and Innovation, Private Bag 0082, Plot 50654, Machel Drive, Gaborone, Botswana c Advanced Materials Division, Nanoscience and Nanotechnology, 200 Malibongwe Drive 2125, Randburg, South Africa
b
Abstract
An easily prepared solid state colorimetric probe for detecting the neurotransmitter dopamine (DA) was developed. The probe, in the form of an electrospun Nylon−6 (N6) nanofibre with embedded un−functionalized gold nanoparticles (UF−AuNPs) produces a clear colour change in the presence of a DA that is detectable by the naked eye. Characterisation of the nanofibre using UV/vis spectroscopy and electron microscopy (TEM) confirmed the formation of the AuNPs in the polymer solution, and that the AuNPs were completely encapsulated within the composite nanofibres before exposure to the analytes. The probe exhibited very high sensitivity towards DA resulting in colour change of the composite fibres from purple to navy blue/black even under low concentrations of DA. The probe was also selective to DA since the colour remained unchanged in the presence of commonly encountered interfering species such as ascorbic acid, uric acid, catechol, epinephrine and norepinephrine. Moreover, the colour change was observed rapid, occurring either immediately on contact with higher concentrations (5 x10−4 M) or within about 3−5 min for the lower concentrations (e.g. 5 x10−7 M). Since this probe does not require the use of any instruments, and is both rapid and stable over time, it can be applied in the field by an inexperienced person. * Corresponding author. Tel.:+27-46-603-7030; fax:+27-46-622-5109.
E-mail address: [email protected] © 2015 2014Published Elsevier by Ltd. All rights © Elsevier Ltd. reserved. Selectionand andpeer-review peer-review under responsibility the Conference Committee Members of 7th International Symposium on Selection under responsibility of theofConference Committee Members of 7th International Symposium on MacroArchitectures and Materials. Macro-and andSupramolecular Supramolecular Architectures and Materials. Keywords: Dopamine; gold nanoparticles; electrospun nanofibres; colorimetric probe; neurotransmitters
2214-7853 © 2015 Published by Elsevier Ltd. Selection and peer-review under responsibility of the Conference Committee Members of 7th International Symposium on Macro- and Supramolecular Architectures and Materials. doi:10.1016/j.matpr.2015.08.036
Nokuthula Ngomane et al. / Materials Today: Proceedings 2 (2015) 4060 – 4069
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1.
Introduction Dopamine (DA) is a biogenic catecholamine neurotransmitter [1] that is intimately involved in the functions of the central nervous, hormonal and cardiovascular systems. In addition, various neurological disorders and their treatment such as the Parkinson’s disease and Schizophrenia are connected to DA concentrations [2-4]. For easy and fast detection of DA, many researchers have focused on developing colorimetric probes [5-7]. The advantage of using the colorimetric technique over other methods (e.g. electrochemical and spectroscopic) is that it excludes the use of expensive instruments that may require technically competent personnel to operate. To date, however, the reported colorimetric probes for DA are mostly solution based, thus requiring the measurement and handling of liquids. This makes it difficult to transport and use in the field. Moreover, the synthesis of most of the colorimetric probes involve following of complicated and long procedures and the use of complex functional groups [8-10].
As such, this paper reports for the first time a solid state colorimetric probe for DA in the form of an electrospun nanofibre containing embedded un−functionalized gold nanoparticles (UF−AuNPs). AuNPs are extremely sensitive owing to their higher extinction coeffients compared with traditional organic chromophores [11], hence they were chosen for colorimetric response. The nanofibres were prepared using a technique known as electrospinning, which is a simple technique that uses high voltage power to induce the formation of ultrafine fibres with diameters that range from few nanometres to several microns depending on conditions [12]. Nylon-6 (N6) was used as the solid support polymer for the UF−AuNPs owing to its good mechanical strength and chemical stability [13]. This allows us to cut the electrospun fibre mats into small strips for ease of use in matrices such as urine. The performance and mechanism of action of the probe was studied. The results showed that the composite N6 and UF-AuNPs probe could be a potential candidate in the detection of DA without the worry of interferences. 2.
Experimental
2.1. Reagents and materials Gold(III)chloride hydrate (99.999%) (HAuCl4•xH2O), sodium borohydride (98.0%) (NaBH 4), dopamine hydrochloride, uric acid (99.0%), ascorbic acid, (−)−epinephrine (EPI), (−)−norepinephrine (NORE), 3,4-dihydroxyL-phenylalanine (CAT), trisodium citrate dehydrate, 3−mercaptopropionic acid and mercaptosuccinic acid were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Hydrochloric acid (32.0%) and nitric acid (55.0%) were from B & M Scientific CC (Western Cape, RSA). Formic acid (85.0%) was purchased from Minema (Gauteng, RSA). Sodium dihydrogen phosphate was purchased from May & Baker (Dagenham, England) and sodium hydroxide was from Merck (Gauteng, RSA). All chemicals were of analytical grade and used as received. All the glassware and magnetic stirrer bar used were washed with freshly prepared aqua regia (1:3 HNO3: HCl) and rinsed thoroughly with water before use. 2.2. Instrumentation Absorbance measurements were performed using a Lambda 25 Perkin−Elmer UV spectrophotometer in 1 cm cuvettes (Santa Clara, CA, USA). Transmission electron microscopy (TEM) images were acquired using a Zeiss Libra® 120 Plus energy filter transmission electron microscope (Germany) with a megaview iTEM camera. High resolution scanning electron microscopy (HRSEM) analysis was conducted using JOEL JSM−700IF. The HRSEM, samples were carbon coated prior analysis. Ultrapure water obtained from a Millipore RiOS™ 16 and Milli−Q Academic® A10 system (Milford, MA, USA) was used throughout the study. 2.3. Preparation of the AuNPs−electrospun nylon−6 nanofibre composite 1.08 g of nylon−6 (N6) was dissolved in a mixture of acetic acid and formic acid (1:1) to make a 16 wt% solution. The mixture was stirred over night for complete dissolution of the polymer. About 0.115 mmol of HAuCl 4 • xH2O was dissolved in the polymer solution and vigorously stirred for 15 min at room temperature. The resulting
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solution was yellow (from whitish) in colour. On addition of 0.00793 mmol of NaBH4 (reducing agent), the solution turned black for a few seconds then wine red. After stirring for 2 min the solution became purple.
2.4. Electrospinning The electrospinning of the purple solution was conducted at an optimal voltage of 25 kV and a flow rate of 0.5 ml/h was controlled using a syringe pump. The continuous N6 fibres were collected on a stationary collector covered with an aluminium foil. The distance from the tip of the spinneret to the collector was 10 cm. 2.5. Colorimetric detection of DA employing the AuNPs + N6 nanofibres at pH 7.4 Portions of the UF−AuNPs +N6 nanofibre mat were cut and introduced to 2 mL of the interfering species (AA, UA, EPI, NORE and catechol, 50 μM) and 5 μM DA. Photo graphs of the fibres were taken within 5 min. Other portions of the nanofibre mat were introduced to various concentrations of DA to determine the visual LOD at pH 7.4. 3.
Results and discussion
3.1. UV/vis characterization of the polymer solution and TEM of the UF–AuNPs + N6 composite nanofibre The polymer composite solution was characterized using UV/vis spectroscopy before electrospinning to determine if nanoparticles had formed. Abroad absorption peak at 572 nm (Fig. 1), which is within the known range for AuNPs (500–600 nm) [14] confirmed the success of their formation in situ. Broad absorption peaks are usually associated with a wide range of particle sizes or aggregation of nanoparticles [6,15]. However, in this case the TEM image of the nanofibres obtained after electrospinning the polymer solution showed that before introduction of DA the AuNPs were spherically shaped and well dispersed within the nanofibre matrix and the average diameter of the AuNPs was estimated to be 8 nm (Fig. 2). Since in our situation we do not have a wide range of particle sizes nor aggregation before introduction of the analyte, we suggest that the broad peak is an indicator of the blending process of the UF–AuNPs and the N6. A similar process was observed in the synthesis of a composite of polycarbonate film with thiol AuNPs [16]. The dispersion of the UF–AuNPs suggested that the AuNPs were stabilized by the functional groups of N6.The nanofibre mat, which was purple in colour, was stored in a dark cupboard at room temperature until needed.
Fig. 1. The UV/vis spectrum of the N6 + AuNPs solution prior to electrospinning.
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Fig. 2. TEM images of the N6 + AuNPs composite nanofibres before DA.
3.2. Colorimetric detection of dopamine The purple fibres were evaluated as probes by introducing strips of the nanofibre mat to various analytes. In aqueous solutions of DA (5 μM) a colour change from the purple to navy blue/black was observed (Fig. 3). However, when the same fibres was dipped in solutions (50 μM each) of other compounds that are known to interfere with DA detection such as uric acid (UA), ascorbic acid (AA), catechol (CAT), norepinephrine (NORE) and epinephrine (EPI) the nanofibre mats remained almost unchanged. No matter how high the concentrations of AA and UA were made, colour change was not observed. The concentration of the catecholamines in urine and blood samples of healthy subjects is normally lower than those for DA for example, in urine NORE is ~0.088–0.47 μmol/24 h and EPI is ~0.002–0.11 μM/24 h [17]. On the other hand, the concentrations of AA and UA are normally found to be higher than those of DA (1.48–4.43 mmol/24 h for UA [18] and AA is ~100–1000 times the concentration of DA [19]).
Fig. 3. The UF−AuNPs + N6 composite nanofibres in DA (5 μM) and other interfering substances (50 μM) at pH 7.4.
3.3. Characterization using high resolution scanning electron microscopy (HRSEM) We have previously investigated the aggregation of nanoparticles in solution to explain colour change in nanoparticles, but this case presents gold nanoparticles in a solid composite. In order therefore to understand what could be happening to the AuNPs to result in such colour change, the fibres were subjected to high resolution scanning electron microscopy analysis was conducted (HRSEM) (Fig. 4). In
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the presence of DA, more AuNPs were observed on the surface of the nanofibres (Fig. 4C&D).There was no significant difference between the fibres that were in the other analytes and that of the control (Fig. 4A&B). The fibres in AA were used as a representative for the interfering substances.
Fig. 4. HRSEM image of (A) the UF−AuNPs +N6 composite nanofibre, (B) the fibres in AA and (C&D) in DA. C and D are the same just different magnifications.
3.4. Evaluation of the detection limit of the N6−AuNPs nanofibre composite probe The fibres were next placed in contact with solutions containing various concentrations of DA to investigate the detection limit for this process (Fig. 5). The colour change was evident already from 5 x 10 -7 M (the normal content of DA in healthy people was in the range of 1.3–2.6 μM [20]) displaying high sensitivity towards DA. The intensity of the colour increased with increasing concentrations of DA, and the development of the final colour was most rapid in DA concentrations over 5 x 10-7 M.
Fig. 5. The UF−AuNPs + N6 composite in various concentrations of DA.
For quantification of DA using the solid state probe (nanofiber mat), open–source software imageJ was used to assist in the analysis of the fibre colours (Fig. 6). It was observed that the intensity of the colour increased with the increase in concentration of DA in a linear fashion. The use of imageJ can be a great alternative where the colour changes are not so clear or for visually impaired people.
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Relative intensity
30 25 20 15 10 5 0 0M
5E-6 M 5E-5 M 5E-4 M 5E-3 M 5E-2 M
Fig. 6. Bar graph showing the relationship between concentration of DA and relative intensities of the colour of the fibres in the various concentration of DA.
In order to understand the mechanism of detection by the probe, further characterization of the materials was necessary.
3.5. Fourier transforms infrared characterization of the composite fibres and TEM image of the fibres in the presence of DA FTIR studies were conducted to investigate any possible interactions between the UF−AuNPs and the functional groups of N6 (Fig. 7). The FTIR spectrum of the N6+UF−AuNPs (Fig. 7a) showed an increase in % transmittance while the intensity of all the peaks remained similar to that of the clean N6 nanofibres (Fig. 7b). The absorption bands at 3299 cm−1 for the clean N6 nanofibres has shifted to 3301 cm−1, while the band at 1639 cm-1 shifted to 1642 cm-1 and the band at 690 cm−1 shifted to 698 cm−1 in the case of the N6 + UF−AuNPs nanofibres. When the N6+UF–AuNPs composite nanofibres were introduced to DA, a decrease in percentage transmittance was observed and the intensity of all the peaks increased significantly (Fig. 5c). In addition to these changes, the peak at 3301 cm−1 of the N6+UF–AuNPs fibres shifted to 3299 cm−1, whereas the peak at 1642 cm−1 moved to 1638 cm−1 and the peak at 698 cm−1 shifted to 687 cm−1 . The shifting of wave numbers from lower to higher numbers has been associated with the presence of interactions between functional groups of polymers and nanoparticles [21-23].
Fig. 7. FTIR spectra of (A) N6 and AuNPs, (B) clean N6 and (C) N6 and AuNPs fibres in DA.
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In this case, since the major shifts are observed at 1639 cm–1 and 690 cm–1 the results implies the existence of molecular interaction between the amide (I) and amide (V) groups of N6 [24] respectively and the AuNPs in the N6 + UF−AuNPs nanofibre composite. These data showed that the AuNPs were stabilized by the functional groups of N6. The shoulder around 3483 cm–1 was assigned to the free NH [25]. Since the bond formed by the interaction between the amide (I) and amide (V) of N6 and the AuNPs are not strong, they can be broken easily. Owing to the strong affinity of DA for AuNPs, putting the nanofibres in a solution with DA result in the breaking of those bonds and the AuNPs become free to interact with DA to form aggregates that can no longer be embedded by the nanofibres. Hence, the shifting of bands from higher to lower wave numbers was assumed to be indicative of broken interactions between the AuNPs and the N6. The TEM image in Fig. 8C confirmed the formation of aggregates which was also in agreement with the HRSEM results. N6 nanofibres have pores that are interconnected, this morphology enables DA to penetrate the nanofibre and interact with the embedded AuNPs. The interactions causes change in the shape of the nanoparticles from the spherical to hexagonal and star shapes, growth and formation of aggregates that can no longer be impregnated by the nanofibres hence they diffuse to the surface (Fig. 8A & B). The change is especially evident from Fig. 8B and D, which are images of the same nanofibre taken at difference magnifications.
Fig. 8. TEM images of the N6 + AuNPs composite nanofibres after DA.
3.6. X-ray photoelectron spectroscopy (XPS) characterization The Au4f XPS spectrum of the probe before introduction to DA (Fig. 9A) showed two peaks at 82.7 eV and 86.3 eV. The two peaks shifted slightly to 81.7 eV and 85.4 eV in the presence of DA (Fig. 9B). The latter peak around 86.3 eV has been assigned to Au3+ species, while the peak at 82.7 eV has been attributed to Au 0. Since there was an excess of gold salt used, a plausible mechanism for the formation of nanoparticles on the surface of the fibres that would be responsible for the colour change is further reduction of gold cations. This is possible since DA is a reducing agent, but unlikely in this case since we would have expected a third peak around 86.3 eV or even a small shoulder after introducing the probe to DA. The third peak together with a significant change to the peak at 85.4 eV would have meant that in the presence of DA excess Au3+ ions was reduced to Au0 or Au1+.
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Fig. 9. Au 4f XPS spectra of the UF–AuNPs + N6 composite fibres before (A) and after (B) DA.
These results implied that the mechanism of detection is based on a direct interaction of the UF−AuNPs and DA, resulting in an unexpected aggregation of nanoparticles near the surface of the nanofibers. In this case it should result in a change in the interaction of functional groups with the surface of the nanoparticles, which we sought to investigate by XPS analysis of nitrogen and oxygen. From the XPS analysis for the binding energy of nitrogen (Fig. 10) it is evident that the relative contributions of all the binding modes that make up the main peak are approximately the same. It is feasible that there is only a very slight decrease of the contribution from the 400 eV peak after exposure to DA. The observation suggests that there was no change in the binding mode with respect to nitrogen. Since the binding energy of different compounds vary and overlap quite a bit, one should expect that any significant variation would be reflected in a large change in the nitrogen binding energy. In our case, this is not seen which fits well with other data and our suggestion that the N6 nanofibre backbone stabilises the nanoparticles.
Fig. 10. XPS high resolution nitrogen 1s spectra of the UF–AuNPs + N6 composite fibres before (A) and after (B) DA.
The oxygen binding energy, on the other hand, showed quite a significant shift to favour the 531.0 eV contribution over the others (Fig. 11). The binding energy at 529.4 eV shifted to 529.0, 532.7 to 533.0, 534.6 to 535.3 and 536.7 to 538.0 eV after DA. This signifies that there is one specific binding mode of oxygen that changes significantly after exposure to DA. According to Beamson and Briggs, the 531.0 eV peak is likely to be that of a carbonyl. Since DA does not have a carbonyl this binding mode could be that of the ortho diphenol moiety (or its easily oxidised dicarbonyl or ortho-quinone form), or could reflect a different binding mode with the polymer backbone. This aspect is being investigated further.
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Fig. 11. XPS high resolution oxygen 1s spectra of the UF–AuNPs + N6 composite fibres before (A) and after (B) DA.
The interaction of the AuNPs with quinone carbonyl justifies the reason there could be no colour change in the other compounds more especially in epinephrine and norepinephrine. The two catecholamines form unstable protonated species that easily oxidize to form quinone derivatives at acidic pH [26,27]. Since the solutions were alkaline (pH 7.4), we do not expect epinephrine and norepinephrine to induce any colour change. 4.
Conclusion
In summary, we have successfully developed a solid state colorimetric probe for DA. The probe is based on un−functionalized AuNPs hosted in electropun N6 nanofibres. The probe does not need any additional functionality to be selective to DA as it displayed very high specificity and sensitivity for DA only and was not responsive in other interfering compounds. The simplicity at which the probe works makes it to have great potential to find application in clinical diagnosis of DA related neurological disorders and diseases. Any individual can be able to use it and take the necessary precautions. Acknowledgement Financial support was provided by the National Research Foundation (NRF) of South Africa, DST/Mintek Nanotechnology Innovation Centre and the Henderson postgraduate scholarship. References [1] N. Civjan, Chemical Biology: Approaches to Drug Discovery and Development to Targeting Disease, John Wiley & Sons, 2012. [2] Z. Guo, M.-L. Seol, M.-S. Kim, J.-H. Ahn, Y.-K. Choi, J.-H. Liu, X.-J. Huang, Analyst (Cambridge, U. K.) 138 (2013) 2683-2690. [3] Y.-R. Kim, S. Bong, Y.-J. Kang, Y. Yang, R.K. Mahajan, J.S. Kim, H. Kim, Biosens. Bioelectron. 25 (2010) 2366-2369. [4] T. Peik-See, A. Pandikumar, H. Nay-Ming, L. Hong-Ngee, Y. Sulaiman, Sensors 14 (2014) 15227-15243. [5] Y. Lin, C. Chen, C. Wang, F. Pu, J. Ren, X. Qu, Chem. Commun. (Cambridge, U. K.) 47 (2011) 1181-1183. [6] L. Liu, S. Li, L. Liu, D. Deng, N. Xia, Analyst (Cambridge, U. K.) 137 (2012) 3794-3799 [7] B. Kong, A. Zhu, Y. Luo, Y. Tian, Y. Yu, G. Shi, Angewandte Chemie 123 (2011) 1877-1880. [8] Y. Zheng, Y. Wang, X. Yang, Sensors and Actuators B: Chemical 156 (2011) 95-99. [9] J.-J. Feng, H. Guo, Y.-F. Li, Y.-H. Wang, W.-Y. Chen, A.-J. Wang, ACS Applied Materials & Interfaces 5 (2013) 1226 -1233. [10] H.-C. Lee, T.-H. Chen, W.-L. Tseng, C.-H. Lin, Analyst 137 (2012) 5352-5357. [11] Y. Zhang, B. Li, X. Chen, Microchimica Acta 168 (2010) 107-113. [12] Y. Qin, Micromanufacturing engineering and technology, William Andrew, 2010. [13] H.R. Pant, C.S. Kim, Polym. Int. 62 (2013) 1008-1013. [14] K. Patel, S. Kapoor, D.P. Dave, T. Mukherjee, Research on Chemical Intermediates 32 (2006) 103-108. [15] J. Polte, R. Erler, A.F. Thuenemann, S. Sokolov, T.T. Ahner, K. Rademann, F. Emmerling, R. Kraehnert, ACS Nano 4 (2010) 1076-1082. [16] C. Larosa, E. Stura, R. Eggenhoffner, C. Nicolini, Materials 2 (2009) 1193-1204.
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