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Cysteine-stabilized silver nanoparticles as a colorimetric probe for the selective detection of cysteamine
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 215 (2019) 203–208
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Cysteine-stabilized silver nanoparticles as a colorimetric probe for the selective detection of cysteamine Siewdorlang Diamai, Devendra P.S. Negi ⁎ Department of Chemistry, North-Eastern Hill University, Shillong 793022, India
a r t i c l e
i n f o
Article history: Received 15 November 2018 Received in revised form 19 February 2019 Accepted 24 February 2019 Available online 25 February 2019 Keywords: Surface plasmon resonance Colorimetric Absorption Detection Silver nanoparticles
a b s t r a c t The purpose of the present research was to design a method for the colorimetric determination of cysteamine. We have employed cysteine-stabilized silver nanoparticles (AgNPs) as a probe. The addition of cysteamine resulted in the quenching of the 400 nm surface plasmon resonance (SPR) band of the AgNPs. It was accompanied by the appearance of a new absorption band at 560 nm. The colour of the colloidal AgNPs changed from yellow to dark brown within a few seconds. The change in colour of the AgNPs was due to their aggregation induced by the addition of cysteamine. Significantly, other biomolecules such as arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutathione, glycine, methionine and 6-mercaptopurine did not cause any change in the colour of the AgNPs. The limit of detection (LOD) of the method was 0.37 μM. The mechanism of the aggregation of the AgNPs induced by cysteamine has also been described. The method has been applied for the detection of cysteamine in human blood serum. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Cysteamine is a naturally occurring aminothiol which is widely applied as an important building block for many natural products. It is used as a therapeutic for the treatment of nephropathic cystinosis [1]. Cysteamine is amongst the important chelating ligands in coordination chemistry and binds preferentially to soft metal ions [2]. The sensing of cysteamine has attracted much interest recently. Various approaches such as fluorescence [3,4], electrochemical [5,6] and colorimetry [7] have been employed for its detection. Recently, Chekin et al. have reported a MoS 2 /reduced graphene oxide nanocomposite for the sensing of cysteamine in the presence of uric acid in human plasma [6]. Upon optimization, the anodic peak current of cysteamine showed a linear relationship with concentration between 0.01 and 20 μM. The limit of detection (LOD) of the method was reported to be 7 nM [6]. Noble metals such as gold and silver in their colloidal form display colourful solutions. The physical origin of light absorption by such particles may be attributed to the coherent oscillation of the conduction band electrons induced by the interacting electromagnetic field. These resonances are called surface plasmons which depend on the shape and size of the particles. The surface plasmon phenomenon is a small particle effect since it is absent in individual atoms and in the bulk [8]. If the addition of an analyte could trigger the aggregation of such particles there will be a change in the surface plasmon resonance (SPR) band ⁎ Corresponding author. E-mail address: [email protected] (D.P.S. Negi).
https://doi.org/10.1016/j.saa.2019.02.101 1386-1425/© 2019 Elsevier B.V. All rights reserved.
of the gold or silver particles. Consequently, there will be a change in the colour of the colloidal solution of the particles. This strategy may be utilized for the colorimetric sensing of biologically important molecules. Recently, several reports have been published on the sensing of metal ions [9–11] and biomolecules [12,13] using silver nanoparticles. In the present work, we have used cysteine-stabilized colloidal silver nanoparticles (AgNPs) as a SPR based probe for the determination of cysteamine. 2. Materials and methods 2.1. Materials Silver nitrate, arginine, asparagine, aspartic acid, methionine, glutamic acid, glycine, and glutathione were obtained from Himedia (India). Cysteine, 6-mercaptopurine and sodium borohydride were obtained from Sigma Aldrich (India). 2-Amino ethane thiol (cysteamine) was obtained from TCI Chemicals (India). All the reagents were of analytical grade and were used without further purification. The water used for preparing the solutions was purified via distillation. 2.2. Instrumentation The UV–visible spectra were acquired on a PerkinElmer Lambda 25 (Singapore) spectrophotometer. A 10 mm path length quartz cuvette was used for the measurements. The TEM measurements were performed using a JEOL 100 CX (USA) transmission electron microscope operating at 100 kV. A drop of the sample was placed on a copper
204
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0.6 cysteine cysteine-capped AgNPs
Transmittance
0.4
0.2 2548
0.0
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm)-1 Fig. 1. UV–visible spectrum of the AgNPs at pH 9.5.
Fig. 3. FT-IR spectra of cysteine in the absence and presence of the AgNPs.
grid. The grid was dried in an oven to remove the water. Infrared spectra were obtained on an Alpha II Bruker (Germany) FT-IR spectrophotometer. Zeta potential measurements were carried out on a Malvern Zetasizer Nano ZS90 (UK) instrument.
undertaking the spectral studies. The pH of the colloidal solution was adjusted to 9.5.
2.3. Synthesis of the cysteine-stabilized AgNPs
Typically, 5 ml aliquots of the synthesized AgNPs at pH 9.5 were taken in different vials. Subsequently, various concentrations (2–100 μM) of the target analytes were added to the vials. The samples were kept undisturbed for 2–5 min at room temperature before performing the UV–Vis absorption measurements.
The cysteine-stabilized AgNPs were synthesized using the method reported by Mandal and coworkers [14]. Typically, 0.1 ml of a 0.1 M silver nitrate solution was added dropwise to 100 ml distilled water under continuous stirring. Subsequently, 0.01 g of sodium borohydride was added to the solution. It resulted in the formation of a yellow coloured solution. The solution was kept undisturbed for 10 min. Subsequently, 2 ml of a 1 mM cysteine solution was added dropwise under stirring to obtain well dispersed AgNPs. The solution was kept overnight before
Fig. 2. TEM image of the AgNPs.
2.4. Preparation of samples for the UV-visible spectral measurements
2.5. Preparation of cysteamine spiked human blood serum samples The blood sample collected from a Pathology laboratory was centrifuged to obtain the blood serum. Then, 40 μl of 0.1 M cysteamine solution was added to the blood serum to obtain 50 mM stock solution of the analyte. Subsequently, different volumes of the as-prepared stock solution were added to the AgNPs to obtain different concentrations of cysteamine in blood serum medium.
Fig. 4. UV–visible spectra of the AgNPs in the presence of various concentrations of cysteamine at pH 9.5 as indicated in the inset.
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3. Results and discussion 3.1. Characterization of the cysteine-stabilized AgNPs The UV–visible spectrum of the AgNPs has been displayed in Fig. 1. A narrow absorption band centred at ~400 nm may be attributed to the SPR phenomenon of the silver metal nanoparticles [14]. The location of this band was found to be within ±5 nm during various preparations of the AgNPs. TEM measurements were carried out to determine the shape and size of the AgNPs. The TEM image is displayed in Fig. 2. The particles were found to be 4 to 15 nm in diameter. The selected area electron diffraction (SAED) is a technique to ascertain the crystalline nature of a sample. The SAED pattern of the AgNPs has been displayed in Fig. S1 of the Supplementary data. The concentric rings seen in the image confirmed the crystalline nature of the material [15]. The binding of cysteine on the surface of the AgNPs was confirmed by FT-IR measurements. The FT-IR spectra of free cysteine and cysteine-capped AgNPs have been displayed in Fig. 3. The band observed at 2548 cm−1 in the figure may be attributed to the S\\H vibration of cysteine [16]. However, this band was not observed for cysteine-capped AgNPs (Fig. 3). It indicates the binding of cysteine to the surface of the AgNPs via the sulfur atom. Fig. 5. TEM image of the AgNPs in the presence of 2 μM cysteamine.
3.2. Effect of the addition of cysteamine on the SPR band of the AgNPs
0.3
Transmittance
Cysteamine Cysteamine+ AgNPs
0.2
0.1 2508
0.0 4000 3500 3000 2500 2000 1500 1000
500
-1
Wavenumber (cm)
Fig. 6. FT-IR spectra of cysteamine in the absence and presence of the AgNPs.
The representative UV–visible spectra of the AgNPs in the presence of cysteamine have been displayed in Fig. 4. The SPR band was quenched and a broad band centred at 560 nm was observed upon increasing the concentration of cysteamine. There were no further significant changes to the absorption spectra upon increasing the concentration beyond 100 μM. The colour of the colloidal AgNPs changed from yellow to dark brown within a few seconds after the addition of cysteamine. These observations indicated the aggregation of the AgNPs in the presence of cysteamine. The role of pH in the aggregation process was also investigated. The experiments were performed at higher pH since the AgNPs were not stable at pH below 9.5. The UV–visible spectra of the AgNPs in the presence of different concentrations of cysteamine at pH 10.5 and 11.5 have been displayed in Figs. S2 and S3 of the Supplementary data. From these figures it is observed that the detection of cysteine was most sensitive at pH 9.5. Therefore, the pH of the AgNPs was kept at 9.5 for all the experiments. The aggregation of the AgNPs was confirmed by performing the TEM measurements. The TEM images of the AgNPs upon addition of 2 μM cysteamine have been displayed in Fig. 5. The AgNPs appeared in an aggregated state compared to the well dispersed particles observed in Fig. 2. The aggregation of the AgNPs was irreversible.
Fig. 7. Photograph of the AgNPs in the absence and presence of 2 μM concentrations of arginine (Arg), asparagine (Aspn), aspartic acid (Asp), cysteamine (Cys), cysteine (L-Cys), glutamic acid (Glu), glutathione (glut), glycine (Gly), 6-mercaptopurine (Mercap) and methionine (Meth) at pH 9.5.
S. Diamai, D.P.S. Negi / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 215 (2019) 203–208
The interaction of cysteamine with the AgNPs was also probed by using FT-IR spectroscopy. The FT-IR spectra of cysteamine in the absence and presence of the AgNPs have been displayed in Fig. 6. The S\\H vibration of cysteamine at 2508 cm−1 was found to disappear in the presence of the AgNPs. It indicated the interaction of cysteamine with the AgNPs via the sulfur atom. 3.3. Selectivity of the SPR based probe for cysteamine The selectivity of the proposed method was investigated by monitoring the effect of the addition of various biomolecules such as arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutathione, glycine, methionine and 6-mercaptopurine on the SPR band of the AgNPs. The absorption spectra of the AgNPs in the absence and presence of the above-mentioned biomolecules have been displayed in Figs. S4 to S12 of the Supplementary data. It was evident form the absorption spectra that there was negligible change in the SPR band of the AgNPs upon the addition of the micromolar concentrations of the biomolecules. It suggested that none of the biomolecules could induce the aggregation of the AgNPs under the reported experimental conditions. The photograph of the AgNPs in the absence and presence of 2 μM concentration of various biomolecules have been displayed in Fig. 7. It is seen from this image that only the addition of cysteamine resulted in a colour change of the AgNPs from yellow to dark brown. It must be pointed out here that the aggregation of the AgNPs in the presence of cysteamine was not affected by the addition of the other biomolecules. Therefore, the cysteine-stabilized AgNPs may be used as a colorimetric probe for the selective detection of cysteamine.
0.0710 0.0705
Avg (A560/397)
206
0.0695 0.0690 0.0685 0.0680 0.6
0.8
1.0
1.2
[Cysteamine]
1.4
1.6
1.8
M)
Fig. 9. Plot of the response versus the concentration of cysteamine.
from Table 1 that the LOD of the present method was much higher than that of the fluorescence and electrochemical methods. However, it was comparable to that reported by the colorimetric method (reference [7]). 3.5. Aggregation mechanism for the AgNPs in the presence of cysteamine Cysteamine is known to ionize in aqueous solution as follows:
3.4. Sensitivity of the AgNP based probe for cysteamine The ratio of absorbance of the red shifted absorption band to the SPR band of the AgNPs is a yardstick to quantify the response of the probe towards an analyte. The response of the AgNPs for the various biomolecules could be quantified by comparing the ratio of their absorbance at 560 nm and 397 nm [17]. Fig. 8 displays that the response of the AgNPs was maximum for cysteamine. The response was found to be linear in the concentration range 0.6–1.8 μM (Fig. 9). The LOD was calculated by using the relation 3σ/S [17]. Here, σ is the standard deviation of the response of the blank while S denotes the slope of the calibration curve. The LOD was found to be 0.37 μM for cysteamine. The LOD for cysteamine determination by the other methods reported during the last six years have been summarized in Table 1. It is evident
R2 = 0.961
0.0700
þ
H3 NCH2 CH2 SH ⇌ þ H3 NCH2 CH2 S− þ Hþ
ð1Þ
þ
H3 NCH2 CH2 S− ⇌ H2 NCH2 CH2 S− þ Hþ
ð2Þ
The pka for steps represented by Eqs. (1) and (2) are reported to be 8.32 and 10.81 respectively [18]. Therefore, at pH 9.5, cysteamine mainly exists in the form represented by the right-hand side of Eq. (1). The cysteine-stabilized AgNPs bear a net negative charge at pH 9.5 since the isoelectric point (pI) of cysteine is known to be 5.02 [14]. The AgNPs were stabilized in the solution due to the electrostatic repulsion between the negatively charged particles. Upon addition of cysteamine to the AgNPs, the cysteamine molecule binds to the surface via its sulfur atom like the case of cysteine. The binding of cysteamine on the surface of the AgNPs was confirmed by carrying out the zeta potential measurements. The zeta potential values of the AgNPs in the absence and presence of cysteamine were found to be 0.787 and −1.15 mV respectively. The decrease in the zeta potential may be attributed to the presence of the negatively charged sulfur atom of cysteamine on the surface of the AgNPs. Upon binding of cysteamine on the surface of a silver particle, its free end (NH3+) bears a positive charge. Sudeep and coworkers had reported the aggregation of gold NPs due to the electrostatic interaction between the zwitter ionic form of the cysteine molecule attached to their surface [19]. The pH of the solution in their work was reported to be 5.6 which
Table 1 Comparison of the various analytical methods for the determination of cysteamine.
Fig. 8. Response of the AgNPs in the absence and presence of 2 μM concentration of various biomolecules at pH 9.5.
Analytical method
LOD
Reference
Fluorescence Fluorescence Electrochemical Electrochemical Colorimetry Colorimetry
150 nM 30.6 nM 9 nM 7 nM 0.13 μM 0.37 μM
[3] [4] [5] [6] [7] Present work
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Fig. 10. Proposed mechanism for the cysteamine induced aggregation of the AgNPs at pH 9.5.
was close to the isoelectric point of cysteine (pI = 5.02). In the present work, the cysteine-stabilized AgNPs would not undergo aggregation since the solution pH was 9.5. Under the experimental conditions, the cysteine molecules attached to the surface of the AgNPs bear a net negative charge. We propose an electrostatic interaction between the positive charge of the cysteamine (NH3+) attached to a silver particle and the negative charge on another such particle. Many such electrostatic interaction result in the aggregation of the AgNPs in the solution. The pictorial representation of the aggregation process has been displayed in Fig. 10.
4. Conclusions The cysteine-stabilized AgNPs have been demonstrated as a colorimetric probe for the selective determination of cysteamine. The change in colour of the colloidal AgNPs from yellow to dark brown was due to the aggregation of the particles in the presence of cysteamine. The aggregation was a result of the electrostatic interaction between the positively charged NH3+ moiety of cysteamine and the negatively charged surface of the AgNPs. The colorimetric method was applied for the qualitative determination of cysteamine in human blood serum. Acknowledgments
3.6. Analysis of cysteamine in human blood serum The colorimetric method described herein was applied for the detection of cysteamine in human blood serum. There was no significant change in the SPR band of the AgNPs upon addition of microlitre quantities of human blood serum (Fig. S13, Supplementary data). It suggests that cysteamine present in blood serum might not be available in a free state to bind to the surface of the AgNPs. However, the SPR band of the AgNPs was found to be quenched upon addition of human blood serum spiked with cysteamine (Fig. 11).
This research was financially sponsored by the University Grants Commission, New Delhi. We acknowledge the Sophisticated Analytical Instrumentation Facility of the North-Eastern Hill University, for the TEM analyses. Declaration of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.02.101.
1.0 Blank 20µM 40µM 60µM 80µM 100µM
Absorbance
0.8
0.6
References
0.4
0.2
0.0 300
400
500
600
700
800
Wavelength (nm) Fig. 11. UV–visible spectra of the AgNPs in the presence of various concentration of cysteamine in spiked human blood serum.
[1] S. Sinha, G. Dey, S. Kumar, J. Mathew, T. Mukherjee, S. Ghosh, Cysteamine-based cell-permeable Zn2+-specific molecular bioimaging materials: from animals to plant cells, ACS Appl. Mater. Interfaces 5 (2013) 11730–11740. [2] H. Fleischer, Y. Dienes, B. Mathiasch, V. Schmitt, D. Schollmeyer, Cysteamine and its homoleptic complexes with group 12 metal ions. Differences in the coordination chemistry of ZnII, CdII, and HgII with a small N,S-donor ligand, Inorg. Chem. 44 (2005) 8087–8096. [3] T. Shu, L. Su, J. Wang, C. Li, X. Zhang, Chemical etching of bovine serum albuminprotected Au25 nanoclusters for label-free and separation-free detection of cysteamine, Biosens. Bioelectron. 66 (2015) 155–161. [4] S. Sarkar, S. Roy, R.N. Saha, S.S. Panja, Thiophene appended dual fluorescent sensor for detection of Hg2+ and cysteamine, J. Fluoresc. 28 (2018) 427–437. [5] V. Arabali, H. Karimi-Maleh, Electrochemical determination of cysteamine in the presence of guanine and adenine using a carbon paste electrode modified with N(4-hydroxyphenyl)-3,5-dinitrobenzamide and magnesium oxide nanoparticles, Anal. Methods 8 (2016) 5604–5610. [6] F. Chekin, R. Boukherroub, S. Szunerits, MoS2/reduced graphene oxide nanocomposite for sensitive sensing of cysteamine in presence of uric acid in human plasma, Mater. Sci. Eng. C 73 (2017) 627–632. [7] V.V. Apyari, S.G. Dmitrienko, V.V. Arkhipova, A.G. Atnagulov, Y.A. Zolotov, Determination of cysteamine using label-free gold nanoparticles, Anal. Methods 4 (2012) 3193–3199.
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[8] S. Link, M.A. El-Sayed, Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods, J. Phys. Chem. B 103 (1999) 8410–8426. [9] H. Yu, Z. Wang, W. Huang, Highly stable and sensitive colorimetric visualization of trivalent chromium using amido black 10B-stabilized silver nanoparticles, Plasmonics 13 (2018) 1459–1465. [10] A. Amirjani, D.F. Haghshenas, Ag nanostructures as the surface plasmon resonance (SPR)-based sensors: a mechanistic study with emphasis on heavy metallic ions detection, Sensors Actuators B Chem. 273 (2018) 1768–1779. [11] D.K. Ban, S. Paul, Rapid colorimetric and spectroscopy based sensing of heavy metal and cellular free oxygen radical by surface functionalized silver nanoparticles, Appl. Surf. Sci. 458 (2018) 245–251. [12] J.K. Rajput Jigyasa, Bio-polyphenols promoted green synthesis of silver nanoparticles for facile and ultra-sensitive colorimetric detection of melamine in milk, Biosens. Bioelectron. 120 (2018) 153–159. [13] M.T. Alula, L. Karamchand, N.R. Hendricks, J.M. Blackburn, Citrate-capped silver nanoparticles as a probe for sensitive and selective colorimetric and spectrophotometric sensing of creatinine in human urine, Anal. Chim. Acta 1007 (2018) 40–49.
[14] S. Mandal, A. Gole, N. Lala, R. Gonnade, V. Ganvir, M. Sastry, Studies on the reversible aggregation of cysteine-capped colloidal silver particles interconnected via hydrogen bonds, Langmuir 17 (2001) 6262–6268. [15] K. Jyoti, M. Baunthiyal, A. Singh, Characterizarion of silver nanoparticles synthesized using Urtica dioica Linn. leaves and their synergistic effects with antibiotics, J. Radiat. Res. Appl. Sci. 9 (2016) 217–227. [16] S. Devi, B. Singh, A.K. Paul, S. Tyagi, Highly sensitive and selective detection of trinitrotoluene using cysteine-capped gold nanoparticles, Anal. Methods 8 (2016) 4398–4405. [17] S. Diamai, W. Warjri, D. Saha, D.P.S. Negi, Sensitive determination of 6mercaptopurine based on the aggregation of phenylalanine-capped gold nanoparticles, Colloids Surf. A Physicochem. Eng. Asp. 538 (2018) 593–599. [18] L. Riauba, G. Niaura, O. Eicher-Lorka, E. Butkus, A study of cysteamine ionization in solution by Raman spectroscopy and theoretical modelling, J. Phys. Chem. A 110 (2006) 13394–13404. [19] P.K. Sudeep, S.T.S. Joseph, K.G. Thomas, Selective detection of cysteine and glutathione using gold nanorods, J. Am. Chem. Soc. 127 (2005) 6516–6517.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Cysteine-stabilized silver nanoparticles as a colorimetric probe for the selective detection of cysteamine Siewdorlang Diamai, Devendra P.S. Negi ⁎ Department of Chemistry, North-Eastern Hill University, Shillong 793022, India
a r t i c l e
i n f o
Article history: Received 15 November 2018 Received in revised form 19 February 2019 Accepted 24 February 2019 Available online 25 February 2019 Keywords: Surface plasmon resonance Colorimetric Absorption Detection Silver nanoparticles
a b s t r a c t The purpose of the present research was to design a method for the colorimetric determination of cysteamine. We have employed cysteine-stabilized silver nanoparticles (AgNPs) as a probe. The addition of cysteamine resulted in the quenching of the 400 nm surface plasmon resonance (SPR) band of the AgNPs. It was accompanied by the appearance of a new absorption band at 560 nm. The colour of the colloidal AgNPs changed from yellow to dark brown within a few seconds. The change in colour of the AgNPs was due to their aggregation induced by the addition of cysteamine. Significantly, other biomolecules such as arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutathione, glycine, methionine and 6-mercaptopurine did not cause any change in the colour of the AgNPs. The limit of detection (LOD) of the method was 0.37 μM. The mechanism of the aggregation of the AgNPs induced by cysteamine has also been described. The method has been applied for the detection of cysteamine in human blood serum. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Cysteamine is a naturally occurring aminothiol which is widely applied as an important building block for many natural products. It is used as a therapeutic for the treatment of nephropathic cystinosis [1]. Cysteamine is amongst the important chelating ligands in coordination chemistry and binds preferentially to soft metal ions [2]. The sensing of cysteamine has attracted much interest recently. Various approaches such as fluorescence [3,4], electrochemical [5,6] and colorimetry [7] have been employed for its detection. Recently, Chekin et al. have reported a MoS 2 /reduced graphene oxide nanocomposite for the sensing of cysteamine in the presence of uric acid in human plasma [6]. Upon optimization, the anodic peak current of cysteamine showed a linear relationship with concentration between 0.01 and 20 μM. The limit of detection (LOD) of the method was reported to be 7 nM [6]. Noble metals such as gold and silver in their colloidal form display colourful solutions. The physical origin of light absorption by such particles may be attributed to the coherent oscillation of the conduction band electrons induced by the interacting electromagnetic field. These resonances are called surface plasmons which depend on the shape and size of the particles. The surface plasmon phenomenon is a small particle effect since it is absent in individual atoms and in the bulk [8]. If the addition of an analyte could trigger the aggregation of such particles there will be a change in the surface plasmon resonance (SPR) band ⁎ Corresponding author. E-mail address: [email protected] (D.P.S. Negi).
https://doi.org/10.1016/j.saa.2019.02.101 1386-1425/© 2019 Elsevier B.V. All rights reserved.
of the gold or silver particles. Consequently, there will be a change in the colour of the colloidal solution of the particles. This strategy may be utilized for the colorimetric sensing of biologically important molecules. Recently, several reports have been published on the sensing of metal ions [9–11] and biomolecules [12,13] using silver nanoparticles. In the present work, we have used cysteine-stabilized colloidal silver nanoparticles (AgNPs) as a SPR based probe for the determination of cysteamine. 2. Materials and methods 2.1. Materials Silver nitrate, arginine, asparagine, aspartic acid, methionine, glutamic acid, glycine, and glutathione were obtained from Himedia (India). Cysteine, 6-mercaptopurine and sodium borohydride were obtained from Sigma Aldrich (India). 2-Amino ethane thiol (cysteamine) was obtained from TCI Chemicals (India). All the reagents were of analytical grade and were used without further purification. The water used for preparing the solutions was purified via distillation. 2.2. Instrumentation The UV–visible spectra were acquired on a PerkinElmer Lambda 25 (Singapore) spectrophotometer. A 10 mm path length quartz cuvette was used for the measurements. The TEM measurements were performed using a JEOL 100 CX (USA) transmission electron microscope operating at 100 kV. A drop of the sample was placed on a copper
204
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0.6 cysteine cysteine-capped AgNPs
Transmittance
0.4
0.2 2548
0.0
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm)-1 Fig. 1. UV–visible spectrum of the AgNPs at pH 9.5.
Fig. 3. FT-IR spectra of cysteine in the absence and presence of the AgNPs.
grid. The grid was dried in an oven to remove the water. Infrared spectra were obtained on an Alpha II Bruker (Germany) FT-IR spectrophotometer. Zeta potential measurements were carried out on a Malvern Zetasizer Nano ZS90 (UK) instrument.
undertaking the spectral studies. The pH of the colloidal solution was adjusted to 9.5.
2.3. Synthesis of the cysteine-stabilized AgNPs
Typically, 5 ml aliquots of the synthesized AgNPs at pH 9.5 were taken in different vials. Subsequently, various concentrations (2–100 μM) of the target analytes were added to the vials. The samples were kept undisturbed for 2–5 min at room temperature before performing the UV–Vis absorption measurements.
The cysteine-stabilized AgNPs were synthesized using the method reported by Mandal and coworkers [14]. Typically, 0.1 ml of a 0.1 M silver nitrate solution was added dropwise to 100 ml distilled water under continuous stirring. Subsequently, 0.01 g of sodium borohydride was added to the solution. It resulted in the formation of a yellow coloured solution. The solution was kept undisturbed for 10 min. Subsequently, 2 ml of a 1 mM cysteine solution was added dropwise under stirring to obtain well dispersed AgNPs. The solution was kept overnight before
Fig. 2. TEM image of the AgNPs.
2.4. Preparation of samples for the UV-visible spectral measurements
2.5. Preparation of cysteamine spiked human blood serum samples The blood sample collected from a Pathology laboratory was centrifuged to obtain the blood serum. Then, 40 μl of 0.1 M cysteamine solution was added to the blood serum to obtain 50 mM stock solution of the analyte. Subsequently, different volumes of the as-prepared stock solution were added to the AgNPs to obtain different concentrations of cysteamine in blood serum medium.
Fig. 4. UV–visible spectra of the AgNPs in the presence of various concentrations of cysteamine at pH 9.5 as indicated in the inset.
S. Diamai, D.P.S. Negi / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 215 (2019) 203–208
205
3. Results and discussion 3.1. Characterization of the cysteine-stabilized AgNPs The UV–visible spectrum of the AgNPs has been displayed in Fig. 1. A narrow absorption band centred at ~400 nm may be attributed to the SPR phenomenon of the silver metal nanoparticles [14]. The location of this band was found to be within ±5 nm during various preparations of the AgNPs. TEM measurements were carried out to determine the shape and size of the AgNPs. The TEM image is displayed in Fig. 2. The particles were found to be 4 to 15 nm in diameter. The selected area electron diffraction (SAED) is a technique to ascertain the crystalline nature of a sample. The SAED pattern of the AgNPs has been displayed in Fig. S1 of the Supplementary data. The concentric rings seen in the image confirmed the crystalline nature of the material [15]. The binding of cysteine on the surface of the AgNPs was confirmed by FT-IR measurements. The FT-IR spectra of free cysteine and cysteine-capped AgNPs have been displayed in Fig. 3. The band observed at 2548 cm−1 in the figure may be attributed to the S\\H vibration of cysteine [16]. However, this band was not observed for cysteine-capped AgNPs (Fig. 3). It indicates the binding of cysteine to the surface of the AgNPs via the sulfur atom. Fig. 5. TEM image of the AgNPs in the presence of 2 μM cysteamine.
3.2. Effect of the addition of cysteamine on the SPR band of the AgNPs
0.3
Transmittance
Cysteamine Cysteamine+ AgNPs
0.2
0.1 2508
0.0 4000 3500 3000 2500 2000 1500 1000
500
-1
Wavenumber (cm)
Fig. 6. FT-IR spectra of cysteamine in the absence and presence of the AgNPs.
The representative UV–visible spectra of the AgNPs in the presence of cysteamine have been displayed in Fig. 4. The SPR band was quenched and a broad band centred at 560 nm was observed upon increasing the concentration of cysteamine. There were no further significant changes to the absorption spectra upon increasing the concentration beyond 100 μM. The colour of the colloidal AgNPs changed from yellow to dark brown within a few seconds after the addition of cysteamine. These observations indicated the aggregation of the AgNPs in the presence of cysteamine. The role of pH in the aggregation process was also investigated. The experiments were performed at higher pH since the AgNPs were not stable at pH below 9.5. The UV–visible spectra of the AgNPs in the presence of different concentrations of cysteamine at pH 10.5 and 11.5 have been displayed in Figs. S2 and S3 of the Supplementary data. From these figures it is observed that the detection of cysteine was most sensitive at pH 9.5. Therefore, the pH of the AgNPs was kept at 9.5 for all the experiments. The aggregation of the AgNPs was confirmed by performing the TEM measurements. The TEM images of the AgNPs upon addition of 2 μM cysteamine have been displayed in Fig. 5. The AgNPs appeared in an aggregated state compared to the well dispersed particles observed in Fig. 2. The aggregation of the AgNPs was irreversible.
Fig. 7. Photograph of the AgNPs in the absence and presence of 2 μM concentrations of arginine (Arg), asparagine (Aspn), aspartic acid (Asp), cysteamine (Cys), cysteine (L-Cys), glutamic acid (Glu), glutathione (glut), glycine (Gly), 6-mercaptopurine (Mercap) and methionine (Meth) at pH 9.5.
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The interaction of cysteamine with the AgNPs was also probed by using FT-IR spectroscopy. The FT-IR spectra of cysteamine in the absence and presence of the AgNPs have been displayed in Fig. 6. The S\\H vibration of cysteamine at 2508 cm−1 was found to disappear in the presence of the AgNPs. It indicated the interaction of cysteamine with the AgNPs via the sulfur atom. 3.3. Selectivity of the SPR based probe for cysteamine The selectivity of the proposed method was investigated by monitoring the effect of the addition of various biomolecules such as arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutathione, glycine, methionine and 6-mercaptopurine on the SPR band of the AgNPs. The absorption spectra of the AgNPs in the absence and presence of the above-mentioned biomolecules have been displayed in Figs. S4 to S12 of the Supplementary data. It was evident form the absorption spectra that there was negligible change in the SPR band of the AgNPs upon the addition of the micromolar concentrations of the biomolecules. It suggested that none of the biomolecules could induce the aggregation of the AgNPs under the reported experimental conditions. The photograph of the AgNPs in the absence and presence of 2 μM concentration of various biomolecules have been displayed in Fig. 7. It is seen from this image that only the addition of cysteamine resulted in a colour change of the AgNPs from yellow to dark brown. It must be pointed out here that the aggregation of the AgNPs in the presence of cysteamine was not affected by the addition of the other biomolecules. Therefore, the cysteine-stabilized AgNPs may be used as a colorimetric probe for the selective detection of cysteamine.
0.0710 0.0705
Avg (A560/397)
206
0.0695 0.0690 0.0685 0.0680 0.6
0.8
1.0
1.2
[Cysteamine]
1.4
1.6
1.8
M)
Fig. 9. Plot of the response versus the concentration of cysteamine.
from Table 1 that the LOD of the present method was much higher than that of the fluorescence and electrochemical methods. However, it was comparable to that reported by the colorimetric method (reference [7]). 3.5. Aggregation mechanism for the AgNPs in the presence of cysteamine Cysteamine is known to ionize in aqueous solution as follows:
3.4. Sensitivity of the AgNP based probe for cysteamine The ratio of absorbance of the red shifted absorption band to the SPR band of the AgNPs is a yardstick to quantify the response of the probe towards an analyte. The response of the AgNPs for the various biomolecules could be quantified by comparing the ratio of their absorbance at 560 nm and 397 nm [17]. Fig. 8 displays that the response of the AgNPs was maximum for cysteamine. The response was found to be linear in the concentration range 0.6–1.8 μM (Fig. 9). The LOD was calculated by using the relation 3σ/S [17]. Here, σ is the standard deviation of the response of the blank while S denotes the slope of the calibration curve. The LOD was found to be 0.37 μM for cysteamine. The LOD for cysteamine determination by the other methods reported during the last six years have been summarized in Table 1. It is evident
R2 = 0.961
0.0700
þ
H3 NCH2 CH2 SH ⇌ þ H3 NCH2 CH2 S− þ Hþ
ð1Þ
þ
H3 NCH2 CH2 S− ⇌ H2 NCH2 CH2 S− þ Hþ
ð2Þ
The pka for steps represented by Eqs. (1) and (2) are reported to be 8.32 and 10.81 respectively [18]. Therefore, at pH 9.5, cysteamine mainly exists in the form represented by the right-hand side of Eq. (1). The cysteine-stabilized AgNPs bear a net negative charge at pH 9.5 since the isoelectric point (pI) of cysteine is known to be 5.02 [14]. The AgNPs were stabilized in the solution due to the electrostatic repulsion between the negatively charged particles. Upon addition of cysteamine to the AgNPs, the cysteamine molecule binds to the surface via its sulfur atom like the case of cysteine. The binding of cysteamine on the surface of the AgNPs was confirmed by carrying out the zeta potential measurements. The zeta potential values of the AgNPs in the absence and presence of cysteamine were found to be 0.787 and −1.15 mV respectively. The decrease in the zeta potential may be attributed to the presence of the negatively charged sulfur atom of cysteamine on the surface of the AgNPs. Upon binding of cysteamine on the surface of a silver particle, its free end (NH3+) bears a positive charge. Sudeep and coworkers had reported the aggregation of gold NPs due to the electrostatic interaction between the zwitter ionic form of the cysteine molecule attached to their surface [19]. The pH of the solution in their work was reported to be 5.6 which
Table 1 Comparison of the various analytical methods for the determination of cysteamine.
Fig. 8. Response of the AgNPs in the absence and presence of 2 μM concentration of various biomolecules at pH 9.5.
Analytical method
LOD
Reference
Fluorescence Fluorescence Electrochemical Electrochemical Colorimetry Colorimetry
150 nM 30.6 nM 9 nM 7 nM 0.13 μM 0.37 μM
[3] [4] [5] [6] [7] Present work
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207
Fig. 10. Proposed mechanism for the cysteamine induced aggregation of the AgNPs at pH 9.5.
was close to the isoelectric point of cysteine (pI = 5.02). In the present work, the cysteine-stabilized AgNPs would not undergo aggregation since the solution pH was 9.5. Under the experimental conditions, the cysteine molecules attached to the surface of the AgNPs bear a net negative charge. We propose an electrostatic interaction between the positive charge of the cysteamine (NH3+) attached to a silver particle and the negative charge on another such particle. Many such electrostatic interaction result in the aggregation of the AgNPs in the solution. The pictorial representation of the aggregation process has been displayed in Fig. 10.
4. Conclusions The cysteine-stabilized AgNPs have been demonstrated as a colorimetric probe for the selective determination of cysteamine. The change in colour of the colloidal AgNPs from yellow to dark brown was due to the aggregation of the particles in the presence of cysteamine. The aggregation was a result of the electrostatic interaction between the positively charged NH3+ moiety of cysteamine and the negatively charged surface of the AgNPs. The colorimetric method was applied for the qualitative determination of cysteamine in human blood serum. Acknowledgments
3.6. Analysis of cysteamine in human blood serum The colorimetric method described herein was applied for the detection of cysteamine in human blood serum. There was no significant change in the SPR band of the AgNPs upon addition of microlitre quantities of human blood serum (Fig. S13, Supplementary data). It suggests that cysteamine present in blood serum might not be available in a free state to bind to the surface of the AgNPs. However, the SPR band of the AgNPs was found to be quenched upon addition of human blood serum spiked with cysteamine (Fig. 11).
This research was financially sponsored by the University Grants Commission, New Delhi. We acknowledge the Sophisticated Analytical Instrumentation Facility of the North-Eastern Hill University, for the TEM analyses. Declaration of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.02.101.
1.0 Blank 20µM 40µM 60µM 80µM 100µM
Absorbance
0.8
0.6
References
0.4
0.2
0.0 300
400
500
600
700
800
Wavelength (nm) Fig. 11. UV–visible spectra of the AgNPs in the presence of various concentration of cysteamine in spiked human blood serum.
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