Appl Biochem Biotechnol DOI 10.1007/s12010-015-1519-0
Bio-Functionalized Silver Nanoparticles: a Novel Colorimetric Probe for Cysteine Detection Hemant P. Borase & Chandrashekhar D. Patil & Rahul B. Salunkhe & Rahul K. Suryawanshi & Beom S. Kim & Vishwas A. Bapat & Satish V. Patil
Received: 16 September 2014 / Accepted: 21 January 2015 # Springer Science+Business Media New York 2015
Abstract Chemical interactions between nanoparticles and biomolecules are vital for applying nanoparticles in medicine and life science. Development of sensitive, rapid, low-cost, and eco-friendly sensors for the detection of molecules acting as disease indicator is need of an hour. In the present investigation, a green trend for silver nanoparticle synthesis was followed using leaf extract of Calotropis procera. Silver nanoparticles exhibited surface plasmon absorption peak at 421 nm, spherical shape with average size of 10 nm, and zeta potential of −22.4 mV. The as-synthesized silver nanoparticles were used for selective and sensitive detection of cysteine. Cysteine induces aggregation in stable silver nanoparticles owing to selective and strong interaction of –SH group of cysteine with silver nanoparticle surface. Cysteine-induced silver nanoparticle aggregation can be observed visually by change in color of silver nanoparticles from yellow to pink. Cysteine concentration was estimated colorimetrically by measuring absorption at surface plasmon wavelength. Limit of detection for cysteine using silver nanoparticles is ultralow, i.e., 100 nM. The mechanistic insight into cysteine detection by silver nanoparticles was investigated using FT-IR, TEM, DLS, and TLC analysis. Proposed method can be applied for the detection of cysteine in blood plasma and may give rise to a new insight into development of eco-friendly fabricated nanodiagnostic device in future.
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1519-0) contains supplementary material, which is available to authorized users.
H. P. Borase : C. D. Patil : R. B. Salunkhe : R. K. Suryawanshi : S. V. Patil School of Life Sciences, North Maharashtra University, Post Box 80, Jalgaon 425001 Maharashtra, India B. S. Kim Department of Chemical Engineering, Chungbuk National University, Chungbuk 361-763, Cheongju, Republic of Korea V. A. Bapat Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur 416004 Maharashtra, India S. V. Patil (*) North Maharashtra Microbial Culture Collection Centre (NMCC), North Maharashtra University, Post Box 80, Jalgaon 425001 Maharashtra, India e-mail: [email protected]
Appl Biochem Biotechnol
Keywords Silver nanoparticles . Cysteine . Colorimetric sensor . FT-IR . Blood plasma
Introduction Cysteine, a –SH group containing α-amino acid having molar mass of 121.16 g mol−1 play a vital role in physiological processes like protein folding and metabolism, posttranslational modification, detoxification, and affinity for heavy metals [1–3]. Cysteine has an important role in physiological developments of herbivores and plants [4–6]. Apart from its role in cellular processes, cysteine is a biomarker for many pathological ailments such as liver damage, heart diseases, skin lesions, hair depigmentation, sepsis, AIDS, cystinuria, and Parkinson’s and Alzheimer’s diseases [3, 7–9]. Recently, cysteine was researched regarding its role in obesity [10]. Cysteine is widely used in pharma products like antibiotics and skin treatment formulations [11]. Considering above multiple roles of cysteine, its analysis and detection in biological and pharmaceutical samples was attempted by devising different methods using HPLC [12], fluorescence spectroscopy [13], capillary electrophoresis [14], and flow injection [15]. The abovementioned methods can detect low levels of cysteine, but they are expensive, time consuming, required skilled labor, and are of limited on-field application. With above background, there is an urgent need to develop simple, rapid, selective, and low-cost method of cysteine detection. Nanotechnology is now arising as a promising tool in new generation disease diagnosis system that has been termed Bnanodiagnostics^ [16, 17]. Silver nanoparticles are gaining worldwide attention as sensors for molecules that have medical and environmental relevance such as mercury [18], tricyclazole [19], melamine [20], and glucose [21]. The characteristics such as surface plasmon resonance, chemical stability, biocompatibility, ease of preparation, low cost, easy end point detection, and high sensitivity and selectivity makes silver nanoparticles a potent alternative to currently used approaches in chemosensing and disease diagnosis. Moreover, the on-field detection of molecules is possible with use of silver nanoparticles. Physicochemical methods currently used for silver nanoparticle synthesis and their use in chemosensing have several disadvantages like use of toxic chemicals in synthesis, harsh synthesis parameters, prone to aggregation, tedious ligand capping process, and concern for the use in biomedical applications [18, 19]. Vital goal of nanosensor technology is eco-friendly production of nanodevices with low environmental pollution and toxicity to nontargets. Then, how chemically synthesized nanodevices will answer above important question? Moreover, apart from application of nanoparticles, the route of nanosynthesis is also important. The synthesis of silver nanoparticles using biological entities such as plant extract and microorganisms arise as new green trend in nanotechnology for nanoparticle synthesis and applications [22]. Bionanosynthesis avoid the above discussed shortcomings associated with chemico-physical methods of nanosynthesis. The earlier reports on use of silver nanoparticles in cysteine detection cannot avoid use of toxic chemicals in silver nanoparticle preparation such as cetyltrimethyl ammonium bromide [23], sodium borohydride, tri-sodium citrate [24-– 25] and are suffer from long synthesis time, separation of uncapped chemicals from silver nanoparticles, potential environmental pollution, and toxicity to nontarget species [18]. Taking these points in consideration, use of eco-friendly synthesized silver nanoparticles in cysteine sensing can be more beneficial for development of clean nanoanlytical methods in cysteine detection. Calotropis procera (family Asclepidaceae) is used in Ayurvedic system of medicine for treatment of conditions such as migraine [26], cough [27], diarrhea, dysentery [28], and cancer
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[29]. Apart from common biochemicals like proteins, glycosides, flavonoids, and saponins, it contains unique metabolites such as calotropenol, proceragenin, and calotoxin [29–31]. These metabolites act as reducing and stabilizing agents in silver nanoparticle synthesis avoiding use of toxic chemicals. As a part of our ongoing work on development of metallic nanoparticles for chemosensing [18], the present report describes use of Calotropis procera leaf extract synthesized silver nanoparticles in cysteine detection for the first time. The biofunctionalized monodispersed silver nanoparticles are yellow in color, but in the presence of cysteine, the yellow color of silver nanoparticles change to pink because cysteine causes aggregation of silver nanoparticles by specific interaction of its –SH group with silver nanoparticles. Interaction of cysteine with silver nanoparticles is then analyzed colorimetrically and by transmission electron microscopy (TEM), dynamic light scattering (DLS), thin layer chromatography (TLC), and Fourier transform infrared (FT-IR) studies. The practical feasibility of present method was checked by detecting cysteine in biological fluids such as blood plasma. The proposed method is rapid, allowing 100 nM cysteine detection at room temperature and is free from use of toxic chemicals. The most important point is that end point using proposed approach is color change and aggregation which can be analyzed via naked eye (without use of costly instrument) by layman.
Experimental Chemicals All glassware used for experiment were washed with 3:1 HNO3/HCl solution, followed by rinsing with water and air drying. Silver nitrate and cysteine were purchased from SDFCL, India; different amino acids were obtained from HiMedia Limited, India. Acetonitrile was procured from Merck, Germany. Biosynthesis of Silver Nanoparticles from C. procera Leaf Extract Fresh leaves of C. procera were washed with deionized water. About 5 g leaves were crushed in mortar and pestle and diluted to 50 ml with deionized water (pH of solution 7.3). The mixture was centrifuged at 10,000 rpm for 10 min at room temperature. The supernatant was separated and used for silver nanoparticle synthesis as reducing and capping agents [18, 22]. Five milliliters of leaf extract was added to 50 ml of AgNO3 solution (1 mM) and incubated at room temperature with stirring at 100 rpm for 10 min. Solutions of AgNO3 and leaf extract were incubated at similar conditions as control. Characterization of Silver Nanoparticles Surface plasmon resonance of silver nanoparticles was analyzed on UV–vis spectrophotometer at wavelength range of 200–700 nm (Shimadzu 1601, Japan) [24, 25], and FT-IR analysis of C. procera leaf extract and silver nanoparticles were done on FT-IR (Shimadzu IR Prestige 21, Japan) to investigate the possible molecules involved in synthesis and stabilization of silver nanoparticles [19]. Morphology and topography of silver nanoparticles were estimated by TEM (Technai-20, Phillips, Netherland). Crystalline property of silver nanoparticles was studied using X-ray powder diffraction (XRD) analysis in 20 to 80 2θ angles, operating at
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40 kV and current of 30 mA (K=1.5406 Ǻ) at 0.045° min−1 continuous speed (Brucker D8 Advance, Germany) [23]. Stability and percent distribution of silver nanoparticles were analyzed using zeta potential (ZP) and dynamic light scattering (DLS) analysis (Zetasizer Nano S90, Malvern Instruments, USA). Sensitivity and Selectivity of Silver Nanoparticles to Cysteine Detection of Cysteine by Visual and Colorimetric Analysis Stock solution of cysteine (1,000 μM) was prepared in deionized water. Different dilutions of stock solution were made to obtain cysteine concentration ranging from 0.01 to 100 μM. Fifty milliliters of cysteine solution was added to 350 μl silver nanoparticles, followed by 15-min incubation. Presence of cysteine was visualized by naked eye observation of color change in silver nanoparticles, i.e., yellow to pink, and aggregation of colloidal silver nanoparticles after cysteine addition [24]. Concentration of cysteine was estimated by monitoring decrease in surface plasmon absorption wavelength of silver nanoparticles (421 nm) after cysteine addition using 1-cm path length cell in wavelength range of 300–700 nm (Shimadzu 1601, Japan). Selectivity of silver nanoparticles to cysteine was verified by adding other amino acid at the concentration of 10 μM while cysteine was added at concentration of 0.1 μM in silver nanoparticles in similar manner as discussed above. Detection of Cysteine in Biological Fluid—Blood Plasma Blood plasma was collected from three healthy adult male and diluted to 20-fold with water. Fifty microliters of blood plasma without cysteine (control) and blood plasma spiked with different concentration of cysteine (test) were allowed to react with 350 μl silver nanoparticles. Absorption spectrum of above mixture was recorded at 421 nm after 15-min incubation [24]. Investigation of Mechanism Behind Cysteine Detection TEM and DLS Analysis of Cysteine-Silver Nanoparticle Complex The aggregation of monodisperse silver nanoparticles after interaction with cysteine was analyzed on TEM (Technai-20, Phillips, Netherland). While change in size of silver nanoparticles after its interface with cysteine was studied by DLS analysis (Zetasizer Nano S90, Malvern Instruments, USA). FT-IR Analysis of Cysteine-Silver Nanoparticle Complex To get insight into mechanism of cysteine detection by silver nanoparticles, FT-IR analysis was performed. In FT-IR analysis, approximately 3 mg of cysteine and cysteine-silver nanoparticle complex were separately mixed with 300 mg KBr and transmission spectra were recorded at 400–4,000 cm−1 in FT-IR (Shimadzu, IR-prestige-21). Difference in wave number, appearance, and disappearance of peaks were analyzed. TLC Analysis of Cysteine-Silver Nanoparticle Complex A mobile phase of butanol/acetic acid/water (4:1:5) was allowed to saturate in beaker. Stationary phases consisted of silicacoated plates (Merck, Germany). Cysteine (control), silver nanoparticles (control), and cysteine + silver nanoparticles (test) were spotted (0.02 ml) separately on silica plate. The plate was air-dried and allowed to run with mobile phase and then spread with 1 % ninhydrin reagent [32]. Observation was made regarding appearance of purple color spot of cysteine in control and test sample.
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Results and Discussion Biosynthesis of Silver Nanoparticles Colorless solution of AgNO3 (1 mM) instantly transformed into yellow color after reaction with aqueous C. procera leaf extract (Fig. 1a). The acquisition of yellow color is an indication of silver nanoparticle fabrication and termed as surface plasmon absorbance, arise due to conduction of free electrons around nanosurface [18, 19]. It was observed that the yellow color of silver nanoparticles exist for period up to 4 months without aggregation, suggesting that C. procera metabolites such as proteins, flavonoids, and saponins, reduce and effectively stabilize the formed silver nanoparticles. The diagrammatic representation of silver nanoparticles synthesis is given in Fig. 1b. Characterizations of Silver Nanoparticles UV–vis Spectrophotometric Analysis of Silver Nanoparticles UV–vis spectrophotometry is widely used to confirm the nanoparticles synthesis by analyzing surface plasmon absorption [19, 24, 33, 34]. C. procera-synthesized silver nanoparticles exhibit sharp absorption peak having maximum absorption (λ max) at 421 nm, and the same surface plasmon absorption was used in colorimetric assay for cysteine. Progressive increase in absorption at 421 nm with increasing incubation time indicates catalytic reduction of silver salt to silver nanoparticles by plant metabolites (Fig. 2a) [18]. The control solutions, C. procera leaf extract and AgNO3 did not show λ max around 421 nm. The pH 6 was found to be optimum for silver nanoparticle synthesis as extreme acidic and alkaline condition changes
Fig. 1 a Photograph showing Eppendorf tubes containing 1 mM AgNO3 solution (1), 100 μM cysteine solution (2), aqueous leaf extract solution of C. procera (3), solution of silver nanoparticles (4), mixture of 0.1 μM cysteine + silver nanoparticles (5), mixture of 500 μM cysteine + silver nanoparticles (6). b Schematic diagram showing process of silver nanoparticles synthesis and cysteine detection
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Fig. 2 a UV–vis absorption spectra of silver nanoparticles, silver nitrate, and C. procera leaf extract solution. b Effect of pH and temperature on silver nanoparticle synthesis
charge of biomolecules, alter activity of enzymes resulting in no synthesis of silver nanoparticles or the synthesized silver nanoparticles are of not desired quality such as small size and stability. At 600 C, maximum synthesis of silver nanoparticles occurred as extreme lower and higher temperature may inactivate thermolabile species (Fig. 2b), thereby reducing yield and quality of silver nanoparticles [22]. TEM, DLS, and ZP Analyses of Silver Nanoparticles Monodispersed nanoparticles of silver with pseudospherical and triangular shape was observed in TEM analysis (Fig. 3a). A zeta potential of −22.4 mV pointed toward stability of silver nanoparticles (Fig. 3b). Most importantly, all nanoparticles displayed size below 100 nm. In DLS analysis, it was found that more than 80 % nanoparticles have size range between 7 and 15 nm. The results of DLS analysis comply with the TEM results of silver nanoparticles (Fig. 4a). XRD and FT-IR Analysis of Silver Nanoparticles X-ray diffraction analysis of silver nanoparticles give reflection planes at 38.11°, 44.30°, 64.48°, and 77.42°, respectively, corresponding to Bragg’s reflections of (111), (200), (220), and (311). Result of XRD confirmed face-centered cubic (fcc) crystal nature (Fig. 3c). The result of XRD analysis matches with that of standard database (JCPDS file no. 783). Some unassigned peaks (*) at 27.93° and 29.28° may be of leafmetabolites of C. procera, and as they are amorphous, they do not show any sharp peaks in the XRD [35]. FT-IR analysis of C. procera leaf extract and silver nanoparticles synthesized using the extract was performed to understand role of C. procera metabolites in nanosynthesis. Several peaks in spectrum of silver nanoparticles such as 732.97, 826.53, 1071.49, 1129.36, and 1632.60 cm−1 correspond to N–H functional groups of amines and amides [18]. Carboxylic acid functional group (RCO–OH) found in proteins and phenols were denoted by peaks at 987.59, 3121.34, 3386.15 cm−1. It was observed that the relative transmission of silver nanoparticles spectrum is lower as compared to spectrum of C. procera. Spectrum of C. procera explains presence functional groups like –N–H, −O–H, C–C, C=C, and C–O, denoting presence and involvement of variety of biomolecules like proteins, phenols, flavonoids, and saponins as reducing and stabilizing agents (Fig. 3d). Shifting of peaks in silver
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Fig. 3 Characterization of silver nanoparticles: a A representative TEM image of silver nanoparticles synthesized from C. procera leaf extract; b zeta potential, c XRD, and d FT-IR
Fig. 4 DLS analysis of silver nanoparticles a Silver nanoparticles without cysteine and b silver nanoparticles with cysteine
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nanoparticles from 1133.22 to 1129.36 cm−1, 1324.18 to 1382.04 cm−1, and 3365.90 to 3386.15 cm−1 was observed. Sensitivity and Selectivity of Silver Nanoparticles to Cysteine—Limit of Detection Yellow color monodispersed silver nanoparticles rapidly transformed into pink color within 5 min of cysteine addition at room temperature. Figure 1b describes the analytical process of cysteine detection using eco-friendly synthesized silver nanoparticles. Addition of increasing concentration of cysteine (0.10 to 1 μM) in silver nanoparticle solution resulted in gradual change of yellow color of silver nanoparticles to pink. Even, addition of cysteine at very low concentration (100 nM) in silver nanoparticles result in pink color which was easily distinguished from initial yellow color silver nanoparticles without cysteine (Figs. 1a and 5). Further increase in cysteine concentration (100 to 1,000 μM) cause rapid aggregation of silver nanoparticles, and nanoaggregates can be clearly visible; furthermore, there was no pink color formation at higher concentration (100 to 1,000 μM), i.e., yellow color of silver nanoparticles directly turned into colorless solution with visible nanoaggregates (Figs. 1a and 5). Surface plasmon absorption band is typical characteristic of silver nanoparticles used to study shape, size, and stability of nanoparticles [33, 34]. Cysteine-induced aggregation in silver nanoparticles was further analyzed by measuring surface plasmon absorption band using UV–vis spectrophotometer. It was observed that the absorption at surface plasmon wavelength of silver nanoparticles (421 nm) decreases linearly with increase in incubation time and cysteine concentration (Fig. 6a–d) indicating rapid aggregation of silver nanoparticles with increase in incubation and cysteine concentration. According to Li and Li [11], nanoparticles can bind cysteine molecules through the Au–S bond. The red shift in surface plasmon absorption wavelength from 421 to 434 nm occurred with increasing cysteine concentration from 0.1 to 1 μM (Fig. 6c). Red shift in surface plasmon absorption of nanoparticles is an indication of increasing size of nanoparticles and aggregation. The limit of detection (LOD) for cysteine using the present approach was as low as 0.10 μM (100 nM). The absorption spectra of cysteine did not show any absorbance due to spectroscopic inertness. Cysteine binds tightly to heavy metals like mercury and cadmium [36]. The monodispersed silver nanoparticles are yellow in color but after aggregation they acquire pink color. Silver nanoparticles aggregated in the presence of cysteine due to covalent interaction of –SH group
Fig. 5 a, b Photographs showing color change in silver nanoparticles from red to pink and aggregation of stable silver nanoparticles upon interaction with different concentrations of cysteine
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Fig. 6 Colorimetric analyses for cysteine detection using silver nanoparticles. a UV–vis spectra of silver nanoparticles and silver nanoparticles + cysteine. b Effect of time on UV–vis spectra and color change of silver nanoparticles after cysteine addition. c Effect of different concentrations of cysteine (0.1 to 10 μM) on UV–vis spectra of silver nanoparticles. d Linear relation between absorption at 421 nm and cysteine concentration
of cysteine with silver nanoparticles surface and via chemisorption type of interaction [11, 37–39]. The spectroscopic methods previously used for cysteine detection (Table 1) requires chromophore that can react with free –SH group and give colored end point because cysteine showed spectroscopic inertness. Earlier reports on cysteine detection using nanoparticles involved functionalization of nanoparticles with specific chemical entity such as sodium carboxymethyl cellulose [1] and cetyltrimethyl ammonium bromide [23]; current approach avoid above tedious protocol of ligand capping and are synthesized by single-step green synthesis protocol. Comparative account of different approaches used for cysteine detection using gold nanoparticles (AuNPs) and silver nanoparticles with respect to present approach is given in Table 1. In Table 1, it was observed that proposed methods has comparable LOD with earlier reports and in addition the proposed method has advantage of use of one step green synthesis which appeared more eco-friendly and biocompatible. Above features are not found in chemically synthesized and capped nanoparticles. Effects of Interfering Compounds Selectivity of silver nanoparticles to cysteine was examined by reacting silver nanoparticles with other amino acids (alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
Appl Biochem Biotechnol Table 1 Comparison of different methods used for cysteine detection using gold nanoparticles and silver nanoparticles Sr. Reducing and stabilizing agents used for silver nanoparticle LOD for Linearity no. (AgNP) and gold nanoparticle (AuNP) syntheses cysteine (μM) (μM)
References
1
Carboxymethyl cellulose (AuNPs)
[1]
2
Trisodium citrate, aspartic acid (AuNPs)
0.10
0.166–1.67 [38]
3
Single-stranded DNA (AuNPs)
0.10
0.1–5
[39]
4 5
Sodium borohydride, trisodium citrate (AgNPs) Calotropis procera leaves extract (AgNPs)
0.10 0.10
0.1–1,000 0.1–10
[24] Present work
40
10–100
threonine, tryptophan, tyrosine, valine) having concentration of 10 μM while cysteine was added at concentration of 0.1 μM. No change in yellow color of silver nanoparticles occurred after interaction with different amino acids except cysteine in which yellow color of silver nanoparticles changed to pink. Similarly, in colorimetric analysis, no significant decrease in surface plasmon absorption of silver nanoparticles happened after addition of different amino acids except in cysteine in which there is drastic decrease in surface plasmon absorption of silver nanoparticles. Methionine showed higher absorbance at 421 nm, but the absorbance was very small as compared to cysteine although the concentration of methionine was hundredfold more than cysteine. Visual and colorimetric analysis revealed selectivity of silver nanoparticles to cysteine over other amino acids (Figs. 7 and S1). Amino acids without thiol group cannot bind to silver nanoparticles under optimized sensing parameters [24]. Optimization of Sensing Parameter As the end point of current cysteine detection method using silver nanoparticles is color change and nanoparticle aggregation, therefore, pH of media plays a vital role. It was found that at extreme acidic and alkaline pH silver nanoparticles get aggregated without cysteine, and the yellow color of silver nanoparticles was also get faded with lower absorbance at 421 nm. These conditions are detrimental for detection purpose; therefore, pH 6 was found to be optimum for stable silver nanoparticles and cysteine detection. We also studied the interaction time optimum for color change in silver nanoparticles after cysteine addition and observed that intensity of pink color reaches to maximum around 15-min interaction time. After that, the reaction mixture tends to aggregate with change in color from pink to colorless (Fig. 6b). The
Fig. 7 Effect of other amino acids on cysteine detection by silver nanoparticles. a UV–vis spectra of silver nanoparticles + other amino acid at concentration of 10 μM while cysteine was of concentration of 0.1 μM
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Fig. 8 Representative result of detection of cysteine in blood plasma by UV–vis spectrophotometric analysis
binding of nanoparticles to cysteine is very fast [40, 41]. Most of the cysteine molecules present in reaction mixture capped on silver nanoparticles surface around 15 min, resulting in transformation of yellow color silver nanoparticles to pink color. Further increase in incubation time creates more nanoaggregates, resulting colorless solution with visual aggregates (Figs. 1b and 5). Detection of Cysteine in Biological Fluid—Blood Plasma Silver nanoparticle sensor exhibits good selectivity on spiking of 50 μM cysteine in blood plasma as reflected by color change in silver nanoparticles from yellow to pink and drastic decrease in surface plasmon absorption (Fig. 8). Recovery experiments were performed by spiking 25, 50, and 100 μM cysteine, and the recovery was in the range of 85–95 % (Table 2). The formula used for calculation of cysteine recovery is a s follows: Recovery ð%Þ ¼ ðcalculated cysteine = added cysteineÞ 100 % Matrix Effect To study the effect of other constituents of blood plasma on cysteine detection (matrix effect), plasma without cysteine spiking was added in silver nanoparticles. No significant difference in color, surface plasmon absorption, and stability of silver nanoparticles was observed after reaction of plasma with silver nanoparticles, suggesting compatibility of silver nanoparticles to Table 2 Recovery results for cysteine in blood plasma Sr. no.
Actual conc. (μM)
Detected conc. (μM)
Recovery (RSD±SD), n=3
1
25
21.33±0.44
85.3 %
2
50
46.31±1.11
92.6 %
3
100
95.33±2.22
95.3 %
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these biological fluids (Fig. 8). Earlier reports also suggest compatibility of nanoparticles to various biofluids [18, 42]. Investigation of Mechanism Behind Cysteine Detection TEM and DLS Analysis of Cysteine-Silver Nanoparticle Complex In TEM micrograph, silver nanoparticles in absence of cysteine are monodispersed with average particle size of 10 nm. After cysteine-silver nanoparticle interaction, silver nanoparticle aggregation takes place with increase in silver nanoparticle size (Fig. 9). DLS measurement also proved cysteine-induced aggregation in silver nanoparticles. In DLS, the average particle size of silver nanoparticles was found to be 10 nm (Fig. 4a). After cysteine-silver nanoparticle interaction, the size of silver nanoparticles was abruptly increased to 79 nm (Fig. 4b). Results of TEM and DLS analyses comply with the results obtained on UV–vis spectroscopy, showing decreased surface plasmon absorption due to silver nanoparticle aggregation and change in yellow color of silver nanoparticles to pink after cysteine addition (Figs. 1 and 4). Change in zeta potential of silver nanoparticles from −22.4 to −3.16 mV indicates less stability of cysteine-silver nanoparticle complex as compared to silver nanoparticles without cysteine (data not shown). Different parameters were verified during cysteinesilver nanoparticles interaction like color, size, and aggregation as tabulated in S2. The reason behind cysteine induced silver nanoparticles aggregation is binding of cysteine onto silver nanoparticles surface making silver nanoparticles prone to aggregation and acquisition of pink color. FT-IR Analysis of Cysteine-Silver Nanoparticle Complex Binding of cysteine on silver nanoparticles surface was analyzed by FT-IR analysis (Fig. 10). The spectrum of cysteine showed bands at 1059.92, 1209.41, and 1,609 cm−1 correspond to the asymmetric and symmetric stretching of COO−. Bands at 1136.11, 1,514, and 3403.51 cm−1 correspond to N–H group. Weak band near 2,550 cm−1 denotes S–H group present in cysteine molecule [37]. After binding of cysteine on silver nanoparticles surface, shifting of bands in the position of carboxyl and amino groups is observed due to a change in dipole moment when cysteine binds on metal surface with high electron density [37].
Fig. 9 A representative TEM image of a silver nanoparticles without cysteine and b cysteine-silver nanoparticle complex
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Fig. 10 FT-IR of a cysteine and b silver nanoparticles + cysteine
Interestingly, S–H band disappeared in the spectra of cysteine-silver nanoparticles confirming S-silver nanoparticle interaction. TLC Analysis of Cysteine-Silver Nanoparticle Complex In TLC, cysteine exhibit purple color spot with Rf 0.40. However, spot of cysteine was not observed in cysteine-silver nanoparticle complex due to attachment of cysteine on nanosurface, and due to attachment, no free groups was available to interact with ninhydrin reagent (S3).
Conclusion In the present investigation, stable, small-sized silver nanoparticles were prepared by ecofriendly synthesis protocol and utilized for cysteine detection. The end point of present assay is simple color change or UV–vis spectroscopic analysis. Due to specific and strong interaction between SH group of cysteine with silver nanoparticles, there was drastic decrease in surface plasmon absorption, change in color, and aggregation in silver nanoparticles. Under optimized parameters, the present system has potential to detect as low as 100 nM of cysteine and is nearly nonresponsive to other amino acids. Moreover, cysteine detection in biological fluids can be done using present system. Apart from green synthesis protocol, the proposed cysteine sensor has some benefits like low cost, on site application. The findings of the current study suggest that nanoparticles may potentially be used to quantify other physicochemical parameters (e.g., chaotropicity) and other substances such as microbial toxins, pesticide residues, and food contamination. Acknowledgments Present research was supported by the Department of Science and Technology, Government of India, under the DST INSPIRE Ph.D. fellowship to Mr. Hemant P. Borase. Authors are thankful to anonymous reviewers for critical evaluation of the paper.
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Bio-Functionalized Silver Nanoparticles: a Novel Colorimetric Probe for Cysteine Detection Hemant P. Borase & Chandrashekhar D. Patil & Rahul B. Salunkhe & Rahul K. Suryawanshi & Beom S. Kim & Vishwas A. Bapat & Satish V. Patil
Received: 16 September 2014 / Accepted: 21 January 2015 # Springer Science+Business Media New York 2015
Abstract Chemical interactions between nanoparticles and biomolecules are vital for applying nanoparticles in medicine and life science. Development of sensitive, rapid, low-cost, and eco-friendly sensors for the detection of molecules acting as disease indicator is need of an hour. In the present investigation, a green trend for silver nanoparticle synthesis was followed using leaf extract of Calotropis procera. Silver nanoparticles exhibited surface plasmon absorption peak at 421 nm, spherical shape with average size of 10 nm, and zeta potential of −22.4 mV. The as-synthesized silver nanoparticles were used for selective and sensitive detection of cysteine. Cysteine induces aggregation in stable silver nanoparticles owing to selective and strong interaction of –SH group of cysteine with silver nanoparticle surface. Cysteine-induced silver nanoparticle aggregation can be observed visually by change in color of silver nanoparticles from yellow to pink. Cysteine concentration was estimated colorimetrically by measuring absorption at surface plasmon wavelength. Limit of detection for cysteine using silver nanoparticles is ultralow, i.e., 100 nM. The mechanistic insight into cysteine detection by silver nanoparticles was investigated using FT-IR, TEM, DLS, and TLC analysis. Proposed method can be applied for the detection of cysteine in blood plasma and may give rise to a new insight into development of eco-friendly fabricated nanodiagnostic device in future.
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1519-0) contains supplementary material, which is available to authorized users.
H. P. Borase : C. D. Patil : R. B. Salunkhe : R. K. Suryawanshi : S. V. Patil School of Life Sciences, North Maharashtra University, Post Box 80, Jalgaon 425001 Maharashtra, India B. S. Kim Department of Chemical Engineering, Chungbuk National University, Chungbuk 361-763, Cheongju, Republic of Korea V. A. Bapat Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur 416004 Maharashtra, India S. V. Patil (*) North Maharashtra Microbial Culture Collection Centre (NMCC), North Maharashtra University, Post Box 80, Jalgaon 425001 Maharashtra, India e-mail: [email protected]
Appl Biochem Biotechnol
Keywords Silver nanoparticles . Cysteine . Colorimetric sensor . FT-IR . Blood plasma
Introduction Cysteine, a –SH group containing α-amino acid having molar mass of 121.16 g mol−1 play a vital role in physiological processes like protein folding and metabolism, posttranslational modification, detoxification, and affinity for heavy metals [1–3]. Cysteine has an important role in physiological developments of herbivores and plants [4–6]. Apart from its role in cellular processes, cysteine is a biomarker for many pathological ailments such as liver damage, heart diseases, skin lesions, hair depigmentation, sepsis, AIDS, cystinuria, and Parkinson’s and Alzheimer’s diseases [3, 7–9]. Recently, cysteine was researched regarding its role in obesity [10]. Cysteine is widely used in pharma products like antibiotics and skin treatment formulations [11]. Considering above multiple roles of cysteine, its analysis and detection in biological and pharmaceutical samples was attempted by devising different methods using HPLC [12], fluorescence spectroscopy [13], capillary electrophoresis [14], and flow injection [15]. The abovementioned methods can detect low levels of cysteine, but they are expensive, time consuming, required skilled labor, and are of limited on-field application. With above background, there is an urgent need to develop simple, rapid, selective, and low-cost method of cysteine detection. Nanotechnology is now arising as a promising tool in new generation disease diagnosis system that has been termed Bnanodiagnostics^ [16, 17]. Silver nanoparticles are gaining worldwide attention as sensors for molecules that have medical and environmental relevance such as mercury [18], tricyclazole [19], melamine [20], and glucose [21]. The characteristics such as surface plasmon resonance, chemical stability, biocompatibility, ease of preparation, low cost, easy end point detection, and high sensitivity and selectivity makes silver nanoparticles a potent alternative to currently used approaches in chemosensing and disease diagnosis. Moreover, the on-field detection of molecules is possible with use of silver nanoparticles. Physicochemical methods currently used for silver nanoparticle synthesis and their use in chemosensing have several disadvantages like use of toxic chemicals in synthesis, harsh synthesis parameters, prone to aggregation, tedious ligand capping process, and concern for the use in biomedical applications [18, 19]. Vital goal of nanosensor technology is eco-friendly production of nanodevices with low environmental pollution and toxicity to nontargets. Then, how chemically synthesized nanodevices will answer above important question? Moreover, apart from application of nanoparticles, the route of nanosynthesis is also important. The synthesis of silver nanoparticles using biological entities such as plant extract and microorganisms arise as new green trend in nanotechnology for nanoparticle synthesis and applications [22]. Bionanosynthesis avoid the above discussed shortcomings associated with chemico-physical methods of nanosynthesis. The earlier reports on use of silver nanoparticles in cysteine detection cannot avoid use of toxic chemicals in silver nanoparticle preparation such as cetyltrimethyl ammonium bromide [23], sodium borohydride, tri-sodium citrate [24-– 25] and are suffer from long synthesis time, separation of uncapped chemicals from silver nanoparticles, potential environmental pollution, and toxicity to nontarget species [18]. Taking these points in consideration, use of eco-friendly synthesized silver nanoparticles in cysteine sensing can be more beneficial for development of clean nanoanlytical methods in cysteine detection. Calotropis procera (family Asclepidaceae) is used in Ayurvedic system of medicine for treatment of conditions such as migraine [26], cough [27], diarrhea, dysentery [28], and cancer
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[29]. Apart from common biochemicals like proteins, glycosides, flavonoids, and saponins, it contains unique metabolites such as calotropenol, proceragenin, and calotoxin [29–31]. These metabolites act as reducing and stabilizing agents in silver nanoparticle synthesis avoiding use of toxic chemicals. As a part of our ongoing work on development of metallic nanoparticles for chemosensing [18], the present report describes use of Calotropis procera leaf extract synthesized silver nanoparticles in cysteine detection for the first time. The biofunctionalized monodispersed silver nanoparticles are yellow in color, but in the presence of cysteine, the yellow color of silver nanoparticles change to pink because cysteine causes aggregation of silver nanoparticles by specific interaction of its –SH group with silver nanoparticles. Interaction of cysteine with silver nanoparticles is then analyzed colorimetrically and by transmission electron microscopy (TEM), dynamic light scattering (DLS), thin layer chromatography (TLC), and Fourier transform infrared (FT-IR) studies. The practical feasibility of present method was checked by detecting cysteine in biological fluids such as blood plasma. The proposed method is rapid, allowing 100 nM cysteine detection at room temperature and is free from use of toxic chemicals. The most important point is that end point using proposed approach is color change and aggregation which can be analyzed via naked eye (without use of costly instrument) by layman.
Experimental Chemicals All glassware used for experiment were washed with 3:1 HNO3/HCl solution, followed by rinsing with water and air drying. Silver nitrate and cysteine were purchased from SDFCL, India; different amino acids were obtained from HiMedia Limited, India. Acetonitrile was procured from Merck, Germany. Biosynthesis of Silver Nanoparticles from C. procera Leaf Extract Fresh leaves of C. procera were washed with deionized water. About 5 g leaves were crushed in mortar and pestle and diluted to 50 ml with deionized water (pH of solution 7.3). The mixture was centrifuged at 10,000 rpm for 10 min at room temperature. The supernatant was separated and used for silver nanoparticle synthesis as reducing and capping agents [18, 22]. Five milliliters of leaf extract was added to 50 ml of AgNO3 solution (1 mM) and incubated at room temperature with stirring at 100 rpm for 10 min. Solutions of AgNO3 and leaf extract were incubated at similar conditions as control. Characterization of Silver Nanoparticles Surface plasmon resonance of silver nanoparticles was analyzed on UV–vis spectrophotometer at wavelength range of 200–700 nm (Shimadzu 1601, Japan) [24, 25], and FT-IR analysis of C. procera leaf extract and silver nanoparticles were done on FT-IR (Shimadzu IR Prestige 21, Japan) to investigate the possible molecules involved in synthesis and stabilization of silver nanoparticles [19]. Morphology and topography of silver nanoparticles were estimated by TEM (Technai-20, Phillips, Netherland). Crystalline property of silver nanoparticles was studied using X-ray powder diffraction (XRD) analysis in 20 to 80 2θ angles, operating at
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40 kV and current of 30 mA (K=1.5406 Ǻ) at 0.045° min−1 continuous speed (Brucker D8 Advance, Germany) [23]. Stability and percent distribution of silver nanoparticles were analyzed using zeta potential (ZP) and dynamic light scattering (DLS) analysis (Zetasizer Nano S90, Malvern Instruments, USA). Sensitivity and Selectivity of Silver Nanoparticles to Cysteine Detection of Cysteine by Visual and Colorimetric Analysis Stock solution of cysteine (1,000 μM) was prepared in deionized water. Different dilutions of stock solution were made to obtain cysteine concentration ranging from 0.01 to 100 μM. Fifty milliliters of cysteine solution was added to 350 μl silver nanoparticles, followed by 15-min incubation. Presence of cysteine was visualized by naked eye observation of color change in silver nanoparticles, i.e., yellow to pink, and aggregation of colloidal silver nanoparticles after cysteine addition [24]. Concentration of cysteine was estimated by monitoring decrease in surface plasmon absorption wavelength of silver nanoparticles (421 nm) after cysteine addition using 1-cm path length cell in wavelength range of 300–700 nm (Shimadzu 1601, Japan). Selectivity of silver nanoparticles to cysteine was verified by adding other amino acid at the concentration of 10 μM while cysteine was added at concentration of 0.1 μM in silver nanoparticles in similar manner as discussed above. Detection of Cysteine in Biological Fluid—Blood Plasma Blood plasma was collected from three healthy adult male and diluted to 20-fold with water. Fifty microliters of blood plasma without cysteine (control) and blood plasma spiked with different concentration of cysteine (test) were allowed to react with 350 μl silver nanoparticles. Absorption spectrum of above mixture was recorded at 421 nm after 15-min incubation [24]. Investigation of Mechanism Behind Cysteine Detection TEM and DLS Analysis of Cysteine-Silver Nanoparticle Complex The aggregation of monodisperse silver nanoparticles after interaction with cysteine was analyzed on TEM (Technai-20, Phillips, Netherland). While change in size of silver nanoparticles after its interface with cysteine was studied by DLS analysis (Zetasizer Nano S90, Malvern Instruments, USA). FT-IR Analysis of Cysteine-Silver Nanoparticle Complex To get insight into mechanism of cysteine detection by silver nanoparticles, FT-IR analysis was performed. In FT-IR analysis, approximately 3 mg of cysteine and cysteine-silver nanoparticle complex were separately mixed with 300 mg KBr and transmission spectra were recorded at 400–4,000 cm−1 in FT-IR (Shimadzu, IR-prestige-21). Difference in wave number, appearance, and disappearance of peaks were analyzed. TLC Analysis of Cysteine-Silver Nanoparticle Complex A mobile phase of butanol/acetic acid/water (4:1:5) was allowed to saturate in beaker. Stationary phases consisted of silicacoated plates (Merck, Germany). Cysteine (control), silver nanoparticles (control), and cysteine + silver nanoparticles (test) were spotted (0.02 ml) separately on silica plate. The plate was air-dried and allowed to run with mobile phase and then spread with 1 % ninhydrin reagent [32]. Observation was made regarding appearance of purple color spot of cysteine in control and test sample.
Appl Biochem Biotechnol
Results and Discussion Biosynthesis of Silver Nanoparticles Colorless solution of AgNO3 (1 mM) instantly transformed into yellow color after reaction with aqueous C. procera leaf extract (Fig. 1a). The acquisition of yellow color is an indication of silver nanoparticle fabrication and termed as surface plasmon absorbance, arise due to conduction of free electrons around nanosurface [18, 19]. It was observed that the yellow color of silver nanoparticles exist for period up to 4 months without aggregation, suggesting that C. procera metabolites such as proteins, flavonoids, and saponins, reduce and effectively stabilize the formed silver nanoparticles. The diagrammatic representation of silver nanoparticles synthesis is given in Fig. 1b. Characterizations of Silver Nanoparticles UV–vis Spectrophotometric Analysis of Silver Nanoparticles UV–vis spectrophotometry is widely used to confirm the nanoparticles synthesis by analyzing surface plasmon absorption [19, 24, 33, 34]. C. procera-synthesized silver nanoparticles exhibit sharp absorption peak having maximum absorption (λ max) at 421 nm, and the same surface plasmon absorption was used in colorimetric assay for cysteine. Progressive increase in absorption at 421 nm with increasing incubation time indicates catalytic reduction of silver salt to silver nanoparticles by plant metabolites (Fig. 2a) [18]. The control solutions, C. procera leaf extract and AgNO3 did not show λ max around 421 nm. The pH 6 was found to be optimum for silver nanoparticle synthesis as extreme acidic and alkaline condition changes
Fig. 1 a Photograph showing Eppendorf tubes containing 1 mM AgNO3 solution (1), 100 μM cysteine solution (2), aqueous leaf extract solution of C. procera (3), solution of silver nanoparticles (4), mixture of 0.1 μM cysteine + silver nanoparticles (5), mixture of 500 μM cysteine + silver nanoparticles (6). b Schematic diagram showing process of silver nanoparticles synthesis and cysteine detection
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Fig. 2 a UV–vis absorption spectra of silver nanoparticles, silver nitrate, and C. procera leaf extract solution. b Effect of pH and temperature on silver nanoparticle synthesis
charge of biomolecules, alter activity of enzymes resulting in no synthesis of silver nanoparticles or the synthesized silver nanoparticles are of not desired quality such as small size and stability. At 600 C, maximum synthesis of silver nanoparticles occurred as extreme lower and higher temperature may inactivate thermolabile species (Fig. 2b), thereby reducing yield and quality of silver nanoparticles [22]. TEM, DLS, and ZP Analyses of Silver Nanoparticles Monodispersed nanoparticles of silver with pseudospherical and triangular shape was observed in TEM analysis (Fig. 3a). A zeta potential of −22.4 mV pointed toward stability of silver nanoparticles (Fig. 3b). Most importantly, all nanoparticles displayed size below 100 nm. In DLS analysis, it was found that more than 80 % nanoparticles have size range between 7 and 15 nm. The results of DLS analysis comply with the TEM results of silver nanoparticles (Fig. 4a). XRD and FT-IR Analysis of Silver Nanoparticles X-ray diffraction analysis of silver nanoparticles give reflection planes at 38.11°, 44.30°, 64.48°, and 77.42°, respectively, corresponding to Bragg’s reflections of (111), (200), (220), and (311). Result of XRD confirmed face-centered cubic (fcc) crystal nature (Fig. 3c). The result of XRD analysis matches with that of standard database (JCPDS file no. 783). Some unassigned peaks (*) at 27.93° and 29.28° may be of leafmetabolites of C. procera, and as they are amorphous, they do not show any sharp peaks in the XRD [35]. FT-IR analysis of C. procera leaf extract and silver nanoparticles synthesized using the extract was performed to understand role of C. procera metabolites in nanosynthesis. Several peaks in spectrum of silver nanoparticles such as 732.97, 826.53, 1071.49, 1129.36, and 1632.60 cm−1 correspond to N–H functional groups of amines and amides [18]. Carboxylic acid functional group (RCO–OH) found in proteins and phenols were denoted by peaks at 987.59, 3121.34, 3386.15 cm−1. It was observed that the relative transmission of silver nanoparticles spectrum is lower as compared to spectrum of C. procera. Spectrum of C. procera explains presence functional groups like –N–H, −O–H, C–C, C=C, and C–O, denoting presence and involvement of variety of biomolecules like proteins, phenols, flavonoids, and saponins as reducing and stabilizing agents (Fig. 3d). Shifting of peaks in silver
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Fig. 3 Characterization of silver nanoparticles: a A representative TEM image of silver nanoparticles synthesized from C. procera leaf extract; b zeta potential, c XRD, and d FT-IR
Fig. 4 DLS analysis of silver nanoparticles a Silver nanoparticles without cysteine and b silver nanoparticles with cysteine
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nanoparticles from 1133.22 to 1129.36 cm−1, 1324.18 to 1382.04 cm−1, and 3365.90 to 3386.15 cm−1 was observed. Sensitivity and Selectivity of Silver Nanoparticles to Cysteine—Limit of Detection Yellow color monodispersed silver nanoparticles rapidly transformed into pink color within 5 min of cysteine addition at room temperature. Figure 1b describes the analytical process of cysteine detection using eco-friendly synthesized silver nanoparticles. Addition of increasing concentration of cysteine (0.10 to 1 μM) in silver nanoparticle solution resulted in gradual change of yellow color of silver nanoparticles to pink. Even, addition of cysteine at very low concentration (100 nM) in silver nanoparticles result in pink color which was easily distinguished from initial yellow color silver nanoparticles without cysteine (Figs. 1a and 5). Further increase in cysteine concentration (100 to 1,000 μM) cause rapid aggregation of silver nanoparticles, and nanoaggregates can be clearly visible; furthermore, there was no pink color formation at higher concentration (100 to 1,000 μM), i.e., yellow color of silver nanoparticles directly turned into colorless solution with visible nanoaggregates (Figs. 1a and 5). Surface plasmon absorption band is typical characteristic of silver nanoparticles used to study shape, size, and stability of nanoparticles [33, 34]. Cysteine-induced aggregation in silver nanoparticles was further analyzed by measuring surface plasmon absorption band using UV–vis spectrophotometer. It was observed that the absorption at surface plasmon wavelength of silver nanoparticles (421 nm) decreases linearly with increase in incubation time and cysteine concentration (Fig. 6a–d) indicating rapid aggregation of silver nanoparticles with increase in incubation and cysteine concentration. According to Li and Li [11], nanoparticles can bind cysteine molecules through the Au–S bond. The red shift in surface plasmon absorption wavelength from 421 to 434 nm occurred with increasing cysteine concentration from 0.1 to 1 μM (Fig. 6c). Red shift in surface plasmon absorption of nanoparticles is an indication of increasing size of nanoparticles and aggregation. The limit of detection (LOD) for cysteine using the present approach was as low as 0.10 μM (100 nM). The absorption spectra of cysteine did not show any absorbance due to spectroscopic inertness. Cysteine binds tightly to heavy metals like mercury and cadmium [36]. The monodispersed silver nanoparticles are yellow in color but after aggregation they acquire pink color. Silver nanoparticles aggregated in the presence of cysteine due to covalent interaction of –SH group
Fig. 5 a, b Photographs showing color change in silver nanoparticles from red to pink and aggregation of stable silver nanoparticles upon interaction with different concentrations of cysteine
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Fig. 6 Colorimetric analyses for cysteine detection using silver nanoparticles. a UV–vis spectra of silver nanoparticles and silver nanoparticles + cysteine. b Effect of time on UV–vis spectra and color change of silver nanoparticles after cysteine addition. c Effect of different concentrations of cysteine (0.1 to 10 μM) on UV–vis spectra of silver nanoparticles. d Linear relation between absorption at 421 nm and cysteine concentration
of cysteine with silver nanoparticles surface and via chemisorption type of interaction [11, 37–39]. The spectroscopic methods previously used for cysteine detection (Table 1) requires chromophore that can react with free –SH group and give colored end point because cysteine showed spectroscopic inertness. Earlier reports on cysteine detection using nanoparticles involved functionalization of nanoparticles with specific chemical entity such as sodium carboxymethyl cellulose [1] and cetyltrimethyl ammonium bromide [23]; current approach avoid above tedious protocol of ligand capping and are synthesized by single-step green synthesis protocol. Comparative account of different approaches used for cysteine detection using gold nanoparticles (AuNPs) and silver nanoparticles with respect to present approach is given in Table 1. In Table 1, it was observed that proposed methods has comparable LOD with earlier reports and in addition the proposed method has advantage of use of one step green synthesis which appeared more eco-friendly and biocompatible. Above features are not found in chemically synthesized and capped nanoparticles. Effects of Interfering Compounds Selectivity of silver nanoparticles to cysteine was examined by reacting silver nanoparticles with other amino acids (alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
Appl Biochem Biotechnol Table 1 Comparison of different methods used for cysteine detection using gold nanoparticles and silver nanoparticles Sr. Reducing and stabilizing agents used for silver nanoparticle LOD for Linearity no. (AgNP) and gold nanoparticle (AuNP) syntheses cysteine (μM) (μM)
References
1
Carboxymethyl cellulose (AuNPs)
[1]
2
Trisodium citrate, aspartic acid (AuNPs)
0.10
0.166–1.67 [38]
3
Single-stranded DNA (AuNPs)
0.10
0.1–5
[39]
4 5
Sodium borohydride, trisodium citrate (AgNPs) Calotropis procera leaves extract (AgNPs)
0.10 0.10
0.1–1,000 0.1–10
[24] Present work
40
10–100
threonine, tryptophan, tyrosine, valine) having concentration of 10 μM while cysteine was added at concentration of 0.1 μM. No change in yellow color of silver nanoparticles occurred after interaction with different amino acids except cysteine in which yellow color of silver nanoparticles changed to pink. Similarly, in colorimetric analysis, no significant decrease in surface plasmon absorption of silver nanoparticles happened after addition of different amino acids except in cysteine in which there is drastic decrease in surface plasmon absorption of silver nanoparticles. Methionine showed higher absorbance at 421 nm, but the absorbance was very small as compared to cysteine although the concentration of methionine was hundredfold more than cysteine. Visual and colorimetric analysis revealed selectivity of silver nanoparticles to cysteine over other amino acids (Figs. 7 and S1). Amino acids without thiol group cannot bind to silver nanoparticles under optimized sensing parameters [24]. Optimization of Sensing Parameter As the end point of current cysteine detection method using silver nanoparticles is color change and nanoparticle aggregation, therefore, pH of media plays a vital role. It was found that at extreme acidic and alkaline pH silver nanoparticles get aggregated without cysteine, and the yellow color of silver nanoparticles was also get faded with lower absorbance at 421 nm. These conditions are detrimental for detection purpose; therefore, pH 6 was found to be optimum for stable silver nanoparticles and cysteine detection. We also studied the interaction time optimum for color change in silver nanoparticles after cysteine addition and observed that intensity of pink color reaches to maximum around 15-min interaction time. After that, the reaction mixture tends to aggregate with change in color from pink to colorless (Fig. 6b). The
Fig. 7 Effect of other amino acids on cysteine detection by silver nanoparticles. a UV–vis spectra of silver nanoparticles + other amino acid at concentration of 10 μM while cysteine was of concentration of 0.1 μM
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Fig. 8 Representative result of detection of cysteine in blood plasma by UV–vis spectrophotometric analysis
binding of nanoparticles to cysteine is very fast [40, 41]. Most of the cysteine molecules present in reaction mixture capped on silver nanoparticles surface around 15 min, resulting in transformation of yellow color silver nanoparticles to pink color. Further increase in incubation time creates more nanoaggregates, resulting colorless solution with visual aggregates (Figs. 1b and 5). Detection of Cysteine in Biological Fluid—Blood Plasma Silver nanoparticle sensor exhibits good selectivity on spiking of 50 μM cysteine in blood plasma as reflected by color change in silver nanoparticles from yellow to pink and drastic decrease in surface plasmon absorption (Fig. 8). Recovery experiments were performed by spiking 25, 50, and 100 μM cysteine, and the recovery was in the range of 85–95 % (Table 2). The formula used for calculation of cysteine recovery is a s follows: Recovery ð%Þ ¼ ðcalculated cysteine = added cysteineÞ 100 % Matrix Effect To study the effect of other constituents of blood plasma on cysteine detection (matrix effect), plasma without cysteine spiking was added in silver nanoparticles. No significant difference in color, surface plasmon absorption, and stability of silver nanoparticles was observed after reaction of plasma with silver nanoparticles, suggesting compatibility of silver nanoparticles to Table 2 Recovery results for cysteine in blood plasma Sr. no.
Actual conc. (μM)
Detected conc. (μM)
Recovery (RSD±SD), n=3
1
25
21.33±0.44
85.3 %
2
50
46.31±1.11
92.6 %
3
100
95.33±2.22
95.3 %
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these biological fluids (Fig. 8). Earlier reports also suggest compatibility of nanoparticles to various biofluids [18, 42]. Investigation of Mechanism Behind Cysteine Detection TEM and DLS Analysis of Cysteine-Silver Nanoparticle Complex In TEM micrograph, silver nanoparticles in absence of cysteine are monodispersed with average particle size of 10 nm. After cysteine-silver nanoparticle interaction, silver nanoparticle aggregation takes place with increase in silver nanoparticle size (Fig. 9). DLS measurement also proved cysteine-induced aggregation in silver nanoparticles. In DLS, the average particle size of silver nanoparticles was found to be 10 nm (Fig. 4a). After cysteine-silver nanoparticle interaction, the size of silver nanoparticles was abruptly increased to 79 nm (Fig. 4b). Results of TEM and DLS analyses comply with the results obtained on UV–vis spectroscopy, showing decreased surface plasmon absorption due to silver nanoparticle aggregation and change in yellow color of silver nanoparticles to pink after cysteine addition (Figs. 1 and 4). Change in zeta potential of silver nanoparticles from −22.4 to −3.16 mV indicates less stability of cysteine-silver nanoparticle complex as compared to silver nanoparticles without cysteine (data not shown). Different parameters were verified during cysteinesilver nanoparticles interaction like color, size, and aggregation as tabulated in S2. The reason behind cysteine induced silver nanoparticles aggregation is binding of cysteine onto silver nanoparticles surface making silver nanoparticles prone to aggregation and acquisition of pink color. FT-IR Analysis of Cysteine-Silver Nanoparticle Complex Binding of cysteine on silver nanoparticles surface was analyzed by FT-IR analysis (Fig. 10). The spectrum of cysteine showed bands at 1059.92, 1209.41, and 1,609 cm−1 correspond to the asymmetric and symmetric stretching of COO−. Bands at 1136.11, 1,514, and 3403.51 cm−1 correspond to N–H group. Weak band near 2,550 cm−1 denotes S–H group present in cysteine molecule [37]. After binding of cysteine on silver nanoparticles surface, shifting of bands in the position of carboxyl and amino groups is observed due to a change in dipole moment when cysteine binds on metal surface with high electron density [37].
Fig. 9 A representative TEM image of a silver nanoparticles without cysteine and b cysteine-silver nanoparticle complex
Appl Biochem Biotechnol
Fig. 10 FT-IR of a cysteine and b silver nanoparticles + cysteine
Interestingly, S–H band disappeared in the spectra of cysteine-silver nanoparticles confirming S-silver nanoparticle interaction. TLC Analysis of Cysteine-Silver Nanoparticle Complex In TLC, cysteine exhibit purple color spot with Rf 0.40. However, spot of cysteine was not observed in cysteine-silver nanoparticle complex due to attachment of cysteine on nanosurface, and due to attachment, no free groups was available to interact with ninhydrin reagent (S3).
Conclusion In the present investigation, stable, small-sized silver nanoparticles were prepared by ecofriendly synthesis protocol and utilized for cysteine detection. The end point of present assay is simple color change or UV–vis spectroscopic analysis. Due to specific and strong interaction between SH group of cysteine with silver nanoparticles, there was drastic decrease in surface plasmon absorption, change in color, and aggregation in silver nanoparticles. Under optimized parameters, the present system has potential to detect as low as 100 nM of cysteine and is nearly nonresponsive to other amino acids. Moreover, cysteine detection in biological fluids can be done using present system. Apart from green synthesis protocol, the proposed cysteine sensor has some benefits like low cost, on site application. The findings of the current study suggest that nanoparticles may potentially be used to quantify other physicochemical parameters (e.g., chaotropicity) and other substances such as microbial toxins, pesticide residues, and food contamination. Acknowledgments Present research was supported by the Department of Science and Technology, Government of India, under the DST INSPIRE Ph.D. fellowship to Mr. Hemant P. Borase. Authors are thankful to anonymous reviewers for critical evaluation of the paper.
Appl Biochem Biotechnol
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