Rapid naked eye detection of arginine by pomegranate peel extract stabilized gold nanoparticles

2.1 Synthesis of gold nanoparticles

Polydispersed, spherical, flower and urchin shaped gold nanoparticles were synthesized by a method already reported by our research group (Kaviya and Prasad, 2015). In brief, the aqueous solution of HAuCl₄ (10 mL) was made in diverse concentration series from 10⁻² M to 10⁻⁵ M. 200 µL of aqueous pomegranate peel extract was mixed to individual precursor solution. After the incorporation of extract, the solution was mixed by simple shaking. The as-prepared AuNPs are represented as: AuNP1 (10⁻² M), AuNP2 (10⁻³ M), AuNP3 (10⁻⁴ M) and AuNP4 (10⁻⁵ M). The synthesized NPs were used for characterization. The detailed characterization has already been published elsewhere (Kaviya and Prasad, 2015). The reproduced results are given in Fig. S1.

2.2 Colorimetric analyse

Colorimetric finding experiments in presence of several amino acids such as L, β and dl-alanine (Ala), aspartic acid (Asp), arginine (Arg), asparagine (Asn), cysteine (Cys), Glutamic acid (Glu), phelylalanine (Phe), glycine (Gly), glutamine (Gln), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), proline (Pro), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr) and Ornithine was accomplished in aqueous solution using biosynthesized AuNPs (1–4) at room temperature. The concentration of arginine was changed from 10⁻¹ M to 10⁻⁶ M. 100 µL of analyte was mixed to 200 µL of AuNPs, together with the addition of ∼1 mL of doubly distilled water. The performance of the analytes were observed through naked eye color change of the NPs and UV–vis absorption spectroscopy to examine the variations in the SPR peak of gold nanoparticles.

2.3 Characterization

UV-3100 Hitachi spectrometer is used for UV–visible spectroscopic measurements. The IR spectra have been recorded by Perkin-Elmer FT-IR spectrometer. Malvern- Zeta sizer is used for the Zeta potential and particle size analysis. The morphology of AuNP was scanned using transmission electron microscopy (TEM) (Philips Tecnai 12). The picture was taken with digital camera (Canon-A3200 IS).

3.1 Detection of arginine by AuNPs

We have used the as-synthesized AuNPs for the finding of amino acids in aqueos solution. The aqueous pomegranet peel extract contains phytochemicals such as as punicalagin and punicalin (Cam and Hisil, 2010; Ismail et al., 2012). These polophenols are responsible for the reduction and the stabilization of AuNPs in solution (Kaviya and Prasad, 2015). We hypothesis that, the function group in the extract attached on the surface of AuNPs (1–4) can also intract with the amino acid through non-covalent interaction which can lead to nanoparticle aggregration and such aggregration leads inter plasmonic oscillation and concomitant color changes in the solution (Kaviya and Prasad, 2014).

Initially, we have treated the four different AuNP systems with twenty-three amino acids in aqueous solution (Figs. S2 and S3). Among them, all the four nanoparticles show immediate color change upon addition of arginine and the color change was visible by naked eye (violet (AuNP1), red (AuNP2), blue (AuNP3) and ash (AuNP4)) changed into brown for AuNP1 and AuNP2; black for AuNP3 and AuNP4) (Inset in Fig. 1). This result indicates the rapid detection of arginine by biosynthesized AuNPs (1–4). We have taken the UV–vis absorption spectra of the AuNPs with the addition of arginine in order to find out the change in the SPR peak of the NPs (Figs. 1, S2 and S3). The intensity of SPR peek of AuNP1 and AuNP2 is declined as the concentration of arginine is increased, along with the formation of new peaks at ∼760 nm and 650 nm, respectively (Fig. 1a and b). Similarly, the intensity of SPR of AuNP3 and AuNP4 were declined and additional peaks appeared at ∼807 and 870 nm, respectively (Fig. 1c and d). Fig. S4 depicts the absorption ratio of AuNP2 between 650 nm and 527 nm was linearly related to the concentration of Arg with the linear range from 1 to 6 µM. The visible finding of arginine was more viable in AuNP2 and AuNP3, compared to AuNP1 and AuNP4, due to the enhanced color contrast.

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Fig. 1

UV-vis absorption spectra of AuNPs (1–4) with different concentration of arginine (10⁻¹ M–10⁻⁶ M). The inset shows the photos indicating the corresponding color changes of the NPs upon addition of arginine. As the concentration of arginine increases, the surface plasmon peak of AuNPs undergoes a shift in the peak, leading to a visible color change.

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Fig. 2 shows the TEM images of AuNPs (1–4) in presence of arginine. The TEM images clearly indicate that, the distance between the two adjacent NP (Fig. S1b–e) is decreased with the addition of arginine. As a result, merging/overlapping of the particle was occurred which effectively shows the aggregation of AuNPs. We have examined the zeta potential of AuNPs in presence and absence (Kaviya and Prasad, 2015) of amino acids (Table S1). The results clearly indicate that, the surface charge on AuNPs is decreased only with the addition of arginine which eventually causes the aggregation of the particles (Kaviya and Prasad, 2014). We measured the variation in the size of the AuNPs with the incorporation of Arg (10⁻¹–10⁻⁶ M) using dynamic light scattering method (Fig. S5). The result shows that the size of the particle is increased with the inclusion of arginine. These results are consistent with the results from TEM analysis (Fig. 2).

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Fig. 2

TEM images of a) AuNP1, b) AuNP2, c) AuNP3 and d) AuNP4 with the addition of arginine [10⁻⁴ M]. The inset is the higher magnification of corresponding AuNP and AuNP aggregates before and after the addition of arginine.

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3.2 Mechanism for the detection of arginine by AuNPs

Among the amino acids, arginine alone contains two primary and secondary amino groups (Johannessen et al., 2005). Hence, it is likely that arginine can have enhanced interaction with the capping biomolecules (punicalagin and punicalin) (Kaviya and Prasad, 2015). This could reduce the distance between the adjacent AuNPs, causing aggregation as well as SPR band shift (Guan et al., 2008) (Scheme 1). The interaction between the functional groups from the pomegranate peel extract which is anchored on the surface of AuNPs and with arginine was studied by FT-IR spectroscopy (Fig. S6). Fig. S6a shows the FT-IR spectrum of aqueous pomegranate peel extract where spectral bands observed at 3342, 2926, 2315, 2138, 1644, 1450, 1211, 1047 and 763 cm⁻¹. The bands are correspond to the stretching vibration of O–H (3342 cm⁻¹), C–H (2926 and 763 cm⁻¹), O—C—O (2315 and 2138 cm⁻¹), C⚌C (1644 and1450 cm⁻¹), C—O (1211 cm⁻¹) and C—OH (1047 cm⁻¹). These are the functional groups responsible for the reduction and formation of AuNPs (Kaviya and Prasad, 2015). FT-IR spectrum of arginine is given in Fig. S6b. A bands appeared at 3151 and 2928 cm⁻¹ are because of the stretching vibrations of NH and CH₂ group, respectively. The peaks at 1680, 1575, 1470, 896 and 796 cm⁻¹ are resemble to bending vibrations of NH₂ (1680 and 896 cm⁻¹), stretching vibrations of CO group (1575 cm⁻¹), bending vibrations of CH₂ group (1470 cm⁻¹) and stretching of CNH group (796 cm⁻¹) (Kumar and Rai, 2010). FT-IR spectrum of pomegranate peel extract capped AuNPs + arginine (Fig. S6c) showed the peaks at 3342, 2932, 2315, 2120, 1646, 1450, 1211, 896 and 763 cm⁻¹ which are the characteristic functional groups of pomegranate peel extract and arginine. The intensity of peaks at 3151, 1575 and 796 cm⁻¹ were decreased which indicates that arginine bound on the surface of biogenic AuNPs either through amine or carboxylic groups (Pu et al., 2013). Further, the presence of gold and nitrogen distribution in the AuNP2 + arginine system is confirmed by EDS analysis and mapping (Fig. S7).

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Scheme 1

Schematic representation for the detection of arginine by pomegranate peel stabilized AuNPs.

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3.3 Effect of pH on the detection of arginine

We have examined the effect of pH on the detection of arginine by AuNP2 (as a model nano system) was carried out at a pH range from 5 to 10. The different media pH was achieved by adjusting different concentration ratios of acids and base. The experimental results showed that pH variation affects the binding affinity between the AuNPs and Arg (Fig. 3a). Lower pH (pH 5 and 6) leads the protonation of nitrogen group in the arginine (Chen et al., 2010) as well as affects the stability of the nanoparticles (Yuan et al., 2014). At higher pH (pH 8 and 9) value cause more deprotonation of the functional group (OH and COOH) on the surface of AuNPs and arginine (Kaviya et al., 2017). As a result, the electrostatic replusion among the AuNPs and arginine (Chen et al., 2010). Self aggregration of nanoparticles were observed when the pH was at 10 due to the fact that shielding effect of ions (Pu et al., 2013). Hence, we choose a neutral pH condition for the ananlysis.

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Fig. 3

a) UV–vis absorption spectra of AuNP2 with the addition of arginine at different pH condition and b) interference study of AuNP2 + arginine in the presence of other amino acid (10⁻² M).

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3.4 Selectivity of AuNPs

Interference studies were carried out in presence of other amino acids. The outcomes indicate that, there is no effect in the SPR absorption of AuNP-arginine system with the addition of other amino acids (Figs. 3b and S8). The UV–vis absorption studies of the peel extract and arginine indicate no color change in the absence of AuNP (Fig. S9). The detection limit for arginine by the biologically synthesized AuNPs falls in the range of 10⁻⁶ M, which is comparable with the existing chemically synthesized AuNPs based sensors (Pu et al., 2013; Rawat and Kailasa, 2014). Table S2 gives the outline assessment of our work with the reported colorimetric detection of Arg by gold nanoparticles. Hence, the biosynthesized nanoparticles can be a suitable candidate for the detection of Arg due to its simple procedure.