Comparative and Selective Interaction of Amino Acid d-Cysteine with Colloidal Gold Nanoparticles in the Presence of a Fluorescent Probe in Aqueous Medium

In this communication, we report the comparative and selective interaction of amino acid d-cysteine (d-Cys) with citrate caped gold nanoparticles (Au NPs) in the presence of a fluorescent dye, rhodamine B (RhB), in aqueous solution. Au NPs of size 27.5 nm could almost fully quench the steady-state fluorescence emission of RhB at their optimum concentrations in the mixed solution. The interactions of d-Cys, l-Cys, all other relevant d- and l-amino acids, neurotransmitters, and other relevant biological compounds with the Au NPs/RhB mixed solution have been explored by monitoring the fluorescence recovery efficiencies from the almost fully quenched state of RhB fluorescence via a simple steady-state spectrofluorometric method. The higher fluorescence recovery for the interaction of d-Cys with the Au NPs/RhB mixed system is accompanied by a distinct color change (red-wine to bluish-black) of the assay medium after the reaction compared to that of all other interfering compounds considered in this work. The sensitivity of this fluorometric response lies in a broad linear range of concentrations of d-Cys and the limit of detection (LOD) is found to be 4.2 nM, which is low compared to many other methods available in the literature. The different degrees of interaction of d-Cys and l-Cys with the Au NPs/RhB mixed sample have been further explored by circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy. The selective interaction of d-Cys with the proposed Au NPs/RhB mixed system is also found to be correlated with interparticle cross-linking and aggregations of nanoparticles by the analysis of ζ potential and dynamic light scattering (DLS) study, transmission electron microscopy (TEM), atomic force microscopy (AFM), UV–vis absorption spectroscopy etc. The proposed interaction mechanism is further studied with a normal human urine sample to elucidate that the optimized combination of Au NPs and RhB may be realized as an efficient platform for detection of the amino acid d-Cys in a real biosample via a simple fluorometric approach.


INTRODUCTION
The fundamental part of antibodies, signaling molecules, enzymes, hormones, receptors, and protein structures present in all living organisms is amino acids. They also act as biochemical rulers in the neurotransmission. 1,2 All of the amino acids present in the nature exist mainly in two forms: dextrorotatory (D) and levorotatory (L) enantiomers. On the basis of the protein-creating efficiency, there are in total 20 amino acids that have been reported. Previously, it was presumed that only L-amino acids were necessary in mammals, including humans, in the formation of components for proteins and peptides. However, various sensitive and selective analytical approaches developed for detecting chiral amino acids 3,4 suggested that diverse D-amino acids are also present in mammalian tissues. Different studies have been performed to explore the physiological functions of D-amino acids. The physiological and the biochemical activities of different D-amino acids such as D-cysteine (D-Cys), D-aspartate (D-Asp), Dalanine (D-Alan), D-serine (D-Ser) etc. in our nerve cells, skin, arterial walls, bones, other tissues, and body fluids, including amniotic fluid, urine, blood plasma, cerebrospinal fluid (CSF), saliva etc., have been systematically studied by various researchers in the last several years. 5,6 The semi-essential, proteinogenic amino acid cysteine (C 3 H 7 NO 2 S) (abbreviated as Cys) contains thiol (−SH), amine (−NH 2 ), and carboxylic (−COOH) functional groups.
The −SH moiety mainly takes part in the nucleophilic enzymatic reactions. 7 Also, the −SH group plays an important structural role in the formation of many proteins by creating the disulfide derivative of Cys. 8 D-Cys creates hydrogen sulfide (H 2 S) and decreases the disulfide bonds present in the receptors. 8 This may act synergistically and increase the responsiveness of neurotransmitters specifically to a particular activity. The deviant activity of receptors causes diseases in the central nervous system (CNS) such as amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia etc. 9,10 In the endocrine gland, H 2 S prevents discharge of insulin due to glucose generation by the pernicious β cells. 11 Tong et al. showed that the biofilm formation of Streptococcus mutans and Streptococcus sanguinis on the surface of teeth could be controlled by free D-Cys. 12 In recent times, the study of the interactions of various proteins and amino acids with nanoparticles (NPs) has attracted great attention in the field of nano-biotechnology. 13 Among the various amino acids, D-Cys is also responsible for the growth of various diseases and disorders in the human body. Thus, the selective and quantitative recognition of D-Cys is very important for the diagnosis and treatments of various diseases. D-Cys can interact and be functionalized with several interesting metal nanoparticles (NPs) 13,14 because NPs possess significant adsorption capabilities due to their large surface area to volume ratio. 15 Sometimes this type of biomolecules can bind with such NPs via selective covalent interactions. 16 Among the various metal-based NPs, 17,18 noble metal NPs such as gold nanoparticles (Au NPs) show significant prospects regarding their potential in various fields of biomedical applications such as drug delivery, 19 biomolecular sensing 20−22 etc. The high extinction coefficient of Au NPs in the visible region renders them as an efficient platform for signaling molecular recognition processes through their color change. 21 The small sizes of these metal NPs with a large surface curvature facilitate their binding with various organic dyes, proteins 16,18 etc. The degree of structural perturbation of the protein molecules due to their interaction with NPs varies for different protein species. 23 As the amino acids are the basic structural units of protein molecules, the study of the interaction of amino acids with Au NPs via a simple spectrofluorometric technique is of particular importance. The −SH-containing amino acid D-Cys is recognized as an important regulator of neurotransmitters and the overall neural cell dynamics in our physiological system. Therefore, a comparative study of the interaction of various forms of Cys and their chiral discrimination while interacting with Au NPs is important for both fundamental and biomedical interests.
In the present study, we have addressed the interaction or reactivities of D-Cys and L-Cys as well as all other relevant amino acids, neurotransmitters etc. with citrate caped Au NPs in the presence of a fluorescent probe, namely rhodamine B (RhB), in aqueous medium. RhB is a cationic organic dye with a high fluorescence quantum yield and has been used as an interesting signaling agent for various biomolecular recognition events. 24,25 The fluorescence emission of the RhB dye is almost fully quenched by the colloidal solution of Au NPs and the mechanism of the quenching has been described in detail in this work. The fully quenched fluorescence emission of this dye due to Au NPs in the aqueous solution is recovered via selective interaction with the amino acid D-Cys through SH− Au bond formation and aggregation of Au NPs. This interaction is also reflected as a distinct color change of the assay medium from red-wine to bluish-black, which is different from the interaction with L-Cys or any other interfering compounds studied in this work. The ratio-metric determination of the fluorescence recovery efficiency 26,27 in the presence of various analytes in the assay medium automatically eliminates the perturbation from any external influences during the assay. However, there are few recent reports on the detection of D-Cys based on various methods such as SRMchromatography, colorimetric, ultra-high-performance liquid chromatography (UHPLC)-URMS-chromatography, electrokinetic chromatography etc. 28−32 However, the proposed fluorometric method, which is free from any direct leveling, offers great advantages for the detection of D-Cys, especially up to the nanomolar level. The aggregations of the Au NPs/RhB system in the presence of both the −SH-containing amino acids, D-and L-Cys, have been visualized by transmission electron microscopy (TEM) and atomic force microscopic (AFM) studies and are correlated with the observed Fourier transform infrared (FTIR), UV−vis absorption, and steadystate fluorescence spectroscopic results. The different extents of interaction of D-Cys and L-Cys with the proposed Au NPs/ RhB mixed system in aqueous medium are also distinguished by the chiral properties using the circular dichroism (CD) spectroscopic technique. Au NPs and the RhB-based fluorescence turn-on sensing platform are also tested with normal human (male) urine as a real biosample to realize the same selective response of the D-Cys present in urine.
2.2. Synthesis of Au NPs. The well-documented Frens' soft chemical reduction method has been used to obtain monodispersed gold nanocolloids with the desired particle size distribution. 21,33 In brief, an aliquot of a 50 mL aqueous solution of HAuCl 4 ·3H 2 O (0.25 M) was heated to boil and 1.2 mL of Na 3 C 6 H 5 O 7 (1%) was added into the solution under vigorous stirring for the formation of gold nanocolloids. In about 100 s, the boiling solution turned faintly blue. After 120 s, the blue color changed to deep red, which indicates the formation of the spherical Au NPs. The synthesized nanocolloidal solution was set aside to cool down to room temperature and stored at 4°C for future use. The final concentration of the aqueous gold nanocolloidal solution was 0.1 mM at pH ∼ 7 in ambient temperature.

Characterization Techniques.
High-resolution transmission electron microscopy (HRTEM) measurements, energy-dispersive spectroscopy (EDS) images, and selected area electron diffraction (SAED) patterns of the Au NPs were collected using a field emission transmission electron microscope (JEM-2010 TEM, JEOL Ltd., Japan) operated at an accelerating voltage of 200 kV. For the HRTEM study, the sample solutions were incubated for 15 min and then a small drop from each solution was spread onto the carbon-coated copper microgrid (PELCO 300 Mesh Grids, Ted Pella Inc.) and subsequently dried at room temperature (25°C). The hydrodynamic nanoparticle size and the effective surface ζ potential of the as-synthesized Au NPs were obtained by a Zetasizer (Zetasizer Nano ZS, Malvern Instruments Ltd., U.K.) via the dynamic light scattering (DLS) method at ambient condition. The UV−vis absorption spectra of the samples were obtained in the wavelength range of 200−800 nm by a doublebeam UV−vis absorption spectrophotometer (UV-1800, Shimadzu, Japan) after proper baseline correction for the solvent background. The steady-state fluorescence emission spectra of the sample solutions were recorded using a spectrofluorometer (Fluoromax-4C, Horiba Instruments Incorporated, USA) under the following conditions: excitation and emission monochromator slit widths of 2 nm each, excitation wavelength of 530 nm, emission wavelength range of 540−700 nm, and experimental temperature of 25°C. Fluorescence-grade quartz cuvettes (path length 1.0 cm, Kozima, Japan) were used in all of the absorption and fluorescence emission measurements. CD spectroscopic measurements were carried out using a JASCO J-815 CD spectrometer (JASCO International Co., Ltd., Japan). The surface morphology and roughness measurement of the samples were performed using a commercially available atomic force microscope (AFM) (NaioAFM, Nanosurf, Germany) in tapping mode in a humidity-controlled environment. For AFM analysis, the freshly prepared Au NPs aqueous colloidal dispersion and a mixture of Au NPs/RhB in the presence of D-Cys and L-Cys, respectively, were deposited onto smooth glass substrates. FTIR spectra of the samples were recorded using a FTIR spectrometer (model: Spectrum Two, make: Perkin Elmer Inc.) in attenuated total reflection (ATR) mode at ambient temperature. The pH of the aqueous solutions was measured using a digital pH meter (FiveEasy Plus, Mettler Toledo, Germany). , and other −SHcontaining compounds like glutathione (GSH) and homocysteine (Hcys) as well as some different biomolecules such as ascorbic acid (AA), uric acid (UA), and neurotransmitters (Na + and K + ) were prepared in the same ambient condition. A 200 μL aqueous solution of each compound was added separately to the Au NPs/RhB mixed solution and incubated for 5 min at ambient condition prior to the spectrofluorometric measurements. The 5 min incubation time used for other analytes except D-Cys was just to confirm whether these other compounds have any effect on the proposed interaction even after a longer time of incubation compared to that of D-Cys.

Characterization of the Au NPs.
The UV−vis absorption spectroscopic data were recorded to explore the optical absorption properties of the as-synthesized citratestabilized colloidal aqueous dispersion of Au NPs and are shown in Figure 1a. The figure shows a distinct absorption band with its maximum at around 528 nm due to the strong characteristic surface plasmon resonance (SPR) of the Au NPs. Also, there are two weak humps observed at 251 and 356 nm, which are mainly attributed to the intraband electronic transitions of gold. 34 The sharp and well-resolved SPR maximum band is originated basically due to both the longitudinal and transverse SPR equivalently, which is an indication of the formation of spherical Au NPs. 35 The average hydrodynamic diameter of the colloidal Au NPs as obtained from the DLS study was about 48.04 nm (as shown in Figure S1a in the Supporting Information). The surface ζ potential of the Au NPs was found to be −30.1 mV (as shown in Figure S1b in the Supporting Information). The high negative ζ potential reveals the homogeneity and stability of the NPs in the aqueous colloidal dispersion. On the other hand, the TEM micrograph as shown in Figure 1b gives the visual evidence that Au NPs were monodispersed with nearly spherical shape and the average particle diameter was ∼27.5 nm, as is evidenced from the lognormal fitting of the particles' size distribution curve (the inset of Figure 1b obtained from Figure S2a in the Supporting Information). The good crystalline nature of the Au NPs was shown by the SAED pattern (as shown in Figure S2b in the Supporting Information). The rings as shown in SAED were indexed as (311), (222), and (400) and correspond to the electron diffractions originated from the planes of the face-centered cubic (FCC) gold crystal. 36 The interplanar spacing of the assynthesized FCC Au NPs was 0.11 nm for the (222) plane as calculated from the HRTEM micrograph (as shown in Figure  S2c in the Supporting Information). The EDS spectrum (as shown in Figure S2d in the Supporting Information) reveals that the nucleation of Au NPs occurred without any impurities and confirms the presence of a large number of NPs in its aqueous colloidal dispersion.

UV−Vis Absorption Spectroscopic Study.
The UV−vis absorption spectroscopic measurements were carried out by adding D-Cys and other D-and L-amino acids, neurotransmitters, AA, UA, including other −SH-containing compounds such as Met, HCys, GSH etc., to the mixed solution of Au NPs and RhB (aqueous mixture of Au NPs (0.1 mM) and RhB (0.1 μM) in 1:1 volume ratio). Figure 2a shows the UV−vis absorption spectra of pure RhB (0.1 μM) (curve 1) along with the mixture of RhB and Au NPs in aqueous solution (curve 2). The concentrations of Au NPs and RhB in the mixed solution were 0.05 mM and 0.05 μM, respectively. The inset of Figure 2a shows the molecular structure of the laser dye RhB. The UV−vis absorption spectrum of pure RhB exhibits a strong absorption band at around 554 nm, which is attributed to the π−π* transition of the dye monomer unit, and a weak hump is observed at around 520 nm, which is originated from the n−π* transition due to the presence of dimeric units. 24 The UV−vis absorption results of the mixed solution further reveal that the monomeric absorption peak of RhB remains unaltered but with decreased intensity, whereas the dimeric band intensity (520 nm) increases. This variation of the absorption intensity may possibly be due to the closure association of the RhB molecules adsorbed onto the NPs' surface. As RhB is a cationic dye and citrate caped Au NPs have a large negative surface ζ potential, the strong electrostatic interaction between Au NPs and RhB is mainly responsible for the adsorption of the dye molecules on the NPs' surface, resulting in the change in the absorption behavior of the dye molecules in their aqueous mixed solution. The number of dye molecules (N dye ) linked with each Au NP is determined using the relation 37 (eq 1)   (1) where n Au is the number of gold atoms in each NP. The number of gold atoms per NP (n Au ) is calculated by using the expression 37,38 n Au = (π/6)(59 nm −3 )(d av )3, where d av is the mean diameter of the Au NPs. Thus, in this case, an Au NP of average diameter 27.5 nm is composed of 642 138 gold atoms.
In the mixed solution of Au NPs (0.1 mM) and RhB (0.1 μM), 642 dye molecules are adsorbed on each Au NP surface.
On the other hand, the UV−vis absorption spectrum of RhB in the presence of D-Cys as shown in Figure S3a Figure 3a, increases with the incubation time in the presence of D-Cys. This is possibly due to the strong cooperative metal ion-ligand interactions. 39 Because of this interaction, the citrate layer on the nanoparticles' surface may be displaced and the interparticle separations of the NPs are possibly reduced due to the subsequent cross-linking through the amino groups of D-Cys. As a consequence, the increased dipole−dipole interactions and the coupling between neighboring plasmons eventually induced the formation of aggregates or clusters 39,40 of Au NPs. This aggregation is manifested as the emergence of the additional band at around 670 nm in the UV−vis absorption spectra. 41,42 In order to have direct visual evidence for the formation of aggregates or clusters of NPs, TEM analysis was also performed. Figure 3b shows the TEM image of the Au NPs/RhB/D-Cys mixed sample and clearly shows very dense and aggregated domains of the NPs when compared to the TEM image of pure Au NPs as shown in Figure 1b. This aggregation eventually induces enhanced dye−dye intermolecular interaction in the mixed solution of Au NPs and RhB. As a result, the RhB molecules might have altered electric transition dipole moments in the microenvironment of Au NPs. 43

Interaction of D-Cys with the Au NPs/RhB Mixed Solution.
To understand the interaction of D-Cys with the Au NPs/RhB mixed ensemble in aqueous medium and the related photophysical properties, steady-state fluorescence emission measurements were performed. It is observed from Figure S3c in the Supporting Information that the steady-state fluorescence emission intensity of the peak at around 574 nm of RhB in the Au NPs/RhB mixed aqueous solution (1:1 volume ratio) is almost quenched for an Au NP concentration of 0.05 mM in the mixed solution at ambient condition. It is important to mention that the electrostatic interaction between the dye molecules and the negatively charged citrate layer on the Au NPs or any formation of H bonds does not change the electronic transition dipole moment of the xanthene moiety of the RhB molecules 44 as there is no shift of the emission band (574 nm) of dye molecules after photoexcitation of the Au NPs/RhB mixed ensemble in the absence or presence of D-Cys. Therefore, the effect of direct molecule on nanoparticle interaction may be excluded from the initial fluorescence quenching of RhB molecules in the presence of Au NPs. Additionally, the possibility of dynamic collisional quenching of fluorescence may be less due to the existence of a strong electrostatic interaction between the RhB molecules and negatively charged citrate caped Au NPs in the studied aqueous medium. Interestingly, there is a sufficient spectral overlap between the absorption spectrum of Au NPs and fluorescence emission spectrum of RhB as shown in Figure S3b in the Supporting Information. So, the observed quenching of fluorescence emission (574 nm band) of RhB in the Au NPs/ RhB mixed solution is mostly due to the efficient nonradiative energy migration through the resonance energy transfer (RET) process (as shown in Figure S3b,c in the Supporting Information) using the dipole−dipole resonance interaction between the excited RhB dye (donor) molecules and Au NP surface (acceptor), as well as the decrease in the radiative rate constant of individual RhB molecules when they are adsorbed onto the NPs' surface. 21,43 This resonance energy transfer process is a distance-dependent phenomenon and therefore the concentration of the Au NPs (acceptor) plays a significant role in controlling the relative distance between the donor and acceptor sites in the mixed solution. The RET efficiency (E) is calculated using the expression 44,45 (eq 2) where F 0 and F are the fluorescence emission intensity of RhB in the absence and presence of Au NPs, respectively. The value of energy transfer efficiency (E) is estimated as 0.9989, considering the fluorescence spectrum of Au NPs/RhB with concentrations of RhB and Au NPs as 0.05 μM and 0.05 mM in the mixed solution, respectively. The value of the binding constant (K b ) to a binding site and the number of binding sites (n) per RhB molecule in the Au NPs/RhB mixed aqueous solution are determined from the fluorescence quenching data of RhB in the presence of Au NPs by using the following eq 3 46 where [Q] is the corresponding quencher (Au NPs) concentration. The values of K b and n are calculated from the intercept and slope of the plot as shown in Figure S3c, where K SV is the Stern−Volmer quenching constant. The deviation from linearity of the Stern−Volmer plot at higher quencher concentration implies that the fluorescence quenching of the RhB dye is a mixture of static and dynamic (diffusion) quenching. 48 Considering the slope of the straightline region of the F 0 /F vs [Q] plot (i.e., at lower concentrations of Au NPs) as shown in Figure S3c,e in the Supporting Information, the value of K SV is calculated as 8.174 × 10 6 M −1 . The bimolecular fluorescence quenching rate constant (K q ) and K SV are related by the relation 49 K q = K SV / τ 0 , where τ 0 is the fluorescence emission lifetime of pure RhB (1.384 ns). 21 The value of K q is calculated as 5.906 × 10 15 M −1 s −1 for this fluorometric titration experiment. The high values of both K SV and K q substantiate the efficient quenching of RhB (donor) fluorescence by gold nanoparticles in the aqueous medium. All of the above calculated photophysical parameters are summarized in Table 1. However, interestingly, after addition of D-Cys to the mixed solution of Au NPs and RhB, the original fluorescence emission (574 nm) of dye molecules is recovered from their quenched state as shown in Figure 4a. The concentrations of RhB and Au NPs for the assay experiments were optimized in such a way that there is an almost complete quenching of the fluorescence emission of RhB in the Au NPs/RhB mixed system before addition of the D-Cys solution when excited with light of wavelength 530 nm and recovery of fluorescence signal in the presence of D-Cys at ambient condition. This "turn-off" and "turn-on" mechanism of fluorescence emission due to the presence of D-Cys in the assay medium is schematically shown in Figure 5. For the assay experiments, the concentrations of RhB and Au NPs in their aqueous mixture were fixed at 0.05 μM and 0.05 mM, respectively. These selected concentrations of RhB and Au NPs in their aqueous solution at ambient condition may be considered as an optimized condition for the fluorescence recovery from the mixed ensemble when exposed to D-Cys in aqueous medium. Here we have studied the recovery of fluorescence emission from the Au NPs/RhB mixed solution in the absence (control) and presence of various concentrations of D-Cys. Also, there is a distinct color change of the mixed final solution from red-wine (control) to bluish-black (after addition of D-Cys) as shown in the inset of  Figure S3f in the Supporting Information, it is further confirmed that there is no significant effect of D-Cys on the fluorescence emission properties of RhB in aqueous solution as the emission spectra of the RhB/D-Cys mixed solution for all concentrations of D-Cys are almost unaltered. Therefore, RhB dye has been considered as a good and efficient fluorescent probe for recognition of the amino acid D-Cys in aqueous medium via a simple spectrofluorometric method in the present work.
It is known that at neutral or under near-physiological pH, citrate caped Au NPs possess three carboxylated (−COO − ) groups, 50 whereas the D-Cys molecule has −SH, −COO − groups, and the amino group (NH 3 + ) in the same aqueous environment. The citrate layer surrounding the Au NPs in aqueous dispersion gives an overall strong negative surface potential as is already confirmed by the ζ potential study (as shown in Figure S1b in the Supporting Information). It is already reported elsewhere that at neutral pH (7.0) cysteine is   neutral, 51 due to which there will be very less possibility of citrate caped Au NPs having direct interaction with D-Cys through its −NH 2 group via electrostatic attraction. 42 However, due to the strong affinity of the −SH moiety of D-Cys towards the Au NPs' surface, they try to encapsulate 41 Au NPs by thiolate−Au bond formation via chemisorption, 39 which eventually displaces the original citrate layer of Au NPs. As a consequence, the RhB molecules that were adsorbed onto the citrate caped nanoparticles' surface by electrostatic interaction 52 now become free in the surrounding environment. This is why the intrinsic fluorescence emission of RhB with peak centered at 574 nm is retrieved after photoexcitation. However, the displacement of the citrate layer of Au NPs should depend upon various factors like the concentration of D-Cys molecules in the assay medium, overall charge distribution, surface potential around the nanoparticle surface, temperature etc. Immediately after the reaction of D-Cys with Au NPs/RhB systems, the presence of amino acid molecules in the encapsulating shell of nanoparticles starts to induce crosslinking (as shown schematically in Figure 5 between the neighboring encapsulated Au NPs) in the mixed solution, resulting in the formation of Au NP aggregates or clusters, which causes a color change of the mixed solution from redwine to bluish-black 53,54 after the reaction. The aggregation of Au NPs in the presence of D-Cys is already evidenced by the UV−vis absorption ( Figure 2b) and TEM study (Figure 3b) as discussed earlier. From the above discussion, it is clear that the fluorescence emission band intensity at 574 nm of the mixed solution of Au NPs and RhB is recovered after addition of D-Cys and this fluorescence recovery is most probably due to the availability of free RhB molecules in the mixed ensemble.
Overall, due to the interaction of D-Cys with the Au NPs/RhB system in solution, the relative distance between the dye molecules and Au NPs might have increased, resulting in the decrease of fluorescence quenching via nonradiative energy transfer pathways.   determination as previously reported in the different studies have been compared with the present work and summarized in Table 2.  Figure S4 in the Supporting Information. From Figure 6a it is observed that the fluorescence emission intensities of the Au NPs/RhB mixed solution in the presence of D-Cys and L-Cys are significantly high compared to those for other interfering compounds, although the concentrations of all of the analytes were the same in these spectrofluorometric measurements. However, when the Au NPs/RhB mixed solution is added with D-Cys, the final solution turns red-wine to bluish-black in color, which is different when mixed with L-Cys solution separately, as shown in Figure S5 in the Supporting Information. The relative fluorescence recovery efficiency [(F − F 0 )/F 0 ] as shown in Figure 6b becomes remarkably high for D-Cys compared to that for all other relevant interfering species or elements. From the above observations it is clear that the interaction of the Au NPs/RhB mixed ensembles with D-Cys in aqueous medium is selective due to the high fluorescence recovery as well as distinct color change of the Au NPs/RhB mixed solution when compared to the interactions with L-Cys and all other D-and L-amino acids, physiologically relevant interfering elements etc. as studied in this experiment. It is important to mention that the difference between fluorescence recovery efficiencies for D-Cys and L-Cys might be attributed to the different chiralities in the molecular structure of these amino acids, as is evidenced from the CD spectra shown in Figure 7a. The high selectivity of the interaction of the Au NPs/RhB mixed solution with D-Cys may be attributed to the change in structural configuration of the amino acid D-Cys, which is manifested as the observed change in the peak position of the CD spectrum. This is because any change in the configuration of the functional group (−SH) while interacting with the Au NPs/RhB mixed ensemble in aqueous medium possibly disrupts the chiral properties in the structure of D-Cys. As a result, there is a blue shift of the peak position at 210 nm in the CD spectrum as shown in Figure 7a. On the other hand, when L-Cys is added to the Au NPs/RhB mixed solution, the peak position observed at 216 nm in the CD spectrum is not altered except for a slight reduction of intensity. These observations clearly indicate that the chiral properties of L-Cys were possibly less affected while interacting with the Au NPs/RhB mixed ensemble compared to those of D-Cys. From the DLS study, as shown in Figures  7b, S8a, and S9a, it is observed that the particle size of Au NPs increases in the presence of both D-and L-Cys. However, for D-Cys, the increase in hydrodynamic particle diameter is higher (91.67 nm) compared to that of L-Cys (67.84 nm). This observation implies the possibility of a high degree of aggregation of Au NPs while interacting with D-Cys when compared to that due to L-Cys. As mentioned earlier, amino acids basically displace the citrate layer of the Au NPs during interaction and induce interparticle cross-linking, which favors rapid aggregation of Au NPs. However, the rate or extent of interparticle cross-linking is possibly higher for D-Cys than for L-Cys. Additionally, the less negative ζ potential (Figures 7b,  S8b, and S9b) of the Au NPs/RhB mixed solution containing D-Cys (−14.03 mV) compared to that with L-Cys (−18.04 mV) confirms the higher aggregation of NPs in the case of D-Cys. 56 To have a better understanding and direct visual evidence of the formation of aggregates or clusters, AFM topographical imaging is performed in a tapping mode. Figure  7c−h shows the AFM topographical image and surface roughness plot of the drop-casted film deposited onto smooth glass substrates from the Au NPs/RhB mixed solution in the presence of D-Cys and L-Cys, respectively. For all of these measurements, Au NPs/RhB mixed solutions were prepared in 1:1 volume ratio and their concentrations in the mixed solution were 0.05 mM and 0.05 μM, respectively. These images show that D-Cys facilitates a higher extent of aggregation or formation of clusters of Au NPs in the mixed solution. The root-mean-square (RMS) surface roughness of the Au NPs/RhB film is estimated as 4.4 nm, whereas for L-Cys and D-Cys it becomes 7.25 and 18.92 nm, respectively. That is, the surface roughness of the film having D-Cys is much higher compared to that of L-Cys. Our AFM analysis revels that D-Cys induces a much greater degree of nanoparticle aggregation. Also, the hydrogen bond between the neighboring D-Cys molecules in the aqueous solution is highly affected by the steric hindrance due to the presence of Au NPs. The high extent of aggregation of NPs in the aqueous mixture of Au NPs/RhB facilitated higher fluorescence recovery from the quenched state due to the release of more number of RhB dye molecules from the Au surface.

Selectivity Study of the Proposed Bio-Nano
Interestingly, our proposed Au NPs/RhB mixed platform does not show any significant or appreciable fluorescence recovery by other aminothiols such as Hcys and GSH in the same aqueous medium as studied in this work. In case of Hcys and GSH, we observe a slight recovery of fluorescence emission from the Au NPs/RhB matrix in aqueous medium. Although Hcys is a homologue of the amino acid Cys, it differs with an additional methylene bridge in its molecular structure. It is also known that 57 nearly at physiological pH, Hcys contains one amino group (−NH 3 + ), one −COO − group, and one −SH group in aqueous medium, whereas citrate caped Au NPs possess three −COO − groups in the same aqueous medium. Therefore, the direct electrostatic binding between Hcys and Au NPs is minimal. Due to the presence of the −SH group in Hcys, there will be still some interaction via thiolate− Au bond formation, but the rate of displacing the citrate layer on which the cationic RhB molecules are bound might be limited due to the difference in the orientation of the amino acid groups or in the interfacial free energies when compared to D-Cys or L-Cys in the studied aqueous environment. 41 As a consequence, the fluorescence emission recovery from the Au NPs/RhB mixed ensemble is less compared to D-Cys and L-Cys. On the other hand, GSH has two −COOH groups associated with glutamic acid and glycine residues and three amino groups (−NH 3 + ) in three amino acids, and one sulfur atom present in the Cys residue. It is expected that at intermediate or higher pH the disassociation of −COOH groups may hinder 58 the binding of GSH with Au nanocolloids via the α amine group. This results in a very little cross-linking between the −SH group and Au colloids. As we have studied the binding interactions at neutral pH (7.0), less interaction between Au and GSH results in lower fluorescence recovery in the Au NPs/RhB aqueous medium. Additionally, the GSH molecule is relatively bulky in size compared to cysteine or Hcys, and therefore, the interfacial reactivity may be less probable due to energetic consideration. However, literature revels that the amino acid Trp can selectively bind with Au NPs. 59 In the present work, we did not find any significant fluorescence recovery from the Au NPs/RhB mixed solution in the presence of D-and L-Trp. This is possibly due to the formation of the nonfluorescent Trp−RhB complex. 60 The binding constant, sometimes referred to as the association constant (K a ), of the Au NPs/RhB system with D-Cys and L-Cys in the aqueous medium has been determined using the Benesi−Hildebrand equation (eq 6) 61,62 where  Table 3. These values confirm that both D-Cys and L-Cys can bind to the Au NPs/RhB system in aqueous solution. But, in the case of D-Cys, this interaction is stronger than that for L-Cys. Therefore, due to the strong binding interaction between D-Cys and citrate caped Au NPs in aqueous medium, the fluorescence recovery efficiency is much higher compared to L-Cys. The values of the Gibbs free energy ΔG and binding constant K a for Au NPs/RhB/D-Cys and Au NPs/RhB/L-Cys complexes have been calculated from the steady-state fluorescence emission spectra and are summarized in Table 3. The negative ΔG indicates that the interactions of both D-Cys and L-Cys with the Au NPs/RhB system are spontaneous. However, the higher negative value of ΔG reveals the faster rate of interaction in the case of D-Cys with the assay medium compared to that for L-Cys. For further investigation on the selectivity and interaction mechanism of Au NPs/RhB with D-and L-Cys, FTIR spectroscopic measurements in ATR mode were performed and are shown in Figure 9. The FTIR spectra of pure D-Cys and L-Cys (curve 1 in (a) and (b), respectively) are very similar and show IR bands with peaks centered at 3166, 1582, and 1390 cm −1 , which are attributed to the stretching of −NH 3 + and the asymmetric and symmetric stretching of the −COO − functional groups of Cys molecules, respectively. In addition, Cys molecules have a weak IR vibrational band near 2550 cm −1 , which is originated due to the characteristic stretching vibration of the S−H bond (thiol group). Other vibrational bands are observed at 2968 cm −1 (CH 2 stretching, asymmetric), 2078 cm −1 (NH 3 bending, asymmetric), 1522 cm −1 (N−H bending), 1340 cm −1 (NH 3 bending, symmetric), 1294 cm −1 (CH 2 wagging), 1195 cm −1 (CH 2 twisting), 1130 cm −1 (SO 2 stretching), 1062 cm −1 (NH 3 rocking), 941 cm −1 (S−H bending), 866 cm −1 (N−O stretching), 635 cm −1 (COOH bending), 536 cm −1 (COOH rocking) etc. These results are consistent with the FTIR spectra of amino acids reported elsewhere. 63−65 Interestingly, for the Au NPs/RhB/D-Cys system, the IR bands corresponding to the stretching of −NH 3 + and −COO − groups of D-Cys are almost absent, possibly due to their cross-linking with other cysteine molecules attached to the neighboring Au NPs. However, for the Au NPs/RhB/L-Cys system, the bands of these stretching vibrations are also abruptly reduced compared to pure L-Cys but the peaks are still visible. These observations may reveal that the interparticle cross-linking is much stronger in the case of D-Cys in the assay medium, resulting in a rapid and greater extent of nanoparticle aggregations, which is reflected in the different color change of the final solution. This may also be due to the change in their dipole moments when binding with a high electron density of the metal surface. But the most significant observation is that the S−H stretching vibration band (2550 cm −1 ) totally disappears for D-Cys after interacting with the Au NPs/RhB mixed system in aqueous medium (Figure 9a curve 3). This indicates that S−H bonds are cleaved in the presence of the metal NPs, which further indicates the strong binding interaction of D-Cys with Au NPs. On the other hand, the S−H stretching vibration band is not totally absent for L-Cys when interacted with the Au NPs/RhB system but it appears as a weak band near 2550 cm −1 as shown in Figure 9b (curve 3). Therefore, it is clear that the interaction of D-Cys with the Au NPs/RhB mixed system is stronger compared to that of L-Cys in the studied aqueous medium. Additionally, the FTIR spectra of the Au NPs/RhB mixed system (curve 2 in Figure 9a,b, respectively) confirm that there is no such significant effect of RhB molecules on the interaction of D-Cys or L-Cys molecules with Au NPs in the assay medium.
3.6. Real Biosample Analysis. The proposed nano-bio interaction is also studied in the presence of normal human urine. The normal urine sample was collected from a healthy laboratory volunteer (male researcher) having no renal complications. In this investigation with the normal urine sample, we maintained the same optimized concentrations of RhB and Au NPs in their mixed solution as discussed earlier, and took extra precautions to avoid any direct contact or any type of contamination with the urine sample. D-Cys may present in both urine and blood plasma. It is already known that diseases like cystinuria leading to the formation of kidney and renal system stone occur due to the free presence of Cys in the urine. 66 Figure S10 in the Supporting Information. The concentrations of D-Cys and all other analytes in the final assay medium containing urine were 2.38 μM, respectively. The corresponding fluorescence recovery efficiency is shown in Figure 10. From this figure it is clear that, the normal urine shows fluorescence recovery efficiency of 42% but mixture of normal urine and D-Cys shows fluorescence recovery efficiency of 73%. Therefore, the usual presence of Cys in the normal  Figure 9. ATR-FTIR spectra of (a) D-Cys (b) and L-Cys in the absence and presence of Au NPs/RhB, respectively.

ACS Omega
http://pubs.acs.org/journal/acsodf Article urine gives the initial fluorescence recovery from Au NPs/RhB system. It is also observed that when all other D and L-amino acids along with the other relevant interfering compounds as considered are mixed with the proposed assay matrix, the fluorescence recovery efficiency is slightly higher than that due to pure normal urine. This may be due to the presence of L-Cys as was added with all other interfering compounds. Additionally, there is also a distinct color change (from redwine to bluish-black) of the assay solution after addition of D-Cys compared to all other analytes including L-Cys as shown in the inset of Figure 10. This study confirms the proposed nanobio interaction is manifested as the selective binding of D-Cys with Au NPs/RhB in in human urine as well. Therefore, the proposed Au NPs/RhB system in aqueous medium under the appropriate and optimized condition may be used as an efficient platform for rapid detection of D-Cys in human urine in a ratio-metric approach to diagnose various renal complications.

CONCLUSIONS
In conclusion, we demonstrate a simple fluorometric method to explore the comparative and selective interaction of the amino acid D-Cys among various other D-and L-amino acids, neurotransmitters, relevant biological compounds etc., with citrate caped gold nanoparticles (27.5 nm) in the presence of the fluorescent dye RhB. Au NPs are found to almost fully quench the fluorescence emission of RhB dye in the optimized concentrations of both Au NPs and RhB via a nonradiative energy transfer pathway from the excited RhB molecules to Au NPs in aqueous solution at neutral pH. This quenched fluorescence is selectively recovered with high efficiency after addition of D-Cys to the Au NPs/RhB mixed solution and there was a distinct color change of the assay medium after reaction with D-Cys when compared to the effect of other interfering compounds studied in this work. The degree of aggregation of Au NPs is greater due to the interaction with D-Cys compared to L-Cys due to strong thiolate−Au bond formation via chemisorption and possibly greater interparticle cross-linking compared to that due to L-Cys and in aqueous solution. The higher value of binding constant and higher negative value of Gibbs free energy for D-Cys interacting with the Au NPs/RhB mixed system compared to those for L-Cys as estimated from the fluorescence recovery results confirm the stronger interaction of D-Cys with the nanoparticle system in the studied aqueous medium. The difference in aggregations of D-Cys and L-Cys while interacting with Au NPs/RhB mixed systems in solution is correlated with the ζ potential results and chiral properties of the amino acids, as evidenced by the ζ potential study and CD spectroscopic characterization, respectively. The ATR-FTIR study further distinguishes the difference of interactions of both D-Cys and L-Cys with the assay matrix through the change in vibrational signatures of the S−H bond as well as other relevant functional groups and is consistent with the observations by the other analytical methods used in this present work. The interaction of D-Cys with the proposed nanoparticle-based platform is highly sensitive and lies in the broad linear range of concentration of 1 nM to 100 μM. The LOD for D-Cys is found to be 4.2 nM, which is lower compared to many available detection methods cited in the literature. The normal human urine sample when mixed with the assay matrix (Au NPs/RhB) and D-Cys exhibits a higher fluorescence recovery and distinct color change (redwine to bluish-black) of the assay medium after reaction compared to that for L-Cys or all other interfering compounds as studied. Therefore, the proposed bio-nano interaction may be realized as an efficient approach for the selective recognition of the amino acid D-Cys in the aqueous environment.