Hidden dynamics of noble-metal-bound thiol monolayers revealed by SERS-monitored entropy-driven exchange of cysteine isotopologues

Vibrational spectroscopy coupled with isotopic labeling provides many insights into dynamic processes within various molecular systems. Here, a newfound utility of surface-enhanced Raman scattering (SERS) spectroscopy as a tool to study noble metal-anchored thiol monolayers is demonstrated for a pair of L-cysteine isotopologues competing to bind the surface of silver nanoparticles (AgNPs). According to our DFT calculations, SERS spectra of L-Cys could be sensitive to 12 C/ 13 C and 14 N/ 15 N isotopic substitutions, which has been experimentally confirmed for the pair of L-Cys isotopologues: Cys-cabn (all 12 C/ 14 N) and Cys-CABN (all 13 C/ 15 N). In the AgNP-anchored state, the two isotopologues reveal distinct Raman shift values (1577 cm -1 / 1633 cm -1 ) of the band assigned to C=O stretching. This characteristic SERS feature has been subsequently employed to probe various exchange scenarios between AgNP-bound and free L-Cys molecules. As the exchange involves two spectrally distinct but chemically identical molecules, the process is exclusively entropy-driven ultimately


Introduction
Controlling the structural physical and chemical properties of the interface can be readily achieved by modifying the metallic surface with organic molecules among which thiol monolayers are of special importance [1][2][3] with a wide range of applications extending from biosensing [4][5][6] , catalysis 7 , to interfacial charge transport [8][9][10][11] .Such monolayers allowed for pivotal advancements in drug delivery 12 , protein adsorption onto surfaces 13,14 , as well as basic research of surfaces and interfaces 15 , and solar cells 16 .Despite immense efforts to understand the physics behind the self-assembly of such monolayers, both theoretically 17,18 and experimentally (including the energetic and kinetic aspects of adsorption [19][20][21] , exchange [22][23][24] and desorption 25,26 , locations of the exchange 27,28 and its mechanism 29,30 ), various aspects of the monolayer dynamics remain obscure.In the context of the potential applications for such monolayers, their stability and dynamics, including the exchange of the surface molecules, are of utmost importance.This naturally extends to establishing appropriate tools to monitor such transformations.
The exchange regime involving isotopologues of a single chemical compound has the advantage of being exclusively entropy driven, i.e. the composition of the equilibrium state is known a priori and is unobscured by varying the chemical affinities of the exchanging thiols with respect to the metal surface 31 .Additionally, since the reduced masses are different between isotopologues, the vibrational fingerprints change accordingly as a result of the isotopic shift.Vibrational spectroscopy thus becomes an obvious choice for the method of study of the exchange between isotopologues.Surface-enhanced Raman scattering (SERS) spectroscopy is particularly appropriate, as the signal acquired with this method originates mainly from the monolayer directly adsorbed onto the plasmonic surface, while the molecules located at a greater distance experience enhancement smaller by orders of magnitude 32 .This relatively short probing range along with extremely high sensitivity 33 and quantitative analysis capability 34,35 make SERS technique particularly advantageous for that application.
Surprisingly, the exchange of isotopologues in the monolayer has not been studied using SERS spectroscopy yet.Some results were reported on the exchange of chemically different ligands 22,[36][37][38][39][40][41][42] , which proved the viability of SERS in this application.Outside of the ligand exchange context, there are scarce examples of works employing various isotopologues of a single compound and SERS spectroscopy.Those were used mainly for unequivocal proof of single-molecule detection [43][44][45] , improvement of the detection limits of SERS-based analytical protocols 46,47 or a facilitated vibrational assignment in a complex system with otherwise overlapping bands [48][49][50] .Perera et al. studied the ability of thiol molecules to exchange citrate ligands, either perdeuterated or not, originating from the synthesis of gold nanoparticles into other adsorbates 40 .Exchange of isotopologues may be tracked with a variety of methods, including radiometric detection 51 , IR spectroscopy 52 , and mass spectrometry 53 , however, the literature lacks analogous reports in which SERS is employed as the detection principle.
To the best of our knowledge, this study describes the first successful implementation of SERS spectroscopy to investigate the dynamic exchange of isotopologues of a thiol compound (cysteine) bound to a metal surface (AgNP).Cysteine was chosen because it can be used to anchor peptides onto the surface of noble metals facilitating the surface modification 54 .Using the isotopically labeled SERS we detected a manifestation of not described earlier phase transition within L-Cys monolayer.We argue that through this novel investigative approach utilizing the high structural and conformational sensitivity of SERS and the straightforward thermodynamics of isotopologue exchange, one may obtain profound insights into the dynamic processes taking place within metal-anchored molecular layers.This knowledge can be further exploited across an extensive range of applications employing monolayer-modified metal nanoparticles, including heterofunctionalized systems.

Chemical reagents and glassware
Hydrochloric and nitric acids, potassium nitrate, and sodium hydroxide were purchased from POCh (Poland).Silver nitrate was obtained from Honeywell (USA).Hydroxylamine hydrochloride, L-Cysteine (Cys-cabn), and L-Cysteine-13 C3, 15 N (Cys-CABN) were delivered by Sigma-Aldrich (USA).Nitrogen gas (99.9999%) was purchased from Air Products (Poland).All reagents were at least of the analytical grade and were used as received without any further purification.
All glassware was cleaned with aqua regia (3:1 HCl:HNO3) and thoroughly rinsed with copious amounts of ultrapure water prior to any experimentation.Ultrapure water (Millipore Milli-Q system, USA, 18.2 MΩ•cm) used for the preparation of all solutions was deaerated beforehand using constant flow of nitrogen for at least 30 minutes.

Synthesis of AgNPs
AgNPs were synthesized using a modified procedure by Leopold and Lendl 55 .All solutions were freshly prepared prior to synthesis.In brief, 300 µL of 1.0 M sodium hydroxide solution was added to 80 mL of 1.87 mM solution of hydroxylamine hydrochloride under vigorous magnetic stirring.After about a minute, a NE-1000 syringe pump (New Era Pump Systems Inc., USA) was used to dispense 10 mL of 8.57 mM AgNO3 solution to the stirred hydroxylamine solution at a constant rate over 30 minutes.The formed gray-brown colloid was kept stirred for 10 additional minutes after which it was moved to a refrigerator (6 °C) until use.
Typically, AgNPs were aged for a week prior to further use, however samples older than three weeks were discarded, as beyond this point the reactivity of the nanoparticles begins to decline, in agreement with reports by Chechik and Ma for Au nanoparticles 56,57 .
Raman activities computed by Gaussian were transformed into respective Raman intensities and presented as described elsewhere 92 .Full-width at half-maximum (FWHM) of the Gaussian-type signals in the calculated spectra were 10, 15, and 25 cm -1 for normal Raman, IR and SERS spectra, respectively.Potential energy distribution (PED) analysis was performed using VEDA4 software 93,94 .Visualizations of the molecular geometry and vibrations were created using GaussView and PyMOL 95 (with PyVibMS plugin 96 ) software.

Isotopic labeling studies
The effects of the isotopic labeling on the vibrational fingerprint of cysteine were studied computationally using DFT calculations.Two ionic forms were considered: the zwitterion for a free molecule and a cysteine molecule adsorbed onto Ag4 + nanocluster via a deprotonated thiol group.The zwitterion was chosen because cysteine exists in this form in the solid state for which Raman spectrum was collected.Thiolate form of cysteine-Ag4 + system was selected as a simple approximation of the conditions representative of the SERS measurement (which has proven to work well for modeling of SERS spectra of short peptides 92 ).The system as a whole had no net charge and the wavefunction was optimized using a spin-unrestricted algorithm with the multiplicity of 2. Both forms had, at first, their geometries optimized and vibrational wavenumbers calculated for the unlabeled isotopologue (Cys-cabn).The energy and optimized atom coordinates are disclosed in the Supporting Information (Chapter 6) for both forms.All vibrational wavenumbers were real proving the optimization to a local minimum.The vibrational wavenumbers were then recalculated for every combination of carbon and/or nitrogen atoms isotopic labeling at the optimized minimal geometry.The naming scheme was based on upper-or lowercase letters c, a, b, and n representing the heavier or lighter isotopologue of carboxyl, alpha, beta carbon atoms and nitrogen atom, respectively.The scheme is detailed further in Table S1 and illustrated in Fig. S1 of the Supporting Information.

IR spectroscopy
Solid-state samples of Cys-cabn and Cys-CABN were studied by Fourier-transform IR spectroscopy using a Nicolet iS50 (Thermo Scientific, USA) spectrometer.Spectra were acquired using attenuated total reflectance (single reflection diamond ATR accessory) in the 400-4000 cm -1 region with the resolution of about 2 cm -1 .

Raman and SERS spectroscopy
Raman and SERS responses were collected using a Labram HR800 (Horiba JobinYvon, France) spectrometer equipped with a diffraction grating of 600 groves/mm and a Peltiercooled CCD detector.All Raman and SERS spectra were excited in a back-scattering mode using a diode-pumped frequency-doubled Nd:YAG laser providing 532 nm radiation.Spectra were acquired at room temperature.The spectrometer was calibrated using the 520 cm -1 Raman band of crystalline silicon prior to each experimental session.

Raman studies of Cys-cabn and Cys-CABN
Solid-state samples of Cys-cabn and Cys-CABN were studied using Raman spectroscopy.
The laser beam was focused on the sample surface using an Olympus BX61 microscope (Japan) with a 50x objective lens.

SERS studies using AgNPs suspensions
Samples for SERS spectroscopy were thermostated prior to spectrum acquisition at a designated temperature using a Biosan TS-100C (Latvia) thermomixer with a SC-24NC sample block.The samples were stirred at 1000 rpm during the whole period of the sample preparation process in order to minimize undesirable effects of diffusion limitations of the ligand in the colloidal solution 97 .
Colloidal suspensions of AgNPs mixed with cysteine aliquots were placed in glass cuvettes with an optical path length of 1 cm.The laser beam was focused through an optical lens permanently mounted in a cuvette holder.Four accumulations (60-second-long each) were averaged for every spectrum.The colloids were activated just before spectrum acquisition by adding one to four successive 40 µL aliquots of 0.5 M KNO3 solution for a satisfactory signal-to-noise ratio.Samples prepared at relatively lower temperatures (e.g. 25 °C or 40 °C) generally changed color from gray-brown to dark green upon addition of the second KNO3 aliquot, indicating AgNPs aggregation.The color change did not occur in samples prepared at temperatures higher than 40 °C which coincided with a worse signal-tonoise ratio in the SERS spectra.Similarly, aggregation was not observed upon addition of cysteine alone under the experimental conditions of this study 98,99 .

Two-step adsorption
1 mL portions of AgNPs colloidal suspensions and further described aliquots of 0.5 mM solutions of Cys-cabn and Cys-CABN were thermostated at a designated temperature.To initialize the adsorption, 10 µL volume of the first isotopologue solution was added to the AgNPs suspension under continuous stirring.After the designated delay time (0, 1, 10, or 29 minutes), a 10 µL portion of the second isotopologue solution was added to the suspension and the adsorption was continued to the total duration of 30 minutes elapsed since the first aliquot had been introduced.SERS spectrum acquisition was started immediately after this period of time.

Reversibility studies
To evaluate the behavior of the system upon temperature changes during the adsorption process, 1 mL volume of AgNPs suspension was thermostated at a designated temperature of 55 °C and a 10 µL aliquot of the Cys-cabn isotopologue 0.5 mM solution was initially added to the suspension.Subsequently, the suspension was cooled down to 40 °C after 1 or 5 minutes from the beginning of Cys-cabn adsorption.10 µL aliquot of Cys-CABN was added after 10 minutes from the beginning of Cys-cabn adsorption, already at a stable temperature of 40 °C.The stirring continued until 30 minutes passed since the first Cys-cabn aliquot was introduced, when the SERS response was collected.

Data analysis
To analyze SERS intensity ratio, curve fitting was performed for all the SERS spectra in the spectral region of interest using two Lorentzian signals of the same width.Linear baseline correction was applied prior to fitting with the baseline parameters obtained by averaging the noise signal within the 5 cm -1 neighborhood of the cutoff values near the bases of the signal.
The fitting was performed using custom software utilizing the SciPy library 100 .
Centroids of the spectral region of interest were also calculated for every spectrum.To account for the small thermal drifts of the Raman instrument calibration throughout the experimentation, data was corrected, as elaborated in detail in Chapter 3 of the Supporting Information and illustrated in Fig. S8.

Isotopic labeling studies
Theoretical calculations of Raman and SERS (cysteine-Ag4 complexes) spectra were performed to evaluate the effect of isotopic substitution of 12 C with 13 C and of 14 N to 15 N on the vibrational signature of cysteine.Results of our DFT calculations (see Fig. 1 and Tables S2-S3) have suggested that cysteine molecules in both forms considered here exhibit a profound change in the energy of vibration at 1664 cm -1 (zwitterion) or 1800 cm -1 (cysteine-Ag4) upon the isotopic substitution of the carboxyl carbon from 12 C to 13   Other substitutions had a comparatively minor effect on this vibrational mode, as all isotopic shifts were smaller than 0.2 cm -1 (see Tables S2-S3).Detailed results of the DFT isotopic analysis are presented in Chapter 1 of the Supporting Information.In particular, a distinct pattern was observed upon increasing the number of atoms substituted for their heavier counterparts, as shown in Fig. 2. The relative changes visualized in Fig. 2 were calculated according to equation (1), presented in Chapter 1 of the Supporting Information.Such a large isotopic shift, as presented by the carboxyl carbon substitution, warranted distinct and well-separated features in the experimental SERS spectra.Although isotopic substitution of the carboxyl carbon alone would be sufficient according to the DFT results, the compound with all 13 C and 15 N atoms, Cys-CABN, was chosen for the experimental part of the study, as it was readily available commercially.S1.

IR and Raman spectroscopy
Vibrational spectra of solid samples were initially collected for the selected Cys isotopologues to verify the vibrational energy differences predicted by the DFT method.IR spectra of solidstate Cys-cabn and Cys-CABN are shown in Fig. S2, together with corresponding computational spectra for the isolated zwitterions.Raman spectra of the compounds, also correlated with corresponding computational spectra, are shown in Fig. S3.PED analysis of the observed vibrations are shown in Table S4 and S5, for Cys-cabn and Cys-CABN, respectively.The assignments are in line with other works on the subject 101 .Experimental and theoretical spectra computed for the zwitterionic form of cysteine are in a good agreement, taking into account that we did not scale the computed vibrational energies.DFT-predicted shift of the νC-O signal was confirmed experimentally, with the value of -39 cm -1 based on the Raman spectra.The shift was also observed in the IR spectra, however as the bands were not resolved clearly, the estimation of the shift value is not viable.

SERS spectroscopy
The experimental SERS spectra of Cys-CABN (see Fig. 1a for molecular structure) and Cyscabn (Fig. 1b) adsorbed on AgNPs for 30 minutes at 25 °C are juxtaposed to the corresponding calculated Raman spectra of Cys-CABN and Cys-cabn adsorbed on the Ag4 cluster in Fig. 1c.As predicted by the DFT calculations, there is a distinct isotopic shift in the experimental vibrational spectra when comparing the fingerprints of Cys-cabn and Cys-CABN (dark blue and green curves in Fig. 1c).The vibrational mode involving mostly the νC-O modes (visualized in the inset of Fig. 1c), discussed above, was assigned to experimental signals at a Raman shift of 1633 cm -1 for Cys-cabn and at 1577 cm -1 for Cys-CABN in experimental SERS spectra.Such a high energy difference confirms the conclusion drawn from DFT calculations that the shift would ensure the existence of distinct features in the experimental SERS spectra.These two bands, corresponding to the same vibrational mode, were selected as marker bands for Cys-cabn (1633 cm -1 ) and Cys-CABN (1577 cm -1 ), respectively.The detailed PED analysis of the vibrational modes observed in SERS spectra is presented in Tables S6 and S7 of the Supporting Information.

Concentration optimization
As the SERS spectrum of Cys-cabn and Cys-cabn/Cys-CABN mixtures adsorbed onto AgNPs was found to depend on the concentration of cysteine aliquots added to the AgNPs colloidal suspensions, the optimization of the cysteine concentration was necessary prior to further experimentation.SERS spectra were collected for samples containing various concentrations of cysteine aliquots (0.25, 0.50, and 1.00 mM), both for the simultaneous coadsorption of Cys-cabn and Cys-CABN (20 µL of equimolar mixture), as well as the two-step adsorption of Cys-cabn followed by addition of Cys-CABN after a 10-minute delay (10 µL aliquots).The comparison of SERS results for such prepared samples is presented in Fig. S4.The lowest concentration tested (0.25 mM) resulted in both SERS marker bands presenting similar intensity for the two-step adsorption experiment, suggesting lack of competition in binding and, therefore, unsaturated surface coverage.Furthermore, the signal-to-noise ratio of the SERS spectrum was rather unsatisfactory.On the other hand, the concentration of 1 mM resulted in the band arising from Cys-CABN (the second isotopologue added), to be of relatively low intensity compared to Cys-cabn.Thus, the concentration of 0.5 mM was chosen for further experiments, as it displayed a sufficient relative intensity of marker bands in the two-step adsorption experiment, allowing for a more accurate SERS analysis.

Coadsorption experiment
The region of the marker bands of the SERS spectra acquired for the samples prepared at various molar ratios of Cys-cabn and Cys-CABN are presented in Fig. 3a, while the entire examined spectral region is presented in Fig. S5.There is a clear evolution of the relative intensities of marker bands in Fig. 3a, following changes in the composition of the cysteine solution added to AgNPs.This evolution was quantified in two ways.Firstly, the relative  Notably, a similar but much less pronounced evolution of signal shape can be observed for other SERS bands, as shown in Fig. S5, proving that although the isotopic shift is widespread among the spectral features, only the selected marker bands were separated so distinctively.
The dependencies shown in Fig. 3b prove that the ratio of the marker bands and the centroid of the marker bands region can be used to reliably estimate the composition of the surface layer comprising Cys-cabn and Cys-CABN molecules adsorbed onto AgNPs in the selected concentration regime.This approach was further employed to investigate the changes in the composition of the monolayer in the temperature-dependent SERS measurements of the exchange of the two isotopologues at the surface of AgNPs.

Two-step adsorption
The schematic representation of the temperature-controlled two-step adsorption experiment is shown in Fig. 4a.SERS spectra obtained for samples prepared at 25 °C with various examined delays are presented in Fig. S6.A prominent change in the spectral profile can be observed upon the introduction of a delay.Remarkably, a delay as short as 1 minute is sufficient to make the isotopologue added first dominate the surface composition of the adsorbed monolayer (compare the intensity of Cys-cabn and Cys-CABN SERS marker bands in Fig. S6) despite equal amounts of both isotopologues present in the system.This is in line with previous reports showing that homocysteine is able to displace the citrate ions from the surface of Au nanoparticles and create a stable monolayer within 60 s from addition 24 .This memory effect constitutes strong evidence that the system is under kinetic control and that detachment of cysteine molecules bound to the AgNP surface faces significant energy barriers.This behavior is observed regardless of the order in which Cys-cabn/Cys-CABN aliquots are added.Reversing this order yields almost exactly the mirror image of the SERS marker bands, as evidenced by Fig. 4b for samples obtained with a 10-minute delay between aliquots.However, the surface composition of the monolayer changes dramatically with increasing temperature, as evidenced by the spectra presented in Fig. 4c, collected for samples prepared at 60 °C.Such samples exhibit spectra with SERS marker bands of virtually equal intensity, suggesting that surface concentrations of both isotopologues are virtually equal as well.This, in turn, indicates that the barriers to the cysteine exchange at the surface of AgNPs are overcome with the moderate increase in temperature and the exchange accelerates.Cys-cabn and Cys-CABN are chemically identical, thus the enthalpy of the exchange reaction is, by definition, equal to zero and the process is driven solely by the entropy.The equilibrium constant for the exchange reaction between chemically identical isotopologues is 1 (in contrast to other works on the subject, where the constant is often difficult to establish 31,102 ).Thus, since the amounts of both isotopologues in the system are equal after the addition of the second aliquot, the equilibrium state would involve equal amounts of both isotopologues adsorbed onto the AgNPs.That is clearly not the case at 25 °C; however, the system approaches such a state via the exchange already at 60 °C (see Fig. 4c).
The surface composition of samples changes monotonically with temperature, as evidenced with the centroids calculated for each sample, shown in Fig. 4d, as well as the relative intensities of the SERS marker bands, shown in Fig. S7.Fig. 5 presents markerbands-limited regions of SERS spectra collected for samples prepared at 25, 40, 50, 55, and 60 °C with various delays, further illustrating the change in the spectral profiles as a function of temperature.It is evident that the relative intensities of the marker bands approach unity with increasing temperature (Fig. 4c) and, consequently, the ratio of surface concentrations of the isotopologues follows that trend.

Reversibility experiments
The temperature ramps applied throughout the experiment validating the reversibility of the behavior described above are shown in Fig. 6a and the corresponding regions of SERS spectra obtained for thus prepared samples are presented in Fig. 6b.It can be seen that the spectra obtained for samples with a temperature change occurring between addition of cysteine aliquots (two middle curves in Fig. 6b) resemble the spectrum of the sample thermostated continuously at a lower temperature of 40 °C (bottom spectrum in Fig 6b).This is a strong indication that the SERS spectral profile (and by extension: the composition of the surface monolayer) is determined by the temperature at which the second cysteine aliquot was added.These observations along with the results of the two-step adsorption experiments at various temperatures suggest that the cysteine layer on AgNPs may, at a temperature higher than 25 °C, undergo a phase transition to a state in which a rapid exchange with the solution can occur.Such a phase transition seems to be reversible, since the SERS spectrum obtained for the cooled sample resembles that of the sample prepared at a lower temperature (see Fig. 6).In addition, a phase transition is a more likely explanation for the observed behavior than overcoming the kinetic barrier of the exchange by increasing the temperature, as the relative temperature rise required here in order to elicit the exchange is smaller than 12% in the Kelvin scale.This relatively minor increase is unlikely to be the sole reason for the observed abrupt activation of the monolayer for the exchange between the surface and the solution, especially as the thiolate monolayers tend not to desorb at that temperature range 103 .Reported experiments performed for cysteine adsorbed in ultrahigh vacuum (UHV) on Au crystalline surfaces showed that such layers are stable up to at least 80 °C104,105 .Desorption of cysteine multilayers from Ag(111) obtained in UHV was observed at 60 °C, however, the first monolayer was stable up to 120 °C106 .Cysteine adsorbed from the solution onto planar gold had negligible desorption at room temperature as well 107 .Computational simulations showed comparable results, as cysteine adsorbed on Au (111), according to DFT, did not desorb upon heating up to 400 K, although it became more mobile 108 .Although some desorption of glutathione from the surface of AgNPs was observed already at the room temperature, it occurred at a timescale of hours rather than minutes 109 .
Temperature-dependent differences in the structure of a cysteine layer were however reported previously.Adsorption of cysteine in UHV onto Au (111) at room temperature led to a heterogenous layer, with both weakly and strongly bound molecules, but adsorption at 330 K led to a uniform layer of chemisorbed molecules instead 110,111 .This proves that even relatively small differences in the temperature can lead to changes in the stability of the monolayer.
Phase transitions in self-assembled alkanethiolate monolayers adsorbed onto metal clusters are generally observed at temperatures below 340 K 112,113 and the process is comparable to melting the adsorbed ligand layer to a state with increased disorder.Short (containing 8 carbon atoms) alkanethiolate layers were observed by NMR spectroscopy to be already in the liquid-like state at 298 K 114 , which means that the transition must occur at lower temperatures.Furthermore, the transition temperature was found to increase with the introduction of polar groups capable of hydrogen bonding 115 .Such hydrogen bonding could reasonably be imagined for the cysteine layer.Although, to the best of our knowledge, such a phase transition has not been reported yet for cysteine, abrupt (but reversible) acceleration of the exchange shown in our results can be satisfactorily explained by a phase transition that introduces disorder to the monolayer.
A similar exchange between the surface-bound molecules and the molecules in the solution was studied in depth by Chechik et al. using electron paramagnetic resonance, spin-labeled ligands, and gold nanoparticles 29,56,57,102,[116][117][118][119][120][121] .However, they observed the exchange at room temperature only, without studying the effect of the temperature change on the mechanism of this reaction.
The secondary observation that can be made from the results of the reversibility experiment (Fig. 6) is that although the temperature changes are not neutral for AgNPs, this influence does not markedly change the behavior of adsorbed cysteine.This is elaborated in detail in Chapter 5 of the Supporting Information.

Conclusions
In this work, we present the viability of SERS spectroscopy coupled with isotopically-labeled ligands as a tool for studying monolayers adsorbed onto plasmonically-active surfaces.In particular, the entropy-driven self-exchange of cysteine isotopologues on AgNPs has been studied using SERS spectroscopy.It was concluded that at 25 °C a delay as small as one minute between the introduction of equimolar aliquots of particular isotopologues is sufficient for the formation of a monolayer composed primarily of the first isotopologue added.However, increasing the temperature during the adsorption process changes the composition of the surface monolayer, as it approaches the equilibrium state (in which the ratio of the surface concentrations of isotopologues is equal to the ratio of the amounts of isotopologues within the whole system) at 60 °C.Temperature ramp experiments revealed that the observed monolayer behavior is reversible.The SERS spectrum is unaffected by any temperature changes prior to addition of the second the shape of the marker-bands region is determined by the temperature at which the second isotopologue was added even if the temperature was higher beforehand.The proposed explanation for such an abrupt but reversible change upon temperature increase is a phase transition that occurs in the monolayer.At an elevated temperature, the number of monolayer defects increases, allowing for a dynamic exchange leading to an equilibrium state that was kinetically restricted at 25 °C.
In summary, this study sheds new light on one of the essential questions concerning the nature of thiol-based ligand exchange on colloidal metal nanoparticles, which has been difficult to address before the use of the isotopically labeled SERS spectroscopy approach proposed here.Our results provide important insight into the exchange dynamics of surfacebound molecules and draw attention to the nanoscale peculiarities of surface-functionalized colloidal metal substrates.The hidden phase transition, revealed here, may prove critical for applications and technologies that require surface displacement of the molecules or knowledge of its temperature-dependent dynamics.

Supporting information
Elaboration of the naming scheme for various isotopologues; detailed results of the DFT calculations for shift of the marker bands for all isotopologues; detailed procedure of calculation of the relative change in spectra between isotopologues; IR and Raman spectra of solid-state Cys-cabn and Cys-CABN compared to relevant DFT-calculated spectra; results of the concentration optimization; full studied range of experimental SERS spectra; details on the algorithm for data correction in centroids calculation; complete PED analysis with assignments of the bands in vibrational spectra of Cys-cabn and Cys-CABN; elaboration of the observed behavior of AgNPs under the experimental conditions; coordinates of atoms and energy for all DFT models utilized throughout the paper.
C, namely -42.0 cm -1 or -45.1 cm -1 , respectively.Potential energy distribution (PED) analysis shows that the vibration involves mainly the νC-O modes.The summary of the results of full PED analysis is shown in Chapter 4 of the Supporting Information.

Figure 1 .
Figure 1.(a)-(b) Structural formulas of the cysteine isotopologues along with their abbreviations used throughout the study.(c) Comparison between the experimental SERS on Ag NPs and calculated Raman spectra of Cys-cabn and Cys-CABN (B3LYP/Aug-cc-pVDZ level of theory for the cysteine molecule in thiolate form interacting with Ag4 + cluster).The vibrational mode corresponding to the isotopic-substitution-sensitive SERS marker bands is highlighted in the inset.The arrows represent the atomic displacements.

Figure 2 .
Figure 2. Relative changes in calculated DFT spectra, as defined in the text, for all combinations of isotopic substitutions within the cysteine molecule.The designations of isotopologues are described in TableS1.
intensities of the Lorentzian signals fitted to the experimental bands showed a linear dependence as a function of the concentration ratio of Cys-CABN and Cys-cabn, visible most clearly in the log-log plot, as shown in Fig. 3b (black squares for the experimental data, red line for the best linear fit).Secondly, centroids were calculated for each examined spectrum, revealing again a very clear dependence on the molar ratio of Cys-CABN and Cys-cabn, as shown in Fig. 3b (green squares).

Figure 3 .
Figure 3. (a) SERS marker bands of mixed Cys-cabn and Cys-CABN layers on Ag NPs coadsorbed from solutions of varying molar ratio of Cys-cabn to Cys-CABN as specified.(b) Plot of the logarithm of the SERS intensity ratio of 1577 and 1633 cm -1 marker bands (black squares) and the spectral centroid (green circles) as a function of the logarithm of Cys-CABN/Cys-cabn solution concentration ratio; the red line corresponds to the best linear fit.

Figure 4 .
Figure 4. (a) Cartoon representation of the two-step adsorption experiment.(b) SERS marker bands of Cys-cabn and Cys-CABN visible for samples prepared at 25°C and (c) at 60°C.(d) Centroid calculated for SERS marker bands of Cys-cabn and Cys-CABN observed in the experimental spectra as a function of temperature.

Figure 5 .
Figure 5. Spectral region of the SERS marker bands of the two isotopologues for samples prepared through the two-step adsorption with various delays at different temperatures (as indicated).E.g. 'cabn 29 min CABN' corresponds to Cys-cabn first mixed with AgNPs, followed by addition of Cys-CABN after 29 minutes.Total time of adsorption prior to SERS measurement was always identical (30 minutes).

Figure 6 .
Figure 6.Schematic diagram of (a) experimental temperature ramp profiles used for (b) SERS characterization of the such prepared samples (view limited to the spectral region showing marker bands).The same color coding is used for the profiles and SERS spectra.