4‐Mercaptopyridine (96%) was purchased from Acros Organics and used without further purification. Methanol was purchased from Fisher Scientific.

A solution of 4-MPy was prepared by dissolving 4-Mpy powder in pure methanol and in deionized (DI) water at a 1 mM concentration. A 1 cm path length cuvette was used for UV-Vis measurements. All absorption spectra were recorded using a Perkin-Elmer Lambda 19 UV-vis-near IR spectrometer. All fluorescence spectra were recorded using a Hitachi F-2500 FL Spectrophotometer. Excitation at 340 nm was used to measure the fluorescence of 4-Mpy dissolved in methanol. Excitation at 320 nm was used to measure the fluorescence of 4-Mpy in water. The Raman spectra of 4-Mpy were investigated using Spectra Pro 2750 (0.75 m Triple Grating Monochromator, 1200 gratings/Spectrograph) at the excitation wavelength of 514.5 nm obtained from an Ar⁺ laser (Spectra Physics). The Raman spectra of liquid 4-Mpy samples were acquired by depositing a few drops of these samples on a glass surface. The Raman spectra of the 4-Mpy powder sample were obtained by placing the solid powder onto a glass surface. The laser was focused on the sample by using a 100× objective lens attached to a confocal microscope, and the power of the laser on stage was 0.56 mW. The laser spot size was 2 µm, and the slit width was 20 µm. The Raman measurement of the sample was taken with 5 accumulations over a 10 s acquisition. To ensure that the obtained spectra were comparable, the settings, including laser power and exposure time, were all kept constant. The silicon line at 520 nm was used to calibrate the observed wavenumbers. The Raman spectra of the droplets represent the bulk solution.

The absorption spectra of 4-Mpy in methanol and water are displayed in Figure 1 . 4-Mpy dissolved in methanol has a maximum absorption (λ_max) at 338 nm, whereas 4-Mpy prepared in water has a maximum absorption (λ_max) at 324 nm.

It is clear from the figure that the maximum absorption band of 4-Mpy shifted to shorter wavelengths (blue shift) as the solvent used to prepare the 4-Mpy solution was changed from methanol to water. This shift in the absorption band can be attributed to the changing polarity of the solvent. However, molecular aggregation through self-assembly due to intermolecular forces cannot be ruled out as a potential cause of this spectral shift.

4-Mpy is highly soluble in methanol but has very low solubility in water. Thus, in methanol, the molecule is expected to remain predominantly in its monomeric form, whereas in water, it is more likely to self-assemble into molecular aggregates due to the unfavorable solvation environment. Indeed, previous studies investigating the aggregation property of 4-Mpy have shown that this molecule aggregates, forming helical chains through self-assembly using hydrogen bonding [25] [26] .

In this study, we examined the formation of 4-Mpy aggregates in water by first preparing an initial solution of 4-Mpy in pure methanol. The solvent composition of this initial solution was then changed by gradually adding water to the solution. In this study, the spectral shift of the absorption band of 4-Mpy was monitored as the water content in the initial solution gradually increased. The results of this study are presented in Figure 2 .

The results show a blue shift in the absorption band of 4-Mpy from 338 nm to 324 nm as the solution composition gradually changed from pure methanol to predominantly water.

According to the molecular exciton model, the observed blue shift in the absorption spectra of 4-Mpy in water is due to the formation of H-type molecular aggregates through intermolecular interactions of this molecule. Previous X-ray diffraction studies on 4-Mpy aggregates have shown that the molecule can self-assemble into elongated helical chains through intermolecular N-H…S hydrogen bonding between pyridine rings [25] . These helical chains further organize into three-dimensional structures stabilized by C-H…S hydrogen bonds, C-H…π interactions, and π-π (van der Waals) stacking. It is therefore possible that, in aqueous solution, 4-Mpy molecules form H-aggregates through a similar self-assembly mechanism, leading to the observed spectral blue shift. In addition, Figure 2 shows a reduction in the intensity of the 4-Mpy absorption band along with the blue shift. This reduction in spectral intensity has been attributed to the formation of molecular aggregates in solution (hypochromic effect) by the exciton model. It is also possible that, as 4-Mpy aggregation increased, the resulting aggregates precipitated out of the solution, thereby contributing to the reduction in intensity of the 4-Mpy absorption band.

To further examine the aggregation of 4-Mpy in water, we conducted a fluorescence study of this molecule in water and methanol. Our study shows that 4-Mpy in water exhibits a relatively strong fluorescence band at 514 nm ( Figure 3(a) ), whereas in methanol this molecule does not show any noticeable fluorescence ( Figure 3(b) ). Previous fluorescence studies on mero-cyanine and hemi-cyanine dyes have reported enhanced fluorescence emission from the H-aggregated structures of these dyes [27] - [29] . The authors of these studies attributed the enhanced fluorescence to the rigidified structure of these cyanine dyes induced by their aggregation. Their studies showed that the monomeric forms of these dyes exhibit very weak fluorescence due to a bond-twisting mechanism that activates non-radiative decay pathways, leading to reduced fluorescence intensity.

The authors concluded that these nonradiative deactivation channels are suppressed due to the formation of the cyanine dye aggregates, causing the aggregates to fluoresce. The fluorescence characteristic of the 4-Mpy aggregates in this study is consistent with that previously observed for cyanine dye aggregates. Results from this study provide further evidence that 4-Mpy molecules are likely to form H-aggregates in aqueous solutions, while in methanol they primarily remain as free molecules.

A comparison of the Raman spectra of 4-Mpy molecules in water and methanol is presented in Figure 4 . A Raman spectrum of the 4-Mpy solid powder is also included in the figure for reference purposes. The figure shows that the Raman spectrum of 4-Mpy molecules in water is dominated by strong enhancements of three bands at 155, 220, and 475 cm⁻¹. Upon close examination of the figure, we can see that the two enhanced bands at 155 and 220 cm⁻¹ are new bands which are absent in the Raman spectra of 4-Mpy in methanol and in solid powder. According to the theory of AERS, these newly enhanced low-frequency Raman bands (lattice modes)

arise from intermolecular vibrations, indicating the formation of 4-Mpy aggregates in water. The enhancements of the low-frequency Raman bands are enabled by the increased polarizability of the aggregated structure and the coupling between excitonic states formed upon aggregation. The appearance of these new low-frequency Raman bands confirms the prediction proposed by the theory of AERS.

In addition, the Raman spectrum of 4-Mpy in water also shows bands above 400 cm⁻¹ associated with the vibrations of the 4-Mpy monomers. These high-frequency bands can also be seen in the Raman spectrum of 4-Mpy in methanol and in solid powder. The correlated Raman band positions and their vibrational assignments are shown in Table 1 .

Among the high-frequency bands, the most enhanced band appears at 475 cm⁻¹, followed by the band at 1530 cm⁻¹. Both bands appear very weak in the Raman spectra of 4-Mpy in methanol and in solid powder. The enhancement of these bands is attributed to Herzberg-Teller intensity borrowing, through which vibrational modes of the monomer gain increased intensity.

The figure also shows reduced intensity of some of the Raman bands at 721, 989, 1041, and 1615 cm⁻¹ in the Raman spectrum of the 4-Mpy in water. These bands appear to have strong intensity in the Raman spectrum of 4-Mpy in solid powder. It is reasonable to expect that the molecular geometry of 4-Mpy aggregates in water differs from that of 4-Mpy monomers in methanol and in solid powder. As a result, following the theory of AERS, the symmetry elements of the aggregates follow selection rules that differ from those of the monomeric species, contributing to variations in Raman intensity. Similar conclusions have also been drawn in analyses of Raman intensity differences between cyanine aggregates and their monomeric forms [30] [31] .

The results of this study strongly suggest the formation of 4-Mpy aggregates in water. In previous studies of the Cyanine dye aggregates, the emergence of two intense low-frequency Raman bands was attributed to the existence of two different types of aggregates [31] . The fact that the Raman spectrum of 4-Mpy shows enhancements of two low-frequency bands indicates the existence of two types of H-aggregates. Indeed, previous studies of 4-Mpy aggregates reported the existence of two types of helical chain conformations—a left-handed helix and a right-handed helix [25] . Therefore, it is possible that 4-Mpy in water aggregates to form two different helical structures, resulting in the enhancements of two intense low-frequency Raman bands.