Investigating the Role of pH and Counterions in the Intrinsic Fluorescence of Solid-State L-Lysine

TL;DR

This work investigates the origins of intrinsic fluorescence in solid-state, non-aromatic L-Lysine aggregates by combining pH- and counterion-controlled experiments with atomistic simulations. Experiments show that increasing pH and changing counterions modulate aggregate morphology and enhance fluorescence, with EQE reaching several percent at neutral to basic pH. Theoretical analysis uses non-adiabatic molecular dynamics on crystal models representing distinct protonation states to reveal that acidic conditions promote non-radiative decay via proton-transfer pathways, while basic conditions favor radiative decay; vibrational modes involving CO stretch and HB fluctuations funnel energy to conical intersections. Collectively, the results establish pH and counterions as key levers to tune non-aromatic solid-state photophysics, offering design principles for pH-responsive optical materials based on amino acids.

Abstract

There is currently a growing interest in understanding the origins of intrinsic fluorescence as a way to design non-invasive probes for biophysical processes. In this regard, understanding how pH influences fluorescence in non-aromatic biomolecular assemblies is key to controlling their optical properties in realistic cellular conditions. Here, we combine experiments and theory to investigate the pH-dependent emission of solid-state L-Lysine (Lys). Lys aggregates prepared at different pH values using HCl and H$_2$SO$_4$ exhibit protonation- and counterion-dependent morphology and fluorescence, as shown by microscopy and steady-state measurements. We find an enhancement in the fluorescence moving from acidic to basic conditions. To uncover the molecular origin of these trends, we performed non-adiabatic molecular dynamics simulations on three Lys crystal models representing distinct protonation states. Our simulations indicate that enhanced protonation under acidic conditions facilitates non-radiative decay via proton transfer, whereas basic conditions favor radiative decay. Our combined experimental-theoretical work highlights pH and counterion identity as key factors tuning fluorescence in Lys assemblies, offering insights for designing pH responsive optical materials based on non-aromatic amino acids.

Figures (24)

• Figure 1: Mole fractions of the four protonation states of L-lysine as a function of pH. The fully protonated cationic species (Lys$^{2+}$) and fully deprotonated anionic species (Lys$^{-}$) each reach near-complete population under highly acidic or basic conditions, respectively. The zwitterionic form (Lys$^0$) peaks at approximately 75$\%$ near the isoelectric point (pH 9.74), reflecting the similar pK$_\text{a}$ values of the $\alpha$- and $\varepsilon$-amino groups.
• Figure 2: Experimental morphological characterization of solid-state L-Lysine (Lys) aggregates prepared at pH 1, 4, 7, and 10 using HCl (panels a–d) and H$_2$SO$_4$ (panels e–h) as acidifying agents. (a, e) Optical microscopy images of crystalline Lys aggregates formed by drop-casting 6.8 mol/L solutions adjusted to pH 1. (b–d, f–h) Scanning electron microscopy (SEM) images of Lys aggregates at pH 4, 7, and 10 for HCl (b–d) and H$_2$SO$_4$ (f–h) conditions. All SEM images were acquired at 3000x magnification with a scale bar of 50 $\mu$m.
• Figure 3: Confocal and fluorescence microscopy of solid-state L-Lysine (Lys) aggregates prepared at various pH values. Aggregates were deposited on clean coverslip glass and air-dried at room temperature. Confocal microscopy images of Lys aggregates formed at pH 1, 4, 7, and 10 using H$_2$SO$_4$ as the acidifying agent are shown in panel a, b, c, and d, respectively. For each pH, images are arranged left to right as follows: panel a begins with an optical microscopy image (dark-field data unavailable), followed by fluorescence images in the blue ($\lambda{\text{exc}}$ = 402 nm, $\lambda{\text{emi}}$ = 421 nm), green ($\lambda_{\text{exc}}$ = 495 nm, $\lambda_{\text{emi}}$ = 519 nm), and red ($\lambda_{\text{exc}}$ = 590 nm, $\lambda_{\text{emi}}$ = 617 nm) channels; panels (b–d) begin with dark-field images followed by the same fluorescence channels.
• Figure 4: Quantitative optical characterization of solid-state L-Lysine (Lys) aggregates prepared at different pH values. Panels a and b show normalized steady-state fluorescence spectra ($\lambda_\text{exc}$ = 330 nm, magenta vertical line) of solid Lys samples prepared with HCl and H$_2$SO$_4$, respectively. Emission spectra are displayed in red, orange, green, and cyan for pH 1, 4, 7, and 10. Corresponding vertical dashed lines in the same colors indicate the emission maxima. Panel c shows the corresponding external quantum efficiency (Ext. Quant. Eff.) measurements: green for HCl and purple for H$_2$SO$_4$.
• Figure 5: Crystal structures used to model the different protonation states of L-lysine: (a) L-lysine sulfate (LLS), (b) L-lysine monohydrochloride dihydrate (LLMHCl), and (c) L-lysine hemihydrate (LLH).