The Dual Use of the Pyranine (HPTS) Fluorescent Probe: A Ground-State pH Indicator and an Excited-State Proton Transfer Probe

Abstract

Conspectus Molecular fluorescent probes are an essential experimental tool in many fields, ranging from biology to chemistry and materials science, to study the localization and other environmental properties surrounding the fluorescent probe. Thousands of different molecular fluorescent probes can be grouped into different families according to their photophysical properties. This Account focuses on a unique class of fluorescent probes that distinguishes itself from all other probes. This class is termed photoacids, which are molecules exhibiting a change in their acid–base transition between the ground and excited states, resulting in a large change in their p K a values between these two states, which is thermodynamically described using the Förster cycle. While there are many different photoacids, we focus only on pyranine, which is the most used photoacid, with p K a values of ∼7.4 and ∼0.4 for its ground and excited states, respectively. Such a difference between the p K a values is the basis for the dual use of the pyranine fluorescent probe. Furthermore, the protonated and deprotonated states of pyranine absorb and emit at different wavelengths, making it easy to focus on a specific state. Pyranine has been used for decades as a fluorescent pH indicator for physiological pH values, which is based on its acid–base equilibrium in the ground state. While the unique excited-state proton transfer (ESPT) properties of photoacids have been explored for more than a half-century, it is only recently that photoacids and especially pyranine have been used as fluorescent probes for the local environment of the probe, especially the hydration layer surrounding it and related proton diffusion properties. Such use of photoacids is based on their capability for ESPT from the photoacid to a nearby proton acceptor, which is usually, but not necessarily, water. In this Account, we detail the photophysical properties of pyranine, distinguishing between the processes in the ground state and the ones in the excited state. We further review the different utilization of pyranine for probing different properties of the environment. Our main perspective is on the emerging use of the ESPT process for deciphering the hydration layer around the probe and other parameters related to proton diffusion taking place while the molecule is in the excited state, focusing primarily on bio-related materials. Special attention is given to how to perform the experiments and, most importantly, how to interpret their results. We also briefly discuss the breadth of possibilities in making pyranine derivatives and the use of pyranine for controlling dynamic reactions.

A regenerable electrochemical sensor for electro-inactive cyclovirobuxine D detection in biological samples

“…We have actually tried 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) for CVB-D electrochemical sensing because the structure of HPTS comprises one phenolate group and three anionic sulfonate groups under physiological conditions. 42 We speculated that CVB-D could interact with HPTS through the electrostatic and hydrogen-bonding interactions between protonated amine groups of CVB-D and sulfonates and phenolate of HPTS. However, the electrochemical signals of HPTS did not show any obvious changes when CVB-D was introduced.…”

Sensitive and specific detection of saccharide species based on fluorescence: update from 2016

“…Because of its satisfactory water solubility and unique photophysical properties [ 29 , 30 ], the anionic dye 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) is extensively utilized as an optical indicator in saccharide sensing [ 23 , 26 , 31 ]. In this process, HPTS reversibly binds to a synthetic receptor (quencher) via electrostatic interaction and π-π stacking interaction.…”

Photoacidity of Indolinospirobenzopyrans in Water

“…Prominent examples are phenols, [50] naphthols [51] and the pyranine dye, HPTS. [52] Their actuation is reversible, but proton recombination is so fast that modest pH changes are difficult to be observed even using intense light sources. [53] On the contrary, metastable-state photoacids are compounds which, after photoexcitation with light of moderate intensity, can access a dissociated state that is relatively long-lived.…”

“…Excited‐state photoacids are compounds displaying extreme acidity differences between their ground state and excited state (the Δp K a is comprised from 6 to 13 units [50] ). Prominent examples are phenols, [50] naphthols [51] and the pyranine dye, HPTS [52] . Their actuation is reversible, but proton recombination is so fast that modest pH changes are difficult to be observed even using intense light sources [53] .…”