Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications

Abstract

Significance: The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various biochemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules.

Critical issues: In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes.

Future directions: The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 29, 518-540.

Keywords: fluorescent probes; glutathione; hydrogen peroxide; ratiometric; reversible reactions; sensing and imaging.

FIG. 1.

Desired features for small-molecule-based fluorescent probes.

FIG. 2.

Two strategies for designing reversible fluorescent probes. Route A is based on a direct chemical equilibrium between the probe and the analyte, such as in the case of RealThiol glutathione probe. The reversibility of Route B is based on two different chemical reactions. Usually, the reverse reaction is catalyzed by an enzyme, such as in the case of the Hyper hydrogen peroxide probe. Grx, glutaredoxin; GSH, glutathione; H₂O₂, hydrogen peroxide.

FIG. 3.

Equations for quantification of analyte concentration using ratiometric probes in different scenarios. (A) Equation 1 defines the relationship between the concentrations of analyte, probe, and reacted probe and the dissociation equilibrium constant K. Equation 2 is used when there is spectral overlap between the unreacted probe and the reacted probe. R is the signal ratio between reacted probe and unreacted probe. R_min and R_max are the R values at the corresponding 0 and saturated analyte concentrations. Equation 3 is used when there is no spectral overlap between the probe and the reacted probe. (B) The relationship between R and a hypothetical analyte concentration. In general, plotting R versus analyte concentration affords a sigmoidal relationship. In contrast, (R-R_min)/(R_max-R) is linear when plotted against the analyte concentration.

FIG. 4.

Two strategies for achieving organelle specificity for small-molecule fluorescent probes. (A) Chemical target strategy. A chemical targeting group, such as TPP for mitochondria targeting, can be conjugated with the probe. The accumulation of the probe-TPP conjugate in mitochondria is driven by the negative mitochondrial membrane potential. (B) Protein tag targeting strategy. This is a two-step strategy. Protein tagging systems, such as Halo and SNAP, can be fused with a POI that is specifically expressed in certain organelles. Then, a conjugate between the probe and the protein tag substrate will react with the organelle-specific protein tag to achieve organelle specificity. TPP, triphenlyphosphonium; POI, protein-of-interest.