A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

Abstract

Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging. Often, a small probe-product spectral separation leads to crosstalk, but the rational development of ratiometric probes with zero probe-product crosstalk remains challenging. Harnessing the recent progress on photophysical modulation of xanthenoid fluorochromes, we propose a powerful and versatile probe design principle, that is, "bridging-group modification," and developed totalROX, a robust probe for monitoring the total cellular oxidative capacity. First, totalRox affirmatively detected the complete set of biorelevant highly oxidative species: per-acids (2 e − ), radicals (1 e − ), nitrosative (NO + ) species, and singlet oxygen ( 1 O 2 ). Nonoxidative or mildly oxidative pro-oxidants, for example, O 2• -, H 2 O 2 , NO, ONOO -, and ClOwere not detected. Second, the absorption/fluorescence maxima of the probe (total-ROX, λ abs /λ em = 425/525 nm) and the detection product (Ox670, λ abs /λ em = 650/675 nm) were separated by ca. 225/150 nm, respectively, which eliminated probe-product crosstalk. Third, it renders the ratiometric signal with a single chromophore and is structurally succinct. TotalROX allowed quantitative analysis and was more sensitive than Amplex Red and CellROX Deep Red, two commercial probes for cell oxidative species. Bioimaging potentials of total-ROX for monitoring cell redox status were exemplified in three different cell lines.

From Spontaneous to Photo‐Triggered and Photo‐Calibrated Nitric Oxide Donors

“…Since he was still in Eric Anslyn's research group as a postdoc, Youjun Yang started to develop molecular tools for chemical biology related to cell redox, especially fluorescent probes. This endeavour has been tremendously fruitful and led to the design of a number of high‐performance probes for NO, [45] ONOO − , [46] oxidative radical, [47] and total oxidative capacity [48] …”

Dual-Emitting Carbonized Polymer Dots Synthesized at Room Temperature for Ratiometric Fluorescence Sensing of Vitamin B12

“…The superior optical probes not only require high sensitivity to analytes but also have high signal-to-noise ratios, especially in complex samples. − The single-fluorescent signal-based sensing system for detecting analytes is usually problematic because of unavoidable interferences including light scattering from the sample, the detection environment, unstable excitation light, and local concentration fluctuation of the optical probe . Considering these drawbacks, a ratiometric fluorescence (FL) sensing system with intrinsic correction is desirable for improving signal-to-noise ratios and the accuracy of determination …”

“…4 Considering these drawbacks, a ratiometric fluorescence (FL) sensing system with intrinsic correction is desirable for improving signal-to-noise ratios and the accuracy of determination. 5 Carbonized polymer dots (CPDs) consist of cross-linked monomers or linear polymers by conjugation or nonconjugation interaction. 6,7 In general, CPDs are mainly prepared by hydrothermal cross-linking polymerization.…”

Polymethine Molecular Platform for Ratiometric Fluorescent Probes in the Second near-Infrared Window

“…27−30 Remarkable NIR-II fluorescence images have been obtained with these fluorophores, and some fluorophores have been pushed to preclinical and clinical in vivo imaging. 31,32 Compared with those short-wavelength fluorophores (<800 nm) for which fluorescence can be conveniently regulated to develop off-on or ratiometric fluorescent probes, 33 most NIR-II fluorophores are always fluorescence on due to the lack of fluorescence regulatory sites. 20,34−36 Only a minority can be modified to construct offon type fluorescent probes.…”