Abstract
The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants.
Highlights
Intracellular sensing by using fluorescent probes is a well-established approach to monitor relevant biological processes at the cellular level
To achieve the objectives showed in the introduction, our new probe must be sensitive to both biothiol and phosphate concentrations in an independent but simultaneous way and exhibit emission in the red spectral range
Free cysteine and homo-cysteine may be found in the cell cytoplasm, their relative abundance is usually lower than 10% that of GSH; may be found in the cell cytoplasm, their relative abundance is usually lower than 10% that of we focused our experiments in solution on the response of the probe to GSH
Summary
Intracellular sensing by using fluorescent probes is a well-established approach to monitor relevant biological processes at the cellular level. Understanding cellular function in terms of metabolism, differentiation, homeostasis, gene expression, or inter-cellular communication is a major aim for many interdisciplinary research branches, since these processes may be strongly affected by pathological states, such as neurodegenerative diseases or cancer [1,2]. A thorough understanding of how diseases work at the molecular and cellular levels will provide an invaluable background to set the basis for new therapeutic tools. The widespread use of fluorescent protein mutants [4] and advanced microscopy techniques, such as super-resolution nanoscopy [5], has resulted in a substantial boost of fluorescence-based cellular sensing.
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