Ratiometric sensors

A significant number of fluorogenic sensors are based purely on an increase or decrease of fluorescence. As discussed above, these probes, though often very useful, lack the internal reference that ratiometric sensors provide and require therefore difficult calibration. Especially hydrolytic enzymes are easily monitored by a fluor-ophore that is held in a non-fluorescent stage until the enzyme of interest removes the quencher. Certainly, it is beneficial if the... 

Frigoli M, Ouadahi K, Larpent C (2009) A cascade FRET-mediated ratiometric sensor for Cu2+ ions based on dual fluorescent ligand-coated polymer nanoparticles. Chemistry 15 8319-30... 

Metcalfe has recently used substituents near the Ca binding site in 28 to modulate the sensing range of ratiometric sensors. 

Fig. 19 A PAMAM dendrimer ratiometric sensor synthesised by conjugation of PAMAM den-drimers to a chloride-sensitive probe (BAC) and to a chloride-insensitive dye (TMR)...

In addition, a genetically encoded ratiometric sensor for H2O2 is now available. This probe, named HyPer, incorporates a YEP variant into the prokaryotic H2O2-sensing protein (OxyR) and was used successfully to monitor the formation of H2O2 in single mitochondria within a living cell [8]. 

A ratiometric sensor 4 for Cd " and Zn " based on calix[4]arene bearing 1,2,3-triazole-linked pyrene units has been reported (Fig. 28.2) [30]. Probe 4 showed strong excimer and weak monomer emissions, due to face-to-face jt-interactions. Addition of Cd " or Zn produced a ratiometric fluorescence change, with a decrease in excimer emission and an increase in monomer enussion. This ratiometric behavior is due to the complexation of Cd " and Zn " by the triazole nitrogen atoms, which causes a change in the conformation of the pyrene arms. No other metal ions produced such ratiometric response, confirming the high selectivity of probe 4. 

Chen, Y Zhu, C. Cen, J. Li, J. He, W Jiao, Y Guo, Z. A reversible ratiometric sensor for intracellular Cu2-i- imaging metal coordination-altered FRET in a dual fluorophore hybrid. Chem. Commun. 2013,49,7632-7634. 

Long, L. Zhang, D. Li, X. Zhang, J. Zhang, C. Zhou, L. A fluorescence ratiometric sensor for hypochlorite based on a novel dual-fluorophore... 

Chang CJ, Javorski J, Nolan EM, Shaeng M, Lippard SJ (2004) A tautomeric zinc sensor for ratiometric fluorescence imaging application to nitric oxide-release of intracellular zinc. Proc Natl Acad Sci USA 101 1129-1134... 

Li H, Xu J, Yan H (2009) Ratiometric fluorescent determination of cysteine based on organic nanoparticles of naphthalene-thiourea-thiadiazole-linked molecule. Sensor Actuat B-Chem... 

With further understanding how molecular rotors interact with their environment and with application-specific chemical modifications, a more widespread use of molecular rotors in biological and chemical studies can be expected. Ratiometric dyes and lifetime imaging will enable accurate viscosity measurements in cells where concentration gradients exist. The examination of polymerization dynamics benefits from the use of molecular rotors because of their real-time response rates. Presently, the reaction may force the reporters into specific areas of the polymer matrix, for example, into water pockets, but targeted molecular rotors that integrate with the matrix could prevent this behavior. With their relationship to free volume, the field of fluid dynamics can benefit from molecular rotors, because the applicability of viscosity models (DSE, Gierer-Wirtz, free volume, and WLF models) can be elucidated. Lastly, an important field of development is the surface-immobilization of molecular rotors, which promises new solid-state sensors for microviscosity [145]. 

Haidekker MA, Brady TP, Lichlyter D, Theodorakis EA (2006) A ratiometric fluorescent viscosity sensor. J Am Chem Soc 128 398-399... 

Milich KN, Akers W, Haidekker MA (2005) A ratiometric fluorophotometer for fluorescence-based viscosity measurement with molecular rotors. Sensor Lett 3 237-243... 

Flanson GT, McAnaney TB, Park ES, Rendell ME, Yarbrough DK, Chu S, Xi L, Boxer SG, Montrose MH, Remington SJ (2002) Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application. Biochemistry 41 15477-15488... 

Center for Healthcare Technologies at Lawrence Livermore National Laboratory in Livermore, potentially capable to measure pH at or near the stroke site29. The probe is the distal end of a 125 pm fibre tapered up to a diameter of 50 pm. A fluorescent pH-indicator, seminaphthorhodamine-1-carboxylate, is embedded inside a silica sol-gel matrix which is fixed to the fibre tip. Excitation of the dye takes place at 533 nm and the emission in correspondence of the acid (580 nm) and basic (640 nm) bands are separately detected. The use of this ratiometric technique obviates worrying about source fluctuations, which have the same effects on the two detected signals. The pH sensor developed was first characterised in the laboratory, where it showed fast response time (of the order of tens of seconds) and an accuracy of 0.05 pH units, well below the limit of detection necessary for this clinical application (0.1 pH units). The pH sensor was also tested in vivo on rats, by placing the pH sensor in the brain of a Spraque-Dawley rat at a depth of approximately 5 mm30. 

The chromophore environment can affect the spectral position of the absorption and emission bands, the absorption and emission intensity (eM, r), and the fluorescence lifetime as well as the emission anisotropy, e.g., in the case of rigid matrices or hydrogen bonding. Changes in temperature typically result only in small spectral shifts, yet in considerable changes in the fluorescence quantum yield and lifetime. This sensitivity can be favorably exploited for the design of fluorescent sensors and probes [24, 51], though it can unfortunately also hamper quantification from simple measurements of fluorescence intensity [116], The latter can be, e.g., circumvented by ratiometric measurements [24, 115],... 

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