Search

The phenolic p[K.sub.a] of fluorescein varies depending on its environment. The fluorescence of the dye varies likewise. Accordingly, a change in fuorescence can report on the association of a fluorescein conjugate to another molecule. Here, we demonstrate how to optimize this process with chemical synthesis. The fluorescence of fluorescein-labeled model protein, bovine pancreatic ribonuclease (RNase A), decreases upon binding to its cognate inhibitor protein (RI). Free and RI-bound fluorescein-RNase A have p[K.sub.a] values of 6.35 and 6.70, respectively, leaving the fluorescein moiety largely unprotonated at physiological pH and thus limiting the sensitivity of the assay. To increase the fluorescein p[K.sub.a] and, hence, the assay sensitivity, we installed an electron-donating alkyl group ortho to each phenol group. 2',7'-Diethylfluorescein (DEF) has spectral properties similar to those of fluorescein but a higher phenolic p[K.sub.a]. Most importantly, free and RI-bound DEF-RNase A have pKa values of 6.68 and 7.29, respectively, resulting in a substantial increase in the sensitivity of the assay. Using DEF-RNase A rather than fluorescein-RNase A in a microplate assay at pH 7.12 increased the Z'-factor from -0.17 to 0.69. We propose that synthetic 'tuning' of the pKa of fluorescein and other pH-sensitive fluorophores provides a general means to optimize binding assays.

We report an online, optical injection interface for capillary zone electrophoresis (CZE) based upon photophysical activation of a caged, fluorogenic label covalently attached to the target analyte. This injection interface allows online analysis of biomolecular systems with high temporal resolution and high sensitivity. Samples are injected onto the separation capillary by photolysis of a caged-fluorescein label using the 351-364 nm irradiation of an [Ar.sup.+] laser. Following injection, the sample is separated and detected via laser-induced fluorescence detection at 488 nm. Detection limits for online analysis of arginine, glutamic acid, and aspartic acid were less than 1 nM with separation times less than 5 s and separation efficiencies exceeding 1 000 000 plates/m. Rapid injection of proteins was demonstrated with migration times less than 500 ms and 0.5 nM detection limits. Online monitoring was performed with response times less than 20 s, suggesting the feasibility of this approach for online, in vivo analysis for a range of biologically relevant analytes.

This work describes the fabrication and evaluation of a poly(dimethyl)siloxane (PDMS)-based device that enables the discrete injection of a sample plug from a continuous-flow stream into a microchannel for subsequent analysis by electrophoresis. Devices were fabricated by aligning valving and flow channel layers followed by plasma sealing the combined layers onto a glass plate that contained fittings for the introduction of liquid sample and nitrogen gas. The design incorporates a reduced-volume pneumatic valve that actuates (on the order of hundreds of milliseconds) to allow analyte from a continuously flowing sampling channel to be injected into a separation channel for electrophoresis. The injector design was optimized to include a pushback channel to flush away stagnant sample associated with the injector dead volume. The effect of the valve actuation time, the pushback voltage, and the sampling stream flow rate on the performance of the device was characterized. Using the optimized design and an injection frequency of 0.64 Hz showed that the injection process is reproducible (RSD of 1.77%, n = 15). Concentration change experiments using fluorescein as the analyte showed that the device could achieve a lag time as small as 14 s. Finally, to demonstrate the potential uses of this device, the microchip was coupled to a microdialysis probe to monitor a concentration change and sample a fluorescein dye mixture.

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(super 2+) sensors, are described. It was found that by combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes.

The comparison of the cellular uptake and localization of a Ru-octaarginine conjugate with and without an appended fluorescein is presented. Attaching fluorescein tag to Ru-octaarginine has lowered the threshold concentration needed for diffusing cytoplasmic labeling and nuclear entry and hence the localization of the fluorophore-bound peptide cannot serve as a proxy for that of the free peptide.

A spectrophotometrical study was conducted to determine the four overlapping p[K.sub.a] values of carboxyfluorescein (CF) in water by using both absorbance diagrams and chemometric analysis. The p[K.sub.a] values obtained are consistent with values for fluorescein, while the additional p[K.sub.a]3 = 4.9 [plusmn] 0.1, very close to that of the less acidic carboxy group of terephthalic acid.

Steady-state and time-resolved fluorescence spectroscopy are used for studying the excitation energy transfer between the dye pair acriflavine to rhodamine-6G in various polymers. The study of acceptor dynamics under energy transfer has shown that the rise time of the acceptor has depended on the nature of the polymer microenvironment.

The development of a mild two-step synthesis of asymmetric 2'-carboxyethyl-substituted fluoresceins and other fluorescein analogues is reported. The reaction involved substituted phthalic anhydride and a carboxyethyl substituted resorcinol analogue followed by a condensation with a second resorcinol analogue.

A frequency domain approach is used for proving the existence of single-molecule surface-enhanced Raman spectroscopy (SMSERS). The existence is demonstrated by using two isotopologues of Rhodamine 6G offering unique vibrational signatures.

Various experiments and methods are used for examining Rhodamine 6G (2-[3'-(ethylamino)-6'-(ethylimino)-2',7'-dimethyl-6'H-xanthen-9'-yl]-benzoic acid) bound to the 5'-end of a 20 base pair long DNA duplex. These two methods have provided a consistent and detailed molecular description of the structural and dynamical heterogeneity.

The influence of PDC-109 on the integrity of differently composed lipid vesicles is investigated. The result presents the oppositional effect of PDC-109 during sperm cell genesis.

Fluorescence correlation spectroscopy (FCS) is used to investigate the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. The micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, with respect to the dye-exchange dynamics.

A comprehensive study of the short and intermediate time scale dynamics of the photoacid pyranine is presented using visible pump-probe spectroscopy in various solvents to elucidate the nature of its proton-kinetics in water. The results are consistent with the earlier reports but unambiguously identify and quantitatively measure the Stokes shift as a separate and distinct phenomenon from the deprotonation process.

A facile synthetic route utilizing readily available reagents is used in a study to develop a series of regiosomerically pure xanthene dye derivatives. Advantages of the method include relatively mild conditions and good to excellent yields.