Absorption molecular probes

The squaraine rotaxanes based on the macrocycle 16b exhibit intense NIR absorption and emission maxima, and it should be possible to develop them into molecular probes for many types of photonic and bioimaging applications. In contrast, the squaraine fluorescence intensity is greatly diminished when the dye is encapsulated with macrocycle 18. The fluorescence is restored when a suitable anionic guest is used to displace the squaraine dye from a pseudorotaxane complex, which indicates that the multicomponent system might be applicable as a fluorescent anion sensor. 

In order to prepare successful NIR molecular probe dyes, NIR dyes must meet the following criteria adequate response to analytes, high lipophilicity and/or reactive functional groups, absorbance maxima compatible with available laser diodes, high fluorescence quantum yield, molar absorptivity, and high photostability. 

The absorption and emission maxima from this table will provide clues to the spectral ranges that are useful for excitation and for fluorescence detection with a particular fluorochrome. However, the absorption and emission spectra have breadth, with slopes and shoulders and secondary peaks (see Fig. 5.6). With efficient fluorochromes, excitation and fluorescence detection at wavelengths distant from the maxima may be possible. Therefore, inspection of the full, detailed spectra is necessary to get the full story. In addition, spectra may shift in different chemical environments (this will explain why maxima vary in different reference tables from different sources). Values in this table are derived primarily from the Molecular Probes Handbook and the article by Alan Waggoner (Chapter 12) in Melamed et al. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

Recent advances in the development of non-invasive, in situ spectroscopic scanned-probe and microscopy techniques have been applied successfully to study mineral particles in aqueous suspension (Hawthorne, 1988 Hochella and White, 1990). In situ spectroscopic methods often utilise molecular probes that have diagnostic properties sensitive to changes in short-range molecular environments. At the particle-solution interface, the molecular environment around a probe species is perturbed, and the diagnostic properties of the probe, which can be either optical or magnetic, then report back on surface molecular structure. Examples of in situ probe approaches that have been used fruitfully include electron spin resonance (ESR) and nuclear magnetic resonance (NMR) spin-probe studies perturbed vibrational probe (Raman and Fourier-transform IR) studies and X-ray absorption (Hawthorne, 1988 Hochella and White, 1990 Charletand Manceau, 1993 Johnston et al., 1993). 

Recently, BOCILLIN FL, a fluorescent penicillin, was synthesized for the detection and characterization of PBPs [34]. BOCILLIN FL, a derivative of penicillin V, is an orange solid with extinction coefficient of 68,000 M 1 cm1 and a maximal absorption at 504 nm (Molecular Probes, Inc.). It fluoresces at 511 nm upon excitation at 504 nm. BOCILLIN FL has been used to detect PBPs from the membrane preparations of several bacterial species, including S. pneumoniae, E. coli, and P. aeruginosa (Fig. 3). A typical procedure of using BOCILLIN FL for the detection of PBPs involves preparation of the bacterial cytoplasmic membranes, incubation of the membrane preparations with BOCILLIN FL, and visualization of PBPs by a UV transilluminator or with the aid of a... 

The excitation spectrum of bound ethidium bromide published by Molecular Probes (Figure 12.5) indicates that, at saturation, fluorophore in the presence of DNA does not absorb in the region between 400 and 420 nm. Also, we can see from the absorption spectra of ethidium bromide recorded at different concentrations of DNA (Figure 12.3) that the OD... 

The extreme sensitivity of the visible absorption spectrum to small changes in the surrounding medium has made this betaine dye a useful molecular probe in the study of micellar systems [298, 299, 443-445], mieroemulsions and phospholipid bilayers [299], model liquid membranes [300], polymers [301, 446], organic-inorganie polymer hybrids [447], sol-gel matrices [448], surfaee polarities [449-451], and the retention behaviour in reversed-phase liquid chromatography [302]. Using polymer membranes with embedded betaine dyes, even an optical alcohol sensor has been developed [452]. 

It has been suggested that the first step of reaction (6) may be the formation of a carboxylic species COOHads. Carboxyl radicals have indeed been observed by Zhu et al." for potentials lower than 0.65 V using Fourier Transform infrared Reflectance Absorption Spectroscopy with the Attenuated Total Reflection mode (ATR-FTtR). Moreover Anderson et al." made numerical simulation which indicated that the formation of an adsorbed carboxylic species was energetically more favorable. Here, it has to be noted that the electro-oxidation of CO being a stracture sensitive reaction (sensitive to the superficial stracture symmehy" and to the presence of surface defects) this species can be used to study the activity of a catalyst but also as a molecular probe to characterize the catalytic surface. ... 

The main focus of this study is to utilize excimer formation between pyrene groups attached to PEG chain ends as a molecular probe of intermolecular complex formation. The change upon complexation was monitored by UV-vlsible absorption, excitation, and fluorescence spectroscopies as well as by fluorescence lifetime measurements. 

Scheme 3.2. Molecular probes used for UV absorption and fluorescence monitoring of tbe reactions for network formation.

One molecular probe, which, in theory, has the specificity and low frequency transitional resolution desired is infrared (IR) spectroscopy. Although this technique has been used to observe polymer transitions (21-42), it has not been used as often or with the success of NMR and ESR. An IR absorption s intensity, band shape, and frequency may change with temperature. There are two extremes of interpretation of the spectroscopy results. Either thermal expansion affects the inherent nature of the dipole moment change, or the concentration of the absorbing species changes with temperature. These two effects can occur simultaneously. This often precludes a straightforward analysis. 

Quasi-elastic Laser Scattering (QELS) provides a versatile tool for monitoring in situ the frequency of thermally induced capillary waves at the Uquid/hquid junction [26, 27]. Unlike the previous techniques based on an absorption/fluores-cence signal arising from molecular probes at the interface, QELS is not sensitive to specific molecules but to the interfacial tension of the molecular junc-... 

Molecular probes are also useful. These are small molecules with a measurable physical property such as fluorescence, visible absorption, n.m.r., or... 

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