Fluorescence extrinsic fluorescent labels

Fluoroimmunoassays comprise a subclass of extrinsic labehng methods where various selective antigen (Ag)- antibody (Ab) immunoassay fluorescent labeling schemes yield a emission signal. One common scheme involves an enzyme-linked immunosorbent assay (ELISA) depicted in Figure 11.2 where the free Ab is tagged with a fluorophore. Numerous analytes can be detected via these types of selective lock-and-key methods. ... 

Fluorescence measurements on proteins require both an appropriate fluorescence technique and the presence of a suitable fluorophore. The techniques used for the application of fluorescence to proteins are described later in this article. In this section, we briefly consider three classes of fluorophores that are used widely to study proteins native fluorophores including fluorescent amino acids, extrinsic fluorescent labels, and auto-fluorescent proteins. Each has advantages for probing proteins and has distinct drawbacks No perfect fluorophore exists for studying proteins. 

Fig. 5.4. Optical Fiber biosensor, (a) Extrinsic optical fiber is used for the guiding the light to and from the sensor area, (b) Intrinsic the receptor molecules are immobilized on the fiber core after decladding of the fiber. The detection is based on fluorescence labels.

Among nonisotopic techniques, fluorescence (both intrinsic and extrinsic) offers a convenient mode of detection, and the sensitivity of some fluorescent labels is comparable to that of radiolabeled iodine. Recent innovations include the use of polarized light for excitation, such that the degree of polarization of the emission as well as its intensity can provide information about the concentration and size-related behavior (e.g., rotational diffusion) of the fluorescent-labeled molecule. One disadvantage of steady-state fluorescence techniques is that many analytical samples either autofluoresce or quench the fluorescence of the substance of interest. A recent development that circumvents this problem utilizes long-lived fluorophores such as the lanthanide metal ions as labels. Detection is time resolved and data are collected after the decay of spurious or otherwise unwanted fluorescence, i.e., after 100-200 psec. 

Elsewhere in this volume Millhauser et al. have discussed the application of nitroxide electron paramagnetic resonance (EPR) spin labels to the study of the structure and dynamics of biopolymers. Another type of EPR spin label that also is useful for investigating biopolymer systems is provided by the photoexcited triplet state of an intrinsic chromophore, because a triplet state carries electronic paramagnetism. A major advantage of the photoexcited triplet state of an intrinsic chromophore over an extrinsic spin label such as a nitroxide adduct is the relatively small structural perturbation caused by the former, which consists only of a localized electronic excitation. Although not as widely exploited as fluorescence, the phosphorescence of proteins, originating from the photoexcited triplet state, has received a great deal of attention. EPR afficionados have a natural attraction to photoexcited triplet states that dates back to the... 

Fluorescein isothiocyanate (FITC) and dansyl-chloride were among the first extrinsic fluorescent labels for proteins used for immunofluorescence microscopy and polarization measurements. Fluorescein and rho-damine labels were extensively employed due to their bright emission in the visible range. These probes have drawbacks including hydrophobicity, small Stokes shifts, and sensitivity to pH and photobleaching, which led to the development of new dyes such as Alexa, Bodipy, and cyanine dye families (see Table 1). These dyes cover a broad... 

Extrinsic probes must be used when the system under study has no useffil intrinsic reporter groups or a reaction produces no fluorescence change from intrinsic reporter groups. In such cases one may use an appropriate fluorescent group which is added to the system to report the reaction. Extrinsic reporter groups come in many forms such as noncovalently bound fluorescent labels. Many of the latter can be covalently attached to the protein of interest at a specific site. 

Fluorescent probes are relatively small molecules that are used to label biomolecules such as proteins, antibodies and nucleic acids. They contain functional groups and specific physical and chemical characteristics that confer suitability for their use as detection moieties. To date, thousands of fluorescent probes are known each with varying spectral properties. Fluorophores may be intrinsic or extrinsic in nature. Intrinsic fluorophores are naturally occurring whereas extrinsic fluorophores are added to generate a fluorescence signal to facilitate measurement of a specific target molecule. Fluorescent labels have provided excellent sensitivity for a range of assay systems that can be applied to the determination of almost any analyte. 

As nonconjugated and nonaromatic polymers are nonfluorescent, studies of their phase behavior using fluorescence spectroscopy require the use of extrinsic fluorescent labels [38-40]. These can be either selectively dissolved into the polymer phases [41] or, most commonly, attached covalently to the polymer chains [42-47]. A criticism that is often made of using extrinsic fluorescent probes is the possible local perturbation induced by the probe itself on the nanoenvironment to be probed. In order to minimize such perturbation, the size and shape of the probe should be chosen so as to cause the minimum possible perturbation on the probed region. 

Extrinsic fluorescent labels have also been used in fluorescence studies of diblock copolymers, as they tend to self assemble into a diversity of microphase-separated ordered structures such as lamellae, spheres, and cylinders [52-57]. 

Fluorescence spectroscopy is also particularly well-suited to clarify many aspects of polymer/surfactant interactions on a molecular scale. The technique provides information on the mean aggregation numbers of the complexes formed and measures of the polarity and internal fluidity of these structures. Such interactions may be monitored by fluorescence not only with extrinsic probes or labelled polymers, but also by using fluorescent surfactants. Schild and Tirrell [154] have reported the use of sodium 2-(V-dodecylamino) naphthalene-6-sulfonate (SDN6S) to study the interactions between ionic surfactants and poly(V-isopropylacrylamide). 

If the soluble protein that specifically adsorbs to the fiber can be extrinsi-cally labeled, the background problem can be avoided. Of course, in vivo proteins cannot be labeled. However, it is conceivable that a protein labeled with a bulky extrinsic group (e.g., fluorescent dextrans) could be confined by a molecular sieve membrane (e.g., a dialysis membrane) within a closed volume surrounding the specifically derivatized optical fiber. When exposed to the (unlabeled) protein in the biological fluid under investigation, the membrane-clad fiber would allow some unlabeled protein to permeate in and... 

Both the physics and the chemistry of proximity to a surface can alter the excited-state lifetime and rotational motion of a fluorescent molecule. An extrinsic label attached to BSA has been found to reduce its fluorescence lifetime upon BSA adsorption to fused silica.(95) The decrease is too large to arise from the physical near-field proximity effects discussed in Section 7.3 ... 

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

Femtosecond spectroscopy has an ideal temporal resolution for the study of ultrafast water motions from femtosecond to picosecond time scales [33-36]. Femtosecond solvation dynamics is sensitive to both time and length scales and can be a good probe for protein hydration dynamics [16, 37-50]. Recent femtosecond studies by an extrinsic labeling of a protein with a dye molecule showed certain ultrafast water motions [37-42]. This kind of labeling usually relies on hydrophobic interactions, and the probe is typically located in the hydrophobic crevice. The resulting dynamics mostly reflects bound water behavior. The recent success of incorporating a synthetic fluorescent amino acid into the protein showed another way to probe protein electrostatic interactions [43, 48]. 

The selection of extrinsic fluorescent probe is driven by the consideration of which biological macromolecule or lipid is to be labelled, the requirement for compatibility between the intended fluorescent probe (in terms of solubility in water, pH sensitivity and so on) and the properties of the molecule to be labelled. Also, choice of the fluorescent probe should be consistent with experimental objectives. For instance, FRET experiments require that extrinsic donor and acceptor fluorophores should be properly matched for their capacity to participate in the FRET effect (see Section 4.5.4). 

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