Dye displacement assay

There are several other techniques Uke the fluorescent dye displacement assays, footprinting, Fourier transform infrared spectroscopy. X-ray crystallography, electron microscopy, confocal microscopy, atomic force microscopy, surface plasmon resonance etc used for hgand-DNA interactions that are not discussed here. 

Reversible dye binding systems can be developed into colourimetric and fluorescent assays for analytical detection. As shown in Figure 11.3, two different binding schemes can be envisioned, (a) competitive dye/analyte inclusion, and (b) cooperative dye/analyte inclusion. The competitive inclusion process is the basis for the dye displacement assay where the analyte displaces the dye from the container molecule. For this process to be visualized, the properties of complexed and uncomplexed dyes must be markedly dif-... 

Recent work involves the use of a 2,2 6,2" terpyridine scaffold with a metal center and pendant guanidinium groupsJ A dye displacement assay was employed in the determination of binding affinities for amino acids to the cavity of 30. The receptor was selective for aspartate with an affinity 1.5 x 10 M in 1 1 water/methanol. Studies with the control host in which the guanidinium groups are absent indicate that the selectivity for aspartate is due to the interactions with the Zn(II) center and the guanidinium moieties. 

A dye displacement assay is primarily a colorimetric competitive binding sensor system where an analyte displaces a dye from a receptor this displacement results in some color change that can be related to the amount of analyte present. Pioneering reports in this arena include the seminal protocols of Anslyn " and Buryak and Severin. ... 

Ehnas had detailed a temperature-sensitive copolymer, which contained boronic acid units its interaction with ARS and loss of flnorescence upon exposure to a series of analytes was discnssed. " However, boronic acid containing hydrogels that had been developed for electrophoresis applications (discnssed later) were reasoned to be snit-able in the stationary phase for a similar supported system. Thus, this platform allows a dye displacement assay to be performed. " ... 

Indicator displacement assays (IDAs) - or, in the specific case of fluorescent indicators, F-IDAs - are based on the next alternative concept described here. A receptor with an affinity for a given analyte is loaded with an indicator, usually a fluorescent or colored dye, whose spectral properties undergo a change upon complexation with the receptor. Treatment of this indicator-receptor complex with the analyte results in the displacement of the indicator from the receptor and a restoration of the indicator s original spectral properties, indirectly reporting analyte coordination (Fig. 27). For effective detection, two main requirements have to be fulfilled (1) the receptor/indicator interaction must be reversible and weaker than the interaction of the receptor with the designated analyte and (2) the indicator must show significantly different optical properties when bound to the receptor and when freely dissolved in solution. 

Nau WM, Ghale G, Hennig A et al (2009) Substrate-selective supramolecular tandem assays monitoring enzyme inhibition of arginase and diamine oxidase by fluorescent dye displacement from calixarene and cucurbituril macrocycles. J Am Chem Soc 131 11558-11570... 

Greene and Shimizu [77] have reported a displacement assay using a colorimetric dye f/V,/V-dimethyI-/V./V-("7-nitro-2.1,3-benzoxadiazol-4-yl)-l, 2-ethanediamine) with absorption maximum at 460 nm. This assay allowed the determination of seven different aromatic amines. Nevertheless, although the dye seems to be more sensitive in the polymer than in solution, after thorough optimization of the assay conditions, the displacement is only significant for the mmol L 1 amine concentration range. 

The binding of citrate by 4.28 has been investigated by the indicator displacement assay (IDA) method (cf. EDTA titrations, Section 3.1.3). Binding of an organic dye indicator with comparable structure to citrate competes with the substrate for the same binding site. The binding of the indicator... 

The Anslyn group has pioneered supramolecular displacement assays as a distinctive method of analysis. A typical example used a tripodal molecule, or podand, to determine the amount of citrate in drinks and the quality of wine made from Pinot Noir grapes [7], The initial work on citrate utilized a complex between a tris(guanidinium) derivative of 1,3,5-triethylbenzene with a fluorescent dye, car-boxyfluorescein. In the presence of citrate the dye is ejected and replaced with the... 

Artificial receptors can be converted into sensors by covalent attachment of a signaling unit such as a fluorescent dye. An interesting alternative are soindicator displacement assays (IDAs) [9]. These assays are based on receptors that are bound to dyes (or fluorescent ligands) via noncovalent interactions. Upon addition of an analyte, the dyes are displaced, which results in a change of their optical properties. These changes can be used to identify and/or quantify the analyte. 

Figure 23 Structures of (a) resin-bound receptors 73a-73e, (b) (phosphorylated) peptide guests, and (c) dyes employed in the indicator displacement assays.

A competitive assay (indicator displacement assay, IDA) was reported by Anslyn using the Zn(II) complex 49 shown in Figure 21. This 2,2 6,2"-terpyridine complex forms a chemosensing ensemble with pyrocatechol violet because of the ability of the dye to chelate the Zn(II) cation. On addition of a series of unprotected amino acids to a solution of the chemosensing ensemble, the release of the dye was visually verified and spectroscopically measured (1 1... 

Artificial binding pockets with receptors can also be used in selective colorimetric displacement assays (Figure 25). After functionalization of the porous host with adequate binding sites, the latter can be loaded with a dye that coordinates to these anchored binding sites. In the presence of an analyte that forms a stronger complex with those inner binding sites, a displacement of the dye and diffusion into the bulk solution takes place, thns lesnlting in the colori-... 

Figure 25 The principle of a colorimetric displacement assay with functionahzed mesoporous materials (a) mesoporous material with (b) pores covalently modified with receptor groups binds dye with (c) complementary charge to form (d) the hybrid receptor-dye sensory material (e) a competent analyte can displace the dye to form (f) an analyte-receptor complex (g) the appearance of the desorbed dye serves as an output signal. (Redrawn from Ref. 106. Royal Society of Chemistry, 2008.)...

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