Overview of Strategies in Fluorescence Sensing
The first step to any successful fluorescence sensing technology is the proper choice of optimal strategy. In this Chapter we focus on different strategies for obtaining the quantitative information on sensor-target interaction. There are different possib
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Overview of Strategies in Fluorescence Sensing
The first step to any successful fluorescence sensing technology is the proper choice of optimal strategy. In this Chapter we focus on different strategies for obtaining the quantitative information on sensor-target interaction. There are different possibilities for that, and the technologies that are currently in use are critically analyzed. They include (a) the labeling of all pool of potential targets as it is commonly used in DNA hybridization assays, (b) competitor displacement assays that can be applied to a broader range of sensor-target affinities, (c) sandwich assays, the most developed of which are the immunoassays and (d) direct reagent-independent sensing. The latter is the most advanced strategy that still is difficult to realize. Finally, the reader will find the Section “Sensing and thinking” with the list of questions and problems addressed to the readers. The specific interaction between the sensor and its target can proceed in solutions and also when one of the partners is immobilized on solid support. The problem is to visualize this interaction in the most convenient and efficient way. In previous Chapter we introduced the basic concepts of sensing, the background of which is the molecular recognition and which can be realized in heterogeneous and homogeneous formats. A general sensor design was presented, the functional elements of which are the receptor, transducer and emitter. Among different technologies of molecular sensing that use different physical principles of detection we outlined the fluorescence technology and highlighted the broad range of its possibilities. Here we focus on different strategies for extracting the information on sensor-target interaction and on the possibilities for its transformation into quantitative target assay.
2.1 Labeling Targets in Fluorescence Assays Historically, fluorescence sensing came into play to substitute the radioactive isotope detection technique and to a large extent borrowed its methodology. Radioisotopes are insensitive to intermolecular interactions and all the methods based on their © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_2
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2 Overview of Strategies in Fluorescence Sensing
usage allow only the quantitative detection of their concentration in the desired locations. Their substitution by fluorescent dyes made the analysis cheaper, safer and much more sensitive. Many fluorescence technologies were developed following this line. Here the fluorescence emission of the dyes indicates the presence of a given target compound in the analyzed system and provides quantitative measure of this compound. Such dyes are in fact a part of the target system but not of the sensor system. In this technology, the whole pool of potential targets has to be labeled (Fig. 2.1). Then, after reacting with the receptor, the receptor-target complexes should be isolated from the pool. If the receptor is immobilized on th
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