Turn-on fluorescence detection

Chart 13.2 Chemical structure of 2,4,6-trinitrotoluene, TNT, an explosive constituent of landmines. 

Adapted from Chen et al. [19] with permission from the National Academy of Sciences USA. 

a protein functionalized with a quencher, Q, is linked to the polymer by electrostatic interaction so that initially fluorescence is quenched. When added protease cleaves a specific bond in the peptide chain, the quencher is released into solution and fluorescence is restored. 

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Addition of the bioagent should result in removal of the QTL from the polymer and a turning on of its fluorescence. Thus, the biosensing using this approach would result in a turn-on fluorescence detection. 

Dodani SC, He QW, Chang CJ (2009) A turn-on fluorescent sensor for detecting nickel in living cells. J Am Chem Soc 131 18020-18021... 

Another probe (25) for the discrimination of Cys from Hey and GSH was also designed by utilizing remarkable difference in reactivity toward Cys, Hey and GSH. The reaction between 25 and Cys produces an amino-substituted BODIPY, exhibiting a yellow turn-on fluorescence response. In contrast, the response of 25 to Hey or GSH introduces a red turn-on fluorescence signal, due to the formation of sulfenyl-substituted BODIPY. These distinguishable fluorescence turn-on responses allow the differentiation of Cys over Hey and GSH (Scheme 7.22b). Moreover, probe 25 was successfully utilized for the detection of Cys in living cells and in monitoring cystathionine y-lyase activity in vitro. 

The fundamental fluorescence turn-on sensor described above for biomolecule detection can be used in many different formats for homogeneous assays. For example, one attractive formulation is a reverse assay (Fig. 2) wherein a molecule similar in structure and function to the ligand is sensed by having the fluorescent polymer and the QTL bioagent complex together. In this case,... 

Near-field scanning microwave microscopy (NSMM) is another system that has been used for label-free detection of both DNA and RNA molecules (42). NSMM monitors the microwave reflectance, a factor that depends on the dielectric permittivity profile across the microarray surface (42). This parameter is, in turn, dependent on the length and surface coverage of the strands, as well as on the hybridization state of the molecules (e.g., unhybridized single-stranded probe vs. hybridized duplex). NSMM technique demonstrated an acceptable resolution (potentially less than 50 pm) and comparable sensitivity to the fluorescent detection (42). 

The ions are electrostatically deflected after passing the interaction/detection region, and detected by a microchannel plate. The time difference between the photon and the ion depends on the position of the ion when emitting the photon. An improvement by imaging the fluorescence on a position sensitive detector, yielding a determination of decay position to less than 1 mm and thus a time resolution of 20ns, made it possible to do measurements on ion beams of less than lOOs T However, this method is limited by the maximal count-rate allowed on the channel plates. An alternative is to have a selective deflection, so that only when a photon has been detected the deflection is turned on and the ion detected. 

If a tube lens is in the fluorescence detection path, the beam configuration may be slightly different than that shown in Fig. 5.90. The microscope may also have additional lenses in the beam path to project an image on a camera, or to increase the light-collection area of direct detection. In any case, there is a simple way to find the image of the microscope lens behind the field lens Turn on the microscope lamp in the transmission beam path, so that the condenser lens fully illuminates the aperture of the microscope lens. The image of the microscope lens can then easily be found by holding a sheet of paper behind the field lens. 

As we go from microfluidics to nanofluidics, the distinction between the scalar-based and particle-based methods becomes obsolete, and in turn, the small dimensions of the microfluidic and nanofluidic channels also allow for increased sensitivity in the fluorescence detections. This, along with the invention of intercalating cyanine dyes, spurs other major applications of fluorescence measurements in microfluidics and nanofluidics the investigation on individual DNA molecules. 

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