Schemes for Fluorescence Sensing

Many probes are available Dependent on probe concentration 

Sensitive to absorption, scatter, and sample autofluoiescence Sensitive to light lasers, photobleaching, etc. 

Requires UV excitation with most present probes, except 

Insensitive to simple absorption and scatter Insensitive to light losses and optical imperfections Simple and robust instrumentation Easy to measure small changes High accuracy 

Probe development simpler than wavelength ratiometric 

---

The various possible schemes for fluorescence sensing are summarized in Figure 1.1. At present, most fluorescence assays are based on the standard intensity-based methods, in which the intensity of the probe molecule changes in response to the analyte of interest. However, there has been the realization that lifetime-based methods possess intrinsic advantages for chemical sensing. (A more detailed description of... 

Figure 1.1. Schemes for fluorescence sensing intensity, intensity ratio, time-domain, and phase-modulation, from left to right.

Figure 1. Schemes for fluorescence sensing. A - single excitation or emission wavelength intensity, B - dual excitation or emission wavelengths intensity ratio C - measurement of intensity decay D -measurement of fluorescence phase angle and/or modulation.

Fig. 10 Dual photomultiplier scheme for fluorescent detection. EP - emitted photon stream from the sensing volume, PS - fiberoptic power splitter, ND -neutral density Alter, PMTx -photomultiplier, El, E2,...

First, the above-mentioned sensors have major drawbacks, as the detection and recognition event is a function of the nature and characteristics of the side chains, and the side chain functionalization of the CP requires advanced synthesis and extensive purification of numerous monomeric and polymeric derivatives. Second, this generation of sensors primarily employed optical absorption as the source for detection, resulting in lower sensitivity when compared with other sensing systems for biological processes. However, the use of fluorescence detection within these sensing systems could justify continued development. More recent examples include a fluorescent polythiophene derivative with carbohydrate functionalized side chains for the detection of different bacteria [15] and novel synthesis schemes for ligand-functionalization of polythiophenes [16]. 

Figure 11.16 Multishell silica nanoparticle for ratiometric fluorescence sensing of Pb2+ ions, (a) Schematic working scheme of the sensor binding of the Pb2+ ions to the thiol groups present on the surface causes the quenching of the emission of the dansylamide dyes (labeled e when unperturbed and q when quenched) in the outer shell but not of the methoxynaphthalene dyes in the core (labeled r). (b) TEM micrograph of the particles (inset) and ratiometric behavior of the fluorescence emission at different Pb2+ concentrations.83 (Reprinted with permission from M. Arduini et al., Langmuir, 2007, 23, 8632—8636. Copyright 2007 American Chemical Society.)...

Zeller PN, Voirin G, Kunz RE (2000) Single-pad scheme for integrated optical fluorescence sensing. Biosens Bioelectron 15 591-595... 

The functionalized boronic acids (15 and 16) are useful for sensing fluoride ion in aqueous solution. Electrochemical redox is used for the detection of fluoride ion with ferrocenyl-boronic acid (15) [8] and for fluorescence detection with aminoboronic acid (16) [9]. Molecule (16) can effectively detect concentration of fluoride ion in the range of 5-30 mM, where the fluoride adduct is stabilized by the additional hydrogen bonding with protonated amine at pH 5.5 as shown in structure 17 (Scheme 3.35). 

Scheme 5.5 Proposed mechanism for glucose sensing with fluorescent quantum dots.

---