Fluorescence-Based Applications

Fluorescence - the absorption of a photon, followed very rapidly by emission of a photon at longer wavelength - has many practical applications. The intrinsic fluorescent properties of a molecule can be utihsed, or fluorescent tags can be attached. 

Fluorescent sensors have been widely investigated for the detection of many types of compounds and a particularly fruitful field is the selective detection of metal ions for example, a series (Zinpyr) of zinc sensors, based on fluorescein-containing attached chelating ligands, which can be used for quantitative determination of Zn or imaging of zinc-containing biological structures.  

These are added to paper, plastics and detergents to increase the whiteness by absorbing UV light and emitting blue light. Many optical brighteners are stilbene derivatives, but a number of heterocyclic ring types are also important, for example the oxazole and triazole derivatives shown below, 1,3,5-triazines and 

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The group of Blanchard-Desce synthesized a family of molecules based on the general scheme displayed in Fig. 16, in order to deduce wide and comprehensive relationships in structure/physical properties (TPA and fluorescence) in the range 700-900 nm for two-photon excited fluorescence-based applications [98,99]. The influence of the conjugated core, of the length or the nature of linkers on TPA and spectroscopic properties was systematically studied. A TPA cross-section of 4200 GM at 710 nm has been measured, with high two-photon fluorescence properties. 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

Fluorescence lifetime-based applications require probes and labels with environment-sensitive lifetimes, while immunoassays or hybridization-based analysis require fluorescent tracers preferably labeled with a single, mono-reactive fluorescent label. 

Due to their low sensitivity toward the environment, cyanine dyes are perfect candidates as fluorescent labels. Squaraine dyes on the other hand display a highly environment-sensitive response and are therefore not only useful as fluorescent probes and labels but also, in particular, well-suited for lifetime-based applications. 

Fluorescence-based bulk viscosity measurement is one application that advertises itself almost immediately, particularly with julolidine-derived molecular rotors where the quantum yield is widely independent from solvent polarity. Solving (5) for r] and assuming proportionality of quantum yield and emission intensity leads to (13),... 

As it has been discussed above, fluorescent-based sensors have found application to the analysis of every possible species from gases (02, C02,... 

In a second experiment, Cy5-labelled antiBSA antibodies were immobilised on a silanised glass slide precoated with metallic nanoislands using a polydimethylsiloxane (PDMS) flow-cell. The antibody solution was left for 1 hour to attach and then the cell was flushed with deionised water. The slide was then dried with N2. For this experiment, a portion of the slide was not coated with metallic nanoislands, in order to act as a reference. Figure 20 shows the image recorded using the fluorescence laser scanner mentioned previously. The enhancement in fluorescence emission between those areas with and without nanoislands (B and A, respectively) is again evident. For both chips, an enhancement factor of approximately 8 was recorded. There is considerable interest in the elucidation and exploitation of plasmonic effects for fluorescence-based biosensors and other applications. 

As opposed to conventional analytical techniques, optical sensors and biosensors, particularly those employing absorption and fluorescence-based sensing materials potentially allow for measurement through transparent or semi-transparent materials in a non-destructive fashion4, 5> 9 10. Chemical sensor technology has developed rapidly over the past years and a number of systems for food applications have been introduced and evaluated with foods. 

Wolfbeis O.S., Fluorescence-based optical sensors for biomedical applications, In Scheggi A.M.V., Martelluci, S., Chester, A.N., Pratesi, R. (Eds.), Biomedical Optical Instrumentation and laser-Assisted Biotechnology, Kluwer Academic Publishers, 1996, p.327-337. 

Soukka T, Rantanen T, Kuningas K (2008) Photon upconversion in homogeneous fluorescence-based bioanalytical assays. Ann N Y Acad Sci 1130 188-200, Fluorescence Methods and Applications Spectroscopy, Imaging, and Probes... 

Rettig W, Rurack K, Sczepan M (2001) From cyanines to styryl bases - photophysical properties, photochemical mechanisms, and cation sensing abilities of charged and neutral polymethinic dyes. In Valeur B, Brochon JC (eds) New trends in fluorescence spectroscopy applications to chemical and life sciences. Springer, Berlin, pp 125-155... 

An interesting practical application is the detection of the citrate anion in soft drinks, as shown in Box 10.3. The strategy is quite different from that of the preceding examples because the anion receptor is not linked to a fluorophore. The latter simply acts in competition with the citrate anion in a fashion that resembles fluorescence-based immunoassays. 

Neumann M., Herten D.-P. and Sauer M. (2001) New Techniques for DNA Sequencing Based on Diode Laser Excitation and Time-Resolved Fluorescence Detection, in Valeur B. and Brochon J. C. (Eds), New Trends in Fluorescence Spectroscopy. Applications to Chemical and Life Sciences, Springer-Verlag, Berlin, pp. 303-29. 

Perhaps the first detailed discussion of such a technique in fluorescent thermometry (shown in Figure 11.10) was given by Zhang et al. in their work(36) based on both mathematical analysis and experimental simulation. Examples of the electronic design of the corresponding system and the application of the technique in a ruby fluorescence-based fiber-optic sensor system are also listed. This shows that there is no difference in the measurement sensitivity between a system using square-wave modulation and one using sinusoidal modulation. However, the former performs a little better in terms of the measurement resolution. 

The work has shown the diversity of fluorescence-based thermometry schemes, materials, signal processing arrangements, and applications. In addition, a close agreement with theoretical predictions on material performance gives confidence to the assessment of new materials for such uses. Probe sizes are potentially smaller and more convenient due to optical source and detector developments. Thus the future for fluorescence thermometry is bright, with the expectation of a range of new ideas in the field in the future. 

Emission lifetime provides a powerful selectivity dimension as each fluorophore has a unique decay rate. While laboratory fluorescent lifetime measurement systems are widely available and the concept of lifetime LIF sensing has been demonstrated,commercial LIF lifetime sensors are not yet available on the market for PAT and field applications to monitor organic analytes or bioagents. Once available, lifetime LIF sensors could revolutionize fluorescent based monitoring by affording superior detection merits along with sufficient recognition power. 

Currently, there is a need for high-throughput determination of nucleic acid sequences. At present, detection systems most commonly employ fluorescence-based methods. However, wide spread applications of such methods are limited by low speed, high cost, size, and number of incubations steps, among other factors. Application of electrochemical methods in affinity DNA sensors presents likely a promising alternative, allowing miniaturization and cost reduction, and potentially allowing application in point-of-care assays. 

Another redox switchable system is based on dyad 21 in which 2-chloro-1,4-naphthoquinone is covalently attached to 5-dimethyl-aminonaphthalene via a non-conjugated spacer. The intrinsic fluorescence of the dansyl excited state in dyad 21 is strongly quenched, due to the intramolecular electron transfer from the excited dansyl to the adjacent quinone acceptor. However, the fluorescence can be switched on by addition of a reducing agent. Apart from chemical switching, the fluorescence of dyad 21 can also be switched electrochemically. This can be realized using a photoelec -trochemical cell, and the solution starts to fluoresce upon application of a reductive potential.31... 

Methods based on acidic hydrolysis of N—NO bond (e) Fluorescence detection containing organic modifier 20% CH3CN in water NOJ Fluorescence (reduction Applicable onty to 80... 

Williams GJ, Thorson JS (2008) A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution. Nat Protoc 3 357-362... 

The measured signal from an in vivo fluorescence-based sensor depends on both the quantum yield of the fluorophore, which is the ratio of emitted photons to absorbed photons, and the absorption of the excited and emitted light by the surrounding tissue.12-15 While SWNT have a much lower quantum yield than many visible fluorophores, the most important factor for depth of implantation actually turns out to be the absorption coefficient of the surrounding medium.16,17 A one-dimensional absorption-fluorescence model can be used to compare the suitability of fluorophores for in vivo applications ... 

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