Detection by fluorescence measurement

Fluorescence can be used for detection in two different ways. Firstly, the separated compounds could be labeled with a fluorescent label (if not fluorescent as such), or UV absorbing compounds could be visualized as dark bands in matrices to which a fluorescent chromophore was added (fluorescence quenching). A variation of the second method was worked out by Eisinger [201] for polyacrylamide gel the gel was placed between a quartz and a fluorescent glass plate and then illuminated with UV light. Ultraviolet absorbing compounds appear as dark bands on the glass plate. When electrophoresis is done on sihca gel thin layer or cellulose, addition of a fluorescent indicator to the sorbent can be made use of in the conventional way. 

In direct fluorescence of either labeled (dansylated) or unlabeled proteins one has to keep in mind that polyacrylamide gel itself is also a fluorescent material its fluorescence appears when excited at 280 or 340 nm. These wavelengths are very close to the excitation wavelengths of native proteins and dansyl chloride or 

8-anilino-l-naphthalenesulphonic acid-labeled proteins. On the other hand polyacrylamide exhibits maximum fluorescence emission at 340 and 460-520 nm, which is sufficiently far away from fluorescence emission of both unlabeled and labeled proteins. However, the broad fluorescence increases considerably the background. It is also possible to prepare non fluorescent polyacrylamide gels by, e.g., adding dithiothreitol or by replacing TEMED by sulphite [202]. 

Direct measurement of tryptophan fluorescence (280/340), as done by Easton et al. [202] or Isenberg et al. [204], has the sensitivity limit between 1-10 /ng of protein. 

Fluorescence labeling of nucleic acids is limited to double-stranded structures. Both double-stranded DNA and RNA react with ethidium bromide under the formation of a fluorescent product (360/580) [206-208]. The sensitivity of this method is 50 /ig for DNA and 100 jag for RNA. The differentiation between RNA and DNA can be done by using specific nucleases. According to Jovin [209] it is also possible to add ethidium bromide at a low concentration (0.1 relative to DNA phosphorus) before electrophoresis without any effect upon the subsequent separation. 

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Figure 9. Immunological activity of the oriented IgG films detected by fluorescence measurements.

Detection By fluorescence measurements, mass spectrometry, or HPLC. 

In the latter process, rubrene is formed in its excited singlet state (R ) which was detected by fluorescence measurements. 

As far as we know, this is the first molecular probe that includes two different types of reporter units activated upon on a specific stimulus. The other option to achieve dual detection would be to use two separate probes. However, in this case there could be a problem of competitive catalysis (circumstances in which the Km of the two substrate is not identical). In our probe, 6-aminoquinoline and 4-nitrophenol, detected by fluorescence and absorbance spectroscopy, respectively, were used as reporter units. Due to the synthetic flexibility of our approach, other reporter molecules with different types of functional groups, like amine or hydroxyl, can be linked to our molecular probe. The two assays must be orthogonal to each other, in order to prevent disturbances in the detection measurement. Another advantage of the probe is the aqueous solubility... 

Fig. 13.16a. As an atom source, a magneto-optical trap (MOT) for cold Cs-atoms was used. The fluorescence of MOT atoms around the MNF was detected by the measurement of fluorescence photons with an avalanche photodiode connected to one end of the fiber. Signals are accumulated and recorded on a PC using a photon-counting.

Cells are typically concentrated by filtration and extracted into an organic solvent (usually acetone) after which, pigments are detected by fluorescence or absorption spectroscopy, sometimes after chromatographic separation (Bidigare and Trees, 2000). The application of HPLC to phytoplankton pigment analysis has lowered the uncertainty in the measurement of Chi a and accessory carotenoids, since compounds are physically separated and individually quantified. 

Detection of peptides in HPLC can be achieved by measuring natural absorbance of peptide bonds at 200-220 nm. Unfortunately at these wavelengths a lot of food components and also the solvents used for analysis absorb, demanding an intensive sample pretreatment and clean-up [129]. Peptides with aromatic residues can be detected at 254 nm (phenylalanine, tyrosine, and tryptophan) or 280 nm (tyrosine and tryptophan). Taking advantage of the natural fluorescence shown by some amino acids (tyrosine and tryptophan), detection by fluorescence can also be used for peptides containing these amino acids [106]. 

Drug residues in foods that strongly fluoresce can be more efficiently detected by fluorescence detectors. Typically, fluorescence sensitivity is 10-1000 times higher than that offered by a UV detector for strong UV-absorbing materials (125). Using a fluorescence detector, it has been possible to detect the presence of even a single analyte molecule in an LC flow cell. This type of detection is very versatile because of its ability to measure the intensity of the fluorescent radiation emitted from analytes excited by UV. 

A phosphorus-specific thermionic detector was also adapted from GLC (See Section III.3.b) for use with small-bore HPLC columns208,307,330,334. Based on an electrically heated rubidium salt bead, it permits detection limits of 0.2-0.5 ng of phosphorus and its response is linear with the amount of phosphorus over several orders of magnitude. This detector yields good results with phosphates which cannot be detected by UV spectrophotometry or by fluorescence measurements. 

Basic concepts of interaction have been refined as new models could be established. The fields of applications were broadened. Currently, the detection of enantiomers by fluorescence measurements and by direct optical methods are well-investigated topics. By introducing various functional groups to established skeletal structures, highly specific recognition of enantiomers is likely to be achieved. Those methods based on recognition by polymer-bonded selectors will continue to profit from advances in modern chromatographic methods. 

The latter mechanism is met in amine-vinyl monomer systems [41-46] (see Scheme 4). Due to the small n-acceptor ability of normal substituted vinyl monomers, an interaction in the ground-state level does not take place. The exciplexes assumed are detectable in aromatic amine-acrylonitrile (AN) systems by their emission spectra, as is shown in Fig. 1 for typical examples. The emission bands at 350 nm (by JV,JV-dimethyl-p-toluidine (DMT)) and 370 nm (by p-phenylene diamine (TMPD) result from the normal fluorescence of the isolated amine. As can be seen, the intensity of the exciplex emission is much higher in the DMT-AN system. This corresponds to the higher polymerization efficiency of that system (<)>[, by A. = 313 nm and 80 K 0.6 for DMT 0.15 for TMPD [46]). Mainly, the much higher dipole moment of DMT (1.1 D) is responsible for this result. The cation radicals [46] or neutral radicals [42] of the amines formed after PET and proton transfer have been detected by ESR measurements. As expected, the rate of photopolymerization of the systems discussed increases with increasing... 

Besides radionuclides (detected on the basis of beta and gamma radiation), labels detected by the measurement of magnetic resonance have been proposed (spin labels - spin immunoanalysis, abr. SIA). Other labels are fluorescent dyes already... 

Figure 6.5. Fluorescence Detection of Oligonucleotide Fragments Produced by the Dideoxy Method. Each of the four chain-terminating mixtures is primed with a tag that fluoresces at a different wavelength (e.g., blue for A). The sequence determined by fluorescence measurements at four wavelengths is shown at the bottom. [From L. M. Smith, J. Z. Sanders, R. J. Kaiser, P. Hughes, C. Dodd, C. R. Connell, C. Heiner, S. B. H. Kent, and L. E. Hood. Nature 321...

Plasma PLP levels have been used frequently as an indicator of vitamin status. Vitamin compciunds can be separated from each other and measured individually by high-pressure liquid chromatography (UPLC), where, after separation, the vitamins are detected by fluorescence (Tsuge, 1997). Great care needs tt> be taken... 

In these multichromophoric cyclodextrins the fluorophores are randomly oriented. Excitation of one of the naphthoate fluorophores is followed by efficient dipole-dipole excitation energy transfer between the seven fluorophores, with a Forster radius of 14 A. This process is not detectable by fluorescence intensity measurements, as neither the intensity nor the decay law are affected by energy transfer between identical fluorophores (also called homotransfer. The dynamics of energy hopping are on the other hand reflected in the fluorescence anisotropy. To avoid depolarization by rotational motion of the fluorophores, experiments were conducted in a low temperature and optically clear rigid glass (9 1 ethanol-methanol at 110 K). 

Figure 5.5 Fluorescence detection of oligonucleotide fragments produced by the dideoxy method. A sequencing reaction is performed with four chain-terminating dideoxy nucleotides, each labeled with a tag that fluoresces at a different wavelength (e.g., red for T). Each of the four colors represents a different base in a chromatographic trace produced by fluorescence measurements at four wavelengths. [After A. J. F. Griffiths et al., An Introduction to Genetic Analysis, 8th ed. (W. H. Freeman and Company, 2005).]...

Fig. 2 Cell arrays can be applied for screening of protein-protein interactions in mammalian cells. Expression plasmids containing cDNAs for bait, reporter, and prey proteins (A) are spotted on the glass surface (B) and reverse transfected into adhesive cell line (C). After 3 days of incubation, proteins interactions are detected by the measurement of the reporter protein fluorescence (D). Since transfected cell clusters are separated by nontransfected cells, hundreds of different prey constructs can be investigated in a single experiment...

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