Direct fluorescence detection

Inside the dark box main sockets and automation port ensure integration of special probe requirements like incubators. There is provision to insert a fiberoptic-guided light source that accept filters for fluorescent work. The sensitivity of the NightOWL is claimed to enable direct detection of reactive oxygen species, even without enhancers like luminol. 

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

While there are only a few examples that can be used for direct detection of desired analytes, many simple molecules and ions do not have optical activity under regular conditions, a chemical reaction is needed to generate an optically active species. The reactions can be acid-base, ion pairing, complexation reactions, or quenching of fluorescence by 02, paramagnetic molecules, etc. Optical sensors for a few analyte or group analytes are summarized below. 

Measurement of fluorescence intensity can be used for quantitative analysis of fluorescent compounds where the intensity of fluorescence is proportional to the concentration of the compound. Because of their high sensitivity and selectivity, analytical techniques based on fluorescence detection are commonly used. If a target compound is fluorescent then direct detection of the fluorescence emitted is possible using a fluorimeter (Figure 4.7). 

When an analyte is fluorescent, direct fluorometric detection is possible by means of a spectrofluorometer operating at appropriate excitation and observation wavelengths. This is the case for aromatic hydrocarbons (e.g. in crude oils), proteins (e.g. in blood serum, in cow milk), some drugs (e.g. morphine), chlorophylls, etc. Numerous fields of applications have been reported analysis of air and water pollutants, oils, foods, drugs monitoring of industrial processes monitoring of species of clinical relevance criminology etc. 

The detection of flu viruses via a fluorescent sandwich immunoassay was reported by Bucher.(10) However, the method sensitivity was too low for direct detection of the virus. A novel sandwich immunoassay was described by Ogcr((lff7 for the detection of Botulinum Toxin A. Antibodies specific for Clostridium botulinum were covalently attached to the surface of a tapered fiber. After the capture of the antigen, a sandwich was formed with a rhodamine-labeled anti-toxin IgG, and the evanescent wave was measured. The assay was highly specific with detection limits near 5 ppb. 

Figure 4.10 Direct analysis of catecholamines in urine sample. Column, Asahipak ES-502C eluent, 75 mM succinic acid + 25 mM borate buffer (pH 6.10) containing 0.5 mM EDTA flow rate, 1.0 min-1 detection, fluorescence reaction detection Ex. 350 nm. Peaks-. 1, adrenaline-, 2, noradrenaline-, and 3, dopamine.

The high sensitivity and selectivity of fluorescence detection make this the obvious choice for improving detection of proteins. Three approaches have been used direct detection of intrinsic protein fluorescence, indirect fluorescence detection, and protein derivatization for fluorescence detection. 

Compared to absorbance detection, direct detection of proteins rich in aromatic amino acids by the intrinsic fluorescence of tryptophan and tyrosine residues provides enhanced sensitivity without the complexity of pre- or postcolumn derivatization. The optimal excitation wavelengths for these amino acids are in the 270- to 280-nm range. 

A simple alternative to direct detection of intrinsic protein fluorescence detection is the technique of indirect fluorescence detection proposed by Kuhr and Yeung.20 In this approach, the analysis buffer contains a fluorescent anion that produces a high background fluorescence signal. Nonfluorescent analyte anions displace the fluorescent species, producing a zone of reduced signal. Sensitivity in indirect fluorescence detection is determined by the dynamic reserve (ratio of signal... 

Prystay, L., Gagne, A., Kasila, P., Yeh, L.A., and Banks, P., Homogeneous cell-based fluorescence polarization assay for the direct detection of cAMP, /. Biomol. Screen., 6, 75, 2001. 

For high-throughput data collection, sample centring via loop detection is currently the method of choice for placing crystals in the X-ray beam. Alternative methods rely on the direct detection of the crystal itself by monitoring the intensity of either X-ray diffraction from the crystal or X-ray fluorescence from an element in the crystal that is not present in the cryoprotectant (Pohl et al., 2004). Both of these approaches have drawbacks. First, some of the limited X-ray lifetime of the crystal (Section 12.6) must be committed to detection instead of data collection. Second, the loop itself has to be placed close to the X-ray beam prior to crystal detection, followed by step-wise translation of the crystal through the beam, thereby increasing the time required to centre the sample. 

Of all the detection methods applied to the lab-on-a-chip, the most popular by far has been laser-induced fluorescence (LIF) detection. Direct LIF detection benefits... 

The excited-state behavior of 1,1,2,2-tetraphenylethene (TPE) has been studied by means of picosecond fluorescence, absorption, and Raman spectroscopies and picosecond optical calorimetry. It has been shown that, like stilbene, TPE derivatives substituted with minimally perturbing stereochemical labels such as methyl groups undergo efficient photoisomerization. However, unlike stilbene, strong spectroscopic evidence exists for the direct detection of the twisted excited singlet state, 5ip herein but traditionally designated as of TPE. 

The subsequent explosion of array technologies has been sparked by two key inno-vations. The first is the use of non-porous solid support, such as glass, which has facilitated the miniaturization of the array and the development of fluorescence-hybridization detection (16, 17, 18). The second critical iimovation has been the development of methods for high-density spatial synthesis of oligonucleotides, which allows the analysis of thousands of genes at the same time. Because DNA cannot bind directly to the glass, the surface is first treated with silane to covalently attach reactive amine, aldehyde, or epoxies groups that allow stable attachment of DNA, proteins, and other molecules. 

The biggest challenge in the HPLC analysis of biotin is its detection. Biotin does not exhibit UV absorbance or fluorescence, nor is it electrochemically active (180). Refractometry, a notoriously nonspecific technique, is the only means for direct detection of biotin. Fortunately, biotin can be converted to UV absorbing or fluorescent derivatives. 

---