Fluorescence detection principle

Methods HPLC with Fluorescence Detection Principle... 

Many researchers using the lectin microarray take advantage of a confocal fluorescence detection principle, because the method has already been adapted for established systems of DNA microarray [18, 19]. The method is simple, and has therefore been widely used. However, it requires washing of the microarray surface after the binding reaction, because a dried sample is required for the confocal-type detection system. One useful application of the system is multicolor detection using, e.g., Cy3 and Cy5, as described later. 

Principles and Characteristics Many of the planar chromatography methods rely on fluorescence detection to achieve the required identification limits exploitation of sensitive and selective derivatisation reactions is of considerable importance. Most TLC scanning densitometers can be operated in the fluorescence mode and are able to record in situ excitation spectra of TLC... 

In addition, typical methods of sensing are total internal reflection fluorescence or monitoring of fluorescence resonance energy transfer6,7. The second class is a direct optical detection principle which relies either on reflectometry or refractometry. The latter is connected to evanescent field... 

An array or a matrix of nucleic acid probes immobilized at discrete locations on a silicon or glass surface provides a convenient means to simultaneously probe a sample for the presence of many different target sequences. Microarray biochip scanning devices, mostly based on fluorescent labels, are now currently available, and could also be used with CL labels to take advantage of the higher sensitivity of this detection principle. 

The detection of HIV-related proteins is one of the most challenging tasks. This is especially true because AIDS should be diagnosed as early as possible to enable an early and effective therapy of this infection. Pavski and Le (57) used the aptamer strategy to detect reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1). A direct and specific ACE method was proposed using laser-induced fluorescence (ACE/LIF) as detection principle. Single-stranded DNA aptamers as probes fluorescently labeled were synthesized. The resulting aptamer is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-l RT was stable, with calibration curves linear up to 50 nM (6 /xg/mL) HIV-1 RT concentration. Both... 

Laser-induced fluorescence (LIF) has also been utilized as a highly sensitive detection principle for CE [48-51]. However, while the LIF detector is now able to achieve zeptomole (10 21) detection limits, conventional derivatization techniques are inefficient at these exceptional levels [52]. Also, CE has successfully been coupled with mass spectrometry (MS) [53], nuclear magnetic resonance (NMR) [54, 55], near-infrared fluorescence (NIRF) [56, 57], radiometric [58], flame photometric [59], absorption imaging [60], and electrochemical (conductivity, amperometric, and potentiometry) [61-63] detectors. A general overview of the main detection methods is shown is Table 1 [64]. 

Like in RILAs, an advantage of fluorescence detection is the possibility of developing homogeneous FILAs using direct or indirect (competitive or displacement) approaches. The fluorescence polarization immunoassay (PFIA) and their homolog fluorescence polarization immuno-like assay (PFILA) are two of the most widely used procedures in homogeneous fluoroassays. Both are based on the principle that fluorescence polarization gives a direct measure of the bound/free ratio of the labeled analyte (tracer) without the need for their separation [23, 28]. 

Different detection principles can be utilized simultaneously. It is possible, for example, to use a UV detector immediately after the column. A fluorescence-generating reagent can then be added followed by fluorescence detection. This permits detection of substances with a poor UV chromophore or may discriminate interferences. 

The first two points represent a general motivation for miniaturization in separation science independent of the actual fabrication technology. The benefit of a reduction of the consumption of sample, reagents, and mobile phase in chemical and biochemical analysis is self-evident and does not need to be discussed further (reduced consumption of precious samples and reagents, reduced amounts of waste, environmental aspects). This advantage is, however, sharply contrasted by its severe implications on the detection side, as discussed elsewhere in this volume in detail. The detection of the separated zones of very small sample volumes critically depends on the availability of highly sensitive detection methods. It is not surprising that extremely sensitive laser-induced-fluorescence (LIF) has been the mostly used detection principle for chip-based separation systems so far. 

By far most of the work discussed in this review has been based on LIF detection, usually with an 488 nm Ar-ion laser as the excitation source. Only very few other examples exist in the literature where other detection principles were investigated. One of these exceptions is an integrated detection cell for chip CE that has been described by Liang et al. [78]. In combination with the U-shaped separation channel, two additional well aligned channels to take up the excitation and collection fibers where micromachined in a glass plate. The U-cell provides a longitudinal path of 120 -140 pm in length parallel to the flow direction and can be used both for absorption and fluorescence measurements. The absorption detection limit was 0.003 AU ( 6 pM of a fluorescein dye) in the fluorescence mode a detection limit of 3 nM fluorescein (20 000 molecules) was achieved. 

Describe, with a labeled diagram, the principle of confocal fluorescent detection. (4 marks)... 

The most commonly-used detectors are those based on spectrophotometry in the region 184-400nm, visible ultraviolet spectroscopy in the region 185-900nm, post-column derivativisation with fluorescence detection (see below), conductivity and those based on the relatively new technique of multiple wavelength ultraviolet detectors using a diode array system detector (described below). Other types of detectors available are those based on electrochemical principles, refractive index, differential viscosity and mass detection. 

A host of detection principles can be used to sense the presence of double-stranded DNA. Fluorescent detection techniques employ dyes that change fluorescence properties upon binding to DNA (this is also discussed in detail in Chapter 2.5). 

In a protein-binding assay with fluorescence detection a microarray of biotin, HPYPP-peptide and WSHHPQFEK-peptide was screened against streptavidin-Cy3 and avidin-Cy5. By following the same principle an anti-human insulin monoclonal antibody was also screened against a set of different peptides. 

The principle of fluorescent detection systems is that an organic molecule, or in some cases a metal complex, can absorb photons in a particular range and then reemit them at a longer wavelength, illustrated by a Jablonski diagram in Fig. 6.4. For this to be used as a detection method the fluorescent species must have an affinity for a specified target or be attached to a molecule that does. 

From a practical point of view, SFC may allow the use of many different detection principles, including both typical LC detectors (UV-absorbance, fluorescence) and typical GC detectors (flame ionization, mass spectrometry). Also, capillary SFC seems to be well within the posssibilities of current technology, while capillary LC is not. 

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