Label detection techniques

For protein microarrays, several label detection techniques are already used because of their simplicity, broad availability of fluorescence dyes and chemical functional groups for conjugation, and scanning instruments. 

Figure 3.6 Summary diagram of protein microchip labeling/detection techniques and main areas, including (A) Direct labeling. (B) Reversed-phase array (RPA). (C) Labeling with tertiary antibody (D) Sandwich labeling. (E) Bead-based array. (F) Biotin-labeled antibody. (G) Label-free method for mass spectrometry. (H) Whole-cell array. (I) Rolling circle amplification (RCA). (See color insert.)...

Since a relatively small number of analytes of interest have native fluorescent properties, derivatization reactions are frequently employed to enable this detection technique to be extended to a broader range of compounds. This is an excellent means of increasing the detectability for a whole range of molecules, but it is important to realize that there are certain limitations. First, it is difficult to obtain quantitative yields at low analyte concentrations. This implies that in some cases, the obtainable detection limit are not limited by the detector sensitivity, but instead by low yields in the derivatization reaction. Furthermore, to shift the equilibrium toward the product side at low analyte concentrations, as much as 104 times excess of fluorescent label may be necessary. Tow concentrations of impurities in the label can be present at levels greater than the analytes of interest and as a result, numerous interfering peaks in the chromatograms may be observed. These problems are discussed in detail in Ref. 181. 

PAL has been known for 30 years, but a new era of applications has appeared recently through the evolution of more efficient photophores and activation, as well as new high-resolution separation and detection techniques. The photo-covalent modification of the binding site, which has an irreversible effect on activity and places a (radio)label, enables a multilevel analysis and identification on the biological target. 

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

Figure 7.8 Some examples of antibody detection techniques, (a) Direct labelling of the primary antibody, (b) indirect using labelled secondary antibody, (c) indirect using biotinylated secondary antibody and labelled streptavidin.

Three types of detection methods can be used for in situ hybridization radioactive, fluorescent, and chromogenic. Radioactive detection techniques are the most commonly used primarily because they are more sensitive, but also because the other techniques are relatively more recent in terms of the development of the appropriate chemistry and the techniques that enable in vitro labeling of probes by these nonradioactive means. 

The versatility of the SPR technique has been shown by a vast amount of publications in the past decades the method has matured into a well-accepted analytical tool for the characterization of interfaces and thin films as well as for the sensitive detection of interfacial biomolecular interaction. With a significant input from engineering, SPR has reached a decent signal-to-noise level with a lower limit for a reliable signal detection corresponding to an effective layer of about 0.3 A [6], which is sufficient for most thin film studies. However, the intrinsic label-free characteristic of SPR detection technique still imposes limitation on further sensitivity improvement, especially if the analysis involves small molecules. 

The natural substrates for lipases are triglycerides but, because of the complexity of these and the fact that they seldom contain a chromophore or other label to enable ready detection of the products, several synthetic substrates have been developed. These enable different detection techniques such as spectrophotometry, fluorimetry, chromatography, or radiometry to be used. It is important to note that, by definition, true lipases are active only on water-insoluble esters while esterases cleave only water-soluble esters (Jaeger et al., 1994). Thus, it is important that methods used for milk and milk products use substrates, which detect true lipase but not esterases as lipases play a major role in the hydrolysis of milk fat, while the role of esterases is considered insignificant (McKay et al., 1995). 

If sticky labels are attached directly to foodstuffs such as fruit and vegetables, then some of the adhesive may remain on the foodstuff when the label is removed and the food is eaten. It is not uncommon to be able to detect by eye and by touch the presence of a small sticky label residue on fruit. The presence of adhesive residues on the food surface has been investigated by Fourier transform-Raman spectroscopy although at the low levels present on the foodstuff after removal of the label this technique could not be used to provide quantitative data. 

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