Alternative Detection Methodology

Other universal detectors have been suggested as potential replacements for UV in solubility assays including ELSD and CAD, although at present no specific assay has been reported [29]. 

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With the advancing automatization and computerization of CE instruments, the application of micromachining techniques, and the improvement of the devices for coupling CE with CL detection, it is hoped that both techniques may be incorporated in the future as suitable methodology in routine laboratories, being complementary to classical techniques such as HPLC and offering new alternatives to the analytical chemist. 

For systems with moderate-to-low probability, CE might not be the chromatographic quantification method of choice, and other alternatives, such as HPLC and GC, should be considered. However, specific procedures (e.g., off-line concentration, stacking techniques, extended light path capillaries) and detectors may be applied to increase solubility and sensitivity of detection, such as derivatization (e.g., carbohydrates, amino acids, amines, etc.) or the use of a specific detector (e.g., contactless conductivity detection, coupling with mass spectrometry, etc.). However, increasing the complexity of the methodology may be counterproductive if it leads to a lower robustness and transferability of the system. 

An alternative to the avidin-biotin technology, the EnVision +System (Dako) detection method, is recommended for universal use in diagnostic and research studies. It is based on enhanced polymer methodology. In comparison with APAAP, PAP, ChcmMatc , CSA, LABC, and SABC methods, the En Vision +System yields optimal detection (Sabattini et al., 1998). Its sensitivity is at least as good as that of Strept ABC techniques, and its use completely eliminates the problem of endogenous biotin. 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

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