32 May / June 2019
(>4000 nt) deteriorated in PEG or PEO solutions when the concentration was above 1.0% and 0.6%, respectively [46].
Microchips from the Agilent bioanalyser were used to measure the ratio of 28S/18S rRNA in Diamond-Blackfan anaemia patients and could become a diagnostic tool [47].
It can be seen that using CE/LIF is becoming more important in RNA work. However, it is only the beginning of this kind of work, which involves mainly biologists, that are less inclined to perform analytical assays and protocol developments on the CE/LIF instruments than analytical chemists are.
IV. The future.
Figure 6: Example of separation using CE/ LIF of 6-IAF-labelled thiols in a pathological sample (1) homocysteine (Hcy), (2) cysteinyl– glycine (CysGly), (3) N-acetylcysteine, (4) cysteine, (Cys), (5) glutathione (GSH), (6) 6-iodoacetamidofluorescein (IAF), (u) unknown. Pathological sample: Hcy, 32 mM; Cys– Gly, 31 mM; Cys, 280 mM; G-SH, 4.1 mM. Separation buffer 50 mM boric acid, 20 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) pH 10. 30 kV, 50 µm id capillary 85/50 cm are total and effective length [35].
The final question to ask: is LIF or LEDIF really of use in today’s science? What will the future research axes and applications be?
First, in the major developments on DNA sequencing with dedicated instruments and separation kits, it can be seen that CE/LIF has had many successes.
For twenty years the analysis of pharmaceutical recombinant monoclonal antibodies was largely performed using LIF or LEDIF. It has become a significant technique in pharmaceutical companies for analysing the polysaccharide part of glycoproteins.
The main issue now is the continued development of simple applications using CE/LIF. This is now the case for polysaccharides, since it has been seen that the CE/LIF/MS experiments are very important to polysaccharides identification [26]. Labelling and separation kits are available, mainly from Sciex.
For the future, the selection of aptamers is a matter of considerable importance and that CE/LIF/Illumina can be developed to help. The main problem, however, is the development of the required bioinformatic analysis tools to identify the selected aptamers and the kits needed to allow scientists to easily run selection experiments using CE/LIF.
Finally, for use in clinical studies, while homocysteine has shown that it can be easily detected using this device, it still lacks automation and applications on other small molecules. The dimethyl arginines (with CBQCA or napthtalene dialdehyde (NDA) labelling) could be good candidates. Now, though, few clinical laboratories have developed such analysis methods. Generally speaking, clinical biochemistry laboratories need quick, robust and kit-based methods, only a few of their protocols use CE/LIF.
In conclusion, while CE/LIF and CE/LEDIF developments in the literature have been decreasing, its future remains promising.
References:
1. G. Nouadje, M. Nertz, Ph. Verdeguer, F. Couderc, J. Chromatogr. A 717 (1995) 335.
2. A. Rodat-Boutonnet, P. Naccache, A. Morin, J. Fabre, B. Feurer, F. Couderc, Electrophoresis (2012) 33 1709.
3. C. Bayle, N. Siri, V. Poinsot, M. Treilhou, E. Caussé, F. Couderc, J. Chromatogr. A 1013 (2003) 123.
4. A. Rodat, P. Gavard, F. Couderc, Biomed. Chromatogr. 23 (2009) 42.
5. F. Zarrin, N.J. Dovichi, Anal. Chem 57 (1985) 2690.
6. Y.F. Cheng, N.J. Dovichi, Science 242 (1988) 562.
7. O.O. Dada, B.J. Huge, N.J. Dovichi, Analyst 137 (2012) 3099.
8. C.D. Skinner, Electrophoresis 30 (2009) 372.
Figure 7: CE-SDS-nongel sieving media separations of nonreduced and reduced preparations of a
5-TAMRA.SE-labelled IgG. Bio-Rad SDS running buffer capillary, untreated fused silica, 50 µm i.d. effective length, 19.4 cm; injection, 15 s at 417 V/cm, applied electric field, 625 V/cm, temperature, 20°C. Detection was performed with laser-induced fluorescence using a 3.5 mW argon ion laser, 488 nm excitation. Insets show silver-stained SDS-PAGE traces of unlabelled sample preparations [39].
9. R.A. Mathies, K. Peck, L. Stryer, Anal. Chem. 62 (1990).
10. C. Bayle, V. Poinsot, C. Fournier-Noel,
F. Couderc, LIF detection: A summary, Electrokinetic Chromatography, Theory,
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