User:PBR1219/Label-Free HPCE Systems

Label-free HPCE (High Performance Capillary Electrophoresis) systems are used in the analysis of biomolecules, mainly for the detection and quantification of protein, DNA and RNA.

The Peregrine 1 system is the first commercially available label-free HPCE system and is marketed by deltaDOT Limited, a UK company that developed Label-Free Intrinsic Imaging (LFII®). The Peregrine 1 system's data analysis of DNA and proteins is also enabled by deltaDOT's Generalized Separation Transform (GST) and Equiphase Vertexing (EV) algorithms.

GST is a method of combining the data from the Peregrine 1 multiple pixel diode array in a way that preserves the peak shape information of the electropherograms. EV is a technique that uses a space-time constraint to significantly increase the signal-to-noise ratio, to improve resolution and to allow associations of a given band in the electrophoretic window with a given injection. In EV the electropherograms are first analyzed to find local peaks that are then used to determine the origin of the bands through vertexing and then to produce a significantly enhanced signal output.

Peregrine 1’s multipoint detection and label-free photonic imaging have mathematical and physicochemical consequences that provide important advantages. First the geometric method of analysis makes it possible to use powerful signal processing algorithms that are computationally highly efficient and can run in real time on a standard PC. Secondly, the ability to use powerful signal processing algorithms allows the use of very low signal levels which permits the use of photonic imaging rather than label detection. Thirdly, photonic imaging has the advantage over the use of labels that the strength of the signal depends on the absorption of light by the sample. This is in contrast to the fact that the strength of a signal from a label is mathematically proportional to the effective volume of the label. This means that with decreasing sample size, the strength of the signal falls as the inverse cube. Thus the strength of the photonic signal falls much more slowly (linearly, rather than with the inverse cube) as the sample amount is reduced giving LFII®-enabled systems a powerful advantage in detecting even minute amounts of sample. If a labeled system and a LFII®-enabled system sample are both reduced in size by a factor 10 then the signal-to-noise of the labeled system drops 100 times more than that of the LFII®-enabled system.

Because the Peregrine 1 HPCE system uses an approach that is distinctly different from conventional capillary electrophoresis (CE) in which the separated components are detected by a single point detector after separation, deltaDOT claims that Peregrine 1 systems are able to produce much better quality results than established CE systems as well as in a fraction of the time taken by gel-based systems and with a significant savings in cost to competing techniques such as mass spectrometry.