The further miniaturisation of electrophoretic systems has led to the development of microchip electrophoresis (MCE), which has many advantages over conventional electrophoresis methods, allowing very high-speed analyses at very low sample sizes. For example, microchip analysis can often be completed in tens of seconds, whereas capillary electrophoresis (CE) can take 20 min and conventional gel electrophoresis at least 2 h. Using new detection systems, such as laser-induced fluorescence, picomole (10⁻¹² mol) to attomole (10⁻¹⁸ mol) sensitivity can be achieved, which is at least two orders of magnitude greater than for conventional CE. Detection systems for molecules that do not fluoresce include electrochemical detectors, pulsed amperometric detection (PAD) and sinusoidal voltammetry. All these detection techniques offer high sensitivity, are ideally suited to miniaturisation, are very low cost and are highly compatible with advanced micromachining and microfabrication technologies (see below). Finally, a potential gradient of only a few volts is required, which eliminates the need for the high voltage power supplies used by CE.

The manufacturing process that produces microchips is called microfabrication. The process etches precise and reproducible capillary-like channels (typically, 50 μm wide and 10 μm deep; slightly smaller than a strand of human hair) on the surface of sheets of quartz, glass or plastic. A second sheet is then fused on top of the first sheet, turning the etched channels into closed microfluidic channels. The end of each channel connects to a reservoir through which fluids are introduced/removed. Typically, the size of chips can be as small as 2 cm². Basically, the microchip provides an electrophoretic system similar to CE, but with more flexibility.

Current developments of this technology are based on integrating functions other than just separation into the chip. For example, sample extraction, pre-con centration of samples prior to separation, PCR amplification of DNA samples using infrared-mediated thermocycling for rapid on-chip amplification and the extraction of separated molecules using microchamber-bound solid phases are all examples of where further functions have been built into a microchip electrophoresis system. An interface has also been developed for microchip electrophoresis mass spectrometry (MCE-MS) where drugs have been separated by MCE and then identified by MS.

One example of a microfluidic circuit system is the Agilent Bioanalyzer. The sample is applied in one area of a small cassette and driven through microchannels (that are pre-filled with dye-laden agarose immediately before use) under computer-controlled electrophoresis. The channels lead to reservoirs allowing, for example, incubation with other reagents, such as dyes, for a specified time. Electrophoretic separation is thus carried out in a microscale format. The small sample size minimises sample and reagent consumption and the units, being computer controlled, allow data to be captured within a very short timescale. The resulting data are presented as an electropherogram with a digital representation of an agarose gel in an easy to interpret format (Figure 1).

Fig1. Example of a computer-generated image from an Agilent 2100 Bioanalyzer. Here, data from the sample being analysed are transformed into gel-like images similar to a stained agarose gel (electropherogram).

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مواضيع ذات صلة

Electrophoresis of RNA

Pulsed-Field Gel Electrophoresis

3′ Cleavage and Polyadenylation of Pre-mRNAs Are Tightly Coupled

Self-Splicing Group II Introns Provide Clues to the Evolution of snRNAs

SR Proteins Contribute to Exon Definition in Long Pre-mRNAs

DNA Sequencing Gels

Nuclear Bodies Are Functionally Specialized Nuclear Domains

Chain Elongation by RNA Polymerase II Is Coupled to the Presence of RNA-Processing Factors

Spliceosomes, Assembled from snRNPs and a Pre-mRNA, Carry Out Splicing

During Splicing, snRNAs Base-Pair with Pre-mRNA

Transport of mRNA Across the Nuclear Envelope

Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions