- More information about capillary electrophoresis method of analysis
- More information about microchip-based Real-time PCR method of analysis
In capillary electrophoresis (CE), a capillary is usually filled with an appropriate buffer. A sample solution is injected from the inlet end of the capillary, electric field is applied to the capillary ends, and components migrate to the detector point with different velocities due to distinctions in their electrophoretic properties. Consequently, they reach the detector at different times. Results are displayed as a sequence of peaks, where, ideally, each peak represents one compound. Peak area or peak height indicate the initial concentration of the compound in the studied mixture.
The main parameter defining differences in electrophoretic mobility of the compounds in CE is their charge-to-mass (e/m) ratio. Since this ratio is a highly specific property of compounds, CE allows a very high resolution separation and can be used to analyse almost all classes of compounds. The presence of ionizable groups (– Si – OH) on the inner surface of the fused silica capillary gives rise to a phenomenon called electroosmotic flow, which is just a bulk flow of the whole liquid inside the capillary occurring when the voltage is applied. Electroosmotic flow enables simultaneous determination of positively and negatively charged compounds in one run. Neutral compounds can also be analysed along with charged species provided that a surfactant (e.g. sodium dodecyl sulphate) is added to the buffer to form a micellar phase that facilitates their separation. This mode of CE, called micellar electrokinetic chromatography, is a very successful combination of electrophoretic and chromatographic techniques where compounds are separated based on the differences in their charge-to-mass ratio and hydrophobic/hydrophilic properties. Several other modes of CE can be performed on CAPEL instruments depending on the nature of separated solutes and general goals of the analysis. Among them are capillary gel electrophoresis, capillary isoelectric focusing, and capillary electrochromatography.
In most cases an UV lamp is used as the light source at the detector point. A monochromator gives the highest flexibility to the user enabling selection of the most appropriate wavelength for the detection and spectral scanning of the separated solutes. Compounds that do not absorb in the UV light (e.g. inorganic ions) can be analysed in the indirect mode with a certain UV – absorbing additive in the buffer.
Сapillary electrophoresis (CE) method has been generally recognized worldwide and is recommended for routine use by such organisations as British, American, and European Pharmacopoeia, World Health Organization (WHO), and the International Organisation of Vine and Wine (OIV).
Polymerase chain reaction (PCR) is the most widely used method in molecular biology area. PCR process exponentially amplifies DNA via enzymatic replication, capable of producing millions of copies of a DNA fragment from just a few copies. This effect is achieved by sequential heating and cooling of the analyzed sample with reagents and detecting amplified products of the reaction if specific targets are present. Test system reagents define target pieces of DNA to be amplified and also the level of amplification of the products.
Classical PCR thermocycler instruments using plastic tubes, well-plates or glass capillary have shortcomings, limiting further technology development opportunities. These well-known limits for PCR advancement can be summarized as follows: low rates of sample heating and cooling resulting in longer analysis time; high temperature gradient in a sample during heating leading to low reaction specificity; high volumes of reagents consumed (5-50 mL) leading to higher cost per test; labour-intensive test-preparation stage resulting in higher labour costs per test; freeze-chain storage and transportation conditions for the reagents.
To improve the PCR method and instrument efficiency researchers turned to microfluidics technology and its applications for PCR analysis systems. Microfluidics is an area of so-called lab-on-chip systems that use photolithographic technology from microelectronics for developing miniaturized systems for analytical testing of chemical substances. Main advantages of the microfluidic chips for PCR applications are fast analysis, low labor and reagents consumption and convenient process of PCR analysis and reagents storage.
Lumex Instruments focused its research efforts on open-well microfluidic chips, providing all the benefits of microfluidic technology operating in simple PCR thermocycler. As a result of the efforts Lumex created a compact PCR thermocycler AriaDNA that uses simple optical scheme with minimum moving parts, making system almost maintenance free. We have also developed a technology for mass production of the microchips. Application of high heat conductivity materials for microchips and modern technological processes for protective layers deposition allow to reach long storage life of the PCR reagents on the microchips at room temperature conditions. Microchips with reagents are designed for the specific user applications in the areas of medicine, veterinary and agriculture.