Microfluidic Capillary Electrophoresis: Better, Faster, Safer, without Smiling and Frowning

Electrophoresis on ease is described as separation of molecules based on their movement in (direct current) electric charge. The first step after the gel and buffer ready is separation. Biomolecules is loaded into a porous gel then electric charge applied. The bigger molecule size, the slower their movement in the electric field, vice versa. In the case of nucleic acid, the movement direction is from negative pole into positive pole because nucleic acid is negatively charged. Loading dye is involved so we able to acknowledge the progress of the process.

Better, Faster, Safer, without Smiling and Frowning

After the electrophoresis or separation process is done, we enter the step of visualization. Appropriate label will be attached into the separated biomolecules. One of the problems we have with this old method is most of these labels are carcinogenic. It means endanger the operator. Another problem is insufficiency of sample quantity. On certain visualization method, let say Ethidium bromide, certain limit of detection applied, meanwhile in some cases we do not have that much quantity of sample. Frowning and smiling also might occur on gel electrophoresis caused by uneven gel or incompatible amount of electric current with gel strength.

After visualization step, the next step is documentation. Quantification is might possible with some vendor’s software. The whole process might take 2-3 hours length of time.

On nowadays conditions and case demands, those methods are actually outdated for their disadvantages. Can you figure out how danger Ethidium bromide even with proper lab protocol? Not to mention waste management of the substance. Based on problems listed, we need electrophoresis that able to overcome label carcinogenic danger, can be done using small amount of sample (good sensitivity), not frowning or smiling, easily quantified, shorter length time of process. With microfluidic electrophoresis, we can get the solution!

Principle; basically, microfluidic electrophoresis has the same principle with old fashion electrophoresis. The difference is the electrophoresis performed in small quantity of sample and the label using fluorescence. Fluorescence label enable us to do more sensitive analytical separation on nucleic acid. Detection used is laser detection. Data collected is similar like chromatogram but called as electrophoregram. The X axis represent time of “retention” and Y axis represent fluorescence intensity. Time of retention will correlate with size of molecule, the bigger it’s size then the bigger also their time retention. Meanwhile fluorescence intensity correlates with their amount. Molecule with high amount will give high intensity.

There are two standard marker (upper and lower marker) given inside. This upper and lower marker is assigned as limit of size. The standard markers also function as size known molecules in the software quantifying tool. For example upper marker with size 1500 base pair will have certain time retention number and also have certain fluorescence intensity for their concentration (ng/ul). With standard marker inside, quantification is mathematically feasible.

There are three possible macromolecule to be analyzed here; DNA, RNA and Protein. Those macromolecules are using the same principle in separation. The sample needed for this kind of method is only 1 microliters. Limit detection available is down to 0,1 nanogram per microliter. This means sensitive is enhanced. With micro volume electrophoresis, the distance of sample is shortened, this means shortening the time needed for separation process. All in time needed is only 30 minutes including preparation, running and analysis. Electrophoregram and quantitative data is become available after the process without any further effort. Gel like image also produced here to ease interpretation. Since gel like data produce from electrophoregram, there will be no smiling and frowning band.

With advantages mentioned, there is one “disadvantage” on this method. The working area should be clean enough since dust might interfere this micro volume electrophoresis. Finally, slightly higher cost per test should not be obstacle for better quality of analytic method, also seen from operator and environment safety point of view
Bynix Saya hanyalah blogger pemula yang ingin sukses didunia blogging

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