Microgel Electrophoresis and Product Analysis

Gelation and DNA-separation in a microfluidic environment

CFCGE_ionicliquid
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Scheme of “continuous flow” CGE. By use of a pipetting robot samples are selected from a standard microtiter plate. To separate the sample from the running buffer (1x TBE) it is enclosed by two small droplets of ionic liquid (1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imid), Ionic Liquid Solvent Innovation). Using the multiport valve then the sample or the running buffer for the CGE are alternately transported to the CGE-Chip. The CGE capillary is filled with Pluronic F 127 (Invitrogen) that allows separating very short oligonucleotides (8-30 bases). The volume of the samples needed for the analysis is less than 10 µl.

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CGE ligation and nicking
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Product analysis of the two reaction steps of the isothermal enzymatic amplification. To track the product formation by CGE the oligonucleotide A is labelled with an Alexa fluorophore 647 and the oligonucleotide BC is internally labelled with an Alexa fluorophore 488. Alternating excitation of the sample with the red (635nm) and the blue laser (488nm) gives the opportunity to directly analyse the spots that migrate through the capillary while electrophoreses is running.

Based on classical CGE (capillary gel electrophoresis) we invented the so-called “continuous flow” CGE. The opposite figure represents the set-up based on our developed programmable fluidic-microwell interface we used for this measuring method, which allows to analyse products of chemical reactions continuously in one measurement session. For the continuous injection of samples in the capillary, a pipetting robot combined with a Cheminert© multiposition stream selector valve is used to transport alternately either a sample that is enclosed by two small packages of ionic liquid or the buffer (1 x TBE) that is needed for the electrophoretic separation. The ionic liquid that separates the samples from the buffer was chosen as it provides special properties that are needed for the work with microfluidic devices. At first it is completely immiscible with aqueous solutions, second it has a very low viscosity and third it does not lead to swelling of PDMS, as this is typical for other hydrophobic substances (e. g. organic solvents, paraffin and mineral oil etc.). Furthermore it does not influence the activity of the electrodes which is a big advantage concerning the planned integration of CGE in our chemical microprocessor chip.

We have successfully tested the principle using aqueous dye solutions and verified the sample compartmentation by fluorescence measurements at different positions on the chip.


An example for the product analysis using CGE is shown in the lower figure, where the progress of the two step reaction (I. Ligation + II. Nicking) of the isothermal enzymatic amplification is presented. Two CGE spectra after 34 min and 236 min respectively illustrate the progress of the reaction. After 34 min both the intermediate product ABC/A’B’C’ and the final product AB/A’B’C’ of the reaction can be detected. Furthermore the ss-oligonucleotide A and the duplex BC/A’B’C’ are visible. After ca. 4 h reaction time the duplex BC/A’B’C’ is totally consumed and therefore no signal is detected for this duplex anymore. The assignment of the signals results from the colour of the fluorophores as well as the comparison with test mixtures of educts and products without enzymes.


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