(306c) Electrophoretic Transport of Nucleic Acids through Nano Structured Surfaces | AIChE

(306c) Electrophoretic Transport of Nucleic Acids through Nano Structured Surfaces


Bhattacharya, S. - Presenter, Institute of Technology-Kanpur
Ghosh, A. - Presenter, Institute of Technology-Kanpur
Singh, D. - Presenter, Institute of Technology-Kanpur
Gurunath, R. - Presenter, Institute of Technology-Kanpur
Patra, T. K. - Presenter, Institute of Technology-Kanpur

Traditional electrophoresis methods like gel electrophoresis [1], zone electrophoresis [2,3], capillary electrophoresis etc. [4,5] have the inability to separate nucleic acids with higher fragment sizes. This is mostly owing to biased reptations which occur because of the entanglement of longer fragments of nucleic acids while traversing gel matrices. A new approach known as surface electrophoresis was proposed and developed in the early part of this decade which used the electrostatic interaction between nucleic acid molecules and charged surfaces as a basis of performing fractionation. This approach gave an alternative to all the earlier developed separation and fractionation techniques for DNA molecules [6-9]. Although a very useful technique in recent times, not much analysis has been developed with this novel fractionating method. For example the behaviour of negatively charged molecules with nano-structured hydrophobic or hydrophilic surfaces or selective nature of nano-patterned and charged surfaces to such molecules has been seldom explored. Molecular dynamic simulations used earlier to predict the behaviour of charged molecules translating across silicon or oxides have revealed some preliminary level explanation. We have been exploring nano-structured surfaces and their influence on nucleic acid mobility under electrophoretic fields. In this work we enumerate some of our findings on nano-structured porous silica (NPS) films. NPS is prepared by the traditional porogen method using poly(methylsilsesquioxane) as matrix and poly(propylene glycol) as porogen. Figure 1 (a) and (b) shows the preparation methodology for NPS by porogen/template extraction method and a SEM image of a NPS film respectively. The pore sizes obtained in this film are in the range of 2-3 nm. We perform translation of ds-DNA (2Kb and more) in variously patterned surfaces and observe the preferential selection of these surfaces as a function of porogen loading and different structuring schemes as illustrated in figure 2. The arrows indicate the direction of motion of the nucleic acids. The mobility studies are performed in a flow cell specially developed in our laboratory for surface electrophoresis. It contains two metallic electrodes positioned at both flanks of a small holder platform. The electrodes are connected to a waveform generator which supplies the requisite signal to the flow-cell. The nano-structured film is coated on a silicon surface by using spin coating or dip coating protocols. The structuring of these films are done using high energy beams and also by heating them to high temperatures. The solution containing ds-DNA and a fluorescent marker (Ethydium bromide) is physically absorbed over the film surface. The whole operation of electrophoresis is carried out within the field of view of a Nikon Trinocular fluorescence microscope and the stage is moved using a pre-calibrated micrometer lead-screw. Fig. 3 shows some images of the dye stained nucleic acids over nano-porous silica films. The mobility of these stains is calculated by moving the cross-bar of the microscope, with the help of stage movement using the earlier described setup, between different positions of the ds-DNA stains. The mobilities are further validated using molecular dynamic simulations.


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