Associate Professor Old Dominion University, United States
Introduction: The DNA hybridization step is used in many applications such as medical diagnosis, agricultural and environmental analyses, pharmacogenomics, and food safety monitoring. Medical diagnostics has become a critical field due to the demanding need for improvement in personnel health. Among them, cancer diagnostics play a key role in medical diagnostics. Polymerase Chain Reaction (PCR)-Based Hybridization, and Next-Generation Sequencing (NGS)are some applications where DNA hydridization is used. Current hybridization uses temeprature and it time-consuming, expensive, and less specific. These limitations affect the accurate detection of diseases. In this work, we have used alternating current (AC) electric fields to enhance DNA hybridization. The utility of electric fields offers a compelling alternative with the potential for increased efficiency in time, cost, and specificity. AC electric fields can improve hybridization kinetics by promoting the alignment and interaction of complementary strands more effectively than thermal methods alone.
Materials and
Methods: A T-shaped interdigitated array of microelectrodes (TIAMs) was used to generate AC electric fields on samples. TIAMs were fabricated on an N-BK7 glass substrate. Prior to experiments the TIAMs underwent 5-10 minutes of treatment with Piranha solution followed by a thorough rinse with deionized (DI) water. DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). The base DNA sequence was 5’- TTT AAT ATT GAT AAG GAT -3’, and the complementary DNA sequence was 5’- ATC CTT ATC AAT ATT AAA -3’. The DNA molarity was 1 µM, SYBR Green I concentration was 10x, and Tween 20 percentage was 0.01% (V/V). To prepare the control sample, base DNA, SYBR Green I, and Tween 20 were combined. A similar procedure was followed for the actual DNA mixture, which included base DNA, complementary DNA, SYBR Green I, and Tween 20. DNA samples were prepared in a 5 µS/cm TE buffer conductivity. Two sets of additional preparations were performed: one was preheated at 70°C for 5 minutes, while the other remained unheated. A 3 µl of DNA solution was pipetted onto the electrode, with a 70 µl layer of TE buffer added around the droplet to slow down the sample evaporation. Experimental observations were made with and without applying an alternating current (AC) potential of 10 V peak-to-peak at 1 MHz. Fluorescence images were captured using a fluorescence microscope after the samples dried. Finally, images were analyzed using ImageJ software to extract fluorescence intensity data.
Results, Conclusions, and Discussions: Figure 1 shows the variation of fluorescence intensity ratio with various experimental conditions. First, the total fluorescence intensities of all experiment images were calculated using the mean fluorescence intensity and mean sample area using ImageJ software. Then, the fluorescence intensity ratio values were calculated by dividing each experiment's total fluorescence intensity by the control experiment's total fluorescence intensity. This data indicated that both the application of the electric field and preheating contributed to higher fluorescence intensities, signifying a high possibility of effective hybridization, especially in the regions of higher electric field gradients of the electrode array. Figure 2 shows enhanced of fluoroscnce intensity with various experimental conditions. Fluorescence images show distinct differences with and without the application of an electric field. A coffee ring was formed in the samples that were not exposed to the electric field, indicative of DNA molecules aggregating at the edges due to evaporation dynamics. Conversely, when the AC electric field was applied, the sample base and complementary DNA were concentrated in high electric field gradient regions due to the dielectrophoretic effect (Figure 2). These findings can be applied to develop microarray-based DNA hybridization tools using AC electric fields in cancer diagnostics applications.
Acknowledgements (Optional): DN acknowledge the funding from National Science Foundation (NSF, grant numbers: 2310106 and 2300064)
