Electronic biosensors for DNA detection typically utilize immobilized oligonucleotide probes on a signal transducer, which outputs an electronic signal when target molecules bind to probes. However, limitation in probe selectivity and variable levels of non-target material in complex biological samples can lead to nonspecific binding and reduced sensitivity. Here we introduce the integration of 2.8 μm paramagnetic beads with DNA fragments. We apply a custom-made microfluidic chip to detect DNA molecules bound to beads by measuring Impedance Peak Response (IPR) at multiple frequencies. Technical and analytical performance was evaluated using beads containing purified Polymerase Chain Reaction (PCR) products of different lengths (157, 300, 613 bp) with DNA concentration ranging from 0.039 amol to 7.8 fmol. Multi-frequency IPR correlated positively with DNA amounts and was used to calculate a DNA quantification score. The minimum DNA amount of a 300 bp fragment coupled on beads that could be robustly detected was 0.0039 fmol (1.54 fg or 4750 copies/bead).
Fig: Strategy overview. (a) Image of the device in which a soft-lithography made PDMS is integrated with the electrode patterned on the fused silicon wafer. (b) The microscope image of channel and electrodes. (c) The sample preparation includes ① Amplifying target DNA using polymerase chain reaction (PCR). ②Immobilizing biotinylated target DNA with streptavidin coated paramagnetic beads. ③ Washing and resuspending. (d) The schematic diagram of detection. The bead is injected from inlet well using micro pipette. As beads flow through the pore, the impedance change is captured by the lock-in amplifier at multiple frequencies. The data are sent to the PC and analyzed in Matlab. (e) Representative data of bare paramagnetic beads passing through the sensing region measured at 7.5 MHz, 10 MHz, and 15 MHz respectively.
Additionally, our approach allowed distinguishing beads with similar molar concentration DNA fragments of different lengths. Using this impedance sensor, purified PCR products could be analyzed within ten minutes to determine DNA fragment length and quantity based on comparison to a known DNA standard.
Sui, J., Gandotra, N., Xie, P. et al. Multi-frequency impedance sensing for detection and sizing of DNA fragments. Sci Rep 11, 6490 (2021). https://doi.org/10.1038/s41598-021-85755-9