Highly sensitive, label-free quantification of DNA

Darius Michael Saadat-Moghaddam

There is a high demand for tools capable of highly sensitive and label-free quantification of DNA in a sample. Currently available methods are encumbered by a lack of sensitivity, reproducibility, and are generally prone to contamination and human error. Recent research has demonstrated that sensing platforms composed of micro and nano-scale components hold great potential in fulfilling bio-sensing applications requiring high sensitivity. Here, a label free amperometric biosensor composed of a functionalized microneedle is proposed. The microneedle biosensor utilizes an alternating current (AC) electric field to facilitate the capture of DNA and uses a current measurement to quantify the captured DNA. During the non-specific capture phase of this study, the gold substrate of the microneedle was coated with 1% polyethyleneimine (PEI) followed by a layer of single walled carbon nanotubes (SWCNT). Using lambda DNA as a target analyte, the sensitivity was found to be discernable from the negative control down to 100 attomolar (aM), which demonstrates a significant improvement over currently available fluorescence and UV absorbance based methods. To transition the study toward specific detection, the microneedles were functionalized with 1% PEI, SWCNT, streptavidin, and biotinylated oligonucleotide probes whose sequence is complementary to the core region of the rpoB gene of Mycobacterium tuberculosis (MTB). Using the core region of the rpoB as the target analyte, this platform achieved a sensitivity of 10fM. All the aforementioned results were substantiated by fluorescence microscopy. This paper also details studies conducted to both optimize coating layers and increase knowledge of the working principal for the microneedle biosensor. The results of these studies found 1% PEI to be the optimal concentration and that the charge carrying capacity of the microneedle declined with the accumulation of DNA on the surface of the electrode. In comparison to other optical-based quantification methods, a microneedle biosensor demonstrated great capability for rapidly concentrating and detecting small amounts of DNA. This approach can be further refined to make the capture/detection of individual types of DNA fragments possible and, by extension, any diseases that may be associated with those fragments.

Highly sensitive, label-free quantification of DNA

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