| dc.description.abstract | Nucleic acids have emerged as leading biomarkers for diagnostic technologies, as they allow for extremely specific discrimination of targets via base-pair binding. Overcoming the obstacles posed enzymes, non-enzymatic nucleic acid tests (NATs) have the potential to be especially valuable for point-of-care testing. Nonetheless, there are several challenges faced by non-enzymatic NATs which make their application in a clinical setting difficult.
Most non-enzymatic NATs rely on thermodynamics-driven hybridization events to amplify a target-induced signal; as a result, non-specific amplification, or leakage, is unavoidable. The magnitude of this leakage is largely dependent on reaction conditions, requiring it to be independently measured for interpretation of results. Left-handed DNA has identical physical properties as naturally-occurring right-handed DNA, but does not interact with its mirror-image counterpart. Identical left-handed DNA elements were shown to match the leakage of right-handed DNA; this led to the creation of a dual-chirality NAT using both right- and left-handed DNA to generate two distinct signals, one measuring leakage and one measuring both leakage and target-induced amplification. The dual-chirality design was effective across a wide range of sample conditions, demonstrating the potential of this new type of NAT for diagnostic applications.
Another challenge faced by non-enzymatic NATs is sub-optimal sensitivity. One exception is a previously-demonstrated dumbbell DNA amplification scheme; it showed excellent sensitivity, but required an unrealistic amount of manual processing to perform. Here, a diagnostic platform was created to automate this reaction in a self-contained, microfluidic reaction cassette. Automation of the reaction greatly reduced the labor required, as well as the opportunities for human error. Additionally, the incorporation of left-handed DNA was demonstrated to function as a built-in control of non-specific amplification. This new diagnostic platform demonstrated a limit of detection of 3000 copies/µL, a significant improvement over most non-enzymatic NATs. In addition to the development DNA target previously used, S. mansoni DNA was also detected without any observed decrease in performance. Schistosomiasis is an infectious disease which persists in much of the world due to inadequate diagnostic testing; the advancements in non-enzymatic NATs presented here take an important step toward the eventual elimination of infectious diseases such as schistosomiasis. | |
