Abstract
The application of electrochemical DNA biosensors in real genomic sample detection is challenging due to the existence of complex structures and low genomic concentrations, resulting in inconsistent and low current signals. This work highlights strategies for the treatment of non-amplified and amplified genomic dengue virus gene samples based on real samples before they can be used directly in our DNA electrochemical sensing system, using methylene blue (MB) as a redox indicator. The main steps in this study for preparing non-amplified cDNA were cDNA conversion, heat denaturation, and sonication. To prepare amplified cDNA dengue virus genomic samples using an RT-PCR approach, we optimized a few parameters, such as the annealing temperature, sonication time, and reverse to forward (R/F) primer concentration ratio. We discovered that the generated methylene blue (MB) signals during the electrochemical sensing of non-amplified and amplified samples differ due to the different MB binding affinities based on the sequence length and base composition. The findings show that our developed electrochemical DNA biosensor successfully discriminates MB current signals in the presence and absence of the target genomic dengue virus, indicating that both samples were successfully treated. This work also provides interesting information about the critical factors in the preparation of genomic gene samples for developing miniaturized PCR-based electrochemical sensing applications in the future. We also discuss the limitations and provide suggestions related to using redox-indicator-based electrochemical biosensors to detect real genomic nucleic acid genes.
Highlights
Dengue rapid diagnostic tests (RDTs) are becoming highly attractive as they offer the fast diagnosis of the dengue virus due to their ease of use and inexpensiveness
It is worth emphasizing that there are speci c issues related to the direct detection of non-ampli ed genomic dengue virus gene-based hybridization events via a DNA biosensor approach
A novel redox indicator of this type could overcome the limitations of methylene blue (MB) seen in this work, as MB is affected by the length and base composition of the DNA sequences
Summary
Due to all these circumstances, researchers have recently focused on the development of point-of-care quantitative biosensors for dengue virus diagnosis with high selectivity and speci city, portability, and ease of use.[2,3,4,5] In terms of sensor biorecognition elements, nucleic acid or DNA biosensors are preferred over enzyme-, antibody-, or microorganism-based biosensors due to their speci c binding through DNA hybridization events, high stability, ease of synthesis, minimal batch-to-batch variation, and biocompatibility.[6]. Most recent works related to electrochemical DNA biosensors have focused on studying miniaturized electrochemical sensor devices,[22] ampli cation signals,[23,24] biorecognition probes with new detection mechanisms,[25] and the optimization of immobilization and hybridization conditions.[26] far too little attention has been focused on the preparation of genomic DNA samples for electrochemical detection This is due to constraints, as relying on the isolation and ampli cation of genomic genes via PCR-based laboratory procedures could restrict the application of electrochemical DNA biosensors in point-of-care testing. We demonstrate two strategies for the preparation of non-ampli ed and ampli ed genomic dengue gene samples and their application in detection based on an electrochemical DNA biosensor
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