Abstract
Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.
Highlights
Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases
The designed homogeneous target-initiated transcription amplification (HTITA) process eventually could transfer single homogeneous target-binding event into numerous single-stranded RNA products which synchronously hybridized with the biotinylated detection probes and immobilized capture probes on the biosensor surface
The differential pulse voltammetric (DPV) measurement was performed to read out the oxidation of a-naphthyl which was the AP-catalyzed product of α -naphthyl phosphate (α -NP), for ultrasensitive and specific detection of pathogenic gene
Summary
Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. Various isothermal amplification strategies such as rolling circle amplification (RCA)[8,9,10], loop-mediated amplification (LAMP)[11], nicking enzyme signal amplification (NESA)[12], exonuclease III-aided signal amplification[13,14], strand displacement reaction (SDR)[15,16] have been integrated in biosensing systems to explore the excellent detection capability toward nucleic acids and other biomacromolecules These isothermal amplification-based biosensors are mostly designed to implement target binding and www.nature.com/scientificreports/. Signal amplification on the interface of biosensors, which is necessarily accompanied by the relatively low binding efficiency and enzyme kinetics owing to the steric hindrance, variant chemical microenvironment and surface crowding effect at the biosensor surface[17,18,19] These defects of heterogeneous target binding and amplification maybe show little impact on the analysis of synthetic target DNA, but inevitably compromise the sensitivity and reproducibility of the biosensors, especially for direct detection a low abundance target gene from complicated genomic DNA in real sample[20]. T7-based transcription amplification has been attempted to develop new isothermal amplification strategy for homogeneous detection of cell surface molecules, DNA binding proteins and nucleic acids[24,25,26,27]
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