A highly amplified, nanoparticle-based, bio-barcoded electrochemical biosensor for the simultaneous multiplexed detection of the protective antigen A (pagA) gene (accession number = M22589) from Bacillus anthracis and the insertion element (Iel) gene (accession number = Z83734) from Salmonella Enteritidis was developed. The biosensor system is mainly composed of three nanoparticles: gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), and nanoparticle tracers (NTs), such as lead sulfide (PbS) and cadmium sulfide (CdS). The AuNPs are coated with the first target-specific DNA probe (1pDNA), which can recognize one end of the target DNA sequence (tDNA), and many NT-terminated bio-barcode ssDNA (bDNA-NT), which act as signal reporter and amplifier. The MNPs are coated with the second target-specific DNA probe (2pDNA) that can recognize the other end of the target gene. After binding the nanoparticles with the target DNA, the following sandwich structure is formed: MNP-2pDNA/tDNA/1pDNA-AuNP-bDNA-NTs. A magnetic field is applied to separate the sandwich structure from the unreacted materials. Because the AuNPs have a large number of nanoparticle tracers per DNA probe binding event, there is substantial amplification. After the nanoparticle tracer is dissolved in 1 mol/L nitric acid, the NT ions, such as Pb2+ and Cd2+, show distinct non-overlapping stripping curves by square wave anodic stripping voltammetry (SWASV) on screen-printed carbon electrode (SPCE) chips. The oxidation potential of NT ions is unique for each nanoparticle tracer and the peak current is related to the target DNA concentration. The results show that the biosensor has good specificity, and the sensitivity of single detection of pagA gene from Bacillus anthracis using PbS NTs is as low as 0.2 pg/mL. The detection limit of this multiplex bio-barcoded DNA sensor is 50 pg/mL using PbS or CdS NTs. The nanoparticle-based bio-barcoded DNA sensor has potential applications for multiple detections of bioterrorism threat agents, co-infection, and contaminants in the same sample.
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