Label-Free Voltammetric Detection Using Individually Addressable Oligonucleotide Microelectrode Arrays

摘要

The utility and performance of label-free, oligonucleotidenprobes for reagentless detection of dilute target analytesnwas examined using a voltammetric transduction principlenin an array format. Multistep, solid-state fabricationnyielded preproduction arrays of 16 individually addressable,n30 μm diameter microelectrodes in a 30 mm × 6.5nmm × 0.5 mm dipstick disposable device. The specificitynof 16 nucleotide (nt) 2′-O-methylribonucleic acid and 22nnt DNA backbone probes bound through Mg2+-nphosphonate bridges to polypyrrole films on the microelectrodesnwere studied using microbial target RNAs ofnvarious lengths. Probe-specific interactions with Escherichiancoli O157 H7 23S rRNA (2907 nt) and Candidanalbicans 18S rRNA (1788 nt) were detected at 65 andn58 fmol/mL, respectively, in volumes as low as 0.5 mL.nSpecificity studies showed that, for a given probe, “nontarget”ntranscripts can contribute to changes in thenvoltammetric detection signal, though with responses thatnnever exceed 70% of the detection signal acquired fornspecifically designed complementary targets. These resultsnstatistically validate the use of the voltammetricnmicroelectrode array for obtaining a “yes-no” answer onncomplementary specific binding. The study also identifiesnchallenges and pitfalls for the selection strategies ofnoligonucleotide probes.