Ultrasensitive Norovirus Detection Using DNA Aptasensor Technology

Abstract

DNA aptamers were developed against murine norovirus (MNV) using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Nine rounds of SELEX led to the discovery of AG3, a promising aptamer with very high affinity for MNV as well as for lab-synthesized capsids of a common human norovirus (HuNoV) outbreak strain (GII.3). Using fluorescence anisotropy, AG3 was found to bind with MNV with affinity in the low picomolar range. The aptamer could cross-react with HuNoV though it was selected against MNV. As compared to a non-specific DNA control sequence, the norovirus-binding affinity of AG3 was about a million-fold higher. In further tests, the aptamer also showed nearly a million-fold higher affinity for the noroviruses than for the feline calicivirus (FCV), a virus similar in size and structure to noroviruses. AG3 was incorporated into a simple electrochemical sensor using a gold nanoparticle-modified screen-printed carbon electrode (GNPs-SPCE). The aptasensor could detect MNV with a limit of detection of approximately 180 virus particles, for possible on-site applications. The lead aptamer candidate and the aptasensor platform show promise for the rapid detection and identification of noroviruses in environmental and clinical samples.

Miniaturized electrochemical sensor modified with aptamers for rapid norovirus detection

“…Visualization and electrochemical (i.e., CV) evaluation of aptamer immobilization confirmed that MEMS electrochemical aptasensor was successfully fabricated via self-assembly of a thiolated aptamer on the gold working electrode surface following a procedure reported previously [3]. …”

“…Aptamer immobilization on the gold working electrode through the affinity between gold and thiol was carried out according to a previous study [3], with slight modification. Prior to immobilization, the fabricated MEMS sensor base was thoroughly washed with DI water and then dried with nitrogen gas (N 2 ).…”

“…Prior to immobilization, the fabricated MEMS sensor base was thoroughly washed with DI water and then dried with nitrogen gas (N 2 ). The aptamer was immobilized on the electrode by drop casting 500 nM HPLC-purified MNV-specific aptamer (AG3, aptamer nucleotide sequence described in Giamberardino et al [3]) modified at the 5' position with a 6-hydroxyhexyl disulfide group (HS-5'-AG3; Integrated DNA Technologies, USA) in 20 mM Tris-ClO 4 buffer (pH 8.6) and incubating for 5 days at 4 ºC. Finally, the electrode was incubated with 1 mM β-mercaptoethanol (BME) in ethanol for 5 min to back-fill the empty spots of the electrode surface, thus reducing the non-specific adsorption onto the gold working electrode surface.…”

“…Aptamers, capable of specifically binding to selected targets, have shown advantages over antibodies for biosensing applications. The unique threedimensional conformational change of aptamers upon targetanalyte binding enables the aptasensor to detect NoV particles with high specificity and sensitivity [3]. In the present study, we aimed to develop a miniaturized MEMS-based electrochemical aptasensor for rapid norovirus detection.…”

Electrochemical biosensors based on nanomodified screen-printed electrodes: Recent applications in clinical analysis

“…In the period considered in this review several examples have been proposed to develop electrochemical disposable sensors based on the use of DNA aptamers or DNA sequences for the detection of aptamers' targets, proteins, transcription-factors and anti-DNA antibodies [65,[90][91][92][93][94][95].…”