Figure 1.
Schematic diagram of the electrochemical detection protocol adopted in this study.
A thiolated norovirus-specific DNA aptamer was self-assembled onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Binding of the virus to the immobilized aptamer causes a decrease in the redox current, measured via square wave voltammetry.
Figure 2.
Summary of rounds for MNV SELEX.
Target stringencies are outlined for sets of rounds (VP = viral particles). Retained fractions were assessed by fluorescence. DNA retained after counter-selection rounds (8 and 9) represents pool that did not interact with the indicated targets (FCV = feline calicivirus and DMEM = dialyzed components of Dulbecco’s Modified Eagle Medium).
Table 1.
Summary of aptamer candidates.
Figure 3.
A) Binding affinity measurements for 5′-fluorescein-modified AG3 with MNV using a polycarbonate filter binding assay (diamonds) and a fluorescence anisotropy assay (triangles). The binding affinity of 5′-fluorescein tagged AG3 to FCV (circles) as well as a non-specific DNA control with MNV (squares) were also measured by anisotropy. Measurements are fitted using either the Hill or Logistic functions (solid lines). Inset: Much higher concentrations of virus are required in order to show any anisotropy change in the controls. B) Fluorescence anisotropy results from binding of 5′-fluorescein-modified AG3 with varying concentrations of GII.3 HuNoV capsid.
Figure 4.
(A) Cyclic voltammograms of the norovirus aptasensor after each immobilization or binding step. The [Fe(CN)6]3−/2− redox couple was monitored for these experiments and cyclic voltammograms were recorded at a scan rate of 100 mV s,–1 where (a) bare SPGE; (b) after self-assembly of the thiolated norovirus specific aptamer; (c) after back-filling with 1 mM 2-mercaptoethanol. (B) Square wave voltammograms obtained using (a) 20 aM, (b) 40 aM, (c) 60 aM, (d) 80 aM, (e) 100 aM, and (f) 120 aM of norovirus in buffer. (C) Calibration plot of current vs. concentration of norovirus. (D) Selectivity experiments performed using (a) buffer alones, (b) 5000 PFU of vesicular stomatitis virus, (c) 5.1 mg mL−1 HSA, (d) 5000 PFU of vaccinia virus (e) 120 aM of norovirus. All experiments were performed in Dulbecco’s phosphate buffered saline after incubation with the developed aptasensor for 1 hr at 25°C. Square wave voltammograms were carried out in the range of −400 to 800 mV with a step potential of 4 mV, amplitude of 5 mV and frequency of 10 Hz. Electrochemical measurements were performed in 25 mM phosphate buffer (pH 7), containing 4 mM K3[Fe(CN)6] and 10 µM hexaamine ruthenium chloride.