15 research outputs found
Reduction in the viability of FCV and MNV after contact with ethanol.
<p>Virus reduction was observed after 20 s (A) or 30 s (B) contact time with a commercial ABHR (62% ethanol) and an aqueous solution of 75% (v/v) ethanol on the fingerpads of adult subjects (n = 6). Significant difference between FCV and MNV (p<0.01) indicated by asterisks.</p
Reduction of MNV after 20 second contact with ethanol.
<p>MNV reduction was observed after 20 s contact time with one of either a commercial ABHR (62% ethanol), 75% ethanol or 80% aqueous ethanol solution on the fingerpads of adult subjects (n = 6). Significant differences were observed between both 62% and 75% (p<0.01) and 80% (p<0.01) indicated by asterisks (*). A significant difference was also seen between 75% and 80% (p<0.01) indicated by the double dagger (‡).</p
Ultrasensitive Norovirus Detection Using DNA Aptasensor Technology
<div><p>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.</p></div
Binding assays.
<p>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.</p
Schematic diagram of the electrochemical detection protocol adopted in this study.
<p>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 <i>via</i> square wave voltammetry.</p
Summary of rounds for MNV SELEX.
<p>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).</p
Aptasensor results.
<p>(A) Cyclic voltammograms of the norovirus aptasensor after each immobilization or binding step. The [Fe(CN)<sub>6</sub>]<sup>3−/2−</sup> redox couple was monitored for these experiments and cyclic voltammograms were recorded at a scan rate of 100 mV s<sup>,–1</sup> where (<i>a</i>) bare SPGE; (<i>b</i>) after self-assembly of the thiolated norovirus specific aptamer; (<i>c)</i> after back-filling with 1 mM 2-mercaptoethanol. (B) Square wave voltammograms obtained using (<i>a</i>) 20 aM, (<i>b</i>) 40 aM, (<i>c</i>) 60 aM, (<i>d</i>) 80 aM, (<i>e</i>) 100 aM, and (<i>f</i>) 120 aM of norovirus in buffer. (C) Calibration plot of current <i>vs.</i> concentration of norovirus. (D) Selectivity experiments performed using (a) buffer alones, (b) 5000 PFU of vesicular stomatitis virus, (c) 5.1 mg mL<sup>−1</sup> 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 K<sub>3</sub>[Fe(CN)<sub>6</sub>] and 10 µM hexaamine ruthenium chloride.</p
Aptamer-Based Impedimetric Sensor for Bacterial Typing
The development of an aptamer-based impedimetric sensor
for typing
of bacteria (AIST-B) is presented. Highly specific DNA aptamers to <i>Salmonella enteritidis</i> were selected via Cell-SELEX technique.
Twelve rounds of selection were performed; each comprises a positive
selection step against <i>S. enteritidis</i> and a negative
selection step against a mixture of related pathogens, including <i>Salmonella typhimurium</i>, <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i>, to ensure the species-specificity
of the selected aptamers. After sequencing of the pool showing the
highest binding affinity to <i>S. enteritidis</i>, a DNA
sequence of high affinity to the bacteria was integrated into an impedimetric
sensor via self-assembly onto a gold nanoparticles-modified screen-printed
carbon electrode (GNPs-SPCE). Remarkably, this aptasensor is highly
selective and can successfully detect <i>S. enteritidis</i> down to 600 CFU mL<sup>–1</sup> (equivalent to 18 CFU in
30 μL assay volume) in 10 min and distinguish it from other
Salmonella species, including <i>S. typhimurium</i> and <i>S. choleraesuis</i>. This report is envisaged to open a new
venue for the aptamer-based typing of a variety of microorganisms
using a rapid, economic, and label-free electrochemical platform
Aptamer-Based Viability Impedimetric Sensor for Bacteria
The development of an aptamer-based viability impedimetric
sensor
for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers
to live <i>Salmonella typhimurium</i> were selected via
the cell-systematic evolution of ligands by exponential enrichment
(SELEX) technique. Twelve rounds of selection were performed; each
comprises a positive selection step against viable <i>S. typhimurium</i> and a negative selection step against heat killed <i>S. typhimurium</i> and a mixture of related pathogens, including <i>Salmonella
enteritidis</i>, <i>Escherichia coli</i>, <i>Staphylococcus
aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i> to ensure the species specificity of
the selected aptamers. The DNA sequence showing the highest binding
affinity to the bacteria was further integrated into an impedimetric
sensor via self-assembly onto a gold nanoparticle-modified screen-printed
carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly
selective and can successfully detect <i>S. typhimurium</i> down to 600 CFU mL<sup>–1</sup> (equivalent to 18 live cells
in 30 μL of assay volume) and distinguish it from other <i>Salmonella</i> species, including <i>S. enteritidis</i> and <i>S. choleraesuis</i>. This report is envisaged to
open a new venue for the aptamer-based viability sensing of a variety
of microorganisms, particularly viable but nonculturable (VBNC) bacteria,
using a rapid, economic, and label-free electrochemical platform