Development of a DNA aptamer for direct and selective homocysteine detection in human serum

l-Homocysteine has been an amino acid intermediate of interest for over 20 years due to its implication in various adverse health conditions, including cardiovascular disease. Here, we report the first in vitro selection and application of high affinity aptamers for the target l-homocysteine. Two novel aptamer sequences were selected following 8 rounds of selection that displayed high affinity binding and selectivity to homocysteine compared to other amino acids. One of the selected aptamers, Hcy 8 (KD = 600 ± 300 nM), was used to develop a gold-nanoparticle biosensor capable of sensitive and selective homocysteine detection in human serum, with a limit of detection of 0.5 μM and a linear range of 0.5-3.0 μM. This biosensor allows rapid detection of free homocysteine in human serum samples at low cost, with little preparation time and could be adapted to be part of a po

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

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

Aptasensor results.

<p>(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 (<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⁻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.</p

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

Summary of aptamer candidates.

<p>Summary of aptamer candidates.</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

Analysis of In Vitro Aptamer Selection Parameters

Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success