Gold Nanoparticle Assay Research Articles

Interference-resistant gold nanoparticle assay for detecting Enterococcus in fresh and marine waters

Enterococci are common indicators of fecal contamination and are used to assess the quality of fresh and marine water, sand, soil, and sediment. However, samples collected from these environments contain various cells and other factors that can interfere with the assays used to detect enterococci. We developed a novel assay for the sensitive and specific detection of enterococci that is resistant to interference from other cells and environmental factors. Our interference-resistant assay used 30-nm gold nanoparticles (AuNPs), streptavidin, and a biotinylated Enterococcus antibody. Enterococci inhibited the interaction between streptavidin and biotin and led to the disaggregation of AuNPs. The absence of enterococci led to the aggregation of AuNPs, and this difference was easily detected by spectrophotometry. This interference-resistant AuNP assay was able to detect whole cells of Enterococcus in the range of 10 to 107 CFU/mL within 3 h, had high specificity for enterococci, and was unaffected by the presence of other intestinal bacteria, such as Escherichia coli. Our examination of fresh and marine water samples demonstrated no interference from other cells or environmental factors. The interference-resistant AuNP assay described here has the potential to be used as a rapid, simple, and effective method for monitoring enterococci in diverse environmental samples.

Dark-Field Microwells toward High-Throughput Direct miRNA Sensing with Gold Nanoparticles.

MicroRNA (miRNA) is a class of short RNA that is emerging as an ideal biomarker, as its expression level has been found to correlate with different types of diseases including diabetes and cancer. The detection of miRNA is highly beneficial for early diagnostics and disease monitoring. However, miRNA sensing remains difficult because of its small size and low expression levels. Common techniques such as quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization and Northern blotting have been developed to quantify miRNA in a given sample. Nevertheless, these methods face common challenges in point-of-care practice as they either require complicated sample handling and expensive equipment, or suffer from low sensitivity. Here we present a new tool based on dark-field microwells to overcome these challenges in miRNA sensing. This miniaturized device enables the readout of a gold nanoparticle assay without the need of a dark-field microscope. We demonstrate the feasibility of the dark-field microwells to detect miRNA in both buffer solution and cell lysate. The dark-field microwells allow affordable miRNA sensing at a high throughput which make them a promising tool for point-of-care diagnostics.

DnaT is a PriC-binding protein

DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT–PriC changed from red to purple, which indicated the protein–protein interactions due to gold nanoparticle aggregate. In addition, this DnaT–PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10−8 M. These results constitute a pioneering study of the DnaT–PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC–SSB–DNA tricomplex and regulates the PriC-dependent replication restart.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A method for selecting structure-switching aptamers applied to a colorimetric gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol.

The first-known aptamer for the stress biomarker cortisol was selected using a tunable stringency magnetic bead selection strategy. The capture DNA probe immobilized on the beads was systematically lengthened to increase the number of bases bound to the complementary pool primer regions following selection enrichment. This resulted in a single sequence (15-1) dominating the final round 15 pool, where the same sequence was the second-highest copy number candidate in the enriched pool with the shorter capture DNA probe (round 13). A thorough analysis of the next-generation sequencing results showed that a high copy number may only correlate with enhanced affinity under certain stringency and enrichment conditions, in contrast with prior published reports. Aptamer 15-1 demonstrated enhanced binding to cortisol (K(d) = 6.9 ± 2.8 μM by equilibrium dialysis; 16.1 ± 0.6 μM by microscale thermophoresis) when compared with the top sequence from round 13 and the negative control progesterone. Whereas most aptamer selections terminate at the selection round demonstrating the highest enrichment, this work shows that extending the selection with higher stringency conditions leads to lower amounts eluted by the target but higher copy numbers of a sequence with enhanced binding. The structure-switching aptamer was applied to a gold nanoparticle assay in buffer and was shown to discriminate between cortisol and two other stress biomarkers, norepinephrine and epinephrine, and a structurally analogous biomarker of liver dysfunction, cholic acid. We believe this approach enhances aptamer selection and serves as proof-of-principle work toward development of point-of-care diagnostics for medical, combat, or bioterrorism targets.

BiotecVisions 2012, October

BiotecVisions 2012, October

Lectin-based gold nanoparticle assay for glycan differentiation

Biotechnology and BioengineeringVolume 109, Issue 9 p. fmvi-fmvi Spotlight Lectin-based gold nanoparticle assay for glycan differentiation First published: 25 July 2012 https://doi.org/10.1002/bit.24315Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume109, Issue9September 2012Pages fmvi-fmvi RelatedInformation

A lectin‐based gold nanoparticle assay for probing glycosylation of glycoproteins

We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins.

Probing DNA’s Interstrand Orientation with Gold Nanoparticles

The interstrand orientation of a DNA duplex plays a pivotal role in its biological and chemical functions. Therefore, developing an efficient way to determine (control and monitor) the parallel or antiparallel conformation of a DNA duplex is of great significance, which, however, remains a big challenge under some circumstances. In this work, we demonstrate that gold nanoparticles tagged on DNA are especially useful in trapping and detecting a special interstrand orientation of a DNA double helix, based on inherent electrostatic and steric repulsions between nanoparticles which will affect their self-assembly into a large structure. More importantly, some of the conformations revealed by the gold nanoparticle assay may even not be thermodynamically preferred and thus will be hard to detect using currently available methods. This simple, straightforward, and efficient methodology capable of dictating and probing a special DNA duplex structure provides a useful tool for conformational analyses and functional explorations of biomolecules as well as biophysical and nanobiomedical research.

Enterovirus 3C Proteases: Structure-based Discovery of Inhibitors

DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT–PriC changed from red to purple, which indicated the protein–protein interactions due to gold nanoparticle aggregate. In addition, this DnaT–PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10−8 M. These results constitute a pioneering study of the DnaT–PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC–SSB–DNA tricomplex and regulates the PriC-dependent replication restart.