ABSTRACT
DNA origami nanostructures are engineered nanomaterials (ENMs) that possess significant customizability, biocompatibility, and tunable structural and functional properties, making them potentially useful materials in fields, such as medicine, biocomputing, biomedical engineering, and measurement science. Despite the potential of DNA origami as a functional nanomaterial, a major barrier to its applicability is the difficulty associated with obtaining pure, well-folded structures. Therefore, rapid methods of analysis to ensure purity are needed to support the rapid development of this class of nanomaterials. Here, we present the development of capillary electrophoresis (CE) as an analytical tool for DNA origami. CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ctITP separation mode demonstrated superior performance in terms of peak resolution (Rs = 2.05 ± 0.3), peak efficiency (N = 12,200 ± 230), and peak symmetry (As = 1.29 ± 0.032). The SYBR family dyes (Gold, Green I, and Green II) were investigated as highly efficient, noncovalent fluorophores for on-column labeling of DNA origami and detection using laser-induced fluorescence. Finally, ctITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.
Subject(s)
Isotachophoresis , Nanostructures , Nanostructures/chemistry , DNA/chemistry , Gold , Electrophoresis, Capillary/methods , Isotachophoresis/methods , Nucleic Acid Conformation , NanotechnologyABSTRACT
In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a "DNA Aptaomics" approach to systematically design functional aptamers as well as to obtain high affinity aptamers.
Subject(s)
Aptamers, Nucleotide , Electrophoresis, Capillary , High-Throughput Nucleotide Sequencing , Reproducibility of Results , ThrombinABSTRACT
The dissolution of silver nanoparticles (AgNPs) to release Ag(I)(aq) is an important mechanism in potentiating AgNP cytotoxicity and imparting their antibacterial properties. However, AgNPs can undergo other simultaneous biophysicochemical transformations, such as protein adsorption, which can mediate AgNP dissolution behaviors. We report the comprehensive analysis of AgNP dissolution and protein adsorption behaviors with monolayer surface coverage of AgNPs by bovine serum albumin (BSA). AgNP dissolution rate constants, kdissolution, were quantified over several particle sizes (10, 20, and 40 nm) and BSA concentrations (0-2 nM) using linear sweep stripping voltammetry. Across all particle sizes, the dissolution rate constant increased with increasing BSA concentrations. However, protein-enhanced dissolution behaviors were most pronounced for 10 nm AgNPs, which exhibited 3.6-fold and 7.7-fold relative enhancement when compared to 20 and 40 nm AgNPs, respectively. Changes to AgNP surface properties upon interaction with BSA were monitored using dynamic light scattering and zeta potential measurements, while BSA-AgNP complex formation was evaluated using UV-vis spectroscopy and circular dichroism spectroscopy. A subtle increase in the BSA-AgNP association constant was observed with an increase in the AgNP size. Together, these results suggest that the AgNP size dependence of BSA-enhanced dissolution of AgNPs is possibly mediated through both displacement of Ag(I)(aq)-loaded BSA by excess protein in the bulk solution and minimized accessibility of the AgNP surface because of BSA adsorption.
Subject(s)
Metal Nanoparticles/chemistry , Serum Albumin, Bovine/chemistry , Silver/chemistry , Animals , Cattle , Kinetics , Particle Size , Surface PropertiesABSTRACT
Linear sweep stripping voltammetry (LSSV) is demonstrated as a sensitive, rapid, and cost-efficient analytical technique for the quantification of silver nanoparticle (AgNP) dissolution rates in simulated sweat. LSSV does not require the extensive sample preparation (e.g., ultrafiltration or centrifugation) needed by more commonly employed techniques, such as atomic spectroscopy. The limit of detection (LOD) of Ag(I)(aq) was 14 ± 6 µg L-1, and measured dissolution rate constants, kdissolution, varied from 0.0168-0.1524 h-1, depending on solution conditions. These values are comparable and agree well with those determined by others in the literature using atomic spectroscopy. Importantly, LSSV had the necessary sensitivity to distinguish the effects of SSW solution conditions on AgNP dissolution rates. Specifically, enhanced dissolution rates were observed with decreased pH and with increased NaCl concentration. The colloidal stability of AgNPs in SSW solutions was also characterized using dynamic light scattering (DLS), ζ potential, and quantitative UV-vis spectroscopy measurements. An increase in AgNP aggregation rate was observed with increased NaCl concentration in SSW, suggesting that the enhancement in AgNP dissolution is driven by the large Cl/Ag ratio, even as the AgNPs undergo significant aggregation.
Subject(s)
Metal Nanoparticles , Silver , Kinetics , Solubility , SweatABSTRACT
Protein corona formed on nanomaterial surfaces play an important role in the bioavailability and cellular uptake of nanomaterials. Modification of surfaces with oligoethylene glycols (OEG) are a common way to improve the resistivity of nanomaterials to protein adsorption. Short-chain ethylene oxide (EO) oligomers have been shown to improve the protein resistance of planar Au surfaces. We describe the application of these EO oligomers for improved protein resistance of 30 nm spherical gold nanoparticles (AuNPs). Functionalized AuNPs were characterized using UV-Vis spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. Capillary electrophoresis (CE) was used for separation and quantitation of AuNPs and AuNP-protein mixtures. Specifically, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was employed for the determination of equilibrium and rate constants for binding between citrate-stabilized AuNPs and two model proteins, lysozyme and fibrinogen. Semi-quantitative CE analysis was carried out for mixtures of EO-functionalized AuNPs and proteins, and results demonstrated a 2.5-fold to 10-fold increase in protein binding resistance to lysozyme depending on the AuNP surface functionalization and a 15-fold increase in protein binding resistance to fibrinogen for both EO oligomers examined in this study. Graphical abstract Using capillary electrophoresis, the addition of short-chained oligo(ethylene oxide) ligands to gold nanoparticles was shown to improve protein binding resistance up to 15-fold.
Subject(s)
Ethylene Oxide/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Protein Corona/analysis , Adsorption , Animals , Cattle , Chickens , Citric Acid/chemistry , Dynamic Light Scattering , Electrophoresis, Capillary , Fibrinogen/analysis , Models, Molecular , Muramidase/analysis , Sulfhydryl Compounds/chemistry , Surface PropertiesABSTRACT
Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or "aptamers" because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4% aptamer content before selection to >15% aptamer content.
Subject(s)
Aptamers, Nucleotide/chemistry , Electrophoresis, Capillary/methods , High-Throughput Nucleotide Sequencing/methodsABSTRACT
We developed a capillary electrophoresis method to separate polystyrene particles (PSPs) with different sizes or different surface functionalities. Separations were performed in buffer and 100 mg L-1 clay or 100 mg L-1 Suwanee River humic acid. In all solutions, PSPs were baseline or near-baseline resolved in less than 15 minutes.
ABSTRACT
Introduction: Biomolecules bind to and transform nanoparticles, mediating their fate in biological systems. Despite over a decade of research into the protein corona, the role of protein modifications in mediating their interaction with nanomaterials remains poorly understood. In this study, we evaluated how glycation of the most abundant blood protein, human serum albumin (HSA), influences the formation of the protein corona on 40 nm silver nanoparticles (AgNPs) and the toxicity of AgNPs to the HepG2 human liver cell line. Methods: The effects of glycation on AgNP-HSA interactions were quantified using circular dichroism spectroscopy to monitor protein structural changes, dynamic light scattering to assess AgNP colloidal stability, zeta potential measurements to measure AgNP surface charge, and UV-vis spectroscopy and capillary electrophoresis (CE) to evaluate protein binding affinity and kinetics. The effect of the protein corona and HSA glycation on the toxicity of AgNPs to HepG2 cells was measured using the WST cell viability assay and AgNP dissolution was measured using linear sweep stripping voltammetry. Results and Discussion: Results from UV-vis and CE analyses suggest that glycation of HSA had little impact on the formation of the AgNP protein corona with protein-AgNP association constants of ≈2x107 M-1 for both HSA and glycated HSA (gHSA). The formation of the protein corona itself (regardless of whether it was formed from HSA or glycated HSA) caused an approximate 2-fold decrease in cell viability compared to the no protein AgNP control. While the toxicity of AgNPs to cells is often attributed to dissolved Ag(I), dissolution studies showed that the protein coated AgNPs underwent less dissolution than the no protein control, suggesting that the protein corona facilitated a nanoparticle-specific mechanism of toxicity. Overall, this study highlights the importance of protein coronas in mediating AgNP interactions with HepG2 cells and the need for future work to discern how protein coronas and protein modifications (like glycation) may alter AgNP reactivity to cellular organisms.
ABSTRACT
The Primarily Undergraduate Nanomaterials Cooperative (PUNC) is an organization for research-active faculty studying nanomaterials at Primarily Undergraduate Institutions (PUIs), where undergraduate teaching and research go hand-in-hand. In this perspective, we outline the differences in maintaining an active research group at a PUI compared to an R1 institution. We also discuss the work of PUNC, which focuses on community building, instrument sharing, and facilitating new collaborations. Currently consisting of 37 members from across the United States, PUNC has created an online community consisting of its Web site (nanocooperative.org), a weekly online summer group meeting program for faculty and students, and a Discord server for informal conversations. Additionally, in-person symposia at ACS conferences and PUNC-specific conferences are planned for the future. It is our hope that in the years to come PUNC will be seen as a model organization for community building and research support at primarily undergraduate institutions.
ABSTRACT
Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP), were compared for the detection and separation of spherical gold nanoparticles (AuNPs) and gold nanorods (AuNRs). The development of ctITP using two different leading ions is described. Overall, when compared to traditional capillary zone electrophoresis (CZE), ctITP resulted in improved peak shape and peak efficiency. Specifically, the number of theoretical plates for AuNR samples increased by a factor of 2-2.5 depending on the choice of leading ion. Further, using ctITP two AuNRs differing by aspect ratio were baseline resolved, whereas the same AuNRs could not be separated using CZE or other techniques like single particle inductively coupled plasma mass spectrometry (spICP-MS) and asymmetric flow field-flow fractionation (AF4). The results of this study demonstrate that ctITP is an efficient on-line technique for the improved detection and separation of gold nanomaterials in CE.
Subject(s)
Chemistry Techniques, Analytical/methods , Gold/chemistry , Gold/isolation & purification , Isotachophoresis , Metal Nanoparticles/chemistry , Nanotubes/chemistry , Electrolytes , Electrophoresis, Capillary , Particle SizeABSTRACT
Nanomedicine utilizes the remarkable properties of nanomaterials for the diagnosis, treatment, and prevention of disease. Many of these nanomaterials have been shown to have robust antioxidative properties, potentially functioning as strong scavengers of reactive oxygen species. Conversely, several nanomaterials have also been shown to promote the generation of reactive oxygen species, which may precipitate the onset of oxidative stress, a state that is thought to contribute to the development of a variety of adverse conditions. As such, the impacts of nanomaterials on biological entities are often associated with and influenced by their specific redox properties. In this review, we overview several classes of nanomaterials that have been or projected to be used across a wide range of biomedical applications, with discussion focusing on their unique redox properties. Nanomaterials examined include iron, cerium, and titanium metal oxide nanoparticles, gold, silver, and selenium nanoparticles, and various nanoscale carbon allotropes such as graphene, carbon nanotubes, fullerenes, and their derivatives/variations. Principal topics of discussion include the chemical mechanisms by which the nanomaterials directly interact with biological entities and the biological cascades that are thus indirectly impacted. Selected case studies highlighting the redox properties of nanomaterials and how they affect biological responses are used to exemplify the biologically-relevant redox mechanisms for each of the described nanomaterials.
Subject(s)
Nanomedicine , Nanostructures , Free Radical Scavengers/pharmacology , Graphite , Humans , Metal Nanoparticles , Nanotubes, Carbon , Oxidation-Reduction , Reactive Oxygen Species/metabolismABSTRACT
Previously, catalytic cerium oxide nanoparticles (CNPs, nanoceria, CeO2-x NPs) have been widely utilized for chemical mechanical planarization in the semiconductor industry and for reducing harmful emissions and improving fuel combustion efficiency in the automobile industry. Researchers are now harnessing the catalytic repertoire of CNPs to develop potential new treatment modalities for both oxidative- and nitrosative-stress induced disorders and diseases. In order to reach the point where our experimental understanding of the antioxidant activity of CNPs can be translated into useful therapeutics in the clinic, it is necessary to evaluate the most current evidence that supports CNP antioxidant activity in biological systems. Accordingly, the aims of this review are three-fold: (1) To describe the putative reaction mechanisms and physicochemical surface properties that enable CNPs to both scavenge reactive oxygen species (ROS) and to act as antioxidant enzyme-like mimetics in solution; (2) To provide an overview, with commentary, regarding the most robust design and synthesis pathways for preparing CNPs with catalytic antioxidant activity; (3) To provide the reader with the most up-to-date in vitro and in vivo experimental evidence supporting the ROS-scavenging potential of CNPs in biology and medicine.
ABSTRACT
Capillary electrophoresis (CE) was used for the characterization of a variety of polymeric micron and sub-micron particles based on size, surface functionality, and binding properties. First, a robust capillary zone electrophoresis (CZE) method was developed for the baseline separation and quantitation of commercially available polystyrene particles with various surface modifications (including amino, carboxylate, and sulfate functional groups) and various sizes (0.2, 0.5, 1.0, and 3.0µm). The separation of DNA-templated polyacrylamide particles from untemplated particles (as used for the Ion Torrent Personal Genome Machine) was demonstrated. Finally, using the 29-base thrombin aptamer and thrombin protein as a model system, a study was undertaken to determine dissociation constants for the aptamer and protein in free solution and when the aptamer was conjugated to a particle, with the goal of better understanding how the use of solid substrates, like particles, affects selection and binding processes. Dissociation constants were determined and were found to be approximately 5-fold higher for the aptamer conjugated to a particle relative to that in free solution.
Subject(s)
Aptamers, Nucleotide/chemistry , DNA/chemistry , Electrophoresis, Capillary/methods , Polystyrenes/chemistry , Polystyrenes/isolation & purification , Thrombin/chemistry , Acrylic Resins/chemistry , Acrylic Resins/isolation & purification , Particle Size , Protein Binding , Solutions/chemistry , Thermodynamics , Thrombin/analysisABSTRACT
Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10-6mol/l) relative to uncoated plates (2.4 × 10-6 mol/l), or plates coated with the related protein fibronectin (2.1 × 10-6 mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.
ABSTRACT
An efficient separation method that utilizes capillary transient isotachophoresis (ctITP) was developed for the preselection of binding ligands. With the ultimate goal of providing enriched fractions from vast libraries for drug discovery, the preselection process described herein entails three distinct elements, which have been validated using a model thrombin protein (target) and thrombin aptamer (ligand) system. First, a high fidelity, on-column labeling scheme employing the noncovalent, fluorescent reagent SYBR Gold was demonstrated for single-stranded DNA with an 11-fold greater sensitivity than pre-column labeling procedures. Second, this on-column labeling was incorporated into a new ctITP method with laser-induced fluorescence (LIF) detection, which provided greatly enhanced resolution of protein-aptamer complex and free aptamer (in comparison to traditional capillary zone electrophoresis (CZE) methods). Third, this enhanced resolution permitted the subsequent accumulation of bound aptamer fractions via an automated collection method, with the establishment of quantitative measures of DNA accumulation. Preselected aptamer or ligand samples such as these can serve as inputs for subsequent lab-on-bead or next-generation-sequencing technologies, enabling accelerated drug discovery.