Equilibrium interactions of biomimetic DNA aptamers produce intrafibrillar calcium phosphate mineralization of collagen.

Native and biomimetic DNA structures have been demonstrated to impact materials synthesis under a variety of conditions but have only just begun to be explored in this role compared to other biopolymers such as peptides, proteins, polysaccharides, and glycopolymers. One selected DNA aptamer has been explored in calcium phosphate and calcium carbonate mineralization, demonstrating sequence-dependent control of kinetics, morphology, and crystallinity. This aptamer is here applied to a biologically-relevant bone model system that uses collagen hydrogels. In the presence of the aptamer, intrafibrillar collagen mineralization is observed compared to negative controls and a positive control using well-studied poly-aspartic acid. The mechanism of interaction is explored through affinity measurements, kinetics of calcium uptake, and kinetics of aptamer uptake into the forming mineral. There is a marked difference observed between the selected aptamer containing a G-quadruplex secondary structure compared to a control sequence with no G-quadruplex. It is hypothesized that the equilibrium interaction of the aptamer with calcium-phosphate precursors and with the collagen itself leads to slow kinetic mineral formation and a morphology appropriate to bone. This points to new uses for DNA aptamers in biologically-relevant mineralization systems and the possibility of future biomedical applications. STATEMENT OF SIGNIFICANCE: Collagen is the protein structural component that mineralizes with calcium phosphate to form durable bone. Crystalline calcium phosphate must be infused throughout the collagen fiber structure to produce a strong material. This process is assisted by soluble proteins that interact with both calcium phosphate precursors and the collagen protein and has been proposed to follow a polymer-induce liquid precursor (PILP) model. Further understanding of this model and control of the process through synthetic, biomimetic molecules could have significant advantages in biomedical, restorative procedures. For the first time, synthetic DNA aptamers with specific secondary structures are here shown to influence and direct collagen mineralization. The mechanism of this process has been studied to demonstrate an important equilibrium between the DNA aptamer, calcium phosphate precursors, and collagen.

Secondary Structure of DNA Aptamer Influences Biomimetic Mineralization of Calcium Carbonate.

Calcium materials, such as calcium carbonate, are produced in natural and industrial settings that range from oceanic to biomedical. An array of biological and biomimetic template molecules have been employed in controlling and understanding the mineralization reaction but have largely focused on small molecule additives or disordered polyelectrolytes. DNA aptamers are synthetic and programmable biomolecules with polyelectrolyte characteristics but with predictable and controllable secondary structure akin to native extracellular moieties. This work demonstrates for the first time the influence of DNA aptamers with known G-quadruplex structures on calcium carbonate mineralization. Aptamers demonstrate kinetic inhibition of mineral formation, sequence and pH-dependent uptake into the mineral, and morphological control of the primarily calcite material in controlled solution conditions. In reactions initiated from the complex matrix of ocean water, DNA aptamers demonstrated enhancement of mineralization kinetics and resulting amorphous material. This work provides new biomimetic tools to employ in controlled mineralization and demonstrates the influence that template secondary structure can have in material formation.

Selected DNA aptamers as hydroxyapatite affinity reagents.

DNA aptamers were selected for their ability to bind specifically and quickly to crystalline hydroxyapatite (Ca10(PO4)6(OH)2; HAP), the primary mineral component of enamel and bone. Aptamers were found to have an enhanced percent of G-nucleotides and a propensity for forming a G-quadruplex secondary structure. One aptamer was studied in comparison to control sequences and was found to bind with high affinity and at high loading capacity, with enhanced binding kinetics, and with specificity for crystalline HAP material over amorphous calcium phosphate (ACP) and ß-tricalcium phosphate (TCP). The fluorescently-functionalized aptamer was demonstrated to specifically label HAP in a surface binding experiment and suggests the usefulness of this selected aptamer in biomedical or biotechnology fields where the labeling of specific calcium phosphate materials is required.

Selected DNA Aptamers Influence Kinetics and Morphology in Calcium Phosphate Mineralization.

DNA aptamers previously selected as calcium phosphate mineralization templates were modified and analyzed to better understand structure-function relationships and to explore the mechanism of templated mineralization. Aptamers were created to strengthen or remove a prevalent G-quadruplex structure and were analyzed for structural stability, affinity to calcium phosphate, influence on homogeneous and heterogeneous calcium phosphate mineralization, and influence on mineral crystallinity and morphology. Aptamers were found to modulate mineralization kinetics in a concentration-dependent manner. Changes to the G-quadruplex structure affected affinity to hydroxyapatite (Ca10(PO4)6(OH)2, HAP) and had a substantial impact on the mineral crystallinity. We propose a model for aptamer-directed mineralization and anticipate the usefulness of these aptamer sequences in future biomimetic and biomedical applications.

Solution rheological parameters modulate calcium phosphate mineralization in a microfluidic device.

Mineralization of calcium phosphate and other materials in vivo and in natural water sources occurs in solutions that are not stagnant, but are flowing. Flow conditions could influence solution mixing and, therefore, mineralization kinetics or mechanism. This work describes the design and characterization of a multi-stream parallel flow microfluidic device that allows for controlled solution mixing and indirect control of laminar flow by altering the microfluidic device width, shape, length, flow rate, and flow velocity. Measurement of solution mixing was accomplished using the protonation of quinine to produce a fluorescent molecule and the rate of calcium phosphate mineralization was monitored by optical microscopy and analysis with Image J software. Experiments were designed to hold the flow rate constant, allowing the solution velocity to vary and to hold the velocity constant, allowing the flow rate to vary. It was found that small changes in laminar flow conditions do not correlate to mineral growth, but solution velocity and flow rate have a substantial effect on calcium phosphate mineralization. AFM and SEM characterization of the mineral produced shows an amorphous material and varying degrees of mineralization possibly due to variation in supersaturation conditions across the solution mixing area. This microfluidic device and analysis procedure allows for improved study of mineralization and the effect of flow conditions relevant to those seen in biological settings.

Supplemental Learning in the Laboratory: An Innovative Approach for Evaluating Knowledge and Method Transfer.

The Multi-Rule Quality Control System (MRQCS) is a tool currently employed by the Centers for Disease Control and Prevention (CDC) to evaluate and compare laboratory performance. We have applied the MRQCS to a comparison of instructor and computer-led pre-laboratory lectures for a supplemental learning experiment. Students in general chemistry and analytical chemistry from both two- and four-year institutions performed two laboratory experiments as part of their normal laboratory curriculum. The first laboratory experiment was a foundational learning experiment in which all the students were introduced to Beer-Lambert's Law and spectrophotometric light absorbance measurements. The foundational learning experiment was instructor-led only, and participant performance was evaluated against a mean characterized value. The second laboratory experiment was a supplemental learning experiment in which students were asked to build upon the methodology they learned in the foundational learning experiment and apply it to a different analyte. The instruction type was varied randomly into two delivery modes, participants receiving either instructor-led or computer-led pre-laboratory instruction. The MRQCS was applied and determined that no statistical difference was found to exist in the QC (quality control) passing rates between the participants in the instructor-led instruction and the participants in the computer-led instruction. These findings demonstrate the successful application of the MRQCS to evaluate knowledge and technology transfer.

Dynamic interactions of amelogenin with hydroxyapatite surfaces are dependent on protein phosphorylation and solution pH.

Amelogenin, the predominant extracellular matrix protein secreted by ameloblasts, has been shown to be essential for proper tooth enamel formation. In this study, amelogenin adsorption to hydroxyapatite (HAP) surfaces, a prototype for enamel mineral, has been studied using a quartz crystal microbalance (QCM) to interrogate effects of protein phosphorylation and solution pH. Dynamic flow-based experiments were conducted at pH 7.4 and 8.0 using native phosphorylated porcine amelogenin (P173) and recombinant non-phosphorylated porcine amelogenin (rP172). Loading capacities (µmol/m2) on HAP surfaces were calculated under all conditions and adsorption affinities (Kad) were calculated when Langmuir isotherm conditions appeared to be met. At pH 8.0, binding characteristics were remarkably similar for the two proteins. However, at pH 7.4 a higher affinity and lower surface loading for the phosphorylated P173 was found compared to any other set of conditions. This suggests that phosphorylated P173 adopts a more extended conformation than non-phosphorylated full-length amelogenin, occupying a larger footprint on the HAP surface. This surface-induced structural difference may help explain why P173 is a more effective inhibitor of spontaneous HAP formation in vitro than rP172. Differences in the viscoelastic properties of P173 and rP172 in the adsorbed state were also observed, consistent with noted differences in HAP binding. These collective findings provide new insight into the important role of amelogenin phosphorylation in the mechanism by which amelogenin regulates enamel crystal formation.

Quartz crystal microbalance analysis of DNA-templated calcium phosphate mineralization.

A quartz crystal microbalance (QCM) sensor was developed for the quantitation of calcium phosphate mineralization and the assessment of DNA as a template molecule. Inherent advantages of QCM, such as nanogram sensitivity, temporal resolution, surface-based measurements, and flow capabilities, were leveraged in the design of this sensor, and in-line fluidic mixing was used to control precursor reaction. This research shows that DNA, a highly programmable anionic polymer, is able to template and control mineralization of calcium phosphate, with nucleation occurring in less than 15 min and initial rates ranging from 4 to 8 ng/min. FT-IR measurements show mineralized material to be calcium phosphate resembling hydroxyapatite (HAP) when a DNA template is used. DNA is a promising mineralization template, and the QCM proves to be a dynamic technique for a broad range of heterogeneous mineralization experiments in complement to classic, diffusion-limited, end-point analysis techniques.

Unexpected toxicity of monolayer protected gold clusters eliminated by PEG-thiol place exchange reactions.

Monolayer protected clusters (MPCs) are small, metal nanoparticles capped with thiolate ligands that have been widely studied for their size-dependent properties and for their ability to be functionalized for biological applications. Common water-soluble MPCs, functionalized by N-(2-Mercaptopropionyl)-glycine (tiopronin) or glutathione, have been used previously to interface with biological systems. These MPCs are ideal for biological applications not only due to their water-solubility but also their small size (<5 nm). These characteristics are expected to enable easy biodistribution and clearance. In this article, we show an unexpected toxicity is associated with the tiopronin monolayer protected cluster (TMPC), making it incompatible for potential in vivo applications. This toxicity is linked to significant histological damage to the renal tubules, causing mortality at concentrations above 20 µM. We further show how the incorporation of poly ethylene glycol (PEG) by a simple place-exchange reaction eliminates this toxicity. We analyzed gold content within blood and urine and found an increased lifetime of the particle within the bloodstream due to the creation of the mixed monolayer. Also shown was the elimination of kidney damage with the use of the mixed-monolayer particle via Multistix analysis, MALDI-TOF MS analysis, and histological examination. Final immunological analysis showed no effect on white blood cell (WBC) count for the unmodified particle and a surprising increase in WBC count with the injection of mixed monolayer particles at concentrations higher than 30 µM, suggesting that there may be an immune response to these mixed monolayer nanoparticles at high concentrations; therefore, special attention should be focused on selecting the best capping ligands for use in vivo. These findings make the mixed monolayer an excellent candidate for further biological applications using water-soluble nanoparticles.

Micromagnetic selection of aptamers in microfluidic channels.

Aptamers are nucleic acid molecules that have been selected in vitro to bind to their molecular targets with high affinity and specificity. Typically, the systematic evolution of ligands by exponential enrichment (SELEX) process is used for the isolation of specific, high-affinity aptamers. SELEX, however, is an iterative process requiring multiple rounds of selection and amplification that demand significant time and labor. Here, we describe an aptamer discovery system that is rapid, highly efficient, automatable, and applicable to a wide range of targets, based on the integration of magnetic bead-based SELEX process with microfluidics technology. Our microfluidic SELEX (M-SELEX) method exploits a number of unique phenomena that occur at the microscale and implements a design that enables it to manipulate small numbers of beads precisely and isolate high-affinity aptamers rapidly. As a model to demonstrate the efficiency of the M-SELEX process, we describe here the isolation of DNA aptamers that tightly bind to the light chain of recombinant Botulinum neurotoxin type A (with low-nanomolar dissociation constant) after a single round of selection.

Electrospray mass spectrometry study of tiopronin monolayer-protected gold nanoclusters.

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) and gel permeation chromatography (GPC) were used to study the synthesis of a series of tiopronin monolayer-protected gold nanoclusters (MPCs) and to monitor their postsynthesis peptide ligand place-exchange reactions. All mass spectra identified the presence of cyclic gold(I)-thiolates with a strong preference for tetrameric species. During the synthesis of pre-monolayer-protected nanoclusters (pre-MPCs), esterified gold(I)-thiolate tetramers were initially observed in minor abundance (with respect to disulfide bridged tiopronin species) before dramatically increasing in abundance and precipitating from solution. After conversion of pre-MPCs to MPCs, ESI-TOF mass spectra demonstrated an overall predominance of tetrameric species with conversion from ester-terminated end groups to carboxyl-terminated end groups. Further modifications were performed through postsynthesis ligand place-exchange reactions to validate the existence of the tetramers. This work suggests that monolayer protection is accomplished by cyclized gold(I)-thiolate tetramers on the gold core surface, and/or that gold(I)-thiolates are a basic building block within the nanoparticles.

Piezoelectric ink jet processing of materials for medical and biological applications.

Many advanced medical and biological devices require microscale patterning of cells, proteins, and other biological materials. This article describes the use of piezoelectric ink jet processing in the fabrication of biosensors, cell-based assays, and other microscale medical devices. A microelectromechanical system-based piezoelectric transducer was used to develop uniform fluid flow through nozzles and to prepare well-defined microscale patterns of proteins, monofunctional acrylate ester, sinapinic acid, deoxyribonucleic acid (DNA), and DNA scaffolds on relevant substrates. Our results demonstrate that piezoelectric ink jet deposition is a powerful non-contact, non-destructive additive process for developing biosensors, cell culture systems, and other devices for medical and biological applications.

Detection of Ebola virus envelope using monoclonal and polyclonal antibodies in ELISA, surface plasmon resonance and a quartz crystal microbalance immunosensor.

Ebola virus (EBOV) Zaire, Sudan, as well as Ivory Coast are virulent human EBOV species. Both polyclonal and monoclonal antibodies (MAbs) were developed against soluble EBOV envelope glycoprotein (GP) for the study of EBOV envelope diversity and development of diagnostic reagents. Three EBOV Sudan-Gulu GP peptides, from the N-terminus, mid-GP, and C-terminus regions were used to immunize rabbits for the generation of anti-EBOV polyclonal antibodies. Polyclonal antisera raised against the C-terminus peptide could detect both Sudan-Gulu as well as Zaire GPs, while anti-N and mid-region peptide polyclonal sera recognized only EBOV Sudan-Gulu GP. Of the three anti-EBOV GP mouse MAbs produced, MAb 15H10 recognized all human EBOV GP species tested (Zaire, Sudan and Ivory Coast), and as well as reacted with the Reston non-human primate EBOV GPs. In addition, MAb 15H10 bound virion-associated GP of all known EBOV species. MAb 17A3 recognized GPs of both EBOV Sudan-Gulu and Zaire, while MAb 6D11 recognized only EBOV Sudan-Gulu GP. To detect EBOV GP, these antibody reagents were used in ELISA, surface plasmon resonance and in a quartz crystal microbalance immunosensor. Thus, polyclonal and monoclonal antibodies can be used in combination to identify and differentiate both human and non-human primate EBOV GPs.

Hemagglutinin linear epitope presentation on monolayer-protected clusters elicits strong antibody binding.

Immunoreactive, multicomponent nanoclusters were assembled through the controlled presentation of a known, synthetic peptide epitope. The epitope comes from the hemagglutanin protein of influenza and is known to bind to a monoclonal anti-HA antibody. Antibody affinity for the immunoreactive MPC was compared to the affinity for traditionally used peptide arrays using the quartz crystal microbalance. The two systems had comparable affinities (Ka), ranging from 0.41 x 10(7) M(-1) to 1.8 x 10(7) M(-1), though the nanocluster used a much lower density of peptide relative to that of the peptide array. These results suggest that functionalized nanoclusters have potential in nanostructure assembly and medical applications. Water-soluble nanoparticles that present known neutralizing peptide epitopes of protein antigens might be used in antiviral influenza vaccines.

Quartz crystal microbalance detection of glutathione-protected nanoclusters using antibody recognition.

A quartz crystal microbalance (QCM) immunosensor was developed for the quantitative detection of glutathione-protected nanoclusters. Advantages intrinsic to QCM were employed to make it an attractive alternative to other immunosensing techniques. We have addressed challenges in the area of QCM mass sensing through experimental correlation between damping resistance and frequency change for a reliable mass measurement. Electrode functionalization was optimized with the use of protein A to immobilize and present polyclonal IgG for antigen binding. This method was developed for the detection of glutathione (antigen)-protected clusters of nanometer size with high surface area and thiolate valency. Quantitation of glutathione-nanocluster binding to immobilized polyclonal antibody provides equilibrium constants (K(a) = (3.6 +/- 0.2) x 10(5) M(-1)) and kinetic rate constants (k(f) = (5.4 +/- 0.7) x 10(1) M(-1) s(-1) and k(r) = (1.5 +/- 0.4) x10(-4) s(-1)) comparable to literature reports. These observations further imply that immunoreactive nanoparticles have potential in medical diagnostics and materials assembly.