Finite element modeling to analyze TEER values across silicon nanomembranes.

Silicon nanomembranes are ultrathin, highly permeable, optically transparent and biocompatible substrates for the construction of barrier tissue models. Trans-epithelial/endothelial electrical resistance (TEER) is often used as a non-invasive, sensitive and quantitative technique to assess barrier function. The current study characterizes the electrical behavior of devices featuring silicon nanomembranes to facilitate their application in TEER studies. In conventional practice with commercial systems, raw resistance values are multiplied by the area of the membrane supporting cell growth to normalize TEER measurements. We demonstrate that under most circumstances, this multiplication does not 'normalize' TEER values as is assumed, and that the assumption is worse if applied to nanomembrane chips with a limited active area. To compare the TEER values from nanomembrane devices to those obtained from conventional polymer track-etched (TE) membranes, we develop finite element models (FEM) of the electrical behavior of the two membrane systems. Using FEM and parallel cell-culture experiments on both types of membranes, we successfully model the evolution of resistance values during the growth of endothelial monolayers. Further, by exploring the relationship between the models we develop a 'correction' function, which when applied to nanomembrane TEER, maps to experiments on conventional TE membranes. In summary, our work advances the the utility of silicon nanomembranes as substrates for barrier tissue models by developing an interpretation of TEER values compatible with conventional systems.

A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.

Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays, and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microscale benefits of rapid magnetic separation. This system comprises magnetic nanoparticles and polymer-antibody (Ab) conjugates that transition from hydrophilic nanoscale reagents to microscale aggregates in response to temperature stimuli. The binary reagent system was benchmarked against Ab-labeled Dynabeads in terms of biomarker isolation kinetics, assay speed, and reagent needs. Surface plasmon resonance (SPR) measurements showed that polymer conjugation did not significantly alter the Ab's binding affinity or kinetics. ELISA analysis showed that the unconjugated Ab, polymer-Ab conjugates, and Ab-labeled Dynabeads exhibited similar equilibrium dissociation constants (Kd), ∼2 nM. However, the binary reagent system isolated HIV p24 antigen from spiked serum specimens (150 pg/mL) much more quickly than Dynabeads, which resulted in shorter binding times by tens of minutes, or about 30-50% shorter overall assay times. The binary reagent system showed improved performance because the Ab molecules were not conjugated to large, solid microparticle surfaces. This stimuli-responsive binary reagent system illustrates the potential advantages of nanoscale reagents in molecule and cell isolations for both research and clinical applications.

Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System.

PURPOSE: In order to improve the detection limit of existing HIV diagnostic assays, we explored the use of a temperature-responsive magnetic nanoparticle reagent system in conjunction with cyanovirin-N for HIV recognition to rapidly and efficiently concentrate viral particles from larger sample volumes, ~ 1 ml. METHODS: Cyanovirin-N (CVN) mutant, Q62C, was expressed, biotinylated, and then complexed with a thermally responsive polymer-streptavidin conjugate. Confirmation of protein expression/activity was performed using matrix assisted laser desorption/ionization (MALDI) and a TZM-bl HIV inhibition assay. Biotinylated CVN mutant recognition with gp120 was characterized using surface plasmon resonance (SPR). Virus separation and enrichment using a thermoresponsive magnetic nanoparticle reagent system were measured using RT-PCR. RESULTS: Biotinylated Q62C exhibited a KD of 0.6 nM to gp120. The temperature-responsive binary reagent system achieved a maximum viral capture of nearly 100% HIV, 1 × 10(5) virus copies in 100 µl, using pNIPAAm-Q62C within 30 minutes. Additionally, the same reagent system achieved nearly 9-fold enrichment by processing a 10-times larger sample of 1000 µl (Fig. 3). CONCLUSION: This work demonstrated a temperature-responsive reagent system that provides enrichment of HIV using antiviral lectin CVN for recognition, which is potentially amenable for use in point-of-care settings.

Purified and surfactant-free coenzyme Q10-loaded biodegradable nanoparticles.

The intent of this work was to synthesize and comprehensively characterize ubiquinone-loaded, surfactant-free biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles in vitro. Surfactant-free, empty and ubiquinone (CoQ10)-loaded biodegradable nanoparticles were synthesized by nanoprecipitation, and the physicochemical properties of these nanoparticles were analyzed with a variety of techniques. Nanoprecipitation consistently yielded individual, sub-200nm, surfactant-free empty and CoQ10-loaded nanoparticles, where the physical and drug encapsulation characteristics were controlled by varying the formulation parameters. CoQ10 release was sustained for 2 weeks but then plateaued before 100% CoQ10 release. A novel, nondestructive purification protocol involving transient sodium dodecyl sulfate (SDS) adsorption to nanoparticles followed by centrifugation and dialysis was developed to yield purified, surfactant-free, CoQ10-loaded nanoparticles. This protocol permitted removal of unencapsulated CoQ10, prevented centrifugation-induced nanoparticle aggregation and preserved the surfactant-free and drug encapsulation properties of the nanoparticles. These CoQ10-loaded nanoparticles are promising as sustained drug delivery devices due to their extended CoQ10 release. Importantly, a surfactant-free nanoprecipitation procedure is presented that in combination with a novel purification step enables the synthesis of individual and purified CoQ10-loaded nanoparticles.

Stoichiometry-dependent formation of quantum dot-antibody bioconjugates: a complementary atomic force microscopy and agarose gel electrophoresis study.

The unique advantages of quantum dot (QD) bioconjugates have motivated their application in biological assays. However, physical characterization of bioconjugated QDs after surface modification has often been overlooked. Here, biotinylated antibodies (biotin-IgG) were attached to commercial streptavidin-conjugated quantum dots (strep-QDs) at different stoichiometric ratios, and these QD bioconjugates were characterized with atomic force microscopy and discontinuous sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE). The results from these complementary analytical techniques showed that the molar ratio determined the relative sizes, molecular weights and morphologies of the QD bioconjugates. Additionally, the novel discontinuous SDS-AGE analysis confirmed specific binding between biotin-IgG and strep-QDs. Researchers who design QD bioconjugates for cell-based assays should consider stoichiometry-dependent differences in the physical properties of their QD bioconjugates.

Endothelial vacuolization induced by highly permeable silicon membranes.

Assays for initiating, controlling and studying endothelial cell behavior and blood vessel formation have applications in developmental biology, cancer and tissue engineering. In vitro vasculogenesis models typically combine complex three-dimensional gels of extracellular matrix proteins with other stimuli like growth factor supplements. Biomaterials with unique micro- and nanoscale features may provide simpler substrates to study endothelial cell morphogenesis. In this work, patterns of nanoporous, nanothin silicon membranes (porous nanocrystalline silicon, or pnc-Si) are fabricated to control the permeability of an endothelial cell culture substrate. Permeability on the basal surface of primary and immortalized endothelial cells causes vacuole formation and endothelial organization into capillary-like structures. This phenomenon is repeatable, robust and controlled entirely by patterns of free-standing, highly permeable pnc-Si membranes. Pnc-Si is a new biomaterial with precisely defined micro- and nanoscale features that can be used as a unique in vitro platform to study endothelial cell behavior and vasculogenesis.

Stimuli-Responsive Polymer-Antibody Conjugates via RAFT and Tetrafluorophenyl Active Ester Chemistry.

Highly efficient polymer-antibody conjugations were demonstrated via a tetrafluorophenyl active ester. A well-defined diblock copolymer was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with a temperature-responsive block, poly(N-isopropylacrylamide), and a block of N,N-dimethylacrylamide and 2,3,5,6-tetrafluorophenyl acrylate active ester. The polymer was conjugated to anti-p24 IgG antibody with about 100% efficiency in as little as 2 h at room temperature in a pH 10.8 buffer. The temperature-responsiveness of the polymer was conferred to the polymer-antibody conjugates after conjugation. The conjugates bound p24 antigen specifically and with binding efficiency comparable to native antibodies. Thus, the active ester diblock copolymer can facilitate the synthesis of temperature-responsive bioconjugates, which may be promising reagents for immunoassays, bioseparations, and specimen-enrichment applications.

Surfactant-free, drug-quantum-dot coloaded poly(lactide-co-glycolide) nanoparticles: towards multifunctional nanoparticles.

Nanoprecipitation was utilized to synthesize biodegradable and surfactant-free nanoparticles loaded with quantum dots. This protocol also yielded nanoparticles coloaded with both quantum dots and hydrophobic drug (Coenzyme Q10) molecules. Importantly, even though surfactants were not utilized during the nanoprecipitation procedure, these loaded nanoparticles did not aggregate. Dialysis efficiently removed unencapsulated quantum dots from nanoparticle suspensions without altering the physical properties of the quantum-dot-loaded nanoparticles. The resultant purified, quantum-dot-loaded nanoparticles were biocompatible in differentiated PC12 cell cultures, which facilitated their use as nanoparticles in microscopy. In fact, confocal imaging studies showed that purified, quantum-dot-loaded nanoparticles were associated with PC12 cells after one day in vitro. These novel and multifunctional coloaded nanoparticles may prove advantageous in future simultaneous drug delivery and imaging applications.

Peptide-conjugated quantum dots activate neuronal receptors and initiate downstream signaling of neurite growth.

Quantum dots (QDs) could serve as fluorescent scaffolds for effecting specific physiological and pharmacological responses in cells. Here, we conjugate the peptide ligand betaNGF to QD surfaces, and confirm surface modification and single QD nanostructure using AFM. We show that betaNGF-QDs retain bioactivity, activate TrkA receptors, and initiate neuronal differentiation in PC12 cells. Receptor-evoked activity of QD-immobilized ligands has wide-ranging implications for the development of molecular tools and therapeutics targeted at understanding and regulating cell function.

Neurotransmitter analog tethered to a silicon platform for neuro-BioMEMS applications.

The design of chemically well-defined, machinable surfaces containing neuroactive molecules offers potential for fundamental neuroscience and clinical neural engineering applications. Here we report the assembly and characterization of silicon platforms containing a tethered form of muscimol. Muscimol, an analog of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), is a potent agonist at postsynaptic GABA(A) and GABA(C) receptors. Surfaces were assembled using covalent avidin conjugation to silanized silicon followed by high-affinity avidin-biotin binding of a biotinylated derivative of muscimol (muscimol-biotin). Contact angle measurements, ellipsometry, and X-ray photoelectron spectroscopy (XPS) were conducted to characterize the wettability, thickness, and chemical composition of progressively deposited surface layers. The data demonstrate successful incorporation of a neurotransmitter analog as part of a layered, silicon-based structure possessing robust and specific biomolecular composition. These findings represent a step toward the design of platforms for applications involving control and modulation of neural signaling.