Modifying Polydiacetylene Vesicle Compositions to Reduce Non-Specific Interactions.

Polydiacetylene (PDA) vesicles provide useful stimuli-responsive behavior as well as by the modular structure afford a means for the design of sensing and delivery systems with tunable target specificity. To reduce inherent non-specific interaction with either anionic or cationic formulations of polydiacetylene vesicles, we explored the use of various lengths of poly(ethylene glycol) (PEG) amphiphiles for integration and polymerization within PDA vesicles. Our results established that as little as 1% of polyethylene glycol amphiphile integration into anionic vesicles was sufficient to significantly reduce non-specific association with mammalian cells. Similarly integrating a low percent of PEG amphiphile content within cationic vesicles could also significantly reduce non-specific cell association, and moreover reduced cytotoxicity. These results may be prove useful in augmenting PDA vesicles formulations for reduced non-specific interaction which is of particularly interest to enhancing selectivity in vesicles designed with integrated targeting moieties for sensing and drug delivery applications.

Self-Assembled Peptide-Labeled Probes for Agglutination-Based Sensing.

The use of polydiacetylene (PDA) vesicles in sensing systems are wide-spread due to the interesting optical properties of this stimuli-responsive material; however, agglutination based sensing with PDA have been relatively underutilized. To demonstrate the means for rapidly generating an agglutination probe based on peptide-displaying polydiacetylene vesicles, we implement here the use of a biotin mimetic peptide functionalized to a diacetylene amphiphile for proof-of-concept detection of a multivalent target, specifically streptavidin. Tuning of the vesicle composition revealed a distinct limit in the surface density of peptide amphiphile that could be displayed for this particular peptide sequence. A wide operational detection range was demonstrated, and the result also revealed an effective agglutination response of the PDA-based probe to streptavidin suggesting possible use of future formulations in profiling other multivalent targets.

Progression in the Fountain Pen Approach: From 2D Writing to 3D Free-Form Micro/Nanofabrication.

The fountain pen approach, as a means for transferring materials to substrates, has shown numerous incarnations in recent years for creating 2D micro/nanopatterns and even generating 3D free-form nanostructures using a variety of material "inks". While the idea of filled reservoirs used to deliver material to a substrate via a capillary remains unchanged since antiquity, the advent of precise micromanipulation systems and functional material "inks" allows the extension of this mechanism to more high-tech applications. Herein, the recent growth in meniscus guided fountain pen approaches for benchtop micro/nanofabrication, which has occurred in the last decade, is discussed. Particular attention is given to the theory, equipment, and experimentation encompassing this unique direct writing approach. A detailed exploration of the diverse ink systems and functional device applications borne from this strategy is put forth to reveal its rapid expansion to a broad range of scientific and engineering disciplines. As such, this informative review is provided for researchers considering adoption of this recent advancement of a familiar technology.

Microfabrication of Custom Collagen Structures Capable of Guiding Cell Morphology and Alignment.

The patterning of biological components into structural analogues of native tissues to simulate an environment for directing cell growth is one important strategy in biomaterials fabrication. It is widely accepted that chemical, mechanical, and topological cues from the extracellular matrix (ECM) provide important signals for guiding cells to exhibit characteristic polarity, orientation, and morphology. To fully understand the delicate relationship between cell behavior and ECM features, biomaterials fabrication requires improved techniques for tailoring nano/microstructured patterns from relevant biological building blocks rather than using nonbiological materials. Here we reveal a unique approach for the nano/microfabrication of custom patterned biomaterials using collagen as the sole building material. With this new fabrication technique, we further revealed that custom collagen patterns could direct the orientation and morphology of fibroblast growth as a function of vertex density and pattern spacing. Our findings suggest that this technique may be readily adopted for the free form fabrication of custom cell scaffolds purely from natural biological molecules including collagen, among other relevant ECM components.

Chiral arrangement of achiral Au nanoparticles by supramolecular assembly of helical nanofiber templates.

Chiral materials composed of organized nanoparticle superstructures have promising applications to photonics and sensing. Reliable customization of the chiroptical properties of these materials remains an important goal; hence, we report a customizable scheme making use of modular gelator components for controlling the helicity and formation of nanofibers over long length scales resulting in hydrogel templates. Controlled growth of gold nanoparticles at spatially arranged locations along the nanofiber is achieved by UV reduction of Au(I) ions on the supramolecular templates. The resulting materials were found to have significant interparticle interactions and well-defined helicity to provide high quality, chiroptically active materials. With this novel approach, the tailored assembly of nanoparticle superstructures with predictable chiroptical properties can be realized in high yield, which we expect to allow rapid advancement of chiral nanomaterials research.

Luminescent calix[4]arene-based metallogel formed at different solvent composition.

We have synthesized a calix[4]arene derivative (1) containing terpyridine and showed that gelation occurred in the presence of Pt(2+) in DMSO/H2O of varying compositions. Gelation was presumably mediated by the Pt-Pt and π-π stacking interactions. The scanning electron microscopy image of the xerogel showed a spherical structure with diameter of 1.8-2.1 µm. Interestingly, the metallogel showed strong luminescence enhancement, which was dependent on the DMSO/H2O ratio of the solvent. We examined the effects of concentration, temperature, and time resolution on the luminescence emission of both the gel 1-Pt(2+) and the sol 1-Pt(2+). The luminescence lifetimes of the metallogel were particularly long, on the order of several microseconds. The luminescence lifetimes were also strongly dependent on the solvent composition. We also determined the thermodynamic parameters for the self-assembly of the gel by the Birks kinetic scheme. Furthermore, the rheological properties of the metallogels in the presence of more than 4.0 equiv of Pt(2+) were independent of the concentration of Pt(2+) applied.

Instant visual detection of picogram levels of trinitrotoluene by using luminescent metal-organic framework gel-coated filter paper.

There is an ongoing need for explosive detection strategies to uncover threats to human security including illegal transport and terrorist activities. The widespread military use of the explosive trinitrotoluene (TNT) for landmines poses another particular threat to human health in the form of contamination of the surrounding environment and groundwater. The detection of explosives, particularly at low picogram levels, by using a molecular sensor is seen as an important challenge. Herein, we report on the use of a fluorescent metal-organic framework hydrogel that exhibits a higher detection capability for TNT in the gel state compared with that in the solution state. A portable sensor prepared from filter paper coated by the hydrogel was able to detect TNT at the picogram level with a detection limit of 1.82 ppt (parts per trillon). Our results present a simple and new means to provide selective detection of TNT on a surface or in aqueous solution, as afforded by the unique molecular packing through the metal-organic framework structure in the gel formation and the associated photophysical properties. Furthermore, the rheological properties of the MOF-based gel were similar to those of a typical hydrogel.

Controlling and assessing the surface display of cell-binding domains on magnetite conjugated fluorescent liposomes.

Biological systems provide us with a diverse source of peptide-based ligands for cellular adhesion. Controlling and assessing the ligand surface density as well as tailoring the surface chemistry to have specific cellular adhesion properties are important in biomaterials design. In the following work, we provide a means for displaying peptide-based ligands on magnetic liposomes in which the surface density and chemistry may be controlled. Simultaneously, the conjugated vesicles provide a fluorescent signal for examining steric hindrance among surface ligands. In addition, the inherent magnetic and fluorescence features of this system revealed potential for magnet-based cell isolation and fluorescent labeling of adhered cells, respectively. Adhered cells were found to remain viable and proliferative, thereby allowing them to be used for subsequent evaluation. In a specific demonstration, we control the density of fibronectin-mimetic ligands on the polydiacetylene liposome surfaces. We find that steric limitation occurring at over 20% surface density result in decreased cell adhesion, in accord with related techniques. The magnetic-liposome system offers the means for not only separating cells adhered to the biomaterial, but also providing the ability to control and assess the biomaterial surface. This may prove particularly useful for examining combinations of peptide-based ligands or for evaluating the molecular-level ligand accessibility and its effect on cell attachment to a biomaterial surface.

Reinforcement of a sugar-based bolaamphiphile/functionalized graphene oxide composite gel: rheological and electrochemical properties.

A sugar-based bolaamphiphile/graphene oxide composite hydrogel has been prepared using simple mixing. Unlike the corresponding sugar-based native gel, the composite gel exhibits a fibrillar structure with a 10-20 nm fiber diameter. The composite gel forms an interdigitated bilayer structure incorporating intermolecular hydrogen-bonding interactions. The composite gel formation did not change the beneficial electrical properties of graphene offering the potential for integration of this new material into electronic systems. Interestingly, the mechanical and electrochemical properties of the composite gel are both dramatically enhanced when compared to the native gel, thereby reflecting that the functionalized graphene oxide layers are efficiently intercalated within the composite gel structure.

Implementation of an Experiential Service-Learning Course in Biomedical Engineering Design for Undergraduate Students.

The unique characteristics of the training needed for today's biomedical engineers can represent a challenge in curriculum design. Practical experiential learning for biomedical engineering undergraduates is important to prevent under-developed professional skills. In this teaching tips article, we provide an example of how to incorporate experiential learning into the biomedical engineering curriculum to address the need for undergraduates to gain the desired skillsets to serve as the next generation of leaders in engineering, medicine, and business all through the lens of civic engagement. Here we outline our implementation of a recently developed service-learning course for our sophomore students that allows introduction of biomedical engineering discipline-specific design process early on in their undergraduate studies. Student teams work to design, build, and test novel devices to solve the unmet need of community partners, and in doing so, the course prepares students in developing technologies that not only address public health needs but that are also embraced by the community. This course in team-based design can help train students in analyzing real world problems for needs-based biomedical engineering through projects identified by interaction with community partners. Providing specifics of how this course was implemented as well as our reflection on student learning, we offer an analysis of the areas of success, a discussion of how interactions with community partners benefits the student professional skills development, and considerations regarding implementation. Here we highlight the ability of this course to exercise students' social awareness in the design of technologies to improve society by addressing the genuine needs of community partners.

Spray-On Colorimetric Sensors for Distinguishing the Presence of Lead Ions.

Sprayable stimuli-responsive material coatings represent a new class of detection system which can be quickly implemented to transform a surface into a color-responsive sensor. In this work, we describe a dipicolylamine-terminated diacetylene-containing amphiphile formulation for spray coating on to a simple paper substrate to yield disposable test strips that can be used to detect the presence of lead ions in solution. We find the response to be very selective to only lead ions and that the sensitivity can be modulated by altering the UV curing time after spraying. Sensitive detection to at least 0.1 mM revealed a clear color change from a blue to red phase. This represents the first demonstration of a spray-on sensor system capable of detection of lead ions in solution.

Comparison of the Strengths and Weaknesses of Machine Learning Algorithms and Feature Selection on KEGG Database Microbial Gene Pathway Annotation and Its Effects on Reconstructed Network Topology.

The development of tools for the annotation of genes from newly sequenced species has not evolved much from homologous alignment to prior annotated species. While the quality of gene annotations continues to decline as we sequence and assemble more evolutionary distant gut microbiome species, machine learning presents a high quality alternative to traditional techniques. In this study, we investigate the relative performance of common classical and nonclassical machine learning algorithms in the problem of gene annotation using human microbiome-associated species genes from the KEGG database. The majority of the ensemble, clustering, and deep learning algorithms that we investigated showed higher prediction accuracy than CD-Hit in predicting partial KEGG function. Motif-based, machine-learning methods of annotation in new species were faster and had higher precision-recall than methods of homologous alignment or orthologous gene clustering. Gradient boosted ensemble methods and neural networks also predicted higher connectivity in reconstructed KEGG pathways, finding twice as many new pathway interactions than blast alignment. The use of motif-based, machine-learning algorithms in annotation software will allow researchers to develop powerful tools to interact with bacterial microbiomes in ways previously unachievable through homologous sequence alignment alone.

Fluorescent composite hydrogels of metal-organic frameworks and functionalized graphene oxide.

GO MOFs! Azobenzoic acid functionalized graphene (A-GO) can act as a structure-directing template that influences hydrogel formation together with metal-organic frameworks (MOFs). Zn(2+) MOFs of pyridine derivatives work as framework linkers between the A-GO sheets (MOF-A-GO, see figure). MOF-A-GO exhibits a strong fluorescence enhancement upon gel formation. In addition, MOF-A-GO selectively recognizes trinitrotoluene.

A BODIPY-functionalized bimetallic probe for sensitive and selective color-fluorometric chemosensing of Hg2+.

A new BODIPY dye conjugate has demonstrated selective quenching by mercury over other metal ions. Coupling of this probe to Au-Fe(3)O(4) nanoparticles as well as platinum electrodes offered sensitive systems for suspension and surface based sensing, respectively.

Peptide Linked Diacetylene Amphiphiles for Detection of Epitope Specific Antibodies.

Antibodies produced in response to adaptive immunity provide a receptor with multiple sites for binding to a distinct epitope of an antigen. Determining antibody levels to specific antigens has important clinical applications in assessing immune status or deficiency, monitoring infectious or autoimmune diseases, and diagnosing allergies. Leveraging that a specific antibody will bind to a distinct small peptide epitope without requiring the entire antigen to be present, we demonstrate in this work a proof-of-concept assay to detect the presence of an antibody by using peptide epitopes linked to an amphiphile to generate a vesicle-based sensing system. By affording multiple copies of the epitope site on the vesicle, we revealed that the vesicles visibly aggregate in response to an antibody specific for that epitope due to multivalent binding provided by the antibody. We also uncovered the role of peptide surface density in providing accessible epitopes on the vesicles for antibody binding. In summary, using a peptide derived from the coat protein of human influenza virus directly linked to a diacetylene-containing amphiphile afforded peptide-laden vesicles that proved capable of detecting the presence of antibodies specific for human influenza hemagglutinin.

Engineering Escherichia coli for Conversion of Dietary Isoflavones in the Gut.

Introducing metabolic pathways to the gut is important to tailor the biochemical components ultimately absorbed by the host. Given identical diets, hosts possessing different consortia of gut bacteria can exhibit distinct health outcomes regulated by metabolic capabilities of the gut microbiota. The disparate competency of the population to metabolize isoflavones, such as dietary daidzein, has shown health benefits for those individuals possessing gut bacteria capable of producing equol from daidzein-rich diets. To begin addressing health inequalities due to gut metabolic pathway deficiencies, we developed a probiotic that allows metabolism of isoflavones to provide a gut phenotype paralleling that of natural equol producers. Toward this goal, we engineered Escherichia coli to produce the enzymes necessary for conversion of daidzein to equol, and as demonstrated in a murine model, these bacteria enabled elevated serum equol levels to dietary daidzein, thus serving as a starting point for more sophisticated systems.

Supramolecular Peptide Nanofiber/PLGA Nanocomposites for Enhancing Pulmonary Drug Delivery.

Effective drug delivery to pulmonary sites will benefit from the design and synthesis of novel drug delivery systems that can overcome various tissue and cellular barriers. Cell penetrating peptides (CPPs) have shown promise for intracellular delivery of various imaging probes and therapeutics. Although CPPs improve delivery efficacy to a certain extent, they still lack the scope of engineering to improve the payload capacity and protect the payload from the physiological environment in drug delivery applications. Inspired by recent advances of CPPs and CPP-functionalized nanoparticles, in this work, we demonstrate a novel nanocomposite consisting of fiber-forming supramolecular CPPs that are coated onto polylactic-glycolic acid (PLGA) nanoparticles to enhance pulmonary drug delivery. These nanocomposites show a threefold higher intracellular delivery of nanoparticles in various cells including primary lung epithelial cells, macrophages, and a 10-fold increase in endothelial cells compared to naked PLGA nanoparticles or a twofold increase compared to nanoparticles modified with traditional monomeric CPPs. Cell uptake studies suggest that nanocomposites likely enter cells through mixed macropinocytosis and passive energy-independent mechanisms, which is followed by endosomal escape within 24 h. Nanocomposites also showed potent mucus permeation. More importantly, freeze-drying and nebulizing formulated nanocomposite powder did not affect their physiochemical and biological activity, which further highlights the translative potential for use as a stable drug carrier for pulmonary drug delivery. We expect nanocomposites based on peptide nanofibers, and PLGA nanoparticles can be custom designed to encapsulate and deliver a wide range of therapeutics including nucleic acids, proteins, and small-molecule drugs when employed in inhalable systems to treat various pulmonary diseases.

Introduction of Plasmid to the Murine Gut via Consumption of an Escherichia coli Carrier and Examining the Impact of Bacterial Dosing and Antibiotics on Persistence.

Purpose: We examine the impacts of dosing strategies of plasmids on bacterial communities in the murine gut by measuring the quantity of plasmids in mouse feces. Methods: We fed mice carrier bacteria, E. coli, that contain plasmids with both a reporter gene and an antibiotic resistant gene. We varied the quantity of the plasmid-carrying bacteria and the length of time the mice consumed the bacteria. We also pretreated the gut with broad-spectrum antibiotics and used continuous antibiotic treatment to investigate selection pressure. We collected bacteria from fecal pellets to quantify the number of plasmid-carrying bacteria via plate assay. Results: Dosing regimens with plasmid-carrying bacteria resulted in a significantly increased duration of persistence of the plasmid within the gut when supplemented continuously with kanamycin during as well as after completion of bacterial dosing. The carrier bacteria concentration influenced the short-term abundance of carrier bacteria. Conclusion: We evaluated the persistence of plasmid-carrying bacteria in the murine gut over time using varying dosage strategies. In future work, we will study how bacterial diversity in the gut impacts the degree of plasmid transfer and the prevalence of plasmid-carrying bacteria over time. Lay Summary: Observing how plasmids persist within the gut can help us understand how newly introduced genes, including antibiotic resistance, are transmitted within the gut microbiome. In our experiments, mice were given bacteria containing a genetically engineered plasmid and were examined for the persistence of the plasmid in the gut. We found long-term persistence of the plasmid in the gut when administering antibiotics during and following dosing of the mice with bacteria carrying the plasmid. The use of higher concentrations of carrier bacteria influenced the short-term abundance of the plasmid-carrying bacteria in the gut. Description of Future Works: Building on evidence from these initial studies that persistence of plasmids within the gut can be regulated by the dosage strategy, we will explore future studies and models of gene uptake in the context of spatial and taxonomic control and further determine if dosing strategies alter the compositional diversity of the gut microbiome.

Polydiacetylene incorporated with peptide receptors for the detection of trinitrotoluene explosives.

Because of their unique optical and stimuli-response properties, polydiacetylene-based platforms have been explored as an alternative to complex mechanical and electrical sensing systems. We linked chromic responsive polydiacetylene (PDA) onto a peptide-based molecular recognition element for trinitrotoluene (TNT) molecules in order to provide a system capable of responding to the presence of a TNT target. We first identified the trimer peptide receptor that could induce chromic changes on a PDA backbone. We then investigated the multivalent interactions between TNT and our peptide-based receptor by nuclear magnetic resonance (NMR) spectroscopy. We further characterized various parameters that affected the conjugated PDA system and hence the chromic response, including the size of end-group motifs, the surface density of receptors, and the length of alkane side chains. Taking these necessary design parameters into account, we demonstrated a modular system capable of transducing small-molecule TNT binding into a detectable signal. Our conjugated PDA-based sensor coupled with molecular recognition elements has already proven useful recently in the development of another sensitive and selective electronic sensor, though we expect that our results will also be valuable in the design of colorimetric sensors for small-molecule detection.

Engineering a reporter cell line to mimic the high oligomannose presenting surface immunoglobulin of follicular lymphoma B cells.

Subtypes of B cell non-Hodgkin's lymphomas, including follicular lymphomas, have shown a unique high oligomannose presentation on their immunoglobulins that will interact with natural receptors of the innate immunity, reportedly causing stimulation and proliferation. From deep sequencing of the variable heavy and light chain sequences of follicular lymphoma involved tissue sections, we identified the consensus variable sequences possessing glycosylation sites at the complementarity determining region. Using this information, we developed a cell line, referred to here as BZ, which displays the consensus variable segments as part of a surface antibody (IgM) and confirmed its presentation of high oligomannose on the heavy chain both in vitro and in vivo. An mCherry expressing variant provided a reporter cell line displaying the high oligomannose surface biomarker while affording clear fluorescent signals for FACS screening as well as for fluorescent in vivo imaging of ectopic xenograft tumors. In developing this reporter cell line that displays the biomarker glycan of follicular lymphoma, we provide a tool that may be used for future screening and validation of receptive moieties for selectively binding high oligomannose for development of targeted diagnostics or therapeutics to such B cell malignancies that display this unique glycan.