Virus-Based Pyroelectricity.

The first observation of heat-induced electrical potential generation on a virus and its detection through pyroelectricity are presented. Specifically, the authors investigate the pyroelectric properties of the M13 phage, which possesses inherent dipole structures derived from the noncentrosymmetric arrangement of the major coat protein (pVIII) with an α-helical conformation. Unidirectional polarization of the phage is achieved through genetic engineering of the tail protein (pIII) and template-assisted self-assembly techniques. By modifying the pVIII proteins with varying numbers of glutamate residues, the structure-dependent tunable pyroelectric properties of the phage are explored. The most polarized phage exhibits a pyroelectric coefficient of 0.13 µC m-2 °C-1 . Computational modeling and circular dichroism (CD) spectroscopy analysis confirm that the unfolding of α-helices within the pVIII proteins leads to changes in phage polarization upon heating. Moreover, the phage is genetically modified to enable its pyroelectric function in diverse chemical environments. This phage-based approach not only provides valuable insights into bio-pyroelectricity but also opens up new opportunities for the detection of various viral particles. Furthermore, it holds great potential for the development of novel biomaterials for future applications in biosensors and bioelectric materials.

Elastic Fluorescent Protein-Based Down-Converting Optical Films for Flexible Display.

Protein-based material design provides great advantages to developing smart biomaterials with tunable structures and desired functions. They have been widely used in many biomedical applications including tissue engineering and drug delivery. However, protein-based materials are not yet widely used in optoelectronic materials despite their excellent optical and tunable mechanical properties. Here, we synthesized engineered fluorescent proteins (FPs) fused with elastic protein for the development of optoelectrical down-converting optical filters for flexible display materials. We synthesized sequence-specific FPs to tune blue, green, yellow, and red colors and fused them with elastic protein to tune mechanical properties. We fabricated flexible self-supporting film materials and characterized mechanical properties and down-converting optical properties. We also fabricated a hybrid light-emitting diode (LED) to down convert blue to desired green, red, and white colors. Furthermore, we constructed a flexible white LED using organic LED as a flexible substrate. Our modular synthesis approach of tunable bio-optoelectrical material approaches will be useful to design future biocompatible and flexible display materials and technologies.

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.

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.

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.

Tuning the Surface Charge of Self-Assembled Polydiacetylene Vesicles to Control Aggregation and Cell Binding.

Polydiacetylene vesicles of various compositions were assembled using a two-part mixture of 10,12-pentacosadiynoic acid (PCDA) and ethylenedioxy-bis-ethylamine (EDEA)-labeled PCDA in order to control surface charge and stability within a desired pH range. Investigation of the interaction of the vesicles with mammalian cells as a function of surface charge was carried out and identified a clear correlation in cell-vesicle association and corresponding cell death for vesicles with positive surface charge. The binding behavior of the vesicles was found to be tunable by regulating the proportion of anionic PCDA relative to cationic PCDA-EDEA content within vesicles as to control the surface charge as a function of pH. Association of vesicles with cells thus depended on the corresponding charge of the vesicles and cell surface. The prospect of this work may serve as a step toward future vesicle designs to allow triggered uptake of vesicles locally within low pH tumor microenvironments.

A Peptide-Lectin Fusion Strategy for Developing a Glycan Probe for Use in Various Assay Formats.

While nucleic acid and protein analysis approaches continue to see significant breakthroughs, analytical strategies for glycan determination have by comparison seen slower technological advances. Here we provide a strategy for glycan probe development using an engineered lectin fusion that can be incorporated into various common pathology lab assay formats including Western blot and agglutination assays. In this proof of concept, we use the natural lectin, Pseudomonas fluorescens agglutinin (PFA), capable of binding core Man alpha(1-3)-Man alpha(1-6)-Man units, where this lectin has previously been shown to bind to the glycans presented by the gp120 coat protein of (HIV) Human Immunodeficiency Virus. In our strategy, we engineered the lectin to possess a fusion of the biotin mimetic tag equence of amino acids V-S-H-P-Q-A-P-F. With the glycan receptive PFA directly linked to the biotin mimic, we could facilitate a probe for various standard clinical assay formats by virtue of coupling to streptavidin-HRP (horseradish peroxidase) or streptavidin beads for Western blot and agglutination assays respectively. We found the PFA fusion retained low nanomolar affinity for gp120 by ELISA (Enzyme Linked Immunosorbent Assay) and microscale thermophoresis. This probe engineering strategy proved effective in the relevant assay formats that may now allow detection for the presence of glycans containing the core Man alpha(1-3)-Man alpha(1-6)-Man units recognized by PFA.

IR-783 Labeling of a Peptide Receptor for 'Turn-On' Fluorescence Based Sensing.

In this study, we examine a means for developing near-IR fluorescent sensors through streamlined, site-specific coupling with peptide-based receptors. As the penultimate step of solid-phase synthesis of a peptide-based receptor, we show a simple means of labeling the N' terminus with the near IR fluorophore IR-783 to afford a viable fluorescent sensor after cleavage from the resin. The proof-of-concept probe utilized a biotin mimetic peptide sequence as the receptive moiety. Here we revealed a "turn-on" fluorescence enhancement upon binding of the biotin mimetic probe to its intended streptavidin target. Not all peptide-receptive moieties tested were able to generate such an enhancement upon target binding, and as such, the rationale for the observed fluorescence response properties is discussed.

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity.

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity.

Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.

We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (µIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the µIEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by µIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.

Surface-Tunable Bioluminescence Resonance Energy Transfer via Geometry-Controlled ZnO Nanorod Coordination.

The use of ZnO nanorods (NRs) as an effective coordinator and biosensing platform to create bioluminescence resonance energy transfer (BRET) is reported. Herein, a hydrothermal approach is applied to obtain morphologically controlled ZnO NRs, which are directly bound to luciferase (Luc) and carboxy-modified quantum dot (QD) acting as a donor-acceptor pair for BRET. BRET efficiency varies significantly with the geometry of ZnO NRs, which modulates the coordination between hexahistidine-tagged Luc (Luc-His6 ) and QD, owing to the combined effect of the total surface area consisting of (001) and (100) planes and their surface polarities. Unlike typical QD-BRET reactions with metal ions (e.g., zinc ions), a geometry-controlled ZnO NR platform can facilitate the design of surface-initiated BRET sensors without being supplemented by copious metal ions: the geometry-controlled ZnO NR platform can therefore pave the way for nanostructure-based biosensors with enhanced analytical performance.

Rapid detection of protein phosphatase activity using Zn(II)-coordinated gold nanosensors based on His-tagged phosphopeptides.

We report a rapid colorimetric assay to detect protein phosphatase (PP) activity based on the controlled assembly and disassembly of gold nanoparticles (AuNPs) via Zn(II)-specific coordination in the presence of His6-tagged phosphopeptides. Among divalent metal ions including Ni(II), Cu(II), Co(II), Mg(II), Mn(II), and Zn(II), only Zn(II) triggered a strong association between phosphopeptides with hexahistidine at a single end and nitrilotriacetic acid (NTA)-modified AuNPs (21.3 nm in core diameter), leading to the self-assembly of AuNPs and consequently changes in color of the AuNP solution. In contrast, unphosphorylated peptides and His6-deficient phosphopeptides did not change the color of the AuNP solution. As a result, protein phosphatase 1 (PP1) activity and its inhibition were easily quantified with high sensitivity by determining the extinction ratio (E520/E700) of colloidal AuNPs. Most importantly, this method was capable of detecting protein phosphatase 2A (PP2A) activity in immunoprecipitated plant extracts. Because PPs play pivotal roles in mediating diverse signal transduction pathways as primary effectors of protein dephosphorylation, we anticipate that our method will be applied as a rapid format method to analyze the activities of various PPs and their inhibition.

Enzymatic glucose biosensors based on nanomaterials.

: Glucose biosensors have an important place in the diagnosis of diabetes as well as in various food and biotechnological processes. Recent advances in nanomaterials have directly improved enzymatic glucose biosensors owing to their distinguished structural and physiochemical properties. Here, we review the recent developments in electrochemical and fluorescent glucose biosensors based on nanomaterials. New technologies that combine nanomaterials with glucose-sensing enzymes will result in promising glucose biosensors with high specificity and sensitivity.