Table_1_Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges.docx

Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.</p

Supraparticle Assemblies of Magnetic Nanoparticles and Quantum Dots for Selective Cell Isolation and Counting on a Smartphone-Based Imaging Platform

There are numerous diagnostic and therapeutic applications for the detection and enumeration of specific cell types. Flow cytometry is the gold standard technique for this purpose but is poorly suited to point-of-need assays. The ideal platform for these assays would combine the immunocytochemical capabilities of flow cytometry with low-cost, portable instrumentation, and a simple and rapid assay workflow. Here, we present a smartphone-based imaging platform (SIP) in tandem with magnetic-fluorescent suprananoparticle assemblies as a step toward these ideal criteria. The assemblies (MNP@QD) are magnetic iron oxide nanoparticles surrounded by a dense corona of many brightly luminescent semiconductor quantum dots (QDs), where both the assemblies and their immunoconjugates are prepared by self-assembly. As proof of concept, we show that the MNP@QD and SIP pairing is able to selectively isolate, fluorescently immunolabel, and count breast cancer cells that are positive for human epidermal growth factor receptor 2 (HER2). These results are an important foundation for future point-of-need diagnostics capable of multiplexed isolation, counting, and immunoprofiling of cells on a smartphone, enabled by the highly advantageous optical properties of QDs

Microfabricated Two-Dimensional Electrophoresis Device for Differential Protein Expression Profiling

A microfluidic separation system is developed to perform two-dimensional differential gel electrophoretic (DIGE) separations of complex, cellular protein mixtures produced by induced protein expression in E. coli. The micro-DIGE analyzer is a two-layer borosilicate glass microdevice consisting of a single 3.75 cm long channel for isoelectric focusing, which is sampled in parallel by 20 channels effecting a second-dimension separation by native electrophoresis. The connection between the orthogonal separation systems is accomplished by smaller channels comprising a microfluidic interface (MFI) that prevents media leakage between the two dimensions and enables facile loading of discontinuous gel systems in each dimension. Proteins are covalently labeled with Cy2 and Cy3 DIGE and detected simultaneously with a rotary confocal fluorescence scanner. Reproducible two-dimensional separations of both purified proteins and complex protein mixtures are performed with minimal run-to-run variation by including 7 M urea in the second-dimension separation matrix. The capabilities of the micro-DIGE analyzer are demonstrated by following the induced expression of maltose binding protein in E. coli. Although the absence of sodium dodecyl sulfate (SDS) in the second-dimension sizing separation limits the orthogonality and peak capacity of the separation, this analyzer is a significant first step toward the reproducible two-dimensional analysis of complex protein samples in microfabricated devices. Furthermore, the microchannel interface structures developed here will facilitate other multidimensional separations in microdevices

Peptides for Specifically Targeting Nanoparticles to Cellular Organelles: <i>Quo Vadis</i>?

ConspectusThe interfacing of nanomaterials and especially nanoparticles within all aspects of biological research continues to grow at a nearly unabated pace with projected applications focusing on powerful new tools for cellular labeling, imaging, and sensing, theranostic materials, and drug delivery. At the most fundamental level, many of these nanoparticles are meant to target not only very specific cell-types, regardless of whether they are in a culture, tissue, an animal model, or ultimately a patient, but also in many cases a specific subcellular organelle. During this process, these materials will undergo a complex journey that must first find the target cell of interest, then be taken up by those cells across the extracellular membrane, and ultimately localize to a desired subcellular organelle, which may include the nucleus, plasma membrane, endolysosomal system, mitochondria, cytosol, or endoplasmic reticulum. To accomplish these complex tasks in the correct sequence, researchers are increasingly interested in selecting for and exploiting targeting peptides that can impart the requisite capabilities to a given nanoparticle construct. There are also a number of related criteria that need careful consideration for this undertaking centering on the nature and properties of the peptide vector itself, the peptide–nanoparticle conjugate characteristics, and the target cell.Here, we highlight some important issues and key research areas related to this burgeoning field. We begin by providing a brief overview of some criteria for optimal attachment of peptides to nanoparticles, the predominant methods by which nanoparticles enter cells, and some of the peptide sequences that have been utilized to facilitate nanoparticle delivery to cells focusing on those that engender the initial targeting and uptake. Because almost all materials delivered to cells by peptides utilize the endosomal system of vesicular transport and in many cases remain sequestered within the vesicles, we critically evaluate the issue of endosomal escape in the context of some recently reported successes in this regard. Following from this, peptides that have been reported to deliver nanoparticles to specific subcellular compartments are examined with a focus on what they delivered and the putative mechanisms by which they were able to accomplish this. The last section focuses on two areas that are critical to realizing this overall approach in the long term. The first is how to select for peptidyl sequences capable of improved or more specific cellular or subcellular targeting based upon principles commonly associated with drug discovery. The second looks at what has been done to create modular peptides that incorporate multiple desirable functionalities within a single, contiguous sequence. This provides a viable alternative to either the almost insurmountable challenge of finding one sequence capable of all functions or, alternatively, attaching different peptides with different functionalities to the same nanoparticle in different ratios when trying to orchestrate their net effects. Finally, we conclude with a brief perspective on the future evolution and broader impact of this growing area of bionanoscience

Time-Gated DNA Photonic Wires with Förster Resonance Energy Transfer Cascades Initiated by a Luminescent Terbium Donor

Functional DNA nanotechnology is a rapidly growing area of research with many prospective photonic applications, including roles as wires and switches, logic operators, and smart biological probes and delivery vectors. Photonic wire constructs are one such example and comprise a Förster resonance energy transfer (FRET) cascade between fluorescent dyes arranged periodically along a DNA scaffold. To date, the majority of research on photonic wires has focused on setting new benchmarks for efficient energy transfer over more steps and across longer distances, using almost exclusively organic fluorescent dyes and strictly DNA structures. Here, we expand the range of materials utilized with DNA photonic wires by demonstrating the use of a luminescent terbium complex (Tb) as an initial donor for a four-step FRET cascade along a ∼15 nm long DNA/locked nucleic acid (LNA) photonic wire. The inclusion of LNA nucleotides increases the thermal stability of the photonic wires while the Tb affords time-gated emission measurements and other optical benefits. Time-gating minimizes unwanted background emission, whether from direct excitation of fluorescent dyes along the length of the photonic wire, from excess dye-labeled DNA strands in the sample, or from a biological sample matrix. Observed efficiencies for Tb-to-dye energy transfer are also closer to the predicted values than those for dye-to-dye energy transfer, and the Tb can be used as an initial FRET donor for a variety of next-in-line acceptors at different spectral positions. We show that the key to using the Tb as an effective initial donor is to optimally position the next-in-line acceptor dye in a so-called “sweet spot” where the FRET efficiency is sufficiently high for practicality, but not so high as to suppress time-gated emission by shortening the Tb emission lifetime to within the instrument lag or delay time necessary for measurements. Overall, the initiation of a time-gated FRET cascade with a Tb donor is a very promising strategy for the design, characterization, and application of DNA-based photonic wires and other functional DNA nanostructures

Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs

Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated “sweet spot” for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective strategy for developing FRET probes for bioassays

Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

Methods for the detection, enumeration, and typing of cells are important in many areas of research and healthcare. In this context, flow cytometers are a widely used research and clinical tool but are also an example of a large and expensive instrument that is limited to specialized laboratories. Smartphones have been shown to have excellent potential to serve as portable and lower-cost platforms for analyses that would normally be done in a laboratory. Here, we developed a prototype smartphone-based flow cytometer (FC). This compact 3D-printed device incorporated a laser diode and a microfluidic flow cell and used the built-in camera of a smartphone to track immunofluorescently labeled cells in suspension and measure their color. This capability was enabled by high-brightness supra-nanoparticle assemblies of colloidal semiconductor quantum dots (SiO2@QDs) as well as a support vector machine (SVM) classification algorithm. The smartphone-based FC device detected and enumerated target cells against a background of other cells, simultaneously and selectively counted two different cell types in a mixture, and used multiple colors of SiO2@QD-antibody conjugates to screen for and identify a particular cell type. The potential limits of multicolor detection are discussed alongside ideas for further development. Our results suggest that innovations in materials and engineering should enable eventual smartphone-based FC assays for clinical applications

Intracellular Bioconjugation of Targeted Proteins with Semiconductor Quantum Dots

Intracellular Bioconjugation of Targeted Proteins with Semiconductor Quantum Dot

Detection of HIV-1 Specific Monoclonal Antibodies Using Enhancement of Dye-Labeled Antigenic Peptides

A simple bifunctional colorimetric/fluorescent sensing assay is demonstrated for the detection of HIV-1 specific antibodies. This assay makes use of a short peptide sequence coupled to an environmentally sensitive dye that absorbs and emits in the visible portion of the spectrum. The core peptide sequence is derived from the highly antigenic six-residue epitope of the HIV-1 p17 protein and is situated adjacent to a terminal cysteine residue which enables site-specific fluorescent labeling with Cy3 cyanine dye. Interaction of the Cy3-labeled p17 peptide with monoclonal anti-p17 antibody resulted in an up to 4-fold increase in dye absorption and greater than 5-fold increase in fluorescent emission, yielding a limit of detection as low as 73 pM for the target antibody. This initial study demonstrates both proof-of-concept for this approach and suggests that the resulting sensor could potentially be used as a rapid screening method for HIV-1 infection while requiring minimal equipment and reagents. The potential for utilizing this assay in simple field-portable point-of-care and diagnostic devices is discussed

Solution-Phase Single Quantum Dot Fluorescence Resonance Energy Transfer

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe−ZnS core−shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor−acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD−protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements