Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms.

Delivering CRISPR: a review of the challenges and approaches.

Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation.

Control over Silica Particle Growth and Particle-Biomolecule Interactions Facilitates Silica Encapsulation of Mammalian Cells with Thickness Control.

Over the last twenty years, many strategies utilizing sol-gel chemistry to integrate biological cells into silica-based materials have been reported. One such strategy, Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition, shows promise as an efficient encapsulation technique due to the ability to vary the silica encapsulation morphology obtained by this process through variation of SG-CViL reaction conditions. In this report, we develop SG-CViL as a tunable, multi-purpose silica encapsulation strategy by investigating the mechanisms governing both silica particle generation and subsequent interaction with phospholipid assemblies (liposomes and living cells).

Volatile Metabolites Emission by In Vivo Microalgae-An Overlooked Opportunity?

Fragrances and malodors are ubiquitous in the environment, arising from natural and artificial processes, by the generation of volatile organic compounds (VOCs). Although VOCs constitute only a fraction of the metabolites produced by an organism, the detection of VOCs has a broad range of civilian, industrial, military, medical, and national security applications. The VOC metabolic profile of an organism has been referred to as its 'volatilome' (or 'volatome') and the study of volatilome/volatome is characterized as 'volatilomics', a relatively new category in the 'omics' arena.

Three-Dimensional Encapsulation of Saccharomyces cerevisiae in Silicate Matrices Creates Distinct Metabolic States as Revealed by Gene Chip Analysis.

In order to design hybrid cellular/synthetic devices such as sensors and vaccines, it is important to understand how the metabolic state of living cells changes upon physical confinement within three-dimensional (3D) matrices. We analyze the gene expression patterns of stationary phase Saccharomyces cerevisiae (S. cerevisiae) cells encapsulated within three distinct nanostructured silica matrices and relate those patterns to known naturally occurring metabolic states.

Magnetic-adhesive based valves for microfluidic devices used in low-resource settings.

Since the introduction of micro total analytical systems (μTASs), significant advances have been made toward development of lab-on-a-chip platforms capable of performing complex biological assays that can revolutionize public health, among other applications. However, use of these platforms in low-resource environments (e.g.

Influence of Silica Matrix Composition and Functional Component Additives on the Bioactivity and Viability of Encapsulated Living Cells.

The remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells is reported. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. These matrices are used to encapsulate living cells engineered with a synthetic riboswitch for cell-based biosensing.

Sol-Generating Chemical Vapor into Liquid (SG-CViL) Deposition- A Facile Method for Encapsulation of Diverse Cell Types in Silica Matrices.

In nature, cells perform a variety of complex functions such as sensing, catalysis, and energy conversion which hold great potential for biotechnological device construction. However, cellular sensitivity to environments necessitates development of bio-nano interfaces which allow integration of cells into devices and maintain their desired functionality. In order to develop such an interface, the use of a novel Sol Generating Chemical Vapor into Liquid (SG-CViL) deposition process for whole cell encapsulation in silica was explored.

Biocompatible microfabrication of 3D isolation chambers for targeted confinement of individual cells and their progeny.

We describe a technique to physically isolate single/individual cells from their surrounding environment by fabricating three-dimensional microchambers around selected cells under biocompatible conditions. Isolation of targeted cells is achieved via rapid fabrication of protein hydrogels from a biocompatible precursor solution using multiphoton lithography, an intrinsically 3D laser direct write microfabrication technique. Cells remain chemically accessible to environmental cues enabling their propagation into well-defined, high density populations.

High-throughput screening of transglutaminase activity using plasmonic fluorescent nanocomposites.

We describe a high-throughput screening (HTS) assay for transglutaminase (TG) enzyme activity using plasmonic fluorescent nanocomposites. We used TG to covalently crosslink 500 μM solution of 5'-biotinamidopentylamine (BP) to N,N'-dimethylcasein (DMC) which was adsorbed onto 384-well microplates. We then bound 0.

Rational redesign of glucose oxidase for improved catalytic function and stability.

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A.

Plasmonic fluorescent nanocomposites of cyanines self-assembled upon gold nanoparticle scaffolds.

Plasmonic fluorescent nanocomposites are difficult to prepare due to strong quenching effects on fluorophores in the vicinity of noble metal nanoparticles such as gold (AuNPs). We successfully prepared plasmonic fluorescent nanocomposites of two cyanines (1 and 2) aggregating upon 2 - 40 nm AuNPs or streptavidin-conjugated 10 nm AuNPs. We used high throughput screening (HTS) for the first time to characterize the spectral properties, aggregation kinetics, aggregation density and photostability of the nanocomposites.

Orthogonal cell-based biosensing: fluorescent, electrochemical, and colorimetric detection with silica-immobilized cellular communities integrated with an ITO-glass/plastic laminate cartridge.

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensor's output.

Multiphoton lithography of nanocrystalline platinum and palladium for site-specific catalysis in 3D microenvironments.

Integration of catalytic nanostructured platinum and palladium within 3D microscale structures or fluidic environments is important for systems ranging from micropumps to microfluidic chemical reactors and energy converters. We report a straightforward procedure to fabricate microscale patterns of nanocrystalline platinum and palladium using multiphoton lithography. These materials display excellent catalytic, electrical, and electrochemical properties, and we demonstrate high-resolution integration of catalysts within 3D defined microenvironments to generate directed autonomous particle and fluid transport.

A parallel microfluidic channel fixture fabricated using laser ablated plastic laminates for electrochemical and chemiluminescent biodetection of DNA.

Herein is described the fabrication and use of a plastic multilayer 3-channel microfluidic fixture. Multilayer devices were produced by laser machining of plastic polymethylmethacrylate and polyethyleneterapthalate laminates by ablation. The fixture consisted of an array of nine individually addressable gold or gold/ITO working electrodes, and a resistive platinum heating element.

Supramolecular self-assembled chaos: polyphenolic lignin's barrier to cost-effective lignocellulosic biofuels.

Phenylpropanoid metabolism yields a mixture of monolignols that undergo chaotic, non-enzymatic reactions such as free radical polymerization and spontaneous self-assembly in order to form the polyphenolic lignin which is a barrier to cost-effective lignocellulosic biofuels. Post-synthesis lignin integration into the plant cell wall is unclear, including how the hydrophobic lignin incorporates into the wall in an initially hydrophilic milieu. Self-assembly, self-organization and aggregation give rise to a complex, 3D network of lignin that displays randomly branched topology and fractal properties.

Cell-directed integration into three-dimensional lipid-silica nanostructured matrices.

We report a unique approach in which living cells direct their integration into 3D solid-state nanostructures. Yeast cells deposited on a weakly condensed lipid/silica thin film mesophase actively reconstruct the surface to create a fully 3D bio/nano interface, composed of localized lipid bilayers enveloped by a lipid/silica mesophase, through a self-catalyzed silica condensation process. Remarkably, this integration process selects exclusively for living cells over the corresponding apoptotic cells (those undergoing programmed cell death), via the development of a pH gradient, which catalyzes silica deposition and the formation of a coherent interface between the cell and surrounding silica matrix.

Fabrication and testing of a microneedles sensor array for p-cresol detection with potential biofuel applications.

We present a miniaturized high-throughput sensor array that will augment biofuel technology by facilitating in situ biochemical measurements upon micrometer-scale surfaces of leaves, stems, or petals. We used semiconductor processing to photopattern Foturan glass wafers and fabricated gold-plated microscopic electrode needles (ElectroNeedles) that pierced 125-mum-thick surfaces without deformation. The 5 x 5 or 10 x 10 arrays of ElectroNeedles can analyze 25 or 100 samples simultaneously, increasing throughput.

Cell-directed localization and orientation of a functional foreign transmembrane protein within a silica nanostructure.

A simple procedure for introducing functional exogenous membrane-bound proteins to viable cells encapsulated within a lipid templated silica nanostructure is described. In one method, bacteriorhodopsin (bR) was added directly to a Saccharomyces cerevisiae solution along with short zwitterionic diacylphosphatidylcholines (diC(6) PC) and mixed with equal volumes of a sol precursor solution. Alternatively, bR was first incorporated into liposomes (bR-proteoliposomes) and then added to an S.

A multifunctional thin film Au electrode surface formed by consecutive electrochemical reduction of aryl diazonium salts.

A multifunctional thin film surface capable of immobilizing two diverse molecules on a single gold electrode was prepared by consecutive electrodeposition of nitrophenyl and phenylboronic acid pinacol ester (PBA-PE) diazonium salts. Activation of the stacked film toward binding platinum nanoparticles (PtNPs) and yeast cells occurred via chemical deprotection of the pinacol ester followed by electroreduction of nitro to amino groups. FTIR spectral analysis was used to study and verify film composition at each stage of preparation.

Maleimide-activated aryl diazonium salts for electrode surface functionalization with biological and redox-active molecules.

A versatile and simple method is introduced for formation of maleimide-functionalized surfaces using maleimide-activated aryl diazonium salts. We show for the first time electrodeposition of N-(4-diazophenyl)maleimide tetrafluoroborate on gold and carbon electrodes which was characterized via voltammetry, grazing angle FTIR, and ellipsometry. Electrodeposition conditions were used to control film thickness and yielded submonolayer-to-multilayer grafting.

Electrically addressable diazonium-functionalized antibodies for multianalyte electrochemical sensor applications.

The direct electrically addressable deposition of diazonium-modified antibodies is examined for electrochemical immunosensing applications. The immobilized antibodies can be detected by the use of electroactive enzyme tags and nanoparticle-gold labeling. Control over antibody functionalization density and minimal spontaneous grafting of diazonium-antibody adducts is shown.

Selective immobilization of DNA and antibody probes on electrode arrays: simultaneous electrochemical detection of DNA and protein on a single platform.

A proof of concept procedure for the electroaddressable covalent immobilization of DNA and protein on arrayed electrodes along with simultaneous detection of multiple bioagents in the same sample solution is described. Carboxyphenyldiazonium was selectively deposited onto five of nine individually addressable electrodes in an array via bias assisted assembly. Amine functionalized DNA probes were covalently coupled to the carboxyl surface via carbodiimide chemistry.