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.

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. We demonstrate this methodology on gram negative (E. coli), gram positive (S. aureus), and eukaryotic (S. cerevisiae) cells. The opportunities to confine viable, single/individual-cells and small populations within user-defined microenvironments afforded by this approach should facilitate the study of cell behaviors across multiple generations.

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. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.

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. cerevisiae solution with diC(6) PC, and this was followed by mixing with sol precursor solution. Films prepared from bR added directly to diC(6) PC resulted in bR localization near S. cerevisiae cells in a disordered and diffuse fashion, while films prepared from bR-proteoliposomes added to the diC(6) PC/yeast solution resulted in preferential localization of bR near yeast cell surfaces, forming bR-containing multilayer vesicles. Importantly, bR introduced via proteoliposomes was observed to modulate pH gradients developed at the cell surface, demonstrating both retained functionality and preferential orientation. Localization of liposome lipid or bR did not occur around neutrally charged latex beads acting as cell surrogates, demonstrating that living cells actively organize the multilayered lipid during evaporation-induced self-assembly. We expect this simple procedure for introducing functional and oriented membrane-bound proteins to the surface of cells to be general and adaptable to other membrane-bound proteins. This advance may prove useful in fundamental studies of membrane protein function and cell-cell signaling and in imparting non-native characteristics to arbitrary cells.

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. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.

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. The utility of the technique for a sandwich immunoassay as well as the ability to individually and selectively address closely spaced microelectrodes for multi-target protein detection in an array format is demonstrated.

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. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.

Reagentless electrochemical immunoassay using electrocatalytic nanoparticle-modified antibodies.

We describe a new approach for reagentless electrochemical immunoassay sensing in which Au/Pd NPs can be "loaded" onto antibodies to create an electrocatalytic antibody that is sensitive to the oxygen reduction reaction.

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). Using Dynamic Light Scattering (DLS) measurements, linear and exponential silica particle growth dynamics were observed which were dependent on deposition buffer ion constituents and ion concentration. Silica particle growth followed a cluster-cluster growth mechanism at acidic pH, and a monomer-cluster growth mechanism at neutral to basic pH. Increasing silica sol aging temperature resulted in higher rates of particle growth and larger particles. DLS measurements employing PEG coated liposomes and cationic liposomes, serving as model phospholipid assemblies, revealed electrostatic interactions promote more stable liposome-silica interactions than hydrogen bonding and facilitate silica coating on suspension cells. However, continued silica reactivity leads to aggregation of silica coated suspensions cells, revealing the need for cell isolation to tune deposited silica thickness. Utilizing these mechanistic study insights, silica was deposited onto adherent HeLa cells under biocompatible conditions with micron scale control over silica thickness, minimal cell manipulation steps, and retained cell viability over several days.

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. There is considerable literature on VOCs extracted destructively from microalgae for applications such as food, natural products chemistry, and biofuels. VOC emissions from living (in vivo) microalgae too are being increasingly appreciated as potential real-time indicators of the organism's state of health (SoH) along with their contributions to the environment and ecology. This review summarizes VOC emissions from in vivo microalgae; tools and techniques for the collection, storage, transport, detection, and pattern analysis of VOC emissions; linking certain VOCs to biosynthetic/metabolic pathways; and the role of VOCs in microalgae growth, infochemical activities, predator-prey interactions, and general SoH.

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. Silica encapsulation methods employed were lipid-templated mesophase silica thin films formed by cell-directed assembly (CDA), lipid-templated mesophase silica particles formed by spray drying (SD), and glycerol-doped silica gel monoliths prepared from an aqueous silicate (AqS+g) precursor solution. It was found that the cells for all three-encapsulated methods enter quiescent states characteristic of response to stress, albeit to different degrees and with differences in detail. By the measure of enrichment of stress-related gene ontology categories, we find that the AqS+g encapsulation is more amenable to the cells than CDA and SD encapsulation. We hypothesize that this differential response in the AqS+g encapsulation is related to four properties of the encapsulating gel: (1) oxygen permeability, (2) relative softness of the material, (3) development of a protective sheath around individual cells (visible in TEM micrographs vide infra), and (4) the presence of glycerol in the gel, which has been previously noted to serve as a protectant for encapsulated cells and can serve as the sole carbon source for S. cerevisiae under aerobic conditions. This work represents a combination of experiment and analysis aimed at the design and development of 3D encapsulation procedures to induce, and perhaps control, well-defined physiological behaviors.

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. developing countries) has yet to be realized as the majority of technologies used to control microfluidic flow rely on off-device hardware with non-negligible size, cost, power requirements and skill/training to operate. In this paper we describe a magnetic-adhesive based valve that is simple to construct and operate, and can be used to control fluid flow and store reagents within a microfluidic device. The design consists of a port connecting two chambers on different planes in the device that is closed by a neodymium disk magnet seated on a thin ring of adhesive. Bringing an external magnet into contact with the outer surface of the device unseats and displaces the valve magnet from the adhesive ring, exposing the port. Using this configuration, we demonstrate on-device reagent storage and on-demand transport and reaction of contents between chambers. This design requires no power or external instrumentation to operate, is extremely low cost ($0.20 materials cost per valve), can be used by individuals with no technical training, and requires only a hand-held magnet to actuate. Additionally, valve actuation does not compromise the integrity of the completely sealed microfluidic device, increasing safety for the operator when toxic or harmful substances are contained within. This valve concept has the potential to simplify design of µTASs, facilitating development of lab-on-a-chip systems that may be practical for use in point-of-care and low-resource settings.

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 ex-vivo 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. In SG-CViL, the high vapor pressure of tetramethyl orthosilicate (TMOS) is utilized to deliver silica into an aqueous medium, creating a silica sol. Cells are then mixed with the resulting silica sol, facilitating encapsulation of cells in silica while minimizing cell contact with the cytotoxic products of silica generating reactions (i.e. methanol), and reduce exposure of cells to compressive stresses induced from silica condensation reactions. Using SG-CVIL, Saccharomyces cerevisiae (S. cerevisiae) engineered with an inducible beta galactosidase system were encapsulated in silica solids and remained both viable and responsive 29 days post encapsulation. By tuning SG-CViL parameters thin layer silica deposition on mammalian HeLa and U87 human cancer cells was also achieved. The ability to encapsulate various cell types in either a multi cell (S. cerevisiae) or a thin layer (HeLa and U87 cells) fashion shows the promise of SG-CViL as an encapsulation strategy for generating cell-silica constructs with diverse functions for incorporation into devices for sensing, bioelectronics, biocatalysis, and biofuel applications.

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 E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. coli cells encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. This unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.

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 0.2 - 2.0 × 10(11)/mL of 10 nm gold nanoparticles-streptavidin conjugate (10 nm AuNPs-SA) to BP via biotin-streptavidin interactions. Finally, J-aggregation of cyanine 1 (25 µM) or 2 (10 µM) upon the 10 nm AuNPs elicited absorption and fluorescence signaling of TG catalysis. The cyanines could be added sequentially to elicit green (590 nm) and red (700 nm) spectral responses from the same set of reactions. Catalysis was linear (r(2) > 0.98) up to 10 min within a linear dynamic range (LDR) of 0.1 - 5 µg/mL enzyme. The multi-wavelength interrogation offered fast results (< 5 min), sensitivity (limit of detection, LOD of 5 ng or 64 fmol TG) and intermediate precision (relative standard deviation, RSD of < 20% over 42 days). Plasmonic fluorescent nanocomposites offer new ways of interrogating biomolecules in HTS format.

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. Fluorescence from nanocomposites declined inversely with AuNPs size: 40 nm ≥ 20 nm > 10 nm > 5 nm > 2 nm. Sensitivity (limit of detection, LOD, 10(5) - 10(11) AuNPs/mL), brightness of the nanocomposites and surface coverage of AuNPs by cyanine aggregates were all influenced by five factors: 1) AuNPs size; 2) cyanine type (1 or 2); 3) aggregate density; 4) distance between aggregates and AuNPs surface; and 5) streptavidin protein conjugation to AuNPs. We propose a model for plasmonic fluorescent nanocomposites based on these observations. Our plasmonic fluorescent nanocomposites have applications in chemical and biological assays.

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. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.

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. Laser machining of both the fluidic pathways in the plastic laminates, and the stencil masks used for thermal evaporation to form electrode regions on the plastic laminates, enabled rapid and inexpensive implementation of design changes. Electrochemiluminescence reactions in the fixture were achieved and monitored through ITO electrodes. Electroaddressable aryl diazonium chemistry was employed to selectively pattern gold electrodes for electrochemical multianalyte DNA detection from double stranded DNA (dsDNA) samples. Electrochemical detection of dsDNA was achieved by melting of dsDNA molecules in solution with the integrated heater, allowing detection of DNA sequences specific to breast and colorectal cancers with a non-specific binding control. Following detection, the array surface could be renewed via high temperature (95 °C) stripping using the integrated heating element. This versatile and simple method for prototyping devices shows potential for further development of highly integrated, multi-functional bioanalytical devices.