Water-Soluble Ionic Metal-Organic Polyhedra as a Versatile Platform for Enzyme Bio-immobilization.

Metal-organic polyhedra (MOPs) can act as elementary structural units for the design of modular porous materials; however, their association with biological systems remains greatly restricted by their typically low stabilities and solubilities in water. Herein, we describe the preparation of novel MOPs bearing either anionic or cationic groups and exhibiting a high affinity for proteins. Simple mixing of the protein bovine serum albumin (BSA) and ionic MOP aqueous solutions resulted in the spontaneous formation of MOP-protein assemblies, in a colloidal state or as solid precipitates depending on the initial mixing ratio. The versatility of the method was further illustrated using two enzymes, catalase and cytochrome c, with different sizes and isoelectric points (pI's) below and above 7. This mode of assembly led to the high retention of catalytic activity and enabled recyclability. Furthermore, the co-immobilization of cytochrome c with highly charged MOPs resulted in a substantial 44-fold increase of its catalytic activity.

In Situ Enzyme Immobilization by Covalent Organic Frameworks.

Enzyme immobilization is a widely reported method to favor the applicability of enzymes by enhancing their stability and re-usability. Among the various existing solid supports and immobilization strategies, the in situ encapsulation of enzymes within crystalline porous matrices is a powerful tool to design biohybrids with a stable and protected catalytic activity. However, to date, only a few metal-organic frameworks (MOFs) and hydrogen-bonded organic frameworks (HOFs) have been reported. Excitingly, for the first time, Y. Chen and co-workers expanded the in situ bio-encapsulation to a new class of crystalline porous materials, namely covalent organic frameworks (COFs). The enzyme@COF materials not only exhibited high enzyme loading with minimal leaching, high catalytic activity and selectivity, chemical and long-term stability and recyclability but could also be scaled up to a few grams. Undoubtedly, this work opens new striking opportunities for enzymatic immobilization and will stimulate new research on COF-based matrices.

Formation of a Single-Crystal Aluminum-Based MOF Nanowire with Graphene Oxide Nanoscrolls as Structure-Directing Agents.

An innovative strategy is proposed to synthesize single-crystal nanowires (NWs) of the Al3+ dicarboxylate MIL-69(Al) MOF by using graphene oxide nanoscrolls as structure-directing agents. MIL-69(Al) NWs with an average diameter of 70±20 nm and lengths up to 2 µm were found to preferentially grow along the [001] crystallographic direction. Advanced characterization methods (electron diffraction, TEM, STEM-HAADF, SEM, XPS) and molecular modeling revealed the mechanism of formation of MIL-69(Al) NWs involving size-confinement and templating effects. The formation of MIL-69(Al) seeds and the self-scroll of GO sheets followed by the anisotropic growth of MIL-69(Al) crystals are mediated by specific GO sheets/MOF interactions. This study delivers an unprecedented approach to control the design of 1D MOF nanostructures and superstructures.

Influence of Filler Pore Structure and Polymer on the Performance of MOF-Based Mixed-Matrix Membranes for CO2 Capture.

To gain insight into the influence of metal-organic framework (MOF) fillers and polymers on membrane performance, eight different composites were studied by combining four MOFs and two polymers. MOF materials (NH2 -MIL-53(Al), MIL-69(Al), MIL-96(Al) and ZIF-94) with various chemical functionalities, topologies, and dimensionalities of porosity were employed as fillers, and two typical polymers with different permeability-selectivity properties (6FDA-DAM and Pebax) were selected as matrices. The best-performing MOF-polymer composites were prepared by loading 25 wt % of MIL-96(Al) as filler, which improved the permeability and selectivity of 6FDA-DAM to 32 and 10 %, while for Pebax they were enhanced to 25 and 18 %, respectively. The observed differences in membrane performance in the separation of CO2 from N2 are explained on the basis of gas solubility, diffusivity properties, and compatibility between the filler and polymer phases.

Enzyme Encapsulation in Mesoporous Metal-Organic Frameworks for Selective Biodegradation of Harmful Dye Molecules.

Microperoxidase-8, a small, peroxidase-type enzyme was immobilized into nanoparticles of the mesoporous and ultra-stable metal-organic framework (MOF) MIL-101(Cr). The immobilized enzyme fully retained its catalytic activity and exhibited enhanced resistance to acidic conditions. The biocatalyst was reusable and showed a long-term stability. By exploiting the properties of the MOF's framework, we demonstrated, for the first time, that the MOF matrix could act in synergy with the enzyme (Microperoxidase-8) and enhance selectivity the oxidation reaction of dyes. The oxidation rate of the harmful negatively charged dye (methyl orange) was significantly increased after enzyme immobilization, probably as a result of the pre-concentration of the methyl orange reactant owing to a charge matching between this dye and the MOF.

Evaporation-Directed Crack-Patterning of Metal-Organic Framework Colloidal Films and Their Application as Photonic Sensors.

A straightforward crack-patterning method is reported allowing the direct formation of periodic cracks in metal-organic framework (MOF) nanoparticle films during dip-coating deposition. The crack propagation and periodicity can be easily tailored by controlling the evaporation front and the withdrawal speed. Several MOF-patterned films can be fabricated on large surfaces and on several substrates (flat, curved or flexible) including the inner surface of a tube, not achievable by other lithographic techniques. We demonstrate that the periodic cracked arrays diffract light and, due to the MOF sorption properties, photonic vapor sensors are fabricated. A new concept of "in-tube", MOF-based diffraction grating sensors is proposed with outstanding sensitivity that can be easily tuned "on-demand" as function of the desired detection range.

Poly(oligoethylene glycol methacrylate) dip-coating: turning cellulose paper into a protein-repellent platform for biosensors.

The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

Printing silicone-based hydrophobic barriers on paper for microfluidic assays using low-cost ink jet printers.

Paper-based microfluidic devices exhibit many advantages for biological assays. Normally, the assays are restricted to certain areas of the paper by hydrophobic barriers comprised of wax or alkyl ketene dimers (AKD). Neither hydrophobic barrier is able to constrain aqueous solutions of surfactants, which are frequently used in biological assays. We demonstrate that rapidly curing silicone resins can be inkjet printed onto pure cellulose paper using inexpensive thermal ink-jet printers. The Piers-Rubinsztajn (PR) reaction dominates the cure chemistry leading to cellulose fibers that are surface coated with a silicone resin. The resulting barriers are able to resist penetration by surfactant solutions and even by the lower surface energy solvents DMF and DMSO. The utility of the barrier was demonstrated using a coliform assay based on detection of ß-galactosidase.

A rapid and sensitive fluorimetric ß-galactosidase assay for coliform detection using chlorophenol red-ß-D-galactopyranoside.

We report on a new fluorimetric assay for ß-galactosidase (ß-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-ß-D-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-L-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-ß-D-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both ß-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.

Multiplexed paper test strip for quantitative bacterial detection.

Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (ß-galactosidase (B-GAL) or ß-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 × 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-ß-D: -glucuronide sodium salt (XG), chlorophenol red ß-galactopyranoside (CPRG) or both and FeCl(3) were entrapped using sol-gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl(3) zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.

Micro-algal biosensors.

Fighting against water pollution requires the ability to detect pollutants for example herbicides or heavy metals. Micro-algae that live in marine and fresh water offer a versatile solution for the construction of novel biosensors. These photosynthetic microorganisms are very sensitive to changes in their environment, enabling the detection of traces of pollutants. Three groups of micro-algae are described in this paper: chlorophyta, cyanobacteria, and diatoms.

Ultrathin hydrophobic films based on the metal organic framework UiO-66-COOH(Zr).

This work reports on the fabrication, optimization and characterization of ultrathin films containing submicrometer particles (sMPs) of the hydrophilic and water stable UiO-66-COOH(Zr) metal organic framework (MOF). MOF particles of ≈200 nm have been synthesized and assembled at the air-water interface by the Langmuir-Blodgett technique. The use of different solvents, mixtures of solvents and surfactants has been investigated in order to improve the stability of MOF dispersions and reduce particle aggregation. The compact MOF/surfactant films containing 10 wt % octadecylphoshonic acid (ODP) have been deposited on substrates of different nature by Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) methods, showing that the presence of even only one MOF/ODP monolayer can increase the water contact angle of highly hydrophilic substrates such as mica or glass up to 120°. These films were characterized by scanning electron microscopy, grazing incidence X-ray diffraction, Fourier transform infrared spectroscopy and atomic force microscopy, revealing the formation of a continuous film where ODP molecules adopt an almost vertical position and cover MOF particles. Moreover, the presence of MOF particles significantly enhances the surface roughness and allows ultrathin, hydrophobic coverage to be obtained. Finally, it has been shown that the crystallinity and the porosity of the MOF remains almost unaltered in MOF/ODP films.

The physics and chemistry of silica-in-silicates nanocomposite hydrogels and their phycocompatibility.

Silicates-in-silica nanocomposite hydrogels obtained from sodium silicates/colloidal silica mixtures have previously been found to be useful for bacterial encapsulation. However the extension of synthesis conditions and the understanding of their impact on the silica matrix would widen the applicability of this process in terms of encapsulated organisms and the host properties. Here the influence of silicates and the colloidal silica concentration as well as pH conditions on the gel time, the optical properties, the structural and mechanical properties of silica matrices was studied. We show that gel formation is driven by silicate condensation but that the aggregation of silica colloids also has a major influence on the transparency and structure of the nanocomposites. Three different photosynthetic organisms, cyanobacteria Anabaena flos-aquae and two microalgae Chorella vulgaris and Euglena gracilis, were used as probes of the phycocompatibility of the process. The three organisms were highly sensitive to the silicate concentration, which impacts both the gelation time and ionic strength conditions. The Ludox content was crucial for cyanobacteria as it strongly impacts the Young's modulus of the matrices. The detrimental effect of acidic pH on cell suspension was compensated by the silica network. Overall, it is now possible to select optimal encapsulation conditions based on the physiology of the targeted cells, opening wide perspectives for the design of biosensors and bioreactors.

Silica@proton-alginate microreactors: a versatile platform for cell encapsulation.

As an alternative approach to the well known Ca(ii)-alginate encapsulation process within silica hydrogels, proton-driven alginate gelation was investigated in order to establish its capacity as a culture carrier, both isolated and embedded in an inorganic matrix. Control over the velocity of the proton-gelation front allows the formation of a hydrogel shell while the core remains liquid, allowing bacteria and microalgae to survive the strongly acidic encapsulation process. Once inside the inorganic host, synthesized by a sol-gel process, the capsules spontaneously redissolve without the aid of external complexing agents. The entrapped cells survive the two-step process to a significant extent; culture's growth restores the initial cell count in less than two weeks. Biosynthesis of Au nanoparticles mediated by the entrapped microalgae illustrates the preservation of the biosynthetic abilities supported by this platform.

Design of metal organic framework-enzyme based bioelectrodes as a novel and highly sensitive biosensing platform.

Nanocomposites combining the mesoporous iron(iii) trimesate MIL-100(Fe) (MIL: Matériaux Institut Lavoisier) and platinum nanoparticles (Pt-NPs) have been used as immobilization matrices of glucose oxidase (GOx). Due to the physico-chemical properties of Pt-NPs (electroactivity) and MIL-100(Fe) (high specific surface area and pore volume, biocompatibility), the resulting GOx-MIL-100(Fe)-PtNP bioelectrode exhibits excellent electrocatalytic performances for glucose detection. This novel glucose biosensor presents a high sensitivity of 71 mA M-1 cm-2 under optimum conditions and a low limit of detection of 5 µM with low response time (<5 s). In contrast, substitution of iron by chromium or aluminum in MIL-100 leads to a much lower sensitivity and higher response time values, suggesting that the iron centres of MIL-100(Fe) may be involved in a synergistic effect which indeed enhances the catalytic oxidation of glucose and biosensor activity. Thus, this work extends the scope of MOF nanoparticles with engineered cores and surface to the field of highly sensitive, durable glucose biosensors.

Tools for water quality monitoring and mapping using paper-based sensors and cell phones.

In this paper we describe a combination of paper-based sensors and a novel smart-phone application for on-site quantification of colorimetric readouts as an ultra-low cost solution to monitoring water quality. The system utilizes a paper-based analytical device (µPAD) that produces a colorimetric signal that is dependent on the concentration of a specific target; a cell phone equipped with a camera for capturing images of two µPADs - one tested with a water sample and the other tested with clean water that is used as a control; and an on-site image processing app that uses a novel algorithm for quantifying color intensity and relating this to contaminant concentration. The cell phone app utilizes a pixel counting algorithm that performs with less bias and user subjectivity than the typically used lab-based software, ImageJ. The use of a test and control strip reduces bias from variations in ambient lighting, making it possible to acquire and process images on-site. The cell phone is also able to GPS tag the location of the test, and transmit results to a newly developed website, WaterMap.ca, that displays the quantitative results from the water samples on a map. We demonstrate our approach using a previously developed µPAD that detects the presence of organophosphate pesticides based on the inhibition of immobilized acetylcholinesterase by these contaminants. The objective of this paper is to highlight the importance and potential of developing and integrated monitoring system consisting of µPADs, cell-phones and a centralized web portal for low-cost monitoring environmental contaminants at a large-scale.

Solid-phase biological assays for drug discovery.

In the past 30 years, there has been a significant growth in the use of solid-phase assays in the area of drug discovery, with a range of new assays being used for both soluble and membrane-bound targets. In this review, we provide some basic background to typical drug targets and immobilization protocols used in solid-phase biological assays (SPBAs) for drug discovery, with emphasis on particularly labile biomolecular targets such as kinases and membrane-bound receptors, and highlight some of the more recent approaches for producing protein microarrays, bioaffinity columns, and other devices that are central to small molecule screening by SPBA. We then discuss key applications of such assays to identify drug leads, with an emphasis on the screening of mixtures. We conclude by highlighting specific advantages and potential disadvantages of SPBAs, particularly as they relate to particular assay formats.

Paper-based microfluidics with an erodible polymeric bridge giving controlled release and timed flow shutoff.

Water soluble pullulan films were formatted into paper-based microfluidic devices, serving as a controlled time shutoff valve. The utility of the valve was demonstrated by a one-step, fully automatic implementation of a complex pesticide assay requiring timed, sequential exposure of an immobilized enzyme layer to separate liquid streams. Pullulan film dissolution and the capillary wicking of aqueous solutions through the device were measured and modeled providing valve design criteria. The films dissolve mainly by surface erosion, meaning the film thickness mainly controls the shutoff time. This method can also provide time-dependent sequential release of reagents without compromising the simplicity and low cost of paper-based devices.

Creating fast flow channels in paper fluidic devices to control timing of sequential reactions.

This paper reports the development of a method to control the flow rate of fluids within paper-based microfluidic analytical devices. We demonstrate that by simply sandwiching paper channels between two flexible films, it is possible to accelerate the flow of water through paper by over 10-fold. The dynamics of this process are such that the height of the liquid is dependent on time to the power of 1/3. This dependence was validated using three different flexible films (with markedly different contact angles) and three different fluids (water and two silicon oils with different viscosities). These covered channels provide a low-cost method for controlling the flow rate of fluid in paper channels, and can be added following printing of reagents to control fluid flow in selected fluidic channels. Using this method, we redesigned a previously published bidirectional lateral flow pesticide sensor to allow more rapid detection of pesticides while eliminating the need to run the assay in two stages. The sensor is fabricated with sol-gel entrapped reagents (indoxyl acetate in a substrate zone and acetylcholinesterase, AChE, in a sensing zone) present in an uncovered "slow" flow channel, with a second, covered "fast" channel used to transport pesticide samples to the sensing region through a simple paper-flap valve. In this manner, pesticides reach the sensing region first to allow preincubation, followed by delivery of the substrate to generate a colorimetric signal. This format results in a uni-directional device that detects the presence of pesticides two times faster than the original bidirectional sensors.

Stabilization of neodymium oxide nanoparticles via soft adsorption of charged polymers.

In this work, two synthetic polyelectrolytes, PSS and PAH, are employed as strong adsorbed surfactants to disperse and stabilize neodymium oxide nanoparticles. The acid-base equilibria of the oxide surfaces of the particles were investigated under different pH conditions in the absence and presence of polyelectrolytes, to optimize particle stabilization through enhancement of the effective repulsive surface charges. Surface charge amplification of a 3:5 ratio was achieved to permit improved particle transparency of 100-fold in visible wavelengths in neutral and acidic pH regimes, and a stable 10-fold surface charge amplification was achieved under basic pH conditions. The potential of polyelectrolytes as stabilizing agents for neodymium oxide NPs in large-scale particle physics experiments requiring extremely high optical transparency over long path length is evaluated based on optical absorbance and particle stability.