The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

We developed a quantum-dot-based fluorescence resonance energy transfer (QD-FRET) nanosensor to visualize the activity of matrix metalloproteinase (MT1-MMP) at cell membrane. A bended peptide with multiple motifs was engineered to position the FRET pair at a close proximity to allow energy transfer, which can be cleaved by active MT1-MMP to result in FRET changes and the exposure of cell penetrating sequence. Via FRET and penetrated QD signals, the nanosensor can profile cancer cells.

Single-layer membrane valves are simple to fabricate and to integrate into microfluidic devices. However, due to their rectangular flow channel geometry, they do not fully seal. Here, we show that liquid flow can be reduced by over 3 orders of magnitude, enabling the contents of forming droplets to be dynamically modulated. We use this precision control to perform combinatorial DNA synthesis in the droplets.

A new imaging system allows visualizing oxygen distributions in 2D. Combining sensor foils with microfluidic devices enables online monitoring of cellular oxygen consumption in whole chip areas. Furthermore, suitable device materials depending on application and cell line can be determined. Numerically simulated oxygen consumption of rat lung microvascular endothelial cells and rat hepatocytes was experimentally validated.

Microfluidic devices fabricated from cyclic olefin copolymer (COC) and polydimethylsiloxane (PDMS) were combined with VisiSens sensor foils to enable monitoring of oxygen tensions in the devices. The oxygen consumption of rat lung microvascular endothelial cells (EC) and rat hepatocytes (Hep) was numerically simulated and experimentally validated using the VisiSens set-up. The VisiSens system enabled determining oxygen levels in the microfluidic devices and allows suggesting suitable culture device material depending on the application and cell line of interest. Furthermore, a previously characterized hypoxia microfluidic chip was evaluated and VisiSens was found to exhibit superior sensing capabilities when compared to similar products.

Tetraphenylethene (TPE) shows a significant increase of fluorescence intensity when the rotational freedom of its phenyl groups is restricted. This special property allows the use of TPE in sensor applications, which have been previously described for the liquid phase only. However, some applications utilising arrays require the immobilisation of TPE dyes on solid surfaces. In this work, we synthesised and investigated the fluorescence behaviour of TPE derivatives on silica particles and quartz slides and suggest ways to employ the dye's properties in solid phase biosensor applications. 2014 Published by Elsevier B.V.

We developed a computational model to predict oxygen levels in microfluidic plastic devices during cell culture. This model is based on experimental evaluation of oxygen levels. Conditions are determined that provide adequate oxygen supply to two cell types, hepatocytes and endothelial cells, either by diffusion through the plastic device, or by supplying a low flow rate of medium.

Using "Generally Recognized as Safe" (GRAS) materials for assembly, edible polyelectrolytes (EPL/PGA) are coated on sugar (maltotriose)–dextran particles and crosslinked (left). Cargo-loaded hollow capsules are created by dissolution of the sugar template (right).

Structure-adjustable capsules are fabricated from inorganic components by using a self-template dissolution-regrowth mechanism to give flake-shell silica microcapsules. The capsules shrink under thermal stimulus and their structures can be adjusted by treatment at different pH values. Tuning of shell pore diameters leads to tailored drug release over prolonged periods.

Low oxygen tensions experienced in various pathological and physiological conditions are a major stimulus for angiogenesis. Hypoxic conditions play a critical role in regulating cellular behaviour including migration, proliferation and differentiation. This study introduces the use of a microfluidic device that allows for the control of oxygen tension for the study of different three-dimensional (3D) cell cultures for various applications. The device has a central 3D gel region acting as an external cellular matrix, flanked by media channels. On each side, there is a peripheral gas channel through which suitable gas mixtures are supplied to establish a uniform oxygen tension or gradient within the device. The effects of various parameters, such as gas and media flow rates, device thickness, and diffusion coefficients of oxygen were examined using numerical simulations to determine the characteristics of the microfluidic device. A polycarbonate (PC) film with a low oxygen diffusion coefficient was embedded in the device in proximity above the channels to prevent oxygen diffusion from the incubator environment into the polydimethylsiloxane (PDMS) device. The oxygen tension in the device was then validated experimentally using a ruthenium-coated (Ru-coated) oxygen-sensing glass cover slip which confirmed the establishment of low uniform oxygen tensions (<3%) or an oxygen gradient across the gel region. To demonstrate the utility of the microfluidic device for cellular experiments under hypoxic conditions, migratory studies of MDA-MB-231 human breast cancer cells were performed. The microfluidic device allowed for imaging cellular migration with high-resolution, exhibiting an enhanced migration in hypoxia in comparison to normoxia. This microfluidic device presents itself as a promising platform for the investigation of cellular behaviour in a 3D gel scaffold under varying hypoxic conditions.

We report a facile approach to immobilize pH-cleavable polymer drug conjugates in mussel inspired polydopamine (PDA) capsules for intracellular drug delivery. Our design takes advantage of the facile PDA coating to form capsules, the chemical reactivity of PDA films, and the acid-labile groups in polymer side chains for sustained pH-induced drug release. The anticancer drug doxorubicin (Dox) was conjugated to thiolated poly(methacrylic acid) (PMA(SH)) with a PH-cleavable hydrazone bond, and then immobilized in PDA capsules via robust thiol-catechol reactions between the polymer-drug conjugate and capsule walls. The loaded Dox showed limited release at physiological pH but significant release (over 85%) at endosomal/lysosomal pH. Cell viability assays showed that Dox-loaded PDA capsules enhanced the efficacy of eradicating HeLa cancer cells compared with free drug under the same assay conditions. The reported method provides a new platform for the application of stimuli responsive PDA capsules as drug delivery systems.

PGA-dopamine conjugates (PGAPDA) were synthesized and assembled onto silica particles to yield biodegradable capsules with tunable degradation properties. Increasing the dopamine content results in thicker films with a concomitant reduction in the degradation kinetics. These tailored capsules are expected to find application in diverse areas, including drug and gene delivery.

A highly generalizable, surface-confined, continuous polymer assembly process, amenable to various substrates, reaction conditions and macromolecules, enables the synthesis of a range of nanoscale supported and freestanding cross-linked polymer films. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The continuous assembly of polymers (CAP) via atom transfer radical polymerisation (ATRP) is reported as an efficient approach for the preparation of dense, cross-linked, nanoscale engineered films as surface coatings, hollow capsules and replica particles. These films can be reinitiated to allow the preparation of thicker films without loss of film growth efficiency while maintaining similar film density.

Biodegradable polymer capsules with drug-loaded multilayers were prepared through click chemistry via layer-by-layer assembly. The capsules can efficiently release incorporated drug by intracellular enzymatic degradation in both sensitive and resistant colorectal cancer cells in vitro. Endocytosis of drug-loaded capsules leads to bypass of Pgp-mediated multidrug resistance in cancer cells.

The inside cover illustrates a pathway to bypass Pgp-mediated multidrug resistance in cancer cells through endocytosis of microcapsules (right), compared with drug effluxing (left), as reported in work by Frank Caruso and co-workers on p. 5398. Biodegradable polymer capsules conjugated with drug were prepared through click chemistry via layer-by-layer assembly. The incorporated drug is subsequently released after the internalization of microcapsules by intracellular enzymatic environment. This approach opens a generic way to challenge Pgp-mediated multidrug resistance.

We report the modular assembly of a polymer-drug conjugate into covalently stabilized, responsive, biodegradable, and drug-loaded capsules with control over drug dose and position in the multilayer film. The cancer therapeutic, doxorubicin hydrochloride (DOX), was conjugated to alkyne-functionalized poly(l-glutamic acid) (PGA(Alk)) via amide bond formation. PGA(Alk) and PGA(Alk+DOX) were assembled via hydrogen bonding with poly(N-vinyl pyrrolidone) (PVPON) on planar and colloidal silica templates. The films were subsequently covalently stabilized using diazide cross-linkers, and PVPON was released from the multilayers by altering the solution pH to disrupt hydrogen bonding. After removal of the sacrificial template, single-component PGA(Alk) capsules were obtained and analyzed by optical microscopy, transmission electron microscopy, and atomic force microscopy. The PGA(Alk) capsules were stable in the pH range between 2 and 11 and exhibited reversible swelling/shrinking behavior. PGA(Alk+DOX) was assembled to form drug-loaded polymer capsules with control over drug dose and position in the multilayer system (e.g., DOX in every layer or exclusively in layer 3). The drug-loaded capsules could be degraded enzymatically, resulting in the sustained release of active DOX over approximately 2 h. Cellular uptake studies demonstrate that the viability of cells incubated with DOX-loaded PGA(Alk) capsules significantly decreased. The general applicability of this modular approach, in terms of incorporation of polymer-drug conjugates in other click multilayer systems, was also demonstrated. Biodegradable click capsules with drug-loaded multilayers are promising candidates as carrier systems for biomedical applications.

Herein we report the preparation of layer-by-layer (LbL) assembled, biodegradable, covalently stabilized capsules with tunable degradation properties. Poly(L-glutamic acid) modified with alkyne moieties (PGA(Alk)) was alternately assembled with poly(N-vinyl pyrrolidone) (PVPON) on silica particles via hydrogen-bonding. The films were cross-linked with a bis-azide linker, followed by removal of the sacrificial template and PVPON at physiological pH through hydrogen bond disruption, yielding one-component PGA(Alk) capsules. To control the kinetics and location of capsule degradation, a number of approaches were investigated. First, a degradable bis-azide cross-linker was incorporated into the inherently enzymatically degradable capsules. Second, we assembled low-fouling capsules composed of nondegradable poly(N-vinyl pyrrolidone-ran-propargyl acrylate) (PVPON(Alk)) via hydrogen bonding with poly(methacrylic acid) (PMA) and combined this with the aforementioned system (PGA(Alk)/PVPON) to produce stratified hybrid capsules. The degradation profiles of these stratified capsules can be closely controlled by the number as well as the position of nondegradable barrier layers in the systems. The facile tailoring of the degradation kinetics makes this stratified LbL approach promising for the design of tailored drug-delivery vehicles.