A Multiplex Assay to Assess the Transaminase Activity toward Chemically Diverse Amine Donors.

The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work presents a novel and multiplex fluorescence-based kinetic assay to assess ATA activity using 4-dimethylamino-1-naphthaldehyde as an amine acceptor. The developed assay allowed us to screen a battery of amine donors using free and immobilized ATAs from different microbial sources as biocatalysts. As a result, using chromatographic methods, 4-hydroxybenzylamine was identified as the best amine donor for the amination of 5-(hydroxymethyl)furfural. Finally, we adapted this method to determine the apparent Michaelis-Menten parameters of a model immobilized ATA at the microscopic (single-particle) level. Our studies promote the use of this multiplex, multidimensional assay to screen ATAs for further improvement.

On-line microfluidic immobilized-enzyme reactors: A new tool for characterizing synthetic polymers.

Biodegradable polymeric materials may eventually replace biostable materials for medical applications, including therapeutic devices, scaffolds for tissue engineering, and drug-delivery vehicles. To further develop such materials, a more fundamental understanding is necessary to correlate parameters including chemical-composition distribution within a macromolecular structure with the final properties of the material, including particle-size. A wide variety of analytical techniques have been applied for the characterization of polymer materials, including hyphenated techniques such as comprehensive two-dimensional liquid chromatography (LC × LC). In this context, we have investigated enzymatic degradation of polyester-based nanoparticles, both in-solution and by the use of an immobilized-enzyme reactor (IMER). We have demonstrated for the first time the implementation of such an IMER in a size-exclusion chromatography system for on-line degradation and subsequent analysis of the polymer degradation products. The effect of residence times ranging from 12 s to 4 min on polymer degradation was assessed. IMER-assisted degradation is much faster compared to in-solution degradation, which requires several hours to days, and opens the possibility to use such reactors in LC × LC modulation interfaces.

Fully-Polymeric pH Sensor Realized by Means of a Single-Step Soft Embossing Technique.

We present here an electrochemical sensor microsystem for the monitoring of pH. The all-polymeric device is comprised of a cyclic olefin copolymer substrate, a 200 nm-thin patterned layer of conductive polymer (PEDOT), and a 70 nm electropolymerized layer of a pH sensitive conductive polymer (polyaniline). The patterning of the fluidic (microfluidic channels) and conductive (wiring and electrodes) functional elements was achieved with a single soft PDMS mold via a single embossing step process. A post-processing treatment with ethylene glycol assured the functional enhancement of the electrodes, as demonstrated via an electrical and electrochemical characterization. A surface modification of the electrodes was carried out, based on voltammetric electropolymerization, to obtain a thin layer of polyaniline. The mechanism for pH sensing is based on the redox reactions of the polyaniline layer caused by protonation. The sensing performance of the microsystem was finally validated by monitoring its potentiometric response upon exposure to a relevant range of pH.

Implementing oxygen control in chip-based cell and tissue culture systems.

Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

Mapping the complex morphology of cell interactions with nanowire substrates using FIB-SEM.

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered.

Cell motility, morphology, viability and proliferation in response to nanotopography on silicon black.

Knowledge of cells' interactions with nanostructured materials is fundamental for bio-nanotechnology. We present results for how individual mouse fibroblasts from cell line NIH3T3 respond to highly spiked surfaces of silicon black that were fabricated by maskless reactive ion etching (RIE). We did standard measurements of cell viability, proliferation, and morphology on various surfaces. We also analyzed the motility of cells on the same surfaces, as recorded in time lapse movies of sparsely populated cell cultures. We find that motility and morphology vary strongly with nano-patterns, while viability and proliferation show little dependence on substrate type. We conclude that motility analysis can show a wide range of cell responses e.g. over a factor of two in cell speed to different nano-topographies, where standard assays, such as viability or proliferation, in the tested cases show much less variation of the order 10-20%.

A microfluidic device with fluorimetric detection for intracellular components analysis.

An integrated microfluidic system that coupled lysis of two cell lines: L929 fibroblasts and A549 epithelial cells, with fluorescence-based enzyme assay was developed to determine ß-glucocerebrosidase activity. The microdevice fabricated in poly(dimethylsiloxane) consists of three main parts: a chemical cell lysis zone based on the sheath flow geometry, a micromeander and an optical fibers detection zone. Unlike many methods described in literature that are designed to analyse intracellular components, the presented system enables to perform enzyme assays just after cell lysis process. It reduces the effect of proteases released in lysis process on determined enzymes. Glucocerebrosidase activity, the diagnostic marker for Gaucher's disease, is the most commonly measured in leukocytes and fibroblasts using 4-methylumbelliferyl-ß-D-glucopyranoside as synthetic ß-glucoside. The enzyme cleavage releases the fluorescent product, i.e. 4-methylumbelliferone, and its fluorescence is measured as a function of time. The method of enzyme activity determination described in this paper was adapted for flow measurements in the microdevice. The curve of the enzymatic reaction advancement was prepared for three reaction times obtained from application of different flow rates of solutions introduced to the microsystem. Afterwards, determined ß-glucocerebrosidase activity was recalculated with regard to 10(5) cells present in samples used for the tests. The obtained results were compared with a cuvette-based measurements. The lysosomal ß-glucosidase activities determined in the microsystem were in good correlation with the values determined during macro-scale measurements.

Microfluidic dissolved oxygen gradient generator biochip as a useful tool in bacterial biofilm studies.

A microfluidic chip for generation of gradients of dissolved oxygen was designed, fabricated and tested. The novel way of active oxygen depletion through a gas permeable membrane was applied. Numerical simulations for generation of O(2) gradients were correlated with measured oxygen concentrations. The developed microsystem was used to study growth patterns of the bacterium Pseudomonas aeruginosa in medium with different oxygen concentrations. The results showed that attachment of Pseudomonas aeruginosa to the substrate changed with oxygen concentration. This demonstrates that the device can be used for studies requiring controlled oxygen levels and for future studies of microaerobic and anaerobic conditions.

Miniaturized tools and devices for bioanalytical applications: an overview.

This article presents an overview of various miniaturized devices and technologies developed by our group. Innovative, fast and cheap procedures for the fabrication of laboratory microsystems based on commercially available materials are reported and compared with well-established microfabrication techniques. The modules fabricated and tested in our laboratory can be used independently or they can be set up in different configurations to form functional measurement systems. We also report further applications of the presented modules e.g. disposable poly(dimethylsiloxane) (PDMS) microcuvettes, fibre optic detectors, potentiometric sensors platforms, microreactors and capillary electrophoresis (CE) microchips as well as integrated microsystems e.g. double detection microanalytical systems, devices for studying enzymatic reactions and a microsystem for cell culture and lysis.