Ana C. Fernandes

Lab-on-Chip Cytometry Based on Magnetoresistive Sensors for Bacteria Detection in Milk

Flow cytometers have been optimized for use in portable platforms, where cell separation, identification and counting can be achieved in a compact and modular format. This feature can be combined with magnetic detection, where magnetoresistive sensors can be integrated within microfluidic channels to detect magnetically labelled cells. This work describes a platform for in-flow detection of magnetically labelled cells with a magneto-resistive based cell cytometer. In particular, we present an example for the validation of the platform as a magnetic counter that identifies and quantifies Streptococcus agalactiae in milk.

Magnetic Counter for Group B Streptococci Detection in Milk

Identification of bovine mastitis pathogens is necessary to control the disease, reduce the risk of chronic infections, and target the antimicrobial therapy to be prescribed. Development prospects for new bovine mastitis diagnosis methodologies go also through rapid and efficient devices that can offer a “cow-side” use, meaning that raw milk collected for analysis should have limited pretreatment. This paper aims at developing a magnetic counter that identifies the presence of Streptococcus agalactiae (a Group B Streptococci) in raw milk. The detection is done with an integrated microfluidic platform, where 50 nm magnetic beads attached to Streptococcus agalactiae are dynamically detected by magnetoresistive sensors. This device allows the analysis of raw milk without bridging the microfluidic channels, making this integrated platform very attractive for fast bacteriological contamination screening.

Multi-function microfluidic platform for sensor integration

The limited availability of metabolite-specific sensors for continuous sampling and monitoring is one of the main bottlenecks contributing to failures in bioprocess development. Furthermore, only a limited number of approaches exist to connect currently available measurement systems with high throughput reactor units. This is especially relevant in the biocatalyst screening and characterization stage of process development. In this work, a strategy for sensor integration in microfluidic platforms is demonstrated, to address the need for rapid, cost-effective and high-throughput screening in bioprocesses. This platform is compatible with different sensor formats by enabling their replacement and was built in order to be highly flexible and thus suitable for a wide range of applications. Moreover, this re-usable platform can easily be connected to analytical equipment, such as HPLC, laboratory scale reactors or other microfluidic chips through the use of standardized fittings. In addition, the developed platform includes a two-sensor system interspersed with a mixing channel, which allows the detection of samples that might be outside the first sensors range of detection, through dilution of the sample solution up to 10 times. In order to highlight the features of the proposed platform, inline monitoring of glucose levels is presented and discussed. Glucose was chosen due to its importance in biotechnology as a relevant substrate. The platform demonstrated continuous measurement of substrate solutions for up to 12 h. Furthermore, the influence of the fluid velocity on substrate diffusion was observed, indicating the need for in-flow calibration to achieve a good quantitative output.

Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants

Selective oxidative functionalization of molecules is a highly relevant and often demanding reaction in organic chemistry. The use of biocatalysts allows the stereo- and regioselective introduction of oxygen molecules in organic compounds at milder conditions and avoids the use of complex group-protection schemes and toxic compounds usually applied in conventional organic chemistry. The identification of enzymes with the adequate properties for the target reaction and/or substrate requires better and faster screening strategies. In this manuscript, a microchannel with integrated oxygen sensors was applied to the screening of wild-type and site-directed mutated variants of naphthalene dioxygenase (NDO) from Pseudomonas sp. NICB 9816-4. The oxygen sensors were used to measure the oxygen consumption rate of several variants during the conversion of styrene to 1-phenylethanediol. The oxygen consumption rate allowed the distinguishing of endogenous respiration of the cell host from the oxygen consumed in the reaction. Furthermore, it was possible to identify the higher activity and different reaction rate of two variants, relative to the wild-type NDO. The meander microchannel with integrated oxygen sensors can therefore be used as a simple and fast screening platform for the selection of dioxygenase mutants, in terms of their ability to convert styrene, and potentially in terms of substrate specificity.

Caught in-between: System for in-flow inactivation of enzymes as an intermediary step in “plug-and-play” microfluidic platforms

The need for fast and comprehensive characterization of biocatalysts has pushed the development of new screening platforms based on microfluidics, capable of monitoring several parameters simultaneously, with new configurations of liquid handling, sample treatment and sensing. Modular microfluidics allows the integration of these newly developed approaches in a more flexible way towards increasing applicability of the microfluidic chips to different types of biocatalysts and reactions. A highly relevant operation in such a system is biocatalyst inactivation, which can enable the precise control of reaction time by avoiding the continuation of the reaction in another module or connecting tubes. Such control is important when different modules of reactors and/or sensing units are used and changed frequently. Here we describe the development, characterization and application of a module for rapid enzyme inactivation. The thermal inactivation platform developed is compared with a standard benchtop ThermoMixer in terms of inactivation efficiency for glucose oxidase and catalase. A higher activity loss was observed for enzyme inactivation under flow conditions (inactivation achieved at 120 s residence time at 338 K and 20 s residence time at 353 K) which indicated a high heat transfer to the fluid under dynamic conditions. Moreover, partial deactivation of the enzymes was observed for the continuous thermal inactivation module, when activity measurements were performed after 1 and 2 days following inactivation. The thermal inactivation unit presented can be easily integrated into modular microfluidic platforms and can be a useful addition for enzyme characterization and screening.

An Efficient Experimental Design Strategy for Modelling and Characterization of Processes

Abstract Designing robust, efficient and economic processes is a main challenge for the biotech industries. To achieve a well-designed bioprocess, understanding the ongoing phenomena and the involved reaction kinetics is crucial. By development of advanced miniaturized reactors, a promising opportunity arises for parallel screening of multiple processes in reduced volumes within high throughput platforms. However, the level of accessible information from each set of experimental design remains to be one of the main issues particularly in the case of complex biosystems. This work introduces a novel generic Model-based Design of Experiments (M-DoE) routine with its main target being model development and system characterization. With the new M-DoE strategy, an improved set of informative experiments are suggested, which consequently reduces the demand for physical resources and analysis. The routine proposes a set of optimum experimental settings to support structural model definition, kinetic order estimation and parameter estimation during a model building procedure and process characterization.