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Innovative High-Throughput SAXS Methodologies Based on Photonic Lab-on-a-Chip Sensors: Application to Macromolecular Studies.

ABSTRACT: The relevance of coupling droplet-based Photonic Lab-on-a-Chip (PhLoC) platforms and Small-Angle X-Ray Scattering (SAXS) technique is here highlighted for the performance of high throughput investigations, related to the study of protein macromolecular interactions. With this configuration, minute amounts of sample are required to obtain reliable statistical data. The PhLoC platforms presented in this work are designed to allow and control an effective mixing of precise amounts of proteins, crystallization reagents and buffer in nanoliter volumes, and the subsequent generation of nanodroplets by means of a two-phase flow. Spectrophotometric sensing permits a fine control on droplet generation frequency and stability as well as on concentration conditions, and finally the droplet flow is synchronized to perform synchrotron radiation SAXS measurements in individual droplets (each one acting as an isolated microreactor) to probe protein interactions. With this configuration, droplet physic-chemical conditions can be reproducibly and finely tuned, and monitored without cross-contamination, allowing for the screening of a substantial number of saturation conditions with a small amount of biological material. The setup was tested and validated using lysozyme as a model of study. By means of SAXS experiments, the proteins gyration radius and structure envelope were calculated as a function of protein concentration. The obtained values were found to be in good agreement with previously reported data, but with a dramatic reduction of sample volume requirements compared to studies reported in the literature.

SUBMITTER: Rodriguez-Ruiz I

PROVIDER: S-EPMC5492703 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Innovative High-Throughput SAXS Methodologies Based on Photonic Lab-on-a-Chip Sensors: Application to Macromolecular Studies.

Rodríguez-Ruiz Isaac I Radajewski Dimitri D Charton Sophie S Phamvan Nhat N Brennich Martha M Pernot Petra P Bonneté Françoise F Teychené Sébastien S

Sensors (Basel, Switzerland) 20170602 6

The relevance of coupling droplet-based Photonic Lab-on-a-Chip (PhLoC) platforms and Small-Angle X-Ray Scattering (SAXS) technique is here highlighted for the performance of high throughput investigations, related to the study of protein macromolecular interactions. With this configuration, minute amounts of sample are required to obtain reliable statistical data. The PhLoC platforms presented in this work are designed to allow and control an effective mixing of precise amounts of proteins, crys ...[more]

PMID: 28574461

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Project description:This paper presents a vertically positioned microfluidic system made of poly(dimethylsiloxane) (PDMS) and glass, which can be applied as a microbubble column (μBC) for biotechnological screening in suspension. In this μBC, microbubbles are produced in a cultivation chamber through an integrated nozzle structure. Thus, homogeneous suspension of biomass is achieved in the cultivation chamber without requiring additional mixing elements. Moreover, blockage due to produced carbon dioxide by the microorganisms-a problem predominant in common, horizontally positioned microbioreactors (MBRs)-is avoided, as the gas bubbles are released by buoyancy at the upper part of the microsystem. The patterned PDMS layer is based on an optimized two-lithographic process. Since the naturally hydrophobic PDMS causes problems for the sufficient production of microbubbles, a method based on polyelectrolyte multilayers is applied in order to allow continuous hydrophilization of the already bonded PDMS-glass-system. The μBC comprises various microelements, including stabilization of temperature, control of continuous bubble formation, and two optical configurations for measurement of optical density with two different sensitivities. In addition, the simple and robust application and handling of the μBC is achieved via a custom-made modular plug-in adapter. To validate the scalability from laboratory scale to microscale, and thus to demonstrate the successful application of the μBC as a screening instrument, a batch cultivation of Saccharomyces cerevisiae is performed in the μBC and compared to shake flask cultivation. Monitoring of the biomass growth in the μBC with the integrated online analytics resulted in a specific growth rate of 0.32 h(-1), which is almost identical to the one achieved in the shake flask cultivation (0.31 h(-1)). Therefore, the validity of the μBC as an alternative screening tool compared to other conventional laboratory scale systems in bioprocess development is proven. In addition, vertically positioned microbioreactors show high potential in comparison to conventional screening tools, since they allow for high density of integrated online analytics and therefore minimize time and cost for screening and guarantee improved control and analysis of cultivation parameters.

Dataset Information

Innovative High-Throughput SAXS Methodologies Based on Photonic Lab-on-a-Chip Sensors: Application to Macromolecular Studies.

Publications

Innovative High-Throughput SAXS Methodologies Based on Photonic Lab-on-a-Chip Sensors: Application to Macromolecular Studies.

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