Metodología avanzada para la captura de LPS en biofluidos

ABSTRACT: Lipopolysaccharide (LPS), or endotoxin, is the main component of the outer membrane of Gram-negative bacteria where lipid A is the responsible segment for its toxicity. It poses a serious risk when detected both in different industries and environments and when is present in human bloodstream as it can lead to sepsis, an exaggerated response to LPS that triggers immune suppression, organ dysfunction or even death. Unfortunately, alternative methods for contaminant removal through extracorporeal blood detoxification processes present drawbacks that make endotoxin detection/removal a crucial challenge to achieve safe and effective detoxification processes. In this regard, magnetofluidic devices deserve special attention and involve two main steps: the sequestration of LPS on suitably functionalized magnetic nanoparticles (MNPs) and, the removal of the MNPs-LPS complex from the biological fluid. Consequently, this dissertation provides an integrated methodology to advance the design of the LPS sequestration step to promote its separation from biofluids through the synthesis of an antilipopolysaccharide (LALF) protein from Limulus polyphemus species using genetic engineering techniques in addition to the quantification of the binding strength of the LALF protein to LPS through a newly approach and addressing the variables that affect the formation of the complex. In addition, in order to contribute to the development of an application for continuous LPS capture, as a first approach, homogeneous and heterogeneous L-L separation of aqueous anions (chromate) in microdevices is addressed experimentally and by means of a theoretical model developed with ANSYS FLUENT, laying the foundations to continue with the microfluidic design for L-S separation and finally, its application to LPS capture.RESUMEN: El lipopolisacárido (LPS), o endotoxina, es el principal componente de la membrana externa de las bacterias Gram negativas donde el lípido A es el segmento responsable de su toxicidad. Su presencia supone un grave riesgo tanto en diferentes industrias y entornos como cuando llega al torrente sanguíneo pudiendo conducir a la sepsis, una respuesta exagerada al LPS que desencadena una supresión inmunitaria, una disfunción orgánica o incluso la muerte. Lamentablemente, los métodos alternativos para la eliminación de contaminantes a través de procesos de detoxificación extracorpórea de la sangre presentan inconvenientes que hacen que la detección/eliminación de endotoxinas sea un reto crucial para lograr procesos de detoxificación seguros y eficaces. En este sentido, los dispositivos magnetofluídicos merecen especial atención e implican dos etapas principales; el secuestro de LPS en nanopartículas magnéticas (MNPs) convenientemente funcionalizadas y, la eliminación del complejo MNPs-LPS del fluido biológico. En consecuencia, esta disertación aporta una metodología integrada para avanzar en el diseño de la etapa de secuestro de LPS para promover su separación de los biofluidos a través de la síntesis de una proteína antilipopolisacáridos (LALF) procedente de la especie Limulus polyphemus mediante técnicas de ingeniería genética además de la cuantificación de la fuerza de unión de la proteína LALF al LPS mediante un nuevo enfoque y teniendo en cuenta las variables que afectan a la formación del complejo. Además, con el objeto de contribuir al desarrollo de una aplicación para la captura de LPS en continuo, como primera aproximación, se aborda para la separación homogénea y heterogénea L-L de aniones acuosos (cromato) en microdispositivos experimentalmente y mediante un modelo teórico desarrollado con ANSYS FLUENT, sentando las bases para continuar con el diseño microfluidico para la separación L-S y finalmente, su aplicación a la captura de LPS.The research described in this dissertation was conducted at the Advanced Separation Processes research group of the Department of Chemical and Biomolecular Engineering at the University of Cantabria. The research was financially supported by the Ministry of Economy and Competitiveness of the Spanish Government through the R&D project RTI2018-093310-B-I00 (MINECO / FEDER, UE)

Fighting against bacterial lipopolysaccharide-caused infections through molecular dynamics simulations: a review

Lipopolysaccharide (LPS) is the primary component of the outer leaflet of Gram-negative bacterial outer membranes. LPS elicits an overwhelming immune response during infection, which can lead to life-threatening sepsis or septic shock for which no suitable treatment is available so far. As a result of the worldwide expanding multidrug-resistant bacteria, the occurrence and frequency of sepsis are expected to increase; thus, there is an urge to develop novel strategies for treating bacterial infections. In this regard, gaining an in-depth understanding about the ability of LPS to both stimulate the host immune system and interact with several molecules is crucial for fighting against LPS-caused infections and allowing for the rational design of novel antisepsis drugs, vaccines and LPS sequestration and detection methods. Molecular dynamics (MD) simulations, which are understood as being a computational microscope, have proven to be of significant value to understand LPS-related phenomena, driving and optimizing experimental research studies. In this work, a comprehensive review on the methods that can be combined with MD simulations, recently applied in LPS research, is provided. We focus especially on both enhanced sampling methods, which enable the exploration of more complex systems and access to larger time scales, and free energy calculation approaches. Thereby, apart from outlining several strategies for surmounting LPS-caused infections, this work reports the current state-of-the-art of the methods applied with MD simulations for moving a step forward in the development of such strategies.Financial support from the Spanish Ministry of Science, Innovation and Universities under the project RTI2018- 093310-B-I00 is gratefully acknowledged. C.G.F. and A.B. are also thankful for the FPU (FPU18/03525) and FPI (BES-2016-077206) postgraduate research grants, respectively

Continuous-flow separation of magnetic particles from biofluids: how does the microdevice geometry determine the separation performance?

The use of functionalized magnetic particles for the detection or separation of multiple chemicals and biomolecules from biofluids continues to attract significant attention. After their incubation with the targeted substances, the beads can be magnetically recovered to perform analysis or diagnostic tests. Particle recovery with permanent magnets in continuous-flow microdevices has gathered great attention in the last decade due to the multiple advantages of microfluidics. As such, great efforts have been made to determine the magnetic and fluidic conditions for achieving complete particle capture; however, less attention has been paid to the effect of the channel geometry on the system performance, although it is key for designing systems that simultaneously provide high particle recovery and flow rates. Herein, we address the optimization of Y-Y-shaped microchannels, where magnetic beads are separated from blood and collected into a buffer stream by applying an external magnetic field. The influence of several geometrical features (namely cross section shape, thickness, length, and volume) on both bead recovery and system throughput is studied. For that purpose, we employ an experimentally validated Computational Fluid Dynamics (CFD) numerical model that considers the dominant forces acting on the beads during separation. Our results indicate that rectangular, long devices display the best performance as they deliver high particle recovery and high throughput. Thus, this methodology could be applied to the rational design of lab-on-a-chip devices for any magnetically driven purification, enrichment or isolation.This research was funded by the Spanish Ministry of Science, Innovation and Universities under the project RTI2018-093310-B-I00, and the FPU and FPI postgraduate research grants (FPU18/03525; BES-2016-077206). Financial support from the National Heart, Lung, and Blood Institute from the United States National Institutes of Health (1R01HL131720-01A1) has also been receive