A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15-1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing

Biological Particle Control and Separation using Active Forces in Microfluidic Environments

Exploration of active manipulation of bioparticles has been impacted by the development of micro-/nanofluidic technologies, enabling evident observation of particle responses by means of applied tunable external force field, namely, dielectrophoresis (DEP), magnetophoresis (MAG), acoustophoresis (ACT), thermophoresis (THM), and optical tweezing or trapping (OPT). In this chapter, each mechanism is presented in brief yet concise, for broad range of readers, as strong foundation for amateur as well as brainstorming source for experts. The discussion covers the fundamental mechanism that underlying the phenomenon, presenting the theoretical and schematic description; how the response being tuned; and utmost practical, the understanding by specific implementation into bioparticles manipulation engaging from micron-sized material down to molecular level particles

Roadmap for optofluidics

Optofluidics, nominally the research area where optics and fluidics merge, is a relatively new research field and it is only in the last decade that there has been a large increase in the number of optofluidic. applications, as well as in the number of research groups, devoted to the topic. Nowadays optofluidics applications include, without being limited to, lab-on-a-chip devices, fluid-based and controlled lenses, optical sensors for fluids and for suspended particles, biosensors, imaging tools, etc. The long list of potential optofluidics applications, which have been recently demonstrated, suggests that optofluidic technologies will become more and more common in everyday life in the future, causing a significant impact on many aspects of our society. A characteristic of this research field, deriving from both its interdisciplinary origin and applications, is that in order to develop suitable solutions a. combination of a deep knowledge in different fields, ranging from materials science to photonics, from microfluidics to molecular biology and biophysics,. is often required. As a direct consequence, also being able to understand the long-term evolution of optofluidics research is not. easy. In this article, we report several expert contributions on different topics. so as to provide guidance for young scientists. At the same time, we hope that this document will also prove useful for funding institutions and stakeholders. to better understand the perspectives and opportunities offered by this research field

Droplet Lasers:A Review of Current Progress

© 2017 IOP Publishing Ltd. It is perhaps surprising that something as fragile as a microscopic droplet could possibly form a laser. In this article we will review some of the underpinning physics as to how this might be possible, and then examine the state of the art in the field. The technology to create and manipulate droplets will be examined, as will the different classes of droplet lasers. We discuss the rapidly developing fields of droplet biolasers, liquid crystal laser droplets and explore how droplet lasers could give rise to new bio and chemical sensing and analysis. The challenges that droplet lasers face in becoming robust devices, either as sensors or as photonic components in the lab on chip devices, is assessed

Engineering mechanobiology: the bacterial exclusively-mechanosensitive ion channel MscL as a future tool for neuronal stimulation technology

The development of novel approaches to stimulate neuronal circuits is crucial to understand the physiology of neuronal networks, and to provide new strategies to treat neurological disorders. Nowadays, chemical, electrical or optical approaches are the main exploited strategies to interrogate and dissect neuronal circuit functions. However, although all these methods have contributed to achieve important insights into neuroscience research field, they all present relevant limitations for their use in in-vivo studies or clinical applications. For example, while chemical stimulation does not require invasive surgical procedures, it is difficult to control the pharmacokinetics and the spatial selectivity of the stimulus; electrical stimulation provides high temporal bandwidth, but it has low spatial resolution and it requires implantation of electrodes; optical stimulation provides subcellular resolution but the low depth penetration in dense tissue still requires the invasive insertion of stimulating probes. Due to all these drawbacks, there is still a strong need to develop new stimulation strategies to remotely activate neuronal circuits as deep as possible. The development of remote stimulation techniques would allow the combination of functional and behavioral studies, and the design of novel and minimally invasive prosthetic approaches. Alternative approaches to circumvent surgical implantation of probes include transcranial electrical, thermal, magnetic, and ultrasound stimulation. Among v these methods, the use of magnetic and ultrasound (US) fields represents the most promising vector to remotely convey information to the brain tissue. Both magnetic and low-intensity US fields provide an efficient mean for delicate and reversible alteration of cells and tissues through the generation of local mechanical perturbations. In this regard, advances in the mechanobiology research field have led to the discovery, design and engineering of cellular transduction pathways to perform stimulation of cellular activity. Furthermore, the use of US pressure fields is attracting considerable interest due to its potential for the development of miniaturized, portable and implantation-free US stimulation devices. The purpose of my PhD research activity was the establishment of a novel neuronal stimulation paradigm adding a cellular selectivity to the US stimulation technology through the selective mechano-sensitization of neuronal cells, in analogy to the well-established optogenetic approach. In order to achieve the above mentioned goal, we propose the cellular overexpression of mechanosensitive (MS) ion channels, which could then be gated upon the application of an US generated pressure field. Therefore, we selected the bacterial large conductance mechanosensitive ion channel (MscL), an exclusively-MS ion channel, as ideal tool to develop a mechanogenetic approach. Indeed, the MscL with its extensive characterization represents a malleable nano-valve that could be further engineered with respect to channel sensitivity, conductance and gating mechanism, in order to obtain the desired biophysical properties to achieve reliable and efficient remote mechanical stimulation of neuronal activity. In the first part of the work, we report the development of an engineered MscL construct, called eMscL, to induce the heterologous expression of the bacterial protein in rodent primary neuronal cultures. Furthermore, we report the structural and functional characterization of neuronal cells expressing the eMscL channel, at both single-cell and network levels, in order to show that the functional expression of the engineered MscL channel induces an effective vi neuronal sensitization to mechanical stimulation, which does not affect the physiological development of the neuronal itself. In the second part of the work, we report the design and development of a water tank-free ultrasound delivery system integrated to a custom inverted fluorescence microscope, which allows the simultaneous US stimulation and monitoring of neuronal network activity at single resolution. Overall, this work represents the first development of a genetically mechanosensitized neuronal in-vitro model. Moreover, the developed US delivery system provides the platform to perform high-throughput and reliable investigation, testing and calibration of the stimulation protocols. In this respect, we propose, and envisage in the near future, the exploitation of the engineered MscL ion channel as a mature tool for novel neuro-technological applications

Cell Separations and Sorting

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.9b05357.NIBIB Grant P41-EB020594COBRE Grant 5P20GM13042

Sonocytology: dynamic acoustic manipulation of particles and cells

Separating and sorting cells and micro-organisms from a heterogeneous mixture is a fundamental step in biological, chemical and clinical studies, enabling regenerative medicine, stem cell research, clinical sample preparation and improved food safety. Particle and cell manipulation by ultrasound acoustic waves provides the capability of separation of cells on the basis of their size and physical properties. Offering the advantages of relatively large microfluidic volumes in a label-free, contactless and biocompatible manner. Consequently, the discovery of alternative methods for precise manipulation of cells and particles is of highly demand. This thesis describes a novel approach of ultrasound acoustic manipulation of particles and cells. The principle of operation of the dynamic acoustic field method is described accompanied with acoustic separation simulations. Furthermore, the complete fabrication and characterisation of two types of ultrasound devices is given. The first one is a bulk acoustic wave (BAW) device and the second is a surface acoustic wave (SAW) device. Successful experiments using the BAW device for sorting different diameter particles with a range from 5 to 45 μm are demonstrated, also experiments for sorting particles depending on their density are presented. Moreover, experiments of the proposed method for sorting porcine dorcal root ganglion (DRG) cells from a heterogeneous mixture of myelin debris depending on their size are displayed. Experimental results of sorting cells depending on their stiffness are demonstrated. Experiments using the fabricated SAW device for sorting different diameter particles in a constant flow with a range from 1 μm to 10 μm are presented. Furthermore, experiments of the proposed method for sorting live from dead Htert cells depending on their mechanical properties, i.e. stiffness are displayed. As a side project a new idea for dynamic acoustic manipulation by rotating the acoustic field is demonstrated. The basic principles of this method and the simulations for verifying this concept are displayed. Experiments for sorting 10 μm from 3 μm polystyrene particles are presented, with two different types of the dynamic acoustic rotating field being examined