Lab‐on‐a‐chip biophotonics: its application to assisted reproductive technologies

With the benefits of automation, sensitivity and precision, microfluidics has enabled complex and otherwise tedious experiments. Lately, lab‐on‐a‐chip (LOC) has proven to be a useful tool for enhancing non‐invasive assisted reproductive technology (ART). Non‐invasive gamete and embryo assessment has largely been through periodic morpohological assessment using optical microscopy and early LOC ART was the same. As we realize that morphological assessment is a poor indication of gamete or embryo health, more advanced biophotonics has emerged in LOC ART to assay for metabolites or gamete separation via optoelectrical tweezers. Off‐chip, even more advanced biophotonics with broad spectrum analysis of metabolites and secretomes has been developed that show even higher accuracy to predicting reproductive potential. The integration of broad spectrum metabolite analysis into LOC ART is an exciting future that merges automation and sensitivity with the already highly accurate and strong predictive power of biophotonics. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92358/1/650_ftp.pd

3D correlative single-cell imaging utilizing fluorescence and refractive index tomography

Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, quantifying the intracellular RI distribution and colocalizing it with fluorescence in 3D. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.Comment: 15 pages, 5 figure

Effects of centrifugal stress on cell disruption and glycerol leakage from Dunaliella salina

Dunaliella salina accumulates large amounts of intracellular glycerol in response to the increases in salt concentration, thus is a potential source for producing fuel grade glycerol as an alternative to biodiesel-derived crude glycerol. D. salina lacks a cell wall; therefore the mode of harvesting Dunaliella cells is critical to avoid cell disruption caused by extreme engineering conditions. This study explored cell disruption and glycerol leakage of D. salina under various centrifugal stresses during cell harvesting. Results show a centrifugal g-force lower than 5000 g caused little cell disruption, while a g-force higher than 9000 g led to ~40 % loss of the intact cells and glycerol yields from the recovered algal pellets. Theoretical calculations of the centrifugal stresses that could rupture Dunaliella cells were in agreement with the experimental results, indicating optimisation of centrifugation conditions is important for recovering intact cells of from D. salina enriched in glycerol

Nanoparticle mediated toxicity and antimicrobial action

Nanomaterials are either inorganic or organic nanosized particles which have many industrial and biological applications such as in cosmetics, environmental remediation, electronics, biosensing and imaging and in drug delivery. Some have toxic effect upon release to the environment causing death of microorganisms and others are biodegradable and nontoxic to the living beings. In this work, two types of nanoparticles were investigated: inorganic titania nanoparticles which have been shown to have toxic effects and organic Carbopol Aqua SF1 microgel particles which were shown to be nontoxic and biodegradable organic nanoparticles for drug delivery.Chapter one explores the current literature relating to nanoparticles. The types, chemical and physical properties, methods of synthesis, characterization and functionalization are discussed along with general applications and the toxicity of titania nanoparticles. The role of nanomaterials as drug delivery systems and their design in terms of stability, swelling studies, encapsulation, drug loading and release, response to stimuli and targeting is also discussed. Finally the use of microfluidics for screening nanoparticle activity is discussed including microfabrications of chips cell trapping methods and microfluidic cell based assay methods.Chapter two is the experimental chapter describing the chemicals and instrumentation used. It also includes the methods used for the synthesis of titania nanoparticles and effects of pH on the zeta potential measurement. In addition to that, methods for the testing of the cytotoxic effects of uncoated and coated titania nanoparticles are described. Finally the methods employed for studying the optimization of the encapsulation of berberine and chlorhexidine into Carbopol Aqua SF1 are also included.Chapter three describe the synthesis of titania nanoparticles (TiO2NPs) and their characterization, including crystallite size, particle size distribution, surface area measurement and zeta potential. It was found that as the temperature increased from 100oC to 800oC, the crystallite size and particle size increased while the surface area decreased. At 100oC, the crystallite size, particle size, surface area and zeta potential of the titania nanoparticles were 5 nm, 25±20 nm, 163 m2 g-1 and +40±9 mV, respectively with anatase as the dominant phase. However, the phase changed to rutile at the annealing temperature of 800oC with the crystallite size, particle size, surface area and zeta potential becoming 142 nm, 145±60 nm, 7.5 m2 g-1 and -26±8 mVrespectively. In addition to that, the zeta potential of the titania nanoparticles at 25 nm size was affected by changing the pH of the suspension, at low pH, the zeta potential was +40 mV giving high stability and fully dispersed particles while the nanoparticles flocculated in the basic medium with a zeta potential of -25 mV, with the isoelectric point of titania nanoparticles being pH 6.7. Changing the pH of the solution for titania nanoparticles caused an irreversible process as it was not possibility to convert the aggregated titania nanoparticles from microscale to nanoscale.Chapter four describes the investigation into the the nanotoxicity of the titania nanoparticles (TiO2NPs) at various hydrodynamic diameters and crystallite size on C. reinhardtii microalgae and S. cerevisiae (yeast) upon illumination with UV and visible light. The cell viability was assessed for a range of nanoparticle concentrations and incubation times. It was found that uncoated TiO2NPs affect the C. reinhardtii cell viability at a much lower particle concentrations than for yeast. It was also observed that the TiO2NPs toxicity increased upon illumination with UV light compared to dark conditions due to the oxidative stress of the reactive oxygen species produced. It was also found that TiO2NPs nanotoxicity increased upon illumination with visible light which indicated that the nanoparticles might also interfere with the microalgae photosynthetic system leading to decreased chlorophyll content upon exposure to TiO2NPs. The results showed that the larger the hydrodynamic diameter of the TiO2NPs the lower their nanotoxicity, with anatase TiO2NPs generally being more toxic than rutile TiO2NPs. A range of polyelectrolyte-coated TiO2NPs were also prepared using the layer by-layer method and their nanotoxicity on yeast and microalgae was studied. It was found that the toxicity of the coated TiO2NPs alternates with their surface charge. TiO2NPs coated with cationic polyeletrolyte as an outer layer exhibited much higher nanotoxicity than the ones with an outer layer of anionic polyelectrolyte. TEM images of sectioned microalgae and yeast cells exposed to different polyelectrolyte-coated TiO2NPs confirmed the formation of a significant build-up of nanoparticles on the cell surface for bare and cationic polyelectrolyte-coated TiO2NPs. The effect came from the increased adhesion of cationic nanoparticles to the cell walls. Significantly, coating the TiO2NPs with anionic polyelectrolyte as an outer layer led to a reduced adhesion and much lower nanotoxicity due to electrostatic repulsion with the cell walls.Chapter five describes the development and characterisation of berberine-loaded and chlorhexidine-loaded polyacrylic acid based microgels. The procedure for loading the Carbopol microgels with both berberine and chlorhexidine was developed using a swelling-deswelling cycle dependent on pH. The result of this protocol was a colloidal suspension of collapsed microgel particles loaded with fixed percentage of the antimicrobial agents, berberine and chlorhexidine, respectively. The initial microgel particle concentration, as well as the initial concentrations of berberine and chlorhexidine, were optimized to allow for maximum encapsulation efficiency of the loaded reagent in the microgel while maintaining the colloidal stability of the Carbopol microgel suspension. It was determined that 0.15 wt% berberine and 0.1% chlorhexidine could be successfully incubated with 0.1 wt% Carbopol microgel while the pH was varied from 8 to 5.5 with a measurable increase of the collapsed microgel due to electrostatic conjugation of these cationic antimicrobial agents with the carboxylic groups of the microgel.While for berberine, only 10% encapsulation efficiency was achieved, for chlorhexidine over 90% encapsulation efficiency was obtained without significant impact on the colloidal stability of the microgel. The zeta potential of the loaded microgels remained negative in the range of -35 mV - -40 mV with very moderate increase of the collapsed (and loaded) microgel particle size. The release of berberine and chlorhexidine from these microgel materials was studied and sustained release from the formulations was demonstrated upon dilution over the period of up to 6 hours. The berberine- and chlorhexidine-loaded microgel particles were then further coated with cationic polyelectrolytes, PAH and PDAC. This carried out to increase the adhesion of these antimicrobial particles to the cell membranes. These studies showed a reversal of the zeta-potential of the PDAC coated microgels after their loading with berberine and chlorhexidine, respectively.In chapter six, the antimicrobial activity of both berberine and chlorhexidine loaded Carbopol microgel was studied upon incubation with algae, yeast and E.coli. It was noticed that an increase in the antimicrobial activity of berberine and chlorhexidine Carbopol microgel occurred after 6 hours incubation time for algae and after 24 hours for E.coli while there was no pronounced antimicrobial action for yeast in comparison with the antimicrobial activity of free berberine or chlorhexidine. This was due to the repulsion forces between the anionic microgel and the anionic cell membrane which did not allow the encapsulated berberine or chlorhexidine to be released and diffuse into the cytoplasm causing cell death. In addition to that, the fully anionic charged Carbopol microgel did not allow berberine and chlorhexidine to be released easily at pH 5.5 while the percentage of release increased with pH up to 7.5.The antimicrobial activity of cationic PDAC coated berberine and chlorhexidine loaded-Carbopol microgel was also studied for algae, yeast and E.coli. It was found that cationic PDAC on its own had an acute toxic effect on algae, yeast and E.coli while the toxicity of cationic PDAC reduced upon coating Carbopol microgel with cationic PDAC. Algae and E.coli stayed viable up to 0.0045 wt. % and 0.009 wt. % of PDAC coated Carbopol microgel, respectively. Yeast was resistance to the PDAC coated carbopol for a wide range of concentrations of PDAC coated Carbopol microgel up to 0.018 wt. %. This was due to the different thicknesses of the cell membrane. The Carbopol microgels with encapsulated berberine and chlorhexidine were then coated with cationic PDAC to form PDAC coated particles. The PDAC coated berberine or chlorhexidine loaded carbopol microgel were then incubated with each of algae, yeast, and E.coli. The coating appeared to increase the antimicrobial actions against algae, yeast and E.coli for short incubation times. The increase in the antimicrobial activity was attributed to the electrostatic interaction between the cationic PDAC coated berberine or chlorhexidine loaded carbopol microgel and the anionic cell membrane allowing diffusion of berberine or chlorhexidine easily through cell membrane causing cell death. TEM images showed aggregation of these cationic PDAC coated berberine or chlorhexidine loaded carbopol microgel on the surface of the cell membrane.Chapter seven describes the development of new microfluidics device for cell trapping to achieve a microscreening cell based assay. The idea involved trapping the cells in a micro chamber and then passing over suspensions of the nanomaterials and monitoring the effect. Three design of microfluidic chips were studied with different designs, channel dimensions (depth and width) cell trapping techniques and materials. Initially chemical adhesion was investigated to adhere cells into micro chamber of the microfluidics device using poly-l-lysine bu the cells detached from the surface of microchip because of shear stress forces. Synthesized magnetic yeast cells which were then investigated for trapping other cells into the micro chamber of the chip but the back pressures were too high when liquids were flowed through the system. Magnetic glass beads were then studied to trap the cells, these were synthesized by coating anionic glass beads with cationic and anionic polyelectrolytes such as PAH and PSS, however, they had a low magnetic response towards the magnet. Synthesized magnetic beads were then synthesized using a PDMS based ferrofluid but again a low magnetic response was obtained towards the magnet. Synthesis by flow focusing microfluidics was the tried to generate mono dispersed magnetic beads where SDS with water was the continuous phase and a styrene based ferrofluid was the dispersed phase but the magnetic beads were unstable and they need to be optimized. The continuous phase was then changed to use Hitenol BC20 a polymerisable surfactant, to form hydrophobic magnetic beads and and a mixing serpentine was added to the chip design to give the generated magnetic beads time to be stablilise. Despite these changes the magnetic beads stayed unstable and therefore an emulsification method was used to fabricate poly dispersed magnetic beads which produced 20μm to 50μm magnetic beads. These beads were successfully utilized for trapping cells into the micro chamber of the chip device. A new microfluidic device was designed with suitable channel dimensions which allowed the magnetic beads to move freely inside the micro chamber of the device. These beads were placed into the micro chamber could be controlled using the neodymium magnet easily move the beads

Engineering a Proteoliposome Transporter to Capture Radioactive Cesium from Water

abstract: Radioactive cesium (137Cs), released from nuclear power plants and nuclear accidental releases, is a problem due to difficulties regarding its removal. Efforts have been focused on removing cesium and the remediation of the contaminated environment. Traditional treatment techniques include Prussian blue and nano zero-valent ion (nZVI) and nano-Fe/Cu particles to remove Cs from water; however, they are not efficient at removing Cs when present at low concentrations of about 10 parts-per-billion (ppb), typical of concentrations found in the radioactive contaminated sites. The objective of this study was to develop an innovative and simple method to remove Cs+ present at low concentrations by engineering a proteoliposome transporter composed of an uptake protein reconstituted into a liposome vesicle. To achieve this, the uptake protein, Kup, from E. coli, was isolated through protein extraction and purification procedures. The new and simple extraction methodology developed in this study was highly efficient and resulted in purified Kup at ~1 mg/mL. A new method was also developed to insert purified Kup protein into the bilayers of liposome vesicles. Finally, removal of CsCl (10 and 100 ppb) was demonstrated by spiking the constructed proteoliposome in lab-fortified water, followed by incubation and ultracentrifugation, and measuring Cs+ with inductively coupled plasma mass spectrometry (ICP-MS). The ICP-MS results from testing water contaminated with 100 ppb CsCl, revealed that adding 0.1 – 8 mL of Kup proteoliposome resulted in 0.29 – 12.7% Cs removal. Addition of 0.1 – 2 mL of proteoliposome to water contaminated with 10 ppb CsCl resulted in 0.65 – 3.43% Cs removal. These removal efficiencies were greater than the control, liposome with no protein. A linear relationship was observed between the amount of proteoliposome added to the contaminated water and removal percentage. Consequently, by adding more volumes of proteoliposome, removal can be simply improved. This suggests that with ~ 60-70 mL of proteoliposome, removal of about 90% can be achieved. The novel technique developed herein is a contribution to emerging technologies in the water and wastewater treatment industry.Dissertation/ThesisDoctoral Dissertation Civil, Environmental and Sustainable Engineering 201

Advances in Microfluidics Technology for Diagnostics and Detection

Microfluidics and lab-on-a-chip have, in recent years, come to the forefront in diagnostics and detection. At point-of-care, in the emergency room, and at the hospital bed or GP clinic, lab-on-a-chip offers the potential to rapidly detect time-critical and life-threatening diseases such as sepsis and bacterial meningitis. Furthermore, portable and user-friendly diagnostic platforms can enable disease diagnostics and detection in resource-poor settings where centralised laboratory facilities may not be available. At point-of-use, microfluidics and lab-on-chip can be applied in the field to rapidly identify plant pathogens, thus reducing the need for damaging broad spectrum pesticides while also reducing food losses. Microfluidics can also be applied to the continuous monitoring of water quality and can support policy-makers and protection agencies in protecting the environment. Perhaps most excitingly, microfluidics also offers the potential to enable entirely new diagnostic tests that cannot be implemented using conventional laboratory tools. Examples of microfluidics at the frontier of new medical diagnostic tests include early detection of cancers through circulating tumour cells (CTCs) and highly sensitive genetic tests using droplet-based digital PCR.This Special Issue on “Advances in Microfluidics Technology for Diagnostics and Detection” aims to gather outstanding research and to carry out comprehensive coverage of all aspects related to microfluidics in diagnostics and detection

Event-triggered logical flow control for comprehensive process integration of multi-step assays on centrifugal microfluidic platforms

Content in the UH Research Archive is made available for personal research, educational, and non-commercial purposes only. Unless otherwise stated, all content is protected by copyright, and in the absence of an open license, permissions for further re-use should be sought from the publisher, the author, or other copyright holder.The centrifugal "lab-on-a-disc" concept has proven to have great potential for process integration of bioanalytical assays, in particular where ease-of-use, ruggedness, portability, fast turn-around time and cost efficiency are of paramount importance. Yet, as all liquids residing on the disc are exposed to the same centrifugal field, an inherent challenge of these systems remains the automation of multi-step, multi-liquid sample processing and subsequent detection. In order to orchestrate the underlying bioanalytical protocols, an ample palette of rotationally and externally actuated valving schemes has been developed. While excelling with the level of flow control, externally actuated valves require interaction with peripheral instrumentation, thus compromising the conceptual simplicity of the centrifugal platform. In turn, for rotationally controlled schemes, such as common capillary burst valves, typical manufacturing tolerances tend to limit the number of consecutive laboratory unit operations (LUOs) that can be automated on a single disc. In this paper, a major advancement on recently established dissolvable film (DF) valving is presented; for the very first time, a liquid handling sequence can be controlled in response to completion of preceding liquid transfer event, i.e. completely independent of external stimulus or changes in speed of disc rotation. The basic, event-triggered valve configuration is further adapted to leverage conditional, large-scale process integration. First, we demonstrate a fluidic network on a disc encompassing 10 discrete valving steps including logical relationships such as an AND-conditional as well as serial and parallel flow control. Then we present a disc which is capable of implementing common laboratory unit operations such as metering and selective routing of flows. Finally, as a pilot study, these functions are integrated on a single disc to automate a common, multi-step lab protocol for the extraction of total RNA from mammalian cell homogenate.Peer reviewe

Lab-in-a-Tube: A portable imaging spectrophotometer for cost-effective, high-throughput, and label-free analysis of centrifugation processes

Centrifuges serve as essential instruments in modern experimental sciences, facilitating a wide range of routine sample processing tasks that necessitate material sedimentation. However, the study for real time observation of the dynamical process during centrifugation has remained elusive. In this study, we developed an innovative Lab_in_a_Tube imaging spectrophotometer that incorporates capabilities of real time image analysis and programmable interruption. This portable LIAT device costs less than 30 US dollars. Based on our knowledge, it is the first Wi Fi camera built_in in common lab centrifuges with active closed_loop control. We tested our LIAT imaging spectrophotometer with solute solvent interaction investigation obtained from lab centrifuges with quantitative data plotting in a real time manner. Single re circulating flow was real time observed, forming the ring shaped pattern during centrifugation. To the best of our knowledge, this is the very first observation of similar phenomena. We developed theoretical simulations for the single particle in a rotating reference frame, which correlated well with experimental results. We also demonstrated the first demonstration to visualize the blood sedimentation process in clinical lab centrifuges. This remarkable cost effectiveness opens up exciting opportunities for centrifugation microbiology research and paves the way for the creation of a network of computational imaging spectrometers at an affordable price for large scale and continuous monitoring of centrifugal processes in general.Comment: 21 Pages, 6 Figure

Design and evaluation of a flexible automatic system for 3D cell cultivation

In this work the modification of the Biomek® Cell Workstation was improved. Next to the basically adherent cell cultivation the new integrated devices, ALPs and processes allowed the further cultivation of suspension cells and the manufacturing of 3D cell constructs (alginate beads, spheroid cultures, pellet cultures). The processes were manually and automatically performed to evaluate the automatic translation. The consequently bioscreenings by the High Throughput Screening System enabled the quality control.In dieser Arbeit erfolgt die Modifikation der Biomek® Cell Workstation. Neben der grundlegenden Kultivierung von adhärenten Zellen erlauben neu integrierte Geräte, ALPs und Prozesse die Kultivierung von Suspensionszellen und die Herstellung von 3D-Zellkulturen (Alginatbeads, Sphäroid Kulturen, Pellet Kulturen). Diese Prozesse wurden manuell und automatisiert durchgeführt, um die automatisierte Übersetzung zu untersuchen. Das anschließende Bioscreening wurde mit der Hochdurchsatzuntersuchungsanlage durchgeführt, welche die Qualitätskontrolle ermöglichte