Author Archive

What are the challenges to sample-in-answer-out technologies in clinical settings?

The boom in microfluidic total analysis systems is spurred by the use of microscale fabrication techniques. In 1999, Agilent Technologies introduced a coin-size device called “LabChip” to the market for the analysis of DNA. The timing of the market release of this device was remarkable as it benefited from the hype of the Human Genome Project. The LabChip has drawn considerable attention from both academia and industry. As a result, the shrink of chemistry labs into coin-sized microfluidic devices has evolved more from technological push than from market pull. Technology-driven development pathway brought challenges, especially for the clinical integration of microfluidic devices due to the lack of standards, focus, and communication between academia and clinics.

A recent Lab on a Chip paper from Martyn Boutelle’s Lab addresses this issue. The authors identify the problem from a well-addressed microdialysis perspective. They state that “taking a microfluidic system into clinical environment brings lots of challenges, not least that during setups and developments the very low the flow rates used in combination with microdialysis means that leaks and misdirection of flows are very hard to see.” The authors define the most significant challenge as the development of a device that’s robust enough to be used by and provide enough information to, the clinical team without micromanagement by experts.

The authors attack the problem by creating a sensor-based online system associated with electrochemical measurement, which would be able to analyze the sample in a miniaturized platform continuously. They developed the microfluidic sensors and chip, but they wanted to increase the use of their technology in real-world scenarios by non-experts, so they looked into ways to introduce more precision and control to the platform. The authors combined their technology with LabSmith microfluidic components and constructed breadboard like layouts for typical lab protocols. The authors add that “the main surprise was the ability to bring the rigor of an analytical laboratory into unusual places such as abattoirs, surgical theatres and public transport!”

In this work and the previous work of the authors, the performance of 3-D printed chips was compared to the PDMS ones. The authors found that PDMS material is much more vulnerable than the 3D print outs when frequently handled. The sensor attached to the chip is programmed to calibrate in regular intervals (e.g., every three hours) in single or multiphase flow conditions. Authors describe the advantage of the system that ”remote access to the scripts allows interaction with the system without the requirement of a highly skilled person being right next to it, which in the context of surgical theatres and hospital wards is a distinct advantage. If the codes and scripts are available to less skilled personnel, they are still able to interact and use the system and by making the system more user-friendly a wider audience and more enthusiasm is generated for the product, increasing interest, uptake, and use.”

The authors would like to improve the platform further by making it wearable since it already has grounds for such an operation with wireless sensors. The next thing to be improved in the system is the feed. At this moment, the syringes have to be regularly refilled. This might not be a problem in the laboratory; however, monitoring can last for days in a clinical setting, and periodically refilling the syringes may lead to noise artifacts. Another improvement could be the ease of operation and troubleshooting when, e.g., a tubing becomes blocked in the middle of the measurement when the user is short on space and time.

Lastly, the authors think that this pioneering platform can help shape the future in the market by giving more people access to an area of science that was previously highly skilled, whilst maintaining analytical robustness. This will be the start to break the barrier between academia-made devices and clinical settings.

 

To download the full article for free* click the link below:

Clinical translation of microfluidic sensor devices: focus on calibration and analytical robustness

Sally A. N. Gowers, Michelle L. Rogers, Marsilea A. Booth, Chi L. Leong, Isabelle C. Samper, Tonghathai Phairatana, Sharon L. Jewell, Clemens Pahl, Anthony J. Strong and Martyn G. Boutelle, Lab Chip, 2019, Lab on a Chip Hot Articles

DOI: 10.1039/C9LC00400A

*access is free with an RSC account (free to register)

 

About the Webwriter

Burcu Gumuscu is a researcher in Mesoscale Chemical Systems Group at the University of Twente in the Netherlands. Her research interests include the development of microfluidic devices for quantitative analysis of proteins from single-cells, next-generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

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Through a cheaper ‘book of life’

Mapping the human genome project has been one of the world’s largest scientific collaborations. Completing the full genome sequencing for “the book of life” took more than 10 years with the efforts of 1000’s scientists and a budget of $3 billion. About 20 years after the finalization of this enormous project, it is now possible to complete a full human genome sequencing within 8 days for about $1,000 thanks to more advanced sequencing tools. Further improvements in genome sequencing tools are still warranted today because the genome sequencing field has been embraced by many more applications, including forensics, disease modeling & identification, and personalized medicine (e.g., identifying the genes that cause a medicine to work in one patient but not in another).

Initial sequencing technologies relied on standard DNA electrophoresis techniques such as slab gels and capillaries, allowing for the preparation of only small numbers of samples at a time. The sample preparation limitation was the primary reason for the increased costs and processing duration during the human genome project. Many efforts have been directed towards improving sample preparation techniques in the last decades. As the first step, electrophoresis techniques have been optimized to boost the sample throughput with user-friendly, smaller, and functional platforms. Traditional DNA separation gels, which have been used as the golden standard for many decades, have been replaced by microfabricated post arrays and nanometer-scale deterministic lateral displacement arrays.

A nice example of nanometer-scale deterministic lateral displacement arrays has been demonstrated recently by researchers from IBM T.J. Watson Research Center and Icahn School of Medicine at Mount Sinai in New York, USA. In this work, the researchers fractioned DNA in the range of 100-10.000 base pairs with a size-selective resolution of 200 base pairs. To achieve that, four different microchip configurations were fabricated on silicon wafers, where the array and nanopillar sizes were tuned for each configuration to obtain the optimum separation performance for the selected range of DNA fragments. Each configuration contained several separate arrays to conduct independent runs in a single chip. Instead of applying an electric field, the researchers applied pressure-driven force to separate the fragments. This strategy is particularly useful for separating non-charged species without being affected by buffer conditions (e.g., ionic strength).

The separation mechanism in the nanometer-scale deterministic lateral displacement array is simple: If the size of a DNA fragment is larger than the diameter of the pillars, the fragment is deflected towards the collection wall at a large angle, also called the bump mode. If the size of a DNA fragment is smaller than the diameter of the pillars, the fragment migrates at an angle nominally zero, termed as the zig zag mode. In such a system, diffusion of DNA fragments lead to intermediate migration angles, termed as the partial-bump mode. Different sizes of DNA fragments could be separated in the array since the fragments will follow distinct trajectories thanks to the existence of different modes. Figure 1 summarizes the separation mechanisms and gives an outline for the nanometer-scale deterministic lateral displacement array.

In the nanometer-scale deterministic lateral displacement array, the gap sizes were tuned from microscale to nanoscale only, without application of any other molecules that could change the DNA diffusion behavior, ionic strength (changing the effective gap distances). In such a setting, the researchers identified, for the first time, the flow velocity-dependence of different fragment lengths. Mainly, changing flow velocity caused a transition between bump and zig zag modes for the given size range of different DNA fragments: Slow speeds lead to partial-bump mode, and high speeds lead to the collapse of all DNA fragments to zigzag mode. The nanometer-scale deterministic lateral displacement array could also be used as a purification tool with 75% recovery and 3-fold concentration enhancement of DNA fragments. This tool could be used effectively for preparing next-generation sequencing libraries, on-chip DNA characterization, and circulating DNA characterization applications.

 

Figure 1. The nanometer-scale deterministic lateral displacement array and DNA separation mechanism at different flow velocities.

To download the full article for free* click the link below:

Gel-on-a-chip: continuous, velocity-dependent DNA separation using nanoscale lateral displacement

Benjamin H. Wunsch, Sung-Cheol Kim, Stacey M. Gifford, Yann Astier, Chao Wang, Robert L. Bruce, Jyotica V. Patel, Elizabeth A. Duch, Simon Dawes, Gustavo Stolovitzky and  Joshua T. Smith, Lab Chip, 2018, Lab on a Chip Articles

DOI: 10.1039/c8lc01053f

 

About the Webwriter

Burcu Gumuscu is a researcher in Mesoscale Chemical Systems Group at the University of Twente in the Netherlands. Her research interests include the development of microfluidic devices for quantitative analysis of proteins from single-cells, next-generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

 

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Microfluidics for improving the natural gas extraction process

 

shale rock

Figure 1. Natural gas extraction from shale rock.

Shale is a type of fine-grained rock that contains silt, clay, mineral particles, and pores ranging from meter to nanometer scales. The high organic material content in shale rock is used in natural gas extraction, for which shale reservoirs are mechanically stimulated to create permeability in the pores. A preferred stimulation method is called hydraulic fracturing, where a pressurized fluid fractures shale stone and keeps the fractures open for gas extraction (Figure 1). Natural gas extraction from shale rock is a relatively new process compared to existing energy sources. It has attracted growing interest in America and Asia especially after the 2000s because of being an environmentally friendly alternative to other consumable energy sources. On the other hand, the gas industry currently struggles with optimizing the use of pore space and fractions for efficient extraction of the gas. In a newly opened shale rock reservoir, volatile components vaporize from meter to micrometer-scale pores first, leaving heavier components in hard-to-access nanometer-scale pores. Extraction of the remaining components is necessary for full utilization of the reservoirs but poses a hard-to-solve problem for the industry.

 

 

 

 

In a recent study published in Lab on Chip, David Sinton and co-authors review the current state of the technology and demonstrate a nano-scale physical model of shale with pores. The authors also study the dynamics of gas production in nanopores via imaging the system optically and developing an analytical model for gas vaporization. They first created a microchip model matching shale nanoporous matrix properties (e.g., dominant pore sizes and permeability) (Figure 2). The microchip model contained approximately 5800 pores connected via 23000 throats, where a hydrocarbon mixture was injected. In the model, the number of the small pores (≤10 nm) is designed to be greater than the number of the larger pores (∼100 nm) to store most of the accessible hydrocarbons. This pore size distribution captures the influence of nanoscale throats connecting the larger pores and is relevant to shale production. High, medium, and low superheat was applied to the filled microchip to investigate the spatiotemporal dynamics of vaporization via optical imaging. An analytical model and experimental results showed that phase change (liquid to vapor) in a pore is largely independent of phase change in neighboring pores.

This work supports the hypothesis that the rapid decline in production rates is due to a shift from the large connected features to the nanoporous matrix, as over time the smallest pores become enriched with heavier fractions. The authors reveal that vaporization rate slows down 3000 times thanks to the nanoscale throat bottlenecks at high temperatures, while the rates reduce further with vaporization of light components in large pores at low temperatures. Even the pores with 10 nm and fewer diameters can significantly influence the production from larger pores by severely gating transport. The authors found that this problem can be solved by applying very low pressures, although currently not available in the field, during the later stages of hydraulic fracturing. This finding seems to open a new avenue in the field of shale rock processing for energy.

Figure 2. Close up view of shale rock, the description of how the evaporation works, and the description of the microchip operation.

To download the full article for free* click the link below:

Natural gas vaporization in a nanoscale throat connected model of shale: multi-scale, multicomponent and multi-phase

Arnav Jatukaran, Junjie Zhong, Ali Abedini, Atena Sherbatian, Yinuo Zhao, Zhehui Jin, Farshid Mostowfi and David Sinton

Lab Chip, 2018, Lab on a Chip Articles

DOI: 10.1039/c8lc01053f

*Article free to read until 7th May 2019

About the Webwriter

Burcu Gumuscu is a researcher in Mesoscale Chemical Systems Group at University of Twente in the Netherlands. Her research interests include the development of microfluidic devices for quantitative analysis of proteins from single-cells, next generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

 

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MicroTAS 2018 Highlights

The 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences (aka MicroTAS) was held last year in Kaohsiung, Taiwan. Welcoming more than 1000 participants, MicroTAS 2018 conference brought together several disciplines including microfluidics, microfabrication, nanotechnology, integration, materials& surfaces, analysis & synthesis, and detection technologies for life sciences & chemistry. Besides the exciting scientific program and great presentations, social/networking events (welcome reception, student mixer, women night out, and conference banquet) have made MicroTAS 2018 conference an unforgettable one. In this article, we would like to share some of the conference highlights with our Lab on a Chip blog readers.

 

 

Unraveling endothelial cell phenotypic regulation by spatial hemodynamic flows with microfluidics 

Sarvesh Varma, Guillermo Garcia-Cardena, & Joel Voldman

Did you know that artery bifurcations are prone to atherosclerosis? Blood flow profiles in vessels can help us to gain insights towards atherosclerosis. In this work, the authors fabricated a soft microdevice to study the effects of helical and chaotic flows on endothelial cells located in vein walls. They hypothesized and demonstrated that a helical (uniform) flow profile results in endothelial cells aligning upstream to flow and gain atheroprotective properties, while the chaotic flow results in misalignment of cells that give rise to atherosclerosis.

The figure shows the morphological adaptations of cells in response to distinct spatial flows, scale bars are 0.1 mm.

 

 

glass-like polymer

 

3D printing of microfluidic glass reactors 

Patrick Risch, Frederik Kotz, Dorothea Helmer & Bastian Rapp

Microfluidic devices are mostly made from PDMS, although this material is not always well-suited for thermal, optical, mechanical and chemical changes. In this work, the authors present a new resin formulation to inspire the 3D printing of glass, which is more durable than PDMS. The resin was fabricated using stereolithography printer and this technique is useful for rapid prototyping of microfluidic devices made from glass for optical detection or chemical reaction applications.

A 3D gradient generator is shown in this figure, scale bar is 2 mm.

 

A Tetris-like modular microfluidic platform for mimicking multi-organ interactions 

Louis Ong Jun Ye, Terry Chng, Chong Lor Huai, Seep Li Huan & Toh Yi-Chin

Modularization is undoubtedly on the rise in microfluidics and this work demonstrates an interesting approach. The authors focused on solving ‘limited compatibility with existing devices‘ problem. To achieve that, ring magnets were utilized to connect different parts of PDMS building blocks that were previously fabricated using micro molds. A modular platform assembled using this approach was shown to culture cells as a proof-of-concept study. The platform is expected to allow facile configuration of complex experimental set-ups involving multiple tissues.

The image shows a modular device (left), and its parts (right) connected each other via magnets, scale bars are 1 cm.

 

 

A magneto-switchable superhydrophobic surface for droplet manipulation

Chao Yang & Gang Li

Surface hydrophobicity is an important feature when it comes to bio and chemical applications. In this work, magneto switchable micro-pillars were made from PDMS and carbonyl iron particles. The pillars erect under influence of a magnetic field, resulting in subsequent switching of the wettability and adhesion of the surface between the water-repellent and water-adhesive states. The surface becomes superhydrophobic (water-repellent) when the magnetic field is applied. The authors demonstrated droplet lifting and transportation on a surface using this approach.

The image depicts the effect of an external magnetic field on the stiffness of micro-pillars.

 

About the Web writer

Burcu Gumuscu is a postdoctoral fellow in Herr Lab at UC Berkeley in the United States. Her research interests include the development of microfluidic devices for quantitative analysis of proteins from single-cells, next-generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

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Simple microfluidic cell sorter device to replace manual tissue dissociation protocols

Even a tiny group of cells has the ability to populate a tumor in tissues. Determining cellular diversity and identifying these small cell groups gains importance when it comes to selection of treatment strategies. Tissue samples taken from patients are required to be dissociated into single-cell suspensions, therefore identification can be efficiently done at single-cell level using a powerful suite of technologies including flow cytometry, mass cytometry, and single cell RNA sequencing. However, breaking a tissue down to a single-cell suspension is not an easy task. The old-school way is to cut the tissue sample into small pieces with a blade and mechanically dissociated by vigorous shaking after the application of proteolytic enzymes. Large aggregates are removed by filtering the suspension through a strainer. This technique significantly increases the sample loss, drops the speed of the process and is not ideal for immediate downstream analysis.

In this month’s Lab on a Chip HOT article series, a group of researchers led by Dr Jered Haun at University of California Irvine presented a novel and simple approach that improves the quality of single-cell suspensions obtained from tissue samples using microfluidics. Jeremy Lombardo, a co-author of this article, explains that “the goal of this work was to fully replace manual intensive tissue dissociation protocols by using microfluidic devices.” The developed tool is a microdevice consisting of two nylon membranes, one with 25-50 µm mesh, and the other with 10-15 µm mesh, attached to micron-sized pores and microchannels. The device is made of laser-micromachined hard plastic (PET, aka. polyethylene terephthalate), which enables operation at high flow rates (>10 mL/min) when compared to PDMS (a silicon-based organic material). Also, the chip has multiple layers for connecting nylon mesh membranes at different levels (Figure 1).

cell sorter

Figure 1. Microfluidic cell sorter device for tissue  samples. The sketch shows the inner layers, consisting of two membranes for operating the device in direct or tangential filtration modes. Membrane mesh size can be adjusted to the cell size. Micrographs on the right show lattice network with several pore sizes used in this work. Pore sizes are (top to bottom) 50, 25, 15, 10 µm diameter.

Working principle of the cell sorter device

The inlet of the device connects to a microporous membrane to introduce tissue samples. The Sample passing through the membrane exits through the effluent outlet. It is also possible to direct a portion of the sample along the surface of the membrane that is connected to the cross-flow outlet. The device is either operated in a direct filtration regime to maximize sample recovery and processing speed, or in a tangential filtration regime to sweep larger tissue fragments and cell aggregates away to prevent clogging.

While the researchers initially hypothesized that under pressure-driven flow, cell and tissue aggregates might disaggregate as they pass through the membranes of the device, they were pleasantly surprised by the drastic level of single cell increases seen in the initial testing of these devices, says Jeremy Lombardo and adds “The hardest part in developing and testing this device was to find a combination of membrane pore sizes that could best dissociate cell aggregates and tissue without compromising cell viability. Thorough testing of various pore sizes and combinations were ultimately carried out with both cell line and murine tissue models before we settled on the final 50 and 15 μm pore sizes.”

Advantages, challenges and the future

The authors summarized the advantages of this platform for Lab on a Chip blog readers: “The device is extremely simple to operate as well as inexpensive to fabricate. It can easily be incorporated into many tissue dissociation applications for improved single cell yields as a standalone device but could also be easily integrated with other downstream microfluidic operations (cell sorting, detection etc.).” According to the authors, “in the current format of cell sorter device, cells that are very large in size would likely be difficult to process, as they would likely span multiple pores of the filters and be traditionally filtered away instead of dissociated.” Although seeming like a challenge, this can easily be addressed by adjusting the filter membrane pore sizes to accommodate these larger cell types.

For the future of the device, the authors indicate, “We are also currently working on integrating this device with upstream, larger scale tissue dissociation devices that we have developed previously to create a fully automatable microfluidic tissue dissociation platform.

 

To download the full article for free* click the link below:

Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells
Xiaolong Qiu, Jeremy A. Lombardo, Trisha M. Westerhof, Marissa Pennell, Anita Ng, Hamad Alshetaiwi, Brian M. Luna, Edward L. Nelson, Kai Kessenbrock, Elliot E. Hui, and Jered B. Haun
Lab Chip, 2018, Lab on a Chip Recent Hot Articles
DOI: 10.1039/c8lc00507a

About the Webwriter
Burcu Gumuscu is a postdoctoral fellow in Herr Lab at UC Berkeley in the United States. Her research interests include development of microfluidic devices for quantitative analysis of proteins from single-cells, next generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

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How soil-worms allow for realistic human physiology studies

Mice, fruit fly, zebra fish easily come to mind when thinking of animal models for human physiology studies – but one animal is often forgotten, although it is as functional as the others. Have you guessed already? We are talking about soil-dwelling worms, aka C. elegans! These animals combine the simplicity of single-cell systems with the complexity of animal models, therefore they can provide significant insights into human disorders. Please take a moment to look at our note below summarizing the key features of C. elegans.

Muscular strength is a good example of human physiology studies  and it relies on calcium-initiated muscle contraction, sarcomere composition and organization, and translocation of actin and myosin molecules. Analysis of such parameters can reveal the formation of muscular dystrophy, a muscle degenerative disorder. However, the measurement of these parameters has been a challenge due to the dependence on random animal-behavior that yields irreproducible results. Recently, researchers from Texas Tech University collaborated with Rutgers University and University of Nottingham to study muscular strength in C. elegans. They achieved to obtain results independent of animal behavior and gait in a miniature system consisting of an elastic micropillar forest (Figure 1a and 1b).

The microfluidic system is made of PDMS, and contains bendable micropillars hanging from the microchannel ceiling. The pillars are bent upon the action of the body muscles when C. elegans crawls through the pillar array. Individual pillar bending events can be quantified using a microscope-camera system and image analysis (Figure 1c). The pillar density is designed to create high mechanical resistance to locomotion, therefore maximum exertable force can be measured independent of animal behavior. Here, maximum exertable force corresponds to the peak force exerted by human quadriceps muscle in a standardized knee extension resistance test.

Figure 1. (a) Image of the microfluidic device with the pillar forest and the ports. (b) Schematic demonstration of the C. elegans strength measurement apparatus. Inset shows a scanning electron microscope picture of the pillars. (c) A sketch of interaction with a pillar by the worm body. The pillar is bent due to the action of the body muscles (shown in red and green). Image from Rahman et al.

The authors of this study explain that animals produce strong forces in highly resistive areas and demonstrate different locomotion regimes based on the body size relative to gap between pillars. Besides the body size, body configuration and behavioral characteristics can be the sources to the magnitude of the force exerted on the pillars. Thanks to the probabilistic nature of the parameters sourcing of the force exerted, a reproducible algorithm can be defined for quantifying muscle strength. Using this strategy, researchers showed for the first time that locomotion between microfluidic pillars comprises of three regimes: non-resistive (worm contacts with 1-2 pillars and doesn’t adjust body posture), moderately resistive (worm contacts with >2 pillars and minimally adjust body posture), and highly resistive (worm contacts with multiple pillars and body posture adjustment is disabled). When operated at highly-resistive regime, the microfluidic system suppresses the animal behavior. This system allows for (1) discriminating between the muscle strength or weakness levels of individual worms of different ages, (2) determining body length decrease and muscular contraction levels led by levamisole treatment, (3) comparing the muscular strength in the wild and mutant C. elegans types. According to the researchers, the future studies can help us to obtain deeper understanding in molecular and cellular circuits of neuromuscular function as well as dissection of degenerative processes in disuse, aging, and disease.

To download the full article for free* click the link below:

NemaFlex: a microfluidics-based technology for standardized measurement of muscular strength of C. elegans
Mizanur Rahman, Jennifer E. Hewitt, Frank Van-Bussel, Hunter Edwards, Jerzy Blawzdziewicz, Nathaniel J. Szewczyk, Monica Driscolld, and Siva A. Vanapalli
Lab Chip, 2018, Lab on a Chip Recent Hot Articles
DOI: 10.1039/c8lc00103k

 

About the Webwriter

Burcu Gumuscu is a postdoctoral fellow in Herr Lab at UC Berkeley in the United States. Her research interests include development of microfluidic devices for quantitative analysis of proteins from single-cells, next generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

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What we know about cancer tumors

Cancer tumors are a lot more complex than we think: besides cancer cells, supportive tissue cells, fat, and even immune cells can be found in a tumor. Combined crosstalk in between these cell groups influences the way the tumor develops or responses to drug treatment. On the other hand, the majority of what we know about cancer tumors has been acquired by studying cell ensembles. Recent strides to improve our understanding of cancer revealed that we have long been missing the stochastic interactions and rare events due to ensemble-average measurements. We can unveil how these cell groups work together and how the rare events change the fate of a tumor thanks to single-cell analysis techniques.

Single cells can be identified by extrinsic and intrinsic markers. Extrinsic markers are definitive of genetic and proteomic states of a cell. Flow cytometry and mass spectrometry have been the workhorse of extrinsic marker analysis, where genetic or proteomic materials are often fluorescently labeled for detection. With these techniques, multiplexed analysis of thousands of cells can be employed simultaneously. Intrinsic markers include size, shape, density, optical, mechanical, and electrical properties which do not require labelling. Microfluidic techniques provide with a plethora of different functionalities to sort the cells based on intrinsic markers. Combination of both extrinsic and intrinsic data advances our understanding of how cell heterogeneity is reflected in cell-to-cell variations in tumor development and drug-response. Although many powerful methods are available for determining extrinsic markers, not many techniques can gather information about a panel of different intrinsic markers.

A recent study from Biological Microtechnology and BioMEMS group at MIT represents an important microfluidic approach for the development of multiparameter intrinsic cytometry tool. The approach includes several different microfluidic modules combined with microscope imaging and image processing by machine learning. Separate modules measuring cell size, deformability, and polarization can be combined and organized within the tool (Figure 1). (i) Size module detects the cell size optically in a flow through system. Cell size module is necessary to separate different cell types that can give important cues about disease state. (ii) In deformability module, cells pass through narrow channels, and their transit time defines the deformability. Cell deformability gives cues about cytoskeletal and nuclear changes associated with cancer progression. (iii) In the polarization module, dielectrophoretic force at a fixed frequency is applied on cells driven by opposing hydrodynamic forces. Cells approach coplanar electrodes with different equilibrium positions depending on their polarizability. Cell polarizability allows for distinguishing subtle changes in biological phenotypes. As a proof-of-concept work, drug-induced structural changes in cells were detected for the first time using five different intrinsic markers, including size, deformability, and polarizability at three frequencies. The authors indicate that this powerful tool can further be equipped with visual readout capabilities, such as deterministic lateral displacement array, inertial microfluidics, acoustophoresis, optical techniques.

Figure 1. Multiparameter intrinsic cytometry combines different microfluidic modules on one substrate along with cell tracking to correlate per-cell information across modules for different intrinsic properties including size, polarizability, and deformability.

 

To download the full article for free* click the link below:

Multiparameter cell-tracking intrinsic cytometry for single-cell characterization
Apichitsopa, A. Jaffe, and J. Voldman
Lab Chip, 2018, Lab on a Chip Recent Hot Articles
DOI: 10.1039/C8LC00240A

*Article free to read until 31st August 2018

About the Webwriter

Burcu Gumuscu is a postdoctoral fellow in Herr Lab at UC Berkeley in the United States. Her research interests include development of microfluidic devices for quantitative analysis of proteins from single-cells, next generation sequencing,compartmentalized organ-on-chip studies, and desalination of water on the microscale.

 

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Microparticles: Good things come in small packages

Microparticles were first described in 1967 by Peter Wolf, a physician, as ‘minute particulate materials’ when he investigated the platelet activity in human plasma. They were initially used as drug delivery agents because their size is as small as pollens, which can easily go into the human body. Not long after the great promise of microparticles has been realized, and today we use microparticles in numerous applications including pharmaceuticals, biomedicine, bioengineering, cosmetics, printing, and food science. The widespread use is not a coincidence, they can be synthesized from a multitude of materials, i.e. metal, polymer, gel etc. Especially, polymer microparticles, conferring a great versatility in size, shape, and chemistry, gained more attention in industry. Just like their usage areas, fabrication techniques of microparticles vary a lot. Polymer microparticle production is typically done in two ways, first microfluidics-assisted techniques including droplet-based fabrication, flow-lithography-based fabrication, and microjetting; second other techniques including centrifugation, electrohydrodynamics, and molding. With a focus on microfluidics-assisted techniques, we bring a few remarkable and commercialized studies on high throughput production of spherical-shaped and irregular-shaped microparticles to your attention.

Spherical microparticles

Particle monodispersity has to be compromised for high-througput production when using coaxial microfluidic devices, and both features are highly desired in medical applications and industry. Luckily, as a droplet-based fabrication technique, high-throughput step emulsification of microparticles addresses this fundamental problem. David Weitz’s research group at Harvard University has recently reported a droplet generator microchip with 135 step-emulsifier nozzles that produce monodisperse emulsions of polymers at an exceptional throughput of 10K mL/h (Figure 1a).1 This means, monodisperse microparticle production with this device is thousands times higher than a typical droplet generator microchip with one droplet maker and a throughput of 10 mL/h. The chip is made of PDMS, which is a flexible and inexpensive material. Monodispersity  at high flow rates is maintained using microchannels connected through an array of parallelized nozzles (Figure 1b). Microparticles are formed at the step between each nozzle and the continuous-phase channel. The formation can be explained by the Laplace pressure difference developing between the nozzle and the symmetric polymer bulb, resulting in suction of the dispersed phase into the bulb. The growing polymer bulb increases the pressure gradient and a neck forms between the nozzle and the bulb due to depletion of dispersed phase, resulting in release of a droplet. This geometry can produce spherical-shaped microparticles. The production efficiency scales linearly with droplet diameter (Figure 1c). Weitz demonstrated the production of oil microcapsules in water with the envision of standardizing the process by converting the emulsifier into a pipettor tip. Such a technology can replace the existing pipettor technology tools including multi-well and robots, and this replacement can serve for parallelizing and automation of the encapsulation chemi- and bio-assays. This technology has recently been introduced to the market by a Switzerland-based startup company called Microcaps.

As an alternative concept, in-air microfluidics is based on the idea of producing droplets at higher flow rates without using microfluidic channels. In the research groups of Detlef Lohse and Marcel Karperien at University of Twente, microparticles were generated using two nozzles, and one of the nozzles is mounted on a vibrating piezoelectric element (Figure 1d). The breakup of the liquid jet ejected from the first nozzle leads to formation of monodisperse droplets, which hit onto a continuous liquid jet ejected from the second nozzle. After passing ‘the meeting point’, both liquids react with each other to form physically-encapsulated microparticles. This technique provides with hundreds to thousands times faster microparticle production when compared to coaxial microchip setups. Such constructs can be especially beneficial in tissue engineering, where rapid fabrication of multi-scale materials with multiple cell types is an ongoing challenge. This technology has recently been introduced to the market by a Dutch startup company called IamFluidics.

Figure 1. Up-scaled step-emulsification device producing monodisperse droplets. (a) A schematic of the entire microfluidic chip actively producing oil-in-water droplets. (b) The emulsification process. (c) Maximum production rates per nozzle plotted against drop diameter, scale bars are 400 µm.The image is modified from Stolovicki et al. (see the references below). (d) Chip-based microfluidics comparison with in-air microfluidics.

Irregular-shaped microparticles

Another microfluidics-assisted fabrication concept is stop-flow lithography, introduced by Patrick Doyle’s research group at Massachusetts Institute of Technology.2 In this concept, while two (or more) streams of monomers flow side by side through a microchannel made of PFPE coated PDMS, the streams are exposed to intermittent illumination of ultraviolet light through a photomask, which blocks the light selectively. Due to the chemical reaction initiated by ultraviolet light, the liquid solidifies, and forms an individual microparticle (Figure 2a). Upon polymerization, gel particles do not stick to the PFPE microchannel walls, allowing for the production of free-floating particles by the virtue of oxygen lubrication layers. As the ultraviolet light is projected onto the stream through the photomask, each particle takes on the shape of the mask, making the microparticles customizable (Figure 2b). Microparticles composed of multiple monomers can be fabricated by combining multiple monomer streams. The single-step production is advantageous to reduce the production costs, however the particle shape is limited by the photomask and the microchannel geometry – not allowing for generation of spherical-shaped particles. For a proof-of-concept demonstration, upconverting nanocrystal laden-microparticles were synthesized and emitted homogenous visible spectrum of light. The technique allows for synthesis of striped microparticles without losing their homogeneous emission property. The microparticles were also encoded with multiple dot-patterns (Figure 2c), each specific to a target molecule (such as DNA) reacting with the other ingredients in the particle. Such a reaction leads to the formation of a fluorescent color in the microparticle, so the reaction can be traced by microscopy. This technology has been introduced to the market by Firefly Bioworks (acquired by Abcam in 2015), and Motif Micro (acquired by YPB Systems in 2018) startup companies.

microparticles

Figure 2. Stop Flow Lithography concept. (a) A schematic demonstration the coaxial microchip. (b) Bright-field and fluorescent images show triangle-shaped particles (c) A mask with an array of barcode particle shapes was aligned on three phase laminar flows in the microchip. Bright-field and fluorescent images show the barcoded particles with three distinct compartments with a region coding “2013”. The image is modified from Bong et al. (see the references below).

To download the full articles click the links below:

1Throughput enhancement of parallel step emulsifier devices by shear-free and efficient nozzle clearance
Elad Stolovicki, Roy Ziblat, and David A. Weitz
Lab Chip, 2018.
DOI: 10.1039/C7LC01037K

2Stop flow lithography in perfluoropolyether (PFPE) microfluidic channels
W. Bong, J. Lee, and P. S. Doyle
Lab Chip, 2014.
DOI: 10.1039/C4LC00877D

About the Webwriter

Burcu Gumuscu is a postdoctoral fellow in Herr Lab at UC Berkeley in the United States. Her research interests include development of microfluidic devices for single-cell analysis, next generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

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Why should we use optofluidics for monitoring marine environment?

Phosphorus is found in natural waters and exerts a major influence on the composition and structure of aquatic ecosystems. It is a crucial nutrient for planktons and algae, which feed fish and other marine organisms. However, human activities may result in excess amount of phosphorus, which, in turn, causes harmful algae to bloom in natural waters. The bloom creates a hostile environment to other forms of marine life by consuming the available oxygen in the sea, and producing toxins. Sea organisms such as fish swim away from the blooms, but the ones that cannot swim, such as shellfish, unfortunately die. We do care about this occurrence as it negatively affects natural life and the economy. There is only one way to interpret the effect of continuously changing phosphorus levels on the strength of the biological pump: real-time monitoring of phosphate levels in the marine environment!

Figure 1. The design of the Fabry-Pérot microcavity, consisting of two parallel mirrors (reflectors) fabricated by coating the surface of the optical fibers with a gold layer. The light is reflected by the mirrors multiple times to enhance the signal. Adapted from Zhu et al., 2017.

Conventional vs. optofluidic monitoring instruments

Conventional phosphate monitoring instruments are mostly used for on-site sampling, then the fresh sample is transported to a laboratory for determining the phosphate level. Laboratories complete one round of analysis in 20 min, often using spectrophotometrical measurement tools. Given the conditions, real-time phosphate monitoring easily becomes laborious, time consuming, and costly. To address this challenge, researchers in Chinese Academy of Sciences, Wuhan University, and The First Institute of Oceanography in China collaborated to develop a portable optofluidic phosphate monitoring tool. However, prototyping an optofluidic marine phosphate detection tool is not straightforward because an absorption cell—a component core to the measurement unit—is simply too big to fit in a microchip. Instead of using a bulky absorption cell, researchers considered integrating a Fabry-Pérot cavity in the microsystem. The Fabry-Pérot cavity consists of two parallel optical fibers with a spacing in between. The cross-sectional surface of each optical fiber is coated with a thin layer of gold to create reflector surfaces (Figure 1) in order to enhance the absorption of phosphate. Shortening the spacing between the reflectors decreases the analysis time from minutes to seconds.

 

How it works?

In the microchip, filtered water sample and a chromogenic reagent are injected into a curved microchannel. After the chromogenic reaction, the water-soluble components are transported into the optical section (Figure 2). The probe light is sent into the Fabry-Pérot cavity via one of the fibers, bounces between the reflectors multiple times to increase the optical feedback and then analyzed by the detector. The obtained absorbance value, therefore, increases linearly with increasing phosphate concentration. In this microsystem, phosphate detection range is 0.1-100 µmol per liter (400 times greater than the range of a conventional instrument) and detection time is 4 seconds (300 times shorter than detection time of a conventional instrument). The authors of the paper think that this technology can be applied to detect other nutrient levels as well as pH changes in marine environment.

 

optofluidic phosphate monitoring

Figure 2. A schematic of the optofluidic microchip consisting of two parts: the microfluidics circuit forming the microreactor in the microchannel, and the optical part to provide optical feedback for enhanced absorption analysis. Adapted from Zhu et al., 2017.

To download the full article for free* click the link below:

 

Optofluidic marine phosphate detection with enhanced absorption using a Fabry–Pérot resonator

M. Zhu, Y. Shi, X. Q. Zhu, Y. Yang, F. H. Jiang, C. J. Sun, W. H. Zhaoc, and X. T. Hanc

Lab Chip, 2017, Lab on a Chip Recent Hot Articles

DOI: 10.1039/C7LC01016H

 

About the Webwriter

Burcu Gumuscu is a postdoctoral fellow in Herr Lab at UC Berkeley in the United States. Her research interests include development of microfluidic devices for quantitative analysis of proteins from single-cells, next generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

*until 16th February 2018

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Spotting cancer: one in a billion cells

The human body consists of tens of trillions cells, all of which theoretically should have the same genome. Depending on genetic and environmental factors, some of these cells experience point mutations. Although most of those mutations are cleaned up by DNA repair enzymes, about 0.01% of them stay. A low percentage of the persistent mutations turn out to be ‘cancer’ while others stay recessive. Several genes, including the ones responsible for cell growth cycle, cause the persistent mutations. Uncontrolled growth of cells leads to formation of tumor, which is now prone to experience more mutations due to continuous proliferation. These mutations create heterogeneity among the cell population of a tumor, and eventually some cells leave their original tumor and start a new one in another organ of the body. When a cell leaves its parent tumor, it starts circulating in blood vessels before settling in. Those cells are called circulating tumor cells (CTCs), and around 1-10 CTC can be found in 1 mL of blood (which contains about 1 billion red blood cells, and 1 million white blood cells). Capturing ultra-rare CTCs has enormous implications in early cancer diagnosis. Often times, analysis of at least 10 mL of blood is necessary to capture sufficient CTCs to confirm their presence. The available technology can achieve CTC capturing in about 10 hours leading to the loss of target cells and decay of detection biomarkers.

Microfluidic devices are well known for precise sorting of microscale materials. Parallelizing microscale sorting in microfluidic devices enables high-throughput sample processing. In the light of this principle, David Issadore, Jina Ko, and their fellow researchers at University of Pennsylvania coined CaTCh FISH, a circulating tumor cell fluorescence in situ hybridization platform for rapid detection of CTCs. David Issadore kindly accepted to talk about this exciting lab-on-a-chip device. According to David, “the main strength of the CaTCh FISH is that it preserves the sensitivity and specificity of microfluidic cell sorting and RNA FISH, but through clever engineering allows these normally very slow laboratory-based operations to be performed rapidly and automatically on a chip.”

RNA FISH, high-throughput cell handling

Figure 1. Overview of the CaTCh FISH platform. Whole blood sample is processed in TEMPO step, where magnetic nano particle based cell separation is followed by single-cell RNA analysis (modified from Ko et al., 2017).

Processing a whole blood sample using CaTCh FISH involves three steps (Figure 1). First, white blood cells are labelled with magnetic nanoparticles. Second, whole blood is passed through a magnetic micropore filter to selectively trap magnetically labelled cells. “Our magnetic micropore device rapidly and precisely removes all of the cells that we know are not CTCs”, says David. The operating principle of magnetic micropore filter is based on strong and highly localized microscale field gradients formed at the edge of micropores to enable application of high flow rates. Third, single cell RNA analysis is performed on isolated cells using rapid in-situ hybridization strategy so that CTCs can be identified within the isolated cell population. In this way, targeted CTCs can be isolated from the rest of the cell population regardless of their physical and molecular properties. Analysis of a 10 mL blood sample takes less than an hour.

As a key novelty, the researchers maintained high-throughput processing and high sensitivity at the same time by integrating the FISH technique (hybridization of 20-50 fluorescently-labelled oligonucleotide probes to the target RNA, and subsequent fluorescence-signal based detection to enhance signal-to-noise ratio) in a microfluidic chip. CaTCh FISH has also been tested in patients with pancreatic cancer and detected CTCs in the real patient samples.

“The CaTCH FISH technology can be easily modified to measure other rare cells, for the diagnosis of other cancers or for stem cell research for example, by modifying the RNA FISH probes”, says David. He considers converting the platform to a high-precision hospital-based diagnostic tool and he collaborates with a company in the Bay area for this process. The CaTCh FISH device is poised to have a big impact on the way cancer is diagnosed.

To download the full article for free* click the link below:

A magnetic micropore chip for rapid (<1 hour) unbiased circulating tumor cell isolation and in situ RNA analysis

Jina Ko, Neha Bhagwat, Stephanie S. Yee, Taylor Black, Colleen Redlinger, Janae Romeo, Mark O’Hara, Arjun Raj, Erica L. Carpenter, Ben Z. Stanger and David Issadore

Lab Chip, 2017, Paper

DOI: 10.1039/C7LC00703E

 

About the Webwriter

Burcu Gumuscu is a postdoctoral fellow in Herr Lab at UC Berkeley in the United States. Her research interests include development of microfluidic devices for quantitative analysis of proteins from single-cells, next generation sequencing, compartmentalized organ-on-chip studies, and desalination of water on the microscale.

*until 5th January 2018

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