Archive for the ‘Hot Articles’ Category

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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Screening lipid libraries with microfluidics

The plasma membrane is a key component of living organisms and is essential to all life. It separates the inside of the cell from the outside, compartmentalizes reactions, and selectively allows transport across it. While a lot of research has gone into how different proteins and surface molecules control the functions of the plasma membrane, less is known about how lipid composition gives rise to specific properties. For instance, transmembrane proteins, which are also a large class of drug targets, may have different requirements for the lipid environment or may have their function modified depending on local lipid composition. Recently, researchers from the David Weitz Lab at Harvard, have developed a microfluidic chip which they used to screen the largest lipid library to date, in order to identify which lipid compositions have specificity for certain protein transmembrane domains. This allows researchers to investigate the effect of local lipid concentration on transmembrane proteins.

The plasma membrane is often described as a ‘simple barrier’. But if that’s the case, then “why does nature go through the trouble of making so many different types of lipids?” explained Roy Ziblat, the lead author on the paper. Ziblat believes that the lipid membrane role is far bigger than a mere barrier and it serves as a substrate for accelerating bio-reactions. The role of the lipids composing the membrane is to control which biomolecules participate in these reactions, by their selectivity to membrane proteins. Having limited success with existing techniques, Ziblat turned to microfluidics to try to answer this question.

The microfluidic chip comprises an array of 108 wells in PDMS where lipid films can be deposited and dried before sealing the chip with another layer of PDMS. Liposomes are generated within the wells by swelling in aqueous buffer. These liposomes are then tested to see whether or not transmembrane domain peptides will insert into them. However, because the transmembrane domain peptides are insoluble, they can’t simply be added into the chip. To get around this, Ziblat et al. turned to cell-free protein synthesis. By loading the chip with DNA for the transmembrane domain peptide and PURExpress (a commercial cocktail of ribosomes, enzymes, and nucleotides for transcription and translation), the peptides can be synthesized in close proximity to the liposomes, thus minimizing precipitation and increasing the chance of insertion. The paper by Ziblat et al., which was featured on the cover of the 7th December issue of Lab on a Chip, also includes a helpful video description of these methods. Ziblat said he first made the video to better communicate his methods with his supervisor and colleagues, but it really helps the reader understand a very technical methodology.

Going forward, Ziblat hopes to use the device to study other membrane interactions, such as virus-cell binding. There’s also hope that this new device and method can be used to identify what the authors call “druggable lipids”—peptides that interact with specific lipids and thus better direct drugs toward specific cells or even organelles.


To download the full article, click the link below:

Determining the lipid specificity of insoluble protein transmembrane domains

R. Ziblat, J. C. Weaver, L. R. Arriaga, S. Chong and D. A. Weitz

Lab Chip, 2018, 18, 3561

DOI: 10.1039/c8lc00311d


About the webwriter

Darius Rackus (right) is a postdoctoral researcher in the Dittrich Bionalytics Group at ETH Zürich. His research interests are in developing integrated microfluidic tools for healthcare and bioanalysis

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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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Emerging Investigator Series – Kyle Bishop

Kyle Bishop

 

Introducing Kyle Bishop: Lab on a Chip‘s latest Emerging Investigator

Kyle Bishop received his PhD in Chemical Engineering from Northwestern University under the guidance of Bartosz Grzybowski for work on nanoscale forces in self-assembly. Following his PhD, Dr. Bishop was a post-doctoral fellow with George Whitesides at Harvard University, where he developed new strategies for manipulating flames with electric fields. He started his independent career at Penn State University in the Department of Chemical Engineering. In 2016, Dr. Bishop moved to Columbia University, where he is currently an Associate Professor of Chemical Engineering. Dr. Bishop has been recognized by the 3M Non-tenured Faculty award and the NSF CAREER award. His research seeks to discover, understand, and apply new strategies for organizing and directing colloidal matter through self-assembly and self-organization far-from-equilibrium.

 

 

Read Dr Bishop’s article entitled ‘Measurement and mitigation of free convection in microfluidic gradient generators’ and find out more about him in the interview below:

Your recent Emerging Investigator Series paper focuses on the measurement and mitigation of free convection in microfluidic gradient generators. How has your research evolved from your first article to this most recent article?

Our first article in Lab on a Chip focused on harnessing electric potential gradients to power transport and separations within microfluidic systems. Here, we examine how chemical gradients can drive fluid flows as well as motions of colloidal particles, lipid vesicles, and living cells. These topics are linked by our continued interest in harnessing and directing thermodynamic gradients to perform dynamic functions at small scales.

What aspect of your work are you most excited about at the moment?

Currently, we are excited by our pursuit of colloidal “robots” that organise spontaneously in space and time to perform useful functions, which can be rationally encoded within active soft matter.

In your opinion, what is the future of microfluidic gradient generators? Any new applications you foresee for them?

Our interest in microfluidic gradient generators grew from a desire to quantify the motions of lipid vesicles in osmotic gradients (so-called osmophoresis).  These measurements were plagued by undesired gradient-driven flows.  We thought that our efforts to understand and mitigate these flows would be useful to others studying gradient driven motions (e.g., chemotaxis of living cells).

What do you find most challenging about your research?

Staying focused. The world is filled with many micro-mysteries that may pique your curiosity, but time is limited. Picking problems and following through on their solution is an ever-present challenge.

In which upcoming conferences or events may our readers meet you?

Our group regularly attends the AIChE Annual Meeting and the ACS Colloid and Surface Science Symposium.

How do you spend your spare time?

Exploring New York City with my family and thinking about science.

Which profession would you choose if you were not a scientist?

What a horrible thought…perhaps a lawyer as I value evidence-based reasoning and the rule of law (physical or otherwise).

Can you share one piece of career-related advice or wisdom with other early career scientists?

Think big and collaborate often.

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Quantitative and multiplex microRNA assays from unprocessed cells in isolated nanoliter well arrays

MIT researchers develop a simple new chip that simplifies sample preparation and reduces sample volume for micro-RNA analysis

In recent years, microRNAs (miRNAs) have emerged as potential biomarkers for a range of diseases. These short (~22 nucleotide) sequences of non-coding RNA function by silencing messenger RNA and thus, provide a form of post-transcriptional regulation in the cell. Consequently, their regulation can have a significant impact on cell function. While they are implicated in many diseases, their role in cancer is of particular interest. It is known that multiple miRNAs are dysregulated in tumours compared to healthy tissues and they can be used as biomarkers in cancer diagnosis. Testing for multiple miRNAs provides a better diagnosis, so it is important to be able to test them as a panel. There are many techniques available to test for miRNAs such as gene chips, quantitative RT-PCR, and RNA-seq, but these technologies all have their own shortcomings. What is common to all them is the need for sample preparation, and mainly RNA extraction from the tissue or cells to be tested. In their recent HOT article, Tentori and researchers from the Doyle Group at MIT describe a chip that could overcome this problem and perform multiplexed testing of miRNA all within a minimum volume of just a few nanoliters.

The new chip comprises two glass slides which can be sandwiched together. The bottom slide has an array of 300 µm diameter wells which contain the miRNA sensors and serve to isolate samples into a small reaction volume. The top slide is then used to deliver lysis reagents to the sample. The authors tried a variety of designs for the top plate, but ultimately settled on an array of 30 µm diameter wells. This has two advantages; 1) Reagents are precisely metered and 2) the top and bottom plates can be sandwiched robustly without any need for precision handling or alignment. This last point is really important to Augusto Tentori, a postdoc in the Doyle Group and the lead author of the paper. Tentori wants “to make devices that are simple and robust, so translation is easier.” The miRNA sensors are polyethylene glycol diacrylate (PEGDA) hydrogels that contain complementary DNA probes.  The posts are photopolymerized in the wells and various sizes, shapes, and patterns of posts can be made. Further, a single well can contain a variety of posts, each functionalized with a different DNA probe targeting a different miRNA. In this way, multiplexed assays can be performed with spatial separation. The chip format of the assay is more sensitive than previous formats and can detect miRNA from less than 20 cells.

Tentori is really excited about the prospects of this new chip. His co-authors include pathologists who have been guiding the project to make sure it is clinically useful, and he really wants to see this technology get into the hands of pathologists and diagnostic technicians.

Read the full article by Tentori et al. here “Quantitative and multiplex microRNA assays from unprocessed cells in isolated nanoliter well arrays” that features in Lab on a Chip’HOT article collection

About the webwriter

Darius Rackus (right) is a postdoctoral researcher in the Dittrich Bionalytics Group at ETH Zürich. His research interests are in developing integrated microfluidic tools for healthcare and bioanalysis

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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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Roll-to-roll PDMS-chips for the masses in molecular diagnostics

PDMS microfluidic devices for molecular diagnostics are now produced at scale using roll-to-roll manufacturing

If there is one material that has enabled microfluidic research in academia, poly(dimethylsiloxane) (PDMS) is surely it. PDMS is cheap and easy to prototype with, and its elastomeric properties have led to complicated structures (e.g. valving) in microfluidic channels. Although it is great for rapid prototyping, there is often a disconnect between the prototype and high throughput manufacturing due to a lack of scalable production methods. Researchers at VTT-Technical Research Centre of Finland and the University of California Berkeley have recently reported a roll-to-roll method for fabricating PDMS microfluidic chips.

In roll-to-roll (R2R) processing—common to the paper industry—long sheets of materials are continuously processed, feeding through rollers and modules with different functionalities. To form R2R microfluidic devices, PDMS was applied to an aluminized paper substrate and then embossed by a heated nickel imprinting cylinder which also cured the PDMS. The devices had good reproducibility and channel depths around 100 µm were achieved. Replication from the nickel master was automated and performed at high throughput of 1.5 m/min. Olli-Heikki Huttunen, one of the authors on the paper, said that “although the process required a lot of fine tuning, it was surprisingly simple.” Like other high-throughput manufacturing techniques (e.g. injection moulding), the nickel tool is quite expensive, but these costs can be overcome by the volume of production.

As a proof-of-principle application, the authors demonstrated nucleic acid detection by loop-mediated isothermal amplification (LAMP). Reagents were spotted and dried in the microchannels using a roll-to-roll compatible dispensing machine, and PDMS lids with vias for fluidic and vacuum connections were formed by a roll-to-roll process (though vias were manually punched) and then bonded manually. Huttunen said that the next steps are to figure out how to manufacture the entire device roll-to-roll, but that it should not be too challenging.

Using aluminized paper as the base substrate for the devices offered a couple advantages. One is that the aluminium dramatically reduced the paper’s autofluorescence. Another advantage was the aluminum reflected back both excitation and emission light, resulting in stronger signals. Results from the test could be read within 20 minutes, suggesting that these devices would be useful for low-cost point-of-care testing.

The challenge for the future, says corresponding author Luke Lee, will be “to learn what the new rules of thinking and design are for roll-to-roll microfluidics in order to solve the problem of mass production in integrated molecular diagnostics for all.” This is an exciting new prospect for both PDMS and the microfluidics community.

To read the full paper for free*, click the link below:

PDMS microfluidic devices for molecular diagnostics are now produced at scale using roll-to-roll manufacturing

*article free to read from 06/06/2018 – 06/07/2018

About the Webwriters

Darius Rackus (Right) is a postdoctoral researcher at the University of Toronto working in the Wheeler Lab. His research interests are in combining sensors with digital microfluidics for healthcare applications.

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