A really low-cost field tool to test water contaminants and blood samples

The simple DIY pump made from a balloon and stockings.

A simple pressure pump, made from balloons and nylon stockings, means more people in more places will be able to test water contaminants and blood samples.

The ingenious device unveiled in the prestigious Lab on a Chip journal cost just $2 to make, yet works almost as well as its expensive and cumbersome lab counterparts.

Pumps are used to make biological samples flow through microfluidic devices while their contents are identified beneath a microscope.

This DIY pump came from a collaboration between researchers at RMIT University and the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, who demonstrated its viability in tests to detect aquatic parasites and cancer cells and to study vascular diseases.

Inspired by football

Study lead author and RMIT engineer, Dr Peter Thurgood,said the team took inspiration for the simple invention from footballs, which hold large pressures when reinforced.

“We started with basic latex balloons, then realised that regular stockings made from nylon and elastane could be a perfect match to reinforce them, allowing them to hold significantly higher pressure and function as pumps,” Thurgood said.

“By simply wrapping three layers of stockings around the latex balloon we were able to increase its internal pressure by a factor of 10 – enough to run many water or blood analyses that would usually require large, expensive pumps.”

Experiments showed the reinforced balloon pump could be used to operate microfluidic devices for several hours without a significant pressure loss.

The pump also fits easily within an incubator and can be left overnight.

A low-cost field tool where it’s needed most 

Study co-author and parasitologist at the Walter and Eliza Hall Institute,Associate Professor Aaron Jex, is a leading researcher in global water quality and public health interventions.

He said this simple innovation opened exciting opportunities in field water testing and the ability to test and diagnose patients for infectious pathogens and aquatic micro-organisms at the point-of-care.

“Parasitic micro-organisms have a major impact in impoverished communities in tropical and subtropical regions globally, but also in developed countries including Australia,” Jex said.

“In order to address this there is an urgent need for field-based, low-cost diagnostic tools that work in challenging, sometimes remote and often complex environments very different from a pristine laboratory.”

“As simple as it may look, this device suits those needs really well and could have a big impact.”

Co-author and RMIT biologist, Dr Sara Baratchi, said it also had promising applications for early diagnosis of diseases at home or in the doctor’s surgery.

The balloon pump was tested as a point-of-care diagnostic device for detection of very low concentrations of target cancer cells in liquid samples, and found to work.

“The hydrodynamic force of liquid produced by the reinforced balloon was enough to isolate cells for study, which was really amazing for a $2 pump!”

Baratchi is now working on applying the simplified pump technology to develop organ-on-chip systems that mimic the flow conditions in dysfunctional vessels, to better understand diseases like atherosclerosis that lead to heart attack and stroke.

An opportunity for outreach

RMIT engineer and project leader, Dr Khashayar Khoshmanesh,is a leading researcher in the field of microfluidic based lab-on-a-chip technologies.

He said while microfluidics had made significant progress over the past decade, their widespread application had been limited by the cost and bulk of pumps required to operate them.

“Simplicity is at the heart of our entire research program. By redesigning sophisticated microfluidic devices into simplified ones, we can maximise their outreach and applications for use in teaching or research in the field, not just in sophisticated labs,” he said.

“We envisage these types of pumps also being suitable for student classwork experiments to support capability development in this important area of research from an earlier stage.”

 

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The ability to diagnose sepsis in minutes

An MIT-invented microfluidics device could help doctors diagnose sepsis, a leading cause of death in U.S. hospitals, by automatically detecting elevated levels of a sepsis biomarker in about 25 minutes, using less than a finger prick of blood. Image: Felice Frankel

When time matters in hospitals, automated system can detect an early biomarker for the potentially life-threatening condition.

A novel sensor designed by MIT researchers could dramatically accelerate the process of diagnosing sepsis, a leading cause of death in U.S. hospitals that kills nearly 250,000 patients annually.

Sepsis occurs when the body’s immune response to infection triggers an inflammation chain reaction throughout the body, causing high heart rate, high fever, shortness of breath, and other issues. If left unchecked, it can lead to septic shock, where blood pressure falls and organs shut down. To diagnose sepsis, doctors traditionally rely on various diagnostic tools, including vital signs, blood tests, and other imaging and lab tests.

In recent years, researchers have found protein biomarkers in the blood that are early indicators of sepsis. One promising candidate is interleukin-6 (IL-6), a protein produced in response to inflammation. In sepsis patients, IL-6 levels can rise hours before other symptoms begin to show. But even at these elevated levels, the concentration of this protein in the blood is too low overall for traditional assay devices to detect it quickly.

In a paper being presented this week at the Engineering in Medicine and Biology Conference, MIT researchers describe a microfluidics-based system that automatically detects clinically significant levels of IL-6 for sepsis diagnosis in about 25 minutes, using less than a finger prick of blood.

In one microfluidic channel, microbeads laced with antibodies mix with a blood sample to capture the IL-6 biomarker. In another channel, only beads containing the biomarker attach to an electrode. Running voltage through the electrode produces an electrical signal for each biomarker-laced bead, which is then converted into the biomarker concentration level.

“For an acute disease, such as sepsis, which progresses very rapidly and can be life-threatening, it’s helpful to have a system that rapidly measures these nonabundant biomarkers,” says first author Dan Wu, a PhD student in the Department of Mechanical Engineering. “You can also frequently monitor the disease as it progresses.”

Joining Wu on the paper is Joel Voldman, a professor and associate head of the Department of Electrical Engineering and Computer Science, co-director of the Medical Electronic Device Realization Center, and a principal investigator in the Research Laboratory of Electronics and the Microsystems Technology Laboratories.

Integrated, automated design

Traditional assays that detect protein biomarkers are bulky, expensive machines relegated to labs that require about a milliliter of blood and produce results in hours. In recent years, portable “point-of-care” systems have been developed that use microliters of blood to get similar results in about 30 minutes.

But point-of-care systems can be very expensive since most use pricey optical components to detect the biomarkers. They also capture only a small number of proteins, many of which are among the more abundant ones in blood. Any efforts to decrease the price, shrink down components, or increase protein ranges negatively impacts their sensitivity.

In their work, the researchers wanted to shrink components of the magnetic-bead-based assay, which is often used in labs, onto an automated microfluidics device that’s roughly several square centimeters. That required manipulating beads in micron-sized channels and fabricating a device in the Microsystems Technology Laboratory that automated the movement of fluids.

The beads are coated with an antibody that attracts IL-6, as well as a catalyzing enzyme called horseradish peroxidase. The beads and blood sample are injected into the device, entering into an “analyte-capture zone,” which is basically a loop. Along the loop is a peristaltic pump — commonly used for controlling liquids — with valves automatically controlled by an external circuit. Opening and closing the valves in specific sequences circulates the blood and beads to mix together. After about 10 minutes, the IL-6 proteins have bound to the antibodies on the beads.

Automatically reconfiguring the valves at that time forces the mixture into a smaller loop, called the “detection zone,” where they stay trapped. A tiny magnet collects the beads for a brief wash before releasing them around the loop. After about 10 minutes, many beads have stuck on an electrode coated with a separate antibody that attracts IL-6. At that time, a solution flows into the loop and washes the untethered beads, while the ones with IL-6 protein remain on the electrode.

The solution carries a specific molecule that reacts to the horseradish enzyme to create a compound that responds to electricity. When a voltage is applied to the solution, each remaining bead creates a small current. A common chemistry technique called “amperometry” converts that current into a readable signal. The device counts the signals and calculates the concentration of IL-6.

“On their end, doctors just load in a blood sample using a pipette. Then, they press a button and 25 minutes later they know the IL-6 concentration,” Wu says.

The device uses about 5 microliters of blood, which is about a quarter the volume of blood drawn from a finger prick and a fraction of the 100 microliters required to detect protein biomarkers in lab-based assays. The device captures IL-6 concentrations as low as 16 picograms per milliliter, which is below the concentrations that signal sepsis, meaning the device is sensitive enough to provide clinically relevant detection.

A general platform

The current design has eight separate microfluidics channels to measure as many different biomarkers or blood samples in parallel. Different antibodies and enzymes can be used in separate channels to detect different biomarkers, or different antibodies can be used in the same channel to detect several biomarkers simultaneously.

Next, the researchers plan to create a panel of important sepsis biomarkers for the device to capture, including interleukin-6, interleukin-8, C-reactive protein, and procalcitonin. But there’s really no limit to how many different biomarkers the device can measure, for any disease, Wu says. Notably, more than 200 protein biomarkers for various diseases and conditions have been approved by the U.S. Food and Drug Administration.

“This is a very general platform,” Wu says. “If you want to increase the device’s physical footprint, you can scale up and design more channels to detect as many biomarkers as you want.”

Learn more: Microfluidics device helps diagnose sepsis in minutes

 

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A microfluidic organ chip model of the human blood-brain barrier for development of brain-targeting therapeutics

This illustration shows how In the Blood-Brain-Barrier (BBB), thin endothelial capillaries (red) are wrapped by supporting pericytes (green) and astrocytes (yellow), enabling them to generate a tight barrier with highly selective transport functions for molecules entering the brain fluid from the blood stream. Credit: Wyss Institute at Harvard University

Microfluidic Organ Chip model of human blood-brain barrier that recapitulates in vivo barrier functions offers new preclinical tool for development of brain-targeting therapeutics

Like airport security barriers that either clear authorized or block unauthorized travelers and their luggage from accessing central operation areas, the blood-brain barrier (BBB) tightly controls the transport of essential nutrients and energy metabolites into the brain and staves off unwanted substances circulating in the blood stream. Importantly, its highly organized structure of thin blood vessels and supporting cells is also the major obstacle preventing life-saving drugs from reaching the brain in order to effectively treat cancer, neurodegeneration, and other diseases of the central nervous system. In a number of brain diseases, the BBB can also locally break down, causing neurotoxic substances, blood cells, and pathogens to leak into the brain and wreak irreparable havoc.

To study the BBB and drug transport across it, researchers have mostly relied on animal models such as mice. However, the precise make-up and transport functions of BBBs in those models can significantly differ from those in human patients, which makes them unreliable for the prediction of drug delivery and therapeutic efficacies. Also, in vitro models attempting to recreate the human BBB using primary brain tissue-derived cells thus far have not been able to mimic the BBB’s physical barrier, transport functions, and drug and antibody shuttling activities closely enough to be useful as therapeutic development tools.

Now, a team led by Donald Ingber, M.D.,Ph.D. at Harvard’s Wyss Institute for Biologically Inspired Engineering has overcome these limitations by leveraging its microfluidic Organs-on-Chips (Organ Chips) technology in combination with a developmentally-inspired hypoxia-mimicking approach to differentiate human pluripotent stem (iPS) cells into brain microvascular endothelial cells (BMVECs). The resulting “hypoxia-enhanced BBB Chip” recapitulates cellular organization, tight barrier functions, and transport abilities of the human BBB; and it allows the transport of drugs and therapeutic antibodies in a way that more closely mimics transport across the BBB in vivo than existing in vitro systems. Their study is reported in Nature Communications.

“Our approach to modeling drug and antibody shuttling across the human BBB in vitro with such high and unprecedented fidelity presents a significant advance over existing capabilities in this enormously challenging research area,” said Ingber, who is the Wyss Institute’s Founding Director. “It addresses a critical need in drug development programs throughout the pharma and biotech world that we now aim to help overcome with a dedicated ‘Brain Targeting Program’ at the Wyss Institute using our unique talent and resources.” Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).

Our approach to modeling drug and antibody shuttling across the human BBB in vitro with such high and unprecedented fidelity presents a significant advance over existing capabilities in this enormously challenging research area

DON INGBER

The BBB consists of thin capillary blood vessels formed by BMVECs, multifunctional cells known as pericytes that wrap themselves around the outside of the vessels, and star-shaped astrocytes, which are non-neuronal brain cells that also contact blood vessels with foot-like processes. In the presence of pericytes and astrocytes, endothelial cells can generate the tightly sealed vessel wall barrier typical of the human BBB.

Ingber’s team first differentiated human iPS cells into brain endothelial cells in the culture dish using a method that had been previously developed by co-author Eric Shusta, Ph.D., Professor of Chemical and Biological Engineering at University of Wisconsin-Madison, but with the added power of bioinspiration. “Because in the embryo the BBB forms under low-oxygen conditions (hypoxia), we differentiated iPS cells for an extended time in an atmosphere with only 5% instead of the normal 20% oxygen concentration,” said co-first author Tae-Eun Park, Ph.D. “As a result, the iPS cells initiated a developmental program very similar to that in the embryo, producing BMVECs that exhibited higher functionality than BMVECs generated in normal oxygen conditions.” Park is a former Postdoctoral Fellow on Ingber’s team who is now an Assistant Professor at Ulsan National Institute of Science and Technology in the Republic of Korea.

Building on a previous human BBB model, the researchers next transferred the hypoxia-induced human BMVECs into one of two parallel channels of a microfluidic Organ Chip device that are divided by a porous membrane and continuously perfused with medium. The other channel was populated with a mixture of primary human brain pericytes and astrocytes. Following an additional day of hypoxia treatment, the human BBB Chip could be stably maintained for at least 14 days at normal oxygen concentrations, which is far longer than past in vitro human BBB models attempted in the past.

Under the shear stress of the fluids perfusing the BBB Chip, the BMVECs go on to form a blood vessel, and develop a dense interface with pericytes aligning with them on the other side of the porous membrane, as well as with astrocytes extending processes towards them through small openings in the membrane. “The distinct morphology of the engineered BBB is paralleled by the formation of a tighter barrier containing elevated numbers of selective transport and drug shuttle systems compared to control BBBs that we generated without hypoxia or fluid shear stress, or with endothelium derived from adult brain instead of iPS cells,” said Nur Mustafaoglu, Ph.D., a co-first author on the study and Postdoctoral Fellow working on Ingber’s team. “Moreover, we could emulate effects of treatment strategies in patients in the clinic. For example, we reversibly opened the BBB for a short time by increasing the concentration of a mannitol solute [osmolarity] to allow the passage of large drugs like the anti-cancer antibody Cetuximab.”

To provide additional proof that the hypoxia-enhanced human BBB Chip can be utilized as an effective tool for studying drug delivery to the brain, the team investigated a series of transport mechanisms that either prevent drugs from reaching their targets in the brain by pumping them back into the blood stream (efflux), or that, in contrast, allow the selective transport of nutrients and drugs across the BBB (transcytosis).

“When we specifically blocked the function of P-gp, a key endothelial efflux pump, we could substantially increase the transport of the anti-cancer drug doxorubicin from the vascular channel to the brain channel, very similarly to what has been observed in human patients,” said Park. “Thus, our in vitrosystem could be used to identify new approaches to reduce efflux and thus facilitate drug transport into the brain in the future.”

In other efforts, drug developers are trying to harness “receptor-mediated transcytosis” as a vehicle for shuttling drug-loaded nanoparticles, larger chemical and protein drugs, as well as therapeutic antibodies across the BBB. “The hypoxia-enhanced human BBB Chip recapitulates the function of critical transcytosis pathways, such as those used by the LRP-1 and transferrin receptors responsible for taking up vital lipoproteins and iron from circulating blood and releasing them into the brain on the other side of the BBB. By harnessing those receptors using different preclinical strategies, we can faithfully mimic the previously demonstrated shuttling of therapeutic antibodies that target transferrin receptors in vivo, while maintaining the BBB’s integrity in vitro,” said Mustafaoglu.

We aim to collaborate with multiple biopharmaceutical partners in a pre-competitive relationship to develop shuttles offering exceptional efficacy and engineering flexibility for incorporation into antibody and protein drugs, because this is so badly needed by patients and the whole field.

JAMES GORMAN

Based on these findings, the Wyss Institute has initiated a “Brain Targeting Program.”  “Initially, the BBB Transport Program aims to discover new shuttle targets that are enriched on the BMVEC vascular surface, using novel transcriptomics, proteomics, and iPS cell approaches. In parallel, we are developing fully human antibody shuttles directed against known shuttle targets with enhanced brain-targeting capabilities,” said James Gorman, M.D., Ph.D., the Staff Lead on the Advanced Technology Team for the BBB Transport Program working with Ingber. “We aim to collaborate with multiple biopharmaceutical partners in a pre-competitive relationship to develop shuttles offering exceptional efficacy and engineering flexibility for incorporation into antibody and protein drugs, because this is so badly needed by patients and the whole field.”

The authors think that in addition to drug development studies, the hypoxia-enhanced human BBB Chip can also be used to model aspects of brain diseases that affect the BBB such as Alzheimer’s and Parkinson’s disease, and to advance personalized medicine approaches by using patient-derived iPS cells.

Read more: Enhanced human Blood-Brain Barrier Chip performs in vivo-like drug and antibody transport

 

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A set of modular hydrogel components that could be useful in a variety of soft robotic and biomedical applications

Re-attached
A new kind of hydrogel material developed at Brown has the ability to react dynamically to its environment–bending, twisting and self-adhering on demand. Above, the researchers demonstrated self-adhering behavior on the tail of a 3-D printed hydrogel salamander. The self-adhering behavior was also used to make hydrogel building blocks that fit together like LEGO blocks.
Wong Lab / Brown University

A new type of hydrogel material developed by Brown University researchers could soon make assembling complex microfluidic or soft robotic devices as simple as putting together a LEGO set.

Using a new type of dual polymer material capable of responding dynamically to its environment, Brown University researchers have developed a set of modular hydrogel components that could be useful in a variety of “soft robotic” and biomedical applications.

The components, which are patterned by a 3D printer, are capable of bending, twisting or sticking together in response to treatment with certain chemicals. For a paper published in the journal Polymer Chemistry, the researchers demonstrated a soft gripper capable of actuating on demand to pick up small objects. They also designed LEGO-like hydrogel building blocks that can be carefully assembled then tightly sealed together to form customized microfluidic devices — “lab-on-a-chip” systems used for drug screening, cell cultures and other applications.

The key to the new material’s functionality is its dual polymer composition, the researchers say.

“Essentially, the one polymer provides structural integrity, while the other enables these dynamic behaviors like bending or self-adhesion,” said Thomas Valentin, a recently graduated Ph.D. student in Brown’s School of Engineering and the paper’s lead author. “So putting the two together makes a material that’s greater than the sum of its parts.”

Hydrogels solidify when the polymer strands within them become tethered to each other, a process called crosslinking. There are two types of bonds that hold crosslinked polymers together: covalent and ionic. Covalent bonds are quite strong, but irreversible. Once two strands are linked covalently, it’s easier to break the strand than it is to break the bond. Ionic bonds on the other hand are not quite as strong, but they can be reversed. Adding ions (atoms or molecules with a net positive or negative charge) will cause the bonds to form. Removing ions will cause the bonds to fall apart.

For this new material, the researchers combined one polymer that’s covalently crosslinked, called PEGDA, and one that’s ionically crosslinked, called PAA. The PEGDA’s strong covalent bonds hold the material together, while the PAA’s ionic bonds make it responsive. Putting the material in an ion-rich environment causes the PAA to crosslink, meaning it becomes more rigid and contracts. Take those ions away, and the material softens and swells as the ionic bonds break. The same process also enables the material to be self-adhesive when desired. Put two separate pieces together, add some ions, and the pieces attach tightly together.

A time-lapse image shows the actuation of a soft gripper.

That combination of strength and dynamic behavior enabled the researchers to make their soft gripper. They patterned each of the gripper’s “fingers” to have pure PEGDA on one side and a PEGDA-PAA mixture on the other. Adding ions caused the PEGDA-PAA side to shrink and strengthen, which pulled the two gripper fingers together. The researchers showed that the setup was strong enough to lift small objects weighing about a gram, and hold them against gravity.

“There’s a lot of interest in materials that can change their shapes and automatically adapt to different environments,” said Ian Y. Wong, an assistant professor of engineering and the paper’s corresponding author. “So here we demonstrate a material the can flex and reconfigure itself in response to an external stimulus.”

But potentially a more immediate application is in microfluidics, the researchers say.

Hydrogels are an attractive material for microfluidic devices, especially those used in biomedical testing. They’re soft and flexible like human tissue, and generally nontoxic. The problem is that hydrogels are often difficult to pattern with the complex channels and chambers needed in microfluidics.

But this new material — and the LEGO block concept it enables — offers a potential solution. The 3D printing process allows complex microfluidic architectures to be incorporated into each block. Those blocks can then be assembled using a socket configuration much like that of real LEGO blocks. Adding ions to the assembled blocks makes a water-tight seal.

“The modular LEGO blocks are interesting in that we could create a prefabricated toolbox for microfluidic devices,” Valentin said. “You keep a variety of preset parts with different microfluidic architectures on hand, and then you just grab the ones you need to make your custom microfluidic circuit. Then you heal them together and it’s ready to go.”

And storing the blocks for long periods before use doesn’t appear to be a problem, the researchers say.

“Some of the samples we tested for this study were three or four months old,” said Eric DuBois, a Brown undergraduate and co-author on the paper. “So we think these could remain usable for an extended period.”

The researchers say they’ll continue to work with the material, potentially tweaking the properties of the polymers to get even more durability and functionality.

Learn more: Dynamic hydrogel used to make ‘soft robot’ components and LEGO-like building blocks

 

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Detecting cancer cells in blood with a new microfluidic device

Diagram shows how the microfluidics device separates cancer cells from blood. The green circles represent cancer cells.
(Credit: Ian Papautsky)

Researchers at the University of Illinois at Chicago and Queensland University of Technology of Australia, have developed a device that can isolate individual cancer cells from patient blood samples.

The microfluidic device works by separating the various cell types found in blood by their size. The device may one day enable rapid, cheap liquid biopsies to help detect cancer and develop targeted treatment plans. The findings are reported in the journal Microsystems & Nanoengineering.

“This new microfluidics chip lets us separate cancer cells from whole blood or minimally-diluted blood,” said Ian Papautsky, the Richard and Loan Hill Professor of Bioengineering in the UIC College of Engineering and corresponding author on the paper. “While devices for detecting cancer cells circulating in the blood are becoming available, most are relatively expensive and are out of reach of many research labs or hospitals. Our device is cheap, and doesn’t require much specimen preparation or dilution, making it fast and easy to use.”

The ability to successfully isolate cancer cells is a crucial step in enabling liquid biopsy where cancer could be detected through a simple blood draw. This would eliminate the discomfort and cost of tissue biopsies which use needles or surgical procedures as part of cancer diagnosis. Liquid biopsy could also be useful in tracking the efficacy of chemotherapy over the course of time, and for detecting cancer in organs difficult to access through traditional biopsy techniques, including the brain and lungs.

However, isolating circulating tumor cells from the blood is no easy task, since they are present in extremely small quantities. For many cancers, circulating cells are present at levels close to one per 1 billion blood cells. “A 7.5-milliliter tube of blood, which is a typical volume for a blood draw, might have ten cancer cells and 35-40 billion blood cells,” said Papautsky. “So we are really looking for a needle in a haystack.”

Microfluidic technologies present an alternative to traditional methods of cell detection in fluids. These devices either use markers to capture targeted cells as they float by, or they take advantage of the physical properties of targeted cells — mainly size — to separate them from other cells present in fluids.

Papautsky and his colleagues developed a device that uses size to separate tumor cells from blood. “Using size differences to separate cell types within a fluid is much easier than affinity separation which uses ‘sticky’ tags that capture the right cell type as it goes by,” said Papautsky. “Affinity separation also requires a lot of advanced purification work which size separation techniques don’t need.”

Learn more: New microfluidics device can detect cancer cells in blood

 

 

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