Continuously Sorting Particles According to Shape

There are numerous filters to separate particles in liquid based on their size, which can be enough to isolate them; however, particle shape can be more important, as it distinguishes healthy red blood cells from those affected by sickle-cell disease or malaria. Shape can also be used to determine what stage a cell is in of the cell cycle, which would benefit researchers looking for dividing cells. Recent research by Dino Di Carlo of UCLA looks to separate particles of differing aspect ratios continuously, using inertial fluid-dynamics. His work, “Continuous Inertial Focusing and Separation of Particles by Shape,” featured in Physical Review X reminds me of his previous work to use inertial fluid-dynamics to continuously filter particles according to size.

Particle Separation

Existing methods to separate particles according to shape include hydro-dynamic filtration (HDF) and deterministic lateral displacement (DLD), along with a few others. DLD involves a grid of posts in a channel that is arranged in a way that effectively separates particles according to size. This can be enhanced by controlling particle orientation in order to filter by particle shape or shear stress to separate by particle deformability. HDF uses highly branched channels to separate particles by size according to the fluidic resistance of the side channels. Both of these methods are passive and continuous, but they have highly complex structures and require low flow rates (no greater than a few µL/min). Di Carlo’s method uses fluid inertia to focus particles in channels at high flow rates from 40 to 80 µL/min. This utilizes a shear-gradient lift force and a wall-effect lift force in order to shift particles across streamlines when the Reynolds number of the particle is on the magnitude of 1 or greater. The shear-gradient lift force is directed down the shear gradient and toward the wall, while the wall-effect lift force is caused by the wake of a particle near the wall and is directed away from the wall. With a Reynolds numbers on the order of 1, inertial lift forces dominate the particle behavior while viscous interactions dominate when the particle Reynolds number is << 1. As the particle Reynolds number increases, migration across streamlines, away from the center line, is observed.

Di Carlo attempted to separate spheres and ellipsoids of varying aspect ratios, while conserving volume. Spheres of 3 µm and 6 µm in diameter, and ellipsoids that conserved the volume at 1:3 and 1:5 aspect ratios were used.

Results

Spheres and ellipsoids are sorted using inertial fluid-dynamics. Particles with larger aspect radios reach equilibrium positions (Xeq) 4 cm downstream.

Spheres and ellipsoids are sorted using inertial fluid-dynamics. Particles with larger aspect radios reach equilibrium positions (Xeq) 4 cm downstream.

This work demonstrates that rod-like particles find equilibrium positions close to the center of the channel, while spheres of the same volume end up in streamlines close to the wall. When the major axis of the particles rotates perpendicular to the plane of the wall, the wall-effect lift increases and the particles is pushed away from the wall. Once the major axis has realigned with the direction of the flow, the wall-effect lift decreases and the particles move towards the wall one again. However, particles with higher aspect ratios experience a wall-effect lift greater than the shear-gradient lift and find equilibrium positions closer to the center of the channel. As the Reynolds number of the particle increases (increasing flow rate is one way to increase Reynolds number), the shear-gradient lift force increases faster than the wall-effect lift force. But the particles with higher aspect ratios rotate and experience a greater wall-effect lift force and return to the center. This relationship with increasing particle Reynolds number allows this method to scale with flow rate, while the previous methods do not.

Four different shape-activated particle-sorting (SAPS) devices were designed with varying numbers of outlets, outlet resistances, channel aspect ratios and flow rates. Three of the devices used 6 µm spheres and their derived ellipsoids, while the final device used the 3 µm particles. All of the devices had varying performances, but the researchers selected device C, which had 7 outlets and isolated 88% of the spheres with 87% purity, 49% of 1:5 rods with 78% purity and 77% of 1:3 rods with 80% purity, for sorting yeast cells. Yeast cells are normally spherical, but form a bispherical twin or aggregate when budding. This change in shape is similar to the varying aspect ratios examined previously. It is useful to synchronize cell cycle stages, but this may be achieved with chemicals that alter cell physiology, changes in temperature or size filtration. SAPS C was able to extract nondividing singles with high yield and purity up to 94% and 54% of budded yeast cells were recovered at 31% purity, which increased from 6.6% purity at the inlet. According to my rough measurements from the paper’s figures, the aspect ratio of budding yeast is less than 2:1 which may explain the difference in performance compared to the original ellipsoids. Previously, Sugaya et al. used a 5 outlet HDF system to separate budding yeast cells. In comparison, Sugaya achieved up to 69.4% purity of budding cells in one outlet, up from 39.4% at the inlet. This same outlet recovered 28.8% of budding cells while another outlet recovered 65.2%. There is still room for improvement of Di Carlo’s budding yeast yield, but this operated at 1500 cell/s, compared to 100 cell/s from previous work that utilized dielectrophoretic forces according to the opacity of dividing yeast.

Future Work

Di Carlo has proposed that this work be used to sort shaped particles in other areas to improve cytometry that operates on spherical particles, alignment of barcoded particles, and identification of microalgae that vary in size and shape. Interestingly, he also introduced the capability of this setup for a non-biological process: improving cement. Cement strength and stability are affected by particle shape and size and could benefit from shape based separation. According to Dr. Di Carlo, “… [Cement] particles that are too large may not react completely in internal regions of the particle, while smaller particles with very high surface area to volume ratios can react too quickly and may not be stable.” I’m excited to see microfluidics expand into more established industries and further demonstrating real-world potential to be a more cost effective, accessible technology.

 

References:

ResearchBlogging.org

 

Masaeli, M., Sollier, E., Amini, H., Mao, W., Camacho, K., Doshi, N., Mitragotri, S., Alexeev, A., & Di Carlo, D. (2012). Continuous Inertial Focusing and Separation of Particles by Shape Physical Review X, 2 (3) DOI: 10.1103/PhysRevX.2.031017

Di Carlo, D., Irimia, D., Tompkins, R., & Toner, M. (2007). Continuous inertial focusing, ordering, and separation of particles in microchannels Proceedings of the National Academy of Sciences, 104 (48), 18892-18897 DOI: 10.1073/pnas.0704958104

Sugaya, S., Yamada, M., & Seki, M. (2011). Observation of nonspherical particle behaviors for continuous shape-based separation using hydrodynamic filtration Biomicrofluidics, 5 (2) DOI: 10.1063/1.3580757

Tracking Immune Responses to Food with a Gut on a Chip

Organs on Chips

In an effort to model the complex processes occurring in human bodies, Donald Ingber has pioneered the development of ‘organs-on-chips,’ reproducing the lung and the gut on microfluidic devices. These systems allow researchers to replicate and study organs without the use of human test subjects. While this is one of the best options, there are too many variables to control, understand, and more importantly, manipulate. At the other end of the spectrum is an in vitro study with a cell line and few variables that hardly resemble the real environment. Researchers in Switzerland have developed their own gut-on-a-chip that imitates a human gastrointestinal tract called the Nutrichip. They hope to use this microfluidic device to study the immune-modulatory function of food (with a strong focus on dairy food). This work is detailed in the article “NutriChip: Nutrition Analysis Meets Microfluidics,” which appears in Lab on a Chip.

Device Aims

The Nutrichip is primarily focused on analyzing the presence and kinetics of inflammatory biomarkers after a meal, which should shed insight on how certain foods impact our bodies. For example, milk products not only provide nutrients, but affect our physiological functions via bacteria, proteins and bioactive peptides. In particular these agents may modulate the fabrication of pro-inflammatory cytokines, which the Nutrichip aims to monitor. The Nutrichip mimics the thin layer of epithelial cells of the intestinal tract and the immune system it interacts with. In this system, the epithelial cells transport nutrients into circulation to be metabolized by the body, and the immune system controls responses to any trespassing pathogenic organisms. Not only do epithelial cells try to keep out pathogens, but they must filter what reaches the immune layer in order to maintain immunological tolerance to nutrients.

Device Design

The device is composed of three distinct parts, an apical layer, a basolateral layer, and a membrane which separates them. The apical layer is populated with a culture of intestinal epithelial cells. In this model, the authors used Caco-2 cells to produce a confluent layer of the intestine. This cell line is derived from a human colorectal adenocarcinoma that demonstrates most of the morphological and functional traits of intestinal cells. The basolateral layer contains a culture of monocytic cell line differentiated into macrophages. A chamber downstream of the monocytes contains magnetic beads functionalized with antibodies against the targeted cytokines. These beads allow in situ capture and washing of the cytokines before fluorescent detection.

Experimental Procedure and Results

The researchers treated the apical layer with tumor necrosis alpha (TNFα) and lipopolysaccharide (LPS), which is found in the membrane of Gram-negative bacteria, for 24 hours. This resulted in a significant secretion of the cytokine Interleukin 6 (IL-6) in the basolateral media. However, the Caco-2 cells protected the macrophages very well, as the LPS concentration applied to the apical layer resulting in a response, was three orders of magnitude greater than that would be applied to the basolateral layer directly to produce the same cytokine response. When the macrophages were treated directly with LPS, there was a significant increase in IL-6 which indicates that they may be able to quantify the IL-6 with their proposed magnetic beads. These two experiments indicate that they can partially mimic the intestinal tract and monitor the effect that certain compounds or nutrients have on the body’s immune system.

Thoughts on Future Work

This is all very preliminary work, and the researchers hope to measure the pro-inflammatory activity of meals, anti-inflammatory activity of dairy products as well the bio-availability of nutrients in digested foods. To make that final component a reality, they also intend to include on-chip digestion capabilities. I am sure there are alternatives to a dynamic digestive tract moving bolus through the system, but I’d love to see it anyway. I think this work could be extremely beneficial in understanding food allergies. We seem to have more food allergies these days that may be due to changes in modern life or may have gone undetected previously (or is perhaps just over diagnosed).

Reference:

ResearchBlogging.org

Ramadan, Q., Jafarpoorchekab, H., Huang, C., Silacci, P., Carrara, S., Koklü, G., Ghaye, J., Ramsden, J., Ruffert, C., Vergeres, G., & Gijs, M. (2013). NutriChip: nutrition analysis meets microfluidics Lab on a Chip, 13 (2) DOI: 10.1039/c2lc40845g

A Microsyringe to Take the Pain out of Shots

Back when I was in sixth grade, I remember reading a little blurb in some science magazine at school that in the future we could receive shots via a method that would feel as soft as a banana peel. Although I’m now a champ at taking shots, it’s still not a bad idea. We’ve had transdermal patches (think nicotine and birth control) for some time now, but those release their medicine over a period of time. A syringe is capable of delivering a dose at once, and can take a biological sample too. Researchers from the University of Pisa have developed this ‘syringe of the future’ within ‘A minimally invasive microchip for transdermal injection/sampling applications’ in Lab on a Chip.

Microsyringe Design

Fabricated 0.25 cm2&nbsp;microsyringe showing (a)&nbsp; needles and (b) reservoirs on reverse.

Fabricated 0.25 cm2 microsyringe showing (a)  needles and (b) reservoirs on reverse.

The microsyringe is actually a 0.5 cm x 0.5 cm microchip featuring thousands of hollow silicon-dioxide microneedles. The microneedles are 100 µm long at a density of 1 million needles/cm2. These needles are at least 100 times denser and 10 times smaller than other results reported in the literature. Furthermore, this is not simply a design for microneedles; the researchers have incorporated a reservoir that is meant to store samples from the body and medicine when injecting. The reservoir is comprised of 14 independent volumes adding up to 4.2µL with a sealed plastic cap. The microsyringe would not penetrate as deeply as a normal syringe and would have a smaller total cross-sectional area, resulting in a less painful injection. The researchers intend this microsyringe to be an integral part of an artificial pancreas that is capable of continuously sampling interstitial fluids, measuring glucose levels and releasing insulin to regulate glucose in the blood.

Microsyringe Theoretical Analysis

Schematic of microsryinge illustrates the array of needles connecting to independent reservoirs on reverse.

Schematic of microsryinge illustrates the array of needles connecting to independent reservoirs on reverse.

If the microsyringe is going to be used in the real world, the microneedles need to be able to puncture the outer layers of the skin without breaking. They first tested this theoretically by analyzing the forces acting on the microneedles during insertion. The force required to pierce the skin (derived from a known skin-piercing pressure and the known area of the microsyringe) are compared to the maximum values of the buckling and bending forces that the microneedles would encounter. The buckling force would arise when the microneedle insertion is misaligned with respect to its axis of symmetry (in other words, when the skin isn’t orthogonal to the microneedle cross-section). The bending force is generated by lateral movement between the tissue and needle during the beginning of insertion. The theoretical analysis reveals that the buckling and bending forces are at least 10 times greater than the piercing force, giving it a factor of safety greater than 10.

Microsyringe Mechanical Testing

SEM of fabricated microsyringe illustrating the density and uniformity of the needles.

SEM of fabricated microsyringe illustrating the density and uniformity of the needles.

The researchers followed up this theoretical analysis by simulating insertion into human skin using agarose gel models, which have mechanical properties similar to those of skin. The microsyringes were inserted into the skin at 200 and 500 gram-forces (typical force produced by finger) for 30, 60 and 120 seconds. After repeated insertion tests of the same microneedles, the researchers found that they all penetrated successfully without significant damage to the needles (characterization by SEM). Since this device needs to be able to store liquid, they tested losses due to evaporation and acceleration (ie dropping). Over a 19 day test period, they measured an evaporation rate of 71 nL/min through the microneedles, which would drain the reservoir in an hour. Under acceleration of 80g, the microneedles lost 1320 nL/min, which would drain the reservoir in 3 minutes of falling.

I wonder how the use of this microsyringe would actually work. 4.2 µL is small to begin with, but each individual reservoir is 0.3 µL. It’ll be difficult to load medicine and collect samples. I’m really interested in the group’s plan to use it in an artificial pancreas. A microsyringe by itself is good, but I love the idea of a pancreas. I suppose that one of the most important aspects of the microsyringe is its reservoir size. What’s a relevant medicine payload? What sample size is needed for analysis? I think this is some promising research from this group, and I look forward to reading about their pancreas.

Reference:

ResearchBlogging.org

Strambini LM, Longo A, Diligenti A, & Barillaro G (2012). A minimally invasive microchip for transdermal injection/sampling applications. Lab on a chip, 12 (18), 3370-9 PMID: 22773092

Rolling Out Cell Sorting with Microfluidics

Cell Sorting

Cells are quite valuable, especially when used for regenerative research, diagnostics or research. But harvested cells do not come presorted and need to be separated from a heterogeneous mixture of cells. There are already numerous methods to sort cells according to biophysical properties such as size, density, morphology, and dielectric or magnetic susceptibility. Cell sorting based on labels can have a higher specificity, but introduces extra steps to add and remove labels, which can affect the phenotype of the cell. Rohit Karnik of MIT has developed a cell sorting method based on cell rolling. The continuous, label-free process is described in “Cell sorting by deterministic rolling” in Lab on a Chip.

Cell Rolling

Target cells initiate cell rolling and enter the space between the ridges, which leads them to the gutter side. Non-target cells do not adhere or roll along the surface and maintain their original trajectories.

Target cells initiate cell rolling and enter the space between the ridges, which leads them to the gutter side. Non-target cells do not adhere or roll along the surface and maintain their original trajectories.

Cell rolling is a phenomenon where a cell is constantly forming and releasing adhesive bonds with a surface under fluid flow. The continuous creation and release of bonds by the cell induces rolling and is an integral role in the movement of lymphocytes, platelets, stem cells and metastatic cancer cells. To induce cell rolling, a surface needs to be coated with a ligand specific to the target cell type. The rolling target cells need to be focused, so slanted ridges are added to the bottom of the channel. When the target cell comes into contact with the surface of the coated ridges, it will begin to roll along it and eventually turn the corner into the space between ridges. By following the path of the direction of the ridges, the targeted cells will be focused on one side of the channel known as the gutter. The non-target cells should not adhere and roll along the ridges, allowing them to be spatially differentiated from the target cells. But the ridges actually serve an additional purpose: Acting as mixers, these ridges introduce circulation to the axial flow. This flow would normally be laminar, which would prevent the majority of cells from coming in contact with the surface of the channel and rolling.

Cell Rolling Testing & Optimization

HL60 (target) and K562 (non-target) cells enter through the same inlet. Due to the cell rolling sorting, the cells exit the two outlets highly organized.

HL60 (target) and K562 (non-target) cells enter through the same inlet. Due to the cell rolling sorting, the cells exit the two outlets highly organized.

Karnik validated this cell sorting method by processing the leukemia cell lines HL60 and K562. The surfaces were coated with P-selectin, which is a known ligand for the target HL60. HL60 and K562 were injected in a single inlet at a ratio of 2:3, respectively. Outlet A held 95.0 ± 2.8% HL60 cells, and outlet B had 94.3 ± 0.9% K562 cells. The cell sorting was extremely successful and 87.2 ± 3.7% and 76.7 ± 14.2% of the HL60 and K562 cells were recovered at the end of the process. Cell loss was most likely due to settling in the syringe at the inlet and cells remaining in the channel and dead volumes at the end of the process. Karnik also investigated the effect of the ligand concentration on cell sorting. Higher concentrations generated stronger cell-surface adhesion, but this came at the expense of cell rolling, so an operating point had to be determined for an ideal cell rolling concentration; at a flow rate of 70 µL/min, the channel was incubated with P-selectin concentration of 1.5 µg/mL.

Discussion

I really like this method of cell sorting because it is both passive and label-free. Although an extra section of channel must be added with coated ridges, no other major components are necessary. This method does not need any more equipment or chambers, making it simple to integrate into a project. With its small footprint, it can also be highly parallelized, negating the need for it to operate at high flow rates which could hinder cell rolling. This could either function as a standalone sorting device or integrated into a device processing a mixture of cells. Similar to other cell sorting procedures, widespread usage of this particular method is limited to availability of information: only cell lines for which we’ve characterized the rolling behavior for can be sorted this way.

Reference:

I really like this method of cell sorting because it is both passive and label-free. Although an extra section of channel must be added with coated ridges, no other major components are necessary. This method does not need any more equipment or chambers, making it simple to integrate into a project. With its small footprint, it can also be highly parallelized, negating the need for it to operate at high flow rates which could hinder cell rolling. This could either function as a standalone sorting device or integrated into a device processing a mixture of cells. Similar to other cell sorting procedures, widespread usage of this particular method is limited to availability of information: only cell lines for which we’ve characterized the rolling behavior for can be sorted this way.

 

Reference:

ResearchBlogging.org Choi, S., Karp, J., & Karnik, R. (2012). Cell sorting by deterministic cell rolling Lab on a Chip, 12 (8) DOI: 10.1039/c2lc21225k

Putting the Squeeze on Microfluidics

Microfluidic devices are able to process small volumes of liquid and are comprised of microscale components, but the devices themselves are not often small themselves. These labs-on-chips are often limited to lives in labs instead of the remote areas that could really benefit from their use. The limitation comes in the form of support equipment used to process or analyze assays that are expensive, bulky, energy consuming and/or require trained professional operators. Syringe pumps are often used in labs to drive liquids used in assays at specific flow rates and to ensure that the right volume is used. The need for complicated, external flow equipment was recently addressed by a group from Peking University. The group’s paper, “Squeeze-chip: a finger-controlled microfluidic flow network device and its application to biochemical assays” was recently featured on the cover of Lab on a Chip.

Squeeze-chip Design

The squeeze-chip is comprised of two check valves on either side of a reservoir. Squeezing the reservoir pumps fluid through one check valve. The reservoir is refilled after release through the second check valve.

The squeeze-chip is comprised of two check valves on either side of a reservoir. Squeezing the reservoir pumps fluid through one check valve. The reservoir is refilled after release through the second check valve.

The ‘squeeze-chip’ is based on a system of check valves and finger-operated pumps. Check valves allow fluid to flow in only one direction and, in this case, are fabricated from PDMS and integrated into a microfluidic card. The pump is a fluid reservoir that can be depressed by a finger. Squeezing the reservoir evacuates fluid through one of the check valves oriented to pass fluid away from the reservoir. After releasing the reservoir, it draws fluid in through a second check valve that’s oriented in a different direction so that it can only feed liquid into the reservoir. Alternatively, specially designed squeeze-chips can handle two immiscible fluids so that with each pump, a small plug of one fluid can be inserted into the system and is sandwiched by the other fluid. The displaced volume is not always equal, but the reservoirs feed into metering channels which only accept a specific volume, adding some control to the squeeze-chip. The authors have had success in delivering volumes ranging from nanoliters to microliters. This is the basic setup for a squeeze-chip, which can be combined with other units to create a more complex, sophisticated system.

Squeeze-chip Validation

Alternate design of the squeeze-chip creates liquid sandwiches one fluid in another when immiscible.

Alternate design of the squeeze-chip creates liquid sandwiches one fluid in another when immiscible.

The researchers demonstrated the squeeze-chip’s ability by running colorimetric assays to measure glucose and uric acid at clinically relevant concentrations of 0-10 mM and 0-15 mM respectively.. These assays comprise a system of squeeze-chips that mix solutions, resulting in a 4mm thick readout chamber, allowing the user to see the solution’s color with the naked eye. The researchers were able to detect glucose as low as 1 mM and uric acid as low as 100 µM with initial sample consumption less than 5 µL per test. Limits of detection can be lowered by increasing the readout chamber thickness, which would make the color darker.

Discussion

Sample operation of squeeze-chip used in colorimetric assay.

Sample operation of squeeze-chip used in colorimetric assay.

I think that the squeeze-chip is a great component to make devices more viable outside of the lab, though it may not be suitable for every card. The metering chambers add some volume control, but, again, this may not be enough. More importantly, volumetric flow rate isn’t controlled, which eliminates the squeeze-chip as a viable option for applications requiring more stringent regulation. There are several considerations that need to be kept in mind when designing any lab-on-a-chip for use outside the lab. Despite any microscale magic taking place, the end-user and intended environment need elevated priority, meaning that these devices need to be relatively cheap, free of any tethers to an advanced lab and operable by people with limited education. The squeeze-chip certainly addresses cost and eliminates a connection to an external syringe pump. It can be operated by hand, or even operated by an actuated piston if the chip is predestined to function in some housing. Usability testing results would be interesting to see as well, including performance variation among users, but it looks like devices using the squeeze-chip can be readily used in areas of need.

Reference:

ResearchBlogging.org

Li, W., Chen, T., Chen, Z., Fei, P., Yu, Z., Pang, Y., & Huang, Y. (2012). Squeeze-chip: a finger-controlled microfluidic flow network device and its application to biochemical assays Lab on a Chip, 12 (9) DOI: 10.1039/C2LC40125H

Diagnosing Similar Diseases in Low Resource Settings

Diagnostics in Low-Resource Settings

A lot of excitement surrounding microfluidics has been about its promising use in diagnosis in low-resource settings. Many infectious diseases present in developing countries are manageable or treatable with available medications, but still account for 1/3 of deaths. In these areas, multiple diseases present similar symptoms, leading to misdiagnosis and thus incorrect treatment. Hundreds of blood-based microfluidic immunoassays are available for diagnostic purposes, but they’re not all created equally. They require varying levels of sample processing or analysis that prohibit their deployment in low-resource settings. Further, while some diseases may have similar symptoms, they might require different detection techniques, with varying sample volumes, reagents and processing time, making it difficult to detect multiple diseases within the same system. This is the focus of recent work from Paul Yager of University of Washington. In his Lab on a Chip paper, “Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards,” he and his colleagues develop a system to detect both Typhoid fever and malaria.

Malaria and Typhoid Dual Detection

The proposed card is compact and intended to integrate with the DxBox. All sample processing occurs on-card including IgG filtration. This also depicts the porous nitrocellulose membrane of the FMIA which provides high assay surface area.

The proposed card is compact and intended to integrate with the DxBox. All sample processing occurs on-card including IgG filtration. This also depicts the porous nitrocellulose membrane of the FMIA which provides high assay surface area.

The developed system is intended to integrate with the DxBox, an ongoing project focused on a point-of-care diagnostic device. As I mentioned before, different diseases might require different means of detection. In this case, the researchers decided to detect antigens generated by malaria parasites and IgM antibodies generated by the host in response to the bacteria responsible for typhoid (Salmonella Typhi). The microfluidic card is based on a flow-through membrane immunoassay (FMIA) composed primarily of nitrocellulose, instead of traditional microfluidic channels. Nitrocellulose is essentially paper and provides a lot of surface area, creating shorter assay times. Enzyme-linked immunosorbent assays (ELISA) are standard lab assays and will be replicated using FMIA. However, ELISA can be slow (more than 3 hours) due to the diffusion between the bulk fluid and the capture service, while the FMIA can perform the same task in half an hour due to its high surface area.

Immunoassay Process

The system processes blood from the same sample in different, parallel steps to test for malaria and typhoid.

The system processes blood from the same sample in different, parallel steps to test for malaria and typhoid.

The detections of both analytes are run in parallel and start with the same unfiltered blood. The card extracts the plasma with a filter, eliminating whole blood cells, and this is where the assays for malaria and typhoid diverge. The typhoid assay must filter out any IgG antibodies (which would cause false positives when testing for IgM) and dilute the sample further. This results in a four-fold increase in the sample volume used in the malaria segment. Each analyte is then captured by immobilized reagents and labeled with gold nanoparticles conjugated to antibodies. The entire process is driven by pneumatic pressure and valves. Pneumatics is cheaper than alternatives, plus it doesn’t dilute the sample with an additional liquid, but it comes at the cost of introduced air bubbles. Air vents were incorporated to eliminate bubbles, but they were not totally eradicated and still obstructed the image analysis sometimes. Within the DxBox, analysis is intended to be carried out by a webcam. However, the current design of the system created nonuniform lighting (which can be rectified), and a flatbed scanner was used instead.

Results

This microfluidic card was tested on blood samples with Typhoid or malaria. Unfortunately the researchers did not test on a large enough sample to evaluate clinical utility or determine a limit of detection for the card. Currently lab-based ELISA has a limit of detection near 4 ng/mL, which is clinically relevant. The researchers also ran each sample on ELISA and a bench-top FMIA in addition to the on-card FMIA. Comparing the quantified signal of the on-card FMIA to ELISA resulted in an R2 value of 0.73, and on-card FMIA vs bench-top FMIA had an R2 value of 0.92. These are fine results that demonstrate how closely the on-card FMIA follows the bench-top methods, but it would mean a whole lot more given a limit of detection.

Discussion

The results of this card design seem promising but will mean a lot more with more testing. The pneumatic actuation was a major hindrance to project success. While they could operate at different pressures, the actuators were unable to actually control the liquid velocity. Also, the pneumatics introduced bubbles into the card, which not only affected the assay process but the final image to be analyzed as well. While only two diseases were showcased here, the authors have indicated that there is already work to create a more complete fever symptom panel. They also acknowledged that this format could be applied to other panels aimed at diarrheal diseases and sexually transmitted diseases as well. This format could really be adapted for a variety of diseases, with the disease diagnosis as the limiting factor for card design. 

Reference:

ResearchBlogging.org

Lafleur, L., Stevens, D., McKenzie, K., Ramachandran, S., Spicar-Mihalic, P., Singhal, M., Arjyal, A., Osborn, J., Kauffman, P., Yager, P., & Lutz, B. (2012). Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards Lab on a Chip, 12 (6) DOI: 10.1039/C2LC20751F

You Eat Corn? I Make Microfluidics with It

Recasting Renewable Resources

Some of us are on the search for new renewable materials that can make our lifestyle a bit more sustainable. Although a new resource may be renewable, that doesn’t mean it makes sense to use. For example, many people went crazy over corn-based ethanol because it would eliminate (or at least reduce) America’s dependence on foreign oil. America is the world’s largest producer of corn, but that’s for a reason; we use it for a lot of things already. By diverting a percentage of corn production towards ethanol, we shook up our corn economy, and not necessarily for the better. Yet we find ourselves looking to corn again, but this time as an alternate material for microfluidics. Gang Logan Liu et al. have presented us with a new method for corn-based microfabrication in "Green microfluidic devices made of corn proteins". The group from the University of Illinois Urbana-Champagne uses the protein zein, which is actually a byproduct of ethanol production. An existing product previously treated as waste now has the promise of new value; this is exactly what we’re looking for as a green resource.

Not only would this corn-based device be renewable, its disposal would also be more environmentally friendly. The authors stress that this is important in sectors that perform a high number of tests. This could be particularly important for agriculture, which needs to constantly test for pollutants and diseases (it would also give a nice ‘full circle’ type feeling). I’m normally more interested in medical applications for microfluidics, but there are a ton of areas that run chemical tests that can be optimized.

Zein Film Creation

This is not the first time Zein has been examined as a viable resource; it has been studied for use in coatings, adhesives, food packaging, drug and functional food delivery systems. Zein is a good candidate for microfluidics because it resembles a rod, and self-organizes with other rods into a two-dimensional film. The authors employed a soft lithography fabrication process using PDMS as a master. Once a single layer was molded, the authors examined binding processes with glass slides as well as other zein layers. While it is conceivable that someone would want to use a glass-zein or zein-zein device, it is also necessary to test it with glass because zein is opaque and prevents us from visually evaluating how well it functions.

Zein Film Attachment

Zein film is prepared via soft lithography, using PDMS as a master. It is sealed to another surface by applying it to ethanol solvent or vapor.

Zein film is prepared via soft lithography, using PDMS as a master. It is sealed to another surface by applying it to ethanol solvent or vapor.

Two methods for attachment were considered: solvent and vapor deposition. Both methods involve heating the surfaces and coating with ethanol before mating the sides. In solvent deposition, the zein side is placed on an ethanol-coated surface to pick up a thin layer before sealing. In vapor deposition, the zein side is exposed to ethanol vapor before sealing. The process of heating and exposing to ethanol allows the zein to become more mobile as well as crosslink with either the opposing zein surface or the micro voids in the glass.

Zein Microfabrication Results

The authors tested the zein-structures in a couple different ways, but they basically boiled down to how well the channels were replicated, sealed and able to allow for normal microfluidics operations. The resulting channels deviated slightly from the master, which could be rectified by reducing the elasticity of the PDMS or changing the zein film thickness. When comparing the solvent and vapor depositions, reliable results were obtained using the vapor. When the solvent was deposited, evidently extra ethanol was transferred to the zein film, distorting the geometry of the channels. While the authors demonstrated that zein sealed well, they did note that it is a somewhat permeable material, and this could be manipulated in some controlled way. Also of note is the fact that zein autofluoresces. Zein is excited using wavelengths from 450 to 490 nm, resulting in excitation emissions at 550 nm to 580 nm. While this doesn’t line up with the properties of Green Fluorescent Protein, it might align with others, making fluorescent detection a bit harder.

I think that the use of zein is promising. It utilizes a renewable material in a new way that gives value to a pre-existing process. Not only is zein renewable, but it is biodegradable, which will answer one of the problems that will surely arise when microfluidic devices have worked their way into mass production. I think we’ll have to see what complicated structures can be created with zein and what solvents really shouldn’t be used.

Reference:

ResearchBlogging.org

Luecha, J., Hsiao, A., Brodsky, S., Liu, G., & Kokini, J. (2011). Green microfluidic devices made of corn proteins Lab on a Chip, 11 (20) DOI: 10.1039/C1LC20726A

Detecting Ovarian Cancer with a Cell Phone and a Microfluidic Chip

This post was chosen as an Editor's Selection for ResearchBlogging.org

Author's note: This post was chosen as an Editor's Selection at ResearchBlogging.org. Thanks for the support!

Ovarian Cancer

Ovarian cancer is the fifth leading cause of cancer related mortality among women. Like many diseases, there is a stark difference in survival rates depending on detection times. When ovarian cancer is detected at stage I, there is a 90% 5 year survival rate. Compare that with the 33% 5 year survival rate when the ovarian cancer is detected in stage III and IV. This disease is unfortunately asymptomatic at early stages, drastically eliminating the odds of discovery with enough time to make a difference.

While using traditional diagnostics like imaging, biopsy, and genetic analysis is impractical for regular screening, there are alternative methods used for women who are high-risk for ovarian cancer or who have family history. Transvaginal sonography can be used annually although it has been shown to have limited efficacy. Blood serum can also be tested to indicate ovarian cancer, but this method only has a sensitivity of 72% at specificity of 95%. Sensitivity and specificity are used to measure how well a system can detect something. To calculate specificity in our case, imagine 100 women without ovarian cancer are tested, and only 5 women are incorrectly told that they have ovarian cancer. This would undoubtedly be corrected in a follow up test. But to calculate sensitivity, imagine 100 women with ovarian cancer and 28 women are incorrectly told that they do not have it.

Not only are these tests inconclusive, they are extremely invasive. In the case of transvaginal sonography, an instrument is inserted in the vagina to check the ovaries. With blood serum testing, blood obviously must be drawn. Biochips currently exist to detect ovarian cancer based on protein biomarkers or DNA sequences, but these rely on fluorescence or chemiluminescence and are designed to be used in laboratory settings. None of the previous methods lend themselves to be used in point-of-care (POC) settings. An ideal POC device would not require expensive parts, be usable by limited trained personnel or be too complex. This would allow it to be used in resource-rich and resource-limited settings, especially if it does not need a continuous power source.

Detecting Ovarian Cancer with Urine

Researchers from Harvard Medical School have developed a cell phone system to detect ovarian cancer that should address the lacking areas of diagnosis so far. “Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care” was featured in the 2011 issue 11 of Lab on a Chip. Utkan Demirci et al. demonstrate a method to non-invasively detect ovarian cancer efficiently with urine and a cell phone. At the heart of this system is an enzyme-linked immunosorbent assay (ELISA). ELISA is a very common technique used in protein detection. In this case, a sandwich ELISA is used to detect the ovarian cancer biomarker Human epididymis protein 4 (HE4). Antibodies targeted to HE4 are conjugated to horseradish peroxidase which catalyzes a substrate and causes blue color to develop. We should then be able to ascertain the amount of HE4 originally in solution by quantifying the resulting color. This process takes place in three different microfluidic channels on a microchip the size of a stamp. These three channels allow a sample to be treated in triplicate or for many samples to be tested at once.

After the urine is loaded in the microfluidic chanel, ELISA is performed resulting in a colorimetric change

After the urine is loaded in the microfluidic chanel, ELISA is performed resulting in a colorimetric change

Cell Phone and CCD Imaging

Two methods were used to detect the change in color. The first method utilized a cell phone (more specifically Sony-Ericsson i790). This took advantage of the built in camera and processing power, allowing all processing steps to be carried out on the single device. The second method uses a lensless charge-coupled device (CCD). CCDs are found in digital cameras and have completely changed the way we capture images. In fact, the cell phone used has its own CCD inside. The CCD is used directly with a computer which analyzes the image with MATLAB. Both methods take a picture of the three microfluidic channels on the chip and compare the colors of the channels to previously measured standards.

Cell phone takes an image of ELISA results and compares the color to calibrated curves

Cell phone takes an image of ELISA results and compares the color to calibrated curves

Calibration and Testing

Before this system can be tested on actual samples, it has to be calibrated with known samples. HE4 was evaluated from 1,250 to 19.5 ng/mL, which was its detection limit. I’m unsure how much urine is actually needed. Each sample was diluted twenty times, and each channel can only handle 96.75 µL including the ELISA solutions. In order to make sure that ELISA was occurring correctly on the microchip, the colored solution was transferred to a 96-well microplate and the optical density was measured with a spectrophotometer. This was validated and a strong correlation between HE4 concentration and color was found for the CCD and cell phone with high R2 values above 0.90. After this calibration, the system was used to differentiate between the urine samples of 19 women with ovarian cancer and 20 women without ovarian cancer. The standard microplate technique and the cell phone and CCD methods were able to distinguish between the normal and cancer samples with statistical significance. When operating at a specificity of 90%, the cell phone and CCD tests achieved 89.5% and 84.2% sensitivity respectively. These results indicate that the new methods can efficiently and effectively detect ovarian cancer in urine.

Strengths

  • Both the CCD and cell phone methods demonstrated their ability to distinguish the difference between healthy and ovarian cancer urine.
  • These methods are extremely portable and can be used in a POC setting.
  • No complex machines or techniques are needed, which makes it cost-effective and allows operation by minimally trained personnel.
  • The low price of these tests makes them more accessible to be used to annually screen high-risk women or to check the efficacy of treatment.
  • Urine is an attractive diagnostic fluid because it is non-invasive and not intimidating.
  • It is unclear how well this could work in early detection at stage I of ovarian cancer because the samples used had later stage cancer. It is possible that the current configuration may not be able to differentiate between normal and early stage if HE4 levels vary between stages.
  • This test could be applied to other diseases with established biomarkers and sandwich ELISAs.

Further Development

  • It is unclear how well this could work in early detection at stage I of ovarian cancer because the samples used had later stage cancer. It is possible that the current configuration may not be able to differentiate between normal and early stage if HE4 levels vary between stages.
  • This test could be applied to other diseases with established biomarkers and sandwich ELISAs.

Reference:

ResearchBlogging.org

Wang, S., Zhao, X., Khimji, I., Akbas, R., Qiu, W., Edwards, D., Cramer, D., Ye, B., & Demirci, U. (2011). Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care Lab on a Chip, 11 (20) DOI: 10.1039/C1LC20479C

Clearing Sepsis with Magnetic Microfluidics

Sepsis

Sepsis is a big killer here in the United States. I know that I don’t really think about that in a normal day, but it’s the truth, and we can’t ignore it. As of 2005, it was the 10th leading cause of death and was just one of two infectious conditions listed in the leading 15 causes of death. Sepsis develops in 750,000 Americans annually, and more than 210,000 die. (That’s a mortality rate of 28 %!) Sepsis not only kills, but it’s accountable for $16.7 billion in annual economic burden. You can see why we need to focus on sepsis, but what is it exactly? Well, sepsis is a response by our bodies to systemic microbial infections. A range of pathogens can cause this reaction, and there is still no clear answer for all the effects on the body that are attributed to sepsis. In general, sepsis is believed to be caused by an infectious agent that compromises the immune system, leaving it unable to properly clear microbes. Treatments for sepsis have included antibiotics, recombinant drugs, membrane blood filtration and blood transfusions. However, these therapies don’t work effectively enough, and many patients die. Hemofiltration and hemadsorption have also been used to clear the blood, but these techniques can also non-specifically remove blood proteins such as cytokines, which are necessary to fight infectious agents. Whole blood transfusions are able to remove the pathogens, but at the expense of the patient’s own immune components and cells that are needed to keep fighting the infection. With all this stacked against us, what are we to do? Turn to a microfluidic therapy I guess.

If you have read a few posts here at Microfluidic Future, you know I love when microfluidics can be applied in therapy. The technology lends itself well to diagnostics, so any bit of therapeutics always piques my interests. A microfluidics device to combat sepsis has been outlined in “Micromagnetic-microfluidic blood cleansing device.” Donald Ingber et al. of the Wyss Institute describe a device capable of clearing the fungal pathogen Candida albicans from the blood. This work builds on previous efforts by Ingber and is 1000 times faster. The patient’s bloodstream could be hooked up to the device, which would clear the blood of the targeted pathogens.

Magnetic Opsonins

Magnetic_opsonins_bind_to_pathogen.jpg

At the heart of this system are magnetic opsonins. These are magnetic micro- or nano-beads that are bound to specific antibodies, in our case, they are anti-C. albicans. This device aims to remove the pathogens from the blood using these opsonins. The authors were able to achieve a binding efficiency of 80% with a bead to pathogen ratio of 120. Although 90% binding occurred between 30 and 60 minutes, about 50% binding was achieved after 5 minutes. The authors believed that a shorter incubation time can be achieved if the device were multiplexed. For example, if each of these devices were longer or attached to each other in series, we could overcome the efficiency of a single device. We could also use multiple devices working simultaneously in parallel, so the entire patient's blood could be processed faster.

Magnetic Microfluidic Cleansing

Clearing_sepsis_with_magnetic_microfluidics.jpg

The fundamental unit of this device is a set of layered channels that provides an interface between the patient’s blood and a carrier fluid. The two fluids are stacked vertically, with blood on the bottom. A solenoid electromagnet creates a magnetic field which draws the opsonins and their fungal passengers into the carrier fluid. The voltage of the electromagnet was optimized to provide a magnetic field that did not cause an overly strong attraction of the particles, which would have caused them to accumulate on the side of the channel instead of flowing with the carrier fluid.

According to the authors, this lab-on-a-chip performed relatively well in modularity and effectiveness in clearing C. albicans. . This device doesn’t involve much external infrastructure (besides the included electromagnet) and could easily function well in point-of-care therapeutics. To evaluate its performance, the authors attempted to clear C. albicans from 10 mL of human whole blood. The concentration of pathogens in this experiment was 106 fungi/mL. The system was able to clear 80% of the pathogen in 30 minutes, which is pretty good. This device may require some heparin to prevent clots within the device, but it would not result in lethal clot formations, hypoperfusion, shock and multiple organ failure seen in current therapies. Additionally, 82% of the opsonins that weren’t bound to the pathogen were cleared as well, which is great because I don’t think that you would want that flowing through your system. This actually leads me to some shortcomings I see in this project, and what I’d like to see in the future.

Improvements

  • To start, only 10 mL of blood was processed, and it took 30 min (20mL/h). In a 70 kg male with 4.9 L of blood, it would take about 10 days to process all the blood. But the authors are aware of this, and they’ve proposed additions in serial and in parallel to enhance the clearance efficiency and to do it faster.
  • During the filtration process, the authors noted that the blood and carrier fluid did not split perfectly at the end of the channels and a resulting in 50-60% loss of blood. This is obviously not good for the patient. The carrier fluid in that case was PBS, and the loss was reduced to 13% when the flow of the PBS was increased. With a ratio of flow between the fluids of four, the carrier fluid was able to force the blood to remain in place. But this comes at a cost, because this could imaginably hinder the opsonins’ migration from the blood into the carrier fluid, but the extent of this hindrance has not been quantified by the authors.
Blood loss in carrier stream

Blood loss in carrier stream

  • Although the volume of blood processed can be increased by adding layers to the system, I really don’t know if the authors tackled a clinically relevant level of C. albicans in the blood. I’ve been trying to find out a lethal level of C. albicans, but haven’t been able to find it. This would significantly impact the value of the system and would necessitate further multiplexing to achieve higher pathogen levels.
  • Finally, I’m not sure how the authors intend to deliver the opsonins to the blood. In this paper, the opsonins were simply incubated in the blood. In the graphics of the device on the cover of Lab on a Chip, the opsonins are introduced to the blood flow shortly before entering the device. This is possible according to the authors if proper multiplexing occurs and binding can be achieved within five minutes. Otherwise, it may be necessary to inject the opsonins into the patient and allow them to incubate before hooking them up to the lab-on-a-chip. But I’m not sure if that is such a good idea.
  • On a related note, I’m interested in the biocompatibility of the opsonins. The authors demonstrated that they bound rather specifically well to the pathogen, and the red blood cells were left untouched. Although the opsonins are quite small (which could make it worse) they could end up all over the body, giving Magneto even more control over you!

I mentioned previously that this paper followed up on previous work done by Ingber, so I think it is safe to say that we’ll be seeing the next iteration in some time. Conceivably, this device could be applied to other pathogens if other opsonins are developed, so this device has a lot of room to grow.

 

References:

ResearchBlogging.org

Melamed, A., & Sorvillo, F. (2009). The burden of sepsis-associated mortality in the United States from 1999 to 2005: an analysis of multiple-cause-of-death data Critical Care, 13 (1) DOI: 10.1186/cc7733

Hotchkiss, R., & Karl, I. (2003). The Pathophysiology and Treatment of Sepsis New England Journal of Medicine, 348 (2), 138-150 DOI: 10.1056/NEJMra021333

Yung, C., Fiering, J., Mueller, A., & Ingber, D. (2009). Micromagnetic–microfluidic blood cleansing device Lab on a Chip, 9 (9) DOI: 10.1039/b816986a

MicroTAS 2011 Oct 2-6

MicroTAS_2011.jpg

Tomorrow is the first day of MicroTAS 2011 (Oct 2-6), which is taking place in Seattle, WA this year. When I go to bed tonight, I’ll close my eyes and wish really hard, but I’m afraid I won’t open them to find myself magically transported to Rain City. Unfamiliar with MicroTAS? Let’s start with the name; MicroTAS (µTAS, micro-TAS or micro TAS) stands for micro total analysis system. This is simply another self-describing name for a lab-on-a-chip. A micro TAS should be a micro-sized device that contains all necessary steps to perform analysis of a sample. If you’ve read my other posts, this is nothing new, just a different name. Anyway, MicroTAS is a conference held every year on “Miniaturized Systems for Chemistry and Life Sciences.” µTAS is "the premier forum for reporting research results in microfluidics, microfabrication, nanotechnology, integration, materials and surfaces, analysis and synthesis, and detection technologies for life science and chemistry." The conference is in its 15th year and is back in the United States. It takes place every three years in the US and has been in Jeju, South Korea and Groningen, the Netherlands since it was in San Diego in 2008. This year’s conference is chaired by James Landers of the University of Virginia. His lab focuses on micro TAS topics like integrating functionality, fluidic control, genetic analysis and protein and small molecule analysis.

Like I said, I won’t be there, but I’ll still point you towards some key components of the conference, and will hopefully be able to give you some updates after the conference. There are a ton of different programs at MicroTAS 2011, and I invite you to check them out. This year, the conference will be presenting the following awards:

Art in Science Award

Cell Block 9 &nbsp;by Nicholas Gunn

Cell Block 9 by Nicholas Gunn

This is the award I’m most excited about. I think that images of micro-sized structures are really beautiful (with or without artificial colors), and I love to see them get recognition. Last year’s winner of this award was Nicholas Gunn of UC Irvine, and he had his image put on the cover of Lab on a Chip. His image is “a colorized SEM micrograph showing fibroblast cells cultured on microscale pedestals.” The pedestals prevented migration of the fibroblasts while still allowing the exchange of soluble factors. I can’t wait to see this year’s winner, and you can bet that I’ll post it here.

 

 

G4PZWS5H4TWS