Spin Silk Like a Spider! No Legs Required (Just Microfluidics)

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

Biomimetics

Biomimetics. I love that word. Well, probably not as much as microfluidics, but it’s a close second. If you’re unfamiliar with the word, it basically refers to design that mimics biology. Biological systems have evolved into finely tuned machines, why not mimic them in order to synthesize what we need? Biomimetics isn’t new, it’s been around in one form or another for a long time (my favorite instance is Velcro), but our capabilities are broadening as we are able to manufacture at smaller, micro and nano levels. If you want to learn more about this topic, you should check out the Biomimetic Microsystems Platform at the Wyss Institute. Today I’d like to share biomimetic microfluidic research that mimics the silk-spinning process of spiders from Korea University.

Mimicking Spider Silk

Sang-Hoon Lee et al.’s paper “Digitally tunable physiochemical coding of material composition and topography in continuous microfibers” showcases the process of finely tuned silk-spinning and its benefits in Nature Materials. There has been interest in using microfibers as a scaffold in tissue engineering, but there has been little success in achieving microfibers with varying chemical and morphological properties. The researchers have developed a process to continuously spin microfibers while varying their properties spatiotemporally. With a digitally controlled microfluidic chip, they are able to produce microfibers with varying chemical compositions, structures, gas bubble size and type, live cells and morphologies. The inspiration for this project comes from spiders. They can produce seven different types of silk that originate from different glands within the spider. As the need for one type of silk arises, fluid from that specific gland is used and the produced silk’s composition changes.

Lee’s spider-chip has several pneumatically controlled inlets that meet at a central point. The resulting flow is focused by a coaxial, sheath flow. In this case, the researchers chose to create alginate microfibers, although other materials such as chitosan and polyethylene glycol can be used. Since alginate requires Ca­+ to gel, the sheath contains CaCl2 to convert the stream into a fiber. In addition to focusing the resulting stream, the sheath also acts as a lubricant during the gelation process. Multiple microfibers can be produced continuously in at once and twisted together, which allows for parallel and serial encryption. This encoding could be used in biosensing, high-throughput screening and cell positioning for tissue engineering, but the researchers also wanted to tackle specific structures produced by spiders.

Silk spun by spiders can be mimicked by a microfluidic chip combining different streams. The streams used encode the microfibers in series or parallel when twisted with other microfibers.

Silk spun by spiders can be mimicked by a microfluidic chip combining different streams. The streams used encode the microfibers in series or parallel when twisted with other microfibers.

Water-Collecting Microfibers

By leaching salt originally included in the fibers, structures are created that attract the water on the microfibers

By leaching salt originally included in the fibers, structures are created that attract the water on the microfibers

Have you ever seen a spider web in the morning, laden with dew and wondered why the drops of water are spaced out instead of drenching the whole strand? Apparently there are puffs along the silk that attract the water along the silk. These puffs were mimicked by the spider-chip by alternating two different streams, one of which contained the salt CaCl2 . After the salt was leached from the structures, they had a higher surface energy than the rest of the microfiber, drawing in the water. The amount of water collected is dependent on the size of the puff, which is tunable and the researchers suggest that this approach could be used in water collection and purification.

Lending a Helping Hand to Neurons

Spider eggs are encapsulated with grooved silk, which was also reproduced in the spider-card with grooved channels. When embryonic neurons were placed on the grooved silk, their axons and dendrites ended up more aligned than those found on smooth microfibers. This might be a vital component in tissue regeneration after spinal cord or nerve damage. The researchers also demonstrated their ability to encapsulate gas bubbles within the fibers at different frequencies and bubble sizes. Gas bubbles within the microfiber might provide a temporary source of oxygen for cells encapsulated cells within the microfiber. Finally, Lee et al. encapsulated fibroblasts and hepatocytes and measured their viability. Unfortunately they only investigated cell viability for 5 days, which isn’t long enough to determine its practicality. They also encoded a microfiber with a chemoattractant and put it in a cell culture. The migration of cells toward the chemoattractant is evident, showing that a spatially encoded fiber could be useful in cell culture organization.

Grooved channels produce microfibers similar to the grooved silk that encapsulates spider eggs. The grooves enhance alignment of neuron dendrites and axons.

Grooved channels produce microfibers similar to the grooved silk that encapsulates spider eggs. The grooves enhance alignment of neuron dendrites and axons.

Expanding greatly on the current uses for microfibers, this advancement will hopefully push the use of microfibers forward. The ability to fine tune the properties of segments within a microfiber has its obvious benefits. But this process is also capable of rapid changes in the microfiber in real time. I’m not sure we have the same needs to quickly change the type of silk produced like a spider. But, I wonder what type of system would need to quickly respond to its environment with different microfiber properties. Any ideas?

Reference:

ResearchBlogging.org

Kang, E., Jeong, G., Choi, Y., Lee, K., Khademhosseini, A., & Lee, S. (2011). Digitally tunable physicochemical coding of material composition and topography in continuous microfibres Nature Materials, 10 (11), 877-883 DOI: 10.1038/nmat3108

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

Microfluidics... In Space?

Radins' microfluidic chip will allow astronauts to test for "blood sugar, liver and kidney function, and more." (Image source)

Radins' microfluidic chip will allow astronauts to test for "blood sugar, liver and kidney function, and more." (Image source)

What realm of life is safe from microfluidics? Hopefully none, as the technology continues to work its way into our lives. Although few us can say we have been to space, but those astronauts (or cosmonauts) up there now can look forward to a bit more microscale flow in the world.

I recently came across an article at Popular Science (which just happens to be one of my favorite websites) about the European Space Agency (ESA), which is planning on deploying microfluidics-based diagnostics into space. The ESA is working with an Irish company Radisens Diagnostics that will produce the device capable of diagnosing ailments in low gravity. The device will require a drop of blood and will test for “blood sugar, liver and kidney function, and more.” Apparently the company already has a similar device and will make some adjustments to make it space-ready. Like the best things that work in space, the device spins, which likely helps the flow in low gravity. The device is about the size of a matchbox, which must make ESA happy, as space and weight are tightly spent when planning trips to space. This lab-on-a-chip will keep the astronauts safer, as they often have to diagnose and treat themselves while in space. Out of anyone in the world, I'd say they have the greatest point-of-care needs. I wonder what will come first: My trip to space with microfluidics, or microfluidics finally finding its rightful place in our everyday lives.

Bloodhound Beads Sniffing out Heart Attacks

How would you detect a heart attack? There are some symptoms that might tell you that you are very likely having a heart attack. Although you might feel pain in the chest, shortness of breath or other known physical symptoms, that doesn’t mean you in are actually having one. Conversely, you may not experience these symptoms but an attack is well on its way. In addition to painful symptoms, an electrocardiogram can be used to further indicate if you’re having a heart attack, but it also isn’t always accurate. But what if you could detect a heart attack by monitoring cardiac specific biomarkers in the blood or saliva? Those attempts are well underway.

Detecting anything in bodily fluids is always a good time for our friend microfluidics to enter the scene. Even after the correct biomarker is established, it must be quantified in a quick and demonstrative way. While biomarker levels vary in statistically significant ways during disease, they may still occur at low levels, making detection very hard. John T McDevitt et al. fulfill this need in “A disposable bio-nano-chip using agarose beads for high performance immunoassays.” I actually worked in Dr. McDevitt’s lab while I was at Rice University, so I’m pretty familiar with his work, although I didn’t work on this project.

Agarose Beads

In this paper, Dr. McDevitt develops a system of agarose beads for immunoassays that can be implemented in a microfluidic system and tests it on C-reactive protein (CRP). CRP is a biomarker that is elevated during inflammation or heart attacks, which could make it useful when detecting or confirming a heart attack when used in conjunction with other biomarkers. This process uses a sandwich immunoassay, which we’ve seen before when detecting ovarian cancer. However, in this case, 280 µm agarose beads are used to house the immunoassay process. These beads are very porous, providing a large surface area to volume ratio, which affords many antibody-capturing locations to be stationed on and within the bead. Other beads made of polystyrene would only allow capture antibodies on the surface, and would not be able to detect as much of the biomarker in the same volume.

Protein-sensing Microarray

Microarray fabrication following PDMS mold creation from silicon master

Microarray fabrication following PDMS mold creation from silicon master

While the porosity of the bead allows the detection to be enhanced, the shape of the bead is also important in this immunoassay. This system is comprised of an array of microscale PDMS cups, each containing an agarose bead. The cups are shaped like inverted pyramids and feature a small hole at the bottom that allows for fluid to drain. This setup drives fluid through the beads by convection, as it is the only exit point for the fluid. In its simplest form, the system contains three stacked layers: the middle layer contains the microarray of inverted pyramids, which is surrounded by two PDMS layers containing the microfluidic injection and drainage channels. The middle layer is made of an epoxy, and actually originates from a lithography-produced silicon mold. The silicon mold looks exactly the same as the epoxy, but due to the cost and process to produce the silicon, it doesn’t make sense to use it in each system. Instead, it is used as a mold for PDMS, which itself is used to mold epoxy, giving us a copy of the original silicon. The resulting epoxy layer can be incorporated by irreversible covalent bonding to the PDMS layers.

CRP Detection with Agarose Beads

A CRP capture and flagging B CRP detection penetrating bead C CRP/fluorescence dose response curve

A CRP capture and flagging B CRP detection penetrating bead C CRP/fluorescence dose response curve

The agarose beads are first incubated with CRP specific capture antibodies, capturing the CRP as it is forced through the agarose beads. Detection antibodies are fluorescently tagged and are pumped through beads, specifically attaching to the anchored CRP. The fluorescence of the beads will correspond to CRP concentration in the sample. In order to calibrate the system properly, control beads are also included in the array which contain antibodies for another antigen. This system is able to produce a precise dose-response curve. Its limit of detection is about 1 ng/ml, which is far below the physiological range of CRP, but may be used while processing saliva, which must be diluted several times since it’s highly viscous. You can also see that the fluorescent green signal infiltrates the bead and is not limited to the surface. This demonstrates the porous agarose bead’s ability to capture more CRP and deliver a stronger signal.

Modelling the Microarray with Computational Fluid Dynamics

This system was also simulated using the computational fluid dynamics program COMSOL, and the results were similar to those found in the real world. Interestingly, there are some special considerations to think about when measuring the intensity of the beads. Not all flow through the array is equal. First, there is a bit of a pressure gradient across the beads such that the beads closest to the source experience the greatest pressure and those further away experience slightly lower pressure. The increased pressure causes more CRP and tagged antibodies through the beads resulting in a higher signal. Second, the size of the drain in each pyramid may vary, which would again affect the pressure experienced by each bead and its resulting intensity. Finally, any deviations in the bead shape will alter how it sits in the inverted pyramid. Once again, smaller beads sitting lower in the holes will experience higher pressures and signals.

Computational Fluid Dynamics simulates fluid flow and antigen capture

Computational Fluid Dynamics simulates fluid flow and antigen capture

Final Thoughts

This paper presents the final component in an immunoassay intended for microfluidic chips. While there are many other processes which must be carried out before the biomarker level can be measured, this method can also be used for a range of biomarkers and is only limited by the nature of the fluid and the concentration of the biomarker.

Reference:

ResearchBlogging.org

Du, N., Chou, J., Kulla, E., Floriano, P., Christodoulides, N., & McDevitt, J. (2011). A disposable bio-nano-chip using agarose beads for high performance immunoassays Biosensors and Bioelectronics, 28 (1), 251-256 DOI: 10.1016/j.bios.2011.07.027

PDMS: The Favorite Material of Microfluidics (for now)

Precise microfluidic channel in PDMS

Precise microfluidic channel in PDMS

Whether you’ve been learning about microfluidics here at Microfluidic Future or somewhere else, you’ve undoubtedly come across the elastomer poly(dimethylsiloxane) (PDMS). PDMS has radically changed the capabilities of microfluidics (and its price tag) since it was first brought into microfluidics by George Whitesides in 1998. PDMS has effectively replaced glass and silicon which were borrowed from existing micromachining industries. PDMS has great resolution and can contain sub-0.1 µm features. But how is PDMS used, and what makes it so great? Hopefully you’ll have these answers by the end of this post.

PDMS Fabrication

PDMS fabrication process, beginning with photolithography

PDMS fabrication process, beginning with photolithography

Soft lithography is a widely used technique to create PDMS structures. This process first requires a mold, or a master, generally produced by photolithography. The mold must represent the hollow space of the microfluidic chip, where the fluids will eventually flow. PDMS is formed by mixing one component which contains silicon hydride groups and another component which contains a vinyl group. This mixture is then poured on top of the master. PDMS cures to a solid after an hour at 70o C, and can then be peeled away from the master without damage to either part, allowing the master mold to be reused. The earlier steps can be completed within 24 hours, and replicas can be mass produced after designing and creating a master.

After removing the PDMS replica, we have an open space in the structure from the master, but our channels have no bottom! We next have to seal the PDMS to another surface, such as glass, silicon, or itself. More importantly, PDMS can be sealed reversibly or irreversibly.

Van der Waals forces allow reversible sealing to smooth surfaces. This seal is airtight, but can only withstand pressures around 5 psi. Adhesive silicone or cellophane tapes can also be used to create a reversibly seal that is stronger than PDMS alone. Reversible seals allow for reuse or added functionality. Remember that the PDMS used in the SIMBAS lab-on-a-chip was sealed reversibly to the glass, allowing reuse or future analysis.

In some cases, a lab-on-a-chip may require an irreversible seal, especially if operating under higher pressures. PDMS can be irreversibly sealed by exposing it (and possibly the other surface) to oxygen plasma. However, the two pieces of chip must be aligned within a minute or else the oxidized PDMS surface will reconstruct in the air.

Complex PDMS structure "sandwiching" a membrane between two layers

Complex PDMS structure "sandwiching" a membrane between two layers

Complex structures can be created by “sandwiching” multiple layers of PDMS. Furthermore, additional components like membranes can be included in this sandwich. This can be a bit of a challenge depending on the complexity of the features, and micro-stages have been created to ensure perfect alignment after oxidation. Alternatively, PDMS can be sealed with the aid of polar solvents. A thin film of a polar solvent is placed between PDMS layers and then heated, evaporating the solvent and sealing the layers.

Instead of using photolithography to create the mold, 3D printing can also be used. 3D printing prints small globules of material according to a CAD file to make 3D structures. 3D printing itself has been growing in capabilities due to its extremely quick prototyping speed. It can achieve a resolution between 50 and 100 µm, which is suitable for microfluidic devices. This mold would then be used in the same way to create PDMS replicas.

Microfluidic Components

Microfluidic chips can be very complex, featuring pumps, valves and mixers. These parts can easily be implemented in a PDMS structure. Mixers can be incorporated into the masters, transferring them to the floor of a channel. Other parts that are made separately, like some of the microvalves I discussed previously, can be placed in the PDMS while curing. This allows a single master to be very versatile while moving independent features about the device.

Key Features

Here are some key features of PDMS adapted from “Poly(dimethylsiloxane) as a Material for Fabricating Microfluidic Devices” by Whitesides.

Property

Characteristic

Consequence

Optical

transparent; UV cutoff, 240 nm

optical detection from 240 to 1100 nm

Electrical

insulating; breakdown voltage,

2 x 107 V/m

allows embedded circuits; intentional breakdown to open connections

Mechanical

elastomeric; tunable Young's modulus, typical value of

~750 kPa

conforms to surfaces; allows actuation by reversible deformation; facilitates release from molds

Thermal

insulating; thermal conductivity, 0.2 W/(m*K); coefficient of thermal expansion, 310 µm/(m*°C)

can be used to insulate heated solutions; does not allow dissipation of resistive heating from electrophoretic separation

Interfacial

low surface free energy

~20 erg/cm

replicas release easily from molds; can be reversibly sealed to materials

Permeability

impermeable to liquid water; permeable to gases and nonpolar organic solvents

contains aqueous solutions in channels; allows gas transport through bulk material; incompatible with many organic solvents

Reactivity

inert; can be oxidized by exposure to plasma

unreactive toward most reagents; surface can be etched; can be modified to be hydrophilic and also reactive toward silanes; etching can alter topography of surfaces

Toxicity

nontoxic

can be implanted in vivo; supports mammalian cell growth

Some clear advantages and unique properties can be seen from this table. I would also like to point out some additional advantages and disadvantages.

Advantages

  • Quicker production time than glass and silicon microfluidic chips
  • Cheaper than glass and silicon

Disadvantages

  • Unless oxidized or treated, the surface of PDMS is hydrophobic which can encourage air bubbles to form in channels and protein adsorption
  • Elasticity restricts aspect ratio of structures as sagging/shrinking can occur.

 This is one part of my Microfluidics Beginer’s Guide. Check out the rest of it and keep learning!

Reference:

ResearchBlogging.org McDonald, J., & Whitesides, G. (2002). Poly(dimethylsiloxane) as a Material for Fabricating Microfluidic Devices Accounts of Chemical Research, 35 (7), 491-499 DOI: 10.1021/ar010110q

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 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.

 

 

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The Microvalve: The Traffic Light of Microfluidics

You could say that valves in microfluidics (or microvalves) are like traffic lights that control flow along microfluidic channels. But I’d say that they’re more like police barricades, stopping anyone they want, wherever they want. The sole purpose of microvalves is to control flow within a microfluidics device, allowing them to become very complex and more automated. Without microvalves, all reactions and mixing must occur in the same space, unless they were premixed elsewhere, which might just eliminate the advantage of microfluidics.

Every microvalve that has ever existed seems to be covered in “A review of microvalves” by Kwang W Oh and Chong H Ahn from the Journal of Micromechanics and Microengineering. I’ll be covering three of the valves mentioned, but feel free to check out all the other microvalves that didn’t make the cut. A companion paper for this post, “Incorporation of prefabricated screw, pneumatic, and solenoid valves into microfluidic devices” by George M Whitesides et al. , featured in Lab on a Chip, describes a way to prefabricate these three valves so that they can easily be added to existing lab-on-a-chips.

Solenoid Microvalves

Prefabricated Solenoid Microvalve

Prefabricated Solenoid Microvalve

You guessed it, solenoid microvalves are based on…solenoids! A solenoid is a coil of wire wound in a helix, like a compressed spring. I won’t go into the physics behind it, but a magnetic field is created by passing current through the solenoid. If you put a metallic object within the coil and vary the current, you could move the object. This is the idea behind solenoid microvalves. A solenoid microvalve is simply a solenoid with an actuator inside of it. The actuator is situated above the channel of a microfluidic device and pushes down on the ceiling of the channel to collapse it and obstruct its flow. In this manner, the solenoid microvalve requires an elastomeric lab-on-a-chip, and is rather bulky.

Whitesides’ Prefab:

Whitesides’ prefabrication does not involve a dramatic redesign of solenoid microvalves. Instead, he places the same solenoid in PDMS housing, which can accommodate the solenoid’s large footprint.

Screw Microvalves

Screw Microvalve

Screw Microvalve

This is another one. You guessed it, microvalves are based on…screws! This is an extremely low tech microvalve that requires little more than a screw. The screw is incorporated into the microfluidic device and deflects the membrane of the channel by twisting it. A ball can be placed beneath the screw to prevent damage to the device. This design is great for lab-on-a-chips that are intended to be disposable or for a low-resource setting. It requires no power, has a small profile (although you need access to screw it manually) and is simple to use. Obviously, it’s not completely automated, but I suppose you could incorporate a small electric screwdriver somewhere.

Whitesides’ Prefab:

Whitesides’ prefabrication method doesn’t really differ too much from the conventional screw microvalve design. The screw is essentially embedded in a housing which can be joined above an existing microfluidic channel. One of the key advantages argued by Whitesides is that prefabrication regulates the screw microvalves. All the prefabricated screw valves are created in an identical manner, and would require the same degree of rotation to shut off flow. This is important because it may not be possible to see how well the flow is obstructed, resulting in leakage or damage to the channels. Screw valves created by different people at different times may be placed differently. A half-turn of one person’s valve might not properly close the channel, while a half-turn of someone else’s valve might go too far and damage the device.

Pneumatic Microvalves

Quake Microvalve (source)

Quake Microvalve (source)

I know it says pneumatic, but these are commonly referred to as Quake Valves, named after Stephen Quake. Quake valves require additional channels, often perpendicular to the targeted microfluidic channels. The additional channels share a thin, common membrane with the targeted channels. When air at the right pressure is applied through the pneumatic channels, the shared membrane is deflected and obstructs the flow of fluid. This completely changed the field of microfluidics since its arrival in 2000, allowing such feats as 400 simultaneous PCR reactions.

As cool as these microvalves are, they certainly have their drawbacks. First, they require more planning because you not only need to incorporate an additional layer of pneumatic channels, but you also have to route all your channels so that they don’t overlap where you don’t want them to. A small change in the design of your microfluidic device could require a massive redesign. While the solenoid valve certainly has a large footprint over the lab-on-a-chip due to the large size of the solenoid, the Quake valves only require the inclusion of an additional layer, keeping the area directly around the device cleaner. But this setup requires a tank of pressurized gas near the device, so in reality its footprint isn’t so small. This obviously hinders a device from easily leaving the lab. The pneumatic valves can be controlled electronically, allowing a device consisting of multiple independent valves to become more automatic. For instance, Albert Folch of University of Washington created this video of a Microfluidic Ballet by controlling valves according to the frequency of the music by Dimitri Shostakovich.

Whitesides Prefab:

Prefabricated Quake Microvalve

Prefabricated Quake Microvalve

Whitesides’ prefabrication of Quake valves diverges from the most from the conventional out of all three microvalves shown here. Simply consisting of a chamber attached to an air supply placed over a channel, this method doesn’t require any additional layers of valves. A conventional Quake valve would require an entire redesign of the pneumatic channel layer, while a prefabricated Quake valve can simply be relocated over a new channel. When activated, the chamber presses down on the channel wall and obstructs the flow.

Final Thoughts

We’ve only examined three different microvalves here, and you can see how radically unique they are. As we develop new microfluidic applications and materials, we’ll continually develop novel ways to control the flow. A high concentration of valves indicates greater device design sophistication, but even so, less can be more, and our best solution might just be the simplest one.

This is one part of my Microfluidics Beginer’s Guide. Check out the rest of it and keep learning!

 

References:

 

ResearchBlogging.org

Oh, K., & Ahn, C. (2006). A review of microvalves Journal of Micromechanics and Microengineering, 16 (5) DOI: 10.1088/0960-1317/16/5/R01

Elizabeth Hulme, S., Shevkoplyas, S., & Whitesides, G. (2009). Incorporation of prefabricated screw, pneumatic, and solenoid valves into microfluidic devices Lab on a Chip, 9 (1) DOI: 10.1039/b809673b

SIMBAS, Everything the Blood Touches Is Our Kingdom

SIMBAS_Lab_on_a_Chip_Cover.jpg

Hey, how’s your biotin? What? No it’s not an organic metal, maybe you call it B7? You’re probably fine, but have you been depressed, lethargic or losing your hair lately? Biotin is pretty important; it’s necessary for metabolism within our cells, so I make sure I never leave home without it. It’s rare for someone to have a biotin deficiency, but if you want to know your levels, give me a drop of your blood, and I’ll have an answer from you in 10 minutes. How? Oh just my self-powered integrated microfluidic blood analysis system (but I like to call it SIMBAS for short).

The SIMBAS emerged from Berkeley and was featured on the inside cover of the 2011 Issue 5 of Lab on a Chip. Luke Lee et al. describe a device capable of picomolar detection in “Stand-alone self-powered integrated microfluidic blood analysis system (SIMBAS).” The device can analyze whole blood without a lot of bells and whistles to get in the way. I’m not saying that this doesn’t have a clever design; it just doesn’t have a battery, moving parts or specialized readers. All you need is some blood and a microscope. The device has five independent analysis streams. There are three parts to a stream, the filter, detector and the suction chamber. All of these components are featured in a slab of PDMS sandwiched between two normal glass slides. Before I describe how the features physically work together, it’s important to note the device’s driving force: low pressure. Before use, the SIMBAS is placed in a low-pressure vacuum, degassing it through the single points of entry of each stream, creating a vacuum. When blood is placed at the inlet the vacuum slowly draws it through the system.

Filtration

The degassed chip draws in blood towards the dead-end suction chambers. The wells filter the blood before passing the detection strips.

The degassed chip draws in blood towards the dead-end suction chambers. The wells filter the blood before passing the detection strips.

After the vacuum sucks in the blood, the red and white blood cells must be filtered out. One major strength of this device is that it receives unadulterated blood. However, in order to prevent the cells from muddling the detection region of the device, they are filtered out by sedimentation. The floor of the 80 µm high, 50 µm wide sample channel opens up to a 2 mm wide, 2 mm deep circular well, which acts as a filter. The large, heavy cells sediment out and take up permanent residence at the bottom depending on the flow rate of the sample. The biomarkers of interest are much lighter and will continue through without joining the blood cells. This produces plasma that is 100% blood cell free, ready for detection. Platelets sediment at lower rates, so they will still remain in the plasma, but this doesn’t seem to be an issue.

Detection

Now that the sample has been fully prepared, it is ready for detection. Remember that the PDMS was placed between two glass slides? The authors immobilized streptavidin bars on the underside of the glass roof. This leaves the streptavidin attached to the ceiling, letting it bind with the biotin that flows by. Streptavidin and biotin have one of the strongest known protein-ligand bonds, making it a perfect choice for this device. When tested, blood was spiked with fluorescently-labeled biotin at 1.5 pM. Many other detectors could be immobilized along the same stream or the other four streams. The five parallel streams allow a way to eliminate errors, or test for biomarkers that are not compatible with each other.

Volume Control

However, we’re still missing the last important feature, the suction trough, an empty region after the detectors. The volume of the trough determines how much sample is drawn through the system. Once the trough is full, flow stops, regardless of how much blood is ready to enter. Modifying the trough volume provides a method to increase the sensitivity of the device. A larger sample volume gives the SIMBAS a better chance to detect a biomarker at low concentrations.

Final Thoughts

Detection of biotin at 1.5 pM, 150 pM, 15 nM & 1.5 µM

Detection of biotin at 1.5 pM, 150 pM, 15 nM & 1.5 µM

The current setup only needs 5 µL of whole blood for each stream. To put that into perspective, modern glucometers need 1 µL at most to glucose levels. It is (relatively) a bit more blood, but you wouldn’t be doing this every day. After 10 minutes, biotin at 1.5 pM can be detected by removing the top slide and looking at it under a microscope. That’s a pretty low detection in my book. Crazy low. I’m sitting here trying to think of an accurate needle in a haystack analogy, but it’s not coming. Overall, this is a pretty innovative, yet simple device, and I’ll tell you what I think of its merits and things I’d like to see developed.

Merits:

  • To start, this is has a great design for a point-of-care device, especially in a resource-poor setting. It has no external or moving parts, and requires no power. Sometimes in microfluidics, things can get very complicated with the number of channels, pumps, reagents etc., but this has a very clean and trouble-resistant design. It can be pre-packaged under low pressure so that a user only has to open it to activate the vacuum and use it within a couple of minutes.
  • The fact that it can receive whole blood also makes it great for point-of-care. Some lab-on-a-chips actually depend on many sample preparation steps or external machines. But all the steps that are needed for it to do its job in isolation should be included, just like SIMBAS. The time between filtration and detection is pretty quick, which is important because the proteins found in the plasma change after longer separations.
  • This doesn’t require much sample, and is still able to detect biotin at the low concentration of 1.5 pM. I’m not sure how clinically relevant that number is for biotin, but if you can detect that, you can play with the configuration to bring that down (or raise it much more easily).

What I’d like to see:

  • The authors state that the biotin-streptavidin detection could be replaced by many other couples. I don’t doubt this, but I would like to see what detection levels they could achieve, since biotin-streptavidin has one of the strongest protein-ligand bonds.
  • In the paper, the authors mention that the detection strips coated on the ceiling were applied on the same day the assay was run. I’d like to know what the shelf life of a prepared card would be, which could really impact their usefulness and value.
  • As I mentioned previously, the authors detected fluorescent-tagged biotin in the blood, and examined this under a microscope. You can see the captured biotin fluorescing in their figure. But biotin isn’t naturally tagged with a fluorophore, which makes me wonder how they would normally detect a wild biomarker. Perhaps there is a noticeable difference under the microscope; otherwise they will need to introduce some tag in the blood that will act as a secondary binding agent.
  • The last thing I’d like to see is the microscope. Well, I’d like to see it removed. This microfluidics system would be even greater if it was completely stand-alone. Daniel Fletcher (also from UC Berkeley) has developed a cell phone-powered field microscope capable of fluorescence microscopy, so it isn’t impossible in a resource-poor setting, but it’s just my wishful thinking.

Reference:

ResearchBlogging.org

Dimov, I., Basabe-Desmonts, L., Garcia-Cordero, J., Ross, B., Ricco, A., & Lee, L. (2011). Stand-alone self-powered integrated microfluidic blood analysis system (SIMBAS) Lab on a Chip, 11 (5) DOI: 10.1039/C0LC00403K