Narrowing the Gap to Characterize Sickle Cell Disease

Microfluidic Future is by no means an accurate representation of all the current, ongoing research in microfluidics. Nevertheless, the fact that you won’t be able to find any articles about assays relying on a biophysical marker isn’t too far off the reality in microfluidics. I suppose this is partly due to the incredible amount of previous work on molecular markers when high resolution control hadn’t been realized yet. Regardless, I was happy to come across an article about a microfluidic device that indicates sickle cell disease risk using the disease’s biophysical characteristics. The work “A Biophysical Indicator of Vaso-occusive Risk in Sickle Cell Disease” appeared in Science Translational Medicine this past February and is a result of ongoing sickle research by MIT and Harvard Medical School. My friend originally forwarded me an article about it on Medgadget, which you should also check out, along with the podcast it mentions.

Sickle Cell Disease

Red blood cells with abnormal Hemoglobin (HbS) can form into a sickle shape and occlude blood vessels (image  source )

Red blood cells with abnormal Hemoglobin (HbS) can form into a sickle shape and occlude blood vessels (image source)

Sickle cell disease affects more than 13 million people worldwide and is responsible for $1.1 billion in costs per year in the United States. A mutation in the hemoglobin molecule causes red blood cells to change shape and stiffen when releasing oxygen. This shape change in many red blood cells can occlude a blood vessel, resulting in a crisis. While this fundamental component of the disease is known, there are many factors and processes relating to this event that are still unknown, resulting in an inability to discern the severity of sickle cell disease for a particular patient, besides the fact that they have it. The ability to predict the severity of the sickle cell disease would both aid the development of new therapies and guide clinical intervention.

Characterizing Disease Severity

The authors of this paper have previously demonstrated that they could simulate the vaso-occlusive crisis events by altering the oxygen concentration of sickle cell disease blood flowing through a capillary-sized microchannel. This paper takes it a step further and quantifies how the blood conductance, defined as velocity per unit pressure drop, changes during the events and uses it as a measure of disease severity. When the authors reduced the oxygen content, blood velocity would decrease, despite the constant pressure applied. The authors hypothesized that the conductance would change faster for patients with severe sickle cell disease as opposed to patients with a more benign form of the disease. You can see that the conductance of a patient with benign sickle cell disease (A) and that of a patient with severe sickle cell disease (B) are drastically different.

Blood from a patient with benign sickle cell disease (A) and sever sickle cell disease (B) were both subjected to changes in oxygen concentration, indicated by the top panels. This resulted in drastically different changes in conductance, which could distinguish the two types of patients.

Blood from a patient with benign sickle cell disease (A) and sever sickle cell disease (B) were both subjected to changes in oxygen concentration, indicated by the top panels. This resulted in drastically different changes in conductance, which could distinguish the two types of patients.

Device Value

As I mentioned, this device has potential use in developing therapies for sickle cell disease. The authors demonstrated this with 5-hydroxymethyl furfural (5HMF), which is known to increase hemoglobin oxygen affinity. Hemoglobin with a higher oxygen affinity would retain its ‘safe’ structure as it would release its oxygen less readily. As expected, this molecule caused a fivefold slower reduction in conductance change compared to an untreated, severe blood sample. While this device’s strength originates in its focus on biophysical markers, it could also be utilized to further understand the process of vaso-occlusive events and guide the handling of patients and discovery of effective therapies.

Regardless of the praise this paper has already received, I think it’s rather solid, and I’m not sure what else I would have liked to see addressed. Don’t expect to see this in your local pharmacy any time soon, though, since it can’t predict the occurrence of crises, but instead would indicate what treatment a patient would need.

Reference:

ResearchBlogging.org Wood DK, Soriano A, Mahadevan L, Higgins JM, & Bhatia SN (2012). A Biophysical Indicator of Vaso-occlusive Risk in Sickle Cell Disease Science Translational Medicine, 4 (123), 1-8 PMID: 22378926

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

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

Microfluidics? What's That? A Beginner's Guide

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

 

Microfluidic chemostat used to study microbes

Microfluidic chemostat used to study microbes

I don’t quite have the resources to poll the United States and the rest of the world, but if I did, this is what I’d ask:

  1. Do you know what microfluidics is?
  2. Can you explain it to me?
  3. Do you currently use anything with this technology?

We may never know the results of the poll, but I think I'd hear "No" and "What is microfluidics?" Have no fear, because today you’re lucky enough to read my Beginner’s Guide to Microfluidics.

To start with, let’s look at the word itself. It is the word fluidics with the prefix micro- strapped onto the front. Thus, we’re looking at the manipulation of fluid at the micro scale, often flowing through channels. There isn't a strict definition, but some like to refer to flow with a channel dimension less than 1,000 µm as microfluidics. It's more complicated than that, so to guide us, I’ll be referencing heavily from a review article George M Whitesides wrote in 2006, “The origins and the future of microfluidics.” For those unfamiliar with Dr. Whitesides, he is a legend in many spheres including microfluidics, and if you are reading a paper on microfluidics, it most likely either cites him, or can be traced to something that does. In this particular article, he looks at four “parents” of microfluidics that have brought us to the technology we have today:

  1. Microanalytical methods
  2. Biodefense
  3. Molecular biology
  4. Microelectronics

Microfluidics’ Loving Parents

Microanalysis

Microanalysis is the chemical analysis and identification of small amounts of matter, such as capillary electrophoresis. First developed in the 1960s, it is a method to separate ionic species using their size to charge ratio. Capillary electrophoresis is still used today, showing that microanalysis not only provided needs for microfluidics to meet, but it also provided some techniques.

Biodefense

Awareness of chemical and biological warfare escalated after the Cold War, fueling the United States’ Defense Advanced Research Projects Agency (DARPA) to heavily fund microfluidics research in the 1990s. Biodefense remains a major area or research in homeland defense as demonstrated by the recently unveiled microfluidics-based device that detects anthrax or other suspect DNA. The suitcase-sized system recovers cells from a sample and analyzes the DNA in less than an hour, while it currently takes 1 to 3 days in the lab. 

Molecular Biology

In the past few decades, genomics activity has progressed quickly and has demanded newer technology to grow even further. Microfluidics had the opportunity to process DNA faster, cheaper and more precisely. Stephen Quake, a professor at Stanford University, has been leading cutting-edge research in sequencing. The ability to sequence DNA in weeks rather than months has allowed him to start a company, Helicos, implementing microfluidic methods. There have been several microfluidic applications for sequencing as researchers and companies race to sequence a human genome for only $1,000.

Microelectronics

While the three previous “parents” have provided some tools and needs for microfluidics, the final “parent” helped it find its form and substance. Microelectronics was also developing at the time (although it was further along), and people saw a way to manufacture micro-sized structures precisely. Microfluidic components were first made of silicon and glass with microelectronics techniques such as photolithography. Eventually, silicon was replaced by materials with more suitable properties. Silicon is not durable, cheap nor transparent to visible or ultraviolet light. This is important because light is often used to either determine the results of a test, or to or to provide excitation energy. The most popular substitute material has been PDMS (polydimethylsiloxane). PDMS is a soft, transparent elastic polymer that is permeable to carbon dioxide and oxygen, making it useful for housing cells. Other cheaper, alternative materials have also been developed, such as thermoplastics, paper and string.

Advantages of Microfluidics

These four parents have given birth to a field that focuses on biochemical and medical applications, and with good reason. While everyone loves small things (I’m sure that micro is sad that it never got its time in the sun before the world became obsessed with nano), there’s more to it than that. A microfluidic device does have a smaller footprint than a huge machine in a lab, but it also uses a lot less fluid too. Why would you make a whole pot of soup just to taste and then dump it down the drain? On the other hand, I would make as little as I could and still taste it. So we can see that microfluidics is beneficial because it uses less costly chemicals and reagents, but it's also attractive due to the physics happening at the micro scale. Now, we’re not talking about leaving the world of classical physics and entering quantum physics, but matter does behave differently, most evidently in the way liquids flow.

Turbulent flow vs Laminar flow

Turbulent flow vs Laminar flow

Laminar flow around air bubbles ( source )

Laminar flow around air bubbles (source)

In fluid mechanics, there are two main types of flow: laminar and turbulent. Turbulent flow is just how it sounds, violent and chaotic. Laminar flow is peaceful, and particles will basically maintain their course without mixing. The state of a flowing liquid is determined by its Reynolds number. This is proportional to the dimensions of the channel and the velocity of the liquid. Slower liquids travelling in narrower channels are more likely to be laminar. This not only makes pretty images like those from Albert Folch’s lab, it gives a new level of control to researchers. Two laminar fluids flowing side-by-side will only slowly mix by diffusion, rather than convection. This allows us to do some clever manipulations, like selectively inserting components to induce mixing in some of the streams in a multi-stream channel. Other techniques exist to create droplets, which allow for controlled mixing, and capillary action is utilized to eliminate the need for pumps.

Microfluidic Applications and Issues

Paul Yager  with a lab-on-a-chip that  detects malaria

Paul Yager with a lab-on-a-chip that detects malaria

Microfluidics can be incorporated into a lab-on-a-chip which is used as an assay or diagnostic test and replaces its large, costly full-size lab counterpart. This has three basic components, a sample, a process and a validation method. For example, you might input a fluid such as saliva or blood to be analyzed. Next, you’ll have to process this blood mechanically or chemically. This may mean mixing it or applying filters to isolate what you want, and then introducing a possible chemical reaction. Even so, what good is this reaction if you can’t tell if it happened? You may be able to see a change in color, or look at it under a fluorescent microscope. These three components can be fulfilled in dozens of ways to create whichever test you so desire.

You must be saying to yourself, “Wow! What a technology! So why haven’t I heard more about this if it can do so many magical things?” Well, space travel is cool too, but when was the last time that you were out in space? Microfluidics just hasn’t made its breakthrough to the rest of us consumers. We can create some pretty cool applications with it, but how often can those prototypes be easily mass-produced? And how easy will they be able to use and troubleshoot and fix? Both concerns are even more important for point-of-care applications. Point-of-care refers to a medical application that is being used right where the caregiver meets the patient. In the developed world, this means that your samples don’t have to be sent to a lab somewhere to be processed, and in the developing world, this gives access to life-saving diagnostics that are normally too expensive to purchase and operate. More importantly, the quick response time of the devices would provide results to people who have travelled to a far-away clinic and need to return home again. No matter how elegant or complex a microfluidic device is, it must be easy and cheap to make, simple to use and durable.

Now, I don’t want it to seem like there are no companies producing microfluidics-based technologies. Some relevant ones are Helicos, FluidigmClaros DiagnosticsMicroFluidic Systems, Affymetrix and Advanced Liquid Logic. If you want to see a more complete list, check out the one at FluidicMems. I hope that I’ve helped you to understand the basics of microfluidics, and you can continue to learn about the field on this site. Oh, and if you were wondering how long it will be until you can get your hands on some microfluidic tech, you probably already have: your inkjet printhead incorporates microfluidics!

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

Reference:

ResearchBlogging.org Whitesides, G. (2006). The origins and the future of microfluidics Nature, 442 (7101), 368-373 DOI: 10.1038/nature05058

Decoding Liquids Before Your Very Eyes

Seeing really is believing. How often can we tell what a liquid is by just looking at it? Not too often. Sure, you might be able to tell when you definitely smell something sulfurous, or have a slippery base and I hope you can pick out milk. But we’re not always that lucky, especially if you’re dealing with something you really shouldn’t be touching or directly smelling. There are a ton of tests we can run to pinpoint what it is and you often need a pro to decipher the results. Ideally you could just read it with your eyes. With a batch of research from Harvard University, we’re one step closer.

The paper entitled, “Encoding Complex Wettability Patterns in Chemically Functionalized 3D Photonic Crystals” was featured in the August issue of the Journal of the American Chemical Society. The lead authors Ian B. Burgess and Joanna Aizenberg, of the Wyss Institute, propose a process to functionalize crystals so that they can differentiate between different fluids.

3D_Pores.jpg

First, these aren’t just any of crystals. These are 3D porous photonic Inverse Opal Films (IOF). They were carefully created to maintain a specific structure. The ability to discern between different fluids is possible due to selective application and erasure of different chemicals. First, a functional group is applied to the surface of the IOF. A slab of PDMS (a silicon-based polymer) is sealed to an area of the IOF. O2 plasma is applied and erases the functional group except the area covered by the PDMS. This can then be repated with a second functional group, and so on and so on. There can even be overlapping areas covered to give you exactly what you need.

W-Ink_Decoding.jpeg

But what’s the point of functionalizing the surface? Well the functionalization affects the wettability of the IOF. Given the surface’s wettability and the surface tension of a liquid, the liquid won’t be able to penetrate the channels. There can be a clear change of color in the infiltrated regions. The authors refer to their system as Watermark-Ink (W-Ink) and suggest that it could be used as security measures. But I think I’d rather see it used in microfluidics. While you may toy with the notion that you could selectively choose which fluids flow through certain channels and partake in reactions, I think it is most useful as an indicator. Many microfluidic devices are intended to be used as point-of-care (POC) diagnostics. A sample is analyzed for a certain component that would indicate something about the patient's health. But at the end of the test, you have to be able to tell if the reactions were positive or negative. I think that the test could be designed to produce two fluids with different surface tensions, depending on the outcome. Then, one of two shapes would appear. It would require no confocal microscope or camera, and certainly wouldn’t need translation.

Reference:

ResearchBlogging.org

Burgess, I., Mishchenko, L., Hatton, B., Kolle, M., Lončar, M., & Aizenberg, J. (2011). Encoding Complex Wettability Patterns in Chemically Functionalized 3D Photonic Crystals Journal of the American Chemical Society, 133 (32), 12430-12432 DOI: 10.1021/ja2053013