Diagnosing Similar Diseases in Low Resource Settings

Diagnostics in Low-Resource Settings

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

Malaria and Typhoid Dual Detection

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

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

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

Immunoassay Process

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

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

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

Results

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

Discussion

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

Reference:

ResearchBlogging.org

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

A Case for Oral Diagnostics with Microfluidics

What’s So Great About Oral Diagnostics?

Well, a lot of things, but let’s start with the basics. In order to use a microfluidic device, you need some type of fluid right? Sure if you had some powder or fine material you could suspend it in a fluid, but for simplicity sake, let’s look at fluids as our test material. If you wanted to run a health-related diagnostic, you only have so many bodily fluids available before you have to get creative and very invasive:

  • Blood
  • Urine
  • Saliva
  • Sweat
  • Mucus
  • Tears

Out of all those fluids, blood (or serum) has been the preferred liquid. It is extremely rich in information and can expose a lot about a systemic condition or report on ailments located deep within the body. You have to filter it if you don’t want the blood cells in your sample, but it’s just a needle prick away. Other ‘fluids’ like mucus or saliva require a bit more work because of how thick and viscous they are, plus you need to filter out the debris floating around in your mouth. If blood is so great, why do we need anything else? Although blood is a great global fluid, sometimes you can get more detailed information by going closer to the source of the problem and choosing a more local fluid, but perhaps one of the greatest reasons is because the process to obtain the blood is still invasive. In the ideal microfluidics world of the future, we would need very small sample sizes and pin pricks wouldn’t be that bad. For now, spitting into a cup is still easier than and more enjoyable than getting stuck. Plus, exposed blood is always a health concern, and should definitely be avoided if possible.

Okay, so I guess you can see why you might want to investigate other fluids, but why saliva? Like I said before, there are some analytes from the blood present in saliva in lower concentrations, like C-Reactive Protein (CRP). In fact, tests have been proposed to monitor growth factors, drugs of abuse, steroids and infectious diseases using oral fluid. CRP is a possible indication of inflammation and is released in events like heart attacks. Detection of this protein along with others can be a good indication of an acute myocardial infarction, but you wouldn’t be able to use that alone. CRP may be present for other types of inflammation, especially a local one occurring in the mouth.

Tackling Periodontal Disease Early

But let’s not focus solely on detecting global diseases and problems. It’s still valuable to detect diseases in the mouth. Periodontal disease is a common oral infectious disease that is a leading cause of tooth loss in adults. Currently, clinical practices don’t have the capability to detect the onset of inflammation leading to periodontal disease and can’t identify the patients at the greatest risk for disease progression. A Point-of-Care device to detect this onset would permit earlier detection and could be utilized outside of the dentist’s office. Testing for periodontal disease can become much easier and widespread since you don’t need a highly trained professional to run the test, and it can be done in health care clinics or at home. There are many underserved communities that cannot afford to visit the dentist, but cheap, regular screening for disease can allow them to manage a disease before it gets out of hand. Additionally, preventing oral diseases can go a long way for the rest of the body, as periodontal disease has been connected to cardiovascular disease, stroke and osteoporosis.

Engaging Saliva in Pharmacogenomics

Finally, oral fluids can play a part in pharmacogenomics studies. Pharmacogenomics is the marriage of genetics and pharmacology. While we may think that we understand the processes of diseases, the diseases and their treatments can vary greatly from person to person depending on genetics. In an ideal world, every single drug and treatment we receive would be tailored specifically to our DNA. There is still a lot of work needed to find out which genes have greater effects on both the disease and treatment, but in order to learn more and to eventually provide tailored treatments, we need to understand our own DNA. Oral fluid can be a great source to obtain that DNA. The DNA we use can come from anywhere, so why not easily dislodge some cells in the mouth instead of pricking ourselves with needles?

There are many bodily fluids for us to choose from, but saliva has some key advantages. There are both important local and global diseases it can test. It certainly is less invasive than blood and does not require the same privacy (and planning) as urine collection. But there still needs to be work to determine the ideal biomarkers in the saliva and amplify their signals. There’s some good advice found in “Translational and Clinical Applications of Salivary Diagnostics” that not only applies to saliva POC devices, but to all their POC device brethren:

“While their analysis core is substantially smaller than that of benchtop alternatives, the network of macroscopic laboratory-based infrastructure required for sample processing, analyte detection, data processing, and reagent handling implies that these platforms are best described as ‘chips-in-a-lab’ rather than true ‘labs-on-a-chip’.”

No matter what fluid we’re using, or disease we’re screening, we need to design these devices with the clear motivation for them to be used outside our labs, and in the wild.

 

References:

ResearchBlogging.org

Giannobile, W., McDevitt, J., Niedbala, R., & Malamud, D. (2011). Translational and Clinical Applications of Salivary Diagnostics Advances in Dental Research, 23 (4), 375-380 DOI: 10.1177/0022034511420434

Hart, R., Mauk, M., Liu, C., Qiu, X., Thompson, J., Chen, D., Malamud, D., Abrams, W., & Bau, H. (2011). Point-of-care oral-based diagnostics Oral Diseases, 17 (8), 745-752 DOI: 10.1111/j.1601-0825.2011.01808.x

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

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

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

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