A Microsyringe to Take the Pain out of Shots

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

Microsyringe Design

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

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

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

Microsyringe Theoretical Analysis

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

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

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

Microsyringe Mechanical Testing

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

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

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

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

Reference:

ResearchBlogging.org

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

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

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

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

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

Filtering Blood During Cardiopulmonary Bypass (CPB)

Cardiopulmonary Bypass

Cardiopulmonary Bypass ( source )

Cardiopulmonary Bypass (source)

More than 1,000 adult and 50 pediatric patients undergo a surgery involving cardiopulmonary bypass (CPB) each day in the United States. A CPB is used when performing surgery on the heart or lungs, leaving them unable to perform their normal functions. But CPB introduces a lot of foreign material to the body, creating adverse reactions. The CPB assembly, drugs and surgical processes can each have their own inflammatory effects. Induced inflammatory responses may include "complement, neutrophil, and platelet activation, endothelial dysfunction, and the release of proinflammatory cytokines."  Analyzing the patient’s blood during CPB is necessary to tie an inflammatory response to its origin in order to reduce a systemic inflammatory response syndrome (SIRS). But in order to monitor the patient’s status, at least 3 ml of blood must be drawn from the CPB system each time. This blood must then be centrifuged to access its plasma component (read more about a recent centrifuge-on-a-chip). Three ml of blood isn't needed to get an accurate reading, but it fits into the current operating procedures. This looks like the perfect opportunity to implement a microfluidic device to continuously filter small volumes of plasma from the CPB system to be analyzed, which is exactly what researchers from Rutgers University did.

Microfilter

Filter membrane sandwiched between channels

Filter membrane sandwiched between channels

The article “Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery” by Jeffrey Zahn et al. is featured in the 2011 issue 17 of Lab on a Chip. The authors wanted to create a lab-on-a-chip component to filter plasma from CPB while collecting only 50-100 µl every 15 minutes, which could be used for the duration of a procedure which may last 4 hours. The proposed device is simple and features two microfluidic channels separated by a semipermeable membrane. In order to increase the filtration rate, the device features 32 channels in parallel. The authors chose to use a membrane with a 200 nm pore size, which allows plasma and proteins of interest to cross into the filter channel while stopping the 6-8 µm red blood cells (RBC). Even though the pores are sized so that only proteins and plasma can pass, that doesn’t mean that they’ll always be able to do so. Some proteins and cells naturally adhere to foreign objects, creating a clot. We don’t want this to happen to our membrane, which could become mostly or completely clogged. While our pore size allows proteins and plasma to pass through at a faster rate than smaller pores, it is more likely to ensnare a cell that can’t pass through. In order to prevent the membrane from clogging, we can introduce an anticoagulant, such as heparin.

Anticoagulants like heparin prevent blood from clotting by disrupting a series of reactions that occur in blood (To learn more, check out this video on Coagulation Cascade from Johns Hopkins University). We normally don’t want anticoagulants in our blood because it would stop us from healing, but they are used in surgeries to diminish reactions to tools or processes. Instruments can be coated with heparin, which the authors did for the filtration device, so that heparin doesn’t have to be added system-wide. The blood’s hematocrit (Hct) also affects the need for an anticoagulant. Hct represents the percent of the blood volume that is occupied by RBC. The mathematical maximum value would be 1, which would mean that the blood was entirely composed of cells and there was no plasma, while 0 would indicate that there are no cells in the blood. Therefore a higher Hct would have a higher density of cells passing through the device and would need more anticoagulant.

The final device by the authors was able to deliver cell-free plasma which made up 15% of the blood volume. The authors noted that although the plasma is cell-free, they needed to verify the extent of hemolysis. Hemolysis is simply the destruction of RBC. We don’t want this happening to our filtered blood and need to make sure this isn’t the reason that no cells are entering our filtrate.

I think that this is a simple, yet needed piece of equipment. It is basically a membrane separating two microfluidic streams. Although the channels are small (the largest width is no greater than 600 µm), the channels across the membrane are different sizes so that they will still align when put together by our imperfect hands. The construction of parts of the device must be precise, but the device becomes more accessible if it does not need a robot to assemble it. This still needs another attachable point-of-care device to actually test the plasma, but this is promising.

Reference:

ResearchBlogging.org

Aran, K., Fok, A., Sasso, L., Kamdar, N., Guan, Y., Sun, Q., Ündar, A., & Zahn, J. (2011). Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery Lab on a Chip, 11 (17) DOI: 10.1039/C1LC20080A

Vortices, not Vortexing: Replacing the Centrifuge with a Lab-on-a-Chip

Centrifuge-on-a-Chip_Cover.jpg

In case you didn’t get the first part of my title, let me tell you a little about centrifugation. Centrifugation is a very common research technique. A solution is centrifuged to isolate suspended particles by spinning it around at high speeds. Depending on the weight of the particles and the force of the centrifuge, the heavier particles will form a pellet at the bottom of the container. The rest will still be suspended in fluid. Depending on which particles you’re after, you can continue doing this by removing the fluid and changing the forces in order to manipulate which particles form the next pellet. But in between each step the pellet must be resuspended and is vortexed (with a vortex mixer) to break up the pellet. Now, keep the definition of vortexing and the process of centrifugation in mind.

Researchers from the University of California, Los Angeles have proposed a ‘Centrifuge-on-a-Chip’ that would replace its bench top counterpart. The article, “Automated cellular sample preparation using a Centrifuge-on-a-Chip” by Di Carlo et al. was featured on the cover of the 2011 Issue 17 of Lab on a Chip. The article proposes a streamlined system that requires no external forces which, like I said before, makes it much more powerful. In order to evaluate the competency of new technology we need to compare it to a golden standard that accomplishes its task the best, no matter how long it may take. In this case, it is the centrifuge and we’ll monitor its three main jobs throughout this post:

  • Separation of cells by size or density
  • Concentration of cells
  • Labeling of cells via solution exchange
Centrifuge-on-a-Chip-Schematic.gif

The authors’ forceless Centrifuge-on-a-Chip is defined by its structure. It features a simple channel that suddenly widens. It is this widening that creates vortexes, enabling filtration. When the particles were flowing through the plain channel, they were held in place by a force that pushes them towards the wall and a force by the wall that pushes them away. When the channel widens (the wall effectively disappears) they succumb to the force pushing them away from the center of the channel. The rate that particles move out of the channel area, into the vortex region, is proportional to their size (greater than the square of the diameter). Therefore only particles above a certain threshold can be captured. The particles are able to be selectively released from their orbits by decreasing the flow rate.

Beyond simply organizing different beads, the authors validate the filtration process by separating and concentrating circulating tumor cells (CTCs) from dilute blood. Given that CTCs can be 33% to 900% greater than size than blood cells, they should be able to be separated using the vortices. This was confirmed by processing 10 ml samples with ~500 CTCs and 2.5 billion blood cells. The end result was a volume of 200 µl that recovered about 20% of the CTCs, which constituted 40% of the volume.

Now, so far we’ve been able to see the device filter cells by size and concentrate the cells, both things on our list. The only thing left to do is see if this is able to label the cells like a normal centrifugation process can. In order to traditionally label cells, they must first be incubated with the labels and centrifuged into a pellet. The fluid with extra labels must be removed and then the cells are resuspended. With the Centrifuge-On-A-Chip, the cells merely have to be caught in a vortex, exposed to the labels and rinsed. The traditional and new techniques were used to label intracellular proteins, cell surface proteins, enzyme substrates and DNA. I’m not a labeling expert, but the comparison between the two techniques seems pretty similar. There seems to be more of a difference for the labeling of the cell surface proteins, but I’m not sure if it is significant. They also demonstrated the ability to tag with primary and secondary antibodies, as well as microbeads. After 5 minutes the vortexed cells were labeled with the same number of microbeads as the traditional cells after 30 min (the same amount of labels was used for both techniques in all experiments). Further, after 30 minutes, the vortexed cells had twice the number of microbeads as the traditional cells. Safe to say, this technique can definitely label cells at least as well as the traditional method.

Centrifuge-on-a-Chip_assays.gif

The Centrifuge-On-A-Chip is clearly a viable contender against the traditional bench top centrifuge and its techniques. While there is no given price for each device, or how many devices would be necessary to provide the same output as a centrifuge, it will certainly cost less than thousands of dollars. Additionally, it is portable and can process the tasks in less time. When compared to the Centrifuge-On-A-Chip’s competitors, it still comes off fairly well. Some others are only able to filter cells and not concentrate them. Some immobilize the cells on membranes, which may prevent them from performing assays, or may clog the membranes. There was also no change in viability before and after the vortexing. However, it is necessary to dilute the blood before it can be processed, but I doubt that this would be negative enough to prevent someone from using this entirely. I suppose that we’ll just have to wait and see how quickly this can enter the market, and how cheaply it can be produced.

Reference:

ResearchBlogging.org

Mach, A., Kim, J., Arshi, A., Hur, S., & Di Carlo, D. (2011). Automated cellular sample preparation using a Centrifuge-on-a-Chip Lab on a Chip, 11 (17), 2827-2834 DOI: 10.1039/c1lc20330d