Sepsis is an adverse systemic inflammatory response caused by microbial infection in blood. In this work, we report a simple microfluidic approach for intrinsic, non-specific removal of both microbes and inflammatory cellular components (platelets and leukocytes) from whole blood, inspired by the in vivo phenomenon of leukocyte margination ^[1] . As blood flows through a narrow microchannel (20 × 20 µm), deformable red blood cells (RBCs) migrate axially to the channel center, resulting in margination of other cell types (bacteria, platelets and leukocytes) towards the channel sides (see Figure 1) ^[2] . With the use of a simple cascaded channel design, the blood samples undergo a 2-stage bacteria removal in a single pass through the device, thereby allowing higher bacterial removal efficiency. As an application for sepsis treatment, we demonstrated separation of Escherichia coli and Saccharomyces cerevisiae spiked into whole blood, achieving high removal efficiencies of ~80% and ~90%, respectively (Figure 2A). Inflammatory cellular components were also depleted by >80% in the filtered blood samples, which could help to modulate the host inflammatory response and potentially serve as a blood-cleansing method for sepsis treatment. The developed technique offers significant advantages including high throughput (~1mL/hr per channel) and label-free separation that allows non-specific removal of any blood-borne pathogens (bacteria and fungi). The continuous processing and collection mode potentially enables the return of filtered blood to the patient directly, similar to a simple and complete dialysis circuit setup. Due to design simplicity, further multiplexing is possible by increasing channel parallelization or device stacking to achieve higher throughput comparable to convectional blood dialysis systems used in clinical settings.

1. H. L. Goldsmith and S. Spain, “Margination of leukocytes in blood flow through small tubes,” Microvascular Research, vol. 27, pp. 204-222, 1984.
2. H. W. Hou, H. Y. Gan, A. A. S. Bhagat, L. D. Li, C. T. Lim, and J. Han, “A microfluidics approach towards high-throughput pathogen removal from blood using margination,” Biomicrofluidics, vol. 6, pp. 024115-13, 2012.

The average diameter of human red blood cells (RBCs) is around 8µm. As RBCs circulate in the body and transport oxygen, they have to deform repeatedly in small blood capillaries. RBC deformability is therefore an important mechanical attribute for efficient oxygen delivery. Several blood related diseases such as malaria, sickle cell anemia, and sepsis are marked with significant alterations in RBC deformability ^{[1] [2] [3]} .

This project studies RBC dynamic deformability using a simple, portable microfluidic device ^[4] . The deformability of individual RBCs can be assessed by the average velocity of RBCs passing through narrow microfluidic channels. The repeated deformations to be experienced by RBCs simulate in vivo blood capillary system. Several blood-related diseases are included in our studies.

1. A. M. Dondorp, E. Pongponratn, and N. J. White, “Reduced microcirculatory flow in severe falciparum malaria: Pathophysiology and electron-microscopic pathology, Acta Tropica vol. 89, pp. 309-317, 2004.
2. O. K. Baskurt, D. Gelmont,  and H. J. Meiselman, “Red blood cell deformability in sepsis,” American Journal of Respiratory and Critical Care Medicine,  vol. 157, pp. 421-427, 1998.
3. S. Chien, “Red cell deformability and its relevance to blood flow,” Ann. Rev. Physiol. vol. 49, pp. 177-192, 1987.
4. H. Bow, IV. Pivkin, M. Diez-Silva, S.J. Goldfless, M. Dao, J.C. Niles, S. Suresh, and J. Han, A microfabricated deformability-based flow cytometer with application to malaria, Lab on a Chip, vol. 11, pp. 1065-1073, 2011.

In the stomach, the biological hydrogel known as mucus protects the stomach wall from the damaging effects of strongly acidic digestive juices inside the stomach lumen. Altered mucus function is linked to gastric diseases including ulcers and cancers. The biophysical mechanisms underlying the barrier are not well understood, due partly to a lack of suitable in vitro tools.

In this work, we developed an in vitro microfluidic system designed to mimic mucus secretion in the stomach (see Figure 1) ^[1] . In our system, mucus components are pumped continuously on-chip into an acidic flow, mimicking in vivo mucus secretion into an acidic stomach lumen. A fluorescent pH indicator added to the samples allows optical tracking of acid diffusion. Our microfluidic system is superior to in vitro macroscale techniques currently used to assay mucus function ^[2] . Advantages of our system include study of barrier function under secretion rather than static conditions, ability to optically measure the pH profile inside the mucus layer, and low sample volume requirement enabling experiments using difficult-to-purify mucus components.

With this system, we demonstrate that continuous secretion of mucin glycoprotein, the dominant protein component of mucus, hinders the diffusion of acid (Figure 2) due to the ability of mucins to directly bind and sequester H⁺ (see ^[1] for more details). We further estimate that the barrier function resulting from direct binding of H⁺ to mucin constitutes a significant portion of the in vivo mucus barrier. This “mucus-secretion-on-a-chip” platform may be used to systematically study the barrier function of each mucus layer component, perform diagnostics of mucus function using small amounts of clinical sample, and test mucus-targeted drugs.

1. D. L. Li, O. Lieleg, S. Jang, K. Ribbeck, and J. Han, “Microfludic in vitro system for quantitative study of stomach mucus barrier function,” Lab on a Chip, 2012, to be published. DOI: 10.1039/C2LC40161D.
2. S. Tanaka, H. H. J. Meiselman, E. Engel, P. H. Guth, O. Furukawa, R. B. Wenby, J. Lee, J. D. Kaunitz, “Regional differences of H+, HCO3-, and CO2 diffusion through native porcine gastroduodenal mucus,” Dig. Dis. Sci., vol. 47, no. 5, pp. 967-973, May 2002.

Although inertial force-induced lateral migration has been extensively studied for almost 50 years and has been utilized in various microchannels to perform size-based separation for cell research, the mechanism of inertial focusing is generally described as the interplay between inertial lift force and dean drag force and lacks information on particle behavior in depth direction, leaving several missing pieces from the physical understanding of inertial focusing parameter space ^{[1] [2] [3]} . Here we present an exploratory study of inertial focusing in planar straight microchannels and spiral microchannels with varying geometry to identify the regimes of particle behavior in response to flow rate and channel dimension. To gather accurate information on the depth direction of a straight channel, we fabricated a pair of straight channels with the same cross-sectional dimensions but different orientations and recorded the focusing positions of particles in the top-down images under the same conditions using these two devices, respectively. Combining the data from these two channels provides unambiguous information on the cross-sectional particle focusing positions. We also developed a polymer-casting technique to fabricate PDMS devices with smooth sidewalls through which one can observe the particle positions at the outermost loop of the planar spiral in the channel depth direction. The data gathered for the same spiral channel but from different directions allowed us to map the distribution of particles in cross-section with a simulated velocity field. With accurate information on particle positions in the cross-sections of straight and spiral channels, we would be able to relate the effect of channel dimension on the force field with the related particle- focusing behavior and identify the key parameters for the optimal design of a size-based separation device targeted at specific size range.

1. S. S. Kuntaegowdanahalli, A. A. Bhagat,, G. Kumar, and I. Papautsky, “Inertial microfluidics for continuous particle separation in spiral microchannels,” Lab. Chip, vol. 9, no. 20, pp. 2973-2980, Oct. 2009.
2. D. Di Carlo, J. F. Edd, K. J. Humphry, H. A. Stone, and M. Toner, “Particle segregation and dynamics in confined flows,” Physical Review Letters, vol. 102, no. 9, p. 094503, Mar. 2009.
3. A. J. Mach, and D. Di Carlo, “Continuous scalable blood filtration device using inertial microfluidics,” Biotechnol. Bioeng, vol. 107, no. 2, pp. 302-311, Oct. 2010.

Collaborators

• N.-T. Nguyen, NTU, Singapore
• C. T. Lim, NUS, Singapore
• Sang Ho Kim, NUS, Singapore
• Peter Preiser, NTU, Singapore
• K. van Vliet, MSE, MIT
• Ming Dao, MSE, MIT
• Linda Griffith, BE, MIT
• Doug Lauffenburger, BE, MIT
• Samuel Lin, BIDMC, Boston

Graduate Students

• Leon Li, Research Assistant, EECS / HST
• Aniruddh Sarkar, Research Assistant, EECS
• Sha Huang, Research Assistant, EECS
• Rho-Kyun Kwak, Research Assistant, ME
• Lidan Wu, Research Assistant, BE
• Wong Cheng Lee, Research Assistant, NUS Bioengineerig (SMART)
• Chun Ping Lim, Research Assistant, NTU (SMART)
• Tian Fock Kong, Research Assistant, NTU (SMART)
• Guo Feng Guan, Research Assistant, NUS (SMART)

Research Staff

• Han Wei Hou, Ph. D.
• Majid Ebrahimi, Ph. D. (SMART)
• Weng Kung Peng, Ph.D. (SMART)
• Alvin Koh, Ph.D. (SMART)

Support Staff

• S. Davco, Administrative Assistant

Publications

Aniruddh Sarkar and Jongyoon Han, “Non-Linear and Linear Enhancement of Enzymatic Reaction Kinetics using a Biomolecule Concentrator,” Lab on a Chip, 11(15), 2569-2576, 2011.

Chen, C.-H., A. Sarkar, Y.-A. Song, M. Miller, S.J. Kim, L. Griffith, D. Lauffenburger, and J. Han, “Enhancing Protease Activity Assay in Droplet-Based Microfluidics Using a Biomolecule Concentrator,” Journal of American Chemical Society, 133, 10368-10371, 2011.

Hou, H.W., A.A.S. Bhagat, W.C. Lee, S. Huang, J. Han, and C.T. Lim, “Microfluidic devices for blood fractionation,” Micromachines, 2, 319-343, 2011.

Cheow, L.F. and J. Han, “Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration,” Analytical Chemistry, 83, 7086-7093, 2011.

Kwak, R., S.J. Kim, and J. Han, “Continuous-flow Biomolecule and Cell Concentrator by Ion Concentration Polarization,” Analytical Chemistry, 83, 7348-7355, 2011.

Yong-Ak Song, Rohat Melik, Amr N. Rabie, Ahmed M. S. Ibrahim, David Moses, Ara Tan, Jongyoon Han, Samuel J. Lin, “Electrochemical Activation and Inhibition of Neuromuscular Systems with Modulation of Ion Concentrations Using Ion-Selective Membranes,” Nature Materials, 10, 980–986, 2011.

Kong TF, Peng WK, Luong TD, Nguyen N-T, Han J. “Adhesive-based liquid metal radio-frequency microcoil for magnetic resonance relaxometry measurement,” Lab on a Chip, 12, 287 – 294, 2012.

Li L, Lieleg O, Jang S, Ribbeck K, Han J. “A microfluidic in vitro system for quantitative study of stomach mucus barrier function,” Lab on a Chip, 2012; accepted for publication.

Hou, HW, Gan HY, Bhagat AAS, Li LD, LIm CT, Han J. A microfluidics approach towards high-throughput pathogen removal from blood using margination. Biomicrofluidics. 2012; in press.

Sarkar, A.; Kolitz, S.; Cheow, L. F.; Lauffenburger, D. A.; Han, J., An Integrated Microfluidic Probe for Concentration-Enhanced Selective Cell Kinase Activity Measurement In MicroTAS, Seattle, WA, 2011; pp 1394-1396.

Cheow, L. F.; Sarkar, A.; Kolitz, S.; Lauffenburger, D.; Han, J., Concentration Enhanced Mobility Shift Assay with Applications to Aptamer-based Biomarker Detection and Kinase Profiling. In MIcroTAS, Seattle, WA, 2011; pp 1023-1025.

Chen, C. H.; Sarkar, A.; Song, Y.-A.; Miller, M. A.; Kim, S. J.; Griffith, L. G.; Lauffenburger, D. A.; Han, J. In ENHANCING/MULTIPLEXING PROTEASE ASSAY WITH DROPLET BASED MICROFLUIDICS USING BIOMOLECULE CONCENTRATOR, MicroTAS 2011, Seattle WA, 2011; pp 2080-2082.

Li, L.; Lieleg, O.; Ribbeck, K.; Han, J. In MICROFLUIDIC IN VITRO MODEL FOR QUANTITATIVE STUDY OF STOMACH MUCIN ACID BARRIER FUNCTION, MicroTAS 2011, Seattle, WA, 2011; Seattle, WA, 2011; pp 771-773.

Bhagat, A. A. S.; Hou, H. W.; Li, L. D.; Lim, C. T.; Han, J. In DEAN FLOW FRACTIONATION (DFF) ISOLATION OF CIRCULATING TUMOR CELLS (CTCs) FROM BLOOD, MicroTAS 2011, 2011; 2011; pp 524-526.

Hou, H. W.; Gan, H. Y.; Bhagat, A. A. S.; Li, L. D.; Lim, C. T.; Han, J. In PATHOGEN AND INFLAMMATORY COMPONENTS REMOVAL FROM BLOOD USING CELL MARGINATION, MicroTAS 2011, Seattle, WA, 2011; Seattle, WA, 2011.

Guan, G.; Bhagat, A. A.; Ong, W. K. P. W. C. L. C. J.; Chen, P. C. Y.; Han, J. In SIZE-INDEPENDENT DEFORMABILITY CYTOMETRY WITH ACTIVE FEEDBACK CONTROL OF MICROFLUIDIC CHANNELS, MicroTAS 2011, Seattle, WA, 2011; Seattle, WA, 2011; pp 1053-1055.