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Biomicrofluidics / Volume 6 / Issue 2 / REGULAR ARTICLES

Biomicrofluidics 6, 024115 (2012); http://dx.doi.org/10.1063/1.4710992 (13 pages)

A microfluidics approach towards high-throughput pathogen removal from blood using margination

Han Wei Hou1,2, Hiong Yap Gan1,3, Ali Asgar S. Bhagat2, Leon D. Li1,4, Chwee Teck Lim2,5,6, and Jongyoon Han1,2,7

1Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
2BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore
3Singapore Institute of Manufacturing Technology (SIMTech), A*STAR, Singapore
4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
5Department of Bioengineering, National University of Singapore, Singapore
6Mechanobiology Institute, National University of Singapore, Singapore
7Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

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(Received 22 February 2012; accepted 17 April 2012; published online 1 May 2012)

Sepsis is an adverse systemic inflammatory response caused by microbial infection in blood. This paper reports 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 invivo phenomenon of leukocyte margination. As blood flows through a narrow microchannel (20 × 20 µm), deformable red blood cells (RBCs) migrate axially to the channel centre, resulting in margination of other cell types (bacteria, platelets, and leukocytes) towards the channel sides. By using 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. 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 (∼1 ml/h per channel) and label-free separation which allows non-specific removal of any blood-borne pathogens (bacteria and fungi). The continuous processing and collection mode could potentially enable the return of filtered blood back to the patient directly, similar to a simple and complete dialysis circuit setup. Lastly, we designed and tested a larger filtration device consisting of 6 channels in parallel (∼6 ml/h) and obtained similar filtration performances. 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.

© 2012 American Institute of Physics

Article Outline

  1. INTRODUCTION
  2. MATERIALS AND METHODS
    1. Fabrication
    2. Cell culture
    3. Sample preparation
    4. Device characterization and analysis
    5. Confocal imaging
  3. RESULTS
    1. Microfluidic design and separation principle
    2. Device optimization and confocal analysis
    3. Device characterization of the cascaded design
    4. Whole blood analysis
    5. High throughput multiplexing using parallel design
  4. DISCUSSION
  5. CONCLUSIONS

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KEYWORDS, PACS, and IPC

Keywords

biomechanics, bioMEMS, blood, cellular biophysics, diseases, filters, haemodynamics, haemorheology, microchannel flow, microorganisms

PACS

  • 85.85.+j

    Micro- and nano-electromechanical systems (MEMS/NEMS) and devices

  • 47.60.Dx

    Flows in ducts and channels

  • 87.19.U-

    Hemodynamics

  • 87.80.Ek

    Mechanical and micromechanical techniques

  • 87.85.gf

    Fluid mechanics and rheology

  • 07.10.Cm

    Micromechanical devices and systems

International Patent Classification (IPC)

  • B81B

    Micro-structural devices or systems, e.g. micro-mechanical devices

  • F15D

    Fluid dynamics, i.e. methods or means for influencing the flow of gases or liquids

ARTICLE DATA

Digital Object Identifier
http://dx.doi.org/10.1063/1.4710992

PUBLICATION DATA

ISSN

1932-1058 (online)

Publisher

$abstract.society.value is a Member of CrossRef American Institute of Physics

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    References

    H. Zhao, E. S. G. Shaqfeh, and V. Narsimhan, Phys. Fluids 24(1), 011902–011921 (2012)PHFLE6000024000001011902000001.

    J. B. Freund, Phys. Fluids 19(2), 13 (2007)PHFLE6000019000002023301000001.

    A. Boulbitch, B. Quinn, and D. Pink, Phys. Rev. Lett. 85(24), 5246 (2000).


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Figures (click on thumbnails to view enlargements)

FIG.1
Schematic illustration of the developed microfluidic device for pathogen removal from blood. (a) The design consists of two cascaded straight microchannels (20 × 20 µm (W × H), 5 mm long for first channel and 1 mm long for the cascaded channel) with two bifurcations (1:8:1) in series. The cascaded design allows removal of bacteria at each bifurcation, thereby achieving a 2-stage bacterial removal in a single step. (b) Cross-sectional and top view of the microchannel illustrating the separation principle. As blood flows through the channel (margination region), deformable RBCs migrate axially to the channel centre, resulting in margination of other cell types (bacteria, platelets, and leukocytes) towards the channel walls and subsequently removed from the side outlets while the centre outlet collects the bacteria-depleted blood.

FIG.1 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.2
Effect of microchannel height (aspect ratio) on bacteria margination. (a) Bacteria concentration in the filtered centre outlet with varying channel heights using a single margination channel of 15 mm length and 20 µm width (schematic single channel design in red box). Bacteria concentration was normalized with sample to determine the percentage of bacteria remaining after filtration. (b) Fluorescent intensity Z-profiles at the outlet centre region of 20 µm and 75 µm height channels and corresponding schematic illustration of their bacteria margination. Confocal images at the mid plane of 20 µm and 75 µm height channels indicate less efficient bacteria margination at the mid plane region of high aspect ratio channel.

FIG.2 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.3
Effect of flow rate on bacteria margination. (a) Normalized bacteria concentration in the filtered centre outlet for increasing flow rate. Bacteria separation efficiency improved as flow rate increases but remained approximately constant beyond 10 µl min−1. Averaged composite images of the bifurcation indicate increase in concentration of FITC-conjugated bacteria at the channel sides with increasing flow rates. (b) Optical images illustrating the enhanced cell free layer at the expanded bifurcation. Most of the marginated bacteria reside within the cell free layer next to the channel wall which allows their efficient removal from the side channels, while the densely packed RBCs are filtered into the larger centre outlet with minimal loss.

FIG.3 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.4
Device characterization of the cascaded design. (a) Averaged fluorescence composite images at the margination channel (20 µm × 20 µm) and corresponding intensity linescans illustrating the larger number of FITC-conjugated bacteria found at the channel sides after margination. Dotted lines indicate the approximate position of channel walls. (b) Optical images and plot of bacteria filtration efficiency at different stages. Experimental results were similar to the theoretical separation efficiencies calculated based on the bifurcation ratio (blue region in schematic) and the complete bacteria margination to the four channel walls. A high bacteria separation efficiency of >80% was achieved at the collected centre outlet after two stages of filtration (enhanced online)[URL: http://dx.doi.org/10.1063/1.4710992.1 ].

FIG.4 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.5
Spiked whole blood analysis. (a) Normalized concentration of different cellular components at the filtered centre outlet using human whole blood spiked with E. coli and S. cerevisiae separately. RBCs concentration increased by ∼30% as more RBCs were packed at the centre due to Fahreaus effect. Inflammatory cellular components (platelets and leukocytes) and microbes undergo margination to the channel sides, resulting in >80% decrease in concentration at the centre outlet. Optical images (100× magnification) of each component are indicated at the top of their corresponding histogram bar. (b) Optical images illustrating yeast filtration at different stages. The larger yeast (∼5 µm) undergoes margination to the four corners in the straight channel (red arrows), resulting in complete margination to the sides and thus higher separation efficiency (enhanced online)[URL: http://dx.doi.org/10.1063/1.4710992.2 ].

FIG.5 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

FIG.6
High throughput blood filtration using a parallel system. (a) Schematic layout of the device consisting of an additional filter region to remove clogs and debris and 6 channels with cascaded design in parallel to achieve higher flow rates. (b) Experimental results indicating similar device performances as compared to single channel in removal of different blood components and bacteria using the parallel system at 100 µl/min.

FIG.6 Download High Resolution Image (.zip file) | Export Figure to PowerPoint

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