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Portable, Constriction-Expansion Blood Plasma Separation and Polymerization-Based Malaria Detection Tatyana A. Shatova, Shefali Lathwal, Marissa R. Engle, Hadley D. Sikes, and Klavs F. Jensen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01355 • Publication Date (Web): 01 Jul 2016 Downloaded from http://pubs.acs.org on July 5, 2016
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Analytical Chemistry
Portable, Constriction-Expansion Blood Plasma Separation and Polymerization-Based Malaria Detection Tatyana A. Shatova, Shefali Lathwal, Marissa R. Engle, Hadley D. Sikes*, Klavs F. Jensen* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. ABSTRACT: A portable, microfluidic blood plasma separation device is presented featuring a constriction-expansion design, which produces 100.0% purity for undiluted blood at 9% yield. This level of purity represents an improvement of at least one order of magnitude with increased yield compared to that achieved previously using passive separation. The system features high flow rates, 5-30 μL/min plasma collection, with minimal clogging and biofouling. The simple, portable blood plasma separation design is hand-driven and can easily be incorporated with microfluidic or laboratory scale diagnostic assays. The separation system was applied to a paper-based diagnostic test for malaria that produced an amplified color change in the presence of Plasmodium falciparum histidine-rich protein 2 at a concentration well below clinical relevancy for undiluted whole blood.
Blood plasma and serum are the most widely used biological fluids in biomedical and diagnostic microfluidic and laboratory applications.1 Blood contains a variety of biomarkers that can be used for detection of immune system response to foreign objects, traumas, inflammation during cardiac surgeries, presence of foreign biomarkers due to infectious diseases, as well as for longterm management of autoimmune disease and cancer.2-5 However, a high concentration of cells in blood (109 cells per mL) often limits point-of-care (POC) diagnostics to pretreated blood or serum. The laboratory standard for blood plasma isolation is centrifugation, which requires laboratory equipment, training, a higher blood volume compared to microfluidics, and does not allow for easy incorporation with microfluidic analysis.6 Therefore biological sample handling and plasma separation remains a limiting component of microfluidic biodetection and POC diagnostics.2 In order to minimize the complexity of the system, and to extend the application of the construct to any type of microfluidic detection system, we focused on passive blood separation approaches that do not require the use of an external field. Active separation, which includes acoustic, electric, and magnetic approaches have been described elsewhere.1,2 The simplest of the passive separation methods is sedimentation, which takes advantage of gravity and the difference in density between plasma and blood cells (1030 kg/m3 and 1050-1100 kg/m3, respectively).7,8 Sedimentation requires long time periods and therefore cannot be the sole driver for blood separation. Despite this limitation, it is occasionally used for blood separation in combination with other methods, such as dead-
end filtration, which features a filter perpendicular to flow, or cross-flow filtration, which features a filter(s) that is parallel to the channel and the flow direction. 9,10 However, the high number of red blood cells present and the extreme deformability of these small discoid cells (red blood cells are approximately 2 μm thick, and 8 μm in diameter), lead to the buildup of a fouling layer on the filter in both methods and thus short device lifetimes. Therefore, other components such as a wash buffer, pulsating flow, or most recently a balance of sedimentation and flow rate have been incorporated into these devices to improve the collected fraction volumes.11,12 Instead of fabricating or incorporating filters into a channel, which can increase complexity or lead to leaks around the edges, other efforts have incorporated obstacles to deviate blood cells from plasma collection. Unfortunately, as in filtration, the high cell fraction in human blood as well as the extreme deformability and discoid shape of red blood cells limit these applications in blood separation. Also, similarly to dead-end filtration, most of the obstacledeviated flow devices feature complex fabrication steps requiring exact feature sizes, high dilution numbers, slow flow rates, and are prone to nonspecific binding.2,13 Another passive separation method is based on microfluidic channel hemodynamics that incorporates inertial, viscous, cell-cell interaction, cell-protein interaction, and other forces stemming from the channel geometry and flow for plasma separation. In these systems, both rigid and deformable cells have been found to migrate across stream-lines based on shear gradient lift and wall lift due to the asymmetry of the fluid profile around the cell.14-17 In blood, this creates a cell-free layer, which also depends on
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Analytical Chemistry
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the concentration of cells, cell-cell interactions, and cellprotein interactions.18 This phenomenon has also been studied for plasma skimming approaches, and has been termed the Zweifach-Fung bifurcation effect.19-24 This manuscript focuses on inertially-dominant systems which allow for high throughput of plasma to enable fast sensor readout. Inertial cell sorting in microfluidic devices is often applied for dilute solutions, which eliminate any particle or protein interactions, and allow for separation based on size.16 This approach has also been applied to trapping rare cells through the use of rapid expansions and contractions in the channel.14,17 The geometry creates vortices within the expansion and can trap cells that have contrasting physical parameters from the most concentrated particles. The method takes advantage of the fact that the majority of cells do not follow the streamlines into the expansion traps. The abrupt expansion approach was applied for plasma separation and showed a significant improvement over literature plasma skimming values due to a temporary increase in the plasma layer near the channel expansion, despite the cell vortices for certain flow regimes.18 The approach described in this manuscript focuses on an optimized microfluidic channel design that incorporates a constriction followed by a gradual channel expansion, which minimizes the cell-trapping vortices while still enhancing the cell-free layer volume. These improvements increase the purity of the plasma collected from both diluted and undiluted whole blood beyond what has been demonstrated to date. The lack of complexity allows for the incorporation with a variety of microfluidic sensor constructs and minimizes design features that can promote biofouling. The purpose of this work is to describe an innovative, portable design that can separate blood plasma, for a range of blood dilution values, without the need for external fields or external equipment. We also verified that the plasma separation system could be combined with downstream point-of-care diagnostic testing using a paper-based colorimetric test that requires a cell-free sample. A recently reported paper-based test used polymerization-based signal amplification (PBA) to provide robust detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in small volumes of serum (10 µL per test).25 PfHRP2 is a soluble protein that is released into the blood stream of malarial patients and is used as a biomarker in commercial rapid diagnostic tests.26 The paper-based PBA test could be performed in air, required less than 100 seconds for the photopolymerization reaction in visible light and used inexpensive reagents25,27,28, making it suitable for low-cost diagnostic applications. The analytical capabilities of this approach have not yet been established for whole blood samples. Pairing this test with a device that rapidly prepares plasma from small volumes of whole blood in low-infrastructure settings represents a critical step towards taking diagnostic tests, which require whole blood samples, away from the laboratory settings to where they are needed the most.
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MATERIALS AND METHODS Device Fabrication. The separation devices were fabricated using photolithography techniques.29 A 4” silicon wafer was coated with a 100 μm thick layer of SU-8 2050 (MicroChem) and patterned using a 25400 dpi transparency mask (CAD/Art Services, Inc.). A 1 cm thick layer of PDMS (Sylgard 184, Dow Corning) was cast onto the SU-8 mold and cured for 2 hr at 80°C. The PDMS was then peeled off and the inlet and outlet holes were punched using a 1.5 mm ID × 1.91 mm OD Harris Uni-Core Puncher (Ted Pella, Inc.). The device was bonded to a glass microscope slide with a thin layer of PDMS using oxygen plasma oxidation (model PDC-32G, Harrick, Ithaca, NY) using 30 s exposure time on high level. Idex 1526 FEP 1/16” OD, 0.020” ID tubing (Upchurch Scientific) was used for inlet and outlet connections. For the inlet, Idex P-152 Y-splitter and Idex P-732 two-way valves (Upchurch Scientific) were used. The tubing was attached to luer-lock syringes (BD) using Idex P-658 female luer adapters (Upchurch Scientific). The flow was driven either using a syringe pump (Harvard Apparatus PHD 2000), or pushed by hand. The device design, shown in Figure 1, features 100 µm tall channels, with a 30 µm wide and 3 mm long inlet channel, which then expands at a range of angles (5-45°) to the full 200 µm wide and 2 mm long outlet channel. The plasma channels are each 20 µm wide at the skimming region, but then expand to a 50 µm width for certain designs, depending on the plasma channel resistance of interest. Typically the 20 µm length varied from 0.5 mm to 1.3 mm in length, and the 50 µm length varied from 0 cm to 1.6 mm in length in order to get the full range of yields presented in this work.
Figure 1. (a) Schematic of the experimental setup (not to scale). (b) Schematic of the blood plasma separation device design; device dimensions in mm, Exp < is the expansion angle, defined as deviation from the horizontal axis. (c) An example microscope image of 0.1x blood cell free layer development in the expansion region of the device.
Blood Handling. Human blood (Research Blood Components) was collected in EDTA-coated vacutainer tubes (BD) and stored at 4° C for up to 10 days, while the cell health was monitored visually. ACD-coated tubes (BD) produced comparable blood storage results, but were not
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Analytical Chemistry
used for the final data collection. No other anticoagulant was added. PBS (VWR) was used for blood sample dilutions. As seen in Figure 1, the inlet tubing is connected to both a blood and a buffer syringe through a Y-splitter. For separation reproducibility, the bubble that forms when the syringe is connected to the inlet tubing was washed away prior to experimental run. Briefly, a small amount of blood (
