A MICROFLUIDIC DEVICE TO MEASURE THE SHEAR ELASTIC MODULUS OF SINGLE RED BLOOD CELLS Ninad Mehendale1, Savita Kumari1, Priyanka Naik1, Dhrubaditya Mitra2 and Debjani Paul1 1 Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, India 2 NORDITA, KTH Royal Institute of Technology and Stockholm University, Sweden ABSTRACT Red blood cells become stiffer in response to environmental and physiological cues. We developed a microfluidic device to measure the shear elastic modulus of single red blood cells (RBCs) from their tracks. The device has a straight channel opening into a funnel. A single semi-circular pillar, positioned at the mouth of the funnel, deflects each RBC from its path as it approaches the pillar. The extent of deflection depends on the RBC stiffness. Using a simple numerical model and a knowledge of RBC tracks, we could calculate the effective shear elastic modulus of healthy and chemically stiffened RBCs. KEYWORDS: cell deformability, red blood cell, shear elastic modulus INTRODUCTION Healthy human RBCs are highly deformable (shear elastic modulus ~ 2 – 10 N/m) [1]. As a result, they can flow through narrow blood vessels without rupturing. In certain diseases such as, malaria, sickle cell disease and diabetes, RBCs lose their deformability. Conventional methods for measuring single cell deformability require expensive equipment and have low throughput. While microfluidic devices are faster and cheaper, they require high speed (~ thousands of fps) cameras to track the shape change of cells in real time. Moreover, they often provide a surrogate measure of cell elasticity, such as, the deformation index or the time a cell takes to pass through a constriction [2]. We present a microfluidic device that works with a 25 fps camera, analyzes hundreds of cells within ~ 3 min and yields the shear elastic modulus of RBCs. EXPERIMENTAL The device design (figure 1; left panel) is inspired by Zhu et al. [1]. The device has a flow-focusing design to prevent overlapping cell trajectories and to ensure that the cells approach the pillar along the center of the channel. In the region where the 40 m wide straight channel opens into a funnel (i.e. deformability measurement region), there is a single semicircular pillar of 20 m diameter. As a biconcave RBC passes Figure 1: Device design and working principle. Stiffer RBCs exit the pillar from either side, it deforms. The deformed the device at larger angles. RBC continues to follow the streamline on which its centre of mass lies, and accordingly, it exits the device at a specific angle. Stiffer cells follow larger exit angles compared to the more deformable ones (figure 1). We track the cell paths instead of their shape change, which eliminates the need of a high speed camera. Blood sample from healthy volunteers was diluted with normal saline (NS) for the experiments. RBCs were treated with 0.01%, 0.05% and 0.1% glutaraldehyde (GA) to make them stiffer. Separate experiments were performed with healthy and glutaraldehyde-treated RBCs. The microfluidic Figure 2: Schematic diagram of sim- device was fabricated in PDMS by standard soft lithography. Two syringe ulated RBC: blue represents pillar and pink represents the RBC. The pumps were used to focus the stream containing RBCs. Videos of the symbols are explained in equation 1. experiment were recorded using a 25 fps microscope camera focused above the funnel region. Cell trajectories were obtained from videos using MATLAB image processing toolbox. We then performed a simulation using COMSOL to map the track of each RBC as it exited the device to a specific streamline number. The central streamline corresponding to the undeflected path was numbered 0, with the streamline numbers increasing on each side. We then performed a second simulation (figure 2) with the deflected RBC positioned next to the pillar. The second simulation allowed us to calculate the pressure difference (P1 – P2) across the deformed RBC lying on each streamline [3]. Using the geometrical parameters shown in figure 2 and the pressure difference obtained by the COMSOL simulation, we obtained the effective shear modulus (Gs) of each RBC using eqn. (1). In this way, we were able to map a shear modulus to each streamline in our device. 1 1 1 (1) 𝑃1 − 𝑃2 = 𝐺𝑠 [ + + ] 𝑟1 𝑟 𝑟 + 2𝑟2 RESULTS AND DISCUSSION Figure 3: (Left) streamline distribution for normal RBCs and 0.1% glutaraldehyde-treated RBCs. (Middle) Shear modulus calibration curve corresponding to different streamlines. (Right) Shear modulus distribution for healthy and stiffened RBCs. As expected from our hypothesis, GA-treated RBCs followed higher streamlines compared to normal RBCs (figure 3; left panel) in our device. Figure 3 (middle panel) shows the calibration curve mapping each streamline to a specific value of the shear elastic modulus (Gs). It allows us to determine Gs of any unknown RBC sample that is passed through our device. Figure 3 (right panel) shows the distribution of Gs for healthy (deformable) and stiffened RBCs. Normal RBCs in our experiment have a peak Gs of 4.8 N/m, which matches with the values reported in the literature using AFM, micropipette aspirations, etc. As expected, the peaks of shear modulus shift to higher values as the cell stiffness is increased by increasing the GA concentration. CONCLUSION We measured the effective shear modulus (Gs) of individual RBCs using a simple microfluidic device design and a regular microscope camera. We estimated the shear modulus of healthy and glutaraldehyde-treated RBCs. Our results are consistent with the Gs values reported in the literature. We are currently working on estimating Gs of RBCs in various stages of P. falciparum life cycle during malarial infection. ACKNOWLEDGEMENTS We thank IIT Bombay for a seed funding and the WHEELS Global Foundation (RD/0117-DON00G0-001 and DO/2017-SUMP001-001) for supporting this work. REFERENCES [1] “A microfluidic device to sort capsules by deformability: a numerical study”, L. Zhu, C. Rorai, D. Mitra, L. Brandt, Soft Matter 10, 39 (2014) [2] “Microfluidic analysis of red blood cell deformability”, Q. Guo, S. P. Duffy, K. Matthews, A.T. Santoso, M.D. Scott, H. Ma, Journal of Biomechanics 47, 1767 (2014). [3] “A fast microfluidic device to measure the deformability of red blood cells”, N. Mehendale, D. Mitra, D. Paul, bioRxiv preprint, 644161 (2019) CONTACT * Debjani Paul (Email: debjani.paul@iitb.ac.in; Tel: +912576 7798)
Enter the password to open this PDF file:
-
-
-
-
-
-
-
-
-
-
-
-