Poster J17 - Rare cell proteoform analysis with microfluidic techniques

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Lesslie Montiel (she/her/hers)

Biomedical Engineering Undergraduate and NIH RISE Fellow
Rowan University
Camden, New Jersey, United States

Co-Author(s)

Yuwei Zhang

Research Assistant
The University of Georgia
Athens, Georgia, United States

Primary Investigator(s)

Yang Liu, PhD

Assistant Professor for School of Chemical, Materials, and Biomedical Engineering
University of Georgia
Athens, Georgia, United States

Introduction: Existing technologies for proteomic analyses in single-cells include mass spectroscopy, flow cytometry, and the single-cell western blot (scWB). An advantage of the rare cell device platform proposed by our lab is its ability to analyze proteoforms. Proteoforms are the different forms of protein that arise from a single gene due to changes in genetic variations, alternatively spliced RNA transcripts and posttranslational modifications [1]. The aforementioned technologies are not optimized for rare cell proteoform investigations. Rare cells are a low abundance cell type that exist in quantities of < 1000 cells per milliliter sample [2]. Circulating tumor cells, residual disease cells, and stem cells are among some rare cells relevant for therapeutic use. Proteoform analyses of these cells can quantify and identify proteins that act as indicators for cell state and function. Considering the scarcity of rare cells, sample loss and throughput are major limitations. Flow cytometry requires limited proteform antibodies and large sample sizes with throughput of hundreds to thousands of cells [3]. Top-down mass spectroscopy is limited to small-to-intermediate sized proteins ( < 25 kDa) [4]. The scWB, made of polyacrylamide (PA) gel patterned with microwells for in situ lysis, electrophoresis, photocapture, and antibody probing of single cells, captures 1-4% of a cell population [5]. Our rare cell device is designed to capture rare cells in “trapping areas” patterned on a polydimethylsiloxane (PDMS) layer that is aligned with microwells on a scWB gel layer. Protein profiles of single rare cells can then be obtained on the scWB.

Materials and

Methods: Rare cell device silicon wafer fabrication: A 50-μm layer of SU-8 3050 was spin-coated onto the silicon wafer at 3000 rpm and soft baked at 95℃ for 25 minutes. Then, the wafer was photopatterned with the “rare cell trapping 1000*1000um” photomask, placed in developer and dried with nitrogen gas. Post-exposure hard baked occurred at 150℃ for 12 minutes. Finally, the wafer was coated with dichlorodimethylsilane.

PDMS layer: Sylgard 184 base and curing agent was used in 10:1 ratio and degassed to fabricate the PDMS layer of the rare cell device. The PDMS was poured onto the silicon wafer in a dish and cured in an oven at 60℃ for 4 hours. After curing, the PDMS containing the rare cell trapping pattern was cut and 1-mm diameter inlet/outlet holes were created. The PDMS layer was treated in the plasma cleaner for 45 seconds, bonded to a glass slide (25 mm x 75 mm), and baked at 80℃ for 15 minutes.

Device loading: For preliminary experiments, 15-micron fluorescent beads and MDA-MB-231 cells suspended in PBS were used, respectively, in place of rare cells. For the experiment with beads, the device channels were washed with 0.05% Tween 20 and, for cells, 0.5% (w/v) BSA and 2mM EDTA was used.

COMSOL: Simulation of scWB electrophoresis of BSA and OVA performed in COMSOL with an applied electric field of 40 V/cm. COMSOL was also used to model the fluid flow into the device channels.

Results, Conclusions, and Discussions: Preliminary experiments were separately conducted using 15-micron fluorescent beads and MDA-MB-231 cells and loaded with 1-mL syringe. For these preliminary experiments, the polyacrylamide gel scWB layer was not used. Results show that the rare cell device is capable of capturing both cells and beads in the trapping areas, but difficulties remain. As depicted in figures 1 and 2, we observed both cells and beads occupy trapping areas throughout the device, however not all regions of the device were equally occupied. We hypothesize that this is due to disproportionate fluid flow rates in the inlet/outlet. To test this hypothesis, we simulated fluid flow in our device at 2 μL/min in the inlet using COMSOL and obtained data points for the first 10 rows of trapping areas of the first column on the device. Specifically, we collected the velocity of fluid in the trapping area (V1) and main channel (V2) and calculated the ratio of V1 to V2 as shown in figure 5B. A higher ratio indicates that cells/beads are more likely to be captured in the trapping areas. Our plot (figure 5A) and simulation data suggests that the first 3 rows of the rare cell trapping areas should have the best trapping and this was reflected in our experiments; we observed the most trapping in the first 3 rows of our device. Further optimization of fluid velocity is to be performed in COMSOL. Furthermore, the next steps of this study will include the integration of the scWB to our rare cell capture platform. Using COMSOL, we modeled the 2D electrophoresis of proteins in a microwell within a 300 μm (width) x 1000 μm (length) space. Figures 3-4 are COMSOL simulations of the electrophoresis of OVA and BSA proteins. Figure 3A and 4A show concentration plots at 15, 30, 45 and 60 seconds of OVA and BSA, respectively. Figures 3B and 4B are migration maps of OVA and BSA proteins, respectively, at 4V and 60 seconds. Finally, with the optimization of fluid flow and the integration of the scWB, our studies will transition to experiments with rare cells.

Acknowledgements (Optional): This material is based upon work supported by a National Science Foundation Research Experiences for Undergraduates (REU) site program under Grant No. 1950581.

[1] The Consortium for Top Down Proteomics, L. M. Smith, and N. L. Kelleher, “Proteoform: a single term describing protein complexity,” Nat Methods, vol. 10, no. 3, pp. 186–187, Mar. 2013, doi: 10.1038/nmeth.2369.
[2] Y. Chen et al., “Rare cell isolation and analysis in microfluidics,” J. Lab Chip, vol. 14, pp. 626-645, Jan. 2014, doi: 10.1039/c3lc90136
[3] A. Adan, G. Alizada, Y. Kiraz, Y. Baran, and A. Nalbant, “Flow cytometry: basic principles and applications,” Critical Reviews in Biotechnology, vol. 37, no. 2, pp. 163–176, Feb. 2017, doi: 10.3109/07388551.2015.1128876.
[4] Yang Zhang, Yu Zhang, Ki-Hee Song. Investigating single-molecule fluorescence spectral heterogeneity of rhodamines using high-throughput single-molecule spectroscopy [Preprint]. doi:10.1021/acs.jpclett.1c00192.s001.
[5] FAQs: Single-cell western: Bio-techne (no date) Bio. Available at: https://www.bio-techne.com/cn/instruments/single-cell-western/single-cell-western-faqs (Accessed: 21 May 2024).