Poster J13 - Feasibility of Single Cell Western Blotting Coupled with Surface Enhanced Raman Spectroscopy

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ella Dzialowski

Undergraduate Researcher
Vanderbilt University
Coppell, Texas, United States

Co-Author(s)

Luyao Zhao

PhD Candidate
University of 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: Proteoforms are specific molecular forms of a protein, encompassing all the protein’s variations due to genetic differences, alternative splicing, and post-translational modifications (1). Identification of proteoforms within a single cell is an important process that leads to discoveries in cell function and disease progression. Standard western blotting identifies proteins present within a large group of cells but cannot identify single cell level protein content. Techniques for single cell western blotting (scWB) have been developed (2) but the immunoprobing step is time-intensive, depends on known antibodies, and requires a large amount of protein for detection. To remedy the issues with immunoprobing, we are developing a new technique that combines methods from scWB and surface enhanced Raman spectroscopy (SERS), a powerful analytical technique that detects and identifies molecules at low concentrations, down to a single molecule (3). When paired with scWB this could yield an improved method for identifying proteoforms within a single cell. The first step in this project was to test the feasibility of scWB with SERS to ensure the new technique can be implemented and improve upon current technologies’ shortcomings. Rhodamine B (RhoB) was used as a test target. First, we determined if RhoB can be detected by SERS after diffusing through polyacrylamide gel (PA-gel) onto the SERS substrate. Second, we found the optimal time to perform SERS after protein loading and passive diffusion. Finally, we used COMSOL to simulate and compare passive diffusion and 3D gel electrophoresis as methods for protein travel down to the SERS substrate.

Materials and

Methods: Device fabrication
Wafer microfabrication and silanization were conducted based on established protocol (4). Each microwell had a diameter of 40 µm and a depth of 40 µm. An 8% PA-gel was prepared by mixing 30% (w/w) acrylamide/bis-acrylamide, gel strengthener, 10x Tris-glycine buffer, and ddH2O and was polymerized for 20 minutes using 0.08% (w/v) APS and 0.08% (v/v) TEMED. After polymerization, gels were delaminated from the wafer using a razor blade and stored in DI water. Silver nanorods (AgNR) were deposited onto a glass slide and plasma cleaned with argon for SERS analysis.

Sample preparation
PA-gel was placed onto an AgNR substrate. RhoB (0.01 mg/mL) was pipetted directly onto the PA-gel. A second sample was prepared by directly pipetting RhoB onto AgNR substrate and then covering with PA-gel (Figure 1). Spectra were collected in and out of the microwells. For each sample, 9 SERS spectra were collected with Renishaw inVia confocal Raman microscope and WiRE software using 785 nm laser. OriginPro was used to average spectra and remove the baseline using asymmetric least squared smoothing.
For the second experiment, PA-gel was placed onto AgNR substrate, RhoB (0.01 mg/mL) was pipetted onto PA-gel, and SERS spectra were collected after 10, 15, 20, 45, 60, and 100 minutes of diffusion. The spectra were analyzed for the intensity of 5 different RhoB peaks.

Simulation
BSA was simulated to be moving downwards from a 40 µm diameter microwell. First, we simulated passive diffusion over 60 minutes. Second, we simulated 3D gel electrophoresis over 5 seconds.

Results, Conclusions, and Discussions: The results support the feasibility of using SERS as an effective replacement for immunoprobing in scWB. When comparing loading RhoB on top of the gel against loading RhoB directly on the substrate and covering by the gel, all four spectra contain the same signature peaks for RhoB (Figure 2) (5). Despite differences in intensity between the two loading methods, both give rise to all the signature peaks and can detect the protein while imaging through the PA-gel. We also concluded that the optimal time point to collect data when using passive diffusion of RhoB through the gel is around 45 minutes (Figure 3). After 45 minutes, the peak intensity begins to decrease which may be due to excess diffusion away from the substrate. Finally, our simulation suggests that gel electrophoresis will be a successful method of transferring proteins from the microwells down to the AgNR substrate (Figure 4). The simulation predicts that 3D electrophoresis will result in 50 times more BSA located on the AgNR substrate than relying solely on passive diffusion, providing much higher protein detection.

This data shows the feasibility of single cell western blotting coupled with surface enhanced Raman spectroscopy to identify the protein contents of a single cell. Since the detection of RhoB is possible, the next steps include repeating these experiments with other proteins of different molecular weights (e.g., Bovine Serum Albumin, Immunoglobin, Ovalbumin). Additionally, identification of protein molecules at low concentrations should be further investigated and a physical set up for electrotransfer of proteins onto the AgNR should be developed.

Acknowledgements (Optional): 1. Smith, L., Kelleher, N., & The Consortium for Top Down Proteomics. (2013). Proteoform: A single term describing protein complexity. Nature Methods, 10(2), 186–187. https://doi.org/10.1038/nmeth.2369
2. Hughes, A., Spelke, D., Xu, Z., et al. (2014). Single-cell western blotting. Nature Methods, 11(8), 749–755. https://doi.org/10.1038/nmeth.2992
3. Wang, Y., & Irudayaraj, J. (2013). Surface-enhanced Raman spectroscopy at single-molecule scale and its implications in biology. Philosophical Transactions: Biological Sciences, 368(1611), 1–10. http://www.jstor.org/stable/41740125
4. Rosàs-Canyelles, E., Modzelewski, A. J., Geldert, A., & others. (2021). Multimodal detection of protein isoforms and nucleic acids from mouse pre-implantation embryos. Nature Protocols, 16(4), 1062–1088. https://doi.org/10.1038/s41596-020-00449-2
5. Lin, S., Hasi, W.-L.-J., Lin, X., Han, S.-q., Lou, X.-T., Yang, F., Lin, D.-Y., & Lu, Z.-W. (2015). Rapid and sensitive SERS method for determination of Rhodamine B in chili powder with paper-based substrates. Analytical Methods, 7(14), 5289–5294. https://doi.org/10.1039/C5AY00028A

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