(K-417) Application of Solid-State Nanopores Fabricated by Automated Controlled Breakdown to Hyaluronan Analysis

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row K - Poster # 417

Presenting Author(s)

Lauren E. Washco

REU Student
Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Wake Forest University School of Medicine, Winston-Salem NC
Stafford Township, New Jersey, United States

Co-Author(s)

SP

Suruchi Poddar

Postdoctoral Fellow
Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Wake Forest University School of Medicine, Winston-Salem NC, United States

Primary Investigator(s)

AH

Adam Hall

Associate Professor
Wake Forest University School of Medicine, United States

Introduction:: Hyaluronan (HA) is a prevalent polysaccharide in the human body that can serve as a potent bioindicator for arthritis, urinary tract cancer, and other diseases. The size-dependent function of HA results in long chains having anti-inflammatory properties and short chains signaling pro-inflammatory responses. Analysis of the distribution of HA molecular weights in biofluids can therefore contribute to disease diagnostics. Solid-state nanopores provide a solution to analyze HA with only a small sample of biological fluid [1]. Automated controlled breakdown (CBD) is an emerging method to fabricate solid-state nanopores of a desired size quickly, at low cost, and on demand [2]. This work outlines the application of CBD nanopores to HA analysis.

Materials and Methods:: Solid-state nanopores were produced in 37.5 nm thick silicon nitride membranes (Norcada Inc.) with a commercial instrument (Spark-E2, Northern Nanopore Instruments) utilizing 1M KCl buffer at pH 12.7 for fabrication [3] and pH 8.0 for size tuning (conditioning). Each pore was tested over multiple days to establish stability and optimal settings for fabrication. LabView software was utilized to analyze the event properties. A ladder composed of three HA molecular weights (MWs) was measured consecutively and the signal (event area or ECD) was monitored (Fig. 1a). The ladder allowed for the calibration of each nanopore to determine the molecular weight distribution of a separate polydisperse HA sample (Fig. 1b). Measurements were performed at 20, 23, and 25 days after fabrication. This process was repeated in triplicate for pores ran 1, 4, and 6 days as well as 5, 7, and 11 days after fabrication.

Results, Conclusions, and Discussions:: The use of a pH 12.7 buffer and ramp rate below 4.5 V/min allowed for the reproducible fabrication of pores at an opening voltage of ~14 V. These pores could then be conditioned to create pores 7-9 nm in diameter. CBD pores were found to have a stable baseline and diameter through multiple measurements. While some variation in ECD was observed (Fig. 1a), the ladder calibration was found to yield the same distribution of polydisperse HA at different time points. CBD Fabricated pores performed comparable measurements on clinical samples to pores fabricated using ion-beam methods. Automated CBD provides an efficient method to fabricate solid-state nanopores. This can be used to detect and analyze HA MW distribution. Future work can be done to calibrate HA distributions across different pores and investigate the ability of CBD pores to measure bioindicators other than HA.

Acknowledgements (Optional): : This project was supported in part by the NSF REU Site (Award #1950281) in the Department of Biomedical Engineering at Wake Forest University School of Medicine and NIH (R01 GM134226).

References (Optional): : [1] Rivas F., et al. Sci Rep (2022) 12: 4469 [2] Waugh M., et al. Nat Protoc (2020) 15: 122-143 [3] Yanagi I., et al. Sci Rep (2019) 9: 13143