From single cells to whole organisms, the development of quantitative tools underpin key advances in biology and medicine. In the past decade, novel microfluidic measurement tools have enabled the study of single-cell, and subcellular, systems providing insight into biological heterogeneity often masked by bulk biological measurements. In parallel, many emergency situations, including the decontamination of limited supplies of personal protective equipment (PPE) during the coronavirus 2019 (COVID-19) pandemic, requires the development of robust, quantitative workflows to ensure effective pathogen inactivation and minimize risk to the end user. In this work, we look at the fundamental principles behind quantitative tool development for two broad classes of problems: (1) single-cell immunoblotting, and (2) ultraviolet C (UV-C) decontamination of N95 filtering facepiece respirators (FFRs) during crisis capacity conditions.
First, we consider single-cell immunoblotting (scI), which enables detection of protein isoforms (proteoforms) from single cells via single-cell polyacrylamide gel electrophoresis (PAGE) and immunoblotting in a hydrogel. Several proteoforms, including the truncated HER2 isoform found in breast cancer tissue, often play key roles in disease progression and resistance. In order to detect and understand the mechanism of how these proteoforms occur, we investigate three key principles of scI: first, we assess factors that impact detection of multiple protein targets from the same cell. Specifically, we investigate the fundamental physicochemical principles that govern multiplex target detection in scI, and develop a quantitative system to assess protein target retention in hydrogels upon multiple immunoprobing rounds. Next, we investigate quantification principles for scI and assess ways in which detection of low abundance protein targets is impacted by different quantification methods. Specifically, by investigating segmentation-based quantification of scI, we find that we are able to detect more low-abundance protein targets than state-of-the-art methods. Third, we discuss preliminary investigations for measurements of multiple types of biomolecules from the same cell, including proteoform and RNA detection. In particular, we investigate outstanding challenges in the simultaneous extraction and detection of RNA and protein during scI, and assess avenues for synchronizing the long binding timescales needed for RNA extraction with the short PAGE timescales necessary for single-cell proteoform detection.
Finally, we investigate quantitative principles for UV-C decontamination of N95 FFRs. We begin by surveying the literature for the current understanding of best practices for UV-C decontamination of N95s. We then explore the use of photochromic indicators (PCIs) to validate UV-C dose, and discuss how PCIs may allow end-users to take into account aspects such as respirator geometry and placement in a decontamination system when measuring applied dose. We conclude by discussing best practices for UV-C dose reporting, and highlight which types of common errors can confound dose measurements.
Taken together, we anticipate that the results of this work will advance the fields of both single-cell measurements and pandemic response. In the field of single-cell measurements, we anticipate that the conclusions of this study will provide strategies to aid in the understanding of proteoform dynamics and regulation within heterogeneous cell populations. In the field of pandemic response, we anticipate that the results of our UV-C decontamination work will not only aid in the re-use of N95 respirators during crisis capacity conditions, but will also aid in the development of quantitative workflows to decontaminate other types of PPE.