Generalizable quantitative analysis of cell spheroid models and tissues using a single electrochemical platform with custom pneumatic and microfluidic circuits - Cellular analysis could be revolutionized by a technology which is capable of rapid, specific measurement of a variety of arbitrary biomolecules with instrumentation that is easy to operate. Current methods and technology to quantify analytes such as small molecules, peptides, or proteins are usually focused on a particular target (or class of analytes), lacking generalizability to other types, and the sample or fluid handling is often accomplished with expensive robotic systems. There remains a pressing, unmet need to develop a platform amenable to rapid, simple to use, generalizable, and quantitative readout of multiple classes of biologically relevant targets. Due to their miniaturization capability and simple instrumentation, electrochemical (EC) sensors have long been proven as viable for low-cost sensing of cellular factors, although the most successful sensors are specialized to targets such as glucose or lactate. Recent developments by the principal investigator (PI) and other bioanalytical scientists have focused on nucleic acid-based EC sensors due to their flexibility, robustness, and applicability to complex samples, although these approaches still are lacking in generalizability or have complex, noncovalent structures that are less amenable to surface stability. In this proposal, we describe our further development of an innovative nucleic acid “bowtie” sensor that exhibits unprecedented generalizability, as well as electrochemical proximity assays (ECPA) which are highly sensitive and generalizable to larger proteins. Alongside EC sensor development, the PI has concurrently engineered innovative microfluidic systems which allow manipulation of nanoliter volumes in an automated way using 3D-printed pneumatic components that operate like plug-and-play electrical circuitry. This project seeks to combine the EC sensors with the fluidic control systems toward ease of use for end users studying cell models, spheroids, or tissues. The work is centered around four well defined yet independent projects. In Project 1, we will integrate EC bowtie sensors with automated pneumatic and microfluidic circuits to provide more automated analysis, including time-resolved measurements with a pneumatic multiplexer design. Project 2 seeks to construct a variety of novel EC sensors with the same bowtie nanostructure. Targeted analytes are unique to direct EC analysis, including L-glutamine, niacin, palmitic acid, adiponectin, leptin, TNF-α, and extracellular vesicles (EVs). In Project 3, automated pneumatics and microfluidics will sample cell spheroids and tissues, with downstream quantification of secreted factors using customized EC sensors. Unique biological dynamics will be revealed with each system, while simultaneously developing a generalizable approach applicable to other cells or tissues. In Project 4, a robust and calibration-free binding model will be devised for the bowtie sensors, and the latest active-reset techniques will be applied to enable reusable sensors. This proposed work is significant, enabling a first-of-its-kind assay platform combining generalizable EC sensors with innovative pneumatic circuits for fluid handling. We expect future systems based on this technology to be usable by nonspecialists for cell, spheroid, and tissue analysis.
