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Dielectrophoresis (DEP) is a fast and reliable nanoparticle recovery method that utilizes nonuniform electric fields to manipulate particles based on their material composition and size, enabling recovery of biologically-derived nanoparticles from plasma for diagnostic applications. When applying DEP to undiluted human plasma, collection of endogenous albumin proteins was observed at electric field gradients much lower than predicted by theory to collect molecular proteins. To understand this collection, nanoparticle tracking analysis of bovine serum albumin (BSA) dissolved in 0.5× phosphate-buffered saline was performed and showed that albumin spontaneously formed aggregate nanoparticles with a mean diameter of 237 nm. These aggregates experienced a dielectrophoretic force as a function of aggregate radius rather than the diameter of individual protein molecules which contributed to their collection. In high conductance buffer (6.8 mS/cm), DEP was able to move these aggregates into regions of high electric field gradient, and in lower conductance buffer (0.68 mS/cm), these aggregates could be moved into high or low gradient regions depending on the applied frequency. Disruption of BSA aggregates using a nonionic detergent significantly decreased the particle diameter, resulting in decreased dielectrophoretic collection of albumin which increased the collection consistency of particles of interest. These results provide techniques to manipulate albumin aggregates via DEP, which impacts collection of diagnostic biomarkers., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

Early detection has led to increased survival for multiple cancers; however, the 5-year survival rate of oral carcinoma (OC) has remained at 40% for the last several decades. Screening for OC is routinely done via visual examinations, followed by tissue biopsy and laboratory testing. Point-of-care testing would be a more convenient and widely available alternative for at-risk individuals. Increased lactate production is a hallmark of many head-and-neck tumors, due to the Warburg Effect, where tumor cells favor glycolysis in the place of oxidative phosphorylation. To detect excess lactate, we have modified the commensal bacterium Escherichia coli Nissle 1917 to express fluorescent reporter genes in response to extracellular lactate. Administering this commensal as a mouth wash and subsequently collecting saliva for the detection of the reporter may allow for noninvasive, early detection of cancerous lesions in at-risk individuals. Furthermore, we demonstrate a new on-chip electrokinetic technique to recover these probiotic probes from model saliva fluid to improve the detection of reporter gene activation., (© 2022 Wiley-VCH GmbH.)

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies., (© 2022 Wiley-VCH GmbH.)