Fluorescent visualization of oil displacement in a microfluidic device for enhanced oil recovery applications.

A microfluidic device was developed to mimic the reservoir pore-scale and track the oil/water phases during air flooding. The chip was generated by combining soft-lithography and NOA81 replication. A unique feature of this approach is the inclusion of fluorescent dyes into the oil/water phases, allowing for real-time visualization of oil recovery without altering the phases' surface properties. As a proof of concept, the air was injected into the water/oil-flooded device for enhanced oil recovery applications.

Characterization of PMI-5011 on the Regulation of Deubiquitinating Enzyme Activity in Multiple Myeloma Cell Extracts.

Deubiquitinating enzyme (DUB)-targeted therapeutics have shown promise in recent years as alternative cancer therapeutics, especially when coupled with proteasome-based inhibitors. While a majority of DUB-based therapeutics function by inhibiting DUB enzymes, studies show that positive regulation of these enzymes can stabilize levels of protein degradation. Unfortunately, there are currently no clinically available therapeutics for this purpose. The goal of this work was to understand the effect of a botanical extract from Artemisia dracunculus L called PMI-5011 on DUB activity in cancer cells. Through a series of kinetic analyses and mathematical modeling, it was found that PMI-5011 positively regulated DUB activity in two model multiple myeloma cells line (OPM2 and MM.1S). This suggests that PMI-5011 interacts with the active domains of DUBs to enhance their activity directly or indirectly, without apparently affecting cellular viability. Similar kinetic profiles of DUB activity were observed with three bioactive compounds in PMI-5011 (DMC-1, DMC-2, davidigenin). Interestingly, a differential cell line-independent trend was observed at higher concentrations which suggested variances in inherent gene expressions of UCHL1, UCHL5, USP7, USP15, USP14, and Rpn11 in OPM2 and MM.1S cell lines. These findings highlight the therapeutic potential of PMI-5011 and its selected bioactive compounds in cancer.

CPProtectides: Rapid uptake of well-folded ß-hairpin peptides with enhanced resistance to intracellular degradation.

Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors or drugs to intact cells. One limitation of CPPs is their rapid degradation by intracellular proteases. ß-hairpin "protectides" have previously been demonstrated to be long-lived under cytosolic conditions due to their secondary structure. The goal of this work was to demonstrate that arginine-rich ß-hairpin peptides function as both protectides and as CPPs. Peptides exhibiting a ß-hairpin motif were found to be rapidly internalized into cells with their uptake efficiency dependent on the number of arginine residues in the sequence. Cellular internalization of the ß-hairpin peptides was compared to unstructured, scrambled sequences and to commercially available, arginine-rich CPPs. The unstructured peptides displayed greater uptake kinetics compared to the structured ß-hairpin sequences; however, intracellular stability studies revealed that the ß-hairpin peptides exhibited superior stability under cytosolic conditions with a 16-fold increase in peptide half-life. This study identifies a new class of long-lived CPPs that can overcome the stability limitations of peptide-based reporters or bioactive delivery mechanisms in intact cells.

FluoroCellTrack: An algorithm for automated analysis of high-throughput droplet microfluidic data.

High-throughput droplet microfluidic devices with fluorescence detection systems provide several advantages over conventional end-point cytometric techniques due to their ability to isolate single cells and investigate complex intracellular dynamics. While there have been significant advances in the field of experimental droplet microfluidics, the development of complementary software tools has lagged. Existing quantification tools have limitations including interdependent hardware platforms or challenges analyzing a wide range of high-throughput droplet microfluidic data using a single algorithm. To address these issues, an all-in-one Python algorithm called FluoroCellTrack was developed and its wide-range utility was tested on three different applications including quantification of cellular response to drugs, droplet tracking, and intracellular fluorescence. The algorithm imports all images collected using bright field and fluorescence microscopy and analyzes them to extract useful information. Two parallel steps are performed where droplets are detected using a mathematical Circular Hough Transform (CHT) while single cells (or other contours) are detected by a series of steps defining respective color boundaries involving edge detection, dilation, and erosion. These feature detection steps are strengthened by segmentation and radius/area thresholding for precise detection and removal of false positives. Individually detected droplet and contour center maps are overlaid to obtain encapsulation information for further analyses. FluoroCellTrack demonstrates an average of a ~92-99% similarity with manual analysis and exhibits a significant reduction in analysis time of 30 min to analyze an entire cohort compared to 20 h required for manual quantification.

Population-based analysis of cell-penetrating peptide uptake using a microfluidic droplet trapping array.

Cell-penetrating peptides (CPPs) have garnered significant attention as a method to introduce reporters and therapeutics into intact cells. While numerous studies have been performed identifying new CPP sequences, relatively little is known about their uptake efficiency at the single-cell level. Here, a droplet microfluidic trapping array was used to characterize CPP uptake across a population of single intact cells. The microfluidic device allowed for facile and rapid isolation and analysis of single-cell fluorescence in a 787-member overhead trapping array with > 99% droplet trapping efficiency. The permeability efficiencies of four different CPPs were studied and compared in HeLa cells. Population analysis was performed using linkage hierarchical cluster analysis by R programming to bin cells into subpopulations expressing very low to very high peptide uptake efficiencies. CPP uptake was observed to be heterogeneous across the population of cells with peptide concentration and sequence both playing important roles in the diversity of CPP uptake, the overall peptide uptake efficiency, and the intracellular homogeneity of peptide distribution. This microfluidic-based analytical approach finds application in personalized medicine and provides new insight in the heterogeneity of CPP uptake which has the potential to affect both biosensor and drug internalization in intact cells. Graphical abstract .

Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter.

Deubiquitinating enzymes (DUBs) regulate the removal of the polyubiquitin chain from proteins targeted for degradation. Current approaches to quantify DUB activity are limited to test tube-based assays that incorporate enzymes or cell lysates, but not intact cells. The goal of this work was to develop a novel peptide-based biosensor of DUB activity that is cell permeable, protease-resilient, fluorescent, and specific to DUBs. The biosensor consists of an N-terminal ß-hairpin motif that acts as both a 'protectide' to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The ß-hairpin was conjugated to a C-terminal substrate consisting of the last four amino acids in ubiquitin (LRGG) to facilitate DUB mediated cleavage of a C-terminal fluorophore (AFC). The kinetics of the peptide reporter were characterized in cell lysates by dose response and inhibition enzymology studies. Inhibition studies with an established DUB inhibitor (PR-619) confirmed the specificity of both reporters to DUBs. Fluorometry and fluorescent microscopy experiments followed by mathematical modeling established the capability of the biosensor to measure DUB activity in intact cells while maintaining cellular integrity. The novel reporter introduced here is compatible with high-throughput single cell analysis platforms such as FACS and droplet microfluidics facilitating direct quantification of DUB activity in single intact cells with direct application in point-of-care cancer diagnostics and drug discovery.

Luminescent nanomaterials for droplet tracking in a microfluidic trapping array.

The use of high-throughput multiplexed screening platforms has attracted significant interest in the field of on-site disease detection and diagnostics for their capability to simultaneously interrogate single-cell responses across different populations. However, many of the current approaches are limited by the spectral overlap between tracking materials (e.g., organic dyes) and commonly used fluorophores/biochemical stains, thus restraining their applications in multiplexed studies. This work demonstrates that the downconversion emission spectra offered by rare earth (RE)-doped ß-hexagonal NaYF4 nanoparticles (NPs) can be exploited to address this spectral overlap issue. Compared to organic dyes and other tracking materials where the excitation and emission is separated by tens of nanometers, RE elements have a large gap between excitation and emission which results in their spectral independence from the organic dyes. As a proof of concept, two differently doped NaYF4 NPs (europium: Eu3+, and terbium: Tb3+) were employed on a fluorescent microscopy-based droplet microfluidic trapping array to test their feasibility as spectrally independent droplet trackers. The luminescence tracking properties of Eu3+-doped (red emission) and Tb3+-doped (green emission) NPs were successfully characterized by co-encapsulating with genetically modified cancer cell lines expressing green or red fluorescent proteins (GFP and RFP) in addition to a mixed population of live and dead cells stained with ethidium homodimer. Detailed quantification of the luminescent and fluorescent signals was performed to confirm no overlap between each of the NPs and between NPs and cells. Thus, the spectral independence of Eu3+-doped and Tb3+-doped NPs with each other and with common fluorophores highlights the potential application of this novel technique in multiplexed systems, where many such luminescent NPs (other doped and co-doped NPs) can be used to simultaneously track different input conditions on the same platform. Graphical abstract ᅟ.