Loss of Hormone Receptor Expression after Exposure to Fluid Shear Stress in Breast Cancer Cell Lines.

Following metastatic spread, many hormone receptor positive (HR+) patients develop a more aggressive phenotype with an observed loss of the HRs estrogen receptor (ER) and progesterone receptor (PR). During metastasis, breast cancer cells are exposed to high magnitudes of fluid shear stress (FSS). Unfortunately, the role for FSS on the regulation of HR expression and function during metastasis is not fully understood. This study was designed to elucidate the impact of FSS on HR+ breast cancer. Utilizing a microfluidic platform capable of exposing breast cancer cells to FSS that mimics in situ conditions, we demonstrate the impact of FSS exposure on representative HR+ breast cancer cell lines through protein and gene expression analysis. Proteomics results demonstrated that 540 total proteins and 1473 phospho-proteins significantly changed due to FSS exposure and pathways of interest included early and late estrogen response. The impact of FSS on response to 17ß-estradiol (E2) was next evaluated and gene expression analysis revealed repression of ER and E2-mediated genes (PR and SDF1) following exposure to FSS. Western blot demonstrated enhanced phosphorylation of mTOR following exposure to FSS. Taken together, these studies provide initial insight into the effects of FSS on HR signaling in metastatic breast cancer.

A microfluidic approach to study variations in Chlamydomonas reinhardtii alkaline phosphatase activity in response to phosphate availability.

Algal growth depends strongly on phosphorus (P) as a key nutrient, underscoring the significance of monitoring P levels. Algal species display a sensitive response to fluctuations in P availability, notably through the expression of alkaline phosphatase (AP) when challenged with P-depletion. As such, alkaline phosphatase activity (APA) serves as a valuable metric for P availability, offering insights into how algae utilize and fix available P resources. However, current APA quantification methods lack single cell resolution, while also being time- and reagent consuming. Microfluidics offers a promising cost-effective solution to these limitations, providing a platform for precise single-cell analysis. In this study, a trap-based microfluidic device was integrated with a commercially available AP live stain to study the single cell APA response of a model algae strain, Chlamydomonas reinhardtii, when exposed to different exogenous P levels. A three-step culture-starve-spike process was used to induce APA in cells cultured under two different basal P levels (1 and 21 mM). When challenged with different spiked P levels (ranging from 0.1-41 mM), C. reinhardtii cells demonstrated a highly heterogeneous APA response. Two-way ANOVA confirmed that this response is influenced by both spiked and basal P levels. Utilizing an unsupervised machine learning approach (HDBSCAN), distinct subpopulations of C. reinhardtii cells were identified exhibiting varying levels of APA at the single-cell level. These subpopulations encompass significant groups of individual cells with either notably high or low APA, contributing to the overall behavior of the cohorts. Considerable intrapopulation differences in APA were observed across cohorts with similar average behavior. For instance, while some cohorts exhibited a concentrated distribution around the overall average APA, others displayed subpopulations dispersed across a wider range of APA levels. This underscores the potential bias introduced by analyzing a small number of cells in bulk, which may skew results by overrepresenting extreme behavioral subpopulations. The findings if this study highlight the need for analytical approaches that account for single cell heterogeneity in APA and demonstrate the utility of microfluidics as a well-suited means for such investigations. This study illuminates the complexities of APA regulation at the single cell level, providing crucial insights that advance our understanding of algal phosphorus metabolism and environmental responses.

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells.

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells.

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, a receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 seconds enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

Incorporating a ß-hairpin sequence motif to increase intracellular stability of a peptide-based PROTAC.

Proteolysis targeting chimeras (PROTACs) have emerged as a new class of therapeutics that utilize the ubiquitin-proteasome system (UPS) to facilitate proteasomal degradation of "undruggable" targets. Peptide-based PROTACs contain three essential components: a binding motif for the target protein, a short amino acid sequence recognized by an E3 ligase called a degron, and a cell penetrating peptide to facilitate uptake into intact cells. While peptide-based PROTACs have been shown to successfully degrade numerous targets, they have often been found to exhibit low cell permeability and high protease susceptibility. Prior work identified peptides containing a ß-hairpin sequence motif that function not only as protecting elements, but also as CPPs and degrons. The goal of this study was to investigate if a ß-hairpin sequence could replace commonly used unstructured peptides sequences as the degron and the CPP needed for PROTAC uptake and function. The degradation of the protein Tau was selected as a model system as several published works have identified a Tau binding element that could easily be conjugated to the ß-hairpin sequence. A series of time- and concentration-dependent studies confirmed that the ßhairpin sequence was an adequate alternative CPP and degron to facilitate the proteasomemediated degradation of Tau. Microscopy studies confirmed the time-dependent uptake of the PROTAC and a degradation assay confirmed that the ß-hairpin conjugated PROTAC had a greater lifetime in cells.

Cells collectively migrate during ammonium chemotaxis in Chlamydomonas reinhardtii.

The mechanisms governing chemotaxis in Chlamydomonas reinhardtii are largely unknown compared to those regulating phototaxis despite equal importance on the migratory response in the ciliated microalga. To study chemotaxis, we made a simple modification to a conventional Petri dish assay. Using the assay, a novel mechanism governing Chlamydomonas ammonium chemotaxis was revealed. First, we found that light exposure enhances the chemotactic response of wild-type Chlamydomonas strains, yet phototaxis-incompetent mutant strains, eye3-2 and ptx1, exhibit normal chemotaxis. This suggests that Chlamydomonas transduces the light signal pathway in chemotaxis differently from that in phototaxis. Second, we found that Chlamydomonas collectively migrate during chemotaxis but not phototaxis. Collective migration during chemotaxis is not clearly observed when the assay is conducted in the dark. Third, the Chlamydomonas strain CC-124 carrying agg1-, the AGGREGATE1 gene (AGG1) null mutation, exhibited a more robust collective migratory response than strains carrying the wild-type AGG1 gene. The expression of a recombinant AGG1 protein in the CC-124 strain suppressed this collective migration during chemotaxis. Altogether, these findings suggest a unique mechanism; ammonium chemotaxis in Chlamydomonas is mainly driven by collective cell migration. Furthermore, it is proposed that collective migration is enhanced by light and suppressed by the AGG1 protein.

Fluorometric Characterization of DUB Activity: From Single-Enzyme Reactions to Live Intact Cells.

Fluorescently tagged molecular probes capable of time- and concentration-dependent quantification of deubiquitinating enzyme (DUB) activity allow for precise characterization of both enzyme and DUB inhibitor. These probes are compatible with most plate readers allowing for rapid, facile fluorometric analysis of DUB activity. DUB activity can be measured in purified enzyme reactions, in cell lysates, or in intact cells depending upon the choice of the fluorometric probe. This chapter describes protocols and potential analysis tools to investigate DUB activity in these three scenarios.

Generation of 3D Spheroids Using a Thiol-Acrylate Hydrogel Scaffold to Study Endocrine Response in ER+ Breast Cancer.

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions by mimicking cell-to-cell interactions and mass transfer limitations of metabolites, oxygen, and drugs. Recent drug studies have suggested that a high rate of preclinical and clinical failures results from mass transfer limitations associated with drug entry into solid tumors that 2D model systems cannot predict. Droplet microfluidic devices offer a promising alternative to grow 3D spheroids from a small number of cells to reduce intratumor heterogeneity, which is lacking in other approaches. Spheroids were generated by encapsulating cells in novel thiol-acrylate (TA) hydrogel scaffold droplets followed by on-chip isolation of single droplets in a 990- or 450-member trapping array. The TA hydrogel rapidly (∼35 min) polymerized on-chip to provide an initial scaffold to support spheroid development followed by a time-dependent degradation. Two trapping arrays were fabricated with 150 or 300 µm diameter traps to investigate the effect of droplet size and cell seeding density on spheroid formation and growth. Both trapping arrays were capable of ∼99% droplet trapping efficiency with ∼90% and 55% cellular encapsulation in trapping arrays containing 300 and 150 µm traps, respectively. The oil phase was replaced with media ∼1 h after droplet trapping to initiate long-term spheroid culturing. The growth and viability of MCF-7 3D spheroids were confirmed for 7 days under continuous media flow using a customized gravity-driven system to eliminate the need for syringe pumps. It was found that a minimum of 10 or more encapsulated cells are needed to generate a growing spheroid while fewer than 10 parent cells produced stagnant 3D spheroids. As a proof of concept, a drug susceptibility study was performed treating the spheroids with fulvestrant followed by interrogating the spheroids for proliferation in the presence of estrogen. Following fulvestrant exposure, the spheroids showed significantly less proliferation in the presence of estrogen, confirming drug efficacy.

Co-culture of Two Different Cell Lines in a Two-Layer Microfluidic Device.

Microfluidic devices have become a promising alternative approach for cellular co-culture. Many approaches incorporate a semipermeable barrier to physically separate, yet chemically connect, two cell types; however, the majority of these approaches utilize batch culture conditions which can result in nutrient depletion and waste accumulation. This chapter describes an alternative approach that allows for the continuous infusion of media, relieving the constraints of batch culture. The microfluidic device consists of two separate layers: a bottom layer of 3% (w/v) agarose to facilitate chemical diffusion and a top polydimethylsiloxane (PDMS) layer into which four parallel fluidic channels were imprinted. The microfluidic approach allows for facile visualization of cells with light microscopy and the ability to add (or subtract) drugs or biomolecules to interrogate the system or modulate the cellular response. Finally, the approach allows for terminal immunostaining of either (or both) cell types.

How Cargo Identity Alters the Uptake of Cell-Penetrating Peptide (CPP)/Cargo Complexes: A Study on the Effect of Net Cargo Charge and Length.

Cell-penetrating peptides (CPPs) have emerged as a powerful tool for the delivery of otherwise impermeable cargoes into intact cells. Recent efforts to improve the delivery capability of peptides have mainly focused on the identity of the CPP; however, there is evidence that the identity of the cargo itself affects the uptake. The goal of this work was to investigate how the characteristics of a peptide cargo, including net charge and length, either enhance or diminish the internalization efficiency of the CPP/cargo complex. A small library of CPP/cargo complexes were synthesized consisting of structured and unstructured CPPs with cargoes of net positive, negative, or neutral charge and lengths of 4 or 8 amino acids. Cargoes with a net positive charge were found to enhance the overall uptake of the complexes while net neutral and negatively charged cargoes diminished uptake. Conversely, the net length of the cargo had no significant effect on uptake of the CPP/cargo complexes. Microcopy images confirmed the increased uptake of the positively charged cargoes; however, an increase in punctate regions with the addition of a cargo was also observed. The effects of the net positively charged cargoes were confirmed with both structured and unstructured CPPs, which demonstrated similar trends of an increase in uptake with the addition of positively charged residues. These findings demonstrate that the net charge of cargoes impacts the uptake of the complex, which can be considered in the future when designing peptide-based reporters or therapeutics.

Simultaneous Droplet Generation with In-Series Droplet T-Junctions Induced by Gravity-Induced Flow.

Droplet microfluidics offers a wide range of applications, including high-throughput drug screening and single-cell DNA amplification. However, these platforms are often limited to single-input conditions that prevent them from analyzing multiple input parameters (e.g., combined cellular treatments) in a single experiment. Droplet multiplexing will result in higher overall throughput, lowering cost of fabrication, and cutting down the hands-on time in number of applications such as single-cell analysis. Additionally, while lab-on-a-chip fabrication costs have decreased in recent years, the syringe pumps required for generating droplets of uniform shape and size remain cost-prohibitive for researchers interested in utilizing droplet microfluidics. This work investigates the potential of simultaneously generating droplets from a series of three in-line T-junctions utilizing gravity-driven flow to produce consistent, well-defined droplets. Implementing reservoirs with equal heights produced inconsistent flow rates that increased as a function of the distance between the aqueous inlets and the oil inlet. Optimizing the three reservoir heights identified that taller reservoirs were needed for aqueous inlets closer to the oil inlet. Studying the relationship between the ratio of oil-to-water flow rates (Φ) found that increasing Φ resulted in smaller droplets and an enhanced droplet generation rate. An ANOVA was performed on droplet diameter to confirm no significant difference in droplet size from the three different aqueous inlets. The work described here offers an alternative approach to multiplexed droplet microfluidic devices allowing for the high-throughput interrogation of three sample conditions in a single device. It also has provided an alternative method to induce droplet formation that does not require multiple syringe pumps.

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.

Development of a Flow-free Gradient Generator Using a Self-Adhesive Thiol-acrylate Microfluidic Resin/Hydrogel (TAMR/H) Hybrid System.

Microfluidic gradient generators have been used to study cellular migration, growth, and drug response in numerous biological systems. One type of device combines a hydrogel and polydimethylsiloxane (PDMS) to generate "flow-free" gradients; however, their requirements for either negative flow or external clamps to maintain fluid-tight seals between the two layers have restricted their utility among broader applications. In this work, a two-layer, flow-free microfluidic gradient generator was developed using thiol-ene chemistry. Both rigid thiol-acrylate microfluidic resin (TAMR) and diffusive thiol-acrylate hydrogel (H) layers were synthesized from commercially available monomers at room temperature and pressure using a base-catalyzed Michael addition. The device consisted of three parallel microfluidic channels negatively imprinted in TAMR layered on top of the thiol-acrylate hydrogel to facilitate orthogonal diffusion of chemicals to the direction of flow. Upon contact, these two layers formed fluid-tight channels without any external pressure due to a strong adhesive interaction between the two layers. The diffusion of molecules through the TAMR/H system was confirmed both experimentally (using fluorescent microscopy) and computationally (using COMSOL). The performance of the TAMR/H system was compared to a conventional PDMS/agarose device with a similar geometry by studying the chemorepulsive response of a motile strain of GFP-expressing Escherichia coli. Population-based analysis confirmed a similar migratory response of both wild-type and mutant E. coli in both of the microfluidic devices. This confirmed that the TAMR/H hybrid system is a viable alternative to traditional PDMS-based microfluidic gradient generators and can be used for several different 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.

Catalytic Enhancement of Inductively Heated Fe3 O4 Nanoparticles by Removal of Surface Ligands.

Heat management in catalysis is limited by each material's heat transfer efficiencies, resulting in energy losses despite current thermal engineering strategies. In contrast, induction heating of magnetic nanoparticles (NPs) generates heat at the surface of the catalyst where the reaction occurs, reducing waste heat via dissipation. However, the synthesis of magnetic NPs with optimal heat generation requires interfacial ligands, such as oleic acid, which act as heat sinks. Surface treatments using tetramethylammonium hydroxide (TMAOH) or pyridine are used to remove these ligands before applications in hydrophilic media. In this study, Fe3 O4 NPs are surface treated to study the effect of induction heating on the catalytic oxidation of 1-octanol. Whereas TMAOH was unsuccessful in removing oleic acid, pyridine treatment resulted in a roughly 2.5-fold increase in heat generation and product yield. Therefore, efficient surfactant removal has profound implications in induction heating catalysis by increasing the heat transfer and available surface sites.

Evaluation of intercellular communication between breast cancer cells and adipose-derived stem cells via passive diffusion in a two-layer microfluidic device.

Breast cancer tumorigenesis and response to therapy is regulated by cancer cell interactions with the tumor microenvironment (TME). Breast cancer signaling to the surrounding TME results in a heterogeneous and diverse tumor microenvironment, which includes the production of cancer-associated fibroblasts, macrophages, adipocytes, and stem cells. The secretory profile of these cancer-associated cell types results in elevated chemokines and growth factors that promote cell survival and proliferation within the tumor. Current co-culture approaches mostly rely on transwell chambers to study intercellular signaling between adipose-derived stem cells (ASCs) and cancer cells; however, these methods are limited to endpoint measurements and lack dynamic control. In this study, a 4-channel, "flow-free" microfluidic device was developed to co-culture triple-negative MDA-MB-231 breast cancer cells and ASCs to study intercellular communication between two distinct cell types found in the TME. The device consists of two layers: a top PDMS layer with four imprinted channels coupled with a bottom agarose slab enclosed in a Plexiglas chamber. For dynamic co-culture, the device geometry contained two centered, flow-free channels, which were supplied with media from two outer flow channels via orthogonal diffusion through the agarose. Continuous fresh media was provided to the cell culture channel via passive diffusion without creating any shearing effect on the cells. The device geometry also allowed for the passive diffusion of cytokines and growth factors between the two cell types cultured in parallel channels to initiate cell-to-cell crosstalk. The device was used to show that MDA-MB-231 cells co-cultured with ASCs exhibited enhanced growth, a more aggressive morphology, and polarization toward the ASCs. The MDA-MB-231 cells were found to exhibit a greater degree of resistance to the drug paclitaxel when co-cultured with ASCs when compared to single culture studies. This microfluidic device is an ideal platform to study intercellular communication for many types of cells during co-culture experiments and allows for new investigations into stromal cell-mediated drug resistance in the tumor microenvironment.

Lignin-graft-PLGA drug-delivery system improves efficacy of MEK1/2 inhibitors in triple-negative breast cancer cell line.

Aim: Few targeted therapies are available for triple-negative breast cancer (TNBC) patients. Here, we propose a novel alkaline-lignin-conjugated-poly(lactic-co-glycolic acid) (L-PLGA) nanoparticle drug delivery system to improve the efficacy of targeted therapies. Materials & methods: L-PLGA nanoparticles (NPs) loaded with the MEK1/2 inhibitor GDC-0623 were characterized, tested in vitro on MDA-MB-231 TNBC cell line and compared with loaded PLGA NPs. Results: Loaded L-PLGA NPs were less than half the size of PLGA NPs, had slower drug release and improved the efficacy of GDC-0623 when tested in vitro. We demonstrated that GDC-0623 reversed epithelial-to-mesenchymal transition in TNBC. Conclusion: Our findings indicate that L-PLGA NPs are superior to PLGA NPs in delivering GDC-0623 to cancer cells for improved efficacy in vitro.

Synthesis and characterization of thiol-acrylate hydrogels using a base-catalyzed Michael addition for 3D cell culture applications.

There is significant interest in developing new approaches for culturing mammalian cells in a three-dimensional (3D) environment due to the fact that it better recapitulates the in vivo environment. The goal of this work was to develop thiol-acrylate, biodegradable hydrogels that possess highly tunable properties to support in vitro 3D culture. Six different hydrogel formulations were synthesized using two readily available monomers, a trithiol (ETTMP 1300 [ethoxylated trimethylolpropane tri(3-mercaptopropionate) 1300]) and a diacrylate (PEGDA 700 [polyethylene glycol diacrylate 700]), polymerized by a base-catalyzed Michael addition reaction. The resultant hydrogels were homogeneous, hydrophilic, and biodegradable. Different mechanical properties such as gelation time, storage modulus (or the elasticity G'), swelling ratio, and rate of degradation were tuned by varying the weight percentage of polymer, the molar ratio of thiol-to-acrylate groups, and the pH of the solution. Cytocompatibility was assessed using two model breast cancer cell lines by both 2D and 3D cell culturing approaches. The hydrogel formulations with a thiol-to-acrylate molar ratio of 1.05 were found to be optimal for both 2D and 3D cultures with MDA-MB-231 cellular aggregates found to be viable after 17 days of 3D continuous culture. Finally, MCF7 cells were observed to form 3D spheroids up to 600 µm in diameter as proof of principle for the thiol-acrylate hydrogel to function as a scaffold for in vitro 3D cell culture. A comparison of the different mechanical properties of the six hydrogel formulations coupled with in vitro cell culture results and findings from previously published hydrogels conclude that the thiol-acrylate hydrogels have significant potential as a scaffold for 3D cell culture.

Dipole-Modulated Downconversion Nanoparticles as Label-Free Biological Sensors.

Ultrasensitive detection of proteins and biomolecules has been previously achieved by optical nanoparticles (NPs) using the principles of Förster resonance energy transfer (FRET). However, the inherent need for labeling the target analyte in these assays hinders their applicability in point-of-use (POU) diagnostics. In this work, a label-free NP-based sensor has been developed that utilizes downconversion luminescence and surface electric dipoles as a novel approach for the detection of avidin. The long-lived luminescence of Eu3+-doped biotinylated NPs was effectively quenched in the presence of avidin in a concentration-dependent manner. The NPs exhibited high avidin selectivity and sensitivity with a limit of detection (LOD) of 7.8 nM and a wide dynamic range spanning 1 nM to 10 µM in deionized (DI) water. The application of the assay in a complex biological matrix consisting of cell growth medium supplemented with 10% v/v serum was verified with minor effects on avidin sensitivity exhibited by an LOD of 34.7 nM. The performance of the system was evaluated by comparing the photoluminescence (PL) intensities of known avidin concentration and the values predicted by the generated calibration curve. The new biosensing strategy has the potential to be extended to the detection of other disease biomarkers or pathogens with LOD and limited matrix effects in POU settings.

Static microdroplet array generated by spraying and analyzed with automated microscopy and image processing.

Microdroplets have received increasing interest as practical platforms for high-throughput biochemical analysis. Typically, numerous discrete aqueous microdroplets containing biochemical targets are generated in a continuous oil phase and characterized using a flow-through configuration. Although this approach is capable of extremely high throughput, it is challenging to provide dynamic characterization of time-dependent reaction kinetics. In this paper, we present a practical and affordable method to create and analyze a massive array of static aqueous microdroplets immersed in oil for biochemical analysis. The discrete microdroplets were produced by an air-spray gun, imaged by automated microscopy, and then characterized by image processing. The location, area, and fluorescence intensity of randomly generated individual microdroplets were automatically registered for high-throughput characterization. With this approach, we rapidly produced and characterized a static microdroplet array of over 0.7 million microdroplets with an average volume of 300 fL and a mean population density of 1.5*105 microdroplets/cm2. Using the developed setup, we demonstrated the dependency of the microdroplets' fluorescence intensity on their volume, as well as characterized the time-dependent enzyme reaction kinetics of ß-galactosidase-mediated cleavage of the substrate fluorescein di-ß-d-galactopyranoside (FDG). The new approach described herein provides an inexpensive alternative solution for high-throughput analysis of dynamic biochemical processes.