Differential lipid analysis of oxaliplatin-sensitive and resistant HCT116 cells reveals different levels of drug-induced lipid droplet formation.

Lipid droplets (LDs) are intracellular storage vesicles composed of a neutral lipid core surrounded by a glycerophospholipid membrane. LD accumulation is associated with different stages of cancer progression and stress responses resulting from chemotherapy. In previous work, a novel dual nano-electrospray ionization source and data-dependent acquisition method for measuring the relative abundances of lipid species between two extracts were described and validated. Here, this same source and method were used to determine if oxaliplatin-sensitive and resistant cells undergo similar lipid profile changes, with the goal of identifying potential signatures that could predict the effectiveness of an oxaliplatin-containing treatment. Oxaliplatin is commonly used in the treatment of colorectal cancer. When compared to a no-drug control, oxaliplatin dosing caused significant increases in triglyceride (TG) and cholesterol ester (CE) species. These increases were more pronounced in the oxaliplatin-sensitive cells than in oxaliplatin-resistant cells. The increased neutral lipid abundance correlated with LD formation, as confirmed by confocal micrographs of Nile Red-stained cells. Untargeted proteomic analyses also support LD formation after oxaliplatin treatment, with an increased abundance of LD-associated proteins in both the sensitive and resistant cells.

DiffN Selection of Tandem Mass Spectrometry Precursors.

Current data-dependent acquisition (DDA) approaches select precursor ions for tandem mass spectrometry (MS/MS) characterization based on their absolute intensity, known as a TopN approach. Low-abundance species may not be identified as biomarkers in a TopN approach. Herein, a new DDA approach is proposed, DiffN, which uses the relative differential intensity of ions between two samples to selectively target species undergoing the largest fold changes for MS/MS. Using a dual nano-electrospray (nESI) ionization source which allows samples contained in separate capillaries to be analyzed in parallel, the DiffN approach was developed and validated with well-defined lipid extracts. A dual nESI source and DiffN DDA approach was applied to quantify the differences in lipid abundance between two colorectal cancer cell lines. The SW480 and SW620 lines represent a matched pair from the same patient: the SW480 cells from a primary tumor and the SW620 cells from a metastatic lesion. A comparison of TopN and DiffN DDA approaches on these cancer cell samples highlights the ability of DiffN to increase the likelihood of biomarker discovery and the decreased probability of TopN to efficiently select lipid species that undergo large fold changes. The ability of the DiffN approach to efficiently select precursor ions of interest makes it a strong candidate for lipidomic analyses. This DiffN DDA approach may also apply to other molecule classes (e.g., other metabolites or proteins) that are amenable to shotgun analyses.

Cellular Invasion Assay for the Real-Time Tracking of Individual Cells in Spheroid or Tumor-like Mimics.

Cellular invasion is the gateway to metastasis, with cells moving from a primary tumor into neighboring regions of healthy tissue. Invasion assays provide a tractable experimental platform to quantitatively assess cellular movement in the presence of potential chemokines or inhibitors. Many such assays involve cellular movement from high cell densities to cell-free regions. To improve the physiological relevance of such assays, we developed an assay format to track cellular movement throughout a uniform density of cells. This assay format imparts diffusion-dominated environments along the channel, resulting in oxygen and nutrient gradients found in spheroids or poorly vascularized tumors. By incorporating oxygen- and pH-sensing films, we quantified spatial and temporal changes in the extracellular environment while simultaneously tracking the movement of a subset of cells engineered to express fluorescent proteins constitutively. Our results show the successful invasion into neighboring tissues likely arises from a small population with a highly invasive phenotype. These highly invasive cells continued to move throughout the 48 h experiment, suggesting they have stem-like or persister properties. Surprisingly, the distance these persister cells invaded was unaffected by the density of cells in the channel or the presence or absence of an oxygen gradient. While these datasets cannot determine if the invasive cells are inherent to the population or if diffusion-dominated environments promote them, they highlight the need for further study.

Selecting the appropriate indirect viability assay for 3D paper-based cultures: a data-driven study.

Cellular viability measurements quantify decreased proliferation or increased cytotoxicity caused by drug candidates or potential environmental toxins. Direct viability measures count each cell to provide an accurate readout. This approach can prove analytically challenging and time-consuming when cells are maintained in 3D structures akin to tissues or solid tumors. While less labor-intensive, indirect viability measures can be less accurate due to the heterogeneous structural and chemical microenvironment that arises when cells are maintained in tissue-like architectures and in contact with extracellular matrices. Here we determine the analytical figures of merit of five indirect viability assays in the paper-based cell culture platform we continue to develop in our laboratory: calcein-AM staining, the CellTiter-Glo assay, imaging fluorescent protein expression, propidium iodide staining, and the resazurin assay. We also determined the compatibility of each indirect assay with hypoxic conditions, intra-experimental repeatability, inter-experimental reproducibility, and ability to predict a potency value for a known antineoplastic drug. Our results show that each assay has benefits and drawbacks to consider when choosing the appropriate readout to answer a particular research question. We also highlight that only one indirect readout is unaffected by hypoxia, a commonly overlooked variable in cell culture that likely yields inaccurate viability measures.

Synthesis and Surface Attachment of Molecular Re(I) Complexes Supported by Functionalized Bipyridyl Ligands.

Eleven 2,2'-bipyridine (bpy) ligands functionalized with attachment groups for covalent immobilization on silicon surfaces were prepared. Five of the ligands feature silatrane functional groups for attachment to metal oxide coatings on the silicon surfaces, while six contain either alkene or alkyne functional groups for attachment to hydrogen-terminated silicon surfaces. The bpy ligands were coordinated to Re(CO)5Cl to form complexes of the type Re(bpy)(CO)3Cl, which are related to known catalysts for CO2 reduction. Six of the new complexes were characterized using X-ray crystallography. As proof of principle, four molecular Re complexes were immobilized on either a thin layer of TiO2 on silicon or hydrogen-terminated silicon. The surface-immobilized complexes were characterized using X-ray photoelectron spectroscopy, IR spectroscopy, and cyclic voltammetry (CV) in the dark and for one representative example in the light. The CO stretching frequencies of the attached complexes were similar to those of the pure molecular complexes, but the CVs were less analogous. For two of the complexes, comparison of the electrocatalytic CO2 reduction performance showed lower CO Faradaic efficiencies for the immobilized complexes than the same complex in solution under similar conditions. In particular, a complex containing a silatrane linked to bpy with an amide linker showed poor catalytic performance and control experiments suggest that amide linkers in conjugation with a redox-active ligand are not stable under highly reducing conditions and alkyl linkers are more stable. A conclusion of this work is that understanding the behavior of molecular Re catalysts attached to semiconducting silicon is more complicated than related complexes, which have previously been immobilized on metallic electrodes.

HepaRG cells adopt zonal-like drug-metabolizing phenotypes under physiologically relevant oxygen tensions and Wnt/ß-catenin signaling.

The cellular microenvironment plays an important role in liver zonation, the spatial distribution of metabolic tasks amongst hepatocytes lining the sinusoid. Standard tissue culture practices provide an excess of oxygen and a lack of signaling molecules typically found in the liver. We hypothesized that incorporating physiologically relevant environments would promote post-differentiation patterning of hepatocytes and result in zonal-like characteristics. To test this hypothesis, we evaluated the transcriptional regulation and activity of drug-metabolizing enzymes in HepaRG cells exposed to three different oxygen tensions, in the presence or absence of Wnt/ß-catenin signaling. The drug-metabolizing activity of cells exposed to representative periportal (11% O2) or perivenous (5% O2) oxygen tensions were significantly less than cells exposed to ambient oxygen. A comparison of cytochrome P450 (CYP) 1A2, 2D6, and 3A4 activity at PP and PV oxygen tensions showed significant increases at the lower oxygen tension. The activation of the Wnt/ß-catenin pathway only modestly impacted CYP activity at PV oxygen tension, despite a significant increase in CYP expression under this condition. Our results suggest oxygen tension is the major contributor to zonal patterning in HepaRG cells, with the Wnt/ß-catenin signaling pathway playing a lesser albeit important role. Our datasets also highlight the importance of including activity-based assays, as transcript data alone does not provide an accurate picture of metabolic competence. Significance Statement This work investigates the post-differentiation patterning of HepaRG cells cultured at physiologically relevant oxygen tensions, in the presence and absence of Wnt/ß-catenin signaling. HepaRG cells exposed to periportal (11% O2) or perivenous (5% O2) oxygen tensions display zonation-like patterning of both cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes. These datasets also suggest that oxygen is a primary regulator of post-differentiation patterning, with Wnt/ß-catenin having a lesser effect on activity but a significant effect on transcriptional regulation of these enzymes.

Tissue Papers: Leveraging Paper-Based Microfluidics for the Next Generation of 3D Tissue Models.

Paper-based scaffolds support the three-dimensional culture of mammalian cells in tissue-like environments. These Tissue Papers, a name that highlights the use of materials obtained from (plant) tissue to generate newly functioning (human) tissue structures, are a promising analytical tool to quantify cellular responses in physiologically relevant extracellular gradients and coculture architectures. Here, we highlight current examples of Tissue Papers, commonly used methods of analysis, and current measurement challenges.

Developing a Drug Screening Platform: MALDI-Mass Spectrometry Imaging of Paper-Based Cultures.

Many potential chemotherapeutics fail to reach patients. One of the key reasons is that compounds are tested during the drug discovery stage in two-dimensional (2D) cell cultures, which are often unable to accurately model in vivo outcomes. Three-dimensional (3D) in vitro tumor models are more predictive of chemotherapeutic effectiveness than 2D cultures, and thus, their implementation during the drug screening stage has the potential to more accurately evaluate compounds earlier, saving both time and money. Paper-based cultures (PBCs) are an emerging 3D culture platform in which cells suspended in Matrigel are seeded into paper scaffolds and cultured to generate a tissue-like environment. In this study, we demonstrate the potential of matrix-assisted laser desorption/ionization-mass spectrometry imaging with PBCs (MALDI-MSI-PBC) as a drug screening platform. This method discriminated regions of the PBCs with and without cells and/or drugs, indicating that coupling PBCs with MALDI-MSI has the potential to develop rapid, large-scale, and parallel mass spectrometric drug screens.

Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

Hypoxia is a common feature in solid tumors. Clinical samples show a positive correlation between the expression of the hypoxia-inducible factor HIF-1α and estrogen receptor alpha (ERα) and a negative correlation between HIF-1α and hormone sensitivity. Results from monolayer cultures are in contention with clinical observations, showing that ER (+) cell lines no longer express ERα under hypoxic conditions (1% O2). Here, we compared the impact of hypoxia on the ERα signaling pathway for T47D cells in a 2D and 3D culture format. In the 2D format, the cells were cultured as monolayers. In the 3D format, paper-based scaffolds supported cells suspended in a collagen matrix. Using ELISA, Western blot, and immunofluorescence measurements, we show that hypoxia differentially regulates ERα protein levels in a culture environment-dependent manner. In the 2D format, the protein levels are significantly decreased in hypoxia. In the 3D format, the protein levels are maintained in hypoxia. Hypoxia reduced ERα transcriptional activation in both culture formats. These results highlight the importance of considering tissue dimensionality for in vitro studies. They also show that ERα protein levels in hypoxia are not an accurate indicator of ERα transcriptional activity, and confirm that a positive stain for ERα in a clinical sample may not necessarily indicate hormone sensitivity.

Screening Estrogen Receptor Modulators in a Paper-Based Breast Cancer Model.

The health risks associated with acute and prolonged exposure to estrogen receptor (ER) modulators has led to a concerted effort to identify and prioritize potential disruptors present in the environment. ER agonists and antagonists are identified with end-point assays, quantifying changes in cellular proliferation or gene transactivation in monolayers of estrogen receptor alpha expressing (ER+) cells upon exposure. While these monolayer cultures can be prepared, dosed, and analyzed in a highly parallelized manner, they are unable to predict the potencies of ER modulators in vivo accurately. Physiologically relevant model systems that better predict tissue- or organ-level responses are needed. To address this need, we describe here a screening platform capable of quantitatively assessing ER modulators in 96 chemically isolated 3D cultures. These cultures are supported in wax-patterned paper scaffolds whose design has improved performance and throughput over previously described paper-based setups. To highlight the potential of paper-based cultures for toxicity screens, we measured the potency of known ER modulators with a luciferase-based reporter assay. We also quantified the proliferation and invasion of two ER+ cell lines in the presence of estradiol. Despite the inability of the current setup to better predict in vivo potencies of ER modulators than monolayer cultures, the results demonstrate the potential of this platform to support increasingly complex and physiologically relevant tissue-like structures for environmental chemical risk assessment.

A pH-Sensing Optode for Mapping Spatiotemporal Gradients in 3D Paper-Based Cell Cultures.

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pKa of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models.

Paper-based Transwell assays: an inexpensive alternative to study cellular invasion.

Cellular movement is essential in the formation and maintenance of healthy tissues as well as in disease progression such as tumor metastasis. In this work, we describe a paper-based Transwell assay capable of quantifying cellular invasion through an extracellular matrix. The paper-based Transwell assays generate similar datasets, with equivalent reproducibility, to commercially available Transwell assays. With different culture configurations, we quantify invasion: upon addition of an exogenous factor or in the presence of medium obtained from other cell types, in an indirect or direct co-culture format whose medium composition is dynamically changing, and in a single-zone or parallel (96-zone) format.

Assessing chemotherapeutic effectiveness using a paper-based tumor model.

In vitro models for screening new cancer chemotherapeutics often rely on two-dimensional cultures to predict therapeutic potential. Unfortunately, the predictive power of these models is limited, as they fail to recapitulate the complex three-dimensional environments in tumors that promote a chemoresistant phenotype. In this study, we describe the preparation and characterization of paper-based cultures (PBCs) engineered to assess chemotherapeutic effectiveness in three dimensional, diffusion-limited environments. Similar environments are found in poorly vascularized tumors. Monotonic gradients develop across these cultures, which are assembled by stacking cell-laden paper scaffolds to yield thick tissue-like structures, and provide distinct chemical environments for each scaffold. After prolonged incubation, the scaffolds can simply be peeled apart and analyzed. Through fluorescence imaging, we determined that viable and proliferative cell populations were most abundant in scaffolds close to the nutrient-rich medium. By adjusting the cell density, we modulated the spatiotemporal evolution of oxygen gradients across the cultures and correlated these environmental changes with cellular sensitivity to SN-38 exposure. From these results, we showed that differences in the oxygen gradients produced cellular populations with significantly different chemosensitivities. Through this work, we highlight PBCs ability to serve as an analytical model capable of determining chemotherapeutic effectiveness under a range of chemical environments.

Thiol-Ene Modified Amorphous Carbon Substrates: Surface Patterning and Chemically Modified Electrode Preparation.

Amorphous carbon (aC) films are chemically stable under ambient conditions or when interfaced with aqueous solutions, making them a promising material for preparing biosensors and chemically modified electrodes. There are a number of wet chemical methods capable of tailoring the reactivity and wettability of aC films, but few of these chemistries are compatible with photopatterning. Here, we introduce a method to install thiol groups directly onto the surface of aC films. These terminal thiols are compatible with thiol-ene click reactions, which allowed us to rapidly functionalize and pattern the surface of the aC films. We thoroughly characterized the aC films and confirmed the installation of surface-bound thiols does not significantly oxidize the surface or change its topography. We also determined the conditions needed to selectively attach alkene-containing molecules to these films and show the reaction is proceeding through a thiol-mediated reaction. Lastly, we demonstrate the utility of our approach by photopatterning the aC films and preparing ferrocene-modified aC electrodes. The chemistry described here provides a rapid means of fabricating sensors and preparing photoaddressable arrays of (bio)molecules on stable carbon interfaces.

Oxygen as a chemoattractant: confirming cellular hypoxia in paper-based invasion assays.

Low oxygen tension, or hypoxia, is a common occurrence in solid tumors. Hypoxia is a master regulator of cellular phenotype, and is associated with increased tumor invasion and aggressiveness as well as adverse patient prognosis. Oxygen has recently been linked with the selective movement of different cancer cell types in three-dimensional invasion assays utilizing paper-based scaffolds. It has remained unclear, however, if cells in these paper-based invasion assays are experiencing hypoxia. In this manuscript, we adapted cell-based methods to measure oxygen tension in our 3D invasion assays: the adduction of pimonidazole to free thiols in the cell, indicative of a reducing environment; the localization of hypoxia inducible factors to the nucleus; and the expression of hypoxia-regulated gene products. We utilized each method to compare the oxygen tension in different locations of the paper-based invasion stacks and found an oxygen gradient is indeed forming. Specifically, we found that the extent of pimonidazole binding, as well as the levels and activities of nucleus-localized HIF-α proteins, increase as the distance between the cells and the source of fresh medium increases. These complementary cell-based readouts not only confirm the selective invasion we observe is due to an oxygen gradient, they also show the gradient is temporal in nature and evolves with increasing culture period.

Real-time imaging of cancer cell chemotaxis in paper-based scaffolds.

Cellular migration is the movement of cells, cultured as a monolayer; cellular invasion is similar to migration, but requires the cells to move through a three-dimensional material such as basement membrane extract or a synthetic hydrogel. Migration assays, such as the transwell assay, are widely used to study cellular movement because they are amenable to high-throughput screens with minimal experimental setup. These assays offer limited information about cellular responses to gradients in vivo because they oversimplify the threedimensional (3D) environment of a tissue. There are a number of invasion assays that support 3D cultures, some of which provide experimental control over the spatial and temporal gradients imparted on the culture. These assays, in their current form, are difficult to setup and maintain, and often require specialized laboratory equipment or engineering expertise. Here we describe a paper-based invasion assay in which cellular movement can be monitored in real-time with fluorescence microscopy. These assays are easily prepared and utilize materials commonly found in any laboratory: a single sheet of paper. These sheets are wax patterned to contain channels in which cells suspended in a hydrogel are seeded and cultured. Cell-containing sheets of paper are placed in a custom-built holder that allows gradients to form along the length of the channels. In this work, we compare the invasion of cells cultured in the presence and absence of an oxygen gradient. Our result support previous findings that oxygen is a chemoattractant, and selectively directs cellular movement in a 3D culture environment.

Quantifying oxygen in paper-based cell cultures with luminescent thin film sensors.

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 (-1). We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures.

Interactions between Hofmeister anions and the binding pocket of a protein.

This paper uses the binding pocket of human carbonic anhydrase II (HCAII, EC 4.2.1.1) as a tool to examine the properties of Hofmeister anions that determine (i) where, and how strongly, they associate with concavities on the surfaces of proteins and (ii) how, upon binding, they alter the structure of water within those concavities. Results from X-ray crystallography and isothermal titration calorimetry show that most anions associate with the binding pocket of HCAII by forming inner-sphere ion pairs with the Zn(2+) cofactor. In these ion pairs, the free energy of anion-Zn(2+) association is inversely proportional to the free energetic cost of anion dehydration; this relationship is consistent with the mechanism of ion pair formation suggested by the "law of matching water affinities". Iodide and bromide anions also associate with a hydrophobic declivity in the wall of the binding pocket. Molecular dynamics simulations suggest that anions, upon associating with Zn(2+), trigger rearrangements of water that extend up to 8 Å away from their surfaces. These findings expand the range of interactions previously thought to occur between ions and proteins by suggesting that (i) weakly hydrated anions can bind complementarily shaped hydrophobic declivities, and that (ii) ion-induced rearrangements of water within protein concavities can (in contrast with similar rearrangements in bulk water) extend well beyond the first hydration shells of the ions that trigger them. This study paints a picture of Hofmeister anions as a set of structurally varied ligands that differ in size, shape, and affinity for water and, thus, in their ability to bind to­and to alter the charge and hydration structure of­polar, nonpolar, and topographically complex concavities on the surfaces of proteins.

Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

Rectification in tunneling junctions: 2,2'-bipyridyl-terminated n-alkanethiolates.

Molecular rectification is a particularly attractive phenomenon to examine in studying structure-property relationships in charge transport across molecular junctions, since the tunneling currents across the same molecular junction are measured, with only a change in the sign of the bias, with the same electrodes, molecule(s), and contacts. This type of experiment minimizes the complexities arising from measurements of current densities at one polarity using replicate junctions. This paper describes a new organic molecular rectifier: a junction having the structure Ag(TS)/S(CH2)11-4-methyl-2,2'-bipyridyl//Ga2O3/EGaIn (Ag(TS): template-stripped silver substrate; EGaIn: eutectic gallium-indium alloy) which shows reproducible rectification with a mean r(+) = |J(+1.0 V)|/|J(-1.0 V)| = 85 ± 2. This system is important because rectification occurs at a polarity opposite to that of the analogous but much more extensively studied systems based on ferrocene. It establishes (again) that rectification is due to the SAM, and not to redox reactions involving the Ga2O3 film, and confirms that rectification is not related to the polarity in the junction. Comparisons among SAM-based junctions incorporating the Ga2O3/EGaIn top electrode and a variety of heterocyclic terminal groups indicate that the metal-free bipyridyl group, not other features of the junction, is responsible for the rectification. The paper also describes a structural and mechanistic hypothesis that suggests a partial rationalization of values of rectification available in the literature.