Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.

Facets and scales in river restoration: Nestedness and interdependence of hydrological, geomorphic, ecological, and biogeochemical processes.

Although river restoration has increased rapidly, observations of successful ecological recovery are rare, mostly due to a discrepancy in the spatial scale of the impact and the restoration. Rivers and their ecological communities are a product of four river facets-hydrology, geomorphology, ecology and biogeochemistry-that act and interact on several spatial scales, from the sub-reach to the reach and catchment scales. The four river facets usually affect one another in predictable pathways (e.g., hydrology commonly controls geomorphology), but we show that the order in which they affect each other and can be restored varies depending on ecoregion and hydroclimatic regime. Similarly, processes at different spatial scales can be nested or independent of those at larger scales. Although some restoration practices are dependent of those at higher scales, other reach-scale restoration efforts are independent and can be carried out prior to or concurrently with larger-scale restoration. We introduce a checklist using the four river facets to prioritize restoration at three spatial scales in order to have the largest positive effect on the entire catchment. We apply this checklist to two contrasting regions-in northern Sweden and in southern Brazil-with different anthropogenic effects and interactions between facets and scales. In the case of nested processes that are dependent on larger spatial scales, reach-scale restoration in the absence of restoration of catchment-scale processes can frankly be a waste of money, providing little ecological return. However, depending on the scale-interdependence of processes of the river facets, restoration at smaller scales may be sufficient. This means that the most appropriate government agency should be assigned (i.e., national vs. county) to most effectively oversee river restoration at the appropriate scale; however, this first requires a catchment-scale analysis of feedbacks between facets and spatial scale interdependence.

DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets.

Techniques to detect and quantify DNA and RNA molecules in biological samples have had a central role in genomics research. Over the past decade, several techniques have been developed to improve detection performance and reduce the cost of genetic analysis. In particular, significant advances in label-free methods have been reported. Yet detection of DNA molecules at concentrations below the femtomolar level requires amplified detection schemes. Here we report a unique nanomechanical response of hybridized DNA and RNA molecules that serves as an intrinsic molecular label. Nanomechanical measurements on a microarray surface have sufficient background signal rejection to allow direct detection and counting of hybridized molecules. The digital response of the sensor provides a large dynamic range that is critical for gene expression profiling. We have measured differential expressions of microRNAs in tumour samples; such measurements have been shown to help discriminate between the tissue origins of metastatic tumours. Two hundred picograms of total RNA is found to be sufficient for this analysis. In addition, the limit of detection in pure samples is found to be one attomolar. These results suggest that nanomechanical read-out of microarrays promises attomolar-level sensitivity and large dynamic range for the analysis of gene expression, while eliminating biochemical manipulations, amplification and labelling.

Fluid and highly curved model membranes on vertical nanowire arrays.

Sensing and manipulating living cells using vertical nanowire devices requires a complete understanding of cell behavior on these substrates. Changes in cell function and phenotype are often triggered by events taking place at the plasma membrane, the properties of which are influenced by local curvature. The nanowire topography can therefore be expected to greatly affect the cell membrane, emphasizing the importance of studying membranes on vertical nanowire arrays. Here, we used supported phospholipid bilayers as a model for biomembranes. We demonstrate the formation of fluid supported bilayers on vertical nanowire forests using self-assembly from vesicles in solution. The bilayers were found to follow the contours of the nanowires to form continuous and locally highly curved model membranes. Distinct from standard flat supported lipid bilayers, the high aspect ratio of the nanowires results in a large bilayer surface available for the immobilization and study of biomolecules. We used these bilayers to bind a membrane-anchored protein as well as tethered vesicles on the nanowire substrate. The nanowire-bilayer platform shown here can be expanded from fundamental studies of lipid membranes on controlled curvature substrates to the development of innovative membrane-based nanosensors.

Fluorescent nanowire heterostructures as a versatile tool for biology applications.

Nanowires are increasingly used in biology, as sensors, as injection devices, and as model systems for toxicity studies. Currently, in situ visualization of nanowires in biological media is done using organic dyes, which are prone to photobleaching, or using microscopy methods which either yield poor resolution or require a sophisticated setup. Here we show that inherently fluorescent nanowire axial heterostructures can be used to localize and identify nanowires in cells and tissue. By synthesizing GaP-GaInP nanowire heterostructures, with nonfluorescent GaP segments and fluorescent GaInP segments, we created a barcode labeling system enabling the distinction of the nanowire morphological and chemical properties using fluorescence microscopy. The GaInP photoluminescence stability, combined with the fact that the nanowires can be coated with different materials while retaining their fluorescence, make these nanowires promising tools for biological and nanotoxicological studies.

Sensitivity and Validation of Porous Membrane Electrical Cell Substrate Impedance Spectroscopy (PM-ECIS) for Measuring Endothelial Barrier Properties.

Measurement of endothelial and epithelial barrier integrity is important for a variety of in vitro models, including Transwell assays, cocultures, and organ-on-chip platforms. Barrier resistance is typically measured by trans-endothelial electrical resistance (TEER), but TEER is invasive and cannot accurately measure isolated monolayer resistance in coculture or most organ-on-chip devices. These limitations are addressed by porous membrane electrical cell-substrate impedance sensing (PM-ECIS), which measures barrier integrity in cell monolayers grown directly on permeable membranes patterned with electrodes. Here, we advanced the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation with TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 µm within the same insert. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest being the most sensitive (p < 0.001). Similarly, smaller electrodes were most sensitive to changes in impedance (40 kHz) corresponding to cell spreading and proliferation (p < 0.001). Barrier disruption with both EGTA and thrombin was detectable by all electrode sizes. Resistances measured by PM-ECIS vs TEER for sodium chloride solutions were positively and significantly correlated for all electrode sizes (r > 0.9; p < 0.0001), but only with 750 µm electrodes for endothelial monolayers (r = 0.71; p = 0.058). These data inform the design and selection of PM-ECIS electrodes for specific applications and support PM-ECIS as a promising alternative to conventional TEER for direct, noninvasive, real-time assessment of cells cultured on porous membranes in conventional and organ-on-chip barrier models.

Bridging barriers: advances and challenges in modeling biological barriers and measuring barrier integrity in organ-on-chip systems.

Biological barriers such as the blood-brain barrier, skin, and intestinal mucosal barrier play key roles in homeostasis, disease physiology, and drug delivery - as such, it is important to create representative in vitro models to improve understanding of barrier biology and serve as tools for therapeutic development. Microfluidic cell culture and organ-on-a-chip (OOC) systems enable barrier modelling with greater physiological fidelity than conventional platforms by mimicking key environmental aspects such as fluid shear, accurate microscale dimensions, mechanical cues, extracellular matrix, and geometrically defined co-culture. As the prevalence of barrier-on-chip models increases, so does the importance of tools that can accurately assess barrier integrity and function without disturbing the carefully engineered microenvironment. In this review, we first provide a background on biological barriers and the physiological features that are emulated through in vitro barrier models. Then, we outline molecular permeability and electrical sensing barrier integrity assessment methods, and the related challenges specific to barrier-on-chip implementation. Finally, we discuss future directions in the field, as well important priorities to consider such as fabrication costs, standardization, and bridging gaps between disciplines and stakeholders.

Fibroblasts cultured on nanowires exhibit low motility, impaired cell division, and DNA damage.

Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA damage. These results are important guidelines to the design and interpretation of experiments involving nanowire-based transfection and electrical characterization of living cells.

Nanofluidics in hollow nanowires.

We present a novel scheme for producing nanotube membranes using free-standing hollow nanowires, with easily controllable dimensions. GaAs-AlInP core-shell nanowires were grown by metal-organic vapor phase epitaxy and were partially embedded in a polymer film. The GaAs core and substrate were etched selectively, leaving tubes with open access to both sides of the membrane. Electrophoretic transport of T4-phage DNA through the hollow nanowires was demonstrated using epifluorescence microscopy.

Spatial mapping of affinity changes for the integrin LFA-1 during cell migration using clusters identified based on local density.

Localization microscopy methods like Stochastic Optical Reconstruction Microscopy (STORM) are very well suited for exploring clustering of proteins, as the data inherently provide a list of molecular coordinates. Here we use state-of-art cluster analysis algorithms (DBSCAN) to explore the clustering behaviour of different affinity forms of the integrin LFA-1. It has been suggested that LFA-1 may form clusters, in order to increase the avidity to ICAM-1. However, this hypothesis still seems to be controversial. In this study, we found, variations in clustering behaviour among the different affinity forms of LFA-1 in migrating T-cells. We found that panLFA-1 is located in clusters throughout the polarised cell on ICAM-1, with an increased density of molecules and clusters in the mid area and rear of the cell, whereas the intermediate and high affinity form of LFA-1 showed an increased number in the mid area of a migrating cell and the high affinity form of LFA-1 in the front and rear. Together, these data suggest that, in addition to LFA-1 conformation, protein clustering might play a role in controlling cell-substrate adhesion on ICAM-1.By applying the cluster analysis algorithm DBSCAN to localization microscopy data, integrin clusters could be identified and different cluster parameters could be quantified.

Conformational flexibility of a model protein upon immobilization on self-assembled monolayers.

The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.

Morphology of living cells cultured on nanowire arrays with varying nanowire densities and diameters.

Vertical nanowire arrays are increasingly investigated for their applications in steering cell behavior. The geometry of the array is an important parameter, which influences the morphology and adhesion of cells. Here, we investigate the effects of array geometry on the morphology of MCF7 cancer cells and MCF10A normal-like epithelial cells. Different gallium phosphide nanowire array-geometries were produced by varying the nanowire density and diameter. Our results show that the cell size is smaller on nanowires compared to flat gallium phosphide. The cell area decreases with increasing the nanowire density on the substrate. We observed an effect of the nanowire diameter on MCF10A cells, with a decreased cell area on 40 nm diameter nanowires, compared to 60 and 80 nm diameter nanowires in high-density arrays. The focal adhesion morphology depends on the extent to which cells are contacting the substrate. For low nanowire densities and diameters, cells are lying on the substrate and we observed large focal adhesions at the cell edges. In contrast, for high nanowire densities and diameters, cells are lying on top of the nanowires and we observed point-like focal adhesions distributed over the whole cell. Our results constitute a step towards the ability to fine-tune cell behavior on nanowire arrays.

Cellular traction forces: a useful parameter in cancer research.

The search for new cancer biomarkers is essential for fundamental research, diagnostics, as well as for patient treatment and monitoring. Whereas most cancer biomarkers are biomolecules, an increasing number of studies show that mechanical cues are promising biomarker candidates. Although cell deformability has been shown to be a possible cancer biomarker, cellular forces as cancer biomarkers have been left largely unexplored. Here, we measure traction forces of cancer and normal-like cells at high spatial resolution using a robust method based on dense vertical arrays of nanowires. A force map is created using automated image analysis based on the localization of the fluorescent tips of the nanowires. We show that the force distribution and magnitude differ between MCF7 breast cancer cells and MCF10A normal-like breast epithelial cells, and that monitoring traction forces can be used to investigate the effects of anticancer drugs.

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy.

DNA sequencing by imaging in an electron microscope is an approach that holds promise to deliver long reads with low error rates and without the need for amplification. Earlier work using transmission electron microscopes, which use high electron energies on the order of 100 keV, has shown that low contrast and radiation damage necessitates the use of heavy atom labeling of individual nucleotides, which increases the read error rates. Other prior work using scattering electrons with much lower energy has shown to suppress beam damage on DNA. Here we explore possibilities to increase contrast by employing two methods, X-ray photoelectron and Auger electron spectroscopy. Using bulk DNA samples with monomers of each base, both methods are shown to provide contrast mechanisms that can distinguish individual nucleotides without labels. Both spectroscopic techniques can be readily implemented in a low energy electron microscope, which may enable label-free DNA sequencing by direct imaging.

From immobilized cells to motile cells on a bed-of-nails: effects of vertical nanowire array density on cell behaviour.

The field of vertical nanowire array-based applications in cell biology is growing rapidly and an increasing number of applications are being explored. These applications almost invariably rely on the physical properties of the nanowire arrays, creating a need for a better understanding of how their physical properties affect cell behaviour. Here, we investigate the effects of nanowire density on cell migration, division and morphology for murine fibroblasts. Our results show that few nanowires are sufficient to immobilize cells, while a high nanowire spatial density enables a "bed-of-nails" regime, where cells reside on top of the nanowires and are fully motile. The presence of nanowires decreases the cell proliferation rate, even in the "bed-of-nails" regime. We show that the cell morphology strongly depends on the nanowire density. Cells cultured on low (0.1 µm(-2)) and medium (1 µm(-2)) density substrates exhibit an increased number of multi-nucleated cells and micronuclei. These were not observed in cells cultured on high nanowire density substrates (4 µm(-2)). The results offer important guidelines to minimize cell-function perturbations on nanowire arrays. Moreover, these findings offer the possibility to tune cell proliferation and migration independently by adjusting the nanowire density, which may have applications in drug testing.

Enhanced laminin adsorption on nanowires compared to flat surfaces.

Semiconductor nanowires are widely used to interface living cells, and numerous nanowire-based devices have been developed to manipulate or sense cell behavior. We have, however, little knowledge on the nature of the cell-nanowire interface. Laminin is an extracellular matrix protein promoting cell attachment and growth. Here, we used a method based on fluorescence microscopy and measured the relative amount of laminin adsorbed on nanowires compared to flat surfaces. The amount of adsorbed laminin per surface area is up to 4 times higher on 55nm diameter gallium phosphide nanowires compared to the flat gallium phosphide surface between the nanowires. We show that this enhanced adsorption on nanowires cannot be attributed to electrostatic effects, nor to differences in surface chemistry, but possibly to pure geometrical effects, as increasing the nanowire diameter results in a decreased amount of adsorbed protein. The increased adsorption of laminin on nanowires may explain the exceptionally beneficial properties of nanowire substrates for cellular growth reported in the literature since laminin is often used as surface coating prior to cell cultures in order to promote cell growth, and also because primary cell suspensions contain endogenous laminin.

Progress toward an aberration-corrected low energy electron microscope for DNA sequencing and surface analysis.

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel imaging technique aimed at high resolution imaging of macromolecules, nanoparticles, and surfaces. MAD-LEEM combines three innovative electron-optical concepts in a single tool: a monochromator, a mirror aberration corrector, and dual electron beam illumination. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. The aberration corrector is needed to achieve subnanometer resolution at landing energies of a few hundred electronvolts. The dual flood illumination approach eliminates charging effects generated when a conventional, single-beam LEEM is used to image insulating specimens. The low landing energy of electrons in the range of 0 to a few hundred electronvolts is also critical for avoiding radiation damage, as high energy electrons with kilo-electron-volt kinetic energies cause irreversible damage to many specimens, in particular biological molecules. The performance of the key electron-optical components of MAD-LEEM, the aberration corrector combined with the objective lens and a magnetic beam separator, was simulated. Initial results indicate that an electrostatic electron mirror has negative spherical and chromatic aberration coefficients that can be tuned over a large parameter range. The negative aberrations generated by the electron mirror can be used to compensate the aberrations of the LEEM objective lens for a range of electron energies and provide a path to achieving subnanometer spatial resolution. First experimental results on characterizing DNA molecules immobilized on Au substrates in a LEEM are presented. Images obtained in a spin-polarized LEEM demonstrate that high contrast is achievable at low electron energies in the range of 1-10 eV and show that small changes in landing energy have a strong impact on the achievable contrast. The MAD-LEEM approach promises to significantly improve the performance of a LEEM for a wide range of applications in the biosciences, material sciences, and nanotechnology where nanometer scale resolution and analytical capabilities are required. In particular, the microscope has the potential of delivering images of unlabeled DNA strands with nucleotide-specific contrast. This simplifies specimen preparation and significantly eases the computational complexity needed to assemble the DNA sequence from individual reads.

Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization.

The analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.

Direct electrical detection of DNA synthesis.

Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3'-hydroxyl group of the DNA molecule during phosphodiester bond formation.

DNA-functionalized MFe2O4 (M = Fe, Co, or Mn) nanoparticles and their hybridization to DNA-functionalized surfaces.

Magnetic MFe2O4 (M = Fe, Co, or Mn) nanoparticles with uniform diameters in the 4-20 nm range and with excellent material properties, reported previously, can be rendered soluble in water or aqueous buffers using a combination of alkylphosphonate surfactants and other surfactants such as ethoxylated fatty alcohols or phospholipids. Surfactant-modified oligonucleotides can be incorporated into the particles' organic shell. The particles can withstand salt concentrations up to 0.3 M, temperatures up to 90 degrees C, and various operations such as concentration to dryness, column or membrane separations, and electrophoresis. The particles can be selectively hybridized to DNA-functionalized gold surfaces with high coverages using a two-story monolayer structure. These particles may find valuable applications involving the magnetic detection of small numbers of biomolecules using spin valves, magnetic tunnel junctions, or other sensors.