Spatially resolved analysis of microenvironmental gradient impact on cancer cell phenotypes.

Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology.

Influence of the processing on composition, protein structure and techno-functional properties of mealworm protein concentrates produced by isoelectric precipitation and ultrafiltration/diafiltration.

Edible insects represent a great alternative protein source but food neophobia remains the main barrier to consumption. However, the incorporation of insects as protein-rich ingredients, such as protein concentrates, could increase acceptance. In this study, two methods, isoelectric precipitation and ultrafiltration-diafiltration, were applied to produce mealworm protein concentrates, which were compared in terms of composition, protein structure and techno-functional properties. The results showed that the protein content of the isoelectric precipitation concentrate was higher than ultrafiltration-diafiltration (80 versus 72%) but ash (1.91 versus 3.82%) and soluble sugar (1.43 versus 8.22%) contents were lower. Moreover, the protein structure was affected by the processing method, where the ultrafiltration-diafiltration concentrate exhibited a higher surface hydrophobicity (493.5 versus 106.78 a.u) and a lower denaturation temperature (161.32 versus 181.44 °C). Finally, the ultrafiltration-diafiltration concentrate exhibited higher solubility (87 versus 41%) and emulsifying properties at pH 7 compared to the concentrate obtained by isoelectric precipitation.

Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data.

Enzymatic degradation of cellulosic biomass is a well-established route for the sustainable production of biofuels, chemicals, and materials. A strategy employed by nature and industry to achieve an efficient degradation of cellulose is that cellobiohydrolases (or exocellulases), such as Cel7A, work synergistically with endoglucanases, such as Cel7B, to achieve the complete degradation of cellulose. However, a complete mechanistic understanding of this exo-endo synergy is still lacking. Here, we used single-molecule fluorescence microscopy to quantify the binding kinetics of Cel7A on cellulose when it is acting alone on the cellulose fibrils and in the presence of its synergy partner, the endoglucanase Cel7B. To this end, we used a fluorescently tagged Cel7A and studied its binding in the presence of the unlabeled Cel7B. This provided the single-molecule data necessary for the estimation of the rate constants of association kON and dissociation kOFF of Cel7A for the substrate. We show that the presence of Cel7B does not impact the dissociation rate constant, kOFF. But, the association rate of Cel7A decreases by a factor of 2 when Cel7B is present at a molar proportion of 10:1. This ratio has previously been shown to lead to synergy. This decrease in association rate is observed in a wide range of total enzyme concentrations, from sub nM to µM concentrations. This decrease in kON is consistent with the formation of cellulase clusters recently observed by others using atomic force microscopy.

High-intensity ultrasound treatment on casein: Pea mixed systems: Effect on gelling properties.

This study aimed to investigate the suitability of the application of high-intensity ultrasounds (HIUS) to improve the acid induced gelation of mixed protein systems formed by casein micelles (CMs) and pea. The protein suspensions were prepared in different protein ratios CMs: pea (100:0, 80:20, 50:50, 20:80, 0:100) at 8% (w/w) total protein concentration. In the suspensions, the ultrasound treatment produced an increase in solubility, surface hydrophobicity, and a decrease in the samples' viscosity, with more remarkable differences in protein blends in which pea protein was the major component. However, the replacement of 20% of CMs for pea proteins highly affected the gel elasticity. Hence, the creation of smaller and more hydrophobic building blocks before acidification due to the HIUS treatment increased the elasticity of the gels up to 10 times. Therefore, high-intensity ultrasounds are a suitable green technique to increase the gelling properties of CMs: pea systems.

Quantifying DNA-mediated liposome fusion kinetics with a fluidic trap.

Self-assembly of synthetic lipid vesicles via lipid membrane fusion is a versatile tool for creating biomimetic nano- and micron-sized particles. These so-called liposomes are used in the development of biosensing platforms, design of drug delivery schemes, and for investigating protein-mediated fusion of biological membranes. This work demonstrates DNA-induced liposome fusion in a nanofluidic trap where the reaction occurs in a 15 femtoliter volume at homogeneous mixing. In contrast to current methods for fusion in bulk, we show that the fusion reaction follows second-order kinetics with a fusion rate of (170 ± 30)/(M-1s-1) times the square number of DNA molecules per liposome. The nanofluidic trapping gives a full characterization of the size and charge of the liposomes before and after fusion. The chip-based approach limits the amount of sample (down to 440 vesicles) and can be parallelized for systematic studies in synthetic biology, diagnostics, and drug delivery.

Quantitative live-cell imaging of lipidated peptide transport through an epithelial cell layer.

Oral drug delivery increases patient compliance and is thus the preferred administration route for most drugs. However, for biologics the intestinal barrier greatly limits the absorption and reduces their bioavailability. One strategy employed to improve on this is chemical modification of the biologic through the addition of lipid side chains. While it has been established that lipidation of peptides can increase transport, a mechanistic understanding of this effect remains largely unexplored. To pursue this mechanistic understanding, end-point detection of biopharmaceuticals transported through a monolayer of fully polarized epithelial cells is typically used. However, these methods are time-consuming and tedious. Furthermore, most established methods cannot be combined easily with high-resolution live-cell fluorescence imaging that could provide a mechanistic insight into cellular uptake and transport. Here we address this challenge by developing an axial PSF deconvolution scheme to quantify the transport of peptides through a monolayer of Caco-2 cells using single-cell analysis with live-cell confocal fluorescence microscopy. We then measure the known cross-barrier transport of several compounds in our model and compare the results with results obtained in an established microfluidic model finding similar transport phenotypes. This verifies that already after two days the Caco-2 cells in our model form a tight monolayer and constitute a functional barrier model. We then apply this assay to investigate the effects of side chain lipidation of the model peptide drug salmon calcitonin (sCT) modified with 4­carbon and 8­carbon-long fatty acid chains. Furthermore, we compare that with experiments performed at lower temperature and using inhibitors for some endocytotic pathways to pinpoint how lipidation length modifies the main avenues for the transport. We thus show that increasing the length of the lipid chain increases the transport of the drug significantly but also makes endocytosis the primary transport mechanism in a short-term cell culture model.

Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging.

Unsuccessful clinical translation of orally delivered biological drugs remains a challenge in pharmaceutical development and has been linked to insufficient mechanistic understanding of intestinal drug transport. Live cell imaging could provide such mechanistic insights by directly tracking drug transport across intestinal barriers at subcellular resolution, however traditional intestinal in vitro models are not compatible with the necessary live cell imaging modalities. Here, we employed a novel microfluidic platform to develop an in vitro intestinal epithelial barrier compatible with advanced widefield- and confocal microscopy. We established a quantitative, multiplexed and high-temporal resolution imaging assay for investigating the cellular uptake and cross-barrier transport of biologics while simultaneously monitoring barrier integrity. As a proof-of-principle, we use the generic model to monitor the transport of co-administrated cell penetrating peptide (TAT) and insulin. We show that while TAT displayed a concentration dependent difference in its transport mechanism and efficiency, insulin displayed cellular internalization, but was restricted from transport across the barrier. This illustrates how such a sophisticated imaging based barrier model can facilitate mechanistic studies of drug transport across intestinal barriers and aid in vivo and clinical translation in drug development.

Gelling properties of black soldier fly (Hermetia illucens) larvae protein after ultrasound treatment.

H. illucens, black soldier fly larvae (BSFL) is one of the sustainable sources of protein. However, the research on the functionality of BSFL proteins is limited and need to be explored to increase consumer acceptance. The aim of this study is to create a gel system from BSFL protein and evaluate the impact of ultrasound treatment at different exposure time (5, 15, 30 min) on the physicochemical properties of BSFL protein. The highest values for surface hydrophobicity, size, ζ-potential were obtained after 15 min of ultrasound treatment and the same was found for the elastic modulus. Finally, confocal laser scanning microscopy (CLSM) along with image analysis revealed the lowest pore size after 15 min of treatment. The high protein content of BSFL protein extract and its promising gel system herein created, are important features to be considered for further development of insect-based food.

Interaction between Fish Skin Gelatin and Pea Protein at Air-Water Interface after Ultrasound Treatment.

The interaction between fish skin gelatin (FG) and pea protein isolate (PPI) was investigated at the air-water interface (A-W) before and after a high intensity (275 W, 5 min) ultrasound treatment (US). We analyzed the properties of the single protein suspensions as well as an equal ratio of FG:PPI (MIX), in terms of ζ-potential, particle size, molecular weight, bulk viscosity and interfacial tension. The foaming properties were then evaluated by visual analysis and by Turbiscan Tower. Confocal laser scanning microscopy (CLSM) was employed to explore the role of the proteins on the microstructure of foams. The results showed that the ultrasound treatment slightly influenced physicochemical properties of the proteins, while in general, did not significantly affect their behavior both in bulk and at the air-water interface. In particular, PPI aggregate size was reduced (-48 nm) while their negative charges were increased (-1 mV) after the treatment. However, when the proteins were combined, higher molecular weight of aggregates, higher foam stability values (+14%) and lower interfacial tension (IFT) values (47.2 ± 0.2 mN/m) were obtained, leading us to assume that a weak interaction was developed between them.

Imaging therapeutic peptide transport across intestinal barriers.

Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides, due to the biological barriers associated with the gastrointestinal tract. In this review, we start by summarizing the pharmacological performance of several clinically relevant orally administrated therapeutic peptides, highlighting their low bioavailabilities. Thus, there is a strong need to increase the transport of peptide drugs across the intestinal barrier to realize future treatment needs and further development in the field. Currently, progress is hampered by a lack of understanding of transport mechanisms that govern intestinal absorption and transport of peptide drugs, including the effects of the permeability enhancers commonly used to mediate uptake. We describe how, for the past decades, mechanistic insights have predominantly been gained using functional assays with end-point read-out capabilities, which only allow indirect study of peptide transport mechanisms. We then focus on fluorescence imaging that, on the other hand, provides opportunities to directly visualize and thus follow peptide transport at high spatiotemporal resolution. Consequently, it may provide new and detailed mechanistic understanding of the interplay between the physicochemical properties of peptides and cellular processes; an interplay that determines the efficiency of transport. We review current methodology and state of the art in the field of fluorescence imaging to study intestinal barrier transport of peptides, and provide a comprehensive overview of the imaging-compatible in vitro, ex vivo, and in vivo platforms that currently are being developed to accelerate this emerging field of research.

Selective Direct Laser Writing of Pyrolytic Carbon Microelectrodes in Absorber-Modified SU-8.

Pyrolytic carbon microelectrodes (PCMEs) are a promising alternative to their conventional metallic counterparts for various applications. Thus, methods for the simple and inexpensive patterning of PCMEs are highly sought after. Here, we demonstrate the fabrication of PCMEs through the selective pyrolysis of SU-8 photoresist by irradiation with a low-power, 806 nm, continuous wave, semiconductor-diode laser. The SU-8 was modified by adding Pro-Jet 800NP (FujiFilm) in order to ensure absorbance in the 800 nm range. The SU-8 precursor with absorber was successfully converted into pyrolytic carbon upon laser irradiation, which was not possible without an absorber. We demonstrated that the local laser pyrolysis (LLP) process in an inert nitrogen atmosphere with higher laser power and lower scan speed resulted in higher electrical conductance. The maximum conductivity achieved for a laser-pyrolyzed line was 14.2 ± 3.3 S/cm, with a line width and thickness of 28.3 ± 2.9 µm and 6.0 ± 1.0 µm, respectively, while the narrowest conductive line was just 13.5 ± 0.4 µm wide and 4.9 ± 0.5 µm thick. The LLP process seemed to be self-limiting, as multiple repetitive laser scans did not alter the properties of the carbonized lines. The direct laser writing of adjacent lines with an insulating gap down to ≤5 µm was achieved. Finally, multiple lines were seamlessly joined and intersected, enabling the writing of more complex designs with branching electrodes and the porosity of the carbon lines could be controlled by the scan speed.

Label-Free Sensing of Biorecognition on Liposomes.

Nanometer-sized liposomes decorated with macromolecules are increasingly used as drug delivery vehicles due to their long lifetimes and target cell specificity, but surface characterization methods often change their properties, which leads to incorrect results. Ligand binding is commonly applied for characterizing these surface modifications. Here, we use a nanofluidic-based label-free sensor for real-time sensing of ligands binding to liposomes. The liposomes are trapped in a nanochannel with a salt concentration gradient, and as the trapping position depends on the liposomes' zeta potential, it changes when charged ligands bind to the liposomes. Our sensing method does not require immobilization of the liposomes or labeling of the ligands with fluorophores, which may both affect the sensing. The zeta potential sensing is demonstrated by measuring hybridization of DNA targets with complementary DNA probes on liposome surfaces. DNA hybridization is monitored for both ensembles and individual liposomes, the latter allows for analysis of ensemble heterogeneity, and we demonstrate sensitivity to changes in surface charge down to 1.5%. DNA hybridization is used to demonstrate label-free sensing, but the method also has potential applications within exosome characterization, where biorecognition of, e.g., surface DNA, proteins, and antibodies is a promising candidate for early stage cancer diagnostics.

Physical Stability of Oil-In-Water Emulsion Stabilized by Gelatin from Saithe (Pollachius virens) Skin.

The objective of the present study was to investigate the physical stability of an oil-in-water (O/W) emulsion stabilized with gelatin from saithe (Pollachius virens) skin obtained with three different extraction protocols compared to two commercial fish skin gelatins. We first investigated the gelatin powder composition, and then produced the O/W emulsions at pH 3 by mechanical dispersion followed by an ultrasonication process. Sodium dodecyl sulfate (SDS) profiles for commercial samples indicated that extensive and unspecific hydrolysis of collagen occurred during the production process, whereas gelatin extracted from saithe fish skin showed typical electrophoresis patterns of type I collagen, with the presence of γ- and ß-chains. Emulsions obtained with commercial samples presented high physical stability over 7 days, with particle size of ~200 nm. However, emulsions obtained with saithe fish skin presented particle size between 300 and 450 nm. Slight differences were observed in viscosity, with values between ~1 and ~4 mPa·s. Interfacial tension measurements presented values between 13 and 17 mN·m-1 with three different regimes for all the systems.

Size and surface charge characterization of nanoparticles with a salt gradient.

Exosomes are nanometer-sized lipid vesicles present in liquid biopsies and used as biomarkers for several diseases including cancer, Alzheimer's, and central nervous system diseases. Purification and subsequent size and surface characterization are essential to exosome-based diagnostics. Sample purification is, however, time consuming and potentially damaging, and no current method gives the size and zeta potential from a single measurement. Here, we concentrate exosomes from a dilute solution and measure their size and zeta potential in a one-step measurement with a salt gradient in a capillary channel. The salt gradient causes oppositely directed particle and fluid transport that trap particles. Within minutes, the particle concentration increases more than two orders of magnitude. A fit to the spatial distribution of a single or an ensemble of exosomes returns both their size and surface charge. Our method is applicable for other types of nanoparticles. The capillary is fabricated in a low-cost polymer device.

In-line whole blood fractionation for Raman analysis of blood plasma.

Blood plasma evaluation has high significance in clinical diagnostics. Current schemes involve the preparation of blood plasma by centrifugation of whole blood followed by electrochemical or spectroscopic analysis. However, centrifugation is often too time-consuming for application in clinical emergency and point-of-care settings. We propose to combine microfluidic, instantaneous plasma fractionation with localized spectroscopic methods for in-line analysis. As an example, we present confocal Raman spectroscopy in fractionated plasma domains at two different Raman excitation wavelengths. Resonance Raman spectroscopy with laser excitation at 408 nm allows the specific detection of free hemoglobin in blood plasma at concentrations above 22 mg dl-1 (level of detection). Consequently, we are able to accurately resolve the range of clinical relevance regarding hemolysis. At near-infrared excitation (785 nm) we furthermore demonstrate the acquisition of characteristic Raman spectra of fractionated blood plasma in the microfluidic setting. These spectra can serve as starting point for a multi-parameter regression analysis to quantify a set of blood plasma parameters from a single Raman spectrum. The combined microfluidics and Raman spectroscopy method is non-destructive and has a whole blood consumption of less than 100 µl per hour. It thus allows for continuous in-line blood plasma monitoring.

Single-molecule DNA-mapping and whole-genome sequencing of individual cells.

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

Concentrating and labeling genomic DNA in a nanofluidic array.

Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final concentration can reach up to 100 µg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.

Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing.

Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

Optothermally actuated capillary burst valve.

We demonstrate the optothermal actuation of individual capillary burst valves in an all-polymer microfluidic device. The capillary burst valves are realised in a planar design by introducing a fluidic constriction in a microfluidic channel of constant depth. We show that a capillary burst valve can be burst by raising the temperature due to the temperature dependence of the fluid surface tension. We address individual valves by using a local heating platform based on a thin film of near infrared absorber dye embedded in the lid used to seal the microfluidic device [L. H. Thamdrup et al., Nano Lett. 10, 826-832 (2010)]. An individual valve is burst by focusing the laser in its vicinity. We demonstrate the capture of single polystyrene 7 µm beads in the constriction triggered by the bursting of the valve.