Search

The correlation between nutritional habits and gut health directly impacts the gut-brain axis via a complex and not yet fully disclosed communication network. Establishing a link between our food intake and specific physiological responses as well as a better knowledge of diseases and the gut microbiota involves solving a challenging puzzle of biochemical pathways. Our understanding is limited by the inaccessibility of the gastrointestinal region to routine non-invasive chemical analysis. Here, we move a step further toward the direct assessment of a protein fermentation product, i.e., ammonium ions, via Ion Selective Electrodes (ISEs) in gastrointestinal digesta samples. By modulating the digestible protein content of the diet regimes of two groups of pigs, we discriminate the level of protein fermentation with a straightforward quasi-in vivo detection method which does not require any sample preparation. Our results show more than a 2-fold increase in ammonium ion concentration (from 180 ppm to 400 ppm) in the proximal colon for a diet based on poorly digestible proteins compared with a diet based on easily digestible proteins. Our approach shows good correlation with a standard laboratory technique for the determination of NH4+, i.e., the Indophenol Blue Method. Our results show the direct sensing of a protein fermentation product in a real matrix and demonstrate the great potential of potentiometric sensors to assess ammonium concentration profiles along the gastrointestinal tract for diets varying in protein digestibility and fermentation.

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements, and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.

The repository contains the raw data ("*.tif") files, data from subsequent analysis steps performed in Matlab ("*.mat") and obtained histograms., Readme.txt, 16.07.2021 Fontana et al. “Probing DNA - transcription factor interactions using single-molecule fluorescence detection in nanofluidic devices” pre-print: https://doi.org/10.1101/2021.05.12.443786 (BioRxiv) The repository contains the raw data ("*.tif") files, data from subsequent analysis steps performed in Matlab ("*.mat") and obtained histograms. For further information, please contact dr. Johannes Hohlbein @ Wageningen University & Research (johannes.hohlbein@wur.nl)

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height and a width of several micrometers confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observe DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level.

In vitro digestions are essential for determining the bioavailability of compounds, such as nutrients. We have developed a cell-free, miniaturized enzymatic digestive system, employing three micromixers connected in series to mimic the digestive functions of the mouth, stomach and small intestine. This system continuously processes samples, e.g. containing nutrients, to provide a constant flow of digested materials which may be presented to a subsequent gut-on-a-chip absorption module, containing living human intestinal cells. Our system incorporates three-compartment enzymatic digestion, one of the key functions of the gastrointestinal tract. In each of these compartments, we modify the chemical environment, including pH, buffer, and mineral composition, to closely mimic the local physiological environment and create optimal conditions for digestive processes to take place. It will therefore provide an excellent addition to existing gut-on-a-chip systems, providing the next step in determining the bio-availability of orally administered compounds in a fast and continuous-flow ex vivo system. In this paper, we demonstrate enzymatic digestion in each separate compartment using compounds, starch and casein, as model nutrients. The use of transparent, microfluidic micromixers based on chaotic advection, which can be probed directly with a microscope, enabled enzyme kinetics to be monitored from the very start of a reaction. Furthermore, we have digested lactoferrin in our system, demonstrating complete digestion of this milk protein in much shorter times than achievable with standard in vitro digestions using batch reactors.

Proposed is a nanofluidic device (20) comprising a first microchannel (31 ), a first branch nanochannel (33) branching off the first microchannel (31 ), a second microchannel (32), a second branch nanochannel (34) branching off the second microchannel (32), and a mixing nanochannel (36). The mixing nanochannel hydraulic resistance is at least one thousand times larger than each of the first and second microchannel hydraulic resistance. A junction (35) is provided which connects the first branch channel, the second branch channel and the mixing nanochannel. The junction (35): - receives a first laminar fluid flow from the first branch nanochannel (33) and a second laminar fluid flow from the second branch nanochannel (34), and - contacts the first and second laminar fluid flows with each other to establish diffusion mixing of the first and second fluids, and - discharge the mixed first and second fluids into the mixing nanochannel inlet (36a).

We report a strategy for the fabrication of a new type of electrochemical nanogap transducer. These nanogap devices are based on signal amplification by redox cycling. Using two steps of electron-beam lithography, vertical gold electrodes are fabricated side by side at a 70 nm distance encompassing a 20 attoliter open nanogap volume. We demonstrate a current amplification factor of 2.5 as well as the possibility to detect the signal of only 60 analyte molecules occupying the detection volume. Experimental voltammetry results are compared to calculations from finite element analysis., ChemE/Organic Materials and Interfaces

We report a strategy for the fabrication of a new type of electrochemical nanogap transducer. These nanogap devices are based on signal amplification by redox cycling. Using two steps of electron-beam lithography, vertical gold electrodes are fabricated side by side at a 70 nm distance encompassing a 20 attoliter open nanogap volume. We demonstrate a current amplification factor of 2.5 as well as the possibility to detect the signal of only 60 analyte molecules occupying the detection volume. Experimental voltammetry results are compared to calculations from finite element analysis., OLD ChemE/Organic Materials and Interfaces

We introduce a nanofluidic mixing device entirely fabricated in glass for the fluorescence detection of single molecules. The design consists of a nanochannel T-junction and allows the continuous monitoring of chemical or enzymatic reactions of analytes as they arrive from two independent inlets. The fluorescently labeled molecules are tracked before, during and after they enter the mixing region, and their reactions with each other are observed by means of optical readout such as Förster Resonance Energy Transfer (FRET). Our method can be used for analyzing the kinetics of DNA annealing in a high-parallelized fashion.

This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by bonding it to a complementary poly(dimethylsiloxane) structure. The resulting structures place the nanowires in the region of highest flow, as opposed to the walls, where it approaches zero, and expose their entire surface area to fluid. We demonstrate active functionality, by constructing a hot-wire anemometer to measure flow through determining the change in resistance of the nanowire as a function of heat dissipation at low voltage ( < 5 V). Further, passive functionality is demonstrated by visualizing individual, fluorescently labeled DNA molecules attached to the wires. We measure rates of flow and show that, compared to surface-bound DNA strands, elongation saturates at lower rates of flow and background fluorescence from nonspecific binding is reduced.

This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by bonding it to a complementary poly(dimethylsiloxane) structure. The resulting structures place the nanowires in the region of highest flow, as opposed to the walls, where it approaches zero, and expose their entire surface area to fluid. We demonstrate active functionality, by constructing a hot-wire anemometer to measure flow through determining the change in resistance of the nanowire as a function of heat dissipation at low voltage ( < 5 V). Further, passive functionality is demonstrated by visualizing individual, fluorescently labeled DNA molecules attached to the wires. We measure rates of flow and show that, compared to surface-bound DNA strands, elongation saturates at lower rates of flow and background fluorescence from nonspecific binding is reduced.