Oxygen Management at the Microscale: A Functional Biochip Material with Long-Lasting and Tunable Oxygen Scavenging Properties for Cell Culture Applications.

Oxygen plays a pivotal role in cellular homeostasis, and its partial pressure determines cellular function and fate. Consequently, the ability to control oxygen tension is a critical parameter for recreating physiologically relevant in vitro culture conditions for mammalian cells and microorganisms. Despite its importance, most microdevices and organ-on-a-chip systems to date overlook oxygen gradient parameters because controlling oxygen often requires bulky and expensive external instrumental setups. To overcome this limitation, we have adapted an off-stoichiometric thiol-ene-epoxy polymer to efficiently remove dissolved oxygen to below 1 hPa and also integrated this modified polymer into a functional biochip material. The relevance of using an oxygen scavenging material in microfluidics is that it makes it feasible to readily control oxygen depletion rates inside the biochip by simply changing the surface-to-volume aspect ratio of the microfluidic channel network as well as by changing the temperature and curing times during the fabrication process.

Every Breath You Take: Non-invasive Real-Time Oxygen Biosensing in Two- and Three-Dimensional Microfluidic Cell Models.

Knowledge on the availability of dissolved oxygen inside microfluidic cell culture systems is vital for recreating physiological-relevant microenvironments and for providing reliable and reproducible measurement conditions. It is important to highlight that in vivo cells experience a diverse range of oxygen tensions depending on the resident tissue type, which can also be recreated in vitro using specialized cell culture instruments that regulate external oxygen concentrations. While cell-culture conditions can be readily adjusted using state-of-the-art incubators, the control of physiological-relevant microenvironments within the microfluidic chip, however, requires the integration of oxygen sensors. Although several sensing approaches have been reported to monitor oxygen levels in the presence of cell monolayers, oxygen demands of microfluidic three-dimensional (3D)-cell cultures and spatio-temporal variations of oxygen concentrations inside two-dimensional (2D) and 3D cell culture systems are still largely unknown. To gain a better understanding on available oxygen levels inside organ-on-a-chip systems, we have therefore developed two different microfluidic devices containing embedded sensor arrays to monitor local oxygen levels to investigate (i) oxygen consumption rates of 2D and 3D hydrogel-based cell cultures, (ii) the establishment of oxygen gradients within cell culture chambers, and (iii) influence of microfluidic material (e.g., gas tight vs. gas permeable), surface coatings, cell densities, and medium flow rate on the respiratory activities of four different cell types. We demonstrate how dynamic control of cyclic normoxic-hypoxic cell microenvironments can be readily accomplished using programmable flow profiles employing both gas-impermeable and gas-permeable microfluidic biochips.

Arsenolipid biosynthesis by the unicellular alga Dunaliella tertiolecta is influenced by As/P ratio in culture experiments.

The influence of arsenate and phosphate levels in water on the formation of arsenic-containing lipids (arsenolipids) and water-soluble arsenicals by a unicellular marine alga was investigated by exposing Dunaliella tertiolecta to five regimes of arsenic and phosphate, and determining the biosynthesized organoarsenicals with HPLC/mass spectrometry. Under all conditions, the major arsenolipid produced by D. tertiolecta was the novel phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside (AsSugPhytol546) representing ca. 35-65% of total arsenolipids. The new compound contains a phytol aglycone and a methoxy group replacing a sugar hydroxyl - two structural features not previously observed for arsenolipids. Minor arsenolipids were several previously reported arsenosugar phospholipids (AsSugPLs, in particular AsSugPL958 and the previously unknown AsSugPL978), the relative quantities of which increased with increasing phosphate exposure, and an arsenic-containing hydrocarbon (AsHC360), which remained unaffected by the different treatments. The relative amount of total arsenolipids produced by D. tertiolecta remained remarkably constant (ca. 45% of total As) and independent of the culture conditions. In contrast, with rising As-concentrations we observed an increase of hydrophilic arsenicals, which were dominated by arsenate and arsenosugars. The results highlight a possible major difference in arsenic biochemistry between macroalgae and unicellular algae with potential implications for how various algae handle their natural arsenic exposure in the world's oceans.

Fast pesticide detection inside microfluidic device with integrated optical pH, oxygen sensors and algal fluorescence.

The necessities of developing fast, portable, cheap and easy to handle pesticide detection platforms are getting attention of scientific and industrial communities. Although there are some approaches to develop microchip based pesticide detection platforms, there is no compact microfluidic device for the complementary, fast, cheap, reusable and reliable analysis of different pesticides. In this work, a microfluidic device is developed for in-situ analysis of pesticide concentration detected via metabolism/photosynthesis of Chlamydomonas reinhardtii algal cells (algae) in tap water. Algae are grown in glass based microfluidic chip, which contains integrated optical pH and oxygen sensors in a portable system for on-site detection. In addition, intrinsic algal fluorescence is detected to analyze the pesticide concentration in parallel to pH and oxygen sensors with integrated fluorescence detectors. The response of the algae under the effect of different concentrations of pesticides is evaluated and complementary inhibition effects depending on the pesticide concentration are demonstrated. The three different sensors allow the determination of various pesticide concentrations in the nanomolar concentration range. The miniaturized system provides the fast quantification of pesticides in less than 10min and enables the study of toxic effects of different pesticides on Chlamydomonas reinhardtii green algae. Consequently, the microfluidic device described here provides fast and complementary detection of different pesticides with algae in a novel glass based microfluidic device with integrated optical pH, oxygen sensors and algal fluorescence.

Simultaneous Determination of Oxygen and pH Inside Microfluidic Devices Using Core-Shell Nanosensors.

A powerful online analysis setup for the simultaneous detection of oxygen and pH is presented. It features core-shell nanosensors, which enable contactless and inexpensive read-out using adapted oxygen meters via modified dual lifetime referencing in the frequency domain (phase shift measurements). Lipophilic indicator dyes were incorporated into core-shell structured poly(styrene-block-vinylpyrrolidone) nanoparticles (average diameter = 180 nm) yielding oxygen nanosensors and pH nanosensors by applying different preparation protocols. The oxygen indicator platinum(II) meso-tetra(4-fluorophenyl) tetrabenzoporphyrin (PtTPTBPF) was entrapped into the polystyrene core (oxygen nanosensors) and a pH sensitive BF2-chelated tetraarylazadipyrromethene dye (aza-BODIPY) was incorporated into the polyvinylpyrrolidone shell (pH nanosensors). The brightness of the pH nanoparticles was increased by more than 3 times using a light harvesting system. The nanosensors have several advantages such as being excitable with red light, emitting in the near-infrared spectral region, showing a high stability in aqueous media even at high particle concentrations, high ionic strength, or high protein concentrations and are spectrally compatible with the used read-out device. The resolution for oxygen of the setup is 0.5-2.0 hPa (approximately 0.02-0.08 mg/L of dissolved oxygen) at low oxygen concentrations (<50 hPa) and 4-8 hPa (approximately 0.16-0.32 mg/L of dissolved oxygen) at ambient air oxygen concentrations (approximately 200 hPa at 980 mbar air pressure) at room temperature. The pH resolution is 0.03-0.1 pH units within the dynamic range (apparent pKa 7.23 ± 1.0) of the nanosensors. The sensors were used for online monitoring of pH changes during the enzymatic transformation of Penicillin G to 6-aminopenicillanic acid catalyzed by Penicillin G acylase in miniaturized stirred batch reactors or continuous flow microreactors.

A microfluidically perfused three dimensional human liver model.

Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-2',7'-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation.

Quantification of arsenolipids in the certified reference material NMIJ 7405-a (Hijiki) using HPLC/mass spectrometry after chemical derivatization.

Arsenic-containing lipids (arsenolipids) are novel natural products recently shown to be widespread in marine animals and algae. Research interest in these arsenic compounds lies in their possible role in the membrane chemistry of organisms and, because they occur in many popular seafoods, their human metabolism and toxicology. Progress has been restricted, however, by the lack of standard arsenolipids and of a quantitative method for their analysis. We report that the certified reference material CRM 7405-a (Hijiki) is a rich source of arsenolipids, and we describe a method based on HPLC-ICPMS/ESMS to quantitatively measure seven of the major arsenolipids present. Sample preparation involved extraction with DCM/methanol, a cleanup step with silica, and conversion of the (oxo)arsenolipids originally present to thio analogues by brief treatment with H2S. Compared to their oxo analogues, the thioarsenolipids showed much sharper peaks on reversed-phase HPLC, which facilitated their resolution and quantification. The compounds were determined by HPLC-ICPMS and HPLC-ESMS, which provided both arsenic-selective detection and high resolution molecular mass detection of the arsenolipids. In this way, the concentrations of two arsenic-containing hydrocarbons and five arsenosugar phospholipids are reported in the CRM Hijiki. This material may serve as a convenient source of characterized arsenolipids to delineate the presence of these compounds in seafoods and to facilitate research in a new era of arsenic biochemistry.

Low cost referenced luminescent imaging of oxygen and pH with a 2-CCD colour near infrared camera.

A low cost imaging set-up for optical chemical sensors based on NIR-emitting dyes is presented. It is based on a commercially available 2-CCD colour near infrared camera, LEDs and tailor-made optical sensing materials for oxygen and pH. The set-up extends common ratiometric RGB imaging based on the red, green and blue channels of colour cameras by an additional NIR channel. The hardware and software of the camera were adapted to perform ratiometric imaging. A series of new planar sensing foils were introduced to image oxygen, pH and both parameters simultaneously. The used NIR-emitting indicators are based on benzoporphyrins and aza-BODIPYs for oxygen and pH, respectively. Moreover, a wide dynamic range oxygen sensor is presented. It allows accurate imaging of oxygen from trace levels up to ambient air concentrations. The imaging set-up in combination with the normal range ratiometric oxygen sensor showed a resolution of 4-5 hPa at low oxygen concentrations (<50 hPa) and 10-15 hPa at ambient air oxygen concentrations; the trace range oxygen sensor (<20 hPa) revealed a resolution of about 0.5-1.8 hPa. The working range of the pH-sensor was in the physiological region from pH 6.0 up to pH 8.0 and showed an apparent pKa-value of 7.3 with a resolution of about 0.1 pH units. The performance of the dual parameter oxygen/pH sensor was comparable to the single analyte pH and normal range oxygen sensors.