Establishment of physiologically relevant oxygen gradients in microfluidic organ chips.

In vitro models of human organs must accurately reconstitute oxygen concentrations and gradients that are observed in vivo to mimic gene expression, metabolism, and host-microbiome interactions. Here we describe a simple strategy to achieve physiologically relevant oxygen tension in a two-channel human small intestine-on-a-chip (Intestine Chip) lined with primary human duodenal epithelium and intestinal microvascular endothelium in parallel channels separated by a porous membrane while both channels are perfused with oxygenated medium. This strategy was developed using computer simulations that predicted lowering the oxygen permeability of poly-dimethylsiloxane (PDMS) chips in specified locations using a gas impermeable film will allow the cells to naturally decrease the oxygen concentration through aerobic respiration and reach steady-state oxygen levels <36 mm Hg (<5%) within the epithelial lumen. The approach was experimentally confirmed using chips with embedded oxygen sensors that maintained this stable oxygen gradient. Furthermore, Intestine Chips cultured with this approach supported formation of a villus epithelium interfaced with a continuous endothelium and maintained intestinal barrier integrity for 72 h. This strategy recapitulates in vivo functionality in an efficient, inexpensive, and scalable format that improves the robustness and translatability of Organ Chip technology for studies on microbiome as well as oxygen sensitivity.

Microfluidic Flow Injection Immunoassay System for Algal Toxins Determination: A Case of Study.

A novel flow injection microfluidic immunoassay system for continuous monitoring of saxitoxin, a lethal biotoxin, in seawater samples is presented in this article. The system consists of a preimmobilized G protein immunoaffinity column connected in line with a lab-on-chip setup. The detection of saxitoxin in seawater was carried out in two steps: an offline incubation step (competition reaction) performed between the analyte of interest (saxitoxin or Ag, as standard or seawater sample) and a tracer (an enzyme-conjugated antigen or Ag*) toward a specific polyclonal antibody. Then, the mixture was injected through a "loop" of a few µL using a six-way injection valve into a bioreactor, in line with the valve. The bioreactor consisted of a small glass column, manually filled with resin upon which G protein has been immobilized. When the mixture flowed through the bioreactor, all the antibody-antigen complex, formed during the competition step, is retained by the G protein. The tracer molecules that do not interact with the capture antibody and protein G are eluted out of the column, collected, and mixed with an enzymatic substrate directly within the microfluidic chip, via the use of two peristaltic pumps. When Ag* was present, a color change (absorbance variation, ΔAbs) of the solution is detected at a fixed wavelength (655 nm) by an optical chip docking system and registered by a computer. The amount of saxitoxin, present in the sample (or standard), that generates the variation of the intensity of the color, will be directly proportional to the concentration of the analyte in the analyzed solution. Indeed, the absorbance response increased proportionally to the enzymatic product and to the concentration of saxitoxin in the range of 3.5 × 10-7-2 × 10-5 ng ml-1 with a detection limit of 1 × 10-7 ng ml-1 (RSD% 15, S N-1 equal to 3). The immunoanalytical system has been characterized, optimized, and tested with seawater samples. This analytical approach, combined with the transportable and small-sized instrumentation, allows for easy in situ monitoring of marine water contaminations.

Towards microbioprocess control: an inexpensive 3D printed microbioreactor with integrated online real-time glucose monitoring.

Bioprocessing is of crucial importance in pharmaceutical, biofuel, food and other industries. Miniaturization of bioprocesses into microbioreactors allows multiplexing of experiments as well as reduction of reagent consumption and labour-intensity. A crucial part of the research within microbioreactors is biochemical analysis of product, byproduct and substrate concentrations that currently heavily relies on large analytical equipment. Biosensors are a promising analytical tool, however, integration into a microbioreactor is associated with challenges in ensuring sterility, appropriate sensing range, control of matrix effects and stability. In this work we present a novel biosensor integrated analytical chip that features an internal, actuated buffer flow in contact with a biosensor downstream and a diffusion limiting membrane exposed to the sample upstream. The technology was developed and tested using an electrochemical glucose oxidase biosensor and was found to successfully surmount the aforementioned challenges including the extension of the linear range of sensitivity to more than 20 g L-1 for online, real time monitoring of glucose. The biosensor integration chip with the glucose biosensor was then mounted onto a 3D printed microbioreactor with 1 mL of internal volume. The system successfully monitored the consumption of glucose of Saccharomyces cerevisiae in real time for more than 8 h. The developed technology and measurement methodologies are transferrable to other biosensors and microbioreactors as well as large scale applications.

Disposable electrochemical immunosensor for cortisol determination in human saliva.

Cortisol is a steroidal hormone and an important stress marker. Free serum cortisol concentration has been identified to correlate well with free salivary cortisol. In this present work an electrochemical immunosensor was developed to determine cortisol concentration within the physiological concentration range found in human saliva. The immunosensor is based on a direct competitive enzyme linked immunoassay using a home-made cortisol-alkaline phosphatase (AP) conjugate synthesized in our laboratory with disposable graphite screen-printed electrodes (SPEs). 1-nalphtyl phosphate (1-NP) was used as an enzymatic substrate and a square wave voltammetry (SWV) for electrochemical detection. To study method suitability for use with saliva samples, calibration curves were performed both in buffer and saliva. In buffer standard samples showed a limit of detection (LOD) of 0.6 ng/ml and working range (WR) of 0.2-44.6 ng/ml with good reproducibility (RSD 10%). Saliva matrix effect was removed effectively with Salivette Cortisol collection device (polyethylene) and a calibration curve showed similar characteristics as in buffer with LOD 1.7 ng/ml and WR 0.5-55.1 ng/ml (RSD 8%) demonstrating the possibility to determine human salivary cortisol within the desired human physiological range. Spiked saliva samples were analyzed with the developed immunosensor presenting excellent 92-114% recovery. Comparison to liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method showed strong 0.90 correlation between methods indicating good accuracy of the developed immunosensor.

Determination of stability characteristics for electrochemical biosensors via thermally accelerated ageing.

Biosensors are devices that are prone to ageing; this phenomenon can be characterized as a decrease in signal over time. Biosensor stability is of a crucial importance for commercial success and as biosensors are presently being applied to an increasing and variety of applications. Stability characteristics related to shelf life, reusability and/or continuous use stability are often poorly investigated or unreported in literature, yet are important factors. Instability or ageing can be accelerated at an elevated temperature; Arrhenius (exponential) and linear models were investigated in order to propose a novel method for rapid ageing characteristics determination. Linear correlation proved more suitable with higher coefficients of determination than exponential correlation. Degradation rate is linearly dependent on temperature and by utilizing the proposed models, long term shelf life of a biosensor can be determined in 4 days and continuous use stability in less than 24h. Reusability studies are found to correlate poorly due to the unpredictable nature of biosensor handling. Basic constructed screen printed electrode glucose oxidase biosensors were used as a model biosensor in order to propose models for shelf life, reusability and continuous use stability.

Development of a competitive immunoassay for the determination of cortisol in human saliva.

Two new protein conjugates were prepared and studied to develop and compare two (direct and indirect) competitive enzyme-linked immunosorbent assay (ELISA) formats for the determination of cortisol in human saliva. Toward this goal, ovalbumin was conjugated to cortisol and used for developing an indirect competitive ELISA, while alkaline phosphatase was coupled with the same analyte for a direct competitive assay. The yield of the conjugation reactions was evaluated. The results obtained show that the indirect and direct ELISA formats developed for cortisol had working ranges of 0.5-70 and 2-330 ng/ml and detection limits of 0.5 and 1.2 ng/ml, respectively. Artificial and real saliva samples were spiked with cortisol to study the matrix effect of saliva. The suitability of the assays for quantification of cortisol in saliva was also studied.

Carbohydrates electrocatalytic oxidation using CNT-NiCo-oxide modified electrodes.

A new sensor for an amplified electrochemical detection of carbohydrates is proposed, where carbohydrates are oxidized by CNT-NiCo-oxide composite in basic solutions. Cyclic voltammograms of the modified electrode show a stable and well defined redox couple in alkaline media due to the synergy of Ni(II)/Ni(III) system with Co(II)/Co(III). The modified electrode shows excellent electrocatalytic activity towards monosaccharides oxidation at reduced overpotential in alkaline solutions. Six monosaccharides were determined amperometrically at the surface of this modified electrode with high sensitivity over a wide range of concentrations, from 0.02 up to 12.12 mM. Low detection limit of 5 µM for glucose could be obtained.