A fully online sensor-equipped, disposable multiphase microbioreactor as a screening platform for biotechnological applications.

A new disposable, multiphase, microbioreactor (MBR; with a working volume of 550 µl) equipped with online sensors is presented for biotechnological screening research purposes owing to its high-throughput potential. Its design and fabrication, online sensor integration, and operation are described. During aerobic cultivation, sufficient oxygen supply is the most important factor that influences growth and product formation. The MBR is a microbubble column bioreactor (µBC), and the oxygen supply was realized by active pneumatic bubble aeration, ensuring sufficient volumetric liquid-phase mass transfer (k L a) and proper homogenization of the cultivation broth. The µBC was equipped with miniaturized sensors for the pH, dissolved oxygen, optical density and glucose concentration that allowed real-time online monitoring of these process variables during cultivation. The challenge addressed here was the integration of sensors in the limited available space. The MBR was shown to be a suitable screening platform for the cultivation of biological systems. Batch cultivations of Saccharomyces cerevisiae were performed to observe the variation in the process variables over time and to show the robustness and operability of all the online sensors in the MBR.

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.

ToF-SIMS Depth Profiling of Metal, Metal Oxide, and Alloy Multilayers in Atmospheres of H2, C2H2, CO, and O2.

The influence of the flooding gas during ToF-SIMS depth profiling was studied to reduce the matrix effect and improve the quality of the depth profiles. The profiles were measured on three multilayered samples prepared by PVD. They were composed of metal, metal oxide, and alloy layers. Dual-beam depth profiling was performed with 1 keV Cs+ and 1 keV O2+ sputter beams and analyzed with a Bi+ primary beam. The novelty of this work was the application of H2, C2H2, CO, and O2 atmospheres during SIMS depth profiling. Negative cluster secondary ions, formed from sputtered metals/metal oxides and the flooding gases, were analyzed. A systematic comparison and evaluation of the ToF-SIMS depth profiles were performed regarding the matrix effect, ionization probability, chemical sensitivity, sputtering rate, and depth resolution. We found that depth profiling in the C2H2, CO, and O2 atmospheres has some advantages over UHV depth profiling, but it still lacks some of the information needed for an unambiguous determination of multilayered structures. The ToF-SIMS depth profiles were significantly improved during H2 flooding in terms of matrix-effect reduction. The structures of all the samples were clearly resolved while measuring the intensity of the MnHm-, MnOm-, MnOmH-, and Mn- cluster secondary ions. A further decrease in the matrix effect was obtained by normalization of the measured signals. The use of H2 is proposed for the depth profiling of metal/metal oxide multilayers and alloys.

Comparison Study of PVD Coatings: TiN/AlTiN, TiN and TiAlSiN Used in Wood Machining.

In this paper, we analyze the possibilities of the protection of tools for wood machining with PVD (Physical Vapor Deposition) hard coatings. The nanolayered TiN/AlTiN coating, nanocomposite TiAlSiN coatings, and single layer TiN coating were analyzed in order to use them for protection of tools for wood machining. Both nanostructured coatings were deposited in an industrial magnetron sputtering system on the cutting blades made of sintered carbide WC-Co, while TiN single layer coating was deposited by evaporation using thermionic arc. In the case of TiN/AlTiN nanolayer coatings the thickness of the individual TiN and AlTiN layer was in the 5-10 nm range, depending on the substrate vertical position. The microstructure and chemical composition of coatings were studied by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) method. Additionally, in the case of the TiN/AlTiN coating, which was characterized by the best durability characteristics, the transmission electron microscope (TEM) and X-ray photoelectron spectroscopy (XPS) methods were applied. The coatings adhesion to the substrate was analyzed by scratch test method combined with optical microscopy. Nano-hardness and durability tests were performed with uncoated and coated blades using chipboard. The best results durability characteristics were observed for TiN/AlTiN nanolayered coating. Performance tests of knives protected with TiN and TiAlSiN hard coatings did not show significantly better results compared to uncoated ones.

Influence of Different Types of Cemented Carbide Blades and Coating Thickness on Structure and Properties of TiN/AlTiN and TiAlN/a-C:N Coatings Deposited by PVD Techniques for Machining of Wood-Based Materials.

The influence of different types of cemented carbide blades and thickness of TiAlN/a-C:N and TiN/AlTiN protective coatings used in the wood industry on cutting performance has been studied. Three types of WC-Co cemented carbide blades with different cobalt content were used in the study. The thicknesses of both types of coatings were ~2 and ~5 µm. The structure, chemical and phase composition were studied using transmission and scanning electron microscopy (TEM, SEM), X-ray dispersion spectroscopy (EDX) and X-ray diffraction (XRD), respectively. The adhesion was evaluated by scratch test. Nanohardness and durability tests of uncoated and coated blades were performed. We found that the blades covered with 5 µm TiN/AlTiN coatings exhibited the best durability characteristic. The cutting distances were within the range ~6700-~7080 depending on the substrates in comparison with pure substrates (~4300-~4900) and 2 µm TiN/AlTiN coatings (~5400-~6600). The presence of a thin and soft outer a-C:N layer aggravates the nanohardness and durability of the coated blades.

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.

Development of a folic acid molecularly imprinted polymer and its evaluation as a sorbent for dispersive solid-phase extraction by liquid chromatography coupled to mass spectrometry.

This work shows the development of a molecularly imprinted polymer to determine folic acid (FA) in food extracts by using dispersive solid-phase extraction and liquid chromatography coupled to mass spectrometry (LC-MS). Herewith, combinations of monomers (methacrylic acid (MAA), 4-vinylpyridine (4VPy) and vinylbenzyl trimethylammonium chloride (VBTMAC)) and crosslinkers (ethylene glycol dimethacrylate (EGDMA) and divinyl benzene (DVB)) were tested in appropriate solvents. Isotherm tests revealed that the MIP with the highest affinity was obtained by combining VBTMAC and EGDMA. Having checked the appropriate template-monomer-crosslinker ratio, the FA MIP was analyzed for its kinetic and equilibrium binding properties, proving very high affinity (more than 2.5 mmol g-1) and MIP/NIP ratio (up to 37). The FA MIP was used to selectively isolate the compound of interest from lettuce and cookies matrices using a dispersive solid-phase extraction protocol (which exhibited appropriate recovery and repeatability, ≥79.50% and ≤13.41 (%RSD in terms of area values), respectively, as well as absence of matrix effect); the resulting extracts were analyzed by a rapid and reliable LC-MS method.

Multi-function microfluidic platform for sensor integration.

The limited availability of metabolite-specific sensors for continuous sampling and monitoring is one of the main bottlenecks contributing to failures in bioprocess development. Furthermore, only a limited number of approaches exist to connect currently available measurement systems with high throughput reactor units. This is especially relevant in the biocatalyst screening and characterization stage of process development. In this work, a strategy for sensor integration in microfluidic platforms is demonstrated, to address the need for rapid, cost-effective and high-throughput screening in bioprocesses. This platform is compatible with different sensor formats by enabling their replacement and was built in order to be highly flexible and thus suitable for a wide range of applications. Moreover, this re-usable platform can easily be connected to analytical equipment, such as HPLC, laboratory scale reactors or other microfluidic chips through the use of standardized fittings. In addition, the developed platform includes a two-sensor system interspersed with a mixing channel, which allows the detection of samples that might be outside the first sensor's range of detection, through dilution of the sample solution up to 10 times. In order to highlight the features of the proposed platform, inline monitoring of glucose levels is presented and discussed. Glucose was chosen due to its importance in biotechnology as a relevant substrate. The platform demonstrated continuous measurement of substrate solutions for up to 12 h. Furthermore, the influence of the fluid velocity on substrate diffusion was observed, indicating the need for in-flow calibration to achieve a good quantitative output.

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.