Effect of dendrimer-based interlayers for enzyme immobilization on a model electrochemical sensing system for glutamate.

In this paper, we discuss dendrimer usage in enzyme-based electrochemical biosensors, particularly with respect to biomolecule loading on the sensing surface. A novel approach to design bioactive layers with immobilized enzymes for electrochemical biosensors using the surface plasmon resonance (SPR) method in combination with electrochemical impedance spectroscopy was presented. The gold surface was modified with linear linkers (various mercaptoalkanoic acids and aminoalkanethiols) and poly(amidoamine) dendrimers from the first- to fifth-generation. The best functionalization procedure was established by detailed SPR studies and transferred onto gold electrodes to electrochemically examine the model enzymatic reaction catalysed by glutamate oxidase. In the case of the chronoamperometric method, the determined sensitivity was 3.36 ± 0.08 µA∙mM-1, and the low limit of detection (LOD) was 1.52 µM. Comparing the sensitivity and LOD obtained for CV measurements, the values of these parameters were 2.5 times higher and 4 times lower, respectively, for the fourth-generation dendrimer-based biosensor and the biosensor with a linear linker. The positive impact of the dendrimer interlayer on the long-term enzyme activity was also confirmed. The research results indicate the possibility of a significant increase in the sensor response using the dendrimer itself without enriching it with electrochemical components.

Automatic air volume control system for ventilation of two patients using a single ventilator: a large animal model study.

The COVID-19 pandemic outbreak led to a global ventilator shortage. Hence, various strategies for using a single ventilator to support multiple patients have been considered. A device called Ventil previously validated for independent lung ventilation was used in this study to evaluate its usability for shared ventilation. We performed experiments with a total number of 16 animals. Eight pairs of pigs were ventilated by a ventilator or anesthetic machine and by Ventil for up to 27 h. In one experiment, 200 ml of saline was introduced to one subject's lungs to reduce their compliance. The experiments were analyzed in terms of arterial blood gases and respiratory parameters. In addition to the animal study, we performed a series of laboratory experiments with artificial lungs (ALs). The resistance and compliance of one AL (affected) were altered, while the tidal volume (TV) and peak pressure (Ppeak) in the second (unaffected) AL were analyzed. In addition, to assess the risk of transmission of pathogens between AL respiratory tracts, laboratory tests were performed using phantoms of virus particles. The physiological level of analyzed parameters in ventilated animals was maintained, except for CO2 tension, for which a permissive hypercapnia was indicated. Experiments did not lead to injuries in the animal's lungs except for one subject, as indicated by CT scan analysis. In laboratory experiments, changes in TV and Ppeak in the unaffected AL were less than 11%, except for 2 cases where the TV change was 20%. No cross-contamination was found in simulations of pathogen transmission. We conclude that ventilation using Ventil can be considered safe in patients undergoing deep sedation without spontaneous breathing efforts.

Nanohollow Titanium Oxide Structures on Ti/FTO Glass Formed by Step-Bias Anodic Oxidation for Photoelectrochemical Enhancement.

In this study, a new anodic oxidation with a step-bias increment is proposed to evaluate oxidized titanium (Ti) nanostructures on transparent fluorine-doped tin oxide (FTO) on glass. The optimal Ti thickness was determined to be 130 nm. Compared to the use of a conventional constant bias of 25 V, a bias ranging from 5 V to 20 V with a step size of 5 V for 3 min per period can be used to prepare a titanium oxide (TiOx) layer with nanohollows that shows a large increase in current of 142% under UV illumination provided by a 365 nm LED at a power of 83 mW. Based on AFM and SEM, the TiOx grains formed in the step-bias anodic oxidation were found to lead to nanohollow generation. Results obtained from EDS mapping, HR-TEM and XPS all verified the TiOx composition and supported nanohollow formation. The nanohollows formed in a thin TiOx layer can lead to a high surface roughness and photon absorbance for photocurrent generation. With this step-bias anodic oxidation methodology, TiOx with nanohollows can be obtained easily without any extra cost for realizing a high current under photoelectrochemical measurements that shows potential for electrochemical-based sensing applications.

Biosensor based on coupled enzyme reactions for determination of arginase activity.

The determination of the enzymatic activity requires constant and reproducible measuring conditions, therefore highly stable potentiometric biosensor operating on the basis of coupled enzyme reactions is proposed for arginase activity determination. Glassy carbon electrode was covered with polyazulene ion-to-electron transducing layer, which ensured improved stability of the prepared sensor. The sensor's selectivity was obtained by applying NH4+-selective membrane on the transducing layer, which was further biofunctionalized with urease via covalent immobilization. The immobilized urease served as an auxiliary enzyme in the arginase-urease coupled enzyme assay. Thanks to obtained high stability (low drift coefficient âˆ¼ 0.9 mV/h) and short response time (36 s), the developed urea biosensor enabled continuous monitoring the coupled enzyme reactions indirectly, through measurement the ammonium ion concentration. Arginase activity and Michaelis-Menten constant for arginine-arginase pair under defined experimental conditions, in particular constant concentration of manganese ions, was determined. Mathematical description of the coupled enzyme reactions kinetics is discussed and used to analyse the reactions with auxiliary enzyme immobilized on the electrode surface. The proposed method of determining arginase activity with use of highly stable potentiometric urease-based biosensor, allows obtaining results in the units of absolute arginase activity.

Highly Stable Potentiometric (Bio)Sensor for Urea and Urease Activity Determination.

There is growing interest for bioanalytical tools that might be designed for a specific user, primarily for research purposes. In this perspective, a new, highly stable potentiometric sensor based on glassy carbon/polyazulene/NH4+-selective membrane was developed and utilized for urease activity determination. Urease-urea interaction studies were carried out and the Michaelis-Menten constant was established for this enzymatic reaction. Biofunctionalization of the ammonium ion-selective sensor with urease lead to urea biosensor with remarkably good potential stability (drift coefficient ~0.9 mV/h) and short response time (t95% = 36 s). The prepared biosensor showed the Nernstian response (S = 52.4 ± 0.7 mV/dec) in the urea concentration range from 0.01 to 20 mM, stable for the experimental time of 60 days. In addition, some insights into electrical properties of the ion-to-electron transducing layer resulting from impedance spectroscopy measurements are presented. Based on the RCQ equivalent circuits comparison, it can be drawn that the polyazulene (PAz) layer shows the least capacitive behavior, which might result in good time stability of the sensor in respect to response as well as potential E0. Both the polyazulene-based solid-contact ion selective electrodes and urea biosensors were successfully used in trial studies for determination of ammonium ion and urea in human saliva samples. The accuracy of ammonium ion and urea levels determination by potentiometric method was confirmed by two reference spectrophotometric methods.

Fabrication of Electrochemical Biosensor Based on Titanium Dioxide Nanotubes and Silver Nanoparticles for Heat Shock Protein 70 Detection.

This paper presents the fabrication methodology of an electrochemical biosensor for the detection of heat shock protein 70 (HSP70) as a potential tumor marker with high diagnostic sensitivity. The sensor substrate was a composite based on titanium dioxide nanotubes (TNTs) and silver nanoparticles (AgNPs) produced directly on TNTs by electrodeposition, to which anti-HSP70 antibodies were attached by covalent functionalization. This manuscript contains a detailed description of the production, modification, and the complete characteristics of the material used as a biosensor platform. As-formed TNTs, annealed TNTs, and the final sensor platform-AgNPs/TNTs, were tested using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction analysis (XRD). In addition, open circuit potential (OCP), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV) of these substrates were used to assess the influence of TNTs modification on their electrochemical characteristics. The EIS technique was used to monitor the functionalization steps of the AgNPs/TNTs electrode and the interaction between anti-HSP70 and HSP70. The produced composite was characterized by high purity, and electrical conductivity improved more than twice compared to unmodified TNTs. The linear detection range of HSP70 of the developed biosensor was in the concentration range from 0.1 to 100 ng/mL.

An integrated actuating and sensing system for light-addressable potentiometric sensor (LAPS) and light-actuated AC electroosmosis (LACE) operation.

To develop a lab on a chip (LOC) integrated with both sensor and actuator functions, a novel two-in-one system based on optical-driven manipulation and sensing in a microfluidics setup based on a hydrogenated amorphous silicon (a-Si:H) layer on an indium tin oxide/glass is first realized. A high-intensity discharge xenon lamp functioned as the light source, a chopper functioned as the modulated illumination for a certain frequency, and a self-designed optical path projected on the digital micromirror device controlled by the digital light processing module was established as the illumination input signal with the ability of dynamic movement of projected patterns. For light-addressable potentiometric sensor (LAPS) operation, alternating current (AC)-modulated illumination with a frequency of 800 Hz can be generated by the rotation speed of the chopper for photocurrent vs bias voltage characterization. The pH sensitivity, drift coefficient, and hysteresis width of the Si3N4 LAPS are 52.8 mV/pH, -3.2 mV/h, and 10.5 mV, respectively, which are comparable to the results from the conventional setup. With an identical two-in-one system, direct current illumination without chopper rotation and an AC bias voltage can be provided to an a-Si:H chip with a manipulation speed of 20 µm/s for magnetic beads with a diameter of 1 µm. The collection of magnetic beads by this light-actuated AC electroosmosis (LACE) operation at a frequency of 10 kHz can be easily realized. A fully customized design of an illumination path with less decay can be suggested to obtain a high efficiency of manipulation and a high signal-to-noise ratio of sensing. With this proposed setup, a potential LOC system based on LACE and LAPS is verified with the integration of a sensor and an actuator in a microfluidics setup for future point-of-care testing applications.

Effects of genetically modified human skin fibroblasts, stably overexpressing hepatocyte growth factor, on hepatic functions of cocultured C3A cells.

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver-specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell-cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model-C3A cells.

Comparison of Gold Nanoparticles Deposition Methods and Their Influence on Electrochemical and Adsorption Properties of Titanium Dioxide Nanotubes.

The increasing interest of attachment of gold nanoparticles (AuNPs) on titanium dioxide nanotubes (TNTs) has been devoted to obtaining tremendous properties suitable for biosensor applications. Achieving precise control of the attachment and shape of AuNPs by methods described in the literature are far from satisfactory. This work shows the comparison of physical adsorption (PA), cyclic voltammetry (CV) and chronoamperometry (CA) methods and the parameters of these methods on TNTs properties. The structural, chemical, phase and electrochemical characterizations of TNTs, Au/TNTs, AuNPs/TNTs are carried out using scanning electron microscopy (SEM), electrochemical impedance spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy. The use of PA methods does not allow the deposition of AuNPs on TNTs. CV allows easily obtaining spherical nanoparticles, for which the diameter increases from 20.3 ± 2.9 nm to 182.3 ± 51.7 nm as a concentration of tetrachloroauric acid solution increase from 0.1 mM to 10 mM. Increasing the AuNPs deposition time in the CA method increases the amount of gold, but the AuNPs diameter does not change (35.0 ± 5 nm). Importantly, the CA method also causes the dissolution of the nanotubes layer from 1000 ± 10.0 nm to 823 ± 15.3 nm. Modification of titanium dioxide nanotubes with gold nanoparticles improved the electron transfer and increased the corrosion resistance, as well as promoted the protein adsorption. Importantly, after the deposition of bovine serum albumin, an almost 5.5-fold (324%) increase in real impedance, compared to TNTs (59%) was observed. We found that the Au nanoparticles-especially those with smaller diameter-promoted the stability of bovine serum albumin binding to the TNTs platform. It confirms that the modification of TNTs with gold nanoparticles allows the development of the best platform for biosensing applications.

The Influence of the Parameters of a Gold Nanoparticle Deposition Method on Titanium Dioxide Nanotubes, Their Electrochemical Response, and Protein Adsorption.

The goal of this research was to find the best conditions to prepare titanium dioxide nanotubes (TNTs) modified with gold nanoparticles (AuNPs). This paper, for the first time, reports on the influence of the parameters of cyclic voltammetry process (CV) -based AuNP deposition, i.e., the number of cycles and the concentration of gold salt solution,on corrosion resistance and the capacitance of TNTs. Another innovation was to fabricate AuNPs with well-formed spherical geometry and uniform distribution on TNTs. The AuNPs/TNTs were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy, and open-circuit potential measurement. From the obtained results,the correlation between the deposition process parameters, the AuNP diameters,and the electrical conductivity of the TNTs was found in a range from 14.3 ± 1.8 to 182.3 ± 51.7 nm. The size and amount of the AuNPs could be controlled by the number of deposition cycles and the concentration of the gold salt solution.The modification of TNTs using AuNPs facilitated electron transfer, increased the corrosion resistance, and caused better adsorption properties for bovine serum albumin.

Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay.

This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-µm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems) was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead-protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.

Optimization of urea-EnFET based on Ta2O5 layer with post annealing.

In this study, the urea-enzymatic field effect transistors (EnFETs) were investigated based on pH-ion sensitive field effect transistors (ISFETs) with tantalum pentoxide (Ta(2)O(5)) sensing membranes. In addition, a post N(2) annealing was used to improve the sensing properties. At first, the pH sensitivity, hysteresis, drift, and light induced drift of the ISFETs were evaluated. After the covalent bonding process and urease immobilization, the urea sensitivity of the EnFETs were also investigated and compared with the conventional Si(3)N(4) sensing layer. The ISFETs and EnFETs with annealed Ta(2)O(5) sensing membranes showed the best responses, including the highest pH sensitivity (56.9 mV/pH, from pH 2 to pH 12) and also corresponded to the highest urea sensitivity (61 mV/pC(urea), from 1 mM to 7.5 mM). Besides, the non-ideal factors of pH hysteresis, time drift, and light induced drift of the annealed samples were also lower than the controlled Ta(2)O(5) and Si(3)N(4) sensing membranes.

Optimization of a PVC Membrane for Reference Field Effect Transistors.

For the miniaturization of ISFET sensing systems, the concept of a REFET with low ion sensitivity is proposed to replace the conventional reference electrodes through the arrangement of a quasi reference electrode and a differential readout circuit. In this study, an ion-unblocking membrane was used as the top layer of a REFET. To optimize the REFET performance, the influences of the silylating process, different plasticizers, and the composition of the PVC cocktails were investigated. A low sensitivity (10.4 ± 2.2 mV/pH) and high linearity (99.7 ± 0.3 %) in the range from pH 2.2 to pH 11.6 was obtained for the REFET with a 60 wt.% DNP/(DNP + PVC) membrane. To evaluate the long term stability, the drift coefficient was estimated, and for the best REFET, it was -0.74 mV/h. Two criteria for assessing the lifetime of REFETs were used, namely the increase in pH sensitivity to a value higher than 15 mV/pH and the degradation of linearity below 99 %. For the best REFET, it was approximately 15 days.