Insulin Conformation Changes in Hybrid Alginate-Gelatin Hydrogel Particles.

There is a strong need to develop an insulin delivery system suitable for oral administration and preserving natural (α-helix) insulin conformation. In this work, we fabricated alginate-gelatin hydrogel beads for insulin encapsulation. Altering matrix composition and crosslinking agents has resulted in various surface morphologies and internal spatial organization. The structures of the insulin-loaded matrices were studied using optical and field emission electronic microscopy. We use FTIR spectroscopy to identify insulin conformation changes as affected by the hydrogel matrices. It was found that blended alginate-gelatin matrices demonstrate better encapsulation efficiency and stronger swelling resistance to a simulated gastric environment than sodium alginate beads crosslinked with the CaCl2. FTIR measurements reveal conformation changes in insulin. It is also confirmed that in the presence of gelatin, the process of insulin fibrinogenesis ceases due to intermolecular interaction with the gelatin. Performed molecular modeling shows that dipole-dipole interactions are the dominating mechanism that determines insulin behavior within the fabricated matrix.

Bioanalytical approaches for the detection, characterization, and risk assessment of micro/nanoplastics in agriculture and food systems.

This review discusses the most recent literature (mostly since 2019) on the presence and impact of microplastics (MPs, particle size of 1 µm to 5 mm) and nanoplastics (NPs, particle size of 1 to 1000 nm) throughout the agricultural and food supply chain, focusing on the methods and technologies for the detection and characterization of these materials at key entry points. Methods for the detection of M/NPs include electron and atomic force microscopy, vibrational spectroscopy (FTIR and Raman), hyperspectral (bright field and dark field) and fluorescence imaging, and pyrolysis-gas chromatography coupled to mass spectrometry. Microfluidic biosensors and risk assessment assays of MP/NP for in vitro, in vivo, and in silico models have also been used. Advantages and limitations of each method or approach in specific application scenarios are discussed to highlight the scientific and technological obstacles to be overcome in future research. Although progress in recent years has increased our understanding of the mechanisms and the extent to which MP/NP affects health and the environment, many challenges remain largely due to the lack of standardized and reliable detection and characterization methods. Most of the methods available today are low-throughput, which limits their practical application to food and agricultural samples. Development of rapid and high-throughput field-deployable methods for onsite screening of MP/NPs is therefore a high priority. Based on the current literature, we conclude that detecting the presence and understanding the impact of MP/NP throughout the agricultural and food supply chain require the development of novel deployable analytical methods and sensors, the combination of high-precision lab analysis with rapid onsite screening, and a data hub(s) that hosts and curates data for future analysis.

Susceptibility of Gardnerella vaginalis biofilms to natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl ester.

Bacterial vaginosis is a common vaginal infection associated with numerous gynecological and obstetric complications. This condition is characterized by the presence of thick adherent vaginal biofilms, composed mainly of Gardnerella vaginalis. This organism is thought to be the primary aetiological cause of the infection paving the way for various opportunists to colonize the niche. Previously, we reported that the natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl ester selectively inhibit the growth of this pathogen. In this study, we used plate counts to evaluate the efficacy of these antimicrobials against established biofilms of G. vaginalis. Additionally, we validated and compared two rapid methods (ATP viability and resazurin assays) for the assessment of cell viability in the antimicrobial-treated G. vaginalis biofilms. Out of the tested antimicrobials, lauramide arginine ethyl ester had the strongest bactericidal effect, followed by subtilosin, with clindamycin and polylysine showing the weakest effect. In comparison to plate counts, ATP viability and resazurin assays considerably underestimated the bactericidal effect of some antimicrobials. Our results indicate that these assays should be validated for every new application.

FEAST of biosensors: Food, environmental and agricultural sensing technologies (FEAST) in North America.

We review the challenges and opportunities for biosensor research in North America aimed to accelerate translational research. We call for platform approaches based on: i) tools that can support interoperability between food, environment and agriculture, ii) open-source tools for analytics, iii) algorithms used for data and information arbitrage, and iv) use-inspired sensor design. We summarize select mobile devices and phone-based biosensors that couple analytical systems with biosensors for improving decision support. Over 100 biosensors developed by labs in North America were analyzed, including lab-based and portable devices. The results of this literature review show that nearly one quarter of the manuscripts focused on fundamental platform development or material characterization. Among the biosensors analyzed for food (post-harvest) or environmental applications, most devices were based on optical transduction (whether a lab assay or portable device). Most biosensors for agricultural applications were based on electrochemical transduction and few utilized a mobile platform. Presently, the FEAST of biosensors has produced a wealth of opportunity but faces a famine of actionable information without a platform for analytics.

Label-free toxin detection by means of time-resolved electrochemical impedance spectroscopy.

The real-time detection of trace concentrations of biological toxins requires significant improvement of the detection methods from those reported in the literature. To develop a highly sensitive and selective detection device it is necessary to determine the optimal measuring conditions for the electrochemical sensor in three domains: time, frequency and polarization potential. In this work we utilized a time-resolved electrochemical impedance spectroscopy for the detection of trace concentrations of Staphylococcus enterotoxin B (SEB). An anti-SEB antibody has been attached to the nano-porous aluminum surface using 3-aminopropyltriethoxysilane/glutaraldehyde coupling system. This immobilization method allows fabrication of a highly reproducible and stable sensing device. Using developed immobilization procedure and optimized detection regime, it is possible to determine the presence of SEB at the levels as low as 10 pg/mL in 15 minutes.

Direct detection of the biological toxin in acidic environment by electrochemical impedimetric immunosensor.

This study describes the direct detection of the biological toxin (Ricin) in acidic environment without pH adjustment by hydrophobically modified electrochemical impedance immunosensor (EII). The nano-porous aluminum substrate for EII was hydrophobically modified via self-assembled monolayer (SAM) of APTES. Biosensor for the detection of the Ricin was fabricated by the covalent cross-linking of antibody (Ab) with APTES-SAM. The immunoreactions between the immobilized Ab and the biological toxin in several diagnostic solutions were monitored by the electrochemical impedance spectroscopy (EIS) under the polarization of EII versus reference electrode. EII could detect the presence of the biological toxin in acidic foods in 20 mins without pH adjustment. The negatively charged ions including hydroxides would be adsorbed on the hydrophobic body of APTES-SAMs by the polarization during EIS analysis, and offset the effect of acids on the immunological activity of the immobilized Ab. It suggested that the adsorption of negatively charged ions helped to keep the immunological activities of the immobilized Ab on EII in acidic environment. Proposed mechanism of the localized pH adjustment that makes possible immunoreaction occurrence in low pH sample matrix is briefly discussed.

Electrochemical synthesis and impedance characterization of nano-patterned biosensor substrate.

The nano-porous anodic aluminum oxide has been used as a substrate material for enzymatic biosensor operating in aqueous solutions. Nano-scale porous structure was formed by electrical anodization in an acid solution. By changing anodization conditions, such as electrolyte concentration, temperature, and anodization time, the ordered hexagonal porous structure with well-controlled pore size and depth can be obtained. Nano-porous alumina substrate with adsorbed enzymes was used as an enzyme electrode and pH sensor. The pH changes are driven by the enzymatic reactions, e.g. penicillin G hydrolysis to form penicilloic acid in the presence of penicillinaze. The advantage of physical adsorption used to bound penicillinaze, the model enzyme in this work, to the porous structure, is that usually no reagents are required and only a minimum of "activation" or clean-up steps. Adsorption tends to be less disruptive to enzyme proteins than chemical attachment. Due to the increased active sensor area, the immobilization of enzymes has been enhanced, which in turn improved the electrode's sensitivity. To characterize the interactions of enzymes with nano-porous alumina oxide, electrochemical impedance spectroscopy (EIS) was used.

Electrokinetic mixing vortices due to electrolyte depletion at microchannel junctions.

Due to electric field leakage across sharp corners, the irrotational character of Ohmic electroosmotic flow is violated. Instead, we demonstrate experimentally and theoretically evidence of electrolyte depletion and vortex separation in electroosmotic flow around a junction between wide and narrow channels. When the penetration length of the electric field exceeds the width of the narrow channel and if the electric field is directed from the narrow to the wide channel, the electromigration of ions diminishes significantly at the junction end of the narrow channel due to this leakage. Concentration depletion then develops at that location to maintain current balance but it also increases the corner zeta potential and the local electroosmotic slip velocity. A back pressure gradient hence appears to maintain flow balance and, at a sufficient magnitude, generates a pair of vortices.

Impedimetric characterization of adsorption of Listeria monocytogenes on the surface of an aluminum-based immunosensor.

The impedimetric characteristics of an immunosensor depend on the electrical properties of an immunosensor substrate. The impedimetric characteristics of an immunosensor compared with adsorption of Listeria monocytogenes were investigated on an aluminum surface insulated with an electrically resistive aluminum oxide layer. Antibody for L. monocytogenes (anti-L. monocytogenes) was immobilized on an aluminum surface that was insulated with a native air-formed aluminum oxide layer. The resistance of impedance (R) value of an aluminum-based immunosensor decreased, especially at 10(4) to 10(6) Hz, where the effect of the reactance of impedance (X) was minimal when L. monocytogenes was adsorbed on the immunosensor surface. The R value of the immunosensor at 81 kHz decreased proportionally to the concentration of L. monocytogenes from 1.3 to 4.3 log CFU mL(-1) . The adsorption of L. monocytogenes produced local protrusions on the immunosensor surface, causing physicochemical changes in the ionic layer formed on the immunosensor surface by a sinusoidal electrical signal input, which might help electrical current to flow and cause the R value to decrease.

Thermal gelation of aqueous hydroxypropylmethylcellulose solutions with SDS and hydrophobic drug particles.

The thermal gelation of hydroxypropylmethylcellulose (HPMC) solutions has been studied as a function of sodium dodecyl sulfate (SDS) concentration with and without griseofulvin, a model particulate BCS Class II drug by rheological measurements of gelation temperature (Tgel), steady-state viscosity (η) at 25 °C, and ζ-potential. Polymer adsorption on the drug was demonstrated by a decrease in η and potential in the absence of SDS. Griseofulvin had a synergistic effect on gelation which was attributed to an effective spanning of associated hydrophobic polymeric regions through interactions with the adsorbed polymer. Adding SDS offsets this effect on Tgel shielding hydrophobic interactions. Higher SDS concentrations had no effect on the particles surface as evidenced by constant ζ-potential and Tgel. Yet, polymeric chains are saturated and larger surfactant aggregates account for the increase in viscosity. Understanding the gelation mechanism and complex interactions of HPMC with surfactants and drugs is necessary for the design of pharmaceutical products and optimization of their performance properties.

Formulation optimization and topical delivery of quercetin from solid lipid based nanosystems.

The presence of large amounts of reactive oxygen species (ROS) leads to oxidative stress that can damage cell membranes, lead to DNA breakage and cause inactivation of free radical scavenger enzymes, eventually resulting in skin damage. Quercetin is a natural flavonoid that has been shown to have the highest anti-radical activity, along with the ability to act as a scavenger of free radicals and an inhibitor of lipid peroxidation. In this research work, a solvent-free solid lipid based nanosystem has been developed and evaluated for topical delivery of quercetin. Systematic screening of the formulation and process parameters led to the development of a solid lipid (glyceryl dibehenate) based nanosystem using a probe ultrasonication method. The selected variant demonstrated good physical stability for up to 8 weeks at 2-8 °C. Transmission electron microscopy (TEM) images showed spherical particles in the nanometer range. In vitro release studies showed biphasic release of quercetin from the SLN formulation, with an initial burst release followed by prolonged release for up to 24h. In vitro permeation studies using full thickness human skin showed higher amounts of quercetin to be localized within the skin compared to a control formulation with particles in the micrometer range. Such accumulation of quercetin in the skin is highly desirable since the efficacy of quercetin in delaying ultra-violet radiation mediated cell damage and eventual necrosis mainly occurs in the epidermis.

Automated drop-on-demand system with real-time gravimetric control for precise dosage formulation.

Many of the therapies for personalized medicine have few dosage options, and the successful translation of these therapies to the clinic is significantly dependent on the drug/formulation delivery platform. We have developed a lab-scale integrated system for microdosing of drug formulations with high accuracy and precision that is capable of feedback control. The designed modular drug dispensing system includes a microdispensing valve unit and is fully automated with a LabVIEW-controlled computer interface. The designed system is capable of dispensing drug droplets with volumes ranging from nanoliters to microliters with high accuracy (relative standard deviation <1%). We have determined that the system is capable of accurate dosing and in-line real-time gravimetric control.

Listeria monocytogenes' Step-Like Response to Sub-Lethal Concentrations of Nisin.

Microbial safety of food products is often accomplished by the formulation of food-grade preservatives into the product. Because of the growing consumer demand for natural substances (including preservatives) in the composition of consumed foods, there is also a growing interest in the natural antimicrobial nisin, which has generally recognized as safe (GRAS) status for certain applications. During the products storage time, concentrations of preservative(s) are decreasing, which may eventually cause a serious problem in the food's microbial safety. Here, for the first time we report on the non-linear response of a foodborne pathogen, Listeria monocytogenes, to sub-lethal concentrations of nisin.

Linearized kinetic model of Listeria monocytogenes biofilm growth.

In this study the dynamics of biofilm formation on aluminum has been investigated. The process of cell growth has been observed using fluorescence microscopy. It has been confirmed that the process of biofilm formation can be represented as a sum of two separate processes: cell adhesion and colony proliferation. The derived set of equations describes kinetics of surface population growth and characteristic times for adsorption and combined growth processes, including characteristic time for the nutrient supply depletion. All equations contain variables based on the fundamental characteristics of bacterial population and can be easily determined from the experimental data or estimated theoretically. The developed theoretical model allows obtaining realistic values for population growth time and characteristic time for nutrient limitation occurrence during the biofilm development. Resulting equations qualitatively describe the biofilm formation process, and allow predicting microbial kinetics in the batch reactor system and determining critical values of the process parameters.