Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay.

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods-reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)-combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10-20 min, whereas for RT-LAMP, the LOD was 30-300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.

Versatile and Easily Designable Polyester-Laser Toner Interfaces for Site-Oriented Adsorption of Antibodies.

Laser toners appear as attractive materials for barriers and easily laminated interphases for Lab-on-a-Foil microfluidics, due to the excellent adhesion to paper and various membranes or foils. This work shows for the first time a comprehensive study on the adsorption of antibodies on toner-covered poly(ethylene terephthalate) (PET@toner) substrates, together with assessment of such platforms in rapid prototyping of disposable microdevices and microarrays for immunodiagnostics. In the framework of presented research, the surface properties and antibody binding capacity of PET substrates with varying levels of toner coverage (0-100%) were characterized in detail. It was proven that polystyrene-acrylate copolymer-based toner offers higher antibody adsorption efficiency compared with unmodified polystyrene and PET as well as faster adsorption kinetics. Comparative studies of the influence of pH on the effectiveness of antibodies immobilization as well as measurements of surface ζ-potential of PET, toner, and polystyrene confirmed the dominant role of hydrophobic interactions in adsorption mechanism. The applicability of PET@toner substrates as removable masks for protection of foil against permanent hydrophilization was also shown. It opens up the possibility of precise tuning of wettability and antibody binding capacity. Therefore, PET@toner foils are presented as useful platforms in the construction of immunoarrays or components of microfluidic systems.

Islet-on-a-chip: Biomimetic micropillar-based microfluidic system for three-dimensional pancreatic islet cell culture.

Type 2 diabetes is currently one of the most common metabolic diseases, affecting all ages worldwide. As the incidence of type 2 diabetes increases, a growing number of studies focus on islets of Langerhans. A three-dimensional research model that maps islet morphology and maintains hormonal balance in vivo is still needed. In this work, we present an Islet-on-a-chip system, specifically a micropillar-based microfluidic platform for three-dimensional pancreatic islet cell culture and analysis. The microfluidic system consisted of two culture chambers that were equipped with 15 circular microtraps each, which were built with seven round micropillars each. Micropillars in the structure of microtraps supported cell aggregation by limiting the growth surface and minimizing wall shear stress, thereby ensuring proper medium diffusion and optimal culture conditions for cell aggregates. Our system is compatible with microwell plate readers and confocal laser scanning microscopes. Because of optimization of the immunostaining method, the appropriate cell distribution and high viability and proliferation up to 72 h of culture were confirmed. Enzyme-linked immunosorbent assays were performed to measure insulin and glucagon secretion after stimulation with different glucose concentrations. To our knowledge, this is the first Lab-on-a-chip system which enables the formation and three-dimensional culture of cell aggregates composed of commercially available α and ß pancreatic islet cells. The specific composition and arrangement of cells in the obtained model corresponds to the arrangement of the cells in rodent pancreatic islets in vivo. This Islet-on-a-chip system may be utilized to test pathogenic effectors and future therapeutic agents.

Simulation of hypoxia of myocardial cells in microfluidic systems.

The paper presents a newly designed microfluidic system that allows simulation of myocardial hypoxia by biochemical method. The geometry of the microsystem was designed in such a way, that quantitative fluorescent measurements using a spectrofluorometric plate reader was possible. Biochemical simulation of hypoxia was carried out using potent mitochondrial oxidative phosphorylation uncoupler-Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Two cardiac cell lines were used in the study-rat cardiomyoblasts (H9C2) and human cardiomyocytes. The effectiveness of biochemical simulation of hypoxia was studied using two fluorescent dyes: carbocyanine iodide (JC-1) and Fluo-4. Changes in the mitochondrial membrane potential and concentration of intracellular calcium ions were tested. The major novelty of this research was the applying the microfluidic system to create hypoxia conditions for cardiac cells using the biochemical approach. In further studies, the presented hypoxia model could be used to develop new methods of treatment of ischemic heart disease for example in cell therapy based on stem cells.

Co-delivery of IR-768 and daunorubicin using mPEG-b-PLGA micelles for synergistic enhancement of combination therapy of melanoma.

Malignant melanoma is an emerging problem worldwide due to the high degree of lethalness. Its aggressiveness and the ability to metastasize along with the heterogeneity at the molecular and cellular levels, limit the overall therapeutic efficacy. Despite significant advances in melanoma treatment over the last decade, there is still a need for improved therapeutic modalities. Thus, we demonstrate here a combinatorial approach that targets multiple independent therapeutic pathways, in which polymeric micelles (PMs) were used as efficacious colloidal nanocarriers loaded with both daunorubicin (DRB) as a cytotoxic drug and IR-768 as a photosensitizer. This afforded the dual drug loaded delivery system IR-768 + DRB in PMs. The fabricated mPEG-b-PLGA micelles (hydrodynamic diameters ≈ 25 nm) had a relatively narrow size distribution (PdI > ca. 0.3) with uniform spherical shapes. CLSM study showed that mPEG-b-PLGA micelles were uptaken by mitochondria, which further contributed to excellent singlet oxygen generation capacity for PDT in A375 melanoma cells. Furthermore, the PMs were efficiently internalized by tested cells through endocytosis, resulting in much higher cellular uptake comparing to the free drug. As a result of these properties, IR-768 + DRB in PMs exhibited very potent and synergistically enhanced anticancer activity against A375 cells. Additionally, this combination approach allowed to reduce drug doses and provided low side effects towards normal HaCaT. This study indicates excellent properties of mPEG-b-PLGA micelles resulting in great therapeutic potential possessed by the developed nanoscale drug delivery system for combined chemo-photodynamic therapy of melanoma.

Human mesenchymal stem cell (hMSC) differentiation towards cardiac cells using a new microbioanalytical method.

Stem cells (SCs) are more and more often applied in tissue engineering and cell therapies, e.g. in regenerative medicine. Standard methods of SC differentiation are time consuming and ineffective. Therefore, new bioanalytical methods (i.e. Lab-on-a-Chip systems) are develop to improve such type of studies. Although, microtechnology is a rapidly growing research area, there are so far not too many works which present SC differentiation into cardiomyocytes in the microsystems. Therefore, we present new microbioanalytical method of SC differentiation towards cardiac cells using a newly developed digitally controlled microdispenser integrated with a Heart-on-a-chip system. Seven-day culture of human mesenchymal stem cells (hMSCs) and their differentiation using biochemical factors such as 5-AZA (2 µM, 24 h) and VEGF (20 ng ml-1, 72 h) were investigated in the microsystem which was automatically operated using smartphone software. hMSC differentiation into the cardiac cells was confirmed using immunostaining of cardiac markers (α-actinin and troponin T). The usage of the microsystem allowed shortening the time of hMSC differentiation in comparison to macroscale method. We showed that the microsystem, in which the in vivo microenvironment is mimicked and dynamic conditions are provided by a microdispenser, favorably affect hMSC differentiation towards cardiac cells. Based on the presented research we can conclude that the developed digitally controlled microsystem could be successfully utilized as a new microbioanalytical method for stem cells differentiation and analysis of their function under dynamic conditions. In the future, this could be a helpful tool for scientists working on regenerative medicine.

Heart-on-a-Chip: An Investigation of the Influence of Static and Perfusion Conditions on Cardiac (H9C2) Cell Proliferation, Morphology, and Alignment.

Lab-on-a-chip systems are increasingly used as tools for cultures and investigation of cardiac cells. In this article, we present how the geometry of microsystems and microenvironmental conditions (static and perfusion) influence the proliferation, morphology, and alignment of cardiac cells (rat cardiomyoblasts-H9C2). Additionally, studies of cell growth after incubation with verapamil hydrochloride were performed. For this purpose, poly(dimethylsiloxane) (PDMS)/glass microfluidic systems with three different geometries of microchambers (a circular chamber, a longitudinal channel, and three parallel microchannels separated by two rows of micropillars) were prepared. It was found that static conditions did not enhance the growth of H9C2 cells in the microsystems. On the contrary, perfusion conditions had an influence on division, morphology, and the arrangement of the cells. The highest number of cells, their parallel orientation, and their elongated morphology were obtained in the longitudinal microchannel. It showed that this kind of microsystem can be used to understand processes in heart tissue in detail and to test newly developed compounds applied in the treatment of cardiac diseases.

Role of SAP7-10 and Morphological Regulators (EFG1, CPH1) in Candida albicans' Hypha Formation and Adhesion to Colorectal Carcinoma Caco-2.

Secreted aspartic proteases (Saps) are considered as key virulence factors of Candida albicans. Hopefully our outlook will widen the knowledge of SAP7's role in C. albicans pathogenesis. The goal of our study was to investigate SAP7 expression during C. albicans adhesion to intestinal human cells. Another objective was to study the role of SAP8-10 and transcriptional regulators: EFG1 and CPH1, using the mutants: Δsap, Δefg1, Δcph1 during growth on Caco-2 monolayer. SAP7 expression was analyzed using real time RT-PCR; relative quantification was normalized against ACT1 in cells after growth on Caco-2. Adherence assay of C. albicans to Caco-2 was performed in a 24-well-plate. The results proved that SAP7 can play a role during the initial adaptation of C. albicans to intestinal tract and decreases over time. Up-regulation of SAP7 occured in the absence of SAP8 and SAP10 (genetic alternations dependence). SAP7 can be regulated by the morphogensis' regulators during C. albicans growth on epithelium. Adhesion of the mutants was indistinguishable from SC5314. The lack of neither SAP8-10 nor EFG1/CPH1 influences the adhesive behaviour of C. albicans. Deletion of SAP8-10 resulted in no filamentation defects. The results help better understand the role of SAP7 during adhesion and morphogenesis in C. albicans.

Flow-through sensor array applied to cytotoxicity assessment in cell cultures for drug-testing purposes.

The viability of cells cultured in microsystems for drug screening purposes is usually tested with a variety of colorimetric/fluorescent methods. In this work we propose an alternative way of assessing cell viability-flow-through sensor array that can be connected in series with cell microbioreactors as compatible detection system. It is shown, that the presented device is capable of cytotoxic effect detection and estimation of cell viability after treatment with 1,4-dioxane and 5-fluorouracil, which proves that it can be used for truly non-invasive, fast, reliable, continuous cell culture monitoring in microscale.

Effect of a high surface-to-volume ratio on fluorescence-based assays.

In the work discussed in this paper, the effect of a high surface-to-volume ratio of a microfluidic detection cell on fluorescence quenching was studied. It was found that modification of the geometry of a microchannel can provide a wider linear range. This is a phenomenon which should be taken into consideration when microfluidic systems with fluorescence detection are developed. The dependence of the linear range for fluorescein on the surface-to-volume ratio was determined. Both fluorescence inner-filter effects and concentration self-quenching were taken into consideration. It was found that inner-filter effects have little effect on the extent of the linear range on the microscale.