This study investigates various microfluidic chip fabrication techniques, highlighting their applicability and limitations in the context of urgent diagnostic needs showcased by the COVID-19 pandemic. Through a detailed examination of methods such as computer numerical control milling of a polymethyl methacrylate, soft lithography for polydimethylsiloxane-based devices, xurography for glass-glass chips, and micromachining-based silicon-glass chips, we analyze each technique's strengths and trade-offs. Hence, we discuss the fabrication complexity and chip thermal properties, such as heating and cooling rates, which are essential features of chip utilization for a polymerase chain reaction. Our comparative analysis reveals critical insights into material challenges, design flexibility, and cost-efficiency, aiming to guide the development of robust and reliable microfluidic devices for healthcare and research. This work underscores the importance of selecting appropriate fabrication methods to optimize device functionality, durability, and production efficiency.

• MeSH
• COVID-19 * virologie   MeSH
• design vybavení MeSH
• dimethylpolysiloxany chemie   MeSH
• laboratoř na čipu * MeSH
• lidé MeSH
• mikrofluidika metody   přístrojové vybavení   MeSH
• mikrofluidní analytické techniky přístrojové vybavení   metody   MeSH
• polymethylmethakrylát chemie   MeSH
• SARS-CoV-2 izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Distance-based detection (DbD) on paper-based microfluidic analytical devices (μPADs) has emerged as a promising, cost-effective, simple, and instrumentation-free assay method. Broadening the applicability of a new way of immobilization of reagent for DbD on μPADs (DμPADs) is presented, employing an ion exchange (IE) interaction of an anionic metallochromic reagent, 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS), on the anion-exchange filter paper. The IE DμPADs demonstrate superiority over standard cellulose filter paper in terms of the degree of reagent immobilization, detection sensitivity, and clear detection endpoints due to the strong retention of 5-Br-PAPS. The study investigated various parameters influencing DbD, including 5-Br-PAPS concentrations (0.25-1 mM), buffer types (acetic acid-Tris, MES), buffer concentrations (20-500 mM), and auxiliary complexing agents (acetic, formic, and glycolic acids). Subsequently, the performance of 17 metals (Ag+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Hg2+, La2+, Mn2+, Ni2+, Pb2+, Ti2+, Zn2+, Al3+, As3+, Fe3+, and V4+) was evaluated, with color formation observed for 12 metals. Additionally, the paper surface was examined using SEM and SEM-EDX to verify the suitability of certain areas in the detection channel for reagent immobilization and metal binding. This method demonstrates quantitation limits of metals in the low μg mL-1 range, showing great potential for the rapid screening of toxic metals commonly found in herbal supplements and cosmetics regulated by the Food and Drug Administration (FDA). Thus, it holds promise for enhancing safety and regulatory compliance in product quality assessment. Furthermore, this method offers a cost-effective, environmentally sustainable, and user-friendly approach for the rapid visual quantification of heavy metals for in-field analysis, eliminating the need for complex instrumentation.

• Publikační typ
• časopisecké články MeSH

The review focuses on the design of detection cells, the use of microcontrollers for processing and evaluation of the detection signal, and the development of multi-detection systems for electromigration, liquid chromatography, flow-through and microfluidic techniques. A separate section is the introduction of modern 3D printing techniques and the use of new printing materials for the design of multidetection systems. In addition to traditional utilisation in separation techniques, new versions of contactless conductivity detectors are finding applications in FIA, SIA, portable and paper based analytical systems or as independent sensors. Applicationwise, C4Ds find new use in gas detection, segmented flow monitoring, as part of point of care devices, and in many other biomedical, environmental, agricultural and industrial applications.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography-mass spectrometry (LC-MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC-MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.

• MeSH
• chromatografie kapalinová metody   MeSH
• extrakce na pevné fázi MeSH
• hmotnostní spektrometrie metody   MeSH
• játra * metabolismus   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• knihovny malých molekul analýza   metabolismus   chemie   MeSH
• laboratoř na čipu MeSH
• léčivé přípravky metabolismus   analýza   MeSH
• lidé MeSH
• organoidy * metabolismus   cytologie   MeSH
• tolbutamid metabolismus   analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).

• MeSH
• imunoanalýza přístrojové vybavení   metody   MeSH
• lidé MeSH
• limita detekce MeSH
• mikrofluidní analytické techniky přístrojové vybavení   MeSH
• nádory prostaty diagnóza   krev   MeSH
• nanočástice chemie   MeSH
• prostatický specifický antigen * analýza   krev   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite the significant health risks associated with Dermanyssus gallinae infestations in humans, they are often overlooked. This study investigated a household case of D. gallinae infestation and explored the resulting clinical manifestations and risk of infection in family members. Microfluidic PCR was employed for high-throughput screening of pathogens in collected mites and blood samples from both chickens and family members. Morphological and molecular examinations confirmed the identity of the mites as D. gallinae sensu stricto (s.s.), with evidence indicating recent blood feeding. Results indicated that the mites exclusively harbored various pathogens, including Bartonella spp., Ehrlichia spp., Apicomplexa, and Theileria spp. Blood samples from family members and poultry tested negative for these pathogens, suggesting a potential reservoir role for D. gallinae. The study further identified haplotypes of D. gallinae, classifying them into D. gallinae s.s., cosmopolitan haplogroup A. Serological analysis revealed elevated IgE seroreactivity against mite proteins in the family member with bite lesions. Antibodies against Bartonella spp. were detected in this individual, indicating exposure to the pathogen. In summary, this study sheds light on the clinical manifestations, pathogen detection, and genetic characterization of D. gallinae infestations, underscoring the necessity of adopting comprehensive approaches to manage such infestations effectively.

• Publikační typ
• časopisecké články MeSH

Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.

• MeSH
• infekce papilomavirem * komplikace   MeSH
• lidé MeSH
• lidské papilomaviry MeSH
• nádory děložního čípku * MeSH
• Papillomaviridae genetika   MeSH
• technologie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27kip1. This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications.

• MeSH
• chloroform MeSH
• laboratoř na čipu MeSH
• mikrofluidika * metody   MeSH
• mikrofluidní analytické techniky * MeSH
• polymethylmethakrylát chemie   MeSH
• Publikační typ
• časopisecké články MeSH

The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed "AND gate" multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines.

• MeSH
• adjuvancia imunologická farmakologie   MeSH
• antigeny chemie   MeSH
• CD8-pozitivní T-lymfocyty MeSH
• dendritické buňky MeSH
• koncentrace vodíkových iontů MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nanovakcíny * MeSH
• ovalbumin MeSH
• reaktivní formy kyslíku MeSH
• vakcíny * chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Background: Anthracycline cardiotoxicity is a well-known complication of cancer treatment, and miRNAs have emerged as a key driver in the pathogenesis of cardiovascular diseases. This study aimed to investigate the expression of miRNAs in the myocardium in early and late stages of chronic anthracycline induced cardiotoxicity to determine whether this expression is associated with the severity of cardiac damage. Method: Cardiotoxicity was induced in rabbits via daunorubicin administration (daunorubicin, 3 mg/kg/week; for five and 10 weeks), while the control group received saline solution. Myocardial miRNA expression was first screened using TaqMan Advanced miRNA microfluidic card assays, after which 32 miRNAs were selected for targeted analysis using qRT-PCR. Results: The first subclinical signs of cardiotoxicity (significant increase in plasma cardiac troponin T) were observed after 5 weeks of daunorubicin treatment. At this time point, 10 miRNAs (including members of the miRNA-34 and 21 families) showed significant upregulation relative to the control group, with the most intense change observed for miRNA-1298-5p (29-fold change, p < 0.01). After 10 weeks of daunorubicin treatment, when a further rise in cTnT was accompanied by significant left ventricle systolic dysfunction, only miR-504-5p was significantly (p < 0.01) downregulated, whereas 10 miRNAs were significantly upregulated relative to the control group; at this time-point, the most intense change was observed for miR-34a-5p (76-fold change). Strong correlations were found between the expression of multiple miRNAs (including miR-34 and mir-21 family and miR-1298-5p) and quantitative indices of toxic damage in both the early and late phases of cardiotoxicity development. Furthermore, plasma levels of miR-34a-5p were strongly correlated with the myocardial expression of this miRNA. Conclusion: To the best of our knowledge, this is the first study that describes alterations in miRNA expression in the myocardium during the transition from subclinical, ANT-induced cardiotoxicity to an overt cardiotoxic phenotype; we also revealed how these changes in miRNA expression are strongly correlated with quantitative markers of cardiotoxicity.

• Publikační typ
• časopisecké články MeSH