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.
Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes. While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems, the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts. Only a few commercial microfluidic systems for enzyme analysis are currently available, and the transformation of numerous published prototypes into commercial platforms is still to be streamlined. This review presents the state-of-the-art, recent trends, and perspectives in applying microfluidic tools in the functional and structural analysis of biocatalysts. We discuss the advantages and disadvantages of available technologies, their reproducibility and robustness, and readiness for routine laboratory use. We also highlight the unexplored potential of microfluidics to leverage the power of machine learning for biocatalyst development.
Intestinal milieu disorders are strongly related to the occurrence of inflammatory bowel diseases (IBDs), which results from mucosa destruction, epithelium disruption, and tight junction (TJ) proteins loss. Excess of H2 S in the intestinal milieu produced by the sulfate-reducing bacteria metabolism contributes to development of IBDs via epithelial barrier breakdown. Conventional interventions, such as surgery and anti-inflammatory medications, are considered not completely effective because of frequent recurrence and other complications. Herein, a novel oral delivery system, a hydroxypropyl methylcellulose acetate succinate (HPMCAS)-based polymer-coated Zr-based metal-organic framework (UiO-66) with a Cux -rhodamine B (CR) probe (hereinafter referred to as HUR), is produced via a co-flow microfluidic approach with the ability to reduce H2 S levels, thus restoring the intestinal lumen milieu. HPMCAS serves as an enteric coating that exposes UiO-66@CR at the pH of the intestine but not the acidic pH of the stomach. The synthesized HUR exhibits notable therapeutic efficacy, including mucosa recovery, epithelium integrity restoration, and TJ proteins upregulation via H2 S scavenging to protect against intestinal barrier damage and microbiome dysbiosis. Thus, HUR is verified to be a promising theranostic platform able to decrease the H2 S content for intestinal milieu disorder treatment. The presented study therefore opens the door for further exploitation for IBDs therapy.
Mikrofluidika je inovativní obor, který se zabývá zpracováním malého množství kapaliny v mikrokanálech. V kombinaci s pokročilými analytickými technikami, jako je např. mikrofluidní PCR, nabízí významné výhody nejen pro analýzu genové exprese. Tato metoda využívá mikrokanály a mikroventily k přesnému dávkování a míchání činidel, čímž se minimalizuje spotřeba vzorku a činidla a také čas stráve‐ ný pipetováním. Tyto vlastnosti činí mikrofluidní PCR ideální pro analýzu genové exprese, kde je vyžadováno podrobné monitorování a kvantifikace mRNA. Jedním z přístrojů umožňujícím mikrofluidní PCR je Biomark X. Díky své schopnosti multiplexování a také díky své‐ mu mikrofluidnímu designu umožňuje analýzu mnoha vzorků současně. Tato pokročilá technologie má široké uplatnění v biologickém výzkumu, diagnostice a personalizované medicíně a nabízí nové příležitosti k objevování a pochopení genetických procesů.
Microfluidics is an innovative science that deals with the manipulation of small volumes of fluid in microchannels. In combination with advanced analytical techniques such as microfluidic PCR, it offers significant advantages not only for gene expression analysis. Microflui‐ dic PCR enables PCR reactions to be performed using very small sample volumes, as it utilizes microchannels and microvalves for precise reagent dispensing and mixing. This fact increases both sensitivity and accuracy of the analysis. The Biomark X instrument utilizes micro‐ fluidic PCR for gene expression analysis, as it is ideal for mRNA quantification. With its multiplexing capability and microfluidic design, it enables the analysis of multiple samples simultaneously. This advanced technology finds broad applications in biological research, diagnostics, and provides new opportunities for the discovery and understanding of genetic processes.
There is an increasing interest in acoustics for microfluidic applications. This field, commonly known as acoustofluidics involves the interaction of ultrasonic standing waves with fluids and dispersed microparticles. The combination of microfluidics and the so-called acoustic standing waves (ASWs) led to the development of integrated systems for contact-less on-chip cell and particle manipulation where it is possible to move and spatially localize these particles based on the different acoustophysical properties. While it was initially suggested that the acoustic forces could be harmful to the cells and could impact cell viability, proliferation, or function via phenotypic or even genotypic changes, further studies disproved such claims. This review is summarizing some interesting applications of acoustofluidics in the manipulations of biomaterials, such as cells or subcellular vesicles, in works published mainly within the last 5 years.
In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 μL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15-115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.
- MeSH
- C-reaktivní protein * MeSH
- imunoanalýza metody MeSH
- indikátory a reagencie MeSH
- lidé MeSH
- mikrofluidika * MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mikročástice jsou široce používány v nesčetných oblastech průmyslu, jako jsou farmaceutika, potraviny, kosmetika a další. Ve srovnání s tradičními metodami pro syntézu mikročástic poskytují mikrofluidní techniky výkonné platformy pro vytváření vysoce kontrolovatelných kapek emulze jako šablon pro výrobu uniformních mikročástic s pokročilými strukturami a funkcemi. Mikrofluidní techniky mohou generovat kapky emulze s přesně řízenou velikostí, tvarem a složením. Přesnější proces přípravy je účinným nástroj ke kontrole profilu uvolňování léčiva a přináší také snadno dostupnou reprodukovatelnost. Článek poskytuje informace o základních nastaveních droplet-based techniky a příklady typů mikročástic připravitelných touto metodou.
Microparticles are widely used in myriad fields such as pharmaceuticals, foods, cosmetics, and other industrial fields. Compared with traditional methods for synthesizing microparticles, microfluidic techniques provide very powerful platforms for creating highly controllable emulsion droplets as templates for fabricating uniform microparticles with advanced structures and functions. Microfluidic techniques can generate emulsion droplets with precisely controlled size, shape, and composition. A more precise preparation process brings an effective tool to control the release profile of the drug and introduces an easily accessible reproducibility. The paper gives information about basic droplet-based set-ups and examples of attainable microparticle types preparable by this method.
- Klíčová slova
- metoda odpaření rozpouštědla, mikrokanálky,
- MeSH
- mikrofluidika metody MeSH
- nanočástice * MeSH
- Publikační typ
- práce podpořená grantem MeSH
Cell immunocapture microfluidic devices represent a rapidly developing field with many potential applications in medical diagnostics. The core of such approach lies in the cell binding to antibody coated surfaces through their surface receptors. Here we show, that the small recombinant protein binders (PBs) can be used for this purpose as well, with the advantage of their constructional flexibility, possibility of fusion with range of tags and cheap mass production. For this purpose, two different PBs derived from Albumin Binding Domain (ABDwt) of streptococcal protein G, so called REX and ARS ligands with proved high affinity and selectivity to the human interleukin-23 (IL-23R) and IL-17 receptor A were used. Four PBs variants recognizing two different epitopes on two different receptors and two PBs variants binding to the same epitope on one receptor but having different peptide spacer with Avitag sequence necessary for their immobilization on sensor surface were tested for cell-capture efficiency. The glass microfluidic Y-system with planar immunocapture channel working in so-called stop-flow dynamic regime was designed. Up to 60-74% immunocapture efficiency of model THP-1 cells on REX/ARS surfaces and practically no cell binding on control ABDwt surfaces was achieved. Moreover, the specific immunocapture of THP-1 cells from mixture with IL-17RA negative DU-145 cells was demonstrated. We discuss the role of the epitope, affinity and immobilization spacer of PBs as well as the influence of stop-flow dynamic regime on the effectivity of THP-1 cell immunocapture. Results can be further exploited in design of microfluidic devices for rare cells immunocapture.
- MeSH
- biosenzitivní techniky * MeSH
- lidé MeSH
- mikrofluidika MeSH
- nádorové cirkulující buňky * MeSH
- receptory interleukinu-17 MeSH
- rekombinantní proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Acute intoxication incidents due to neurotoxic organophosphate (OP) insecticides are occasionally reported, related either to suicidal attempts or occupational exposure due to the misuse of protective equipment. Among them, chlorpyrifos is a compound related to great controversy, which is still authorized and easily accessible in many countries around the world. However, to screen for its exposure markers, instrumental methods are commonly applied, which cannot enable rapid monitoring at an early stage of an intoxication. Therefore, in this study, a microfluidic paper-based analytical device (μPAD) able to rapidly screen for chlorpyrifos-oxon, the toxic chlorpyrifos metabolite, in human serum was developed and fully validated. The μPAD combines wax-printed butyrylcholinesterase (BChE) paper sensors, a lab-on-a-chip (LOC) prototype injector and a smartphone as the analytical detector. In principle, the wax-printed strips with adsorbed BChE are embedded into LOC injectors able to deliver samples and reagents on-demand. A smartphone reader was used to monitor the color development on the strips providing binary qualitative results. μPAD method performance characteristics were thoroughly evaluated in terms of specificity, detection capability (CCβ) and ruggedness. The developed analytical platform is rapid (results within 10 min), cost-efficient (0.70 €), potentially applicable at the point-of-need and attained a low CCβ (10 μg L-1 in human serum). Finally, μPAD characteristics were critically compared to well-established methods, namely an in-house BChE microplate assay and liquid chromatography tandem mass spectrometry.
- MeSH
- chytrý telefon MeSH
- dursban * MeSH
- laboratoř na čipu MeSH
- lidé MeSH
- mikrofluidika MeSH
- mikrofluidní analytické techniky * MeSH
- papír MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Bile acids are a group of compounds essential for lipid digestion and absorption with a steroid skeleton and a carboxylate side chain usually conjugated to glycine or taurine. Bile acids are regulatory molecules for a number of metabolic processes and can be used as biomarkers of various disorders. Since the middle of the twentieth century, the detection of bile acids has evolved from simple qualitative analysis to accurate quantification in complicated mixtures. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. This article overviews the literature from the last two decades (2000-2020) and focuses on bile acid analysis in various human biological samples. The methods for sample preparation, including the sample treatment of conventional (blood plasma, blood serum, and urine) and unconventional samples (bile, saliva, duodenal/gastric juice, feces, etc.) are shortly discussed. Eventually, the focus is on novel analytical approaches and methods for each particular biological sample, providing an overview of the microcolumn separation techniques, such as high-performance liquid chromatography, gas chromatography, and capillary electrophoresis, used in their analysis. This is followed by a discussion on selected clinical applications.
- MeSH
- lidé MeSH
- mikrotechnologie MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- žlučové kyseliny a soli analýza chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
