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
- Chromatography, Liquid methods MeSH
- Solid Phase Extraction MeSH
- Mass Spectrometry methods MeSH
- Liver * metabolism MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Small Molecule Libraries analysis metabolism chemistry MeSH
- Lab-On-A-Chip Devices MeSH
- Pharmaceutical Preparations metabolism analysis MeSH
- Humans MeSH
- Organoids * metabolism cytology MeSH
- Tolbutamide metabolism analysis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Validation Study MeSH
Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.
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
- Smartphone MeSH
- Chlorpyrifos * MeSH
- Lab-On-A-Chip Devices MeSH
- Humans MeSH
- Microfluidics MeSH
- Microfluidic Analytical Techniques * MeSH
- Paper MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.
- MeSH
- Equipment Design MeSH
- Metal Nanoparticles chemistry MeSH
- Lab-On-A-Chip Devices * MeSH
- Microfluidic Analytical Techniques instrumentation MeSH
- Drinking Water microbiology MeSH
- Spectrum Analysis, Raman instrumentation MeSH
- Silver chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The fast and efficient detection of foodborne pathogens is a societal priority, given the large number of food-poisoning outbreaks, and a scientific and technological challenge, given the need to detect as little as 1 viable cell in 25 gr of food. Here, we present the first approach that achieves the above goal, thanks to the use of a micro/nano-technology and the detection capability of acoustic wave sensors. Starting from 1 Salmonella cell in 25 ml of milk, we employ immuno-magnetic beads to capture cells after only 3 h of pre-enrichment and subsequently demonstrate efficient DNA amplification using the Loop Mediated Isothermal Amplification method (LAMP) and acoustic detection in an integrated platform, within an additional ½ h. The demonstrated 4 h sample-to-analysis time comes as a huge improvement to the current need of few days to obtain the same result. In addition, the work presents the first reported Lab-on-Chip platform that comprises an acoustic device as the sensing element, exhibiting impressive analytical features, namely, an acoustic limit of detection of 2 cells/μl or 3 aM of the DNA target and ability to detect in a label-free manner dsDNA amplicons in impure samples. The use of food samples together with the incorporation of the necessary pre-enrichment step and ability for multiple analysis with an internal control, make the proposed methodology highly relevant to real-world applications. Moreover, the work suggests that acoustic wave devices can be used as an attractive alternative to electrochemical sensors in integrated platforms for applications in food safety and the point-of-care diagnostics.
- MeSH
- Acoustics instrumentation MeSH
- Food Analysis instrumentation MeSH
- Biosensing Techniques instrumentation MeSH
- Equipment Design MeSH
- DNA, Bacterial analysis genetics MeSH
- Food Contamination analysis MeSH
- Lab-On-A-Chip Devices MeSH
- Humans MeSH
- Limit of Detection MeSH
- Milk microbiology MeSH
- Foodborne Diseases microbiology MeSH
- Food Microbiology MeSH
- Salmonella genetics isolation & purification MeSH
- Salmonella Infections microbiology MeSH
- Animals MeSH
- Sound MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
INTRODUCTION: Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. MATERIALS AND METHODS: A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. RESULTS: The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. CONCLUSIONS: The characteristics established in our study are in concordance with the manufacturer's specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.
- MeSH
- Actins genetics MeSH
- Antigens, Surface genetics MeSH
- DNA, Neoplasm genetics MeSH
- ErbB Receptors genetics MeSH
- Glutamate Carboxypeptidase II genetics MeSH
- Lab-On-A-Chip Devices * MeSH
- Humans MeSH
- Multiplex Polymerase Chain Reaction methods MeSH
- Biomarkers, Tumor genetics MeSH
- Neoplastic Cells, Circulating metabolism MeSH
- Prostatic Neoplasms, Castration-Resistant blood genetics MeSH
- Prostate-Specific Antigen genetics MeSH
- Reagent Kits, Diagnostic MeSH
- Gene Expression Regulation, Neoplastic * MeSH
- Reproducibility of Results MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The development of integrated, fast and affordable platforms for pathogen detection is an emerging area where a multidisciplinary approach is necessary for designing microsystems employing miniaturized devices; these new technologies promise a significant advancement of the current state of analytical testing leading to improved healthcare. In this work, the development of a lab-on-chip microsystem platform for the genetic analysis of Salmonella in milk samples is presented. The heart of the platform is an acoustic detection biochip, integrated with a microfluidic module. This detection platform is combined with a micro-processor, which, alongside with magnetic beads technology and a DNA micro-amplification module, are responsible for performing sample pre-treatment, bacteria lysis, nucleic acid purification and amplification. Automated, multiscale manipulation of fluids in complex microchannel networks is combined with novel sensing principles developed by some of the partners. This system is expected to have a significant impact in food-pathogen detection by providing for the first time an integrated detection test for Salmonella screening in a very short time. Finally, thanks to the low cost and compact technologies involved, the proposed set-up is expected to provide a competitive analytical platform for direct application in field settings.
- MeSH
- DNA, Bacterial analysis MeSH
- Lab-On-A-Chip Devices microbiology MeSH
- Milk microbiology MeSH
- Food Microbiology methods MeSH
- Salmonella genetics isolation & purification MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
We present an integrated and low-cost microfluidic platform capable of extraction of nucleic acids from real biological samples. We demonstrate the application of this platform in pathogen detection and cancer screening. The integrated platform consists of three units including a pretreatment unit for separation of nucleic acids from lysates, a preconcentration unit for concentration of isolated nucleic acids and a sensing unit localized at a designated position on the chip for specific detection of the target nucleic acid. The platform is based on various electrokinetic phenomena exhibited by ion exchange membranes in a DC electrical field that allow them to serve as molecular filters, analyte preconcentrators and sensors. In this manuscript, we describe each unit of the integrated chip separately and show specific detection of a microRNA (miRNA 146a) biomarker associated with oral cancer as a proof-of-concept experiment. This platform technology can easily be extended to other targets of interest by optimizing the properties of the ion exchange membranes and the specific probes functionalized onto the sensors.
- MeSH
- Electric Conductivity * MeSH
- Lab-On-A-Chip Devices * economics MeSH
- MicroRNAs analysis MeSH
- Biomarkers, Tumor analysis MeSH
- Mouth Neoplasms diagnosis MeSH
- Systems Integration MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Microreactor technology is an interdisciplinary field that combines science and engineering. This new concept in production, analysis and research is finding increasing application in many different fields. Benefits of this new technology pose a vital influence on chemical industry, biotechnology, the pharmaceutical industry and medicine, life science, clinical and environmental diagnostic. In the last few years, together with microplant development, a great part of research investigation is focused on integrated micro-systems, the so called micro-total-analysis-systems (μ-TAS) or lab-on-chip (LOC). They are devices that perform sampling, sample preparation, detection and date processing in integrated model. Cell sorting, cell lysis, single cell analysis and non-destructive single cell experiments on just one microreactor, makes the LOC platform possible. Clinical diagnostic devices are also leaning towards completely integrated, multiple sophisticated biochemical analyses (PCR amplification, cell lysis, separation and detection) all on a single platform and in real time. Special attention is also paid to the usage of microdevices in tissue. Tissue engineering is one of the most promising fields that can lead to in vitro tissue and organ reconstruction ready for implantation and microdevices can be used to promote the migration, proliferation and the differentiation of cells in controlled situations.
- MeSH
- Biophysics MeSH
- Biomedical Technology MeSH
- Biomedical Research MeSH
- Equipment Design MeSH
- DNA analysis MeSH
- Financing, Organized MeSH
- Immunoassay methods instrumentation MeSH
- Metabolomics methods MeSH
- Patch-Clamp Techniques instrumentation MeSH
- Microchemistry instrumentation MeSH
- Microchip Analytical Procedures MeSH
- Microfluidics methods instrumentation MeSH
- Microfluidic Analytical Techniques methods MeSH
- Microtechnology methods instrumentation MeSH
- Miniaturization methods instrumentation MeSH
- Polymerase Chain Reaction methods instrumentation MeSH
- Cell Separation methods instrumentation MeSH
- Tissue Engineering methods instrumentation MeSH
- Publication type
- Review MeSH
