RESUMEN
Immunohistochemistry and fluorescence in situ hybridization are the two standard methods for human epidermal growth factor receptor 2 (HER2) assessment. However, they have severe limitations to assess quantitatively intratumoral heterogeneity (ITH) when multiple subclones of tumor cells co-exist. We develop here a high-content, quantitative analysis of breast cancer tissues based on microfluidic experimentation and image processing, to characterize both HER2 protein overexpression and HER2 gene amplification at the cellular level. The technique consists of performing sequential steps on the same tissue slide: an immunofluorescence (IF) assay using a microfluidic protocol, an elution step for removing the IF staining agents, a standard FISH staining protocol, followed by automated quantitative cell-by-cell image processing. Moreover, ITH is accurately detected in both cluster and mosaic form using an analysis of spatial association and a mathematical model that allows discriminating true heterogeneity from artifacts due to the use of thin tissue sections. This study paves the way to evaluate ITH with high accuracy and content while requiring standard staining methods.
Asunto(s)
Neoplasias de la Mama/genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Receptor ErbB-2/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Hibridación Fluorescente in Situ/métodos , Técnicas Analíticas Microfluídicas/métodos , Receptor ErbB-2/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as â¼1 pg (â¼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.
Asunto(s)
Técnicas Biosensibles , ADN Circular/análisis , Farmacorresistencia Microbiana/genética , Dispositivos Laboratorio en un Chip , Microscopía Fluorescente/métodos , Reacción en Cadena de la Polimerasa/métodos , Antibacterianos/farmacología , Carbapenémicos/farmacología , Carbocianinas/química , Sondas de ADN/metabolismo , ADN Circular/genética , ADN Circular/metabolismo , Dimetilpolisiloxanos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Expresión Génica , Humanos , Meticilina/farmacología , Microscopía Fluorescente/economía , Microscopía Fluorescente/instrumentación , Mutación , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa/economía , Reacción en Cadena de la Polimerasa/instrumentación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
This critical review summarizes the developments in the integration of micro-optical elements with microfluidic platforms for facilitating detection and automation of bio-analytical applications. Micro-optical elements, made by a variety of microfabrication techniques, advantageously contribute to the performance of an analytical system, especially when the latter has microfluidic features. Indeed the easy integration of optical control and detection modules with microfluidic technology helps to bridge the gap between the macroscopic world and chip-based analysis, paving the way for automated and high-throughput applications. In our review, we start the discussion with an introduction of microfluidic systems and micro-optical components, as well as aspects of their integration. We continue with a detailed description of different microfluidic and micro-optics technologies and their applications, with an emphasis on the realization of optical waveguides and microlenses. The review continues with specific sections highlighting the advantages of integrated micro-optical components in microfluidic systems for tackling a variety of analytical problems, like cytometry, nucleic acid and protein detection, cell biology, and chemical analysis applications.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentación , Microfluídica/métodos , Óptica y Fotónica/métodos , Animales , Línea Celular , Simulación por Computador , Técnicas Electroquímicas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Microtecnología/métodos , Ácidos Nucleicos/análisis , Proteínas/análisis , Propiedades de SuperficieRESUMEN
As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mitoribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Rotenona/análogos & derivados , Animales , Antihelmínticos/farmacología , Caenorhabditis elegans/genética , Resistencia a Medicamentos/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Rotenona/farmacologíaRESUMEN
This article describes the preparation and characterization of PEDOT-coated paper electrodes. Their specific behavior was investigated, especially the impact of electrode shape on the electrochemical properties. It was found that different electrode geometries promote different results, largely because of a potential drop in the bulk of the electrode material. More importantly, the suitability of these substrates for bio- and neurochemical analyses was investigated. The paper electrodes were found to offer better resistance to both protein and neurotransmitter foulings, in comparison to a planar electrode. Interestingly, long paper electrodes were more stable during sustained oxidation of dopamine and serotonin than short ones, possibly because of the conjunction of surface passivation and potential drop allowing for the site of oxidation to move along the electrode as fouling progresses.
Asunto(s)
Técnicas Electroquímicas , Neurotransmisores/análisis , Papel , Polímeros/química , Bioensayo , Electrodos , Propiedades de SuperficieRESUMEN
High-refractive index dielectric microspheres positioned within the field of view of a microscope objective in a dielectric medium can focus the light into a so-called photonic nanojet. A sample placed in such nanojet can be imaged by the objective with super-resolution, i.e. with a resolution beyond the classical diffraction limit. However, when imaging nanostructures on a substrate, the propagation distance of a light wave in the dielectric medium in between the substrate and the microsphere must be small enough to reveal the sample's nanometric features. Therefore, only the central part of an image obtained through a microsphere shows super-resolution details, which are typically â¼100 nm using white light (peak at λ = 600 nm). We have performed finite element simulations of the role of this critical distance in the super-resolution effect. Super-resolution imaging of a sample placed beneath the microsphere is only possible within a very restricted central area of â¼10 µm2, where the separation distance between the substrate and the microsphere surface is very small (â¼1 µm). To generate super-resolution images over larger areas of the sample, we have fixed a microsphere on a frame attached to the microscope objective, which is automatically scanned over the sample in a step-by-step fashion. This generates a set of image tiles, which are subsequently stitched into a single super-resolution image (with resolution of λ/4-λ/5) of a sample area of up to â¼104 µm2. Scanning a standard optical microscope objective with microsphere therefore enables super-resolution microscopy over the complete field-of-view of the objective.
RESUMEN
Dielectric microspheres with appropriate refractive index can image objects with super-resolution, that is, with a precision well beyond the classical diffraction limit. A microsphere is also known to generate upon illumination a photonic nanojet, which is a scattered beam of light with a high-intensity main lobe and very narrow waist. Here, we report a systematic study of the imaging of water-immersed nanostructures by barium titanate glass microspheres of different size. A numerical study of the light propagation through a microsphere points out the light focusing capability of microspheres of different size and the waist of their photonic nanojet. The former correlates to the magnification factor of the virtual images obtained from linear test nanostructures, the biggest magnification being obtained with microspheres of â¼6-7 µm in size. Analyzing the light intensity distribution of microscopy images allows determining analytically the point spread function of the optical system and thereby quantifies its resolution. We find that the super-resolution imaging of a microsphere is dependent on the waist of its photonic nanojet, the best resolution being obtained with a 6 µm Ø microsphere, which generates the nanojet with the minimum waist. This comparison allows elucidating the super-resolution imaging mechanism.
RESUMEN
Biomarker analysis is playing an essential role in cancer diagnosis, prognosis, and prediction. Quantitative assessment of immunohistochemical biomarker expression on tumor tissues is of clinical relevance when deciding targeted treatments for cancer patients. Here, we report a microfluidic tissue processor that permits accurate quantification of the expression of biomarkers on tissue sections, enabled by the ultra-rapid and uniform fluidic exchange of the device. An important clinical biomarker for invasive breast cancer is human epidermal growth factor receptor 2 [(HER2), also known as neu], a transmembrane tyrosine kinase that connotes adverse prognostic information for the patients concerned and serves as a target for personalized treatment using the humanized antibody trastuzumab. Unfortunately, when using state-of-the-art methods, the intensity of an immunohistochemical signal is not proportional to the extent of biomarker expression, causing ambiguous outcomes. Using our device, we performed tests on 76 invasive breast carcinoma cases expressing various levels of HER2. We eliminated more than 90% of the ambiguous results (n = 27), correctly assigning cases to the amplification status as assessed by in situ hybridization controls, whereas the concordance for HER2-negative (n = 31) and -positive (n = 18) cases was 100%. Our results demonstrate the clinical potential of microfluidics for accurate biomarker expression analysis. We anticipate our technique will be a diagnostic tool that will provide better and more reliable data, onto which future treatment regimes can be based.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Inmunohistoquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Receptor ErbB-2/metabolismo , Biomarcadores de Tumor/genética , Femenino , Fluorescencia , Humanos , Procesamiento de Imagen Asistido por Computador , Receptor ErbB-2/genéticaRESUMEN
We detect by optical microscopy Au and fluorescent nanoparticles (NPs) during their motion in water-based medium, using an array of dielectric microspheres that are patterned in a microwell array template. The microspheres act as lenses focusing the light originating from a microscope objective into so-called photonic nanojets that expose the medium within a microfluidic channel. When a NP is randomly transported through a nanojet, its backscattered light (for a bare Au NP) or its fluorescent emission is instantaneously detected by video microscopy. Au NPs down to 50 nm in size, as well as fluorescent NPs down to 20 nm in size, are observed by using a low magnification/low numerical aperture microscope objective in bright-field or fluorescence mode, respectively. Compared to the NPs present outside of the photonic nanojets, the light scattering or fluorescence intensity of the NPs in the nanojets is typically enhanced by up to a factor of â¼40. The experimental intensity is found to be proportional to the area occupied by the NP in the nanojet. The technique is also used for immunodetection of biomolecules immobilized on Au NPs in buffer and, in future, it may develop into a versatile tool to detect nanometric objects of environmental or biological importance, such as NPs, viruses, or other biological agents.
RESUMEN
Postprandial inflammation is an important factor for human health since chronic low-grade inflammation is associated with chronic diseases. Dairy products have a weak but significant anti-inflammatory effect on postprandial inflammation. The objective of the present study was to compare the effect of a high-fat dairy meal (HFD meal), a high-fat non-dairy meal supplemented with milk (HFM meal) and a high-fat non-dairy control meal (HFC meal) on postprandial inflammatory and metabolic responses in healthy men. A cross-over study was conducted in nineteen male subjects. Blood samples were collected before and 1, 2, 4 and 6 h after consumption of the test meals. Plasma concentrations of insulin, glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, TAG and C-reactive protein (CRP) were measured at each time point. IL-6, TNF-α and endotoxin concentrations were assessed at baseline and endpoint (6 h). Time-dependent curves of these metabolic parameters were plotted, and the net incremental AUC were found to be significantly higher for TAG and lower for CRP after consumption of the HFM meal compared with the HFD meal; however, the HFM and HFD meals were not different from the HFC meal. Alterations in IL-6, TNF-α and endotoxin concentrations were not significantly different between the test meals. The results suggest that full-fat milk and dairy products (cheese and butter) have no significant impact on the inflammatory response to a high-fat meal.
Asunto(s)
Productos Lácteos , Dieta Alta en Grasa/efectos adversos , Inflamación/etiología , Adulto , Animales , Antiinflamatorios , Glucemia/análisis , Índice de Masa Corporal , Proteína C-Reactiva/análisis , Colesterol/sangre , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Estudios Cruzados , Endotoxinas/sangre , Humanos , Inflamación/prevención & control , Insulina/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Leche , Estudios Prospectivos , Triglicéridos/sangre , Factor de Necrosis Tumoral alfa/sangreRESUMEN
We describe the concept of magnetic particle-scanning for on-chip detection of biomolecules: a magnetic particle, carrying a low number of antigens (Ag's) (down to a single molecule), is transported by hydrodynamic forces and is subjected to successive stochastic reorientations in an engineered magnetic energy landscape. The latter consists of a pattern of substrate-bound small magnetic particles that are functionalized with antibodies (Ab's). Subsequationuent counting of the captured Ag-carrying particles provides the detection signal. The magnetic particle-scanning principle is investigated in a custom-built magneto-microfluidic chip and theoretically described by a random walk-based model, in which the trajectory of the contact point between an Ag-carrying particle and the small magnetic particle pattern is described by stochastic moves over the surface of the mobile particle, until this point coincides with the position of an Ag, resulting in the binding of the particle. This model explains the particular behavior of previously reported experimental dose-response curves obtained for two different ligand-receptor systems (biotin/streptavidin and TNF-α) over a wide range of concentrations. Our model shows that magnetic particle-scanning results in a very high probability of immunocomplex formation for very low Ag concentrations, leading to an extremely low limit of detection, down to the single molecule-per-particle level. When compared to other types of magnetic particle-based surface coverage assays, our strategy was found to offer a wider dynamic range (>8 orders of magnitude), as the system does not saturate for concentrations as high as 10(11) Ag molecules in a 5 µL drop. Furthermore, by emphasizing the importance of maximizing the encounter probability between the Ag and the Ab to improve sensitivity, our model also contributes to explaining the behavior of other particle-based heterogeneous immunoassays.
Asunto(s)
Anticuerpos Inmovilizados/química , Inmunoensayo/instrumentación , Magnetismo/instrumentación , Imanes/química , Técnicas Analíticas Microfluídicas/instrumentación , Plata/química , Animales , Biotina/química , Bovinos , Diseño de Equipo , Fenómenos Magnéticos , Ratones , Modelos Químicos , Estreptavidina/análisis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
A dose-response strategy may not only allow investigation of the impact of foods and nutrients on human health but may also reveal differences in the response of individuals to food ingestion based on their metabolic health status. In a randomized crossover study, we challenged 19 normal-weight (BMI: 20-25 kg/m(2)) and 18 obese (BMI: >30 kg/m(2)) men with 500, 1000, and 1500 kcal of a high-fat (HF) meal (60.5% energy from fat). Blood was taken at baseline and up to 6 h postprandially and analyzed for a range of metabolic, inflammatory, and hormonal variables, including plasma glucose, lipids, and C-reactive protein and serum insulin, glucagon-like peptide-1, interleukin-6 (IL-6), and endotoxin. Insulin was the only variable that could differentiate the postprandial response of normal-weight and obese participants at each of the 3 caloric doses. A significant response of the inflammatory marker IL-6 was only observed in the obese group after ingestion of the HF meal containing 1500 kcal [net incremental AUC (iAUC) = 22.9 ± 6.8 pg/mL × 6 h, P = 0.002]. Furthermore, the net iAUC for triglycerides significantly increased from the 1000 to the 1500 kcal meal in the obese group (5.0 ± 0.5 mmol/L × 6 h vs. 6.0 ± 0.5 mmol/L × 6 h; P = 0.015) but not in the normal-weight group (4.3 ± 0.5 mmol/L × 6 h vs. 4.8 ± 0.5 mmol/L × 6 h; P = 0.31). We propose that caloric dose-response studies may contribute to a better understanding of the metabolic impact of food on the human organism. This study was registered at clinicaltrials.gov as NCT01446068.
Asunto(s)
Biomarcadores/sangre , Peso Corporal/fisiología , Dieta Alta en Grasa , Grasas de la Dieta/administración & dosificación , Obesidad/metabolismo , Adulto , Glucemia , Índice de Masa Corporal , Proteína C-Reactiva/metabolismo , Colesterol/sangre , Estudios Cruzados , Endotoxinas/sangre , Ingestión de Energía , Ayuno , Péptido 1 Similar al Glucagón/sangre , Humanos , Insulina/sangre , Interleucina-6/sangre , Masculino , Comidas , Persona de Mediana Edad , Periodo Posprandial , Suiza , Triglicéridos/sangre , Circunferencia de la CinturaRESUMEN
Microorganisms, encompassing both uni- and multicellular entities, exhibit remarkable diversity as omnipresent life forms in nature. They play a pivotal role by supplying essential components for sustaining biological processes across diverse ecosystems, including higher host organisms. The complex interactions within the human gut microbiota are crucial for metabolic functions, immune responses, and biochemical signalling, particularly through the gut-brain axis. Viruses also play important roles in biological processes, for example by increasing genetic diversity through horizontal gene transfer when replicating inside living cells. On the other hand, infection of the human body by microbiological agents may lead to severe physiological disorders and diseases. Infectious diseases pose a significant burden on global healthcare systems, characterized by substantial variations in the epidemiological landscape. Fast spreading antibiotic resistance or uncontrolled outbreaks of communicable diseases are major challenges at present. Furthermore, delivering field-proven point-of-care diagnostic tools to the most severely affected populations in low-resource settings is particularly important and challenging. New paradigms and technological approaches enabling rapid and informed disease management need to be implemented. In this respect, infectious disease diagnostics taking advantage of microfluidic systems combined with integrated biosensor-based pathogen detection offers a host of innovative and promising solutions. In this review, we aim to outline recent activities and progress in the development of microfluidic diagnostic tools. Our literature research mainly covers the last 5 years. We will follow a classification scheme based on the human body systems primarily involved at the clinical level or on specific pathogen transmission modes. Important diseases, such as tuberculosis and malaria, will be addressed more extensively.
Asunto(s)
Enfermedades Transmisibles , Virus , Humanos , Microfluídica , Ecosistema , Enfermedades Transmisibles/diagnóstico , Sistemas de Atención de PuntoRESUMEN
We propose a concept for enhancing the fluorescent detection signal of on-chip immunoassays by using three-dimensional 3 and 8 µm size microtextured structures as substrates for the assay, which allows exploitation of a large number of fluorophores within the focal plane of an optical microscope for detection. Additionally, we demonstrate the use of 3 and 9.75 µm dielectric microparticles, respectively, as in situ lenses on the 3 and 8 µm microstructures, respectively, for further enhancement of the optical signal. In our model system, the fluorescent complex is formed on (3-aminopropyl)triethoxysilane microstructures and we use carboxyl-functionalized melamine microparticles as the in situ lenses. Mouse IgG diluted in phosphate-buffered saline is used as model target antigen and can be easily detected down to a concentration of 2 ng/mL thanks to these approaches. Also, we present a detailed two-dimensional numerical study of the light propagation through a dielectric microparticle using the finite element method, providing key insight into the signal amplification mechanism of a microlens, and point out its advantageous use in microfluidic assays.
Asunto(s)
Inmunoensayo/métodos , Inmunoglobulina G/análisis , Microesferas , Animales , Anticuerpos Inmovilizados/inmunología , Colorantes Fluorescentes/química , Inmunoensayo/instrumentación , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microscopía de Contraste de Fase , Polimetil Metacrilato/química , Propilaminas , Silanos/química , Especificidad por Sustrato , Triazinas/químicaRESUMEN
Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re-scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput-per-footprint (TPFP) in using inertial focusing are explored by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. Inertial particle focusing is demonstrated with a TPFP up to 0.3 L/(min cm²) in high aspect-ratio rectangular microfluidic channels that are readily fabricated with a post-CMOS integratable process, suggesting at least a 100-fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost-effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.
RESUMEN
We study the presence within the worm Caenorhabditis elegans (C. elegans) of a fluorescent strain of the worm's bacterial food (Escherichia coli (E. coli) OP50) during early adulthood. Use of a microfluidic chip based on a thin glass coverslip substrate allows investigation of the intestinal bacterial load using a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution objective (60×). High-resolution z-stack fluorescence images of the gut bacteria in adult worms, which were loaded in the microfluidic chip and subsequently fixed, were analyzed using IMARIS software and 3D reconstructions of the intestinal bacterial load in the worms were obtained. We present an automated bivariate histogram analysis of the volumes and intensities of the bacterial spots for each worm and find that, as the worms age, the bacterial load in their hindguts increases. We show the advantage of single-worm resolution automated analysis for bacterial load studies and anticipate that the methods described in our work can be easily implemented in existing microfluidic solutions to enable thorough studies of bacterial proliferation.
RESUMEN
We investigated the bacterial food digestion and accumulation in wild-type adult Caenorhabditis elegans (C. elegans) worms that have fed on either non-pathogenic RFP-expressing Escherichia coli (E. coli) OP50 or pathogenic-RFP-expressing Pseudomonas aeruginosa (P. aeruginosa) PAO1 during the first 4 days of adulthood. Once the worms had completed their planned feeding cycles, they were loaded on microfluidic chips, where they were fixed to allow high-resolution z-stack fluorescence imaging of their intestines utilizing a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution oil-immersion objective (60×). IMARIS software was used to visualize and analyze the obtained images, resulting in the production of three-dimensional constructs of the intestinal bacterial load. We discovered two distinct patterns for the bacteria-derived fluorescence signal in the intestine: (i) individual fluorescent spots, originating from intact bacteria, were present in the fluorescent E. coli-OP50-fed worms, and (ii) individual fluorescent spots (originating from intact bacteria) were dispersed in large regions of diffuse fluorescence (RDF), originating from disrupted bacteria, in fluorescent P. aeruginosa-PAO1-fed worms. We performed a semi-automated single-worm-resolution quantitative analysis of the intestinal bacterial load, which showed that the intestinal bacterial load generally increases with age of the worms, but more rapidly for the fluorescent P. aeruginosa-PAO1-fed worms.
RESUMEN
The usage of microfluidics for automated and fast immunoassays has gained a lot of interest in the last decades. This integration comes with certain challenges, like the reconciliation of laminar flow patterns of micro-scale systems with diffusion-limited mass transport. Several methods have been investigated to enhance microfluidic mixing in microsystems, including acoustic-based fluidic streaming. Here, we report both by numerical simulation and experiments on the beneficiary effect of acoustic agitation on the uniformity of immunostaining in large-size and thin microfluidic chambers. Moreover, we investigate by numerical simulation the impact of reducing the incubation times and the concentrations of the biochemical detection reagents on the obtained immunoassay signal. Finally, acoustofluidic mixing was successfully used to reduce by 80% the incubation time of the Her2 (human epidermal growth factor receptor 2) and CK (cytokeratins) biomarkers for the spatial immunostaining of breast cancer cell pellets, or reducing their concentration by 66% and achieving a higher signal-to-background ratio than comparable spatially resolved immunostaining with static incubation.
Asunto(s)
Acústica , Microfluídica , Humanos , Microfluídica/métodos , InmunoensayoRESUMEN
Biochemical reaction rates in microfluidic systems are known to be limited by the diffusional transport of reagents, leading often to lowered sensitivity and/or longer detection times in immunoassays. Several methods, including electrically powering electrodes to generate AC electrothermal flow (ACET) on-chip, have been adopted to enhance the mass transport of the reagents and improve microfluidic mixing. Here, we report a novel ACET electrode design concept for generating in-plane microfluidic mixing vortices that act over a large volume close to the reaction surface of interest. This is different from the traditional ACET parallel electrode design that provides rather local vertical mixing vortices directly above the electrodes. Both numerical simulation and experimental studies were performed to validate the new design. Moreover, numerical simulation was carried out to show the effects of experimental factors such as the reaction kinetics (association constant) and the reagent concentration on the ACET-enhanced surface-based assays. As a proof of concept, the new design for the ACET-enhanced immunoassays was used to improve the immunostaining signal of the HER2 (human epidermal growth factor receptor 2) cancer biomarker on breast cancer cells. Finally, the concept of scaling up the design has been validated by experiments (immunoassays on breast cancer cells for different ACET power and different assay times). In particular, we show that larger ACET in-plane designs can agitate and mix the fluid over large microfluidic volumes, which further enhances the immunoassay's output. We have achieved a 6-times enhancement in the assay signal with a 75% reduction in assay time.
Asunto(s)
Neoplasias de la Mama , Microfluídica , Humanos , Femenino , Diseño de Equipo , Microfluídica/métodos , Electrodos , Simulación por ComputadorRESUMEN
Correction for 'Bubble-enhanced ultrasonic microfluidic chip for rapid DNA fragmentation' by Lin Sun et al., Lab Chip, 2022, 22, 560-572, https://doi.org/10.1039/D1LC00933H.