RESUMEN
Angiogenesis, the growth of new blood vessels, is a critical factor of carcinogenesis. Neomycin and neamine, two drugs blocking the nuclear translocation of angiogenin (ANG), have been proven to inhibit tumour growth in vivo. However, the high toxicity of neomycin prevents its therapeutic use, thus indicating that the less toxic neamine may be a better candidate. Endothelial cells were cultured on a biocompatible multiple microelectrode array (MMA). The release of NO evoked by ANG or vascular endothelial growth factor (VEGF) was detected electrochemically. The effects of neomycin and neamine on ANG- and VEGF-induced NO releases have been investigated. Neomycin totally blocks NO release for concentrations down to the pM range, probably through the inhibition of the Akt kinase phosphorylation, as revealed by confocal microscopy. On the other hand, both ANG- and VEGF-induced NO releases were not significantly hindered by the presence of high concentrations of neamine. The inhibition of the Akt pathway and NO release are expected to lead to a severe decrease in tissue growth and repair, thus indicating a possible cause for the toxicity of neomycin. Furthermore, the data presented here show that ANG- and VEGF-induced NO releases are not dependent on the nuclear translocation of angiogenin, as these events were not abolished by the presence of neamine.
Asunto(s)
Inductores de la Angiogénesis , Neomicina , Neomicina/farmacología , Óxido Nítrico , Factor A de Crecimiento Endotelial Vascular/farmacología , Células Endoteliales , Fosforilación , Factores de Crecimiento Endotelial Vascular , Células CultivadasRESUMEN
We report the results of a label-free analysis of ribonuclease activity using droplet-based microfluidics. The ribonucleolytic activity of ribonucleases (RNases) plays a critical role in cellular functions such as development, survival, growth and differentiation. Altered ribonucleolytic activity and/or the expression level of the RNase A family are known to be associated with pancreatic, bladder, ovarian and thyroid cancers among others. For this reason, the RNase A family is a meaningful protein biomarker that can be used in the diagnosis of cancer and as a target for new drug screening. There are some successful traditional methods for analysing the RNase activity, such as radioactive label-based assay, methylene blue-based assay, gel zymography, as well as other more recently developed methods such as electrochemical assay and fluorescence resonance energy transfer (FRET). However, these methods require analytical samples with a volume ranging from microliters to milliliters, and are not suitable for high-throughput analysis. Therefore, we integrated ethidium bromide (EtBr), which intercalates the chemical itself to nucleic acid, to droplet-based microfluidics for a cost-effective, high-throughput analysis. Put simply, this method is dependent on the amount of intercalated EtBr molecules on RNA. Our assay also uses visible light that is harmless to humans, unlike previous methods that used harmful UV rays, to excite the EtBr molecules. Specifically, we monitored the ribonucleolytic activity of less than 10 nM RNase A in droplets of about 330 picoliters. Also, half the maximal inhibitory concentration (IC50) of the RNase inhibitor was successfully measured in the same volume of droplets at a frequency of 40 hertz.
Asunto(s)
Técnicas Analíticas Microfluídicas , Ribonucleasas/análisis , Etidio , Fluorescencia , Humanos , ARNRESUMEN
We report a droplet-based microfluidic immunosensor for the rapid and accurate detection of melamine, an organic base that has been implicated in widescale adulteration of food products such as milk. Our melamine assay is based on the competitive reaction between native melamine and a melamine-fluorescein isothiocyanate (FITC) conjugate against an anti-hapten antibody. The adoption of fluorescence polarization, allows the quantification of melamine in a more direct and rapid manner than established heterogeneous methods based on liquid chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). The detection protocol provides a limit of detection of 300 ppb, which is below the maximum allowable melamine levels (2.5 ppm) defined by the U.S. Food and Drug Administration and the European Commission to a significant extent.
Asunto(s)
Técnicas Biosensibles , Análisis de los Alimentos , Microfluídica , Triazinas/aislamiento & purificación , Animales , Ensayo de Inmunoadsorción Enzimática , Humanos , Leche/química , Triazinas/química , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Glaucoma is a vision-threatening disorder characterized by progressive death of retinal ganglion cells (RGCs), although little is known about therapeutic milestones. Due to its complex and multifactorial pathogenesis, multipronged therapeutic approach is needed. Angiogenin (ANG), now called ribonuclease (RNase) 5, has been previously known as angiogenic factor and more recently its biologic activity is extended to promoting cell survival via its ribonucleolytic activity. Here, we revealed the defect of ANG in human glaucomatous trabecular meshwork (TM) cells and identified novel multiple functions of ANG as an anti-glaucomatous strategy. ANG was highly expressed in normal eyes and normal TM cells compared to glaucomatous TM cells. ANG induced intraocular pressure (IOP) lowering in rat models of both normal and elevated IOP, and as a possible mechanism, activated Akt-mediated signals for nitric oxide (NO) production, an important regulator of IOP in glaucomatous TM cell. Moreover, we demonstrated ANG-induced production of matrix metalloproteinase (MMP)-1 and -3 and rho-kinase inhibition for TM remodeling. For anti-glaucomatous defense optimization, ANG not only elicited immune-modulative pathways via indolamine 2,3-dioxygenase (IDO) activation in TM cells and suppression of Jurkat T cells, but also rescued neural stem cells (NSCs) from apoptosis induced by glaucomatous stress. These results demonstrate that novel multi-functional effects of ANG may have benefits against glaucoma in ocular tissues.
Asunto(s)
Apoptosis , Glaucoma/fisiopatología , Presión Intraocular , Células-Madre Neurales/patología , Ribonucleasa Pancreática/metabolismo , Malla Trabecular/fisiopatología , Animales , Línea Celular , Células Cultivadas , Glaucoma/metabolismo , Glaucoma/patología , Humanos , Células Jurkat , Masculino , Células-Madre Neurales/metabolismo , Ratas Sprague-Dawley , Ribonucleasa Pancreática/análisis , Malla Trabecular/citología , Malla Trabecular/metabolismo , Malla Trabecular/patologíaRESUMEN
Herein, we describe the development of a multilayer droplet microfluidic system for creating concentration gradients and generating microdroplets of varying composition for high-throughput biochemical and cell-based screening applications. The 3D droplet-based microfluidic device consists of multiple PDMS layers, which are used to generate logarithmic concentration gradient reagent profiles. Parallel flow focusing structures are used to form picoliter-sized droplets of defined volumes but of varying composition. As proof of concept, we demonstrate rapid enzymatic activity assays and drug cytotoxicity assays on bacteria. The 3D droplet-based microfluidic platform has the potential to allow for high-efficiency and high-throughput analysis, overcoming the structural limitations of single layer microfluidic systems.
Asunto(s)
Técnicas de Química Analítica/métodos , Técnicas Analíticas Microfluídicas , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/enzimología , Caspasa 3/química , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , CinéticaRESUMEN
Herein, we describe for the first time the integration of pneumatic micro-pumps with droplet-based microfluidic systems as basic platform for the rapid detection and quantitation of biomarkers. Specifically, we combine this microfluidic platform with fluorescence polarization detection to identify and quantify the potent blood vessel inducing protein bovine angiogenin within cow's milk in high-throughput. The droplet-based fluorescence polarization immunoassay is successful in accurately determining the concentration (4.84±1.21 µg/mL) of bovine angiogenin in cow's milk, affords a 10 fold reduction in dead volumes when compared to conventional droplet-based microfluidic experiments and requires an total sample volume of less than 1 nL.
Asunto(s)
Biomarcadores/sangre , Técnicas Biosensibles , Ribonucleasa Pancreática/biosíntesis , Animales , Biomarcadores/química , Vasos Sanguíneos/metabolismo , Bovinos , Inmunoensayo de Polarización Fluorescente , Humanos , Técnicas Analíticas Microfluídicas , Leche/química , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/aislamiento & purificaciónRESUMEN
The heparin complex of rat angiogenin revealed that a heparin strand is fitted into a positively charged groove formed by the dual binding site of rat angiogenin, suggesting that cell adhesion to angiogenin is facilitated by its interaction with substrates on the cell surface and can be inhibited by heparin.
Asunto(s)
Heparina/química , Heparina/metabolismo , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Adhesión Celular , Cristalografía por Rayos X , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , RatasRESUMEN
We report the fast and sensitive trace analysis of diaquat dibromide monohydrate (DQ) in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. This sensor is composed of two compartments: the first one is for droplet generation for fresh silver nanoparticle (AgNP) synthesis and the second for droplet merging for SERS detection. Silver ions were nucleated and grown to large size AgNPs in droplets, and then each droplet was synchronously merged with another droplet containing DQ for SERS detection. This two-phase liquid-liquid segmented flow system prevented memory effects caused by the precipitation of nanoparticle aggregates on channel walls because the aqueous droplets were isolated by a continuous oil phase. The limit of detection (LOD) of DQ in water was determined to be below 5 nM, which satisfies the maximum contaminant level defined by the United States EPA. This method was also validated successfully in DQ-spiked tap water. The SERS-based integrated sensing system is expected to be useful as an in-the-field sensing platform for fast and reproducible trace analysis of environmental pollutants in water.
Asunto(s)
Bromuros/análisis , Diquat/análisis , Monitoreo del Ambiente/métodos , Espectrometría Raman , Contaminantes Químicos del Agua/análisis , Coloides/química , Iones , Límite de Detección , Nanopartículas del Metal/química , Microfluídica , Microesferas , Nanotecnología , Plata/química , Agua/químicaRESUMEN
Angiogenin (ANG) is reportedly multifunctional, with roles in angiogenesis and autoimmune diseases. This protein is involved in the innate immune system and has been implicated in several inflammatory diseases. Although ANG may be involved in the anti-inflammatory response, there is no evidence that it has direct anti-inflammatory effects. In this study we sought to determine whether ANG has an anti-inflammatory effect in human corneal fibroblasts (HCFs) exposed to media containing tumor necrosis factor-alpha (TNF-α). We found that ANG reduced the mRNA expression of interleukin-1 beta (IL-1ß), -6, -8 and TNF-α receptors (TNFR) 1 and 2. In contrast, ANG increased the mRNA expression of IL-4 and -10. Protein levels of TANK-binding kinase 1 (TBK1) were reduced by ANG in HCFs treated with TNF-α. Moreover, ANG diminished the expression of IL-6 and -8 and monocyte chemotactic protein- (MCP-) 1. The protein expression of nuclear factor-κB (NF-κB) was downregulated by ANG treatment. These findings suggest that ANG suppressed the TNF-α-induced inflammatory response in HCFs through inhibition of TBK1-mediated NF-κB nuclear translocation. These novel results are likely to play a significant role in the selection of immune-mediated inflammatory therapeutic targets and may shed light on the pathogenesis of immune-mediated inflammatory diseases.
Asunto(s)
Córnea/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleasa Pancreática/farmacología , Células Cultivadas , Quimiocina CCL2/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We report a SERS-based competitive immunoassay technique for the early diagnosis of acute myocardial infarction (AMI). Simultaneous quantification of the dual cardiac markers, CK-MB and troponin I, was achieved by single wavelength excitation.
Asunto(s)
Forma MB de la Creatina-Quinasa/análisis , Inmunoensayo , Infarto del Miocardio/diagnóstico , Espectrometría Raman , Troponina I/análisis , Enfermedad Aguda , Biomarcadores/análisis , Humanos , MagnetismoRESUMEN
Over the past two decades, the application of microengineered systems in the chemical and biological sciences has transformed the way in which high-throughput experimentation is performed. The ability to fabricate complex microfluidic architectures has allowed scientists to create new experimental formats for processing ultra-small analytical volumes in short periods and with high efficiency. The development of such microfluidic systems has been driven by a range of fundamental features that accompany miniaturization. These include the ability to handle small sample volumes, ultra-low fabrication costs, reduced analysis times, enhanced operational flexibility, facile automation, and the ability to integrate functional components within complex analytical schemes. Herein we discuss the impact of microfluidics in the area of high-throughput screening and drug discovery and highlight some of the most pertinent studies in the recent literature.
Asunto(s)
Descubrimiento de Drogas , Técnicas Analíticas Microfluídicas , Microfluídica/métodos , Animales , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
In this paper, a porous polymer nanostructure has been integrated with droplet-based microfluidics in a single planar format. Monolithic porous polymer (MPP) was formed selectively within a microfluidic channel. The resulting analyte bands were sequentially comartmentalised into droplets. This device reduces band broadening and the effects of post-column dead volume by the combination of the two techniques. Moreover it offers the precise control of nano/picoliter volume samples.
RESUMEN
We present a fully integrated droplet-based microfluidic platform for the high-throughput assessment of photodynamic therapy photosensitizer (PDT) efficacy on Escherichia coli. The described platform is able to controllably encapsulate cells and photosensitizer within pL-volume droplets, incubate the droplets over the course of several days, add predetermined concentrations of viability assay agents, expose droplets to varying doses of electromagnetic radiation, and detect both live and dead cells online to score cell viability. The viability of cells after encapsulation and incubation is assessed in a direct fashion, and the viability scoring method is compared to model live/dead systems for calibration. Final results are validated against conventional colony forming unit assays. In addition, we show that the platform can be used to perform concurrent measurements of light and dark toxicity of the PDT agents and that the platform allows simultaneous measurement of experimental parameters that include dark toxicity, photosensitizer concentration, light dose, and oxygenation levels for the development and testing of PDT agents.
Asunto(s)
Fenómenos Electromagnéticos , Ensayos Analíticos de Alto Rendimiento/métodos , Microfluídica/métodos , Fármacos Fotosensibilizantes/análisis , Agua/análisis , Evaluación Preclínica de Medicamentos/métodos , Fármacos Fotosensibilizantes/metabolismoRESUMEN
We report the rapid and highly sensitive trace analysis of mercury(ii) ions in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. Aptamer-modified Au/Ag core-shell nanoparticles have been fabricated and utilized as highly functional sensing probes. All detection processes for the reaction between mercury(II) ions and aptamer-modified nanoparticles were performed in a specially designed microdroplet channel. Small water droplets that included sample reagents were separated from each other by an oil phase that continuously flowed along the channel. This two-phase liquid-liquid segmented flow system prevented the adsorption of aggregated colloids to the channel walls due to localized reagents within encapsulated droplets. The result was reduced residence time distributions. The limit of detection (LOD) of mercury(II) ions in water was determined by the SERS-based microdroplet sensor to be below 10 pM, which is three orders below the EPA-defined maximum contaminant level. This combination of a SERS-based microfluidic sensor with aptamer-based functional nanoprobes can be used for in-the-field sensing platforms, due to its size and simplicity.
Asunto(s)
Aptámeros de Nucleótidos/química , Oro/química , Mercurio/análisis , Nanopartículas del Metal/química , Plata/química , Espectrometría Raman , Carbocianinas/química , Iones/química , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Aceites/química , Agua/químicaRESUMEN
A novel SERS-based sandwich immunoassay using DNA aptamers, silica-encapsulated hollow gold nanospheres (SEHGNs) and a gold-patterned microarray was developed for sensitive detection of VEGF (vascular endothelial growth factor) angiogenesis protein markers. Here, a DNA aptamer conjugated to SEHGN was used as a highly reproducible SERS-encoding nanoprobe, and a hybrid microarray including hydrophilic gold wells and other hydrophobic areas was used as a SERS substrate. Target specific DNA aptamers that fold into a G-quadruplex structure were used as a target recognition unit instead of VEGF antibodies. The detection sensitivity was increased by 2 or 3 orders of magnitude over the conventional ELISA method. In particular, the dynamic concentration range was 3 or 4 orders of magnitude greater than that of conventional ELISA. The results demonstrate that this sensing strategy using DNA aptamers is a powerful platform for the design of novel immune-sensors with high performance. In particular, SERS-based detection using SEHGNs provides great promise for highly sensitive biomarker sensing with unprecedented advantages.
Asunto(s)
Aptámeros de Nucleótidos/química , Ensayo de Inmunoadsorción Enzimática , Oro/química , Nanosferas/química , Dióxido de Silicio/química , Factor A de Crecimiento Endotelial Vascular/análisis , Biomarcadores de Tumor/análisis , G-Cuádruplex , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Espectrometría RamanRESUMEN
We report the development of a programmable surface-enhanced Raman scattering (SERS)-based micro-network gradient platform to simultaneously detect two different types of DNA oligomer mixtures. The utility of this platform was demonstrated by quantitative analysis of two breast cancer-related (BRCA1) DNA oligomer mixtures. To generate on-demand concentration gradients, the microfluidic circuit was designed using an electric-hydraulic analogy. Then a multi-gradient microfluidic channel was fabricated based on the theoretical design of the concentration control module. These micro-network structures automatically produce a series of different concentration gradients by continuously mixing Cy3-labeled DNA oligomers (BRAC1-Mutation) with TAMRA-labeled DNA oligomer (BRAC1-Wild). The SERS signals for different ratios of duplex DNA oligomer mixtures, adsorbed on the surface of silver nanoparticles, were measured under flowing conditions. Total analysis time from serial mixing to SERS detection takes less than 10 min because all experimental conditions are automatically controlled inside the exquisitely designed microfluidic channel. This novel SERS-based DNA sensing technology in a micro-network gradient channel is expected to be a powerful analytical tool to simultaneously detect multiple DNA oligomer mixtures.
Asunto(s)
ADN/análisis , ADN/química , Técnicas Analíticas Microfluídicas/instrumentación , Oligodesoxirribonucleótidos/análisis , Oligodesoxirribonucleótidos/química , Espectrometría Raman/instrumentación , Proteína BRCA1/genética , Secuencia de Bases , ADN/genética , Mutación , Oligodesoxirribonucleótidos/genética , Propiedades de Superficie , Factores de TiempoRESUMEN
We demonstrate the integration of a droplet-based microfluidic device with high performance liquid chromatography (HPLC) in a monolithic format. Sequential operations of separation, compartmentalisation and concentration counter were conducted on a monolithic chip. This describes the use of droplet-based microfluidics for the preservation of chromatographic separations, and its potential application as a high frequency fraction collector.
Asunto(s)
Cromatografía Líquida de Alta Presión/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Diseño de Equipo , Fluoresceína-5-Isotiocianato/aislamiento & purificación , Colorantes Fluorescentes/aislamiento & purificaciónRESUMEN
Retinal neovascularization is observed in progression of diabetic retinopathy. New vessels grow into the vitreous cavity in proliferative diabetic retinopathy, resulting in traction retinal detachment and vitreous hemorrhage. To overcome the catastrophic visual loss due to these complications, efforts have been focused on the treatment of retinal neovascularization. In this study, we demonstrated the inhibitory effect of recombinant human apolipoprotein(a) kringle V (rhLK8) in an animal model of ischemia-induced retinal neovascularization. rhLK8 induced no definite toxicity on endothelial cells and retinal tissues at the therapeutic dosage. Interestingly, rhLK8 showed antiangiogenic effect, particularly on fibronectin-mediated migration of endothelial cells. Further experiments demonstrated high binding affinity of rhLK8 to α3ß1 integrin, and suppression of it might be the mechanism of antiangiogenic effect of rhLK8. Furthermore, rhLK8 inhibited phosphorylation of focal adhesion kinase, resulting in suppression of activation of consequent p130CAS-Jun NH(2)-terminal kinase. Taken together, our data suggested the possible application of rhLK8 in the treatment of retinal neovascularization by suppression of fibronectin-mediated angiogenesis.
Asunto(s)
Apolipoproteínas A/uso terapéutico , Células Endoteliales/efectos de los fármacos , Fibronectinas/metabolismo , Retina/efectos de los fármacos , Neovascularización Retiniana/tratamiento farmacológico , Animales , Apolipoproteínas A/farmacología , Movimiento Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Fosforilación/efectos de los fármacos , Retina/metabolismo , Neovascularización Retiniana/metabolismoRESUMEN
Droplet-based microfluidic systems have emerged as a powerful platform for performing high-throughput biological experimentation. In addition, fluorescence polarization has been shown to be effective in reporting a diversity of bimolecular events such as protein-protein, DNA-protein, DNA-DNA, receptor-ligand, enzyme-substrate, and protein-drug interactions. Herein, we report the use of fluorescence polarization for high-throughput protein-protein interaction analysis in a droplet-based microfluidic system. To demonstrate the efficacy of the approach, we investigate the interaction between angiogenin (ANG) and antiangiogenin antibody (anti-ANG Ab) and demonstrate the efficient extraction of dissociation constants (K(D) = 10.4 ± 3.3 nM) within short time periods.
Asunto(s)
Polarización de Fluorescencia , Proteínas/química , Anticuerpos Monoclonales/química , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Ribonucleasa Pancreática/químicaRESUMEN
We report a highly sensitive optical imaging technology using surface-enhanced Raman scattering (SERS)-fluorescence dual modal nanoprobes (DMNPs). Fluorescence microscopy is a well-known imaging technique that shows specific protein distributions within cells. However, most currently available fluorescent organic dyes have relatively weak emission intensities and are rapidly photo-bleached. Thus more sensitive and stable probes are needed. In this work we develop DMNPs, which can be used for both SERS and fluorescence detection. SERS detection is a powerful technique that allows ultrasensitive chemical or biochemical analysis through unlimited multiplexing and single molecule sensitivity. Combining advantages of fluorescence and SERS allows these dual modal nanostructures to be used as powerful probes for novel biomedical imaging. In this work, the fabrication and characterization of the SERS-fluorescence DMNPs and application to biological imaging were investigated using markers CD24 and CD44, which are co-expressed in MDA-MB-231 breast cancer cells, as a model system. SERS imaging with DMNPs was found to be a powerful tool to determine the co-localization of CD24 and CD44 in the cell.