RESUMO
96-Well plate has been the traditional method used for screening drug compounds libraries for potential bioactivity. Although this method has been proven successful in testing dose-response analysis, the microliter consumption of expensive reagents and hours of reaction and analysis time call for innovative methods for improvements. This work demonstrates a droplet microfluidic platform that has the potential to significantly reduce the reagent consumption and shorten the reaction and analysis time by utilizing nanoliter-sized droplets as a replacement of wells. This platform is evaluated by applying it to screen drug compounds that inhibit the tau-peptide aggregation, a phenomena related to Alzheimer's disease. In this platform, sample reagents are first dispersed into nanolitre-sized droplets by an immiscible carrier oil and then these droplets are trapped on-demand in the downstream of the microfluidic device. The relative decrease in fluorescence through drug inhibition is characterized using an inverted epifluorescence microscope. Finally, the trapped droplets are released on-demand after each test by manipulating the applied pressures to the channel network which allows continuous processing. The testing results agree well with that obtained from 96-well plates with much lower sample consumption (â¼200 times lower than 96-well plate) and reduced reaction time due to increased surface volume ratio (2.5 min vs 2 h).
Assuntos
Compostos Azo/análise , Avaliação Pré-Clínica de Medicamentos/métodos , Técnicas Analíticas Microfluídicas , Inibidores de Proteínas Quinases/análise , Compostos Azo/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Agregados Proteicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Ecotoxicogenomic approaches to environmental monitoring provide holistic information, offer insight into modes of action, and help to assess the causal agents and potential toxicity of effluents beyond the traditional end points of death and reproduction. Recent investigations of toxicant exposure indicate dose-dependent changes are a key issue in interpreting genomic studies. Additionally, there is interest in developing methods to integrate gene expression studies in environmental monitoring and regulation, and the No Observed Transcriptional Effect Level (NOTEL) has been proposed as a means for screening effluents and unknown chemicals fortoxicity. However, computational methods to determine the NOTEL have yet to be established. Therefore, we examined effects on gene expression in Daphnia magna following exposure to Cu, Cd, and Zn over a range of concentrations including a tolerated, a sublethal, and a nearly acutely toxic concentration. Each concentration produced a distinct gene expression profile. We observed differential expression of a very few genes at tolerated concentrations that were distinct from the expression profiles observed at concentrations associated with toxicity. These results suggest that gene expression analysis may offer a strategy for distinguishing toxic and nontoxic concentrations of metals in the environment and provide support for a NOTEL for metal exposure in D. magna. Mechanistic insights could be inferred from the concentration-dependent gene expression profiles including metal specific effects on disparate metabolic processes such as digestion, immune response, development and reproduction, and less specific stress responses at higher concentrations.