Paper-Based Microfluidics Immunoassay for Detection of Canine Distemper Virus

ABSTRACT Paper-based devices present low-cost and are versatile, making them very attractive for clinical analysis. To manufacture those devices wax patterns are printed on paper surface and upon heating the wax permeates through the entire thickness of the paper, creating hydrophobic barriers that delimit test areas. Antibodies produced in rabbits against canine distemper virus (CDV) were physically adsorbed on the surface of gold nanoparticles (AuNPs) and incubated with CDV viral antigens, forming the immunocomplex. Anti-CDV antibodies were immobilized into the microchannels by physical adsorption, forming the test region. The test solution containing conjugated AuNPs was applied at the bottom of the microchannel and it was eluted with a phosphate buffer solution 0.01 M pH 7.4. When the solution containing the AuNPs reached the test zone the recognition of antigens contained on the immunocomplex occurred with the consequent development of a red line, which represents a positive outcome for the test. This method demonstrated the success of physical immobilization of antibodies on AuNPs and the physical immobilization of antibodies on cellulose's surface. This colorimetric assay brings simplicity and versatility to clinical analyses, presenting potential for CDV diagnosis.

Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.

DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados, normalizados e os dados foram estatisticamente analisados para verificar os genes diferencialmente expressos. De acordo com nossos dados, 104 genes foram diferencialmente expressos para o meio de cultura XDM2 e 30 genes para o BCYE. No presente estudo, nós demonstramos que a técnica de DNA microarrays eficientemente diferencia genes expressos sob diferentes condições de cultivo.