RESUMEN
The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.
Asunto(s)
Anticuerpos/aislamiento & purificación , Técnicas Biosensibles , Técnicas de Diagnóstico Cardiovascular , Inmunoensayo/métodos , Anticuerpos/genética , Proteína C-Reactiva/genética , Proteína C-Reactiva/aislamiento & purificación , Cromatografía de Afinidad , Productos de Degradación de Fibrina-Fibrinógeno/genética , Productos de Degradación de Fibrina-Fibrinógeno/aislamiento & purificación , Oro/química , Humanos , Nanopartículas del Metal/química , Mioglobina/sangre , Mioglobina/aislamiento & purificación , Tiras Reactivas/químicaRESUMEN
A microarray analytical system for performing tests of latex agglutination reaction in microformat with digital image registration was developed. The system allows the application of latex microdrops to the surface of the carrier in the form of a regular microarray and mixing of the latex droplets with the individual samples in each droplet of the microarray. The reaction is performed in a total mixture volume of about 1 microl for each of 30 samples simultaneously with video registration and interpretation of the results using a scanning device and specially developed software. The results of the semi-quantitative determination of C-Reactive Protein, Rheumatoid Factor and Anti-Streptolysin O concentrations by traditional macro- and proposed micro-arrayagglutination method were compared with the immunoturbidimetric measurements used as reference method. It was concluded that the suggested method for performing latex agglutination reactions on the basis of a microarray approach with digital image evaluation of results can provide a high throughput and reliable results and also offers significant advantages to the traditional latex agglutination tests with visual interpretation. Comprehensive documentation and objectification of readouts show a siginificant improvement to the present methodology.