Attomolar sensitivity microRNA detection using real-time digital microarrays.

MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA-451a (miR-451) and miRNA-223-3p (miR-223). We demonstrated improvements in sensitivity in comparison to traditional end-point assays that employ capture on solid phase support, by implementing real-time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low-abundance biomarkers in the presence of low-affinity but high-abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to [Formula: see text] 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.

Immunoassay for HIV Drug Metabolites Tenofovir and Tenofovir Diphosphate.

Poor patient adherence to antiretroviral medication represents a major obstacle for managing disease and reducing rates of new HIV infections. The measurement of patient drug levels is the most objective method of determining adherence. Tenofovir and tenofovir diphosphate are metabolites of some of the most common HIV medications for treatment and prevention and can be quantified by mass spectrometry. Here, we report the development of a competitive enzyme linked immunoassay as a simplified approach for detecting tenofovir and tenofovir diphosphate. Monoclonal antibodies were produced by two tenofovir-hapten conjugates and screened for binding to immobilized tenofovir, and then for competition by tenofovir and tenofovir diphosphate. Antibody specificity was evaluated against adenosine phosphates, which are close structural analogs. We performed numerical simulations of reaction equilibrium to guide assay optimization. When used to evaluate spiked tenofovir in plasma and spiked tenofovir diphosphate in red blood cell lysate, the optimized assay had high sensitivity and specificity.