ABSTRACT
Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most severe infections encountered in immunodepressed individuals. The diagnosis of Pneumocystis pneumonia continues to be challenging due to the absence of specific symptoms in infected patients. Moreover, the standard diagnostic method employed for its diagnosis involves mainly PCR-based techniques, which besides being highly specific and sensitive, require specialized personnel and equipment and are time-consuming. Our aim is to demonstrate an optical biosensor methodology based on surface plasmon resonance to perform such diagnostics in an efficient and decentralized scheme. The biosensor methodology employs poly-purine reverse-Hoogsteen hairpin probes for the detection of the mitochondrial large subunit ribosomal RNA (mtLSU rRNA) gene, related to P. jirovecii detection. The biosensor device performs a real-time and label-free identification of the mtLSU rRNA gene with excellent selectivity and reproducibility, achieving limits of detection of around 2.11 nM. A preliminary evaluation of clinical samples showed rapid, label-free and specific identification of P. jirovecii in human lung fluids such as bronchoalveolar lavages or nasopharyngeal aspirates. These results offer a door for the future deployment of a sensitive diagnostic tool for fast, direct and selective detection of Pneumocystis pneumonia disease.
ABSTRACT
Nanoplasmonic biosensors based on nanogap antenna structures usually demand complex and expensive fabrication processes in order to achieve a good performance and sensitive detection. We here report the fabrication of large-area nanoplasmonic sensor chips based on nanogap antennas by employing a customized, simple and low-cost colloidal lithography process. By precisely controlling the angle for tilted e-beam metal evaporation, an elliptical mask is produced, which defines the total length of the dipole antenna nanostructures while assuring that the plasmonic response is oriented in the same direction along the sensor chip. Large-area sensor chips of nanogap antennas formed by pairs of gold nanodisks separated by gaps with an average size of 11.6 ± 4.7 nm are obtained. The optical characterization of the nanogap antenna structures in an attenuated total reflection (ATR) configuration shows a bulk refractive index sensitivity of 422 nm per RIU, which is in agreement with FDTD numerical simulations. The biosensing potential of the cm2-sized nanostructured plasmonic sensor chips has been evaluated for the detection of miRNA-210, a relevant biomarker for lung cancer diagnosis, through a DNA/miRNA hybridization assay. A limit of detection (LOD) of 0.78 nM (5.1 ng mL-1) was achieved with no need of further amplification steps, demonstrating the high sensitivity of these plasmonic nanogap antennas for the direct and label-free detection of low molecular weight biomolecules such as miRNAs.
