A microfluidic platform combined with bacteriophage receptor binding proteins for multiplex detection of Escherichia coli and Pseudomonas aeruginosa in blood

Bloodstream infections (BSIs) are triggered by the existence of pathogens in blood and are considered a major health burden worldwide, especially when they result in sepsis and septic shock. Common diagnostic methods are time-consuming, present low specificity, or suffer from interference of blood components, which hampers a timely and effective treatment of BSIs. In this work, a novel microfluidic assay was developed combining a bead-based chip and bacteriophage receptor binding proteins (RBPs) as extremely specific and sensitive recognition molecules for the multiplex concentration and detection of Escherichia coli and Pseudomonas aeruginosa, which are highly prevalent bacteria in BSIs. The device comprises a microcolumn in which antibody-functionalized agarose beads were packed allowing the entrapment of the target bacterium from blood, providing its concentration and separation. For bacterial detection, two recombinant RBPs (Gp54 and Gp17) were fused with different fluorescent proteins and used for the identification of P. aeruginosa and E. coli by the measurement of the distinct fluorescent signals obtained. The developed microfluidic-based assay enabled a fast (70min) and highly specific multiplex detection of both pathogens in whole blood, achieving a detection limit of around 103 CFU, without requiring any time-consuming bacterial pre-enrichment step. Furthermore, it provided a quantitative assessment of bacterial loads present in blood. Noteworthy, this miniaturized and inexpensive device presents simple fabrication and operation, showing great potential to be fully automated, demonstrating to be ideal in point-of-care settings.The authors acknowledge the funding from the Portuguese Foundation for Science and Technology (FCT) under the scope of the project “Phages‐on‐chip” PTDC/BTM‐SAL/32442/2017 (POCI‐01-0145‐FEDER‐032442) and the strategic funding of the research units CEB (UIDB/04469/2020) and INESC MN (UID/05367/2020) through the pluriannual BASE and PROGRAMATICO financing and BioTecNorte operation (NORTE‐01-0145‐FEDER‐000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. S.P.C. and C.R.F.C. acknowledge the FCT for the grants SFRH/BD/130098/2017 and PD/BD/135274/2017, respectively.info:eu-repo/semantics/publishedVersio

Pre-miRNA-149 G-quadruplex as a molecular agent to capture nucleolin

PD/BD/142851/2018 PD/00065/2013 MIT-EXPL/BIO/0008/2017 IF/00959/2015One of the most significant challenges in capturing and detecting biomarkers is the choice of an appropriate biomolecular receptor. Recently, RNA G-quadruplexes emerged as plausible receptors due to their ability to recognize with high-affinity proteins. Herein, we have unveiled and characterized the capability of the precursor microRNA 149 to form a G-quadruplex structure and determined the role that some ligands may have in its folding and binding capacity to nucleolin. The G-quadruplex formation was induced by K+ ions and stabilized by ligands, as demonstrated by nuclear magnetic resonance and circular dichroism experiments. Surface plasmon resonance measurements showed a binding affinity of precursor microRNA 149 towards ligands in the micromolar range (10−5–10−6 M) and a strong binding affinity to nucleolin RNA-binding domains 1 and 2 (8.38 × 10−10 M). Even in the presence of the ligand PhenDC3, the binding remains almost identical and in the same order of magnitude (4.46 × 10−10 M). The molecular interactions of the RNA G-quadruplex motif found in precursor miRNA 149 (5′-GGGAGGGAGGGACGGG- 3′) and nucleolin RNA-binding domains 1 and 2 were explored by means of molecular docking and molecular dynamics studies. The results showed that RNA G-quadruplex binds to a cavity between domains 1 and 2 of the protein. Then, complex formation was also evaluated through polyacrylamide gel electrophoresis. The results suggest that precursor microRNA 149/ligands and precursor microRNA 149/nucleolin RNA-binding domains 1 and 2 form stable molecular complexes. The in vitro co-localization of precursor microRNA 149 and nucleolin in PC3 cells was demonstrated using confocal microscopy. Finally, a rapid and straightforward microfluidic strategy was employed to check the ability of precursor microRNA 149 to capture nucleolin RNA-binding domains 1 and 2. The results revealed that precursor microRNA 149 can capture nucleolin RNA-binding domains 1 and 2 labeled with Fluorescein 5-isothiocyanate in a concentration-dependent manner, but PhenDC3 complexation seems to decrease the ability of precursor microRNA 149 to capture the protein. Overall, our results proved the formation of the G-quadruplex structure in the precursor microRNA 149 and the ability to recognize and detect nucleolin. This proof-of-concept study could open up a new framework for developing new strategies to design improved molecular receptors for capture and detection of nucleolin in complex biological samples.publishersversionpublishe

Regenerable Bead-Based Microfluidic Device with integrated THIN-Film Photodiodes for Real Time Monitoring of DNA Detection

Nanoporous microbead-based microfluidic systems for biosensing applications allow enhanced sensitivities, while being low cost and amenable for miniaturization. The regeneration of the microfluidic biosensing system results in a further decrease in costs while the integration of on-chip signal transduction enhances portability. Here, we present a regenerable bead-based microfluidic device, with integrated thin-film photodiodes, for real-time monitoring of molecular recognition between a target DNA and complementary DNA (cDNA). High-sensitivity assay cycles could be performed without significant loss of probe DNA density and activity, demonstrating the potential for reusability, portability and reproducibility of the system