Point-of-care detection of extracellular vesicles: Sensitivity optimization and multiple-target detection.

Extracellular vesicles (EVs) are membrane-bound nanovesicles delivered by different cellular lineages under physiological and pathological conditions. Although these vesicles have shown relevance as biomarkers for a number of diseases, their isolation and detection still has several technical drawbacks, mainly related with problems of sensitivity and time-consumed. Here, we reported a rapid and multiple-targeted lateral flow immunoassay (LFIA) system for the detection of EVs isolated from human plasma. A range of different labels (colloidal gold, carbon black and magnetic nanoparticles) was compared as detection probe in LFIA, being gold nanoparticles that showed better results. Using this platform, we demonstrated that improvements may be carried out by incorporating additional capture lines with different antibodies. The device exhibited a limit of detection (LOD) of 3.4×106EVs/µL when anti-CD81 and anti-CD9 were selected as capture antibodies in a multiple-targeted format, and anti-CD63 labeled with gold nanoparticles was used as detection probe. This LFIA, coupled to EVs isolation kits, could become a rapid and useful tool for the point-of-care detection of EVs, with a total analysis time of two hours.

Silver and gold enhancement methods for lateral flow immunoassays.

Sensitivity is the main concern at the development of rapid test by lateral flow immunoassays. On the other hand, low limits of detection are often required at medical diagnostics and other field of analysis. To overcome this drawback, several enhancement protocols have been described. In this paper, we have selected different silver enhancement methods and one dual gold conjugation, and we critically compared the amplification produced when applied to a gold-nanoparticle based lateral flow immunoassay for the detection of prostate specific antigen (PSA). The highest amplification was obtained by using an immersion method based on a solution of silver nitrate and hydroquinone/citrate buffer in proportion 1:1. Under these conditions, the system is capable of detecting PSA within 20 min at levels as low as 0.1 ng/mL, with a 3-fold sensitivity improvement.

Amperometric fructose sensor based on ferrocyanide modified screen-printed carbon electrode.

The first fructose sensor using a commercial screen-printed ferrocyanide/carbon electrodes (SPFCE) is reported here. The ferrocyanide is included in the carbon ink of the commercial screen-printed carbon electrode. The immobilization of enzyme d-fructose dehydrogenase (FDH) was carried out in an easy way. An aliquot of 10µL FDH was deposited on the electrode surface and left there until dried (approximately 1h) at room temperature. The sensor, so constructed, shows a good sensitivity to fructose (1.25µA/mM) with a slope deviation of ±0.02µA/mM and a linear range comprised between 0.1 and 1mM of fructose, with a limit of detection of 0.05mM. These sensors show good intersensors reproducibility after a previous pretreatment and a high stability. Fructose was determined in real samples as honey, Cola, fruit juices (orange, tomato, apple and pineapple), red wine, red and white grapes, musts and liquor of peach with a good accuracy.