Identifying the pathogen responsible for an infection is a requirement in order to personalize antimicrobial treatments. Detecting bacterial enzymes, such as proteases, lipases, and oxidoreductases, is a winning approach for detecting pathogens at the point of care. In this Article, a new method for detecting urease-producing bacteria rapidly and at ultralow concentrations is reported. In this method, longsome bacteriological culture steps are substituted for a 10 min capture procedure with positively charged magnetic beads. The presence of urease-positive bacteria on the particles is then queried with a plasmonic signal generation step that generates blue-or red-colored nanoparticle suspensions upon addition of the enzyme substrate. These colorimetric signals, which can be easily identified by eye, are generated by the NH 3 -dependent assembly of gold nanoparticles in the presence of bovine serum albumin (BSA). The proposed method can detect Proteus mirabilis with a limit of detection of 10 1 cells mL −1 , with a total assay time of 40 min, even in the presence of a large excess of urease-negative bacteria (Pseudomonas aeruginosa). Furthermore, it does not require bulky equipment, and it can detect P. mirabilis at clinically relevant concentrations within minutes, making it suitable for detecting urease-positive pathogens at the point of care.
Lung-secreted IgG and IgM antibodies are valuable biomarkers for
monitoring the local immune response against respiratory infections.
These biomarkers are found in lower airway secretions that need to
be liquefied prior to analysis. Traditional methods for sample liquefaction
rely on reducing disulfide bonds, which may damage the structure of
the biomarkers and hamper their immunodetection. Here, we propose
an alternative enzymatic method that uses O2 bubbles generated
by endogenous catalase enzymes in order to liquefy respiratory samples.
The proposed method is more efficient for liquefying medium- and high-viscosity
samples and does not fragment the antibodies. This prevents damage
to antigen recognition domains and recognition sites for secondary
antibodies that can decrease the signal of immunodetection techniques.
The suitability of the enzymatic method for detecting antibodies in
respiratory samples is demonstrated by detecting anti-SARS-CoV-2 IgG
and IgM to viral N-protein with gold standard ELISA in bronchial aspirate
specimens from a multicenter cohort of 44 COVID-19 patients. The enzymatic
detection sharply increases the sensitivity toward IgG and IgM detection
compared to the traditional approach based on liquefying samples with
dithiothreitol. This improved performance could reveal new mechanisms
of the early local immune response against respiratory infections
that may have gone unnoticed with current sample treatment methods.
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