A 2D MicrofluidicPaper-Based Analytical Device forDiagnosis of Canine Visceral Leishmaniasis via Mass Spectrometry-BasedImmunoassay

This work presents the first indirect immunoassay performed on a paper-based microfluidic platform for the diagnosis of canine visceral leishmaniasis (CVL). The IgG antibody biomarker, which signifies the presence of this infectious disease, was captured with a recombinant K39 antigen and detected with secondary antibodies that were conjugated with cleavable ionic probes. The use of ionic probes enabled direct analysis of the assay results through an on-chip paper spray mass spectrometry (MS) technology. This MS-based immunoassay was developed to allow for early detection of CVL in asymptomatic dogs. The sensitivity required for such a diagnostic method was demonstrated through internal standard calibration in which sample dilution as low as 1/4000 was achieved. Aside from high sensitivity, the ionic probes are stable, which allowed the paper device to be stored at room temperature and under ambient conditions for 30 days without affecting the diagnostic outcome. Our method was used to analyze 20 clinical canine serum samples, where we detected a 2 orders of magnitude higher signal for CVL-positive samples compared to negative samples. MS signal derived from the 10 CVL-positive serum clinical samples showed a strong correlation with antibody titers determined by immunofluorescence assay. This correlation was confirmed through Pearson’s statistical analysis. Overall, the high sensitivity and positive results from stability tests observed for our platform are expected to enable large-scale CVL screening in asymptomatic dogs in remote areas, especially when combined with portable mass spectrometers.