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. 2024 Jun 4;14(1):12830.
doi: 10.1038/s41598-024-62784-8.

Parallel detection of multiple biomarkers in a point-of-care-competent device for the prediction of exacerbations in chronic inflammatory lung disease

Niels Röckendorf #  1 , Katrin Ramaker #  1 , Karoline Gaede  2   3 , Kristof Tappertzhofen  1 , Lars Lunding  4   3 , Michael Wegmann  4   3 , Peter Horbert  5 , Karina Weber  5 , Andreas Frey  6
Affiliations

Parallel detection of multiple biomarkers in a point-of-care-competent device for the prediction of exacerbations in chronic inflammatory lung disease

Niels Röckendorf et al. Sci Rep. .

Abstract

Sudden aggravations of chronic inflammatory airway diseases are difficult-to-foresee life-threatening episodes for which advanced prognosis-systems are highly desirable. Here we present an experimental chip-based fluidic system designed for the rapid and sensitive measurement of biomarkers prognostic for potentially imminent asthma or COPD exacerbations. As model biomarkers we chose three cytokines (interleukin-6, interleukin-8, tumor necrosis factor alpha), the bacterial infection marker C-reactive protein and the bacterial pathogen Streptococcus pneumoniae-all relevant factors in exacerbation episodes. Assay protocols established in laboratory environments were adapted to 3D-printed fluidic devices with emphasis on short processing times, low reagent consumption and a low limit of detection in order to enable the fluidic system to be used in point-of-care settings. The final device demonstrator was validated with patient sample material for its capability to detect endogenous as well as exogenous biomarkers in parallel.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Design of the microfluidic reaction device. (A) Device version 1: four chambers loaded/processed individually; (B) Scheme of fluid flow and microscopic top view of individual analysis chamber (grayscale image); (C) Device version 2: four chambers loaded/processed in parallel; (D) Demonstrator units of fluidic devices version 1 and 2, equipped with syringes for manual operation.
Figure 2
Figure 2
Detection of endogenous biomarkers by sandwich immunoassay. (A) Slide arrays processed in open well format. (B) Measurement of IL-6, IL-8 and TNF-α at different concentrations in undiluted or diluted saliva (N = 3).
Figure 3
Figure 3
Detection of model pathogen S. pneumoniae. (A) Slide arrays processed in open well format. (B, C) Measurement of increasing numbers of bacteria, captured by different amounts of antibody in individual spots. Analyte was applied either in PBST (B) or in saliva (C) (each N = 3). Graphs in the right panel depict results of linear regression analysis assessing the relationship between fluorescence read-out (relative fluorescence unists, RFU) and analyte concentration in the samples. Note that the highest concentration of bacteria (1 × 108 CFU/ml) has been omitted from all regression analyses.
Figure 4
Figure 4
Detection of markers on glass slides using a microfluidic device. (A) Slide with four reaction arrays carrying duplicate spots of capture antibodies for the detection of four different analytes. (B) Detection of analytes in individually spiked saliva samples (analyte concentration: 10 ng/ml) using a mixture of biotinylated detection antibodies. (C) Detection of analytes in mixed spiked saliva samples (IL-6 & IL-8: 10 ng/ml, TNF-α: 25 ng/ml, S. pneumoniae: 2 × 105 CFU/ml) using individual biotinylated detection antibodies in each reaction chamber.
Figure 5
Figure 5
Measurement of IL-8 in a fluidic device with 4 parallel channels attached to a single sample inlet structure (A). The measurements of IL-8 are not significantly different (1way ANOVA, Kruskal Wallis Test, Dunn’s multiple comparisons test) in the four channels (B).
Figure 6
Figure 6
Detection of endogenous biomarkers IL-6, IL-8 and TNF-α in non-spiked saliva samples of selected asthma- and COPD patients, using the quadriplex fluidic device. One saliva sample spiked with IL-6, IL-8 and TNF-α, at 10 ng/ml each, was used as control.
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