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Review
. 2017 Dec 6;12(1):017103.
doi: 10.1088/1752-7163/aa88e4.

Non-invasive lung disease diagnostics from exhaled microdroplets of lung fluid: perspectives and technical challenges

Victor N Morozov  1 , Andrey Y Mikheev, Yuri M Shlyapnikov, Alexander A Nikolaev, Irina V Lyadova
Affiliations
Review

Non-invasive lung disease diagnostics from exhaled microdroplets of lung fluid: perspectives and technical challenges

Victor N Morozov et al. J Breath Res. .

Abstract

The combination of ultra-sensitive assay techniques and recent improvements in the instrumentation used to collect microdroplets of lung fluid (MLF) from exhaled breath has enabled the development of non-invasive lung disease diagnostics that are based on MLF analysis. In one example of this approach, electrospun nylon filters were used to collect MLFs from patients with pulmonary tuberculosis. The filters were washed to obtain liquid probes, which were then tested for human immunoglobulin A (h-IgA) and fractions of h-IgA specific to ESAT-6 and Psts-1, two antigens secreted by Mycobacterium tuberculosis. Probes collected for 10 min contained 100-1500 fg of h-IgA and, in patients with pulmonary tuberculosis, a portion of these h-IgA molecules showed specificity to the secreted antigens. Separate MLFs and their dry residues were successfully collected using an electrostatic collector and impactor developed especially for this purpose. Visualization of MLF dry residues by atomic force microscopy made it possible to estimate the lipid content in each MLF and revealed mucin molecules in some MLFs. This exciting new approach will likely make it possible to detect biomarkers in individual MLFs. MLFs emerging from an infection site ('hot' microdroplets) are expected to be enriched with infection biomarkers. This paper discusses possible experimental approaches to detecting biomarkers in single MLFs, as well as certain technological problems that need to be resolved in order to develop new non-invasive diagnostics based on analysing biomarkers in separate MLFs.

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Figures

Figure 1.
Figure 1.
Disposable filters used for collecting MLFs from patients in tuberculosis clinics. (a) Collection of exhaled MLFs with a disposable filter. (b) Holder for two filters.
Figure 1.
Figure 1.
Disposable filters used for collecting MLFs from patients in tuberculosis clinics. (a) Collection of exhaled MLFs with a disposable filter. (b) Holder for two filters.
Figure 1.
Figure 1.
Disposable filters used for collecting MLFs from patients in tuberculosis clinics. (a) Collection of exhaled MLFs with a disposable filter. (b) Holder for two filters.
Figure 2.
Figure 2.
(a) Image of a simple impactor with a thermostated aluminum base enabling control of substrate temperature. (b) Dark-field image of dry residues of MLFs collected on a mica surface at a substrate temperature of 45 °C and a flow rate of 2.2 l min−1.
Figure 2.
Figure 2.
(a) Image of a simple impactor with a thermostated aluminum base enabling control of substrate temperature. (b) Dark-field image of dry residues of MLFs collected on a mica surface at a substrate temperature of 45 °C and a flow rate of 2.2 l min−1.
Figure 2.
Figure 2.
(a) Image of a simple impactor with a thermostated aluminum base enabling control of substrate temperature. (b) Dark-field image of dry residues of MLFs collected on a mica surface at a substrate temperature of 45 °C and a flow rate of 2.2 l min−1.
Figure 3.
Figure 3.
Images of dry residues of exhaled MLFs collected on a mica surface under various conditions using the impactor shown in figure 2(a). (a) After collection on mica at 22 °C. (b) After collection on mica heated to 50 °C. (c) and (d) Images of structures after collection at 45 °C followed by briefly wetting the mica surface by breathing on it.
Figure 3.
Figure 3.
Images of dry residues of exhaled MLFs collected on a mica surface under various conditions using the impactor shown in figure 2(a). (a) After collection on mica at 22 °C. (b) After collection on mica heated to 50 °C. (c) and (d) Images of structures after collection at 45 °C followed by briefly wetting the mica surface by breathing on it.
Figure 3.
Figure 3.
Images of dry residues of exhaled MLFs collected on a mica surface under various conditions using the impactor shown in figure 2(a). (a) After collection on mica at 22 °C. (b) After collection on mica heated to 50 °C. (c) and (d) Images of structures after collection at 45 °C followed by briefly wetting the mica surface by breathing on it.
Figure 4.
Figure 4.
Example assay of exhaled biomarkers in a probe collected from a patient with pulmonary tuberculosis using the filter shown in figure 1(a). (a) Structure of the microarray used in the active assay. (b) Dark-field image of a pattern of microarray-bound magnetic beads coated with antibody molecules specific to human IgA after the IgA molecules were electrophoretically captured from the probe by using the procedure described in [48].
Figure 4.
Figure 4.
Example assay of exhaled biomarkers in a probe collected from a patient with pulmonary tuberculosis using the filter shown in figure 1(a). (a) Structure of the microarray used in the active assay. (b) Dark-field image of a pattern of microarray-bound magnetic beads coated with antibody molecules specific to human IgA after the IgA molecules were electrophoretically captured from the probe by using the procedure described in [48].
Figure 4.
Figure 4.
Example assay of exhaled biomarkers in a probe collected from a patient with pulmonary tuberculosis using the filter shown in figure 1(a). (a) Structure of the microarray used in the active assay. (b) Dark-field image of a pattern of microarray-bound magnetic beads coated with antibody molecules specific to human IgA after the IgA molecules were electrophoretically captured from the probe by using the procedure described in [48].
Figure 5.
Figure 5.
Principle of diagnostics based on detection of biomarkers in each individual exhaled microdroplet.
Figure 5.
Figure 5.
Principle of diagnostics based on detection of biomarkers in each individual exhaled microdroplet.
Figure 5.
Figure 5.
Principle of diagnostics based on detection of biomarkers in each individual exhaled microdroplet.
Figure 6.
Figure 6.
(a) Microarray of MLFs deposited on a CMC surface through a metal grid using the impactor shown in figure 2(a). The image was taken under dark-field illumination. (b) Pattern of magnetic beads coated with anti-hIgA left on the surface after the beads contacted the microarray.
Figure 6.
Figure 6.
(a) Microarray of MLFs deposited on a CMC surface through a metal grid using the impactor shown in figure 2(a). The image was taken under dark-field illumination. (b) Pattern of magnetic beads coated with anti-hIgA left on the surface after the beads contacted the microarray.
Figure 6.
Figure 6.
(a) Microarray of MLFs deposited on a CMC surface through a metal grid using the impactor shown in figure 2(a). The image was taken under dark-field illumination. (b) Pattern of magnetic beads coated with anti-hIgA left on the surface after the beads contacted the microarray.
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