Our tears reflect not only our emotions, but they also bear signatures of health and disease. Tears contain nanoscale extracellular vesicles called exosomes that carry proteins and nucleic acids released from living cells. However, collecting exosomes from tears is restricted by low recovery, long processing times, and small sample volumes. These challenges pose technical obstacles in using tears for biomarker discovery and clinical diagnostics.
Scientists have now developed a quick and simple nanotechnology-based method to isolate exosomes from tears and have used it to identify several proteins and micro RNAs in exosomes.1 This could promote the diagnosis and monitoring of different ocular and systemic diseases. The new technology, called iTEARS (incorporated tear-exosomes analysis via rapid isolation system), uses an ultrafast-isolation system called EXODUS2 that is based on negative-pressure oscillation and on-device immunodetection.
Fei Liu, PhD, a research scientist at the School of Ophthalmology and Optometry, at the School of Biomedical Engineering, in Wenzhou Medical University in China and the senior investigator of the study said, “We hope to see the secret of diseases and emotions by isolating, purifying, and identifying these tiny nanoscale biological particles using the EXODUS method together with proteomics and sequencing technologies.”
Tony Jun Huang, PhD, a professor of mechanical engineering at Duke University said, “This work is excellent. The findings of this research allude to the potential for extracellular vesicle separation for noninvasive disease diagnostics. Incorporating downstream extracellular vesicle analysis with extracellular vesicle separation technology will be extremely important for expanding the field of disease diagnostics.” (Huang was not involved in the study).
The iTears technology was developed in the laboratories of Luke Lee, PhD, professor at Harvard Medical School and Fei Liu, PhD, professor at Wenzhou Medical University.
Detecting diseases in a teardrop
“Ironically, we do not recognize that tears contain rich clinical information about different parts of our body,” said Liu.
Tears are secreted by lachrymal glands in our eyelids through filtration of blood. Exosomes that are released by cells and circulate in blood, pass directly into tears, bypassing this filtration, owing to their small size. Studies have shown exosomes carry molecular signatures of immune regulation, angiogenesis, tumor invasion, and cell migration.
Tears provide an ideal sample for clinical diagnostics because they have low potential for contaminant interference, are directly correlated with circulating fluids, and offer the added advantage that their collection can be fast and noninvasive. Exosome-based liquid biopsy technology that uses tear samples offers an attractive alternative to disease classification, patient stratification, prognostic prediction, and point-of-care diagnostics.
Liu’s team has isolated exosomes from as little as 10 µL of tears in a 5-minute protocol using nanoporous membrane-based resonators. The researchers noted that they quantified the exosomes collected from the tear samples using the Schirmer test strip.3 Proteins from the exosomes could be tagged with fluorescent probes while they were still on the device and then transferred to other instruments for further analysis. The researchers ran transcriptomic and proteomic analyses on the RNA and proteins isolated from exosomes to detect and quantify biomarkers.
Liu said, “We obtain more proteins and miRNAs through tear-extracellular vesicles per drop than in urine or blood. Studies of tears are not “a hot topic” when compared with other body fluids (e.g., urine and blood), but it is time to recognize their importance in molecular diagnosis.”
Liu’s team identified the proteins CALML5, KART6A, and S100P to classify different forms of dry eye diseases and the miRNAs miR-145-5p, miR-214-3p, miR-218-5p, and miR-9-5p for the monitoring diabetic retinopathy, by isolating proteins and miRNA from exosomes, respectively.
“We use a novel protocol for sensitive molecular diagnosis due to efficient isolation, purification, and preconcentration of extracellular vesicles from a few drops of human tears within five minutes, which other commercial techniques cannot reach,” said Liu.
Liu and his team believe iTEARS can improve molecular diagnostics using noninvasive tear samples to identify ocular disorders, systemic diseases, neurodegenerative diseases, and cancer.
Next steps
In upcoming projects Liu intends to explore signaling mechanisms that result in pathologies and can be used in precision molecular diagnosis of various diseases. Liu also plans to quantify molecular signatures of emotional states in tears, particularly biomarkers of psychological stress to develop diagnostic tests for neuropsychiatric conditions.
“We hope to provide a clinical translation method for tear-based molecular diagnostics and prognostics not only for eye-related diseases, but also for cancers, diabetes mellitus, neurologic diseases, and the biological functions of emotional tears,” said Liu.
References
- Hu L., Zhang T., Ma H., et. al. Discovering the Secret of Diseases by Incorporated Tear Exosomes Analysis via Rapid-Isolation System: iTEARS. ACS Nano 2022.https://pubs.acs.org/doi/10.1021/acsnano.2c02531
- Chen, Y., Zhu, Q., Cheng, L. et al. Exosome detection via the ultrafast-isolation system: EXODUS. Nat Methods 18, 212–218 (2021). https://doi.org/10.1038/s41592-020-01034-x
- Yao Y.N., Di D., Yuan Z.C. et al. Schirmer Paper Noninvasive Microsampling for Direct Mass Spectrometry Analysis of Human Tears. Anal. Chem. 2020, 92, 6207−6212. https://pubs.acs.org/doi/10.1021/acs.analchem.9b05078
