An official website of the United States government
Exosomes have gained recognition in cancer diagnostics and therapeutics. However, most exosome isolation methods are time-consuming, costly, and require bulky equipment, rendering them unsuitable for point-of-care (POC) settings. Microfluidics can be the key to solving these challenges. Here, we present a double filtration microfluidic device that can rapidly isolate exosomes via size-exclusion principles in POC settings. The device can efficiently isolate exosomes from 50–100 µL of plasma within 50 min. The device was compared against an already established exosome isolation method, polyethylene glycol (PEG)-based precipitation. The findings showed that both methods yield comparable exosome sizes and purity; however, exosomes isolated from the device exhibited an earlier miRNA detection compared to exosomes obtained from the PEG-based isolation. A comparative analysis of exosomes collected from membrane filters with 15 nm and 30 nm pore sizes showed a similarity in exosome size and miRNA detection, with significantly increased sample purity. Finally, TEM images were taken to analyze how the developed devices and PEG-based isolation alter exosome morphology and to analyze exosome sizes. This developed microfluidic device is cost-efficient and time-efficient. Thus, it is ideal for use in low-resourced and POC settings to aid in cancer and disease diagnostics and therapeutics.
more »
« less
Towards nanovesicle-based disease diagnostics: a rapid single-step exosome assay within one hour through in situ immunomagnetic extraction and nanophotonic label-free detection
Exosomes have been considered as high-quality biomarkers for disease diagnosis, as they are secreted by cells into extracellular environments as nanovesicles with rich and unique molecular information, and can be isolated and enriched from clinical samples. However, most existing exosome assays, to date, require time-consuming isolation and purification procedures; the detection specificity and sensitivity are also in need of improvement for the realization of exosome-based disease diagnostics. This paper reports a unique exosome assay technology that enables completing both magnetic nanoparticle (MNP)-based exosome extraction and high-sensitivity photonic crystal (PC)-based label-free exosome detection in a single miniature vessel within one hour, while providing an improved sensitivity and selectivity. High specificity of the assay to membrane antigens is realized by functionalizing both the MNPs and the PC with specific antibodies. A low limit of detection on the order of 10 7 exosome particles per milliliter (volume) is achieved because the conjugated MNP–exosome nanocomplexes offer a larger index change on the PC surface, compared to the exosomes alone without using MNPs. Briefly, the single-step exosome assay involves (i) forming specific MNP–exosome nanocomplexes to enrich exosomes from complex samples directly on the PC surface at the bottom of the vessel, with a >500 enrichment factor, and (ii) subsequently, performing in situ quantification of the nanocomplexes using the PC biosensor. The present exosome assay method is validated in analyzing multiple membrane proteins of exosomes derived from murine macrophage cells with high selectivity and sensitivity, while requiring only about one hour. This assay technology will provide great potential for exosome-based disease diagnostics.
more »
« less
- PAR ID:
- 10308851
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 21
- Issue:
- 18
- ISSN:
- 1473-0197
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Neurotransmitters are crucial for the proper functioning of neural systems, with dopamine playing a pivotal role in cognition, emotions, and motor control. Dysregulated dopamine levels are linked to various disorders, underscoring the need for accurate detection in research and diagnostics. Single-stranded DNA (ssDNA) aptamers are promising bioreceptors for dopamine detection due to their selectivity, improved stability, and synthesis feasibility. However, discrepancies in dopamine specificity have presented challenges. Here, we surface-functionalized a nano-plasmonic biosensing platform with a dopamine-specific ssDNA aptamer for selective detection. The biosensor, featuring narrowband hybrid plasmonic resonances, achieves high specificity through functionalization with aptamers and passivation processes. Sensitivity and selectivity for dopamine detection are demonstrated across a wide range of concentrations, including in diverse biological samples like protein solutions, cerebrospinal fluid, and whole blood. These results highlight the potential of plasmonic “aptasensors” for developing rapid and accurate diagnostic tools for disease monitoring, medical diagnostics, and targeted therapies.more » « less
-
Phyllachora maydis is a fungal plant pathogen that causes tar spot of corn ( Zea mays) in North and South America, causing devastating yield losses under favorable conditions. Although the causal agent is relatively easy to diagnose via macroscopic and microscopic observations, other diseases and conditions, such as insect frass, have been mistaken for tar spot of corn. Furthermore, conidia and ascospores in isolation can be difficult to visually distinguish from other fungi, and the development of signs and symptoms of the disease may not be observed until 12 to 20 days after infection. Therefore, we developed a TaqMan quantitative polymerase chain reaction (qPCR) assay for the detection and quantification of this pathogen to be used for diagnostics and airborne spore quantification. The assay was designed for the internal transcribed spacer region of P. maydis. The specificity of the assay was confirmed and tested against various nontarget Phyllachora species, corn pathogens, endophytes, and P. maydis samples from several states in the Midwest and from Mexico. The detection limit of this assay was determined to be 100 fg of genomic P. maydis DNA. To demonstrate the transferability of this technology, the assay was tested in different labs using various qPCR thermal cyclers. This assay can be used in downstream research involving latency period, disease prediction, and diagnostics. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .more » « less
-
Kalesh, Karunakaran (Ed.)Introduction:Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes. Methods:Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points. Results:HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), (p< 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24 hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein (p< 0.001) and exo-RNA (p< 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections (p< 0.001). Discussion:Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents.more » « less
-
Magnetic nanoparticles (MNPs) are highly versatile nanomaterials in nanomedicine, owing to their diverse magnetic properties, which can be tailored through variations in size, shape, composition, and exposure to inductive magnetic fields. Over four decades of research have led to the clinical approval or ongoing trials of several MNP formulations, fueling continued innovation. Beyond traditional applications in drug delivery, imaging, and cancer hyperthermia, MNPs have increasingly advanced into molecular medicine. Under external magnetic fields, MNPs can generate mechano- or thermal stimuli to modulate individual molecules or cells deep within tissue, offering precise, remote control of biological processes at cellular and molecular levels. These unique capabilities have opened new avenues in emerging fields such as genome editing, cell therapies, and neuroscience, underpinned by a growing understanding of nanomagnetism and the molecular mechanisms responding to mechanical and thermal cues. Research on MNPs as a versatile synthetic material capable of engineering control at the cellular and molecular levels holds great promise for advancing the frontiers of molecular medicine, including areas such as genome editing and synthetic biology. This review summarizes recent clinical studies showcasing the classical applications of MNPs and explores their integration into molecular medicine, with the goal of inspiring the development of next-generation MNP-based platforms for disease treatment.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D1LC00446H
