Exosomes represent the next ?omic? frontier in diagnostic biomarkers. As such, numerous technologies have been developed to exploit detection of exosomal proteins or microRNA (miRNA) cargo for potential biomarker development. In general, these approaches are based on the assumption that detection of a particular exosomal protein or miRNA profile will correlate with a specific disease. Yet, this assumption is undermined by the inherent variability resulting from heterogeneity of exosome populations collected from biological fluids. Further, previous attempts to develop protein and, especially, miRNA-based biomarkers have revealed that a single miRNA sequence can be correlated with numerous diseases even without the confounding heterogeneity of exosomes. Thus, we hypothesize that to achieve appropriate specificity for the discovery of exosomal biomarkers, correlation of exosomal proteins and miRNAs ? simultaneously detected and resolvable at the single-exosome level ? is necessary. Here we propose a highly dense multiplexed microsystem implementing a new isothermal nucleic acid amplification method for digital exosome analysis, enabling the specific correlation of membrane protein markers and miRNA sequences at the single exosome level. A low- cost and easy-to-use thermoplastic chip with integrated amplification reagents enables self-discretization of exosomes for spatially multiplexed analysis, scalable up to one million reactions. For specific detection of miRNA sequences and membrane proteins, we have designed an isothermal reaction optimized for the amplification of short DNA and RNA sequences that identifies miRNA sequences as well as DNA oligonucleotides conjugated to antibodies that label the exosome surface proteins. The reaction, referred to as transcription cycling amplification (TCA), utilizes DNA polymerase with exonuclease activity to degrade donor/quencher-labeled probes (i.e., FRET probes) for specific and real-time quantitative detection. In tandem, RNA polymerase acts on the polymerized oligo for the cyclical generation of RNA that leads to exponential amplification, providing highly sensitive detection. By integrating these two tools, we will develop a biomarker discovery platform that first digitizes the exosomes across a dense reaction array and correlates miRNA sequences with membrane proteins in single exosomes.

Public Health Relevance

Previous attempts to identify disease biomarkers from circulating exosomes and their microRNA have fallen short due to challenges with exosome population heterogeneity, variability in methods, and lack of specificity of circulating microRNA sequences. We propose to develop a tool capable of high throughput analysis of exosomes at the single exosome level, eliminating population and experimental variability. Additionally, our multi-parameter analysis will simultaneously identify exosomal membrane proteins and microRNA sequences, improving the specificity of biomarkers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM130923-03
Application #
10092199
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Sammak, Paul J
Project Start
2019-02-01
Project End
2023-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
3
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Maryland College Park
Department
Biomedical Engineering
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742