Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma

Abstract

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 μL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives

“…For instance, size‐exclusion chromatography, which allows size‐based separation in a single column, with the majority of EVs eluting before soluble components such as proteins and HDL . Moreover, novel isolation protocols have been also used such as precipitation kits (charge‐based precipitation, PROSPR, immune affinity, and others), which can reduce time and the amount of starting sample. Currently, several biotechnological companies are working on developing quick and easy ways to isolate EVs from biological fluids for clinical applications, such as ExoQuick(System Biosciences, Mountain View, CA, USA), which allows the isolation of exosomes .…”

Progress in Microfluidics‐Based Exosome Separation and Detection Technologies for Diagnostic Applications

“…This technique is label‐free, charge‐free, and controllable, which has led DEP to become an alternative and powerful method for particle, cell and protein separation . Recently, this method has been used to separate exosomes from undiluted human plasma samples . In this microfluidic chip, nanoparticles and nanoscale entities are attracted to the high electric field regions, while cells and larger entities migrate into the low electric field areas.…”

The cancer exosomes: Clinical implications, applications and challenges

“…proposed a microfluidic chip based on immune capture, which could extract exosomes with immune capture function from small quantified breast cancer samples and help clinical diagnosis of breast cancer patients. Ibsen et al . designed an alternating current electrokinetic (ACE) microarray device, which allowed the isolation of glioblastoma‐derived exosomes from a plasma sample (30–50 μl), as well as apparently simplifying processing steps and enabling less time required.…”

“…Fang et al 83 proposed a microfluidic chip based on immune capture, which could extract exosomes with immune capture function from small quantified breast cancer samples and help clinical diagnosis of breast cancer patients. Ibsen et al 84 designed an alternating current electrokinetic (ACE) microarray device, which allowed the isolation of glioblastoma-derived exosomes from a plasma sample (30-50 μl), as well as apparently simplifying processing steps and enabling less time required. Another comprehensive exosome analysis tool and potential noninvasive diagnostic platform, ExoPCD-chip, is a two-stage microfluidic platform whose isolation and analysis applied for liver cancer-derived exosomes as low as 4.39 × 10 3 particles/ml from a serum sample (30 μl) within 3.5 hr.…”