Pancreatic ductal adenocarcinoma (PDAC) typically has nonspecific symptoms and is often found too late to treat. Because diagnosis of PDAC involves complex, invasive, and expensive procedures, screening populations at increased risk will depend on developing rapid, sensitive, specific, and cost-effective tests. Exosomes, which are nanoscale vesicles shed into blood from tumors, have come into focus as valuable entities for noninvasive liquid biopsy diagnostics. However, rapid capture and analysis of exosomes with their protein and other biomarkers have proven difficult.
Here, UCSD engineers present a simple method integrating capture and analysis of exosomes and other extracellular vesicles directly from whole blood, plasma, or serum onto an AC electrokinetic microarray chip. In this process, no pretreatment or dilution of sample is required, nor is it necessary to use capture antibodies or other affinity techniques. Subsequent on-chip immunofluorescence analysis permits specific identification and quantification of target biomarkers within as little as 30 min total time. In this initial validation study, the biomarkers glypican-1 and CD63 were found to reflect the presence of PDAC and thus were used to develop a bivariate model for detecting PDAC. Twenty PDAC patient samples could be distinguished from 11 healthy subjects with 99% sensitivity and 82% specificity. In a smaller group of colon cancer patient samples, elevated glypican-1 was observed for metastatic but not for nonmetastatic disease. The speed and simplicity of ACE exosome capture and on-chip biomarker detection, combined with the ability to use whole blood, will enable seamless “sample-to-answer” liquid biopsy screening and improve early stage cancer diagnostics.
Schematic diagram illustrates the ACE (AC electrokinetic) direct immunoassay procedure
Undiluted whole blood, plasma, or serum sample is added directly to the chip. A 10 min application of AC current to the chip effects dielectrophoretic separation and isolation of target nanoscale extracellular vesicles (EVs) and other particulates onto the chip electrodes. Unbound material is washed off the chip with 0.5× phosphate-buffered saline during an additional 10 min of AC current. After the current is stopped, EVs and nucleic acid targets can be analyzed by different methods, including addition of fluorescent reporter antibodies and nucleic acid dyes that are applied to the chip and then incubated for the appropriate times. Following the final wash step, the chip is ready for direct imaging and analysis of the fluorescent signal.