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Microfluidic Chip Could Enable More Efficient Exosome Isolation, Analysis


NEW YORK (GenomeWeb) – Researchers at the University of Kansas have developed a microfluidic chip for the isolation and analysis of tumor-associated exosomes.

According to the scientists, the chip, which uses self-assembled herringbone nanopatterns, can isolate exosomes at levels undetectable using conventional microfluidic platforms and could allow for more sensitive and more rapid analysis of these vesicles.

In a study published this week in Nature Biomedical Engineering, the researchers used to device to collect exosomes from ovarian cancer patients and identify potential protein biomarkers for early detection and monitoring of the disease.

They are now using the technology to explore other applications within oncology, including detection of biomarkers for identifying non-invasive breast cancers that are likely to become invasive cancers, said Yong Zeng, associate professor of chemistry at Kansas and senior author on the study.

Zeng is also co-founder of the firm Clara Biotech, which offers exosome isolation services based on technology developed by him and colleagues including Mei He, an assistant professor of technology at Kansas and cofounder and CSO at the company. He was a coauthor on the Nature Biomedical Engineering study.

The company launched its exosome isolation services in November of 2018 and last month launched an early-access program where customers can apply to get its isolation platform for internal use. Zeng, who does not currently have a role at the firm, said that Clara's technology is based on work done prior to the recent study but it could potentially be brought into the company.

Exosomes are membrane-bound bodies produced by cells and shed into the bloodstream and other bodily fluids. Their molecular make-up reflects that of their cell of origin, which has made them an area of growing interest in liquid biopsy research, the idea being that it might be easier to measure proteins or nucleic acids in exosomes derived from, for instance, cancer cells, than to detect cancer-linked nucleic acids or proteins circulating freely in patient blood or urine.

A number of researchers and companies have identified exosome-bound biomarkers linked to various diseases, with firms like Exosome Diagnostics (acquired last year by Bio-Techne) developing tests to help clinicians diagnose prostate cancer and guide therapy in lung cancer.

A major challenge of exosome work is isolating these vesicles for downstream analysis.

"This is the main headache for people who want to work in this field," Zeng said, noting that conventional methods like differential centrifugation can be very time-consuming. This is particularly an issue for measuring exosome-bound proteins, as conventional immunoassays can require fairly large amounts of sample, he added.

To address this difficulty, some researchers have turned to microfluidic systems which offer faster and more sensitive exosome isolation. However, microfluidic devices have also run into limitations, Zeng said, noting that the challenge is essentially getting the exosomes present in the fluid being analyzed to make contact in an efficient way with the antibodies or other affinity reagents used to functionalize the surfaces of the chip.

To improve this contact, he and his colleagues developed a three-dimensional herringbone patterned chip that helps better move exosomes from the bulk solution to the functionalized chip surface while also providing more surface area and affinity reagent density to improve exosome capture.

The researchers fabricated the chips using a nano self-assembly process that allows them to be produced at low cost and without requiring any specialized fabrication facilities, Zeng said.

He and his colleagues combined this exosome isolation step with an in situ multiplexed protein immunoassay technology they presented in a 2016 study in Lab on a Chip, allowing them to go from sample to readout of protein marker measurements in a few hours, Zeng said. He noted that differential centrifugation combined with a conventional immunoassay could take as long as two days to return results.

"The goal is to combine the [exosome] isolation and downstream analysis in an integrated workflow so that you don't have to worry about the loss of sample or contamination," he said. "You can start with a very small amount of material and go all the way to the answer you want."

Ultimately, Zeng said he and his colleagues aim to develop an automated plug-and-play device where sample could be loaded on a cartridge and then inserted into the instrument which would isolate the exosomes and measure the levels of target proteins or other analytes of interest.

He added that such a system could be particularly useful in the clinical setting, allowing for high throughput and multiplexed measurements.

In the Nature Biomedical Engineering study the researchers used the system to analyze exosomes isolated from 20 ovarian cancer patients and 10 healthy controls, using 2 µl plasma samples and measuring levels of the proteins CD24, epithelial cell adhesion molecule (EpCAM), and folate receptor alpha protein (FRα). While CD24 and EpCAM were detected at high levels in both cases and controls, FRα was present at low levels in the ovarian cancer samples but was "almost undetectable" in the controls, the authors wrote, noting that the results indicate FRα could be a candidate marker for early detection of ovarian cancer.

The study is one of a number of recent efforts looking at exosomes and extracellular vesicles as a source of cancer protein markers. Last month, researchers at the University of Florida and China's National Center for Nanoscience and Technology (NCNST) demonstrated the ability of a thermophoretic isolation approach to distinguish between stage I cancer cases and healthy controls with 95 percent sensitivity and 100 percent specificity.

Purdue University spinout Tymora Analytical Operations is developing extracellular vesicle-based proteomic assays for applications including monitoring bladder cancer recurrence.

Last year, Exosome Dx, which has focused largely on nucleic acid analysis, made its move into protein detection with the first placements of its Shahky exosome analysis instrument.