Researchers isolate intact tumor microRNAs using antibody-conjugated nanowires

A research team in Japan has developed an efficient and minimally invasive cancer detection device that uses high-performance zinc oxide nanowires to selectively capture extracellular vesicles (EVs) from bodily fluids.

Using this device, researchers successfully captured cancer-related EVs from the blood serum of ovarian cancer patients. The EVs' surface membrane proteins and microRNAs remained intact, indicating the potential for sensitive disease analysis. These findings were published in the journal Device.

A liquid biopsy is a procedure that collects disease-related information from bodily fluids, such as blood and urine. Unlike traditional tissue biopsies, it places less physical burden on patients.

EVs are nanoscale vesicles that carry diverse molecular contents such as microRNA and messenger RNA, and display membrane proteins that indicate their cell of origin. EVs reflect disease states and serve as promising diagnostic indicators for liquid biopsy.

Accurate and efficient isolation of EVs from complex biological fluids is essential for identifying disease-associated molecules, but conventional techniques are time-consuming, require large sample volumes, and lack specificity.

A team led by Takao Yasui, a professor at Nagoya University's Graduate School of Engineering, previously achieved efficient EV capture using zinc oxide nanowires they developed.

They are now collaborating with Yasuhide Inokuma, a professor at Hokkaido University, and researchers from the Institute of Science Tokyo, Kyoto University, and the National Institutes for Quantum Science and Technology to develop antibody-conjugated nanowire technology for the selective capture of cancer-derived EVs.

The initial challenge was attaching antibodies to nanowires. Conventional adhesives bind both target and non-specific proteins and require lengthy attachment times.

The team used the synthetic polymer polyketone to create six N-hydroxysuccinimide-functionalized polyketone (pKNHS) variants with different chain lengths. Of these, pKNHS 4.2 showed optimal stability for adsorption onto zinc oxide nanowires and effective antibody immobilization, enabling single-step antibody modification.

Evaluation of the new technology in cultured cell experiments

Researchers evaluated the capture efficiency of antibody-conjugated nanowires for cultured breast cancer cells using pKNHS 4.2. While antibody-free nanowires captured about 65% of CD9-positive EVs, CD9 antibody-conjugated nanowires achieved 90% efficiency. These results demonstrate the technology's effectiveness in selectively recovering target molecules.

Further experiments showed that nanowires modified with antibodies for ovarian cancer markers CLDN3, FOLR1, and TROP2 enabled the selective recovery of EVs from ovarian cancer cells.

Analysis of serum from cancer and non-cancer patients

Researchers isolated EVs using CLDN3, FOLR1, and TROP2 antibody-modified nanowires from the serum of six patients with high-grade serous ovarian carcinoma, an aggressive ovarian cancer subtype, and six non-cancer individuals. Analysis of microRNAs in EVs revealed distinct profiles between the patient and non-cancer groups.

When comparing microRNAs in EVs captured with the three antibodies, researchers identified 126 microRNAs common to all, indicating signals shared by ovarian cancer. They also found microRNAs unique to each antibody: 40 for CLDN3, 37 for FOLR1, and 45 for TROP2. These findings suggest that EVs with different membrane proteins have distinct microRNA profiles.

Significance and future perspectives

"In this study, we developed a nanowire microfluidic device capable of selectively capturing cancer-associated EVs with high efficiency, while suppressing nonspecific adsorption through simple chemical modification," said Yasui, a corresponding author of the study.

We also demonstrated that this approach maintains both EV membrane proteins and internal microRNAs intact, showing strong potential for highly sensitive analysis of cancer states."

Takao Yasui, Nagoya University

Kunanon Chattrairat, an assistant professor and corresponding author, said: "We plan to compare and evaluate this technology against existing clinical methods and expand its application to capture more specific EV subpopulations. In the long run, we aim to apply this technology to non-invasive liquid biopsies and early diagnosis across a variety of cancer types."