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Japanese Team Develops Nanowire Technology for Detecting Cancer Biomarkers in Urine

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NEW YORK (GenomeWeb) – A team of Japanese researchers has developed a new approach for detecting cancer biomarkers in urine.

Using a device constructed of nanowires, the scientists were able to net over a thousand types of microRNAs, which they then transferred to a commercial microarray platform for analysis.

Given the high yield of miRNA markers they were able to isolate, the researchers believe the approach could be commercialized and introduced into routine cancer testing. The nanowire device and methods were described in a new paper published in Science this month.

"Our developed device could allow us to collect thousands of urinary microRNAs from only 1 milliliter of urine," said Takao Yasui, an assistant professor in the department of biomolecular engineering at Nagoya University and corresponding author on the paper.

"In Japan, people collect about 10 milliliters of urine for medical checkups a year at least and a couple milliliters of urine are discarded," said Yasui. "If we could use the discarded urine for new microRNA-based medical checkups using the developed device, it could have a beneficial impact on healthcare."

Yasui developed the new device together with colleagues at Japan's Science and Technology Agency, National Cancer Center Research Institute, and National Institute of Advanced Industrial Science and Technology. Researchers at Kyushu University and Osaka University in Japan, King Mongkut's Institute of Technology Ladkrabang in Bangkok, Thailand, and Kaohsiung Medical University in Taiwan also participated.

The researchers set out to construct a device capable of catching extracellular vesicles transporting miRNAs. Other methods exist for obtaining miRNA-containing EVs, but the concentration of EVs is typically low, meaning that not enough markers are captured to make an effective diagnosis. According to the authors, alternative approaches include ultracentrifugation or differential centrifugation, immunoaffinity-based capture, and size exclusion chromatography. However, as they noted in the paper, it is unclear if the miRNAs absent from urine samples analyzed using these methods are actually present in the patients or if they are simply undetectable due to their low abundance.

The Japanese researchers' new approach relies on playing the forces of negatively charged EVs off of positively charged nanowires to extract the miRNAs from the urine of patients with a variety of diseases. In the case of the new Science study, they looked at samples from patients with pancreatic, liver, bladder, and prostate cancer, in addition to healthy subjects.

The work originated in the lab of Yoshinobu Baba, a professor of applied analytical chemistry in Nagoya University's Graduate School of Engineering, and Yasui's colleague. The effort to create the nanowire device, together with partners across Japan, some of whom have moved on to other appointments elsewhere in Asia, dates back years, Yasui noted.

In 2013, a research team involving Yasui published a paper in ACS Nano that described a nanowire array embedded in a microchannel on a fused silica substrate that could be used to manipulate and separate DNA molecules within seconds. Following the publication of the ACS Nano paper, Yasui and colleagues were encouraged to focus on crafting a device that could collect microRNAs, particularly in urine samples, he said. As a result, in 2015 he and his research team published a paper in Scientific Reports describing the use of 3D nanowire structures embedded in microchannels for the separation of small biomolecules, including DNA, protein, and RNA molecules.

Yasui said his team was moved to focus on analyzing miRNAs within urine EVs because they believed such a breakthrough could "strongly impact cancer-related research studies." He added that the goal of pursuing methods for employing miRNAs for early cancer diagnosis has been emphasized by various national and regional healthcare research authorities, including a project conducted by the Japan Agency for Medical Research and Development.

Other companies and research teams are certainly exploring the idea. Last month, Ymir Genomics announced a partnership with the Knight Cancer Institute at Oregon Health and Science University to develop urinary miRNA biomarkers that will help detect hepatocellular carcinoma in at-risk patient populations. And in August, researchers at the University of Pennsylvania announced a large, multicenter project involving multi-omic profiling of urine samples to search for markers related to kidney transplant outcomes in children and adults.

The result of the Japanese researchers' effort was a 3cm x 1cm device consisting of zinx oxide nanowires embedded in microfluidic, herringbone-structured poly (dimethylsiloxane) microchannels. According to Yasui, the scientists used a syringe pump to flow urine into the device, and then transferred the extracted samples on Toray's 3D-Gene microarray platform for miRNA analysis by pipetting.

Toray, based in Tokyo, has been providing the Japanese market with three-dimensional, expression profiling arrays fabricated using black resin for years. Yasui said the researchers used the chips because competitive offerings often have a large coefficient of variation (CV) value or provide unreliable data. Toray provided the researchers with their chip's CV values and "reliable data," Yasui said, underscoring their decision to use the platform over others.

In the future, Yasui noted, the researchers hope to develop an automated system for carrying out the entire assay.

Using the device, Yasui and colleagues were able to detect 1,106 different types of miRNAs in a single milliliter, versus an average yield of 200 to 400 miRNAs in total obtained using conventional methods, such as centrifugation.

Yasui attributed the success of the team's approach to the mechanical stability of the anchored nanowires during the buffer flow, as well as to the electrostatic collection of EVs onto the nanowires. Because the approach yielded potential cancer-related miRNAs in urine for not only urological malignancies such as prostate cancer and bladder cancer, but also non-urological ones such as liver cancer and pancreatic cancer, the researchers believe it will be widely applicable.

"Our new approach should allow researchers to move closer to the long-term goal of urinary miRNA-based early diagnoses and timely medical checkups for a variety of cancers," Yasui said.

To move toward that goal, the research team has begun to increase the number of experiments it runs on the device, as well as expanding the different types of cancers for which it tests. It has also begun commercialization discussions with a number of companies. He said that one existing partner intends to sell the device in the future, but he declined to name the firm citing a non-disclosure agreement.

As for when it might hit the market in Japan or elsewhere, Yasui that could happen in several years. "The expectation for the timescale is quite difficult at this stage because we need to verify many things," he said. "However, our motivation is that the device should be available within 10 years."

In the paper, the authors acknowledged a need to perform further trials for biomarker recognition. However, they argued that their approach will be a "powerful tool" not only for cancer researchers but for future medical applications.