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Yale-Led Team Develops Nanosensors for Detecting Biomarkers in Whole Blood

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This story originally ran on Jan. 6.

By Tony Fong

A team of researchers led by Yale scientists has created a nanosensor device that they said can detect cancer protein biomarkers in whole blood for the first time, representing a "paradigm shift in label-free electronic sensing of biomolecules."

The researchers have launched a company, Altura Diagnostics, to explore opportunities to commercialize the technology, but the firm is still in the very early stages of development.

In a proof-of-concept study describing their work and the technology published Dec. 13 in Nature Nanotechnology, the authors said that although label-free nanosensors, which were first developed in 2001, can detect biomarkers and "provide point-of-care diagnosis that is low cost, rapid, specific, and sensitive," the complexity of blood has limited the clinical use of such sensors.

The researchers, however, have developed a "capture-and-release" technology that targets specific biomarkers for purification and then detection. In addition to being inexpensive, their technology is quick. In their study, they were able to detect two commonly used biomarkers in whole blood in about 20 minutes, compared to the span of several days that would be required with current technology.

It also requires smaller sample sizes than current technology: 10 microliters compared to milliliters with existing methods.

The new technology comprises two parts: a filtration device that captures the biomarker of interest, and electronic nanosensors that then detect them. The filter, a microfluidic purification chip, is key to the technology.

The chip is avidin-functionalized and treated with antibodies against specific biomarkers "conjugated to biotinylated photocleavable crosslinkers containing a specific 19-mer DNA sequence," the authors wrote.

After the biomarkers are bound to the antibodies, the remaining blood is washed away. The purified solution is then irradiated with ultraviolet light, which cleaves the antibody attachment group. The resulting solution can then be analyzed by nanosensors for biomarker detection.

The researchers demonstrated the utility of their technology by testing its ability to detect prostate-specific antigen and carbohydrate antigen 15.3, biomarkers for prostate cancer and breast cancer, respectively.

"It was actually surprising, once we came up with the idea, how easy it was," Mark Reed, a professor of engineering and applied science at Yale, and a corresponding author on the study, told ProteoMonitor. "The point is that it's pretty darn specific. The capture and release is a very nice way to capture what you want very specifically and to filtrate out the rest of the stuff that you don't want."

An important element is that the purification chip comprises two antibodies, resulting in a "significant improvement in selectivity over previous label-free nanosensing schemes," Reed and his co-authors wrote. This aspect increases the specificity of their technology to that of traditional sandwich assay techniques, they added.

Indeed, they compare the sensitivity of their technology as equivalent to being able to detect a grain of salt in a large swimming pool.

"This new method is much more precise in reading out concentrations and is much less dependent on the individual operator's interpretation," which can compromise current technologies, Tarek Fahmy, an associate professor of biomedical and chemical engineering at Yale, said in a statement.

However, the technology is dependent on antibody quality, and some potential problems and issues may exist. In particular, the quality of commercially available antibodies is questionable, and antibodies don't exist for many proteins. However, Reed said neither limitation posed a problem in the published study. He added that he doesn't believe it will limit the technology moving forward.

"Find me a protein that you want to look at and there's a very good chance that we can find a very good specific antibody for it," he said. Similarly, he said that antibody quality did not present problems in their research.

"For what we were looking at, we got very good signal and no false positives," he said. "We were able to get on the order of less than 10 percent accuracy of measuring these, which is pretty darn good."

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Reed added that because the technology allows for the integration of "many" nanosensors to simultaneously detect for the same biomarkers, it gives "very good signal-to-noise, so the integration aspect is a tremendous advance to be able to eliminate false positives."

In their research, the authors used about eight nanosensors, but in commercial use, Reed said that the number can be increased to "as large as you want to be able to get down to however low a false-positive that you'd like to achieve."

The technology can also be used detect multiple different biomarkers.

Their study looked at two biomarkers, but in ongoing research Reed and his co-authors would like to investigate a "whole panel" of biomarkers on one chip. That would require additional development of the electronics and interfaces to increase the plexing capabilities.

"Once you're able to do the multiplexing and do the various functionalization fluidics to be able to functionalize each of the nanowires," each chip would be able to detect tens or even hundreds of biomarkers, Reed said. "It's not very complicated to do that."

He estimated that the cost for manufacturing the chips was less than a dollar per chip and could be potentially "much less. … Probably the thing that will determine the cost will be how many simultaneous measurements you want to do because you have to do the functionalization chemistry, and those are cost issues," he said.

Commercialization Plans

The Yale researchers recently launched Altura Diagnostics to commercialize the technology. Joe Straight, a biotech veteran, is the CEO. Before joining Altura, he was the CEO of Synscia, which developed therapeutics for eye-related diseases. He was also the co-founder and CEO of Verax Biomedical, a developer of tests for detecting bacterial contaminants in blood components and tissues.

The firm is currently in fundraising mode, Reed said.

Before the technology is ready to hit the market though, a number of capabilities would need to be built in, especially if it is to be used as a point-of-care diagnostic. In addition to increasing the plexing capabilities, the researchers have not looked into whether the technology is capable of detecting modified forms of proteins and differentiating them from unmodified forms of the same proteins. Another area of further research is how other types of binding agents, such as aptamers, may work with the technology. And the researchers plan to test it with other biomarkers, Reed said, though he declined to identify them.

A main priority is identifying an immediate use for the technology, such as an unmet need or an application for which the approach would offer significant improvements. The company is currently concentrating on how best to exploit its speed, low sample-size requirements, and extreme sensitivity.

"We're in the process of trying to figure out what the best application is," Reed said.

While the technology was used for the detection of biomarkers in the Nature Nanotechnology article, Reed said that it also has use for discovery work. Many questions exist about the kinetics of the binding of the various antibody-protein pairs, "and we can access those," he said. One aspect of the researchers' work was to show that the kinetics and the binding corresponded "to give very well quantified levels of detection to determine what the concentrations were," he said.

The technology is "a real scientific tool because you get some real interesting kinetics out of understanding what we're doing." Mass specs, by comparison, are "not good for this intermediate range of proteins that you want to look at. This is true unlabeled detection so you're actually looking at the binding of something onto the antibody."

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