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UPenn Targeting Ultrasensitive Protein Detection at the POC With Microbubbling Digital Assay


NEW YORK – Researchers at the University of Pennsylvania have developed a proof-of-principle platform that they anticipate could enable ultrasensitive protein detection and diagnosis of a broad range of medical conditions at the point of care.

During internal validation of the platform, the microbubbling digital assay achieved sensitivity levels that were 170 times higher than that of a test used in centralized laboratories, the Roche Elecsys Cobas Total PSA test, said Ping Wang, one of its developers and an associate professor of pathology and laboratory medicine at the Hospital of the University of Pennsylvania.

In a study published recently in Angewandte Chemie, the researchers reported using the platform for postprostatectomy surveillance of prostate-specific antigen (PSA), achieving a detection limit of .06 picograms per milliliter. They also used the system for early pregnancy detection using beta human chorionic gonadotropin (βhCG) as a biomarker, achieving a detection limit of 2.84 picograms per milliliter. 

Such limits of detection are the equivalent of those achieved by one of the most sensitive protein detection platforms used for research, the Quanterix Simoa single molecule system, Wang said, noting that the size and expense of the Simoa platform makes it suitable for use in laboratories but not at the point of care.

While point-of-care testing technology can provide convenience and rapid results, its analytical performance falls short of central laboratory tests in many instances, necessitating the need for higher performance POC assays for protein biomarker detection.

Massachusetts Institute of Technology researchers, for example, are developing an amplification-free, massively parallel microfluidic testing platform that could significantly boost protein biomarker test performance and improve molecular testing workflows.

Wang, who oversees point-of-care and core lab clinical testing at the Hospital of the University of Pennsylvania, said that she and her colleagues have been considering developing their own platform for some time. Although commercialization of the UPenn diagnostic test is still a long way off, the proof-of-principal platform that Wang and her colleagues have developed represents a first step that could lead to a test that improves on central lab tests on speed and convenience and competes with them on performance, according to Wang.

The platform employs an array of picoliter-sized microwells and platinum nanoparticle labels to enable the generation of microbubbles on a biochip. A connected smartphone enables recording of images and counting of protein molecules indicative of disease.

"The core discovery here is that we have a novel signaling and detection method for generating high-sensitivity assays," Wang said. "We converted an invisible signal generated by a single or a few protein molecules into a signal that's visible with a smart phone under a low magnification microscope, and that visible signal is a microbubble."

In operation, blood from a patient sample flows into picometer-size wells on a microchip. When target protein molecules are in the blood, they trigger sandwich reactions in the wells. A platinum nanoparticle, part of a single sandwich complex, catalyzes the generation of oxygen from a hydrogen peroxide reagent also present in the well. That leads to the production of microbubbles confined to discrete spaces on the chip.

When a bubble is present, one or more target molecules are present in a well, and when a bubble is not present, no target molecule is in the well. The presence of a bubble represented by '1' and the absence of a bubble represented by '0' is easily interpreted by a smartphone with its specialized app. That led to naming the device a microbubbling digital assay, Wang said.

"The idea of using microbubbles to connect the invisible nanoworld to the visible microworld is intriguing, and its use as an end-point in an assay opens up a new direction for ultrasensitive immunoassays," Larry Kricka, an emeritus professor of pathology and laboratory medicine at the University of Pennsylvania's Perleman School of Medicine, said in an interview.

Kricka, who has not been involved in developing the platform, said that he believes the use of microbubbles as the endpoint in an assay is an entirely new approach. The levels of sensitivity obtained for the assay with the two tested analytes, PSA and βhCG, is impressive, he said. However, it is also important to note that "There's still quite a bit of work to be done [with a view to achieving commercialization], especially for the point-of-care application."

The challenge with point of care may come in making the platform as simple as possible to operate, he said, adding that it will probably not be possible to make it as simple to use as a pregnancy test that can be purchased in the supermarket. However, a slight increase in complexity over the simple lateral flow format used in a pregnancy test "should not be seen as a negative" because the significant increase in sensitivity that the researchers have achieved is important in more specialized applications at the point of care, Kricka said.

Shana Kelley, who runs a laboratory at the University of Toronto focused on developing molecules and devices that enable biological activities to be measured and manipulated, echoed some of Kricka's comments.

"This is a very clever way to read out protein concentration with impressive limits of detection," said Kelley, who is not associated with the development of the UPenn platform. "It appears to have the appropriate performance characteristics for development as a point-of-care assay. However, the readout mechanism may be sensitive to vibration and temperature, so its robustness will need to be evaluated to know whether it is a good fit for testing outside of a well-controlled laboratory setting."

Point of care PSA

Wang and her colleagues at the Hospital of the University of Pennsylvania have had a particular interest in developing a point-of-care test for post-prostatectomy surveillance of prostate specific antigen. For years, the university hospital had been sending requests for post-prostatectomy surveillance testing to an external reference laboratory, but it brought testing in house about a year and half ago, she said.

Hospital urologists needing to monitor their patients for recurrence of prostate cancer were steadily increasing the volume of requests for testing. The group began using the US Food and Drug Administration-cleared Roche Elecsys Cobas Total PSA electrochemistry test for clinical diagnostics and the Quanterix Simoa platform for clinical research.

"At that time, we also raised the question whether we could develop a point-of-care testing device that would offer patients the capability of monitoring their PSA levels at home or in a physician's clinic," Wang said.

They knew that a point-of-care test could enable detecting recurrence earlier because patients could monitor the disease biomarker more frequently, eliminating the need for a patient to go to a clinic to obtain a blood draw and then wait a few days to receive results, Wang said.

"We wanted to see if there was a way to generate high sensitivity at the point of care on a small portable device and at best make the assay compatible with a smartphone, because almost everyone nowadays has a smartphone," she said.

The researchers believe that if they can commercialize the platform it would provide the basis for developing tests not only for prostate cancer recurrence and early pregnancy, but also for a range of other diagnostic applications that require sensitive detection of protein molecules.

The researchers are in discussions with various clinician groups to decide on the applications to target, Wang said. The platform can be adapted for nucleic acid-based detection, and the researchers are exploring that option. Testing for infectious diseases, cardiovascular disease, and cancer are among the applications on their radar, she said.

Next steps

The next steps for the technology's development include more extensive assay testing, clarification of its potential in central and point-of-care testing, and further work to refine its user-friendliness and propel it into the "disruptive" phase of technology development, Kricka said. The platform would need to be manufactured at an affordable price, which seems possible, he added.

Wang said that the biochip that the group has designed can be manufactured for less than a couple of dollars per chip and that its price is likely to fall with mass production. The overall cost of goods for the platform, including reagents, should be between $10 and $20, she said.

Many options are on the table for commercializing the test, including licensing and codevelopment with existing companies, and establishing a startup with support from the Penn Center for Innovation, Wang said.

The research plan is to expand the platform's capability to include "many more biomarkers," she said.

Shivani Passey, a spokesperson for Roche Diagnostics, said that on deadline the firm didn't have an opportunity to review the study in depth, but "if the new technology it describes is clinically validated and approved, [the UPenn platform] could join others on the horizon that hold great promise for improving patient care, which is important to all of us."