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Johns Hopkins Researchers Developing Single-Molecule Microfluidic Platform to Detect Disease Markers

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NEW YORK (360Dx) – Researchers at Johns Hopkins University are developing a new test that uses single molecule imaging to identify protein targets of potential interest to clinicians at a sub-femtomolar limit of detection.

The technique, single-molecule augmented capture (SMAC), operates in a similar way to enzyme-linked immunosorbent assays (ELISAs) that are routinely applied by clinical labs to capture and detect molecules by measuring enzymatic reactions. However, the new device is a microfluidic platform that is 1,000 times more sensitive than a traditional ELISA, according to its lead developer Shih-Chin Wang, a researcher in the Johns Hopkins department of biophysics.

The ability to identify and characterize individual biomarker protein molecules in patient blood samples with that level of accuracy could improve diagnosis of diseases at an earlier stage when treatment is typically more effective, Wang said.

She noted that the platform can be developed to achieve multiplexing and high-throughput while characterizing circulating proteins of interest to clinicians.

Wang this week presented a proof-of-concept study describing the microfluidic platform at the 63rd Biophysical Society Annual Meeting in Baltimore, Maryland.

She said in an interview that the device has the potential to be developed for a range of clinical diagnostic applications, and that she has already used it as a liquid biopsy that separately detected prostate-specific antigen, mutant p53 proteins that are associated with cancer, and programmed death-ligand 1 (PD-L1) biomarkers.

Taekjip Ha, a biophysicist and professor of biophysics and biomedical engineering at Johns Hopkins University, said that the SMAC technique has potential for a broad range of clinical applications.

In addition to the indications for which Wang and her colleagues have already tested the technique, it could eventually be used in the diagnosis of conditions that require analysis of tissue samples, such as cancers, Ha said. Further, it could use urine samples to detect proteins indicative of kidney disease and blood samples to detect transplant failure or pathogens indicative of multiple medical conditions.

Ha is not involved in developing Wang's prototype, but he has assessed her work as a member of her thesis committee and provided advice.

Ha and his colleagues have developed a similar technology, a single-molecule pull-down assay that combines the principles of a conventional pull-down test — used to determine a physical interaction between two or more proteins — with single-molecule fluorescence microscopy.

They described its development and use in 2011 in Nature.

That assay enables direct visualization of individual cellular protein complexes, revealing the number and type of proteins in an in vivo complex. Researchers are using the assay for single-molecule biochemical studies, Ha said.

But, Ha noted in a 2016 review about advances in single-molecule pull-down methods and biological systems, published in Current Opinion in Structural Biology, that adequate passivation of microscope slides is required to prevent false positives resulting from non-specific adsorption from extracts.

"Detecting proteins at the single molecule level is not new, but [Wang] has pushed the approach forward by detecting proteins in a liquid biopsy format that has potential medical applications," Ha said. With single-molecule augmented capture, "they've solved many of the issues associated with properly passivating surfaces to prevent high fluorescence background noise in a microfluidic setting," he said. Further, they have figured out how to capture a high yield of proteins when low amounts are present in various biological sources, he added.

Wang said that with Chih-Ping Mao, a researcher and graduate student in the department of pathology at Johns Hopkins, she used the new technique to find mutant p53, a tumor-specific nuclear protein not previously detected in the blood because current tests cannot detect its extremely low concentrations.

They found mutant p53 in samples from patients with ovarian cancer that were not present in samples from patients without cancer. Using SMAC, they found PD-L1 on the surface of some cancer cells, which could lead to the detection of cancers with PD-L1 levels that are undetectable by standard blood tests, the researchers said.

The design of the microfluidic assay is not complicated, Wang said.

Using a small volume of blood — about 1 microliter ­— the researchers cycle a diluted blood sample multiple times through the microfluidic device and capture the protein targets of interest within the device prior to counting them and obtaining characteristics of the protein molecules by evaluating their fluorescent signals.

In detecting p53, for example, Wang and her colleagues used capture antibodies to adhere p53 proteins to a microfluidic chamber as blood is cycled through it. After the blood was washed away, a fluorescence antibody was attached to the protein allowing it to be detected through fluorescence.

By limiting the amount of space within the microfluidic proof-of-concept device, they have been able to reduce the volume of reagent needed to identify the analytes of interest. "Because we can concentrate lots of signal within a limited space, we can also see a strong signal associated with the single molecules," she said.

The group integrated a high density of biotin molecules on the microfluidic devices' cover glass, and linked biotin-labeled capture antibodies through use of streptavidin. That greatly improves the capture efficiency of target molecules in blood and increases the sensitivity of detecting analytes of interest, Wang said.

If the researchers are successful in commercializing their approach, they will face competition from other firms producing protein detection platforms and assays.

For example, Lexington, Massachusetts-based Quanterix has developed an approach for detecting thousands of single protein molecules simultaneously. According to the firm, its Simoa platform uses similar reagents to a conventional ELISA to measure proteins in a variety of different matrices at femtomolar concentrations. In the first step of the single-molecule immunoassay, antibody capture agents are attached to the surface of paramagnetic beads used to concentrate a dilute solution of molecules.

Wang said that the Johns Hopkins SMAC technology is different from other assays that employ single molecule detection because of the way it concentrates molecules and their fluorescence signals on the platform's surfaces.

She said that in a bid to commercialize the platform, which could be completed within about four years, she is considering launching a startup company and could look to license the technology.

The researchers also are seeking to further automate and simplify the platform so that it is easy to use, Wang said, adding that they will first look to target applications, such as prostate specific antigen and PD-L1 testing, for which there are established markets.

The researchers will also need to validate the clinical utility of the platform to obtain regulatory approvals and reimbursement from payors, and entering the most established markets should present fewer barriers to entry than trying to become a first mover in a new application area, Ha said.

The speed with which clinical trials can be completed will depend on the researchers being able to attract adequate investment, he added.