Tests that look for biomarkers could help physicians diagnose disease before symptoms present themselves. But it’s difficult to find the right protein, metabolite, or other molecule in the body that signals the start of a disease. Now researchers have described a sensitive new assay that generates its own synthetic biomarkers to detect harmful blood clots in mice (J. Am. Chem. Soc. 2014, DOI: 10.1021/ja505676h).
Unfortunately, natural biomarkers that are both specific to a disease and easy to detect are relatively rare. So Sangeeta N. Bhatia of Massachusetts Institute of Technology and David R. Walt of Tufts University decided to develop an assay that caused diseased cells or tissues to produce a synthetic molecule the scientists could easily find.
To create the assay, the scientists combined technologies their two groups had been working on: Bhatia’s group had synthesized worm-shaped iron oxide nanoparticles that they decorated with molecules to home in on diseased cells, while Walt’s team had developed single-molecule arrays (SiMoA) that allowed them to detect extremely low quantities of biological compounds of interest. For the new assay, the two teams decorated the nanoworms with a peptide that can be cleaved by thrombin, an enzyme activated at high levels in clotting disorders. When the nanoparticles bump into active thrombin in a mouse with clotting problems, the enzymes clip off a labeled peptide that the mice then excrete in their urine.
The researchers then treat the urine with an antibody-labeled microbead and a reporter protein conjugated to streptavidin. The antibodies on the beads bind to one end of the excreted peptide, while the streptavidin conjugate binds to the other end. So in the presence of the synthetic biomarker, the microbeads end up decorated with the reporter protein. The scientists then distribute the beads in an array of microwells only large enough for one bead to fit in each. They treat the wells with a compound that glows when cleaved by the reporter protein and then count glowing wells. The team can then relate this number to the degree of clotting in the mouse.
The test is exceptionally sensitive at detecting the synthetic biomarkers, Walt says. SiMoA can detect concentrations as low as 100 femtomolars of a peptide in urine compared with 100 pM for a traditional enzyme-linked immunosorbent assay (ELISA). By adding a large excess of the antibody-labeled beads to the urine sample, the researchers can be confident that each bead contains only one copy of the synthetic biomarker. As a result, they can measure thrombin activity easily at high and low levels.
Ease of detection at low peptide concentrations also could speed clinical testing, Walt says. Dosing patients with nanoparticles or other synthetic molecules typically triggers strict regulatory hurdles because of concerns about toxicity. But because this test involves microdoses of the nanoparticles—less than 100 µg—it may face fewer regulatory challenges.
The team hopes to apply the strategy to other diseases, especially cancer, Walt says. “It’s often difficult to find a biomarker that’s specific to a particular cancer and is present in the bloodstream at an early enough stage to detect it,” he says. “This technology now removes some of those limitations.”
The use of synthetic biomarkers is “a welcome addition to other techniques” for disease diagnostics, says Eleftherios P. Diamandis of Mount Sinai Hospital, in Toronto. The success of the test still depends on the sensitivity and specificity of the enzymes connected to the disease states—thrombin, in this case. So the researchers will need to do more testing to determine how well this strategy compares to existing ones, he says.