Speed, accuracy, and affordability are of the essence in the detection of established and newly emerging pathogens to provide timely care, mitigate transmission, and help lower the financial burden on healthcare systems. Among them, those causing sexually transmitted diseases (STIs), including HIV/AIDS and hepatitis C, cause a major global burden on health care systems. In the U.S. alone, an estimated 68 million people suffer from an STI. Although most of the causative pathogens can be detected in clinical laboratories, cost, transport needs, and social barriers can significantly hinder diagnosis. Thus, there is an urgent need for reliable and affordable point-of-care (PoC) diagnostic tests that could be used at-home or in under-resourced clinical settings.
A multi-disciplinary research team at the Wyss Institute has developed a solution to this challenge by creating an enzyme-free pathogen detection platform that can target specific nucleic acid sequences and proteins. The technology leverages biomolecular nanotechnology to assemble nucleic acid-based “nanoseeds” that only form in the presence of specific pathogen-derived molecules. The nanoseeds then trigger downstream signal amplification via the rapid (within minutes) self-assembly of much larger, ribbon-like DNA structures that can easily be detected using low-cost readout methods. The method, called Crisscross Nanoseed Detection, is highly specific and ultrasensitive due to the suppression of non-specific interactions that cause unwanted false positive results.
Since the nanoseed formation that starts the assembly process can, in principle, be designed to respond to a large variety of target biomarkers, and the isothermal assembly can be carried out in a one-pot reaction in about 15 minutes without the addition of enzymes and further reagents, Crisscross Nanoseed Detection presents a highly adaptable, cost-effective, and robust platform. In addition, it can be multiplexed to allow the parallel detection of several biomarkers at a time. The nanoseed formation process can also detect biomarkers that are prone to mutation – such as viral structural and bacterial resistance molecules – that are often not detectablewith other methods.
The team completed a proof-of-concept for the conversion of ssDNA into strong dsDNA signals using Crisscross Nanoseed Detection, and is now developing assays for the detection of infectious disease-specific biomarkers including protein targets, as well as a simple POC diagnostic device that combines detection of pathogen-specific biomarkers with readouts compatible with a range of at-home and clinical settings. In addition to diagnostic applications, Crisscross Nanoseed Detection also has the potential to replace gold standard PCR-based assays as a research tool by eliminating the requirement for amplifying enzymes in the assembly process.