College of Science Associate Professor of Chemistry Xiyun “Richard” Guan received a $422,500 three-year renewal award from the National Institutes of Health to continue his project entitled “Label-Free Nanopore Biosensor for Rapid, Ultrasensitive, and Multiplex Detection of Protease Activities.”
Guan’s group is working on the development of biosensors for detecting environmental toxins and biomolecules using nanopore sensing. A nanopore is a nanoscale cavity or channel, sometimes created by a pore-forming protein. When a nanopore resides in a membrane, only single molecules can pass through the pore, and as a result the pore acts as a single-molecule detector. Over the last 15-20 years, these nanoscale pores have been used not only to analyze the sequence of DNA, but also to study various types of chemical interactions. They also have been used to investigate biomolecular structure, for example the folding and unfolding (denatured) of proteins, and for other applications.
As an emerging new technique, nanopore sensing has many advantages, including real-time detection. It also does not require the use of fluorescent dyes or radioactive materials; therefore, it is known as a label-free technique. Nanopore sensing has the ability to detect ultra-low concentrations of analytes (targeted species), for example trace amounts of biomolecules as found in human blood samples. Nanopore sensors can detect the concentration and identity of an analyte based on the ionic current modulations in a salt solution. When the molecules of interest, such as peptides, proteins, or DNA (with diameters smaller than the nanopore) pass through a single nanopore, they will produce current modulations for analysis.
One aspect of Guan’s current nanopore research is centered on pioneering a new, highly selective and sensitive technique to measure the activities of proteases, enzymes that break down proteins and peptides (short amino acid chains). Proteases occur naturally in all living organisms and play key roles in diverse biological processes, from cell regeneration and metastasis to cell deterioration and immune defense. Accordingly, alterations in the structure and expression patterns of proteases underlie many human pathological processes including cancer, arthritis, osteoporosis, inflammatory disorders, and neurodegenerative, cardiovascular and autoimmune diseases. Thus, proteases may serve as valuable diagnostic or prognostic markers for disease states, and are becoming increasingly important targets for drug discovery.
Nanopore measurement of the protease activity is achieved by real-time monitoring of the cleavage of a peptide. When there is no presence of a target protease, the peptide molecules pass through the nanopore giving one signal reading. By contrast, if the protease is present in a solution and, acting as a scissors, cuts the peptide molecules, the cleavage products produce entirely different current modulations. A comparison of the readings quantifies the protease’s concentration and enzymatic activity.
The real-time, label-free nanopore sensing technique discovered in this project should find useful application in the detection of proteases of medical or biological importance.
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110632. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.