In April 2025, the journal Small Methods published an article presenting a new bioanalytical method for single-molecule detection. The approach combines DNA nanostructures with optical and molecular amplification to analyze target molecules without enzymatic reactions and without the usual partitioning of the sample. This principle opens the way to highly sensitive and simpler diagnostic tests than those offered by current solutions.
The method is designed for extremely sensitive detection at the level of individual DNA or protein molecules, for example important biomarkers that are often present in samples only at very low concentrations. This is crucial, for instance, for early cancer diagnostics from blood, where even a few target molecules can indicate the presence of an emerging tumour. Conventional methods can indeed detect target analytes down to the single-molecule level, but they require a complex enzyme-based workflow in an environment where the sample is partitioned into a large number of miniature reactors. These aspects increase technical complexity and thus cost. The new approach circumvents these obstacles.
At the heart of the method is the combination of optical amplification and a special tethered catalytic hairpin assembly (tCHA), which generates a detectable optical signal directly on the sensor surface. Once a target molecule is captured on the surface, it triggers a DNA cascade that forms a small cluster of fluorescent labels at the capture site, so that it appears as a bright spot under the microscope. The number of labels can be controlled by means of a nanoscale flexible polymer linker, which defines the perimeter within which the reaction proceeds and whose outcome can be read out.
The second key component is plasmon-enhanced fluorescence (PEF). On the sensor surface, an enhanced electromagnetic field is generated that locally increases the brightness of fluorescent molecules. This makes it possible to readily capture individual tCHA reactions as well-separated bright spots – and each spot represents one original molecule in the sample. Instead of complex evaluation of an average signal, it is thus sufficient to literally “count the bright spots”.
The authors point out, however, that this is only the beginning. For now, the fraction of captured molecules compared to the total amount present in the sample is still low, and the method will need further refinement. If these challenges can be addressed, the approach may become the basis for much faster, more sensitive and simpler diagnostic tests suitable even for routine clinical practice.
The published article is available here: https://onlinelibrary.wiley.com/doi/10.1002/smtd.202500037