Design, Rationalization, and Automation of a Catalytic Sensing Mechanism for Homogeneous SERS Biosensors.

The current pandemic has shown that we need sensitive and deployable diagnostic technologies. Surface-enhanced Raman scattering (SERS) sensors can be an ideal solution for developing such advanced point-of-need (PON) diagnostic tests. Homogeneous (reagentless) SERS sensors work by directly responding to the target without any processing step, making them capable for simple one-pot assays, but their limitation is the achievable sensitivity, insufficient compared to what is needed for sensing of viral biomarkers. Noncovalent DNA catalysis mechanisms have been recently exploited for catalytic amplification in SERS assays. These advances used catalytic hairpin assembly (CHA) and other DNA self-assembly processes to develop sensing mechanisms with improved sensitivities. However, these mechanisms have not been used in OFF-to-ON homogeneous sensors, and they often target the same biomarker, likely due to the complexity of the mechanism design. There is still a strong need for a catalytic SERS sensor with a homogeneous mechanism and a rationalization of the catalytic sensing mechanism to translate this sensing strategy to different targets and applications. We developed and investigated a homogeneous SERS sensing mechanism that uses catalytic amplification based on DNA self-assembly. We systematically investigated the role of three domains in the fuel strand (internal loop, stem, and toehold), which drives the catalytic mechanism. The thermodynamic parameters determined in our studies were used to build an algorithm for automated design of catalytic sensors that we validated on target sequences associated with malaria and SARS-CoV-2 strains. With our mechanism, we were able to achieve an amplification level of 20-fold for conventional DNA and of 36-fold using locked nucleic acids (LNAs), with corresponding improvements observed in the sensor limit of detection (LOD). We also show a single-base sequence specificity for a sensor targeting a sequence associated with the omicron variant, tested against a delta variant target. This work on catalytic amplification of homogeneous SERS sensors has the potential to enable the use of this sensing modality in new applications, such as infectious disease surveillance, by improving the LOD while conserving the sensor's homogeneous character.