Abstract
Majority of protocols for quantitative analysis of biomarkers (including nucleic acids) require calibrations and target standards. In this work, we developed a principle for quantitative analysis that eliminates the need for a standard of a target molecule. The approach is based on stoichiometric reporting. While stoichiometry is a simple and robust analytical platform, its utility toward the analysis of biomolecules is very limited due to the lack of general methodologies for detecting the equivalence point. In this work, we engineer a new target/probe-binding model that enables detecting the equivalence point while maintaining an appropriate level of specificity. We establish the probe design principles through theoretical simulations and experimental confirmation. Further, we demonstrate the utility of the stoichiometric analysis via a proof-of-concept system based on oligonucleotide hybridization. Overall, the approach that requires neither standard nor calibration yields quantitative results with an adequate accuracy (> 90-110%) and a high specificity. The principles established in our work are very general and can extend beyond oligonucleotide targets toward quantitative analysis of many other biomolecules such as antibodies and proteins.