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The transducer molecule is engineered to generate distinct channel blockade signals depending on its interaction with target molecules [2] . Statistical models are trained for each binding mode, bound and unbound, for example, by exposing the transducer molecule to zero or high (excess) concentrations of the target molecule. The transducer molecule is engineered so that these different binding states generate distinct signals with high resolution. Once the signals are characterized, the information can be used in a real‐time setting to determine if trace amounts of the target are present in a sample through a serial, high‐frequency sampling, and pattern recognition, process.

Thus, in Nanoscope applications of the SSA Protocol, due to the molecular dynamics of the captured transducer molecule, a unique reference signal with strongly stationary (or weakly, or approximately stationary) signal statistics is engineered to be generated during transducer blockade, analogous to a carrier signal in standard electrical engineering signal analysis. In these applications a signal is deemed “strongly” stationary if the EM/EVA projection (HMM method from ssss1) on the entire dataset of interest produces a discrete set of separable (non‐fuzzy domain) states. A signal is deemed “weakly” stationary if the EM/EVA projection can only produce a discrete set of states on subsegments (windowed sections) of the data sequence, but where state‐tracking is possible across windows (i.e. the non‐stationarity is sufficiently slow to track states – similar to the adiabatic criterion in statistical mechanics). A signal is approximately stationary, in a general sense, if it is sufficiently stationary to still benefit, to some extent, from the HMM‐based signal processing tools (that assume stationarity).