To use biosensors for in vitro and in vivo applications, there is an undisputable need to improve their analytical sensitivity and specificity inside unprocessed host media. Toward this goal, we have developed a series of strategies founded on differential signal generation to both reduce the background and increase the signals measured in complex media. Inspired by electronic circuits, we have designed electrochemical and photoelectrochemical biosensors that translate biorecognition to multiple separate, but correlated signal transduction events, which are combined to improve the limit-of-detection, sensitivity, and specificity of biosensors. To implement differential biosensing for electrochemical biosensing, we have rationally designed inter-correlated electrodes that are integrated with RNA-cleaving DNAzymes for reagent-less, wash-free, and automated signal generation. This system has been used for culture-free and regent-less bacterial detection. Differential electrochemical signaling is implemented by translating the presence of bacterial targets to two correlated binding events, one on each electrode, and combining the data obtained from both electrodes for improving assay sensitivity and reliability. Evaluation of this new class of differential sensor using more than 40 urine samples has led to clinically-relevant sensitivity and specificity. The idea of differential signaling was also tested with photoelectrochemical biosensors that use optical excitation and electrochemical readout for sensing. These assays use electrodes that are programmed to perform signal-on and signal-off sensing on the same chip. Combining the signals generated from both events has led to limit-of-detections and sensitivities that surpass those of non-differential sensors in buffer and complex media. The data presented in our work demonstrate that differential sensing would be a useful tool for enhancing the performance metrics of biosensors, especially when operated in complex media, for use in in vitro and in vivo applications.

Differential signal generation in biosensing: A sensitivity enhancement strategy

ACS Fall 2021

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