Scalable Nanogap Sensors for Non-Redox Enzyme Assays
Abstract
Clinical diagnostic assays that monitor redox enzyme activity are widely used in small, low-cost readout devices for point-of-care monitoring (e.g., a glucometer); however, monitoring non-redox enzymes in real-time using compact electronic devices remains a challenge. We address this problem by using a highly scalable nanogap sensor array to observe electrochemical signals generated by a model non-redox enzyme system, the DNA polymerase-catalyzed incorporation of four modified, redox-tagged nucleotides. Using deoxynucleoside triphosphates (dNTPs) tagged with para-aminophenyl monophosphate (pAPP) to form pAP-deoxyribonucleoside tetra-phosphates (AP-dN4Ps), incorporation of the nucleotide analogs by DNA polymerase results in the release of redox inactive pAP-triphosphates (pAPP3) that are converted to redox active small molecules para-aminophenol (pAP) in the presence of phosphatase. In this work, cyclic enzymatic reactions that generated many copies of pAP at each base incorporation site of a DNA template in combination with the highly confined nature of the planar nanogap transducers ( z = 50 nm) produced electrochemical signals that were amplified up to 100,000×. We observed that the maximum signal level and amplification level were dependent on a combination of factors including the base structure of the incorporated nucleotide analogs, nanogap electrode materials, and electrode surface coating. In addition, electrochemical signal amplification by redox cycling in the…
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