Scalable Nanogap Sensors for Non-Redox Enzyme Assays

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 nanogap is independent of the in-plane geometry of the transducer, thus allowing the nanogap sensors to be highly scalable. Finally, when the DNA template concentration was constrained, the DNA polymerase assay exhibited different zero-order reaction kinetics for each type of base incorporation reaction, resolving the closely related nucleotide analogs.