A-382 Rapid, amplification-free DNA detection from undiluted plasma on dry-printed CNT electrodes via enzymatic chronoamperometry readout

Abstract

Point-of-care (PoC) facilities need nucleic-acid assays that can shorten turnaround time and simplify workflow while maintaining analytical performance in realistic matrices. We describe a materials-driven electrochemical platform based on floating catalyst chemical vapor deposition-synthesized continuous carbon nanotube network (FCCVD CNT) electrodes (Canatu dry-printed electrodes, DPEs) for amplification-free, sequence-specific DNA detection directly from undiluted human plasma. The aim is to showcase the CNT transducer platform as an enabling component for future lab-developed tests and IVD development in complex media; circulating tumor DNA (ctDNA) is used here as a representative application, though the approach generalizes to other nucleic acid biomarkers (such as cell-free DNA, cfDNA) via probe design. Mass manufacturable Canatu FCCVD CNT electrodes with integrated three-electrode setup were fabricated by sheet-to-sheet dry-printing, and functionalized with sequence-specific capture probes that were utilized in a multi-step assay format, where the hybridization of the target DNA strand (cf/ctDNA) in undiluted human plasma is quantified with a horseradish peroxidase (HRP) reporter and a 3,3',5,5'-Tetramethylbenzidine (TMB) substrate using a chronoamperometric readout. The DNA assay was designed for three different strands, corresponding to three individual mutations observed in breast cancer monitoring (Exon-19, T790M and L858R). Specificity of the assay was evaluated with our previous generation ctDNA biosensor using the same probes, using cross-target and wild-type control yielding negligible signals, indicating single-nucleotide selectivity for the target. The assay achieved a limit of detection in the magnitude of 100fM (Exon-19 stack), an analytical range of 0.1-10pM and time to result of ca. 1 hour from sample application to readout. These analytical characteristics demonstrate that the enzyme-assisted electrochemical readout on Canatu FCCVD CNT electrodes can access sub-picomolar sensitivity in complex matrices without nucleic-acid amplification while preserving a short, laboratory-friendly workflow. The significantly low negative control / blank signal from the chronoamperometric readout of the assay-functionalized CNT electrodes incubated in undiluted plasma suggested extremely good non-fouling behavior and platform specificity. The bare transducer quality and variation of the platform was confirmed using direct electrochemistry-based quality control procedure, resulting in <5% CV in sheet-to-sheet measurements. An HRP-TMB chronoamperometric assay implemented on Canatu dry-printed CNT electrodes delivers amplification-free DNA detection directly in undiluted human plasma with sub-picomolar sensitivity and less than an hour turnaround-time. The combination of (i) scalable and stable production, (ii) low background, and (iii) sub-picomolar sensitivity highlights the suitability of these CNT electrodes as an enabling material platform for rapid, targeted DNA biomarkers in the clinical laboratory setting, possibly at PoC. Ongoing and future work will extend this platform to other assay formats to highlight its versatility in electrochemical detection in complex matrices.