Improved nucleic acid sensing, exemplified using pure target DNA sequences, by MO capture probes compared to DNA capture probes, as revealed from PO2-, PO3- and nucleobase mass peaks

Detection of gene mutations is central to molecular diagnostics. The conventional detection methods that employ DNA capture probes are widely used, yet they may suffer from non-specific signals, difficulties in single nucleobase mismatch discrimination, and nuclease-induced degradation of the DNA capture probe. Herein, a strategy for detection of nucleic acid sequences using the nuclease-resistant morpholino oligonucleotide (MO) capture probe is reported. Since, spherical NPs offer increased surface area, thus increasing the total number of capture probe available for target recognition, MO probes were immobilized onto spherical NPs. Time-of-flight secondary ion mass spectrometry (TOF-SIMS), a sensitive surface analysis method, was employed to assess the status of target binding. Target binding was confirmed by recognizing the differences in chemical structures of MO and DNA, i.e., high-intensity peaks for PO2 - and PO3 - were observed when fully complementary target DNA was introduced to the sensor surface, but not for the non-complementary DNA and MO-only surface. The MO-based detection offered significantly improved performance than the DNA-based system in discriminating complementary and non-complementary DNA sequences, including the sequences having the most prevalent driver mutations in exon 19 and exon 21, from non-small cell lung cancer (NSCLC) patients, with limit of detection of 1 pM, and machine learning validation. Since MO is devoid of PO3 -, the MO capture probe-based detection of DNA sequences was unambiguous.