Electrical Detection Assay Based on Programmable Nucleic Acid Probe for Efficient Single-Nucleotide Polymorphism Identification

Thelarge-scale pandemic and fast evolution of severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2) variants have triggered an urgentneed for an efficient and sensitive on-site nucleic acid testing methodwith single-nucleotide polymorphism (SNP) identification capability.Here, we report a multiplexed electrical detection assay based ona paperclip-shaped nucleic acid probe (PNprobe) functionalized field-effecttransistor (FET) biosensor for highly sensitive and specific detectionand discrimination of SARS-CoV-2 variants. The three-stem structureof the PNprobe significantly amplifies the thermodynamic stabilitydifference between variant RNAs that differ in a single-nucleotidemutation. With the assistance of combinatorial FET detection channels,the assay realizes simultaneously the detection and identificationof key mutations of seven SARS-CoV-2 variants, including nucleotidesubstitutions and deletions at single-nucleotide resolution within15 min. For 70 simulated throat swab samples, the multiplexed electricaldetection assay shows an identification accuracy of 97.1% for thediscrimination of SARS-CoV-2 variants. Our designed multiplexed electricaldetection assay with SNP identification capability provides an efficienttool to achieve scalable pandemic screening.

Automated summary

The development of an efficient and sensitive on-site nucleic acid testing method with SNP identification capability is urgently needed due to the large-scale pandemic and fast evolution of SARS-CoV-2 variants. In this study, a multiplexed electrical detection assay based on a PNprobe functionalized FET biosensor was developed for highly sensitive and specific detection and discrimination of SARS-CoV-2 variants. The assay achieved simultaneous detection and identification of key mutations of seven SARS-CoV-2 variants within 15 minutes, with an identification accuracy of 97.1% for 70 simulated throat swab samples.