MSACL 2023 Abstract

Ultra-Sensitive Molecular Detection of a Covid-19 Nucleotide Sequence Using an Enzyme-Linked Immuno-mass Spectrometric Assay (ELiMSA)

Nan Cheng, John Marshall
Department of Chemistry and Biology, Toronto Metropolitan University, Toronto, Canada

Jaimie Dufresne (Presenter) Toronto Metropolitan University

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: There is an urgent need for sensitive assays for both protein and molecular analytes for the drug development of biologics, pharmacokinetics/pharmacodynamics, minimal residual disease detection, basic research, and diagnostics of low abundance biomolecules. Enzyme-linked Immuno-Mass Spectrometric Assay (ELiMSA) is a technology that combines affinity-based capture with enzyme amplification and detection by liquid chromatography and mass spectrometry to sensitively detect both proteins and oligonucleotides.

OBJECTIVES: The primary objective of this study was to design a molecular ELIMSA for a synthetic Covid-19 oligonucleotide sequence and to determine the sensitivity, range and linearity of the assay. In addition, the assay was performed on PCR products and compared to detection by agarose gel electrophoresis and Gel Red fluorescence staining.

METHODS: A complimentary capture sequence to the COVID-19 target sequence was fixed to a reactive NOS 96 well plate. A biotinylated detector probe and synthetic target in concentrations ranging from 100fM to 10nM were heated, linearized, applied to the capture probes, and allowed to cool and anneal. After washing, the conjugated reporter enzyme alkaline phosphatase - streptavidin (APSA) was applied and bound the biotinylated detector probe. After washing, the substrate adenosine monophosphate was applied and converted to adenosine that is highly ionizable and may be sensitively detected by LC–ESI–MS. In a second experiment, an ELiMSA was performed on the PCR products of the synthetic Covid-19 sequence and compared to PCR with detection by electrophoresis and Gel Red fluorescence staining.

RESULTS: The standard curve of the synthetic Covid-19 sequence by ELiMSA showed the target was safely detected at 100fM, had a linear range over four orders of magnitude from 100fM to 10nM and an R2 of 0.9942. PCR with gel electrophoresis and Gel Red fluorescence staining was able to detect the synthetic Covid-19 sequence at 100 femtograms whereas PCR followed by ELiMSA was able to detect the target at 100 attograms.

CONCLUSION: Complimentary nucleotide sequences coupled to reporter enzymes can now be used to detect and quantify nucleotide sequences using ultra sensitive liquid chromatography and mass spectrometry. Similar to previous protein ELIMSA, the use of mass spectral detection results in greater sensitivity than UV/VIS or fluorescent assays.