Specific Nucleic Acid Detection Using a Nanoparticle Hybridization Assay

TL;DR

This paper presents a nanoparticle hybridization assay for detecting specific nucleic acid sequences, such as SARS-CoV-2 DNA, without the need for amplification, reducing errors associated with traditional methods.

Contribution

The study introduces a gold nanoparticle aggregation method that detects DNA targets via surface plasmon resonance shifts and nanoparticle tracking, avoiding amplification-related errors.

Findings

Detected SARS-CoV-2 DNA with a 6 nm plasmon shift

Observed decreased nanoparticle diffusion in target presence

Confirmed specificity with control DNA experiments

Abstract

Simple methods to detect biomolecules including specific nucleic acid sequences have received renewed attention since the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus pandemic. Notably, biomolecule detection that uses some form of signal amplification will have some form of amplification-related error, which in the polymerase chain reaction involves mispriming and subsequent signal amplification in the no template control, ultimately providing a limit of detection. To demonstrate the feasibility of the detection of a DNA target sequence without molecular or chemical signal amplification that avoids amplification errors, a gold nanoparticle aggregation assay was developed and tested. Two primers bracketing a 94 base pair target sequence from SARS-CoV-2 were conjugated to 10 nm diameter gold nanoparticles by the salt aging method, with conjugation and primer-target hybridization confirmed by agarose gel electrophoresis and nanospectrophotometry. Upon mixing of both conjugated nanoparticles with target, a surface plasmon resonance shift of 6 nm was observed, and lower electrophoretic mobility of a band containing both DNA fluorescence and gold absorption signals. This did not occur in the presence of a control DNA molecule of the same size and composition as the target but with a randomly scrambled base position. Nanoparticle tracking at 30 frames per second using a sensitive darkfield microscope revealed a lower measured diffusion coefficient of scattering objects in the target mixture than in the control mixture or with bare gold nanoparticles.