Multifunctional Nanoparticle Platform for Surface Accumulative Nucleic Acid Amplification and Rapid Electrochemical Detection: An Application to Pathogenic Coronavirus

Abstract

Of various molecular diagnostic assays, the real-time reverse transcription polymerase chain reaction is considered the gold standard for infection diagnosis, despite critical drawbacks that limit rapid detection and accessibility. To confront these issues, several nanoparticle-based molecular detection methods have been developed to a great extent, but still possess several challenges. In this study, a novel nucleic acid amplification method termed nanoparticle-based surface localized amplification (nSLAM) is paired with electrochemical detection (ECD) to develop a nucleic acid biosensor platform that overcomes these limitations. The system uses primer-functionalized Fe 3 O 4 −Au core−shell nanoparticles for nucleic acid amplification, which promotes the production of amplicons that accumulate on the nanoparticle surfaces, inducing significantly amplified currents during ECD that identify the presence of target genetic material. The platform, applying to the COVID-19 model, demonstrates an exceptional sensitivity of ∼1 copy/μL for 35 cycles of amplification, enabling the reduction of amplification cycles to 4 cycles (∼7 min runtime) using 1 fM complementary DNA. The nSLAM acts as an accelerator that actively promotes and participates in the nucleic acid amplification process through direct polymerization and binding of amplicons on the nanoparticle surfaces. This ultrasensitive fast-response system is a promising method for detecting emerging pathogens like the coronavirus and can be extended to detect a wider variety of biomolecules.

Photothermal Nanomaterials: A Powerful Light-to-Heat Converter

“…The amplification process with 40 cycles can be completed in 306 s for SARS-CoV-2 and in 264 s for λ-DNA, together with an amplification efficiency of 91%. In a recent PCR example, the nucleic acid amplification can be reduced to 4 cycles with a runtime of 7.5 min for the detection of SARS-CoV-2 . PCR-based tests have been the gold standard with a high analytical accuracy of 99% for COVID-19 confirmation. , Although the PCR technology has been already commercialized for rapid diagnosis, particularly for COVID-19, the investigation of nanomaterials with better photothermal performances is still an essential pathway for the development of ultrafast, robust, and portable PCR at the point-of-care level. , …”

Electrical Nanobiosensors for Nucleic Acid Based Diagnostics

“…A nanoparticle-based surface localized amplification method (nSLAM) was developed and combined with electrical biosensors for viral RNA detection by Soh and his co-workers (Figure 1d). 57 The biosensors can detect concentrations of RNA as low as ∼1 copy/μL for 35 cycles of amplification and 1 fM complementary DNA with amplification in 4 cycles (∼7 min runtime). In short, electrochemical nanobiosensors are employed for sensitive detection of nucleic acid due to the easy operation, low cost, rapid response, distinctive signal, and potential for multiplexed platforms.…”

“…The biosensor by using MB and Fc as electrochemical indicators could simultaneously detect 5.0 and 6.8 ag/μL of the S and ORF1ab genes, respectively, within an hour. A nanoparticle-based surface localized amplification method (nSLAM) was developed and combined with electrical biosensors for viral RNA detection by Soh and his co-workers (Figure d) . The biosensors can detect concentrations of RNA as low as ∼1 copy/μL for 35 cycles of amplification and 1 fM complementary DNA with amplification in 4 cycles (∼7 min runtime).…”

“…Moreover, the electrical mediators intercalated between biorecognition elements and target nucleic acid are often employed as signal indicators to enhance electrical response at the transduction interface . The electrical mediators, such as methylene blue (MB), , ferrocene (Fc), ruthenium (Ru), metal ion, and metallic nanoparticles, play a key role in enhancing electrical response of biosensors due to the electron transfer process occurring at the sensing interface. MB and Fc are popular electrochemical indicators, while metallic nanoparticles and metal ions can be used as electrical indicators for nucleic acid detection.…”

“…(d) Schematic diagram of the nSLAM electrochemical system for detection of SARS-CoV-2 RNA. Reproduced from ref . Copyright 2023 American Chemical Society.…”