Ultrasensitive Electrochemical Detection of Mutated Viral RNAs with Single-Nucleotide Resolution Using a Nanoporous Electrode Array (NPEA)

Abstract

The detection of nucleic acids and their mutation derivatives is vital for biomedical science and applications. Although many nucleic acid biosensors have been developed, they often require pretreatment processes, such as target amplification and tagging probes to nucleic acids. Moreover, current biosensors typically cannot detect sequence-specific mutations in the targeted nucleic acids. To address the above problems, herein, we developed an electrochemical nanobiosensing system using a phenomenon comprising metal ion intercalation into the targeted mismatched double-stranded nucleic acids and a homogeneous Au nanoporous electrode array (Au NPEA) to obtain (i) sensitive detection of viral RNA without conventional tagging and amplifying processes, (ii) determination of viral mutation occurrence in a simple detection manner, and (iii) multiplexed detection of several RNA targets simultaneously. As a proof-of-concept demonstration, a SARS-CoV-2 viral RNA and its mutation derivative were used in this study. Our developed nanobiosensor exhibited highly sensitive detection of SARS-CoV-2 RNA (∼1 fM detection limit) without tagging and amplifying steps. In addition, a single point mutation of SARS-CoV-2 RNA was detected in a one-step analysis. Furthermore, multiplexed detection of several SARS-CoV-2 RNAs was successfully demonstrated using a single chip with four combinatorial NPEAs generated by a 3D printing technique. Collectively, our developed nanobiosensor provides a promising platform technology capable of detecting various nucleic acids and their mutation derivatives in highly sensitive, simple, and time-effective manners for point-of-care biosensing.

Needs, Challenges and Countermeasures of SARS-CoV-2 Surveillance in Cold-Chain Foods and Packaging to Prevent Possible COVID-19 Resurgence: A Perspective from Advanced Detections

“…Zhong et al integrated primer-encoded microsphere technology and dual fluorescence decoding strategy to establish an encodable, multiple microsphere phase amplification sensing platform, which could identify 10 key single-nucleotide variants in the receptor-binding region simultaneously for the detection of SARS-CoV-2 variants of concern [ 75 ]. Furthermore, an electrochemical nano biosensor system was proposed to detect SARS-CoV-2 RNA and its mutant derivatives, without labelling and amplification, which was combined with 3D printing technology to achieve multi-channel detection [ 76 ]. A light-up CRISPR/Cas13 method was used to detect SARS-CoV-2 and its mutated variants with the sensitivity as low as 82 copies [ 77 ].…”

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

“…The nucleic acid composition, length, and other factors lead to a unique melting curve profile. Metal ion indicators are also used in place of intercalating dyes for SARS-CoV-2 detection [ 88 ].…”

“…This signal is derived by transducing the biological event into an electrical signal, typically with electrodes employed for biomolecule immobilization and electron movement [ 112 , 113 ]. Electrochemical biosensors have been explored for multiplex detection of SARS-CoV-2 RNA by many research groups, being incorporated into different NAATs [ 73 , 114 ], CRISPR-based techniques [ 73 ], and amplification-free tests [ 80 , 86 , 88 ], similar to optical readout assays. However, electrochemical biosensors do not require optical instrumentation.…”

“…Many research groups have shown that electrochemical assays have the potential to detect nucleic acid targets without any amplification or in complex clinical samples without purification, regardless of the amplification techniques available to improve sensitivity [ 80 , 86 , 88 ]. Li et al demonstrated an ultrasensitive biosensing technique utilizing the trans-cleavage mechanism of CRISPR Cas13a and the graphene field-effect transistor (gFET).…”

“…Moreover, Yoon et al developed the Nanoporous Electrode Array (NPEA) that could enhance the redox signal further due to its ability to provide enhanced electron transfer. Sensing probes can be scattered across the NPEA, hybridizing with SARS-CoV-2 RNA of the complementary sequence, immobilizing the nucleic acid sequences of significance, and allowing the metal ions to intercalate [ 88 ].…”