A Homogeneous Multicomponent Nucleic Acid Enzyme Assay for Universal Nucleic Acid Detection by Single-Particle Inductively Coupled Plasma Mass Spectrometry

Abstract

Single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) has great potential for sensitive analysis of nucleic acids; however, it usually requires separation of target-induced nanoparticle reporters, and the sequence of probes on nanoparticle reporters has to be tuned for each target accordingly. Here, we developed a homogeneous multicomponent nucleic acid enzyme (MNAzyme) assay for universal nucleic acid detection. The two components of MNAzyme contain target recognition sites, substrate binding sites, and a catalytic core. Only in the presence of a specific nucleic acid target, the MNAzyme will assemble to trigger its nucleic acid enzyme activity and cleave its substrate (Linker DNA). The Linker DNA could link gold nanoparticle (AuNP) probes to form a larger assembled particle, while the cleavage of Linker DNA will disturb the linkage between probes, inducing a smaller assembled particle. The assembled particles with different sizes could be differentiated and sensitively detected in SP-ICP-MS, which also enables the tolerance of a complex matrix. By simply altering the sequences of the target recognition sites in MNAzyme, we applied the assay for two types of nucleic acids (long strand DNA and short strand RNA), malaria DNA and miRNA-10b. With increasing the target concentration, the signal intensity of each assembled particle decreases, but the frequency of assembled particle pulse increases. Both targets could be quantitatively detected from 0.1 to 25 pmol L–1 with high specificity in serum samples. The developed MNAzyme–SP-ICP-MS assay possesses simple operation in a homogeneous reaction, easy tunability for multiple types of nucleic acid targets, and good compatibility with clinic samples.

A homogeneous nucleic acid assay for simultaneous detection of SARS-CoV-2 and influenza A (H3N2) by single-particle inductively coupled plasma mass spectrometry

“…The LOQs and linear range of this method were comparable with those SP-ICP-MS methods. Specifically, since no nucleic acid amplification method was introduced in this method, the LOQs of this method were at the same level as DNA detection assay based on SP-ICP-MS methods [ 20 , 23 ], but higher than that of other SP-ICP-MS methods [ 26 , 27 ] which introduced nucleic acid amplification. Compared with electrochemical methods, the LOQs of the developed method was lower than the method reported in reference [ 31 , 32 ], but slightly higher than the work with nucleic acid amplification involved [ 33 ].…”

“…The preparation of Au NPs and Ag NPs probes was based on our previous work [ 26 ] with slight modifications. Briefly, 10 μL of ammonium acetate buffer solution (500 mmol L -1 with 10 mg mL -1 Tris(2-carboxyethyl)phosphine (TCEP), pH 6.8) was added into 10 μL of SH-CoV-1 and SH-CoV-2 solutions (100 μmol L -1 ), respectively.…”

“…Later on, they [ 25 ] extended the SP-ICP-MS homogeneous immunoassay to simultaneous analysis of multiple cancer related biomarkers. Yin et al [ 26 ] established a universal detection method for different kinds of nucleic acids (such as DNA, RNA, and microRNA) by combining SP-ICP-MS homogeneous detection with MNAzyme amplification. Li et al [ 27 ] used target-induced hybridization chain reaction (HCR) to generate a corresponding number of Au NP aggregates for SP-ICP-MS detection, realizing analysis of DNA at fM level.…”

Atomic spectrometry update: review of advances in the analysis of clinical and biological materials, foods and beverages

“…Disease-related microRNAs (miRNAs) are an emerging class of biomarkers for which various ICP-MS based detection methodologies have been described using different amplication strategies and elemental tags. [186][187][188][189] Sandwich hybridization on streptavidin coated microtitre plates was used to detect three target miRNAs with three NP probes (Ag, Au and Pt), quantied by ICP-MS. 186 Improved selectivity, sensitivity and dynamic range was achieved in a multiple-metal-NP-tagging method with hybridization chain reaction amplication. 187 Here, three metal NP probes (Ag, Au and Pd) interacted with one target miRNA and ICP-MS response patterns were interrogated to enhance selectivity compared with other miRNA detection methods.…”

“…Kang et al 188 presented a multiplexed assay using three MNAzymes (multicomponent nucleic acid enzyme) paired with three target miRNAs and three lanthanide tags with MB-DNA probes. In a different MNAzyme-based approach, cleavage of linker DNA by MNAzyme in the presence of target miRNA resulted in smaller AuNP aggregate size detected by spICP-MS. 189 In addition to solution-based immunoassay platforms, elemental tagging with LA-ICP-MS and LIBS has demonstrated potential for quantitative biomarker imaging in different sample types. 96,155,157,190 Quantication and localization of dystrophin in muscle tissue and amyloid beta in mouse brain tissue were achieved using Gd-labelled Abs and AuNP conjugates, respectively, with LA-ICP-MS detection.…”