A high catalytic efficiency associated with a robust chemical structure are among the ultimate goals when developing new biocatalytic systems for biosensing applications. To get ever closer to these goals, we report here on a combination of metal-organic framework (MOF)-based nanozymes and a G-quadruplex (G4)-based catalytic system known as G4-DNAzyme. This approach aims at combining the advantages of both partners (chiefly, the robustness of the former and the modularity of the latter). To this end, we used MIL-53(Fe) MOF and linked it covalently to a G4-forming sequence (F3TC), itself covalently linked to its cofactor hemin. The resulting complex (referred to as MIL-53(Fe)/G4-hemin) exhibited exquisite peroxidase-mimicking oxidation activity and an excellent robustness (being stored in water for weeks). These properties were exploited to devise a new biosensing system based on a cascade of reactions catalyzed by the nanozyme (ABTS oxidation) and an enzyme, the alkaline phosphatase (or ALP, ascorbic acid 2-phosphate dephosphorylation). The product of the latter poisoning the former, we thus designed a biosensor for ALP (a marker of bone diseases and cancers), with a very low limit of detection (LOD, 0.02 U L-1), which is operative in human plasma samples.

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Efficient Biocatalytic System for Biosensing by Combining Metal-Organic Framework (MOF)-Based Nanozymes and G-Quadruplex (G4)-DNAzymes.

Rolling circle amplification (RCA) is a simple and isothermal DNA amplification technique that is used to generate thousands of repeating DNA sequences using circular templates under the catalysis of DNA polymerase. 

Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications

Functional nucleic acids as modular components against SARS-CoV-2: From diagnosis to therapeutics

This review focuses on critical scientific barriers that the field of point-of-care (POC) testing of SARS-CoV-2 is facing and possible solutions to overcome these barriers using functional nucleic acid (FNA)-based technology. Beyond the summary of recent advances in FNA-based sensors for COVID-19 diagnostics, our goal is to outline how FNA might serve to overcome the scientific barriers that currently available diagnostic approaches are suffering. The first introductory section on the operationalization of the COVID-19 pandemic in historical view and its clinical features contextualizes essential SARS-CoV-2-specific biomarkers. The second part highlights three major scientific barriers for POC COVID-19 diagnosis, that is, the lack of a general method for (1) designing receptors of SARS-CoV-2 variants; (2) improving sensitivity to overcome false negatives; and (3) signal readout in resource-limited settings. The subsequent part provides fundamental insights into FNA and technical tricks to successfully achieve effective COVID-19 diagnosis by using in vitro selection of FNA to overcome receptor design barriers, combining FNA with multiple DNA signal amplification strategies to improve sensitivity, and interfacing FNA with portable analyzers to overcome signal readout barriers. This review concludes with an overview of further opportunities and emerging applications for FNA-based sensors against COVID-19. 

https://link.springer.com/content/pdf/10.1007/s00604-022-05242-4.pdf

Recent advances of functional nucleic acid-based sensors for point-of-care detection of SARS-CoV-2

Dengue caused by dengue virus (DENV) is a highly pathogenic viral disease. Early detection of pathogens is very important for patient treatment and pathogen control. This work designed a dual signal amplification strategy for sensitive fluorescent detection of DENV RNA. The dual signal amplification was achieved by using a target-triggered hybridization chain reaction (HCR) to connect multiplex proximity ligation activated CRISPR-Cas12a (pCRISPR-Cas12a). The HCR was triggered by the recognition of DENV RNA to a hairpin probe (H3), which led to a product to initiate the continuous hybridization of a couple of hairpins. Here one of the hairpins (H2) was designed to contain two domains ( a and b ), and domains a and b from two H2 formed a proximity ligation sequence (PLS) to connect the multiplex pCRISPR-Cas12a onto the HCR product, in which domain a activated the Cas12a, and domain b enhanced the enzymatic activity by about 5 times. Upon the enzymatic cleavage of single-stranded DNA labeled with a fluorophore and quencher at each end (ssDNA-FQ), a strong fluorescent signal was produced for DENV RNA analysis, which showed a linear range from 1 pM to 10 nM and a detection limit of 51 fM. The excellent performance of the proposed homogenous fluorescent assay with high sensitivity and superior accuracy endowed this strategy broad application prospects in clinical disease diagnosis. • A universal fluorescence biosensing strategy was developed for highly sensitive detection of Dengue virus RNA. • The parallel connection of proximity sequence enhanced CRISPR-Cas12a was achieved through target-triggered HCR. • This proposed homogenous detection method showed a wide detection range and 50 fM-level detection limit. • This proposed strategy possesses good accuracy, high precision, and good extendability for target analysis. 

Proximity sequence enhanced CRISPR-Cas12a connected through hybridization chain reaction for sensitive biosensing of dengue virus

Sensitive detection of the SARS-CoV-2 protein remains a great research interest in clinical screening and diagnosis owing to the coronavirus epidemic. Here, an ultrasensitive chemiluminescence (CL) imaging strategy was developed through proximity hybridization to trigger the formation of a rolling circle-amplified G-quadruplex/hemin DNAzyme for the detection of the SARS-CoV-2 protein. The target protein was first recognized by a pair of DNA-antibody conjugates, Ab-1 and Ab-2, to form a proximity-ligated complex, Ab-1/SARS-CoV-2/Ab-2, which contained a DNA sequence complemental to block DNA and thus induced a strand displacement reaction to release the primer from a block/primer complex. The released primer then triggered a rolling circle amplification to form abundant DNAzyme units in the presence of hemin, which produced a strong chemiluminescent signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide. The proposed assay showed a detectable concentration range over 5 orders of magnitude with the detection limit down to 6.46 fg/mL. The excellent selectivity, simple procedure, acceptable accuracy, and intrinsic high throughput of the imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis.

A Rolling Circle-Amplified G-Quadruplex/Hemin DNAzyme for Chemiluminescence Immunoassay of the SARS-CoV-2 Protein.

A universal and highly sensitive electrochemical biosensing strategy was developed by using a triplet nanostructure-mediated dendritic hybridization chain reaction (HCR) for the analysis of Dengue virus (DENV) nucleic acid. A locked dsDNA was firstly recognized by the target DENV nucleic acid fragment to release the initiator, which was then captured by a capture DNA modified gold electrode to initiate the HCR in the presence of biotin labeled substrates A and B, and two helpers. After the recognition reactions of the captured initiator with substrate A and then helper 1, one DNA structure was formed to react with substrate B, which produced a triplet nanostructure to trigger the dendritic HCR by the toehold of the hybridization structure. After the dendritic product was bound with avidin labeled horseradish peroxidase (avidin-HRP), an amperometric signal could be obtained to achieve ultrasensitive electrochemical detection of DENV. The non-enzymatic isothermal amplification was driven only by entropy. The stable three-dimensional dendritic nanostructure with rigid double helix could assure the excellent performance of the designed electrochemical biosensing strategy. The proposed method showed a detection range of 1.6–1000 pM with a detection limit of 188 fM and ability to distinguish single-base mutation. By changing the recognition sequence of the initiator, the detection of different DENV nucleic acid fragments could be achieved with the same performance. Thus, this method had good extendability for other nucleic acids, providing a promising candidate for nucleic acid detection in early clinical diagnosis.

Jinling Fu

Papers

A Rolling Circle-Amplified G-Quadruplex/Hemin DNAzyme for Chemiluminescence Immunoassay of the SARS-CoV-2 Protein.

Electrochemical biosensing of DENV nucleic acid amplified with triplet nanostructure-mediated dendritic hybridization chain reaction