Electrochemical nucleic acid sensors: Competent pathways for mobile molecular diagnostics.

Electrochemical nucleic acid biosensor has demonstrated great promise in clinical diagnostic tests, mainly because of its flexibility, high efficiency, low cost, and easy integration for analytical applications. Numerous nucleic acid hybridization-based strategies have been developed for the design and construction of novel electrochemical biosensors for diagnosing genetic-related diseases. This review describes the advances, challenges, and prospects of electrochemical nucleic acid biosensors for mobile molecular diagnosis. Specifically, the basic principles, sensing elements, applications in diagnosis of cancer and infectious diseases, integration with microfluidic technology and commercialization are mainly included in this review, aiming to provide new insights and directions for the future development of electrochemical nucleic acid biosensors.

Hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons for spatial-controlled genome editing and precise cancer therapy.

CRISPR-Cas13a holds enormous potential for developing precise RNA editing. However, spatial manipulation of CRISPR-Cas13a activity remains a daunting challenge for elaborately regulating localized RNase function. Here, we designed hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons (RNCOs-D), featuring tumor-specific recognition and spatial-controlled activation of Cas13a, for precise cancer synergistic therapy. RNCOs-D consists of programmable RNA nanosponges (RNSs) capable of targeted delivery and caging chemotherapeutic drug, and nanocapsules (NCs) anchored on RNSs for cloaking Cas13a/crRNA ribonucleoprotein (Cas13a RNP) activity. The acidic endo/lysosomal microenvironment stimulates the outer decomposition of NCs with concomitant Cas13a RNP activity revitalization, while the inner disassembly through trans-cleavage of RNSs initiated by cis-recognition and cleavage of EGFR variant III (EGFRvIII) mRNA. RNCOs-D demonstrates the effective EGFRvIII mRNA silencing for synergistic therapy of glioblastoma cancer cells in vitro and in vivo. The engineering of RNSs, together with efficient Cas13a activity regulation, holds immense prospect for multimodal and synergistic cancer therapy.

CeO2/MXene heterojunction-based ultrasensitive electrochemiluminescence biosensing for BCR-ABL fusion gene detection combined with dual-toehold strand displacement reaction for signal amplification.

An "on-off" nonenzymatic and ultrasensitive electrochemiluminescence (ECL) biosensing platform has been constructed to detect BCR-ABL fusion gene based on CeO2/MXene heterojunction and configuration-entropy driven dual-toehold strand displacement reaction (DT-SDR) for signal amplification. The CeO2/MXene heterojunction were prepared via one-step hydrothermal method through in situ synthesis of CeO2 nanocubes on the surface of Ti3C2-MXene nanosheets. Surprisingly, the prepared CeO2/MXene heterojunction with good dispersion and excellent conductivity not only significantly enhanced ECL emission of S2O82-/O2 system, but also acted as good electrode modification materials to provide massive active sites for three-stranded ST/AS/BK complex immobilization. In the presence of target BCR-ABL fusion gene and Bio-FS, target BCR-ABL fusion gene bound to dual-toehold exposed at the ends of ST, replacing AS and BK and obtaining ST/target with a loop. Subsequently, Bio-FS bound to the loop (as toehold) in ST strand of ST/target to form ST/Bio-FS, replacing the target to further trigger a new SDA cycle. This configuration-entropy driven DT-SDR made three-stranded ST/AS/BK complex transform into dual-stranded ST/Bio-FS in the electrode interface. Ultimately, the quenching labels of streptavidin modified Pt nanoparticles functionalized polydopamine composites (SA-Pt@PDA) were introduced via biotin and streptavidin recognition, realizing ECL emission quenching of S2O82-/O2 system for "on-off" detection of BCR-ABL fusion gene. The developed ECL biosensor for BCR-ABL fusion gene detection achieves the wide concentration variation from 1 fM to 100 pM with low limit of detection down to 0.27 fM, which provides new enlightenment and basis for molecular diagnosis of chronic myelogenous leukemia in clinical practice.

Janus wireframe DNA cube-based 3D nanomachine for rapid and stable fluorescence detection of exosomal microRNA.

Exosomal microRNAs (miRNAs) have been demonstrated to be credible biomarkers for the diagnosis and monitoring of tumors. Nevertheless, developing simple, rapid, and stable biosensing strategies that are capable of accurately detecting exosomal miRNAs remains a challenge. Herein, an accelerated and biostable three-dimensional (3D) nanomachine based on Janus wireframe DNA cube was constructed for sensitive fluorescence measurement of exosomal miRNA. The Janus wireframe DNA cube could propel target exosomal miRNA-21 rapid movement on its surface by catalytic hairpin assembly (CHA), releasing a massive fluorescence signal. Benefiting from the Janus wireframe DNA cube, the developed 3D nanomachine exhibited significantly improved reaction rate and enhanced biostability in complex biofluids compared to conventional CHA. As a result, this fluorescence biosensing strategy achieved rapid, stable, and single-step detection of exosomal miRNA-21 with the detection limit down to the picomole level. Therefore, this work offers a brief sensing tool for nucleic acid biomarkers detection, which has great application potential in tumor diagnosis.

Dispersion-to-localization of catalytic hairpin assembly for sensitive sensing and imaging microRNAs in living cells from whole blood.

Localized DNA circuits have shown good performance regarding reaction rate and sensitivity for sensing intracellular microRNAs (miRNAs). However, these methods reported recently require large kinds of DNA strands and suffer from low signal-to-background (S/B) ratio, which hinder their clinical application. To circumvent these issues, we herein developed a novel strategy for sensitive sensing and imaging miRNAs in living cells based on dispersion-to-localization of catalytic hairpin assembly (DL-CHA). This strategy consists of only three classes of DNA strands (two hairpins and a linker strand), which largely reduces sequence design complexity. Additionally, owing to the unique engineering of the substrate transformation from dispersion to localization, the DL-CHA exhibits not only minimal background leakage but also intensive signal amplification, thus significantly improving the S/B ratio. In particular, the simple sensing method is capable of imaging miRNAs in cells from clinical blood samples for the diagnosis of breast cancer. Therefore, this work provides a powerful tool for intracellular molecules detection and gives a much broader design space for constructing high-performance DNA circuits.

Femtomolar and locus-specific detection of N6-methyladenine in DNA by integrating double-hindered replication and nucleic acid-functionalized MB@Zr-MOF.

In this study, a novel electrochemical biosensor was constructed for ultrasensitive and locus-specific detection of N6-Methyladenine (m6A) in DNA using double-hindered replication and nucleic acid-coated methylene blue (MB)@Zr-MOF. Based on the combination of m6A-impeded replication and AgI-mediated mismatch replication, this mode could effectively stop the extension of the strand once DNA polymerase encountered m6A site, which specifically distinguish the m6A site from natural A site in DNA. Also, Zr-MOF with high porosity and negative surface potential features was carefully chose to load cationic MB, resulting a stable and robust MB@Zr-MOF electrochemical tag. As a result, the developed biosensor exhibited a wide linear range from 1 fM to 1 nM with detection limit down to 0.89 fM. Profiting from the high sensitivity and selectivity, the biosensing strategy revealed good applicability, which had been demonstrated by quantitating m6A DNA at specific site in biological matrix. Thus, the biosensor provides a promising platform for locus-specific m6A DNA analysis.

Surface plasmon resonance biosensor for exosome detection based on reformative tyramine signal amplification activated by molecular aptamer beacon.

Human epidermal growth factor receptor 2 (HER2)-positive exosomes play an extremely important role in the diagnosis and treatment options of breast cancers. Herein, based on the reformative tyramine signal amplification (TSA) enabled by molecular aptamer beacon (MAB) conversion, a label-free surface plasmon resonance (SPR) biosensor was proposed for highly sensitive and specific detection of HER2-positive exosomes. The exosomes were captured by the HER2 aptamer region of MAB immobilized on the chip surface, which enabled the exposure of the G-quadruplex DNA (G4 DNA) that could form peroxidase-like G4-hemin. In turn, the formed G4-hemin catalyzed the deposition of plentiful tyramine-coated gold nanoparticles (AuNPs-Ty) on the exosome membrane with the help of H2O2, generating a significantly enhanced SPR signal. In the reformative TSA system, the horseradish peroxidase (HRP) as a major component was replaced with nonenzymic G4-hemin, bypassing the defects of natural enzymes. Moreover, the dual-recognition of the surface proteins and lipid membrane of the desired exosomes endowed the sensing strategy with high specificity without the interruption of free proteins. As a result, this developed SPR biosensor exhibited a wide linear range from 1.0 × 104 to 1.0 × 107 particles/mL. Importantly, this strategy was able to accurately distinguish HER2-positive breast cancer patients from healthy individuals, exhibiting great potential clinical application.