Ultrasensitive Detection Of microRNA Research Articles

Ultrahigh-Speed 3D DNA Walker with Dual Self-Protected DNAzymes for Ultrasensitive Fluorescence Detection and Intracellular Imaging of microRNA.

Herein, a dual self-protected DNAzyme-based 3D DNA walker (dSPD walker), composed of activated dual self-protected walking particles (ac-dSPWPs) and track particles (TPs), was constructed for ultrasensitive and ultrahigh-speed fluorescence detection and imaging of microRNAs (miRNAs) in living cells. Impressively, compared with the defect that "one" target miRNA only initiates "one" walking arm of the conventional single self-protected DNAzyme walker, the dSPD walker benefits from the secondary amplification and spatial confinement effect and could guide "one" target miRNA to generate "n" secondary targets, thereby initiating "n" nearby walking strands immediately, realizing the initial rate over one-magnitude-order faster than that of the conventional one. Moreover, in the process of relative motion between ac-dSPWPs and TPs, the ac-dSPWPs could cleave multiple substrate strands simultaneously to speed up movement and reduce the derailment rate, as well as combine with successive TPs to facilitate a large amount of continuous signal accumulation, achieving an ultrafast detection of miRNA-221 within 10 min in vitro and high sensitivity with a low detection limit of 0.84 pM. In addition, the DNA nanospheres obtained by the rolling circle amplification reaction can capture the Cy5 fluorescence dispersed in liquids, which achieves the high-contrast imaging of miRNA-221, resulting in further ultrasensitive imaging of miRNA-221 in cancer cells. The proposed strategy has made a bold innovation in the rapid and sensitive detection as well as intracellular imaging of low-abundance biomarkers, offering promising application in early diagnosis and relevant research of cancer and tumors.

Dual-Ligand Europium-Organic Gels as a Highly Efficient Anodic Annihilation Electrochemiluminescence Emitter for Ultrasensitive Detection of MicroRNA.

In this study, a novel europium dual-ligand metal-organic gel (Eu-D-MOGs) with high-efficient anodic annihilation electrochemiluminescence (ECL) was synthesized as an ECL emitter to construct a biosensor for ultrasensitive detection of microRNA-221 (miR-221). Impressively, compared to the ECL signal of europium single-ligand metal-organic gels (Eu-S-MOGs), the ECL signal of Eu-D-MOGs was significantly improved since the two organic ligands could jointly replace the H2O and coordinate with Eu3+, which could remarkably reduce the nonradiative vibrational energy transfer caused by the coordination between H2O and Eu3+ with a high coordination demand. In addition, Eu-D-MOGs could be electrochemically oxidized to Eu-D-MOGs•+ at 1.45 V and reduced to Eu-D-MOGs•- at 0.65 V to achieve effective annihilation of ECL, which overcame the side reaction brought by the remaining emitters at negative potential. This benefited from the annihilation ECL performance of the central ion Eu3+ caused by its redox in the electrochemical process. Furthermore, the annihilation ECL signal of Eu3+ could be improved by sensitizing Eu3+ via the antenna effect. In addition, combined with the improved rolling circle amplification-assisted strand displacement amplification strategy (RCA-SDA), a sensitive biosensor was constructed for the sensitive detection of miR-221 with a low detection limit of 5.12 aM and could be successfully applied for the detection of miR-221 in the lysate of cancer cells. This strategy offered a unique approach to synthesizing metal-organic gels as ECL emitters without a coreactant for the construction of ECL biosensing platforms in biomarker detection and disease diagnosis.

Target-driven cascade amplified assembly of covalent organic frameworks on tetrahedral DNA nanostructure with multiplex recognition domains for ultrasensitive detection of microRNA

BackgroundMicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis. ResultsIn this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the “freeze-drying-reduction” method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn2+. Without the addition of exogenous Mn2+, the dynamically-generated Mn2+-powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM. Significance and noveltyTaking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn2+-powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening.

Ferroelectric Bi2O2Te-Based Plasmonic Biosensor for Ultrasensitive Biomolecular Detection.

Ultrasensitive detection of biomarkers, particularly proteins, and microRNA, is critical for disease early diagnosis. Although surface plasmon resonance biosensors offer label-free, real-time detection, it is challenging to detect biomolecules at low concentrations that only induce a minor mass or refractive index change on the analyte molecules. Here an ultrasensitive plasmonic biosensor strategy is reported by utilizing the ferroelectric properties of Bi2O2Te as a sensitive-layer material. The polarization alteration of ferroelectric Bi2O2Te produces a significant plasmonic biosensing response, enabling the detection of charged biomolecules even at ultralow concentrations. An extraordinary ultralow detection limit of 1fm is achieved for protein molecules and an unprecedented 0.1fm for miRNA molecules, demonstrating exceptional specificity. The finding opens a promising avenue for the integration of 2D ferroelectric materials into plasmonic biosensors, with potential applications spanning a wide range.

Flexible-Arranged Biomimetic Array Integrated with Parallel Entropy-Driven Circuits for Ultrasensitive, Multiple, and Reliable Detection of Cancer-Related MicroRNAs.

With the emergence of microRNA (miRNA) as a promising biomarker in cancer diagnosis, it is significant to develop multiple analyses of miRNAs. However, it still faces difficulties in ensuring the sensitivity and accuracy during multiplex detection owing to the low abundance and experimental deviation of miRNAs. In this work, a flexible-arranged biomimetic array integrated with parallel entropy-driven circuits (EDCs) was developed for ultrasensitive, multiplex, reliable, and high-throughput detection of miRNAs. The biomimetic array was fabricated by arrangement of various photonic crystals (PCs) for adjustable photonic band gaps (PBGs) and specific fluorescence enhancement. Meanwhile, two cancer-related miRNAs and one reference miRNA were introduced as multiple analytes as a proof-of-concept. The parallel EDCs with negligible crosstalk were designed based on the modular property. Because of the one-to-one match between the emitted fluorescence of parallel EDCs and the PBGs of the flexible-arranged biomimetic array, the generated fluorescence signal triggered by target miRNAs can be enhanced on the corresponding domain of the array. Furthermore, the amplified signal of the array was detected with high-throughput scanning, which could reveal specific information on cancer-related miRNAs as well as reference miRNA, enhancing the abundance and reliability of the analysis. The proposed array has the merits of a modular design, flexible deployment, simple operation (nonenzymatic and isothermal), improved accuracy, high sensitivity, and multiplex analysis, showing potential in disease diagnosis.

A versatile upconversion-based multimode lateral flow platform for rapid and ultrasensitive detection of microRNA towards health monitoring

MicroRNAs are small single-stranded RNA molecules associated with gene expression and immune response, suggesting their potential as biomarkers for health monitoring. Herein, we designed a novel upconversion-based multimode lateral flow assay (LFA) system to detect microRNAs in body fluids by simultaneously producing three unique signals within a detection strip. The core-shell Au-DTNB@Ag nanoparticles act as both the Raman reporters and acceptors, quenching fluorescence from upconversion nanoparticles (UCNPs, NaYF4: Yb3+, Er3+) via the Förster resonance energy transfer mechanism. Using microRNA-21 as a representative analyte, the LFA system offers remarkable detection range from 2 nM to 1 fM, comparable to outcomes from signal amplification methods, due to the successful single-layer self-assembly of UCNPs on the NC membrane, which greatly enhances both the convenience and sensitivity of the LFA technique. Additionally, our proprietary fluorescence-Raman detection platform simplifies result acquisition by reducing procedural intricacies. The biosensor, when evaluated with diverse bodily fluids, showed remarkable selectivity and sustained stability. Importantly, our LFA biosensor effectively identified periodontitis and lung cancer patients from healthy subjects in genuine samples, indicating significant potential for disease prediction, early diagnosis, and progression tracking. This system holds promise as a multifunctional tool for various biomarker assays.

3D walkable DNA gears for ultrasensitive detection of multiple microRNAs in lung cancer cell lysates

As a tumor biomarker with therapeutic application potential, microRNA (miRNA) was crucial for the accurate and sensitive detection of early-stage tumors. Herein, a unique three dimensional (3D) DNA nanomachine (DNM) was created, which was capable detecting lung cancer-related biomarkers miRNA-21, miRNA-205 and miRNA-125b in lung cancer cell lysates with extreme sensitivity. The 3D DNM was composed of DNA scissors and three flexible walkable DNA gears modified with various species of silver nanoclusters (AgNCs). Based on the flexibility of DNA scissors and the walkability of DNA gears, neighboring DNA gears closed the distance between different species of AgNCs by walking in the presence of targets, generating fluorescence resonance energy transfer (FRET) effect and emitting different kinds of fluorescence to complete the highly sensitive detection of single targets and multiple targets. The findings demonstrated that a linear model provided an excellent match for the association between fluorescence signal and target miRNAs. For miRNA-21, miRNA-205, and miRNA-125b, the limits of detection (LODs) (signal/noise = 3) were 4.2 pmol/L (pM), 6.3 pM, and 10.2 pM, respectively. Their recoveries in A549 cell lysate samples ranged from 95.3 to 108.8 % with relative standard deviations of 1.26 %–4.88 %. Satisfactorily, the 3D DNM displayed exceptional analytical performance with high sensitivity and stability, strong specificity and reproducibility, which was triumphantly employed to identify miRNAs in tumor cell lysates, providing a workable technique in creating adaptable nanostructure for dependable bioanalysis and clinical diagnosis of cancer biomarkers.

An Ultrasensitive and Efficient microRNA Nanosensor Empowered by the CRISPR/Cas Confined in a Nanopore.

This work presents a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-nanopipette nano-electrochemistry (Cas = CRISPR-associated proteins) capable of ultrasensitive microRNA detection. Nanoconfinement of the CRISPR/Cas13a within a nanopipette leads to a high catalytic efficacy of ca. 169 times higher than that in bulk electrolyte, contributing to the amplified electrochemical responses. CRISPR/Cas13a-enabled detection of representative microRNA-25 achieves a low limit of detection down to 10 aM. Practical application of this method is further demonstrated for single-cell and real human serum detection. Its general applicability is validated by addressing microRNA-141 and the SARS-CoV-2 RNA gene fragment. This work introduces a new CRISPR/Cas-empowered nanotechnology for ultrasensitive nano-electrochemistry and bioanalysis.

Ultrasensitive detection of microRNA-21 in human serum based on the confinement effect enhanced chemical etching of gold nanorods

Natural enzymes, such as horseradish peroxidase (HRP), are a class of important biocatalysts with the high specificity, but their catalytic efficiency is usually unsatisfactory. Thus, the higher catalytic efficiency induced by the confinement effect is promising in optical sensing systems. In this work, a dark-field light scattering sensing platform was fabricated by the confinement effect of HRP from hybridization chain reaction (HCR) and then released to solution by the toehold-mediated strand displacement reaction (TSDR). Then, HRP catalyzed the 3,3′,5,5′-tetramethylbenzidine (TMB) to TMB2+ with the assistance of hydrogen peroxide, which etched the gold nanorods (AuNRs) with the weakened light scattering. The single-particle assay was established based on the decreased light scattering intensity of AuNRs under dark-field microscope. The proposed assay revealed excellent analytical performance within a linear range from 25 pmol/L to 600 pmol/L, and a low limit of detection of 3.12 pmol/L. Additionally, it also manifested satisfactory recovery of miRNA-21 in human serum samples. The high sensitivity, excellent specificity, and universal applicability make this sensing platform promising for disease diagnosis.

Triple cycling integrating with primer exchange reaction (TCiPER) for ultra-sensitive and label-free detection of MicroRNA

Primer exchange reaction (PER), a novel and effective isothermal amplification method, is extensively employed in the development of microRNAs (miRNAs) detection methods. Nevertheless, the PER-based miRNA detection methods necessitate enhanced sensitivity and interference resistance. Here, we propose a fluorescence biosensor, referred to as Triple Cycling integrating with Primer Exchange Reaction (TCiPER), for miRNA detection with straightforward operation, reduced interferences, and significantly increased sensitivity. This method has a high sensitivity with a low limit of detection of 56 aM because the target miRNA recognizes the detection probe and initiates triple signal cycles. PER template (PT) is initially occupied by the hairpin probe (HP), which reduces the possibility of fault initiation of PER by the primer similar sequences, and ii) the produced G-quadruplex sequences combine with thioflavin T (ThT) to produce significant fluorescence, which is superior to traditional fluorescent methods that are developed on the basis of costly and carefully selected fluorescent probes. In conclusion, this strategy substantially enhanced the convenience of sensors fabricated using PER strategies and provided a new perspective for the future development of extensive and sensitive nucleic acid detection analysis tools.

Enzyme-free electrochemical biosensor based on bio-barcode amplification for ultra-sensitive detection of microRNA.

The rapid and accurate detection of miRNAs is of great significance for early diagnosis and treatment of cancer. Hence, a novel enzyme-free and label-free electrochemical biosensor based on bio-barcode amplification for detecting miRNAs was presented. Sandwich structures constructed of magnetic nanoparticles modified with DNA probes, gold nanoparticles with numerous barcoded DNA strands that hybridized with target miRNAs were fabricated as the amplifier. The released barcoded DNA strands then acted as the secondary targets and triggered the electrochemical sensor with a significant electrochemical response. A highly sensitive (detection limit of 0.24 fM) and selective electrochemical miRNA detection was realized, which has great potential for application in miRNA-related clinical diagnosis and biochemical research.

A novel cascade catalytic electrochemical biosensor based on bifunctional Mn3O4@AuNPs with self-supplied O2 property and glucose oxidase-like activity for ultrasensitive detection of microRNA

Herein, a bifunctional nanomaterial Mn3O4@AuNPs with self-supplied oxygen (O2) property and glucose oxidase-like activity was exploited to construct a novel cascade catalytic electrochemical biosensor for ultrasensitive detection of microRNA-499 (miRNA-499). Impressively, compared with the traditional AuNPs nanozyme with single glucose oxidase-like activity, the Mn3O4@AuNPs could not only act as a glucose oxidative mimetic enzyme to catalyze the oxidation of glucose to gluconic acid and the reduction of dissolved O2 to low concentration of hydrogen peroxide (H2O2) to obtain a electrochemical signal in the presence of hemin/G-quadruplex DNAzyme with peroxidase-like activity, but also electrocatalyze water splitting to obtain abundant dissolved O2 to further assist in the production of high concentration of H2O2 for a significantly improved electrochemical response. Meanwhile, with the aid of the target-induced dual Mg2+-assisted DNAzyme cleavage recycling amplification for the increasement of target miRNA-499 conversion efficiency, the proposed electrochemical biosensor achieved the ultrasensitive detection of miRNA-499 with a detection limit as low as 6.2 aM. As a result, this strategy might provide a potential method to upgrade the catalytic efficiency of mimic enzyme cascade reaction with eventual applications in bioanalysis and disease diagnosis.

Plasmonic Nanoparticle-Enhanced Optical Techniques for Cancer Biomarker Sensing.

This review summarizes recent advances in leveraging localized surface plasmon resonance (LSPR) nanotechnology for sensitive cancer biomarker detection. LSPR arising from noble metal nanoparticles under light excitation enables the enhancement of various optical techniques, including surface-enhanced Raman spectroscopy (SERS), dark-field microscopy (DFM), photothermal imaging, and photoacoustic imaging. Nanoparticle engineering strategies are discussed to optimize LSPR for maximum signal amplification. SERS utilizes electromagnetic enhancement from plasmonic nanostructures to boost inherently weak Raman signals, enabling single-molecule sensitivity for detecting proteins, nucleic acids, and exosomes. DFM visualizes LSPR nanoparticles based on scattered light color, allowing for the ultrasensitive detection of cancer cells, microRNAs, and proteins. Photothermal imaging employs LSPR nanoparticles as contrast agents that convert light to heat, producing thermal images that highlight cancerous tissues. Photoacoustic imaging detects ultrasonic waves generated by LSPR nanoparticle photothermal expansion for deep-tissue imaging. The multiplexing capabilities of LSPR techniques and integration with microfluidics and point-of-care devices are reviewed. Remaining challenges, such as toxicity, standardization, and clinical sample analysis, are examined. Overall, LSPR nanotechnology shows tremendous potential for advancing cancer screening, diagnosis, and treatment monitoring through the integration of nanoparticle engineering, optical techniques, and microscale device platforms.

Tailoring high-energy self-powered sensing system by Walker-mediated CRISPR/Cas12a cascade signal amplification and hybridization chain reaction for ultrasensitive microRNA detection

MicroRNAs (miRNAs) are important cancer-associated biomarkers for early detection. However, accurately measuring miRNAs is challenging due to their low abundance and high homogeneity in human serum. CRISPR technology can greatly improve the specificity and sensitivity of detection in biosensor construction. In this study, we develop a remarkably responsive self-powered biosensing platform for detecting microRNA-21 (miRNA-21) using hybridization chain reaction (HCR) and CRISPR/Cas12a amplification cascade mediated by 3D DNA walkers. Carbon-coated molybdenum disulfide nanotubes with a large surface area and excellent conductivity are fabricated on flexible carbon paper as the anode and cathode for biological fuel cell construction. Glucose oxidase is loaded onto the anode surface, while serving as the biological cathode, it initiated HCR, generating abundant double-stranded DNA. The negatively charged DNA backbone captures electron acceptors [Ru(NH3)6]3+ through electrostatic adsorption, resulting in a significant open circuit voltage (EOCV) signal. Through CRISPR/Cas12a amplification cascade mediated by 3D DNA walkers, a smaller EOCV is produced, enabling sensitive target detection. The method exhibits a detection range spanning from 0.0001 to 10,000 pM and capable of detecting as low as 19.0 aM (S/N = 3). This highly selective and sensitive method provides an effective approach for accurately detecting tumor markers in clinical applications.

Self-Powered Biosensor Based on DNA Walkers for Ultrasensitive MicroRNA Detection.

A novel self-powered biosensor is fabricated for ultrasensitive microRNA-21 (miRNA-21) detection, which includes an enzymatic biofuel cell (EBFC), DNA walkers, a digital multimeter (DMM), and a capacitor. As a novel strategy for signal amplification, DNA walkers are designed in the cathode, while the capacitor stores electrochemical energy from the EBFC to further boost the instantaneous current displayed by the DMM. When miRNA-21 is present, the DNA walkers are provoked to walk from as-opened hairpin structures to other hairpin structures, generating double-strand DNA structures, which stimulate [Ru(NH3)6]3+ to be adsorbed on the cathode surface by electrostatic interaction. Afterward, [Ru(NH3)6]3+ is reduced to [Ru(NH3)6]2+, and the open circuit voltage (EOCV) is significantly increased. Depending on the approach of signal amplification from DNA walkers, this biosensor displays an ultrasensitive assay toward miRNA-21 in the range of 0.5 to 104 fM, with a detection limit of 0.15 fM. In addition, this self-powered biosensor displays high selectivity for miRNA-21 assay in human serum samples.

Smart Target-Initiated Catalytic DNA Junction Circuit Amplification Strategy for the Ultrasensitive Electrochemiluminescence Detection of MicroRNA.

One of the highly attractive research directions in the electrochemiluminescence (ECL) field is how to regulate and improve ECL efficiency. Quantum dots (QDs) are highly promising ECL materials due to their adjustable luminescence size and strong luminous efficiency. MoS2 NSs@QDs, an ECL emitter, is synthesized via hydrothermal methods, and its ECL mechanism is investigated using cyclic voltammetry and ECL-potential curves. Then, a stable and vertical attachment of a triplex DNA (tsDNA) probe to the MoS2 nanosheets (NSs) is applied to the electrode. Next, an innovative ECL sensor is courageously empoldered for precise and ultrasensitive detection of target miRNA-199a through the agency of ECL-resonance energy transfer (RET) strategy and a dextrous target-initiated catalytic three-arm DNA junction assembly (CTDJA) based on a toehold strand displacement reaction (TSDR) signal amplification approach. Impressively, the ingenious system not only precisely regulates the distance between energy donor-acceptor pairs leave energy less loss and more ECL-RET efficiency, but also simplifies the operational procedure and verifies the feasibility of this self-assembly process without human intervention. This study can expand MoS2 NSs@QDs utilization in ECL biosensing applications, and the proposed nucleic acid amplification strategy can become a miracle cure for ultrasensitive detecting diverse biomarkers, which helps researchers to better study the tumor mechanism, thereby unambiguously increasing cancer cure rates and reducing the risk of recurrence.

Near-Infrared-Driven Nanorocket for Rapid and Ultrasensitive Detection of MicroRNA.

Herein, by introducing gold nanostars (AuNSs) as fuel core, a near-infrared-driven nanorocket (NIDNR) with pretty fast walking was exploited for ultrasensitive miRNA detection. Compared with traditional nanomaterials-comprised nanomachines (NMs), the NIDNR possesses much better kinetic and thermodynamic performance owing to the extra photothermal driving force from localized surface plasmon (LSP). Impressively, the whole reaction time of NIDNR down to 15 min was realized, which is almost more than 8 times beyond those of conventional DNA-based NMs. This way, the inherent obstacle of traditional NMs, including long reaction time and low efficiency, could be easily addressed. As a proof of concept, the NIDNR was successfully applied to develop an electrochemical biosensing platform for rapid and sensitive detection of miRNA with an LOD down to 2.95 aM and achieved the real-time assay of real biological samples from human hepatocellular carcinoma cells (MHCC97L) and HeLa, thus providing an innovative insight to design more versatile DNA nanomachines for ultimate application in biosensing platform construction and clinical sample detection.

Dual-mode DNA walker-based optical fiber biosensor for ultrasensitive detection of microRNAs

We present a novel dual-mode DNA-walker based optical fiber biosensor (DMDW-Opt biosensor) for sensitive assay of micro-RNAs in bio-samples. In the sensor system, we develop a new strategy for the cascade amplification, DNA-walker/rolling cycle amplification (RCA), by the use of the residue track of the walker. The strategy can significantly improve the response of the sensor and avoid any tedious operation procedure. Dual-mode readouts, i.e., fluorescence and chemiluminescence, are measured independently without interfering with each other to achieve reliable and accurate analysis. Optical fibers with the surface modified by gold nanoparticles are utilized as the support for fabrication of the sensor, which would be benefit for developing miniaturized and portable sensing devices. The performance of the proposed method is evaluated by using micro-RNAs (MiR-155 and MiR-21) as the analytical target. The method is successfully applied for accurate determination of micoRNAs in human serum and MCF-7 cells. Our method can perform sensitive assays of MiR-155 with limit-of-detection as low as 97.72 fM and 11.22 fM, MiR-21 with limit-of-detection as low as 107.15 fM and 8.32 fM for the fluorescence- and the chemiluminescence-readout respectively, and the biosensor exhibits excellent specificity, reproducibility and storage stability, indicating its valuable potential applications for sensing trace-amount targets in complicated real samples.

Selenium and nitrogen co-doped carbon dots with highly efficient electrochemiluminescence for ultrasensitive detection of microRNA

In this work, selenium and nitrogen co-doped carbon dots (SeN-CDs) possessing highly efficient electrochemiluminescence (ECL) and excellent biocompatibility were synthesized as a new emitter with S2O82− as a coreactant for constructing a biosensor to detect microRNA-221 (miRNA-221) sensitively. Notably, the SeN-CDs exhibited superior ECL performance compared with the N-doped CDs, in which selenium with excellent redox activity served as a coreaction accelerator for facilitating the electroreduction of S2O82− to significantly improve ECL efficiency. Furthermore, target-induced T7 exonuclease (T7 Exo)-assisted double cycle amplification strategy could convert traces of target miRNA-221 into large amounts of output DNA to capture three-dimensional (3D) nanostructures (DTN-Au NPs-DOX-Fc) loaded with large amounts of ECL signal quencher. The constructed biosensor could realize ultrasensitive detection of miRNA-221 and has a low detection limit reaching 2.3 aM, with a successful application to detect miRNA-221 in lysate of Hela and MHCC97-L cancer cell. This work explored a novel method to strengthen the ECL performance of CDs to construct an ECL biosensing platform with sensitive detecting of biomarkers and disease diagnosis.

A sodium-dependent split DNAzyme fluorescent sensor under butanol acceleration for ultrasensitive detection of PIWI-interacting RNAs and microRNAs

Split DNAzymes have been designed as multifunctional biosensors. However, their limited sensitivity and low resistance to nuclease hinder their applications in biological samples. Herein, we developed a sodium-dependent and butanol accelerated split DNAzyme fluorescent sensor named Na+-BAS DNAzyme which has an ultra-sensitivity for quantifying PIWI-interacting RNAs/microRNAs and can be directly applied in blood biopsy to detect nucleic acids within the extracellular vesicles (EVs). The Na+-BAS DNAzyme consists of two split DNA strands based on NaA43 DNAzyme, a starting hairpin DNA for binding target to initiate the combining of the split enzymes, and a hairpin RNA-embedded DNA substrate labeled a quencher and a fluorophore for signal readout after its cleavage by the assembled DNAzyme complex. To achieve ultra-sensitivity, we employed a simple but key approach of butanol dehydration to highly accelerate the measuring reaction. The sensor has achieved an ultra-low detection limit of 12 × 10−18 M miR-21 after 30 min reaction, and 3.98 or 2.69 particles μL−1 MCF-7 EVs through miR-21 or piR-20365 detection. With these high performances, the sensor can detect extremely trace abundance of miR-21 in a single MCF-10a or MCF-7 EV, quantifying it as 5.93 × 10−3 or 0.12 copies per EV. Without any chemical structure modification, the sensor can be directly used in the plasma sample for breast cancer diagnosis which has obtained both 100% sensitivity and specificity using the biomarker of piR-20365. The Na+-BAS DNAzyme sensor’s ultra-sensitivity, high efficiency, universality, and simplicity make it a great potential in the clinical diagnostic application.