Herein, an ultrasensitive and versatile electrochemical biosensor was developed through the target-controlled capture and release of signal probe-loaded DNA nanotube for the ultrasensitive detection of two different types of cancer-related biomarkers, microRNA-21 (miRNA-21) and glutathione (GSH). In this system, target 1 (miRNA-21) first triggered duplex-specific nuclease (DSN)-assisted recycle amplification to generate numerous disulfide-linked DNA strands (DL), which could effectively capture DNA nanotube to immobilize methylene blue (MB) to produce remarkable electrochemical signals and achieve the ultrasensitive detection of miRNA-21 with a detection limit down to 32.6 aM. Furthermore, in the presence of target 2 (GSH), the electrochemical signal was significantly reduced by a thiol-disulfide bond exchange reaction on DL to release MB-immobilized DNA nanotubes away from the sensing interface, which enabled the sensitive analysis of GSH with a detection limit of 0.379 nM. Impressively, this strategy could achieve ultrasensitive detection of different types of biomarkers to prominently lessen false-positive responses from the current sensing methods toward a single biomarker or the same type of biomarker and remarkably heighten the accuracy and precision of early cancer diagnosis. Meanwhile, the proposed electrochemical biosensor made it possible to realize the regenerative analysis of targets over four times without extra fuel, which could conspicuously improve the analytical efficiency compared with that of traditional biosensing assays. As a result, this study might open up novel insights to design a versatile and multifunctional sensing platform and encourage deeper exploration for detecting different types of biomarkers in the fields of early disease diagnosis and biochemical research.

Versatile Electrochemical Biosensor Based on the Target-Controlled Capture and Release of DNA Nanotubes for the Ultrasensitive Detection of Multiplexed Biomarkers.

Herein, a long period liner DNA tandem (Lr-DNT) was intelligently designed as DNA track for quadruped DNA walker (q-walker) to run in an orderly and efficient manner, which could be applied to construct an electrochemical biosensor for rapid and ultrasensitive detection of microRNA-21 (miRNA-21). Impressively, benefiting from the orderliness and equidistance of Lr-DNT, the q-walker could be endowed with a high controllability, directionality as well as a quite short reaction time down to 20 min compared with those of traditional DNA walkers walked on the stochastic tracks. Once the target miRNA-21 interacted with the locked q-walker, the q-walker could be activated to expeditiously cleave Lr-DNT for releasing amounts of signal probes ferrocene (Fc) with the assistance of the Nt.BbvCI enzyme. This way, the developed q-walker could not only readily overcome the problem of low reaction efficiency but also address the drawback of time consumption in a previous strategy. As a proof of concept, the prepared biosensor could accomplish sensitive detection of target miRNA-21 with a detection limit down to 31 aM. As a result, this tactic gave impetus to design high-performance sensing platform with ultimate application in clinical sample analysis and nucleic acid based cancer diagnostics.

Highly Efficient Quadruped DNA Walker Guided by Ordered DNA Tracks for Rapid and Ultrasensitive Electrochemical Detection of miRNA-21.

MicroRNA (miRNA) is an ideal biomarker, and the sensitive monitoring of low-abundance miRNA is of great significance for the treatment of cancer at the onset stage. Herein, a novel Y-shaped DNA-mediated photoelectrochemical (PEC) sensing platform based on the Bi2S3@CdIn2S4 photoanode is proposed for the detection of miRNA-21. First, an n-n type Bi2S3@CdIn2S4 heterojunction with tight interface is formed for the first time through a layer-by-layer assembly strategy due to the matched band structures of Bi2S3 and CdIn2S4. The photocurrent intensity of Bi2S3@CdIn2S4/GCE electrode is 1.7-fold higher than that of Bi2S3/GCE electrode and 2.5-fold higher than that of CdIn2S4/GCE electrode under visible light irradiation. Additionally, miRNA-21-triggered catalytic hairpin assembly facilitates the formation of Y-shaped DNA structure with CdS quantum dots (CdS QDs). As the photosensitizer of Bi2S3@CdIn2S4 heterojunction, CdS QDs accelerate the separation of electron-hole pairs in the sensing system and realizes photocurrent signal enhancement, thus realizing the “signal on” detection of the miRNA-21. The proposed PEC biosensor exhibits a superior sensitivity of 0.18 fM and the recoveries of human serum range from 98.0% to 104% with a relative standard deviation of 5.8–8.9%. Therefore, the constructed PEC biosensor has potential application value in the early diagnosis and treatment of miRNA-related diseases. 

A “signal on” photoelectrochemical biosensor for miRNA-21 detection based on layer-by-layer assembled n-n type Bi2S3@CdIn2S4 heterojunction

Since the first discovery in 1994, DNAzymes have been extensively applied in biosensing and therapy that acting as recognition elements and signal generators with the outstanding properties of good stability, simple synthesis and high sensitivity. One subset, RNA-cleaving DNAzymes, are widely employed for diverse applications, including as reporters capable of transmitting detectable signals. In this review, we focus on the recent advances of RNA-cleaving DNAzyme-based amplification strategies in scaled-up biosensing, the application in diagnosis and disease treatment are also disscussed. We highlight two major types of RNA-cleaving DNAzyme-based amplification strategies, namely direct response amplification strategies and combinational response amplification strategies. The direct response amplification strategies refer to those based on novel designed single-stranded DNAzyme, and the combinational response amplification strategies mainly include two-part assembled DNAzyme, cascade reactions, CHA/HCR/RCA, DNA walker, CRISPR-Cas12a and aptamer. Finally, we present the current status of DNAzymes, the challenges and prospects of DNAzyme-based biosensors. This article is protected by copyright. All rights reserved.

RNA-Cleaving DNAzyme-based Amplification Strategies for Biosensing and Therapy.

Probing endogenous molecular profiles in living entities is of fundamental significance to decipher biological functions and exploit novel theranostics. Despite programmable nucleic acid-based aptasensing systems across the breadth of molecular imaging, an aptasensing system enabling in vivo imaging with high sensitivity, accuracy, and adaptability is highly required yet is still in its infancy. Artificial catalytic DNA circuits that can modularly integrate to generate multiple outputs from a single input in an isothermal autonomous manner, have supplemented powerful toolkits for intracellular biosensing research. Herein, a multilayer nonenzymatic catalytic DNA circuits-based aptasensing system is devised for in situ imaging of a bioactive molecule in living mice by assembling branched DNA copolymers with high-molecular-weight and high-signal-gain based on avalanche-mimicking hybridization chain reactions (HCRs). The HCRs aptasensing circuit performs as a general and powerful sensing platform for precise analysis of a series of bioactive molecules due to its inherent rich recognition repertoire and hierarchical reaction accelerations. With tumor-targeting capsule encapsulation, the HCRs aptasensing circuit is specifically delivered into tumor cells and allowed the high-contrast imaging of intracellular adenosine triphosphate in living mice, highlighting its potential for visualizing these clinically important biomolecules and for studying the associated physiological processes.

Modular Assembly of a Concatenated DNA Circuit for In Vivo Amplified Aptasensing.

DNA-based nanoprobes have attracted extensive interest in the field of bioanalysis. Notably, engineered DNA nanoprobes that can respond to multiple pathological parameters are desirable to detect targets precisely. Here we design a split aptamer/DNAzyme (aptazyme)-based DNA probe for fluorescence detection of ATP and further develop a cooperatively activatable DNA nanoprobe for tumor-specific imaging of ATP in vivo. The DNA nanoprobes comprising split aptazyme-coated MnO2 nanovectors have high stability and are synergistically activated by multiple biomarkers, GSH and ATP. Upon stimuli by overexpressed GSH in tumor cells, this DNA nanoprobe can release the aptazyme and self-supply cofactor Mn2+ of the DNAzyme. Sequentially, intracellular ATP induces the proper folding of the split ATP aptamer and Mn2+-dependent DNAzyme, which activates the specific cleavage of substrate and generates the optical readout signal. This nanoprobe exhibits remarkable resistance to enzymatic degradation, satisfactory biosafety, identifies ATP specifically within cancer cells, and selectively lights up solid tumors. Our research provides a reliable method for ATP imaging in cancer cells and opens a new avenue for biochemical research and highly accurate disease diagnosis.

A Cooperatively Activatable DNA Nanoprobe for Cancer Cell-Selective Imaging of ATP

Functional DNA nanostructures have been widely used in various bioassay fields. Yet, the programmable assembly of functional DNA nanostructures in living cells still represents a challenging goal for guaranteeing the sensitive and specific biosensing utility. In this work, we report a self-catalytic DNA assembly (SDA) machine by using a feedback deoxyribozyme (DNAzyme)-amplified branched DNA assembly. This SDA system consists of catalytic self-assembly (CSA) and DNAzyme amplification modules for recognizing and amplifying the target analyte. The analyte initiates the CSA reaction, leading to the formation of Y-shaped DNA that carries two RNA-cleaving DNAzymes. One DNAzyme can then successively cleave the corresponding substrate and generate numerous additional inputs to activate new CSA reactions, thus realizing a self-catalytic amplification reaction. Simultaneously, the other DNAzyme is assembled as a versatile signal transducer for cleaving the fluorophore/quencher-modified substrate, leading to the generation of an amplified fluorescence readout. By incorporating a flexible auxiliary sensing module, the SDA system can be converted into a universal sensing platform for detecting cancerous biomarkers, e.g., a well-known oncogene microRNA-21 (miR-21). Moreover, the SDA system realized the precise intracellular miR-21 imaging in living cells, which is attributed to the reciprocal amplification property between CSA reactions and DNAzyme biocatalysis. This compact SDA amplifier machine provides a universal and facile toolbox for the highly efficient identification of cancerous biomarkers and thus holds great potential for early cancer diagnosis.

A Deoxyribozyme-Initiated Self-Catalytic DNA Machine for Amplified Live-Cell Imaging of MicroRNA.

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A Cooperatively Activatable DNA Nanoprobe for Cancer Cell-Selective Imaging of ATP

A Deoxyribozyme-Initiated Self-Catalytic DNA Machine for Amplified Live-Cell Imaging of MicroRNA.