Intracellular MicroRNA Imaging and Specific Discrimination of Prostate Cancer Circulating Tumor Cells Using Multifunctional Gold Nanoprobe-Based Thermophoretic Assay.

Circulating tumor cells (CTCs) have emerged as powerful biomarkers for diagnosis of prostate cancer. However, the effective identification and concurrently accurate imaging of CTCs for early screening of prostate cancer have been rarely explored. Herein, we reported a multifunctional gold nanoprobe-based thermophoretic assay for simultaneous specific distinguishing of prostate cancer CTCs and sensitive imaging of intracellular microRNA (miR-21), achieving the rapid and precise detection of prostate cancer. The multifunctional gold nanoprobe (GNP-DNA/Ab) was modified by two types of prostate-specific antibodies, anti-PSMA and anti-EpCAM, which could effectively recognize the targeting CTCs, and meanwhile linked double-stranded DNA for further visually imaging intracellular miR-21. Upon the specific internalization of GNP-DNA/Ab by PC-3 cells, target aberrant miR-21 could displace the signal strand to recover the fluorescence signal for sensitive detection at the single-cell level, achieving single PC-3 cell imaging benefiting from the thermophoresis-mediated signal amplification procedure. Taking advantage of the sensitive miR-21 imaging performance, GNP-DNA/Ab could be employed to discriminate the PC-3 and Jurkat cells because of the different expression levels of miR-21. Notably, PC-3 cells were efficiently recognized from white blood cells, exhibiting promising potential for the early diagnosis of prostate cancer. Furthermore, GNP-DNA/Ab possessed good biocompatibility and stability. Therefore, this work provides a great tool for aberrant miRNA-related detection and specific discrimination of CTCs, achieving the early and accurate diagnosis of prostate cancer.

Recent Advances in Electrochemiluminescence Biosensors for MicroRNA Detection.

Electrochemiluminescence (ECL) as an analytical technology with a perfect combination of electrochemistry and spectroscopy has received considerable attention in bioanalysis due to its high sensitivity and broad dynamic range. Given the selectivity of bio-recognition elements and the high sensitivity of the ECL analysis technique, ECL biosensors are powerful platforms for the sensitive detection of biomarkers, achieving the accurate prognosis and diagnosis of diseases. MicroRNAs (miRNAs) are crucial biomarkers involved in a variety of physiological and pathological processes, whose aberrant expression is often related to serious diseases, especially cancers. ECL biosensors can fulfill the highly sensitive and selective requirements for accurate miRNA detection, prompting this review. The ECL mechanisms are initially introduced and subsequently categorize the ECL biosensors for miRNA detection in terms of the quenching agents. Furthermore, the work highlights the signal amplification strategies for enhancing ECL signal to improve the sensitivity of miRNA detection and finally concludes by looking at the challenges and opportunities in ECL biosensors for miRNA detection.

Ag-Doped Metal-Organic Frameworks' Heterostructure for Sonodynamic Therapy of Deep-Seated Cancer and Bacterial Infection.

Metal-organic frameworks (MOF) have attracted great potential in sonodynamic therapy (SDT) owing to large sonosensitizers' loading and fast reactive oxygen species' (ROS) diffusion; however, the low ligand-to-metal charge transfer efficiency sharply impairs the SDT effect. Herein, we report the design of MIL@Ag heterostructures with high electron-hole pairs separation efficiency and enhanced diverse ROS generation ability for deep-seated cancer treatment and bacterial infection. The MIL@Ag heterostructure is composed of Ti-based MOFs (named MIL), on which are in situ assembled silver nanoparticles (Ag NPs). The electrochemical experiments and density functional theory calculations verify that the introduction of Ag NPs can significantly improve the electron transfer efficiency and O2 adsorption capacity of MIL. Under ultrasound irradiation, the doped Ag NPs can trap the activated electrons from MIL to reduce surrounding O2 and produce superoxide radicals (•O2-), while the activated holes enable oxidizing H2O to produce hydroxyl radicals (•OH). Thus, they efficiently improve the therapeutic efficiency of SDT. MIL@Ag-PEG-mediated SDT implements A549 cancer cells' killing under a tissue barrier of 2 cm and eradicates the bacterial infection of Staphylococcus aureus, thus promoting wound healing. Therefore, MIL@Ag-PEG provides a promising strategy for augmenting SDT performance by rational heterostructure design of sonosensitizers.

Mitochondria MicroRNA Spatial Imaging via pH-Responsive Exonuclease-Assisted AIE Nanoreporter.

Mitochondrial microRNAs (mitomiRs) critically orchestrate mitochondrial functions. Spatial imaging of mitomiRs is essential to understand its clinical value in diagnosis and prognosis. However, the direct monitoring of mitomiRs in living cells remains a key challenge. Herein, we report an AIE nanoreporter strategy for mitomiRs imaging in living cells through pH-controlled exonuclease (Exo)-assisted target cycle signal amplification. The AIE-labeled DNA detection probes are conjugated on Exo III encapsulated polymeric nanoparticles (NPs) via consecutive adenines (polyA). The amplified sensing functions are off during the cytoplasm delivery process, and it can be spatially switched from off to on when in the alkaline mitochondria (about pH 8) after triphenylphosphonium (TPP)-mediated mitochondrial targeting. Where the NPs degraded to release Exo III and cancer-specific mitomiRs hybridize with AIE-labeled DNA detection probes to expose the cleavage site of released Exo III, enabling spatially restricted mitomiRs imaging. The mitomiRs expression fluctuation was also realized. This study contributes to a facile strategy that could easily extend to a broad application for the understanding of mitomiRs-related pathological processes.

Recent Advances in Near-Infrared-II Fluorescence Imaging for Deep-Tissue Molecular Analysis and Cancer Diagnosis.

Fluorescence imaging with high sensitivity and minimal invasiveness has received tremendous attention, which can accomplish visualized monitoring and evaluation of cancer progression. Compared with the conventional first near-infrared (NIR-I) optical window (650-950 nm), fluorescence imaging in the second NIR optical window (NIR-II, 950-1700 nm) exhibits deeper tissue penetration capability and higher temporal-spatial resolution with lower background interference for achieving deep-tissue in vivo imaging and real-time monitoring of cancer development. Encouraged by the significant preponderances, a variety of multifunctional NIR-II fluorophores have been designed and fabricated for sensitively imaging biomarkers in vivo and visualizing the treatment procedure of cancers. In this review, the differences between NIR-I and NIR-II fluorescence imaging are briefly introduced, especially the advantages of NIR-II fluorescence imaging for the real-time visualization of tumors in vivo and cancer diagnosis. An important focus is to summarize the NIR-II fluorescence imaging for deep-tissue biomarker analysis in vivo and tumor tissue visualization, and a brief introduction of NIR-II fluorescence imaging-guided cancer therapy is also presented. Finally, the significant challenges and reasonable prospects of NIR-II fluorescence imaging for cancer diagnosis in clinical applications are outlined.

Engineering DNA walkers for bioanalysis: A review.

Considerable advances have been made in the design, modularization, functionalization, and regulation of DNA nanostructures over the past 40 years. These advances have accelerated the development of DNA nanomachines such as DNA walkers, dynamic nanomachines with walking feet, tracks, and driven forces, which have highly sensitive detection and signal amplification abilities that can be applied to various bioanalytical contexts and therapeutic strategies. Here, we describe a rational design of the nano-bio interface, the kinetics of DNA walkers and the strategies for improving their efficiency and sensitivity. We also outline the various bioanalytic and imaging applications to which DNA walkers have been applied, such as electrochemical and optical measurements, when integrated with other simulation and activation tools. Finally, we compare the performances of novel DNA walker-based strategies for bioanalysis and propose a method to improve DNA walker design.

Spatially Resolved, Error-Robust Multiplexed MicroRNA Profiling in Single Living Cells.

Simultaneous imaging of multiple microRNAs (miRNAs) in individual living cells is challenging due to the lack of spectrally distinct encoded fluorophores and non-cytotoxic methods. We describe a multiplexed error-robust combinatorial fluorescent label-encoding method, termed fluorophores encoded error-corrected labels (FluoELs), enabling multiplexed miRNA imaging in living cells with error-correcting capability. The FluoELs comprise proportional dual fluorophores for encoding and a constant quantitative single fluorophore for error-corrected quantification. Both are embedded in 260 nm core-shell silica nanoparticles modified with molecular beacon detection probes. The FluoELs are low cytotoxic and could accurately quantify and spatially resolve nine breast-cancer-related miRNAs and evaluate their coordination. The FluoELs enabled a single-cell analysis platform to evaluate miRNA expression profiles and the molecular mechanisms underlying miRNA-associated diseases.

ROS-Activated nanoscale coordination polymers for enhanced ultrasound-mediated therapy for the treatment of cancer.

Stimuli-responsive nanoplatforms for efficient delivery of drugs in an on-demand manner show promising potential for killing cancer cells with high accuracy and minimal invasiveness. Herein, taking advantage of the good tissue-penetrating depth of sonodynamic therapy (SDT), reactive oxygen species (ROS)-responsive nanoscale coordination polymers (NCPs) were designed through self-assembly of porphyrins (PP) and platinum, which contained ROS-cleavable thioketal (TK) linkers to enhance the release of doxorubicin (Dox) during SDT. Upon exposure to the ultrasound (US), the Dox-loaded NCPs (PTK@PEG/Dox) could generate high amounts of cytotoxic ROS and heat, which not only induced the apoptosis of MCF-7 cells but also facilitated the efficient release of Dox due to the decomposition of the ROS-sensitive TK linkers, achieving the synergistic therapy of US-induced therapy and chemotherapy. After being modified with Arg-Gly-Asp (RGD) peptide, RGD/PTK@PEG exhibited a good targeting ability to cancer cells. Importantly, using the multicellular tumor spheroids (MCTS) derived from MCF-7 cells as a model, the RGD/PTK@PEG/Dox exhibited an efficient and controlled release behavior of Dox under the US irradiation, accompanying a tremendous anti-cancer effect for inducing apoptosis in the solid tumor tissues. This work provided a potential strategy to design controllable and stimuli-responsive nanoplatforms for synergistic/enhanced US-induced cancer therapy. STATEMENT OF SIGNIFICANCE: Stimulus-responsive nanoplatforms can deliver drugs efficiently in an on-demand manner, showing the potential to kill cancer cells with high accuracy and minimal invasiveness. Taking advantage of the good penetration ability of ultrasound (US), nanoscale coordination polymers (NCP) composed of porphyrin (PP), thioketal (TK) linkers, and platinum(II) were prepared via a coordination-driven self-assembly procedure. After doxorubicin (Dox) was loaded on the NCP (PTK@PEG/Dox), the nanoplatform responded to reactive oxygen species (ROS) under the stimulation of US, and induced the on-demand release of Dox, thereby achieving the combined therapeutic effect of sonodynamic therapy (SDT) and chemotherapy for cancer. This work provides a potential strategy for the development of controllable and stimuli-responsive nanoplatforms for enhanced ultrasound-induced cancer therapy.

Shedding Light on DNA-Based Nanoprobes for Live-Cell MicroRNA Imaging.

DNA-based nanoprobes integrated with various imaging signals have been employed for fabricating versatile biosensor platforms for the study of intracellular biological process and biomarker detection. The nanoprobes developments also provide opportunities for endogenous microRNA (miRNA) in situ analysis. In this review, the authors are primarily interested in various DNA-based nanoprobes for miRNA biosensors and declare strategies to reveal how to customize the desired nanoplatforms. Initially, various delivery vehicles for nanoprobe architectures transmembrane transport are delineated, and their biosecurity and ability for resisting the complex cellular environment are evaluated. Then, the novel strategies for designing DNA sequences as target miRNA specific recognition and signal amplification modules for miRNA detection are presented. Afterward, recent advances in imaging technologies to accurately respond and produce significant signal output are summarized. Finally, the challenges and future directions in the field are discussed.

DNA Logic Circuits for Multiple Tumor Cells Identification Using Intracellular MicroRNA Molecular Bispecific Recognition.

The aberrant expression level of intracellular microRNAs (miRNAs) holds great promise for differentiating cell types at the molecular level. However, cell subtype discrimination based on a single miRNA molecular level is not sufficient and reliable. Herein, multiple identifiable and reporting modules are integrated into a single DNA circuit for multiple cancer cell subtypes identification based on miRNAs bispecific recognition. The DNA three-dimensional (3D) logic gate nano-hexahedron framework extends three recognition modules and three reporting modules to form three "AND" logic gates. Each Boolean operator "AND" returns an "ON" signal in the presence of bispecific miRNAs, simultaneously enabling three types of cell subtype identification. It not only enables the discrimination of cancer cells A549 and MCF-7 from normal cells NHDF but also successfully distinguishes different types of cancer cells. The bispecific intracellular miRNA controllable DNA circuit, with low signal-to-noise ratio, easily extends to various cell type discrimination by adjusting the miRNA species, provides huge opportunities for accurately differentiating multiple cell types at the molecular level.

Advances in technology and applications of nanoimmunotherapy for cancer.

Host-tumor immune interactions play critical roles in the natural history of tumors, including oncogenesis, progress and metastasis. On the one hand, neoantigens have the potential to drive a tumor-specific immune response. In tumors, immunogenic cell death (ICD) triggered by various inducers can initiate a strong host anti-immune response. On the other hand, the tolerogenic tumor immune microenvironment suppresses host immune responses that eradicate tumor cells and impair the effect of tumor therapy. Therefore, a deeper understanding and more effective manipulation of the intricate host-tumor immune interaction involving the host, tumor cells and the corresponding tumor immune microenvironment are required. Despite the encouraging breakthroughs resulting from tumor immunotherapy, no single strategy has elicited sufficient or sustained antitumor immune responses in most patients with specific malignancies due to limited activation of specific antitumor immune responses and inadequate remodeling of the tolerogenic tumor immune microenvironment. However, nanotechnology provides a unique paradigm to simultaneously tackle all these challenges, including effective "targeted" delivery of tumor antigens, sustained ICD mediation, and "cold" tumor microenvironment remodeling. In this review, we focus on several key concepts in host-tumor immune interactions and discuss the corresponding therapeutic strategy based on the application of nanoparticles.

Target-Cell-Specific Bioorthogonal and Endogenous ATP Control of Signal Amplification for Intracellular MicroRNA Imaging.

A stringent signal amplification method to profile microRNA (miRNA) expression within a specific cell remains a key challenge in biology. To address this issue, we report a target-cell-specific DNA nanosystem for endogenous adenosine-5'-triphosphate (ATP) bioorthogonal activation of the hybridization chain reaction (HCR) to spatiotemporally controlled signal amplification detection of miRNA in vitro and in vivo. The system consists of ATP aptamer-sealed engineered HCR functional units combined with a cancer cell membrane-encapsulated glutathione (GSH)-responsive metal-organic framework (MOF). Once the nanosystem is specifically and efficiently internalized into a cancer cell through membrane-mediated homing targeting, the MOF structure degrades and releases HCR functional units. The endogenous high expressional ATP recognizes the aptamer, allowing the HCR functional units to adopt its active modality. The activated HCR functional units are then able to spatiotemporally and bioorthogonally image miRNA with high sensitivity in vitro and in vivo.

Glutathione-Responsive Biodegradable Nanoplatform with Endogenous Esterase-Triggered Nitric Oxide Release for Gas Therapy and Enhanced Chemotherapy.

The potential therapeutic effect of nitric oxide (NO) for cancers has received considerable attention as a "killer" that causes damage to mitochondria and DNA by oxidation or nitrosation. However, the fabrication of an intelligent and controllable NO release system has remained elusive in the desired location to realize selective cancer therapy. Herein, an intelligent endogenous esterase-triggered nitric oxide (NO) generator for synergetic cancer therapy is fabricated by integrating NO prodrug and doxorubicin (DOX) into a single glutathione (GSH)-responsive mesoporous silica nanoparticle (MPND). When the MPND is internalized into the cancer cell, the rupture of -S-S- bridges and the degradation of MPND occur in the tumor microenvironment with a high level of GSH, inducing the on-demand release of DOX. Importantly, the high endogenic esterase concentration can activate the prodrug to generate abundant NO, which further enhances the release performance of DOX. In vitro results verify that the release profiles of NO and DOX show the stimuli-responsive dependence of endogenic esterase and GSH, respectively, demonstrating the potential for on-demand release in the cancer cells. Consequently, MPND shows a high antitumor efficiency in MCF-7 cancer cells. Furthermore, using multicellular tumor spheroids to mimic in vivo experiment, MPND can enhance the tumor penetration and therapeutic effect for killing the deep tumor tissue at the central location. Therefore, the endogenous esterase-triggered NO nanogenerators may provide a potential alternative strategy to develop NO-relevant platforms for synergistic cancer therapy.

Uniform palladium nanosheets for fluorimetric detection of circulating tumor DNA.

Fluorescence quenching property of two-dimensional (2D) nanosheets (NSs) have received extensively attention in the construction of novel biosensing platform. However, the heterogeneity of the wide-size distribution and inefficient fluorescence quenching capacity limit its wide practical applications. Herein, for the first time, we report a novel fluorescent biosensor based on uniform palladium NSs (Pd NSs) with excellent fluorescence quenching efficiency and differential affinity toward ssDNA versus dsDNA and combination with a pair of DNA detection probes with fluorophore for detecting circulating tumor DNA (ctDNA). The DNA detection probes are facilitated to adsorbed to the surface of Pd NSs, leading to efficient fluorescence quench. In the presence of target DNA, it can be linked by T4 DNA ligase to form long DNA duplex structures, which display weak affinity toward Pd NSs, producing the fluorescence recovery. The remarkable fluorescence quenching efficiency and ssDNA/dsDNA differential affinity of Pd NSs make it have a good detection ability without signal amplification. The result indicates that this facile but cost-effective strategy holds great promise in bioanalysis.

Enhanced cancer therapy by hypoxia-responsive copper metal-organic frameworks nanosystem.

Tumor hypoxia-responsive size-switchable nanosystems for precise delivery of drug into deep tumor show great prospects for killing cancer cells with high specificity and minimal invasiveness. However, the development of versatile nanosystems is still a challenge. Herein, for the first time, we report a novel hypoxia-responsive copper metal-organic framework nanoparticles (Cu-MOF NPs) for chemodynamic therapy and sonodynamic therapy (CDT/SDT). The large size Cu-MOF NPs show good stability under normal oxygen partial pressure and enhance tumor accumulation, and it quickly degraded and released Cu2+ and Ce6 when exposed to the hypoxic tumor microenvironment (TME), significantly reinforced the intratumoral penetration. The internalized Cu2+ reacts with local GSH to deplete GSH and reduce Cu2+ to Cu+, which subsequently reacts with endogenous H2O2 to produce cytotoxic hydroxyl radicals (•OH) through Fenton-like reaction for CDT. The released Ce6 further mediated SDT under US irradiation. The synergistic SDT/CDT efficacy was significantly enhanced owing to the GSH depletion, realizing selective and effective MCF-7 killing with minimal invasiveness. This work presents a novel hypoxia-responsive MOF nanosystem with intrinsic CDT properties, mainly, the MOF nanosystem is flexible to the integration with other therapy approaches. It provides a general strategy to design a hypoxia-responsive MOF nano theranostic platform.

Biodegradable Metal-Organic Frameworks Power DNAzyme for in Vivo Temporal-Spatial Control Fluorescence Imaging of Aberrant MicroRNA and Hypoxic Tumor.

MicroRNAs (miRNAs) are involved in the essential progresses of many diseases and have emerged as therapeutic and diagnostic biomarkers. The combination of miRNA aberrant expression and tumor microenvironment (TME) features holds great potential for precise tumor imaging diagnosis but has been minimally explored. Herein, we rationally design a DNA@Cu-MOF nanosystem containing copper metal-organic frameworks (Cu-MOF) and a DNAzyme-assisted signal amplification procedure for deregulated miRNA-related hypoxic tumor diagnosis. The nanoprobes comprising a signal strand block Cu-specific DNAzyme precursor and a substrate strand are assembled on the surface of the hypoxia-responsive Cu-MOF. Under TME characterized by hypoxia, the DNA@Cu-MOF nanosystem disassociates and accomplishes the release of abundant Cu2+, DNAzyme precursor, and substrate strand. Target aberrant miRNA displaces the signal strand to recover one fluorescence signal for detection. Importantly, it activates the Cu-specific DNAzyme amplification, which produces miRNA aberrant expression-dependent fluorescence signal for hypoxic tumor diagnosis. Both in vitro and in vivo experiments validate its good performance for tumor cell diagnosis. The hypoxia-driven and miRNA-binding-induced self-powered and temporal-spatial fluorescence imaging nanosystem not only provides a great tool for aberrant miRNA-related hypoxic tumor diagnosis but also is readily applied for the control and modulation of biological functions.

Upconversion Nanoparticle-Induced Multimode Photodynamic Therapy Based on a Metal-Organic Framework/Titanium Dioxide Nanocomposite.

Photodynamic therapy (PDT) possesses two pathways depending on the type of high-toxicity reactive oxygen species (ROS), superoxide anion radical (O2·-) and hydroxyl radical (·OH) generated through Type I and singlet oxygen (1O2) generated through Type II, inducing cancer cell apoptosis. However, the low efficiency of ROS generation and poor biocompatibility are the limitations of the traditional photosensitizers for PDT. Herein, inspired by photochemical reactions of titanium dioxide and porphyrin-based metal-organic frameworks, we developed a nanoplatform by covering ultrasmall titanium dioxide nanoparticles on a heterodimer made up of upconversion nanoparticles and metal-organic frameworks, realizing a multimode PDT through Type I and Type II mechanisms. Once irradiated by a near-infrared light, upconversion nanoparticles could generate ultraviolet and visible lights, which were not only able to stimulate different photochemical reactions of titanium dioxide and porphyrin but also accomplish deep penetration photodynamic therapy. Our photosensitive agent exhibited good biocompatibility and an effective multimode PDT performance, which could meet the needs of different situations of photodynamic therapy in the future.

Algae Extraction Controllable Delamination of Vanadium Carbide Nanosheets with Enhanced Near-Infrared Photothermal Performance.

The two-dimensional (2D) vanadium carbide (V2 C) MXene has shown great potential as a photothermal agent (PTA) for photothermal therapy (PTT). However, the use of V2 C in PTT is limited by the harsh synthesis condition and low photothermal conversion efficiency (PTCE). Herein, we report a completely different green delamination method using algae extraction to intercalate and delaminate V2 AlC to produce mass V2 C nanosheets (NSs) with a high yield (90 %). The resulting V2 C NSs demonstrated good structural integrity and remarkably high absorption in near infrared (NIR) region with a PTCE as high as 48 %. Systemic in vitro and in vivo studies demonstrate that the V2 C NSs can serve as efficient PTA for photoacoustic (PA) and magnetic resonance imaging (MRI)-guided PTT of cancer. This work provides a cost-effective, environment-friendly, and high-yielding disassembly approach of MAX, opening a new avenue to develop MXenes with desirable properties for a myriad of applications.

A Bacteriochlorin-Based Metal-Organic Framework Nanosheet Superoxide Radical Generator for Photoacoustic Imaging-Guided Highly Efficient Photodynamic Therapy.

Hypoxic tumor microenvironment is the bottleneck of the conventional photodynamic therapy (PDT) and significantly weakens the overall therapeutic efficiency. Herein, versatile metal-organic framework (MOF) nanosheets (DBBC-UiO) comprised of bacteriochlorin ligand and Hf6(µ3-O)4(µ3-OH)4 clusters to address this tricky issue are designed. The resulting DBBC-UiO enables numerous superoxide anion radical (O2 -•) generation via a type I mechanism with a 750 nm NIR-laser irradiation, part of which transforms to high toxic hydroxyl radical (OH•) and oxygen (O2) through superoxide dismutase (SOD)-mediated catalytic reactions under severe hypoxic microenvironment (2% O2), and the partial recycled O2 enhances O2 -• generation. Owing to the synergistic radicals, it realizes advanced antitumor performance with 91% cell mortality against cancer cells in vitro, and highly efficient hypoxic solid tumor ablation in vivo. It also accomplishes photoacoustic imaging (PAI) for cancer diagnosis. This DBBC-UiO, taking advantage of superb penetration depth of the 750 nm laser and distinct antihypoxia activities, offers new opportunities for PDT against clinically hypoxic cancer.

Functional DNA hexahedron for real-time detection of multiple microRNAs in living cells.

Intracellular microRNA (miRNA) analysis in single cell is highly informative and offers valuable insights to its physiological and pathological state, but it must confront the pivotal challenge of gene probe delivery and conditional release. Herein, we report an assembled DNA mini-hexahedron (DMH) that can selectively package and protect miRNA probe, target-cell-specific delivery and release it based on the target sequence recognition for intracellular miRNA detection. In brief, the DMH is self-assembled from six single-stranded oligonucleotide strands through rational design, one of which containing AS1411 sequence for specific uptake. Two fluorescent dye labeled recognition strands are inserted into two DMH edges with quencher groups through partially complementary hybridization. We find that this DMH possesses great biocompatibility, good trans-membrane ability and are able to protect the gene cargo against enzymatic degradation and protein binding. Fluorescence restoration caused by the target-mediated competitive chain replacement reaction allows to simultaneous detection of two cancer-related intracellular miRNAs with little false-positive signal, providing a powerful tool to discriminate healthy normal cell and cancerous cell. Thus, the construct opens a new avenue to circumvent the challenges in gene delivery, specific delivery and intrinsic interferences resistance.