Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research. Thus far, many signal amplification strategies have been ...developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA). In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency. In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers. As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms. In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence
in situ
hybridization (FISH) imaging, live cell imaging, and targeted drug delivery. In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail. The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples. Finally, the review provides insight into the challenges and future perspectives of HCR.
This review provides a comprehensive overview of the fundamental principles, analysis techniques, and application fields of hybridization chain reaction and its development status.
DNA has become a promising material to construct high-order structures and molecular devices owing to its sequence programmability. Herein, a DNA machine based on branched catalytic hairpin assembly ...(bCHA) is introduced for dynamic self-assembly of DNA dendrimers. For this system, a Y-shaped hairpin trimer tethered with three kinds of hairpins (H1, H2 and H3) is constructed. The introduction of an initiator (I) triggers a cascade of CHA reactions among hairpin trimers, leading to the formation of DNA dendrimers. Through labeling fluorophore/quencher pairs in the hairpin trimers, this catalytic DNA machine is applied as a versatile amplification platform to analyze nucleic acids using microRNA-155 (miR-155) as a model analyte. Benefiting from the "diffusion effect", the proposed bCHA achieves a greatly improved sensitivity in comparison with traditional CHA. This catalytic amplifier exhibits high sensitivity toward miR-155 detection with a dynamic range from 2.5 nM to 500 nM and demonstrates excellent selectivity to distinguish the single-base mismatched sequence from the perfectly complementary one, which is further applied to detect low-abundance miR-155 spiked in complex matrices with minimal interference. This method is further applied for
imaging of miR-155 in different live cells. The bCHA reaction can be specifically triggered by intracellular miR-155, achieving monitoring of the dynamic miRNA expression and distribution. Overall, our proposed enzyme-free dynamic DNA self-assembly strategy provides a versatile approach for the development of DNA nanotechnology in biosensing and bioimaging, and monitoring the cellular miRNA-related biological events.
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•DNA-Au nanomachine activated by tumor microenvironment GSH is constructed.•G-quadruplexes are incorporated to target tumor and modulate tumor hypoxia for enhanced PDT.•Triple ...combination of PDT/PTT/chemotherapy is achieved for cancer treatment.
Multimodal therapy is a combination of different therapeutic modalities within one single platform to enhance treatment efficiency, which holds great promise in biomedical research and clinical practice. Herein, a glutathione (GSH)-activated DNA-Au nanomachine with tumor-targeting capacity has been developed for imaging-guided photodynamic/photothermal/chemo combination therapy of murine breast cancer. In this theranostic nanoplatform, G-quadruplex double-stranded DNA (G-dsDNA) containing AS1411 aptamer is served as not only the targeting unit to specifically recognize murine 4T1 breast cancer cells but also the carrier for loading photosensitizer chlorins e6 (Ce6), iron-containing porphyrin hemin and anticancer drug doxorubicin (Dox). In tumor microenvironment, the excessive GSH triggers the rupture of disulfide bridges and results in the dissociation of DNA-Au nanomachine along with the release of Dox for chemotherapy and the aggregation of AuNPs for photothermal therapy (PTT) as well as in situ imaging. Meanwhile, hemin and Ce6 delivered by G-quadruplexes alleviate the tumor hypoxia, which further promotes the generation of reactive oxygen species (ROS) for enhanced photodynamic therapy (PDT). The in vitro and in vivo assays validate that the combination of PDT/PTT/chemotherapy shows a high efficiency for targeted damage of 4T1 cells and inhibition of tumor growth. Therefore, the proposed multifunctional DNA-Au nanomachine provides a powerful and robust theranostic nanoplatform for imaging-guided combinatorial anticancer therapy.
Toehold-mediated strand displacement-based nanocircuits are developed by integrating catalytic hairpin assembly (CHA) with hybridization chain reaction (HCR), which achieves self-assembly of ...hyperbranched DNA structures and is readily utilized as an enzyme-free amplifier for homogeneous CRET detection of microRNA with high sensitivity and selectivity.
Multifunctional AgNPs@GQDs with nanozyme activity have been applied for dual-mode biosensing and cancer theranostics based on the H2O2-responsive degradation.
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A multifunctional ...nanocomposite of AgNPs@GQDs is prepared by synergistic in-situ growth of silver nanoparticles (AgNPs) on the complex of tannic acid (TA) and graphene quantum dots (GQDs) for the construction of dual-mode biosensing platform and cancer theranostics. The nanocomposite exhibits a hydrogen peroxide (H2O2)-responsive degradation, in which Ag0 is oxidized to Ag+ along with the release of oxidized TA and GQDs. The degradation induces the decreased absorbance and enhanced fluorescence (FL) intensity due to the suppression of Förster resonance energy transfer (FRET) in AgNPs@GQDs, which is employed for colorimetric/fluorescence dual-mode sensing of H2O2. The intrinsic peroxidase-like activity of GQDs nanozyme can effectively catalyze the oxidation reaction, enhancing the detection sensitivity significantly. Based on the generation of H2O2 from the oxidation of glucose with the catalysis of glucose oxidase (GOx), this nanoprobe is versatilely used for the determination of glucose in human serum. Further, through combining the H2O2-responsive degradation of AgNPs@GQDs with high H2O2 level in cancer cells, the nanocomposites exhibit good performance in cancer cell recognition and therapy, in which the synergistic anticancer effect of Ag+ and oxidized TA contribute to effective cell death, and the liberated GQDs are used to monitor the therapeutic effect by cell imaging.
Small interfering RNA (siRNA) has emerged as a kind of promising therapeutic agents for cancer therapy. However, the off-target effect and degradation are the main challenges for siRNAs delivery. ...Herein, an enzyme-free DNA amplification strategy initiated by a specific endogenous microRNA has been developed for in situ generation of siRNAs with enhanced gene therapy effect on cervical carcinoma.
This strategy contains three DNA hairpins (H1, H2/PS and H3) which can be triggered by microRNA-21 (miR-21) for self-assembly of DNA nanowheels (DNWs). Notably, this system is consistent with the operation of a DNA logic circuitry containing cascaded "AND" gates with feedback mechanism. Accordingly, a versatile biosensing and bioimaging platform is fabricated for sensitive and specific analysis of miR-21 in HeLa cells via fluorescence resonance energy transfer (FRET). Meanwhile, since the vascular endothelial growth factor (VEGF) antisense and sense sequences are encoded in hairpin reactants, the performance of this DNA circuit leads to in situ assembly of VEGF siRNAs in DNWs, which can be specifically recognized and cleaved by Dicer for gene therapy of cervical carcinoma.
The proposed isothermal amplification approach exhibits high sensitivity for miR-21 with a detection limit of 0.25 pM and indicates excellent specificity to discriminate target miR-21 from the single-base mismatched sequence. Furthermore, this strategy achieves accurate and sensitive imaging analysis of the expression and distribution of miR-21 in different living cells. To note, compared to naked siRNAs alone, in situ siRNA generation shows a significantly enhanced gene silencing and anti-tumor effect due to the high reaction efficiency of DNA circuit and improved delivery stability of siRNAs.
The endogenous miRNA-activated DNA circuit provides an exciting opportunity to construct a general nanoplatform for precise cancer diagnosis and efficient gene therapy, which has an important significance in clinical translation.
Rolling circle replication (RCR), including rolling circle amplification (RCA) and rolling circle transcription (RCT), is an isothermal enzymatic reaction. Because of its high amplification ...efficiency, RCR is a powerful biosensing tool for detecting biomolecules. In recent years, RCR has also been extended to the field of bioimaging to better understand biological pathways. Furthermore, RCR provides a simple technique to design and generate DNA/RNA structures with unique advantages in delivering drugs and enhanced targeting ability. In this review, we introduce the fundamentals of RCR and describe the most recent advances in RCR-based detection methods and delivery vehicles for biosensing, bioimaging, and biomedicine. Finally, some challenges and further opportunities of RCR-based biotechnology are discussed.
Rolling circle replication (RCR) is an isothermal nucleic acid amplification approach that holds great potential in biotechnology.RCR can serve as a powerful biosensing platform for the detection of various biomolecules in bioanalysis and diagnosis.In bioimaging, RCR can provide a unique perspective in comprehensive understanding of biological processes and effective diagnosis of diseases.Programmable, biodegradable RCR-based assemblies have been used as versatile advanced drug carriers for targeted theranostics.
As a conventional amplification technique, polymerase chain reaction (PCR) has been widely applied to detect a variety of analytes with exponential amplification efficiency. However, the requirement ...of thermocycling procedures largely limits the application of PCR-based methods. Alternatively, several isothermal amplification techniques have been developed since the early 1990s. In particular, according to the reaction kinetics, isothermal exponential amplification techniques possess higher amplification efficiency and detection sensitivity. The isothermal exponential amplification techniques can be mainly divided into two categories: enzyme-based isothermal exponential amplification and enzyme-free isothermal exponential amplification. Considering the advantages of high sensitivity and selectivity, high signal-to-noise ratio, low cost and rapid response time, exponential amplification electrochemical biosensors have attracted considerable attention. In this review, we introduce the basic principles of isothermal exponential amplification techniques and summarize their applications in electrochemical biosensors during the past five years. We also highlighted the present challenges and further perspectives of isothermal exponential amplification-based electrochemical biosensors.
•Isothermal exponential amplification has demonstrated as a powerful tool for bioanalytical applications.•Basic principles of isothermal exponential amplification techniques are introduced.•Their applications in electrochemical biosensors during the past five years are summarized.•The challenges and perspectives of isothermal exponential amplification-based electrochemical biosensors are highlighted.
The fabrication of nickel oxide/carbon nanotube (NiO/CNT) nanocomposites by atomic layer deposition (ALD) and their electrochemical performances for sensing hydroquinone (HQ) and catechol (CC) were ...investigated. NiO nanopaticles with a size of ~4.9nm formed by ALD were highly dispersed on the wall of pristine CNT. Then, the prepared NiO/CNT nanocomposites were used to modify glassy carbon electrode (NiO/CNT/GCE) for electrochemical detection, which presented enhanced electrochemical performances towards detecting HQ and CC. Not only were the redox peak currents of HQ and CC greatly enhanced, but also the oxidization peaks of the both phenolic isomers could be well separated. Under the optimized conditions, the current responses are linear for HQ and CC in the range of 10–500μM and 10–400μM, with detection limit of 2.5μM (S/N=3) for both compounds.
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•Nickel oxide (NiO) nanoparticles were homogeneously deposited on carbon nanotube (CNT) by atomic layer deposition (ALD).•NiO/CNT exhibits great sensitivity, stability and anti-interference ability towards detecting hydroquinone and catechol.•This is the first time that NiO/CNT nanocomposites prepared by ALD have been applied to detect electroactive molecules.•NiO/CNT nanocomposites have extensive application prospects in real water samples.
High throughput analysis of miRNAs is of great significance to clinical and biomedical applications. In this work, we have developed a highly sensitive and selective chemiluminescence imaging array ...(CLIA) for simultaneous detection of three miRNAs with high throughput, easy operation and low cost. In this assay, three kinds of hairpins that can specially recognize respective miRNAs (miR-155, miR-let-7a and miR-141) are parallel modified on the magnetic beads (MBs). The employment of MBs enables quick sorting of multiplex targets in high yield and purity with high throughput. Upon introduction of the target miRNAs, they hybridize to the corresponding hairpins and initiate the DNA machines with the assistance of Klenow fragment exo- DNA polymerase and Nb.BbvCI NEase, achieving exponential amplification of targets. After that, the toehold-mediated strand displacement (TMSD) reactions are performed through sequential addition of displacement probes under magnetic separation, resulting in the release of HRP-tagged DNA hybrids in solution for CL imaging and recovery of MBs for repetitive use. This CLIA method demonstrates ultrahigh sensitivity with detection limits down to fM level, wide linear range over 5 orders of magnitude, excellent selectivity to distinguish one-base mismatched target miRNA, and good performance in real sample analysis. Together with the advantages of high throughput, easy operation, acceptable accuracy, and good recyclability and reproducibility, the proposed multiplex CLIA method holds great potential in practical applications, such as profiling the pattern of miRNA expression, disease screening, biomedical research, and so on.
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•A chemiluminescence imaging array is developed for simultaneous detection of multiple microRNAs.•The operation of DNA machine achieves exponential amplification of targets.•Toehold-mediated strand displacement-based magnetic separation allows spatial and temporal segregation.•The magnetic carriers can be recovered for repetitive use.
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