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.
The monitoring of blood glucose levels is essential to the diagnosis and management of diseases, especially diabetes. Thus, it is of great importance to develop a simple, accurate and sensitive ...method for glucose detection. Herein, the three-dimensional DNA flowers-templated Ag nanoclusters (DFs/AgNCs) with enhanced fluorescent brightness and good stability are in situ synthesized via rolling circle amplification (RCA), which are further successfully applied for the construction of fluorescent/electrochemical dual-mode glucose biosensor. In this assay, the AgNCs encapsulated in DFs can be etched by H2O2 that is generated from the process of glucose oxidase (GOx)-catalyzed glucose oxidation, resulting in the release of Ag+ to facilitate the signal-off fluorescent detection of glucose. Based on the coordination interaction of cytosine-Ag+-cytosine (C-Ag+-C), the liberated Ag+ further induces the conformation switch of ferrocene (Fc)-labeled DNA capture probe (Fc-DNA) with C-rich sequence on Au electrode, leading to the signal-on electrochemical detection of glucose. The proposed DFs/AgNCs-based fluorescent/electrochemical dual-mode biosensor has achieved the sensitive and selective detection of glucose in human serums with good accuracy and reliability, which not only provides a rapid, reliable and accurate method for glucose detection but also opens a new avenue to the development of DNA nanotechnology-based biosensing strategies.
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•Three-dimensional DNA flowers (DFs) are prepared via rolling circle amplification.•Fluorescent silver nanoclusters (AgNCs) are in situ synthesized using DFs as template.•DFs/AgNCs act as both fluorescent indicator and Ag+ generator.•A fluorescent/electrochemical dual-mode biosensor is constructed for glucose detection.
The regulation of biocatalytic cascades in microenvironments for high performance and extended applications is still challenging. Herein, we develop a rolling circle amplification (RCA)-based one-pot ...method to prepare the micron-sized DNA flowers (DFs), which achieve the co-encapsulation and spatial regulation of bi-enzyme molecules, glucose oxidase (GOx) and horseradish peroxidase (HRP). In this system, GOx and HRP are integrated into the DFs simultaneously during RCA with the bridging of magnesium between enzyme residues and phosphate backbones on DFs. The cascade of GOx/HRP is regulated with the formation of highly ordered and hydrogen-bonded water environment in the cavity of DFs, resulting in an enhanced cascade catalytic efficiency compared with that in homogeneous solution. Moreover, the high density of DNA scaffold ensures the encapsulation of GOx/HRP with high efficiency. Accordingly, a glucose electrochemical biosensor with amplified signal response is fabricated using the as-prepared GOx/HRP DFs as biosensing interface, realizing sensitive detection of glucose. Further, through designing the complementary sequence of aptamer into the programmable circular template of RCA, the bi-enzyme co-encapsulated DFs are versatilely applied to sensitive and selective detection of cancerous exosomes and thrombin in “signal-on” and “signal-off” modes, respectively, which are further applied to the analysis of complex biological samples successfully. Overall, the encapsulation of multi-enzyme with DFs proposes a promising strategy to regulate the microenvironment of biocatalytic cascades, which hold great potential in biotechnology, bioanalysis and disease diagnosis.
•A rolling circle amplification-based one-pot method is developed to prepare DNA flowers (DFs).•An enhanced cascade biocatalysis of GOx/HRP is regulated in highly ordered DFs with hydrogen-bonded water environment.•GOx/HRP DFs are versatilely applied to fabricate electrochemical biosensors with signal amplification.•Successful analysis of complex biological samples is demonstrated.
As a kind of promising nanomaterials, metal nanoclusters (MNCs) generally composed of several to hundreds of metal atoms have received increasing interest owing to their unique properties, such as ...ultrasmall size (<2 nm), fascinating physical and chemical properties, and so on. Recently, template-assisted synthesis of MNCs (e.g., Au, Ag, Cu, Pt and Cd) has attracted extensive attention in biological fields. Up to now, various templates (e.g., dendrimers, polymers, DNAs, proteins and peptides) with different configurations and spaces have been applied to prepare MNCs with the advantages of facile preparation, controllable size, good water-solubility and biocompatibility. Herein, we focus on the recent advances in the template-assisted synthesis of MNCs, including the templates used to synthesize MNCs, and their applications in biosensing, bioimaging, and disease theranostics. Finally, the challenges and future perspectives of template-assisted synthesized MNCs are highlighted. We believe that this review could not only arouse more interest in MNCs but also promote their further development and applications by presenting the recent advances in this area to researchers from various fields, such as chemistry, material science, physiology, biomedicine, and so on.
•Recent progress in template-assisted synthesis of metal nanoclusters (MNCs) has been overviewed.•The roles of different templates on the synthesis and physiochemical properties of MNCs are discussed comprehensively.•Applications of template-synthesized MNCs in biosensing, bioimaging and theranostics are introduced systematically.•The challenges and future perspective of templated synthesis of MNCs are highlighted.
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•DNA tetrahedron-mediated branched catalytic hairpin assembly (DTM-bCHA) is defined.•The reaction kinetics and amplification efficiency are significantly improved.•In situ SERS ...imaging of miRNA with high sensitivity and specificity are achieved.•A set of two-input molecular logic gates are constructed based on DTM-bCHA.
Nonenzymatic isothermal amplification strategy has emerged as a versatile tool for microRNA detection. However, the slow kinetics, limited sensitivity and unsatisfactory efficiency often hinder their further practical applications. Herein, we proposed a DNA tetrahedron-mediated branched catalytic hairpin assembly (DTM-bCHA), achieving the dynamic fabrication of three-dimensional hyperbranched DNA structures. Compared with traditional target-catalyzed hairpin assembly, the reaction kinetics of DTM-bCHA is accelerated by 11.1-fold through tethering the hairpin probes on DNA tetrahedron due to the increased collision probability among the reactants. Moreover, the surface enhanced Raman scattering (SERS) hot spots are generated by assembling Raman reporter DTNB-functionalized gold nanoparticles (DTNB@AuNPs) with hyperbranched DNAs, leading to the ultrasensitive detection and in situ imaging of microRNA-21 (miR-21) in different living cells. Moreover, a series of two-input molecular logic gates, including AND, OR, NOR and INHIBIT gates, are constructed in response to miR-21 and miR-155, which achieves the multiplexed analysis of biomarkers in a facile way. Overall, the proposed DTM-bCHA based isothermal amplification strategy expands the avenues for the construction of complex DNA nanostructures, which is of great significance for bioanalysis and clinical diagnosis.
MicroRNAs (miRNAs), serving as the regulators for gene expression and cellular function, have emerged as the important biomarkers for diagnosis of cancers. In this study, a label-free electrochemical ...biosensing platform equipped with metal-organic frameworks (MOFs)-based nanoreactors has been developed by coupling three-dimensional (3D) DNA walker for amplification detection of miRNA. The MOF-based nanoreactors are constructed via the encapsulation of GOx in zeolitic imidazolate framework-8 (ZIF-8) driven by the rapid GOx-triggered nucleation of ZIF-8 with high catalytic activity, which also contributes to preserve the biological activity of GOx even in harsh environments. The gold nanoparticles (AuNPs) are further loaded on the surface of ZIF-8 by electrostatic adsorption, which can be used to not only anchor the orbit of 3D DNA walker by Au–S covalent bond but also promote the electron transfer on electrode interface. In the presence of target miRNA-21, the 3D DNA walker is initiated, resulting in the recycling of targets and the immobilization of numerous fuel DNAs with G-quadruplex/hemin complex on the nanoreactors spontaneously. As a result, a cascade catalysis reaction is triggered in the confined space of ZIF-8 nanoreactors, where the H2O2 as an intermediate is generated with the oxidization of glucose catalyzed by GOx and subsequently decomposed by G-quadruplex/hemin HRP-mimicking DNAzyme for the further oxidation of ABTS to obtain a differential pulse voltammetry (DPV) signal. Under the optimal conditions, the proposed electrochemical biosensor exhibits an excellent performance for amplification detection of miRNA-21 in the dynamic working range from 0.1 nM to 10 μM with a detection limit of 29 pM, which opens a new way for clinical analysis of miRNAs and early diagnosis of cancers.
•GOx@ZIF-8 nanoreactors are prepared by rapid GOx-triggered nucleation of ZIF-8 with high catalytic activity.•AuNPs loaded on GOx@ZIF-8 are served as the support of 3D DNA orbits and the bridge for electron transport.•3D DNA walker triggered by target miRNA facilitates dual-enzyme cascade catalysis.•Successful analysis of human serum samples is demonstrated.
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•DNA addition polymerization (DAP) reaction is proposed for controllable assembly of DNA nanospheres (DNSs).•The DAP paradigm is consistent with the molecular logic operation ...consisted of cascaded AND gates with a feedback mechanism.•The DNSs with co-delivery of co-RNAi and chemo-drugs achieve synergistic therapy of cancer.
Nucleic acid, as a design medium, has become one of the most promising biopolymers to perform the dynamic functions, such as catalytic hybridization, target triggered self-assembly and DNA computing in the applications of biosensing, bioimaging and biomedicine. Herein, a DNA addition polymerization (DAP) reaction is proposed to guide the DNA self-assembly towards the construction of size-controlled DNA nanospheres (DNSs). Analogous to chemical step-growth addition polymerization, our proposed DAP reaction is carried out using DNA dimer and DNA hairpin as monomers, in which the initiator acts as both the stimulus and catalyst and N-arm DNA junctions as the structural units, resulting in the formation of three-dimensional DNSs without the generation of by-product. Notably, the designed DAP paradigm is consistent with the molecular logic operation consisted of cascaded AND gates with a feedback mechanism. Further, a versatile nano-drug delivery system is successfully constructed via functionalization the DNSs with chemo-drug (doxorubicin, Dox) and two different siRNAs (anaplastic lymphoma kinase-specific siRNA (ALK-siRNA) and survivin siRNA (Sur-siRNA)) simultaneously. The multifunctional DNSs with good stability and biocompatibility in vitro and in vivo demonstrate an excellent performance in gene-chemo combination therapy of anaplastic large cell lymphoma (ALCL). Overall, the DAP strategy provides a simple yet practical route to build multifunctional DNA nanomaterials, which sheds new light on the development of DNA nanotechnology and cancer therapy.
HARP gap-filling performance is related with trench profile. In this paper, the influence of STI morphology on HARP gap-filling performance is studied. Both the slight undercut between top SiN and ...active area (AA), and top SiN CD, don't show impact on HARP gap-filling performance for the observed range. The side wall angle is key factor. For side wall angle of 89°, we have proved from both theory and experiment, that even 0.5° reduction of side wall angle can greatly reduce the STI void density.
To improve land use efficiency, urban renewal must also consider urban microclimates and heat islands. Existing research has depended on manual interpretation of high-resolution optical satellite ...imagery to resolve land surface temperature (LST) changes caused by urban renewal; however, the acquired ground time series data tend to be uneven and unique to specific frameworks. The objective of this study was to establish a more general framework to study LST changes caused by urban renewal using multi-source remote sensing data. Specifically, urban renewal areas during 2007–2017 were obtained by integrating Landsat and yearly Phased Array type L-band Synthetic Aperture Radar (PALSAR) images, and LST was retrieved from Landsat thermal infrared data using the generalized single-channel algorithm. Our results showed that urban renewal land (URL) area accounted for 1.88% of urban land area. Relative LST between URL and general urban land (GUL) of Liwan, Yuexiu, Haizhu, and Tianhe districts dropped by 0.88, 0.42, 0.43, and 0.10 K, respectively, whereas those of Baiyun, Huangpu, Panyu, and Luogang districts presented opposite characteristics, with a rise in the LST of 0.98, 1.03, 1.63, and 2.11 K, respectively. These results are attributable to population density, building density, and landscape pattern changes during the urban renewal process.
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•The 3D porous sulfonated polyetheretherhetone (PEEK) was proposed for the application in drug delivery.•Controllable and durable BMP-2 release could be successfully achieved.•The ...osteogenic differentiation activity of BMP-2-loaded PEEK materials was optimized.
Polyetheretherketone (PEEK) is ideal for dental and orthopedic applications because its mechanical properties are similar to cortical bones. However, its inherent inert ability hinders its clinical applications. In this work, bone morphogenetic protein-2 (BMP-2) was immobilized onto the sulfonated PEEK (SPEEK) using lyophilization technology. The surface morphologies of the samples were analyzed by field-emission scanning electron microscopy (FE-SEM), and the chemical compositions were analyzed by energy-dispersive X-ray spectrometry (EDS). The release content of BMP-2 of the samples immersed in the PBS (pH = 7.4) was detected by a human BMP-2 ELISA kit. The results indicated that controllable and durable BMP-2 release was accomplished due to the three-dimensional (3D) network of sulfonated PEEK. The in vitro cellular experiments showed that the BMP-2-immobilized samples significantly enhanced the initial adhesion and spreading of rat bone mesenchymal stem cells (rBMSCs). Moreover, the collagen secretion, extracellular matrix mineralization and ALP activity were also improved. Thus, the BMP-2-immobilized samples greatly promoted the osteogenic differentiation of rBMSCs, which revealed that BMP-2 immobilization paves the way for the use of PEEK in clinical applications.
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