A bimetallic peroxidase-mimicking nanozyme with antifouling property for construction of sensor array to identify phosphoproteins and diagnose cancers.

Protein phosphorylation is a significant post-translational modification that plays a decisive role in the occurrence and development of diseases. However, the rapid and accurate identification of phosphoproteins remains challenging. Herein, a high-throughput sensor array has been constructed based on a magnetic bimetallic nanozyme (Fe3O4@ZNP@UiO-66) for the identification and discrimination of phosphoproteins. Attributing to the formation of Fe-Zr bimetallic dual active centers, the as-prepared Fe3O4@ZNP@UiO-66 exhibits enhanced peroxidase-mimicking catalytic activity, which promotes the electron transfer from Zr center to Fe(II)/Fe(III). The catalytic activity of Fe3O4@ZNP@UiO-66 can be selectively inhibited by phosphoproteins due to the strong interaction between phosphate groups and Zr centers, as well as the ultra-robust antifouling capability of zwitterionic dopamine nanoparticle (ZNP). Considering the diverse binding affinities between various proteins with the nanozyme, the catalytic activity of Fe3O4@ZNP@UiO-66 can be changed to various degree, leading to the different absorption responses at 420 nm in the hydrogen peroxide (H2O2) - 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) system. By simply extracting different absorbance intensities at various time points, a sensor array based on reaction kinetics for the discrimination of phosphoproteins from other proteins is constructed through linear discriminant analysis (LDA). Besides, the quantitative determination of phosphoproteins and identification of protein mixtures have been realized. Further, based on the differential level of phosphoproteins in cells, the differentiation of cancer cells from normal cells can also be implemented by utilizing the proposed sensor array, showing great potential in disease diagnosis.

Dual-mode optical biosensor based on multi-functional DNA structures for detecting bioactive small molecules.

Semiconducting polymer dots and hemin-functionalized DNA nanoflowers with excellent peroxidase-like activity and high fluorescent brightness are prepared for fluorescent/colorimetric dual-mode sensing of dopamine and glutathione as low as nM and µM, respectively. This biosensor is readily applied to the analysis of complicated biological samples with high selectivity and accuracy, which opens up promising prospects in clinical applications.

NIR-driven photoelectrochemical-fluorescent dual-mode biosensor based on bipedal DNA walker for ultrasensitive detection of microRNA.

Optical biosensors have become powerful tools for bioanalysis, but most of them are limited by optic damage, autofluorescence, as well as poor penetration ability of ultraviolet (UV) and visible (Vis) light. Herein, a near-infrared light (NIR)-driven photoelectrochemical (PEC)-fluorescence (FL) dual-mode biosensor has been proposed for ultrasensitive detection of microRNA (miRNA) based on bipedal DNA walker with cascade amplification. Fueled by toehold-mediated strand displacement (TMSD), the bipedal DNA walker triggered by target miRNA-21 is formed through catalytic hairpin assembly (CHA), which can efficiently move along DNA tracks on CdS nanoparticles (CdS NPs)-modified fluorine doped tin oxide (FTO) electrode, resulting in the introduction of upconversion nanoparticles (UCNPs) on electrode surface. Under 980 nm laser irradiation, the UCNPs serve as the energy donor to emit UV/Vis light and excite CdS NPs to generate photocurrent for PEC detection, while the upconversion luminescence (UCL) at 803 nm is monitored for FL detection. This PEC-FL dual-mode biosensor has achieved the ultrasensitive and accurate analysis of miRNA-21 in human serum and different gynecological cancer cells. Overall, the proposed dual-mode biosensor can not only couple the inherent features of each single-mode biosensor but also provide mutual authentication of testing results, which opens up a new avenue for early diagnosis of miRNA-related diseases in clinic.

Pyroelectric-Effect-Assisted Near-Infrared-Driven Photoelectrochemical Biosensor Based on Exponential DNA Amplifier for MicroRNA Detection.

Although near-infrared responsive photoelectrochemical (PEC) biosensors have less damage to biological components compared to UV-visible light, they still reveal an inferior response due to the rapid recombination of photogenerated electron-hole. In this study, a near-infrared-driven PEC biosensor is fabricated for microRNA (miRNA) detection via integrating photoelectricity and pyroelectricity. Upon the introduction of target miRNA-21, the exponential DNA amplifier is triggered based on enzyme-assisted strand displacement amplification (SDA), releasing multiple Ag2S reporter probes to hybridize with capture probes immobilized on a CdS-2-mercaptobenzimidazole (2MBI)-modified photoelectrode. As a result, under the stimulation of NIR, the photoelectric conversion of Ag2S NPs generates the photocurrents. In addition, due to the strong hole acceptor ability of MBI, the pyroelectric effect of CdS-2MBI nanocomposites is enhanced, which generates highly pyroelectro-induced charge separation efficiency and induces the pyroelectric current benefited from the spontaneous polarization of CdS-2MBI caused by the temperature variation under the function of Ag2S nanoheaters. Impressively, this PEC biosensor has achieved the sensitive and selective determination of miRNA-21 with a detection limit as low as 54 fM. Overall, this NIR-driven PEC biosensor based on pyroelectric and photoelectric effects opens up a new horizon for bioanalysis and early disease diagnosis.

Nanoscale Metal-Organic Frameworks Combined with Metal Nanoparticles and Metal Oxide/Peroxide to Relieve Tumor Hypoxia for Enhanced Photodynamic Therapy.

Photodynamic therapy (PDT) is a clinically approved noninvasive tumor therapy that can selectively kill malignant tumor cells, with promising use in the treatment of various cancers. PDT is typically composed of three important parts: the specific wavelength of light, photosensitizer (PS), and oxygen. With the progressing investigation on PDT treatment, the most recent attention has focused on improving photodynamic efficiency. Tumor hypoxia has always been a critical factor hindering the efficacy of PDT. Nanoscale metal-organic frameworks (nMOF), the fourth generation of PS, present great potential in photodynamic therapy. In particular, nMOF combined with metal nanoparticles and metal oxide/peroxide has demonstrated unique properties for enhanced PDT. The metal and metal oxide nanoparticles can catalyze H2O2 to generate oxygen or automatically produces oxygen, alleviating the hypoxia and improving the photodynamic efficiency. Metal peroxide nanoparticles can spontaneously produce oxygen in water or under acidic conditions. Therefore, this Review summarizes the recent development of nMOF combined with metal nanoparticles (platinum nanoparticles and gold nanoparticles) and metal oxide/peroxide (manganese dioxide, ferric oxide, cerium oxide, calcium peroxide, and magnesium peroxide) for enhanced photodynamic therapy by alleviating tumor hypoxia. Finally, future perspectives of nMOF combined nanomaterials in PDT are put forward.

Functional Nucleic Acids-Engineered Bio-Barcode Nanoplatforms for Targeted Synergistic Therapy of Multidrug-Resistant Cancer.

Rational design of multifunctional nanomedicines has revolutionized the therapeutic efficacy of cancers. Herein, we have constructed the functional nucleic acids (FNAs)-engineered nanoplatforms based on the concept of a bio-barcode (BBC) for synergistic targeted therapy of multidrug-resistant (MDR) cancer. In this study, the platinum(IV) prodrug is synthesized to covalently link two kinds of FNAs at a rational ratio to fabricate three-dimensional BBC-like DNA nanoscaffolds, accompanied by the one-pot encapsulation of ZnO nanoparticles (NPs) through electrostatic interaction. The multivalent AS1411 aptamers equipped in ZnO@BBCs facilitate specific and efficient endocytosis into MDR human lung adenocarcinoma cells (A549/DDP). In response to the intracellular environment of A549/DDP cells, such as the lysosome-acidic pH and overexpressed GSH, the ZnO NPs are degraded into Zn2+ ions for generating reactive oxygen species (ROS), while the Pt(IV) prodrugs are reduced into Pt(II) active species by glutathione (GSH), followed by the release of therapeutic DNAzymes for chemotherapy and gene therapy. In particular, the designed system plays an important role in remodeling the intracellular environment to reverse cancer MDR. On the one hand, the depletion of GSH promotes the downregulation of glutathione peroxidase 4 (GPX4) for amplifying oxidative stress and increasing lipid peroxidation (LPO), resulting in the activation of ferroptosis. On the other hand, the silence of early growth response protein 1 (Egr-1) mRNA by Zn2+-dependent DNAzymes directly inhibits the proliferation and migration of MDR cells, which further suppresses the P-glycoprotein (P-gp)-mediated drug efflux. Thus, the proposed nanoplatforms show great promise for the development of versatile therapeutic tools and personalized nanomedicines for MDR cancers.

Hierarchical Porous Metal-Organic Framework as Biocatalytic Microreactor for Enzymatic Biofuel Cell-Based Self-Powered Biosensing of MicroRNA Integrated with Cascade Signal Amplification.

Enzymatic biofuel cells have become powerful tools in biosensing, which however generally suffer from the limited loading efficiency as well as low catalytic activity and poor stability of bioenzymes. Herein, the hierarchical porous metal-organic frameworks (MOFs) are synthesized using tannic acid (TA) for structural etching, which realizes co-encapsulation of glucose dehydrogenase (GDH) and nicotinamide adenine dinucleotide (NAD+ ) cofactor in zeolitic imidazolate framework (ZIF-L) and are further used as the biocatalytic microreactors to modify bioanode. In this work, the TA-controlled etching can not only expand the pore size of microreactors, but also achieve the reorientation of enzymes in their lower surface energy form, therefore enhancing the biocatalysis of cofactor-dependent enzyme. Meanwhile, the topological DNA tetrahedron is assembled on the microreactors, which acts as the microRNA-responsive "lock" to perform the cascade signal amplification of exonuclease III-assisted target recycling on bioanode and hybridization chain reaction (HCR) on biocathode. The proposed self-powered biosensor has achieved a detection limit as low as 2 aM (6 copies miRNA-21 in a 5 µL of sample), which is further successfully applied to identify cancer cells and clinical serums of breast cancer patients based on the different levels of miRNA-21, holding great potential in accurate disease identification and clinical diagnosis.

A colorimetric and photothermal dual-mode biosensing platform based on nanozyme-functionalized flower-like DNA structures for tumor-derived exosome detection.

Tumor-derived exosomes can be served as a kind of promising biomarkers for early diagnosis of cancers. Herein, a colorimetric/photothermal dual-mode exosomes sensing platform is developed for human breast cancer cell (MCF-7)-derived exosomes based on encapsulation of 3,3',5,5'-tetramethylbenzidine-loaded graphene quantum dot nanozymes (TMB-GQDzymes) into DNA flowers (DFs) via rolling circle amplification (RCA). To achieve specific detection, EpCAM aptamer for MCF-7 cell-derived exosomes is immobilized on the well plate, while the complementary sequence of another CD63 aptamer is designed into the circular template to obtain abundant capture probes. Benefitting from the dual-aptamer recognition strategy, a sandwich structure of EpCAM aptamer/exosomes/TMB-GQDzymes@DFs is formed, in which the GQDzymes can catalyze the oxidation of TMB in the presence of H2O2. The resulting products of TMB oxidation (oxTMB) can induce not only the absorption changes but also a near-infrared (NIR) laser-driven photothermal effect, achieving dual-mode detection of exosomes with the limit of detection (LOD) of 1027 particles/µL (colorimetry) and 2170 particles/µL (photothermal detection), respectively. In addition, this sensing platform has demonstrated excellent performance to well distinguish breast cancer patients from healthy individuals in serum samples analysis. Overall, the proposed dual-readout biosensor opens promising prospects for exosome detection in biological study and clinical applications.

Recent Progress in Sensor Arrays: From Construction Principles of Sensing Elements to Applications.

The traditional sensors are designed based on the "lock-and-key" strategy with high selectivity and specificity for detecting specific analytes, which however are not suitable for detecting multiple analytes simultaneously. With the help of pattern recognition technologies, the sensor arrays excel in distinguishing subtle changes caused by multitarget analytes with similar structures in a complex system. To construct a sensor array, the multiple sensing elements are undoubtedly indispensable units that will selectively interact with targets to generate the unique "fingerprints" based on the distinct responses, enabling the identification among various analytes through pattern recognition methods. This comprehensive review mainly focuses on the construction strategies and principles of sensing elements, as well as the applications of sensor array for identification and detection of target analytes in a wide range of fields. Furthermore, the present challenges and further perspectives of sensor arrays are discussed in detail.

Biometric Photoelectrochemical-Visual Multimodal Biosensor Based on 3D Hollow HCdS@Au Nanospheres Coupled with Target-Induced Ion Exchange Reaction for Antigen Detection.

Three-dimensional (3D) hollow photoactive nanomaterials can enhance light capture due to the light scattering benefiting from the unique hollow nanostructures, which contributes to the decrease in energy loss and the electron-hole recombination during the process of photoelectric conversion. Herein, a 3D hollow HCdS@Au nanosphere synthesized by the templated-assisted method and photodeposition is employed to construct a multimodal sensing platform by combining the photoelectrochemical (PEC) biosensor with colorimetric analysis and photothermal imaging. In the presence of target carcinoembryonic antigen (CEA), a sandwich structure is formed on magnetic beads based on the dual-aptamer recognition, followed by the initiation of rolling circle amplification (RCA) to bind numerous CuO-DNA probes. Upon stimulation by chlorhydric acidic, a large number of Cu2+ is released from CuO, which could interact with yellow HCdS@Au on electrode to produce dark CuS by ion exchange. As a result, with increased CEA level, the photocurrent is weakened and the color of electrode interface is changed from yellow to dark, which thus facilitates the PEC and colorimetric detection of CEA. Simultaneously, the formed CuS with highly photothermal effect can achieve qualitative visual analysis of CEA using a portable infrared thermal imager. This work exhibits an excellent performance for sensitive and selective detection of CEA in the dynamic working range from 0.015 to 2.4 ng/mL with a detection limit as low as 3.5 pg/mL. Moreover, the proposed PEC biosensor is successfully applied to CEA determination in human serum, which holds great promise in accurate analysis of biomarkers and early diagnosis of diseases in the clinic.

A nanozyme-based colorimetric sensor array as electronic tongue for thiols discrimination and disease identification.

Thiol analysis is of vital significance due to the essential roles in disease diagnosis, while the highly similar structures of thiols are a major challenge in practical determination. Herein, a nanozyme-based colorimetric sensor array has been proposed as electronic tongue for excellent discrimination and sensitive quantitation of thiols. The sensing units are fabricated by integrating the terephthalic acid modified graphene quantum dots (TPA@GQDs) with three transition metal ions (Fe2+, Cu2+ and Zn2+) via coordination, respectively, which not only provide sufficient substrate binding sites but also form the metal ion-regulated catalytic active centers. In this way, disparate promotion degrees on the peroxidase-like catalytic activity have been achieved in different metal ion-TPA@GQD ensembles. Based on the strong binding affinity between metal ions and thiols, the catalytic active centers are removed from TPA@GQDs, which inhibits the catalytic activity of sensing unit to diverse degrees. Accordingly, using 3, 3', 5, 5'-tetramethylbenzidine (TMB) as chromogenic substrate in the presence of hydrogen peroxide (H2O2), each sensing unit can generate differential colorimetric signals (fingerprints) for six thiol analytes, which can be accurately discriminated through linear discriminant analysis (LDA) with a detection limit of 50 nM. In addition, the discrimination of the same thiol with different concentrations and thiol mixtures have also been achieved. Furthermore, inspired by the distinct levels of thiols in practical samples, the proposed sensor array enables the identification of thiol-associated diseases by means of machine learning algorithm, which makes a positive contribution to medical diagnosis.

Upconversion Nanoparticle@Au Core-Satellite Assemblies for In Situ Amplified Imaging of MicroRNA in Living Cells and Combined Cancer Phototherapy.

Stimuli-responsive therapy of cancer with spatial and temporal control is crucial in improving the treatment efficacy and minimizing the side effects. MicroRNA (miRNA) as an important biomarker has become one of the most promising endogenous stimuli for cancer therapy. However, the therapy efficacy is often impeded by the low expression amount of miRNA. Herein, the upconversion nanoparticle@Au (UCNP@Au) core-satellite nanostructures are rationally fabricated for isothermal amplification detection and in situ imaging of microRNA-21 (miR-21) in living cells based on the toehold-mediated strand displacement (TMSD) reaction, which is further applied to miRNA-responsive combined photothermal and photodynamic therapy of breast cancer. The UCNP@Au are constructed by linking AuNPs to photosensitizers Rose Bengal (RB)-loaded UCNPs through DNA hybridization. The upconversion luminescence (UCL) is quenched by AuNPs, resulting in the attenuation of singlet oxygen generation of RB. Once UCNP@Au are internalized into MCF-7 cells, the overexpressed intracellular miR-21 trigger the cyclic disassembly of UCNP@Au through cascade TMSD reactions, which facilitate the restoration of UCL for in situ imaging of miR-21 with signal amplification. Moreover, the released AuNPs are aggregated for photothermal therapy (PTT), while the singlet oxygen generated by RB is enhanced for photodynamic therapy (PDT). Compared with single-mode therapy, the miRNA-activated combinational phototherapy has demonstrated a greatly improved therapeutic efficacy for breast cancer. Therefore, our proposed core-satellite nanostructures cannot only achieve in situ amplified imaging of endogenous miRNA but also provide an effective nanoplatform for stimuli-responsive combinational phototherapy, which hold great prospects in early diagnosis and treatment of cancers.

Two-Dimensional Quantum Dot-Based Electrochemical Biosensors.

Two-dimensional quantum dots (2D-QDs) derived from two-dimensional sheets have received increasing interest owing to their unique properties, such as large specific surface areas, abundant active sites, good aqueous dispersibility, excellent electrical property, easy functionalization, and so on. A variety of 2D-QDs have been developed based on different materials including graphene, black phosphorus, nitrides, transition metal dichalcogenides, transition metal oxides, and MXenes. These 2D-QDs share some common features due to the quantum confinement effects and they also possess unique properties owing to their structural differences. In this review, we discuss the categories, properties, and synthetic routes of these 2D-QDs and emphasize their applications in electrochemical biosensors. We deeply hope that this review not only stimulates more interest in 2D-QDs, but also promotes further development and applications of 2D-QDs in various research fields.

Metal-organic framework nanoreactor-based electrochemical biosensor coupled with three-dimensional DNA walker for label-free detection of microRNA.

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.

Trimetallic AuPtCo Nanopolyhedrons with Peroxidase- and Catalase-Like Catalytic Activity for Glow-Type Chemiluminescence Bioanalysis.

Chemiluminescence (CL) with stable and glowing light emission is vital for the accurate detection of biomarkers. Moreover, the catalyst plays an important role in CL systems. Herein, the trimetallic AuPtCo nanopolyhedrons with peroxidase- and catalase-like catalytic activities are readily synthesized via a one-step reduction method. After reaction with the substrate ABEI and oxidant H2O2, the AuPtCo nanozyme can catalyze the CL emission in a flash type. Interestingly, it has been found that the biofunctionalization of the AuPtCo surface can endow the catalytic interface with a slow-diffusion effect, thereby prolonging the emission of glow-type CL. On this basis, two biofunctionalized AuPtCo nanocomposites, named as AuPtCo@Cys and AuPtCo@Ab, are prepared, achieving sensitive and selective detection of H2O2 and lipoprotein-associated phospholipase A2 (Lp-PLA2), respectively. Further, the proposed glow-type CL assays are successfully applied for the determination of H2O2 and Lp-PLA2 in female vaginal discharge and human serum samples, respectively, which exhibit good correlation with the clinical results. Overall, the trimetallic AuPtCo nanozyme-based glow-type CL analysis has demonstrated as a powerful and robust tool for biomarker analysis, which holds great promise in clinical applications.

Endogenous microRNA triggered enzyme-free DNA logic self-assembly for amplified bioimaging and enhanced gene therapy via in situ generation of siRNAs.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Dual-mode glucose nanosensor as an activatable theranostic platform for cancer cell recognition and cascades-enhanced synergetic therapy.

Integration of disease diagnosis and therapy is crucial in precise medicine, while the "always on" mode often hinders its clinical applications. Herein, inspired by cascaded catalysis, an integrated dual-mode glucose nanosensor as an activable theranostic platform is developed, which is further exploited for cancer cell recognition and enhanced synergistic therapy of lymph cancer. This nanosensor is prepared through the in-situ growth of silver nanoparticles (AgNPs) with the synergetic reduction of tannic acid (TA) and graphene quantum dots (GQDs), which are further decorated with glucose oxidase (GOx). A cascaded catalytic reaction is triggered by glucose, in which GOx catalyzes the oxidation of glucose into gluconic acid and hydrogen peroxide (H2O2), and hydroxyl radical (•OH) is further produced with the catalysis of GQDs nanozyme with peroxidase-like activity, resulting in the degradation of AgNPs@GQDs-GOx with the release of Ag+. Accordingly, a "turn-off" colorimetric and "turn-on" fluorescence dual-mode glucose nanosensor is fabricated, which is readily applied for cancer cell recognition via fluorescence imaging based on the high glucose level in tumor microenvironment. Moreover, the degradation of AgNPs@GQDs-GOx in response to glucose facilitates the cascades-enhanced synergistic therapy of lymph cancer with the combination of starving-like therapy, metal ion therapy and TA-induce apoptosis. This study highlights a glucose-activated theranostic nanoplatform, which provides a great opportunity for cancer-related biosensing, bioimaging and biomedical applications.

Rolling Circle Replication for Biosensing, Bioimaging, and Biomedicine.

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.

Versatile electrochemical biosensor based on bi-enzyme cascade biocatalysis spatially regulated by DNA architecture.

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.

Recent advances in templated synthesis of metal nanoclusters and their applications in biosensing, bioimaging and theranostics.

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.