Self-Powered Engineering of Cell Membrane Receptors to On-Demand Regulate Cellular Behaviors.

On-demand engineering of cell membrane receptors to nongenetically intervene in cellular behaviors is still a challenge. Herein, a membraneless enzyme biofuel cell-based self-powered biosensor (EBFC-SPB) was developed for autonomously and precisely releasing Zn2+ to initiate DNAzyme-based reprogramming of cell membrane receptors, which further mediates signal transduction to regulate cellular behaviors. The critical component of EBFC-SPB is a hydrogel film on a biocathode which is prepared using a Fe3+-cross-linked alginate hydrogel film loaded with Zn2+ ions. In the working mode in the presence of glucose/O2, the hydrogel is decomposed due to the reduction of Fe3+ to Fe2+, accompanied by rapid release of Zn2+ to specifically activate a Zn2+-responsive DNAzyme nanodevice on the cell surface, leading to the dimerization of homologous or nonhomologous receptors to promote or inhibit cell proliferation and migration. This EBFC-SPB platform provides a powerful "sensing-actuating-treating" tool for chemically regulating cellular behaviors, which holds great promise in precision biomedicine.

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.

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.

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.

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.

One-pot alkali cutting-assisted synthesis of fluorescence tunable amino-functionalized graphene quantum dots as a multifunctional nanosensor for sensing of pH and tannic acid.

Herein, a one-pot alkali cutting-assisted synthesis approach has been developed to gain fluorescence (FL) tunable amino functionalized GQDs (NH2-GQDs), which exhibit concentration- and excitation-dependent FL behaviors, due to the self-assembled J-type aggregation effect and different electronic transitions governed by graphene basal plane and functional groups. While NH2-GQDs possess brighter FL emission than pristine GQDs, owning to the functionalization of amino groups with strong electron withdrawing ability. Particularly, the pH-dependent FL behavior of NH2-GQDs further reflects the FL emission mechanism originated from the intrinsic zigzag sites and introduced amino and carboxylic groups, which is available for pH sensing. Moreover, the NH2-GQDs also show a FL quenching upon reaction with tannic acid (TA), resulting in the construction of a FL turn-off TA sensing platform. A good linear relationship is obtained between logarithm of FL intensity (log F) and TA concentration in a linear dynamic range of 1-40 µM and a limit of detection of 43.3 nM (3σ/s, n = 9) is achieved, with a precision of 0.08% RSD at a concentration level of 5 µM (n = 9). This work features a simple and direct approach to acquire multifunctional nanosensor, providing great potential for further applications in chem/biosensing.

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.

Chemiluminescence and Bioluminescence Imaging for Biosensing and Therapy: In Vitro and In Vivo Perspectives.

Chemiluminescence (CL) and bioluminescence (BL) imaging technologies, which require no external light source so as to avoid the photobleaching, background interference and autoluminescence, have become powerful tools in biochemical analysis and biomedical science with the development of advanced imaging equipment. CL imaging technology has been widely applied to high-throughput detection of a variety of analytes because of its high sensitivity, high efficiency and high signal-to-noise ratio (SNR). Using luciferase and fluorescent proteins as reporters, various BL imaging systems have been developed innovatively for real-time monitoring of diverse molecules in vivo based on the reaction between luciferin and the substrate. Meanwhile, the kinetics of protein interactions even in deep tissues has been studied by BL imaging. In this review, we summarize in vitro and in vivo applications of CL and BL imaging for biosensing and therapy. We first focus on in vitro CL imaging from the view of improving the sensitivity. Then, in vivo CL applications in cells and tissues based on different CL systems are demonstrated. Subsequently, the recent in vitro and in vivo applications of BL imaging are summarized. Finally, we provide the insight into the development trends and future perspectives of CL and BL imaging technologies.

DNA flower-encapsulated horseradish peroxidase with enhanced biocatalytic activity synthesized by an isothermal one-pot method based on rolling circle amplification.

DNA nanotechnology has been developed to construct a variety of functional two- and three-dimensional structures for versatile applications. Rolling circle amplification (RCA) has become prominent in the assembly of DNA-inorganic composites with hierarchical structures and attractive properties. Here, we demonstrate a one-pot method to directly encapsulate horseradish peroxidase (HRP) in DNA flowers (DFs) during RCA. The growing DNA strands and Mg2PPi crystals lead to the construction of porous DFs, which provide sufficient interaction sites for spontaneously incorporating HRP molecules into DFs with high loading capacity and good stability. Furthermore, in comparison with free HRP, the DNA flower-encapsulated HRP (termed HRP-DFs) demonstrate enhanced enzymatic activity, which can efficiently biocatalyze the H2O2-mediated etching of gold nanorods (AuNRs) to generate distinct color changes since the longitudinal localized surface plasmon resonance (LSPR) frequency of AuNRs is highly sensitive to the changes in the AuNR aspect ratio. Through rationally incorporating the complementary thrombin aptamer sequence into the circular template, the synthesized HRP-DF composites are readily used as amplified labels for visual and colorimetric detection of thrombin with ultrahigh sensitivity and excellent selectivity. Therefore, our proposed strategy for direct encapsulation of enzyme molecules into DNA structures shows considerable potential applications in biosensing, biocatalysis, and point-of-care diagnostics.

Exonuclease-assisted target recycling amplification for label-free chemiluminescence assay and molecular logic operations.

A versatile exonuclease III (Exo III)-assisted target recycling amplification (Exo-TRA) strategy is demonstrated to achieve label-free and homogeneous chemiluminescence (CL) detection of target DNA. Inspired by this principle, a series of logic gates are constructed using DNA molecules as "inputs" to activate the Exo-TRA system.

Cross-catalytic hairpin assembly-based exponential signal amplification for CRET assay with low background noise.

A toehold-mediated strand displacement (TMSD)-based cross-catalytic hairpin assembly (C-CHA) is demonstrated in this study, achieving exponential amplification of nucleic acids. Functionally, this system consists of four hairpins (H1, H2, H3 and H4) and one single-stranded initiator (I). Upon the introduction of I, the first CHA reaction (CHA1) is triggered, leading to the self-assembly of hybrid H1·H2 that then initiates the second CHA reaction (CHA2) to obtain the hybrid H3·H4. Since the single-stranded region in H3·H4 is identical to I, a new CHA1 is initiated, which thus achieves cross operation of CHA1 and CHA2 and exponential growth kinetics. Interestingly, because the C-CHA performs in a cascade manner, this system can be considered as multi-level molecular logic circuits with feedback mechanism. Moreover, through incorporating G-quadruplex subunits and fluorescein isothiocyanate (FITC) in the product of H1·H2, this C-CHA is readily utilized to fabricate a chemiluminescence resonance energy transfer (CRET) biosensing platform, achieving sensitive and selective detection of DNA and microRNA in real samples. Since the high background signal induced by FITC in the absence of initiator is greatly reduced through labeling quencher in H1, the signal-to-noise ratio and detection sensitivity are improved significantly. Therefore, our proposed C-CHA protocol holds a great potential for further applications in not only building complex autonomous systems but also the development of biosensing platforms and DNA nanotechnology.