Dual-mode detection of 2,6-pyridinedicarboxylic acid based on the enhanced peroxidase-like activity and fluorescence property of novel Eu-MOFs.

2,6-Pyridinedicarboxylic acid (DPA) is a significant biomarker of anthrax, which is a deadly infectious disease for human beings. However, the development of a convenient anthrax detection method is still a challenge. Herein, we report a novel europium metal-organic framework (Eu-MOF) with an enhanced peroxidase-like activity and fluorescence property for DPA detection. The Eu-MOF was one-step synthesized using Eu3+ ions and 2-methylimidazole. In the presence of DPA, the intrinsic fluorescence of Eu3+ ions is sensitized, the fluorescence intensity linearly increases with an increase in DPA concentration, and the fluorescence color changes from blue to purple. Simultaneously, the peroxide-like activity of the Eu-MOF is enhanced by DPA, which can promote the oxidation of TMB to oxTMB. The absorbance values increase linearly with DPA concentrations, and the colorimetric images change from colorless to blue. The dual-mode detection of DPA has good sensitivity with a colorimetric detection limit of 0.67 µM and a fluorescent detection limit of 16.67 nM. Moreover, a simple detection method for DPA was developed using a smartphone with the RGB analysis system. A portable kit with standard color cards was developed using paper test strips. The proposed methods have good practicability for DPA detection in real samples. In conclusion, the developed Eu-MOF biosensor offers a valuable and general platform for anthrax diagnosis.

A novel metal-organic framework of Co-hemin for portable and visual colorimetric detection of 2,4-dichlorophenoxyacetic acid.

On-site quantitative analysis of 2,4-dichlorophenoxyacetic acid (2,4-D) is of significant importance for addressing increasing concerns about public health and environmental quality. Here, a novel metal-organic framework (MOF) of Co-hemin is synthesized and first used for on-site colorimetric monitoring of 2,4-D. 2,4-D as an inhibitor of alkaline phosphatase could specifically suppress the production of ascorbic acid, which restrained in situ etching of Co-hemin and further triggered the colorimetric response. In the colorimetric assay, Co-hemin displayed good oxidase-like activity without addition of H2O2, which could avoid the shortcomings of H2O2 such as toxicity and instability. The Co-hemin biosensor exhibited a relatively low detection limit of 33 ng mL-1 for 2,4-D by the UV method. Moreover, a smartphone based RGB analysis system for the sensitive detection of 2,4-D was developed, and exhibited a good linear relationship between the RGB model parameter and the concentration of 2,4-D. The operability and accuracy of the Co-hemin biosensor were confirmed by the quantitative determination of 2,4-D in real samples, such as serum and tap water. Also, the Co-hemin based colorimetric biosensor showed good selectivity and specificity. Moreover, the developed assays displayed good application in constructing complex logic gates. This work not only provided a portable and visual platform for on-site monitoring of 2,4-D, but also expanded application prospects in the field of complex biological analysis.

A novel metal-organic framework of Ba-hemin with enhanced cascade activity for sensitive glucose detection.

Early glucose detection is important in both healthy people and diabetic patients. Glucose biosensing based on glucose oxidase (GOX) is a common method. However, native proteins are mostly membrane impermeable and are prone to degradation in complex sample environments. Herein, we report a facile one-step biomineralization method by simply mixing aqueous solutions of hemin and barium nitrate with glucose oxidase (GOX) to form Ba-hemin@GOX composites. Glucose (Glu) is introduced through self-driven sampling to trigger the GOX-catalysed production of hydrogen peroxide, which could help the subsequent 3,3',5,5'-tetramethylbenzidine (TMB) oxidation reaction catalysed by Ba-hemin to yield the blue-coloured product. The sensor exhibited a detection limit as low as 3.08 µM. The operability and accuracy of the Ba-hemin@GOX biosensor were confirmed by the quantitative determination of glucose in real samples, such as tap water, serum and drinks. Moreover, the Ba-hemin@GOX-based colorimetric biosensor showed good selectivity, storage stability and recoverability. The experimental results reveal that a GOX activity of more than 90% was still maintained even after being incubated at 60 °C for 30 minutes, and Ba-hemin@GOX could be reused for glucose detection at least six times. Even after 30 days of storage, the relative activity was still more than 90%. Overall, the developed Ba-hemin@GOX biosensor provides a valuable and general platform for applications in colorimetric biosensing and medical diagnostics.

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection.

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

mRNA-Activated Multifunctional DNAzyme Nanotweezer for Intracellular mRNA Sensing and Gene Therapy.

Deoxyribozyme (DNAzyme) is regarded as a promising gene therapy drug. However, poor cellular uptake efficacy and low biological stability limit the utilization of DNAzyme in gene therapy. Here, we report a well-known programmable DNAzyme-based nanotweezer (DZNT) that provides a new strategy for the detection of TK1 mRNA and survivin mRNA-targeted gene silencing therapy. At the end of the DZNT arm, there are two functionalized single-stranded DNA and each consists of two parts: the segment complementary to TK1 mRNA and the split-DNAzyme segment. The hybridization with intracellular TK1 mRNA enables the imaging of TK1 mRNA. Meanwhile, the hybridization draws the split-DNAzyme close to each other and activates DNAzyme to cleave the survivin mRNA to realize gene silencing therapy. The results demonstrate that the DZNT nanocarrier has excellent cell penetration, good biocompatibility, and noncytotoxicity. DZNT can image intracellular biomolecule TK1 mRNA with a high contrast. Furthermore, the split-DNAzyme can efficiently cleave the survivin mRNA with the aid of TK1 mRNA commonly present in cancer cells, accordingly can selectively kill cancer cells, and has no harm to normal cells. Taken together, the multifunctional programmable DZNT provides a promising platform for the early diagnosis of tumors and gene therapy.

An intelligent nanodevice based on the synergistic effect of telomerase-triggered photodynamic therapy and gene-silencing for precise cancer cell therapy.

The development of intelligent and precise cancer therapy systems that enable accurate diagnosis and specific elimination of cancer cells while protecting normal cells to improve the safety and effectiveness of the treatment is still a challenge. Herein, we report a novel activatable nanodevice for precise cancer therapy. The nanodevice is constructed by adsorbing a DNA duplex probe onto MnO2 nanosheets. After cellular uptake, the DNA duplex probe undergoes telomerase-triggered conformation switching, resulting in a Ce6 "turn-on" signal for the identification of cancer cells. Furthermore, Deoxyribozyme (DNAzyme) is activated to catalyse the cleavage of survivin mRNA, actualizing a precise synergistic therapy in cancer cells involving photodynamic therapy and gene-silencing. The MnO2 nanosheets provide Mn2+ for the DNAzyme and relieve hypoxia to improve the efficiency of the photodynamic therapy. Live cell studies reveal that this nanodevice can diagnose cancer cells and specifically eliminate them without harming normal cells, so making the treatment safer and more effective. The developed DNA-MnO2 nanodevice provides a valuable and general platform for precise cancer therapy.

Improving resolving ability of expansion microscopy by varying crosslinker concentration.

Here, we systematically investigated the performance of expansion microscopy (ExM) with different crosslinker concentrations. We modified ExM with 0.06% N,N'-methylenebisacrylamide (MBAA) (termed 0.06%-MBAA ExM), increased the expansion factor to 5.7 and achieved a lateral resolution of ∼50 nm with a common confocal microscope.

A photocontrolled and self-powered bipedal DNA walking machine for intracellular microRNA imaging.

In this work, we report a photocontrolled and self-powered DNA walking machine with bipedal DNAzyme walkers for intracellular microRNA imaging.

A spatial-confinement hairpin cascade reaction-based DNA tetrahedral amplifier for mRNA imaging in live cells.

The three-dimensional (3D) DNA nanostructure has been got much attention due to its excellent biocompatibility, enhanced structural stability, highly programmable and perfect cell-delivery performance. Here, a novel 3D DNA tetrahedron amplifier (DTA) has been developed for rapid and efficient mRNA imaging in living cells using target catalyzing spatial-confinement hairpin DNA assembly cascade reaction inside the DNA nanostructure. The DTA was constructed by assembling a DNA tetrahedron with four DNA strands at first, and then by assembling two metastable DNA hairpins H1 (Cy5) and H2 (Cy3) at specific locations of the DNA tetrahedron. In the presence of target mRNA, the catalyzed hairpin assembly (CHA) reaction on the DTA could be triggered and a H1-H2 duplexes nanostructure could be formed, which would obtain a significant fluorescence resonance energy transfer (FRET) signal, and release the target mRNA could trigger next H1-H2 duplexes formation. Due to the 3D DNA tetrahedral spatial-confinement effect, the circular reaction of DTA could achieve rapid and efficient amplification detection of target mRNA in living cells. Moreover, the DTA show excellent structural stability and non-cytotoxicity. This strategy presents a versatile method for the ultrasensitive detection of biomarkers in living system and gains a deeper development of the DNA nanostructures in biomedical functions.

Aptamer-Functionalized DNA Origami for Targeted Codelivery of Antisense Oligonucleotides and Doxorubicin to Enhance Therapy in Drug-Resistant Cancer Cells.

Drug resistance is a major obstacle to the efficient therapy of drug-resistant cancer. To overcome this problem, we constructed a multifunctional DNA origami-based nanocarrier for codelivery of a chemotherapeutic drug (doxorubicin, Dox) and two different antisense oligonucleotides (ASOs; B-cell lymphoma 2 (Bcl2) and P-glycoprotein (P-gp)) into drug-resistant cancer cells for enhanced therapy. To increase the targeting ability of origami, staple strands with 5'-end extended MUC1 sequences were used in the preparation of aptamer-functionalized origami carrying ASOs (Apt-origami-ASO). Dox-loaded Apt-origami-ASO (Apt-Dox-origami-ASO) was prepared by electrostatic adsorption of Dox in origami. Atomic force microscopy (AFM) images demonstrated the successful preparation of Apt-origami-ASO. In vitro studies showed that the Apt-Dox-origami-ASO (Apt-DOA) could controllably release Dox in pH 5.0 phosphate-buffered saline (PBS) buffer and release ASOs in response to glutathione. Further experiments revealed that the origami could protect ASOs against nuclease degradation in 10% FBS. Confocal imaging showed that the Apt-DOA nanocarrier could efficiently enter the Hela/adriamycin (ADR) cells and escape from lysosomes for codelivery of Dox and ASOs into the cytoplasm. The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot assays testified the efficient silencing of Bcl2 and P-gp mRNA and downregulation of the corresponding protein expressions by Apt-DOA in Hela/ADR cells. Moreover, with the synergetic effect by codelivery of multi-ASOs and Dox, the anticancer assay showed that Apt-DOA could circumvent multidrug resistance and significantly enhance cancer therapy in Hela/ADR and MCF-7/ADR cells. Hence, this multifunctional origami-based codelivery nanocarrier presents a new strategy for efficient therapy of drug-resistant cancer.

Engineering a Biodegradable Nanocarrier for Enhancing the Response of T98G Cells to Temozolomide.

Temozolomide (TMZ), the most common DNA alkylating agent, is predominantly mediated by O6-methylguanine DNA lesions for the treatment of glioblastoma (GBM). When O6-methylguanine-DNA methyltransferase (MGMT) is present, TMZ-induced O6-methylguanine lesions are repaired, resulting in the emergence of resistance to chemotherapy. Herein, we attempted to enhance the response of T98G cells to TMZ by gene silencing of MGMT. In this work, we developed transition metal manganese (Mn)-doped mesoporous silica nanoparticles (MSNs) as a carrier system for the co-delivery of TMZ and 10-23 DNAzyme, and realized gene silencing to enhance the TMZ sensitivity in T98G cells. The intelligent theranostic platform based on manganese-doped mesoporous silica nanoparticles (Mn-MSNs) can be decomposed and release chemotherapy drugs under acidic pH and reducing conditions. Meanwhile, the produced Mn2+ could act as a cofactor of 10-23 DNAzyme to effectively cleave MGMT mRNA, knock down MGMT protein, and sensitize T98G cells to TMZ-induced apoptosis. By co-delivering TMZ and 10-23 DNAzyme employing Mn-MSNs, the concentrations of TMZ that needed to inhibit cell growth by 50% (IC50 values) decreased (by more than 3.8-fold) compared with free TMZ. This work shows that the designed platform holds great promise for advancing the treatment of drug-resistant cancer.

Biomineralized Metal-Organic Framework Nanoparticles Enable Enzymatic Rolling Circle Amplification in Living Cells for Ultrasensitive MicroRNA Imaging.

The enzymatic amplification strategy in living cells faces challenges of highly efficient intracellular codelivery of amplification reagents including DNA polymerase. In this work, we develop biomineralized metal-organic framework nanoparticles (MOF NPs) as a carrier system for intracellular codelivery of ϕ29 DNA polymerase (ϕ29DP) and nucleic acid probes and realize a polymerization amplification reaction in living cells. A pH-sensitive biodegradable MOF NP of zeolitic imidazolate framework-8 (ZIF-8) is utilized to encapsulate ϕ29DP and adsorb nucleic acid probes. After uptake into cells, the encapsulated ϕ29DP and surface-adsorbed DNA probes are released and escaped from endolysosomes. In the presence of ϕ29DP and deoxyribonucleotide triphosphates (dNTPs), the intracellular miRNA-21 triggers a rolling circle amplification (RCA) reaction and the autonomous synthesized Mg2+-dependent DNAzyme cleaves the fluorogenic substrate, providing a readout fluorescence signal for the monitoring of miRNA-21. This is the first example of the intracellular RCA reaction in living cells. Therefore, the proposed method provides new opportunities for achieving enzymatic amplification reaction in living cells.

A microRNA-triggered self-powered DNAzyme walker operating in living cells.

DNA-based nanomachines have received increasing attention due to their great potential to mimic natural biological motors and create novel modes of motion. Here, we report a DNAzyme-based walking machine, which can operate in living cells after triggered by intracellular miRNA-21. The walking machine is constructed by assembling DNAzyme walking strands and FAM-labeled substrate strands on a single gold nanoparticle (AuNP). The DNAzyme walking strand is first silenced by a blocker strand. After cellular uptake, DNAzyme-based walker can be triggered by intracellular miRNA-21 and autonomously walk along the AuNP-based 3D track fueled by DNAzyme-catalyzed substrate cleavage. Each walking step results in the cleavage of a substrate strand and the release of a FAM-labeled DNA strand, allowing real-time monitoring of the operation of the machine. The DNAzyme-based walking machine has been successfully applied to image and monitor miRNA-21 expression levels in living cells with excellent specificity and reliability. This walking machine would hold great potential in the miRNA associated biological research and disease diagnostics.

In Situ Synthesis of Ultrathin ZIF-8 Film-Coated MSNs for Codelivering Bcl 2 siRNA and Doxorubicin to Enhance Chemotherapeutic Efficacy in Drug-Resistant Cancer Cells.

Multiple drug resistance is a persistent obstacle for efficient chemotherapy of cancer. Herein, we report a novel drug delivery platform. A zeolitic imidazole framework-8 (ZIF-8) film with a few nanometer thickness was in situ synthesized on the surface of carboxylated mesoporous silica (MSN-COOH) nanoparticles (NPs) for pore blocking and efficient loading of small interfering RNAs to fabricate a pH-responsive drug delivery system. The ZIF-8 film could convert the charge of MSN-COOH from negative to positive for efficient loading of siRNA via electrostatic interactions and protect siRNA from nuclease degradation. The positively charged ZIF-8 film facilitates cellular uptake and endo-lysosome escape of the NPs. In addition, the ultrathin ZIF-8 film can decompose in the acidic endo-lysosome and trigger the intracellular release of siRNAs and chemotherapeutic drugs, leading to a significantly enhanced chemotherapeutic efficacy for multidrug-resistant cancer cells including MCF-7/ADR and SKOV-3/ADR cells as demonstrated by the confocal laser scanning microscopy image, cell viability assay, Annexin V&PI staining, and flow cytometry. This approach provides a promising strategy for pH-triggered, stimuli-responsive delivery of nucleic acid drugs and chemotherapeutic agents with remarkably enhanced chemotherapeutic efficacy.

Biomineralized Metal-Organic Framework Nanoparticles Enable Intracellular Delivery and Endo-Lysosomal Release of Native Active Proteins.

Efficient delivery and endo-lysosomal release of active proteins in living cells remain a challenge in protein-based theranostics. We report a novel protein delivery platform using protein-encapsulating biomineralized metal-organic framework (MOF) nanoparticles (NPs). This platform introduces an adapted biomimetic mineralization method for facile synthesis of MOF NPs with high protein encapsulation efficiency and a new polymer coating strategy to confer the NPs with long-term stability. In vitro results show that protein-encapsulating MOF NPs have the advantages of preserving protein activity for months and protecting proteins from enzyme-mediated degradation. Live cell studies reveal that MOF NPs enable rapid cellular uptake, efficient release and escape of proteins from endo-lysosomes, and preservation of protein activity in living cells. Moreover, the developed platform is demonstrated to enable easy encapsulation of multiple proteins in single MOF NPs for efficient protein co-delivery. To our knowledge, it is the first time that protein-encapsulating MOF NPs have been developed as a generally applicable strategy for intracellular delivery of native active proteins. The developed protein-encapsulating biomineralized MOF NPs can provide a valuable platform for protein-based theranostic applications.

A graphene oxide nanosensor enables the co-delivery of aptamer and peptide probes for fluorescence imaging of a cascade reaction in apoptotic signaling.

Cytochrome c (Cyt c) and caspase-3 are the key mediators in apoptotic signaling. As is known to all, the release of Cyt c from mitochondria is a vital caspase activation pathway and defines the point of no-return in cell apoptosis. However, it has not been reported that any fluorescence imaging tools could allow simultaneous visualization of Cyt c translocation and caspase-3 activation in apoptotic cells. Here, we develop a sensitive nanosensor that holds the capability of imaging of the released Cyt c from the mitochondria and a caspase-3 activation cascade reaction in apoptotic signaling. The nanosensor is constructed by the assembly of a fluorophore (Cy5)-tagged DNA aptamer on graphene nanosheets that have been covalently immobilized with a FAM-labeled peptide. After a spatially selective delivery into the cytoplasm, the Cy5-tagged DNA aptamer assembled on the nanosensor can bind with Cyt c released from the mitochondria to the cytoplasm and dissociate from graphene, triggering a red fluorescence signal. In addition, the caspase-3 activated by the Cyt c released to the cytoplasm can cleave the FAM-labeled peptide and result in a green fluorescence output. The nanosensor exhibits rapid response, high sensitivity and selectivity for in vitro assays, and high contrast imaging of Cyt c and caspase-3 in living cells. It also provides the method for the study of the kinetic relationship between the Cyt c translocation and caspase-3 activation through simultaneous imaging of Cyt c and caspase-3. The developed nanosensor described here will be an efficient and potential platform for apoptosis research.

Nanoscale Zeolitic Imidazolate Framework-8 for Ratiometric Fluorescence Imaging of MicroRNA in Living Cells.

MicroRNAs (miRNAs) play important roles in cell differentiation, proliferation, and apoptosis and have been recognized as valuable biomarkers for clinical disease diagnosis. Here, we adopt for the first time zeolitic imidazolate framework-8 (ZIF-8) as a nanocarrier to efficiently deliver a nucleic acid probe to living cells and develop a novel ratiometric fluorescence strategy based on DNAzyme for miRNA-21 imaging. A Cy5-labeled 8-17 DNAzyme strand and a Cy3-labeled substrate strand containing a segment complementary to the target miRNA-21 first form a duplex probe, and fluorescence resonance energy transfer (FRET) takes place. After adsorption on the ZIF-8 surface and cellular uptake, the probe/ZIF-8 nanocomplex degrades in acidic endosome and releases duplex probes and Zn2+, and the latter can act as an effective cofactor for 8-17 DNAzyme. The intracellular miRNA-21 hybridizes with the complementary segment of the substrate strand and results in dissociation from the DNAzyme-substrate duplex probe after DNAzyme cleaves the substrate into two fragments, accompanied by the change in the FRET signal. The proposed method has been applied to image miRNA-21 expression levels in MCF-7, HeLa, and L02 cells with high contrast and reliability. The fluctuation of miRNA-21 expression level induced by miRNA-21 mimic or inhibitor can also be monitored through the obvious imaging color change. Taken together, the proposed method provides a powerful tool for cancer diagnosis and miRNA-associated biological study.