Always positive covalent organic nanosheet enabling pH-independent adsorption and removal of Cr(â ¥).

Rapid and highly effective removal of hexavalent chromium (Cr(Ⅵ)) is extremely vital to water resources restoration and environmental protection. To overcome the pH limitation faced by most ionic absorbents, an always positive covalent organic nanosheet (CON) material was prepared and its Cr(VI) adsorption and removal capability was investigated in detail. As-prepared EB-TFB CON (TFB = 1,3,5-benzaldehyde, EB = ethidium bromide) shows strong electropositivity in the tested pH range of 1 ∼ 10, display a pH-independent Cr(VI) removal ability, and work well for Cr(VI) pollution treatment with good anti-interference capability and reusability in a wide pH range covering almost all Cr(VI)-contaminated real water samples, thus eliminating the requirement for pH adjustment. Moreover, the nanosheet structure, which is obtained by a facile ultrasonic-assisted self-exfoliation, endows EB-TFB CON with fully exposed active sites and shortened mass transfer channels, and the Cr(VI) adsorption equilibrium can be reached within 15 min with a high adsorption capacity of 280.57 mg·g-1. The proposed Cr(VI) removal mechanism, which is attributed to the synergetic contributions of electrostatic adsorption, ion exchange and chemical reduction, is demonstrated by experiments and theoretical calculations. This work not only provides a general Cr(VI) absorbent without pH limitation, but also presents a paradigm to prepare ionic CONs with relatively constant surface charges.

DNA-Functionalized Porphyrinic Metal-Organic Framework-Based Drug Delivery System for Targeted Bimodal Cancer Therapy.

A DNA-functionalized porphyrinic MOF (porMOF) drug delivery system was successfully constructed. porMOF as a photosensitizer and drug delivery carrier can integrate photodynamic therapy (PDT) and chemotherapy. Via the strong coordination interaction between the zirconium cluster of porMOF and the terminal phosphate group of DNA, the stable modification of the DNA layer on the porMOF surface is achieved. Meanwhile, the introduction of C/G-rich base pairs into the DNA double-stranded structure provides more binding sites of chemotherapeutic drug doxorubicin (DOX). AS1411, an aptamer of nucleolin proteins that are overexpressed by cancer cells, is introduced in the double-stranded terminal, which can endow the nanosystem with the ability to selectively recognize cancer cells. C-rich sequences in DNA double strands form an i-motif structure under acidic conditions to promote the highly efficient release of DOX in cancer cells. In vitro and in vivo experiments demonstrate that the synergistic PDT/chemotherapy modality achieves highly efficient cancer cell killing and tumor ablation without undesirable side effects.

Telomerase-activated Au@DNA nanomachine for targeted chemo-photodynamic synergistic therapy.

We successfully designed a smart activatable nanomachine for cancer synergistic therapy. Photodynamic therapy (PDT) and chemotherapy can be activated by intracellular telomerase while anti-cancer drugs can be effectively transported into tumour cells. An Sgc8 aptamer was designed, which can specifically distinguish tumour cells from normal cells and perform targeted therapy. The nanomachine entered the tumour cells by recognising PTK7, which is overexpressed on the surface of cancer cells. Then, the "switch" of the system was opened by TP sequence extension under telomerase stimulus. So, the chemotherapeutic drug DOX was released to achieve the chemotherapy, and the Ce6 labelled Sgc8-apt was released to activate the PDT. It was found that if no telomerase existed, the Ce6 would always be in an "off" state and could not activate the PDT. Telomerase is the key to controlling the activation of the PDT, which effectively reduces the damage photosensitisers cause to normal cells. Using in vitro and in vivo experiments, the nanomachine shows an excellent performance in targeted synergistic therapy, which is expected to be utilised in the future.

Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing.

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 µM, and 8.3 × 10-5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.

Efficient food safety analysis for vegetables by a heteropore covalent organic framework derived silicone tube with flow-through purification.

A heteropore covalent organic framework incorporated silicone tube (S-tube@PDA@COF) was used as adsorbent to purify the matrices in vegetable extracts. The S-tube@PDA@COF was fabricated by a facile in-situ growth method and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and N2 adsorption-desorption. The as-prepared composite exhibited high removal efficiency of phytochromes and recovery (81.13-116.62%) of 15 chemical hazards from 5 representative vegetable samples. This study opens a promising avenue toward the facile synthesis of covalent organic frameworks (COFs)-derived silicone tubes for streamline operation in food sample pretreatment.

Lignin-based covalent organic polymers with improved crystallinity for non-targeted analysis of chemical hazards in food samples.

Lignin, the most abundant source of renewable aromatic compounds derived from natural lignocellulosic biomass, has great potential for various applications as green materials due to its abundant active groups. However, it is still challenging to quickly construct green polymers with a certain crystallinity by utilizing lignin as a building block. Herein, new green lignin-based covalent organic polymers (LIGOPD-COPs) were one-pot fabricated with water as the reaction solvent and natural lignin as the raw material. Furthermore, by using paraformaldehyde as a protector and modulator, the LIGOPD-COPs prepared under optimized conditions displayed better crystallinity than reported lignin-based polymers, demonstrating the feasibility of preparing lignin-based polymers with improved crystallinity. The improved crystallinity confers LIGOPD-COPs with enhanced application performance, which was demonstrated by their excellent performances in sample treatment of non-targeted food safety analysis. Under optimized conditions, phytochromes, the main interfering matrices, were almost completely removed from different phytochromes-rich vegetables by LIGOPD-COPs, accompanied by "full recovery" of 90 chemical hazards. Green, low-cost, and reusable properties, together with improved crystallinity, will accelerate the industrialization and marketization of lignin-based COPs, and promote their applications in many fields.

Easily operated COF-based monolithic sponges as matrix clean-up materials for non-targeted analysis of chemical hazards in oil-rich foods.

Non-targeted analysis of chemical hazards in foods plays a crucial role in controlling food safety. However, because it brings forward high demand for sample pretreatment, materials suitable for the pretreatment of foods, especially animal foods, are rare. Herein, covalent organic frameworks (COF)-based monolithic materials were constructed by three successive steps: preparation of polydimethylsiloxane (PDMS) sponge using sugar cube as a sacrificial template, loading of a heteroporous COF on PDMS sponge via ultrasonic or in-situ growth method, coating of the obtained PDMS@COF by polydopamine (PDA) network. As-prepared PDMS@COF@PDA sponges were demonstrated to work well in sample pretreatment of animal foods for non-targeted analysis of chemical hazards. After a simple vortex treatment for about 2 min, more than 98% triglycerides, the main interfering matrix components in animal foods, could be removed from lard and pork samples, accompanied by "full recovery" (recovery efficiencies: ≥63%) of 44 chemical hazards with different physicochemical properties. Besides providing promising sample pretreatment materials for non-targeted food safety analysis, this work also paves a feasible way to improve COF-based monolithic materials and thus promote their practical applications, because we found that the introduction of PDA network on COF-based monolithic material surface could play a role in "killing three birds with one stone": enhancing the stability of the materials by overcoming the detachment of COF during operations; controllably adjusting hydrophobic and hydrogen-bonding interactions on the material surface to promote the removal of triglycerides; weakening the hydrophobic and π-π interactions between COF and chemical hazards to increase the recoveries of chemical hazards.

A CRISPR/Cas12a-responsive dual-aptamer DNA network for specific capture and controllable release of circulating tumor cells.

The separation and detection of circulating tumor cells (CTCs) have a significant impact on clinical diagnosis and treatment by providing a predictive diagnosis of primary tumors and tumor metastasis. But the responsive release and downstream analysis of live CTCs will provide more valuable information about molecular markers and functional properties. To this end, specific capture and controllable release methods, which can achieve the highly efficient enrichment of CTCs with strong viability, are urgently needed. DNA networks create a flexible, semi-wet three-dimensional (3D) microenvironment for cell culture, and have the potential to minimize the loss of cell viability and molecular integrity. More importantly, responsive DNA networks can be reasonably designed as smart sensors and devices to change shape, color, disassemble, and giving back to external stimuli. Here, a strategy for specifically collecting cells using a dual-aptamer DNA network is designed. The proposed strategy enables effective capture, 3D encapsulation, and responsive release of CTCs with strong viability, which can be used for downstream analysis of live cells. The programmability of CRISPR/Cas12a, a powerful toolbox for genome editing, is used to activate the responsive release of captured CTCs from the DNA network. After activation by a specified double-strand DNA (dsDNA) input, CRISPR/Cas12a cleaves the single-stranded DNA regions in the network, resulting in molecular to macroscopic changes in the network. Accompanied by the deconstruction of the DNA network into fragments, controllable cell release is achieved. The viability of released CTCs is well maintained and downstream cell analysis can be performed. This strategy uses the enzymatic properties of CRISPR/Cas12a to design a platform to improve the programmability and versatility of the DNA network, providing a powerful and effective method for capturing and releasing CTCs from complex physiological samples.

MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a.

Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO2 nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO2 nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO2 nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn2+ as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO2 nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO2 nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.

"RESET" Effect: Random Extending Sequences Enhance the Trans-Cleavage Activity of CRISPR/Cas12a.

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing applications. However, the lack of exploration on the fundamental properties of CRISPR/Cas12a not only discourages further in-depth studies of the CRISPR/Cas12a system but also limits the design space of CRISPR/Cas12a-based applications. Herein, a "RESET" effect (random extending sequences enhance trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, a single-stranded DNA, which is too short to work as the activator, can efficiently activate CRISPR/Cas12a after being extended a random sequence from its 3'-end, even when the random sequence folds into secondary structures. The finding of the "RESET" effect enriches the CRISPR/Cas12a-based sensing strategies. Based on this effect, two CRISPR/Cas12a-based biosensors are designed for the sensitive and specific detection of two biologically important enzymes.

Recent Advances in Constructing Higher-Order DNA Structures.

Molecular self-assembly is widely used in the fields of biosensors, molecular devices, efficient catalytic materials, and medical biomaterials. As the carrier of genetic information, DNA is a kind of biomacromolecule composed of deoxyribonucleotide units. DNA nanotechnology extends DNA of its original properties as a molecule that stores and transmits genetic information from its biological environment by taking advantage of its unique base pairing and inherent biocompatibility to produce structurally-defined supramolecular structures. With the continuously development of DNA technology, the assembly method of DNA nanostructures is not only limited on the basis of DNA hybridization but also other biochemical interactions. In this review, we summarize the latest methods used to construct higher-order DNA structures. The problems of DNA nanostructures are discussed and the future directions in this field are provided.

Controllable synthesis of uniform large-sized spherical covalent organic frameworks for facile sample pretreatment and as naked-eye indicator.

The successful application of covalent organic frameworks (COFs) depends on not only their unique chemical structures but also their morphology, size, and architecture. Spherical COFs (SCOFs) are attracted special attention due to the superiority of spherical materials in many applications. However, the synthesis of uniform large-sized SCOFs remains a challenge. Herein, by carefully optimizing the synthesis of a heteropore COF, we find that solvent type and catalyst concentration play important roles in determining the morphology and size of COFs, and eventually achieve the controllable synthesis of large SCOFs with uniform sizes ranging from 200 µm to 5 mm. The obtained SCOFs keep the dual-pore feature of the heteropore COF and show good stability and high crystallinity. To exhibit the superior application potential of SCOFs, the SCOFs with a size range of 200-300 µm were demonstrated to be promising solid-phase extraction (SPE) fillers. As-prepared SCOFs-packed SPE column could effectively remove ≥99% phytochrome matrix from 6 different vegetable samples in 10 s, accompanied by 72.56-112.37% recoveries of 33 chemical hazards with different physicochemical properties, thus showing greatly promising application prospects in sample pretreatment of nontargeted food safety analysis. By utilizing acid/base-adjusted reversible color change, millimeter-sized SCOFs were developed as an easy-to-operate and reusable naked-eye indicator of acids.

DNA nanolantern-mediated catalytic hairpin assembly nanoamplifiers for simultaneous detection of multiple microRNAs.

Simultaneous detection of multiple microRNAs (miRNAs) with high sensitivity can give accurate and reliable information for clinical applications. By uniformly anchoring hairpin probes on the surface of DNA nanolantern, a three-dimensional DNA nanostructure contains abundant and adjustable modification sites, highly integrated DNA nanoprobes were designed and developed as catalytic hairpin assembly (CHA)-based signal amplifiers for enzyme-free signal amplification detection of target miRNAs. The nanolantern-based CHA (NLC) amplifiers, which were facilely prepared via a simple "one-pot" annealing method, showed enhanced biostability, improved cell internalization efficiency, accelerated CHA reaction kinetics, and increased signal amplification capability compared to the single-stranded DNA hairpin probes used in traditional CHA reaction. By co-assembling multiple hairpin probes on a DNA nanolantern surface, as-prepared NLC amplifiers were demonstrated to work well for highly sensitive and specific imaging, expression level fluctuation analysis of two miRNAs in living cells, and miRNAs-guided tumor imaging in living mice. The proposed DNA nanolantern-based nanoamplifier strategy might provide a feasible way to promote the cellular and in vivo applications of nucleic acid probes.

Oxidative Cleavage-Based Three-Dimensional DNA Biosensor for Ratiometric Detection of Hypochlorous Acid and Myeloperoxidase.

Methods to detect and quantify disease biomarkers with high specificity and sensitivity in biological fluids play a key role in enabling clinical diagnosis, including point-of-care testing. Myeloperoxidase (MPO) is an emerging biomarker for the detection of inflammation, neurodegenerative diseases, and cardiovascular disease, where excess MPO can lead to oxidative damage to biomolecules in homeostatic systems. While numerous methods have been developed for MPO analysis, most techniques are challenging in clinical applications due to the lack of amplification methods, high cost, or other practical drawbacks. Enzyme-linked immunosorbent assays are currently used for the quantification of MPO in clinical practice, which is often limited by the availability of antibodies with high affinity and specificity and the significant nonspecific binding of antibodies to the analytical surface. In contrast, nucleic acid-based biosensors are of interest because of their simplicity, fast response time, low cost, high sensitivity, and low background signal, but detection targets are limited to nucleic acids and non-nucleic acid biomarkers are rare. Recent studies reveal that the modification of a genome in the form of phosphorothioate is specifically sensitive to the oxidative effects of the MPO/H2O2/Cl- system. We developed an oxidative cleavage-based three-dimensional DNA biosensor for rapid, ratiometric detection of HOCl and MPO in a "one-pot" method, which is simple, stable, sensitive, specific, and time-saving and does not require a complex reaction process, such as PCR and enzyme involvement. The constructed biosensor has also been successfully used for MPO detection in complex samples. This strategy is therefore of great value in disease diagnosis and biomedical research.

Signal amplification and output of CRISPR/Cas-based biosensing systems: A review.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) proteins are powerful gene-editing tools because of their ability to accurately recognize and manipulate nucleic acids. Besides gene-editing function, they also show great promise in biosensing applications due to the superiority of easy design and precise targeting. To improve the performance of CRISPR/Cas-based biosensing systems, various nucleic acid-based signal amplification techniques are elaborately incorporated. The incorporation of these amplification techniques not only greatly increases the detection sensitivity and specificity, but also extends the detectable target range, as well as makes the use of various signal output modes possible. Therefore, summarizing the use of signal amplification techniques in sensing systems and elucidating their roles in improving sensing performance are very necessary for the development of more superior CRISPR/Cas-based biosensors for various applications. In this review, CRISPR/Cas-based biosensors are summarized from two aspects: the incorporation of signal amplification techniques in three kinds of CRISPR/Cas-based biosensing systems (Cas9, Cas12 and Cas13-based ones) and the signal output modes used by these biosensors. The challenges and prospects for the future development of CRISPR/Cas-based biosensors are also discussed.

Heteropore covalent organic framework-based composite membrane prepared by in situ growth on non-woven fabric for sample pretreatment of food non-targeted analysis.

A heteropore covalent organic framework (COF)-based composite membrane material was prepared and proved to have a satisfactory effect on the pretreatment of vegetable samples. The composite membrane was fabricated by in situ growth of a dual-pore COF on the surface of polydopamine (PDA)-aminated non-woven (NW) fabric. Due to the difference in the strength of the interaction between the phytochromes/COF and the pesticides/COF, the removal of phytochromes and the recovery of pesticides can be achieved by adjusting the composition of the solution. Through a simple immersion or filtration operation, NW@PDA@COF composite membrane can quickly and almost completely remove interfering phytochromes in the samples. The recovery of pesticides was determined by HPLC-MS/MS, and the recovery efficiencies were 72.3~101.7% and 67.3~106.7% for immersion and filtration modes of five different vegetable samples, respectively; the RSD is between 1.1 and 19% (n = 3). The limits of detection and quantification for the 13 pesticides investigated were 0.08 µg·L-1 and 0.23 µg·L-1, respectively. A wide linear range of 1~1000 µg·L-1 was observed with R2 values from 0.9774 to 0.9998. The membrane can be repeatedly used for at least 10 times by using a facile elution treatment. Compared to other commonly used sample pretreatment materials, heteropore COF-based composite membrane is superior in terms of sorbent amount, treatment time, operation simplicity, and material reusability.

DNA nanostructure-based nucleic acid probes: construction and biological applications.

In recent years, DNA has been widely noted as a kind of material that can be used to construct building blocks for biosensing, in vivo imaging, drug development, and disease therapy because of its advantages of good biocompatibility and programmable properties. However, traditional DNA-based sensing processes are mostly achieved by random diffusion of free DNA probes, which were restricted by limited dynamics and relatively low efficiency. Moreover, in the application of biosystems, single-stranded DNA probes face challenges such as being difficult to internalize into cells and being easily decomposed in the cellular microenvironment. To overcome the above limitations, DNA nanostructure-based probes have attracted intense attention. This kind of probe showed a series of advantages compared to the conventional ones, including increased biostability, enhanced cell internalization efficiency, accelerated reaction rate, and amplified signal output, and thus improved in vitro and in vivo applications. Therefore, reviewing and summarizing the important roles of DNA nanostructures in improving biosensor design is very necessary for the development of DNA nanotechnology and its applications in biology and pharmacology. In this perspective, DNA nanostructure-based probes are reviewed and summarized from several aspects: probe classification according to the dimensions of DNA nanostructures (one, two, and three-dimensional nanostructures), the common connection modes between nucleic acid probes and DNA nanostructures, and the most important advantages of DNA self-assembled nanostructures in the applications of biosensing, imaging analysis, cell assembly, cell capture, and theranostics. Finally, the challenges and prospects for the future development of DNA nanostructure-based nucleic acid probes are also discussed.

Nonenzymatic catalytic assembly of valency-controlled DNA architectures for nanoparticles and live cell assembly.

The precise control over high-order DNA architecture assembly might be challenging due to complicated circuit design and functional unit synthesis. Here, we show an enzyme-free, catalytic assembly to construct nanometer and micrometer architectures in a bottom-up manner and applied them in nanoparticles and cell assembly.

Terminal deoxynucleotidyl transferase combined CRISPR-Cas12a amplification strategy for ultrasensitive detection of uracil-DNA glycosylase with zero background.

A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10-6 U/mL and 1 × 10-4 U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.