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Biodegradation, particularly via enzymatic degradation, has emerged as an efficient and eco-friendly solution for Poly (ethylene terephthalate) (PET) pollution. The production of PET hydrolases plays a role in the large-scale enzymatic degradation. However, an effective variant, 4Mz, derived from Thermobifida fusca cutinase (Tfu_0883), was previously associated with a significant reduction in yield when compared to the wild-type enzyme. In this study, a novel cell membrane protection strategy was developed to enhance the yield of 4Mz. This approach increased the yield of 4Mz by 18.2-fold from shaken flasks to 3-L bioreactors, reaching a yield of 3.1 g·L -1 , the highest yield of a PET hydrolase described thus far. In addition, the raw culture broth from 4Mz was applied directly for the enzymatic degradation of PET bottles, achieving a 91.2 % degradation rate. These advancements render the large-scale enzymatic degradation of PET more feasible, thus contributing to the more sustainable management of plastic waste., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Photodynamic therapy (PDT) is a newly emerged strategy for disease treatment. One challenge of the application of PDT drugs is the side-effect caused by the non-specificity of the photosensitive molecules. Most of the photosensitizers may invade not only the pathogenic cells but also the normal cells. In recent, people tried to use special cargoes to deliver the drugs into target cells. DNA nanoflowers (NFs) are a kind of newly-emerged nanomaterial which constructed through DNA rolling cycle amplification (RCA) reaction. It is reported that the DNA NFs were suitable materials which have been widely applied as nanocargos for drug delivery in cancer chemotherapeutic treatment. In this paper, we have introduced a new multifunctional DNA NF which could be prepared through an one-pot RCA reaction. This proposed DNA NF contained a versatile AS1411 G-quadruplex moiety, which plays key roles not only for specific recognition of cancer cells but also for near-infrared ray based photodynamic therapy when conjugating with a special porphyrin molecule. We demonstrated that the DNA NF showed good selectivity toward cancer cells, leading to highly efficient photo-induced cytotoxicity. Moreover, the in vivo experiment results suggested this DNA NF is a promising nanomaterial for clinical PDT., (© 2024 Wiley-VCH GmbH.)

To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.

Neurological disorders are closely linked to the alterations in cell membrane permeability (CMP) and mitochondrial membrane potential (MMP). Changes in CMP and MMP may lead to damage and death of nerve cells, thus triggering the onset and progression of neurological diseases. Therefore, monitoring the changes of these two physiological parameters not only benefits the accurate assessment of nerve cell health status, but also enables providing key information for the diagnosis and treatment of neurological diseases. However, the simultaneous monitoring of these two cellular physiological parameters is still challenging. Herein, we design and synthesize two quinolinium-carbazole-derivated fluorescent probes (OQ and PQ). As isomers, the only difference in their chemical structures is the linking position of the carbazole unit in quinoline rings. Strikingly, such a subtle difference endows OQ and PQ with significantly different organelle-staining behaviors. PQ mainly targets at the nucleus, OQ can simultaneously stain cell membranes and mitochondria in normal cells, and performs CMP and MMP-dependent translocation from the cell membrane to mitochondria then to the nucleus, thus holding great promise as an intracellular translocation probe to image the changes of CMP and MMP. After unraveling the intrinsic mechanism of their different translocation abilities by combining experiments with molecular dynamics simulations and density functional theory calculations, we successfully used OQ to monitor the continuous changes of CMP and MMP in three neurological disease-related cell models, including oxidative stress-damaged, Parkinson's disease, and virus-infected ones. Besides providing a validated imaging tool for monitoring cellular physiological parameters, this work paves a promising route for designing intracellular translocation probes to analyze cellular physiological parameters associated with various diseases.

The assessment of atherosclerosis (AS) progression has emerged as a prominent area of research. Monitoring various pathological features of foam cell (FC) formation is imperative to comprehensively assess AS progression. Herein, a simple b enzo s piropyran- j uloli d ine-based probe, BSJD , with switchable dual-color imaging ability was developed. This probe can dynamically and reversibly adjust its molecular structure and fluorescent properties in different polar and pH environments. Such a polarity and pH dual-responsive characteristic makes it superior to single-responsive probes in dual-color imaging of lipid droplets (LDs) and lysosomes as well as monitoring their interaction. By simultaneously tracking various pathological features, including LD accumulation and size changes, lysosome dysfunction, and dynamically regulated lipophagy, more comprehensive information can be obtained for multiparameter assessment of FC formation progression. Using BSJD , not only the activation of lipophagy in the early stages and inhibition in the later phases during FC formation are clearly observed but also the important roles of lipophagy in regulating lipid metabolism and alleviating FC formation are demonstrated. Furthermore, BSJD is demonstrated to be capable of rapidly imaging FC plaque sites in AS mice with fast pharmacokinetics. Altogether, BSJD holds great promise as a dual-color organelle-imaging tool for investigating disease-related LD and lysosome changes and their interactions.

Sensors designed based on the trans -cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans -cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin , respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo , allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.

The spacing between cells has a significant impact on cell-cell interactions, which are critical to the fate and function of both individual cells and multicellular organisms. However, accurately measuring the distance between cell membranes and the variations between different membranes has proven to be a challenging task. In this study, we employ metal-induced energy transfer (MIET) imaging/spectroscopy to determine and track the intermembrane distance and variations with nanometer precision. We have developed a DNA-based molecular adhesive called the DNA nanobrush, which serves as a cellular adhesive for connecting the plasma membranes of different cells. By manipulating the number of base pairs within the DNA nanobrush, we can modify various aspects of membrane-membrane interactions such as adhesive directionality, distance, and forces. We demonstrate that such nanometer-level changes can be detected with MIET imaging/spectroscopy. Moreover, we successfully employed MIET to measure distance variations between a cellular plasma membrane and a model membrane. This experiment not only showcases the effectiveness of MIET as a powerful tool for accurately quantifying membrane-membrane interactions but also validates the potential of DNA nanobrushes as cellular adhesives. This innovative method holds significant implications for advancing the study of multicellular interactions.

Objective: To assess the extent of recurrent laryngeal nerve (RLN) and superior laryngeal nerve (SLN) damage in patients with idiopathic vocal cord paralysis (IVCP) exhibiting different paralytic sides., Methods: A total of 84 IVCP cases were evaluated using stroboscopic laryngoscopy, voice analysis, and laryngeal electromyography (LEMG). The results were compared between patients with left-sided paralysis and right-sided paralysis based on different disease courses (less than or more than 3 months)., Results: Initially, the average age and disease progression of IVCP patients were found to be similar regardless of the side of paralysis ( p  > .05). Additionally, there were no significant variations in voice indicators, such as MPT, DSI, and VHI, between IVCP patients with left and right vocal cord paralysis ( p  > .05). Furthermore, no disparities were detected in the latencies and amplitudes of the paralyzed RLN and SLN, as well as the durations and amplitudes of the action potentials in the paralyzed TM and PCM, among IVCP patients with left and right vocal cord paralysis ( p  > .05). Notably, the amplitudes of the left paralytic CM were significantly lower than those of the right paralytic CM (0.45 vs. 0.53, Z  = -2.013, p  = .044). In addition, no disparities were observed in APDs and amplitudes between the ipsilateral PCM and TM, either for patients with left or right vocal fold paralysis ( p  > .05). Finally, all the IVCP patients were subdivided into two subgroups according to different disease course (less than or more than 3 months), and in each subgroup, the comparison of voice indicators and LEMG results in IVCP patients with left or right vocal fold paralysis were similar with the above findings ( p  > .05)., Conclusion: Overall, the degree of RLN and SLN damage appeared to be similar in IVCP patients with left and right vocal cord paralysis, provided that the disease course was comparable., Level of Evidence: 4., Competing Interests: All authors have no conflict of interest., (© 2024 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals LLC on behalf of The Triological Society.)

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 N 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

There have been many analytical methods for natural estrogens in commercial dairy milk samples, but in most of which, only four major estrogens (estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-estradiol (αE2)) were included. This work developed an effective GC-MS analytical method for simultaneous analysis of twelve natural estrogens in commercial dairy milk sample, in which eight far-less well-known natural estrogens (2-hydroxyestone (2OHE1), 4-hydroxyestrone (4OHE1), 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 16-epiestriol (16epiE3), 16α-hydroxyestrone (16αOHE1), 16-ketoestradiol (16ketoE2) and 17epiestriol (17epiE3)) were included besides the four major natural estrogens. With liquid-liquid extraction and solid phase extraction, twelve natural estrogens in commercial dairy milk could be effectively extracted. The established method showed good linearity (R 2  > 0.9991), low limits of detections (LODs, 0.02-0.11 ng/g), as well as excellent recoveries (64-117%) with satisfactory low relative standard deviations (RSDs, 0.8-14.7%). This established method was applied to seven commercial dairy milk samples, and all the twelve natural estrogens were frequently detected except for 4OHE2 without detection in any sample. Our results showed that the concentration contribution ratios of the eight far-less well-known natural estrogens in commercial dairy milk samples contributed to 32-83%, while the corresponding contribution ratios based on estrogen equivalence (EEQ) were 21-62%. This work highlighted the high abundance of the eight far-less well-known natural estrogens in commercial dairy milk based on both concentration and EEQ, which has been neglected for a long time., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

C-C single bond-linked covalent organic frameworks (CSBL-COFs) are extremely needed because of their excellent stabilities and potential applications in harsh conditions. However, strategies to generate CSBL-COFs are limited to the acetylenic self-homocoupling Glaser-Hay reaction or post-synthetic reduction of vinylene-based COFs. Exploring new strategies to expand the realm of CSBL-COFs is urgently needed but extremely challenging. To address the synthetic challenges, we for the first time developed a general approach via the reaction between aromatic aldehydes and active methyl group-involving monomers with enhanced acidity, which realized the successful construction of a series of CSBL-COFs. As expected, the obtained CSBL-COFs exhibited outstanding chemical stability, which can stabilize in 6 M NaOH, 3 M HCl, boiling water, and 100 mg/mL NaBH 4 for at least 3 days. It is important to mention that CSBL-COFs possess a large amount of ionic sites distributed throughout the networks; gentle shaking allowed our COFs to easily self-disperse as nanoparticles and suspend in water for at least 12 h without reprecipitating. As far as we know, such self-dispersed COFs with high water dispersity are rare to date, and few examples are mainly limited to the guanidinium- and pseudorotaxane-based COFs. Our work thus developed a family of self-dispersed COFs for potential applications in different sorts of fields. Our contribution would thus pave a new avenue for constructing a broader class of CSBL-COFs for their wide applications in various fields.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Real-time visualization of individual viral mRNA translation activities in live cells is essential to obtain critical details of viral mRNA dynamics and to detect its transient responses to environmental stress. Fluorogenic RNA aptamers are powerful tools for real-time imaging of mRNA in live cells, but monitoring the translation activity of individual mRNAs remains a challenge due to their intrinsic photophysical properties. Here, we develop a genetically encoded turn-on 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-binding RNA nanozipper with superior brightness and high photostability by in situ self-assembly of multiple nanozippers along single mRNAs. The nanozipper enables real-time imaging of the mobility and dynamic translation of individual viral mRNAs in live cells, providing information on the spatial dynamics and translational elongation rate of viral mRNAs., (© 2023 Wiley-VCH GmbH.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: To analyze the clinical characteristics of patients with overweight acute type A aortic dissection, and to explore the risk factors of acute kidney injury in patients with overweight acute type A aortic dissection., Methods: From March 2019 to February 2022, the clinical data of 71 patients with acute type a aortic dissection diagnosed by CTA and undergoing surgical treatment with BMI > 24 in the First People's Hospital of Yunnan Province were retrospectively analyzed, and analyzed by univariate and logistic multivariate analysis methods., Results: The mean BMI of all included patients was 27.23, The mean surface area of all included human populations was 1.833. The mean age of all patients was (52.06 ± 10.71) years old, and 35 patients developed acute kidney injury after surgery. Multi-factor Logistics regression analysis confirmed the risk factors for postoperative acute kidney injury in overweight patients with acute type A aortic dissection, including gender, CPB transit time and intraoperative infusion of suspended red blood cells. Seven patients in the AKI group died in hospital after surgery and two patients died in the non-AKI group., Conclusions: Among patients with overweight acute Type A aortic dissection, the incidence of AKI is 49.30%. According to multi-factor Logistics regression analysis, gender, CPB transit time and intraoperative suspended red blood cell volume are independent risk factors for postoperative acute kidney injury in patients with overweight acute Type A aortic dissection., (© 2023. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Fluorescent molecular probes are very powerful tools that have been generally applied in cell imaging in the research fields of biology, pathology, pharmacology, biochemistry, and medical science. In the last couple of decades, numerous molecular probes endowed with high specificity to particular organelles have been designed to illustrate intracellular images in more detail at the subcellular level. Nowadays, the development of cell biology has enabled the investigation process to go deeply into cells, even at the molecular level. Therefore, probes that can sketch a particular organelle's location while responding to certain parameters to evaluate intracellular bioprocesses are under urgent demand. It is significant to understand the basic ideas of organelle properties, as well as the vital substances related to each unique organelle, for the design of probes with high specificity and efficiency. In this review, we summarize representative multifunctional fluorescent molecular probes developed in the last decade. We focus on probes that can specially target nuclei, mitochondria, endoplasmic reticulums, and lysosomes. In each section, we first briefly introduce the significance and properties of different organelles. We then discuss how probes are designed to make them highly organelle-specific. Finally, we also consider how probes are constructed to endow them with additional functions to recognize particular physical/chemical signals of targeted organelles. Moreover, a perspective on the challenges in future applications of highly specific molecular probes in cell imaging is also proposed. We hope that this review can provide researchers with additional conceptual information about developing probes for cell imaging, assisting scientists interested in molecular biology, cell biology, and biochemistry to accelerate their scientific studies.

A nitrogen (N), oxygen (O)-rich porphyrin-based covalent organic framework (COF), in which interlayer porphyrin molecules are vertically stacked, is prepared and characterized. As-prepared N,O-rich TpTph COF shows a high adsorption capacity for Cd 2+ due to the abundant coordination sites. More interesting, it is found that the formation of COF enlarges the porphyrin ring center space, thus facilitating the Cd 2+ coordination, and the resulting optical signal changes make the ratiometric detection of Cd 2+ possible. Furthermore, using carbon fiber (CF) filaments, which are obtained from low cost and easy-to-obtain actived carbon mask, as support, porphyrin COF-based CF@TpTph membrane is prepared through in-situ growth of COF on the support followed by simple mechanical pressing. The CF@TpTph membrane is demonstrated to work well for both Cd 2+ removal and enrichment from soil and water samples, and shows the advantages of ease of handling, robust stability, reduced secondary pollution risk to samples, and good reusability. This work provides a powerful tool for Cd 2+ removal and enrichment, exhibits that preparing porphyrin-based COFs is a feasible way to promote the interactions between porphyrin ring and Cd 2+ , and demonstrates that mechanical pressing is a promising strategy for the design of COF-based monolithic materials to promote the practical applications of COFs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Poly (ethylene terephthalate) (PET) is a widely used type of general plastic that produces a significant amount of waste due to its non-degradable properties. We propose a novel directional-path modification (DPM) strategy, involving positive charge amino acid introduction and binding groove remodeling, and apply it to Thermobifida fusca cutinase to enhance PET degradation. The highest value of PET degradation (90%) was achieved in variant 4Mz (H184S/Q92G/F209I/I213K), exhibiting values almost 30-fold that of the wild-type. We employed molecular docking, molecular dynamics simulations, and QM/MM MD for the degradation process of PET, accompanied by acylation and deacylation. We found that the distance of nucleophilic attack was reduced from about 4.6 Å in the wild type to 3.8 Å in 4Mz, and the free energy barrier of 4Mz dropped from 14.3 kcal/mol to 7.1 kcal/mol at the acylation which was the rate-limiting step. Subsequently, the high efficiency and universality of the DPM strategy were successfully demonstrated in LCC, Est119, and BhrPETase enhancing the degradation activity of PET. Finally, the highest degradation rate of the pretreated commercial plastic bottles had reached to 73%. The present study provides insight into the molecular binding mechanism of PET into the PET hydrolases structure and proposes a novel DPM strategy that will be useful for the engineering of more efficient enzymes for PET degradation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

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 MnO 2 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 MnO 2 nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO 2 nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn 2+ 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, MnO 2 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 MnO 2 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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., (© 2022 Wiley-VCH GmbH.)

The molecular biomarkers are molecules that are closely related to specific physiological states. Numerous molecular biomarkers have been identified as targets for disease diagnosis and biological research. To date, developing highly efficient probes for the precise detection of biomarkers has become an attractive research field which is very important for biological and biochemical studies. During the past decades, not only the small chemical probe molecules but also the biomacromolecules such as enzymes, antibodies, and nucleic acids have been introduced to construct of biosensor platform to achieve the detection of biomarkers in a highly specific and highly efficient way. Nevertheless, improving the performance of the biosensors, especially in clinical applications, is still in urgent demand in this field. A noteworthy example is the Corona Virus Disease 2019 (COVID-19) that breaks out globally in a short time in 2020. The COVID-19 was caused by the virus called SARS-CoV-2. Early diagnosis is very important to block the infection of the virus. Therefore, during these months scientists have developed dozens of methods to achieve rapid and sensitive detection of the virus. Nowadays some of these new methods have been applied for producing the commercial detection kit and help people against the disease worldwide. DNA-based biosensors are useful tools that have been widely applied in the detection of molecular biomarkers. The good stability, high specificity, and excellent biocompatibility make the DNA-based biosensors versatile in application both in vitro and in vivo. In this paper, we will review the major methods that emerged in recent years on the design of DNA-based biosensors and their applications. Moreover, we will also briefly discuss the possible future direction of DNA-based biosensors design. We believe this is helpful for people interested in not only the biosensor field but also in the field of analytical chemistry, DNA nanotechnology, biology, and disease diagnosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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/H 2 O 2 /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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

Cytochrome c (Cyt c ), one of the most significant proteins acting as an electron transporter, plays an important role during the transferring process of the energy in cells. Apoptosis, one of the major forms of cell death, has been associated with various physiological regularity and pathological mechanisms. It was found that Cyt c can be released from mitochondria to cytosol under different pathological conditions, triggering subsequent cell apoptosis. Herein, we developed a fluorescence nanoprobe based on negatively charged CuInS 2 -ZnS-GSH quantum dots (QDs) for the sensitive determination of Cyt c . CuInS 2 -ZnS-GSH QDs with high photochemical stability and favorable hydrophilicity were prepared by a simple hot reflux method and emit a bright orange-red light. The electron-deficient heme group in Cyt c is affiliated with the electron-rich CuInS 2 -ZnS-GSH QDs through the photo-induced electron transfer process, resulting in a large decrease in fluorescence intensity of QDs. A good linearity for concentration of Cyt c in the range of 0.01-7 μmol L -1 is obtained, and the detection limit of Cyt c is as low as 1.1 nM. The performance on the detection of Cyt c in spiked human serum and fetal bovine serum samples showed good recoveries from 85.5% to 95.0%. Furthermore, CuInS 2 -ZnS-GSH QDs were applied for the intracellular imaging in HeLa cells showing an extremely lower toxicity and excellent biocompatibility., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

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 R 2 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.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Accurate and specific analysis of adenosine triphosphate (ATP) expression levels in living cells can provide valuable information for understanding cell metabolism, physiological activities and pathologic mechanisms. Herein, DNA nanolantern-based split aptamer nanoprobes are prepared and demonstrated to work well for in situ analysis of ATP expression in living cells. The nanoprobes, which carry multiple split aptamer units on the surface, are easily and inexpensively prepared by a "one-pot" assembly reaction of four short oligonucleotide strands. A series of characterization experiments verify that the nanoprobes have good monodispersity, strong biostability, high cell internalization efficiency, and fluorescence resonance energy transfer (FRET)-based ratiometric response to ATP in the concentration range covering the entire intracellular ATP expression level. By changing the intracellular ATP level via different treatments, the nanoprobes are demonstrated to show excellent performance in intracellular ATP expression analysis, giving a highly ATP concentration-dependent ratiometric fluorescence signal output. ATP-induced formation of large-sized DNA aggregates not only amplifies the FRET signal output, but also makes in situ ATP-imaging analysis in living cells possible. In situ responsive crosslinking of nanoprobes also makes them capable of lighting up the mitochondria of living cells. By simply changing the split aptamer sequence, the proposed DNA nanolantern-based split aptamer strategy might be easily extended to other targets.

Peritrophic membrane (PM) refers to a vital physical barrier enabling shrimp to resist pathogen invasion. It primarily consists of chitin and proteins, mostly chitin-binding protein (CBP). CBPs have been identified from microorganisms to higher organisms. In the present study, a CBP, designated MjCBP, was reported from Marsupenaeus japonicus. The open reading frame of MjCBP was 1854 bp, encoding a protein with 618 amino acids (MH544098). To be specific, the theoretical pI and molecular mass of mature MjCBP reached 5.43 and 66064.00 Da, respectively. MjCBP consisted of seven type Ⅱ chitin-binding domains (ChtB D2), which was up-regulated after being challenged with Vibrio anguillarum and then agglutinating several bacteria. In addition, MjCBP and the first chitin-binding domain (CBD1) could bind to several Gram-positive and Gram-negative bacteria via the binding process to lipopolysaccharides and peptidoglycans, whereas CBD1 was not capable of agglutinating bacteria. Moreover, the anterior and posterior segments of CBD1 were synthesized in vitro, and the posterior segment could bind to lipopolysaccharides. However, both segments fail to agglutinate bacteria. Furthermore, MjCBP and CBD1 facilitated the clearance of V. anguillarum in vivo, and the silencing of MjCBP via RNA interference reduced the ability of bacterial clearance. As revealed from the mentioned results, MjCBP acts as an opsonin or pattern recognition receptor to achieve antibacterial immune response in shrimp., (Copyright © 2020. Published by Elsevier Ltd.)

Eastern Cooperative Oncology Group (ECOG) performance status and Gleason score are commonly investigated factors for overall survival (OS) in men with castration-resistant prostate cancer (CRPC). However, there is a lack of consistency regarding their prognostic or predictive value for OS. Therefore, we performed this meta-analysis to assess the associations of ECOG performance status and Gleason score with OS in CRPC patients and compare the two markers in patients under different treatment regimens or with different chemotherapy histories. A systematic literature review of monotherapy studies in CRPC patients was conducted in the PubMed database until May 2019. The data from 8247 patients in 34 studies, including clinical trials and real-world data, were included in our meta-analysis. Of these, twenty studies reported multivariate results and were included in our main analysis. CRPC patients with higher ECOG performance statuses (≥ 2) had a significantly increased mortality risk than those with lower ECOG performance statuses (<2), hazard ratio (HR): 2.10, 95% confidence interval (CI): 1.68-2.62, and P < 0.001. The synthesized HR of OS stratified by Gleason score was 1.01, with a 95% CI of 0.62-1.67 (Gleason score ≥ 8 vs <8). Subgroup analysis showed that there was no significant difference in pooled HRs for patients administered taxane chemotherapy (docetaxel and cabazitaxel) and androgen-targeting therapy (abiraterone acetate and enzalutamide) or for patients with different chemotherapy histories. ECOG performance status was identified as a significant prognostic factor in CRPC patients, while Gleason score showed a weak prognostic value for OS based on the available data in our meta-analysis., Competing Interests: None

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection. The proposed strategy showed a wide linear detection range (more than 3 orders of magnitude ranging from 1× 10 -5 to 2.5 × 10 -2 U/mL T4 PNKP) and a detection limit as low as 3.3 × 10 -6 U/mL. It was successfully used for the PNKP activity detection in cell extracts with high fidelity and displayed great potential for enzyme inhibitor screening and inhibitory capability evaluation. This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes and provides a way to improve the sensitivity by introducing an isothermal signal amplification step. Such an isothermal DNA amplification-CRISPR/Cas-combined biosensor design concept might expand CRISPR/Cas-based sensing systems and promote their applications in various fields such as disease diagnosis and drug screening., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel nucleic acid-based isothermal signal amplification strategy, named cross-boosting extension-nicking reaction (CBENR) is developed and successfully used for rapid and ultrasensitive detection of polynucleotide kinase (PNK) activity. Only two simple oligonucleotides (recognition substrate (RS) and TaqMan probe) are applied to construct the PNK-sensing platform. In the presence of PNK, the 3'-phosphate end of RS will be converted to the 3'-hydroxyl one, and then extended to a long poly-adenine (poly-A) sequence under the catalysis of terminal deoxynucleotidyl transferase (TdT). The poly-A sequence provides multiple binding sites for the TaqMan probe to form multiple DNA duplexes. Subsequently, ribonuclease HII (RNase HII) cuts the TaqMan probe into two parts at the pre-set uracil site, generating a fluorescence signal and providing new substrates for TdT elongation. The TdT-catalyzed substrate extension and RNase HII-catalyzed probe nicking are boosted by each other, resulting in persistent enlargement of these two reactions and thus giving ultrahigh signal amplification efficiency. Utilizing the CBENR-based PNK sensor, ultrasensitive detection of PNK activity was achieved with a detection limit as low as 3.0 × 10 -6 U mL -1 . Quantification of endogenous PNK activity at the single-cell level and the screening/evaluation of PNK inhibitors were also achieved.

Nontargeted analysis of food safety requires selective removal of interference matrices and highly efficient recovery of chemical hazards. Porous materials such as covalent organic frameworks (COFs) show great promise in selective adsorption of matrix molecules via size selectivity. Considering the complexity of interference matrices, we prepared crystalline heteropore COFs whose two kinds of pores have comparable sizes to those of several common phytochromes, main interference matrices in vegetable sample analysis. By controlling the growth of COFs on the surface of Fe 3 O 4 nanoparticles or by utilizing a facile co-electrospinning method, heteropore COF-based magnetic nanospheres or electrospun nanofiber films were prepared, respectively. Both the nanospheres and the films maintain the dual-pore structures of COFs and show good stability and excellent reusability. Via simple magnetic separation or immersion operation, respectively, they were successfully used for the complete removal of phytochromes and highly efficient recovery of 15 pesticides from the extracts of four vegetable samples, and the recoveries are in the range of 83.10-114.00 and 60.52-107.35%, respectively. Film-based immersion operation gives better sample pretreatment performance than the film-based filtration one. This work highlights the great application potentials of heteropore COFs in sample pretreatment for nontargeted analysis, thus opening up a new way to achieve high-performance sample preparation in many fields such as food safety analysis, environment monitoring, and so on.

Nucleic acid aptamers have been widely used in various fields such as biosensing, DNA chip, and medical diagnosis. However, the high susceptibility of nucleic acids to ubiquitous nucleases reduces the biostability of aptamers and limits their applications in biological contexts. Therefore, improving the biostability of aptamers becomes an urgent need. Herein, we present a simple strategy to resolve this problem by directly replacing the d-DNA-based aptamers with left-handed l-DNA. By testing several reported aptamers against respective targets, we found that our proposed strategy stood up well for nonchiral small molecule targets (e.g., Hemin and cationic porphyrin) and chiral targets whose interactions with aptamers are chirality-independent (e.g., ATP). We also found that the l-DNA aptamers were indeed endowed with greatly improved biostability due to the extraordinary resistance of l-DNA to nuclease digestion. With respect to other small-molecule targets whose interactions with aptamers are chirality-dependent (e.g., kanamycin) and biomacromolecules (e.g., tyrosine kinase-7), however, the proposed strategy was not entirely effective likely due to the participation of the DNA backbone chirality into the target recognition. In spite of this limitation, this strategy indeed paves an easy way to screen highly biostable aptamers important for the applications in many fields.

Pesticides are among the most widespread organic contaminants in aquatic environments. In this work, a new green fluorescence application was proposed for the simultaneous determination of four widely employed pesticides in environmental water samples. To overcome the highly overlapped spectra within the analytes, and with the tissue matrix interferences in complex solutions, we have used the multivariate calibration methods such as parallel factor analysis (PARAFAC) and unfolded partial least squares coupled to residual bilinearization (U-PLS/RBL). These four pesticides can be identified simultaneously, and the correlation coefficients between resolved and actual spectra are all above 0.95. The second-order advantage allowed the determination of four pesticides at the ng mL - 1 level, even in the presence of humic acid (HA). The best results were obtained with the limits of detection of 1.72-18.69 for Carbendazim (CBZ), 0.30-5.19 for carbaryl (CAR), 0.35-6.32 for chlorothalonil (CHL), and 4.92-29.96 for tsumacide (TSU) (ng mL - 1 ), which can fully meet the quantitative detection and analysis requirements of trace pesticides in water samples. The real water sample of Bohai Seawater was used to check the performance of this approach in practical applications, which have achieved good prediction results of U-PLS/RBL. This study demonstrated the proposed method is rapid, accurate, sensitive, low detection limit, and environmentally friendly to determinate multi-pesticide residues in environmental water samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

L-type lectins (LTLs) belong to the lectin family and are characterized by a conserved structural motif in their carbohydrate recognition domain. LTLs are homologous to leguminous lectins. In this study, we identified and functionally characterized an LTL from kuruma shrimp Marsupenaeus japonicus. We designated this LTL as MjLTL2. MjLTL2 contains a signal peptide, a Lectin&#95;leg domain, a coiled coil, and transmembrane domain. MjLTL2 is distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestine; higher expression levels are seen in hemocytes and the hepatopancreas than in other tissues. MjLTL2 was upregulated following challenge of shrimp with Vibrio anguillarum and white spot syndrome virus (WSSV). MjLTL2 can agglutinate several bacteria without Ca 2+ . In addition, MjLTL2 could bind to several Gram-positive and -negative bacteria by binding to their lipopolysaccharide and peptidoglycan. However, MjLTL2 could not enhance the clearance of V. anguillarum in vivo. In the presence of WSSV infection, MjLTL2 knockdown by RNA interference resulted in a 7-day lower cumulative mortality of M. japonicus. Moreover, less VP19, VP24, VP26, and VP28 mRNAs were extracted from the hemocytes of MjLTL2 knockdown shrimp than from the control. These results suggest that MjLTL2 is involved in immune responses in shrimp., (Copyright © 2020. Published by Elsevier Ltd.)

A simple and green layer-by-layer assembly strategy is developed for the preparation of a highly bioavailable nanocomposite photosensitizer by assembling near-infrared (NIR) light-sensitive porphyrin/G-quadruplex complexes on the surface of a highly biocompatible nanoparticle that is prepared via Zn 2+ -assisted coordination self-assembly of an amphiphilic amino acid. After being efficiently delivered to the target site and internalized into tumor cells via enhanced permeability and retention effect and interactions between aptamers and tumor markers, the as-prepared nanoassembly can be directly used as an NIR light-responsive photosensitizer for tumor photodynamic therapy (PDT) since the porphyrin/G-quadruplex complexes are exposed on the nanoassembly surface and kept in an active state. It can also disassemble under the synergistic stimuli of an acidic pH environment and overexpressed glutathione, leasing more efficient porphyrin/G-quadruplex composite photosensitizers while reducing the interference caused by glutathione-dependent 1 O 2 consumption. Since the nanoassembly can work no matter if it is disassembled or not, the compulsory requirement for in vivo photosensitizer release is eliminated, thus resulting in the great improvement of the bioavailability of the photosensitizer. The PDT applications of the nanoassembly were well demonstrated in both in vitro cell and in vivo animal experiments.