A translocation fluorescent probe for analyzing cellular physiological parameters in neurological disease models.

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.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

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.

Multiparameter Assessment of Foam Cell Formation Progression Using a Dual-Color Switchable Fluorescence Probe.

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 benzospiropyran-julolidine-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.

Photoactivatable CRISPR/Cas12a Sensors for Biomarkers Imaging and Point-of-Care Diagnostics.

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.

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.

Metal-Induced Energy Transfer (MIET) Imaging of Cell Surface Engineering with Multivalent DNA Nanobrushes.

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.

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.

Simultaneous determination of twelve natural estrogens in dairy milk using liquid-liquid extraction and solid-phase extraction coupled with gas chromatography-mass spectrometry.

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), 17ß-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 (R2 > 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.

General Approach to Construct C-C Single Bond-Linked Covalent Organic Frameworks.

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 NaBH4 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.

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.

In Situ Self-Assembly of Fluorogenic RNA Nanozipper Enables Real-Time Imaging of Single Viral mRNA Translation.

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.

Recent Development of Advanced Fluorescent Molecular Probes for Organelle-Targeted Cell Imaging.

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.

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.

Porphyrin COF and its mechanical pressing-prepared carbon fiber hybrid membrane for ratiometric detection, removal and enrichment of Cd2.

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 Cd2+ 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 Cd2+coordination, and the resulting optical signal changes make the ratiometric detection of Cd2+ 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 Cd2+ 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 Cd2+ removal and enrichment, exhibits that preparing porphyrin-based COFs is a feasible way to promote the interactions between porphyrin ring and Cd2+, and demonstrates that mechanical pressing is a promising strategy for the design of COF-based monolithic materials to promote the practical applications of COFs.

"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.

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.