The in vitro detection of low abundance biomolecules via nonenzymatic signal amplification is an attractive strategy. However, it remains a challenge to monitor targets of interest in situ in living cells by low-background interference and visualized enzyme-free signal amplification strategies. Taking advantage of the single-molecule imaging and dynamic DNA nanotechnologies, we have achieved the target-triggered self-assembly of nanostructure-based dual-color fluorescent probes (NDFPs) by an enzyme-free toehold-mediated strand displacement cascade. NDFPs will facilitate the simple and visualized monitoring of microRNA (miRNA) at the femtomolar level. The recycled miRNA can be considered as the catalyst for the assembly of multiple H1/H2 duplexes. This generated the fluorescence signal of the enhanced target expression, indicating both in vitro and in vivo signal-amplified imaging. Moreover, the NDFPs improved the measurement accuracy by dual-color colocalization imaging to greatly avoid false-positive signals and enabled the successful in situ imaging of miRNA in living cells in real time. This work provides a strategy to visually monitor and study the integration of signal amplification detection and single-molecule imaging. NDFPs may be an important step toward the enzyme-free amplified monitoring and imaging of various biomolecules in living cells at the single-molecule level.

HSP may present with acute abdomen without typical skin manifestations, and gastroscopy and colonoscopy can be helpful in the early diagnosis of HSP in these patients. Typical endoscopic findings include diffuse mucosal oedema, erythema, petechia or multiple irregular ulcers, especially in the second portion of the duodenum or in the terminal ileum.

Carbon dots (CDs) are extensively studied to investigate their unique optical properties such as undergoing electron transfer in different scenarios. This study presents an in-depth investigation to study the ensemble-averaged state/bulk state and single-particle level photophysical properties of CDs that have been passivated with electron-accepting (CD-A) and electron-donating molecules (CD-D) on their surface. Our ensemble-averaged state experiments including UV-Vis absorbance titrations, time-resolved photoluminescence (PL) spectra, and 2D emission studies depicted that both CD-A or CD-D had a blue-shift in emission, with a drastic increase in emission intensity, and consequently higher quantum yields, and that acceptor populations (CD-A) dominate overall photophysical properties. Interestingly, transmission electron microscopy and atomic force microscopy revealed that the mixing of donor and acceptor particles (CD-A+ CD-D) leads to a formation of at least two associated geometries, which was dependent on time, concentration, intramolecular electron/charge transfer and hydrogen bonding. On the other hand, single-particle studies revealed that the instantaneous intensity of CDs was comparable, but that CD-A and CD-D have a larger on-time duty cycle, attributed to an increase of blinking frequency. On-and off-time power-law analysis further revealed that CD-A has a larger off-time distribution slope than bare CDs, This article is protected by copyright. All rights reserved. 3 while their on-time distribution is similar. CD-D exhibits an increase in both on-and off-time distribution slope compared to bare CDs. These results indicate that the electron donor provides additional bright and dark states, while the electron acceptor primarily provides bright states, which can explain the increased blinking frequency in CD-A and CD-D at the single-particle level. Singleparticle studies, however, did not reveal an "acceptor-dominating" scenario based on analysis of instantaneous intensity, bleaching kinetics, and photoblinking, indicating that the direct interaction of CD-A and CD-D may affect their photophysical properties in the bulk state due to formation of hierarchical structural assemblies. We anticipate that these fundamental results will further provide insights towards our understanding of the complex mechanism associated with CD emission, which is one of the key contributors to their successful application. As an immediate application of these CDs, we have shown that they can be used as a sensing array for metal ions and can serve as a powerful toolbox for the technological application of CDs.

Direct imaging of single‐molecule and its movement is of fundamental importance in biology, but challenging. Herein, aided by the nanoconfinement effect and resultant high reaction activity within metal–organic frameworks (MOFs), the designed Ru(bpy)32+ embedded MOF complex (RuMOFs) exhibits bright electrochemiluminescence (ECL) emission permitting high‐quality imaging of ECL events at single molecule level. By labeling individual proteins of living cells with single RuMOFs, the distribution of membrane tyrosine‐protein‐kinase‐like7 (PTK7) proteins at low‐expressing cells is imaged via ECL. More importantly, the efficient capture of ECL photons generated inside the MOFs results in a stable ECL emission up to 1 h, allowing the in operando visualization of protein movements at the cellular membrane. As compared with the fluorescence observation, near‐zero ECL background surrounding the target protein with the ECL emitter gives a better contrast for the dynamic imaging of discrete protein movement. This achievement of single molecule ECL dynamic imaging using RuMOFs will provide a more effective nanoemitter to observe the distribution and motion of individual proteins at living cells.

A single-molecule assay for multiple microRNA detection.

Direct imaging of electrochemical reactions at the single-molecule level is of potential interest in materials, diagnostic, and catalysis applications. Electrochemiluminescence (ECL) offers the opportunity to convert redox events into photons. However, it is challenging to capture single photons emitted from a single-molecule ECL reaction at a specific location, thus limiting high-quality imaging applications. We developed the nanoreactors based on Ru(bpy)3 2+-doped nanoporous zeolite nanoparticles (Ru@zeolite) for direct visualization of nanoconfinement-enhanced ECL reactions. Each nanoreactor not only acts as a matrix to host Ru(bpy)3 2+ molecules but also provides a nanoconfined environment for the collision reactions of Ru(bpy)3 2+ and co-reactant radicals to realize efficient in situ ECL reactions. The nanoscale confinement resulted in enhanced ECL. Using such nanoreactors as ECL probes, a dual-signal sensing protocol for visual tracking of a single biomolecule was performed. High-resolution imaging of single membrane proteins on heterogeneous cells was effectively addressed with near-zero backgrounds. This could provide a more sensitive tool for imaging individual biomolecules and significantly advance ECL imaging in biological applications.

Linaridins are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. In this study, a genome mining approach led to the identification of a novel linaridin, mononaridin (MON), from Streptomyces monomycini. In-frame deletion genetic knockout studies showed that, in addition to many genes essential for MON biosynthesis, monM encodes an S-adenosyl methionine (SAM)-dependent α-N-methyltransferase that is responsible for installing two methyl groups in the MON N-terminus. Besides SAM, MonM also accepts ethyl-SAM and allyl-SAM, in which the methyl of SAM is replaced by an ethyl and an allyl, respectively. We showed that ethyl-SAM and allyl-SAM have distinct reactivities in MonM catalysis, and this observation was further investigated in detail by density functional theory (DFT) calculations. Remarkably, MonM acts efficiently on nisin, a prototypic lantibiotic that is structurally very different from the native substrate, and the ability of MonM to transfer an allyl group to the nisin N-terminus allowed production of a fluorescently labeled nisin, which can be further used in microscopic cell analysis. Our studies provide new insights into linaridin biosynthesis and demonstrate the potential of linaridin methyltransferases in bioengineering applications.

Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single-molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently, the streptavidin-labeled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labeled methylated dsDNA molecules, the single-molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. SssI concentration in the concentration range of 0.001–1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.