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A high-affinity fluorescence probe for copper(II) ions and its application in fluorescence lifetime correlation spectroscopy
摘要： Copper is one of the most important transition metals in many organisms where it catalyzes a manifold of different processes. As a result of copper’s redox activity, organisms have to avoid unbound ions, and a dysfunctional copper homeostasis may lead to multifarious pathological processes in cells with very severe ramifications for the affected organisms. In many neurodegenerative diseases, however, the exact role of copper ions is still not completely clarified. In this work, a high-affinity and highly selective copper probe molecule, based on the naturally occurring tetrapeptide DAHK is synthesized. The sensor (log KD = ? 12.8 ± 0.1) is tagged with a fluorescent BODIPY dye whose fluorescence lifetime distinctly decreases from 5.8 ns ± 0.2 ns to 0.4 ns ± 0.1 ns on binding to copper(II) cations. It is shown by using fluorescence lifetime correlation spectroscopy that the concentration of both probe and probe-copper complex can be simultaneously measured even at nanomolar concentration levels. This work presents a possible starting point for a new type of probe and method for future in vivo studies to further reveal the exact role of copper ions in organisms.
关键词： BODIPY,Single molecule detection,FLIM,Alzheimer,ATCUN motif,Parkinson,DAHK
更新于2025-11-21 11:24:58
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Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope
摘要： Prism-based total internal re?ection ?uorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent ?eld of a con?ned region of space, which is bene?cial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research.
关键词： single-molecule detection,single-molecule FRET,?uorescence microscope,pTIRF
更新于2025-09-23 15:21:01
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Optoplasmonic characterisation of reversible disulfide interactions at single thiol sites in the attomolar regime
摘要： Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.
关键词： Optoplasmonic,Label-free sensing,Attomolar regime,Thiol-disulfide exchange,Single-molecule detection
更新于2025-09-23 15:21:01
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Atomic Force Microscopy in Molecular and Cell Biology || In Situ Single Molecule Detection on Cell Membrane and Label Molecule Distributions Using AFM/NSOM
摘要： Consisting of viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, cell membranes have proven to play very important roles in ensuring the stability of the intracellular environment and order of cellular signal transductions. The developments of modern cell biology, immunology, and medicine urge us to explore more precise cell membrane structures and detailed functions of biomolecules on cell membranes. Due to the minuscule size of biomolecules and their clusters on cell membranes (varying from several nanometers to hundreds of nanometers), a high resolution microscopy is needed to explore the cell membrane biomolecule distribution.
关键词： single molecule detection,biomolecule distribution,AFM,cell membrane,NSOM
更新于2025-09-23 15:21:01
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Construction of single quantum dot nanosensor with the capability of sensing methylcytosine sites for sensitive quantification of methyltransferase
摘要： The CpG island methylation plays an important role in diverse biological processes including the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. Due to the dependence of DNA methylation upon DNA methyltransferase (MTase) activity, DNA MTases have become the potential targets in anticancer therapy. Herein we demonstrate for the first time the construction of a single quantum dot (QD) nanosensor with the capability of sensing methylcytosine sites for sensitive quantification of M.SssI CpG methyltransferase (M.SssI MTase). We design a biotin-/phosphate-modified double-stranded DNA (dsDNA) substrate with a 5’-G-C-G-mC-3’/3’-mC-G-mC-G-5’ site for sensing M.SssI MTase. In the presence of M.SssI MTase, the methylation-responsive sequence of dsDNA substrate is methylated and cleaved by GlaI endonuclease, producing two dsDNA fragments with free 3’-OH terminus. In the presence of terminal deoxynucleotidyl transferase (TdT), multiple Cy5-dATPs can be sequentially added to the free 3’-OH terminus of dsDNA fragments to obtain the biotin-/multiple Cy5-labeled dsDNAs. The resultant biotin-/multiple Cy5-labeled dsDNAs can assemble on the surface of the streptavidin-coated QD to obtain a QD-dsDNA-Cy5 nanostructure in which the fluorescence resonance energy transfer (FRET) from the QD to Cy5 can occur. The emission of Cy5 can be simply quantified by single-molecule detection. By the integration of sensing methylcytosine sites and enzymatic polymerization, the sensitivity of this nanosensor have been significantly enhanced. This nanosensor can detect as low as 2.1 × 10-7 U/μL M.SssI MTase with good selectivity against other cytosine MTases, and it can be further applied for the screening of MTase inhibitors and complex biological sample analysis, holding great potential in clinic diagnosis and drug discovery.
关键词： methyltransferase,quantum dot,methylcytosine,nanosensor,single-molecule detection
更新于2025-09-16 10:30:52
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High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices
摘要： Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height and a width of several micrometers confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observe DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level.
关键词： fluorescence microscopy,DNA,nanofluidic devices,single-molecule detection,biomolecular interactions
更新于2025-09-04 15:30:14