Surface Plasmon Resonance Microscopy: From Single-Molecule Sensing to Single-Cell Imaging

Surface plasmon resonance microscopy (SPRM) is a versatile platform for chemical and biological sensing and imaging. Great progress in exploring its applications, ranging from single-molecule sensing to single-cell imaging, has been made. In this Minireview, we introduce the principles and instrumentation of SPRM. We also summarize the broad and exciting applications of SPRM to the analysis of single entities. Finally, we discuss the challenges and limitations associated with SPRM and potential solutions.     

Nanopore‐Based Confined Spaces for Single‐Molecular Analysis

The field of nanopore sensing at the single‐molecular level is in a “boom” period. Such nanopores, which are either composed of biological materials or are fabricated from solid‐state substrates, offer a unique confined space that is compatible with the single‐molecular scale. Under the influence of an electrical field, such single‐biomolecular interfaces can read single‐molecular information and, if appropriately fine‐tuned, each molecule plays its individual ionic rhythm to compose a “molecular symphony”. Over the past few decades, many research groups have worked on nanopore‐based single‐molecular sensors for a range of thrilling chemical and clinical applications. Furthermore, for the past decade, we have also focused on nanopore‐based sensors. In this Minireview, we summarize the recent developments in fundamental research and applications in this area, along with data algorithms and advances in hardware, which act as infrastructure for the electrochemical analysis.     

When Nanozymes Meet Single‐Atom Catalysis

Nanomaterials with enzyme‐like activities, coined nanozymes, have been researched widely as they offer unparalleled advantages in terms of low cost, superior activity, and high stability. The complex structure and composition of nanozymes has led to extensive investigation of their catalytic sites at an atomic scale, and to an in‐depth understanding of the biocatalysis occurring. Single‐atom catalysts (SACs), characterized by atomically dispersed active sites, have provided opportunities for mimicking metalloprotease and for bridging the gap between natural enzymes and nanozymes. In this Minireview, we illustrate the unique properties of nanozymes and we discuss recent advances in the synthesis, characterization, and applications of SACs. Subsequently, we outline the impressive progress made in single‐atom nanozymes and we discuss their applications in sensing, degradation of organic pollutants, and in therapeutic roles. Finally, we present the major challenges and opportunities remaining for a successful marriage of nanozymes and SACs.     

Redox‐Filled Carbon‐Fiber Microelectrodes for Single‐Cell Exocytosis

Carbon‐fiber microelectrodes (CFEs) are the primary electroanalytical tool in single‐cell exocytosis and in vivo studies. Here we report a new study on the kinetic properties of electrolyte‐filled CFEs in single‐cell measurements and demonstrate that the addition of outer sphere redox species, such as Fe(CN)₆^3? and Ru(NH₃)₆³⁺, in the backfill electrolyte solution can greatly enhance the kinetic response of CFEs. We show that at 750 mV, a voltage normally applied for detection of dopamine, the presence of fast outer sphere redox species in the backfilling solution significantly enhances the kinetic response of CFEs toward fast dopamine detection at single PC12 cells. Moreover, we also demonstrate that the use of Fe(CN)₆^3? in the backfilling solution has enabled direct measurement of dopamine at applied voltages as low as 200 mV. This kinetic enhancement is believed to be due to faster electron‐transfer kinetics on the coupling pole as compared to the sluggish reduction of oxygen. We anticipate that such redox‐filled CFE ultramicroelectrodes will find many useful applications in single cell exocytosis and in vivo sensing.     

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

There is a persistent need for small‐molecule fluorescent labels optimized for single‐molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red‐shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed “DCDHF” fluorophores, for use in live‐cell imaging based on the push–pull design: an amine donor group and a 2‐dicyanomethylene‐3‐cyano‐2,5‐dihydrofuran (DCDHF) acceptor group, separated by a π‐rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red‐emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low‐intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small‐molecule fluorophores, which are needed for super‐resolution imaging schemes that require active control (here turning‐on) of single‐molecule emission.     

Single‐Molecule Observation of Intermediates in Bioorthogonal 2‐Cyanobenzothiazole Chemistry

We report a single‐molecule mechanistic investigation into 2‐cyanobenzothiazole (CBT) chemistry within a protein nanoreactor. When simple thiols reacted reversibly with CBT, the thioimidate monoadduct was approximately 80‐fold longer‐lived than the tetrahedral bisadduct, with important implications for the design of molecular walkers. Irreversible condensation between CBT derivatives and N‐terminal cysteine residues has been established as a biocompatible reaction for site‐selective biomolecular labeling and imaging. During the reaction between CBT and aminothiols, we resolved two transient intermediates, the thioimidate and the cyclic precursor of the thiazoline product, and determined the rate constants associated with the stepwise condensation, thereby providing critical information for a variety of applications, including the covalent inhibition of protein targets and dynamic combinatorial chemistry.     

Single‐Molecule Mechanochemical Sensing Using DNA Origami Nanostructures

While single‐molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single‐molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7‐tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet‐derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single‐molecule sensing with improved throughput.     

Highly Multiplexed Single‐Cell In Situ Protein Analysis with Cleavable Fluorescent Antibodies

Limitations on the number of proteins that can be quantified in single cells in situ impede advances in our deep understanding of normal cell physiology and disease pathogenesis. Herein, we present a highly multiplexed single‐cell in situ protein analysis approach that is based on chemically cleavable fluorescent antibodies. In this method, antibodies tethered to fluorophores through a novel azide‐based cleavable linker are utilized to detect their protein targets. After fluorescence imaging and data storage, the fluorophores coupled to the antibodies are efficiently cleaved without loss of protein target antigenicity. Upon continuous cycles of target recognition, fluorescence imaging, and fluorophore cleavage, this approach has the potential to quantify over 100 different proteins in individual cells at optical resolution. This single‐cell in situ protein profiling technology will have wide applications in signaling network analysis, molecular diagnosis, and cellular targeted therapies.     

Addressing the Requirements of High‐Sensitivity Single‐Molecule Imaging of Low‐Copy‐Number Proteins in Bacteria

Single‐molecule fluorescence super‐resolution imaging and tracking provide nanometer‐scale information about subcellular protein positions and dynamics. These single‐molecule imaging experiments can be very powerful, but they are best suited to high‐copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single‐molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single‐molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.     

Dual‐Polarization Imaging of a Dual‐Fluorophore Ion Sensor: A Single‐Molecule Study

The fluorescence emission of the dual‐fluorophore Ca²⁺ ion sensor molecule, calcium‐green 2 (CG‐2), has been characterized using dual‐polarization imaging at the single‐molecule level. By comparing the fluorescence intensity of individual CG‐2 molecules in two mutually orthogonal polarization image channels, information about the relative orientation of the two constituent fluorophores in the molecule is obtained. Experimental results from polarization measurements are compared with those predicted from a geometric model based on coupled‐fluorophores that are randomly distributed in space. The results confirm previous optical spectroscopy‐based predictions of the orientation of CG‐2′s fluorophores, and the general applications of this dual‐polarization imaging approach for characterizing the optical properties of molecules containing multiple fluorophores is discussed.     

Non‐Interpenetrated Single‐Crystal Covalent Organic Frameworks

Growth of covalent organic frameworks (COFs) as single crystals is extremely challenging. Inaccessibility of open‐structured single‐crystal COFs prevents the exploration of structure‐oriented applications. Herein we report for the first time a non‐interpenetrated single‐crystal COF, LZU‐306, which possesses the open structure constructed exclusively via covalent assembly. With a high void volume of 80 %, LZU‐306 was applied to investigate the intrinsic dynamics of reticulated tetraphenylethylene (TPE) as the individual aggregation‐induced‐emission moiety. Solid‐state ²H NMR investigation has determined that the rotation of benzene rings in TPE, being the freest among the reported cases, is as fast as 1.0×10⁴ Hz at 203 K to 1.5×10⁷ Hz at 293 K. This research not only explores a new paradigm for single‐crystal growth of open frameworks, but also provides a unique matrix‐isolation platform to reticulate functional moieties into a well‐defined and isolated state.     

Auger Ionization Beats Photo‐Oxidation of Semiconductor Quantum Dots: Extended Stability of Single‐Molecule Photoluminescence

Despite the bright and tuneable photoluminescence (PL) of semiconductor quantum dots (QDs), the PL instability induced by Auger recombination and oxidation poses a major challenge in single‐molecule applications of QDs. The incomplete information about Auger recombination and oxidation is an obstacle in the resolution of this challenge. Here, we report for the first time that Auger‐ionized QDs beat self‐sensitized oxidation and the non‐digitized PL intensity loss. Although high‐intensity photoactivation insistently induces PL blinking, the transient escape of QDs into the ultrafast Auger recombination cycle prevents generation of singlet oxygen (¹O₂) and preserves the PL intensity. By the detection of the NIR phosphorescence of ¹O₂ and evaluation of the photostability of single QDs in aerobic, anaerobic, and ¹O₂ scavenger‐enriched environments, we disclose relations of Auger ionization and ¹O₂‐mediated oxidation to the PL stability of single QDs, which will be useful during the formulation of QD‐based single‐molecule imaging tools and single‐photon devices.     

Single‐Atom Iron Boosts Electrochemiluminescence

The traditional luminol–H₂O₂ electrochemiluminescence (ECL) sensing platform suffers from self‐decomposition of H₂O₂ at room temperature, hampering its application for quantitative analysis. In this work, for the first time we employ iron single‐atom catalysts (Fe‐N‐C SACs) as an advanced co‐reactant accelerator to directly reduce the dissolved oxygen (O₂) to reactive oxygen species (ROS). Owing to the unique electronic structure and catalytic activity of Fe‐N‐C SACs, large amounts of ROS are efficiently produced, which then react with the luminol anion radical and significantly amplify the luminol ECL emission. Under the optimum conditions, a Fe‐N‐C SACs–luminol ECL sensor for antioxidant capacity measurement was developed with a good linear range from 0.8 μm to 1.0 mm of Trolox.     

Single‐Atom Electrocatalysts

Recent years have witnessed a dramatic increase in the production of sustainable and renewable energy. However, the electrochemical performances of the various systems are limited, and there is an intensive search for highly efficient electrocatalysts by more rational control over the size, shape, composition, and structure. Of particular interest are the studies on single‐atom catalysts (SACs), which have sparked new interests in electrocatalysis because of their high catalytic activity, stability, selectivity, and 100 % atom utilization. In this Review, we introduce innovative syntheses and characterization techniques for SACs, with a focus on their electrochemical applications in the oxygen reduction/evolution reaction, hydrogen evolution reaction, and hydrocarbon conversion reactions for fuel cells (electrooxidation of methanol, ethanol, and formic acid). The electrocatalytic performance is further considered at an atomic level and the underlying mechanisms are discussed. The ultimate goal is the tailoring of single atoms for electrochemical applications.     

Chemical and Topographical Single‐Cell Imaging by Near‐Field Desorption Mass Spectrometry

Simultaneously acquiring chemical and topographical information within a single cell at nanoscale resolutions is vital to cellular biology, yet it remains a great challenge due to limited lateral resolutions and detection sensitivities. Herein, the development of near‐field desorption mass spectrometry for correlated chemical and topographical imaging is reported, thereby bridging the gap between laser‐based mass spectrometry (MS) methods and multimodal single‐cell imaging. Using this integrated platform, an imaging resolution of 250 nm and 3D topographically reconstructed chemical single‐cell imaging were achieved. This technique offers more in‐depth cellular information than micrometer‐range laser‐based MS imaging methods. Considering the simplicity and compact size of the near‐field device, this technique can be introduced to MALDI‐MS, expanding the multimodal abilities of MS at nanoscale resolutions.     

A Single‐Atom Nanozyme for Wound Disinfection Applications

Single‐atom catalysts (SACs), as homogeneous catalysts, have been widely explored for chemical catalysis. However, few studies focus on the applications of SACs in enzymatic catalysis. Herein, we report that a zinc‐based zeolitic‐imidazolate‐framework (ZIF‐8)‐derived carbon nanomaterial containing atomically dispersed zinc atoms can serve as a highly efficient single‐atom peroxidase mimic. To reveal its structure–activity relationship, the structural evolution of the single‐atom nanozyme (SAzyme) was systematically investigated. Furthermore, the coordinatively unsaturated active zinc sites and catalytic mechanism of the SAzyme are disclosed using density functional theory (DFT) calculations. The SAzyme, with high therapeutic effect and biosafety, shows great promises for wound antibacterial applications.     

Flexible Viologen‐Based Porous Framework Showing X‐ray Induced Photochromism with Single‐Crystal‐to‐Single‐Crystal Transformation

A viologen‐based Borromean entangled porous framework was found to be sensitive to both Cu_Kα and Mo_Kα X‐ray sources, showing rapid photochromic response and recovery within one minute. The X‐ray‐induced photochromic process is accompanied by a reversible single‐crystal‐to‐single‐crystal (SC‐SC) structural transformation, an unprecedented phenomenon for X‐ray sensitive materials. The complex can be further processed into portable thin films for detecting the dose of the X‐ray exposure. Moreover, the photochromism can occur over a broad temperature range of 100–333 K, both in the form of single crystals and thin films, making it a potential candidate for practical indoor and outdoor applications.     

Optimizing the Acquisition and Analysis of Confocal Images for Quantitative Single‐Mobile‐Particle Detection

Quantification of the fluorescence properties of diffusing particles in solution is an invaluable source of information for characterizing the interactions, stoichiometry, or conformation of molecules directly in their native environment. In the case of heterogeneous populations, single‐particle detection should be the method of choice and it can, in principle, be achieved by using confocal imaging. However, the detection of single mobile particles in confocal images presents specific challenges. In particular, it requires an adapted set of imaging parameters for capturing the confocal images and an adapted event‐detection scheme for analyzing the image. Herein, we report a theoretical framework that allows a prediction of the properties of a homogenous particle population. This model assumes that the particles have linear trajectories with reference to the confocal volume, which holds true for particles with moderate mobility. We compare the predictions of our model to the results as obtained by analyzing the confocal images of solutions of fluorescently labeled liposomes. Based on this comparison, we propose improvements to the simple line‐by‐line thresholding event‐detection scheme, which is commonly used for single‐mobile‐particle detection. We show that an optimal combination of imaging and analysis parameters allows the reliable detection of fluorescent liposomes for concentrations between 1 and 100 pM . This result confirms the importance of confocal single‐particle detection as a complementary technique to ensemble fluorescence‐correlation techniques for the studies of mobile particle.     

Light‐Harvesting Nanoparticle Probes for FRET‐Based Detection of Oligonucleotides with Single‐Molecule Sensitivity

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20‐nm light‐harvesting nanoantenna particles, built of a tailor‐made hydrophobic charged polymer poly(ethyl methacrylate‐co‐methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87‐fold brighter than quantum dots QDots 605 in single‐particle microscopy (with 550‐nm excitation), these DNA‐functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250‐fold signal amplification. The obtained FRET nanoprobes enable single‐molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi‐fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.     

A Photoisomerization‐Activated Intramolecular Charge‐Transfer Process for Broadband‐Tunable Single‐Mode Microlasers

Miniaturized lasers with high spectral purity and wide wavelength tunability are crucial for various photonic applications. Here we propose a strategy to realize broadband‐tunable single‐mode lasing based on a photoisomerization‐activated intramolecular charge‐transfer (ICT) process in coupled polymer microdisk cavities. The photoisomerizable molecules doped in the polymer microdisks can be quantitatively transformed into a kind of laser dye with strong ICT character by photoexcitation. The gain region was tailored over a wide range through the self‐modulation of the optically activated ICT isomers. Meanwhile, the resonant modes shifted with the photoisomerization because of a change in the effective refractive index of the polymer microdisk cavity. Based on the synergetic modulation of the optical gain and microcavity, we realized the broadband tuning of the single‐mode laser. These results offer a promising route to fabricate broadband‐tunable microlasers towards practical photonic integrations.