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. 2025 Oct 7;4(3):256–273. doi: 10.1021/cbmi.5c00112

The Fantastic Single-Molecule Techniques

Huang Tang †,‡, Shuting Liu †,‡, Chenyue Kang †,‡, Xiang Wang †,‡,§, Xi Zhang †,‡, Kun Li †,‡, Gege Duan †,‡, Zheng Li ∥,⊥,*, Boyang Hua †,‡,*, Xing-Hua Xia †,‡
PMCID: PMC13014335  PMID: 41889465

Abstract

In the past 50 years, single-molecule techniques have been rapidly developed and widely applied in numerous fields of biology, offering insights that conventional biochemical assays cannot discover. In this review, to help fully appreciate the effectiveness of single-molecule methods, we systematically summarize the various advantages of performing biochemical assays at the single-molecule level. To demonstrate that this strategy is not limited to the few representative cases discussed here, we propose a single-molecule polysome profiling technique, taking inspiration from previous examples. Finally, we point out a possibility in the future of unifying different biochemical assays on the platform of single-molecule microscopy, which will reduce the cost of laboratory instrumentation and inevitably promote the adoptability and automation of a variety of biochemical and biophysical methods.

Keywords: single-molecule techniques, sequencing technologies, biomarker detection, biochemical and biophysical assays, super-resolution chemical imaging, biomolecular interactions, fluorescence, electrochemiluminescence, plasmonic scattering

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1. Introduction

The modern-day single-molecule techniques encompass a huge family of methods and tools that can be loosely divided into three broad categories, i.e., single-molecule electrical techniques (such as patch-clamps, nanopores, , and scanning tunneling microscopy), optical techniques (such as fluorescence, − Raman, , infrared spectroscopy, − and electrochemiluminescence), and force techniques (such as atomic force microscopy, − optical tweezers, − and magnetic tweezers , ). It is a star-studded collection that bears not one but several Nobel prize-winning inventions. Although these techniques are built upon vastly different instrument principles and measure very different physical properties, the one thing they all have in common is that they all carry the modifier “single-molecule”. This begs the question: why “single-molecule”? The short answer to this question often goes as follows: unlike bulk measurements, single-molecule measurements avoid ensemble-averaging the observables, such that heterogeneous subpopulations within an ensemble can be revealed, and transition dynamics between different system states observed without the need of bulk synchronization. In addition to these intrinsic features, there are more “perks” that these single-molecule methods can offer. In this review, we will introduce several representatives of single-molecule techniques, compare and contrast these techniques with their conventional bulk counterparts, and in doing so, parse out the unique advantages that researchers are able to gain by choosing the single-molecule versions of the conventional methods. Among the various single-molecule detection modalities, fluorescence is perhaps the most commonly used in biology thanks to its exceptionally strong signal, very low background, and excellent molecular specificity. This review will therefore focus on the single-molecule fluorescence methods, while mentioning examples of other optical (e.g., electrochemiluminescence and surface plasmon resonance , ) and electrical (e.g., nanopores) techniques to illustrate that these advantages are not exclusive to fluorescence.

We first give a brief introduction of the detection platforms and readout principles that are commonly used in these single-molecule optical techniques. Combining the optical detection platforms with these readout principles, one can build a variety of assays to extract important molecular information at the single-molecule level, which is the focus of this review. The assays that are discussed in this review include (1) single-molecule real-time sequencing methods, which utilize fluorescence signals to report the nucleotide identity information; (2) single-molecule array technologies, which utilize fluorescence signals to reveal the absolute analyte quantity information; (3) single-molecule pull-down assays and fluorescence intensity shift assays, which report a combination of identity, quantity, and molecular interaction information; (4) a single-molecule electrochemiluminescence imaging technique that reveals chemical reactivity information with super-resolution; (5) a plasmonic scattering imaging technique that reveals molecular weight, size, and interaction information. We keep a balanced selection of assays including both well-established commercial techniques and proof-of-concept in-house techniques to demonstrate the broad impact of single-molecule methodologies.

2. The Basics of Single-Molecule Optical Techniques

2.1. Single-Molecule Optical Detection Platforms

A single-molecule optical detection platform (e.g., a microscope) typically consists of several key parts, i.e., a high-sensitivity photon detector, a high-numerical-aperture objective (except for near-field microscopy), and a high-intensity light source, if excitation is required. Connecting these parts together is a light path, the design of which distinguishes the different types of optical platforms. In general, there are two broad categories of microscopy: (1) point scanning microscopy, such as (far-field) confocal microscopy and near-field scanning optical microscopy (NSOM); (2) wide-field microscopy, such as total internal reflection fluorescence (TIRF) microscopy and light-sheet microscopy. Although the early “proof-of-concept” work in the field was demonstrated using confocal − or NSOM, nowadays it is more common for researchers to employ TIRF microscopy for single-molecule fluorescence detection due to its noninvasive, high-throughput, and highly sensitive natures. In TIRF microscopy, an inclined incident laser beam forms total internal reflection at the glass–water interface and generates an evanescent field for illuminating the samples (Figure a–c). By this means, the excitation volume of TIRF microscopy is confined within ∼100 nm of the imaging surface and the background fluorescence signal from the bulk solution is largely suppressed. The application of TIRF microscopy in single-molecule fluorescence detection was first achieved by the Yanagida group in their seminal paper to study the individual ATP turnover events of immobilized myosin motors, where hundreds to thousands of single molecules can be detected in parallel with an excellent signal-to-noise ratio (SNR). TIRF microscopy can be configurated via either the objective-type, the prism-type, or the micromirror-type instrumentations; − the incident angle and the imaging depth can be tuned to adapt for thicker samples such as cells (highly inclined and laminated optical sheet, or HILO), making TIRF microscopy a versatile platform for various single-molecule assays (Figure d). In this sense, an adjustable angle laser illumination microscope is a more generalized platform that supports the TIRF, HILO, as well as epi-fluorescence modes. In this review, all the fluorescence-based methods can be performed on such a microscope, with only the single-molecule real-time sequencing technologies requiring an additional microfabricated imaging chip, whereas (1) the optical platform for electrochemiluminescence shares the light emission and collection portion with TIRF microscopy; and (2) the plasmonic scattering imaging technique utilizes an illumination mechanism similar to the total internal reflection configuration.

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Schematic diagrams of different TIRF configurations. (a) The objective-type, (b) the prism-type, and (c) the micromirror-type TIRF. (d) The HILO illumination configuration.

2.2. Single-Molecule Optical Readout Principles

The common information that can be read out with fluorescence detection is the molecular presence and identity (in combination with high-specificity labeling methods), quantity, and spatial localization and distance. For reporting molecular identities, conventional fluorescence techniques typically rely on a color-based approach. However, due to the relatively broad spectra of fluorophores, the limitation in spectral multiplexing capability means only a few different identities can be simultaneously marked. To alleviate this limitation, a sequential labeling strategy has been developed (Figure a), as demonstrated in multiplexed error-robust fluorescence in situ hybridization (MERFISH), sequential barcode fluorescence in situ hybridization (seqFISH), and DNA points accumulation for imaging in nanoscale topography (DNA-PAINT), , which trade-off assay rounds to resolve overlapping fluorescence spectra.

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Schematic representation of how (a) molecular identity, (b) quantity, and (c) localization and distance information is extracted.

There are several approaches for quantification in single-molecule fluorescence techniques (Figure b), such as the intensity-based analog approach for relative quantification, and the counting-based digital approaches for absolute quantification, such as the spot counting, photobleaching step counting, and kinetics counting (e.g., quantitative PAINT, or qPAINT).

Finally, thanks to the development of super-resolution optical microscopy and algorithms, the localization accuracy now reaches the nanometer range, far beyond the diffraction-limited length scale. Once the localizations of two or more objects are determined, one can easily calculate the distance among the objects (e.g., colocalization). In addition, the molecular distance can be measured through fluorescence spectroscopy and intensity methods, such as Förster resonance energy transfer (FRET), − and photoisomerization-related fluorescence enhancement (PIFE). − Combined together, colocalization (micrometers to nanometers), FRET (3–7) nm, and PIFE (0–4) nm cover a wide range of distance measurements (Figure c). The spatial localization and distance are such fundamental physical properties that many derivative information can be obtained through their measurements, such as molecular conformations and structures, interactions, activities, and movements (e.g., velocity). Moreover, other optical detection modalities complement fluorescence in terms of the information that can be measured. For instance, the chemical reactivity can be inferred via the electrochemiluminescence imaging, while the molecular weight and size via the plasmonic scattering mode.

Now that we have covered both the optical detection platforms and the readout principles, we discuss the various assays that are built on the foundations.

3. Representative Single-Molecule Tools

3.1. Single-Molecule Real-Time Sequencing Technologies

Sequencing technologies for nucleic acids have propelled the advancement of biology, biomedical research, and disease diagnostics. The first-generation sequencing technology, represented by Sanger sequencing (Figure a), employs the dideoxy chain-termination method, where the incorporation of dideoxynucleoside triphosphates (ddNTPs) terminates DNA polymerase-mediated primer extension reactions, resulting to truncated DNA products that are sequenced via polyacrylamide gel electrophoresis. , Despite its high accuracy and a relatively long read length of up to a kilobase, its low-throughput nature limits its application in solving large-scale, omics-level questions. To overcome these constraints, next-generation sequencing technology (NGS) was invented and rapidly developed during the last 20 years. NGS employs DNA random fragmentation, adapter ligation, and solid-phase amplification to generate high-density monoclonal sequence clusters. Each cluster represents a unique DNA fragment to be sequenced, thus drastically improving the sequencing throughput. In combination with the sequencing-by-synthesis (SBS) chemistry using reversible polymerization terminators, NGS captures the fluorescence signals generated during the polymerase-driven incorporation of labeled nucleotides and achieves round-by-round base calling from billions of clusters in parallel. , Although the template amplification and cluster formation strategy used in NGS significantly increases the SNR and improves the base calling accuracy, this step introduces amplification bias and, more critically, leads to signal decay due to the “phasing” issue (progressive loss of synchronization among molecules within a cluster during successive synthesis cycles, Figure b). This decay in base-calling accuracy gradually occurs after hundreds of sequencing cycles and significantly compromises the read length.

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Schematic representation of three generations of sequencing technologies. (a) Sanger sequencing: the process involves (1) DNA template amplification with DNA polymerase and dNTPs, (2) four separate reactions with ddNTPs (ddATP, ddCTP, ddTTP, or ddGTP) for chain termination, and (3) fragment separation by gel electrophoresis for (4) sequence readouts. (b) Next-generation sequencing: the short-read technology (100–300 nt) using fluorescently labeled nucleotides, showing progressive loss of synchronization among cluster molecules after each round of synthesis. (c) Single-molecule sequencing: employing ZMW nanostructures (inset) or nanopores to detect the individual nucleotides traversing through the detection zone, generating fluorescence or electrical signals that facilitate the sequence determination. Adapted with permission from ref . Copyright © 2003 The American Association for the Advancement of Science. (d) Design of the circular consensus sequencing template. Adapted with permission from ref . Copyright © 2010 The Oxford University Press.

Since the key issue that limits NGS’s read length is the progressive loss of synchronization among cluster molecules, conducting sequencing experiments at the single-molecule level can circumvent the need for synchronization and fundamentally address the root problem. Building upon this principle, third-generation sequencing technology (TGS) has emerged and bloomed in recent years. By directly detecting signals from individual nucleic acid molecules (Figure c), these methods not only eliminate the systemic biases inherent in NGS due to template amplification, but also enable ultralong reads (>10 kb) while preserving the base modification information on the original copies of the nucleic acid molecules. − For example, Helicos is an early single-molecule sequencing technology based on fluorescence detection and SBS. Similar to NGS, this technology fragments DNA to prepare sequencing libraries and uses polymerases to add fluorescently labeled nucleotides to primers for sequencing. Different from NGS, Helicos directly sequences single DNA molecules instead of clusters, thus avoiding the errors and biases introduced during amplification. One of the common technical difficulties that fluorescence-based single-molecule sequencing technologies face is the detection of single-molecule signals in the presence of high fluorescence background. Typically, polymerase reactions require micromolar concentrations of fluorescently labeled dNTPs, creating a high fluorescence background that renders conventional single-molecule detection methods inadequate. To address this challenge, Turner et al. developed the zero-mode waveguide (ZMW) nanostructures, fabricating ∼100 nm diameter aluminum-clad apertures on quartz substrates (Figure c, inset). In comparison to the relatively large excitation volumes around picoliters for confocal and TIRF microscopy, these nanostructures in ZMW reduce the excitation volume to the attoliter scale by leveraging the evanescent wave confinement, thus achieving real-time single-molecule observation even at high concentrations of fluorescently labeled dNTPs. , Using dNTPs with fluorophores covalently attached to the terminal phosphate group, the technology successfully tracked continuous DNA polymerase synthesis and revealed polymerase kinetic states.

In spite of an improved read length, TGS only managed to achieve a modest base calling accuracy in its early stages. To address this issue, Turner et al. developed a novel approach termed circular consensus sequencing (CCS), in which the target DNA fragments are ligated to hairpin-shaped sequencing adapters, forming a unique “dumbbell-shaped” circular structure (Figure d). In this configuration, DNA polymerase performs multiple rounds of rolling-circle amplification around the same DNA molecule. This design ensures each base to be sequenced for multiple times, allowing the generation of high-accuracy consensus sequences from individual reads via redundant observations. To further reduce the error rate, Michael et al. optimized CCS by increasing the sequencing coverages, refining the base-calling algorithms, and leveraging an improved polymerase chemistry to enhance the number of passes on the same molecule. These approaches achieved high-fidelity long reads with an accuracy exceeding 99.8%, overcoming the trade-off between read length and accuracy in TGS technologies.

It is worth mentioning that in addition to the fluorescence-based single-molecule sequencing technologies, the nanopore-based sequencing technologies also achieved an excellent base calling accuracy and ultralong read lengths beyond the limitations of NGS. − Due to the above-mentioned technical advancements, these single-molecule sequencing technologies have been commercialized, with representative platforms including PacBio’s single-molecule real-time (SMRT) sequencing technology and Oxford Nanopore’s nanopore sequencing technology (such as the MinION system), significantly driving the paradigm shift in genomics research. For instance, in the field of genome integrity analysis, Sergey et al. combined the PacBio HiFi system with nanopore sequencing to complete telomere-to-telomere (T2T) sequencing of the human genome, filling 8% of the sequence gaps. , In summary, combining the single-molecule perspective with sequencing technologies provides longer reads and more accurate measurement results and, when paired with more streamlined library preparation kits and workflows, opens up brand-new analytical dimensions for life science research.

3.2. Single-Molecule Array Technologies

The accurate quantification of ultralow-concentration biomarkers (e.g., proteins, nucleic acids, enzymes, and metabolites) is critical for diverse applications in clinical diagnostics, drug discovery, and environmental monitoring. Conventional methods such as PCR (Figure a) and ELISA serve as the gold standard in many diagnostics fields. However, as the conventional methods face limitations in sensitivity, matrix interference, and inability to resolve trace-level mixtures in many cases, they have proven inadequate for accurate biomarker sensing at low concentrations, thereby creating an imperative for ultrasensitive detection techniques. − Furthermore, traditional molecular assays typically offer relative quantification and require spike-in controls to achieve absolute quantification.

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Some typical demonstrations of conventional and Simoa technologies. (a) Schematic of the conventional quantitative PCR (qPCR) method. (b) Schematic of a prototype Simoa assay for single-molecule detection of target molecules by dispensing samples into thousands of individual microcompartments and quantifying the proportion of units with positive (“1”) readout. Adapted with permission from ref . Copyright © 2020 The American Association for the Advancement of Science. (c) Schematic illustration of a dSimoa assay for quick imaging of attomolar proteins from a droplet-based sample loading. Adapted with permission from ref . Copyright © 2020 American Chemical Society. (d) Schematic illustration of a ddEXPAR assay in hydrogel network for absolute determination of ternary miRNAs (miR-223, miR-143, and let-7a) extracted from human urine exosomes on a microfluidic chip. Adapted with permission from ref . Copyright © 2024 American Chemical Society.

One way to circumvent these drawbacks is to introduce digital assays, which presents a transformative alternative by enabling single-molecule counting in partitioned samples. Taking advantage of eliminating normalization dependencies and realizing absolute quantification without external calibration, this single-molecule array (Simoa) technology stands out as an ultrasensitive digital platform capable of detecting target moleculesprimarily proteins but also nucleic acids and other biomolecules, with single-molecule sensitivity. For example, Simoa platforms, such as digital enzyme-linked immunosorbent assay (dELISA), have enhanced measurement sensitivities for various proteins by up to 1000-fold compared to traditional ELISA. ,, In the Simoa prototype system, target analytes are captured on biofunctionalized paramagnetic beads (MBs), with a large excess of beads ensuring Poisson-distributed single-molecule binding (0 or 1 molecule per bead). The fluorophore-labeled microbeads are then isolated in an array of femtoliter-sized microwells designed for single-bead occupancy. After sealing the Simoa substrate with oil to facilitate reagent reactions, enzyme-catalyzed fluorescent products remain confined within individual micron-sized compartments. Only wells containing one target molecule generate discrete, localized fluorescence outputs that can be easily detected by an optical imaging system (Figure b). , This approach therefore enables background-free detection with femtomolar to attomolar sensitivity through digital counting statistics.

While Simoa achieves subfemtomolar detection limits and remains the gold standard for ultrasensitive protein analysis, its sensitivity is fundamentally constrained by low sampling efficiencies. To address this limitation, bead encapsulation in water-in-oil droplets has emerged as a promising strategy to enhance sampling efficiency in digital bioassays. Recent advances in digital droplet-based immunoassays demonstrate bead loading efficiency of up to 60%, achieving sensitivities that rival or even surpass (by up to an order of magnitude) conventional Simoa technology. , However, their integration into point-of-care (POC) systems is complicated by the requirement for precisely controlled droplet generation. Besides, a major challenge for POC implementation lies in the throughput limitation caused by the requirement to image numerous bead-free droplets. Walt et al. addressed these limitations by developing dropcast single-molecule assays (dSimoa), an innovative approach that combines on-bead signal generation with bead dropcasting into monolayer films for single-molecule counting (Figure c). This platform offers several key advantages: (i) by localizing a nondiffusible fluorescent signal to each target-carrying bead, it eliminates the need for bead loading into microwells or droplets for signal compartmentalization; (ii) it enables analysis of significantly more beads, thereby improving sampling efficiency and enhancing sensitivity; (iii) the template-free readout only requires a basic optical setup for signal transduction, thus offering improved cost-effectiveness for POC integration. These advancements allow the dSimoa platform to achieve attomolar detection limits, with an up to 25-fold sensitivity improvement over traditional Simoa technology, and represent the current state of the art for ultrasensitive protein detection.

The Simoa platform has also shown promise for nucleic acid detection, including microRNAs (miRNAs), which serve as valuable noninvasive biomarkers despite their extremely low abundance in urine (2–4 orders of magnitude lower than in blood). , While exponential isothermal amplification (EXPAR) enables rapid miRNA quantification, its multiplexing capability is limited by spurious amplification, poor sensitivity, and reproducibility. , Li et al. developed a droplet-based digital EXPAR (ddEXPAR) platform that combines immunomagnetic exosome capture with compartmentalized amplification, enabling femtomolar-sensitive quantification of miRNAs in microwells (Figure d). This approach generates background-free, heterogeneous fluorescence profiles that differ from the conventional homogeneous solution-based assays for nucleic acid amplification and distinguished healthy individuals from primary urethral carcinoma patients and further classified disease subtypes. Compared to traditional solution-based EXPAR, this digital assay significantly reduces nonspecific amplification common to occur in bulk solution and improves sensitivity by over 2 orders of magnitude. While there are other methods for digital analysis of nucleic acids using quantitative polymerase chain reaction (qPCR) , or isothermal amplification strategies, ,, the exponential nature of such amplifications renders it prone to leaky reactions and inevitably induce spurious, nonspecific amplification reactions. One solution is to embed amplification reagents in a nanoporous hydrogel matrix that provides ideal permeability to allow free diffusion of nucleic acids and easy removal of unreacted primers.

In summary, Simoa technology demonstrates pioneering advancement in bioanalytical detection by achieving absolute quantification at an attomolar sensitivity through its single-molecule counting methodology. Concurrent technological innovations have progressively improved the sensitivity, simplicity, and adaptability of such platforms: the streamlined dSimoa platform is able to reach attomolar sensitivity for common protein determination; ddEXPAR as a useful complement to digital PCR significantly enhances specificity for multiplexed miRNA analysis in human bodily fluids. These novelties collectively improve the clinical precision of biomarker analysis and optimize early disease diagnosis. In addition, Simoa technology exhibits superior biocompatibility with complex biological matrices and enables robust multiplexed detection, positioning it as a viable standardized platform for translational medicine applications. Despite the diverse applications in ultrasensitive protein and nucleic acid detection, Simoa technologies still suffer from a few drawbacks including complicated assay optimization, sensitivity to contamination, limited multiplexing capability, and data analysis complexity. With continued development to overcome the above obstacles, Simoa is poised to play an increasingly critical role in early disease diagnosis and post-therapeutic recurrence monitoring.

3.3. Single-Molecule Pull-Down Assays

Protein–protein interactions (PPIs) are crucial in understanding many biological processes and have long been a central topic in biological mechanism studies. − To study the direct or indirect PPIs, pull-down assays are the gold standard widely used in the field (Figure a). In pull-down assays, bait proteins carrying affinity tags specifically bind to the antibodies or substrates immobilized on beads, capturing the bait proteins and their interacting partners. With many types of orthogonal affinity tags and their corresponding beads (e.g., GST, FLAG, HA tags) developed to facilitate the applicability, complexes of interest are routinely pulled down, and the captured proteins are easily eluted for downstream analysis with Western blot , or mass spectrometry methods. Despite its simplicity and versatility, pull-down experiments face certain limitations, such as the inability to distinguish mixed complex subpopulations and to determine the complex stoichiometry, the difficulty in capturing weak or transient interactions, , and the incompatibility with large-scale PPI screening.

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Schematic diagrams of the bulk and single-molecule pull-down experiments. (a) Conventional pull-down assays are performed in test tubes and use bulk detection modality, such as Western blots and mass spectrometry analysis. (b) Single-molecule pull-down assays are performed in imaging flow channels under fluorescence microscopy. Subpopulations of complexes can be distinguished (e.g., AB + AC vs ABC), and stoichiometric information can be provided via photobleaching trajectory analysis. Adapted with permission from ref . Copyright © 2011 Springer Nature.

To overcome these drawbacks, the Ha group developed an ingenious, yet simple, method for studying cellular protein complexes at the single-molecule level (Figure b). In this method, named the single-molecule pull-down assays (SiMPull), , physiological macromolecular complexes are directly pulled down from cell or tissue extracts onto the imaging surface on a single-molecule fluorescence microscope, which is precoated with antibodies that specifically recognize the tag-of-choice. Typically, the antibody coating is achieved using the PEG-biotin–streptavidin system, where the surface is functionalized with a PEG-based polymer layer (e.g., linear or branched PEG, , or PEG-containing detergents) that also sparsely carries biotin groups for attachment; on top of this layer, streptavidin (or NeutrAvidin) acts as “glue” to tether the biotinylated antibodies. Besides, the PEG coating also serves the purpose of passivation to prevent the adsorption and interference of nonspecific proteins. Flow channels are constructed on the imaging surface to facilitate buffer exchange. Cell lysates that contain the fluorescently labeled bait proteins are then introduced on the imaging surface, leading to the coimmunoprecipitation of the interacting partners, which are also labeled with fluorophores for detection. After washing out the unbound components, the target complexes are observed and analyzed through the TIRF microscopy, generating single-molecule signals with a high SNR. ,

As an excellent example for the effectiveness of SiMPull, Jain et al. studied the mTOR kinase-related complexes and confirmed that both mTORC1 and mTORC2 are dimeric in composition. Furthermore, the study revealed that although mTORC1 and mTORC2 are dimeric overall, their individual components (such as mTOR, raptor, rictor, etc.) primarily exist as monomers when expressed separately. This finding indicates that the dimerization of mTORC1 and mTORC2 is not driven by interactions between any single component but rather is the result of multiple subunits cooperating to form a composite surface. The authors then investigated the effects of different physiological conditions (such as nutrient deprivation and energy stress) and pharmaceutical treatments (such as rapamycin) on the mTOR complex assembly and stoichiometry. The results showed that most of the conditions and treatments do not lead to the alteration of the dimeric nature of the mTOR complexes; only rapamycin partially disrupts mTORC1, leading to transient monomeric mTOR intermediates, while mTORC2 remains unaffected.

Compared to the traditional pull-down techniques, performing pull-down experiments at the single-molecule level has several obvious advantages. (1) To measure the copy number of fluorescently labeled molecules in the complex, SiMPull experiments record the photobleaching trajectories of the fluorescent labels (i.e., the number of sudden stepwise drops in the fluorescence intensity). After accounting for the maturation and labeling efficiency of the fluorescent labels, the stoichiometry of the target proteins in the complex can be inferred based on the distribution of photobleaching steps. (2) SiMPull can distinguish complex subpopulations in a heterogeneous mixture that are often obscured under ensemble measurements. For example, a 1:1 mixture of two complexes, AB3 and AB, could be mistakenly determined as AB2 in stoichiometry if not measured at the single-molecule level. (3) SiMPull allows for the real-time observation of dynamic changes in protein complexes and the determination of protein–protein interaction kinetics. (4) SiMPull has extremely high sensitivity, capable of detecting and quantifying low-abundance complexes. (5) Finally, SiMPull requires a significantly lower sample volume than bulk methods, making it possible to study cells or tissues that are difficult to culture in large quantities. In spite of the various advantages, the sensitivity and specificity of SiMPull still depend on the antibody quality for capturing and detecting the target complexes, which poses a major bottleneck in the assay.

3.4. Fluorescence Intensity Shift Assays

Electrophoretic mobility shift assay (EMSA) is a widely used technique in molecular biology to study nucleic acid-binding proteins, such as transcription factors, chromatin remodelers, and ribonucleoprotein complexes. − The basic principle of EMSA involves the electrophoresis separation of protein–DNA complexes from free DNA under nondenaturing conditions. The DNA band on the gel is shifted when it is bound to a protein, hence the name “mobility shift” (Figure a). Based on the protein and nucleic acid concentrations used, reaction times, and mobility shift patterns observed, one can deduce a rich set of information from EMSA, such as the binding affinity, kinetics, and stoichiometry. , Moreover, EMSA has been used in combination with microfluidic and nanomaterial-enhanced detection, and applied in disease-related research. Despite its widespread utility in detecting protein-nucleic acid interactions, EMSA has several critical limitations. Most notably, due to a relatively long separation time on gels, transient biomolecular complexes can dissociate during electrophoresis. Therefore, EMSA suffers a poor sensitivity for studying such interactions, even when radiolabeled nucleic acids are used for detection. In addition, once complexes are separated on gels, it is difficult to sequentially add or remove reagents (e.g., another protein or nucleic acid) to study dissociation and the sequential multicomponent interactions.

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Schematic diagrams of conventional EMSA and single-molecule FISA for studying protein-nucleic acid interactions. (a) EMSA detects protein-nucleic acid complexes through gel electrophoresis separation, where the bound DNA (e.g., CtsR-bound DNA) migrates slower than DNA alone (i.e., shifted bands). EMSA has limited sensitivity for weak or transient interactions due to complex dissociation during electrophoresis. (b) FISA overcomes the limitations by utilizing the single-molecule PIFE effect. A fluorophore placed near the protein-binding site on the DNA exhibits intensity changes upon protein binding. MscB binding to the CtsR-DNA complex further enhances the fluorescence intensity, as shown via the increased PIFE effect. The increases in fluorescence intensities reflect real-time protein binding, offering a higher sensitivity for dynamic or low-affinity interactions as compared to EMSA.

Inspired by the development of SiMPull, Cai et al. developed a fluorescence intensity shift assay (FISA) that effectively overcomes the aforementioned limitations of EMSA. Based on the PIFE principle, FISA detects the nucleic acid–protein interactions by positioning a fluorophore (such as sulfonated Cy3) in the vicinity of the protein binding sites on the nucleic acids and monitoring the fluorescence intensity shifts due to protein binding (Figure b). To achieve a strong PIFE effect while minimizing interference with the molecular interactions, the choice and labeling positions of the fluorophore are empirically tested and carefully validated during pilot experiments. As an example, using single-molecule PIFE-FISA, Cai et al. investigated the binding kinetics between the bacterial heat-shock regulator CtsR and its target DNA in real time, as well as the modulation mechanism of the CtsR-DNA complex by the arginine kinase McsB. , The quantitative FISA measurements showed that CtsR binds rapidly and stably to the target DNA with K D and Hill coefficients of 10.4 nM and 2.0, respectively. Besides, it is revealed that McsB transiently interacts with the CtsR-DNA complex, with the k on and k off rates of 0.75 μM–1 s–1 and 0.34 s–1, respectively. The McsB treatments render the CtsR-DNA complex more sensitive to elevated temperatures, as evidenced by the fluorescence intensity downshift in FISA, indicating rapid CtsR dissociation upon heated buffer washes. This kinetic information obtained by FISA, in combination with mass spectrometry and molecular simulation results, together suggest a new hierarchical phosphorylation mechanism of McsB, which highlights the functional importance of periphery arginine residues on CtsR regulation.

Several clear advantages of FISA stand out in comparison to those of EMSA. (1) FISA provides dynamic information on weak and transient interactions that would likely be lost during gel electrophoresis (e.g., the interaction between McsB and the CtsR-DNA complex with a K D around 400 nM). (2) FISA allows a low sample volume and easy buffer exchange thanks to the incorporation with flow channel configuration. This benefit is particularly evident when FISA is used to determine the temperature sensitivity of the CtsR-DNA complex, where a rapid introduction of heated buffers and quick washout of dissociated proteins is necessary. (3) Compared to other single-molecule methods that study protein-nucleic acid interactions (e.g., colocalization and FRET , ), PIFE-FISA bypasses the need of labeling proteins, which not only avoids the nonspecific binding of fluorescently labeled proteins but also overcomes the “concentration barrier” of detecting single-molecule signals in the presence of high concentrations of labeled species in solution. As a result, the researchers can readily use a protein concentration of 200 nM to observe the transient binding events of McsB to the CtsR-DNA complex. However, the need for optimization of the fluorophores and strict control of the illumination conditions remain limitations to the broader application of the assay.

3.5. Single-Molecule Electrochemiluminescence

Electrochemiluminescence (ECL) is an optoelectronic phenomenon where luminophores are electrochemically excited on the electrodes at controlled potentials, which leads to light emission facilitated by coreactants. − Since ECL is induced by electrochemical reactions rather than external light, this technique exhibits virtually no background signal, therefore enabling ultrasensitive optical signal readout and affording an excellent SNR. , Thanks to these desirable characteristics, ECL has been widely employed as a readout method in a broad set of high-sensitivity bioimaging and bioanalytic diagnostic techniques, including ECL-based antigen imaging (Figure a), viral protein identification, and ultrasensitive immunoassays. , These methods are distinguished by their simplicity, low detection limits, and good reproducibility, and have been extensively integrated into commercial automated analysis platforms. However, conventional ECL measurements yield only averaged signals from an ensemble of luminophores, posing significant challenges in observing individual molecular differences, transient kinetic behaviors, and spatial heterogeneity during the reaction process. , This averaging effect tends to obscure the intrinsic complexity of the reaction systems, thereby limiting the understanding of the underlying microscopic mechanisms. , Although the development of ECL imaging has improved spatial resolution to some extent (Figure b), there remains a significant gap in the ECL field compared to that achieved by single-molecule super-resolution fluorescence imaging techniques.

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Schematic diagrams of conventional and single-molecule ECL measurements. (a) Schematic representation of T cell recognition and discrimination via label-free ECL imaging on an electrode surface modified with different stimuli. Adapted with permission from ref . Copyright © 2023 Wiley-VCH GmbH, Weinheim. (b) Conventional (diffraction-limited) ECL images. (c) Design of the single-molecule ECL microscopy setup integrating wide-field optical imaging and electrochemical recording for observing individual stochastic electrochemical reactions in solution. (d) Single-molecule super-resolution ECL images. Adapted with permission from ref . Copyright © 2021 Springer Nature.

Detecting single ECL reactions with high spatial resolution has long been a goal in the field of single-molecule imaging. − However, unlike most single-molecule fluorescence techniques, where the SNR of detecting a single fluorophore can be boosted by the hundreds to thousands of photons the fluorophore generates, it is extremely difficult to detect a single ECL luminophore because only a few photons are generated each time the luminophore is excited. , To tackle these obstacles, the Feng group has pioneered the development of a highly sensitive ECL microscopy platform (Figure c), capable of achieving synchronized electrochemical control with wide-field optical detection. Specifically, this platform integrates a standard three-electrode configuration with an inverted optical microscope and utilizes a custom-designed transparent indium tin oxide (ITO) electrode, which ensures the electrochemical reactivity and optical transparency, both required in this experiment. By applying precisely controlled potentials and diluting the reactant concentrations to reduce the molecular collision frequency, single-molecule events of ECL reactions were successfully isolated in both space and time. Facilitated by a high-numerical-aperture (NA) objective lens and an electron-multiplying charge-coupled device (EMCCD) detector, the platform achieves high-sensitivity detection of single-photon emission events characteristic of single ECL reactions in solution.

Additionally, Feng et al. further exploited the stochastic spatiotemporal distribution of single-molecule ECL signals and applied an image reconstruction strategy to achieve super-resolution imaging. Although individual ECL events generate only a few photons, constraining the precision of conventional single-molecule localization algorithms, repeated emissions at the same reaction site can form multiple localization clusters in adjacent regions, thereby substantially enhancing the spatial resolution of image reconstruction. As a result, the platform achieves a spatial resolution of approximately 22 nm in the Ru­(bpy)3 2+/TPrA system and was successfully applied to the dynamic super-resolution imaging of live cells (Figure d), thus establishing a label-free, laser-free, and real-time imaging methodology based on the single-molecule ECL microscopy. Specifically, during live-cell imaging, the system demonstrated a spatial and temporal resolution of 150 nm and 12 s, respectively, which are comparable to those achieved by optimized super-resolution fluorescence microscopy. Furthermore, employing the single-molecule ECL microscopy, researchers achieved super-resolution imaging of Ru­(bpy)3 2+-mediated reactions on gold electrode surfaces and investigated the underlying mechanisms of electrocatalytic reactions. This research revealed that structural reorganization of the catalyst during reactions plays a pivotal role in determining its activity, providing a new insight into the dynamic relationship between surface structures and catalytic performance.

Compared to the conventional ECL techniques, single-molecule ECL imaging overcomes the intrinsic limitations of ensemble-averaged measurements, enabling ultrasensitive and super-resolution detection of individual chemical reaction events. − This method offers direct molecular-level insights into catalytic reactions, , molecular recognition, and electron transfer processes, , revealing critical details that are often obscured in traditional ensemble measurements. With broad application prospects in areas such as single-cell analysis, − tracking membrane protein dynamics, and investigating interfacial reactions in materials chemistry, single-molecule ECL lays a solid foundation for the development of next-generation functional imaging technologies, and opens new frontiers for understanding chemical processes at the molecular level, especially when equipped with a multichannel multiplexed imaging capability.

3.6. Other Single-Molecule Versions of Ensemble Biochemical Assays

The strategy of augmenting ensemble biochemical assays with single-molecule capability is definitely not limited to the several examples discussed above. For example, as the single-molecule counterpart of the surface plasmon resonance (SPR, Figure a,b) method, the Tao group developed the plasmonic scattering microscopy (PSM, Figure c,d), which is able to achieve digital kinetic and equilibrium analysis of individual protein interactions while obtaining the size information on the binding proteins, , as demonstrated via detecting single molecules of human immunoglobulin M (IgM) and immunoglobulin A (IgA). In addition, a super-resolution single-molecule fluorescence binding assay has also been developed by the Ha group, where the accuracy of multicolor colocalization between ligands and receptors is enhanced via the application of the centroid localization algorithm. This innovation effectively corrects for common artifacts including nearby nonspecific binding events and optically overlapping binding sites at the diffraction-limited resolution (Figure e,f). Once again, various advantages are amply demonstrated in these examples.

8.

8

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Schematic diagrams of conventional and two general single-molecule kinetic assays. (a) Schematic of the conventional SPR method. (b) A representative SPR result of the IgA-anti-IgA interaction. (c) In PSM, surface plasmonic waves are excited by directing light from the objective at the bottom, on which a gold-coated glass slide is placed. Scattering of the plasmonic waves by a particle or protein (E s) and by the gold surface (E b) is collected from the objective at the top. (d) Representative PSM image of individual IgM molecules and the corresponding intensity histogram. The protein size can be inferred from the intensity value. Adapted with permission from ref . Copyright © 2020 Springer Nature. (e, f) Application of the single-molecule centroid localization algorithm in multicolor colocalization microscopy can detect and correct two major artifacts in this type of assay, i.e., the nonspecific binding events and optically overlapping receptors. Adapted with permission from ref . Copyright © 2018 American Chemical Society.

4. The Limitations of Single-Molecule Optical Techniques

Although providing a rich set of molecular information, single-molecule techniques typically examine only a few distinct samples per experiment, which limits the single-molecule techniques’ capability to explore large sequence spaces. Two recently developed techniques, i.e., multiplexed single-molecule characterization at the library scale (MUSCLE), and single-molecule parallel analysis for rapid exploration of sequence space (SPARXS), combined single-molecule fluorescence detection with high-throughput sequencing, so that the functions, dynamics, and corresponding sequences of thousands of distinct biomacromolecules were measured. This enhanced capacity in multiplexing enables the investigation and understanding of the fundamental “sequence-structure-function” relationship of biomacromolecules. Further developments of these methods and their applications in a broader set of biological systems are a field of active research.

5. Conclusion and Perspectives

In summary, through the examples discussed, we have seen many attractive features of the single-molecule tools and methods, from negating the need for bulk synchronization to observe nucleotide sequences in SMRT sequencing to digital counting and absolute quantification of biomolecules in Simoa, from unmasking molecular heterogeneity and determining stoichiometry in SiMPull, to easy integration with flow channel configuration and in situ imaging in FISA, and finally to the capability of spatial super-resolution in single-molecule kinetic assays and ECL (Figure ). The superior performance of these methods, such as ultralong reads, richer information, and fewer artifacts, as well as the ability to work with weak or transient interactions, are arguably the direct results of their unique technical advantages.

9.

9

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Various technical advantages of single-molecule techniques.

Can more ensemble biochemical assays be converted to their single-molecule versions? The answer is probably yes. Here, we propose a new single-molecule assay, named “single-molecule polysome profiling”. The conventional polysome profiling technique, which sediments and separates polysomes in a density gradient under ultracentrifugation conditions, is the standard (and perhaps the only) method available to determine the overall distribution of translating ribosomes in cells. Though being broadly applied, this technique barely resolves heavy polysomes containing more than ten ribosomes and only provides a semiquantitative estimate of the amounts of different polysomes, not mentioning the lengthy operation it demands on an ultracentrifuge. Inspired by the subunit counting method in SiMPull, it is reasonable to assume that one can probably count the number of ribosomes in a polysome complex if each ribosome is fluorescently labeled and the polysome complex immobilized on the imaging surface. Since the number of resolvable fluorescence intensity levels is already around three or four in FISA, and can reach up to 20, as demonstrated by the Ha group, it is foreseeable that the single-molecule polysome profiling assay could not only detect larger polysome complexes in a more quantitative way, but also significantly reduce the sample amount and assay time. In addition, one can further augment the fluorescence assay with scattering imaging to obtain the total mass of the polysome complexes.

While assays like ELISA, pull-down experiments, and EMSA are routinely carried out in many biochemistry laboratories, each assay requires its own set of apparatus and has a different operation protocol with specific steps that are not transferrable among assays; some of the instruments required for these assays (e.g., a plate reader) can be rather expensive. We cannot help but wonder what would happen if the imaging-based single-molecule versions of many common assays were fully developed and adopted as the “bread-and-butter” tools in laboratories. Not only could one access all the benefits of single-molecule experiments discussed above, but also the number of different lab instruments would drastically decrease; perhaps one carefully designed multipurpose microscope could suffice. As a result, all these experiments could be performed on the same microscope platform and the difference in operating different assays could be as trivial as just adding different buffers in the flow channels. These efforts would inevitably promote a wider accessibility and applicability of new biochemical methods and tools.

Acknowledgments

This work was supported by the Fundamental Research Funds for the Central Universities 2024300410 (to B.H.), the State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University 5431ZZXM2403 (to B.H.), and the National Natural Science Foundation of China (Nos. 22404079, 22076125, U24A20503).

Glossary

Vocabulary

Ensemble average

the average of a property (structure, activity, etc.) over a large number of molecules, rather than that of an individual molecule

Heterogenous subpopulations

distinct subgroups of molecules within a sample, whose certain property is different from the average

Bulk synchronization

methods that force a population of molecules to exist in the same state; often required to study state transitions and reaction kinetics

Diffraction-limited resolution

the optical resolution limit due to diffraction, i.e., λ/2NA, where λ is the observation wavelength and NA is the numerical aperture

Numerical aperture

describes the portion of emitted light from a point source that an objective lens can collect, i.e., nsinθ, where n is the refractive index and θ is the half an gle of the widest cone of light that the objective lens collects

Point spread functions (PSFs)

describes how a point light source is imaged by an optical system; due to optical diffraction, point spread functions have a finite size

#.

H.T. and S.L. contributed equally to this work.

The authors declare no competing financial interest.

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