High-Throughput Identification of Single Nanoparticles via Electrochemically Assisted High-Resolution Plasmonic Scattering Interferometric Microscopy.

The identification of nanoparticles within heterogeneous mixtures poses significant challenges due to the similarity in physical properties among different nanomaterials. Here, we present electrochemically assisted high-resolution plasmonic scattering interferometric microscopy (HR-PSIM). This technique allows for the high-throughput identification of nanoparticles by accurately measuring the refractive index of individual nanoparticles without interference from background signals. Through elimination of parabolic scattering interference and employing electrochemical modulation, HR-PSIM demonstrates high spatial resolution and stability against background noise, enabling the differentiation of nanoparticles with closely matched refractive indices, such as Au and Ag nanoparticles. The efficacy of this method is demonstrated through its application in real-time, label-free imaging of nanoparticle electrochemical activity, providing a platform for the precise and high-throughput characterization of nanomaterials. The robustness of our approach against electrochemical interference and its high spatial resolution mark a significant advancement in the field of nanomaterial analysis, promising wide-ranging applications in nanoparticle research and beyond.

Ferroelectric Bi2O2Te-Based Plasmonic Biosensor for Ultrasensitive Biomolecular Detection.

Ultrasensitive detection of biomarkers, particularly proteins, and microRNA, is critical for disease early diagnosis. Although surface plasmon resonance biosensors offer label-free, real-time detection, it is challenging to detect biomolecules at low concentrations that only induce a minor mass or refractive index change on the analyte molecules. Here an ultrasensitive plasmonic biosensor strategy is reported by utilizing the ferroelectric properties of Bi2O2Te as a sensitive-layer material. The polarization alteration of ferroelectric Bi2O2Te produces a significant plasmonic biosensing response, enabling the detection of charged biomolecules even at ultralow concentrations. An extraordinary ultralow detection limit of 1 fm is achieved for protein molecules and an unprecedented 0.1 fm for miRNA molecules, demonstrating exceptional specificity. The finding opens a promising avenue for the integration of 2D ferroelectric materials into plasmonic biosensors, with potential applications spanning a wide range.

Single-molecule calorimeter and free energy landscape.

The precise measurement of thermodynamic and kinetic properties for biomolecules provides the detailed information for a multitude of applications in biochemistry, biosensing, and health care. However, sensitivity in characterizing the thermodynamic binding affinity down to a single molecule, such as the Gibbs free energy ([Formula: see text]), enthalpy ([Formula: see text]), and entropy ([Formula: see text]), has not materialized. Here, we develop a nanoparticle-based technique to probe the energetic contributions of single-molecule binding events, which introduces a focused laser of optical tweezer to an optical path of plasmonic imaging to accumulate and monitor the transient local heating. This single-molecule calorimeter uncovers the complex nature of molecular interactions and binding characterizations, which can be employed to identify the thermodynamic equilibrium state and determine the energetic components and complete thermodynamic profile of the free energy landscape. This sensing platform promises a breakthrough in measuring thermal effect at the single-molecule level and provides a thorough description of biomolecular specific interactions.

An Ultrasensitive Plasmonic Sensor Based on 2D Ferroelectric Bi2 O2 Se.

Plasmonic biosensing is a label-free detection method that is commonly used to measure various biomolecular interactions. However, one of the main challenges in this approach is the ability to detect biomolecules at low concentrations with sufficient sensitivity and detection limits. Here, 2D ferroelectric materials are employed to address the issues with sensitivity in biosensor design. A plasmonic sensor based on Bi2 O2 Se nanosheets, a ferroelectric 2D material, is presented for the ultrasensitive detection of the protein molecule. Through imaging the surface charge density of Bi2 O2 Se, a detection limit of 1 fM is achieved for bovine serum albumin (BSA). These findings underscore the potential of ferroelectric 2D materials as critical building blocks for future biosensor and biomaterial architectures.

Plasmonic probing of the adhesion strength of single microbial cells.

Probing the binding between a microbe and surface is critical for understanding biofilm formation processes, developing biosensors, and designing biomaterials, but it remains a challenge. Here, we demonstrate a method to measure the interfacial forces of bacteria attached to the surface. We tracked the intrinsic fluctuations of individual bacterial cells using an interferometric plasmonic imaging technique. Unlike the existing methods, this approach determined the potential energy profile and quantified the adhesion strength of single cells by analyzing the fluctuations. This method provides insights into biofilm formation and can also serve as a promising platform for investigating biological entity/surface interactions, such as pathogenicity, microbial cell capture and detection, and antimicrobial interface screening.

Real-Time Plasmonic Imaging of the Compositional Evolution of Single Nanoparticles in Electrochemical Reactions.

Real-time probing of the compositional evolution of single nanoparticles during an electrochemical reaction is crucial for understanding the structure-performance relationship and rationally designing nanomaterials for desirable applications; however, it is consistently challenging to achieve high-throughput real-time tracking. Here, we present an optical imaging method, termed plasmonic scattering interferometry microscopy (PSIM), which is capable of imaging the compositional evolution of single nanoparticles during an aqueous electrochemical reaction in real time. By quantifying the plasmonic scattering interferometric pattern of nanoparticles, we establish the relationship between the pattern and composition of single nanoparticles. Using PSIM, we have successfully probed the compositional transformation dynamics of multiple individual nanoparticles during electrochemical reactions. PSIM could be used as a universal platform for exploring the compositional evolution of nanomaterials at the single-nanoparticle level and offers great potentials for addressing the extensive fundamental questions in nanoscience and nanotechnology.

Transient Plasmonic Imaging of Ion Migration on Single Nanoparticles and Insight for Double Layer Dynamics.

Single-nanoparticle electrochemistry offers electrochemical behaviors of individual entities beyond the ensemble system. An electric double layer (EDL) exists on any charged particle-liquid interface because of counter-ion accumulation, while direct measuring of the interfacial ion migration remains a challenge. Herein, a plasmonic-based transient microscopic method, with a temporal resolution of 1-2 µs, was demonstrated to directly track the ion migration dynamics on single charged nanoparticles. We found that the dynamics of EDL formation might deviate significantly from the prediction made by using the classical resistance-capacitance (RC) model under nanoscale and transient conditions. Under ultrafast charging, due to the limit migration rate of ions in the solution, the actual time scale of the EDL formation could be up to 5 times slower than the predicted value from the RC model. We then proposed a new theoretical model to describe the transient dynamics of EDL formation. These results may expand our current knowledge about nano-electrochemistry and transient electrochemistry.

Unraveling Cell-Type-Specific Targeted Delivery of Membrane-Camouflaged Nanoparticles with Plasmonic Imaging.

Cell-membrane-camouflaged nanoparticles (CMC-NPs) have been increasingly exploited to develop various therapeutic tools due to their high biocompatibility and cell-type-specific tumor-targeting properties. However, the molecular mechanism of CMC-NPs for homotypic targeting remains elusive. Here, we develop a plasmonic imaging method by coating gold nanoparticles (AuNPs) with cancer cell membranes and perform plasmonic imaging of the interactions between CMC-NPs and living cells at the single-cell level. Quantitative analysis of CMC-NPs in a different clustering status reveals that the presence of cell membranes on CMC-NPs results in a 7-fold increase in homotypic cell delivery and nearly 2 orders of magnitude acceleration of the intracellular agglomeration process. Significantly, we identify that integrin αvß3, a cell surface receptor abundantly expressed in tumor cells, is critical for the selective cell recognition of CMC-NPs. We thus establish a single-cell plasmonic imaging platform for probing NP-cell interactions, which sheds new light on the therapeutic applications of CMC-NPs.

Distinguishing cancer cell lines at a single living cell level via detection of sialic acid by dual-channel plasmonic imaging and by using a SERS-microfluidic droplet platform.

A high-throughput, dual-channel single cell analytical method is described for the detection of sialic acid (SA) on single cell based on the use of microfluidic droplets integrated with plasmonic imaging and surface-enhanced Raman spectroscopy (SERS) with the assistance of a multifunctional metal nanoparticle-based probe. The multifunctional plasmonic nanoprobe was prepared by modifying silver nanoparticles (AgNPs) with 4-mercaptophenylboronic acid (MPBA) that both warrants SA recognition and acts as a Raman reporter. This nanoprobe is a high-contrast indicator under bright field imaging due to the strong energy loss feature of AgNPs, and also owns possesses a strong SERS enhancement capability toward MPBA. Cells incubated with the plasmonic nanoprobes were isolated in water-in-oil droplets and then were re-dispersed in a chamber array chip. High-precision profiles of SA on a single cell in one droplet were obtained by the bright field imaging and image processing. The SA expression levels on different cell lines (MCF-7, HepG2, SGC and BNL.CL2) traced by SERS spectroscopy were compared. The statistical data among different cell lines confirm that the SA expression levels on cancer cells are much higher than that on normal cells. Single cell analysis further revealed that the cell-to-cell variations are more obvious in cancer cell lines. This study provides a valuable tool for understanding glycan-related biochemical processes. Graphical abstract A high-throughput, dual-channel microfluidic droplet platform succeeded in distinguishing different cancer cell lines at single living cell level integrated with plasmonic imaging and surface-enhanced Raman spectroscopy with assistance of a multifunctional metal nanoparticle-based probe.

Identification of Nanoparticles via Plasmonic Scattering Interferometry.

The development of optical imaging techniques has led to significant advancements in single-nanoparticle tracking and analysis, but these techniques are incapable of label-free selective nanoparticle recognition. A label-free plasmonic imaging technology that is able to identify different kinds of nanoparticles in water is now presented. It quantifies the plasmonic interferometric scattering patterns of nanoparticles and establishes relationships among the refractive index, particle size, and pattern both numerically and experimentally. Using this approach, metallic and metallic oxide particles with different radii were distinguished without any calibration. The ability to optically identify and size different kinds of nanoparticles can provide a promising platform for investigating nanoparticles in complex environments to facilitate nanoscience studies, such as single-nanoparticle catalysis and nanoparticle-based drug delivery.

Dual-Targeting Nanovesicles for In†Situ Intracellular Imaging of and Discrimination between Wild-type and Mutant p53.

p53 is a tumor-suppressor protein related to the cell cycle and programmed cell apoptosis. Herein, dual-targeting nanovesicles are designed for in situ imaging of intracellular wild-type p53 (WTp53) and mutant p53 (MUp53). Nanovesicle-encapsulated plasmonic gold nanoparticles (AuNPs) were functionalized with consensus DNA duplexes, and a fluorescein isothiocyanate (FITC)-marked anti-MUp53 antibody was conjugated to the nanovesicle surface. After entering the cytoplasm, the released AuNPs aggregated through recognition of WTp53 and the double-stranded DNA. The color changes of AuNPs were observed using dark-field microscopy, which showed the intracellular WTp53 distribution. The MUp53 location was detected though the immunological recognition between FITC-labeled anti-MUp53 and MUp53. Thus, a one-step incubation method for the in situ imaging of intracellular WTp53 and MUp53 was obtained; this was used to monitor the p53 level under a drug treatment.

Label-Free Tracking of Single Organelle Transportation in Cells with Nanometer Precision Using a Plasmonic Imaging Technique.

Imaging and tracking of nano- and micrometer-sized organelles in cells with nanometer precision is crucial for understanding cellular behaviors at the molecular scale. Because of the fast intracellular dynamic processes, the imaging and tracking method must also be fast. In addition, to ensure that the observed dynamics is relevant to the native functions, it is critical to keep the cells under their native states. Here, a plasmonics-based imaging technique is demonstrated for studying the dynamics of organelles in 3D with high localization precision (5 nm) and temporal (10 ms) resolution. The technique is label-free and can track subcellular structures in the native state of the cells. Using the technique, nanometer steps of organelle (e.g., mitochondria) transportation are observed along neurite microtubules in primary neurons, and the 3D structure of neurite microtubule bundles is reconstructed at the nanometer scale from the tracks of the moving organelles.

Real-time monitoring of phosphorylation kinetics with self-assembled nano-oscillators.

Phosphorylation is a post-translational modification that is involved in many basic cellular processes and diseases, but is difficult to detect in real time with existing technologies. A label-free detection of phosphorylation is reported in real time with self-assembled nano-oscillators. Each nano-oscillator consists of a gold nanoparticle tethered to a gold surface with a molecular linker. When the nanoparticle is charged, the nano-oscillator can be driven into oscillation with an electric field and detected with a plasmonic imaging approach. The nano-oscillators measure charge change associated with phosphorylation of peptides attached onto a single nanoparticle, allowing us to study the dynamic process of phosphorylation in real time without antibodies down to a few molecules, from which Michaelis and catalytic rate constants are determined.

Deep-Learning Driven, High-Precision Plasmonic Scattering Interferometry for Single-Particle Identification.

Label-free probing of the material composition of (bio)nano-objects directly in solution at the single-particle level is crucial in various fields, including colloid analysis and medical diagnostics. However, it remains challenging to decipher the constituents of heterogeneous mixtures of nano-objects with high sensitivity and resolution. Here, we present deep-learning plasmonic scattering interferometric microscopy, which is capable of identifying the composition of nanoparticles automatically with high throughput at the single-particle level. By employing deep learning to decode the quantitative relationship between the interferometric scattering patterns of nanoparticles and their intrinsic material properties, this technique is capable of high-throughput, label-free identification of diverse nanoparticle types. We demonstrate its versatility in analyzing dynamic surface chemical reactions on single nanoparticles, revealing its potential as a universal platform for nanoparticle imaging and reaction analysis. This technique not only streamlines the process of nanoparticle characterization, but also proposes a methodology for a deeper understanding of nanoscale dynamics, holding great potential for addressing extensive fundamental questions in nanoscience and nanotechnology.

Nanoscale Spatiotemporal Dynamics of Microbial Adhesion: Unveiling Stepwise Transitions with Plasmonic Imaging.

Understanding bacterial adhesion at the nanoscale is crucial for elucidating biofilm formation, enhancing biosensor performance, and designing advanced biomaterials. However, the dynamics of the critical transition from reversible to irreversible adhesion has remained elusive due to analytical constraints. Here, we probed this adhesion transition, unveiling nanoscale, step-like bacterial approaches to substrates using a plasmonic imaging technique. This method reveals the discontinuous nature of adhesion, emphasizing the complex interplay between bacterial extracellular polymeric substances (EPS) and substrates. Our findings not only deepen our understanding of bacterial adhesion but also have significant implications for the development of theoretical models for biofilm management. By elucidating these nanoscale step-like adhesion processes, our work provides avenues for the application of nanotechnology in biosensing, biofilm control, and the creation of biomimetic materials.

Plasmonic Scattering Imaging of Surface-Bonded Nanoparticles at the Solution-Solid Interface.

Imaging nanoscale objects at interfaces is essential for revealing surface-tuned mechanisms in chemistry, physics, and life science. Plasmonic-based imaging, a label-free and surface-sensitive technique, has been widely used for studying the chemical and biological behavior of nanoscale objects at interfaces. However, direct imaging of surface-bonded nanoscale objects remains challenging due to uneven image backgrounds. Here, we present a new surface-bonded nanoscale object detection microscopy that eliminates strong background interference by reconstructing accurate scattering patterns at different positions. Our method effectively functions at low signal-to-background ratios, allowing for optical scattering detection of surface-bonded polystyrene nanoparticles and severe acute respiratory syndrome coronavirus 2 pseudovirus. It is also compatible with other imaging configurations, such as bright-field imaging. This technique complements existing methods for dynamic scattering imaging and broadens the applications of plasmonic imaging techniques for high-throughput sensing of surface-bonded nanoscale objects, enhancing our understanding of the properties, composition, and morphology of nanoparticles and surfaces at the nanoscale.

Quick and Mild Isolation of Intact Lysosomes Using Magnetic-Plasmonic Hybrid Nanoparticles.

Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic-plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (Rt). Detailed analysis of time variations of Rt for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (tdelay) and temperature (T), the influences of tdelay and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, tdelay = 30 min and T = 4 °C were optimal settings.

Plasmonic Probing Single-Cell Bio-Current Waves with a Shrinking Magnetite Nanoprobe.

Probing of the single-cell level extracellular electron transfer highlights the maximum output current for microbial fuel cells (MFCs) at hundreds of femtoampere per cell, which is difficult to achieve by existing devices. Past studies focus on the external factors for boosting charge-extraction efficiency from bacteria. Here, we elucidate the intracellular factors that determine this output limit by monitoring the respiratory-driven shrinking kinetics of a single magnetite nanoprobe immobilized on a single Shewanella oneidensis MR-1 cell with plasmonic imaging. Quantified dissolving of nanoprobes unveils a previously undescribed bio-current fluctuation between 0 and 2.7 fA on a ∼40 min cycle. Simultaneously tracing of endogenous oscillations indicates that the bio-current waves are correlated with the periodic cellular electrokinesis. The unsynchronized electron transfer capability in the cell population results in the mean current of 0.24 fA per cell, significantly smaller than in single cells. It explains why the averaged output current of MFCs cannot reach the measured single-cell currents. This work offers a different perspective to improve the power output by extending the active episodes of the bio-current waves.

A Robust Nanoparticle-based Magnetic Separation Method for Intact Lysosomes.

Lysosome isolation is a preresiquite for identifying lysosomal protein composition by mass spectroscopic analysis, to reveal lysosome functions, and their involvement in some diseases. Magnetic nanoparticle-based fractionation has received great attention for lysosome isolation, owing to its high efficiency, purity, and preservation of lysosomal structures. Understanding the intracellular trafficking of magnetic probes is the key point of this technique, to determine the appropriate time for magnetic isolation of lysosomes, because this parameter changes depending on different cell lines used. The traditional magnetic probes, such as superparamagnetic iron oxide nanoparticles (SPIONs), require surface modification by fluorescent dyes to enable the investigation of their intracellular trafficking, which has some disadvantages, including the possible alternation of their bio-interaction, and the instability of fluorescence properties in the lysosomal environment. To overcome those limitations, we present a protocol that employs magnetic-plasmonic nanoparticles (MPNPs) to investigate intracellular trafficking using their intrinsic imaging capability, followed by quick lysosome isolation using a magnetic column. This protocol can be easily applied to isolate the intact lysosomes of any adherent cell lines. Graphical abstract.

Single-Particle Electrochemical Imaging Provides Insights into Silver Nanoparticle Dissolution at the Solution-Solid Interface.

Dissolution of nanoparticles is an environmental interfacial process that affects the transformation of nanoparticles. Understanding the dissolution processes of nanoparticles is important to predict their fate in the aquatic environment. However, studying nanoparticle dissolution kinetics is still challenging since dissolution is usually coupled with nanoparticle aggregation. Here, we probed the dissolution process of Ag nanoparticles at the single-particle level by surface plasmon resonance microscopy. The single-particle imaging capability enabled us to classify Ag nanoparticles, measure the dissolution dynamics of single nanoparticles, and correlate the aggregation size with oxidation activity. Moreover, we studied the dual effect of natural organic matter on the dissolution of Ag nanoparticles and validated this result with real natural freshwater. Our study provides new insights into the dissolution of Ag nanoparticles, and this technique can be extended for other nanomaterials to evaluate their fate in aquatic environments.