Real‐Time Label‐Free Monitoring of Nanoparticle Cell Uptake

The surface plasmon resonance technique in combination with whole cell sensing is used for the first time for real‐time label‐free monitoring of nanoparticle cell uptake. The uptake kinetics of several types of nanoparticles relevant to drug delivery applications into HeLa cells is determined. The cell uptake of the nanoparticles is confirmed by confocal microscopy. The cell uptake of silica nanoparticles and polyethylenimine–plasmid DNA polyplexes is studied as a function of temperature, and the uptake energies are determined by Arrhenius plots. The phase transition temperature of the HeLa cell membrane is detected when monitoring cell uptake of silica nanoparticles at different temperatures. The HeLa cell uptake of the mesoporous silica nanoparticles is energy‐independent at temperatures slightly higher than the phase transition temperature of the HeLa cell membrane, while the uptake of polyethylenimine–DNA polyplexes is energy‐dependent and linear as a function of temperature with an activation energy of Ea = 62 ± 7 kJ mol^?1 = 15 ± 2 kcal mol^?1. The HeLa cell uptake of red blood cell derived extracellular vesicles is also studied as a function of the extracellular vesicle concentration. The results show a concentration dependent behavior reaching a saturation level of the extracellular vesicle uptake by HeLa cells.     

Design of Hierarchical Beads for Efficient Label‐Free Cell Capture

Defined hierarchical materials promise cell analysis and call for application‐driven design in practical use. The further issue is to develop advanced materials and devices for efficient label‐free cell capture with minimum instrumentation. Herein, the design of hierarchical beads is reported for efficient label‐free cell capture. Silica nanoparticles (size of ≈15 nm) are coated onto silica spheres (size of ≈200 nm) to achieve nanoscale surface roughness, and then the rough silica spheres are combined with microbeads (≈150–1000 µm in diameter) to assemble hierarchical structures. These hierarchical beads are built via electrostatic interaction, covalent bonding, and nanoparticle adherence. Further, after functionalization by hyaluronic acid (HA), the hierarchical beads display desirable surface hydrophilicity, biocompatibility, and chemical/structural stability. Due to the controlled surface topology and chemistry, HA‐functionalized hierarchical beads afford high cell capture efficiency up to 98.7% in a facile label‐free manner. This work guides the development of label‐free cell capture techniques and contributes to the construction of smart interfaces in bio‐systems.     

High Surface‐Enhanced Raman Scattering Performance of Individual Gold Nanoflowers and Their Application in Live Cell Imaging

Molecular imaging techniques based on surface‐enhanced Raman scattering (SERS) face a lack of reproducibility and reliability, thus hampering its practical application. Flower‐like gold nanoparticles have strong SERS enhancement performance due to having plenty of hot‐spots on their surfaces, and this enhancement is not dependent on the aggregation of the particles. These features make this kind of particle an ideal SERS substrate to improve the reproducibility in SERS imaging. Here, the SERS properties of individual flower‐like gold nanoparticles are systematically investigated. The measurements reveal that the enhancement of a single gold nanoparticle is independent of the polarization of the excitation laser with an enhancement factor as high as 10⁸. After capping with Raman signal molecules and folic acid, the gold nanoflowers show strong Raman signal in the living cells, excellent targeting properties, and a high signal‐to‐noise ratio for SERS imaging.     

Superficial‐Layer‐Enhanced Raman Scattering (SLERS) for Depth Detection of Noncontact Molecules

Although the strength of Raman signals can be increased by many orders of magnitude on noble metal nanoparticles, this enhancement is confined to an extremely short distance from the Raman‐active surface. The key to the development of Raman spectroscopy for applications in diagnosis and detection of cancer and inflammatory diseases, and in pharmacology, relies on the capability of detecting analytes that are noninteractive with Raman‐active surfaces. Here, a new Raman enhancement system is constructed, superficial‐layer‐enhanced Raman scattering (SLERS), by covering elongated tetrahexahedral gold nanoparticle arrays with a superficial perovskite (CH₃NH₃PbBr₃) film. Plasmonic decay is depressed along the vertical direction away from the noble metal surface and the penetration depth is increased in the perovskite media. The vertical penetration of SLERS is verified by the spatial distribution of the analytes via Raman imaging in layer‐scanning mode.     

A New Approach to Assess Gold Nanoparticle Uptake by Mammalian Cells: Combining Optical Dark‐Field and Transmission Electron Microscopy

Toxicological effects of nanoparticles are associated with their internalization into cells. Hence, there is a strong need for techniques revealing the interaction between particles and cells as well as quantifying the uptake at the same time. For that reason, herein optical dark‐field microscopy is used in conjunction with transmission electron microscopy to investigate the uptake of gold nanoparticles into epithelial cells with respect to shape, stabilizing agent, and surface charge. The number of internalized particles is strongly dependent on the stabilizing agent, but not on the particle shape. A test of metabolic activity shows no direct correlation with the number of internalized particles. Therefore, particle properties besides coating and shape are suspected to contribute to the observed toxicity.     

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.     

Label‐Free Optofluidic Nanobiosensor Enables Real‐Time Analysis of Single‐Cell Cytokine Secretion

Single‐cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single‐cell resolution reveals the real‐time functional status of individual cells. Fluorescent and colorimetric‐based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label‐free optofluidic nanoplasmonic biosensor is introduced for single‐cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin‐2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single‐cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single‐cell signaling for basic and clinical research.     

Smart‐Dust‐Nanorice for Enhancement of Endogenous Raman Signal,Contrast in Photoacoustic Imaging,and T2‐Shortening in Magnetic Resonance Imaging

Raman microspectroscopy provides chemo‐selective image contrast, sub‐micrometer resolution, and multiplexing capabilities. However, it suffers from weak signals resulting in image‐acquisition times of up to several hours. Surface‐enhanced Raman scattering (SERS) can dramatically enhance signals of molecules in close vicinity of metallic surfaces and overcome this limitation. Multimodal, SERS‐active nanoparticles are usually labeled with Raman marker molecules, limiting SERS to the coating material. In order to realize multimodal imaging while acquiring the rich endogenous vibronic information of the specimen, a core–shell particle based on “Nanorice”, where a spindle‐shaped iron oxide core is encapsulated by a closed gold shell, is developed. An ultrathin layer of silica prevents agglomeration and unwanted chemical interaction with the specimen. This approach provides Raman signal enhancement due to plasmon resonance effects of the shell while the optical absorption in the near‐infrared spectral region provides contrast in photoacoustic tomography. Finally, T2‐relaxation of a magnetic resonance imaging (MRI) experiment is altered by taking advantage of the iron oxide core. The feasibility for Raman imaging is evaluated by nearfield simulations and experimental studies on the primate cell line COS1. MRI and photoacoustics are demonstrated in agarose phantoms illustrating the promising translational nature of this strategy for clinical applications in radiology.     

Volume‐Enhanced Raman Scattering Detection of Viruses

Virus detection and analysis are of critical importance in biological fields and medicine. Surface‐enhanced Raman scattering (SERS) has shown great promise in small molecule and even single molecule detection, and can provide fingerprint signals of molecules. Despite the powerful detection capabilities of SERS, the size discrepancy between the SERS “hot spots” (generally, <10 nm) and viruses (usually, sub‐100 nm) yields poor detection reliability of viruses. Inspired by the concept of molecular imprinting, a volume‐enhanced Raman scattering (VERS) substrate composed of hollow nanocones at the bottom of microbowls (HNCMB) is developed. The hollow nanocones of the resulting VERS substrates serve a twofold purpose: 1) extending the region of Raman signal enhancement from the nanocone surface (e.g., surface “hot spots”) to the hollow area within the cone (e.g., volume “hot spots”)—a novel method of Raman signal enhancement, and 2) directing analyte such as viruses of a wide range of sizes to those VERS “hot spots” while simultaneously increasing the surface area contributing to SERS. Using HNCMB VERS substrates, greatly improved Raman signals of single viruses are demonstrated, an achievement with important implications in disease diagnostics and monitoring, biomedical fields, as well as in clinical treatment.     

Antibody‐Mimetic Peptoid Nanosheet for Label‐Free Serum‐Based Diagnosis of Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia characterized by progressive cognitive decline. Current diagnosis of AD is based on symptoms, neuropsychological tests, and neuroimaging, and is usually evident years after the pathological process. Early assessment at the preclinical or prodromal stage is in a great demand since treatment after the onset can hardly stop or reverse the disease progress. However, early diagnosis of AD is challenging due to the lack of reliable noninvasive approaches. Here, an antibody‐mimetic self‐assembling peptoid nanosheet containing surface‐exposed Aβ42‐recognizing loops is constructed, and a label‐free sensor for the detection of AD serum is developed. The loop‐displaying peptoid nanosheet is demonstrated to have high affinity to serum Aβ42, and to be able to identify AD sera with high sensitivity. The dense distribution of molecular recognition loops on the robust peptoid nanosheet scaffold not only mimics the architecture of antibodies, but also reduces the nonspecific binding in detecting multicomponent samples. This antibody‐mimetic 2D material holds great potential toward the blood‐based diagnosis of AD, and meanwhile provides novel insights into the antibody alternative engineering and the universal application in biological and chemical sensors.     

Correct Spectral Conversion between Surface‐Enhanced Raman and Plasmon Resonance Scattering from Nanoparticle Dimers for Single‐Molecule Detection

Simultaneous measurement of surface‐enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR) in nanoparticle dimers presents outstanding opportunities in molecular identification and in the elucidation of physical properties, such as the size, distance, and deformation of target species. SERS–LSPR instrumentation exists and has been used under limited conditions, but the extraction of SERS and LSPR readouts from a single measurement is still a challenge. Herein, the extraction of LSPR spectra from SERS signals is reported and a tool for measuring the interparticle distance from Raman enhancement data by the standardization of the SERS signal is proposed. The SERS nanoruler mechanism incorporates two important aspects (the LSPR scattering peak shift and the Raman shift for measuring interparticle distance), and signifies their exact one‐to‐one correspondence after spectral correction. The developed methodology is applied to calculate the interparticle distance between nanoparticle dimers from SERS signals, to detect and quantify DNA at the single‐molecule level in a base‐pair‐specific manner. It is also shown that the SERS nanoruler concept can be used in structural analysis for the specific detection of the interaction of immunoglobulin G (IgG) with its target from bianalyte Raman signals with identical shaping at single‐molecule resolution. The SERS profile shaping approach not only offers a new detection mechanism for single molecules, but also has excellent potential for studying protein interactions and the intracellular detection of mRNA.