Transmission Electron Microscopy Grids Research Articles

Label-free detection of platelet-derived growth factor-BB via liquid crystal/aqueous interface functionalized using a double-stranded DNA probe

A liquid crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with cationic surfactant octyltrimethylammonium bromide (OTAB) and adsorbed with a double-stranded deoxyribonucleic acid (dsDNA) probe at the LC/aqueous interface (TEMOTAB/DNA) was utilized for the highly specific detection of platelet-derived growth factor (PDGF-BB). Upon adsorption of the OTAB-coated 4-cyano-4′-pentylbiphenyl (5CB) on the TEM grid (TEMOTAB) and subsequent adsorption of the dsDNA probe, a homeotropic orientation was observed. Subsequently, the TEMOTAB/DNA biosensor was exposed to PDGF-BB, which induced a homeotropic-to-planar configurational change in 5CB, as observed using polarized optical microscopy with crossed polarizers. The optimized adsorption density of the dsDNA probe facilitated the detection of 2.63 pg mL−1 PDGF-BB in the detection range of 5–200 pg mL−1. Remarkably, this LC-based biosensor discriminated PDGF-BB from bovine serum albumin, immunoglobulin G, hemoglobin, carcinoembryonic antigen, epidermal growth factor, PDGF-AA, and PDGF-AB. The application potential of this sensor was further demonstrated by its effective performance in detecting PDGF-BB in both human serum and synthetic urine samples. Because of its high specificity, sensitivity, and label-free detection capabilities, this biosensor has potential applications in cancer detection.

Liquid crystal assisted optical DNA biosensor for the selective detection of bacterium Neisseria gonorrhoeae

ABSTRACT A highly selective and sensitive label-free liquid crystal (LC)-based optical DNA biosensor has been reported for the detection of bacterial disease. A sensing platform is constructed using nematic LC (4-cyano-4’−pentyl biphenyl) filled in a confined mesh area of transmission electron microscope (TEM) grid placed within a Polydimethylsiloxane (PDMS) well of a fixed dimension for the detection of bacterial infection caused by Neisseria gonorrhoeae. The optical response associated with LC orientation was investigated by using N. gonorrhoeae nucleotide-specific oligonucleotide probe interacting with both the synthetic complementary ssDNA as well as genomic DNA isolated from N. gonorrhoeae under polarised optical microscope. The grey scale area of the resultant POM images was quantised using Image J software and monitored as a function of DNA concentration. The LC-cell has been able to detect the genomic DNA of N. gonorrhoeae bacteria down to the lowest detectable concentration of 10 pM. The optical response studies of LC-biosensor with genomic DNAs isolated from different bacteria showed high specificity and selectivity exclusively for N. gonorrhoeae pathogen and its potential as a convenient, rapid, and reliable label-free detection for gonorrhoea disease.

Atomic-Scale Characterization of Microscale Battery Particles Enabled by a High-Throughput Focused Ion Beam Milling Technique.

The cathode materials in lithium-ion batteries (LIBs) require improvements to address issues such as surface degradation, short-circuiting, and the formation of dendrites. One such method for addressing these issues is using surface coatings. Coatings can be sought to improve the durability of cathode materials, but the characterization of the uniformity and stability of the coating is important to assess the performance and lifetime of these materials. For microscale particles, there are, however, challenges associated with characterizing their surface modifications by transmission electron microscopy (TEM) techniques due to the size of these particles. Often, techniques such as focused ion beam (FIB)-assisted lift-out can be used to prepare thin cross sections to enable TEM analysis, but these techniques are very time-consuming and have a relatively low throughput. The work outlined herein demonstrates a FIB technique with direct support of microscale cathode materials on a TEM grid that increases sample throughput and reduces the processing time by 60-80% (i.e., from >5 to ∼1.5 h). The demonstrated workflow incorporates an air-liquid particle assembly followed by direct particle transfer to a TEM grid, FIB milling, and subsequent TEM analysis, which was illustrated with lithium nickel cobalt aluminum oxide particles and lithium manganese nickel oxide particles. These TEM analyses included mapping the elemental composition of cross sections of the microscale particles using energy-dispersive X-ray spectroscopy. The methods developed in this study can be extended to high-throughput characterization of additional LIB cathode materials (e.g., new compositions, coating, end-of-life studies), as well as to other microparticles and their coatings as prepared for a variety of applications.

Optimizing Atomic Layer Deposition Processes with Nanowire‐Assisted TEM Analysis

AbstractAtomic layer deposition (ALD) is one of the premier methods to synthesize ultra‐thin materials on complex surfaces. The technique allows for precise control of the thickness down to single atomic layers, while at the same time providing uniform coverage even for structures with extreme aspect ratios such as deep trenches or wires. While many materials can be readily deposited using ALD there is still a lot of research going on to make other materials more accessible. When establishing a new process or adapting an existing process to a new reactor, precise optimization of the deposition parameters is necessary. However, characterizing the parameters of deposition rate, uniformity, composition, and structure is a challenging and time‐consuming task. Here a method is presented to optimize these process parameters during ALD deposition using high‐aspect ratio nanowires and transmission electron microscopy (TEM). Nanowire samples are prepared directly on TEM grids that are put into the ALD reactor during deposition. Within min of the process the coated nanowires can be analyzed by TEM to obtain the thickness of the layers, chemical composition, crystallinity, and conformality of the coating. This allows for a high testing throughput and subsequently a rapid optimization of deposition parameters.

A simple coordinate transformation method for quickly locating the features of interest in TEM samples.

We developed a simple coordinate transformation method for quickly locating features of interest (FOIs) of samples in transmission electron microscope (TEM). The method is well suited for conducting sample searches in aberration-corrected scanning/transmission electron microscopes (S/TEM), where the survey can be very time-consuming because of the limited field of view imposed by the highly excited objective lens after fine-tuning the aberration correctors. For implementation, a digital image of the sample and the TEM holder was captured using a simple stereo-optical microscope. Naturally presented geometric patterns on the holder were referenced to construct a projective transformation between the electron and optical coordinate systems. The test results demonstrated that the method was accurate and required no electron microscope or specimen holder modifications. Additionally, it eliminated the need to mount the sample onto specific patterned TEM grids or deposit markers, resulting in universal applications for most TEM samples, holders and electron microscopes for fast FOI identification. Furthermore, we implemented the method into a Gatan script for graphical-user-interface-based step-by-step instructions. Through online communication, the script enabled real-time navigation and tracking of the motion of samples in TEM on enlarged optical images with a panoramic view.

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar.

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM.

Spatial constraints and stochastic seeding subvert microbial arms race.

Surface attached communities of microbes grow in a wide variety of environments. Often, the size of these microbial community is constrained by their physical surroundings. However, little is known about how size constraints of a colony impact the outcome of microbial competitions. Here, we use individual-based models to simulate contact killing between two bacterial strains with different killing rates in a wide range of community sizes. We found that community size has a substantial impact on outcomes; in fact, in some competitions the identity of the most fit strain differs in large and small environments. Specifically, when at a numerical disadvantage, the strain with the slow killing rate is more successful in smaller environments than in large environments. The improved performance in small spaces comes from finite size effects; stochastic fluctuations in the initial relative abundance of each strain in small environments lead to dramatically different outcomes. However, when the slow killing strain has a numerical advantage, it performs better in large spaces than in small spaces, where stochastic fluctuations now aid the fast killing strain in small communities. Finally, we experimentally validate these results by confining contact killing strains of Vibrio cholerae in transmission electron microscopy grids. The outcomes of these experiments are consistent with our simulations. When rare, the slow killing strain does better in small environments; when common, the slow killing strain does better in large environments. Together, this work demonstrates that finite size effects can substantially modify antagonistic competitions, suggesting that colony size may, at least in part, subvert the microbial arms race.

Analyzing the cellular plasma membrane by fast and efficient correlative STED and platinum replica EM

The plasma membrane of mammalian cells links transmembrane receptors, various structural components, and membrane-binding proteins to subcellular processes, allowing inter- and intracellular communication. Therefore, membrane-binding proteins, together with structural components such as actin filaments, modulate the cell membrane in their flexibility, stiffness, and curvature. Investigating membrane components and curvature in cells remains challenging due to the diffraction limit in light microscopy. Preparation of 5–15-nm-thin plasma membrane sheets and subsequent inspection by metal replica transmission electron microscopy (TEM) reveal detailed information about the cellular membrane topology, including the structure and curvature. However, electron microscopy cannot identify proteins associated with specific plasma membrane domains. Here, we describe a novel adaptation of correlative super-resolution light microscopy and platinum replica TEM (CLEM-PREM), allowing the analysis of plasma membrane sheets with respect to their structural details, curvature, and associated protein composition. We suggest a number of shortcuts and troubleshooting solutions to contemporary PREM protocols. Thus, implementation of super-resolution stimulated emission depletion (STED) microscopy offers significant reduction in sample preparation time and reduced technical challenges for imaging and analysis. Additionally, highly technical challenges associated with replica preparation and transfer on a TEM grid can be overcome by scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides the highest spatial resolution of plasma membrane proteins and their underlying membrane and is, therefore, a suitable method to study cellular events like endocytosis, membrane trafficking, or membrane tension adaptations.

Liquid‐Phase Transfer of Organic–Inorganic Halide Perovskite Films for TEM Investigation and Planar Heterojunction Fabrication

AbstractOrganic–inorganic halide perovskites (OIHPs) show high promise in optical and electronic applications such as solar cells, light‐emitting diodes, and nonlinear optics. However, the fundamental knowledge of the atomic‐scale microstructures in OIHP thin films is limited due to the challenge in characterizing them using transmission electron microscopy (TEM). Here a solution‐phase “release‐and‐transfer” method is demonstrated, which entails the lifting of OIHP films from their original substrates while maintaining the film integrity, followed by a sequential transfer onto a TEM grid. The freestanding nature of the OIHP films with a nanoscale thickness, prepared as such, allows a direct TEM observation in the plan view, complementing those typical cross‐sectional views enabled by focus‐ion‐beam specimen fabrication. Using low‐dose scanning TEM, the atomic‐scale microstructure of transferred OIHP films is confirmed to be generally maintained, while the microstrain existing in original films is largely relaxed. This “release‐and‐transfer” method is generic to both standard 3D and low‐dimensional OIHPs. Based on a simple layer‐by‐layer transfer, the fabrication of a 2D–3D planar heterojunction with a good interfacial contact and optoelectronic properties is achieved. This unique methodology offers new opportunities to accelerate the fundamental and practical developments of OIHP materials and devices.

Transmission through graphene of electrons in the 30 – 900 eV range

Here, we report on accurate transmission measurements of electrons below 1 keV through suspended monolayer graphene. Monolayer graphene was grown via chemical vapor deposition and transferred onto transmission electron microscopy (TEM) grids. A monochromatic electron gun has been employed to perform the measurements in the 30 – 900 eV range in ultra-high vacuum. The graphene transparency is obtained from the absolute measurement of the direct beam current and the transmitted one, by means of a Faraday cup. We observed a transmission going from ∼20 to ∼80% for monolayer graphene within the experimental electron energy range. The high quality and the grid coverage of the suspended graphene has been proved via micro-Raman, X-ray photoemission, electron energy loss spectroscopies and field-emission scanning electron microscopy. After a 550 °C in-vacuum annealing of the samples, the main contribution to the C 1s spectrum is due to the sp2 component and the evidence of suspended monolayer graphene has been observed through the π-plasmon excitation.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, the use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Furthermore, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. We conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Use of Boron Doped Diamond Corrosion Free Supports to Evaluate Fuel Cell Electrocatalyst Stability Under Accelerated Stress Testing

Proton exchange membrane fuel cell (PEMFC) electrocatalysts are typically Pt or Pt alloy nanoparticles (NPs) supported on high surface area carbon black powders, the latter which contain a significant sp2 carbon component. One of the barriers to PEMFC commercialisation is the lifetime of the catalyst-support system, which should be at least 5,000 hours for automotive applications.[1] During the start-up and shut-down of the PEMFC, the cathode is subjected to significant oxidative potentials (greater than 1 V vs RHE) which can lead to corrosion of the predominantly sp2 bonded carbon support, a key factor which can limit the lifetime of PEMFCs. [2] High quality boron doped diamond (BDD) is an sp3 bonded network with above 1:1000 B:C ratio, which is required for metal-like electronic conductivity. BDD has many favourable properties for electrochemical experiments, most notably: higher electrochemical corrosion resistance with respect to sp2 carbons, low background currents, and a wide aqueous solvent window.[3] In this work we first explore the electrochemical corrosion stability of BDD when undergoing electrochemical cycling in acid solutions. To achieve this BDD substrates (BDD TEM grid) suitable for both electrochemical experiments and high magnification (single atom resolution) transmission electron microscopy (TEM) experiments were produced using precision ion polishing (Gatan PIPS-II). The surface was characterised using X-ray photoelectron spectroscopy to ensure the polishing process did not significantly alter the diamond substrate. Under aggressive electrochemical potential cycling in perchloric acid and sulfuric acid solutions, both TEM morphological analysis and Electron Energy Loss Spectroscopy (EELS) thickness measurements showed no evidence of BDD corrosion. [4] The BDD-TEM substrate was then used as a platform for investigating the degradation of a PEMFC electrocatalyst (Pt NPs) under accelerated stress testing (AST) [1] conditions on an atomic level, using the corrosion free carbon support. This enables the influence of other degradation pathways [2] (such as aggregation, Ostwald Ripening & direct dissolution) to be explored in more detail, free from issues associated with corrosion of the support. Pt NPs, in the size range 1 – 4 nm, were sputter coated onto the BDD TEM grid. Identical location ex-situ TEM (IL-TEM)[5] in combination with image analysis was used to probe NP changes e.g. size, shape, position, on an individual basis before, and after, AST (Fig. 1, which contains c.a. 200 NPs for analysis). Such measurements were complemented by Inductively Coupled Plasma – Optical Emission Spectroscopy analysis for any dissolved Pt, and electrochemical cyclic voltammetry measurements of the Pt to highlight changes in electrocatalytic behaviour (hydrogen evolution reaction and oxygen reduction reaction) due to AST cycling.Figure 1 – High magnification annular dark field (ADF) identical location images of Pt NPs, a) Pt/BDD before AST, b) Pt NPs after AST, c) Cyclic voltammogram of the 1000 cycle AST from 0.8 to 1.4 V vs Ag|AgCl (1.6 to 1.6 V vs RHE).

Characterization of Emissions from Carbon Dioxide Laser Cutting Acrylic Plastics.

Carbon dioxide laser cutters are used to cut and engrave on various types of materials, including metals, wood, and plastics. Although many are equipped with fume extractors for removing airborne substances generated during laser cutting, gases and particulate matter can be released upon opening the lid after completion. This study focused on investigating laser cutting acrylic sheets and associated emissions. Real-time instruments were utilized to monitor both particulate concentrations and size distributions, while the patented Tsai diffusion sampler was used to collect particulate samples on a polycarbonate membrane and transmission electron microscopy (TEM) grid. Identification of released gases consisted of the use of gas sampling with Teflon gas bags followed by analysis using gas chromatography-mass spectrometry (GC-MS). A portable ambient infrared air analyzer was used to quantify the concentrations of the chemicals released by laser cutting activities. The results of the study found that a significant concentration of particulate matter, including nanoplastic particles ranging 15.4-86 nm in particle sizes, and microplastics with agglomerates were released each time the laser cutter lid was opened and were observed to gradually increase in concentration for a period of at least 20 min after the completion of a cut. The GC-MS gaseous samples primarily contained methyl methacrylate at a low level close to the detection limit of the infrared air analyzer.

A simple experiment to study the array theorem using Fraunhofer diffraction of a two-dimensional grating

A transmission electron microscopy (TEM) grid comprises an array of identical square apertures. In this study, the Fraunhofer diffraction pattern of a TEM grid was obtained by a 532 nm wavelength diode laser. It was compared with the Fraunhofer diffraction pattern computed by using the array theorem and employed to estimate the mesh size of the TEM grid.

Novel Focused Ion Beam Liftouts for Spatial Characterization of Spherical Biominerals With Transmission Electron Microscopy.

Focused ion beam (FIB) is frequently used to prepare electron- and X-ray-beam-transparent thin sections of samples, called lamellae. Typically, lamellae are prepared from only a subregion of a sample. In this paper, we present a novel approach for FIB lamella preparation of microscopic samples, wherein the entire cross-section of the whole sample can be investigated. The approach was demonstrated using spherical, porous, and often hollow microprecipitates of biologically precipitated calcium carbonate. The microprecipitate morphology made these biogenic samples more fragile and challenging than materials commonly investigated using FIB lamellae. Our method enables the appropriate orientation of the lamellae required for further electron/X-ray analyses after attachment to the transmission electron microscopy (TEM) grid post and facilitates more secure adhesion onto the grid post. We present evidence of autofluorescence in bacterially precipitated vaterite using this lamella preparation method coupled with TEM selected area diffraction. This innovative approach allows studying biomineralization at the micro to nano scales, which can provide novel insights into bacterial responses to microenvironmental conditions.

"Nano Lab" Advanced Characterization Platform for Studying Electrocatalytic Iridium Nanoparticles Dispersed on TiOxNy Supports Prepared on Ti Transmission Electron Microscopy Grids.

Aiming at speeding up the discovery and understanding of promising electrocatalysts, a novel experimental platform, i.e., the Nano Lab, is introduced. It is based on state-of-the-art physicochemical characterization and atomic-scale tracking of individual synthesis steps as well as subsequent electrochemical treatments targeting nanostructured composites. This is provided by having the entire experimental setup on a transmission electron microscopy (TEM) grid. Herein, the oxygen evolution reaction nanocomposite electrocatalyst, i.e., iridium nanoparticles dispersed on a high-surface-area TiOxNy support prepared on the Ti TEM grid, is investigated. By combining electrochemical concepts such as anodic oxidation of TEM grids, floating electrode-based electrochemical characterization, and identical location TEM analysis, relevant information from the entire composite's cycle, i.e., from the initial synthesis step to electrochemical operation, can be studied. We reveal that Ir nanoparticles as well as the TiOxNy support undergo dynamic changes during all steps. The most interesting findings made possible by the Nano Lab concept are the formation of Ir single atoms and only a small decrease in the N/O ratio of the TiOxNy-Ir catalyst during the electrochemical treatment. In this way, we show that the precise influence of the nanoscale structure, composition, morphology, and electrocatalyst's locally resolved surface sites can be deciphered on the atomic level. Furthermore, the Nano Lab's experimental setup is compatible with ex situ characterization and other analytical methods, such as Raman spectroscopy, X-ray photoelectron spectroscopy, and identical location scanning electron microscopy, hence providing a comprehensive understanding of structural changes and their effects. Overall, an experimental toolbox for the systematic development of supported electrocatalysts is now at hand.

Fracture Toughness Characteristics of Stacked Bilayer Graphene via Far-Field Displacement Measurements.

Mechanical properties of graphene, e.g., strength, modulus, and fracture toughness are extremely sensitive to flaws. Here the fracture properties of stacked bilayer graphene sheets (SBLG) are reported, obtained via stacking two individually grown graphene sheets. The SBLG is presented here as a building block for flaw-resilient nanomaterials. The fracture properties of freestanding SBLG sheets, suspended on transmission electron microscope (TEM) grids, are characterized by stretching the TEM grid inside an scanning electron microscope (SEM) chamber and monitoring the local displacements in real-time. The fracture toughness is measured and expressed as a function of the critical displacement required to propagate existing cracks in the experiment via computational models. This approach decouples force and displacements measurements, and utilizes the known elastic modulus along with the known displacement boundary conditions at the onset of crack growth to estimate the far field force and stress. This strategy represents a breakthrough in nanoscale fracture mechanics for statistical analysis and high throughput experimens on multiple samples at a time. Results demonstrate that the SBLG is markedly tougher than as-grown single or multilayer graphene, with a mode I fracture toughness of ≈28.06±7.5 MPa . The mechanisms leading to a higher toughness of SBLG are also analyzed and discussed.

Review on High-Throughput Micro-Combinatorial Characterization of Binary and Ternary Layers towards Databases.

The novel, single-sample concept combinatorial method, the so-called micro-combinatory technique, has been shown to be suitable for the high-throughput and complex characterization of multicomponent thin films over an entire composition range. This review focuses on recent results regarding the characteristics of different binary and ternary films prepared by direct current (DC) and radiofrequency (RF) sputtering using the micro-combinatorial technique. In addition to the 3 mm diameter TEM grid used for microstructural analysis, by scaling up the substrate size to 10 × 25 mm, this novel approach has allowed for a comprehensive study of the properties of the materials as a function of their composition, which has been determined via transmission electron microscopy (TEM), scanning electron microscopy (SEM), Rutherford backscattering spectrometry (RBS), X-ray diffraction analysis (XRD), atomic force microscopy (AFM), spectroscopic ellipsometry, and nanoindentation studies. Thanks to the micro-combinatory technique, the characterization of multicomponent layers can be studied in greater detail and efficiency than before, which is beneficial for both research and practical applications. In addition to new scientific advances, we will briefly explore the potential for innovation with respect to this new high-throughput concept, including the creation of two- and three-component thin film databases.

Ultralow-Loss Substrate for Nanophotonic Dark-Field Microscopy.

For the colloidal nanophotonic structures, a transmission electron microscope (TEM) grid has been widely used as a substrate of dark-field microscopy because a nanometer-scale feature can be effectively determined by TEM imaging following dark-field microscopic studies. However, an optically lossy carbon layer has been implemented in conventional TEM grids. A broadband scattering from the edges of the TEM grid further restricted an accessible signal-to-noise ratio. Herein, we demonstrate that the freely suspended, ultrathin, and wide-scale transparent nanomembrane can address such challenges. We developed a 1 mm by 600 μm scale and 20 nm thick poly(vinyl formal) nanomembrane, whose area is around 180 times wider than a conventional TEM grid, so that the possible broadband scattering at the edges of the grid was effectively excluded. Also, such nanomembranes can be formed without the assistance of carbon support; allowing us to achieve the highest signal-to-background ratio of scattering among other substrates.

Thiol-End-Group Dendrons Decorated with Gold Nanoparticles Immobilized on Amino-Functionalized Graphene Oxide for SERS Detection

Surface-enhanced Raman scattering (SERS) has abundant chemical fingerprint information to analyze complex analytes under aqueous conditions. However, SERS requires uniform metal particles to generate a hotspot effect and improve sensitivity and enhancement by localized surface plasmon resonance. Therefore, in this study, SERS detection chips have been developed using uniform gold nanoparticle (AuNP) arrays embedded on thiol-end-group dendritic polymers immobilized on amino-functionalized graphene oxide (AGO) thin films of transmission electron microscopy (TEM) grids. The graphene oxide (GO) thin film was fabricated by low-damage plasma with mixed gas (O2/H2) and then further functionalized by diethylenetriamine to form AGO substrates. Based on the efficient building block of dual-functional 4-isocyanato-4′-(3,3-dimethyl-2,4-dioxo-azetidino)-diphenylmethane (IDD), the 0.5 and 1.5 generations of thiol-end-functionalized poly(urea/malonamide) dendrons (DG0.5-SH and DG1.5-SH) were precisely synthesized and grafted onto AGO substrates. The interparticle gap of AuNPs could be well controlled and decorated on these thiol-end-group dendrons to achieve the optimal SERS enhancement effect by manipulating the sizes of AuNPs and generating dendrons. The results indicate that 31 nm AuNPs immobilized on the DG0.5-SH@AGO substrate exhibited reproducible and strong SERS enhancement compared with free AuNPs. Graphene-based SERS substrates provide rapid and stable SERS detection in solutions, which offers great potential for practical applications in detecting biomolecules and environmental pollutants.