Influence of albumin concentration on surface characteristics and cellular responses in the pre-incubation of multi-walled carbon nanotubes.

Reliable characterization of protein coronas (PCs) that form when nanomaterials are introduced into biological fluids is a critical step in the development of safe and efficient nanomedicine. We observed that bovine serum albumin (BSA)-coated multi-walled carbon nanotubes (MWCNTs) do not induce cytotoxicity, but have different cellular uptake rates depending on the BSA pretreatment concentration. To determine how these slight differences affect A549 cell responses and intracellular changes, we conducted spectroscopic (circular dichroism and Fourier-transform infrared) and spectrometric (nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry) analyses. The various characterization techniques conducted in this study reveal the following. (i) The composition ratio of PCs on MWCNTs differs depending on the BSA concentration. (ii) Analysis of the secondary structure of the proteins revealed that the α-helix structure increased with increasing BSA concentration. (iii) Proteomic analysis showed that different biological pathways were activated at levels higher and lower than 5 mg mL-1. Such combined spectroscopic and spectrometric approaches provide an integrated understanding of PC composition as well as how nano/bio-interface states are linked to cellular-level responses. Our results can support reliable and practical applications of nanomedicine development.

Long-Term Culture of Human Pluripotent Stem Cells in Xeno-Free Condition Using Functional Polymer Films.

Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies.

Role of Chalcogenides in Sensitive Therapeutic Drug Monitoring Using Laser Desorption and Ionization.

This study investigates the applicability of six transition metal dichalcogenides to efficient therapeutic drug monitoring of ten antiepileptic drugs using laser desorption/ionization-mass spectrometry. We found that molybdenum ditelluride and tungsten ditelluride are suitable for the sensitive quantification of therapeutic drugs. The contribution of tellurium to the enhanced efficiency of laser desorption ionization was validated through theoretical calculations utilizing an integrated model that incorporates transition-metal dichalcogenides and antiepileptic drugs. The results of our theoretical calculations suggest that the relatively low surface electron density for the tellurium-containing transition metal dichalcogenides induces stronger Coulombic interactions, which results in enhanced laser desorption and ionization efficiency. To demonstrate applicability, up to 120 patient samples were analyzed to determine drug concentrations, and the results were compared with those of immunoassay and liquid chromatography-tandem mass spectrometry. Agreements among these methods were statistically evaluated using the Passing-Bablok regression and Bland-Altman analysis. Furthermore, our method has been shown to be applicable to the simultaneous detection and multiplexed quantification of antiepileptic drugs.

Hydrophobic surface induced pro-metastatic cancer cells for in vitro extravasation models.

In vitro vascularized cancer models utilizing microfluidics have emerged as a promising tool for mechanism study and drug screening. However, the lack of consideration and preparation methods for cancer cellular sources that are capable of adequately replicating the metastatic features of circulating tumor cells contributed to low relevancy with in vivo experimental results. Here, we show that the properties of cancer cellular sources have a considerable impact on the validity of the in vitro metastasis model. Notably, with a hydrophobic surface, we can create highly metastatic spheroids equipped with aggressive invasion, endothelium adhesion capabilities, and activated metabolic features. Combining these metastatic spheroids with the well-constructed microfluidic-based extravasation model, we validate that these metastatic spheroids exhibited a distinct extravasation response to epidermal growth factor (EGF) and normal human lung fibroblasts compared to the 2D cultured cancer cells, which is consistent with the previously reported results of in vivo experiments. Furthermore, the applicability of the developed model as a therapeutic screening platform for cancer extravasation is validated through profiling and inhibition of cytokines. We believe this model incorporating hydrophobic surface-cultured 3D cancer cells provides reliable experimental data in a clear and concise manner, bridging the gap between the conventional in vitro models and in vivo experiments.

Monitoring lipid alterations in Drosophila heads in an amyotrophic lateral sclerosis model with time-of-flight secondary ion mass spectrometry.

Lipid alterations in the brain are well-documented in disease and aging, but our understanding of their pathogenic implications remains incomplete. Recent technological advances in assessing lipid profiles have enabled us to intricately examine the spatiotemporal variations in lipid compositions within the complex brain characterized by diverse cell types and intricate neural networks. In this study, we coupled time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an amyotrophic lateral sclerosis (ALS) Drosophila model, for the first time, to elucidate changes in the lipid landscape and investigate their potential role in the disease process, serving as a methodological and analytical complement to our prior approach that utilized matrix-assisted laser desorption/ionization mass spectrometry. The expansion of G4C2 repeats in the C9orf72 gene is the most prevalent genetic factor in ALS. Our findings indicate that expressing these repeats in fly brains elevates the levels of fatty acids, diacylglycerols, and ceramides during the early stages (day 5) of disease progression, preceding motor dysfunction. Using RNAi-based genetic screening targeting lipid regulators, we found that reducing fatty acid transport protein 1 (FATP1) and Acyl-CoA-binding protein (ACBP) alleviates the retinal degeneration caused by G4C2 repeat expression and also markedly restores the G4C2-dependent alterations in lipid profiles. Significantly, the expression of FATP1 and ACBP is upregulated in G4C2-expressing flies, suggesting their contribution to lipid dysregulation. Collectively, our novel use of ToF-SIMS with the ALS Drosophila model, alongside methodological and analytical improvements, successfully identifies crucial lipids and related genetic factors in ALS pathogenesis.

Time-sequential fibroblast-to-myofibroblast transition in elastin-variable 3D hydrogel environments by collagen networks.

BACKGROUND: Fibrosis plays an important role in both normal physiological and pathological phenomena as fibroblasts differentiate to myofibroblasts. The activation of fibroblasts is determined through interactions with the surrounding extracellular matrix (ECM). However, how this fibroblast-to-myofibroblast transition (FMT) is regulated and affected by elastin concentration in a three-dimensional (3D) microenvironment has not been investigated. METHODS: We developed an insoluble elastin-gradient 3D hydrogel system for long-lasting cell culture and studied the molecular mechanisms of the FMT in embedded cells by nanoflow LC-MS/MS analysis along with validation through real-time PCR and immunofluorescence staining. RESULTS: By optimizing pH and temperature, four 3D hydrogels containing fibroblasts were successfully fabricated having elastin concentrations of 0, 20, 50, and 80% in collagen. At the low elastin level (20%), fibroblast proliferation was significantly increased compared to others, and in particular, the FMT was clearly observed in this condition. Moreover, through mass spectrometry of the hydrogel environment, it was confirmed that differentiation proceeded in two stages. In the early stage, calcium-dependent proteins including calmodulin and S100A4 were highly associated. On the other hand, in the late stage after several passages of cells, distinct markers of myofibroblasts were presented such as morphological changes, increased production of ECM, and increased α-SMA expression. We also demonstrated that the low level of elastin concentration induced some cancer-associated fibroblast (CAF) markers, including PDGFR-ß, and fibrosis-related disease markers, including THY-1. CONCLUSION: Using our developed 3D elastin-gradient hydrogel system, we evaluated the effect of different elastin concentrations on the FMT. The FMT was induced even at a low concentration of elastin with increasing CAF level via calcium signaling. With this system, we were able to analyze varying protein expressions in the overall FMT process over several cellular passages. Our results suggest that the elastin-gradient system employing nonlinear optics imaging provides a good platform to study activated fibroblasts interacting with the microenvironment, where the ECM plays a pivotal role.

Comparison study of mouse brain tissue by using ToF-SIMS within static limits and hybrid SIMS beyond static limits (dynamic mode).

In the study of degenerative brain diseases, changes in lipids, the main component of neurons, are particularly important because they are used as indicators of pathological changes. One method for the sensitive measurement of biomolecules, especially lipids, is time-of-flight secondary ion mass spectrometry (ToF-SIMS) using pulsed argon cluster ions. In this study, biomolecules including various lipids present in normal mouse brain tissue were measured using ToF-SIMS equipped with pulsed argon cluster primary ions. Based on the ToF-SIMS measurement results, hybrid SIMS (OrbiSIMS), which is a ToF-SIMS system with the addition of an orbitrap mass analyzer, was used to directly identify the biomolecules by the region in the real tissue samples. For this, the results of ToF-SIMS, which measured the tissue samples from a single mouse brain within static limits, were compared with those from OrbiSIMS measured beyond the static limits in terms of the differences in molecular profiling. From this analysis, two types of positive and negative ions were selected for identification, with the OrbiSIMS MS/MS results indicating that the positive ions were glycerophosphocholine and the negative ions were glycerophosphoinositol and sulfatide, a sphingolipid. Then, to confirm the identification of the molecular candidates, lipids were extracted from mirror image tissue samples, and LC-MS/MS also using an orbitrap mass analyzer was performed. As a result, the direct identification of molecular candidate groups distributed in particular regions of the tissue samples via OrbiSIMS was found to be consistent with the identification results by LC-MS/MS for extracted samples.

Quantitative Analysis of Immunosuppressive Drugs Using Tungsten Disulfide Nanosheet-Assisted Laser Desorption Ionization Mass Spectrometry.

For organ transplantation patients, the therapeutic drug monitoring (TDM) of immunosuppressive drugs is essential to prevent the toxicity or rejection of the organ. Currently, TDM is done by immunoassays or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods; however, these methods lack specificity or are expensive, require high levels of skill, and offer limited sample throughput. Although matrix-assisted (MA) laser desorption ionization (LDI) mass spectrometry (MS) can provide enhanced throughput and cost-effectiveness, its application in TDM is limited due to the limitations of the matrixes such as a lack of sensitivity and reproducibility. Here, we present an alternative quantification method for the TDM of the immunosuppressive drugs in the blood of organ transplant patients by utilizing laser desorption ionization mass spectrometry (LDI-MS) based on a tungsten disulfide nanosheet, which is well-known for its excellent physicochemical properties such as a strong UV absorbance and high electron mobility. By adopting a microliquid inkjet printing system, a high-throughput analysis of the blood samples with enhanced sensitivity and reproducibility was achieved. Furthermore, up to 80 cases of patient samples were analyzed and the results were compared with those of LC-MS/MS by using Passing-Bablok regression and Bland-Altman analysis to demonstrate that our LDI-MS platform is suitable to replace current TDM techniques. Our approach will facilitate the rapid and accurate analysis of blood samples from a large number of patients for immunosuppressive drug prescriptions.

Global Proteomics to Study Silica Nanoparticle-Induced Cytotoxicity and Its Mechanisms in HepG2 Cells.

Silica nanoparticles (SiO2 NPs) are commonly used in medical and pharmaceutical fields. Research into the cytotoxicity and overall proteomic changes occurring during initial exposure to SiO2 NPs is limited. We investigated the mechanism of toxicity in human liver cells according to exposure time [0, 4, 10, and 16 h (h)] to SiO2 NPs through proteomic analysis using mass spectrometry. SiO2 NP-induced cytotoxicity through various pathways in HepG2 cells. Interestingly, when cells were exposed to SiO2 NPs for 4 h, the morphology of the cells remained intact, while the expression of proteins involved in mRNA splicing, cell cycle, and mitochondrial function was significantly downregulated. These results show that the toxicity of the nanoparticles affects protein expression even if there is no change in cell morphology at the beginning of exposure to SiO2 NPs. The levels of reactive oxygen species changed significantly after 10 h of exposure to SiO2 NPs, and the expression of proteins associated with oxidative phosphorylation, as well as the immune system, was upregulated. Eventually, these changes in protein expression induced HepG2 cell death. This study provides insights into cytotoxicity evaluation at early stages of exposure to SiO2 NPs through in vitro experiments.

Comparative study on formation of protein coronas under three different serum origins.

Nanomaterials form a complex called "protein corona" by contacting with protein-containing biological fluids such as serum when they are exposed to physiological environments. The characteristics of these proteins, which are one of the substantial factors in cellular response, are affected by the interactions between the nanomaterials and the biological systems. Many studies have investigated the biological behaviors of nanomaterials by conducting experiments in vitro and in vivo; however, the origin of the biological materials used is rather inconsistent. This is due to the fact that the composition of the protein coronas may differ depending on the animal origin, not on the composition or size of the nanoparticles. The resulting differences in the composition of the protein coronas can lead to different conclusions. To identify the differences in protein corona formation among sera of different species, we investigated protein coronas of gold and silica nanoparticles in serum obtained from various species. Using comparative proteomic analysis, common proteins adsorbed onto each nanoparticle among the three different sera were identified as highly abundant proteins in the serum. These findings indicate that protein corona formation is dependent on the serum population rather than the size or type of the nanoparticles. Additionally, in the physiological classification of protein coronas, human serum (HS) was found to be rich in apolipoproteins. In conclusion, our data indicate that HS components are different from those of bovine or mouse, indicating that the serum species origin should be carefully considered when selecting a biological fluid.

Electro-inductive effect: Electrodes as functional groups with tunable electronic properties.

In place of functional groups that impose different inductive effects, we immobilize molecules carrying thiol groups on a gold electrode. By applying different voltages, the properties of the immobilized molecules can be tuned. The base-catalyzed saponification of benzoic esters is fully inhibited by applying a mildly negative voltage of -0.25 volt versus open circuit potential. Furthermore, the rate of a Suzuki-Miyaura cross-coupling reaction can be changed by applying a voltage when the arylhalide substrate is immobilized on a gold electrode. Finally, a two-step carboxylic acid amidation is shown to benefit from a switch in applied voltage between addition of a carbodiimide coupling reagent and introduction of the amine.

Direct Chemical Imaging of Ligand-Functionalized Single Nanoparticles by Photoinduced Force Microscopy.

Chemical characterizations of biochemically functionalized single nanoparticles are necessary to optimize the nanoparticle surface functionality in recently advanced nanobiological applications but have not yet been fully explored because of technical difficulties. Exploiting the photoinduced force exerted on a light-illuminated nanoscale tip, nanoscale mid-infrared hyperspectral images with a 10 nm spatial resolution of a monolayer ligand-functionalized single gold nanoparticle under ambient and environmental conditions are presented. We extend our study to the diagnosis of nanoscale heterogeneous chemical contaminants which come from a particle functionalization process but are undetectable in conventional ensemble-averaged imaging technique. High sensitivity and high spatial resolution are achieved via the strongly localized tip-enhanced force at the junction between the gold-coated tip and the functionalized nanoparticle in photoinduced force microscopy, which far exceeds the capability of the conventional methods. The present study paves a new way to directly detect heterogeneous nanochemicals at the single-component level, which is necessary to evaluate nanomaterial safety in biomedical applications.

Numerical evaluation of polyethylene glycol ligand conjugation to gold nanoparticle surface using ToF-SIMS and statistical analysis.

Nanoparticles (NPs) are substances between 1 and 100 nm in size. They have been the subject of numerous studies because of their potential applications in a wide range of fields such as cosmetics, electronics, medicine, and food. For biological applications of nanoparticles, they are usually coated with a substance capable of preventing agglomeration of the nanoparticles and nonspecific binding and exhibiting water-solubility characteristics with specific immobilized (bio)molecules. In order to evaluate the chemical properties of the surface-modified nanoparticles for bioapplications, including drug delivery, a simple and reliable method for the analysis of the presence of the surface chemicals and the ligand states of the nanoparticles is necessary. In this study, the authors numerically evaluated the extent of polyethylene glycol (PEG) ligand conjugation on AuNPs by concurrently adopting a microliquid inkjet printing system for sampling of the PEGylated AuNPs solution and ToF-SIMS imaging together with statistical analysis. The statistical correlation values calculated from the signals of PEG and Au measured by ToF-SIMS imaging on the sample spots made by a microliquid inkjet printing system showed better reproducibility and improved correlation values compared to the pipet spotting. Their improved method will be useful to evaluate ligand-conjugated nanoparticles for quality control of each conjugation process.

Ar-gas cluster ion beam in ToF-SIMS for peptide and protein analysis.

Since Ar-gas cluster ion beams (Ar-GCIBs) have been introduced into time-of-flight secondary ion mass spectrometry (ToF-SIMS), there have been various attempts to analyze organic materials and biomolecules that require low-damage analysis and high sensitivity, because Ar-GCIBs allow soft ionization of large molecules such as peptides and proteins due to the low energy per atom. Here, the authors adopted the Ar-GCIB as a primary beam to detect proteins including human insulin, ubiquitin, and cytochrome C (molecular weights are 5808, 8564, and 12 327 Da, respectively). They have confirmed that the detection of the intact proteins was possible when the Ar-GCIB was used as a primary ion beam. In addition, they successfully identified each protein by analyzing the trypsin-digested peptides in myoglobin, cytochrome C, and bovine serum albumin. They also attempted on-surface enzymatic digestion to identify proteins on the surface of the Si wafer and obtained results identical to those of in-solution digestion. It is expected that the authors' on-surface digestion method can enable the application of ToF-SIMS for the analysis of proteins present in biological tissues.

Comparison between thaw-mounting and use of conductive tape for sample preparation in ToF-SIMS imaging of lipids in Drosophila microRNA-14 model.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging elucidates molecular distributions in tissue sections, providing useful information about the metabolic pathways linked to diseases. However, delocalization of the analytes and inadequate tissue adherence during sample preparation are among some of the unfortunate phenomena associated with this technique due to their role in the reduction of the quality, reliability, and spatial resolution of the ToF-SIMS images. For these reasons, ToF-SIMS imaging requires a more rigorous sample preparation method in order to preserve the natural state of the tissues. The traditional thaw-mounting method is particularly vulnerable to altered distributions of the analytes due to thermal effects, as well as to tissue shrinkage. In the present study, the authors made comparisons of different tissue mounting methods, including the thaw-mounting method. The authors used conductive tape as the tissue-mounting material on the substrate because it does not require heat from the finger for the tissue section to adhere to the substrate and can reduce charge accumulation during data acquisition. With the conductive-tape sampling method, they were able to acquire reproducible tissue sections and high-quality images without redistribution of the molecules. Also, the authors were successful in preserving the natural states and chemical distributions of the different components of fat metabolites such as diacylglycerol and fatty acids by using the tape-supported sampling in microRNA-14 (miR-14) deleted Drosophila models. The method highlighted here shows an improvement in the accuracy of mass spectrometric imaging of tissue samples.

On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase Inhibitor Screening.

Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix. In our method, cysteine-tethered peptides adsorbed onto a gold surface produced changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kinase activity. Using mass spectrometry imaging of multiple compartments on the gold surface in the presence of a peptide substrate, we screened 13,727 inhibitors, of which seven were initially found to have inhibitor efficiencies that surpassed 50%. Of these, we were able to identify a new breakpoint cluster region-abelson (BCR-ABL)T315I kinase inhibitor, henceforth referred to as KR135861. KR135861 showed no cytotoxicity and was subsequently confirmed to be superior to imatinib, a commercial drug marketed as Gleevec. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABLT315I tyrosine kinase, with an IC50 value as low as 1.3 µM. In in vitro experiments, KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCR-ABLT315I, in contrast to imatinib's inhibitory effects only on Ba/F3 cells expressing wild-type BCR-ABL. Due to the surface sensitivity and selectivity of SIMS imaging, it is anticipated that our approach will make it easier to validate the small modifications of a substrate in relation to enzyme activity as well as for drug discovery. This mass spectrometry imaging analysis enables efficient screening for protein kinase inhibitors, thus permitting high-throughput drug screening with high accuracy, sensitivity, and specificity.

Nanoparticle-protein complexes mimicking corona formation in ocular environment.

Nanoparticles adsorb biomolecules to form corona upon entering the biological environment. In this study, tissue-specific corona formation is provided as a way of controlling protein interaction with nanoparticles in vivo. In the vitreous, the composition of the corona was determined by the electrostatic and hydrophobic properties of the associated proteins, regardless of the material (gold and silica) or size (20- and 100-nm diameter) of the nanoparticles. To control protein adsorption, we pre-incubate 20-nm gold nanoparticles with 5 selectively enriched proteins from the corona, formed in the vitreous, to produce nanoparticle-protein complexes. Compared to bare nanoparticles, nanoparticle-protein complexes demonstrate improved binding to vascular endothelial growth factor (VEGF) in the vitreous. Furthermore, nanoparticle-protein complexes retain in vitro anti-angiogenic properties of bare nanoparticles. In particular, priming the nanoparticles (gold and silica) with tissue-specific corona proteins allows nanoparticle-protein complexes to exert better in vivo therapeutic effects by higher binding to VEGF than bare nanoparticles. These results suggest that controlled corona formation that mimics in vivo processes may be useful in the therapeutic use of nanomaterials in local environment.

Probing organic ligands and their binding schemes on nanocrystals by mass spectrometric and FT-IR spectroscopic imaging.

We report an analysis method to identify conjugated ligands and their binding states on semiconductor nanocrystals based on their molecular information. Surface science techniques, such as time-of-flight secondary-ion mass spectrometry (ToF-SIMS) and FT-IR spectroscopy, are adopted based on the micro-aggregated sampling method. Typical trioctylphosphine oxide-based synthesis methods of CdSe/ZnS quantum dots (QDs) have been criticized because of the peculiar effects of impurities on the synthesis processes. Because the ToF-SIMS technique provides molecular composition evidence on the existence of certain ligands, we were able to clearly identify n-octylphosphonic acid (OPA) as a surface ligand on CdSe/ZnS QDs. Furthermore, the complementary use of the ToF-SIMS technique with the FT-IR technique could reveal the OPA ligands' binding state as bidentate complexes.

A Facile Route for Patterned Growth of Metal-Insulator Carbon Lateral Junction through One-Pot Synthesis.

Precise graphene patterning is of critical importance for tailor-made and sophisticated two-dimensional nanoelectronic and optical devices. However, graphene-based heterostructures have been grown by delicate multistep chemical vapor deposition methods, limiting preparation of versatile heterostructures. Here, we report one-pot synthesis of graphene/amorphous carbon (a-C) heterostructures from a solid source of polystyrene via selective photo-cross-linking process. Graphene is successfully grown from neat polystyrene regions, while patterned cross-linked polystyrene regions turn into a-C because of a large difference in their thermal stability. Since the electrical resistance of a-C is at least 2 orders of magnitude higher than that for graphene, the charge transport in graphene/a-C heterostructure occurs through the graphene region. Measurement of the quantum Hall effect in graphene/a-C lateral heterostructures clearly confirms the reliable quality of graphene and well-defined graphene/a-C interface. The direct synthesis of patterned graphene from polymer pattern could be further exploited to prepare versatile heterostructures.

Guided formation of sub-5 nm interstitial gaps between plasmonic nanodisks.

To achieve a reliable formation of a surface-enhanced Raman scattering (SERS) sensor with evenly distributed hot spots on a wafer scale substrate, we propose a hybrid approach combining physical nanolithography for preparing Au nanodisks and chemical Au reduction for growing them. During the chemical growth, the interstitial distance between the nanodisks decreased from 60 nm to sub-5 nm. The resulting patterns of the nanogap-rich Au nanodisks successfully enhance the SERS signal, and its intensity map shows only a 5% or less signal variation on the entire sample.