Dual-Function Janus Nanozymes for Performance Evaluation and Application in a Surrogate Virus Neutralization Test with Vaccinated Samples.

The need exists for biosensing technologies capable of sensitively and accurately detecting various biomarkers. In response, the development of nanozymes is actively underway; they have advantages in stability, cost, performance, and functionalization over natural enzymes commonly used for signal amplification in sensing technologies. However, the performance of nanozymes is interdependent with factors such as shape, size, and surface functional moiety, making it challenging to perform quantitative performance comparisons based on the nanozyme material. In this study, we propose a physical synthetic approach to fabricate double-layered bimetallic nanozymes with identical shapes, sizes, and surfaces but different material compositions. These Janus nanozymes consist of a nanozymatic layer responsible for catalytic activity and a gold layer responsible for quantification and efficient surface modification. Based on their identical physicochemical properties, the synthesized double-layered bimetallic nanozymes allow, for the first time, a quantitative comparison of nanozymatic activities in terms of various kinetic parameters. We compared several candidates and found that the Ir-Au nanozyme exhibited the best performance. Subsequently, we applied this nanozyme to detect neutralizing antibodies against SARS-CoV-2 based on a surrogate virus neutralization test. The results demonstrated a limit of detection as low as 2 pg/mL and selectivity specifically toward MERS-CoV. The performance of this assay was further validated using vaccinated samples, demonstrating the potential of our approach as a cost-effective, rapid, and sensitive diagnostic tool for neutralizing antibody detection against viruses such as SARS-CoV-2.

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.

Simultaneous Multiplexed Imaging of Biomolecules in Transgenic Mouse Brain Tissues Using Mass Spectrometry Imaging: A Multi-omic Approach.

The importance of multi-omic-based approaches to better understand diverse pathological mechanisms including neurodegenerative diseases has emerged. Spatial information can be of great help in understanding how biomolecules interact pathologically and in elucidating target biomarkers for developing therapeutics. While various analytical methods have been attempted for imaging-based biomolecule analysis, a multi-omic approach to imaging remains challenging due to the different characteristics of biomolecules. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool due to its sensitivity, chemical specificity, and high spatial resolution in visualizing chemical information in cells and tissues. In this paper, we suggest a new strategy to simultaneously obtain the spatial information of various kinds of biomolecules that includes both labeled and label-free approaches using ToF-SIMS. The enzyme-assisted labeling strategy for the targets of interest enables the sensitive and specific imaging of large molecules such as peptides, proteins, and mRNA, a task that has been, to date, difficult for any MS analysis. Together with the strength of the analytical performance of ToF-SIMS in the label-free tissue imaging of small biomolecules, the proposed strategy allows one to simultaneously obtain integrated information of spatial distribution of metabolites, lipids, peptides, proteins, and mRNA at a high resolution in a single measurement. As part of the suggested strategy, we present a sample preparation method suitable for MS imaging. Because a comprehensive method to examine the spatial distribution of multiple biomolecules in tissues has remained elusive, our strategy can be a useful tool to support the understanding of the interactions of biomolecules in tissues as well as pathological mechanisms.

Evaluation of Time-of-Flight Secondary Ion Mass Spectrometry Spectra of Peptides by Random Forest with Amino Acid Labels: Results from a Versailles Project on Advanced Materials and Standards Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

Utilization of chromogenic enzyme substrates for signal amplification in multiplexed detection of biomolecules using surface mass spectrometry.

MicroRNAs (miRNAs) are important post-transcriptional gene regulators and can serve as potential biomarkers for many diseases. Most of the current miRNA detection techniques require purification from biological samples, amplification, labeling, or tagging, which makes quantitative analysis of clinically relevant samples challenging. Here we present a new strategy for the detection of miRNAs with uniformity over a large area based on signal amplification using enzymatic reactions and measurements using time-of-flight secondary ion mass spectrometry (ToF-SIMS), a sensitive surface analysis tool. This technique has high sequence specificity through hybridization with a hairpin DNA probe and allows the identification of single-base mismatches that are difficult to distinguish by conventional mass spectrometry. We successfully detected target miRNAs in biological samples without purification, amplification, or labeling of target molecules. In addition, by adopting a well-known chromogenic enzymatic reaction from the field of biotechnology, we extended the use of enzyme-amplified signal enhancement ToF (EASE-ToF) to protein detection. Our strategy has advantages with respect to scope, quantification, and throughput over the currently available methods, and is amenable to multiplexing based on the outstanding molecular specificity of mass spectrometry (MS). Therefore, our technique not only has the potential for use in clinical diagnosis, but also provides evidence that MS can serve as a useful readout for biosensing to perform multiplexed analysis extending beyond the limitations of existing technology.

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.

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.

Molecular depth profiling on rat brain tissue sections prepared using different sampling methods.

Brain imaging using time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been reported to produce the distorted biomolecular distributions due to the cholesterol-induced matrix effect when cholesterol migrates to the surface, particularly in white matter, which contains a high level of cholesterol. Frozen-hydrated analysis has been used to inhibit the movement of cholesterol in the brain. In this paper, the authors propose new sample preparation and drying methods that can be used to obtain accurate biomolecular images at room temperature, instead of frozen-hydrated analysis using liquid-nitrogen, which must be continuously supplied to maintain the sample at -160 °C during the experiment. The rat brain prepared by the tape-supporting method on a precooled (-20 °C) stainless steel plate was freeze-dried in a load-lock chamber of ToF-SIMS for about an hour and moved directly to the main chamber. Using this preparation method, the authors found that cholesterol did not migrate to the surface in the corpus callosum (white matter) of the rat brain and sulfatide-related signals obtained from the cerebellum were not reduced in white matter. Our tape-supporting and freeze-drying sampling method for brain tissues could be a useful tool to study important metabolites of neurodegenerative diseases.

TOF-SIMS analysis of an isocitrate dehydrogenase 1 mutation-associated oncometabolite in cancer cells.

The development of analytical tools for accurate and sensitive detection of intracellular metabolites associated with mutated metabolic enzymes is important in cancer diagnosis and staging. The gene encoding the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) is mutated in various cancers, and mutant IDH1 could represent a good biomarker and potent target for cancer therapy. Owing to a mutation in an important arginine residue in the catalytic pocket, mutant IDH1 catalyzes the production of 2-hydroxyglutarate (2-HG) instead of its wild type product α-ketoglutarate (α-KG), which is involved in multiple cellular pathways involving the hydroxylation of proteins, ribonucleic acid, and deoxyribose nucleic acid (DNA). Since 2-HG is an α-KG antagonist, inhibiting normal α-KG-dependent metabolism, high intracellular levels of 2-HG result in abnormal histone and DNA methylation. Therefore, accurate and sensitive analytical tools for the direct detection of 2-HG in cancer cells expressing mutant IDH1 would benefit this field, as it would minimize the need both for complicated experimental procedures and for large amounts of biological samples. Here, the authors describe a useful analytical method for the direct detection of 2-HG in lysates from a mutant IDH1-expressing cell line by time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis, a powerful surface analysis tool. In addition, the authors verified the efficacy of the specific mutant IDH1 inhibitor AGI-5198 by tracking the intracellular 2-HG concentration, which decreased in a dose-dependent manner. Our results demonstrate the large potential of TOF-SIMS as an analytical tool for the simple, direct detection of oncometabolites during cancer diagnosis, and for verifying the efficiency of the targeted cancer drugs.

Improved mass resolution and mass accuracy in TOF-SIMS spectra and images using argon gas cluster ion beams.

The popularity of argon gas cluster ion beams (Ar-GCIB) as primary ion beams in time-of-flight secondary ion mass spectrometry (TOF-SIMS) has increased because the molecular ions of large organic- and biomolecules can be detected with less damage to the sample surfaces. However, Ar-GCIB is limited by poor mass resolution as well as poor mass accuracy. The inferior quality of the mass resolution in a TOF-SIMS spectrum obtained by using Ar-GCIB compared to the one obtained by a bismuth liquid metal cluster ion beam and others makes it difficult to identify unknown peaks because of the mass interference from the neighboring peaks. However, in this study, the authors demonstrate improved mass resolution in TOF-SIMS using Ar-GCIB through the delayed extraction of secondary ions, a method typically used in TOF mass spectrometry to increase mass resolution. As for poor mass accuracy, although mass calibration using internal peaks with low mass such as hydrogen and carbon is a common approach in TOF-SIMS, it is unsuited to the present study because of the disappearance of the low-mass peaks in the delayed extraction mode. To resolve this issue, external mass calibration, another regularly used method in TOF-MS, was adapted to enhance mass accuracy in the spectrum and image generated by TOF-SIMS using Ar-GCIB in the delayed extraction mode. By producing spectra analyses of a peptide mixture and bovine serum albumin protein digested with trypsin, along with image analyses of rat brain samples, the authors demonstrate for the first time the enhancement of mass resolution and mass accuracy for the purpose of analyzing large biomolecules in TOF-SIMS using Ar-GCIB through the use of delayed extraction and external mass calibration.

Measuring Compositions in Organic Depth Profiling: Results from a VAMAS Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the measurement of composition in organic depth profiling. Layered samples with known binary compositions of Irganox 1010 and either Irganox 1098 or Fmoc-pentafluoro-l-phenylalanine in each layer were manufactured in a single batch and distributed to more than 20 participating laboratories. The samples were analyzed using argon cluster ion sputtering and either X-ray photoelectron spectroscopy (XPS) or time-of-flight secondary ion mass spectrometry (ToF-SIMS) to generate depth profiles. Participants were asked to estimate the volume fractions in two of the layers and were provided with the compositions of all other layers. Participants using XPS provided volume fractions within 0.03 of the nominal values. Participants using ToF-SIMS either made no attempt, or used various methods that gave results ranging in error from 0.02 to over 0.10 in volume fraction, the latter representing a 50% relative error for a nominal volume fraction of 0.2. Error was predominantly caused by inadequacy in the ability to compensate for primary ion intensity variations and the matrix effect in SIMS. Matrix effects in these materials appear to be more pronounced as the number of atoms in both the primary analytical ion and the secondary ion increase. Using the participants' data we show that organic SIMS matrix effects can be measured and are remarkably consistent between instruments. We provide recommendations for identifying and compensating for matrix effects. Finally, we demonstrate, using a simple normalization method, that virtually all ToF-SIMS participants could have obtained estimates of volume fraction that were at least as accurate and consistent as XPS.

Probing nanoparticles and nanoparticle-conjugated biomolecules using time-of-flight secondary ion mass spectrometry.

Bio-conjugated nanoparticles have emerged as novel molecular probes in nano-biotechnology and nanomedicine and chemical analyses of their surfaces have become challenges. The time-of-flight (TOF) secondary ion mass spectrometry (SIMS) has been one of the most powerful surface characterization techniques for both nanoparticles and biomolecules. When combined with various nanoparticle-based signal enhancing strategies, TOF-SIMS can probe the functionalization of nanoparticles as well as their locations and interactions in biological systems. Especially, nanoparticle-based SIMS is an attractive approach for label-free drug screening because signal-enhancing nanoparticles can be designed to directly measure the enzyme activity. The chemical-specific imaging analysis using SIMS is also well suited to screen nanoparticles and nanoparticle-biomolecule conjugates in complex environments. This review presents some recent applications of nanoparticle-based TOF-SIMS to the chemical analysis of complex biological systems.

Bioinspired, cytocompatible mineralization of silica-titania composites: thermoprotective nanoshell formation for individual chlorella cells.

Hard-shell case: Using a (RKK)4 D8 peptide allows mineralization to occur under cytocompatible conditions. Thus individual Chlorella cells could be encapsulated within a SiO2 -TiO2 nanoshell with high cell viability (87 %). The encapsulated Chlorella showed an almost threefold increase in their thermo-tolerance after 2 h at 45 °C.

b2 Peaks in SERS Spectra of 4-Aminobenzenethiol: A Photochemical Artifact or a Real Chemical Enhancement?

Strong b2 peaks (1142, 1391, 1438, and 1583 cm(-1)) in the SERS spectra of 4-aminobenzenethiol (ABT) have been regarded by many as a textbook example of chemically enhanced SERS signals. However, this interpretation is in serious doubt after the recent claim that they arise from 4,4'-dimercaptoazobenzenes (DMAB) photogenerated during the acquisition of SERS, not the genuine chemically enhanced signals of ABT. Subsequent attempts to prove or disprove this claim have failed to provide any decisive verdict. Here we present spectroscopic and mass spectrometric evidence that further support the photogeneration of DMABs from ABTs on an Ag surface. Furthermore, we show that the amount of the DMAB is sufficient to explain the b2 intensities of ABT.

The regional ratio of cholesteryl palmitate to cholesteryl oleate measured by ToF-SIMS as a key parameter of atherosclerosis.

OBJECTIVE: Changes in cholesterol ester (CE) content regulate the progression of atherosclerosis. However, the spatial dynamics of CE subsets and their quantitative changes during lesion progression are not well understood due to a lack of appropriate imaging techniques. In this study, we developed an imaging-based analysis method to map the distribution of CE subsets using time-of-flight secondary ion mass spectrometry (ToF-SIMS). METHODS: Serial sections of atherosclerotic aortic sinuses from apolipoprotein E knock-out mice (n = 15) fed a 0.15% high-fat diet for 12-20 weeks were examined by ToF-SIMS. RESULTS AND CONCLUSION: We found that the ratio of cholesteryl palmitate (Ch-PA) to cholesteryl oleate (Ch-OA) increased by approximately 99% (p = 0.02) as atherosclerosis progressed, whereas the ratios of cholesteryl linoleate (p = 0.09) and cholesteryl stearate (p = 0.22) to Ch-OA did not change significantly. In advanced atherosclerotic plaques, in situ and in-vitro cell death assays showed that local Ch-PA densities were highly correlated with an increase in the number of apoptotic cells. These results suggest that increased Ch-PA may contribute to the formation of a necrotic core by increasing cell death. Our results indicate that the regional ratio of CEs as measured by ToF-SIMS might be a valuable new marker of atherosclerotic progression.

Electrochemical release of amine molecules from carbamate-based, electroactive self-assembled monolayers.

In this paper, carbamate-based self-assembled monolayers (SAMs) of alkanethiolates on gold were suggested as a versatile platform for release of amine-bearing molecules in response to the electrical signal. The designed SAMs underwent the electrochemical oxidation on the gold surface with simultaneous release of the amine molecules. The synthesis of the thiol compounds was achieved by coupling isocyanate-containing compounds with hydroquinone. The electroactive thiol was mixed with 11-mercaptoundecanol [HS(CH(2))(11)OH] to form a mixed monolayer, and cyclic votammetry was used for the characterization of the release behaviors. The mixed SAMs showed a first oxidation peak at +540 mV (versus Ag/AgCl reference electrode), indicating the irreversible conversion from carbamate to hydroquinone groups with simultaneous release of the amine molecules. The analysis of ToF-SIMS further indicated that the electrochemical reaction on the gold surface successfully released amine molecules.

Microscale mechanisms of agarose-induced disruption of collagen remodeling.

Cells are strongly influenced by the local structure and mechanics of the extracellular matrix (ECM). We recently showed that adding agarose to soft collagen ECMs can mechanically stiffen these hydrogels by two orders of magnitude while limiting 3D cell motility, which we speculated might derive from agarose-mediated inhibition of collagen fiber deformation and remodeling. Here, we directly address this hypothesis by investigating the effects of agarose on cell-collagen interactions at the microscale. Addition of agarose progressively restricts cell spreading, reduces stress fiber and focal adhesion assembly, and inhibits macroscopic gel compaction. While time-of-flight secondary ion mass spectrometry and scanning electron microscopy fail to reveal agarose-induced alterations in collagen ligand presentation, the latter modality shows that agarose strongly impairs cell-directed assembly of large collagen bundles. Agarose-mediated inhibition of cell spreading and cytoarchitecture can be rescued by ß-agarase digestion or by covalently crosslinking the matrix with glutaraldehyde. Based on these results, we argue that cell spreading and motility on collagen requires local matrix stiffening, which can be achieved via cell-mediated fiber remodeling or by chemically crosslinking the fibers. These findings provide new mechanistic insights into the regulatory function of agarose and bear general implications for cell adhesion and motility in fibrous ECMs.

Mass imaging of iron oxide nanoparticles inside cells for in vitro cytotoxicity.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging analysis was performed on murine macrophage cells treated with various concentrations of iron oxide (Fe3O4) nanoparticles, which are used as MRI contrast agents. First, murine macrophage cells were seeded on a slide glass for 24 hrs and treated with varying concentrations of Fe3O4 nanoparticles for 24 hrs. To expose a cross section of each cell and obtain a distribution of the nanoparticles inside the cells, the cells were sputtered using Bi ions after which the cross section of each cell was scanned and imaged using the focused cluster ion beam with a spatial resolution of 300 nm. Fe3O4 nanoparticles were found mainly in the cytoplasm region of the cells, not in the nucleus region of cells, suggesting that the uptake of the Fe3O4 nanoparticles were into the cytoplasm of cell, not into the nucleus of cell. Based on these observations, our protocol using mass imaging analysis would be a useful addition to the study of in vitro nanoparticle cytotoxicity.