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Amyotrophic lateral sclerosis (ALS) is a degenerative disease caused by motor neuron damage in the central nervous system, and it is difficult to diagnose early. Drosophila melanogaster is widely used to investigate disease mechanisms and discover biomarkers because it is easy to induce disease in Drosophila through genetic engineering. We performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to investigate changes in phospholipid distribution in the brain tissue of an ALS-induced Drosophila model. Fly brain tissues of several hundred micrometers or less were sampled using a fly collar to obtain reproducible tissue sections of similar sizes. MSI of brain tissues of Drosophila cultured for 1 or 10 days showed that the distribution of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI), was significantly different between the control group and the ALS group. In addition, the lipid profile according to phospholipids differed as the culture time increased from 1 to 10 days. These results suggest that disease indicators based on lipid metabolites can be discovered by performing MALDI-MSI on very small brain tissue samples from the Drosophila disease model to ultimately assess the phospholipid changes that occur in early-stage ALS.

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.

A potential method to tune reactivity In organic chemistry, reaction rates are often sensitive to substituents that either donate or withdraw electron density from a reactive carbon center. Heo et al. now report that when reactants are tethered to an electrode, the polarization effect of an applied potential can exert an analogous influence, essentially acting as a tunable functional group. The authors demonstrate rate modulations in ester hydrolysis and arene cross-coupling, as well as a two-step acid-to-amide conversion that benefits from inversion of the polarization midway through the reaction. Science , this issue p. 214

Reliable quantification of nanoparticle uptake in mammalian cells is essential to study the effects of nanoparticles in the fields of medicine and environmental science. Most conventional quantification methods, such as electron microscopy or confocal imaging, are laborious and semi-quantitative and therefore not readily applicable to routine analyses. Here, we developed assays to quantify fluorescently labelled nanoparticle uptake in mammalian cells using a flow cytometer. The first approach was to measure the percentage of nanoparticle-containing cells based on a cutoff fluorescence intensity as set from a histogram of control cells, which is a quick and easy way to relatively compare nanoparticle uptake in the same set of experiments. The second approach was to measure the calibrated fluorescence intensity of the nanoparticle-treated cells in molecules of equivalent soluble fluorophore (MESF) values using calibration beads, which allows for comparisons between different sets of experiments. We successfully applied the developed assays to more readily measure fluorescence-labelled silica nanoparticle uptake in A549 lung carcinoma cells in a quantitative rather than semi-quantitative manner. We further tested the assays with nine different types of mammalian cells and investigated the correlation between cell type/size and nanoparticle uptake.

Fulminant hepatitis is a serious inflammatory condition of the liver characterized by massive necrosis of liver parenchyma following excessive immune cell infiltration into the liver, and possibly causing sudden hepatic failure and medical emergency. However, the underlying mechanisms are not fully understood. Here, we investigated the role of cyclic AMP-responsive element-binding protein, hepatocyte specific (CREBH) in concanavalin A (ConA)-driven hepatitis-evoked liver injury. C57BL/6J (WT) and Crebh knockout (KO) mice injected with ConA (7.5 or 25 mg/kg) and bone marrow (BM) chimeric mice, generated by injection of BM cells into sub-lethally irradiated recipients followed by ConA injection (22.5 or 27.5 mg/kg) 8 weeks later, were used for in vivo study. Primary mouse hepatocytes and HEK293T cells were used for a comparative in vitro study. Crebh KO mice are highly susceptible to ConA-induced liver injury and prone to death due to increased neutrophil infiltration driven by enhanced hepatic expression of neutrophil-attracting chemokines. Notably, BM chimera experiment demonstrated that Crebh-deficient hepatocytes have an enhanced ability of recruiting neutrophils to the liver, thereby promoting hepatotoxicity by ConA. Intriguingly, in vitro assays showed that p65, a subunit of NF-κB and common transcription factor for various chemokines, dependent transactivation was inhibited by CREBH. Furthermore, p65 expression was inversely correlated with CREBH level in ConA-treated mice liver and TNFα-stimulated primary mouse hepatocytes. This is the first demonstration that CREBH deficiency aggravates inflammatory liver injury following chemokine-dependent neutrophil infiltration via NF-κB p65 upregulation. CREBH is suggested to be a novel therapeutic target for treatment of fulminant hepatitis.

Appropriate surface designs and precise characterizations of nanoparticles (NPs) are important but as of yet unrealized in biomedical applications of nanomaterials. We report a facile synthetic approach to the preparation of iron oxide (Fe3O4) NPs with different surface charges through exchange with polymeric ligands. An accurate analysis method for characterization of the surface-modified Fe3O4 NPs using a combination of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) and Fourier transform infrared spectroscopy (FT-IR) techniques is demonstrated. A comparison of the spatial distribution of the ligand and core particles with ToF-SIMS and FT-IR verified the successful modification and purification. In addition, the surface was shown to play an important role in interactions with live cells in terms of cytotoxicity, cellular uptake, and even gene transfection. Interestingly, precise control over the surface chemistry of NP enabled efficient transfection of siRNA into suspension type cell, the Jurkat human leukemic T cell line, achieving successful down regulation of NOTCH1 whose signaling pathway is known to be involved in acute leukemia. Our report presents an in-depth study of the interactions between the NPs and cells, as well as their synthesis, characterization, and surface-dependent performance properties.

We report polyethylene glycol (PEG)-grafting antifouling surfaces using a plasma copolymerized (PcP) technique to monitor protease activity in complex media. By varying the mixing ratio of the PEG and ethylenediamine (EDA) precursors, the PcP-PEG-EDA (PcP-PE) film was able to easily control surface amine density with good preservation of the internal PEG structure. We found that nonspecific protein adsorption was dramatically reduced in serum-containing media on the PcP-PE films, as opposed to that on plasma polymerized-EDA (PP-E) films without PEG. When SPR sensor chips coated with PcP-PE film were employed to detect protease activity, biotinylated luciferase probes (luciferase-peptide-biotin) on streptavidin-conjugated SPR chips enabled real-time and label-free measurement of matrix metalloproteinase activity in cell culture media. Owing to its excellent antifouling ability, this newly developed method boasts minimal nonspecific binding and can serve as a biochip platform to promote a wide range of applications in the biological field.

Titanium dioxide (TiO2) nanoparticles are used in a wide variety of products, such as renewable energy resources, cosmetics, foods, packaging materials, and inks. However, large quantities of surfactants are used to prepare waterborne TiO2 nanoparticles with long-term dispersion stability, and very few studies have investigated the development of pure water dispersion technology without the use of surfactants and synthetic auxiliaries. This study investigated the use of focused ultrasound to prepare surfactant-free waterborne TiO2 nanoparticles to determine the optimal conditions for dispersion of TiO2 nanoparticles in water. Under 395–400 kHz and 100–105 W conditions, 1 wt% TiO2 colloids were prepared. Even in the absence of a surfactant, in the water dispersion state, the nanoparticles were dispersed with a particle size distribution of ≤100 nm and did not re-agglomerate for up to 30 days, demonstrating their excellent dispersion stability.

The increasingly widespread use of engineered nanoparticles in medical, industrial, and food applications has raised concerns regarding their potential toxicity to humans and the environment. Silicon dioxide nanoparticles (SiO2 NPs), which have relatively low direct toxicity, have been increasingly applied in both consumer products and biomedical applications, leading to significantly higher exposure for humans and the environment. We carried out a toxicity assessment of SiO2 NPs using the common water flea D. magna by focusing on physiological and behavioral indicators such as heart rate, swimming performance, and growth. Exposure to SiO2 NPs did not produce acute or chronic toxicity at limited concentrations (&lt, 100 μg/mL), but did have statistically significant negative effects on heart rate, swimming distance, and body size. The use of fluorescein isothiocyanate in a silica matrix allowed the tracing and visualization of clear SiO2 NP accumulation in D. magna, which was confirmed by ICP-MS. Although exposure to SiO2 NPs seemed to affect cardiac and swimming performance, such end-point experiments may be insufficient to fully understand the toxicity of these nanoparticles. However, the physiological and behavioral changes shown here suggest potential adverse effects on the aquatic environment by substances previously considered nontoxic.

The direct in situ synthesis of cadmium sulfide (CdS) nanowires (NWs) was presented by direct synthesis of CdS NWs on the gold surface of an interdigitated electrode (IDE). In this work, we investigated the effect of a strong oxidant on the surfaces of the CdS NWs using X-ray photoelectron spectroscopy, transmission electron microscopy, and time-of-flight secondary ion mass spectrometry. We also fabricated a parylene-C film as a surface passivation layer for in situ-synthesized CdS NW photosensors and investigated the influence of the parylene-C passivation layer on the photoresponse during the coating of parylene-C under vacuum using a quartz crystal microbalance and a photoanalyzer. Finally, we used the in situ-synthesized CdS NW photosensor with the parylene-C passivation layer to detect the chemiluminescence of horseradish peroxidase and luminol and applied it to medical detection of carcinoembryonic antigen.

A new general method of controlling the chemical reactivity of molecules is proposed. Instead of tuning the electronic properties of reactive molecules by employing functional groups that impose different inductive effects to a molecule, we propose to immobilize the parent molecule onto an electrode, for example by installing a thiol group to the molecule and forming a self-assembled monolayer on a gold surface. By applying a voltage the electronic property of the immobilized molecule can be tuned, as is commonly done by decorating the molecule with electron-donating and electron-withdrawing groups. As a proof of principle, it is shown that the base-catalyzed saponification of benzoic ester can be shut down completely by applying a negative voltage, while it can be accelerated by using a positive voltage. Furthermore it is shown that the Suzuki-Miyaura cross-coupling reaction can be affected by the voltage when the arylhalide substrate of the reaction is immobilized on a gold electrode.

In this study, the soft-type and hard-type lead zirconate titanate (PZT) ceramics were compared in order to create an optimal system for ultrasonic dispersion of nanoparticles, and sound pressure energy for each PZT ceramic was analyzed and closely examined with ultrasonic energy. TiO2 was water-dispersed using the soft-type and hard-type PZT transducer, possessing different characteristics, and its suspension particle size and distribution, polydispersity index (PDI), zeta potential, and dispersion were evaluated for 180 days. Furthermore, it was confirmed that the particles dispersed using the hard-type PZT transducer were smaller than the particles dispersed using the soft-type PZT by 15 nm or more. Because the hard-type PZT transducer had a lower PDI, uniform particle size distribution was also confirmed. In addition, by measuring the zeta potential over time, it was found that the hard-type PZT transducer has higher dispersion safety. In addition, it was confirmed that the ultrasonically dispersed TiO2 suspension using a hard-type PZT transducer maintained constant particle size distribution for 180 days, whereas the suspension from the soft-type PZT aggregated 30 days later. Therefore, the hard-type PZT is more suitable for ultrasonic dispersion of nanoparticles.

Reduced graphene oxide (rGO) is a graphene-like material that exhibits high productivity for a wide range of industrial applications. To promote the application of rGO, it is important to not only produce high-quality rGO but also precisely evaluate the output. The intensity ratio of the D to G band in the Raman scattering is commonly used to assess the defect density of the carbon materials; however, this ratio is limited to evaluate the reduction degree of rGO because of the ambiguity arising from the superposition of the bands. In this study, we investigate the relationship between the intensity ratio of D* to G band and the reduction of graphene oxide (GO) to evaluate the degree of reduction of rGO. The spectral analysis of GO and rGO, along with systematic research of the thermally reduced GO synthesized via thermal treatment (100–900 °C) revealed a strong linkage between the D*/G intensity ratio and the C/O atomic ratio. The atomic vibrational relationships were elucidated by the assignment of the D* band, based on the density functional perturbation theory calculations. These findings explain the atomic vibrational properties of rGO and provide an indicator of the quality of rGO to optimize its performance for applications.

Fabrication methods to produce an amine-functionalized surface have been widely investigated as it is a promising substrate for biological studies, owing to its ability to stably immobilize various biomolecules. In this study, a biocompatible amine-functionalized polyethylene glycol (aPEG) thin film was fabricated by using plasma-enhanced chemical vapor deposition (PECVD) to overcome the disadvantages of the conventional wet process. A functionalized PEG diamine was used as a single precursor to simplify the fabrication process, and the relationship between the surface functional group density and the plasma power was investigated. Also, the surfaces of the functionalized thin films were quantitatively analyzed to determine the optimal fabrication condition that yields the most excellent surface amine density. The non-fouling resistance of the aPEG thin film and its applicability for biological applications was demonstrated by successfully immobilizing the matrix metalloproteinase-7 (MMP-7) enzyme, which is a well-known biomarker for cancers, with minimal non-specific adsorption (NSA). Our fabrication method was further applied to the modification of rather challenging substrates, including carbon paper, polyethylene terephthalate (PET) film, silicon wafer, polyvinylidene fluoride (PVDF), and successful modification was confirmed by the results of time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis. It is expected that our fabrication method can enable applications of the functionalized thin film in bioscience by presenting a versatile surface modification technique for deposition of biocompatible material onto various substrates, which can be potentially used in disease diagnosis, medical implants, and bioengineering.

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.

Biopolymer adsorption onto a membrane is a significant issue in the reliability of solid-state nanopore devices, since it degrades the device performance or promotes device failure. In this work, a poly(ethylene glycol) (PEG) layer was coated on a silicon nitride (SiNx) membrane by plasma-polymerized vapor deposition to inhibit biopolymer adsorption. From optical observations, the deposited PEG layer demonstrated increased hydrophilicity and anti-adsorption property compared to the SiNx surface. Electrical properties of the PEG/SiNx nanopore were characterized, showing Ohmic behavior and a 6.3 times higher flicker noise power due to the flexible conformation of PEG in water. Antifouling performance of each surface was analyzed by measuring the average time from voltage bias to the first adsorption during DNA translocation experiments, where the modified surface enabled two times prolonged device operation. The time to adsorption was dependent on the applied voltage, implying adsorption probability was dominated by the electrophoretic DNA approach to the nanopore. DNA translocation behaviors on each surface were identified from translocation signals, as the PEG layer promoted unfolded and fast movement of DNA through the nanopore. This work successfully analyzed the effect of the PEG layer on DNA adsorption and translocation in solid-state nanopore experiments.

We integrate the adhesive properties of marine mussels, the lubricating properties of pitcher plants, and the nonfouling properties of diatoms into nanostructured surfaces to develop a device called a micro-omnifluidic (μ-OF) system to solve the existing challenges in microfluidic systems. Unlike conventional poly(dimethylsiloxane)-based fluidic systems that are incompatible with most organic solvents, the μ-OF system utilizes a variety of solvents such as water, ethanol, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, n-hexane, 1,2-dichloroethane, acetic acid, 2-propanol, acetone, toluene, diesel oil, dioxane, gasoline oil, hexadecane, and xylene. The μ-OF system is based on a phenomenon called microchannel induction that spontaneously occurs when virtually all droplets of solvents are applied on omniphilically micropatterned regions of a slippery liquid-infused porous surface. Any solvents with surface tension greater than that of the lubricant (17.1 mN/m, Fluorinert FC-70) are able to repel the infused lubricant located on top of the omniphilic microlines, triggering controlled movement of the droplet by gravity along the microlines. We also demonstrated that the μ-OF system is reusable by the nonadsorption properties of the silicified layer. Due to the organic solvent compatibility, we were able to perform organic reactions with high portability and energy efficiency in operation.

Local application requires fewer nanoparticles than systemic delivery to achieve effective concentration. In this study, we investigated the potential toxicity and efficacy of bare titanium dioxide (TiO 2 ) nanoparticles by local administration into the eye. Mono-disperse, 20 nm-size TiO 2 nanoparticles did not affect the viability of retinal constituent cells within certain range of concentrations (~ 1.30 μg/mL). Furthermore, local delivery of TiO 2 nanoparticles did not induce any significant toxicity at the level of gene expression and histologic integrity in the retina of C57BL/6 mice. Interestingly, at the low concentration (130 ng/mL) without definite toxicity, these nanoparticles suppressed in vitro angiogenesis processes and in vivo retinal neovascularization in oxygen-induced retinopathy mice when they are administered intravitreally. Taken together, our results demonstrate that even TiO 2 nanoparticles can be safely utilized for the treatment of retinal diseases at the adequate concentration levels, especially through local administration. From the Clinical Editor In this paper the local application of titanium dioxide is described as a local treatment for retinal diseases associated with neovascularization. While these nanoparticles have known systemic toxicity, this work demonstrates that when applied locally in a mouse model, they can be used without observable toxicity even in their native forms.

We present a comparative study of the time-of-flight-secondary ion mass spectrometry (ToF-SIMS), Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS) techniques on a mixed self-assembled monolayer (SAM) of biotinylated polyethylene glycol alkane thiol (BPT) and 11-mercaptoundecanol (MUD) as a model system. This mixed SAM was employed to maximize specific binding between the biotin groups in the mixed SAM and the streptavidin, while minimizing nonspecific adsorption of the streptavidin. For a better quantitative analysis of our mixed SAM by using ToF-SIMS, we performed a principal component analyses (PCA) with various peak sets and found that the negative ions of the MUD complementary peak set showed good correlation between the XPS and FT-IR quantification results and the scores for principal component 1 from a PCA of the ToF-SIMS data. These findings show that ToF-SIMS analysis along with PCA would make a good surface analysis technique for identifying intact molecules of adsorbates and for quantifying at once each adsorbate in a mixed SAM. Copyright © 2014 John Wiley & Sons, Ltd.

Nanoparticles can be involved in biological activities such as apoptosis, angiogenesis, and oxidative stress by themselves. In particular, inorganic nanoparticles such as gold and silica nanoparticles are known to inhibit vascular endothelial growth factor (VEGF)-mediated pathological angiogenesis. In this study, we show that anti-angiogenic effect of inorganic nanospheres is determined by their sizes. We demonstrate that 20 nm size gold and silica nanospheres suppress VEGF-induced activation of VEGF receptor-2, in vitro angiogenesis, and in vivo pathological angiogenesis more efficiently than their 100 nm size counterparts. Our results suggest that modulation of the size of gold and silica nanospheres determines their inhibitory activity to VEGF-mediated angiogenesis.

To develop a methodology for absolute determination of the surface areal density of functional groups on organic and bio thin films, medium energy ion scattering (MEIS) spectroscopy was utilized to provide references for calibration of X-ray photoelectron spectroscopy (XPS) or Fourier transformation-infrared (FTIR) intensities. By using the MEIS, XPS, and FT-IR techniques, we were able to analyze the organic thin film of a Ru dye compound (C58H86O8N8S2Ru), which consists of one Ru atom and various stoichiometric functional groups. From the MEIS analysis, the absolute surface areal density of Ru atoms (or Ru dye molecules) was determined. The surface areal densities of stoichiometric functional groups in the Ru dye compound were used as references for the calibration of XPS and FT-IR intensities for each functional group. The complementary use of MEIS, XPS, and FT-IR to determine the absolute surface areal density of functional groups on organic and bio thin films will be useful for more reliable development of applications based on organic thin films in areas such as flexible displays, solar cells, organic sensors, biomaterials, and biochips.

Optical coherence tomography (OCT) angiography (OCTA) has been actively studied as a noninvasive imaging technology to generate retinal blood vessel network maps for the diagnoses of retinal diseases. Given that the uses of OCT and OCTA have increased in the field of ophthalmology, it is necessary to develop retinal phantoms for clinical OCT for product development, performance evaluation, calibration, certification, medical device licensing, and production processes. We developed a retinal layer-mimicking phantom with microfluidic channels based on microfluidic fabrication technology using polydimethylsiloxane (PDMS) and titanium dioxide (TiO(2)) powder. We implemented superficial and deep retinal vessels using microfluidic channels. In addition, multilayered thin films were synthesized with multiple spin-coating processes that comprised layers that corresponded to the retinal layers, including the ganglion cell layer (GCL), inner plexiform layer (IPL), and inner nuclear layer (INL). The phantom was formed by merging the multilayered thin film, and microfluidic channels were assembled with an optical lens, water chamber, and an aluminum tube case. Finally, we obtained cross-sectional OCT images and en-face OCTA images of the retinal phantom using lab-made ophthalmic OCT. From the cross-sectional OCT image, we could compare each of the layer thicknesses of the phantom with the corresponding layer thicknesses of the human retina. In addition, we obtained en-face OCTA images with injections of intralipid solutions. It is shown that this phantom will be able to be potentially used as a convenient tool to evaluate and standardize the quality and accuracy of OCT and OCTA images.

primary ion beams did not. A PCA of the TOF-SIMSspectra was initially performed to identify characteristic secondary ions of streptavidin. Chemical images ofeach characteristic ion were reconstructed from the raw data and used in the second PCA run, which resultedin a contrasted – and corrected – image of the micropatterns of streptavidin by the Ga

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.

A newly fabricated plasma-polymerized poly(ethylene glycol) (PP-PEG) film shows extremely low toxicity, low fouling, good durability, and chemical similarity to typical PEG polymers, enabling live cell patterning as well as various bioapplications using bioincompatible materials. The PP-PEG film can be overlaid on any materials via the capacitively coupled plasma chemical vapor deposition (CCP-CVD) method using nontoxic PEG200 as a precursor. The biocompatibility of the PP-PEG-coated surface is confirmed by whole blood flow experiments where no thrombi and less serum protein adsorption are observed when compared with bare glass, polyethylene (PE), and polyethylene terephthalate (PET) surfaces. Furthermore, unlike bare PE films, less fibrosis and inflammation are observed when the PP-PEG-coated PE film is implanted into subcutaneous pockets of mice groin areas. The cell-repellent property of PP-PEG is also verified via patterning of mammalian cells, such as fibroblasts and hippocampal neurons. These results show that our PP-PEG film, generated by the CCP-CVD method, is a biocompatible material that can be considered for broad applications in biomedical and functional materials fields.

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.

Angiogenesis-related blindness indicates the spectrum of retinal diseases that are caused by pathological angiogenesis, resulting in catastrophic vision loss. We aimed to demonstrate the antiangiogenic effect of silicate nanoparticles (SiNPs) on the retinal neovascularization. No direct toxicity of SiNPs was observed on retinal neuronal or endothelial cells, nor on the retinal tissue. Furthermore, intravitreal injection of SiNPs effectively reduced anomalous retinal angiogenesis in oxygen-induced retinopathy mice. SiNPs also effectively inhibited in vitro vascular endothelial growth factor (VEGF)-induced angiogenesis. Via suppression of VEGF receptor-2 phosphorylation induced by VEGF, SiNPs blocked ERK 1/2 activation. SiNPs could be an inhibitor of the potency and safety of retinal neovascularization that is mediated by VEGF and utilized in the treatment of angiogenesis-related blindness. From the Clinical Editor In this important preclinical study, silicate NP-s are studied to address retinal neovascularization, an important pathomechanism of different retinal diseases that could lead to catastrophic vision loss. The authors conclude that SiNP-s could be utilized as inhibitors of retinal neovascularization mediated by VEGF and propose future applications in the treatment of angiogenesis-related blindness.

Polyethylene glycol (PEG) is a well-known non-fouling material used for protein adsorption and cellular adhesion. Recently, we successfully fabricated plasma-polymerized PEG (PP-PEG) thin films on various substrates by using a plasma enhanced chemical vapor deposition method and PEG200 as a precursor. The structure of our PP-PEG thin film was found to be very similar to the chemical structure of a PEG polymer and also bore a non-fouling characteristic for numerous proteins and cells without in vitro or in vivo toxicity. In this study, we modified this PP-PEG thin film with a simple H2/He plasma treatment to change the non-fouling surface property to a fouling one for proteins and cells. To make the patterned surfaces for proteins or cells on the protein- or cell-resistant areas, depositing a new fouling PP thin film was unnecessary since it was possible to perform a simple H2/He plasma treatment through a metal shadow mask on the PP-PEG thin film. Various surface analysis techniques such as water contact angle, atomic force microscopy, X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry were used to confirm the drastic changes in the surface chemical properties resulting from the plasma treatment. These results show that a PP-PEG film, together with its plasma treatment through a shadow mask, is a very simple and useful patterning technique for various biological applications. Copyright © 2012 John Wiley & Sons, Ltd.

Gold nanoparticles (AuNPs) and poly(ethylene glycol) (PEG) are known to be safe, biocompatible materials. In this study, AuNPs were conjugated with PEG ligands, and the suspensions of AuNPs with and without free PEG ligands were characterized using UV-visible spectroscopy, hydrodynamic size measurement, and time-of-flight SIMS (ToF-SIMS) imaging analysis. The two AuNPs solutions were each dropped onto a hydrophilic surface (bare Si wafer) and a hydrophobic surface (a plasma-polymerized cyclohexane-coated Si wafer), then dried to produce self-aggregated micropatterns, from which ToF-SIMS images were obtained. From these ToF-SIMS images, we calculated the Pearson product–moment correlation coefficient (PMCC) of the AuNPs and PEG intensities to quantitatively measure the coexistence of both materials in the solution. Good coexistence between AuNPs and PEG ligands was obtained only from the sample film prepared by dropping the suspension of PEG-conjugated AuNPs without free ligands onto a hydrophobic surface. The degrees of coexistence (i.e. PMCC value) seem to correlate with the amount of free PEG ligands in suspension to affect the stability of organic conjugated nanoparticles in solution. In addition, it was found that minimal interaction between the conjugated ligands and the substrate used for the sample film preparation led to an optimum PMCC statistical analysis. Our results lead us to believe that this new approach could simplify the method by which to quantitatively study the degrees of coexistence between nanoparticles and ligands, the amount of free ligands, and the stability of ligand-conjugated nanoparticles in suspension. Copyright © 2012 John Wiley & Sons, Ltd.

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.

Simultaneous measurement of growth morphology and electronic energy level alignment for pentacene layers on silicon oxide before and after the octadecyltrichlorosilane (OTS) self-assembled monolayer treatment has been carried out by photoemission electron microscopy (PEEM) and ultraviolet photoelectron spectroscopy (UPS). On the bare silicon oxide, the change of the highest occupied molecular orbital (HOMO) position with the pentacene thickness is marginal because of relatively strong interaction between the substrate and pentacene from the beginning. On the OTS-covered surface, due to the weak interaction between the substrate and pentacene, the diffusion of pentacene is enhanced and domain size gradually grows, leading to more or less standing-up orientation of the pentacene molecules. This comparison explains well the substrate-dependent growth morphology and interfacial dipole change of pentacene thin films.

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 β-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.

The pathological angiogenesis in the retina is the major cause of vision loss at all ages. In particular, retinopathy of prematurity (ROP) is a leading cause of blindness in children. This study investigated whether gold nanoparticle (GNP) could inhibit retinal neovascularization in the animal model of ROP. Intravitreal injection of GNP significantly inhibited retinal neovascularization in the mouse model of ROP. In addition, GNP effectively suppressed VEGF-induced in vitro angiogenesis of retinal microvascular endothelial cells including proliferation, migration and capillary-like networks formation. GNP blocked VEGF-induced auto-phosphorylation of VEGFR-2 to inhibit consequently ERK 1/2 activation. GNP never affected on the cellular viability of retinal microvascular endothelial cells and induced no retinal toxicity. Our data suggest that GNP could be a potent inhibitor to retinal neovascularization without retinal toxicity. Furthermore, GNP could be extensively applied to variable vaso-proliferative retinopathies mediated by VEGF.

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.

Poly(ethylene glycol) (PEG) has been widely used in biomedical applications because of its protein-resistant and nontoxic properties. In this study, a plasma-polymerized ethylene glycol (PPEG) thin film was deposited on a substrate using a capacitively coupled plasma chemical vapour deposition (CCP-CVD) method with various plasma powers and ethylene glycol as a precursor. The surfaces of the PPEG thin films were characterized using time-of-flight secondary ion mass spectrometry (ToF-SIMS) along with a principal component analysis (PCA), and XPS. We confirmed that the PPEG thin film deposited at low plasma power closely resembles PEG polymers in terms of surface chemical composition and protein-resistant property. To make patterned surfaces with protein-resistant property for proteins, a PPEG thin film was first uniformly deposited on a glass slide and patterns were produced on the PPEG coated surface by depositing a plasma-polymerized amine film through a metal shadow mask. When we spotted proteins on the patterned surface, they were immobilized only onto the plasma-polymerized amine areas but not onto the PPEG areas. These results show that PPEG surfaces would be useful for the construction of various protein arrays.

Heart tissues from a normal rat, myocardial infarcted rat, and mesenchymal stem cell (MSC)-treated rat after myocardial infarction (MI) were studied using ToF-SIMS, with the assistance of principal component analysis (PCA). To our knowledge, this is the first such published study. In PCA results obtained from both positive and negative ion spectra of normal (control), MI, and MSC-treated MI tissues, the normal and MSC-treated tissues could be clearly distinguished from the MI tissue in terms of chemical composition. For the MI tissue, fatty acid signals from inside the cell and phosphocholine signals from the cell membrane decreased while glucose signals increased. In the MSC-treated MI tissue, the signal levels of phosphocholine and fatty acids returned to those for normal tissue. Our observations for myocardium tissues are consistent with previous biological studies, which show that the loss of reversibility of cell injury associated with MI could cause degradation to the cell membrane, and that MI could slow fatty acid uptake and oxidation and increase glucose uptake and utilization for ATP synthesis in myocardium. This is, of course, because fatty acids need oxygen for β-oxidation. We anticipate that this label-free ToF-SIMS method partnered with PCA will be helpful to the investigation of myocardial disease and MSC therapy since it is based on chemical information at the tissue level.

Mixed self-assembled monolayers (SAMs) consisting of different molecules have been used to modify surface wettability, functionalize end groups for chemical binding, and control adsorption of proteins, DNA, or cells. In these applications, control over the quantification of the surface composition of a mixed SAM is especially important when designing biochips and biosensors. Previously, we presented a method by which ToF-SIMS can be used to quantify the composition of two adsorbates with acetylene and propene functional groups from their secondary ion yield ratio in mixed SAMs on gold substrates. We concluded that although the ion yield ratio of the two adsorbates in mixed SAM was not equal to the mole fraction of Diyne (-acetylene) and Diene (-propene) in a solution, this was due to a different secondary ion formation, and not to the difference in the amount of adsorbates on the surface. In this study, we revisit the same sample system, this time applying a FT-IR technique to test the accuracy of our previous findings. We found that the absorbances of the acetylene and propene functional groups neatly and linearly correlated with the mole fraction in the solution, which were consistent with our previous conclusions. We also used this methodology to quantify mixed silane SAMs on a silica surface. Our study shows that the ToF-SIMS and FT-IR techniques can complement one another and be useful to quantify mixed SAMs on various substrates.

Oxidation-induced stress evolutions in Ta thin films were investigated using ex situ microstructure analyses and in situ wafer curvature measurements. It was revealed that Ta thin films are oxidized to a crystalline TaO2 layer, which is subsequently oxidized to an amorphous tantalum pentoxide (a-Ta2O5) layer. Initial layered oxidation from Ta to TaO2 phases abruptly induces high compressive stress up to about 3.5 GPa with fast diffusion of oxygen through the Ta layer. Subsequently, it is followed by stress relaxation with the oxidation time, which is related to the slow oxidation from TaO2 to Ta2O5 phases. The initial compressive stress originates from the molar volume expansion during the layered formation of TaO2 from the Ta layer, while the relaxation of the compressive stresses is ascribed to the amorphous character of the a-Ta2O5 layer. According to Kissinger's analysis of the stress evolution during an isochronic heating process, the oxygen diffusion process through the a-Ta2O5 layer is the rate-controlling stage in the layered oxidation process of forming a a-Ta2O5/TaO2/Ta multilayer and has an activation energy of about 190.8 kJ/mol.

Gold nanoparticles (AuNPs) and poly(ethylene glycol) (PEG) are known safe materials to be used in vivo, and so, are becoming increasingly important in biomedical applications. Here, the stability of PEG-conjugated AuNPs in an aqueous solution was monitored as a function of time using several techniques, namely dynamic light scattering (DLS)-size analysis, UV-visible absorption spectrometry and ToF-SIMS. Roughly 15-nm-sized AuNPs were synthesized and pegylated with mPEG (MW = 5 kDa). Four different samples were synthesized, depending on whether or not a dialysis procedure was performed right after synthesis of the AuNPs or pegylation of the AuNPs. The DLS results show that size of mPEG-conjugated AuNPs and standard deviation increased in cases where a dialysis procedure was performed after pegylation of the AuNPs, indicating aggregation of the AuNPs due to the detachment of mPEG ligands from the surface of the AuNPs. Also, results from UV-visible absorption spectra and ToF-SIMS images point to the likelihood that free mPEG ligands in solution help prevent aggregation by maintaining a dynamic equilibrium between the conjugated ligands on the surface of the AuNPs and the free ligands in the aqueous solution. Conversely, once free, mPEG ligands were eliminated from the solution by dialysis and conjugated mPEG ligands detached from the surface of AuNPs into the solution, suspension stability lowered and the AuNPs showed signs of aggregation. We believe that the ToF-SIMS imaging analysis with DLS and UV-visible absorption analyses would be useful to study the stability of PEG-conjugated AuNPs in different environmental conditions.

Complementary use of medium-energy ion-scattering spectroscopy (MEIS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analyses was proposed as a new methodology for absolute quantification of functional groups in organic ultrathin films. First, MEIS analysis was used to determine the absolute areal densities of Ru dye molecules in organic ultrathin films, which were made by spin coating Ru 535-bis TBA (C 58 H 86 O 8 N 8 S 2 Ru) solutions with varying concentrations. The thicknesses of the films were less than 2 nm each and the densities of these ultrathin layers were linearly correlated with the solution concentrations. The determined absolute areal densities of the Ru organic molecules were then used to calculate the areal densities of the stoichiometric functional groups. Secondly, the ToF-SIMS intensities of the characteristic peaks such as Ru + , C 16 H 35 N + , CO 2 - , CSN - , CSNRu - , etc, associated with the functional groups of a Ru 535-bis TBA molecule were correlated by the densities of the Ru organic molecules. Our results show that the correlation curves obtained through the complementary use of MEIS and ToF-SIMS techniques could be useful to quantify functional groups on organic ultrathin films.

Reagents Abl tyrosine kinase and cAMP-dependent protein kinase (PKA) were purchased from New England Biolab. Monoclonal antiphosphotyrosine antibody (anti-pY, mouse IgG2b isotype from PY20) and mouse immunoglobulin G (mIgG) were from Sigma. Two kinase inhibitors, stautosporin for Abl and 4-cyano-3-methylisoquinoline (CMIQ) for PKA, were purchased from Sigma and Calbiochem, respectively. Amine-functionalized glass (Nexterion Slide A+) was obtained from Schott (Germany), and poly (ethylene glycol)-succinimidyl α-methylbutanoate (mPEG-SMB, MW 2000) was purchased from Nektar Inc (USA). Three kinds of peptide substrates used in this study were synthesized from Peptron Inc. (Korea): Ac-IYAAPKKC, AcIYAAPKK(G)4C and Ac-LRRASL(G)4C. Hydrogenetrachloroaurate(III) trihydrate (99.9% HAuCl4⋅3H2O), sodium citrate dihydrate (99.9%, 2-hydroxy-1,2,3-propanetricarboxylic acid trisodium salt, C6H5Na3O7⋅2H2O) and sodium borohydride (99%, NaBH4) were purchased from Sigma-Aldrich. A chambered silicon coverslip (50 wells, 3mm × 1mm, sterile) was also from Sigma-Aldrich.

A quantitative analysis of the surface density of amine groups on a plasma-polymerized ethylenediamine thin film deposited on a platinum surface using inductively coupled plasma chemical vapor deposition method is described. UV–visible spectroscopy together with a chemical derivatization technique using Fourier transform infrared (FT-IR) spectroscopy was used to obtain the quantitative information. Chemical tags of pentafluorobenzaldehyde were hybridized with the surface amine groups and were easily detected due to the characteristic absorption bands of C–F stretching, aromatic ring and C N stretching vibrations in the reflection–absorption FT-IR spectra. The surface amine density was reproducibly controlled as a function of deposition plasma power and quantified using UV–visible spectroscopy. A good linear correlation was observed between the FT-IR intensities of the characteristic absorption bands and the surface amine densities, suggesting the possibility of using this chemical derivatization technique to quantify the surface densities of specific functional groups on an organic surface. Chemical derivatization was also used with X-ray photoelectron spectroscopy on the same samples, and the results were compared with those obtained from FT-IR and time-of-flight secondary ion mass spectrometry. Although each analysis technique has different probing depths from the surface, the three different data sets obtained from the chemical tags correlated well with each other since each analysis technique measured the chemical tags on the sample surface.

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.

A chemical derivatization technique in ToF-SIMS along with principal component analysis (PCA) were used to perform a quantitative study of the surface amine density of the plasma-polymerized ethylenediamine (PPEDA) thin film. We used the scores on principal component (PC) 1 from a PCA of ToF-SIMS data for the PPEDA films and their chemical-derivatized surfaces for comparison with the surface amine densities. These surface amine densities were independently determined by UV–visible spectroscopy. Our work found a good linear relationship between the surface amine densities and the scores on PC 1 from a PCA of the ToF-SIMS data for the chemical-derivatized PPEDA surfaces, but not for the PPEDA thin films themselves. In addition to quantification, our PCA results provided insights into the surface chemical composition of each surface.

ToF-SIMS analysis with principal component analysis (PCA) has been used for quantitatively studying the interaction between streptavidin and biotin on a dendrimer surface. A poly(amidoamine) dendrimer surface was used as a model amine surface for biotinylation. The surface streptavidin density was systematically varied and independently quantified using the surface plasmon resonance (SPR) technique. A good linear correlation of streptavidin density was observed between the ToF-SIMS and SPR results.

A bismuth cluster ion-beam-based time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been successfully used to image micropatterns of streptavidin and Chinese hamster ovary (CHO-k1) cells, as received and without any labeling. Three different analysis ion beams (Bi+, Bi3+, and Bi32+) were compared to obtain label-free TOF-SIMS chemical images of micropatterns of streptavidin, which were subsequently used for generating biotinylated cell patterns. Unlike using a Bi+ ion beam, using a Bi3+ or Bi32+ primary analysis ion beam yielded well-contrasted-TOF-SIMS images of streptavidin characteristic secondary ions. A principal component analysis of TOF-SIMS data was performed to generate a chemical image of the streptavidin itself. A chemical specific TOF-SIMS image analysis gave us a better understanding of the localization of cells at the outer boundaries of the streptavidin-patterned circles. Our work suggests that using cluster-ion analysis beams together with multivariate data analysis for TOF-SIMS...

A chemical derivatization technique in time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been developed to quantify the surface density of amine groups of plasma-polymerized ethylenediamine thin film deposited on a glass surface by inductively coupled plasma chemical vapor deposition. Chemical tags of 4-nitrobenzaldehyde or pentafluorobenzaldehyde were hybridized with the surface amine groups and were detected in TOF-SIMS spectra as characteristic molecular secondary ions. The surface amine density was controlled in a reproducible manner as a function of deposition plasma power and was also quantified using UV-visible spectroscopy. A good linear correlation was observed between the results of TOF-SIMS and UV-visible measurements as a function of plasma power. This shows that the chemical derivatization technique in TOF-SIMS analysis would be useful in quantifying the surface density of specific functional groups that exist on the organic surface.

We developed a label-free and positive-readout surface enhanced Raman scattering (SERS) assay using reverse-hairpin molecular beacons (RHBs) for the detection of RNA genetic markers associated with a high pathogenicity influenza (HPAI) virus. The structure of RHBs flexibly changed from a linear configuration (open state) to hairpin (closed state) upon targeting, such that the Raman label was closed on the SERS substrate and induced an increase of SERS intensity (OFF-to-ON). By improving sequence-specific RNA/DNA hybridization efficiency, we adjusted the stem-loop ratio of RHB, which was efficient at values of less than 1. The optimized RHBs exhibited dramatic changes in signal based on a fluorescence system in which the target was present. We demonstrated that the OFF-to-ON SERS system using RHB immobilized on silver-coated gold nanobowls permitted rapid hybridization. This proof-of-concept could provide a potential diagnostic tool for point-of-care influenza virus detection.

Interaction between streptavidin and biotin on poly(amidoamine) (PAMAM) dendrimer-activated surfaces and on self-assembled monolayers (SAMs) was quantitatively studied by using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface protein density was systematically varied as a function of protein concentration and independently quantified using the ellipsometry technique. Principal component analysis (PCA) and principal component regression (PCR) were used to identify a correlation between the intensities of the secondary ion peaks and the surface protein densities. From the ToF-SIMS and ellipsometry results, a good linear correlation of protein density was found. Our study shows that surface protein densities are higher on dendrimer-activated surfaces than on SAMs surfaces due to the spherical property of the dendrimer, and that these surface protein densities can be easily quantified with high sensitivity in a label-free manner by ToF-SIMS.

The spacing of chemical functional groups on self-assembled monolayers (SAMs) plays an important role in controlling the density of biomolecules in biochips and biosensors. In this sense, a mixed SAM made of two different terminal groups is a useful organic surface since spacing can be easily controlled by changing a relative mole fraction in a mixture solution. In this study, an acetylene–OCH2O(EG)3(CH2)11S–S(CH2)11(EG)3OCH2O–propene (Eneyne) SAM and mixed SAMs made by a mixture of (S(CH2)11(EG)3OCH2O–acetylene)2 (Diyne) and (S(CH2)11(EG)3OCH2O–propene)2 (Diene) were produced on gold substrates and measured by using ToF-SIMS. The secondary ion yield ratio of [Au·S(CH2)11(EG)3OCH2O–acetylene]− to [Au·S(CH2)11(EG)3OCH2O–propene]− was measured for each mixed SAM and plotted as a function of the mole fraction of Diyne to Diene in a SAM solution. The ion yield ratio of a mixed SAM produced from a solution with a mole fraction of 0.5 (i.e., 1:1 mixture) was 0.3, which corresponded well to the ion yield ratio measured from an Eneyne SAM. A time-dependent experiment of Eneyne SAM formation and immersion experiment of Eneyne SAM into Diyne solution or into Diene solution were performed. The relative ion yield ratio of 0.3 was due to a different secondary ion formation and not due to the difference in the amount of adsorbates on the surface, nor to the different binding strengths onto the gold surface. Our study shows that a mixed SAM with well-controlled spacing can be produced and quantified by using the ToF-SIMS technique.