Development of Novel Dengue NS1 Multiplex Lateral Flow Immunoassay to Differentiate Serotypes in Serum of Acute Phase Patients and Infected Mosquitoes.

Dengue is among the most rapidly spreading arboviral disease in the world. A low-cost, easy to use point-of-care diagnostic tool for the detection and differentiation of dengue virus serotypes could improve clinical management, disease prevention, epidemiological surveillance, and outbreak monitoring, particularly in regions where multiple serotypes co-circulate. Despite widespread deployment, no commercial dengue antigen diagnostic test has proven effective in differentiating among dengue virus serotypes. In the current study, we first established mAb pairs and developed a multiplex lateral flow immunoassay for the simultaneous detection of the dengue viral NS1 antigen and identification of serotype. The proposed system, called Dengue serotype NS1 Multiplex LFIA, provides high sensitivity and specificity. In testing for JEV, ZIKV, YFV, WNV, and CHIKV, the multiplex LFIA gave no indication of cross- reactivity with cell culture supernatants of other flaviviruses or chikungunya virus. In analyzing 187 samples from patients suspected of dengue infection, the detection sensitivity for serotype D1 to D4 was 90.0%, 88.24%, 82.61%, and 83.33% and serotype specificity was 98.74%, 96.13%, 99.39%, and 97.04%, respectively. Our multiplex LFIA can also identify mono- and co-infection of different serotype of dengue viruses in mosquitoes. The proposed Multiplex LFIA provides a simple tool for the rapid detection of dengue serotypes and in the differential diagnosis of fever patients in regions where medical resources are limited and/or multiple DENVs co-circulate.

Nanoparticles assembled from fucoidan and trimethylchitosan as anthrax vaccine adjuvant: In vitro and in vivo efficacy in comparison to CpG.

Fucoidan/trimethylchitosan nanoparticles (FUC-TMC-NPs) have the potential to improve the immunostimulating efficiency of anthrax vaccine adsorbed (AVA). FUC-TMC-NPs with positive (+) or negative (-) surface charges were prepared via polyelectrolyte complexation, both charged NP types permitted high viability and presented no cytotoxicity on L929, A549 and JAWS II dendritic cells. Flow cytometry measurements indicated lower (+)-FUC-TMC-NPs internalization levels than (-)-FUC-TMC-NPs, yet produced high levels of pro-inflammatory cytokines IFN-γ, IL12p40, and IL-4. Moreover, fluorescence microscope images proved that both charged NP could deliver drugs into the nucleus. In vivo studies on A/J mice showed that (+)-FUC-TMC-NPs carrying AVA triggered an efficient response with a higher IgG anti-PA antibody titer than AVA with CpG oligodeoxynucleotides, and yielded 100 % protection when challenged with the anthracis spores. Furthermore, PA-specific IgG1 and IgG2a analysis confirmed that (+)-FUC-TMC-NPs strongly stimulated humoral immunity. In conclusion, (+)-FUC-TMC-NP is promising anthrax vaccine adjuvant as an alternative to CpG.

A fucoidan-quaternary chitosan nanoparticle adjuvant for anthrax vaccine as an alternative to CpG oligodeoxynucleotides.

We examined the efficacy of fucoidan-N-(2-hydroxy-3-trimethylammonium)propylchitosan nanoparticles (FUC-HTCC NPs) as adjuvants for anthrax vaccine adsorbed (AVA). Positively and negatively surface-charged FUC-HTCC NPs were prepared via polyelectrolyte complexation by varying the mass ratio of FUC and HTCC. When cultured with L929 cells or JAWS II dendritic cells, both charged NPs showed high cell viability and low cytotoxicity, observed via MTT assay and lactate dehydrogenase release assay, respectively. In addition, we have monitored excellent NPs uptake efficacy by dendritic cells and observed that combining FUC-HTCC NPs with AVA significantly increases the magnitude of IgG-anti-protective antigen titers in A/J mice compared to that by CpG oligodeoxynucleotides plus AVA or AVA alone, and PA-specific IgG1 and IgG2a analysis confirmed that FUC-HTCC NPs strongly stimulated humoral immunity. Furthermore, FUC-HTCC NPs plus AVA provided a superior survival rate (100%) of A/J mice compared to CpG oligodeoxynucleotides plus AVA (75%) or AVA alone (50%) following anthrax lethal toxin challenge. The findings support FUC-HTCC NPs as a potential adjuvant of AVA for rapid induction of protective immunity.

Development of an Enzyme-Linked Immunosorbent Assay for Rapid Detection of Dengue Virus (DENV) NS1 and Differentiation of DENV Serotypes during Early Infection.

Dengue fever, caused by infections with the dengue virus (DENV), affects nearly 400 million people globally every year. Early diagnosis and management can reduce the morbidity and mortality rates of severe forms of dengue disease as well as decrease the risk of wider outbreaks. Although the early diagnosis of dengue can be achieved using a number of commercial NS1 detection kits, none of these can differentiate among the four dengue virus serotypes. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for the detection of dengue virus (DENV) NS1 by pairing a serotype-cross-reactive monoclonal antibody (MAb) with one of four serotype-specific MAbs in order to facilitate the rapid detection of NS1 antigens and the simultaneous differentiation of DENV serotypes. A total of 146 serum samples obtained from patients suspected to be in the acute phase of DENV infection were used to evaluate the clinical application of our novel test for the detection and serotyping of DENV. The overall sensitivity rate of our test was 84.85%, and the sensitivity rates for serotyping were as follows: 88.2% (15/17) for DENV serotype 1 (DENV1), 94.7% (18/19) for DENV2, 75% (12/16) for DENV3, and 66.6% (6/9) for DENV4. Moreover, there was no cross-reactivity among serotypes, and no cross-reactivity was observed in sera from nondengue patients. Thus, our test not only enables the rapid detection of the dengue virus but also can distinguish among the specific serotypes during the early stages of infection. These results indicate that our ELISA for DENV NS1 is a convenient tool that may help elucidate the epidemiology of DENV outbreaks and facilitate the clinical management of DENV infections.

Effects of the temperature and beam parameters on depth profiles in X-ray photoelectron spectrometry and secondary ion mass spectrometry under C60(+)-Ar(+) cosputtering.

Polymethylmethacrylate (PMMA) is widely used in various fields, including the semiconductor, biomaterial and microelectronic fields. Obtaining the correct depth profiles of PMMA is essential, especially when it is used as a thin-film. There have been many studies that have used earlier generation of cluster ion (SF5(+)) as the sputtering source to profile PMMA films, but few reports have discussed the use of the more recently developed C60(+) in the PMMA sputtering process. In this study, X-ray photoelectron spectroscopy (XPS) and dynamic secondary ion mass spectroscopy (D-SIMS) were used concurrently to monitor the depth profiles of PMMA under C60(+) bombardment. Additionally, the cosputtering technique (C60(+) sputtering with auxiliary, low-kinetic-energy Ar(+)) was introduced to improve the analytical results. The proper cosputtering conditions could eliminate the signal enhancement near the interface that occurred with C60(+) sputtering and enhance the sputtering yield of the characteristic signals. Atomic force microscopy (AFM) was also used to measure the ion-induced topography. Furthermore, the effect of the specimen temperature on the PMMA depth profile was also examined. At higher temperatures (+120°C), the depolymerization reaction that corresponded to main-chain scission dominated the sputtering process. At lower temperatures (-120°C), the cross-linking mechanism was retarded significantly due to the immobilization of free radicals. Both the higher and lower sample temperatures were found to further improve the resulting depth profiles.

Electron tomography of HEK293T cells using scanning electron microscope-based scanning transmission electron microscopy.

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-µm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

Effect of cosputtering and sample rotation on improving C60(+) depth profiling of materials.

In the past decade, buckminsterfullerene (C(60))-based ion beams have been utilized in surface analysis instruments to expand their application to profiling organic materials. Although it had excellent performance for many organic and biological materials, its drawbacks, including carbon deposition, carbon penetration, continuous decay of the sputtering rate, and a rough sputtered surface, hindered its application. Cosputtering with C(60)(+) and auxiliary Ar(+) simultaneously and sample rotation during sputtering were proposed as methods to reduce the above-mentioned phenomena. However, the improvement from these methods has not been compared or studied under identical conditions; thus, the pros and cons of these methods are not yet known experimentally. In this work, a series of specimens including bulk materials and thin films were used to explore the differences between cosputtering and sample rotation on the analytical results. The results show that both of these methods can alleviate the problems associated with C(60)(+) sputtering, but each method showed better improvement in different situations. The cosputtering technique better suppressed carbon deposition, and could be used to generally improve results, especially with continuous spectra acquisition during sputtering (e.g., dynamic secondary ion mass spectrometry (SIMS) depth profiling). In contrast, for the scheme of sputter-then-acquire (e.g., alternative X-ray photoelectron spectrometry or dual-beam static SIMS depth profiling), a better result was achieved by sample rotation because it resulted in a flatter sputtered surface. Therefore, depending on the analytical scheme, a different method should be used to optimize the experimental conditions.

Dramatically enhanced oxygen uptake and ionization yield of positive secondary ions with C60+ sputtering.

To explore C(60)(+) sputtering beyond low-damage depth profiling of organic materials, X-ray photoelectron spectrometry (XPS) and secondary ion mass spectrometry (SIMS) were used to examine metallic surfaces during and after C(60)(+) sputtering. During C(60)(+) sputtering, XPS spectra indicated that the degrees of carbon deposition were different for different metallic surfaces. Moreover, for some metals (e.g., Al, W, Ta, Ti, and Mo), the intensity of the O 1s photoelectron increased significantly during C(60)(+) sputtering, even though the instrument was under ultrahigh vacuum (<5 × 10(-7) Pa). This result indicated that the rate of oxygen uptake was greater than the rate of C(60)(+) sputtering. This behavior was not observed with the commonly used Ar(+) sputtering. To measure the oxygen uptake kinetics, pure oxygen was leaked into the chamber to maintain a 5 × 10(-6) Pa oxygen environment. The C(60)(+)-sputtered surface had a clearly increased rate of oxygen uptake than the Ar(+)-sputtered surface, even for moderately reactive metals such as Fe and Ni. For relatively nonreactive metals such as Cu and Au, a small amount of carbon was implanted and no oxygen uptake was observed. High-resolution XPS spectra revealed the formation of metal carbides on these reactive metals, and the carbon deposition and enhanced uptake of oxygen correlated to the carbide formation. Because oxygen enhances the secondary ion yield through surface passivation, the enhanced oxygen uptake due to C(60)(+) sputtering could be beneficial for SIMS analysis. To examine this hypothesis, C(60)(+) and Ar(+) were used as primary ions, and it was found that the intensity enhancement (because of the oxygen flooding at 5 × 10(-6) Pa) was much higher with C(60)(+) than with Ar(+). Therefore, oxygen flooding during C(60)(+) sputtering has a great potential for enhancing the detection limit due to the enhanced oxygen uptake.

Parallel detection, quantification, and depth profiling of peptides with dynamic-secondary ion mass spectrometry (D-SIMS) ionized by C60(+)-Ar(+) co-sputtering.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) using pulsed C(60)(+) primary ions is a promising technique for analyzing biological specimens with high surface sensitivities. With molecular secondary ions of high masses, multiple molecules can be identified simultaneously without prior separation or isotope labeling. Previous reports using the C(60)(+) primary ion have been based on static-SIMS, which makes depth profiling complicated. Therefore, a dynamic-SIMS technique is reported here. Mixed peptides in the cryoprotectant trehalose were used as a model for evaluating the parameters that lead to the parallel detection and quantification of biomaterials. Trehalose was mixed separately with different concentrations of peptides. The peptide secondary ion intensities (normalized with respect to those of trehalose) were directly proportional to their concentration in the matrix (0.01-2.5 mol%). Quantification curves for each peptide were generated by plotting the percentage of peptides in trehalose versus the normalized SIMS intensities. Using these curves, the parallel detection, identification, and quantification of multiple peptides was achieved. Low energy Ar(+) was used to co-sputter and ionize the peptide-doped trehalose sample to suppress the carbon deposition associated with C(60)(+) bombardment, which suppressed the ion intensities during the depth profiling. This co-sputtering technique yielded steadier molecular ion intensities than when using a single C(60)(+) beam. In other words, co-sputtering is suitable for the depth profiling of thick specimens. In addition, the smoother surface generated by co-sputtering yielded greater depth resolution than C(60)(+) sputtering. Furthermore, because C(60)(+) is responsible for generating the molecular ions, the dosage of the auxiliary Ar(+) does not significantly affect the quantification curves.

Effect of the chemical composition on the work function of gold substrates modified by binary self-assembled monolayers.

This study demonstrated that the work function (Φ) of Au substrates can be fine-tuned by using series ratios of binary self-assembled monolayers (SAMs). By using pure amine- and carboxylic acid-bearing alkanethiol SAM on gold substrates, Φ of Au changed from 5.10 to 5.16 and 5.83, respectively, as determined by ultra-violet photoelectron spectrometry (UPS). The shift in Φ due to the use of different functional groups was rationalized by considering the dipole moments of the molecules anchored on the Au surface. A series of binary SAMs were fabricated by mixing carboxylic acid- and amine-terminated alkanethiols in the deposition solution. By mixing these functional groups in SAMs, a linear correlation between Φ with respect to chemical composition (hence the effective dipole moment on the Au surface) was observed. It was found that arbitrary Φ between extremes (5.16 and 5.83) controlled by respective functional groups can be obtained by changing the chemical composition of SAMs. The Scanning Kelvin Probe (SKP) was also used to measure the contact potential difference (CPD) between SAMs and referencing Au on a patterned substrate prepared by photo-lithography. It was found that the CPD of SAMs with different chemical compositions correlates to their Φ. However, the magnitude of the CPD was smaller than the difference in Φ measured by UPS that was possibly due to the adsorption of contaminants in air.