Wafer-Scale LSPR Substrate: Oblique Deposition of Gold on a Patterned Sapphire Substrate.

Label-free detection of biomolecules using localized surface plasmon resonance (LSPR) substrates is a highly attractive method for point-of-care (POC) testing. One of the remaining challenges to developing LSPR-based POC devices is to fabricate the LSPR substrates with large-scale, reproducible, and high-throughput. Herein, a fabrication strategy for wafer-scale LSPR substrates is demonstrated using reproducible, high-throughput techniques, such as nanoimprint lithography, wet-etching, and thin film deposition. A transparent sapphire wafer, on which SiO2-nanodot hard masks were formed via nanoimprint lithography, was anisotropically etched by a mixed solution of H2SO4 and H3PO4, resulting in a patterned sapphire substrate (PSS). An LSPR substrate was finally fabricated by oblique deposition of Au onto the PSS, which was then applied to label-free detection of the binding events of biomolecules. To the best of our knowledge, this paper is the first report on the application of the PSS used as an LSPR template by obliquely depositing a metal.

Establishment of a developmental neurotoxicity test by Sox1-GFP mouse embryonic stem cells.

Developmental toxicity tests have been generated by applying the embryonic stem cell tests at the European Centre for the Validation of Alternative Methods, or by using the embryoid body test in our laboratory. This study was undertaken to explore novel developmental neurotoxicity (DNT) assay, using a Sox1-GFP cell line (mouse embryonic stem cells with an endogenous Sox1-GFP reporter). The expression of Sox1, a marker for neuroepithelial cells, is detected by green fluorescence, and the fluorescence intensity is a critical factor for achieving neuronal differentiation. Sox1-GFP cells cultured for 24 h were exposed to eleven neurotoxicants and four non-neurotoxicants. CCK-8 assays were performed to determine IC50 values after 48 h of chemical treatment. The fluorescence intensity of GFP was measured 4 days after treating the cells, and it was observed to decrease after exposure to neurotoxicants at higher concentrations, thereby indicating that the neuronal differentiation of Sox1-GFP cells is inhibited by the chemicals. Taken together, the results obtained in this study provide a model for DNT using embryonic stem cells, which may be applied to evaluate the toxicity of new chemicals or new drug candidates.

Enhancing biosensing sensitivity of metal nanostructures through site-selective binding.

The localised surface plasmon resonance (LSPR) at the surface of metal nanostructures can induce a highly intense electromagnetic (EM) field, which is confined to the edges with big curvature or at narrow gaps between nanostructures. Therefore, the localisation of target molecules at these sites is crucial to achieve high sensitivity in LSPR-based biosensors. To this end, we fabricated a 40 nm high gold nano-truncated cone (GNTC) array using thermal nanoimprint lithography. As the EM field is most intense at the side surface and relatively weak at the top surface of GNTC, we improved the detection sensitivity by blocking the top surface with oxides to limit adsorption of antibodies and antigens to the top surface. We observed the difference in sensitivity by detecting α-fetoprotein (AFP) on the oxide-capped and uncapped GNTC arrays through sandwich immunoassay and enzymatic precipitation. The capped GNTC array exhibited higher detection sensitivity than the uncapped one. Particularly, six-fold enhancement of sensitivity was achieved in the serum sample. We used atomic force microscopy and electron microscopy to validate that the deposition of the oxides on the top surface of GNTC effectively blocked the adsorption of the biomolecules and the target molecules were preferentially adsorbed on the side surfaces.

Pre-validation study of alternative developmental toxicity test using mouse embryonic stem cell-derived embryoid bodies.

The embryoid body test (EBT) is a developmental toxicity test method that assesses the half inhibitory concentrations of substances in the area of embryoid bodies (EBs), and in the viability of mouse embryonic stem cells (ESCs) and fibroblasts (3T3 cells) following chemical exposure for three and four days, respectively. In the previous study, the EBT showed more advanced than the embryonic stem cell test (EST) from the European Centre for the Validation of Alternative Methods (ECVAM) applying cardiac differentiation of mouse ESCs, because the EBT greatly reduced the exposure time, labor, and amount of materials required, and misclassification of embryotoxic potential. This pre-validation study evaluated the predictive accuracy of the EBT using 26 coded test substances by two steps: intra-laboratory and inter-laboratory reproducibility tests. Since some substances have different embryotoxic potentials at different pregnancy periods, in this study, a new prediction model consisting of non-toxic and toxic classes was used, instead of the existing prediction model assessing embryotoxicants in four classes. The results of the intra- and inter-laboratory tests were highly accurate (above 80%) when substances were classified using the predictive model. In conclusion, EBT can accurately classify various embryotoxicants in a short time with less effort and greater validation.

Advanced developmental toxicity test method based on embryoid body's area.

Embryonic stem cell test (EST) evaluates the embryotoxic potential of substances and measures the half inhibition in viability of mouse embryonic stem cells (ESCs), fibroblasts (3T3 cells) and in cardiac differentiation of ESC. In this study, we suggest the developmental toxicity test method (termed EBT) applying area of embryoid bodies (EBs) instead of cardiac differentiation of EST. In the assessment of 21 substances, EB area was logarithmically decreased in dose-dependent manner. Decline in EB area resulted in decrease of beating ratio during differentiation of ESCs. In classification by the EBT-based prediction model reflecting decline in cell viability and EB area, toxicity for 21 chemicals showed 90.5% accuracy. In the results of next generation sequencing, reduction in EB area resulted from cell cycle arrest mediated by HDAC2 and CDKN2A. Conclusively, EBT is advanced and is a useful tool to assess and classify various embryotoxicants in a short time with less effort.

Enzyme-coupled nanoplasmonic biosensing of cancer markers in human serum.

As the use of biosensors for early in-vitro diagnosis of malignant diseases has expanded, issues associated with ultra-sensitivity and wide dynamic range have become paramount. In this study, we designed a sub-zeptomolar sensor for detecting the alpha-feto protein (AFP) that utilizes localized surface plasmon resonance (LSPR) assisted by bio-catalytic reaction and a self-controlled detection scheme. A gold nanodot array (GNA), serving as a plasmonic material, was fabricated using an improved UV nanoimprint lithography (NIL) method that employs a sacrificial layer. In the new approach, LSPR observation is highly stable because it employs a back reflection mode, which avoids passing the signal through the sample solution. An enzyme-precipitation reaction was conducted on the AFP antigen-antibody complex on the surface of the gold nanodot (nano-ELISA) using a procedure that was described previously. To extend the AFP detection limit below femtomolar concentrations, a scheme involving self-controlled detection was employed to lower the limit. In this method, the sample including the target simultaneously plays the role of both sample and negative control. Using this scheme, AFP can be detected at concentrations as low as 14 aM (0.7 zeptomole in 50µL serum) and with the wide dynamic range of 10fgmL(-1)-10ngmL(-1). Importantly, the new method provides a platform for studying and monitoring interactions between biochemically active substances that are present in very low concentrations. Finally, the general strategy used to design the detection method can be easily adapted to the ultra-sensitive detection of other biomarkers and pathogens.