On-site detection of methicillin-resistant Staphylococcus aureus (MRSA) utilizing G-quadruplex based isothermal exponential amplification reaction (GQ-EXPAR).

Methicillin-resistant Staphylococcus aureus (MRSA) has a high incidence in infectious hospitals and communities, highlighting the need for early on-site detection due to its resistance to methicillin antibiotics. The present study introduces a highly sensitive detection system for mecA, a crucial methicillin marker, utilizing an RCA-based isothermal exponential amplification reaction. The G-quadruplex-based isothermal exponential amplification reaction (GQ-EXPAR) method designs probes to establish G-quadruplex secondary structures incorporating thioflavin T for fluorescence. The system, unlike conventional genetic detection methods, works with portable isothermal PCR devices (isoQuark), facilitating on-site detection. A detection limit of 0.1 fmol was demonstrated using synthetic DNA, and effective detection was proven using thermal lysis. The study also validated the detection of targets swabbed from surfaces within bacterial 3D nanostructures using the GQ-EXPAR method. After applying complementary sequences to the padlock probe for the target, the GQ-EXPAR method can be used on various targets. The developed method could facilitate rapid and accurate diagnostics within MRSA strains.

Determinants of wearer satisfaction factors for harnesses in upper-limb assistive wearable robots.

Wearable robots are increasingly being deployed for use in industrial fields. However, only a few studies have focused on the usability of wearable robots. The present study evaluated the factors affecting the usability of a harness in securing a wearable robot to the body because the harness directly affects the work efficiency, and thus its design and use require careful consideration. A comparative evaluation of the arrangement of the Vest Exoskeleton before and after improvements was conducted, in which participants performed a benchmark assembly task while wearing the robot. Results showed that wearability decreased after the improvements due to the additional straps and buckles used, but the overall wearing satisfaction improved as a result of increased stability. Stability and convenience were the main factors affecting the overall wearing satisfaction, while sub-indicators included wearing comfort and tactile sensation. Therefore, improvements in stability, such as those related to fixation strength and tactile sensation, had a direct positive impact on the overall wearing satisfaction.

Correction: Investigation of craquelure patterns in oil paintings using precise 3D morphological analysis for art authentication.

[This corrects the article DOI: 10.1371/journal.pone.0272078.].

Plasmonic Nanopillars-A Brief Investigation of Fabrication Techniques and Biological Applications.

Nanopillars (NPs) are submicron-sized pillars composed of dielectrics, semiconductors, or metals. They have been employed to develop advanced optical components such as solar cells, light-emitting diodes, and biophotonic devices. To integrate localized surface plasmon resonance (LSPR) with NPs, plasmonic NPs consisting of dielectric nanoscale pillars with metal capping have been developed and used for plasmonic optical sensing and imaging applications. In this study, we studied plasmonic NPs in terms of their fabrication techniques and applications in biophotonics. We briefly described three methods for fabricating NPs, namely etching, nanoimprinting, and growing NPs on a substrate. Furthermore, we explored the role of metal capping in plasmonic enhancement. Then, we presented the biophotonic applications of high-sensitivity LSPR sensors, enhanced Raman spectroscopy, and high-resolution plasmonic optical imaging. After exploring plasmonic NPs, we determined that they had sufficient potential for advanced biophotonic instruments and biomedical applications.

Quantitative comparison of EGFR expression levels of optically trapped individual cells using a capacitance biosensor.

Cellular endocytosis is an essential phenomenon which induces cellular reactions, such as waste removal, nutrient absorption, and drug delivery, in the process of cell growth, division, and proliferation. To observe capacitance responses upon endocytosis on a single-cell scale, this study combined an optical tweezer that can optically place a single cell on a desired location with a capacitance sensor and a cell incubation chamber. Single HeLa cancer cell was captured and moved to a desired location through optical trapping, and the single-cell capacitance change generated during the epidermal growth factor (EGF) molecule endocytosis was measured in real time. It was found that single HeLa cells showed a larger increase in capacitance values compared to that of the single NIH3T3 cells when exposed to varying EGF concentrations. In addition, the capacitance change was in proportion to the cell's EGF receptor (EGFR) level when cells of different levels of EGFR expression were tested. An equation derived from these results was able to estimate the EGFR expression level of a blind-tested cell. The biosensor developed in this research can not only quickly move a single cell to a desired location in a non-invasive manner but also distinguish specific responses between cancer and normal cells by continuous measurement of real-time interactions of a single cell in culture to the external ligands.

Resolution-enhanced optical inspection system to examine metallic nanostructures using structured illumination.

We developed a structured illumination-based optical inspection system to inspect metallic nanostructures in real time. To address this, we used post-image-processing techniques to enhance the image resolution. To examine the fabricated metallic nanostructures in real time, a compact and highly resolved optical inspection system was designed for practical industrial use. Structured illumination microscopy yields multiple images with various linear illumination patterns, which can be used to reconstruct resolution-enhanced images. Images of nanosized posts and complex structures reflected in the structured illumination were reconstructed into images with improved resolution. A comparison with wide-field images demonstrates that the optical inspection system exhibits high performance and is available as a real-time nanostructure inspection platform. Because it does not require special environmental conditions and enables multiple systems to be covered in arrays, the developed system is expected to provide real-time and noninvasive inspections during the production of large-area nanostructured components.

Investigation of craquelure patterns in oil paintings using precise 3D morphological analysis for art authentication.

The development of scientific technology for art authentication has elicited multidimensional evidence to distinguish forgeries from original artwork. Here, we analyzed the three-dimensional morphology of cracks that contain information, such as the painting features of artworks, using optical coherence tomography. The forgeries were produced by an expert from original oil paintings with cracks that occur owing to paint drying, canvas aging, and physical damage. Parameters, such as shape, width, and depth, were compared based on the cross-sectional images of the original and fake cracks. The original cracks were rectangular and inverted, but the fake cracks were relatively simple inverted triangles. The original cracks were as deep as the thickness of the upper layer and mostly were "thin/deep" or "wide/shallow". The fake cracks were observed to be "'thin/shallow" or "wide/deep". This study aims to improve the understanding of crack characteristics and promote the development of techniques for determining art authenticity.

Isothermal amplification-mediated lateral flow biosensors for in vitro diagnosis of gastric cancer-related microRNAs.

MicroRNAs (miRNAs) are important diagnostic and prognostic biomarkers for various tumors. Currently, many diagnostic systems have been developed to detect miRNAs, but simple techniques for detecting miRNAs are still required. Recently, we reported that the expression of miRNA-135b is upregulated in gastric epithelial cells during gastric inflammation and carcinogenesis. Our aim was to develop an in vitro diagnostic platform to analyze the expression of gastric cancer-related biomarkers in the blood. The diagnostic platform comprised an isothermal amplification-based lateral flow biosensor (IA-LFB) that enables easy diagnosis of gastric cancer through visual observation. In this platform, trace amounts of biomarkers are isothermally amplified through rolling circle amplification (RCA), and the amplified product is grafted to the LFB. The performance of the IA-LFB was confirmed using RNAs extracted from in vitro and in vivo models. The platform could detect target miRNAs within 3 h with excellent sensitivity and selectivity. In particular, the IA-LFB could detect the overexpression of gastric cancer-related markers (miRNA-135b and miRNA-21) in RNAs extracted from the blood of patients with various stages (stages 1-4) of gastric cancer compared to that in healthy volunteers. Therefore, IA-LFB is a simple and sensitive in vitro diagnostic system for detecting gastric cancer-related biomarkers and can contribute to the early diagnosis and prognosis monitoring of gastric cancer. Furthermore, this technology can be applied to systems that can detect multiple biomarkers related to various diseases (such as infectious and genetic diseases).

A Review of Advanced Impedance Biosensors with Microfluidic Chips for Single-Cell Analysis.

Electrical impedance biosensors combined with microfluidic devices can be used to analyze fundamental biological processes for high-throughput analysis at the single-cell scale. These specialized analytical tools can determine the effectiveness and toxicity of drugs with high sensitivity and demonstrate biological functions on a single-cell scale. Because the various parameters of the cells can be measured depending on methods of single-cell trapping, technological development ultimately determine the efficiency and performance of the sensors. Identifying the latest trends in single-cell trapping technologies afford opportunities such as new structural design and combination with other technologies. This will lead to more advanced applications towards improving measurement sensitivity to the desired target. In this review, we examined the basic principles of impedance sensors and their applications in various biological fields. In the next step, we introduced the latest trend of microfluidic chip technology for trapping single cells and summarized the important findings on the characteristics of single cells in impedance biosensor systems that successfully trapped single cells. This is expected to be used as a leading technology in cell biology, pathology, and pharmacological fields, promoting the further understanding of complex functions and mechanisms within individual cells with numerous data sampling and accurate analysis capabilities.

Enhancing Electrical Outputs of Piezoelectric Nanogenerators by Controlling the Dielectric Constant of ZnO/PDMS Composite.

Structural optimizations of the piezoelectric layer in nanogenerators have been predicted to enhance the output performance in terms of the figure of merit. Here, we report the effect of dielectric constant on electrical outputs of piezoelectric nanogenerator using ZnO/PDMS composites with varied ZnO coverages. The dielectric constant of piezoelectric layers was adjusted from 3.37 to 6.75. The electrical output voltage of 9 mV was achieved in the nanogenerator containing the ZnO/PDMS composite with the dielectric constant of 3.46, which is an 11.3-fold enhancement compared to the value of the nanogenerator featuring the composite with high dielectric constants. Significantly, lowering the dielectric constant of the piezoelectric layer improves the electrical output performance of piezoelectric nanogenerators.

Plasmonic sensing, imaging, and stimulation techniques for neuron studies.

Studies to understand the structure, functions, and electrophysiological properties of neurons have been conducted at the frontmost end of neuroscience. Such studies have led to the active development of high-performance research tools for exploring the neurobiology at the cellular and molecular level. Following this trend, research and application of plasmonics, which is a technology employed in high-sensitivity optical biosensors and high-resolution imaging, is essential for studying neurons, as plasmonic nanoprobes can be used to stimulate specific areas of cells. In this study, three plasmonic modalities were explored as tools to study neurons and their responses: (1) plasmonic sensing of neuronal activities and neuron-related chemicals; (2) performance-improved optical imaging of neurons using plasmonic enhancements; and (3) plasmonic neuromodulations. Through a detailed investigation of these plasmonic modalities and research subjects that can be combined with them, it was confirmed that plasmonic sensing, imaging, and stimulation techniques have the potential to be effectively employed for the study of neurons and understanding their specific molecular activities.

Fiber-Optic Localized Surface Plasmon Resonance Sensors Based on Nanomaterials.

Applying fiber-optics on surface plasmon resonance (SPR) sensors is aimed at practical usability over conventional SPR sensors. Recently, field localization techniques using nanostructures or nanoparticles have been investigated on optical fibers for further sensitivity enhancement and significant target selectivity. In this review article, we explored varied recent research approaches of fiber-optics based localized surface plasmon resonance (LSPR) sensors. The article contains interesting experimental results using fiber-optic LSPR sensors for three different application categories: (1) chemical reactions measurements, (2) physical properties measurements, and (3) biological events monitoring. In addition, novel techniques which can create synergy combined with fiber-optic LSPR sensors were introduced. The review article suggests fiber-optic LSPR sensors have lots of potential for measurements of varied targets with high sensitivity. Moreover, the previous results show that the sensitivity enhancements which can be applied with creative varied plasmonic nanomaterials make it possible to detect minute changes including quick chemical reactions and tiny molecular activities.

Injection-seeded high-repetition-rate short-pulse micro-laser based on upconversion nanoparticles.

We demonstrate a high repetition-rate upconversion green pulsed micro-laser, which is prepared by the fast thermal quenching of lanthanide-doped upconversion nanoparticles (UCNPs) via femtosecond-laser direct writing. The outer rim of the prepared upconversion hemi-ellipsoidal microstructure works as a whispering-gallery-mode (WGM) optical resonator for the coherent photon build-up of third-harmonic ultra-short seed pulses. When near-infrared (NIR) femtosecond laser pulses of wavelength 1545 nm are focused onto the upconversion WGM resonator, the optical third-harmonic is generated at 515 nm together with the upconversion luminescence. The weak third-harmonic (TH) seed pulses are coherently amplified in the hemi-ellipsoidal upconversion resonator as a result of the resonant interaction between the incident femtosecond laser field, the TH, the upconversion luminescence and the WGM. This upconversion lasing preserves the original repetition rate of the NIR pump laser and the output polarization state is also coherently aligned to the pump laser polarization. Because of the isotropic nature of the upconversion micro-ellipsoids, the upconversion lasing shows maximum intensity with a linearly polarized pump beam and minimum intensity with a circularly polarized pump beam. Our scheme devised for realizing high-repetition-rate lasing at higher photon energies in a compact micro platform will open up new ways for on-chip optical information processing, high-throughput microfluidic sensing, and localized micro light sources for optical memories.

Nanochannel-driven rapid capture of sub-nanogram level biomarkers for painless preeclampsia diagnosis.

Preeclampsia (PE) is a pregnancy-specific hypertensive syndrome recognized as the leading cause of maternal and fetal morbidity and mortality worldwide. Painful blood-collection procedures or low accuracy of non-invasive approaches require faster, patient-friendly, and more sensitive diagnostic technologies. Here we report a painless, highly sensitive detection platform using nanoporous microneedles (nMNs) that enables rapid capture of biomarkers present at sub-nanogram levels. The highly porous nanostructures on the nMN surface were prepared by anodization of aluminum MN and then functionalized by immobilization of capture antibodies to detect target biomarkers based on an immunoassay method. The immuno-functionalized nMN array demonstrated rapid capture of an estrogen (E2) biomarker for PE following a 1-min incubation and exhibited a concentration-dependent change in fluorescence intensity over the E2 range of 0.5 ng mL-1 to 1000 ng mL-1 after treatment with fluorescence-detection antibodies. Remarkably, the nMN patch selectively detected sub-nanogram-levels of E2 in subcutaneous interstitial fluid from rats with increased diagnostic accuracy as compared with commercial immunoassay kits. This bio-functionalized nMN platform showed improved biosensing capability for multiple PE-related biomarkers, including hormones and proteins. Furthermore, this painless method demonstrated efficacy as a point-of-need diagnostic platform using portable smartphone-based fluorescence microscope to obtain fluorescence images of biomarker-captured nMN arrays.

Experimental confirmation of plasmonic field cancellation under specific conditions of trapezoidal nanopatterns.

In this study, we investigated plasmonic field localization with trapezoidal nanopatterns under normal incident light excitation to find optimum structures for sensing and imaging. A finite element method was used to calculate the fundamental characteristics of the localized surface plasmon with varied trapezoidal nanopatterns. First, we describe how to localize the plasmonic fields on the trapezoidal patterns and then report our results from the investigation of the optimum properties of the nanopatterns for maximized field intensity. Initially, we expected that maximized field localization would lead to enhancement of the sensing sensitivity or imaging resolution in plasmon-based sensing and imaging systems. However, more interestingly, we found a field cancellation effect under specific modality conditions through the simulation. Thus, we thoroughly investigated the principle of the effect and extracted the modality conditions that induced field cancellation. In addition, specific modality conditions of nanopatterns that could be fabricated with conventional lithographic methods were numerically determined. Then, the field cancellation effect was experimentally verified using scanning nearfield optical microscopy. The results indicate that trapezoidal nanopatterns bring about enhanced field localization at the shaper edge of nanopatterns than do conventional rectangular nanopatterns and that plasmonic field cancellation can be observed under specific modality conditions of nanopatterns, even for conventional rectangular nanopatterns. Thus, it is suggested that careful fabrication and maintenance are needed to obtain strong plasmonic localization. Finally, the feasibility of providing a novel sensing platform using the field cancellation effect is suggested.

Improvement of High Affinity and Selectivity on Biosensors Using Genetically Engineered Phage by Binding Isotherm Screening.

The genetically engineered M13 bacteriophage (M13 phage), developed via directed evolutionary screening process, can improve the sensitivity of sensors because of its selective binding to a target material. Herein, we propose a screening method to develop a selective and sensitive bioreporter for toxic material based on genetically engineered M13 phage. The paraquat (PQ)-binding M13 phage, developed by directed evolution, was used. The binding affinities of the PQ-binding M13 phage to PQ and similar molecules were analyzed using isothermal titration calorimetry (ITC). Based on the isotherms measured by ITC, binding affinities were calculated using the one-site binding model. The binding affinity was 5.161 × 10-7 for PQ, and 3.043 × 10-7 for diquat (DQ). The isotherm and raw ITC data show that the PQ-binding M13 phage does not selectively bind to difenzoquat (DIF). The phage biofilter experiment confirmed the ability of PQ-binding M13 bacteriophage to bind PQ. The surface-enhanced Raman scattering (SERS) platform based on the bioreporter, PQ-binding M13 phage, exhibited 3.7 times the signal intensity as compared with the wild-type-M13-phage-coated platform.

Intermolecular distance measurement with TNT suppressor on the M13 bacteriophage-based Förster resonance energy transfer system.

An M13 bacteriophage-based Förster resonance energy transfer (FRET) system is developed to estimate intermolecular distance at the nanoscale using a complex of CdSSe/ZnS nanocrystal quantum dots, genetically engineered M13 bacteriophages labeled with fluorescein isothiocyanate and trinitrotoluene (TNT) as an inhibitor. In the absence of trinitrotoluene, it is observed that a significant spectral shift from blue to green occur, which represents efficient energy transfer through dipole-dipole coupling between donor and acceptor, or FRET-on mode. On the other hand, in the presence of trinitrotoluene, the energy transfer is suppressed, since the donor-to-acceptor intermolecular distance is detuned by the specific capturing of TNT by the M13 bacteriophage, denoted as FRET-off mode. These noble features are confirmed by changes in the fluorescence intensity and the fluorescence decay curve. TNT addition to our system results in reducing the total energy transfer efficiency considerably from 16.1% to 7.6% compared to that in the non-TNT condition, while the exciton decay rate is significantly enhanced. In particular, we confirm that the energy transfer efficiency satisfies the original intermolecular distance dependence of FRET. The relative donor-to-acceptor distance is changed from 70.03 Å to 80.61 Å by inclusion of TNT.

Plasmon-Coupled Whispering Gallery Modes on Nanodisk Arrays for Signal Enhancements.

Metallic nanostructures including single and double nanodisks are successfully used to enhance the localized electric field in vicinity of microcavity in whispering gallery mode (WGM) sensor. We demonstrate numerical calculations of plasmonic coupling of WGMs to single and double nanodisk arrays on a planar substrate. We then experimentally confirmed that the resonance wavelength of WGM sensor was dramatically shifted by adoption of single and double nanodisks on the surface of microcavity in the WGM sensor. Thus, our approach provides the tunable sensitivity of WGM sensor, and has a great potential to be used in numerous areas where the single biomolecule, protein-protein folding and biomolecular interactions are involved.

Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling.

BACKGROUND: Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR. METHODS: EGFR expression was assessed using Western blotting, immunofluorescence and capacitance assays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stably transfected with a CUG2 expression vector (A549-CUG2; BEAS-CUG2) or an empty control vector (A549-Vec; BEAS-Vec). After siRNA-mediated EGFR, Stat1 and HDAC4 silencing, antioxidant and multidrug resistance protein and mRNA levels were assessed using Western blotting and RT-PCR. In addition, the respective cells were treated with doxorubicin after which apoptosis and reactive oxygen species (ROS) levels were measured. Stat1 acetylation was assessed by immunoprecipitation. RESULTS: We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFR silencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR. CONCLUSION: Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.

Bioinspired M-13 bacteriophage-based photonic nose for differential cell recognition.

A bioinspired M-13 bacteriophage-based photonic nose was developed for differential cell recognition. The M-13 bacteriophage-based photonic nose exhibits characteristic color patterns when phage bundle nanostructures, which were genetically modified to selectively capture vapor phase molecules, are structurally deformed. We characterized the color patterns of the phage bundle nanostructure in response to cell proliferation via several biomarkers differentially produced by cells, including hydrazine, o-xylene, ethylbenzene, ethanol and toluene. A specific color enables the successful identification of different types of molecular and cellular species. Our sensing technique utilized the versatile M-13 bacteriophage as a building block for fabricating bioinspired photonic crystals, which enables ease of fabrication and tunable selectivity through genetic engineering. Our simple and versatile bioinspired photonic nose could have possible applications in sensors for human health and national security, food discrimination, environmental monitoring, and portable and wearable sensors.