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True random numbers are essential for ensuring information security and supporting simulations across various industries. With the exponential growth of data driven by advancements in artificial intelligence, robust encryption for communications has become increasingly important. While software-based deterministic random number algorithms are cost-effective and easy to use, they are vulnerable to attacks by powerful supercomputers, highlighting the need for more secure alternatives. As portable electronic devices and information-gathering sensors proliferate, portable true random number generators (TRNGs) are critical for maintaining security. In this work, hybrid material-based photodetectors composed of anionic polymers and perovskites that maximize stochastic photogeneration for TRNG applications are presented. By integrating perovskite photodetectors with simple electronic circuits, compact, low-power TRNG devices have been developed that are versatile and resilient to environmental factors. These devices generate 10 000 bits s -1 without resets or delays, achieving significant miniaturization. The generated 10 Mbit random number is validated through US National Institute of Standards and Technology (NIST) testing. Using a 480 000-bit random sequence, perfect image encryption, ensuring protection against hacking are demonstrated. Additionally, the perovskite TRNG can operate under external light even when embedded in pork skin, realizing its potential as an implantable device for personal security and authentication., (© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Selective spectral discrimination of visible and near-infrared light, which accurately distinguishes different light wavelengths, holds considerable promise in various fields, such as automobiles, defense, and environmental monitoring. However, conventional imaging technologies suffer from various issues, including insufficient spatial optimization, low definition, and optical loss. Herein, a groundbreaking advancement is demonstrated in the form of a dual-band photodiode with distinct near-infrared- and visible-light discrimination obtained via simple voltage control. The approach involves the monolithic stacking integration of methylammonium lead iodide (MAPbI 3 ) and Si semiconductors, resulting in a p-Si/n-phenyl-C 61 -butyric acid methyl ester/i-MAPbI 3 /p-spiro-MeOTAD (PNIP) device. Remarkably, the PNIP configuration can independently detect the visible and near-infrared regions without traditional optical filters under a voltage range of 3 to -3 V. In addition, an imaging system for a prototype autonomous vehicle confirms the capability of the device to separate visible and near-infrared light via an electrical bias and practicality of this mechanism. Therefore, this study pushes the boundaries of image sensor development and sets the stage for fabricating compact and power-efficient photonic devices with superior performance and diverse functionality., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

It is shown that structural disorder-in the form of anisotropic, picoscale atomic displacements-modulates the refractive index tensor and results in the giant optical anisotropy observed in BaTiS 3 , a quasi-1D hexagonal chalcogenide. Single-crystal X-ray diffraction studies reveal the presence of antipolar displacements of Ti atoms within adjacent TiS 6 chains along the c-axis, and threefold degenerate Ti displacements in the a-b plane. 47/49 Ti solid-state NMR provides additional evidence for those Ti displacements in the form of a three-horned NMR lineshape resulting from a low symmetry local environment around Ti atoms. Scanning transmission electron microscopy is used to directly observe the globally disordered Ti a-b plane displacements and find them to be ordered locally over a few unit cells. First-principles calculations show that the Ti a-b plane displacements selectively reduce the refractive index along the ab-plane, while having minimal impact on the refractive index along the chain direction, thus resulting in a giant enhancement in the optical anisotropy. By showing a strong connection between structural disorder with picoscale displacements and the optical response in BaTiS 3 , this study opens a pathway for designing optical materials with high refractive index and functionalities such as large optical anisotropy and nonlinearity., (© 2024 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

As one of the most fundamental physical phenomena, charge density wave (CDW) order predominantly occurs in metallic systems such as quasi-1D metals, doped cuprates, and transition metal dichalcogenides, where it is well understood in terms of Fermi surface nesting and electron-phonon coupling mechanisms. On the other hand, CDW phenomena in semiconducting systems, particularly at the low carrier concentration limit, are less common and feature intricate characteristics, which often necessitate the exploration of novel mechanisms, such as electron-hole coupling or Mott physics, to explain. In this study, an approach combining electrical transport, synchrotron X-ray diffraction, and density-functional theory calculations is used to investigate CDW order and a series of hysteretic phase transitions in a dilute d-band semiconductor, BaTiS 3 . These experimental and theoretical findings suggest that the observed CDW order and phase transitions in BaTiS 3 may be attributed to both electron-phonon coupling and non-negligible electron-electron interactions in the system. This work highlights BaTiS 3 as a unique platform to explore CDW physics and novel electronic phases in the dilute filling limit and opens new opportunities for developing novel electronic devices., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Materials with large birefringence (Δn, where n is the refractive index) are sought after for polarization control (e.g., in wave plates, polarizing beam splitters, etc.), nonlinear optics, micromanipulation, and as a platform for unconventional light-matter coupling, such as hyperbolic phonon polaritons. Layered 2D materials can feature some of the largest optical anisotropy; however, their use in most optical systems is limited because their optical axis is out of the plane of the layers and the layers are weakly attached. This work demonstrates that a bulk crystal with subtle periodic modulations in its structure-Sr 9/8 TiS 3 -is transparent and positive-uniaxial, with extraordinary index n e = 4.5 and ordinary index n o = 2.4 in the mid- to far-infrared. The excess Sr, compared to stoichiometric SrTiS 3 , results in the formation of TiS 6 trigonal-prismatic units that break the chains of face-sharing TiS 6 octahedra in SrTiS 3 into periodic blocks of five TiS 6 octahedral units. The additional electrons introduced by the excess Sr form highly oriented electron clouds, which selectively boost the extraordinary index n e and result in record birefringence (Δn > 2.1 with low loss). The connection between subtle structural modulations and large changes in refractive index suggests new categories of anisotropic materials and also tunable optical materials with large refractive-index modulation., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

As biomolecules essential for sustaining life, proteins are generated from long chains of 20 different α-amino acids that are folded into unique 3D structures. In particular, many proteins have molecular recognition functions owing to their binding pockets, which have complementary shapes, charges, and polarities for specific targets, making these biopolymers unique and highly valuable for biomedical and biocatalytic applications. Based on the understanding of protein structures and microenvironments, molecular complementarity can be exhibited by synthesizable and modifiable materials. This has prompted researchers to explore the proteomimetic potentials of a diverse range of materials, including biologically available peptides and oligonucleotides, synthetic supramolecules, inorganic molecules, and related coordination networks. To fully resemble a protein, proteomimetic materials perform the molecular recognition to mediate complex molecular functions, such as allosteric regulation, signal transduction, enzymatic reactions, and stimuli-responsive motions; this can also expand the landscape of their potential bio-applications. This review focuses on the recognitive aspects of proteomimetic designs derived for individual materials and their conformations. Recent progress provides insights to help guide the development of advanced protein mimicry with material heterogeneity, design modularity, and tailored functionality. The perspectives and challenges of current proteomimetic designs and tools are also discussed in relation to future applications., (© 2023 Wiley-VCH GmbH.)

Perovskite quantum dot (QD) light-emitting diodes (PeLEDs) are ideal for next-generation display applications because of their excellent color purity, high efficiency, and cost-effective fabrication. However, developing a technology for high-resolution multicolor patterning of perovskite QDs remains challenging, owing to the chemical instability of these materials. To overcome these issues, in this work, the generation of surface defects is prevented by controlling the ligand-binding kinetics using a stable ligand system (Stable LS). The crystalline reconstruction of perovskite QDs after addition of the Stable LS results in an ≈18% increase in their photoluminescence quantum yield in solution and it also improves the ambient stability of the perovskite QD solution. Moreover, the perovskite QDs with Stable LS can undergo cross-linking under UV irradiation. The tightly bridged perovskite QDs effectively prevent moisture-assisted ligand dissociation in film state due to the increased hydrophobicity and restricted movement of the cross-linked surface ligands. Thus, the cross-linked perovskite QD film shows improved chemical/environmental stability without substantial deterioration in optoelectrical properties. As a result, a white electroluminescent device with high resolution (≈1 μm) is successfully fabricated by inkjet printing using green and red perovskite QDs., (© 2021 Wiley-VCH GmbH.)

Antiphase boundaries (APBs) in 2D transition metal dichalcogenides have attracted wide interest as 1D metallic wires embedded in a semiconducting matrix, which could be exploited in fully 2D-integrated circuits. Here, the anisotropic morphologies of APBs (i.e., linear and saw-toothed APBs) in the nanoscale are investigated. The experimental and computational results show that despite their anisotropic nanoscale morphologies, all APBs adopt a predominantly chalcogen-oriented dense structure to maintain the energetically most stable atomic configuration. Moreover, the effect of the nanoscale morphology of an APB on electron transport from two-probe field effect transistor measurements is investigated. A saw-toothed APB has a considerably lower electron mobility than a linear APB, indicating that kinks between facets are the main factors of scattering. The observations contribute to the systematical understanding of the faceted APBs and its impact on electrical transport behavior and it could potentially extend the applications of 2D materials through defect engineering to achieve the desired properties., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transition metal dichalcogenides, especially MoS 2 , are considered as promising electrocatalysts for hydrogen evolution reaction (HER). Since the physicochemical properties of MoS 2 and electrode morphology are highly sensitive factor for HER performance, designed synthesis is highly pursued. Here, an in situ method to prepare a 3D carbon/MoS 2 hybrid catalyst, motivated by the graphene ribbon synthesis process, is reported. By rational design strategies, the hybrid electrocatalysts with cross-connected porous structure are obtained, and they show a high HER activity even comparable to the state-of-the-art MoS 2 catalyst without appreciable activity loss in long-term operations. Based on various physicochemical techniques, it is demonstrated that the synthetic procedure can effectively guide the formation of active site and 3D structure with a distinctive feature; increased exposure of active sites by decreased domain size and intrinsically high activity through controlling the number of stacking layers. Moreover, the importance of structural properties of the MoS 2 -based catalysts is verified by controlled experiments, validating the effectiveness of the designed synthesis approach., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

2D molybdenum disulfide (MoS 2 ) possesses excellent optoelectronic properties that make it a promising candidate for use in high-performance photodetectors. Yet, to meet the growing demand for practical and reliable MoS 2 photodetectors, the critical issue of defect introduction to the interface between the exfoliated MoS 2 and the electrode metal during fabrication must be addressed, because defects deteriorate the device performance. To achieve this objective, the use of an atomic layer-deposited TiO 2 interlayer (between exfoliated MoS 2 and electrode) is reported in this work, for the first time, to enhance the performance of MoS 2 photodetectors. The TiO 2 interlayer is inserted through 20 atomic layer deposition cycles before depositing the electrode metal on MoS 2 /SiO 2 substrate, leading to significantly enhanced photoresponsivity and response speed. These results pave the way for practical applications and provide a novel direction for optimizing the interlayer material., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost-effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation-dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error-prone hybridization-based detection, the multiplex assay process is complicated because of the size-based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high-resolution CE-SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar-sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three-color fluorescence-labeled probes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture-based methods require relatively longer time to identify drug-resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug-resistant Gram-positive pathogens, we developed a method by using stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with high-resolution CE single-strand conformation polymorphisms (CE-SSCP) system. We designed three probe sets for genes specific to Gram-positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram-positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain-specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target-specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug-resistant Gram-positive pathogens from sepsis patients' blood., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although the resolution of CE-SSCP has been significantly improved by using a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO; Pluronic(®)) triblock copolymer as a separation medium, CE-SSCP on a microchip format is not widely applicable because their resolution is limited by short channel length. Therefore, a strategy to improve the resolution in channels of limited lengths is highly required for enabling microchip-based CE-SSCP. In this study, we developed a high-resolution CE-SSCP microchip system by controlling the width of the pluronic-filled channel. We tested four different channel widths of 180, 240, 300, and 400 μm, and found that 300 μm showed the highest resolution in the separation of two pathogen specific markers. Potential applications of our method in various genetic analyses were also shown by using SNP markers for spinal muscular atrophy., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A lithographically aligned palladium nano-ribbon (Pd-NRB) array with gaps of less than 40 nm is fabricated on a poly(ethylene terephthalate) substrate using the direct metal transfer method. The 200 μm Pd-NRB hydrogen gas sensor exhibits an unprecedented sensitivity of 10(9) % after bending treatment, along with fast sensing behavior (80% response time of 3.6 s and 80% recovery time of 8.7 s) at room temperature., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel cell-based biosensing platform is developed using a combination of sequential laser interference lithography and electrochemical deposition methods. This enables the sensitive discrimination of dopaminergic cells from other types of neural cells in a completely nondestructive manner. This platform and detection strategy may become an effective noninvasive in situ monitoring tool that can be used to determine stem cell fate for various regenerative applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Liquid-phase sensing of various organic solvents is performed for the first time, using a solvent-resistant organic-transistor platform. Sensors composed of a cross-linked poly(3-hexylthiophene) (P3HT)-azide co-polymer and a calixarene derivative exhibit highly sensitive and selective sensing behavior, owing to the selective binding effects of the liquid analytes with the calixarene-functionalized P3HT-azide, extending the range of their use in practical applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Antibody-conjugated magnetic nanoparticles (Ab-MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab-MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab-MNPs as a pathogen detection method requires accurate characterization of Ab-MNP capture ability and standardization of all handling processes. In this study, we used high-resolution CE-single strand conformational polymorphism coupled with a stuffer-free multiplex ligation-dependent probe amplification system to characterize Ab-MNPs. The capture ability of Ab-MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab-MNPs was also assessed. The results showed that the stuffer-free multiplex ligation-dependent probe amplification system has the potential to serve as a standard characterization method for Ab-MNPs. Moreover, the precise characterization of Ab-MNPs facilitated robust pathogen detection in various applications., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple and precise diagnostic method for spinal muscular atrophy (SMA) using high-resolution CE-based single-strand conformation polymorphism (CE-SSCP) was developed in this study. SMA is a common genetic disorder caused by an abnormality in the relative copy numbers of SMN1 and its centromeric copy SMN2, which differ only in two nucleotides, namely at exons 7 and 8. Therefore, the precise discrimination of the differences in sequence as well as their relative quantities is crucial for the diagnosis of SMA. Multiplex ligation-dependent probe amplification and sequence-sensitive DNA separation using hydroxyethyl cellulose and hydroxypropyl cellulose blended polymer matrix are currently the available methods used in the diagnosis of SMA. However, these methods are limited by their extended hybridization step and low resolution. In this study, the simultaneous discrimination of SMN exons 7 and 8 was successfully demonstrated using high-resolution CE-SSCP. Unlike the previously reported alternative method, single base differing amplicons were baseline-separated because of its extraordinary resolution, thus providing accurate and precise quantification of each paralog., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CE-SSCP analysis is a well-established DNA separation method that is based on variations in mobility caused by sequence-induced differences in the conformation of single-stranded DNA. The resolution of CE-SSCP analysis was improved by using a Pluronic polymer matrix, and it has been successfully applied in various genetic analyses. Because the Pluronic polymer forms a micellar cubic structure in the capillary, it provides a stable internal structure for high-resolution CE-SSCP analysis. We hypothesized that formation of micellar cubic structure is influenced by the end hydroxyl group of the Pluronic polymer, which affords structural stability through hydrogen bonding. To test this hypothesis, the hydroxyl group was halogenated to eliminate the hydrogen bonding without disturbing the polarity of polymer matrix. CE-SSCP resolution of two DNA fragments with a single base difference was significantly worse in the halogenated polymer matrices due to band broadening. The viscoelastic properties of control (which has hydroxyl group), chlorinated, and brominated F108 solution upon heating were also investigated by rheological experiments, and we found that gelation was significantly associated with resolution. In this series of experiments, the effect of the hydroxyl group in Pluronic polymer matrix on separation resolution of CE-SSCP analysis was demonstrated., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

By combining nanoimprint lithography technique and a two-step lift-off process, a Si nanotube array is fabricated and applied as a light absorber for n-Si/PEDOT:PSS hybrid solar cells. The light is effectively trapped within the nanotubes and the device reveals a Jsc of 29.9 mA · cm(-2) and a power conversion efficiency of 10.03%, which is an enhancement of 13.4% compared to the cell having the best-known Si architecture of nanocones as a light absorber to date., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Accuracy, simplicity, and cost-effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase-based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray-based detection, the design process and multiplex assay procedure are complicated because of the DNA size-dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition-optimized ligation and high-resolution CE-based SSCP. With this high-resolution CE-SSCP system, we are able to use similar-sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single-base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this genomic era, the ability to assay multiple genomic hot spots that have strong clinical implications is greatly desired. Conventional PCR-based methods suffer from frequent false-positive detections, particularly when a multiplex analysis is desirable. As an alternative to the error-prone conventional methods, multiplex ligase-based genotyping methods combined with CE have a strong potential. In this review, both previously developed methods and emerging methods are described to reveal the specificity, sensitivity, and simplicity of the ligase-based methods. For each step (ligation, amplification, and separation), the principles of several alternative methods are discussed along with their applications to explore the future development of ligase-based diagnostic methods., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lower respiratory tract infection is one of the most common infectious diseases. However, conventional methods for detecting infectious pathogens are time-consuming, and generally have a limited impact on early therapeutic decisions. We previously reported a rapid and sensitive method for detecting such pathogens using stuffer-free multiplex ligation-dependent probe amplification coupled with high-resolution CE-SSCP. In this study, we report an application of this method to the detection of respiratory pathogens. As originally configured, this method was capable of simultaneously detecting seven bacterial species responsible for lower respiratory tract infections, but its detection limit and assay time were insufficient to provide useful information for early therapeutic decisions. To improve sensitivity and shorten assay time, we added a target-specific preamplification step, improving the detection limit from 50 pg of genomic DNA to 500 fg. We further decreased time requirements by optimizing the hybridization step, enabling the entire assay to be completed within 7 h while maintaining the same detection limit. Taken together, these improvements enable the rapid detection of infectious doses of pathogens (i.e. a few dozen cells), establishing the strong potential of the refined method, particularly for aiding early treatment decisions., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

All-solution-processed transparent thin film transistors (TTFTs) are demonstrated with silver grid source/drain electrodes, which are fabricated by printing and subsequent silver nanoparticles solution coating, which allows continuous processing without using high vacuum systems. The silver grid electrode shows a reasonable transmittance in visible range, moderate electrical conductance and mechanical strength. The TTFTs are employed to drive liquid crystal cells and demonstrate a successful switching operation., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pluronic F108 block copolymers have shown a great promise to achieve the desirable high resolution in the conformation-sensitive separation of ssDNA using CE-SSCP. However, fundamental understanding of the structures and properties of Pluronic matrix affecting the resolution is still limited. Unlike conventional gel-forming homopolymers, Pluronic F108 block copolymers are amphiphilic macromolecules consisting of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers, which are capable of forming a highly ordered micellar structure in aqueous solution. In this study, we have performed a series of experiments by blending different types of Pluronic polymers to control the formation of micelles and to study the correlation between separation and rheological characteristics of Pluronic gels affecting the resolution of CE-SSCP. Our experiments have been specifically designed to elucidate how the micellar structure affects the resolution of CE-SSCP upon altering the size uniformity and constituent homogeneity of the micelles. Our results suggest that uniformly sized micelle packing is the primary structural feature of Pluronic gel matrix for the high-resolution separation, while the size and constituent of the micelle themselves need to be considered as secondary factors., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A periodically aligned array of graphene nanorings (GRNRs) with a sub-15 nm linewidth at a pitch of 450 nm is fabricated with a large area, 9 cm(2) , through conventional nanoimprint lithography coupled with sophisticated metal deposition and plasma-etching processes. The existence of the single-layer GRNRs is verified by various techniques., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The success of antimicrobial therapy depends on effective prescription of antibiotics. Assessment of clinical isolates using rapid antimicrobial susceptibility tests allows effective microbiological therapy to be commenced in a timely manner. However, conventional antimicrobial susceptibility testing is time-consuming and laborious. In the present study, we employed stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with analysis of single-strand conformation polymorphisms, via high-resolution CE, to develop a multiplex antibiotic susceptibility test. Using this method, parallel analysis of specific genetic markers was employed to determine minimal inhibitory concentration values. The values derived using the stuffer-free MLPA method agreed with those estimated using a conventional broth dilution method. These findings indicate that the stuffer-free MLPA-based approach is a viable alternative to the conventional method., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent improvements in the multiplex ligation-dependent probe amplification (MLPA) method promise successful multiplex analysis of various genetic markers. In particular, it has been demonstrated that elimination of the stuffer sequence included in MLPA probes for length-dependent analysis substantially simplifies the probe design process and improves the accuracy of the analysis. As is the case for other CE-based methods, MLPA could be further developed on a microchip platform. However, high-resolution analysis of short MLPA probes requires careful microchip operation. In this study, we developed a microchip device for the multiplex analysis of five food-borne pathogens using a stuffer-free probe set. Microchip channel design and electrophoresis operating conditions were first optimized for reproducible analysis, after which two sieving matrices were tested. Finally, the method was validated using DNA samples isolated from intentionally infected milk., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen-specific genes are optimal markers. Although multiplex ligation-dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length-dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer-free MLPA using high-resolution CE-SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE-SSCP analysis was designed for species-specific detection. After careful optimization of each MLPA step, to ensure that CE-SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5-5 pg of genomic DNA, and more than 100-fold increase could be quantitatively determined. Thus, MLPA-CE-SSCP is a sensitive and reliable technique for pathogen detection., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Structural variation of human genome such as duplications and deletions, collectively termed copy number variation (CNV), is one of the major genetic variations. Reliable and efficient measurement of CNV will be essential to develop diagnostic tools for CNV-related diseases. We established a strategy based on multiplex PCR and capillary electrophoresis (CE) for reliable CNV assay. Multiplex-PCR was performed using five primer sets for target loci and a diploid control (DC). We designed primers satisfying three conditions: different size of each PCR product for CE separation, unified annealing temperature for multiplex PCR, and suitability for quantitative PCR (qPCR). We defined the accurate PCR cycles for quantification of copy numbers at which the amplifications for all targets were supposed to be exponential, named maximum doubling cycle. CE was carried out with PCR product and the ratio of the peak areas (target/diploid control) was calculated. Our multiplex PCR-CE analysis reliably determined copy numbers of X chromosome with variable copies ranging from 1 to 5 and showed higher reliability than qPCR (correlation coefficient 0.996 versus 0.898). When measuring the six randomly selected autosomal CNV targets using our multiplex PCR-CE, the results agreed with those from qPCR. In addition, our strategy was validated for the broad application to commonly used CE devices. Taken together, this assay will be useful for accurate analysis of multiple disease-associated CNVs in a clinical setting., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several methods based on screening for a 16S ribosomal RNA gene marker have been developed for rapid and sensitive detection of pathogenic microorganisms. One such method, CE-based SSCP (CE-SSCP), has enormous potential because the technique can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S ribosomal RNA gene-specific markers unless combined with additional modification steps. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this study, we developed a method based on a high-resolution CE-SSCP system using a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer that is capable of simultaneous and quantitative detection of 12 clinically important pathogens. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing easy identification of pathogens. A series of experiments using different amounts of genomic pathogen DNA showed that the method had a limit of detection of 0.31-1.56 pg and a dynamic range of approximately 10(2). These results indicate that high-resolution CE-SSCP systems have considerable potential in the clinical diagnosis of bacteria-induced diseases.

Rapid and sensitive detection of food-borne pathogens is critical for food safety from the viewpoint of both the public health professionals and the food industry. Conventional method is, however, known to be labor-intensive, time-consuming, and expensive due to the separate cultivation and biochemical assay. Many relevant technologies, such as flow cytometry, MALDI-MS, ESI-MS, DNA microarray, and CE, have been intensively developed to date. Among them, CE is considered to be the most efficient and reproducible because of low sample loss and simple automation. CE-based pathogen detection methods can be classified into three categories based on the separation targets: cell separation, nucleic-acid-based identification, and protein separation coupled with characterization. In this review, recent developments in each sphere of CE-based technology are discussed. Additionally, the critical features of each approach and necessary future technical improvements are also reviewed.

Although CE-SSCP analysis combined with 16S ribosomal RNA gene-specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE-SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE-SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE-SSCP analysis. A poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE-SSCP analysis using the PEO-PPO-PEO triblock copolymer as the polymer matrix and four same-sized DNA fragments of similar sequence content. Among 48 commercially available PEO-PPO-PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO(137)PPO(43)PEO(137) exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE-SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO-PPO-PEO triblock copolymer may serve as an ideal matrix for high-resolution CE-SSCP analysis.

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE-SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer as a sieving matrix for CE-SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO-PPO-PEO copolymers, 255-bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO-PPO-PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan gel. Due to enhanced dynamic coating and sieving ability, PEO-PPO-PEO copolymer displayed fourfold enhancement of resolving power in the CE-SSCP to separate same-sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high-resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO-PPO-PEO triblock copolymer an excellent matrix in the CE-SSCP analysis on the microdevice.

Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria-induced disease. Among the existing techniques for identifying microbial, CE-SSCP combined with 16S ribosomal RNA gene-specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE-SSCP can separate PCR products with high-resolution, multiplex detection and quantification are complicated by primer-dimer formation and non-specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template-tagging followed by multiplex asymmetric PCR and subsequent CE-SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia-inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE-SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.

Conventional antimicrobial activity analyses such as the broth dilution method and disk diffusion test are considerably demanding processes for new antimicrobial agent discovery and sensitive diagnosis of infectious diseases. Here, we developed a new antimicrobial activity analysis system using CE-based SSCP (CE-SSCP) combined with 16S rRNA gene-specific PCR (PCR/CE-SSCP). Using this method, the population change in the microbial community in response to specific antimicrobial agents could be quantified with a high sensitivity and accuracy from a small sample amount. Using a mixture of microorganisms comprising Escherichia coli, Corynebacterium glutamicum, Acinetobacter calcoaceticus, and Staphylococcus aureus as a model system, the linear correlation between the genomic DNA concentrations and peak areas in 16S rRNA gene-specific PCR/CE-SSCP was determined; consequently, quantification of cell concentrations could be demonstrated using this method. Compared to the minimum inhibitory concentration (MIC) values from the conventional broth dilution method, this new system provided almost the same MIC values for popular antimicrobial agents such as kanamycin, spectinomycin, and streptomycin. The results demonstrated that the newly developed method can be a substitute for the conventional antimicrobial analysis method and highlighted its high potential in the areas of new antimicrobial agent discovery and clinical diagnosis.

Even though mRNA quantification provides significant information for biological analysis, current methods such as Northern blot analysis and real-time PCR are known to be laborious and lacking in precision. In this study, we demonstrate a new precise mRNA quantification method using CE based on SSCP (CE-SSCP) coupled with reverse transcription. mRNA samples could be simply analyzed for the quantification directly with reverse transcript obtained from a single reaction. This helps to avoid considerable errors generated by a series of the tedious manual steps. Also, unlike real-time PCR, reverse transcripts can be directly quantified by CE-SSCP in this method without further data estimation. Reproducibility and accuracy of CE-SSCP for mRNA quantification was examined using enhanced green fluorescent protein (eGFP) mRNA transcribed in vitro. Specific reverse transcription primer was determined for the accurate quantification of eGFP mRNA from total RNA obtained from the recombinant Escherichia coli. Using elongation factor Tu mRNA as an internal standard, it was shown that sample-to-sample variation could be minimized. Expression kinetics at both mRNA level and protein level was studied and the potential of CE-SSCP in expression analysis was demonstrated by comparison with the eGFP activity assay.

Psychrophilicity of Gram-positive bacterium, Bacillus psychrosaccharolyticus was investigated in a proteomic approach. One hundred and thirty-one protein spots were analyzed by electrospray ionization-quadrupole-time of flight-tandem mass spectrometry and identified using an unpublished translated contig database as well as a nonredundant Gram-positive bacteria protein database from NCBI because of the lack of a genome sequence of this organism. Results focused on proteomic behavior of cold-response show that global up-regulation of metabolic functions and protective mechanism by stress responses might play a major role in psychrophilicity of B. psychrosaccharolyticus.