Search

Cordyceps militaris, an entomopathogenic Cordyceps mushroom, is a crucial ethnopharmacological agricultural product with applications in traditional oriental remedies in East Asia. Since lipases are reported to serve as key enzymatic equipment for entomopathogenic fungi during the host infection, the presence of various lipases with different biochemical features in C. militaris was elucidated. Three lipases from C. militaris (CML) of 60-70 kDa were isolated according to protein hydrophobicity; isoform relationships were identified by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry. The CML isoforms exhibited distinct substrate specificities, which were related to the hydrophobicity of each isoform. Furthermore, the integral stereoselectivity of each lipase towards trioleoylglycerol diverged into two classes (sn-1,3 and sn-2 regioselectivity) that are rare in canonical fungal lipases. Overall, our results demonstrate that C. militaris secretes lipase isoforms with cocktail-like enzyme functions that may contribute to the entomopathogenic life cycle of C. militaris. Each CML isoform has distinct advantages for biocatalyst applications in the food and oleochemical industries.

UiO-66-NH2 was synthesized in situ and analyzed as a reactive sorbent for nerve agent removal in the solid-state environment. UiO-66-NH2 had the smallest pore sizes, centered at 7 Å with added pores at 13 and 15 Å, indicating a hierarchically microporous structure. The in situ reaction was examined using phosphorus-31 solid state-magic angle spinning nuclear magnetic resonance (31P SS-MAS NMR) and further confirmed by gas chromatography – mass spectrometry (GC-MS). The results show that the nerve agents were decomposed via hydrolysis by UiO-66-NH2 and the overall rates were reduced for solid-phase reactions. Of the characterized components O-pinacolyl methylphosphonofluoridate (GD), N, N-dimethylphosphoramidocyanidate (GA), and O-ethyl S-diisopropylaminomethyl methylphosphonothioate (VX), GD was decomposed faster than GA and VX by UiO-66-NH2. Specifically, GD was decomposed to O-pinacolyl-methylphosphonic acid (PMPA) with a half-life of approximately 430 min in the presence of neat UiO-66-NH2. However, GA and VX were slowly hydrolyzed, forming reaction products such as ethylphosphate and dimethylphosphoramidic (DMPA) acid monoethylester from GA, and ethyl methylphosphonic acid (EMPA), methylphosphonic acid (MPA), and 2-(Diisopropylamino)ethyldisulfide (DES)2 from VX, respectively. This work describes the processes involved for these materials when they are deployed to protect against a nerve agent release, implying the practical application of UiO-66-NH2 for a broad range of filtration applications in the field.

We demonstrated protein and cellular patterning with a soft lithography technique using poly[2-aminoethyl methacrylate-co-oligo(ethylene glycol) methacrylate] films on gold surfaces without employing a backfilling process. The backfilling process plays an important role in successfully generating biopatterns; however, it has potential disadvantages in several interesting research and technical applications. To overcome the issue, a copolymer system having highly reactive functional groups and bioinert properties was introduced through a surface-initiated controlled radical polymerization with 2-aminoethyl methacrylate hydrochloride (AMA) and oligo(ethylene glycol) methacrylate (OEGMA). The prepared poly(AMA-co-OEGMA) film was fully characterized, and among the films having different thicknesses, the 35 nm-thick biotinylated, poly(AMA-co-OEGMA) film exhibited an optimum performance, such as the lowest nonspecific adsorption and the highest specific binding capability toward proteins.

Nature has developed a fascinating strategy of cryptobiosis ("secret life") for counteracting the stressful, and often lethal, environmental conditions that fluctuate sporadically over time. For example, certain bacteria sporulate to transform from a metabolically active, vegetative state to an ametabolic endospore state. The bacterial endospores, encased within tough biomolecular shells, withstand the extremes of harmful stressors, such as radiation, desiccation, and malnutrition, for extended periods of time and return to a vegetative state by breaking their protective shells apart when their environment becomes hospitable for living. Certain ciliates and even higher organisms, for example, tardigrades, and others are also found to adopt a cryptobiotic strategy for survival. A common feature of cryptobiosis is the structural presence of tough sheaths on cellular structures. However, most cells and cellular assemblies are not "spore-forming" and are vulnerable to the outside threats. In particular, mammalian cells, enclosed with labile lipid bilayers, are highly susceptible to in vitro conditions in the laboratory and daily life settings, making manipulation and preservation difficult outside of specialized conditions. The instability of living cells has been a main bottleneck to the advanced development of cell-based applications, such as cell therapy and cell-based sensors. A judicious question arises: can cellular tolerance against harmful stresses be enhanced by simply forming cell-in-shell hybrid structures? Experimental results suggest that the answer is yes. A micrometer-sized "Iron Man" can be generated by chemically forming an ultrathin (100 nm) but durable shell on a "non-spore-forming" cell. Since the report on silica nanoencapsulation of yeast cells, in which cytoprotective yeast-in-silica hybrids were formed, several synthetic strategies have been developed to encapsulate individual cells in a cytocompatible fashion, mimicking the cryptobiotic cell-in-shell structures found in nature, for example, bacterial endospores. Bioinspired silicification and phenolics-based coatings are, so far, the main approaches to the formation of cytoprotective cell-in-shell hybrids, because they ensure cell viability during encapsulations and also generate durable nanoshells on cell surfaces. The resulting cell-in-shell hybrids extrinsically possess enhanced resistance to external aggressors, and more intriguingly, the encapsulation alters their metabolic activity, exemplified by retarded or suppressed cell cycle progression. In addition, recent developments in the field have further advanced the synthetic tools available to the stage of chemical sporulation and germination of mammalian cells, where cytoprotective shells are formed on labile mammalian cells and broken apart on demand. For example, individual HeLa cells are coated with a metal-organic complex of ferric ion and tannic acid, and cellular adherence and proliferation are controlled by the programmed shell formation and degradation. Based on these demonstrations, the (degradable) cell-in-shell hybrids are anticipated to find their applications in various biomedical and bionanotechnological areas, such as cytotherapeutics, high-throughput screening, sensors, and biocatalysis, as well as providing a versatile research platform for single-cell biology.

Using a lotus leaf as our model, we fabricated an extremely low surface energy micro/nanostructured coating for textiles that repel oil, water, and chemical warfare agents (CWAs) using a simple process that is suitable for large scale production. This coating, called “OmniBlock”, consisted of approximately 200-nm silica nanoparticles, tetraethylorthosilicate, 3-glycidoxypropyl trimethoxysilane, and a perfluorooctanoic acid-free fluoropolymer (Fluorolink S10) that was cross-linked between Si-O-Si groups via a sol-gel process. The perfluorooctanoic acid-free fluoropolymer-coated silica nanoparticles were simply applied to the surface of a cotton fabric by a dip–dry–cure process, forming dense, continuous, and uniform layers of OmniBlock coating. OmniBlock modified the surface of the cotton fibers, creating a rough, high surface area uniform coating with many micro-crevasses. As a result, n-dodecane, water, and CWAs beaded up without wetting the surface, exhibiting large contact angles of 154° for water and 121° for n-dodecane, with a small shedding angle of 5° and contact angle hysteresis of 3.2° for water. The designed coating showed excellent liquid repellence properties against three types of CWAs: 129°, 72°, and 87° for sulfur mustard (HD), soman (GD), and VX nerve agents, respectively. Furthermore, OmniBlock coating shows good mechanical properties under tensile strength and wash tests. This remarkable ability to repel CWAs is likely to have potential military applications in personal protective equipment systems requiring self-cleaning functions.

Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-Fe(III) nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-Fe(III) nanocoat, mimicking the sporulation and germination processes found in nature.

The cytoprotective coating of physicochemically labile mammalian cells with a durable material has potential applications in cell-based sensors, cell therapy, and regenerative medicine, as well as providing a platform for fundamental single-cell studies in cell biology. In this work, HeLa cells in suspension were individually coated with silica in a cytocompatible fashion through bioinspired silicification. The silica coating greatly enhanced the resistance of the HeLa cells to enzymatic attack by trypsin and the toxic compound poly(allylamine hydrochloride), while suppressing cell division in a controlled fashion. This bioinspired cytocompatible strategy for single-cell coating was also applied to NIH 3T3 fibroblasts and Jurkat cells.

In this Opinion we discuss the development of artificial spores and their maturation as an independent field of research. The robust cell-in-shell structures have displayed unprecedented characteristics, which include the retardation of cell division and extensive cytoprotective capabilities that encompass exposure to osmotic pressure, shear force, heat, UV radiation, and lytic enzymes. Additionally, the nanothin shells act as highly versatile scaffolds for chemical functionalization to equip cells for implementation in tissue engineering, biosensors, cell therapy, or other biotechnological applications. We also explore the future direction of this emerging field and dictate that the next phase of research should focus on attaining more intricate engineering to achieve stimulus-responsive shell-degradation, multilayer casings with orthogonal functions, and the encapsulation of multiple cells for multicellular artificial spores.

Department of Chemistry, KAIST, Daejeon 305-701, Korea. *E-mail: ischoi@kaist.ac.kr Department of Marine Bio-Materials & Aquaculture, Pukyong National University, Busan 608-737, Korea Department of BioNanomaterials, Bio-Campus of Korea Polytechnic, Chung-nam 320-905, Korea Department of Science Education, Kyungnam University, Masan 631-701, Korea Department of Chemistry, Kyungpook National University, Daegu 701-702, Korea. *E-mail: jkl@knu.ac.kr Received January 28, 2013, Accepted February 24, 2013

Cells are encapsulated individually within thin and tough shells in a cytocompatible way, by mimicking the structure of bacterial endospores that survive under hostile conditions. The 3D 'cell-in-shell' structures-coined as 'artificial spores'-enable modulation and control over cellular metabolism, such as control of cell division, resistance to external stresses, and surface-functionalizability, providing a useful platform for applications, including cell-based sensors, cell therapy, regenerative medicine, as well as for fundamental studies on cellular metabolism at the single-cell level and cell-to-cell communications. This Concept focuses on chemical approaches to single-cell encapsulation with artificial shells for creating artificial spores, including cross-linked layer-by-layer assembly, bioinspired mineralization, and mussel-inspired polymerization. The current status and future prospects of this emerging field are also discussed.

In this study, pool boiling CHF experiments under atmospheric pressure with SA508 test heater was performed. SA508 is the material of the reactor pressure vessel in a nuclear power plant. Therefore, the CHF behavior of SA508 material is important for the reactor pressure vessel integrity at accident, but there are few studies about that. SA508 material showed very different CHF behavior from other cases (stainless steel). It showed very higher CHF value and the test heater surface was changed significantly. This test heater surface change is due to corrosion of SA508, and the rate of corrosion increases with boiling time. The corrosion product is analyzed as magnetite (Fe3O4), and magnetite has been used as one of nanofluid for CHF enhancement. The size of magnetite produced on the test heater surface is 20–140 nm, that is, it is nanoscale. Therefore, the CHF enhancement mechanism of SA508 material is similar to the case using other test heater (like stainless steel) with magnetite nanofluid. Additives like TSP, Al2O3 and CNT nanoparticles are also tested, but they did not show the CHF enhancement effect. In case of TSP, it showed CHF decrease effect due to preventing effect on SA508 corrosion.

E-mail: sunghoyang@knue.ac.krReceived June 16, 2014, Accepted July 2, 2014Key Words : Silica, Thin film, Histidine, Self-assembled monolayer, SilicificationThere exists a great deal of interest in silica thin films,which potentially allow us to develop various applicationsincluding heterogeneous catalysis

In this study, the effects of tri-sodium phosphate (TSP) and boric acid on CHF enhancement were studied. Both TSP and boric acid are used to control pH in nuclear power plants. TSP is a kind of surfactant, and several surfactants, include TSP, have been reported to have an effect on enhancement of heat transfer. Nothing has yet been reported for the case of boric acid. CHF experiments were performed with mass flux ranging from 100-500 kg/m2 s and inlet subcooling temperature of 50 °C under atmospheric pressure. The test section was a vertical circular SS316 tube having an inner diameter of 10.98 mm. Its heated length was 224 mm, and it was heated by a heat flux control system using DC electricity. Fluids in the test loop were plain water, TSP solutions, and boric acid solutions. TSP solutions had three concentrations (0.2, 0.4, 0.6%), and boric acid solutions had four concentrations (0.2, 0.4, 0.6, 0.8%). In the case of TSP, 21.4% enhancement of CHF was observed at the inlet subcooling temperature of 50 °C and extremely low mass flux (100 kg/m2 s). In the case of boric acid, 12.4% enhancement of CHF was observed at inlet subcooling temperature of 50 °C and extremely low mass flux 100 kg/m 2 s.

In this work, we report that high-density, vertically grown silicon nanowires (vg-SiNWs) direct a new in vitro developmental pathway of primary hippocampal neurons. Neurons on vg-SiNWs formed a single, extremely elongated major neurite earlier than minor neurites, which led to accelerated polarization. Additionally, the development of lamellipodia, which generally occurs on 2D culture coverslips, was absent on vg-SiNWs. The results indicate that surface topography is an important factor that influences neuronal development and also provide implications for the role of topography in neuronal development in vivo.

Although the programmed degradation of biocompatible films finds applications in various fields including biomedical and bionanotechnological areas, coating methods have generally been limited to be substrate-specific, not applicable to any kinds of substrates. In this paper, we report a dopamine derivative, which allows for both universal coating of various substrates and stimuli-responsive film degradation, inspired by mussel-adhesive proteins. Two dopamine moieties are linked together by the disulfide bond, the cleavage of which enables the programmed film degradation. Mechanistic analysis of the degradable films indicates that the initial cleavage of the disulfide linkage causes rapid uptake of water molecules, hydrating the films, which leads to rapid degradation. Our substrate-independent coating of degradable films provides an advanced tool for drug delivery systems, tissue engineering, and anti-fouling strategies.

The design of stimuli-responsive self-assembled molecular systems capable of undergoing mechanical work is one of the most important challenges in synthetic chemistry and materials science. Here we report that foldectures, that is, self-assembled molecular architectures of β-peptide foldamers, uniformly align with respect to an applied static magnetic field, and also show instantaneous orientational motion in a dynamic magnetic field. This response is explained by the amplified anisotropy of the diamagnetic susceptibilities as a result of the well-ordered molecular packing of the foldectures. In addition, the motions of foldectures at the microscale can be translated into magnetotactic behaviour at the macroscopic scale in a way reminiscent to that of magnetosomes in magnetotactic bacteria. This study will provide significant inspiration for designing the next generation of biocompatible peptide-based molecular machines with applications in biological systems., Controlling organic materials with magnetic fields in a dynamic fashion is a challenging task. Here, the authors show that synthetic ß-peptide foldamers can be rotated at will under a dynamic magnetic field and that this can be extended to macroscopic scale objects containing these materials.

A highly cytocompatible LbL process was developed, based on the thiol–exchange reaction, for forming in situ cross-linked and degradable films under physiologically mild conditions., Formation of both mechanically durable and programmably degradable layer-by-layer (LbL) films in a biocompatible fashion has potential applications in cell therapy, tissue engineering, and drug-delivery systems, where the films are interfaced with living cells. In this work, we developed a simple but versatile method for generating in situ cross-linked and responsively degradable LbL films, based on the thiol–exchange reaction, under highly cytocompatible conditions (aqueous solution at pH 7.4 and room temperature). The cytocompatibility of the processes was confirmed by coating individual yeast cells with the cross-linked LbL films and breaking the films on demand, while maintaining the cell viability. In addition, the processes were applied to the controlled release of an anticancer drug in the HeLa cells.

Formation of both mechanically durable and programmably degradable layer-by-layer (LbL) films in a biocompatible fashion has potential applications in cell therapy, tissue engineering, and drug-delivery systems, where the films are interfaced with living cells. In this work, we developed a simple but versatile method for generating in situ cross-linked and responsively degradable LbL films, based on the thiol–exchange reaction, under highly cytocompatible conditions (aqueous solution at pH 7.4 and room temperature). The cytocompatibility of the processes was confirmed by coating individual yeast cells with the cross-linked LbL films and breaking the films on demand, while maintaining the cell viability. In addition, the processes were applied to the controlled release of an anticancer drug in the HeLa cells.

The cytoprotection of individual living cells under in vitro and daily-life conditions is a prerequisite for various cell-based applications including cell therapy, cell-based sensors, regenerative medicine, and even the food industry. In this work, we use a cytocompatible two-step process to encapsulate Saccharomyces cerevisiae in a highly uniform nanometric (

Single-cell encapsulation promises the cytoprotection of the encased cells against lethal stressors, reminiscent of the sporulation process in nature. However, the development of a cytocompatible method for chemically mimicking the germination process (i.e., shell degradation on-demand) has been elusive, despite the shell degradation being pivotal for the practical use of functional cells as well as for single cell-based biology. We report that an artificial shell, composed of tannic acid (TA) and Fe(III) , on individual Saccharomyces cerevisiae controllably degrades on-demand, while protecting the yeast from multiple external aggressors, including UV-C irradiation, lytic enzymes, and silver nanoparticles. Cell division is suppressed by the TA-Fe(III) shell, but restored fully upon shell degradation. The formation of a TA-Fe(III) shell would provide a versatile tool for achieving the chemical version of "sporulation and germination".

In this chapter, we describe methods for encapsulating individual cells within inorganic nanoshells. Inspired by the biomineralization that occurs in nature, living cells have been encapsulated within various inorganic shells made of gold, calcium phosphate, calcium carbonate, silica, or titania. The viability and metabolic activities of the cells were ensured by the careful selection of biocompatible materials and processes. Importantly, inorganic shells have been useful for enhancing long-term cell viability, controlling cell division, protecting the cytoplasm against foreign aggression, and functionalizing cell surfaces. This chapter focuses on the chemical reactions used to generate inorganic shells, categorized as direct reduction, in situ crystallization, and in situ condensation, and on the artificial control of the cellular behaviors conferred by the inorganic shells. The future prospects in the field of cell encapsulation within inorganic materials are also discussed.

The chronological progresses in single-cell nanocoating are described. The historical developments in the field are divided into biotemplating, cytocompatible nanocoating, and cells in nano-nutshells, depending on the main research focuses. Each subfield is discussed in conjunction with the others, regarding how and why to manipulate living cells by nanocoating at the single-cell level.

The chemical control of cell division has attracted much attention in the areas of single cell-based biology and high-throughput screening platforms. A mussel-inspired cytocompatible encapsulation method for achieving a "cell-division control" with cross-linked layer-by-layer (LbL) shells is developed. Catechol-grafted polyethyleneimine and hyaluronic acid are chosen as polyelectrolytes for the LbL process, and the cross-linking of polyelectrolytes is performed at pH 8.5. Cell division is controlled by the number of the LbL nanolayers and cross-linking reaction. We also suggest a new measuring unit, t-2.0 OD 600, for quantifying "cell-division timing" based on microbial growth kinetics.

Catalytic natures of organometallic catalysts are modulated by coordinating organic ligands with proper steric and electronic properties to metal centers. Carbon-based nanomaterials such as graphene nanoplatelets are used with and without N-doping and multiwalled carbon nanotube as a ligand for ethylene polymerizations. Zirconocenes or titanocenes are immobilized on such nanomaterials. Polyethylenes (PEs) produced by such hybrids show a great increase in molecular weight relative to those produced by free catalysts. Specially, ultra-high-molecular-weight PEs are produced from the polymerizations at low temperature using the hybrid with N-doped graphene nanoplatelets. This result shows that such nanomaterials act a crucial role to tune the catalytic natures of metallocenes.

Overlay requirements for semiconductor devices are getting more demanding as the design rule shrinks. According to ITRS expectation[1], on product overlay budget is less than 8nm for the DRAM 40nm technology node. In order to meet this requirement, all overlay error sources have to be analyzed and controlled which include systematic, random, even intrafield high order errors. In this paper, we studied the possibility of achieving

In this paper, we propose a novel anti-collision algorithm for generation 2 RFID system to estimate the number of tags and to use the optimal frame size. Depending on the number of tags, there exists the optimal frame size in the sense that the system efficiency is maximized. The number of tags can be estimated from the collision ratio in terms of collided slots per frame. In addition, we compare our proposed algorithm with other existing schemes by simulation. The proposed scheme is easy to implement, and the simulation results show that its performance improves significantly compared to other schemes in terms of the identification time and the number of consumed slots.

Acute renal failure induced by methotrexate (MTX) can be lethal because renal excretion of the drug can be delayed. Pre-existing renal impairment, abstention, or underdosage of folinic acid and inadequate hydration facilitate toxicity. the prolonged high serum levels of MTX result in severe mucositis and pancytopenia, but strategies useful to accelerate MTX removal have not been universally accepted. We report a case of a 13-year-old girl with osteosarcoma who was treated with high-dose MTX because of thoracic tumor recurrence. No side effects were observed after 2 cycles of high-dose MTX; however, after the third cycle there was a delayed MTX elimination followed by clinical toxicity. Forty hours post-MTX infusion the serum level of MTX was 5.39 x 10(-4) mol/L. Treatment was based on symptomatic measures, such as maintenance of an abundant and alkaline diuresis and parenteral administration of folinic acid. Concomitantly, plasma exchange was employed to accelerate MTX removal and reduce its toxicity. After 24 days, she was discharged from the hospital, and her renal function recovered gradually. (C) 2003 Wiley-Liss, Inc. Universidade Federal de São Paulo, Hematol & Transfus Med Serv, Escola Paulista Med, BR-04024002 São Paulo, Brazil GRAAC, IOP, Pediat Oncol Inst, São Paulo, Brazil Universidade Federal de São Paulo, Hematol & Transfus Med Serv, Escola Paulista Med, BR-04024002 São Paulo, Brazil Web of Science

The selective and sensitive detection of target DNA is an important issue for clinical diagnostics, including identification of single nucleotide polymorphism (SNP), as well as for fundamental studies in molecular biology. The commonly used DNA-detection techniques are mainly based on the DNA microarrays, which present probe-DNA molecules immobilized onto a solid surface in an array format for the bio-specific hybridization with the unknown target-DNA fragments. Although the DNA microarrays have been utilized as a basic platform for the clinical detection, selectivity and sensitivity are still limited toward the singlenucleotide discrimination, which precludes the accurate analysis of SNP. In addition, duplexes between the target and probe DNAs exist in a stable form only in the presence of salt because of the electrostatic repulsion between negatively charged ribose-phosphate backbones of DNA; these salt conditions also induce the secondary and tertiary structures of DNA oligomers themselves, which could disturb the hybridization between the target and probe DNAs. On the other hand, peptide nucleic acid (PNA), an artificial nucleic acid analog, has been introduced to overcome the drawbacks of natural DNA in the aspects of specificity, sensitivity, and stability, and been as a molecular tool for the identification of SNP. The neutral backbone of PNA enhances the stability of PNA-DNA duplexes due to the absence of interstrand repulsion between the negatively charged phosphate groups of DNA. In addition, the unnatural structure makes PNA not degraded by enzymatic reactions. In this work, we generated a PNA-array platform to detect the SNP of cytochrome P450 2C19 (CYP2C19) gene. The CYP2C19 gene is known to play an important role in the metabolism of therapeutic drugs, including diazepam, proguanil, lansoprazole, and omeprazole. It is noteworthy that 15-20% of the Asians are genetically deficient in CYP2C19, failing to produce enzymes for the metabolism, compared with the occurrence of 3-5% of Caucasian populations. Specifically, there exist two variant alleles leading to the mutant types. The first varient is CYP2C19*2, which has a G → A nucleotide substitution at position 681, resulting in an aberrant splicing site; the second one, CYP2C19*3, which does a G → A substitution at position 636, resulting in a premature termination codon. According to the previous report, the frequency of CYP2C19*2 and CYP2C19*3 for Koreans is 0.21 and 0.12, respectively. Onto the silicon oxide surface were aldehyde groups introduced via imine formation between amine-terminated self-assembled monolayers (SAMs) and glutaldehyde. Another imine linkage was used for immobilizing the PNA probes, and unreacted aldehyde groups were passivated with 2(2aminoethoxy)ethanol. We designed four probe-PNAs based on the clinically important SNPs of CYP2C19, and denoted them 636W and 636M for the wild and mutant types of CYP2C19*3, and 681W and 681M for CYP2C19*2, respectively (Fig. 1). The linker of the PNA probes contained repetitive ethylene glycol groups and ended with several lysine groups. The immobilization of the probe PNAs was confirmed by hybridization of target DNAs. The target DNAs (T-636W, T-636M, T-681W, and T-681M), complementary to each of the PNA probes, were tagged with Cy3 (green) for wild type and Cy5 (red) for mutant type, respectively. The hybridization was performed for 1 h at 40 C with the DNA solution containing both T-636W and T636M (100 nM), and the same conditions were used for hybridization of T-681W/M. As shown in Fig. 2a, each DNA was selectively hybridized with its complementary probe