Geometric and electronic structure of Au on Au/CeO2 catalysts during the CO oxidation: Deactivation by reaction induced particle growth

Changes of the geometric and electronic structure of gold on Au/CeO2 catalysts induced by different pre-treatments (oxidative and reductive) and by the CO oxidation reaction at 80°C were followed by operando XANES / EXAFS measurements. The results showed that i) oxidative pre-treatment (O2) leads to larger Au nanoparticles than reductive pre-treatment (CO), that ii) Au is predominantly metallic during CO oxidation, irrespective of the preceding pre-treatment, and that iii) there is a reaction induced Au particle growth. Correlations with the activity of the respective catalysts and its temporal evolution give insights into the origin of deactivation of these catalysts under reaction conditions, in particular on reaction induced changes in the Au particle size.

Introduction
Supported Au catalysts have attracted enormous interest in recent years because of their exceptionally high activity in a number of oxidation and reduction reactions [1]. One of the most prominent examples is the (preferential) CO oxidation reaction. Despite extensive efforts many important aspects of this reaction are still under debate, such as the active Au species or the physical origin of the pronounced deactivation during the reaction [1], which is relevant also for practical applications.
In the meantime there has been increasing evidence that the exact nature and working principle of Au catalysts depends strongly both on the nature of the support and the reaction and catalyst pretreatment conditions [2,3]. While for CO oxidation on Au/TiO 2 at temperatures around 80 °C and higher Au is exclusively present in form of Au 0 nanoparticles (NPs), which are also the active Au species [3,4], this may be different for other supports and reaction conditions. Furthermore, detailed information on the geometric structure and on the electronic structure and state of the gold species in these catalysts has so far mainly been provided by ex situ techniques such as transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) measurements. Considering that these properties may be different under reaction conditions, in situ information is highly desirable.
For the CO oxidation on highly active Au/CeO 2 catalysts, previous results led to contradicting conclusions on the nature of the active Au species. While some groups claimed that gold in ionic form (Au + , Au 3+ ) is predominantly responsible for the activity of the Au catalysts [5], other groups suggested that metallic Au 0 NPs represent the active species during reaction [6]. Likewise, deactivation of Au/CeO 2 catalysts during CO oxidation had been proposed to mainly originate from (i) effects associated with changes of the formal oxidation state of Au [5,7], (ii) the build-up of surface poisoning carbon containing species at the perimeter of the Au-support interface [8], and/or (iii) sintering of Au NPs during reaction [7,9]. Due to the considerable differences in the pre-treatment and reaction conditions in the different studies, a more general understanding of the active Au species and its role in the reaction and deactivation process can hardly be derived from these data.
We here present results of a combined kinetic and in situ XAS (XANES / EXAFS) study where we investigated the structural and electronic properties of the Au species in a Au/CeO 2 catalyst upon pretreatment in either oxidative (O 2 /N 2 ) or reductive (CO/N 2 ) atmosphere and during subsequent CO oxidation at 80°C, and their correlation with the catalyst activity and its evolution with time, to unravel the role of changes in the Au particle size and in its electronic state in the deactivation of the catalyst.

Experimental Section
The operando XANES / EXAFS measurements were performed on a highly dispersed 4.5 wt.% Au/CeO 2 catalyst with a surface area of 188 m 2 g -1 , at the XAFS beamline at the Elettra Synchrotron (Trieste) using a Si(111) double crystal monochromator [10] and a specially designed reaction cell [11]. The catalyst was prepared by a deposition-precipitation procedure described elsewhere [12]. EXAFS/XANES data were recorded before and after the pre-treatment as well as during reaction at the Au L 3 -edge (11919 eV) in the fluorescence mode, using a Si drift diode detector (KETEK GmbH). The pre-edge region was measured from 11616 -11885 eV, followed by the XANES (11885 -11970 eV) and the EXAFS region (11970 -12500 eV). The EXAFS spectra were evaluated using XDAP software. The theoretical references were calculated by FEFF 8.0, and calibrated by measured reference spectra (Au foil and Au 2 O 3 powder) [13]. About 20 mg of the diluted catalyst (1:3 with SiO 2 ) were placed in the reaction cell (catalyst bed size: 2 x 10 mm). Next, the catalyst was pre-treated either in oxidative (O400: 10% O 2 in N 2 ) or in reductive (CO400: 10% CO in N 2 ) atmosphere. This included first heating up to 400°C in N 2 , then holding the catalyst for 30 min in the respective gas mixture at 400°C, and afterwards cool-down in N 2 to the reaction temperature of 80°C (all at 1.0 atm). Subsequently, the CO oxidation was performed in an idealized gas mixture (1% CO, 1% O 2 , 98% N 2 ) at 1 atm and 80°C. Kinetic measurements were performed under identical conditions.

Influence of pre-treatment on the nature and dispersion of Au nanoparticles
The influence of the pre-treatment on the nature of the Au species as well as on the initial metal dispersion was determined by recording XANES/EXAFS at the Au L 3 -edge in N 2 at 80°C after the respective pre-treatments (see above) and in N 2 at 25°C for the fresh sample (N25). A comparison of the normalized XANE spectra (see Fig.1) clearly demonstrates that the fresh Au/CeO 2 catalyst shows almost identical features as the Au 2 O 3 reference (inflection point: 11918 eV; peak: 11921 eV). Hence, it largely consists of ionic Au 3+ species. The strong oxide peak visible on the fresh sample, however, disappeared almost completely after CO400 pre-treatment, and instead a fully metallic Au 0 species is formed. After the O400 pre-treatment, in contrast, a significant fraction of oxidic species is still present, although to a much lower extent compared to the fresh sample (N25). These results agree well with EXAFS data obtained after the different pre-treatments. After CO400 pre-treatment, the spectrum can be fitted with only metallic Au-Au scatterers, while for O400 pre-treated catalyst it has to be fitted with a model containing both metallic (Au-Au) and oxidic (Au-O) shells (see Table 1 and Fig. 2) [14]. These findings confirm the presence of oxidic Au species after O400. Hence, the ionic gold species present on the fresh Au/CeO 2 catalyst sample are reduced to Au 0 , either partly (O400) or completely (CO400). Furthermore, the pre-treatment procedure results in remarkable differences in the Au-Au CN and, hence, in the Au NP size, with the higher dispersion / smaller Au particle size for CO400. While the Au-Au CN is 6.3 after O400, it is only 3.9 after CO400. Assuming hemispherical Au NPs one can also calculate the corresponding dispersion, which is much higher after CO400 (100%) as compared to O400 (54%), and the Au particle size, which is much smaller after a CO400 pre-treatment (table 1).

Reaction induced changes: Au oxidation state and Au particle size
During kinetic measurements (see Fig. 3) the CO oxidation activity over Au/CeO 2 catalysts was found to be i) lower after CO400 than after O400 and ii) strongly decreasing with time on stream for both pre-treatments (deactivation after 1000 min on stream: 36% for O400 and 46% for CO400).
To obtain more information on the Au oxidation state of the Au/CeO 2 catalysts, XANES data were recorded at different times on stream during the CO oxidation. While for the CO400 pre-treated catalyst (Fig. S1) they did not show significant changes during the reaction (only metallic Au species), the respective data after O400 pre-treatment (Fig. S2) revealed a fast decrease of the oxidic peak upon changing from N 2 to the reaction gas mixture, leading to complete disappearance in less than 5 min. Thus, independent of the pre-treatment, we always find mainly Au 0 species after a few minutes on stream. Similar observations of Au 0 NPs as dominant Au species during CO oxidation had been reported earlier for other Au catalysts, e.g., for Au/TiO 2 [4]. This strong decrease of ionic Au species in the O400 pre-treated sample occurs on a rather short time scale compared to the catalyst deactivation, which is less than 5% within the first 10 min. In contradiction to previous reports [5,7], who claimed that cationic gold species are responsible for the high CO oxidation activity of Au/CeO 2 , we conclude that the decrease in activity under present reaction conditions is not mainly related to changes in the Au oxidation state, e.g., from Au 3+ to Au 0 . This is also supported by the respective data for the CO400 pre-treated catalyst, which equally showed a significant decrease in activity.
Additional information on the Au-Au CN and Au NP size under reaction conditions were derived from EXAFS measurements (see Table S1). These data show a pronounced increase of the Au-Au CN, from 6.3 to 8.0 and from 4.1 to 5.9 for O400 and CO400 pre-treated samples, respectively, within 175 min of reaction. For the first series, the corresponding k 2 -weighted Fourier Transformations of EXAFS data are shown in Fig. 2 after different time intervals. This increase in CN reflects a decrease in the dispersion of the Au NPs from 54% to 34% (O400) and from 100% to 60% (CO400), indicating a reaction-induced particle growth (see Fig. 4). Although a direct comparison of the activities after O400 and CO400 is not possible due to the rather different catalyst state after both pre-treatments, it is obvious that for both samples the activity decreases with increasing Au particle size, similar to other Au catalysts [8]. Moreover, also on a quantitative scale the loss of activity and the loss of Au dispersion are of similar order of magnitude. The dispersion decreases by a factor of ~0.6 for both pretreatments within 175 min on stream, while the decrease in activity (within the same time) is 0.81 and 0.57 for O400 and CO400, respectively. Considering the (comparable) time scales of deactivation and Au particle sintering (in contrast to changes in the Au oxidation state) and their similar magnitudes, we conclude that under present reaction conditions reaction-induced Au particle size growth contributes significantly to the catalyst deactivation. Nevertheless, especially in the beginning of the reaction other factors such as changes in the catalyst oxidation state or site blocking by adsorbed surface species e.g., by carbonates or formate species, may contribute to the initial fast deactivation.
In summary, different pre-treatments lead to different initial states of the Au/CeO 2 catalyst. While Au NPs after O400 pre-treatment contain a significant amount of oxidic species, only metallic Au species could be detected after CO400 pre-treatment. During CO oxidation only metallic Au NPs are present, which grow in size for both pre-treatments. Accordingly, the relatively strong deactivation of Au/CeO 2 catalysts with time on stream is mainly attributed to reaction-induced Au particle size growth.


extfieldsofstudy: Chemistry Medicine

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Endophytic fungal isolate mediated biosynthesis of silver nanoparticles and their free radical scavenging activity and anti microbial studies

The present study reports that the biosynthesis of AgNPs using an endophytic fungus isolated from the ethnomedicinal plant Centella asiatica. The endophytic fungus was identified as Aspergillus versicolor ENT7 based on 18S rRNA gene sequencing (NCBI Accession number KF493864). The AgNPs synthesized were characterized by UV–visible spectroscopy, Fourier transform infra-red spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), particle size analyzer, and zeta potential measurements. The UV–Vis absorption spectra showed the peak at 429 nm which confirmed the synthesis of AgNPs. TEM analysis revealed that the AgNPs were spherical in shape with 3–40 nm in size; similar results were also obtained by Horiba particle size analyzer with 5–40 nm in size. The synthesized AgNPs were highly stable due to their high negative zeta potential value of −38.2 mV. XRD studies showed (111), (200), (220), (311), and (222) planes of the face-centered cubic (FCC) lattice, indicating the crystalline nature of the AgNPs. Selected area electron diffraction (SAED) pattern of the AgNPs showed five circular fringes which were in accordance with XRD data and confirmed the formation of high crystalline nature of AgNPs. FTIR measurements indicated the peaks at 3273, 2925, 1629, 1320, and 1020 cm−1 corresponding to different functional groups possibly involved in the synthesis and stabilization of AgNPs. The synthesized AgNPs exhibited effective free radical scavenging activity with the IC50 value of 60.64 µg/ml. The synthesized AgNPs were found to be highly toxic against both gram-positive and gram-negative bacteria and also showed a very good antifungal activity.

Introduction
Over the last decade majority of the researchers shifted their focus to nanometer-sized particles, particularly metal nanoparticles because of their controllable size and shape, ease of synthesis, and strong optical properties. The unique size-and shape-dependent properties of metal nanoparticles have significantly impacted all spheres of human life and making nanobiotechnology a promising field (Ravindran et al. 2013). Nanobiotechnology find innumerable applications due to strong optical absorbance and surface bioconjugation with molecular biomarkers related to surface plasmon resonance effect (Kreibig and Vollmer 1995). Noble metal nanoparticles particularly silver nanoparticles (AgNPs) have gained a significant interest in the recent years, owing to their remarkable optical, optoelectronic, magnetic, catalytic, and thermal properties and thus find applications as optical receptors (Karimzadeh and Mansour 2010), sensors (Cobley et al. 2009), catalysts in chemical reactions (Edison and Sethuraman 2012), signal enhancers in SERS-based enzyme Immunoassay (Chen et al. 2009), DNA detection and delivery, bio-analyzers (Ravindran et al. 2013), Cytotoxic (Asad et al. 2013), free radical scavenging (Ramamurthy et al. 2013), and antimicrobial agents (Netala et al. 2014(Netala et al. , 2015aKotakadi et al. 2013).
Several physical and chemical approaches have been reported for the biosynthesis of silver nanoparticles, including thermal decomposition (Plante et al. 2010), laser ablations (Simakin et al. 2004), gamma radiation assisted (Chol et al. 2005), polyol assisted, and chemical reduction methods (Frattine et al. 2005). Irradiation and other physical methods are not environmental friendly and also possess health risks. Hazardous chemicals, such as hydroxylamine, poly-N-vinyl pyrrolidine, polyvinyl alcohol, and sodium borohydride, are involved in the chemical approaches. The application of chemical approaches is not eco-friendly and possesses biological risks, and the toxic chemicals on the surface limit the applications of AgNPs in pharmaceutical and biomedical fields. Hence, the biological methods for the synthesis of AgNPs have gained a significant interest in the field of nanotechnology. Supramolecular complexes of biomolecules and their topographic and electrostatic properties are being widely employed in biomedical and pharmaceutical fields. Peptides to proteins, sugars to polysaccharides, isoprenes to terpenoids, polyphenols, glycosides, plant and microbial derived compounds, viral particles, etc., are being constantly explored for the biosynthesis of metal nanoparticles and novel carriers. Biological approaches are simple, rapid, and cost effective and involve the synthesis of non-toxic, clean, and biocompatible AgNPs. Fungal mediated synthesis of AgNPs has recently emerged as a new avenue in the field of nanobiotechnology. Compared to bacteria, fungi are more advantageous, because they grow at fast rates and very easy to culture, and maintain in the laboratory. Fungi being eukaryotes produce high amounts of proteins and other biomolecules. These proteins and biomolecules will often associate with nanoparticles that must be preventing the agglomeration and stabilize nanoparticles. Various fungi have been reported to synthesize AgNPs, including Alternaria alternate (Monali et al. 2009), Amylomyces rouxii (Javed et al. 2010), Aspergillus niger (Netala et al. 2015b), Coriolus versicolor (Rashmi and Varma 2009), Fusarium solani (Rafie et al. 2012), Humicola sp (Asad et al. 2013), Neurospora crossa (Longoria et al. 2011), Schizophyllum radiatum (Metuku et al. 2014), and Trichoderma viride (Fayaz et al. 2009) have been reported.
Endophytic fungi are the intriguing group of fungal species which colonise living and healthy tissues of plants. Endophytic fungi produce natural bioactive compounds which are considered to be alternative sources for plant producing bioactive compounds (Strobel et al. 2002). Exploration of endophytic fungi for the biosynthesis of AgNPs is considered as their another important application in the pharmaceutical and biomedical field. In the present study, we report the biosynthesis of AgNPs using extracellular filtrate of endophytic fungus Aspergillus versicolor ENT7 strain isolated from the healthy leaf tissues of Centella asiatica, an important medicinal plant which harbors many endophytic fungi. The AgNPs were characterized using different measurements which include UV-Vis, FTIR, XRD, TEM, particle size analyzer, and zeta potential measurements. Biomedical importance of the AgNPs was evaluated by checking for free radical scavenging and antimicrobial activities.

Isolation of endophytic fungi
Mature leaves of Centella asiatica were collected from plants grown in the Sri Venkateswara University campus, Tirupati, A.P. India. Leaves were washed under running tap water and then with teepol solution to ensure for dust free and clean. Then, leaves were washed thoroughly with sterile double distilled water (SDDW). Under aseptic conditions, leaves were surface sterilized with 10 % H2O2 and then with 80 % alcohol followed by thorough rinsing with SDDW for three to four times. Leaves were dried on sterile blotting paper and then cut into small segments. Leaf segments were placed on solidified potato dextrose agar (PDA) plates. PDA plates were incubated at 24 ± 2°C for 10 days. After 10 days, fungal mycelia were harvested and transferred onto fresh PDA plates. Pure fungal cultures were then identified by 18S rRNA gene amplification and sequencing.

DNA extraction
For the extraction of genomic DNA, 50 mg of fungal mycelia was frozen in liquid nitrogen and mechanically disrupted. The extraction of genomic DNA was carried out using Qiagen kit (USA) according to manufacturer's instructions.

PCR amplification and sequencing of 18S rRNA gene
The ITS region (ITS 1-5.8S-ITS 2) of 18S rRNA gene was amplified using fungal domain specific primers ITS1-5 0 -T CCGTAGGTGAACCTGCGG-3 0 (forward primer) and ITS4-5 0 -TCCTCCGCTTATTGATATGC-3 0 (reverse primer). Amplification was performed in a 50 ul reaction mixture containing 50 ng of template DNA, 200 lM each dNTP, 1.5 mM MgCl 2 , 20 pmol of each primer, and 0.4 U of Taq DNA polymerase in a CG palm cycler (Genetix biotech asia). The amplification cycles consisted of the initial denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 45 s, 55°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 7 min. The amplification was confirmed by running the amplified product in 1.2 % w/v agarose gel electrophoresis with ethidium bromide staining and documented by gel documentation system (Major science, UVDI). The PCR products were purified using gel extraction kit. Sequencing was carried out at MWGAG Biotech, Bangalore, India. The sequence obtained was analyzed by BLASTn and NCBI and was identified by homology search for closely related sequence. Multiple sequence alignment was carried out using Clus-talW2, and phylogenetic tree was constructed using the neighbor-joining (NJ) method.

Biosynthesis of AgNPs
The synthesis of silver nanoparticles was carried out according to the method described earlier (Jaidev and Narasimha 2010). The fungal isolate Aspergillus versicolor was cultured in 100 ml of potato dextrose broth and was incubated at 26 ± 2°C in shaking incubator (LABLINE) at a speed of 100 rpm. After 7 days of growth, the fungal biomass was harvested and washed thoroughly with SDDW to prevent the contamination of medium components. 10 g of fungal biomass was taken in 250 ml Erlenmeyer flask containing 100 ml SDDW and incubated at 28 ± 4°C for 72 h in shaking incubator at the speed of 100 rpm. After incubation, the aqueous solution was separated by filtration through Whatmann No.1 filter paper. This solution, namely, fungal filtrate, used for the synthesis of AgNPs. 100 ml of 1 mM of silver nitrate was then added to 100 ml of fungal filtrate and incubated at 28 ± 4°C for 24 h in the dark condition.

Characterization of AgNPs
The synthesis of AgNPs was confirmed by the absorbance spectrum using UV-Vis spectrometer (Analytical Technologies Ltd, India). The spectrum was recorded with a resolution of 1 nm between 200 and 700 nm. The synthesized AgNPs were purified by the centrifugation of solution of AgNPs at 15,000 rpm for 15 min thrice with continuous washing the pellet with sterile Milli Q water. The final pellet that obtained was dried in hot air oven at 50°C for 24 h, and the pure powder obtained was used for FTIR, XRD, TEM, and other studies. FTIR analysis was carried out to study the functional groups possibly involved in the synthesis and stabilization of AgNPs. FTIR spectrum (Model ALPHA interferometer) was recorded in the range of 500-4000 cm -1 with the resolution of 2 cm -1 . XRD patterns were obtained on an Ultima IV X-ray powder diffractometer (Rigaku, Tokyo, Japan) using CuKa radiation (k = 1.5406). The shape and size of the AgNPs were determined using TEM (FEI Tecnai F12, Philips Electron Optics, Holland) operated at 100 kV. Particle size and zeta potential experiments were carried out using a Horiba Nanopartica instrument.

Free radical scavenging activity
The free radical scavenging activity of the AgNPs was measured in vitro by 2, 2 0 -diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay according to the method described earlier (Mittal et al. 2006). The stock solution was prepared by dissolving 4 mg of DPPH in 100 ml of methanol and stored at 20°C. 2 ml of this solution was added to 1 ml of methanol solution containing tested samples of different concentrations (25, 50, 75, and 100 lg/ml). Ascorbic acid was used as standard. DPPH radical scavenging activity (RSA) was measured by the absorbance at 517 nm RSA % ð Þ ¼ ½ðcontrol absorbanceÀsample absorbanceÞ= control absorbance ð Þ Â 100:

Antimicrobial activity
Antimicrobial activity of the biosynthesized AgNPs was checked against both bacterial and fungal pathogens. Antibacterial activity of the AgNPs was checked against most pathogenic Staphylococcus aureus, Streptococcus pneumonia (gram positive), Pseudomonas aeruginosa, and Klebsiella pneumonia (gram negative) by employing the disc diffusion method using 24 h active cultures (Pulicherla et al. 2015). In the present study, bacterial cultures showed that the optical density (OD) value of 0.2 at 600 nm wavelength, according to McFarland calculation bacterial cell density, in this study is determined as 6 9 10 7 CFU/ml. 200 ll of bacterial inoculum was spread on nutrient agar (NA) plates. Sterile paper disc dipped in 25 ll (1 mg/1 ml) of AgNPs solution was placed on agar plates. Streptomycin was used as the standard antibiotic. NA plates were incubated at 37°C for 24 h and observed for the zone of inhibition (ZOI). Antifungal activity of the AgNPs was tested against the fungus Candida albicans and Candida nonalbicans using the disc diffusion method (Pulicherla et al. 2015). 200 ll of fungal inoculum was spread on the potato dextrose agar (PDA) plates. Sterile paper disc impregnated with 25 lL of AgNPs solution (1 mg/1 ml) was placed on the PDA plate.
Voriconazole was used as positive control. PDA plates were incubated at 25°C for 72 h to observe the inhibition zones.

Results and discussion
Fungal identification using ITS rRNA gene sequencing and phylogenetic analysis The isolated endophytic fungus appeared as whitish brown in color (Fig. 1) and was characterized by the PCR amplification of 18S rRNA gene using ITS primers. The amplified PCR product was around the size of 500bps. The Sanger's dideoxy nucleotide sequencing of amplified ITS region (ITS 1-5.8S-ITS 2) of 18 s rRNA gene resulted in 517 bp nucleotide sequence. The Blastn analyses, pairwise and multiple sequence alignment revealed 98-100 % identity with the sequences of A. versicolor strains and is designated as Aspergillus versicolor ENT 7 and has been deposited in NCBI GenBank (Accession Number KF493864). Multiple sequence alignment was carried out using ClustalW2 with default parameters. Phylogenetic tree was constructed by the neighbour-joining (NJ) method with nucleotide pairwise genetic distance corrections. Bootstrap test of phylogeny was carried out to check the reliability of tree topology as a percentage of 1000 replications. All branches with \70 % bootstrap support were collapsed (Fig. 2).

Biosynthesis of AgNPs
The synthesis of AgNPs by fungal filtrate of A. versicolor ENT 7 was investigated. After 24 h of incubation, light yellow color of the reaction mixture turned to dark brown color (Fig. 3). The color change indicated the reduction of silver ions (Ag ? ) into AgNPs (Ag 0 ). It is well known that AgNPs exhibit a dark brown color in aqueous solution due to surface plasmon resonance (SPR). The fascinating and bright colors of metal nanoparticles are related to the localized surface plasmon resonance (SPR). The applications of metal nanoparticles have broadened due to surface bioconjugation with molecular biomarkers strong optical absorbance related to SPR (Kreibig and Vollmer 1995;Kelly et al. 2002).

UV-visible spectra analysis
The UV-visible spectra of synthesized AgNPs (Fig. 4) showed characteristic SPR peak at 429 nm. SPR peak at 429 nm confirmed the synthesis of AgNPs and is also responsible for the spherical shape of AgNPs synthesized. The shape of the synthesized AgNPs is further confirmed by the TEM analysis. Different metabolites and proteins present in the extracellular fungal filtrate could be responsible for the synthesis and stabilization of AgNPs, which is further confirmed by the FTIR analysis.

FTIR spectra analysis
The FTIR analysis (Fig. 5) showed intensive peaks at 3273, 2925, 1629, 1320, and 1020 cm -1 . The peak at 3273 cm -1 corresponding to N-H stretching of the secondary amide of the protein and the peak at 2925 cm -1 corresponding to C-H stretching of methylene groups of the protein (Bozanic et al. 2010). Peak at 1629 cm -1 corresponding to -CO stretch of amide I band of proteins (Fayaz et al. 2009). The sharp peak at 1320 cm -1 can be assigned to C-N stretching vibrations of aromatic amines (Rashmi and Varma 2009). The extracellular proteins present in the fungal filtrate could be responsible for the reduction of silver ions, Ag ? into nanosize silver particles, Ag 0 . These proteins have strong ability to bind silver nanoparticles, acts as capping agents and thus provided the stability to them. These results are consisted with the earlier reports for the fungal mediated synthesis of AgNPs (Jaidev and Narasimha 2010;Rafie et al. 2012: Asad et al. 2013. The peak at 1020 cm -1 can be assigned to C-OH of the phenols, supporting the participation of polyphenols, such as flavanoids and triterpenoids, in the reduction of Ag ? into Ag 0 (Litvin et al. 2012;Litvin and Minaev 2013). FTIR analysis revealed that the polyphenols could act as bioreducing agents, while proteins could play a dual role as bioreducing and stabilizing agents.

XRD analysis
The XRD pattern of the biosynthesized AgNPs was shown in Fig. 6. XRD pattern revealed four diffraction peaks at 38.31°, 44.58°, 64.71°, 77.71°, and 81.92°could be indexed to (111), (200), (220), (311), and (222) planes, respectively. All the peaks corresponding to face-centered cubic (FCC) lattice phase of silver and were consisted with the standard JCPDS (File No 87-0719) data. Thus, XRD pattern obtained for the AgNPs that revealed the crystalline nature of AgNPs with FCC phase and consisted with many earlier reports of the AgNPs synthesized by fungal extracts (Rafie et al. 2012;Asad et al. 2013).

TEM analysis
TEM analysis was performed to determine the size and shape of the biosynthesized AgNPs. TEM micrographs were represented at different resolutions (Fig. 7a, c). TEM micrographs obtained shows that AgNPs were roughly

Particle size determination
The particle size of the biosynthesized AgNPs was detected by intensity and laser diffraction. After the analysis of AgNPs, it has been revealed that the particles synthesized with extracellular fungal filtrate are in the range of 5-40 nm in size. The average size (hydrodynamic radius) of AgNPs was found to be 15.5 nm (Fig. 8). The results are similar to that of TEM results indicating that the size of the AgNPs is uniform in nature. The biosynthesized AgNPs are poly-dispersed in nature. The stability was further confirmed by zeta potential of the particles.

Zeta potential of AgNPs
The electrostatic repulsive force between the nanoparticles depends on the charge which is present on the surface of the nanoparticles. In the present study, the very high negative value of zeta potential confirms the repulsion among the particles and thereby increases the stability of the formulation, and prevents the nanoparticles from agglomeration in the medium, leading to the long-term stability. The zeta potential of the AgNPs of fungal filtrate was found to be -38.2 mV (Fig. 9). Based on the above results, it is concluded that the AgNPs synthesized with A. versicolor fungal filtrate were very stable.

Free radical scavenging activity
Free radical scavenging activity of the AgNPs was checked by DPPH radical scavenging assay. This method is dependent on the reduction of DPPH radical to the nonradical form DPPH-H in the presence of a hydrogen-donating antioxidant. The radical scavenging activity and IC 50 values of all the tested samples were represented in Table 1. The radical scavenging activity was increased with the increasing concentrations of tested samples. The radical scavenging activity for the AgNPs at 100 lg/ml was determined as 60.04 % which is close to 68.52 % obtained for the standard ascorbic acid at the same concentration. However, the radical scavenging activity for the fungal filtrate was found to be 41.19 %. Thus, the biosynthesized AgNPs were proved to be very effective scavengers. The radical scavenging activity was also expressed as IC 50 (inhibitory concentration of the test sample to scavenge 50 % radicals). IC 50 value for the AgNPs is found to be 60.64 lg/ml, while same for the fungal filtrate was found to be 144.84 lg/mL. The lower IC 50 value means high radical scavenging activity of the sample. Thus, the biosynthesized AgNPs found to be very good antioxidants. Free radical scavenging activity of the AgNPs is mainly due to hydrogen-donating molecules, such as polyphenols, flavonoids, terpenoids, proteins, and other biomolecules, that are present in the colloidal solution of AgNPs or the secondary metabolites, proteins, and other biomolecules that are involved in the synthesis and stabilization of AgNPs. Silver nitrate could not show any free radical scavenging activity in our study. Silver nitrate does not have any hydrogen to participate in the scavenging of free radical DPPH. There were no reports also for the free radical scavenging activity of silver nitrate. Free radical scavenging activity studies of AgNPs biosynthesized in this study are in line with many earlier reports (Ramamurthy et al. 2013). Antioxidants are the compounds which protect cells against the cellular damaging effects of reactive oxygen species (ROS), such as hydroxyl radicals, peroxy radicals, peroxylnitrile, super oxide, and singlet oxygen. The lower concentration of antioxidants or the imbalance between antioxidants and ROS results in oxidative stress which could be linked to inflammation, neurodegenerative disorders, atherosclerosis, aging, and cancer (Ramamurthy et al. 2013). Based on our results, it is revealed that the AgNPs can be used as ingredients in the antioxidant formulations in pharmaceutical or biomedical field. The excellent radical scavenging activity of the AgNPs allowed them to be employed in the numerous applications, such as food preparation ingredients, food

Antimicrobial activity
After incubation for 24 h, growth inhibition (ZOI) was observed around discs impregnated with AgNPs and streptomycin. Figure 10 shows the strong antibacterial activity of AgNPs against Staphylococcus aureus, Streptococcus pneumonia (gram negative), Pseudomonas aeruginosa, and Klebsiella pneumonia (gram negative); maximum ZOI was observed against Pseudomonas aeruginosa (18.5 mm) followed by Klebsiella pneumonia (13.6 mm), Staphylococcus aureus (12.1 mm), and Streptococcus pneumonia (11.8 mm). The results revealed that AgNPs showed strong inhibition against gram-negative  bacteria compared to gram-positive bacteria. Not only antibacterial activity, AgNPs also showed a very good antifungal activity against most pathogenic Candida albicans and Candida nonalbicans and formed the ZOI values of 12.2 and 13.6 mm, respectively. The results revealed that the growth inhibitory activity of AgNPs against fungal pathogens can be increased further by increasing the concentration of AgNPs as these are clean, non-toxic, and biocompatible, but the drugs will be toxic and non-biocompatible at higher concentrations. The results of the antifungal activity were consisted with many earlier reports (Kim et al. 2009;Rafie et al. 2012). The antimicrobial activity of AgNPs is probably the sum of distinct mechanisms. AgNPs react with thiol (-SH) groups of vital enzymes/proteins and inactivates them, in turn stop the progress of cellular metabolisms which leads to cell death (Morones et al. 2005;Netala et al. 2015b). AgNPs form strong interaction with proteins of the proton pump and phospholipid portion of the bacterial membrane resulted in the dissipation of membrane proton gradient causing disruption of bacterial membranes (Kotakadi et al. 2014). AgNPs form strong interaction with respiratory chain enzymes which causes uncoupling of electron transport chain (ETC) and oxidative phosphorylation (OP) as a result cell death occurs (Morones et al. 2005;Feng et al. 2000). Proteomic analysis revealed that the exposure of bacteria to nanosilver resulted in alterations in the expression of envelope and HSPs, as a result AgNPs can penetrate and can disrupt the membranes and leads to leakage of protons, potassium ions and reducing sugars and eventually leads to death (Lok et al. 2006). The excellent antimicrobial activity of the AgNPs allows them to be used in ingredients of topical ointments to prevent bacterial and fungal infections against open and burn wounds, AgNPs embedded water purifier equipments, preparation of injection molded plastic products, and employed in coating-based applications, including air ducts and counter tops. The important advantages of AgNPs-based antimicrobial agents are their biocompatibility, health and environmental safety, and their excellent stability.

Conclusion
An endophytic fungal isolate, ENT7 was isolated from the leaves of Centella asiatica and was identified as Aspergillus versicolor based on the molecular characterization of 18 s rRNA gene. A simple and environmental benign approach for the biosynthesis of AgNPs using extracellular filtrate of Aspergillus Versicolor ENT7 as reducing agent has been reported. The synthesized AgNPs were well dispersed with the average size of 15.5 nm, spherical in shape, high crystalline in nature, and highly stable with zeta potential of -38.2 mV. The biosynthesized AgNPs exhibited a very good antioxidant and antimicrobial activity and clearly indicated their pharmaceutical and biomedical importance. Fig. 10 Antibacterial activity of the AgNPs against S. aureus (a), S. pneumonia (b), P. aeruginosa and d K.
Pneumonia. No inhibition zones formed by fungal filtrate (i) Very well inhibition zones were observed by Streptomycin (ii) and by AgNPs (iii)


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Practical Synthesis of Axially Chiral Dicarboxylates via Pd-Catalyzed External-CO-Free Carbonylation

We have developed a safe and practical synthetic method for preparing axially chiral diphenyl dicarboxylates using Pd-cat-alyzed external-CO-free carbonylation with phenyl formate as a CO surrogate. Optimized conditions consisted of axially chiral [1,1 ′ -binaphthalene]-2,2 ′ -diyl ditriﬂate and its conge -ners, each easily prepared from commercially available enantiomerically pure diols, Pd(OAc) 2 , 1,3-bis(diphenylphosphino)-propane, ethyldiisopropylamine, and no solvent. To demon-strate the potential utility of these products, this method was conducted on gram-scale and the phenyl ester products were converted to other useful compounds, and both processes were carried out without difﬁculty.

Axially chiral compounds have found wide applications as ligands and organocatalysts in asymmetric organic reactions. [1][2][3][4][5] Their chiral backbones effectively construct an asymmetric environment around the molecules, enabling catalytic transformations, often with high enantioselectivity. A number of biaryl and spirocyclic chiral ligands are commercially available from multiple suppliers, and many recent papers mentioning the development of novel axially chiral ligands and organocatalysts indicate increasing demand and potential of axially chiral compounds for asymmetric catalysis.
To circumvent the use of toxic CO gas, alternative methods using CO surrogates have attracted much attention in recent years. [24][25][26][27] We recently reported a series of external-CO-free carbonylation of haloarenes utilizing phenyl formate or other formic acid derivatives as CO surrogates. [28][29][30][31][32] These CO surrogates can generate CO under mild conditions in a closed reaction vessel, and this CO is consumed during carbonylation in a highly safe and efficient manner. We hypothesized that this external-CO-free carbonylation would be a powerful tool to directly afford not only [1,1′-binaphthalene]-2,2′dicarboxylates, but also other axially chiral dicarboxylates from the corresponding ditriflates without handling toxic CO gas or strongly basic compounds. We report herein a practical synthetic method for various axially chiral dicarboxylates in an enantiomerically pure form using phenyl formate as a CO surrogate.
We first optimized the reaction conditions for Pd-catalyzed phenoxycarbonylation of (R)-[1,1′-binaphthalene]-2,2′-diyl ditriflate (1), which was easily prepared from enantiomerically pure (R)-1,1′-bi-2-naphthol, 33) using phenyl formate as a CO surrogate, as shown in Table 1. Following Takaya's procedure with CO gas, 19) we chose Pd(OAc) 2 as a Pd source, 1,3-bis(diphenylphosphino) propane (DPPP) as a ligand, ethyldiisopropylamine as a base, and dimethyl sulfoxide (DMSO) as a solvent. However, only a trace amount of the desired phenyl ester 2 was obtained under the external-CO-free conditions (entry 1). Solvent screening (entries 2-5) yielded N,Ndimethylformamide (DMF) as the solvent of choice (entry 5), though a certain amount of monocarbonylated by-product 3 was still present. A review of other phosphine ligands at slightly raised temperatures (entries [6][7][8][9][10][11] showed that DPPP was the best ligand. 34) Notably, subtle differences in the ligand structure significantly influenced the outcome of the reaction. Xantphos and tri(tert-butyl) phosphine, which were effective in the aryloxycarbonylation of haloarenes, 28,29,31,32) were both not suitable for the reaction of 1 with phenyl formate, probably owing to steric repulsion between the substrate and the substituents of these ligands. Decreasing of the amount of DPPP improved the yield (entries 12, 13), probably because   of the suppression of excess ligation to the Pd center. We also tested other bases (entries [14][15][16][17][18][19][20]; bulky amines such as ethyldiisopropylamine (entry 12) and 2,2,6,6-tetramethylpiperidine (TMP) (entry 16) were found to be most effective. This tendency is similar to that found in Takaya's studies. 19) Further studies revealed that the yield could be improved by removing the solvent (entry 21), increasing the amounts of phenyl formate and base, raising the temperature to 120°C (entry 22), and increasing the reaction time (entry 23). It was confirmed that no racemization occurred during this carbonylation: enantiomeric purity of 2 obtained from ditriflate 1 (>99% ee) was measured to be >99% ee by chiral HPLC.
Under the optimized conditions (Table 1, entry 23), various enantiomerically pure ditriflates bearing axial chirality were examined for phenoxycarbonylation ( Table 2). Substituents at the 6 and 6′ positions of the 2,2′-binaphthyl scaffold did not adversely affect the carbonylation reaction; they instead afforded the desired dicarboxylates 4-7 in good yield. Notably, base-sensitive ester moieties tolerated the reaction conditions to give 7. The only exception was the derivative bearing nitro groups (for compound 8). The reaction proceeded smoothly with a partially saturated substrate to give 9 in high yield. Unfortunately, ditriflate with methyl groups on the 3 and 3′ positions failed to afford product 10. Instead, the monophenoxycarbonylated compound was obtained in modest yield. This carbonylation method was also applicable to the synthesis of spirocyclic dicarboxylate 11, which was produced in good yield. Methyl-substituted biphenyl 12, albeit in racemic form, was also synthesized by this phenoxycarbonylation.
The reaction was tested on gram-scale to evaluate its practicality (Chart 2). To our delight, more than 1 g of 2 was obtained in a single reaction which was conducted in a simple two-necked flask equipped with an empty balloon without any difficulties.
Owing to their relative electrophilicity when compared with alkyl esters, the phenyl ester groups could be easily derivatized to other functional groups using nucleophiles (Chart 3). Hydrolysis of 2 afforded dicarboxylic acid 13, and reduction using lithium aluminum hydride gave diol 14 in high yield. Bis(2-(trimethylsilyl) ethyl) ester 15, a useful protected analogue of dicarboxylic acid, 8,35) was obtained via ester exchange. Moreover, 2 was converted into diamide 16 in good yield.
In summary, a novel and practical synthetic method to produce axially chiral dicarboxylates was developed using Pdcatalyzed external-CO-free carbonylation with phenyl formate as a CO surrogate. Binaphthyl and other ditriflates, which were readily prepared from the corresponding enantiomerically pure diols, were directly converted to dicarboxylates, and they can be used as potential intermediates of chiral ligands and organocatalysts. Without using a strong base or CO gas from a cylinder, the reaction can be conducted in a safe and practical manner. Furthermore, gram-scale synthesis of dicarboxylates and subsequent derivatization are readily feasible. Since various axially chiral diols are commercially available in the enantiomerically pure form, the present reaction can offer a general, rapid access to axially chiral dicarboxylates that would help accelerate the development of more efficient asymmetric transformations.


extfieldsofstudy:
Chemistry
Medicine
Computer Science

s2fieldsofstudy: Chemistry Biology

text:
Label-Free Detection of Human Glycoprotein (CgA) Using an Extended-Gated Organic Transistor-Based Immunosensor

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.

Introduction
Chromogranin A (CgA), which is one of the glycoproteins, exists in secretory granules [1]. CgA is processed into small peptides, several of which affect the secretory function of the parent cells [2]. CgA is co-stored and co-released with catecholamines from storage granules in the adrenal medulla, or with the parathyroid gland [2]. Serum CgA is generally applied as an early biomarker for monitoring in several diseases, such as endocrine tumors, heart failure, hypertension, and neurodegenerative/neuropsychiatric diseases [3][4][5][6]. In addition, salivary CgA is also known as a marker of psychophysical stress [7,8], such as exposure to situations of anxiety [9][10][11] and depressive mood [12,13]. For these reasons, the monitoring of CgA levels is very important for the management of our health conditions.
In general, immunoassays, such as radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), fluorescent and chemiluminescent immunoassays, have been developed for protein determination [14]. Although such immunoassays have high selectivity and sensitivity, they are relatively complicated due to the necessity of labels and relatively large-sized equipment. Therefore, these methods are not amenable to easy and low-cost testing of glycoproteins. Among biosensor platforms such as quartz crystal microbalance (QCM), field-effect transistor (FET) and surface plasmon resonance (SPR), FET-based sensors are one of the promising approaches owing to their simplicity and capability of highly sensitive detection based on portable/compact sensor systems. Accordingly, carbon nanomaterial field-effect transistor (FET)-based biosensors for the detection of CgA were preliminary studied [15]. However, the FET-based biosensors for CgA detection are in their early stages.
Organic field-effect transistors (OFETs) are one of the best candidates for chemical/biosensor devices because of their low-cost processability and mechanical flexibility [16]. The development of OFET-based biosensors has just begun to bloom in recent years. For example, OFET-based sensors have been applied to the detection of biologically active substances such as proteins [17][18][19][20], saccharides [21], etc. Especially, we have developed an OFET-based immunoassay to detect immunoglobulin [19,20]. Toward that end, we herein demonstrate the label-free immunosensor device based on an OFET for glycoproteins such as human CgA (hCgA).

Materials and Methods
The OFET-based immunosensor was composed of two components; the stable operation of the fabricated device was achieved by isolating of the drive unit (the OFET) and the detection portion (the extended-gate electrode). The details of the device fabrication were described in our previous report [22].
PBTTT (poly{2,5-bis(3-hexadecylthiophene-2-yl)thieno[3,2-b]thiophene} [23])-based FET with the tetradecylphosphonic acid (C 14 -PA)/AlOx dielectric layer [24] was designed for an accurate electrical detection of CgA under ambient conditions ( Figure 1). All electrodes were deposited on a substrate via thermal vacuum evaporation (30 nm in thickness). To improve the device stability, the OFET part was fully covered with an amorphous fluorinated polymer (Cytop ® CTL-809M, Asahi Glass, Tokyo, Japan) by spin-coating. Additionally, an extended-gate electrode consists of the deposited Au (50 nm in thickness) modified with a self-assembled monolayer (SAM) of 5-carboxy-1-pentanethiol (CPT, Dojindo Laboratories, Kumamoto, Japan). these methods are not amenable to easy and low-cost testing of glycoproteins. Among biosensor platforms such as quartz crystal microbalance (QCM), field-effect transistor (FET) and surface plasmon resonance (SPR), FET-based sensors are one of the promising approaches owing to their simplicity and capability of highly sensitive detection based on portable/compact sensor systems. Accordingly, carbon nanomaterial field-effect transistor (FET)-based biosensors for the detection of CgA were preliminary studied [15]. However, the FET-based biosensors for CgA detection are in their early stages. Organic field-effect transistors (OFETs) are one of the best candidates for chemical/biosensor devices because of their low-cost processability and mechanical flexibility [16]. The development of OFET-based biosensors has just begun to bloom in recent years. For example, OFET-based sensors have been applied to the detection of biologically active substances such as proteins [17][18][19][20], saccharides [21], etc. Especially, we have developed an OFET-based immunoassay to detect immunoglobulin [19,20]. Toward that end, we herein demonstrate the label-free immunosensor device based on an OFET for glycoproteins such as human CgA (hCgA).

Materials and Methods
The OFET-based immunosensor was composed of two components; the stable operation of the fabricated device was achieved by isolating of the drive unit (the OFET) and the detection portion (the extended-gate electrode). The details of the device fabrication were described in our previous report [22].

Characterization of the Fabricated Device
In a previous report, we characterized the modification of the Au extended-gate electrode using various techniques [19]. We also measured the electrical characteristics of the OFET device. The fabricated OFET can be operated under low voltages (<3 V) [22], indicating that the device can be applied to immunosensing in aqueous media.

Label-Free Electrical Detection of hCgA
After immobilizing the biotin-tagged anti-CgA antibody on the extended-gate electrode, we immersed the electrode into the PBS solution with the hCgA, and then we measured the electrical characteristics of the connected OFET. The electrical characteristics of the fabricated OFET in titration experiments were measured by a source meter (2636B, Keithley, Cleveland, OH, USA). To estimate the output signal (i.e., the threshold voltage, V TH ) from transfer characteristics, the following equation [25] was used: where I DS is the drain current, W and L are the channel width (1000 µm) and length (50 µm), µ is the field-effect mobility, C is the capacitance of the gate dielectric (~0.8 µF/cm 2 ) [26], and V GS is the gate voltage. In addition, the electrical response (∆V TH ) induced by the charged analyte (the charge density, Q) can be expressed in the following equation: meaning that the shift of the transfer characteristics can be affected by the charge where the captured analyte is bound on the extended-gate electrode [27,28]. Figure 2a shows that the transfer characteristics of the OFET device were positively shifted upon the addition of the increased concentration of the hCgA. The reason for the positive shift of the extended-gated FET is generally explained by the interfacial potential shift at the extended-gate/electrolyte interface [29]; these results suggest that the negatively charged hCgA was captured on the extended-gate electrode [30]. In Figure 2b, we plotted the relationship between the concentration of the hCgA and the electrical responses of the OFET device (changes in the threshold voltage). Because an extended-gated FET is one of the potentiometric-based electrochemical sensors [31], the threshold voltage is directly reflected in the interfacial potential shift at the extended-gate/electrolyte interface.
The addition of hCgA over 5.0 µg/mL induced a saturated response, most likely due to the anti-CgA being fully bound to the hCgA (Figure 2b). The limit of detection (LOD) [32] and the limit of quantification (LOQ) [32] were estimated to be 0.31 µg/mL (~6 nM) and 1.0 µg/mL (~19 nM), respectively, meaning that the sensitivity was comparable to that of a carbon nanotube (CNT)-based FET sensor device for CgA [15]. More importantly, the fabrication process for our designed OFET was much simpler than that for the photolithographic technology-based CNT-FET (see Materials and Methods section). Although the sensitivity for the hCgA in conventional immunoassays (e.g., ELISA) was higher than our fabricated immunosensor [33], the protocol for the label-free detection based on the OFET is much simpler. Additionally, the assay time of the fabricated OFET immunosensor (~0.5 h) is quicker than an ELISA method (~2.5 h) [34]. Furthermore, the sensitivity of our fabricated OFET could be improved by connecting the device with OFET-based amplifier circuits [35]. the OFET is much simpler. Additionally, the assay time of the fabricated OFET immunosensor (~0.5 h) is quicker than an ELISA method (~2.5 h) [34]. Furthermore, the sensitivity of our fabricated OFET could be improved by connecting the device with OFET-based amplifier circuits [35].

Selectivity
To investigate the selectivity of the fabricated OFET immunosensor, we also measured various analytes using the same device. We chose IgA [36], amylase [37,38], and myeloperoxidase [39] as the analytes (they are regularly contained in human saliva). As a result, a selective response to the hCgA was observed (Figure 3). Because of its relatively large protein size, the observed weak response to the addition of IgA might be derived from a physical adsorption onto the extended-gate electrode. The utilized monoclonal antibody (clone code: LK2H10) has high specificity for the hCgA [40], meaning that the response to hCgA is derived from the immune interaction between the hCgA and the extended-gate electrode immobilized with the anti-CgA antibody.

CgA Detection in Artificial Saliva
Based on previous reports [7,8], the CgA level in human saliva might be applied as a psychological stress marker. Therefore, for evaluation of the feasibility of the OFET-based immunosensor in practical applications, we finally attempted the detection of hCgA in a commercially available artificial saliva (Saliveht ® Aerosol, purchased from Teijin Pharma Co. Ltd., Tokyo, Japan) containing many chemical species (NaCl, KCl, CaCl2, MgCl2, and K2HPO4) for medication. As a result, the threshold voltage of the fabricated OFET device was positively shifted by increasing the hCgA level (Figure 4), suggesting that the fabricated OFET could be used for the

Selectivity
To investigate the selectivity of the fabricated OFET immunosensor, we also measured various analytes using the same device. We chose IgA [36], amylase [37,38], and myeloperoxidase [39] as the analytes (they are regularly contained in human saliva). As a result, a selective response to the hCgA was observed (Figure 3). Because of its relatively large protein size, the observed weak response to the addition of IgA might be derived from a physical adsorption onto the extended-gate electrode. The utilized monoclonal antibody (clone code: LK2H10) has high specificity for the hCgA [40], meaning that the response to hCgA is derived from the immune interaction between the hCgA and the extended-gate electrode immobilized with the anti-CgA antibody. the OFET is much simpler. Additionally, the assay time of the fabricated OFET immunosensor (~0.5 h) is quicker than an ELISA method (~2.5 h) [34]. Furthermore, the sensitivity of our fabricated OFET could be improved by connecting the device with OFET-based amplifier circuits [35].

Selectivity
To investigate the selectivity of the fabricated OFET immunosensor, we also measured various analytes using the same device. We chose IgA [36], amylase [37,38], and myeloperoxidase [39] as the analytes (they are regularly contained in human saliva). As a result, a selective response to the hCgA was observed (Figure 3). Because of its relatively large protein size, the observed weak response to the addition of IgA might be derived from a physical adsorption onto the extended-gate electrode. The utilized monoclonal antibody (clone code: LK2H10) has high specificity for the hCgA [40], meaning that the response to hCgA is derived from the immune interaction between the hCgA and the extended-gate electrode immobilized with the anti-CgA antibody.

CgA Detection in Artificial Saliva
Based on previous reports [7,8], the CgA level in human saliva might be applied as a psychological stress marker. Therefore, for evaluation of the feasibility of the OFET-based immunosensor in practical applications, we finally attempted the detection of hCgA in a commercially available artificial saliva (Saliveht ® Aerosol, purchased from Teijin Pharma Co. Ltd., Tokyo, Japan) containing many chemical species (NaCl, KCl, CaCl2, MgCl2, and K2HPO4) for medication. As a result, the threshold voltage of the fabricated OFET device was positively shifted by increasing the hCgA level (Figure 4), suggesting that the fabricated OFET could be used for the

CgA Detection in Artificial Saliva
Based on previous reports [7,8], the CgA level in human saliva might be applied as a psychological stress marker. Therefore, for evaluation of the feasibility of the OFET-based immunosensor in practical applications, we finally attempted the detection of hCgA in a commercially available artificial saliva (Saliveht ® Aerosol, purchased from Teijin Pharma Co. Ltd., Tokyo, Japan) containing many chemical species (NaCl, KCl, CaCl 2 , MgCl 2 , and K 2 HPO 4 ) for medication. As a result, the threshold voltage of the fabricated OFET device was positively shifted by increasing the hCgA level (Figure 4), suggesting that the fabricated OFET could be used for the selective detection of the hCgA contained in biological fluids such as saliva. The observed response denotes a similar tendency as the titration experiment in a PBS solution (Figure 2). In an artificial saliva experiment, the LOD and the LOQ were estimated to be 0.11 µg/mL (~2 nM) and 0.38 µg/mL (~7 nM), respectively. selective detection of the hCgA contained in biological fluids such as saliva. The observed response denotes a similar tendency as the titration experiment in a PBS solution (Figure 2). In an artificial saliva experiment, the LOD and the LOQ were estimated to be 0.11 µg/mL (~2 nM) and 0.38 µg/mL (~7 nM), respectively.

Conclusions
In conclusion, we have successfully demonstrated an electrical label-free immunosensing of hCgA using an extended-gated OFET device immobilized with anti-CgA antibody. The detection limit (0.11 µg/mL in the artificial saliva) was comparable to that of the reported FET-based sensor [15]. Although the estimated LOD value (~2 nM) is seven times higher than the physiological level of hCgA (~0.3 nM [41]), the sensitivity could be improved by FET-based electrical circuits [35,42]. Additionally, our fabricated device is much simpler and more rapid than the conventional immunoassay (e.g., ELISA) for hCgA detection (the assay time is ~30 min). In previous research, we succeeded in decreasing the assay time (~1 min) for a phosphoprotein using by an OFET sensor functionalized with an "artificial receptor" [43]. Therefore, further improvement of the assay time for hCgA could be achieved with similar approaches (i.e., an OFET sensor functionalized with an artificial receptor for hCgA). Hence, we believe that our preliminary results on the OFET-based immunosensors could pave the way for a new approach in the monitoring of hCgA levels for human health conditions in daily life.

Conclusions
In conclusion, we have successfully demonstrated an electrical label-free immunosensing of hCgA using an extended-gated OFET device immobilized with anti-CgA antibody. The detection limit (0.11 µg/mL in the artificial saliva) was comparable to that of the reported FET-based sensor [15]. Although the estimated LOD value (~2 nM) is seven times higher than the physiological level of hCgA (~0.3 nM [41]), the sensitivity could be improved by FET-based electrical circuits [35,42]. Additionally, our fabricated device is much simpler and more rapid than the conventional immunoassay (e.g., ELISA) for hCgA detection (the assay time is~30 min). In previous research, we succeeded in decreasing the assay time (~1 min) for a phosphoprotein using by an OFET sensor functionalized with an "artificial receptor" [43]. Therefore, further improvement of the assay time for hCgA could be achieved with similar approaches (i.e., an OFET sensor functionalized with an artificial receptor for hCgA). Hence, we believe that our preliminary results on the OFET-based immunosensors could pave the way for a new approach in the monitoring of hCgA levels for human health conditions in daily life.