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Materials Science
Medicine

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Thermally stable hybrid polyarylidene(azomethine-ether)s polymers (PAAP): an ultrasensitive arsenic(III) sensor approach

ABSTRACT A new category of thermally stable hybrid polyarylidene(azomethine-ether)s and copolyarylidene(azomethine-ether)s (PAAP) based on diarylidenecycloalkanones has been synthesized using solution polycondensation method. For potential cationic sensor development, a thin layer of PAAP onto a flat glassy carbon electrode (GCE, surface area: 0.0316 cm2) was prepared with conducting nafion (5%) coating agent to fabricate a sensitive and selective arsenic (III) [As3+] ion in short response time in neutral buffer system. The fabricated cationic sensor was measured the analytical performances such as higher sensitivity, linear dynamic range, detection limit, reproducibility, and long-term stability towards As3+ ions. The sensitivity and detection limit were calculated as 2.714 μAμM−1cm−2 and 6.8 ± 0.1 nM (SNR of 3; 3N/S) respectively from the calibration curve. This novel approach can be initiated a well-organized route of an efficient development of heavy selective arsenic sensor for hazardous pollutants in biological, environmental, and health care fields. Real sample analysis for various environmental sample was performed with PAAP-modified-GCE.

Introduction
Hybrid materials have been attracted researchers from chemistry and engineering back ground last decade for the fascinating properties related to the combination of both inorganic and organic building blocks via blending or covalent bonding [1]. Amazing and interesting new behaviors have been generated due to the combination of two or more building blocks through the mechanical blending and bonding interaction. Hybrid polymers are a well-known class of polymers that can be designed by carrying two or more different functional groups which related to specific groups of normal types of polymers such as polyamines, polyesters, polyketones, polyethers, polyurethanes, polystyrene, PMMA, polysilazane, polyacrylamide, polyarylidenes, etc. Last few decades, many research works related to the synthesis and characterization of different types of hybrid polymers have been reported [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Two types of hybrid polymers are reported till now, the first type is organic to organic hybridizations while the second type is inorganic to organic hybridizations. Moreover, hybrid polymers are considered as one of the most popular types of synthetic polymers and copolymers in recent years due to of their new characteristics and specifications that are completely different from both original polymers characterizations. Besides that, the properties of hybrid polymers are also investigated by the chemical capability of the singular components and their way of interaction [16,17]. The target hybrid aromatic polyarylidene(azomethine-ether) is an important type of such polymers. These polymers and copolymers can carry characters of features of components such as polyarylidenes, polyazomrthines, and polyethers.
Polyazomethines base on aromatic or heteroaromatic nucleus have been comprehensively examined in the literature long time ago due to its fantastic advantages. These include:, nonlinear optical properties, semiconducting properties, high thermal & mechanical stabilities over wide range of applications, environmentally stable and a moderate antibacterial effect. In addition to polyazomethines have a great ability to change its structure onto complex formation with different metal ions and protonated form with different acids [18][19][20]. Polyazomethines can easily fabricated as convenient materials for electronic devices and micro chips. Despite all of these above-mentioned features, polyazomethines still suffer from high melting temperatures and pour solubility in common organic solvents. Such problems make them obstinate for industrial diversion using traditional methods [21][22][23]. CONTACT  Polyarylidenes are another class of synthetic polymers that can be produced through the formation of arylidene linkage in the polymers main chains. These kind of polymers almost have enjoyable properties which can be applied in a variety of industrial fields. Adequate number of arylidene polymers are found in the literature holding conducting ability, liquid crystal properties, inhibitive corrosion protection, thermal stabilities, attractive morphology, biological screening in some cases and other properties [24][25][26][27][28][29][30][31][32]. So far, aromatic polyethers are very common high performance class of synthetic polymers which can be easily prepared through the formation of aryl ether linkage as a corner stone in the polymer chain as well as the new ether bond can also treated as the main polymer formulation response. As reported aromatic nucleophilic substitution reaction is the common procedure that extremely applied for such polymerization. Aromatic dihalide molecules invigorated by a high electron deficiency groups are chaired with dihydroxy molecules in order to form the targeted polyethers [33,34]. The presence of ether bonds into the macromolecular back bone with aromatic substituents afford polymers easily treated. Consequently, the polymers display superior melt processing behaviors, solubility, adhesion, hydrolytic stability and thermal oxidative. Furthermore, it can also show moderate high glass transition temperatures and tough mechanical properties as well as liquid crystalline properties which may lead them to be applied in microelectronics fabrications and other applications [35][36][37]. Polyethers show also great ability for film production that can be used in ulta-filtration membranes gas separation and aerospace vehicles [38].
Arsenic pollution affects regions in all corners of the world. Among others, contaminated areas include Argentina, Bangladesh, China, India, Mexico, Myanmar, Nepal, Pakistan, Vietnam, and parts of the USA [39]. Specifically, contamination of aquifers in Bangladesh is deemed most serious; consequently, a great deal of research efforts and field studies concerning arsenic contamination and mitigation have been focused on this region. Arsenic poisoning incurred from chronic exposure to high levels of arsenic is referred to as arsenicosis. Symptoms of this condition include skin lesions and hard patches on the palms of hands and soles of feet; skin and internal organ cancers; diseases of blood vessels in the legs; and also diabetes, high blood pressure, and reproductive disorders [40]. The current World Health Organization (WHO) maximum contamination limit (MCL) for arsenic in drinking water, defined in 1993, is 10 μg/L [41]. In solution, arsenic is readily converted from one species to another via chemical and biological redox pathways. Since arsenic speciation determines both its bioavailability and its potency as a toxin, there is much interest in the literature for speciation studies of arsenic. Generally speaking, these studies are difficult and expensive to carry out, and so they are not 'first response measures' [42]. Various nanostructure materials modified electrode has been used to enhance the sensing performance of detection to As(III) which has an obvious connection with skin pigmentation and thickening and various types of cancers of skin, lungs, bladder, kidney, etc [43]. Sensors based on organic molecules, DNA, oligonucleotide, and protein have been developed and tested for heavy metal ions. But many of these techniques have limitations such as elevated working temperatures, lower-water solubility, nitrogen-purge buffer, time consuming, and uneconomical that cause inconvenience for As detection in biological, and environmental samples. So it is necessary to develop a simple, sensitive, and reliable technique for the identification of toxic metallic ions for environmental safety, food quality control, protection, and human being health [44]. At present, development of detection methods such as capillary electrophoresis, fluorimetry, gas and liquid chromatography, mass spectrometry, and spectrophotometry are usually consistent and perceptive, but possess limitations, expensive including time consuming and extraction, and requiring pre-concentration steps which increase the risk generation of other hazardous byproducts, and sample loss [45]. Electrochemical sensing of toxic As molecules represents a promising approach that can be used to complement already existing techniques owing to collective features such as low cost, easy instrumentation, high selective and sensitive, and significant for miniaturization [46]. Arsenic is a toxic substance, which caused various cancers and other serious diseases after longterm exposure [47]. Generally, As contamination in drinking water and groundwater is becoming a threat to global health, and as many as 140 million people worldwide may have been exposed to drinking water with As contamination levels higher than the World Health Organization's guideline of 10 ppb [48]. As(III) is one of the most harmful substances in water to human health, and its toxicity is at least as 60 times as of As(V) or organic arsenic compounds [49]. Very recently, different analytical techniques for the detection of As(III) at the trace level have been developed, such as high performance liquid chromatography (HPLC), atomic fluorescence spectrometry (AFS), inductively coupled plasma-mass spectrometry (ICP-MS) [47,50] etc. Although these analytic methods might be achieved low detection limits, but still they have some limitations such as expenses, time consuming and high skill requirements for operation. Thus, there is still a necessity to develop a simple, fast and highly sensitive analytic technique for the detection of As(III) in environment. Toxic As 3+ ion is generally severe to health and environment, therefore it is immediately required the detection by using a reliable cationic-sensor method with PAAP/GCE. The As 3+ ion by thin PAAP films on GCE was prepared and studied in details of the cationic sensors by electrochemical approaches. The easy-coating method for the construction of PAAP thin-film within binding-agents is executed for preparation of films onto GCE. In this approach, PAAP fabricated films with conducting binders (5% nafion) was utilized towards the target As 3+ analytes using reliable Current-vs-Voltage method. It was confirmed that the fabricated cationic sensor is unique and noble research work for ultra-sensitive recognition of As 3+ ions with PAAP/GCE in short response-time.

Instrumentation
All melting points reported for the monomers, premonomers and model compound are uncorrected and determined on a Gallen-kamp Melting Point apparatus with a digital thermometer type MFB-595-010M. Elemental analyses were estimated by an Elemental Analyses system GmbH, VARIOEL, V 2.3 July 1998 CHNS Mode. IR spectra were determined on IR-470, Infrared spectrophotometer, Shimadzu using the KBr pellet technique. Room temperature 1 H-NMR spectra were carried out on a varian EM-390-NMR (90 MHz) spectrometer and a GNM-LA 400-MHz NMR spectrophotometer using DMSO or CDCl 3 as deuterated solvents and in the presence of TMS as an internal reference. Mass spectra were investigated on a Jeol JMS600 mass spectrometer. I-V method (two electrodes composed onto fabricated GCE) was measured for toxic arsenic ions for PAAP/GCE by using Keithley-Electrometer from USA.

Characterization techniques
(0.5% w/v) of polymer solutions in DMSO at 30 ºC were investigated in order to measure the inherent viscosities using an Ubbelohde suspended level viscometer. Solubility character of polymers and copolymers was determined at room temperature using 0.02 g of polymer in 3-5 ml of solvent using DMSO, DMF, THF, DMA, CHCl 3 : CH 3 COCH 3 (1:1), CH 2 Cl 2 , H 2 SO 4 concentrated and HCOOH as solvents. The X-ray diffractographs of the polymers were carried out with a Philips X-ray PW1710 diffractometer, and Ni -filtered CuKα radiations. Thermogravimetric analysis (TGA) and differential thermal gravimetric (DTG) were obtained in air with TA 2000 thermal analyzer at heating rate of 10°C/min. in air. The morphological features for polymers and copolymers were estimated by a Scanning electron microscope (SEM) using a Jeol-JSM-5400 LV-SEM. The SEM sample was prepared by putting a smooth part of polymer powder on a copper holder and then coating it with a goldpalladium alloy. SEM micrographs were picked up using a Pentax Z-50P Camera with Ilford film. The images obtained using a low dose technique at accelerating voltage of 15kV. Detailed experimental procedures for monomers (1 a,b , 2 a,b & 3 a,b their sodium salts 4 a,b & 5 a,b ), model compound (6) and polymers (7 a-d & 8 a-f ) synthesis are studied and described elaborately in the Electronic Supplementary Information file (Ω).

Fabrication of GCE with PAAP
Phosphate buffer solution (PBS, 0.1 M) at pH 7.0 is prepared by mixing of equi-molar concentration of 0.2 M Na 2 HPO 4 and 0.2 M NaH 2 PO 4 solution in 100.0 mL deionize water at room conditions. GCE is fabricated with PAAP using 5% ethanolic nafion solution as a conducting binder. Then it is kept in the oven at 30.0°C for 1 hour until the film is completely dried, stable, and smooth. A cell is assembled with PAAP/Nafion/GCE and Pd-wire as a working and counter electrodes respectively. As received As 3+ solution (1.0 M) is diluted to make various concentrations (0.5 M~10.0 nM) in DI water and used as a target analyte. The ratio of current versus concentration (slope of calibration curve) is used to calculate the As 3+ sensitivity. Detection limit is evaluated from the ratio of 3N/S (ratio of Noise×3 vs. Sensitivity) from the linear dynamic range of calibration curve. Electrometer is used as a constant voltage sources for I-V measurement in simple two electrode system. Amount of 0.1M PBS was kept constant in the beaker as 5.0 mL throughout the chemical investigation. The PAAP is fabricated and employed for the detection of As 3+ ions in liquid phase. I-V response was measured with PAAP/Nafion/GCE film.

Results and discussion
The main target in this research work is to synthesize and characterize a new class polymers in the form of polyarylidene(azomethine-ether)s and copolyarylidene-(azomethine-ether)s through solution polycondensation method and to shed light on certain characteristics, such as solubility, thermal stability, crystallinity and morphology. This type of polymers can be applied in different high performance applications. However, that needs special characters such as high thermal stability and high T g as well according to the application applied for selective and sensitive arsenic sensor development by electrochemical approaches. However, these new polymers and copolymers necessitate the synthesis of required monomers as harbingers.

Monomers synthesis
Two azomethine monomers 1 a,b are synthesized by two different methods, the first method which is considered as acidcatalyzed condensation. In this method 4,4`-diaminodiphenyl ether can easily interact with 4-halobenzaldehyde in 1:2 molar ratio using DMF as a solvent and in the presence of few drops of glacial acetic acid. The second method is accomplished using basiccatalyzed condensation of 4,4`-diaminodiphenyl ether with 4-halobenzaldehyde in absolute ethanol and in the presence of few drops of dry piperidine [51][52][53][54][55] as shown in Figure 1.
The chemical structure of these azomethine monomers 1 a,b are elucidated by both correct elemental and spectral data. The IR spectra showed the disappearance of absorption bands due to primary amino groups, displayed characteristic absorption bands at 1610-620 cm −1 due to C = N and absorption band at 1240-1275 cm −1 due to C-O-C bonds (ether linkage) (cf. figure S1 as an example). As well as 1 H-NMR spectra showed distinctive absorption beaks that attributed to certain groups are found as illustrated in the experimental part (cf. figure  S2 as an example). Whereas, diarylidenecycloalkanone monomers 2 a,b and 3 a,b are prepared by forthright condensation of one mole of cycloalkanone with double moles of p-hydroxybenzaldehyde or 4-hydroxy-3-methoxybenzaldehyde in the presence of absolute ethanol and catalytic amount of dry HCl gas as a catalyst. The reaction these monomers with sodium ethoxide afforded readily their sodium salt 4 a,b and 5 a,b as shown in Figure 2. The structure of these monomers are elucidated by correct elemental and spectral analyses as described in our previous work [56,57] .

Synthesis of model compound
Before accomplishing the polymerization process, the aimed model compound 6 are prepared by interacting one mole of 4,4`-oxo-bis(4``-halobenzylideneimino-phenylene) 1 a,b with double moles of sodium phenoxide in DMSO as well as in the existence of potassium carbonate anhydrous according to Figure 3. The chemical structure of the resulted model compound 6 is investigated by correct elemental data and spectral analyses. The IR spectrum shows the feature absorption band at 1265 cm −1 due to ether connection. Beside, other characteristic peaks are due to other groups. 1 H-NMR spectrum of model compound 6 show an increase in the aromatic protons and finally the mass spectrum exhibited a molecular ion peak (m/z) in accordance with its molecular structure as shown in the experimental part (cf. figures S3, S4, S5).

Synthesis of polyarylidene(azomethineether)s 7 a-d
Our aim is to produce a new category of polyarylidene (azomethineether)s using solution polycondensation method [25][26][27][28][29][30]. These new polymers are produced by means of condensation reaction of azomethine monomers 1 a,b with diarylidenecycloalkanone monomers 4 a,b and 5 a,b in DMSO as well as in the presence of potassium carbonate anhydrous [58] as illustrated in Figure 4 . The chemical structure of these new polymers is confirmed by elemental and spectral data. The elemental analyses of all the polymers synchronize with the distinctive periodical units of each polymer; (see experimental part). The elemental analyses of such polymers deviated up to 0.90% from the theoretical amounts.
The IR spectra in KBr pellets for all the formed polymers show the new absorption beaks at 1660-1645 cm −1 which due to carbonyl group of the cycloalkanone moiety, absorption beaks at 1250-1275 cm −1 due to ether linkage. More particularly, other distinctive beaks which are attributed to the other common functional groups present in the polymers structures are also examined such as: at 3090-3030 cm −1 for CH stretching of aromatic  ring, at 2940-2900 cm −1 for CH stretching of aliphatic groups, at 1620-1600 cm −1 for C = N azomethine groups (cf. figure S6 as selected example).

Synthesis of copolyarylidene(azomethineether)s 8 a-f
The titled copolymers are also synthesized by solution polycondensation technique [25][26][27][28][29][30] using the procedure described in the literature [57] by the interaction of two moles of two varied diarylidenecycloalkanone monomers 4 a,b and 5 a,b (1:1 molar ratio) together with two moles of azomethine monomers 1 a,b in suitable amount of DMSO and in the presence of potassium carbonate anhydrous as well. (cf. Figure 5).
The structure of these polymers is estimated by elemental analyses and spectral data. As well as the elemental analyses of all the copolymers synchronize with the distinctive periodical units of each copolymer; the data are given in the experimental part. Deviation up to 1.1% from the theoretical values is found. The IR spectra in KBr pellets for all the resulting copolymers show the absorption beaks at 1665-1640 cm −1 due to carbonyl groups of the cycloalkanone moieties, absorption beaks at 1245-1265 cm −1 due to ether linkage. at 3040-3020 cm −1 for CH stretching of aromatic ring, at 2940-2910 cm −1 for CH stretching of aliphatic groups and at 1620-1595 cm −1 for azomethine groups (cf. figure S7 as selected example).

Polymers and copolymers characterization
Various characterization techniques are applied for the obtained polymers and their corresponding copolymers are determined and the data are discussed below. Ordinary temperature solubility test for polyarylidene (azomethine-ether)s 7 a-d and copolyarylidene(azomethine-ether)s 8 a-f , is examined using variety of solvents: DMF, DMSO, DMA, THF, CHCl 3 -CH 3 COCH 3 (1:1), CH 2 Cl 2 , HCOOH and conc. H 2 SO 4 . A standard of 5% (w/v) solution is taken for each sample. It can be clarified from Table 1 that all the polymers and copolymers, are insoluble in alcohols, benzene, and acetone as examples for common organic solvents, but completely soluble in HCOOH and conc. H 2 SO 4 acids giving an orange to red colors for polymers 7 a-d and giving a red to deep red colors for copolymers 8 a-f . As it can be also clarified from Table 1 that, the plurality of the polymers and copolymers are also soluble in DMSO exclude polymers, 7 a,b which are slightly soluble. Whereas, in polar aprotic solvents such as DMF, DMA it is mentioned that all polymers are partially soluble in both solvents. Whereas, the majority of the copolymer is also soluble in similar solvent except copolymer 8 a , which is partially soluble. Moreover, all the polymers as well as copolymers are completely insoluble in CHCl 3 -CH 3 COCH 3 mixture and CH 2 Cl 2 . Furthermore, diarylidenecyclohexanone based polymers are slightly more soluble than on diarylidenecyclopentanone based polymers. Such behavior is clearly noted in aprotic solvents (eg. DMSO, DMF, and THF) than the former polymers. The main reason for this observation is due to the greater flexibility of the cyclohexanone moieties along the chains as detected in our previous studies [13,28]. As well as the solubility character of the synthesized copolymers is slightly higher than the corresponding polymers, which appeared clearly in (DMF, DMA and THF) except copolymer 8 a which has partial solubility effect in the same solvents.
The inherent viscosities (η inh ) of polymers 7 a-d , and copolymers 8 a-f , are tested in DMSO at 30°C with an Ubbelohde suspended level viscometer. The test is measured only for the completely soluble polymers and copolymers. The inherent viscosity values are calculated according to the following equation: The solution concentration C is 0.5g/100 ml, η/η o = relative viscosity (or viscosity ratio). The data are also listed in Table 1. On comparison the inherent viscosity (η inh ) values for all polymers and copolymers, it was found that the polymer 7 c has high viscosity value (0.43 dL/g), and this may be attributed to high molecular weight of this polymer within the series. Whereas, copolymers 7 a,c have high viscosity values (0.65, 0.70 dL/g respectively) and this also may be attributed to high molecular weight of these copolymers within the series. Moreover, copolymers 7 b,d have low viscosities (0.49 and 0.50 dL/g respectively), which may be attributed to low molecular weight of these copolymers within the series . On comparison between the viscosity values of the resulting polymers and their corresponding copolymers, it was found that, mainly the copolymers have higher viscosity values than the polymers, which may be attributed to the high molecular weight of those copolymers than the corresponding polymers.
The XRD of selected examples of polyarylidene(azomethine-ether)s 7 b,d and copolyarylidene(azomethineether)s 8 b,f are represented in Figure 6. XRD patterns of  -3  3  3  1  1  1  5  5  7 b  -3  3  3  1  1  1  5  5  7 c  0.43  3  5  3  1  1  1  5  5  7 d  0.38  3  5  3  2  1  1  5  5  8 a  0.65  3  5  3  2  1  1  5   polymers 7 b,d and copolymer 8 f indicate that these polymers are semi-crystalline, where the diffractograms show an amorphous halo beak in the region 2θ = 5-60°a nd this indicates a low degree of crystallinity. As well as a few reflection beaks that are intermediates between amorphous and crystalline interferences are found. The low crystallinity behavior is exhibited, which might be due to the existence of methoxy groups as pendent groups along the polymer backbone which decrease the crystallinity. Methoxy group adjudicate the chains from each others, cases an efficient obstruction between the repeating units and compel it to unsymmetrical direction [57]. Whereas, XRD patterns of copolymer 8 b show that many reflection peaks that are ranging in crystalline phase within the same region of 2θ. It is so easy to say that this copolymer is completely crystalline. This signalizes that there is a major class of structures that are moderate in the ordered states between crystalline and amorphous phases with respect long-range order in the configuration of their atoms and molecules.
The morphology of the synthesized polymers 7 a-d and copolymers 8 a-f , is tested by scanning electron microscopy (SEM) and the micrographs are given in Figure 7. The samples are prepared as described in the experimental part. The study of selected polymers and copolymers shows that the surface of polyarylidene(azomethineether)s 7 c (Figure 7a, magnification X = 500) consists of accumulative grain, with higher magnification X = 15,000 ( Figure 7b) shows merged spherical particles. Whereas, SEM images show the surface of copolyarylidene(azomethineether)s 8 e (Figure 7c, magnification X = 1500) consists of porous particles seems to be like spongy shape, with higher magnification X = 3500 (Figure 7d) shows, more porous merged particles with cavity structure.
The thermal properties of the polyarylidene(azomethine-ether)s 7 a-d , and copolyarylidene(azomethineether)s 8 a-f , are determined by TGA, DTG DTA techniques in air at a heating rate of 10°C min −1 . As mentioned earlier in the results and discussion part and according to the thermal behavior of the new polymers and copolymers. Therefore, it is so important to investigate the thermal degradation for these new polymers and copolymers as well in correlation with their structure in order to explain the better characteristics for such selected applications. TG thermographs of the synthesized polymers and copolymers are illustrated in (figures S8, S9 and S10) and Table 2 shows the temperatures for 10, 25 and 50% of weight losses. In most cases T 10 (temperatures for 10% weight loss) are found to be the polymers degradation temperatures (PDT); which means that no significant decomposition are examined before such temperatures [24]. For the polyarylidene(azomethine-ether)s 7 a,c the curves show a little weight loss within the range 2-5% (onset at 90°C to 140°C), which is due to loss of imbibed humidity and entrapped solvents. The curves also indicate that the polymers decompose in two steps for polymer 7 c and in three steps for polymer 7 a . The predictable nature of degradation in the first step is ranged between 245 and 376°C; seems to be slow and count on the origin of these polymers. The degradation in this step is due to a pyrolytic oxidation of carbon-carbon double bonds that come from arylidene moieties followed by cleavage of many other weak bonds. Meanwhile, the second step is ranged between 470 and 575°C and consider as the faster degradation step. The prospective nature of degradation in this step is attribute to cleavage of ether linkage and scission of many other bonds as well. At the end, many burnt segment will be produced as an indicator for the formation of end product. The rate of degradation becomes maximal in the range 530 − 550°C and is almost finished at around 650°C, Polymer 7 a show lower T 50% value compared with other T 50% values of other tested samples which is referred to the lower stability of that polymer. For the copolyarylidene(azomethineether)s 8 a,b,d,e the thermographs of these copolymers also display a little weight loss within the range 2-4% (onset at 110°C to 180°C), which may be also referred to loss of imbibed humidity and entrapped solvents. The curves also mention that the majority of the copolymers decompose in two stages except copolymer 8 b in three steps. The prospective nature of degradation in the first and second steps is ranged between 200 and 370°C and 380 and 570°C respectively. Whereas, the third step for polymer 8 b is ranged between 580 and 670°C. No significant changes are found in the decomposition behaviors for these copolymers than previously mentioned polymers; nearly the same explanation is utilized. All the selected polymers and copolymers have PDT in the range of 145~340°C. Consequently, the results in Table 2 announce that the order of thermal stability of these samples is 7 c > 8 d > 8 e > 8 a > 8 b > 7 a . The abbreviation (FDT) is related to the final decomposition temperature and it is defined as the temperature at which the rate of decomposition is almost finished. So far, the given TG micrographs also display that all the tested polymers and copolymers have very high FDT values which are ranged between 572 and > 650°C. This is of course another good indication for the higher thermal stability for the desired polymers and copolymers even for polymer 7 a . Polymer 7 a has lower thermally stable values at T 10 , T 25 and T 50 while compared with other substituents. Whereas, it has higher FDT value nearly like the other derivatives. Which mean that the decomposition can be completed at very high temperature.
The determination of glass transition temperature T g by using DTA measurements are well known technique. Big number of synthetic polymers with long chain show distinctive succession of changes when they are heated. DTA curves for polymers 7 a,c , and of copolymers 8 d,e as selected examples are given in (figures S11 and S12). It can be seen that the majority of these polymers show a broad endothermic peaks become clear above 190°C. Since, the TGA hints does not show thermal decomposition at this temperature except for polymer 7 a , which shows a distinct weak glass transition temperature T g at 57.08°C, which in agreement with its thermal degradation at 10%. Furthermore, polymer 7 c shows T g at 215.56°C and no T m more has been estimated. It should be noted that the disappearance of T m of these polymers may be attributed to the rapid decomposition. As well as, copolymer 8 d and copolymer 8 e show T g at 194. 21°C & 225.19°C and T m at 397.75°C & T m at 403.7°C respectively. It is easily noticed from the above results that, all of tested polymers and copolymers have high T g values which is attributed to the high rigidity of those polymers chains.

Application: detection of As 3+ ions using PAAP/GCE by I-V method
The potential application of PAAP assembled onto GCE as a heavy and toxic arsenic sensor (especially As 3+ analyte in buffer system) has been investigated for measuring and detecting target As 3+ ions. The PAAP/GCE sensors have facile advantages such as stability in air, non-toxicity, chemical inertness, electro-chemical activity, simplicity to assemble, ease in fabrication, and chemosafe characteristics. As in the case of As 3+ ions sensor, the current response in I-V method of PAAP/GCE considerably changes when aqueous metallic analyte is adsorbed. The PAAP/GCE was applied for fabrication of ionic-sensor, where heavy metallic As 3+ ion was measured as target analyte. The fabricated PAAP/GCE electrode was put into the oven at low temperature (30.0°C) Table 2. Thermal behavior of poly(arylidene-ether)s 7 a,c and copoly(arylidene-ether)s 8 b,d. for 1.0 hrs to make it dry, stable, and uniform the surface totally. I-V signals of As 3+ ion chemical sensor are anticipated having PAAP/GCE on thin-film as a function of current versus potential. The resultant electrical responses of target As 3+ ion are investigated by simple and reliable I-V technique using PAAP/Nafion/GCE assembly. The holding time of electrometer was set for 1.0 sec. A significant amplification in the current response with applied potential is noticeably confirmed. I-V characteristic of the PAAP/Nafion/GCE is activated as a function of As 3+ ions concentration at room conditions, where improved current response is presented (Scheme 1). For a low concentration of As 3+ ions in liquid medium, there is a smaller surface coverage of As 3+ ions molecules on PAAP/Nafion/GCE film and hence the surface reaction proceeds steadily. By increasing the As 3+ ions concentration, the surface reaction is increased significantly (gradually increased the response as well) owing to surface area (assembly of PAAP/Nafion/GCE) contacted with As 3+ ions molecules (Scheme 1a). Further increase of As 3+ ions concentration on PAAP/ Nafion/GCE surface, it is exhibited a more rapid increased the current responses, due to larger area covered by As 3+ ions and the interaction of the nitrogen and oxygen containing functional groups (Scheme 1b). The molecular interaction could be approaches as inter-molecular and intra-molecular interactions of the PAAP polymer chains. Usually, the surface coverage of As 3+ ions on PAAP/binders/GCE surface is reached to saturation, based on the regular enhancement of current responses, which shown in Scheme 1c. The PAAP/Nafion/GCE was employed for the detection of As 3+ ion in liquid phase. I-V responses were measured with PAAP/GCE coated thin-film (in two electrodes system). The concentration of As 3+ ion was varied from 10.0 nM to 0.5 M by adding de-ionized water at different proportions. It is studied the control experiment about the uncoated and PAAP-coated electrode using I-V method and presented in Figure 8. Here, Figure 8(a) is represented the I-V responses for bare-GCE (grey-dotted) and PAAP/GCE (green-dotted) electrodes. In PBS system, the PAAP/GCE electrode shows that the reaction is enhanced slightly due to the presence of PAAP coated onto GCE surface. A considerable enhancement of current value with applied potential is demonstrated with fabricated PAAP/GCE in presence of target As 3+ ion analyte, which is presented in Figure 8 Analytical parameters (such as sensitivity, detection limit, linearity, and linear dynamic range etc) were investigated from the calibration curve (current vs. concentration), which was presented in Figure 8(d). A wide range of As 3+ concentration was selected to study the possible detection limit (from calibration curve), which was examined in 10.0 nM to 0.5 M. The sensitivity was calculated from the calibration curve, which was close to 2.714 µAcm −2 µM −1 . The linear dynamic range of the PAAP/ GCE sensor was employed from 10.0 nM to 0.1 M Scheme 1. Mechanism of the probable interaction of As 3+ with PAAP polymer with conducting nafion binders embedded onto GCE. (a) Fabricated GCE electrode, (b) inter-or intra-molecular interactions between lone-pair of nitrogen and oxygen (PAAP) and As 3+ , and (c) current responses in presence of As 3+ ions by I-V method.
(linearly, r 2 = 0.9923), where the detection limit was calculated about 6.8 ± 0.1 nM (ratio, 3N / S ). In presence of PAAP/Nafion/GCE layer on electrode, the electrical resistance is decreased under with the target As 3+ in PBS phase. The film-resistance was decreased gradually (increasing the resultant current) upon increasing the As 3+ concentration in bulk system.
In two-electrode system, I-V characteristic of the PAAP/GCE is activated as a function of As 3+ concentration at room conditions, where the improved current response is observed. As obtained, the current response of the PAAP/Nafion/GCE is increased with the increasing concentration of As 3+ ; however similar phenomena for cationic detection have also been reported earlier [59][60][61]. For a low concentration of As 3+ in liquid medium, there is a smaller surface coverage of As 3+ ions on PAAP/GCE film and hence the surface reaction proceeds steadily. By increasing the As 3+ concentration, the surface reaction is increased significantly (gradually increased the response as well) owing to large surface area contacted with As 3+ molecules. Further increase of As 3+ concentration on PAAP/GCE surface, it is exhibited a more rapid increased the current responses, due to larger surface covered by As 3+ chemical. Usually, the surface coverage of As 3+ molecules on PAAP/GCE surface is reached to saturation, based on the regular enhancement of current responses. Selectivity was studied for sensor in presence other chemicals like Mg 2+ , Sn 2+ , As 3+ , Au 3+ , Al 3+ , Zn 2+ , Ce 2+ , Sb 3+ , Co 2+ , Cd 2+ , Ba 2+ , Ca 2+ and Y 3+ using the PAAP/Nafion/GCE, which is presented in Figure 5(a). It was observed the highest current response for As 3+ by I-V measurement compared to other cations in the solution. The concentrations of heavy metallic analytes are kept constant at 0.1 µM level in PBS system. Therefore, As 3+ was selectively detection with PAAP/GCE surface in this approach.
To check the reproducibly and storage stabilities, I-V response for PAAP/GCE sensor was examined and presented in Figure 9(a). After each experiment (each runs), the fabricated PAAP/GCE substrate was washed thoroughly with the phosphate buffer solution and observed that the current response was not significantly decreased.
Here it is observed the current loss in each reading is negligible compared to initial response of sensors using PAAP/GCE. A series of seven successive measurements of 0.1µM As 3+ in 0.1 M PBS yielded a good reproducible signal at PAAP/GCE sensor with a relative standard deviation (RSD) of 2.58%. The sensitivity was retained almost same of initial sensitivity up to seven days, after that the response of the fabricated PAAP/GCE electrode gradually decreased. The As 3+ sensor based on PAAP/GCE is displayed good reproducibility and stability for over week and no major changes in sensor responses are found. After a week, the chemical sensor response with PAAP/ GCE was slowly decreased, which may be due to the weak-interaction between fabricated PAAP/GCE active surfaces and As 3+ ions. I-V responses were also measured for all derivatives of R in PAAP polymer embedded GCE electrodes, such as 7(a), 7(b),7(c), and 7(d). It is observed that the current response was exhibited significantly higher for PAAP [R: 7(a)] compared to other composition (Figure 9c) due to the enhancement of large surface area into the polymer. Based on the current responses on various compositions, the details heavy metallic As 3+ sensor development is approached for PAAP/GCE [7(a)] in terms of analytical parameters such as sensitivity, detection limit, linearity, reproducibility, selectivity etc. The higher current response of the fabricated PAAP/ Nafion/GCE could be attributed to the excellent absorption (porous surfaces in PAAP/binders/GCE) and adsorption ability, high catalytic-decomposition activity, and good biocompatibility of the conduction PAAP polymers.
The significant result was achieved by PAAP/GCE, which can be employed as proficient electron mediators for the development of efficient cationic sensors. Actually the response time was around 10.0 sec for the fabricated PAAP/GCE to reach the saturated steady-state level [62]. The higher sensitivity of the fabricated PAAP/GCE could be attributed to the excellent absorption (porous surfaces in PAAP/Nafion/GCE) and adsorption ability and high catalytic-activity of branches derivative of PAAP. The estimated sensitivity of the fabricated sensor is relatively higher and detection limit is comparatively lower than previously reported cationic sensors based on other nano-composites or nano-materials modified electrodes measured by I-V systems [63][64][65][66]. Due to high specific surface area, PAAP provides a favorable nano-environment for the As 3+ detection with good quantity. The high sensitivity of PAAP/Nafion/GCE provides high electron communication features which enhanced the direct electron transfer between the active sites of PAAP and coated-GCE. The PAAP/GCE system is demonstrated a simple and reliable approach for the detection of toxic heavy cationic species. It is also compared the performance of the PAAP/Nafion/GCE electrode for the selective detection of As 3+ depending on various compounds or materials by electrochemical approaches [67][68][69][70][71][72][73][74][75][76][77][78] and compared to other published reports and presented in Table 3 As per the observation, the sensor probe of PAAP/ Nafion/GCE exhibited the highest sensitivity compared with other values. It is also revealed that the significant access to a large group of cations for wide-range of ecological, environmental and health-care fields.
The real samples such as industrial effluent, tape and sea water were analyzed in order to validate the proposed I-V technique using PAAP/Nafion/GCE for arsenic determination. A standard addition method had been applied to estimate the concentration of As 3+ in real samples. A fixed amount (~25.0 µL) of each real sample was analyzed in 10.0 mL bulk solution (PBS, 0.1 M) using the fabricated PAAP/Nafion/GCE. The results have been included regarding the measurement of As 3+ in industrial effluent, tap and sea water samples, which apparently confirmed that the proposed I-V technique is satisfactory, reliable, and suitable for analyzing real samples with assembled of PAAP/Nafion/GCE (Table 4).

Conclusion
Thermally stable hybrid polyarylidene(azomethine-ether) s and copolyarylidene-(azomethineether)s containing diarylidenecycloalkanone moieties as cornerstones are synthesized by solution poly-condensation method. The required Monomers, pre-monomers and model compound are also synthesized and their chemical framework is established by right elemental and spectral analyses. Variable characterization tools are also tested and are studied for the desired compounds. All the synthesized polymers and copolymers are completely insoluble in most simple organic solvents. The PDT thermal stabilities for all the tested polymers and copolymers are in the order 7 c > 8 d > 8 e > 8 a > 8 b > 7 a . Although polymer 7 a is considered as the lower thermally stable polymer at T 10 , T 25 and T 50 , even it still has higher FDT value like the other stable derivatives. Furthermore, all the polymers and copolymers have high T g values except for polymer 7 a which has a distinct weak value (57.08°C). Later, PAAP was fabricated for detecting selective cation with the conducting coating binders onto flat GCE by electrochemical approaches, which displayed higher sensitivity and selective towards As 3+ ions. The analytical performances of the fabricated As 3+ sensors are excellent in terms of sensitivity, detection limit, linear dynamic ranges, selectivity, and in short response time. PAAP/ Nafion/GCE assembly is exhibited higher-sensitivity 2.714 µAcm −2 µM −1 ) and lower-detection limit (6.8 ± 0.1 nM) with good linearity in short response time, which efficiently utilized as cationic-sensor for selective As 3+ onto PAAP/Nafion/GCE. This novel approach is introduced a well-organized route of efficient heavy metallic sensor development for environmental pollutants and health-care fields in broad scales.


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Engaging with Raman Spectroscopy to Investigate Antibody Aggregation

In the last decade, a number of studies have successfully demonstrated Raman spectroscopy as an emerging analytical technique for monitoring antibody aggregation, especially in the context of drug development and formulation. Raman spectroscopy is a robust method for investigating protein conformational changes, even in highly concentrated antibody solutions. It is non-destructive, reproducible and can probe samples in an aqueous environment. In this review, we focus on the application and challenges associated with using Raman spectroscopy as a tool to study antibody aggregates.

Introduction
The past 30 years have seen increasingly rapid growth in the number of approved antibody-based biopharmaceutical products. Over 60 products have been approved for therapeutic use and approximately 50 more are currently in late stage clinical development [1][2][3][4]. Despite this success, antibody production is still laden with challenges that impact the time and cost of development [5][6][7][8].
One key issue is the control and understanding of aggregation, which can compromise not only product quality but also safety; in particular, the propensity of aggregates to trigger immune responses [7][8][9][10]. Antibody aggregates can occur in various forms and there is currently no standard definition for an aggregate [11]. Those with altered higher order structures (non-native aggregates) are often defined as a net irreversible cluster of protein monomers that have lost some or all of their native folded structure [12][13][14][15][16]. One key advantage of Raman spectroscopy is that spectral variations including peak intensities, peak position or shape, can be directly related to changes in protein secondary and tertiary structure. This unique sensitivity to structural changes in unfolded or aggregated proteins makes Raman Spectroscopy a powerful technique for the prediction and monitoring of antibody aggregation at all stages of drug production [6,[17][18][19][20][21].
Raman spectroscopy is non-invasive, non-destructive and can be used to study samples in solid and liquid forms. There is also flexibility with the choice of laser excitation wavelength, making it possible to obtain selective information from the samples. More importantly, there is no specific requirement to dilute samples to lower concentrations; therefore, Raman spectroscopy is suitable for high or low sample concentrations. Unlike more frequently utilized spectroscopic techniques such as circular dichroism, ultra-violet absorption and fluorescence where high concentrations can saturate spectral signals, in Raman spectroscopy, high concentrations give stronger signals and can actually improve the quality of the spectra [22,23]. High-quality spectral data offers the opportunity to monitor structural perturbations in test conditions that are relevant to practical usage rather than extrapolating from dilute solutions that may have a different behavior [13,24]. The current understanding of the degree to which protein structural changes influence and define aggregation is limited [12,[25][26][27]. Developing and employing analytical approaches, such as Raman spectroscopy, which overcome existing limitations, is an effective way to bridge this gap and expand the knowledge of aggregation mechanisms from a structural perspective [6,8,11,28].

Raman Theory
Raman spectroscopy is the inelastic scattering of light from molecules in the presence of a monochromatic light source [29,30]. When light (or photons) interact with a molecule, it can induce a transition in energy state that leaves the molecule in an excited vibrational state. The majority of scattered photons from the molecule are scattered without any change in energy (elastic or Rayleigh scattering). A small fraction of photons however, will be scattered at a different energy from that of the incident light. This effect is referred to as inelastic or Raman scattering. This difference in scattered frequencies, or Raman shift, is measured in units of cm −1 (wavenumber which is equal to 1/wavelength of the Raman shift). It is a relatively weak process, with a probability of 1 in 10 6 −10 8 photons being scattered this way [29,30]. The frequency of the Raman scattered photon can be either less than the incident frequency (Stokes scattering) or greater than the incident frequency (anti-Stokes scattering) ( Figure 1). Before spectra collection, the instrument is usually calibrated using materials that have strong Raman peaks such as silicon (520 cm −1 ) and diamond (1332 cm −1 ) [30]. These materials are either integrated into the instrument or may be external. Ideally, the choice of material should have Raman peaks in the same wavenumber range of the sample to be examined. The process of collection is optimized using spectral parameters such as laser power, exposure time, and number of accumulations [30,31].
Antibodies 2018, 7, x FOR PEER REVIEW 2 of 12 which overcome existing limitations, is an effective way to bridge this gap and expand the knowledge of aggregation mechanisms from a structural perspective [6,8,11,28].

Raman Theory
Raman spectroscopy is the inelastic scattering of light from molecules in the presence of a monochromatic light source [29,30]. When light (or photons) interact with a molecule, it can induce a transition in energy state that leaves the molecule in an excited vibrational state. The majority of scattered photons from the molecule are scattered without any change in energy (elastic or Rayleigh scattering). A small fraction of photons however, will be scattered at a different energy from that of the incident light. This effect is referred to as inelastic or Raman scattering. This difference in scattered frequencies, or Raman shift, is measured in units of cm −1 (wavenumber which is equal to 1/wavelength of the Raman shift). It is a relatively weak process, with a probability of 1 in 10 6 −10 8 photons being scattered this way [29,30]. The frequency of the Raman scattered photon can be either less than the incident frequency (Stokes scattering) or greater than the incident frequency (anti-Stokes scattering) ( Figure 1). Before spectra collection, the instrument is usually calibrated using materials that have strong Raman peaks such as silicon (520 cm −1 ) and diamond (1332 cm −1 ) [30]. These materials are either integrated into the instrument or may be external. Ideally, the choice of material should have Raman peaks in the same wavenumber range of the sample to be examined. The process of collection is optimized using spectral parameters such as laser power, exposure time, and number of accumulations [30,31]. The Raman shift results in a unique spectrum for each molecule according to the chemical bonds within it, with peaks corresponding to the vibrational frequencies of different functional groups. In the case of proteins, including antibodies, the Raman spectrum contains large amounts of structural information as there are 3N-6 vibrational modes, where N is the number of atoms, for a nonlinear molecule [30]. A typical Raman spectrum of a protein can be divided into regions identifying backbone structure, side chain environments as well as specific secondary structural elements. These regions can be further divided into the amide I region (~1630-1700 cm −1 ) assigned to C=O stretching of carbonyl groups, the amide III region (~1230-1340 cm −1 ) assigned to NH bending and Cα-N stretching modes, while the backbone skeletal stretch (~870-1150 cm −1 ) is assigned to Cα-C, Cα-Cβ, Cα-N stretches [32][33][34]. An example of a Raman spectrum of a monoclonal antibody is shown in Figure 2, demonstrating the wide range of structural information available from Raman spectra of proteins with the potential to provide unique insights into the aggregation process. The Raman shift results in a unique spectrum for each molecule according to the chemical bonds within it, with peaks corresponding to the vibrational frequencies of different functional groups. In the case of proteins, including antibodies, the Raman spectrum contains large amounts of structural information as there are 3N-6 vibrational modes, where N is the number of atoms, for a nonlinear molecule [30]. A typical Raman spectrum of a protein can be divided into regions identifying backbone structure, side chain environments as well as specific secondary structural elements. These regions can be further divided into the amide I region (~1630-1700 cm −1 ) assigned to C=O stretching of carbonyl groups, the amide III region (~1230-1340 cm −1 ) assigned to NH bending and C α -N stretching modes, while the backbone skeletal stretch (~870-1150 cm −1 ) is assigned to C α -C, C α -C β , C α -N stretches [32][33][34]. An example of a Raman spectrum of a monoclonal antibody is shown in Figure 2, demonstrating the wide range of structural information available from Raman spectra of proteins with the potential to provide unique insights into the aggregation process. However, two well-known limitations of the Raman effect are its intrinsic weakness and the interference of fluorescence. The drive to overcome these challenges has largely influenced significant technological advances that has transformed modern day Raman instrumentation. Essentially there has been an increase in the capability, efficiency and variety of lasers, low noise detectors, high quality optical systems including confocal microscopes that seek to maximize the scattering process [35][36][37]. As a consequence of advances in instrumentation, there are a number of variant Raman techniques, which apply the same basic principles as shown in Figure 1, but have instrument settings or adaptations that are designed to enhance the signal in a specific way or provide specific information [20,30,31]. Two such techniques that have been distinctly applied to aggregation studies include Raman optical activity (ROA) and deep UV-resonance Raman spectroscopy (DUVRR).These techniques are discussed in greater detail in later sections. Key advantages of DUVRR include low fluorescence and enhanced signals [20,30]. However, photodegradation may occur, but is usually reduced through measures that limit constant exposure of the sample to the laser beam such as spinning the sample during spectra collection as well as by reducing exposure time [30]. Although ROA has an extremely weak Raman signal and requires farlonger acquisition times than conventional Raman techniques, it uniquely provides structural information about the conformation of chiral molecules, including proteins [38,39].
Regardless of the Raman technique applied in the study of protein aggregates, a large amount of information can be contained within the spectral data set. In addition, heterogeneous samples like formulations and proteins in the presence of buffers could swamp Raman signals or create a complex spectrum with overlapping peaks, making it difficult to interpret. Therefore, chemometric methods are often required to extract and analyze the volume of information that may exist in the acquired spectra. In antibody aggregation studies, common methods include difference spectra, principal component analysis (PCA), partial least squares (PLS) and multivariate curve resolution (MCR) [22,23,40]. The application of chemometrics to Raman data is a broad area of research. It generally includes aspects such as spectral variance analysis, data pretreatment, sample variance analysis, classification, spectral deconvolution and quantitative regression analysis [20,41]. Several other methods are available for these processes, including pre-processing methods (baseline correction, cosmic ray removal, smoothing, de-noising and normalization); feature extraction methods such as linear discriminant analysis (LDA) and classification methods including linear discriminant classification (LDC), and hierarchical clustering analysis (HCA) [31,42]. For more extensive reviews on chemometric approaches as well as available Raman instrumentation aimed at biological samples, the reader is directed to the listed references [30,31,37,41,42].

Secondary and Tertiary Structure
A key cause of protein aggregation is the formation of non-native, partially unfolded intermediates with exposed regions capable of inter-molecular interactions [21,26,43]. The propensity of a protein to unfold, thereby exposing regions of residues, is not only influenced by changes in physiochemical conditions (e.g., temperature, pressure, pH and excipients) but can also be due to the intrinsic conformational properties of the protein (e.g., primary sequence, secondary and tertiary structure) [16,21,26]. Consequently, in order to understand and reduce antibody aggregation, it is necessary to evaluate even the most subtle of structural changes. Figure 3 highlights the key Raman spectral features associated with either α-helical or β-sheet structure, as well as an example of pH-induced loss of secondary and tertiary structure in α-lactalbumin as the native structure unfolds [44]. Subtle differences can be observed in the spectra of α-lactalbumin as the unfolding protein forms a series of intermediate species observed between the pH ranges 0.8-3.6, 3.6-4.6 and 4.6-7.8 [44].  Figure 3. (a) pH-induced changes in the Raman spectra of lactalalbumin from pH 0.8-7.8, (b) Shaded peaks in the Raman spectra of selected polypeptides and phenylalanine, assigned to specific protein structure: -α-helix (purple), β-sheet (yellow) and aromatic residues (blue) [44].
As previously demonstrated in Figure 2, the Raman spectrum of a typical antibody has numerous features that can be easily assigned to both secondary and tertiary structure conformation. The Raman peaks observed at~960, 1245 and 1674 cm −1 assigned to β-sheet indicate that this is the major secondary structure of the antibody, whilst features in the regions of~500-550 cm −1 assigned to disulfide conformations, 800-860 cm −1 assigned to hydrogen bonding state of tyrosine residues and the peak at~1550 cm −1 assigned to tryptophan are all markers of tertiary structure [17,24,43]. Raman spectroscopy is therefore an ideal tool for the characterisation of not only the native antibody but also the partially or fully-unfolded intermediates that lead to the formation of aggregates, including providing insight into the actual aggregation mechanisms [13,40,43].

Aggregation Mechanisms
The ratio of native to less-ordered or non-native intermediates can vary depending on the propensity of a protein to unfold and the specific aggregation mechanism, which in turn controls aggregate population size and concentrations [12,14,28]. Three reported aggregation mechanisms include nucleation dominated (ND) where new aggregates are formed directly from the monomeric proteins, chain polymerization (CP) where partially unfolded monomers add onto existing aggregates and associated polymerization (AP), which occurs when existing aggregates combine to form larger ones [28,[45][46][47]. By using a combination of Raman spectroscopy, circular dichroism (CD) and dynamic light scattering (DLS), Barnett et al. [13] were able to demonstrate differences in the extent of structural changes of anti-streptavidin IgG1 when ND aggregation was induced compared to CP or AD. Significant Raman spectral changes during ND where observed in the amide I region, in the tryptophan assigned peak at~1550 cm −1 and in the 510-540 cm −1 region. Raman peaks in this latter region have been extensively used to determine changes in S-S stretching of the disulfide bonds as peak position is directly related to the gauche/trans conformers. The Raman peaks at~508, 520 and 540 cm −1 are associated with GGG, TGG and TGT, respectively [48][49][50]. Through the comparative use of DLS, Barnett's study also indicated that aggregates formed by ND were relatively smaller than those formed via the CP and CP/AP mechanisms, indicating that the most significant structural changes are related to the change from monomer to dimer to trimer, rather than being observed as aggregates grow in size by combining aggregates [13].
The combination of Raman spectroscopy and DLS was also applied to the investigation of aggregation mechanisms in the model protein lysozyme, providing complementary information that determined the degree of influence of aggregation on the reversibility of protein unfolding and refolding [24]. By relating changes in protein size to shifts in position of peaks observed in the amide I region alongside changes in the tyrosine Fermi doublet at 850 and 830 cm −1 , they were able to determine that during heating, at pH 4, changes in lysozyme tertiary structure occurred before significant changes in either secondary structure or size [24]. The ratio of the two tyrosine peaks observed at~830 and 850 cm −1 (Intensity 850 /Intensity 830 ) in a protein spectrum is another well-established Raman marker of hydrophobicity and side chain solvent exposure. A drop in the ratio of the tyrosine Fermi doublet indicates a decrease in exposure and increased involvement of tyrosine residues as strong hydrogen bond donors, whilst an increase in ratio indicates the opposite effect [51,52]. A lack of significant temperature-induced changes in secondary structure of lysozyme at pH 4 was attributed to the formation of small aggregates as opposed to more substantial changes observed at pH 7 where large lysozyme aggregates were recorded [24].

Perturbation-Induced Aggregation
One of the key advantages of Raman spectroscopy is the ability to monitor perturbation-induced structural changes as shown in Table 1. This potentially provides insight into antibody stability of not only newly engineered antibodies but throughout the whole production process, right through to the formulated product.
Gómez et al. [40] demonstrated the sensitivity of Raman spectroscopy to differences in heat-induced conformational transitions in IgG4 variants. By comparing Raman spectral data sets of each heated IgG4 variant against the native stable antibody, it could be determined if unfolding only, aggregation only or both processes occurred. In particular, the Raman regions of~760-770 and 875-880 cm −1 assigned to tryptophan, displayed the most significant changes indicating the extent of solvent exposure [53,54]. During heating, variations in Raman peak intensity indicated that tryptophan residues became initially less exposed as the F ab domain of the antibody unfolded, followed by increased solvent exposure as the F c domain unfolded; however, if aggregation occurred, there was further loss of exposure, suggesting changes in orientation of tryptophan residues within the F c domain depending on the IgG4 thermal response [40].
Zhou et al. [22] also investigated temperature-induced spectral variation but their study investigated a formulated IVIG stored in a 250 nM glycine buffer. Although a key advantage of Raman spectroscopy is its sensitivity to structural and chemical composition, this also means that any recorded spectra will contain signals from all components of the sample; therefore, the spectrum of a protein in a buffer will contain signal from the protein and buffer, with buffer peaks potentially masking important protein peaks. In order to overcome this problem with the IVIG in glycine, the difference spectrum was determined by subtracting the spectrum acquired at 20 • C from the protein spectra acquired at different temperatures during heating [22]. From the difference spectra, they were able to resolve increased intensities in the β-sheet assigned peak at 1668 and 1686 cm −1 with heating as well as substantial peak shifts from the tyrosine Fermi doublet and tryptophan peak at~1550 cm −1 , again identifying specific changes in both secondary and tertiary structure during aggregation of the antibody [22]. This approach using the difference spectra was also successfully used by Webster et al. [55] to analyze Raman spectra acquired of insulin subjected to shear flow for 90 min. By subtracting the unsheared insulin spectrum from the sheared ones recorded, they were able to determine changes in marker peaks at 1678, 1630 and 1625 cm −1 , indicating an increase in intermolecular H-bonded β-sheet structure of the protein [55]. Insulin aggregates were also investigated in formulated products using difference spectra, where spectral contributions of both glycerol and insulin were subtracted from the Raman spectrum of the heat stressed insulin [56]. The most significant spectral changes with heating were observed at 1675 cm −1 suggesting that this Raman peak is an important marker for protein aggregation.  [43] Increased perturbation in (I 510 / 540 ) for ND aggregates Anti-streptavidin IgG1(temperature) ND aggregation pathway showed greater sensitivity to this marker. [13] 850/830 (Tyr) No change rhuMab * (Lyophilisation) - [25] Downshift IVIG * (temperature) Change in tertiary structure [22] Increased ratio Rabbit IgG (concentration) Increased molecular interaction [23] 770/875 (Trp) Increased/decreased intensity IgG4 variant (temperature) Less exposure to solvent/more exposure to solvent [40] 1000 (Phe) Increased intensity Rabbit IgG (storage) Pre-aggregation [23] 1230 (β-sheet) Broadening and shift to 1245 cm −1 Human IgG1 (pH) Formation of intermolecular β-sheet [43] I 1340 /I 1360 (Trp) Decreased ratio IVIG (temperature) Reduced hydrophobicity (change in tertiary structure) [22] 1555 (Trp) Downshift IVIG/temperature Change in tertiary structure [22] Redshift Anti-streptavidin IgG1(temperature) Change in tertiary structure [13] 1630 (β-structure) Increased/decreased intensity IgG4 variant (temperature) Increase in intermolecular H-bonding of β-structure/Loss of bonding or loss of secondary structure [40] 1669 (β-sheet) Decreased intensity Human IgG1(pH) Loss of Intramolecular β-sheet [43] 1686 (β-sheet) Increased intensity Human IgG1(pH) Intermolecular β-sheet formation [43] * Recombinant humanized monoclonal antibody (rhuMab), intravenous immunoglobulin (IVIG).

Raman Optical Activity
Although Raman spectra has been extensively demonstrated as a sensitive tool for determining structural changes, some secondary and tertiary structural changes in proteins can be hard to determine in antibodies due to the dominance of the β-structure features in the spectrum, masking very subtle changes [22,25]. In a pH-induced study of IgG1 using FT-Raman spectroscopy, no variations in either the amide I or the amide III regions were observed, suggesting that secondary structure remained stable; however, when the alternative Raman technique of Raman optical activity (ROA) was applied, differences in these regions could be observed in the ROA spectra ( Figure 4) [43]. ROA measures a small difference in intensity of Raman scattering from chiral molecules in right-and left-circularly polarized light and/or a small circularly polarized component in the Raman scattered light. It is therefore extremely sensitive to the most rigid parts of the protein [39,57]. It has been extensively used to investigate a range of biomolecules, including unfolding and aggregation in proteins [58][59][60][61][62][63][64]. In a pH-induced study of human IgG, variations observed in the ROA spectra of IgG1 and IgG2 at pH 7 and pH 3 revealed that whilst the secondary structure of the antibody remains relatively unchanged, significant changes in tertiary structure could be determined. Significant spectral differences were observed for the tyrosine and phenylalanine peak at 1622 cm −1 , the tryptophan assigned peaks at 1556, 1455 and 1345 cm −1 and the tyrosine peak at 1220 cm −1 , suggesting that the aromatic side chain environment changes at acidic pH with unfolding of the tertiary structure [43]. ROA has also been used to investigate temperature-induced aggregation in IgG4 in the presence of formulation excipients [65]. In the same way that buffers and excipients can mask Raman spectral features arising from the protein, ROA spectral features from the formulation placebo were also observed to dominate the ROA spectrum of the IgG4 [65]. As with the previously discussed Raman studies, the formulation placebo spectrum was subtracted from the spectra of the heated IgG4 revealing significant peaks in the amide I region assigned to β-sheet, the positive peaks at 1300 and 1320 cm −1 and negative peaks at 1200 and 1250 cm −1 ( Figure 5). As the sample was heat-stressed over four weeks, there was a loss of intensity in the ROA peaks, indicating a loss of both secondary and tertiary structure [65].

Deep UV Resonance Raman
Another alternative Raman approach that has been used in the study of protein conformation is deep UV resonance Raman spectroscopy (DUVRR) spectroscopy. DUVRR spectroscopy has excellent sensitivity to structural changes in proteins and by tuning the laser within the UV region (180-260 nm), very specific information can be achieved. UV excitation also has the advantage of enhancing the Raman signal by a factor of 10 3 -10 5 over conventional Raman scattering through the resonance effect [30]. In resonance spectroscopy, if the energy of the incident laser is within the molecular absorption peaks of chromophores or aromatics in the molecules, the signal can be enhanced, in the case of proteins resulting in a spectrum dominated by amide vibrations and aromatic amino acid peaks. One form of aggregation that has been extensively studied by DUVRR is the formation of fibrils [66][67][68][69][70][71]. By using a combination of 204 and 198 nm, Xiong et al. were able to assess changes in hydrogen bonding during polypeptide aggregation and fibril formation due to peak shifts observed in the amide I and amide II regions [70]. The excitation wavelength of 220 nm has also been used to determine two distinct stages of aggregation and fibril formation in the Tau protein by identifying variations in the DUVRR spectral features associated with an increase in β-sheet and loss of less-ordered structure, including a shift in peak position of the 1238 cm −1 to 1230 cm −1 [72]. In another study of fibrils, the sensitivity of the phenylalanine peak at~1000 cm −1 to resonance enhancement was used to monitor spectral differences in the DUVRR spectra of hen egg white lysozyme (HEWL) fibrils compared to HEWL in its native conformation [67]. The fibril spectra showed a significant decrease in the peak intensity at~1000 cm −1 compared to native lysozyme [67]. Further studies demonstrated that the loss of intensity observed with the fibrils was greater than the loss of intensity that was observed for the soluble phase of incubated lysozyme [66,68]. This distinction was linked to a greater exposure of the fibrils to water, highlighting the capacity of this Phe peak to probe solvent exposure as well as aid sample identification.
Although to date DUVRR spectroscopy has been under-utilized in the investigation of antibody aggregation, Bueno et al. investigated thermal unfolding of Rituximab using DUVRR spectroscopy at an excitation wavelength of 205 nm [73]. Rituximab is an IgG1 and was studied in a formulation containing polysorbate 80; however, unlike other Raman techniques, the resonance enhancement of the DUVRR spectra resulted in a spectrum dominated by the protein rather than the excipient. In particular, a peak shift, along with a broadening of the peak, was observed from~1670 to 1660 cm −1 with heating indicating an increase in less-ordered structure [73].

Conclusions
As demonstrated, Raman spectroscopy is a powerful technique for the analysis of antibody aggregates, providing dynamic information about secondary structure, tertiary structure, and aggregation mechanisms. Raman features can clearly be used to identify changes, including solvent exposure of residues, conformation, molecular interaction and hydrogen bonding. The interpretation of these parameters can facilitate the elucidation of structural variations associated with aggregated antibodies or the aggregation process itself when measurements are made in-situ. This information can be readily obtained with minimal sample preparation from liquids samples, even at high concentrations as well as solid samples, thus making it feasible to directly compare and consistently monitor different states of an antibody sample.
The capacity of Raman spectroscopy for the structural analysis of antibodies and their aggregates throughout the development and formulation process, including end-use conditions, has the potential to generate valid predictions for candidate selection and optimization during drug development. In addition, during the manufacturing process, spectral markers of aggregation could be used to assure and monitor product quality.
A number of alternative Raman spectroscopic techniques, including ROA and DUVRR variant techniques, have intrinsic qualities that seek to overcome some of the challenges of conventional Raman spectroscopy in addition to providing tailored or specific information. A wider application and targeted development of Raman techniques can facilitate rigorous benchmarking with other established techniques and consequently their acceptance for routine use in the analysis of antibody aggregates.
Author Contributions: Both authors have made significant contributions to the writing of this paper and approve the submitted version of this manuscript. Both authors also agree to be personally accountable for own contributions as well as resolving any questions or concerns about this work.
Funding: This research received no external funding.


PRELIMINARY PHYTOCHEMICAL SCREENING, GC-MS PROFILING AND IN VITRO EVALUATION OF BIOLOGICAL ACTIVITIES OF GARCINIA ATROVIRIDIS ROOT EXTRACTS

Nur Salsabila Ahmad Roslan, Seema Zareen, Normaiza Zamri and Muhammad Nadeem Akhtar Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, 26300 Gambang, Malaysia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 01 November 2018 Final Accepted: 03 December 2018 Published: January 2019

…………………………………………………………………………………………………….... Introduction:-
Garcinia atroviridis is commonly known as 'asam gelugur' in Malaysia, India, Myanmar, and Indo-China. This plant species specifically belongs to the Guttiferae. This plant is endemic species in Peninsular Malaysia. It grows wildly in lowland and hill forest up to 600-meter altitude. It was also planted by the locals for its economic and medicinal purpose (MacKeen et al., 2000). The dried G. atroviridis fruits known as 'asam keping' are sold commercially as seasoning. It is sour and is used to season curry, dressing fish and others. G. atroviridis have medicinal values as traditionally, it has been used to treat a cough, the decoction of its leaf with roots can be used to treat an earache, and the juice of the leaf is given to female after delivery (Tisdale et al., 2003;Burkill, 1966). In the previous study (MacKeen et al., 2000), it was reported that the crude methanolic extract of the different parts of the G. atroviridis such as fruit, leaf, stem, and trunk barks showed it possessed antibacterial, antifungal, antioxidant, and antitumorpromoting properties. This study analyses the chemical compositions of the different extracts of G. atroviridis roots via preliminary phytochemical screening and GC-MS. The antibacterial activities of all extracts were determined by the disc diffusion method while antioxidant activities were evaluated based on the DPPH radical scavenging activities.

Solvent-solvent extraction
The dried roots were ground into a coarse powder using a pulverizer. Fine ground pulverized material was dissolved in different solvents as described in Figure 1. Powdered plant material was soaked in methanol for three days at room temperature and the solvent was filtered by using a sieve. This was repeated 3-4 times until the extract gave no coloration. The extract was distilled and concentrated under reduced pressure in the Buchi rotavapor yielding a gumlike residue (45.45 g). The same was repeated with organic solvents and distilled water of increasing polarity (starting with lipophilic solvent n-Hexane, ending with the more hydrophilic n-Butanol). The solvent from each extract was filtered and concentrated under reduced pressure in Buchi rotavapour. Finally, the extracts of n-hexane, dichloromethane, ethyl acetate, and n-butanol were collected, weighed (8.72 g, 4.45 g, 9.79 g and 1.08 g, respectively) and stored in the refrigerator at 4 o C for further phytochemical analysis.

Phytochemical Screening
The extract was subjected to preliminary phytochemical tests to determine the group of secondary metabolites present in the sample. The screening of the phytochemicals such as alkaloids, carbohydrates, anthraquinones, glycosides, saponin, protein and amino acids, phytosteroids, oils and fats, and phenols and flavonoids was carried out by following the standard procedure as mentioned by Sofowora (1993), Trease and Evans (1989) and Harborne (1984).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was carried out by Agilent 7980A series GC instrument. The DB-1MS column with the dimensions of 30 m Χ 0.25 mm capillary column was used for the analysis. The initial temperature was kept at 50 °C for 3 minutes and the maximum temperature was up to 250 °C. Helium was used as a carrier gas at a flow rate of 1.0 mL/min and the total run time was 60 minutes. Interpretation of mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology MS library (NIST-MS library).

Antibacterial Test Test microorganisms
The procedure was according to MacKeen et al., 2000. Four bacterial strains, i.e. Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 10536) and Proteus vulgaris (ATCC 33420) was used in this study. The nutrient broth was prepared, and the microorganism was cultured and incubated in the incubator shaker at 30°C for 24 hours. The concentration of the cultures was adjusted turbidometrically at a wavelength of 600 nm to 10 8 colony forming units (CFU) per mL.

Disc Diffusion Method
The bacterial cultures (10 8 CFU/mL) were inoculated on the prepared nutrient agar. 15 μl of samples with a concentration of 20 mg/mL was loaded onto blank filter paper discs. The loaded disc was placed on the previously inoculated agar. The plates were inverted and incubated in the incubator for 24 hours at 30 °C. The presence of clear inhibition zones around the discs showed the antibacterial activity of the extracts (MacKeen et al., 2000). The disc diffusion tests were presented in triplicate and the antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by the extracts. Commercially available antibiotics discs were used as a positive control whilst the solvent used to dissolve the extracts acted as a negative control.

Antioxidant Assay α, α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging method
The scavenging effects of samples for DPPH radical was determined according to the method by Hui et al. (2017), with slight modification. Briefly, the initial concentration of the sample was prepared at 1 mg/mL as well as concentration for ascorbic acid which acted as standard in this assay. 0.2mM DPPH was prepared freshly and must be protected from light throughout the experiment. 100 μL of DPPH was added to 100 μL of sample with different concentrations. After incubated for 30 minutes in the dark at room temperature, the absorbance of the solution was read at 517 nm. The solution without extract; only methanol and DPPH were used as a control. All test was performed in triplicates. The data were expressed as mean ± standard deviation (SD). The calculation of the percentage of radical scavenging activity was as followed: Abs C, Absorbance of control at t= 30 minutes Abs S, Absorbance of sample t= 30 minutes

Results and Discussion:-Preliminary Phytochemical Screening
The results of the presence of important metabolites in hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), butanol (BuOH) and methanol (MeOH) extracts of Garcinia atroviridis roots are tabulated in Table 1.

GC-MS Analysis
The GC-Mass Spectrometry analysis of all extracts was given in Table 2         0.00 0.00 0.00 0.00 Tests were carried out in triplicate. Data was expressed as mean ± standard deviation (SD) ND: not determine; the particular antibiotic disc was not tested on the microorganisms +ve: positive control 59 -ve: negative control Antioxidant test DPPH assay DPPH radical scavenging method was used to evaluate the antioxidant activity of the root extracts of G. atroviridis. DPPH assay is a fast and efficient method to determine the free radical scavenging activity. The color changed from purple to yellow signifies the decline in absorbance of the DPPH radical (Jadid et al., 2017). In this study, all extracts showed a similar increasing trend in antioxidant activity with an increase in their concentrations (Table 9).
IC means inhibition concentration and IC 50 is the concentration of the sample or antioxidant that is required to inhibit 50% of DPPH radicals (Jadid et al., 2017). Hence, the lower the IC 50 , the better the antioxidant activity. Phongpaichit et al., 2007, considered the IC 50 range from 10 to 50 mg/mL to display strong antioxidant activity, while 50 to 100 mg/mL is considered intermediate and weak antioxidant activity for IC 50 value that more than 100 mg/mL. According to the IC 50 of the extracts (Table 10), butanol (50.59 μg/mL) showed better antioxidant activity followed by ethyl acetate (51.7 μg/mL) and dichloromethane (53.17 μg/mL). Meanwhile, methanol (124.7 μg/mL) and hexane (131.8 μg/mL) extracts displayed weak activity. However, none of the extracts were comparable to the standard, ascorbic acid (AA) with IC 50 of 13.21 μg/mL. Previous study (MacKeen et al., 2000), methanolic roots extracts of G. atroviridis showed significant antioxidant activity.

Conclusion:-
This paper emphasizes the phytochemical profiling of G. atroviridis roots extracts using preliminary phytochemical screening and GC-MS analysis. In this study, it was shown that the G. atroviridis roots extract contained many biologically active constituents, hence correlates with previous studies. The extracts of the sample were then tested for its antibacterial and antioxidant activity where promising results can be observed, though, further study such as bioprospecting is important to support its biological properties.


extfieldsofstudy:
Medicine
Materials Science

text:
Direct methanol synthesis from methane in a plasma-catalyst hybrid system at low temperature using metal oxide-coated glass beads

A plasma-catalyst hybrid system was used to synthesize methanol directly from methane. A dielectric barrier discharge (DBD) plasma combined with the catalyst was introduced in order to overcome the difficulties of catalyst-only batch reactions such as high reaction pressure and separation of liquid product. Of the transition metal oxides, Mn2O3-coated glass bead showed the highest methanol yield of about 12.3% in the plasma-catalyst hybrid system. The reaction temperature was maintained below 100 °C due to the low plasma input power (from 1.3 kJ/L to 4.5 kJ/L). Furthermore, the reactivity of the catalyst was maintained for 10 hr without changing the selectivity. The results indicated that the plasma-induced OH radical might be produced on the Mn2O3 catalyst, which led to methanol synthesis.

Direct methanol synthesis from methane in a plasma-catalyst hybrid system at low temperature using metal oxide-coated glass beads

Heesoo Lee & Do Heui Kim
A plasma-catalyst hybrid system was used to synthesize methanol directly from methane. A dielectric barrier discharge (DBD) plasma combined with the catalyst was introduced in order to overcome the difficulties of catalyst-only batch reactions such as high reaction pressure and separation of liquid product. Of the transition metal oxides, Mn 2 O 3 -coated glass bead showed the highest methanol yield of about 12.3% in the plasma-catalyst hybrid system. The reaction temperature was maintained below 100 °C due to the low plasma input power (from 1.3 kJ/L to 4.5 kJ/L). Furthermore, the reactivity of the catalyst was maintained for 10 hr without changing the selectivity. The results indicated that the plasma-induced OH radical might be produced on the Mn 2 O 3 catalyst, which led to methanol synthesis.
Methane is a main component of natural gas, which has large reserves spread all over the world 1 . Therefore, methane conversion to value-added products has drawn much attention because of scientific and industrial significance 2 . Currently, the production of methanol from natural gas should go through the route of synthesis gas, which is considered to be energetically inefficient. Thus, direct methanol synthesis from methane has been steadily investigated by many researchers [3][4][5] . However, direct methanol synthesis through catalytic reactions has limitations given that a large quantity of thermal energy is required to selectively activate the C-H bonds. Unfortunately, introducing thermal energy results in unwanted oxidation of methane into CO or CO 2 . To prevent this, a batch reaction has been carried out under a high pressure using liquid-phase reactants with expensive oxidants such as H 2 O 2 [6][7][8][9][10] . Though this could enhance the methanol selectivity, the methanol yield remains low (<10%), and separation and regeneration processes are needed 11 .
This study introduces plasma to overcome such difficulties. Plasma can activate methane to produce a methyl radical at lower temperatures 12 . The plasma can be divided into thermal plasma and non-thermal plasma by the degree of thermal equilibrium 13 . The temperatures of electron, ion, and radical become identical in the thermal plasma condition. On the contrary, while electron temperature is high, the temperature of bulk gas can be maintained at low temperature in the non-thermal plasma condition 14 . This is because the mass of electron that is accelerated by plasma is very light, which leads to increase of the gas temperature by only a few degrees 15,16 . Accordingly, partially activated gas including ion and radical can be generated in a simple reactor configuration 17 . At this point, the reaction atmosphere can remain isothermal condition. Because of this circumstance, structural variation of the catalyst can be hardly created in non-thermal plasma condition using a simple reactor configuration 18 . There are diverse plasma sources such as corona, microwave, arc, spark, radio frequency, and dielectric barrier discharge (DBD) 19 . Among these various plasma sources, dielectric barrier discharge (DBD) plasma has been generally applied by former researchers because it can be formed at atmospheric pressure where other non-thermal plasmas are hardly generated [20][21][22][23][24] . Therefore, DBD plasma was selected as a plasma source in this study. The system was designed with the plasma activated methane and catalyst that play a role in improving the methanol selectivity in a continuous packed bed reactor which does not require separation and regeneration processes.

Results and Discussion
Plasma only reaction. First, the experiment was conducted under plasma only condition as the baseline for the plasma-catalyst hybrid reaction. In the plasma only reaction, the methane conversion and methanol selectivity were measured in the range of plasma input voltage from 6.5 kV P-P to 8.5 kV P-P at intervals of 0.5 kV P-P at low temperature. Due to the plasma property, methane was able to activate and oxidize without adding any thermal energy. Aside from CO, CO 2 , and methanol, oxygenated products were hardly detected. Among the products, CO was the most abundant during the reaction. The plasma power calculated from V-Q Lissajous figure changed from 2.3 W to 7.2 W (from 1.4 kJ/L to 4.3 kJ/L), depending on the input voltage that led to variations in the reaction temperature from 61 °C to 108 °C. As the input voltage increased, the methanol selectivity decreased since the methanol was more oxidized to the products such as CO and CO 2 . However, the methane conversion also increased due to an increase in the plasma power. Consequently, the overall methanol yield increased from 5.1% to 8.7% while the input voltage varied from 6.5 kV P-P to 8.5 kV P-P ( Table 1). The methanol yield was confirmed to steadily increase although the methanol selectivity decreased as the input voltage went up in this reaction condition. According to Zhou et al., the DBD plasma can provide electrons with high energy enough to dissociate CH 4 and O 2 25 . Under the plasma condition, the oxygen radical, methyl radical, and hydrogen radical can be produced. In addition, excited oxygen atom can react with CH 4 to produce CH 3 and OH. Possible reaction pathways for methanol synthesis under plasma condition are described in Table 2.
Glass bead with plasma reaction. To improve the methanol productivity, glass beads were loaded into the reactor as a packing material. Before loading, the glass beads were etched using a 5 M NaOH solution to improve the adhesion of the metal oxide. After inserting the glass beads, the plasma configuration became much more stable, as shown in Fig. 1, due to the spherical shape and low dielectric constant of the glass beads. In addition, packing material like glass bead is helpful for expanding the discharge reaction since the streamers or microdischarges tend to propagate along the solid surface 26 . The electric field would be enhanced by loading packing material because of the short distance in the adjacency of contact points. The plasma energy is apt to be consumed by the electron-impact dissociation and ionization reactions by the enhanced electric field. At all plasma conditions from 6.5 kV P-P to 8.5 kV P-P at low temperature, the reaction of the glass beads with plasma resulted in a higher methanol yield than with the plasma only reaction. The methanol yield increased from 6.3% to 9.6% in the presence of the glass beads (Table 3). However, the methanol selectivity decreased as the input voltage increased higher. The methanol yield was confirmed to hardly increase above 7.5 kV P-P . Furthermore, the increase rate, which is the division of the methanol yield in the presence of the glass bead by that of plasma only, decreased from 124.4% to 110.8%. The plasma power varied from 2.2 W to 7.6 W (from 1.3 kJ/L to 4.5 kJ/L) according to the input voltage, which was similar to the plasma only reaction. Considering the reactivity and specific input energy, 7.5 kV P-P was selected as the optimum plasma condition. were coated to improve the activity for methanol synthesis. The surface area of all catalysts was below 1 m 2 /g. As displayed in Table 4, MnO, Mn 2 O 3 , and Fe 2 O 3 showed a higher activity than the glass beads with plasma. In addition, some catalyst with plasma in the current study demonstrated higher performance than the previous reports which applied plasma only or catalyst only reaction. The catalysts displayed a similar plasma configuration and power as that of the glass beads. Hence, this synergistic effect can be inferred to result not from the plasma alteration but from a catalytic reaction. The ratio of CH 3 OH yield and specific input energy is shown in Table 4. The ratio underlined that the reaction in this study required lower energy to gain same methanol yield than the references. Furthermore, most of the products were CO, CO 2 , and CH 3 OH which were easy to separate. On the other hand, MnO 2 , NiO, and Co 3 O 4 demonstrated poor activity that was even lower than that of the plasma only condition. In the case of MnO 2 , NiO, and Co 3 O 4 , the plasma configuration became conductive according to the Lissajous figure (Fig. 2). Due to the increased conductivity, the plasma configuration altered significantly, and the plasma was nearly extinguished, which gave rise to a sharp decrease in the methane conversion. Of these, MnO 2 showed a high methanol selectivity even though it indicated a low methane conversion since plasma hardly formed. After the reaction, all catalysts did not appear to undergo an alteration in structure as shown in Fig. 3. The formation of OH radicals is crucial for direct methanol synthesis from methane 27,28 . According to Guo et al., the OH radical can be adsorbed well into the MnO x catalyst 29 . This result suggested that the Mn 2 O 3 catalyst could increase the possibility of the OH radical to participate in methanol synthesis reaction. The long term activity of the catalyst was explored to confirm the durability of the catalyst and to apply these in a practical application. Figure 4 shows the methane conversion, methanol selectivity, and methanol yield of the Mn 2 O 3 /glass beads showing the highest methanol yield in the plasma-catalyst hybrid system with the    time-on-stream (TOS). As shown in Fig. 4, during a reaction for the first one hour, the methane conversion increased, and the methanol selectivity decreased, resulting in a slight increase in the methanol yield. After a reaction for 10 hr, the methanol yield was stabilized at about 12%. This indicated that the plasma-catalyst hybrid system was active and stable for direct methanol synthesis from methane.

Conclusion
In summary, the plasma-catalyst hybrid system was applied into a direct methanol synthesis from methane. Under the plasma only condition, methane started to become activated to produce methanol below 100 °C. By loading the glass beads, the plasma configuration was stabilized, leading to an increase in the methanol yield. Some transition metal oxides, such as MnO x , Fe 2 O 3 , Co 3 O 4 , NiO were coated on the glass beads to improve the activity. Among the catalysts, the Mn 2 O 3 showed the highest methanol yield of 12.3% when combined with plasma below 100 °C, at an ambient pressure. From the results, it can be implied that the Mn 2 O 3 catalyst could enhance the possibility of the reaction between OH radical and methane more significantly, which resulted in the highest methanol yield. Furthermore, the stability of the catalyst was maintained for 10 hr, presenting a plasma-catalyst hybrid system that could be a potentially efficient process for direct methanol synthesis from methane.

Methods
Reaction system. Figure 5 indicates the schematic view of overall plasma-catalyst hybrid system. This system was originated and slightly modified from the previous study 30 . Besides, this system is well delineated in the past paper 31 Table 4. Methane conversion and product selectivity of the metal oxide-coated glass beads under a plasma condition (7.5 kV P-P , 4 kHz). a P and GB represent the plasma and glass beads, respectively. b The specific input energy (SEI) is 30 kJ/L without a catalyst at 50 °C. c Temperature: 630 °C, contact time: 2.5 s, oxygen: 15.5 vol%, Si/Fe ratio: 22, without plasma. In the middle of the reactor, there was plasma discharge zone. Within the plasma discharge zone, the packing materials such as glass bead and metal oxide-coated glass bead (Φ = 2.0 mm) which have spherical shape were loaded on the quartz wool. In order to pack the plasma discharge gap, the height of catalyst bed exceeded 20.0 mm. K type thermocouple was utilized to measure and record the catalyst bed temperature. Because the thermocouple could function as a ground electrode and be affected by plasma, thermocouple was protected by  another quartz tube and located just below the quartz wool though it might not be exact temperature of catalyst bed.
Shape of the plasma-catalyst hybrid reactor is shown in Fig. 6. The catalyst zone and the plasma zone were combined into one reaction region, which led to the overlap of them at the same position in this reactor. Thickness of the stainless steel rod that was located at the middle of reactor was 4.0 mm. Stainless steel plate that had a length of 20.0 mm and a thickness of 0.2 mm surrounded the outer surface of the reactor. Stainless steel rod acted as high voltage electrode and stainless steel plate did as ground electrode. Accordingly, the length of plasma zone was 20.0 mm, and the distance of discharge gap was 4.0 mm. As a result, the reaction volume was about 2.0 cm 3 in plasma only condition. However, the packing material occupied the space where the plasma was generated in case of plasma-catalyst hybrid reaction, which led to the diminution of the plasma reaction volume. An arbitrary function generator (GW INSTEK AFG-2012) and high voltage amplifier (TREK 20/20C-HS) generated an AC high voltage with maximum of 8.5 kV P-P . This resulted in the creation of the plasma discharge. Except the input voltage, the experiments in this study were conducted under the same plasma conditions including sinusoidal waveform and driving frequency (4 kHz).
Catalyst preparation. Commercial 2.0 mm borosilicate glass bead (Sigma Aldrich) was utilized as a support under DBD plasma condition. Then, various metal oxides were loaded on the spherical glass bead. The catalyst was prepared by following procedure 32 . The glass beads were etched with 5 M NaOH (≥98%, Sigma Aldrich) solution at 100 °C, followed by immersing it in a suspension of metal oxide. Next, the mixture was dried at 19 120 °C for 1 h and washed with distilled water. The operation was repeated several times until the glass beads were no longer transparent with desired content of metal oxide. MnO (99%, Sigma Aldrich), Mn 2 O 3 (99.9%, Sigma Aldrich), MnO 2 (≥99%, Sigma Aldrich), Fe 2 O 3 (≥99%, Sigma Aldrich), NiO (99%, Sigma Aldrich), and Co 3 O 4 (99.5%, Sigma Aldrich) were used as metal oxides. Fixed amount (1 wt%) of metal oxides were loaded to each of the catalysts. 4.0 g of catalysts were used in the reaction. X-Ray Diffraction (RIGAKU SMARTLAB) with a Cu Kα radiation operated at 40 kV and 50 mA was utilized to verify the solid-state phases of the catalysts. In addition, the  Activity measurement. The plasma only reaction and the plasma-catalyst hybrid reaction were carried out at atmospheric pressure utilizing the same α-Al 2 O 3 reactor. The reactants were 20% CH 4 , 10% O 2 , balanced in Ar. In addition to Ar, He can be used as balance gas or discharge gas. However, the Ar plasma is known to have better energy transfer efficiency compared to the He plasma under same conditions 33 . Thus, Ar selected as balance gas. The total gas flow was maintained at 100 ml/min. Mass flow controllers (SIERRA) controlled the flow of each gas. Gas chromatography (AGILENT GC 6890 N) equipped with a thermal conductivity detector (TCD) and a flame ionization detector (TCD) detected the product gases after the reactant gases went through the discharge region. The line from the latter part of the reactor to the gas chromatography was heated up to 120 °C, so all products were vaporized. The columns inside the GC were CP-7429 capillary column for TCD, and DB-5 capillary column for FID. CP-7429 column was composed of two parallel columns (CP-Molsieve 5 Å and PoraBOND Q) which allowed us to detect H 2 , O 2 , CO, CH 4 , CO 2 , C 2 H 2 , C 2 H 4 , C 2 H 6 , C 3 H 6 , and C 3 H 8 . By DB-5 column, CH 3 OH and HCHO were identified. Methane conversion and methanol yield were obtained at low temperature. However, the reaction temperature was changed from room temperature to about 100 °C depending on the plasma condition. Methane conversion, and selectivity and yield of CO, CO 2 , and CH 3 OH are acquired in the following: