Cupric chloride

Antioxidant (DPPH and ABTS radical scavenging, reducing power (CUPRAC and FRAP), phosphomolybdenum, and metal chelating (ferrozine method)) and enzyme inhibitory activities [cholinesterase (ChE) Elmann’s method], tyrosinase (dopachrome method), α-amylase (iodine/potassium iodide method), and α -glucosidase (chromogenic PNPG method)) were determined using the methods previously described by Zengin et al. (2014) (link) and Dezsi et al. (2015) (link).
For the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay: Sample solution (1 mg/mL; 1 mL) was added to 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was read at 517 nm after a 30 min incubation at room temperature in the dark. DPPH radical scavenging activity was expressed as millimoles of trolox equivalents (mg TE/g extract).
For ABTS (2,2′-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid) radical scavenging assay: Briefly, ABTS+ was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12–16 in the dark at room temperature. Prior to beginning the assay, ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution (1 mg/mL; 1 mL) was added to ABTS solution (2 mL) and mixed. The sample absorbance was read at 734 nm after a 30 min incubation at room temperature. The ABTS radical scavenging activity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016a (link)).
For CUPRAC (cupric ion reducing activity) activity assay: Sample solution (1 mg/mL; 0.5 mL) was added to premixed reaction mixture containing CuCl₂ (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH₄Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to premixed reaction mixture (3 mL) without CuCl₂. Then, the sample and blank absorbances were read at 450 nm after a 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. CUPRAC activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For FRAP (ferric reducing antioxidant power) activity assay: Sample solution (1 mg/mL; 0.1 mL) was added to premixed FRAP reagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a ratio of 10:1:1 (v/v/v). Then, the sample absorbance was read at 593 nm after a 30 min incubation at room temperature. FRAP activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For phosphomolybdenum method: Sample solution (1 mg/mL; 0.3 mL) was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90 min incubation at 95°C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016c (link)).
For metal chelating activity assay: Briefly, sample solution (1 mg/mL; 2 mL) was added to FeCl₂ solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl₂ solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was sub-tracted from that of the sample. The metal chelating activity was expressed as milligrams of EDTA (disodium edetate) equivalents (mg EDTAE/g extract).
For ChE inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with DTNB (5,5-dithio-bis(2-nitrobenzoic) acid, Sigma, St. Louis, MO, United States) (125 μL) and AChE [acetylcholines-terase (Electric ell AChE, Type-VI-S, EC 3.1.1.7, Sigma)], or BChE [BChE (horse serum BChE, EC 3.1.1.8, Sigma)] solution (25 μL) in Tris–HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI, Sigma) or butyrylthiocholine chloride (BTCl, Sigma) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as galanthamine equivalents (mgGALAE/g extract) (Mocan et al., 2016b (link)).
For Tyrosinase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with tyrosinase solution (40 μL, Sigma) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of L-DOPA (40 μL, Sigma). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after a 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as kojic acid equivalents (mgKAE/g extract) (Mocan et al., 2017 (link)).
For α-amylase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with α-amylase solution (ex-porcine pancreas, EC 3.2.1.1, Sigma) (50 μL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37°C. After pre-incubation, the reaction was initiated with the addition of starch solution (50 μL, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated 10 min at 37°C. The reaction was then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Savran et al., 2016 (link)).
For α-glucosidase inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with glutathione (50 μL), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50 μL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside, Sigma) (50 μL) in a 96-well microplate and incubated for 15 min at 37°C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 μL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Llorent-Martínez et al., 2016 (link)).
All the assays were carried out in triplicate. The results are expressed as mean values and standard deviation (SD). The differences between the different extracts were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test with α = 0.05. This treatment was carried out using SPSS v. 14.0 program.

Tg mouse production. The transgene construct contained the gene cassette encoding IL-2Rα/mVenus followed by the SV40 early-gene polyadenylation signal in the place of the initiation codon in exon 3 of the ChAT gene30 (link). The construct was linearized by SfiI digestion, purified by pulse field gel electrophoresis, and microinjected into fertilized C57BL/6J mouse eggs, which were then implanted into pseudopregnant females. The ChAT-IL-2Rα/mVenus Tg mice were identified by Southern blot hybridization or PCR with genomic DNA prepared from tail clips. Tg and non-Tg littermates were used for the following experiments. All animal experiments were approved and performed in accordance with the guidelines for the care and use of laboratory animals established by the Animal Experiments Committee of Fukushima Medical University and Hiroshima University.
Intracranial surgery. Mice (8 weeks old) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and subjected to bilateral intracranial injection of IT solution [20 μg/ml anti-Tac(Fv)-PE38 in PBS containing 0.1% mouse serum albumin]. For targeting of cholinergic neurons in the MS/vDB and NBM, solution was injected into 12 sites (0.2 μl/site) and 6 sites (0.3 μl/site), respectively, through a glass micropipette that was stereotaxically introduced by using the coordinates from an atlas of the mouse brain52 . The anteroposterior, mediolateral and dorsoventral coordinates (mm) from bregma and dura were (1.1, ±0.1, −3.7), (1.1, ±0.1, −4.1), (0.8, ±0.1, −3.8), (0.8, ±0.3, −4.7), (0.6, ±0.1, −3.7), and (0.6, ±0.1, −4.2) for injection into the MS/vDB; and (−0.4, ±1.6, −3.7), (−0.7, ±1.8, −3.8), and (−0.9, ±2.0, −3.8) for injection into the NBM. Injection was carried out at a constant flow rate of 0.1 μl/min with a microinfusion pump, and the micropipette was left in situ for 2 min after each infusion.
Drug treatment. Donepezil hydrochloride (Sequoia Research Products Ltd.) and rivastigmine hydrogen tartrate (provided by Novartis Pharma AG, Basel, Switzerland) were dissolved into saline at a concentration of 0.2 or 0.4 mM. Mice received the intraperitoneal treatment of drug solution (2 or 4 μmol/kg) 30 min before the behavioural testing.
Histology. Fixed brains were cut into sections, and the sections were incubated with primary antibodies for GFP (rabbit, 1:2,000, Life Technologies), ChAT (mouse, 1:1,000, Millipore), parvalbumin (rabbit, 1:1000, Sigma-Aldrich), and then with fluorescein isothiocyanate-conjugated or biotinylated secondary antibodies. The immunoreactive signals were visualized by using a Vectastain Elite ABC kit. For double immunofluorescence histochemistry, the sections were incubated with anti-GFP and anti-ChAT antibodies, and then with species-specific secondary antibodies conjugated to Alexa488 (Molecular Probes) and Cy3 (Jackson ImmunoResearch). 4,6-Diamidino-2-phenylindole (DAPI, 1:1,000, Molecular Probes) was used to label nuclei. For cell counts, the number of immunopositive cells in each area was counted in the representative four sections through the MS/VDB or NBM (the anteroposterior coordinates from bregma: 1.3, 0.9, 0.7, and 0.5 mm for the MS/VDB; and −0.3, −0.5, −0.8, and −1.0 mm for the NBM), and the total number of immunopositive cells was calculated. Cresyl violet staining was processed to check for nonspecific damage on the brain tissue around the injection sites. For AChE staining, brain sections were rinsed in 0.1 M maleic acid buffer (pH = 6.0) and incubated for 10 min in 0.1 M maleic acid buffer containing 340 μM acetylthiocholine iodide, 50 μM sodium citrate, 30 μM cupric sulphate, and 5 μM potassium ferricyanide. To inhibit non-acetylcholinesterases, 10 nM ethopropazine was added to the solution. After the incubation, sections were washed with 50 mM Tris-HCl buffer (pH = 7.6) and soaked in 50 mM Tris-HCl buffer containing 620 nM cobalt chloride. Sections were then incubated for 7 min in 50 mM Tris-HCl buffer containing 190 μM diaminobenzidine, 0.003% H₂O₂, and 0.1% nickel ammonium sulphate.
Behavioural analysis. Adult naïve male mice were housed in standard lab Plexiglas cages (225 × 338 × 140 mm, length × width × height, four mice per cage) on a 12-h light/12-h dark cycle. The experiments were conducted during the light period. After the surgery, mice were given a 1-week recovery period, followed by the serial object exploration task32 (link) or one-trial object exploration task33 (link)34 (link). Different mice were used for the serial object exploration task, one-trial object recognition task, and the experiment with drug treatments. The open fields for these tasks were positioned in the centere of a room that had overhead lighting and contained various visual cues, including a computer, monitor, and shelves and posters on the wall. The animals’ behaviour was monitored using an overhead colour CCD camera (AVC-636SN; ITS, Co. Ltd.) connected to a digital video cassette recorder. During the tasks, the number of times the mouse snout made contact with an object (i.e., number of contacts) was manually counted. The counted data were confirmed by the video-recorded behaviour. The measurements of exploration were scored by an observer who was blinded to the animal groups and drug treatments.
For the serial object exploration task, a circular, polyvinylchloride open field (70-cm in diameter, 40-cm high) was used, and four positions in the open field were marked as north (N), south (S), east (E), and west (W) (see Fig. 2a). The wall was equipped with a striped board composed of 2.5-cm-wide vertical black and white lines in the N position. The base of the open field was divided into four quadrants (NW, NE, SW, and SE), and further subdivided into 16 equal-sized areas for the measurement of locomotor activity. All of the objects (A–F) used for the task had different visual and haptic features. The task contained seven successive sessions (S1–S7; 6 min for each session) with an intersession interval of 3 min. These sessions consisted of four different phases for familiarization (S1), object exploration (S2–S4), displaced object exploration (S5/S6), and novel object exploration (S7). During S1, a mouse was placed in the empty open field and familiarized with it. During S2–S4, five different objects (A–E) were used, and four objects (A–D) were placed in the middle of each quadrant, while another object (E) was positioned in the centre of the field. Two objects (B and E) were displaced during S5, and no objects were moved during S6. Another object (A) was replaced by a novel object (F) during S7. A duplicate in the case of a repeating object was used for each phase within a complete trial. The mouse was allowed to explore freely in the open field during S2–S7, and the number of contacts with the objects was counted. In each session, the mouse began its exploration from one of four release points (N, S, E, and W) in a pseudo-random manner. Mice were placed back in their home cages during the 3-min intersession interval. Locomotor activity was assessed by counting the number of unit crossings with the subdivided areas in the base. During S5, the average of number of contacts with non-displaced objects (A, C, and D) or displaced objects (B and D) was calculated. During S7, the average number of contacts with non-displaced objects (C and D) or displaced objects (B and E) and the number of contacts with the novel object (F) were determined. All objects and the open field were washed with 70% ethanol after each trial.
For the one-trial object exploration task, a square, polyvinylchloride open field (35 × 35 cm and 30 cm high) was used (see Fig. 3a). The task consisted of two sessions (3 min for each session) for the object exploration and displaced/novel object exploration with an intersession delay period of 3 or 30 min. The sequencing of 3-min and 30-min delays after the object exploration was counterbalanced among the mice. During the object exploration session, two identical objects were placed in the open field in a line-shaped spatial configuration, and during the displaced/novel object exploration session one object was displaced or exchanged with a novel one (B). A duplicate in the case of a repeating object was used for each phase within a complete trial. The mice were allowed to explore freely in the open field during the sessions, and the number of contacts with the objects was counted. In each session, the mice began their exploration from one of two release points in a pseudo-random manner and were placed back in their home cages during the 3- or 30-min intersession delay period. The left–right positions of the displaced and non-displaced/novel objects were counterbalanced. The open field and objects were washed in 70% ethanol after each trial.
Statistical analysis. For statistical comparisons, the ANOVA and post hoc Bonferroni test were used with significance set at P < 0.05. All values were expressed as the mean ± s.e.m. of the data. Repeated ANOVA was used for the analysis of within-subjects design.

The preparation technique and the thickness of the PA template substantially define the result of metal electrodeposition. Therefore, in spite of the fact that this procedure became almost standard, the technology for the formation of the PA template is constantly improved by researchers. Generally, before the deposition of NWs, the thick alumina film is detached from the Al substrate after removing the barrier layer at the bottom of the pores. Next, a conductive layer is formed by means of sputtering a metal usually onto the back side of the template with continuous nanochannels [2 (link),19 (link),33 (link)–34 (link)].
In our work, the custom-made PAs were prepared by dc anodization of Al foil, as described in details elsewhere [35 ]. First, commercial aluminium foil (99.995%) with a size of 60 × 48 mm and a thickness of ca. 100 µm is annealed at 350 °C for 1 h. Then, the samples were electropolished in a mixture of chloric acid and acetic acid 1:4 (volumetric ratio) at T ≈ 8 °C and a voltage of 25 ± 2 V for 1–2 min to reduce the surface roughness. Next, the samples were washed in distilled water and dried in a dry air stream. Before anodization, the technological frame has been formed along the perimeter and in the center of the substrate. It is necessary to strengthen the mechanical stability of a free-standing membrane and to restrict certain zones with identical surface area. The frame destination and its formation procedure are described in more detail in [35 ]. Thick porous alumina films with ordered structure of pores have been prepared by two-step anodization in aqueous solution of oxalic acid (H₂C₂O₄, 0.3 M) at 15 °C. The first stage of anodization was performed under a constant voltage of 50 ± 5 V for 25 min. After the first anodization, the preformed oxide film was removed by wet chemical etching in a mixture of phosphoric acid (H₃PO₄, 0.5 M) and chromic acid (H₂Cr₂O₇, 0.2 M) at 80 ± 5 °C for 5 min. The second stage of anodization was performed under the same conditions for 1 to 4 h.
Then, electrochemical etching of the barrier layer at the bottom of the pores was carried out by gradual reduction of the forming voltage down to 15 ± 2 V. Further, the detachment of alumina from the substrate was performed by Al dissolution in a saturated solution of hydrochloric acid and cupric chloride (HCl + CuCl₂). Chemical dissolution of the rest of a barrier layer at the pore bottom and chemical pore widening was performed in 4 wt % Н₃РО₄ (30 °C) for 15 min. Finally, an electric contact metal (Ta 300 nm + Ni 300 nm or Ta 300 nm + Cu 300 nm) layer was sputtered onto the back side of PA, and a protective coating of chemically resistant varnish HSL (perchlorovinyl lacquer) was applied. As a result, the alumina template with a 30–90 µm thick ordered structure (Figure 1) with pore diameters of 50 ± 5 nm has been fabricated.

Reagent-grade chemicals were purchased in their pure form and used as received: titanium (IV) oxide (TiO₂, 99.0%, with particle size = 10μm), polyacrylonitrile (PAN, MW = 150.000), N,N–dimethylformamide (DMF, 99.0%), benzene (C₆H₆, 99.0%), aluminum chloride (AlCl₃, 98.0%), cupric chloride (CuCl_2, 99.0%), hydrochloric acid (HCl, 37.0%), sodium hydroxide (NaOH, 99.0%), polymeric perfluorosulfonic acid (nafion, (5.0%), and potassium hydroxide (KOH, 98.0%).

tert-Butyl acrylate (TBA, 99%), cupric bromide (CuBr₂, 99.9%), tris(2-pyridylmethyl)amine (TPMA, 98%), N,N-dimethylformamide (DMF, 99.8%), methanol (MeOH, 99.9%), butyl methacrylate (BMA, 99%), cupric chloride (CuCl₂, 98%), pentamethyldiethylenetriamine (PMDETA, 98%), anisole (99%), stannous octoate (95%), tetrahydrofuran (THF, 99.5%), dichloromethane (DCM, 99.5%), trifluoroacetic acid (TFA, 99.5%), methylparaben (99%), triethylamine (99.5%), 2-bromoisobutyryl bromide (98%), 4-phenylphenol (99%), 2-naphthol (99%), 1,8-naphthalic anhydride (99%), ethanolamine (99%), ethanol (99.9%), and 1-hydroxypyrene (98%) were purchased from commercial sources without further purification unless otherwise stated. All the purchased monomers were purified by passing through a column of basic alumina to remove inhibitors. For flash column chromatography, silica gel with 200–300 mesh was used.