Potassium hexacyanoferrate

HBF₄ (48% in H₂O), 4-dodecylaniline (97%), 4-ethylaniline (98%), NaNO₂ (97.0%), acetic acid (≥99.7%), propionic acid (≥99.5%), acetonitrile (≥99.9%), tetrabutylammonium tetrafluoroborate (>99%), dihexadecyl phosphate (DHP), bis(2-ethylhexyl) phosphate (BEHP), potassium hexacyanoferrate(III) K₃[Fe(CN)₆], potassium hexacyanoferrate (II) trihydrate, K₂[Fe(CN)₆], (≥99.5%), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, ≥99.9%), ruthenium hexamine [Ru(NH₃)₆]³⁺, and phosphate buffered saline (PBS) tablets were obtained from Sigma-Aldrich (Oakville, ON, Canada). Hydrochloric acid (Reagent grade) was obtained from Caledon Laboratories Ltd. (Georgetown, ON, Canada). All solutions were prepared with deionized water obtained with a Millipore system (Woodbine, ON, Canada) after filtration with 0.4 µm filters. Nitrogen gas (99.999% purity) for electrochemical experiments was purchased from Air Liquide (Mississauga, ON, Canada).

Thiolated MPT64 aptamer in the form of HS-(CH₆)₆-OP(O)₂O-(CH₂CH₂O)₆-5′-TTTTT-aptamer-3′ were synthesized by Eurogentec (Belgium)^{11 (link)–13} . Magnesium chloride (MgCl₂, ≥99%)_, sodium chloride (NaCl, ≥99.5%), sulfuric acid (H₂SO₄, 96%), potassium hexacyanoferrate (III), potassium hexacyanoferrate (II) trihydrate, and 6-mercapto-1-hexanol (MCH) used in the sample preparation were of analytical grade and purchased from Sigma-Aldrich, UK. Tris base (≥99.0%), isopropanol (propan-2-ol, HPLC grade) were purchased from Fisher Scientific. Absolute ethanol was purchased from Aidabul Distillery (Kazakhstan). Target immunogenic protein MPT64 (46 kDa) (Rv1980c) (Gene ID: 581375; Accession#: CAA53143) of Mtb H37Rv strain with a concentration of 1 mg/ml was obtained from EnoGene Biotech Co Ltd (Nanjing, China). His and Trx tagged MPT64 protein was purified from recombinant Escherichia coli strain by an affinity chromatography with a >90% purity. According to the manufacturer, the protein was suitable for the use in multiple immunoassay formats, including ELISA, Western Blot, and rapid tests. Non-target ESAT-6 (p463-1) and CFP-10 proteins were purchased from Sunny lab (USA). All aqueous solutions were prepared using 18.2 MΩcm ultra-pure water with a Pyrogard filter (Millipore, UK).

Tetrahydrofuran (THF), poly(vinyl chloride)
(PVC), bis(2-ethylhexyl) sebacate (DOS), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate
(NaTFPB), valinomycin, regioregular poly(3-octylthiophene-2,5-diyl)
(POT), tris(hydroxymethyl)-aminomethane (Tris), potassium hexacyanoferrate(II),
and potassium hexacyanoferrate(III) were from Aldrich (Germany).
Other chemicals used, including hydrochloric acid, were of analytical
grade and were obtained from POCh (Gliwice, Poland). Doubly distilled
and freshly deionized water (resistivity 18.2 MΩ cm, Milli-Qplus,
Millipore, Austria) was used throughout this work.
Unless otherwise
stated, the buffer used was 0.1 M Tris (adjusted
with HCl) to pH 7.3; for a control experiment, 0.1 M Tris buffer (adjusted
with NaOH) to pH 9.0 was used.

The reducing power of the aqueous PCD–β-Lg mixtures was studied with a modified method described by Oyaizu (1986) [32 (link)]. First, the β-Lg solution (0.1 wt.%) was incubated with the PCD in a molar ration of 1:3 (β-Lg:PCD) at pH 6.0 or pH 9.0 for one hour while stirring the mixture. A volume of 2 mL of the aqueous PCD–β-Lg mixtures, or an ascorbic acid (99.7%; Merck KGaA, Darmstadt, Germany) calibration standard (10–60 mg/L), were mixed with 2 mL of the phosphate buffer (0.2 M, pH 6.6; Merck KGaA, Darmstadt, Germany) and 2 mL of potassium hexacyanoferrate (1%; Merck KGaA, Darmstadt, Germany). The samples were incubated in a water bath at 50 °C for 20 min. Then, 2 mL of trichloroacetic acid (10%; Carl Roth GmbH & Co. KG, Karlsruhe, Germany) were added and the samples were centrifuged at 1,500× g for 10 min. For the photometric measurement, 1.3 mL of the supernatant was mixed with 0.26 mL of iron (III) chloride (0.1 %; neoLab^® Migge GmbH, Heidelberg, Germany) and 1.3 mL of distilled water. The photometric measurement was, then, performed exactly 10 min after mixing the reagents in the cuvette at 700 nm [33 (link)].

Glucose oxidase (GOx) (EC 1.1.3.4, from Aspergillus niger, specific activity 210 U mg⁻¹), potassium hexacyanoferrate(III), luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) sodium salt, hydrogen peroxide solution 30% (w/w), glucose, pullulan (from Aureobasidium pullulans) and Whatman CHR 1 chromatographic paper (20 × 20 cm² sheets) were purchased from Merck KGaA (Darmstadt, Germany). All other reagents were of the highest purity available.
potassium hexacyanoferrate(III) (0.02 mol L⁻¹) and luminol (0.2 mol L⁻¹) solutions for the fabrication of the origami µPAD were prepared in deionized water and stored at 4 °C in the dark until use. Stock solutions of GOx (1000 U mL⁻¹) were prepared in 0.1 mol L⁻¹ phosphate buffer (pH 5.5) and stored at −20 °C until use. For loading in the biosensor, the GOx stock solution was diluted to the working concentration (50 U mL⁻¹) with 0.01 mol L⁻¹ phosphate buffer (pH 5.5) and supplemented by 50 mg mL⁻¹ of pullulan as a stabilizing agent. Hydrogen peroxide and glucose standard solutions were prepared in 0.01 mol L⁻¹ phosphate buffer (pH 5.5).
An enzymatic colorimetric assay in the 96-well microplate format (Invitrogen Glucose Colorimetric Detection Kit, Thermo Fisher Scientific, Waltham, MA, USA) was used as a reference method for assessing glucose concentration of real samples.

After the incubation of the cells with VSOP, the cells were washed two times with water prior to incubation with Nuclear Fast Red Stain solution for 30 min (Roth, Karlruhe, Germany). After washing with water, the cells were treated with a solution consisting of a 1:1 mixture of 2% HCl and 2% potassium hexacyanoferrate (Merck). The cells were mounted with coverslips and were photographed within 15 min using an inverse DMi1 microscope equipped with a MC 120 HD microscope camera (Leica, Wetzlar, Germany).

Fresh cut chervil was received from Frøvoll Farm, Randaberg, located in Southwestern Norway (59°0′56.3″ North, 5°37′25.8″ East). Experimental research and field studies on the plants, including the collection of plant material complied with relevant institutional, national, and international guidelines and legislation. Ferric chloride hexahydrate (Fluka), potassium hexacyanoferrate, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium peroxodisulfate, gum Arabic, hydrochloric acid, 85% phosphoric acid, gallic acid, Trolox, potassium phosphate, sodium chloride, chlorogenic acid and formic acid were provided from Merck, Norway. Methanol (Rathburn) and acetonitrile (Rathburn) were provided from Teknolab AS, Norway.