Potentiometry

ATP levels were determined with CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, USA) according to the manufacturer’s specifications. Intracellular NAD(P)H levels were measured by auto-fluorescence using specific excitation and emission wavelengths of 340/428 nm as described^{26 (link)}. Mitochondrial membrane potential (∆Ψm) and intracellular ROS levels in intact cells were determined by flow cytometry using the potentiometric probe tetramethylrhodamine methyl ester (TMRM, Molecular Probe) and dihydroethidium (DHE) probe, respectively. Details are provided in the supplementary document.

4-Chloromethylstyrene (1a) and all the other reagents and solvents were from Merck (formerly Sigma-Aldrich, Darmstadt, Germany) and were purified by standard procedures. Azo-bis-isobutyronitrile (AIBN) was crystallized from methanol (MeOH). 4-(2-bromoethyl)-styrene (1b) was prepared by a known procedure [16 (link)]. The organic solutions were dried over anhydrous magnesium sulphate and were evaporated using a rotatory evaporator operating at a reduced pressure of about 10–20 mmHg. The melting ranges of the solid compounds in this study were determined on a 360 D melting point device with a resolution of 0.1° C (MICROTECH S.R.L., Pozzuoli, Naples, Italy). The melting points and boiling points are uncorrected. The FTIR spectra were recorded as films or KBr pellets on a Perkin Elmer System 2000 instrument (PerkinElmer, Inc., Waltham, MA, USA), while ATR-FTIR analyses were carried out using a Spectrum Two FT-IR Spectrometer (PerkinElmer, Inc., Waltham, MA, USA) ¹H and ¹³C NMR spectra were acquired on a Bruker DPX spectrometer (Bruker Italia S.r.l., Milan, Italy) at 300 and 75.5 MHz, respectively. Fully decoupled ¹³C NMR spectra were reported. Chemical shifts were reported in ppm (parts per million) units relative to the internal standard tetra-methyl-silane (TMS = 0.00 ppm), and the splitting patterns were described as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad signal). Mass spectra were obtained with a GC-MS Ion Trap Varian Saturn 2000 instrument (Varian, Inc., Palo Alto, CA, USA; EI or CI mode; filament current: 10 mA) equipped with a DB-5MS (J&W) capillary column. Elemental analyses were performed with an EA1110 Elemental Analyzer (Fison Instruments Ltd., Farnborough, Hampshire, England). The UV-Vis spectra were acquired using a UV-Vis spectrophotometer (HP 8453, Hewlett Packard, Palo Alto, CA, USA) equipped with a 3 mL cuvette. HPLC analyses were performed on a Jasco model PU-980 instrument (JASCO Corporation, Hachioji, Tokyo, Japan), equipped with a Jasco Model UV-970/975 intelligent UV/Vis detector (JASCO Corporation, Hachioji, Tokyo, Japan) at room temperature. A constant flow rate (1 mL/min), UV detection at 254 nm, a 25 × 0.46 cm Hypersil ODS 5 mm column, and a mixture of acetonitrile/water 6/4 as an eluent were employed for the acquisitions. GC-FID analyses were performed on a Perkin Elmer Autosystem (Varian, Inc., CA, USA), using a DB-5, 30 m, diameter 0.32 mm, and a film 1 mm capillary column. Column chromatography was performed on Merck silica gel (70–230 mesh). Viscosity measurements were performed with an Ubbelhode micro viscosimeter (SI Analytics, Hattenbergstr, Germany) at 30 °C in MeOH. Dynamic Light Scattering (DLS) and Z-potential (ζ-p) determinations were performed using a Malvern Nano ZS90 light scattering apparatus (Malvern Instruments Ltd., Worcestershire, UK). Potentiometric titrations were carried out using a Hanna Micro-processor Bench pH Meter (Hanna Instruments Italia srl, Ronchi di Villafranca Padovana, Padova, Italy), which was calibrated using standard solutions at pH =4, 7, and 10 before titrations. Lyophilizations were performed using a freeze–dry system (Labconco, Kansas City, MI, USA). Thin layer chromatography (TLC) was carried out using aluminum-backed silica gel plates (Merck DC-Alufolien Kieselgel 60 F254, Merck, Washington, DC, USA), and detection of spots was made by UV light (254 nm) using a Handheld UV Lamp, LW/SW, 6W, UVGL-58 (Science Company^®, Lakewood, CO, USA).

Milli-Q water was used for preparation of stock solutions and samples. Exact concentrations of stock solutions of the ligand and the organometallic cations were determined by pH-potentiometric titrations according to procedure published in our former work [50 (link)]. Organometallic complexes were obtained by mixing the corresponding ligand and metal precursor ([Rh(η⁵-C₅Me₅)(µ-Cl)Cl]₂, [Ru(η⁶-p-cymene)(µ-Cl)Cl]₂) in 1:0.5 molar ratio in water; solutions were equilibrated for 24 h. HSA stock solutions were prepared in modified phosphate buffered saline (PBS′), pH 7.40 containing 12 mM Na₂HPO₄, 3 mM KH₂PO₄, 1.5 mM KCl and 100.5 mM NaCl. Concentration of the K⁺, Na⁺ and Cl^‒ ions corresponds approximately to that of the human blood serum. Residual citrate content of HSA was removed by repeated ultrafiltration of the protein stock solution, and its concentration was calculated from its UV absorption: λ_{280 nm}(HSA) = 36,850 M⁻¹ cm⁻¹ [51 (link)]. Stock solutions of WF and DG were prepared as described previously [31 (link)]. Samples containing RhCp* and RuCym compounds were incubated for 2 and 24 h, respectively, prior to the measurements, or the reaction kinetic was followed. All samples were prepared in PBS’ and incubated at 25 ± 0.1 °C and measurements were carried out at this temperature.

The printed circuit board was designed and wired by using the AutoCAD designer, and it was printed and aligned for soldering. After connecting the electrodes through the sensor interface, the board was powered by a battery module to collect potentiometric signals from the surface of the ion-sensing microneedle electrode. The signals were processed through a two-stage differential circuit to reduce signal distortion and common-mode noise interference. Subsequently, the collected analog signal was converted into a digital signal using the STM32 chip through analog-to-digital conversion, and a gradient conversion was performed according to the calibration curve obtained from in vitro experiments to obtain the corresponding ion concentration. The concentration was transmitted to the Bluetooth module via a serial port to send the concentration data of different ions to the mobile terminal that displays the trend graph of ion changes through the interface that was designed using LabVIEW software (NI, USA).