A Novel Boranil-Based Colorimetric and Fluorescence Quenching Probe for Selective Detection and Bioimaging of the Fe³⁺ ion

Abstract

A novel, readily synthesizable boranil-based chemosensor, HSR, was structurally and spectroscopically characterized by single-crystal X-ray diffraction, IR spectroscopy, ¹H, ¹³C, and ¹¹B NMR spectroscopy, and high-resolution mass spectrometry (HRMS). HSR exhibited highly selective colorimetric and fluorescence responses toward the Fe³⁺ ion over a wide range of competing metal ions, including Na⁺, K⁺, Ba²⁺, Ca²⁺, Mg²⁺, Al³⁺, Zn²⁺, Ni²⁺, Cu²⁺, Co³⁺, Pb²⁺, Cd²⁺, Hg²⁺, and Fe²⁺. Upon Fe³⁺ addition, the solution color changes from pale yellow to red-brown, accompanied by pronounced quenching of fluorescence signals. Concentration-dependent Fe³⁺ ion titrations revealed significant modulation of the photoluminescence and UV–vis absorption profiles. The binding stoichiometry and association constant between HSR and the Fe³⁺ ion were determined by UV–vis and fluorescence titrations, Job’s plot analysis, and HRMS, confirming strong complex formation. Notably, no interference from biologically relevant or toxic metal ions was observed. The limit of detection (LOD), calculated using the 3σ method, was 7.5 × 10^–6 M. The association constant (K _a), obtained from a Benesi–Hildebrand analysis, was 2.35 × 10¹⁵ M^–1, indicating exceptionally strong Fe³⁺ ion binding. Furthermore, HSR displayed good solubility in mixed EtOH/H₂O media and efficient cellular uptake with no cytotoxicity, highlighting its suitability for the detection of the Fe³⁺ ion and bioimaging applications.

Introduction

The pervasive roles of metal ions in biological systems highlight the critical need for sensitive, selective, and reliable metal sensing in biological samples. − Some known metals, such as iron, zinc, copper, and others, are extremely beneficial to living organisms, whereas others, such as chromium, mercury, and cadmium, are extremely detrimental to living cells. The imbalance in the metal-to-mineral ratio in the human body is primarily caused by an unhealthy lifestyle and uncontrolled eating habits. As a result, the accurate and sensitive measurement of these ions is an imperative necessity of time. Metal detection in biological samples is a crucial procedure that can be accomplished by quantification. A good metal sensor should react with very small amounts of metals and provide a very quick response by incubating the metals in their ionic form. A sensitive sensor is able to measure very low concentrations, even micro to nanomolar concentrations of metals. The direct incubation of metal ions in fluorescent solutions at varying concentrations and the alteration of luminescence characteristics with an increase in metal ion concentrations are the foundation of this analysis among known metal sensing techniques.

The reactivity of iron depends upon its two oxidation states, ferric (Fe³⁺ ion) and ferrous (Fe²⁺ ion). Ferric ions play a substantial role in the regulation of several biochemical and cellular processes, and together, their deficiency as well as excess amounts are reported to cause various diseases. An excess amount of Fe³⁺ ions in the body has been allied with an upgraded propensity of certain cancers and abnormal functioning of organs such as the heart, pancreas, and liver, and diseases such as anemia, Alzheimer’s disease, epilepsy, and hemochromatosis. Hence, maintaining proper iron balance is essential for maintaining homeostasis and health.

Therefore, it is of inordinate importance to precisely determine the concentration of the Fe³⁺ ion in living cells. Fluorescent probes are a useful tool for tracking metal ions ,− in living cells because of their sensitivity, ease of use, excellent selectivity, and real-time monitoring capabilities.

Fluorescent probes have been extensively employed in the imaging of living cells and the detection of trace iron ions, offering a potent way to investigate the function of the Fe³⁺ ion in biological processes. For fluorescent probes, in order to better distinguish the Fe³⁺ ion, the design of recognition groups is crucial. Formerly reported probes , used to sense Fe³⁺ primarily use two recognition approaches.

Based on the fact that the Fe³⁺ ion contributes to several oxidation and hydrolysis reactions, such as the oxidation of catechol, ferrocene, and hydroxylamine, the hydrolysis of acetal and Schiff base, and the chelation of the Fe³⁺ ion with fluorophores, the first mode is the probe design. However, is it possible for the addition of additional heteroatoms to probe molecules to interact with an iron ion and alter their fluorescence?

The second mode is the probe chelated with an iron ion, which commonly bears groups containing N, S, and O elements into the probe. The coordination of the iron ion with elements present can lead to variations in fluorescence behavior and, hence, will help in sensing the iron ion. The most prevalent fluorophores in terms of the makeup of fluorescent probes are coumarin, rhodamine, cyanine, boron-dipyrromethene (BODIPY), 1,8-naphthalimide, and boranils. , Among them, the borate complexes based on salicylideneimine ligands, named by the abbreviation boranils, appear as the most auspicious, owing to their facile synthesis and dual-state emission properties. Boranil dyes have been used in bioimaging, bioconjugation, and biosubstrate detection, and they have shown themselves to be good substitutes for BODIPY dyes.

Leveraging this principle, we report for the first time the synthesis of a fluorescent probe incorporating o-catechol and N-(2-methylphenyl)­salicylideneimine (HL) motifs and establish its application for Fe³⁺-ion detection. This probe offers a safe, efficient, cost-effective, and highly sensitive strategy for monitoring iron ions in living systems.

Experimental Section

Materials and Methods

Rigorous precautions were taken to exclude moisture from glassware, reactants, and solvents during the synthesis of the boron­(III) compound. Solvents were dried and purified by standard procedures. N-(2-Methylphenyl)­salicylideneimine (HL) was prepared by the published procedure. Salicylaldehyde and o-toluidine from TCI America were used without any purification. Triisopropyl borate and o-catechol (Sigma-Aldrich) were distilled before use. Boron was estimated as methyl borate. Isopropyl alcohol in an azeotrope was determined oxidimetrically. The salts Fe­(NO₃)₃·9H₂O, CuCl₂·2H₂O, NaCl, BaCl₂, CaCl₂·2H₂O, KCl, Pb­(CH₃COO)₂·3H₂O, MgCl₂·6H₂O, Cd­(CH₃COO)₂·2H₂O, KNO₃, Al­(NO₃)₃·9H₂O, ZnCl₂, HgCl₂, Ni­(NO₃)₂·6H₂O, Co­(NO₃)₂·6H₂O, FeSO₄, and FeCl₃ were purchased from Sigma-Aldrich, USA, with 99.99% purity. The above-mentioned salts were dissolved in ultrapure water to prepare concentrated (0.1 M) solutions of each compound. The stock solution (1 × 10^–4 M) of HSR was prepared in an ethanol medium and employed for spectroscopic studies.

Physical Measurement

Melting points of the complexes were determined by the open capillary method using a Gallenkamp melting point apparatus and are reported uncorrected. Elemental analyses were carried out on a EuroVector EA 3000 elemental analyzer. FT-IR spectra were recorded in the range of 4000–400 cm^–1 using KBr pellets on a PerkinElmer FT-IR spectrophotometer. ¹H, ¹³C, and ¹¹B NMR spectra were acquired in CDCl₃ on a JEOL ECZ-500 MHz NMR spectrometer, with tetramethylsilane (TMS) as the internal standard. High-resolution mass spectra were obtained using a SCIEX X500R QTOF mass spectrometer. UV–vis absorption spectra were recorded on an Infinite 200 PRO plate reader (Tecan, Manne Dorf, Switzerland). Fluorescence emission spectra were measured using a PerkinElmer LS-55 fluorescence spectrometer.

Crystallographic Studies

Single-crystal X-ray data of the synthesized compounds were recorded on a Rigaku Oxford diffractometer using graphite monochromated Mo Kα radiation (λ = 0.7107 Å). Data collections for all complexes were performed at room temperature. The collected data was analyzed with the help of CrysAlisPro CCD software. The structures were solved with the SHELXL-2018 program for crystal structure solution and full-matrix least-squares refinement against F ² using SHELXL-2018 with the WinGX2 package of programs. Final refinement included the atomic positions for all atoms, anisotropic thermal parameters for all the non-hydrogen atoms, and isotropic thermal parameters for all the hydrogen atoms. Crystallographic data of the boron complex [(L)­B­(OC₆H₄O)] (HSR) were deposited in the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK (CCDC 2423865).

Cell Culture

The HEK-293 (a non-malignant cell line) cell line was obtained from the National Centre for Cell Science (NCCS), Pune, India. HEK-293 cells were grown in DMEM (complete DMEM containing 10% FBS) supplemented with penicillin (100 IU) and streptomycin (100 μg/mL) at 37 °C in a humidified incubator containing 5% CO₂.

MTT Assay

The effect of HSR on the survival of non-malignant HEK-293 cells was examined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) as described by Jaiswara et al. and Liu et al. , HEK-293 (1 × 10⁵ cells/mL) cells were seeded in a 96-well plate, followed by treatment with different concentrations of HSR (1, 5, 10, 20, 50, and 100 μM) for 24 h. Consequently, 50 μL of MTT solution (5 mg/mL in PBS) was added to each well of a 96-well cell culture plate, followed by 4 h incubation in a CO₂ incubator. Further, the culture supernatant was carefully discarded from each well without disturbing the pellet. The formazan crystals were dissolved in 50 μL of acidified isopropanol. Absorbance was measured at 540 nm by an ELISA plate reader (ERBA LISA SCAN, Germany).

Bioimaging

HEK-293 (1 × 10⁵ cells/mL) cells were seeded in a six-well plate and incubated with 20 μM HSR (M.W. 329.15) with or without 10 μM Fe­(NO₃)₃·9H₂O (M.W. 404.00) at 37 °C, followed by analysis of Fe³⁺ ion detection by a fluorescence microscope. For the detection of the Fe³⁺ ion, cells were pretreated with 20 μM HSR for 1 h with the subsequent addition of 10 μM Fe­(NO₃)₃·9H₂O for 30 min. Thereafter, cells were washed thrice with PBS and observed under a fluorescence microscope (Olympus, Japan) upon excitation at 405 nm.

Synthesis of N-(2-Methylphenyl)­salicylideneimine (HL)

An equimolar amount of salicylaldehyde (5.85 g, 48 mmol) and 2-methyl-1-aminobenzene (4.37 g, 48 mmol) in methanol (30 mL) was refluxed for ∼4 h, during which the solution turned yellow. The excess of solvents was removed by distillation to obtain a yellow solid. It was further recrystallized from methanol and dried under reduced pressure to get a yellow-colored pure crystalline compound. Yield: 8.80 g, 87%; m.p.: 46 °C; Anal. Found: C 79.45, H 6.14, N 6.58%; Calc. For C₁₄H₁₃NO: C 79.59; H 6.20; N 6.63%; IR (KBr Pellets, cm^–1): 3468m, 3064w, 2820w, ν_(CN) 1629m, 1560m, 1490s, 1357m, ν_(CO) 1274s, 1106m, 1037w, 912m, 835s, 758s, 654w; ¹H NMR (500.16 MHz, CDCl₃): δ_H = 13.47 (1H, s, –OH), 8.57 (1H, s, –CHN), 7.40 (2H, m, Ph-H), 7.27 (2H, q, Ph-H, J = 6.5 Hz), 7.20 (1H, t, Ph-H, J = 8 Hz), 7.11 (2H, dd, Ph-H, J = 8, 8 Hz), 6.95­(1H, t, Ph-H, J = 8 Hz), 2.41 (3H, s, o-CH₃); ¹³C NMR (125.76 MHz, CDCl₃): δ_C = 162.35 (C1), 161.34 (C7), 147.61 (C8), 133.21 (C3), 132.43 (C5), 132.30 (C12), 130.82 (C10), 127.13 (C11), 126.96 (C4), 119.46 (C6), 119.13 (C2), 117.84 (C9), 117.33 (C13), 18.33 (o-CH₃); HRMS (ESI-TOF) m/z: [M + H]⁺ calc for C₁₄H₁₃NO 212.1075; found 212.1192. The IR, ¹H NMR, ¹³C NMR, and HRMS (ESI-TOF) spectra of HL are provided in Figures S1–S4.

Synthesis of [(L)­B­(OC₆H₄O)] (HSR)

An equimolar amount of triisopropyl borate (0.9359 g, 4.98 mmol), o-catechol (0.5479 g, 4.98 mmol), and HL (1.0504 g, 4.98 mmol) were dissolved in benzene (60 mL). The reaction mixture was refluxed, and 2-propanol thus liberated was fractionated azeotropically with benzene over a period of 14 h. The progress of the reaction was followed by estimating the 2-propanol in the azeotrope (found, 0.91 g; calculated, 0.90 g). After completion of the reaction, excess solvent was removed under reduced pressure, and the yellow solid product was recrystallized from a benzene/n-hexane mixture. Yield: 1.60 g, 98%; m.p.: 150 °C; Anal. Found: C, 72.88; H, 4.82; N, 4.22; B, 3.21%; Calc. For C₂₀H₁₆BNO₃: C, 72.98; H, 4.90; N, 4.26; B, 3.38%; IR (KBr Pellets, cm^–1): 3056w, 2966w, ν_(CN) 1615m, 1559m, 1483s, ν_(BO) 1378w, ν_(CO) 1292m, 1267w, 1155w, 1086m, 940m, 856m, ν_(BN) 807m, 752S, 598w;¹H NMR (500.16 MHz, CDCl₃): δ_H = 8.19 (1H, s, –CHN), 7.69 (1H, t, Ph-H, J = 7.5 Hz), 7.45 (1H, d, Ph-H, J = 8 Hz), 7.28 (1H, dd, Ph-H, J = 7.5 Hz), 7.18 (3H, q, Ph-H, J = 8.5 Hz), 7.06 (2H, m, Ph-H), 6.59­(4H, s, Ph-H), 2.34 (3H, s, o-CH₃); ¹³C NMR (125.76 MHz, CDCl₃): δ_C = 165.11 (C1), 159.29 (C7), 150.57 (C14), 141.27 (C8), 139.51 (C3), 132.41 (C5), 131.71 (C12), 131.03 (C10), 128.82 (C11), 126.38 (C4), 125.70 (C2), 120.98 (C19), 120.41 (C18), 120.03 (C6), 119.20 (C9), 108.91 (C13), 17.77 (o-CH₃); ¹¹B NMR (160.47 MHz, CDCl₃): 7.42; HRMS (ESI-TOF) m/z: [M + H]⁺ calc for C₂₀H₁₇BNO₃ 330.1301; found, 329.0096. The IR, ¹H NMR, ¹³C NMR, ¹¹B NMR, and HRMS (ESI-TOF) spectra of the HSR synthesized in benzene are provided in Figures S5–S9.

Alternative Synthetic Route for HSR

Equimolar quantities of triisopropyl borate (1.5123 g, 8.04 mmol), o-catechol (0.8854 g, 8.04 mmol), and HL (1.6974 g, 8.04 mmol) were dissolved in 70 mL of toluene. The resulting reaction mixture was refluxed, during which its color gradually changed to dark yellow. The 2-propanol formed during the reaction was continuously removed by azeotropic distillation with toluene over a period of 16 h. The progress of the reaction was monitored by measuring the amount of 2-propanol collected in the azeotrope (found, 1.46 g; calculated, 1.45 g). Upon completion of the reaction, the excess solvent was removed under reduced pressure, and the resulting yellow crystalline solid was purified by recrystallization from a toluene/n-hexane mixture. Yield: 2.57 g, 97%; m.p.: 150 °C; IR (KBr Pellets, cm^–1): 3041w, 2967w, ν_(CN) 1630m, 1557w, 1489s, ν_(BO) 1366w, 1311m, ν_(CO) 1232m, 1096m, 1023m, 949m, 857w, ν_(BN) 808s, 728w, 581w, 465w; ¹H NMR (500.16 MHz, CDCl₃): δ_H = 8.09 (1H, s, –CHN), 7.59 (1H, t, Ph-H, J = 7.5 Hz), 7.35 (1H, d, Ph-H, J = 8 Hz), 7.20 (1H, dd, Ph-H, J = 8 Hz), 7.08 (3H, m, Ph-H), 6.96 (2H, m, Ph-H), 6.47 (4H, s, Ph-H), 2.28 (3H, s, o-CH₃); ¹³C NMR (125.76 MHz, CDCl₃): δ_C = 165.12 (C1), 159.22 (C7), 150.59 (C14), 141.24 (C8), 139.46 (C3), 132.36 (C5), 131.74 (C12), 130.98 (C10), 128.77 (C11), 126.31 (C4), 125.65 (C2), 120.36 (C19), 119.93 (C18), 119.13 (C6), 114.99 (C9), 108.86 (C13), 17.73 (o-CH₃). ¹¹B NMR (160.47 MHz, CDCl₃): 7.43 ppm. The IR, ¹H NMR, ¹³C NMR, and ¹¹B NMR spectra of the HSR synthesized in toluene are provided in Figures S10–S13.

Theoretical Calculations

Density functional theory (DFT) was employed to investigate the electronic structure, geometry, and stability of the compound. Calculations were carried out using the B3LYP functional with the 6-311G+(d,p) basis set in Gaussian 09, and the optimized structure was visualized in Gauss View 6.0 (Figure S14).

Frontier molecular orbital (FMO) analysis was performed to understand the compound’s electronic and optical properties. HOMO–LUMO energies were obtained using TD-DFT (B3LYP/6-311+G­(d, p)) with Gaussian 09. From these energy values, several quantum chemical descriptors were derived, including ionization potential (IP), electron affinity (EA), electronegativity (χ), hardness (η), softness (S), chemical potential (μ), and electrophilicity index (ω). Based on Koopmans’ theorem, IP and EA were estimated as IP = −E _HOMO and EA = −E _LUMO. For the studied compound, the calculated HOMO and LUMO energies were −5.460 eV and −2.869 eV, respectively, giving an energy gap of 2.59 eV, as shown in Figure S15.

The computed parameters (Table S1) reveal that the compound possesses a moderate HOMO–LUMO gap, balanced IP/EA values, and a high electrophilicity index, confirming the feasibility of intramolecular charge-transfer interactions and indicating potential biological activity.

Results and Discussion

N-(2-Methylphenyl)­salicylideneimine (HL) was prepared (Scheme ) by the condensation of salicylaldehyde with o-toluidine in an equimolar ratio. The boron­(III) compound [(L)­B­(OC₆H₄O)] (HSR) was synthesized by the reaction of triisopropyl borate, o-catechol, and HL, Scheme .

1. Synthetic Route of HL and HSR.

In benzene, the newly synthesized yellow crystalline solid compound HSR was obtained in 98% yield and soluble in common organic solvents, i.e., methanol, ethanol, isopropanol, chloroform, DMSO, DMF, etc.

Spectroscopic Studies

IR Spectra

The infrared spectrum of HL shows (Experimental Section) the following features: IR studies of the Schiff base ligand exhibit important IR absorptions at 3468 ν­(OH), 1629 ν­(CN), and 1274 ν­(C–O) cm^–1. The following significant characteristics were observed when the IR spectra of HL and HSR (Experimental Section) were compared: (i) the broad band of ν­(OH) was found to be absent in the complex; (ii) the stretching frequency due to the azomethine ν­(CHN) group upon coordination with the boron atom just slightly decreased from 1629 to 1615 cm^–1 in the complex; (iii) the stretching frequency of phenolate ν­(C–O) for the HL ligand at 1274 cm^–1 was found relocated at a higher frequency at 1292 cm^–1 in HSR; and (iv) an emergence of a new weak band in HSR at 1378 cm^–1 can be due to the ν­(B–O) stretching frequency and those at 807 cm^–1 ascribed to ν­(B ← N).

¹H NMR: HL and HSR exhibit the following characteristic peaks in the ¹H NMR spectra: (i) HL shows a sharp singlet at 13.42 ppm due to the hydroxyl group (O–H), found to be absent in boron complex HSR, endorsing the coordination through the oxygen atom; (ii) HL shows a singlet signal at 8.57 ppm attributed to the azomethine (CHN) proton, which is shifted upfield to 8.19 ppm in the HSR, possibly due to nitrogen–boron coordination; and (iii) signals due to aromatic protons as well as o-CH₃ are found almost unchanged in HSR with respect to HL.

¹³C NMR: A comparison of the ¹³C NMR spectra of HL with HSR has revealed the following important features: (i) HSR exhibits a signal due to the azomethine carbon at 160.5 ppm, shifted compared to HL at 156.5 ppm, indicating coordination of the azomethine nitrogen to boron; and (ii) HSR shows a signal at 163.8 ppm, supporting the formation of a B–O–C bond.

¹¹B NMR: HSR exhibits a signal at 7.42 ppm, which is characteristic of tetra-coordinated boron compounds. ,

Mass Studies: The HRMS TOF-MS ES⁺ spectra of HL and HSR ratify the formation of the purported compounds. In the HRMS TOF-MS ES⁺ spectrum of HL, the molecular ion peak is observed as [M + H]⁺ at 212.1192 [C₁₄H₁₃NO; calculated mass = 212.1075], corresponding to the molecular mass of the Schiff base. However, in HSR, the molecular peak ion peak observed as [M + H]⁺ at 329.0096 [C₂₀H₁₇BNO₃; calculated mass = 330.1301] corresponds to the molecular composition of newly synthesized compound HSR.

Crystal Data Analysis

The HSR has been characterized by a single-crystal X-ray diffractometer. The relevant crystallographic data are listed in Table . The selected bond lengths and angles are given in Table . The compound is crystallized into the monoclinic system with space group P2₁/c. The 2-methyl phenyl ring of the Schiff base ligand is disordered. Disordered atoms were split into two parts and then refined using appropriate restraints. The boron atom has tetrahedral geometry and is bonded with three oxygen atoms and one nitrogen atom (Figure ). The bond lengths (C–O, 1.468(3), 1.458(3), 1.437(3), and B–N, 1.594(3) Å) are analogous to reported data. The bond angles are slightly deviated from regular tetrahedral values. The N(1)–C(1) bond distance of 1.287(3) Å indicates the presence of an imine double bond, whereas the neighboring N(1)–C(12) bond length is 1.457(6) Å, typical for a N–C single bond.

1. Crystallographic Data Collection Parameters for HSR.

parameter	value
empirical formula	C20H16BNO3
formula weight	329.15
T (K)	293(2)
λ (Å)	1.54184
crystal system	monoclinic
space group	P21/c
a (Å)	15.5189(4)
b (Å)	7.6561(2)
c (Å)	14.5494(3)
α (deg)	90
β (deg)	100.787(2)
γ (deg)	90
V (Å3)	1698.13(7)
Z	4
μ(Μο – Κα)/mm–1	0.691
reflections collected/unique	15,977/3053
R (int)	0.0392
final R indices [I > 2σ(I)]	R 1 = 0.0540, wR 2 = 0.1026
R indices (all data)	R 1 = 0.0780, wR 2 = 0.1103
GOF on F2	1.096

2. Selected Bond Lengths (Å) and Bond Angles (deg) for HSR.

bonds	HSR
O(1)–B(1)	1.468(3)
O(2)–B(1)	1.458(3)
O(3)–B(1)	1.437(3)
N(1)–B(1)	1.594(3)
N(1)–C(1)	1.287(3)
N(1)–C(12)	1.457(6)
O(1)–B(1)–O(2)	106.1(2)
O(1)–B(1)–O(3)	112.68(19)
O(1)–B(1)–N(1)	107.17(17)
O(2)–B(1)–O(3)	112.26(19)
O(2)–B(1)–N(1)	109.20(18)
O(3)–B(1)–N(1)	109.26(19)

1.

Thermal ellipsoid for HSR with a 30% probability level.

The presence of C–H···O and C–H···π interactions (2.804 and 2.649 Å) between the oxygen of coordinated catechol and the ligand phenyl ring as well as the ligand and catechol phenyl ring results in a trimeric cyclic structure (Figure a). The presence of π···π interactions with a distance of 3.399 Å between phenyl rings of the ligand and C–H···π interactions (2.804 Å) between the catechol phenyl ring and the ligand phenyl ring generates a two-dimensional supramolecular sheet (Figure b).

2.

(a) Trimeric structure due to C–H···O and C–H···π interactions and (b) 2D sheets due to C–H···π and π···π interactions.

Absorption and Emission Study: The photophysical properties of the probe [(L)­B­(OC₆H₄O)] HSR were studied in ethanol medium using UV–vis and fluorescence spectroscopy. The ethanolic solution of HSR (1 × 10^–4 M) exhibits two absorbance peaks at 341 and 270 nm as shown in Figure . The absorption below 250 nm was observed due to base compound of HSR. In Figure , the effect of the addition of the Fe³⁺ (1 × 10^–1 M) ion is shown. Under the effect of the Fe³⁺ ion over HSR, the absorption peak at 341 nm shows no change, but we have an apparent shift in the absorption peak around 270 nm toward a lower wavelength, known as a blue shift, due to the addition of the Fe³⁺ ion (Figure ). Here, this peak has completely disappeared, but some unsmoothness around the 250 nm wavelength was observed. This result indicates the metal-sensing potential of the prepared HSR compound. By the concentration variation of other ions, likewise Na⁺, Ba²⁺, Ca²⁺, Al³⁺, Zn²⁺, Ni²⁺, Cu²⁺, Co³⁺, K⁺, Pb²⁺, Cd²⁺, Mg²⁺, Hg²⁺, and Fe²⁺, no change in the absorption peak appeared in Figure S16. The reason for the change/shift in the particular absorption peaks at 270 nm due to addition of the Fe³⁺ ion is due to some structural changes in HSR that occurred. This result is an important proof of the ability of the prepared HSR probe for Fe³⁺ ion sensing. To evaluate the stability of HSR as an iron-sensing probe, its absorption behavior was examined across a pH range of 2, 4, 6, 8, 10, and 12. The results are summarized in Figure S17. The analysis revealed that the absorption intensity is significantly higher at pH 4, while at the other pH values, it remains nearly constant. Based on these observations, pH 4 was selected as the optimal condition for subsequent sensing studies. The reversibility of the HSR probe was investigated using EDTA; however, no significant changes were observed (Figure ).

3.

The UV–vis analysis for the pure HSR probe and the Fe³⁺ ion-doped HSR probe.

4.

Reversibility of HSR probe sensing using EDTA.

The photoluminescence emission studies of prepared HSR were done through 370 nm excitation, as shown in Figure a. Through this excitation, we observed a broad peak in the range of 380–550 nm. HSR shows dual emission spectra at 404 and 428 nm when excited at 370 nm. The emission properties of the HSR were studied at an excitation wavelength of 370 nm in ethanol, as shown in Figure a. The observation of broad peaks in organic ligands is very common. The pure HSR was incubated with different toxic and nontoxic metal ions at constant concentrations, such as Na⁺, Ba²⁺, Ca²⁺, Al³⁺, Zn²⁺, Ni²⁺, Cu²⁺, Co³⁺, K⁺, Pb²⁺, Cd²⁺, Mg²⁺, Hg²⁺, and Fe²⁺ ions, in an ethanol medium. The prepared samples were excited at 370 nm to observe the fluorescence emission intensity around 425 nm changing due to the presence of these ions. This analysis has shown that apart from the Fe³⁺ ion, no metal ion has this capability to change the fluorescence intensity of pure HSR as observed and shown in Figure b.

5.

(a) The emission spectra of pure HSR due to 370 nm excitation are observed (inset: the photograph of emission color is shown). (b) The photoluminescence intensity variation at the same concentration of different metal ions confirming that HSR is clearly a potential probe for the detection of Fe³⁺ ions. (c) shows a comparative change in intensity of photoluminescence in the presence of Fe³⁺ ions (inset: photograph of comparative change of photoluminescence is shown). (d) The ratiometric concentration variation effect on photoluminescence efficiency of HSR.

Colorimetric Analysis

Apart from fluorescence intensity variation, we also observed a colorimetric change upon the addition of the Fe³⁺ ion; the color of HSR changed from pale yellow to red-brown as shown in Figure a,b. This colorimetric change did not appear with other metal ions such as Na⁺, Ba²⁺, Ca²⁺, Al³⁺, Zn²⁺, Ni²⁺, Cu²⁺, Co³⁺, K⁺, Pb²⁺, Cd²⁺, Mg²⁺, Hg²⁺, and Fe²⁺, as observed and shown in Figure a,b. Through this analysis, we have obtained the result that the Fe³⁺ ions have a significant effect on the optical properties of HSR in different optical analyses, such as UV–vis absorption analysis, fluorescence emission, and colorimetric analysis. Here, it is interesting that only the Fe³⁺ ion can make the UV–vis absorption peak around 270 nm disappear and weaken the fluorescence of the probe, leading to quenching of the fluorescence intensity of HSR and the color of HSR. In addition, with the above-mentioned metal cations, a similar analysis was carried out by varying the anions, namely, CH₃COO^–, F^–, Cl^–, CO₃ ^2–, and SO₄ ^2–. The results are summarized in the Supporting Information (Figure S18). The analysis indicates that, consistent with most cations, the anions do not significantly influence the fluorescence emission intensity of the HSR probe; in contrast, the Fe³⁺ ion exhibits a distinct effect.

6.

(a,b) Images demonstrating the color change of HSR with different metal ions under visible light (a) and ultraviolet light of wavelength 370 nm (b).

Quantification of the Fe³⁺ ion concentration variation. The Fe³⁺ ion effect on the fluorescence emission intensity of HSR is studied through the concentration variation of the Fe³⁺ ion in an ethanol medium. The HSR-Fe³⁺ complexes were prepared with different concentrations of Fe³⁺ ions from 1 equiv to 10 equiv. Here, a total of 20 samples are prepared with a difference of 0.5 equiv of Fe³⁺ ion. The effect of coexisting metal ions on the HSR-Fe³⁺ complex was studied through emission spectra. As shown in Figure d, a comparative study of the change in fluorescence intensity of HSR with the HSR-Fe³⁺ complex is done. This analysis clearly indicates that with an increase in the concentration of Fe³⁺ ions, the fluorescence intensity of HSR is quenched very fast up to the 5 equiv concentration of Fe³⁺ ions. The further increase in Fe³⁺ ion concentration still reduces the fluorescence intensity of HSR, but this reduction in fluorescence intensity is very slow. At 10 equiv concentration of Fe³⁺ ion, it was observed that the Fe³⁺ ion completely quenched the fluorescence intensity of HSR as shown in Figure c.

In Figure , the variation of quenching of fluorescence intensity with increasing concentration of Fe³⁺ ion is shown. Here, the quenching effect due to an increasing concentration of Fe³⁺ ions is very high. This result shows that the developed HSR sensor is very sensitive. Here, until the concentration of 5 equiv, the fluorescence intensity is quenching linearly, but beyond this concentration, the quenching is very slow, meaning it achieves a saturation in the ground of intensity.

7.

(a) Fe³⁺ ion concentration variation-based luminescence intensity variation. (b) I ₀ – I vs concentration of Fe³⁺ ions in HSR.

Binding Mode Analysis of the HSR-Fe³⁺ Complex: Because Fe³⁺ is strongly oxophilic, the initially formed boronate interaction is expected to weaken − upon metal coordination, resulting in the formation of a more stable Fe­(III) complex featuring N,O- and O,O-chelation. Accordingly, the proposed binding mode shown in Scheme S1 involves coordination through the imine nitrogen and phenolic oxygen atoms, as well as through the phenolic and catecholic oxygen donor atoms. − To investigate the binding mode of the HSR-Fe³⁺ complex, Job’s plot was created between fluorescence intensity and [Fe³⁺]/[Fe³⁺] + [HSR] as shown in Figure a. Through this analysis, the maximum relative intensity is observed at 0.6, indicating that the HSR-Fe³⁺ complex has a 2:1 (M/L) molar ratio. The binding constant of the HSR-Fe³⁺ complex was determined using modified Benesi–Hildebrand equations for a 2:1 (M/L) stoichiometry (eq ):

$$\frac{I_0}{I_0-\mathit{I}}=\left(\frac{\mathit{a}}{\mathit{b}-\mathit{a}}\right)(\frac{1}{K_{\mathrm{B}}{[\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}]}^2}+1)$$ 1

where I ₀ and I are the fluorescence intensities of HSR at 390 nm in the absence and presence of various concentrations of Fe³⁺ ion; a and b are constants; and [substrate] is the concentration of Fe³⁺ ion. The linear fit observed in the I ₀/(I ₀ – I) vs 1/[Fe³⁺]² plot provides a binding constant K _B = 2.35 × 10¹⁵ M^–1, supporting the 2:1 (M/L) complex stoichiometry (Figure c). The high binding constant is consistent with the highly selective sensing of the Fe³⁺ ion by HSR, without interference from other metal ions (Figure d). The formation of the 1:2 complex was confirmed by HRMS (TOF-MS, ES⁺) analysis (Figure S19), which exhibited a molecular ion peak at m/z 583.2609 ([M + H]⁺, (C₂₀H₁₆BCl₄Fe₂NO₃); calculated mass = 582.6520).

8.

(a–d) (a) Job’s plot has been shown. (b) The relative intensity variation with respect to different concentrations of Fe³⁺ ion is shown. (c) The relative intensity vs 1/[Fe³⁺]² is plotted. (d) The interference study due to the presence of different metal ions has been done.

The detection limit (LOD) of HSR was calculated using eq :

$$\mathrm{LOD}=3\sigma /\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}$$ 2

where the standard deviation (σ) was calculated by recording the fluorescence intensity of HSR without the Fe³⁺ ion over ten measurements.

In Figure b, the relative intensity variation with respect to different concentrations of Fe³⁺ ions is shown. A plot of fluorescence intensity as a function of the concentration of the added Fe³⁺ ion was used to determine the slope. The detection limit of HSR for the Fe³⁺ ion was found to be 7.5 × 10^–6 M.

HSR is non-toxic to HEK-293 cells: The effect of HSR on cell viability was examined on a non-malignant cell line, HEK-293. HEK-293 (1 × 10⁵ cells/mL) cells were seeded in a 96-well culture plate, followed by treatment with different concentrations of HSR (1, 5, 10, 20, 50, and 100 μM) for 24 h and estimation of cell viability as described in Materials and Methods. As shown in Figure , HSR did not exhibit any cytotoxicity on HEK-293 cells.

9.

Effect of HSR treatment on HEK-293 cells. HEK-293 (1 × 10⁵ cells/mL) cells were treated with different concentrations of HSR (1, 5, 10, 20, 50, and 100 μM) for 24 h, followed by estimation of percent viability by the MTT assay as described in the Experimental Section. The values shown are the mean ± SD of three independent experiments done in triplicate. For the determination of the mean difference between each group, we performed one-way ANOVA and found a p-value <0.05, showing a significant difference between the control and treated groups in respect of viability.

Fe³⁺ ion sensing by the HSR: HEK-293 (1 × 10⁵ cells/mL) cells were incubated with HSR, Fe­(NO₃)₃·9H₂O, and HSR and Fe­(NO₃)₃·9H₂O. In the presence of HSR, HEK-293 cells exhibited a bright blue fluorescence, whereas a decreased fluorescence was observed upon exposure to HSR and Fe­(NO₃)₃·9H₂O (Figure ). However, untreated control cells or cells treated with Fe­(NO₃)₃·9H₂O only did not show any fluorescence. Interestingly, we observed significantly decreased HSR-mediated fluorescence at pH 3.5, which might be due to higher uptake of Fe³⁺ ions at lower pH. This clearly indicates that the accumulation of the Fe³⁺ ion can be sensed in the cytoplasm of living cells by HSR. −

10.

Analysis of HSR-Fe­(NO₃)₃·9H₂O-mediated fluorescence in HEK-293 cells. HEK-293 (1 × 10⁵ cells/mL) cells were incubated in the medium containing 20 μM HSR, 10 μM Fe­(NO₃)₃·9H₂O, and 20 μM HSR + Fe­(NO₃)₃·9H₂O, followed by fluorescence analysis for detection of the Fe³⁺ ion as described in Materials and Methods. Pictures in panel 2 show detection of fluorescence in cells incubated with HSR, Fe­(NO₃)₃·9H₂O, or HSR + Fe­(NO₃)₃·9H₂O. The photographs shown are form a representative experiment done in triplicate. Arrows indicate cells exhibiting fluorescence under a fluorescence microscope.

Table shows a comparative study of HSR with previously reported literature. Compared to previously reported probes, the present work exhibits the lowest detection limit (7.5 × 10^–6 M) and the highest binding constant (2.35 × 10¹⁵ M^–1), demonstrating superior sensitivity and strong binding affinity, along with successful application in cell imaging.

3. Comparison of Reported Probes with the Present Work.

Conclusion

We have reported a novel boranil-based chemosensor. The probe can be used for detection and imaging of the Fe³⁺ ion through distinct colorimetric and fluorescence quenching responses. Detailed spectroscopic investigations confirmed strong complexation with the Fe³⁺ ion, characterized by a well-defined binding stoichiometry, an exceptionally high association constant, and a low limit of detection. The sensing performance of HSR remains unaffected by competing biologically relevant and toxic metal ions, which underscores its robustness and selectivity. In addition, HSR exhibits good aqueous compatibility, no cytotoxicity, and efficient cellular uptake, which supports its applicability in biological environments. Collectively, these results establish HSR as a promising molecular platform for Fe³⁺ ion sensing and fluorescence-based bioimaging with potential relevance to environmental and biomedical applications.

Glossary

Abbreviations

TMS

tetramethyl silane

HL

N-(2-methylphenyl)­salicylideneimine

IP

ionization potential

EA

electron affinity

χ

electronegativity

η

hardness

S

softness

μ

chemical potential

ω

electrophilicity index

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c01069.

• Crystallographic data for HSR (CIF)

• Crystal data and experimental details for HSR (PDF)

• Schematic illustration of synthesis of HL and HSR, IR spectrum of HL, ¹H NMR spectrum of HL, ¹³C NMR spectrum of HL, HRMS (ESI-TOF) spectrum of HL, IR spectrum of HSR synthesized in benzene, ¹H NMR spectrum of HSR synthesized in benzene, ¹³C NMR spectrum of HSR synthesized in benzene,¹¹B NMR spectrum of HSR synthesized in benzene, HRMS (ESI-TOF) spectrum of HSR synthesized in benzene, IR spectrum of HSR synthesized in toluene, ¹H NMR spectrum of HSR synthesized in toluene, ¹³C NMR spectrum of HSR synthesized in toluene,¹¹B NMR spectrum of HSR synthesized in toluene, optimized structure of HSR, HOMO–LUMO of HSR with band gap energy, table of the energy values of HSR calculated using the B3LYP/6-311+G­(d,p) method, absorption spectra of HSR with different metal ions, effect of pH on UV–vis absorption intensity, photoluminescence intensity variation at the same concentration of different anions, HRMS (ESI-TOF) spectrum of the complex, UV–vis spectra of a dilute solution of HSR (1 × 10^–5 M) with and without Fe³⁺, and proposed binding mode of Fe³⁺ ions with the HSR probe (PDF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

This work is dedicated to Late Prof. Anirudh Singh on his 87th birthday.