The remarkable tendency of toxic cyanide ions to be bioaccumulated in human cells is a serious human health hazard


The remarkable tendency of toxic cyanide ions to be bioaccumulated in human cells is a serious human health hazard. Thus, visualizing, monitoring, and determining the ultra-trace concentrations of toxic cyanide ions with ultrasensitive and ultra-selective approaches in biological cells are of great interest. We report the manufacture of branching molecular architectures (BMAs) for colorant tracking and biocompatible cyanide monitoring of ultra-trace concentrations (? 88 ppt, parts per trillion) of toxic CN? ions in living cells, i.e., HeLa cells, in the order of seconds. The decoration of colorant (dendritic branch) aggregates into the thin-layered crust around and within the molecular building cavities of geode-shelled nanorods of porous organic–inorganic aluminum frameworks provided building blocks for BMAs. We used simple, batch-contact colorant-tracking assays to assess the suitability of the BMA geodes for the monitoring/assessment/inhibition of toxic cyanide compounds in biological cells. The BMAs showed the potential of a wide-range detection and continuous monitoring for CN? ions in real biological and environmental samples. Our result provided sufficient evidence for the biocompatibility and low cytotoxicity of BMAs during the continuous monitoring and exposure to HeLa cells. Under physiological conditions, the BMAs were used for the in vitro fluorescence tracking/sensing of cyanide ions in HeLa cells. Our BMA geodes may have the potential to diminish the health threats associated with toxicant exposure in human cells.
Keywords: cyanide; monitoring; tracking; in vitro; biocompatible.
1. Introduction
Cyanide usage in the global industry is inevitable. The material is extensively used in metallurgy, mining, synthetic fibers, resins, dyes, and electroplating 1. However, cyanide is extremely poisonous at very low doses in humans 2,3. The cyanide anions supplied by the decomposition of toxic cyanide in the human body can quickly combine with the hemoglobin in red blood cells, prevent oxygen transfer, and promote organism death. The World Health Organization recommends 1.9 ?M as the threshold safety level of cyanide in drinking water 3,4. In this regard, cyanide anion detection has attracted scientific concern worldwide.
Over the past decades, many approaches, such as electrochemical, potentiometric, and voltammetry, have been explored to determine cyanide anion levels in aqueous solutions 5–7. However, the use of optical chemosensors in cyanide detection involves a short response time and simple procedures relative to those of other methods 8–10. Thus, research on this field has attracted increased attention given the rapid and accurate cyanide sensing in aqueous media via this strategy and the evidence-based confirmation of its sensing effectiveness by comprehensive reviews. Novel cyanide-sensing technologies have been developed. Examples include new cyanide receptors, such as glucoconjugated o-(carboxamido) aldehyde hydrazine-linked azo dye 11 and 4,4-bis-3-(4-nitrophenyl) thiourea diphenylmethane (or ether); 12 dual-mode probes based on coumarin and malonyl urea derivative dyes;13 aggregation-induced emissive hexaphenylbenzene 14 and 7,7,8,8-tetracyanoquinodimethane; 15 and near-infrared chemodosimeters based on 5,10-dihexyl-5,10-dihydrophenazine. 16
The formation of optical chemosensors by immobilizing fluorophore or chromophore organic probes onto mesoporous 17-21 or microporous surfaces is a significant development in practical applications. In general, physical 22-26 and chemical 27 immobilizations are the two major processes in fabricating optical chemosensors. The physical trapping of colorant j-aggregate receptors is uncomplicated. However, unfavorable orientations and diminished functionality are expected in the colorant j-aggregate receptors. The optical chemosensors grafted via chemical immobilization procedures have short lifetimes because of the outflow of the organic probe in the solution. 28 The chemical binding approach of optical chemosensors onto a mesoporous or microporous scaffold is the most efficient technique for constructing optical chemosensors with a highly reproducible response and a long lifetime. 29 One disadvantage of chemical immobilization is the unregulated covalent binding of optical receptors to a surface. This drawback may restrict the carrier’s active site.
To quantify and track the ultra-trace toxicant species in biological cells, numerous analytical techniques were applied. The monitoring/tracking of toxicants in living cells can be studied on either fixed or live models. These analytical techniques including spatially resolved mass spectrometry techniques 30 such as, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), electron spectroscopy imaging (ESI) and secondary ion mass spectrometry (SIMS), combined with electron energy loss spectroscopy (EELS), 31,32 and X-ray fluorescence microscopy (XRFM) significantly enabled the distribution and concentration of toxicants in static biological specimens.33 However, these analytical protocols often associated with practical training, skillfully technical demands in their handling and working may lead to high operating cost of analysis and difficulty for daily routine monitoring of species.
In the last several years, metal–organic frameworks (MOFs) have attracted research attention. 34 Highly crystalline and highly porous MOFs are compounds composed of metal ions or clusters coordinated to organic linkers to form 1D, 2D, or 3D structures. The organic linker structure arrangement controls the fabrication of the porous structure and augments the specific surface area. 34–37 MOFs possess improved potential compared with other sensors for various applications, such as selective gas adsorption/storage, 38 heterogeneous catalysis, 39 carriers for nanomaterials, 40 and luminescence. 41 Furthermore, MOFs can serve nanomaterial platforms, which are loaded with optical and fluorescent probes, in designing optical and fluorescent chemosensors for detecting heavy-metal and toxic anions in different water sources. 42
In the present work, the branching molecular architectures (BMAs) showed potential applications in colorant tracking and biocompatible cyanide monitoring of ultra-trace concentrations (up to 88 parts per trillion ppt) of toxic CN? ions in living cells, i.e., HeLa cells, in the order of seconds. We investigated the micropore geometry, stability, particle morphology, and shape of the wrapping-colorant buildings and their potential as selective platforms for accommodating dendritic colorant (branch) aggregates (L1). The decoration of dendritic colorant (branch) aggregates into thin-layered crust films around and within the molecular building voids of inorganic–organic framework carriers allowed for the hierarchical engineering of BMAs and afforded continuous monitoring of toxic CN? ions in living cells (Scheme 1). This study revealed the superior performance of BMAs specifically tailored for detecting target CN? ions in biological cells. Our finding provided a cutoff evidence for the biocompatibility and low cytotoxicity of BMAs during the continuous monitoring and exposure to HeLa cells. Moreover, our result provided a new direction in the application of organic–inorganic framework carriers to environmental and biological sample analyses.
2. Experimental Section
2.1. Material
Most of chemicals were purchased from Aldrich and used without further purification Benzohydrazide, 2-Hydroxy-5-methyl-1,3-benzenedicarboxaldehyde 97% were bought from Wako chemicals, Tokyo, japan. The selectivity of the sensing system toward cyanide (CN?) was also evaluated by using another anion including F ?, Br ?, Cl?, I-, NO3?, CH3COO ?, H2PO4?, SO42?, ClO4-, HSO4-. The salt solutions were prepared in distilled water and the concentration was kept as 10 ppm. Ethanol for spectral detection was HPLC reagent without fluorescent impurity and H2O was Milli-Q water. All solvents were analytical reagents.
2.2. Fabrication design of branching molecular architectures (BMA)
A simple one-pot solvothermal approach was performed to fabricate supermicropores Al-based metal-organic frameworks involving N, N-dimethylformamide (DMF) and water as solvent. Starting reactants were Al(NO3)3. 9H2O (0.51 g), 2-amino terephthalic acid (0.56 g) in mixed solvent of N,N-dimethylformamide(DMF) with Milli-Q water and stirred for only 5mins. The reactants were placed in a Teflon-lined autoclave and heated for 24 h at 160?C under static conditions. The prepared white powder was filtered under vacuum and washed with DMF. The materials were activated in boiling methanol overnight and stored for 24 h at 80?C, to remove organic species stuck within the micropores. The branching molecular architectures (BMA) were constructed via a direct immobilization method process. The ethanolic solution of 10 mg of the L1 receptor with 0.5 g of carrier with stirring at room temperature till saturation. The solvent is removed gently using rotatory evaporator at ambient temperature, leading to change in carrier color from white to pale yellow which indicates that the direct attachment stacking of L1 receptor into the surface of the carrier. The process repeated for several times to ensure the filling of micropores and adsorption capacity of the carrier reached the equilibrium. The BMA was washed with deionized water until no elution of L1 color was observed and dried to 60oC for 12h. Field emission scanning electron microscopy (FE-SEM), scanning transmission electron microscopy coupled with energy dispersive X-ray spectroscopy for elemental mapping (FESEM-EDS), Small- and wide-angle powder X-ray diffraction (SAXRD, and WAXRD, respectively), and N2 adsorption-desorption isotherms (see supporting information) were used to characterize the surface morphology of the microporous (BMA) structures. The adsorption capacity (Qt, mmol g-1) of L1 into aluminum organic-inorganic frameworks carrier at saturation was determined by the following equation: Qt = (Co-C) V/m, where Qt is the adsorbed amount at saturation time t, V is the solution volume (L), m is the mass of the carriers (g), and Co and C are the initial and saturation concentrations at time t, respectively. Our finding shows that the loading capacity of L1 into carrier is 0.055 mmol g-1.
2.3. Cell culture and in vitro study
The Hela cell line was obtained from Hela (ATCC® CRL1721™) and was cultured by incubation under 5% CO2 at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10% horse serum. The culture medium was replaced every 3 to 4 days.
2.4. Cytotoxicity studies
The cell viability of the probe L1, and BMA were tested against HeLa cell lines using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were seeded into a well plate at a density of 50 × 104 cells per well and incubated in medium containing the probe L1, and BMA at concentrations ranging from 0- 50 ?g/ml for 24 h. To each well, 100?L of MTT was added and the plates were incubated at 37 °C for 4 h to allow MTT to form formazan crystals by reacting with metabolically active cells. The medium with MTT was removed from the wells. Intracellular formazan crystals were dissolved by adding 100 ?L of DMSO to each well and the plates were shaken for 10 min. The absorbance was recorded using microplate reader.
2.5. Flow Cytometry Measurements.
The cell concentration was adjusted in 12-well and the Hela cells sample without BMA and CN- ions act as control. After exposure to 20µg/ml of BMA and 20µg/ml BMA + 100 ppb CN-, cells were washed three times with phosphate buffered saline (PBS), trypsinized, centrifuged at 3 000 rpm for 3 min, and resuspended in PBS and stained with 200 ?l of propidium iodide (50 ?g ml?1). Then, these cells were analyzed using a flow cytometer.
2.6. Confocal microscopy measurements
The cell concentration was adjusted in 6-well and the untreated cells act as a control. After that a desired amount of BMA (20 ?g/mL) and 20µg/ml BMA + 100 ppb CN-, were added to each well, incubated in a humid chamber for an additional 30 min under 5% CO2 at 37 °C followed by thoroughly washing using DPBS. The Nuclear counter-staining was performed using 0.1 ?g/mL of DAPI in PBS for 5 min. Finally, confocal microscopy measurements were observed using a Leica TCS SPE5 X machine.
2.7. Fluorescence monitoring of CN- ions in living cells
The cell concentration was adjusted in black 96-well as previously mentioned. The cells were seeded into a well plate at a density of 50 × 104 cells per well and incubated in medium containing BMA at concentrations ranging from 0-50 ?g/ml the corresponding fluorescence spectrum (denoted by Fo). The CN- ions were added to the BMA design to adjust the final concentrations to (10, 25, 50, 100, 200) ppb the corresponding fluorescence spectrum (denoted by F). The fluorescence intensity was recorded with microplate reader for different time of incubation (0, 24, 48, 72) hours. Consequently, the concentration of the CN- ions was estimated qualitatively and quantitatively to predict the detection and monitoring limit.