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Islam M. El-Sewify, [Mohamed A. Shenashen](https://orcid.org/0000-0003-1592-5877), Rasha F. El-Agamy, Mohammed S. Selim, Norah F. Alqahtani, Ahmed Elmarakbi, [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350), Mahmoud M. Selim, Mostafa M.H. Khalil, [Sherif A. El-Safty](https://orcid.org/0000-0001-5992-9744)

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[Ultrasensitive Visual Tracking of Toxic Cyanide Ions in Biological Samples Using Biocompatible Metal–Organic Frameworks Architectures](https://mdr.nims.go.jp/datasets/867294d6-8046-4439-8025-cd6138c76c93)

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Template for Electronic Submission to ACS Journals1Ultrasensitive Visual Tracking of Toxic Cyanide Ions in Biological Samples1Using Biocompatible Metal–organic Frameworks Architectures2I.M. El-Sewify a,b, M.A. Shenashena,c, R.F. ElAgamyd, M.S. Selimc, N.F. Alqahtanie, A.3Elmarakbi f, M. Ebaraa, M.M. Selimg, M.M.H. Khalilb, S.A. El-Saftya,∗.4a Research Center for Macromolecules and Biomaterials, National Institute for Materials Science5(NIMS), 1-2-1 Sengen, Tsukubashi, Ibaraki-ken 305-0047, Japan6b Department of Chemistry, Faculty of Science, Ain Shams University, 11566 Cairo, Abbassia,7Egypt8c Department of Petrochemical, Egyptian Petroleum Research Institute (EPRI), Nasr City, 117279Cairo, Egypt10d College of Computer Science and Engineering, Taibah University, Yanbu, 966144 Saudi11Arabia12e Department of Chemistry, College of Science, University of Jeddah, Jeddah 21589, Saudi13Arabia14f Faculty of Engineering and Environment, Northumbria University, Newcastle Upon Tyne, NE1158ST, UK.16g Al-Aflaj College of Science and Human Studies, Prince Sattam Bin Abdulaziz University, Al-17Aflaj 710-11912, Saudi Arabia18E-mail: sherif.elsafty@nims.go.jp; & sherif.el-safty@sunderland.ac.uk19Homepage: https://samurai.nims.go.jp/profiles/sherif_elsafty202122mailto:amailto:sherif.elsafty@nims.go.jpmailto:sherif.el-safty@sunderland.ac.ukhttps://samurai.nims.go.jp/profiles/sherif_elsafty2ABSTRACT23The extraordinary accumulation of cyanide ions within biological cells is a severe health risk.24Detecting and tracking toxic cyanide ions within these cells by simple and ultrasensitive25methodologies are of immense curiosity. Here, continuous tracking of ultimate levels of CN−-26ions in HeLa cells was reported employing biocompatible branching molecular architectures27(BMAs). These BMAs were engineered by decorating colorant-laden dendritic branch within and28around the molecular building hollows of the geode-shelled nanorods of organic–inorganic Al-29frameworks. Batch-contact methods were utilized to assess the potential of hollow-nest30architecture for inhibition/evaluation of toxicant CN--ions within HeLa cells. The nanorod BMAs31revealed significant potential capabilities in monitoring and tracking of CN− ions (88 parts per32trillion) in biological trials within seconds. These results demonstrated sufficient evidence for the33compatibility of BMAs during HeLa cell exposure. Under specific conditions, the BMAs were34utilized for in-vitro fluorescence tracking/sensing of CN− in HeLa cells. The cliff swallow nest35with massive mouths may have the potential to reduce the health hazards associated with36toxicant exposure in biological cells.3738KEYWORDS: Visual Monitoring; Cyanide; HeLa cells; In-vitro; Biocompatible; Al-MOF39Nanorods.4041Environmental Implication:42Aquatic ecosystems and human health are seriously threatened by cyanide. This study43describes the creation of biocompatible materials for monitoring/tracking CN--ions in water and44biological samples.4531. INTRODUCTION46The manipulation of cyanide in global industry is foreseeable. Raw materials containing47cyanide are significantly utilized in dyes, electroplating, and fibers [1]. The toxic cyanide can be48promptly combined with hemoglobin and promote organism death via preventing oxygen49transfer in the human body, and minimal cyanide doses is enormously poisonous [2,3]. The50World Health Organization proposes the threshold safety for cyanide concentration (1.9 μM) in51drinking water [3,4]. In this regard, the recognition of CN− has attracted worldwide scientific52concern.53Many methodologies, such as potentiometric, voltammetry, and electrochemical have54been studied to determine CN− ions [5-7]. Novel technologies have been utilized for detecting55ultra-trace concentrations of cyanide ions [8-16]. The creation of chemosensors by confining56sensitive and selective probes into porous structures has earned widespread acclaim in the field57of tracking and detecting many targets [17-21]. Chemical and physical decorations are the two58foremost procedures in designing chemosensors [22- 27]. The trapping of colorant receptors by59using physical process is uncomplicated. However, critical alignments of colorant receptors over60the porous platform are expected due to the organic receptor discharge in tested solution. In61addition, the drawbacks of the designed chemosensors using chemical methods are instability62and one-time use [28-39]. The decoration procedures of chromophores onto nanoporous platform63are an extremely effective technique for creating sensors with long-term stability [40].64The common weakness of grafting process is the unorganized binding events of organic probes65into the porous carrier surface, and this drawback may restrict the carrier’s active site. Numerous66analytical techniques were applied in biological cells to track and enumerate ultra-trace toxic67pollutants and monitor common pollutants. The conventional approaches [41- 43] can684significantly identify the concentration and distribution of toxic species in living samples [44].69However, these classical approaches associated with intensive handling and training and70expensive operating procedures may lead to dull detection of toxic pollutants.71Inorganic–organic frameworks have recently received research attention due to the high porosity72and crystallinity of metal–organic frameworks (MOFs) [45]. They are composed of metal ions73and organic linkers to design uniform structures. The linker structure arrangement enhances the74surface area and controls the construction of organic–inorganic frameworks [46-49]. The optical75chemosensors using MOFs as carrier improved the sensing potential compared with the reported76chemosensors for various purposes, such as catalysis, adsorption, and carriers for luminescence77[50-53]. Moreover, the inorganic–organic framework platforms, which are stacked with78chromophores, have been applied for monitoring toxicants in different applications [54].79In this study, the fabricated biocompatible branching molecular architectures (BMAs) revealed80prospective functions in cyanide tracking in HeLa cells within seconds. The BMA stability and81structural morphology were examined. The organic aggregates crust layer within and around the82inorganic–organic framework cavities approved for fabricating BMAs geodes and offered83continuous tracking of CN− ions in HeLa cells. The findings demonstrated evidence for84monitoring/tracking CN− ions in HeLa cells by using BMA hierarchal geodes (Scheme 1) and for85the high biocompatibility of the designed optical chemosensors during exposure and monitoring86of CN− ions in HeLa cells. This study offers a novel method for utilizing metal–organic87frameworks in biological and environmental evaluations.88589Scheme 1. Schematic representation of tracking and capturing of CN- ions in living cell via using90BMA geodes. The bright field images of the contaminated HeLa cell (A) and the treated HeLa91show high fluorescence enhancement after formation of the complex [BMA-CN-](B). After92incubation of HeLa cells for 24 hours, a 20 µg /ml of [BMA] 100 ppb of CN- ions were added93and incubated for 24 hours. The BMA geodes were fabricated via immobilization of organic94receptors into the microporous surface of BMA geodes (C). The FE-SEM images (D) show the95high morphological stability of nanorods with geodes shells crystals of BMA.9662. EXPERIMENTAL SECTION972.1. Chemicals and Materials98For preparation of organic chromophore, 2-hydroxy-5-methylisophthalaldehyde, Benzohydrazide99were purchased from Wako chemicals, Tokyo, Japan. To fabricate inorganic -organic100frameworks, Aluminum nitrate nonahydrate (Al(NO3)3·9H2O), the organic linker 2-terephthalic101acid in mixed solvent N,N-dimethylformamide chemicals were obtained from Sigma-Aldrich. To102adjust the pH of the solution, (HEPES) buffer and disodium hydrogen phosphate were used. All103used solvents were analytical reagents and ethanol was used for spectral recognition. To perform104the selectivity toward cyanide (CN−), we evaluated using common interfering anions including F105−, Br −, Cl−, I-, NO3−, CH3COO−, H2PO4−, SO42−, ClO4-, HSO4-.1062.2. Fabrication of BMA107A one-pot solvothermal approach was applied using a mixed solvent containing N,N-108dimethylformamide (DMF) and water to fabricate supermicroporous Al-MOF nanorods. 1,4-109Benzenedicarboxylic acid (0.56 g) and Al(NO3)3.9H2O (0.51 g) were mixed with the solvent.110After the mixture was stirred for 5 mins, it was heated in an autoclave at 160 °C overnight. The111white powder obtained was purified using the mixed solvent. The engineered Al-MOF nanorods112were stimulated by boiling with methanol overnight at 80 °C to remove unreacted and trapped113organic linkers within the Al-MOF micropores. Direct decoration was performed to design114optical chemosensors. The Al-MOF nanorods (0.5 g) were stirred with organic chromophore115ethanolic solution (0.1 g) until saturation. The removal of solvent resulted in the discoloration of116the Al-MOF nanorods, indicating the stacking of the prepared organic receptor L1 into the117supermicroporous cavities. The decoration process was repeated several times, and the nanorods1187were dried to 60 °C for 12 h to verify the loading of supermicroporous cavities in the Al-MOF119nanorods.1202.3. Cytotoxicity Studies121The cell feasibility of the receptors and BMA were examined on HeLa cell lines by using122methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The cells were seeded into a well123plate at 50 × 104 cells per well and incubated in a medium containing the receptor and BMA at124concentrations ranging from 0 μg/ml to 50 μg/ml for 24 h. MTT (100 μL) was added to each well125and incubated by reacting with metabolically active HeLa cells. The MTT and media were126discarded from the well plate. Dimethyl sulfoxide was added to each well to suspend127intracellular formazan crystals, and the microplates were shaken for 10 mins. A microplate128reader was used to record the change in absorbance intensity.1293. RESULTS AND DISCUSSION1303.1 Fabrication of Decorated Nanorod Architectures131One-pot, simple, and template-free assays were conducted to examine the construction of geode-132shelled inorganic–organic frameworks. The engineering of BMA geodes was successfully133created using the direct decoration process of dendritic branch aggregates into super-134microporous scaffold for tracking and monitoring of CN− ions in biological samples. The results135revealed uniform nanorod branches along swirled caves with multifunctional surfaces. The136fabricated BMAs were designed via physical immobilization, and the organic linker showed a137significant characteristic in designing uniform construction with high-surface-area carriers under138solvothermal condition [55- 63]. The direct immobilization process resulted in homogenous139BMAs with outer and interior nanorod surface. Moreover, the binding between the receptors and140the active centers in the carrier surface at room temperature is achieved by van der Waals and1418hydrogen bonding interactions (Scheme 2). The swirled caves along the branches and organic142nature of the Al-MOF carriers augmented the physical interactions between the multifunction143surface of nanorods and the j-aggregate probes. Moreover, the binding affinity of BMA144chemosensors with CN− ions was enhanced, and fast recognition was observed under optimal145requirements.146147Scheme 2. (A & B) Structural formation and optical images of the colorant and fluorescent J-148aggregates of L1 receptor and its [L1-CN] complex with dendritic spine stacking arrangement of149parallel alignment of L1 colorant fiber in ethanol (B-a) and dendritic branches (B-b) molecular1509directional structure of L1 colorant at pH 8 and during the formation of [L1-CN] complex with151addition of CN- ions, respectively.1523.2. Structure Characterization153Field-emission scanning electron microscopy (FESEM) were conducted to investigate the154development of nest-shaped carriers, which were combined and distributed as hierarchal geodes155(Figures 1A and 1B). The results showed that the 50–100 nm Al-MOF geode-shelled nanorod156structures were ordered of asymmetrical open-pore systems that linked across the entire and with157the cage cavities. As shown in Figure 1, the data showed the formation of a chaotically shaped158window, well-arranged nanorods, and a cliff swallow nest with massive mouths. The FESEM159images revealed advancement of supermicroporous cavities to confine the organic probes and160hallow interior structure (Figures 1C–1E). The polyrods of Al-MOFs uniformly accumulated and161aggerated around the massive mouths.162The well-organized Al-MOF stability with hierarchal geode shells was explained using the163atomic force microscopic images in Figure 1G. The TEM images confirmed the formation of164external crust layers via simple immobilization of dendritic branch around the BMAs. The165components of BMA hierarchical engineering were as follows:166(i) Randomly shaped channels and active facet {101} of inorganic–organic-Al167framework cliff swallow nest with massive mouths structures enabled trapping of168receptors.169(ii) Uniform virtual and dense sheath colorant constructions were formed.170(iii) The aggerates inside the microchannels allowed for the hierarchical engineering of171BMA geodes.17210(iv) The crust layer of organic chromophores showed simultaneous tracking, fast response173time, and binding of toxic CN− compounds.174(v) Retention of BAMs' multifunctional binding sites during electron or charge transport175led to an extensive range of cyanide colorant tracking in biological cells.176177Figure 1. FE-SEM images (A) of aluminum organic-inorganic nanorods with geodes shells178morphology b) High-magnification FE-SEM images of nanorods with geodes shells. Low- and17911high-magnification STEM (C-F) of hierarchal geodes. G) AFM micrograph of high-order180nanorods with geodes shells multiple vast-mouth caves and controllable entrance sizes as vesicle181traps. H) the molecular building of crystal structure the tower-like structure of aluminum182organic-inorganic frameworks carriers via binding interaction of aluminum salt with terephthalic183acid.184A N2 adsorption–desorption isotherm was used to investigate the surface morphology of185the carrier before and after decoration. The measurements confirmed the microporous cage of the186carrier and BMAs (Figure S3A). The isotherms of the carrier and BMAs exhibited an unveiled187type (I) of adsorption behavior. However, considering the dense decoration of organic colorant188L1 on the carrier channels, the N2 isotherm supported the uniform pore openings (< 2 nm) and189microporous cavity (∼1.8 nm). The results showed a reduction in the carrier surface area. The190surface area of BMAs (683 m2/g) was less than that of the Al inorganic–organic framework191carrier. Moreover, the volume of BMAs pore (0.0964 cm3/g) was less than that of Al inorganic–192organic frameworks (0.16 cm3/g). Therefore, the remarkable reductions in carrier pore volume193and surface area confirmed that the organic colorant receptors successfully shielded the organic–194inorganic framework carrier surfaces. The NLDFT/GCMC data were used to examine the195scaffold pore type [32]. Figure S3B displays an exceptional peak located at ∼1.7 nm, confirming196the supermicropore diameters of the inorganic–organic framework carriers and the BMA geodes197were distributed within this range. The results revealed the uniform decoration of organic198receptor L1 into the microporous cavities of the carriers.199The crystal structure and stability of the supermicroporous carriers and BMAs geodes200was confirmed by X-ray diffraction patterns. The results showed well-defined diffraction peaks,201revealing the well-organized structure assembly of the carrier and BMA geodes. The highly20212ordered peaks situated at 2θ values of 8.1° and 9.1° which matched with the Al inorganic–203organic frameworks.51 These results proved the successful construction of the microporous Al204organic–inorganic framework carriers. Furthermore, the retention in Bragg peaks confirmed the205colorant j-aggregate receptors of L1 decoration and accumulation into the supermicroporous206cavities of the carriers with high crystal-structure stability (Figures S3C and S3D).207The molecular arrangement of the colorant aggregate (L1) in the inorganic–organic208architectures were confirmed using optical images (Scheme 3), which showed a branching209dendrite molecular aggregation. This optical microscopic observation demonstrated that the210spatial orientation of L1 transitioned from H-aggregates in the ethanol solution to form j-211aggregates in the solid phase within the trapping of L1 into the surface of the geode-shelled212nanorods of the organic–inorganic framework carrier. The BMAs sustaining the dendritic213arrangement of colorant aggregates into the carrier surface minimized the formation of H-214aggregates as a result of trapping of the j-aggregates’ molecular dimensionality. The j-aggregates215may form a highly ordered molecular alignment at the exterior surface of the colorant-wrapping216architecture nanorods for rapid monitoring of toxic cyanide compounds.21713218Scheme 3. (A, B) building of molecular structures of colorant and fluorescent j-aggerates of L1219receptor and its [L1-CN] complex into the nanorods with geodes shells surface of inorganic-220organic design in the formation BMA and BMA-CN- design respectively. (C) Schematic design221of the interaction and binding of CN- ion onto nanorods with geodes shells multi-facets222interaction surfaces during CN- ion sensing at pH=3 and recovery of cyanide toxic compounds in223living cells and water. (D) The optical microscopic images of the colorant (D-a) and fluorescent22414(D-b& D-C) J-aggregates of L1 receptor and its complex with dendritic t (end-to-end) stacking225arrangement.2263.3. Exclusive Colorant Tracking of CN− Ions in Water227The absorption and fluorescence spectra of the organic receptors of L1 were obtained to assess228the suitability of the fabricated colorant receptor of L1 for monitoring CN− ions in homogenous229liquid assays. L1 was titrated with CN− at pH 8.0. The enhancement in absorption intensity of the230organic probe L1 was observed at 454 nm, and then it decreased at 363 nm. The isosbestic point231at 392 nm was then obtained (Figure 2A). Scheme S1 shows that the yellowish-green color of the232organic probe L1 was produced as the CN− ion concentration gradually increased in the range of2330–2000 parts per billion (ppb). In the fluorometric titration profiles (Figure 2B), the fluorescence234intensity at λem = 540 nm (λex = 440 nm) of L1 (20 μM) was augmented upon the successive235addition of CN− ions in ethanol at pH 8.0 (phosphate buffer). The findings showed that the236fluorescence intensity was enhanced as the CN− ion concentration increased and thus confirmed237the sensing ability of the colorant aggregates of L1 for CN− ions via naked-eye inspection under238visible light or UV (365 nm, Scheme S1). Under optimal sensing conditions, the absorption and239emission spectra were obtained to show the high selectivity of the organic probe L1 to CN− ions240in the presence of various interfering ions, such as F−, Br−, Cl−, I−, NO3−, CH3COO−, H2PO4−,241SO42−, ClO4−, and HSO4−. The results revealed the high stability of the L1–CN− complex in the242presence of common interfering anions. The colorant j-aggregate receptors of L1 discriminated243between CN− and chemically close ions (Figures 2C and 2D).244Under optimal workability conditions, fluorometric titrations were carried out to assess245the sensitivity of organic receptors in monitoring CN− ions (Figures 2F). The linear calibration24615curves between the relative colorant j-aggregate receptors of L1 fluorescence intensity of the247CN− ion concentration (ppm) were observed, with a high correlation coefficient (R2 = 0.998).24824916Figure 2. (A) Changes in the UV-vis absorption spectra and (B) fluorescence spectra of L1250solutions in ethanol upon titration with standardized CN- ions under optimal sensing conditions251(pH of 8, staying time of 3 min, the volume of 20 mL, and temperature of 25 °C). Effect of252anions as interfering ions on the colorimetric (C) fluorescence spectra (D) of L1. Calibration253plots for L1 with absorbance (E) and fluorescence spectra (F) measured for the CN- ions at λ460,254λ540 respectively, linear-fit line are inserted in the linear concentration range before the saturation255of the calibration plots for the colorimetric and fluorescence spectra of L1 measured at λ460, λ540256respectively with different [CN-] concentrations.2572583.4. BMA Detection of CN− Ions259The sensor amount, contact time or response time, and reaction pH for CN− ions were260investigated to assess the fluorometric sensing assay parameters of BMAs in water. The261homogeneity of the BMAs and the ability to detect CN− ions were then observed using262fluorometric sensing assay. The selectivity of the BMAs for CN− ions was estimated in a mixture263of common interfering and multiple anions. With λex = 400 nm, no significant change was264observed in the fluorescence intensity of the BMAs. However, a considerable change was found265in the emission intensity and color of the BMAs under a UV lamp at 365 nm after adding CN−266ions. This observation confirmed the high sensitivity and selectivity of the BMAs for monitoring267ultra-trace concentrations of CN− ions (Figure 3).268In the fluorescence sensing assays of CN− ions (Figure 3), a significant enhancement in269fluorescence intensity was observed at λem = 460 nm with increasing CN− ion concentration.270Furthermore, the linear calibration curves allowed for detecting CN− ions with high sensitivity at271optimal conditions. Scheme S2 demonstrates the charge transfer mechanism that occurred in the27217CN−–L1 complex at pH 3.0. The remarkable improvement in the sensitivity and selectivity for273CN− ions through the use of BMAs was investigated, as shown in Table 1. Meanwhile, Table 2274shows that the calculated LOD for monitoring CN− targets by using single BMA geode was up to27588 ppt, which is lower than that in recently reported approaches.276277Figure 3. A) Fluorometric spectra of the BMA during the addition of various interfering anions278under optimal sensing conditions (pH of 3, staying time of 5 min, BMA amount of 5 mg, volume279of 20 mL, and temperature of 25 °C). B) Effect of common interfering anions on fluorescence280spectra of BMA in the absence and presence of CN- ions (2 ppm) under optimal sensing281conditions (pH of 3, staying time of 5 min, BMA amount of 5 mg, volume of 20 mL, and282temperature of 25 °C). fluorescence spectra (C) and calibration plots (D) of the BMA for the CN-283ions at λ453. A linear-fit line is inserted in the linear concentration range before the saturation of284the calibration plots for fluorescence spectra of BMA measured at λ453 with different [CN-]28518concentrations under optimal sensing conditions (pH of 3, staying time of 5 min, RFC amount of2865 mg, volume of 20 mL and temperature of 25 °C).287Table 1: Fluorometric sensing of CN- ions parameters for the organic probe (L1) and BMA.288289290Table 2: Comparison between the prepared BMA and the reported methods in sensitivity of CN-291ions.292Parameter Fluorescent probe (S1) BMASolvent Ethanol Milli-Q waterExcitation wavelength 440 nm 400 nmEmission wavelength 540 nm 453 nmLOD 13 ppb 0.088 ppbLOQ 40 ppb 0.2 ppbLinear rang 2 ppb- 2000ppb 0.5 ppb – 10 ppbResidual square 0.999 0.999Specific pH 8 3Time of response (seconds) 20 15Optical SensorsDetection limit(ppb)RefHAPQA 15 [64]IR 786 perchlorate 13 [65](Z)-1-((benzo[d]thiazol-ylimino)methyl)naphthalen-2-ol1560 [66]192933.5.Exclusive Colorant Tracking of CN− Ions in Biological Cells294Continuous monitoring and visualization of toxic cyanide compounds are important in reducing295the health threats associated with toxicant exposure in human cells, particularly in developing296countries. Therefore, novel approaches for the detection and colorant tracking of CN− in297contaminated biological cells are imperatively needed. By using MTT protocols, the cell viability298and cytotoxicity of the colorant probe L1 and BMAs were investigated in this study. The cells299were incubated with different colorant aggregates of L1 and BMA concentrations for 24 h at30037 °C. The metabolic activity of the HeLa cells decreased with increasing cytotoxicity of the301organic receptors of L1 and the BMA concentration (Figure 4B). The untreated HeLa cells302served as the control for all measurements. The colorant aggregates of L1 and BMAs exhibited303low cytotoxicity to HeLa cells. For example, 24% of the cells were damaged at a high BMA304concentration (50 μg/ml), and 4% of the cells were lost at 10 μg/ml. This result revealed that the305cytotoxicity of the organic probe L1 and BMAs did not significantly affect the cell population306and led to a low cytotoxicity and cell viability, confirming that the BMAs can be applied for307monitoring/tracking/inhibition of CN− ions in biological cells.3083.6.Visualization of CN− ions in HeLa cells309Confocal fluorescence microscopy was performed to investigate the monitoring and colorant310tracking of CN− ions in HeLa cells. Such capability can be attributed to the high biocompatibility311of the BMAs. First, the HeLa cells were incubated with BMAs (20.0 μg/ml) in PBS buffer for 30312imidazo-anthraquinones 93.6 [67]pyrazine-derived chemosensor 496 [68]BMA 0.088 Present work20min at 37 °C and visualized using confocal fluorescence microscopy with an excitation313wavelength of 488 nm (Figure 4A). The results revealed that the cells incubated with BMAs314displayed very weak fluorescence. Moreover, the high biocompatibility and low toxicity were315investigated via confocal microscopic visualization of cells incubated with the BMAs (20.0316μg/ml) and the control. No shrinkage nor damage was visualized on the cell membrane, and the317microunit of the BMAs was clearly obtained by cell imaging. The extracellular medium of the318HeLa cells was washed off with buffer several times to remove excessive BMAs. The BMA-319treated cells were supplemented with 100 ppb CN− in a medium with polyethersulphone buffer320for 30 min at 37 °C. The results showed that the intracellular area fluorescence intensity was321enhanced. Throughout the imaging experiments, the HeLa cells were viable, as demonstrated by322the bright-field transmission images and confocal microscopic images of the HeLa cells treated323with BMA geodes and CN− ions (Scheme 1). Thus, BMAs can be applied as biocompatible324fluorescent nanomonitors for visualizing/tracking CN− ions in living cells (Figure 4C).32521326Figure 4. A) Schematic representation illustrates the fluorescence enhancement after the binding327interaction between CN- ions and the biocompatible (BMA) in HeLa cells. B) Percentage (%) of328cell viability and (%) of cell inhibition of HeLa cells treated with different concentrations (10-50329μg/ml) of L1 (a), (10-50 μg/ml) of BMA (B). C) Confocal microscope images recorded at330excitation wavelength of 488 nm and emission wavelength at 540 nm of control and HeLa cells33122with incubated 10 μg/mL BMA with 10μM CN- for 30 min and HeLa cells with incubated 10332μM of organic probe L1 with 10μM [CN-] for 30 min.3333.7.Fluorescence-activated Cell Sorting334The cellular volume in flow cytometry was estimated via forward scattering (FSC) intensity,335which was obtained for the HeLa cells treated with BMAs. The findings for the BMAs with CN−336ions were close to those for the control cells. Figure 5 shows that the FSC intensity was nearly337constant. The change in FSC intensity may be ascribed to the swelling or shrinking of cells as a338result of the cell death process. The flow cytometry results revealed that the monitoring of CN−339ions in living cells were not due to the total cell death. Overall, the data confirmed the high340biocompatibility features of BMAs under optimal conditions, matching the results obtained from341the cytotoxicity studies. The majority of the untreated control cells were viable (99.14%). By342contrast, 3.04% of the pierced cells with BMAs were fluorescein isothiocyanate (FITC)-positive343apoptotic cells. The data demonstrated that the BMAs did not drastically enhance the percentage344of FITC-positive apoptotic cells in comparison with the untreated cells (Figure 5). The low BMA345concentration allowed for the improved monitoring of CN− ions at optimal cellular conditions.34623347Figure 5. The flow cytometry of the examined BMA geodes in presence and in absence of CN-348ions. HeLa cells were incubated for 24h without any material act as a control (A, A1) followed349by incubation for 4 hours with 20 μg/mL [BMA] (B, B1) and with 20 μg/mL [BMA] +100 ppb350of [CN-] (C, C1). (D) statistical results of the examined BMA in presence and in absence of CN-.351243.8. Tracking/Monitoring of CN− Ions in HeLa Cells352The HeLa cell concentration in a 96-well black dish was adjusted to quantify CN− ions and353detect them in HeLa cells. The concentration of BMAs was adjusted, with no remarkable effect354on the HeLa cells. The untreated HeLa cells initially incubated for 24 h served as the control.355The fluorescence enhancement depended on the concentration of the CN− ions. Figure 6A shows356that time did not remarkably affect the recorded fluorescence intensity of CN− ion monitoring.357The emission intensity enhanced with increasing CN− levels (Figure 6B). The enhanced emission358intensity was then recorded using a spectrofluorometer and a microplate reader.359360361Figure 6. A) Time dependent fluorescence enhancement of living cell line stimulated by 20362µg/ml [BMA] with different concentration of CN- ions. B) Calibration curve of 10, 20, 30, 40363µg/ml [BMA] against different concentration of CN- that were induced in PBS solution at364pH=7.4.365366367253684. CONCLUSIONS369Contaminated cyanide compounds in the ecosystem are a critical concern owing to the370growing of urbanization. The significant health problems caused by toxic cyanide compounds371are of interest because of their immutability in the ecosystem. Therefore, ultrasensitive372approaches for detection/recovery of CN− ions in HeLa Cells are urgently needed. BMA geodes373were successfully fabricated for tracking and monitoring of toxic CN− ion concentrations in374HeLa cells within few seconds. The BMAs were decorated via direct dressing approaches by375wrapping the hydrophobic colorant L1 onto the geode-shelled nanorods of porous organic–376inorganic Al frameworks. The BMA geodes exhibited high biocompatibility and cell viability377through CN− ion detection and monitoring in biological trials. Moreover, the fluorescence378intensity of the intracellular area enhanced after CN− ions were added. Under specific conditions,379the results confirmed the high applicability of BMA geodes for in-vitro fluorescence of CN− ion380tracking/sensing in HeLa cells, indicating the BMA geode-shelled nanorods’ capability in381reducing health hazards due to toxicant exposure.382383REFERENCES384[1] S. Malkondu, S. Erdemir, S. Karakurt, Red and blue emitting fluorescent probe for cyanide385and hypochlorite ions: Biological sensing and environmental analysis. 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