Brassica napus roots to characterize the effects of six individual macronutrient deprivations . Our results showed that before any visual phenotypic response, all macronutrient deprivations led to a large modulation of the transcriptome and metabolome involved in various metabolic pathways, and some were common to all macronutrient deprivations. Significantly, comparative transcriptomic analysis allowed the definition of a subset of 3282, 2011, 6325, 1384, 439, and 5157 differentially expressed genes (DEGs) specific to N, Mg, P, S, K, and Ca deprivations, respectively. Surprisingly, gene ontology term enrichment analysis performed on this subset of specific DEGs highlighted biological processes that are common to a number of these macronutrient deprivations, illustrating the complexity of nutrient interactions. In addition, a set of 38 biochemical compounds that discriminated the macronutrient deprivations was identified using a metabolic approach. The opportunity to use these specific DEGs and/or biochemical compounds as potential molecular indicators to diagnose macronutrient deficiency is discussed.The early and specific diagnosis of a macronutrient deficiency is challenging when seeking to better manage fertilizer inputs in the context of sustainable agriculture. Consequently, this study explored the potential for transcriptomic and metabolomic analysis of  Nitrogen (N), magnesium (Mg), phosphorus (P), sulfur (S), potassium (K), and calcium (Ca) are macronutrients present in large amounts in plant tissues (more than 0.1% of dry weight) and are essential for plant growth, seed yield, and nutritional quality . In highSince macronutrients are involved in many crucial metabolic pathways ,13, all Citrus sinensis adapts to Mg privation in association with DEGs implicated in cell cycle regulation, signal transduction, photosynthesis, cell wall remodeling, and the antioxidant system, as well as phosphorylation control 66] so thw/v), pH 8) were added and the mixture was vortexed for 40 s. Then, 750 μL of chloroform:isoamylalcohol (24/1: v/v) was added before centrifugation at 15,000× g for 5 min at 4 °C. The supernatant was recovered and 750 µL of a 4M LiCl solution (w/v) were added for nucleic acid precipitation overnight at 4 °C. The mixture was then centrifuged at 15,000× g for 20 min at 4 °C, the supernatant was removed, and 100 μL of sterile water was used to suspend the pellet. Extracted RNAs were purified by DNAse digestion using RNA Clean & Concentrator kits . Total RNA quantification was evaluated by spectrophotometry at 260 nm  before Reverse Transcription (RT). A 1 µg quantity of total RNAs was converted to cDNAs using an iScript cDNA synthesis kit .Total RNAs were extracted from 200 mg of fresh root samples previously powdered using a mortar containing liquid nitrogen, according to the protocols of ,68. BrieFor qPCR, 4 µL of 100× diluted cDNAs were added to 11 µL of 1X SYBR Green Master Mix  containing 0.5 µM of specific primers. Amplification reactions were performed with a real-time thermocycler  using the following three step program: an activation step at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 10 s, and finally, an extending step at 60 °C for 40 s. For q-PCR amplifications, the primers used are provided in −ΔΔCtRelative expression = 2WithUsing this method, root relative expression of the target gene in the control sample (without nutrient deprivation) was equal to 1 .®, California, CA, USA) with an average size of 260 bp and were sequenced in paired-end (PE) mode with a read length of 75 bases on the NextSeq500 with approximately 25 million PE reads per sample. To remove poor quality sequences, classical trimming  was performed and the STAR_2.5.2a mapper was used to align reads against the Brassica napus transcriptome . The abundance of each of the (annotation V5 from Genoscope accessed in February 2021: http://www.genoscope.cns.fr/brassicanapus/data/) was evaluated by unequivocal mapping of the PE reads to each gene. According to this method, 25% of reads were unmapped and 10% of reads with multi-hits were removed. Finally, 65% of reads may have combined with a gene without ambiguity.The RNA-seq samples were obtained with Illumina NexSeq500 from POPS platform of Institute of Plant Science (IPS2) in Paris-Saclay (France). RNA-seq libraries were generated with the TruSeq Stranded mRNA protocol . We chose to consider a gene as differentially expressed (DEG) for an adjusted p-value ≤ 0.05, whatever the absolute value of the “Log2 fold change”  was performed with gene ontology (GO) information slimmed to plants using g:Profiler (version e103_eg50_p15_eadf141) with the g:SCS multiple testing correction method  and RevBrassica napus DEGs was generated by filtering for the occurrence of the “transport” term in the annotation V5 from Genoscope. This list was supplemented with a set of DEGs associated with the GO term “ion transport” (GO:0006811) extracted from Amigo2 and unambiguously related to the transport of known elements . This set of 302 DEGs encoding ion transporters is presented in Among genes that are differentially expressed for at least one macronutrient deprivation, a set of TM, Thermo Fisher Scientific, Bremen, Germany) following the procedure described in [As previously described by , dried sribed in .Total N measurement was analyzed with 1.5 mg of fine powder placed in tin capsules before analysis with an isotope-ratio mass spectrometer  linked to a C/N/S analyzer .n = 5 tissues  and Q = quantity of each element. This calculation was performed with consideration of all random combinations between the set of five replicates of Day 0 and Day 10. Thus, 10-day net uptake is indicated as the mean ± S.E for n = 25.Whole plant quantity  was determined before the 10-day net uptake calculation as follow:2O  and 1% formic acid . After extraction, samples were centrifuged, and the supernatant was collected for UPLC-MS/MS (ultra-performance liquid chromatography-tandem mass spectrometry) analysis. For the UPLC-MS/MS analysis, the separation and the detection were accomplished using an Acquity UPLC system  coupled to a Xevo G2-S QTof mass spectrometer (Waters) equipped with an LockSpray electrospray ionization (ESI) source. Sample separation was carried out by injecting 10 µL into an HSS T3 C18, 2.1 × 100 mm, 1.8 µm column (Waters) at a flow rate of 0.5 mL min−1, and the column oven was maintained at 40 °C. The mobile phases were composed of solvent A (Milli-Q water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The separation was achieved by the following gradient: 0–1 min at 98% A, 1–7 min from 98% to 0% A, maintained at 0% A to 9 min, 9–10 min from 0% to 98% A, maintained at 98% until 12 min for column regeneration. The MS analysis was carried out in positive and negative ionization modes with the following parameters: source voltage 0.5 kV (pos) and 2.5 kV (neg); cone voltage 40 V; source temperature 130 °C; desolvation gas temperature 550 °C; desolvation gas flow 900 L/h. Mass spectra were acquired in MSE mode from 50 to 1200 m/z at 0.1 s scan−1. The ramp collision energy was set at 25 to 40 V. For each macronutrient deprivation, all metabolites significantly modulated compared to control are listed in p-value of each enriched group is given by the Kolmogorov-Smirnov test.Fifty milligrams of ground frozen root tissues were weighed and extracted with 1 mL of 70% MeOH , 29% Hn = 5, while net nutrient uptake was given as the mean ± S.E. for n = 25. Statistical analyses were performed using R software (v4.0.3) [t-test. Heatmaps and clustering were generated with R Commander (v2.7-1) from the heatmap 2 package or the Morpheus-Broad Institute (https://software.broadinstitute.org). Regarding the metabolomics data, multivariate analysis was performed and allowed us to assign a variable importance in projection (VIP) score to each metabolite in order to determine the relative contribution of each metabolite to discriminate each macronutrient deficiency from the other.Statistical analysis was based on five independent replicates each consisting of a pool of two individual plants, except for transcriptomic data, for which three independent replicates were used. Thus, plant biomass and photosynthesis are indicated as the mean ± S.E for (v4.0.3)  and R Co


E6/E7 carcinogenic process, and are characterized by effects on the host genome, which result in the inactivation of tumor suppressors  and the activation of oncogenes , thus activating oncogenic signaling pathways . This study could enhance our understanding of HPV integration and pave the way for subsequent HPV studies in PSCC.Penile squamous cell carcinoma (PSCC) has been regarded as an HPV-related cancer for a long time. However, the integration pattern and carcinogenic pathways of HPV in PSCC remain unclear. The results of this study provide insights into the HPV-related carcinogenic mechanism in PSCC, which may be less prone to involvement in the traditional E2 oncogene was not prone to involvement in integration. Eleven of the 35 (31.4%) HPV-integrated PSCCs harbored intact HPV E2; these tumors had lower HPV E6 and E7 expression and higher expression of p53 and pRb proteins than those with disrupted E2 did (p < 0.001 and p = 0.024). Integration breakpoints are preferentially distributed in or near host genes, including previously reported hotspots  and newly identified hotspots , which are mainly involved in oncogenic signaling pathways . Regarding the phosphorylation levels of JNK, p38 was higher in HPV-positive tumors with MAPK-associated integration than those in HPV-positive tumors with other integration and those in HPV-negative tumors. In vitro, KLF5 knockdown inhibited proliferation and invasion of PSCC cells, while silencing CADM2 promoted migration and invasion. In conclusion, this study enhances our understanding of HPV-induced carcinogenesis in PSCC, which may not only rely on the E6/E7 oncogenes, but mat also affect the expression of critical genes and thus activate oncogenic pathways.Human papillomavirus (HPV) is a significant etiologic driver of penile squamous cell carcinoma (PSCC). The integration pattern of HPV and its carcinogenic mechanism in PSCC remain largely unclear. We retrospectively reviewed 108 PSCC cases who received surgery between 2008 and 2017. Using high-throughput viral integration detection, we identified 35 HPV-integrated PSCCs. Unlike cervical cancer, the HPV  E6/E7-induced carcinogenesis triggered by disruption of the HPV E2 oncogene: The disruption of E2 results in a loss of negative feedback control of the expression of the E6 and E7 viral oncoproteins that are involved in the inactivation of p53 and pRb proteins [E6/E7-induced carcinogenesis, in some cases, HPV integration also induces carcinogenesis through directly activating oncogenes or inactivating tumor suppressors in the human genome [Penile squamous cell carcinoma (PSCC) is a rare malignancy worldwide, but its incidence is much higher in developing areas such as Africa, South America, and Southeast Asia. Human papillomavirus (HPV) infection is a significant etiologic driver of PSCC . In mostproteins ,5. Besidn genome ,9. HowevAPOBEC mutation [The integration pattern and characteristics of HPV in the PSCC genome are also unclear. Several studies have indicated that HPV-positive PSCC harbors a different genomic profile ,11,12,13mutation ; HPV-posE6 and E7 expression); 35 of the 41 PSCCs were verified to harbor HPV integration using HIVID analysis and further analysis of HPV integration was performed. Moreover, 67 HPV-negative cases were used to compare the clinicopathological characteristics and the expression of key genes with 35 HPV-integrated cases. The study design is shown in In this study, we used tumor specimens from 108 PSCC patients who underwent surgical intervention at the Sun Yat-sen University Cancer Center between 2008 and 2017. The inclusion criteria were as follows: (I) Histologically confirmed PSCC; (II) no anti-cancer treatments received before surgery; (III) no history or concurrence of other malignant tumors; (IV) complete clinicopathological data. Histologic subtypes of PSCC were reviewed by two pathologists according to the fourth edition of the World Health Organization’s classification . Tumor s+ PSCC samples were subjected to library preparation as previously reported  covering the full-length HPV genomes of 18 subtypes , namely,Adaptor, low-quality, and short sequence (less than 40 bp) data were first removed to obtain clean data, which were then mapped to 314 genomes of 32 HPV subtypes  using BuNext, the initial integration sites were obtained by SVdetect analysis , high-coq-value was <0.05.Subsequently, the genomic regions/elements and genes involved in HPV integration were analyzed. High-confidence results were annotated using ANNOVAR , referriAfter routine deparaffinization, rehydration, antigen retrieval, and endogenous peroxidase inactivation, FFPE specimen slides (4.0 μm-thick) were subjected to immunohistochemistry (IHC) staining using primary antibodies against phospho-(p-)JNK , Danvers, MA, USA), p-ERK , p-p38 , p-STAT3 , β-catenin , CEP19 , NRROS , CADM2  or KLF5 , p53 , pRb , and the Dako REAL™ EnVision™ Detection System  according to the manufacturer’s instructions. To score the IHC staining, 3–5 (depending on the size of the tumor area) independent microscopic fields (100×) of each tumor specimen were examined in terms of the integrated optical density (IOD) via Image Pro Plus 6.0  . The sta450nm) [Two PSCC cell lines previously established by our group, Penl2 (RRID: CVCL_VF95) and 149RCa (RRID: CVCL_VF96) , were cu450nm) .The migration ability of PSCC cells was assessed using a wound healing assay ,25. Brie5 149RCa cells or 1 × 105 Penl2 cells were seeded into Boyden chambers (8 µm pore) with Matrigel coating . After 24 or 36 h of incubation, the cells on the lower surface of the filter were fixed, stained, and counted.The invasion ability of PSCC cells was evaluated using a Transwell assay as described previously ,26. Brie® Premix Ex TaqTM (TaKaRa) with specific primers  after being normalized to the control .Total RNA was extracted from PSCC cells or RNA later-preserved tissues using Trizol reagent , and then reverse-transcribed into cDNA using HiScript II . qPCR assay was performed using SYBR primers . The relχ2 test, Fisher’s exact test, and Pearson’s correlation analysis were employed for correlation analysis, the Mann–Whitney and Kruskal–Wallis tests were used for rank-sum testing, and the Kaplan–Meier method with the log-rank test was used for survival analysis. Disease-specific survival was defined as the time from surgery to either death caused by disease or last follow-up. The difference between two or more groups was analyzed by Student’s t-test or analysis of variance (ANOVA). Statistical significance was set at p < 0.05.All statistical analyses were performed using SPSS 22.0 . The We performed an HIVID assay in 41 HPV-positive PSCC previously identified by qPCR . HIVID dp < 0.001)  were observed HPV integration, while the rest three tumors only harbored episomal HPV DNA. In total, 2252 HPV integration breakpoints were identified in 35 PSCC specimens  . Among t< 0.001) .p = 6.6 × 10−62), common fragile sites (p = 8.5 × 10−25), SINE-Alu (p = 3.9 × 10−43), and satellites (p = 6.9 × 10−9), but were relatively scarce in areas without those elements (p = 5.1 × 10−217).We deciphered the pattern of HPV integration in the human genome and found that although the integration event distributed in all chromosomes in the human genome a, they wp = 3.1 × 10−10), but not in intergenic regions (p = 6.7 × 10−10) (p = 3.2 × 10−5) and 3′ untranslated regions (p = 7.8 × 10−3), but not in 5′ untranslated regions (p = 1.7 × 10−4); in intergenic regions, breakpoints were clustered in regions that were <100 kb from genes , but were scarcer in regions >700 kb from genes (p = 2.2 × 10−16)  d. In int× 10−16) e.E6/E7 carcinogenic process often triggered by HPV E2 disruption. The integration of E2 causes the disruption of E2, thus increasing the level of HPV E6/E7 protein and decreasing the level of p53 and pRb [E2 was significantly less prone to be involved in HPV integration than expected (p = 1.25 × 10−5)  of them harbored <3 HPV E2 integration events. Although the number of cases is relatively small, patients with intact E2 had higher disease-specific survival rates than those with disrupted E2 (p = 0.094)   (E1 (p = 1.36 × 10−32) was more prone to involve in integration, while several other regions, such as E6 (p = 3.42 × 10−4), E7 (p = 7.06 × 10−6), and the LCR region (p = 7.39 × 10−4) were less prone to involvement in integration  b, which = 0.094) c. In tum< 0.001) d. Moreovctively) e. Other egration b. The whng of L2 .3) than in those with grade G1–2 .As HPV integration could also induce carcinogenesis by activating oncogenes or inactivating tumor suppressors in the human genome, to investigate which gene and signaling pathway play a key role in HPV-induced PSCC, we identified 2277 genes that were likely affected by HPV integration . These gp = 6.44 × 10−9; p = 0.042, 0.030, and 0.323, respectively) and HPV-negative tumors  (p = 0.018) and HPV-negative tumors (p < 0.001)   b–d. Next< 0.001) e. Howevectively) . Taken tKLF5, LRP1B, and KLF12) have previously been described in cervical cancer [CADM2, CEP19, CSMD1, and NRROS) are reported for the first time in the present study. Moreover, some hotspot genes were located in closer proximity, such as KLF5–LINC00392–KLF12, CADM2–LINC02070, TACR3–CXXC4, NRROS–CEP19, and LINC02170–CMC2, which may have been affected by shared integrations  to examine their expression in PSCC by IHC staining    a, KLF5 lctively) a. Howevectively) .CADM2, and another occurred in the downstream region. We report CADM2 as an HPV-integration hotspot gene in this study. We preliminarily investigated the in vitro function of CADM2, and found that knock down of CADM2  , p-JNK , and p-p38   than that in HPV-negative tumors ( <0.001) a, we sup< 0.001) d. These In the present study, we determined the pattern of HPV integration in PSCC and investigated the impact of HPV integration on penile carcinogenesis. Several findings may contribute a lot to our current knowledge of HPV-induced PSCCs.E2 were observed than expected in PSCC, whereas no such tendency exists in cervical cancer [E2, indicating that these tumors are not triggered by HPV E2 gene disruption. Even in 24 tumors with disrupted E2, half (12/24) of them only harbored less than three E2 integration sites, suggesting that E2 disruption may not play a critical role in those tumors. The similar pattern was also observed in other HPV-related squamous cell carcinomas, such as oropharynx squamous cell carcinoma [E2 harbor lower expression of HPV E6 and E7, and higher expression of p53 and pRb than tumors integrating HPV E2 DNA. These results remind us that many HPV-induced PSCCs are highly possibly generated by directly affecting critical host genes. Meanwhile, patients with intact E2 had marginally better survival than those with disrupted E2 (p = 0.094). A similar result also exists in oropharynx squamous cell carcinoma [First, we identified a carcinogenic mechanism in PSCC that is uncommon in cervical cancer. Significantly fewer integration breakpoints in HPV l cancer . Eleven arcinoma ,29,30. MKLF5 and KLF12 (13q22.1), MYC (8q24.21), and TP63 and TPRG1 (3q28) [+ tumors, including in chromosomal regions 2q33.2-q33.3 and 2q35. Macedo et al. [Second, we first depicted the integration pattern of HPV in PSCC in a direct way. Previous studies in other HPV-related cancers have revealed that HPV integration events are more likely to occur inside or near gene regions and in within genome instability elements. Loci at 13q22.1, 8q24.21, 3q28, 17q23.1, and 17q23.2 are the top five hotspot cytoband regions. Specifically, integration-affected hotspot genes include 1 (3q28)  found thCADM2, which has never been reported to be hotspot gene in other HPV-related tumors. All of the integrations in the CADM2 gene were caused by the HPV16 subtype in our study. CADM2 has been reported to be a tumor suppressor in hepatocellular carcinoma and in glioma [Third, we identified many hotspot genes and pathways that were associated with HPV-induced carcinogenesis in PSCC. One of the hotspot genes was n glioma ,32, and n glioma . In thisn glioma ,35,36,37n glioma . Thus, Hn glioma .CADM2 gene and the activation of the oncogene KLF5, as well as the MAPK and JAK/STAT signaling pathways, other effects induced by HPV integration may also exist. As several new HPV integration clustered regions  were observed in PSCC, we plan to investigate hotspot genes in these regions to further clarify the additional carcinogenic mechanisms involved in PSCC.The major limitation of this study is the relatively small sample size (35 integrated tumors), which limits making a definite conclusion. As PSCC is a rare tumor, a large-scale study would be difficult to carry out. In addition to the inactivation of the tumor-suppressor E6/E7 carcinogenic process, but also characterized by affecting the host genome, which resulted in the inactivation of tumor suppressors  and the activation of oncogenes  and oncogenic signaling pathways . Our findings enhance our understanding of HPV integration and pave the way for subsequent HPV studies in PSCC. Further large-scale multicenter studies and mechanical studies of KLF5 and CADM2 are warranted.We described a whole genome-wide study of HPV integration in PSCC, and identified an HPV-related carcinogenic mechanism not only through the traditional


Yersinia enterocolitica in a co-cultivation approach using a bacterial biosensor carrying a special sensor plasmid is described. We combined selective enrichment with the simultaneous detection of pathogens by recording autoinducer-1-induced bioluminescent response of the biosensor. This new approach enables real-time detection with a calculated sensitivity of one initial cell in a sample after 15.3 h of co-cultivation, while higher levels of initial contamination can be detected within less than half of the time. Our new method is substantially faster than conventional microbial cultivation and should be transferrable to other zoonotic foodborne pathogens. As we could demonstrate, quorum sensing is a promising platform for the development of sensitive assays in the area of food quality, safety, and hygiene.Foodborne zoonotic pathogens have a severe impact on food safety. The demand for animal-based food products  is increasing, and therefore faster methods are necessary to detect infected animals or contaminated food before products enter the market. However, conventional detection is based on time-consuming microbial cultivation methods. Here, the establishment of a quorum sensing-based method for detection of foodborne pathogens as  Escherichia coli (STEC) (8000 cases), and yersiniosis (7000 cases) [Yersinia enterocolitica [In 1959, the World Health Organization (WHO) defined zoonoses as diseases that are naturally transmittable between humans and other vertebrates . Due to 0 cases) . In 99% Yersinia (family Enterobacteriaceae) comprises 19 species, including the human and animal pathogens Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis [Y. enterocolitica is a Gram-negative bacillus, with a coccoid to rod-shaped morphology. This psychrophilic species can grow at temperatures within the range 0–45 °C, with optimal growth at 25–28 °C. Y. enterocolitica is motile at 25 °C, but loses motility at 37 °C [Y. enterocolitica are assigned to six biotypes on the basis of their biochemical features  and comprise more than 70 serotypes depending on their O-lipopolysaccharide determinants [The genus rculosis . Y. enterminants . Generalrminants . Neverthrminants .Y. enterocolitica is usually transferred to humans via the consumption of contaminated raw or undercooked pork, or by direct contact with contaminated carcasses [arcasses . Yersiniarcasses . Pigs ararcasses ,13. EspeY. enterocolitica requires microbial cultivation. An isolation step is necessary, consisting of selective enrichment followed by plating onto selective agar medium. Presumptive colonies are identified by biochemical and pathogenic phenotype. Depending on the choice of enrichment strategy, isolation may take from 48 h up to several weeks [The conventional detection of al weeks ,15,16. TY. enterocolitica by amplifying the virulence-associated genes. Multiplex PCRs have been developed, and more sensitive detection can be achieved by real-time PCR [Y. enterocolitica directly are usually serotype-specific and are not well suited for sensitive detection of a bacterial infection [Y. enterocolitica in animal blood and fecal samples [Y. enterocolitica-specific antibodies, which only permits a conclusion about (a possible past) yersiniosis. Hence, these methods are not applicable for the detection of an actual bacterial contamination, e.g., in food samples. By this, current commercially available ELISA-based methods are not feasible for efficient analysis and detection of food contamination with Y. enterocolitica.The polymerase chain reaction (PCR) allows the genotypic characterization of time PCR ,17,18,19time PCR . Immuno- samples ,21. HoweTo address the limitations of conventional detection methods and current molecular assays, we developed a new approach for the rapid and sensitive detection of bacteria using quorum sensing (QS) signaling molecules. QS is generally known as a bacterial cell-cell communication process to coordinate gene expression depending on population density. In contrast to low cell density (LCD) populations, at which individual gene expression is favored, the synchronous expression of multiple genes is beneficial at high cell density (HCD) populations. QS-controlled processes include antibiotic production, sporulation, bioluminescence, biofilm formation, and virulence factor secretion. The QS system is based on signaling molecules called autoinducers (AIs), which are produced and secreted by cells. After reaching a local threshold concentration at HCD, the AIs trigger a signal cascade that modulates gene expression ,23.N-Acyl homoserine lactone-based molecules (AHLs), consisting of a homoserine lactone ring moiety and an acyl side chain, used for intraspecies communication in Gram-negative bacteria [Y. enterocolitica produces the AHLs N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) [The AIs are divided into three major groups: AI-1 (AI-1), AI-2 (AI-2), and peptide-based AIs (AIPs) . AIPs arbacteria . AHL spebacteria . Y. entee (OHHL) . TherefoVibrio fischeri. The generation of bioluminescence is regulated by the LuxI and LuxR proteins. LuxI is an AHL synthase that produces OHHL. The luxI gene is normally expressed at a basal level, resulting in the production of small quantities of OHHL [luxI and the lux operon (luxCDABEG). LuxI therefore initiates a positive feedback loop by synthesizing the ligand that binds to LuxR and induces the synthesis of more LuxI. The increase in OHHL levels also triggers the expression of the lux operon in adjacent cells [lux operon generate bioluminescence via a luciferase-catalyzed oxidation reaction [The best-characterized QS system as a paradigm of intraspecies communication among Gram-negative bacteria is the LuxI/R system of the bioluminescent bacterium  of OHHL . AHLs ar of OHHL . As the nt cells ,31. The reaction .luxR gene or the gene of a LuxR homologue, the cognate promoter, as well as a reporter gene for the detection. The presence of a compatible AHL finally induces the expression of the reporter gene whose product is detectable by, for example, bioluminescence [luxR together with the promoter luxI’, or homologues as lasR and rhlR with corresponding promoters, to the lux operon (luxCDABE) of Photorhabdus luminescens [lux operon offers a non-destructive real-time detection as a reporter gene and a higher temperature stability than the lux operon of V. fischeri, which is relevant for expression in recombinant E. coli at 37 °C [Within the last decades, the application of bacterial biosensor techniques has advanced the study of AHLs (AI-1) in QS systems and enables a simple method for qualitative and quantitative detection of AHLs ,34,35. Tnescence . In a prinescens . This luat 37 °C ,37. Addiat 37 °C .In contrast, physico-chemical methods such as GC-MS or LC-MS/MS are frequently used for identification and quantification of AHLs due to their high specificity. However, these devices are highly expensive and need highly trained staff for handling, running, and maintaining the equipment. Furthermore, for method development highly trained staff are required, and sensitivity can vary greatly depending on method parameters, machine settings, and sample preparation. As demonstrated by Bauer and colleagues (2016), GC-MS methods, e.g., showed LODs of just 3.2 to 6.2 µM of single AHLs . TargeteY. enterocolitica based on QS. Therefore, we used the V. fischeri LuxI/R system for detection of Y. enterocolitica by monitoring the production of its AHLs using a bacterial biosensor carrying a modified sensor plasmid derived from pSB401 [lux operon by binding the LuxR protein, inducing bioluminescence and indicating whether the sample is contaminated with Y. enterocolitica . It was based on the sequence of pSB401 , which elication .Escherichia coli strain BL21 (DE3) cells  according to the manufacturer’s protocol. Aliquots of 50 µL and 100 µL and the pellet were plated on lysogeny broth (LB) selection plates  containing 100 µg mL−1 ampicillin. After an overnight incubation at 37 °C, successfully transformed cells were isolated on further selection plates and incubated overnight (37 °C). A colony was then used for preparation of fresh overnight cultures of the biosensor. The pMA-RQ_luxR-lux sensor plasmid was transformed into chemically competent 600 = 0.4. The culture was directly used for the luminescence assay. We mixed 20 µL of sample (synthetic AI standard concentrations or sterile culture supernatants) with 180 µL of the biosensor culture in C8 LockWell Lumi white 96-well plates . For background measurement, the corresponding buffer or medium was used as blank. Kinetic measurements were carried out for 5 h at 30 °C recording luminescence signals every 20 min using a CLARIOstar plate reader  with the following set parameters: top optic, 11.0 mm focal height, 10 s measuring interval time, 10 s orbital shaking at 200 rpm before measurement. An overnight culture in 5 mL selective LB was prepared with a clone from the master plate and incubated overnight at 37 °C, shaking at 160 rpm. On the next day, 25 mL of selective LB broth was inoculated with the overnight culture at a 1:1000 ratio. The biosensor culture was cultivated at 37 °C, shaking at 160 rpm, until the culture reached OD2HPO4, 1.5 mM KH2PO4; pH 7.4) to concentrations ranging from 0.3 nM up to 200 nM and used as standard concentrations for calibration in the luminescence bioassay.HHL and OHHL were purchased from Merck . Stock solutions with a concentration of 20 mM were prepared in DMSO and stored at −20 °C. On the day of the bioassay, the stock was freshly diluted with sterile phosphate-buffered saline  was obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures . Overnight cultures were prepared by inoculating 5 mL tryptic soy broth  glucose, 5 g L−1 NaCl, 2.5 g L−1 K2HPO4; pH 7.3) with 5 µL of a thawed Y. enterocolitica glycerol stock. For experimental cultures, selective CIN medium was inoculated with the overnight culture at a ratio of 1:1000. The selective medium was derived from the CIN agar formulation as previously described [−1 gelatin peptone, 1.5 g L−1 casein peptone, 1.5 g L−1 animal tissue peptone, 2 g L−1 yeast extract, 20 g L−1 mannitol, 0.5 g L−1 sodium deoxycholate, 0.5 g L−1 sodium cholate, 1 g L−1 sodium chloride, 2 g L−1 sodium pyruvate, 0.01 g L−1 magnesium sulfate, 0.004 g L−1 irgasan, 0.4 g L−1 cefsulodin, 0.25 g L−1 novobiocin; pH 7.4). All cultures were incubated at 28 °C, with shaking at 160 rpm.escribed . Agar anY. enterocolitica was recorded by inoculating 300 mL selective CIN medium with an overnight culture (1:1000) and taking 12 mL samples at regular intervals, starting at t = 0 and ending at 24 h. The optical density was measured at 600 nm (OD600) to determine cell growth. Culture supernatants were generated from 10 mL aliquots µ=lnXg for 5 min). The supernatants were filtered using a 0.2 µm syringe sterile filter  and subsequently stored at −80 °C.Sterile culture supernatants were prepared from 10 mL samples taken during the growth curve experiment . Bacteriv/v) analytical grade anhydrous acetic acid . For this, acidified ethyl acetate was added to collected supernatant (1:3 v/v) and vortexed for 1 min. Afterwards, the organic phase was collected, and the remaining aqueous phase was extracted again as described. In total, five extraction cycles were performed. Pooled organic phases were concentrated by evaporation before drying using a SpeedVac vacuum concentrator  for 1 h at RT. For LC-MS/MS analysis, the residue was reconstituted in 200 µL acetonitrile . To determine the performance and recovery rates achieved using the LLE protocol, we spiked the culture supernatants with 100 ng of OHHL and HHL as additional samples.AHLs were extracted from sterile culture supernatants by liquid-liquid extraction (LLE) in pro-analysis-grade ethyl acetate  containing 1%  into autosampler vials with inserts. The samples were separated by HPLC on a Curosil-PFP 3 µm 150 × 3.0 mm column  using the Agilent 1200 HPLC system with a diode array detector set to 190–400 nm. The mobile phases were (A) degassed ddH2O containing 5 mM LiChropur ammonium acetate (Merck), 0.1% (v/v) HPLC-grade trifluoroacetic acid , and 0.1% (v/v) ACS-grade formic acid (Carl Roth) and (B) ultra-LC-MS/MS-grade acetonitrile  containing 0.1% (v/v) trifluoroacetic acid. We injected 10 µL samples and separated them by gradient flow at a constant flow rate of 0.4 mL min−1. The gradient was held at 100% A for 2 min before changing to 100% B over a 12 min linear gradient, followed by a 2 min hold step at 100% B before returning to 100% A over 4 min, followed by a final re-equilibration for 5 min at 100% A. For calibration, stock solutions of OHHL and HHL  were thawed and diluted with acetonitrile to the following concentrations: 0.01, 0.05, 0.1, 0.15, 0.3, 0.5 µg mL−1.AHL extracts were passed through a Claristep 0.2 µm regenerated cellulose filter with an area of 0.7 cm2  into autosampler vials with inserts. The samples were separated by HPLC on a Curosil-PFP 3 µm 150 × 3.0 mm column  using the Agilent 1200 HPLC system with a diode array detector set to 190–400 nm. The mobile phases were (A) degassed ddH2O containing 5 mM LiChropur ammonium acetate (Merck), 0.1% (v/v) HPLC-grade trifluoroacetic acid , and 0.1% (v/v) ACS-grade formic acid (Carl Roth) and (B) ultra-LC-MS/MS-grade acetonitrile  containing 0.1% (v/v) trifluoroacetic acid. We injected 10 µL samples and separated them by gradient flow at a constant flow rate of 0.4 mL min−1. The gradient was held at 100% A for 2 min before changing to 100% B over a 12 min linear gradient, followed by a 2 min hold step at 100% B before returning to 100% A over 4 min, followed by a final re-equilibration for 5 min at 100% A. For calibration, stock solutions of OHHL and HHL  were thawed and diluted with acetonitrile to the following concentrations: 0.01, 0.05, 0.1, 0.15, 0.3, 0.5 µg mL−1.AHL extracts were passed through a Claristep 0.2 µm regenerated cellulose filter with an area of 0.7 cmThe HPLC fractions were analyzed on an API 3000/3200 Q TRAP device  equipped with an electro spray ionization probe. Mass scans were carried out in positive ion mode with the following parameters: curtain gas (30 AU), collision gas (medium), ion spray voltage (5500 V), source temperature (550 °C), ion source gas 1 (40), ion source gas 2 (50). AHLs were detected in two multiple reaction monitoring (MRM) experiments, one based upon the selective 102.1 Da lactone ring fragment shown previously to be well suited for AHL detection , and theY. enterocolitica were prepared as described in Yersinia overnight culture at the same ratio. The biosensor culture was incubated at 37 °C, shaking at 160 rpm, until an OD600 reached 0.4, at which point the culture was directly used in the bioassay. The Yersinia culture was incubated at 28 °C, shaking at 160 rpm, and was diluted to defined OD600 values once the OD600 had reached or slightly exceeded 0.05. These defined values were based on cell numbers previously selected for this study. The corresponding OD600 was estimated using the specific correlation factor (Overnight cultures of the biosensor and n factor .Similar to the method described in min was determined on the basis of Equation (3) [For evaluation of pMA biosensor functionality, a kinetic luminescence assay for five hours using standard concentrations of the synthetic OHHL AI in combination with pMA biosensor was performed a. PBS wition (3) :(3)Lummimin into corresponding regression function, the lowest detectable concentration of OHHL  could be calculated. Averaging the corresponding LoDs of three independent experiments, a LoD of 0.50 nM ± 0.29 nM OHHL could be calculated after 20 min of incubation. After further 20 min of incubation, the sensitivity had more than doubled, with a LoD of 0.21 nM ± 0.02 nM OHHL, while extension of the assay time to 60 min or 120 min produced even higher sensitivities of LoD = 0.20 nM ± 0.03 nM or 0.17 nM ± 0.05 nM, respectively.By inserting LumY. enterocolitica, producing OHHL and HHL, was cultivated for 24 h in selective CIN medium. Several samples throughout cultivation were collected for quantifying cell density and analyzing the presence of OHHL by using previously described kinetic luminescence assay. The growth curve of Y. enterocolitica is plotted in correlation with the luminescence signals obtained from the sterile filtered supernatant samples after 120 min of kinetic measurement . AHL concentrations are compared to the corresponding growth curve in As the luminescence signal could be induced by both types of AHLs, OHHL and HHL, the culture supernatants were analyzed regarding the specific AHL concentrations using LC-MS/MS. AHLs were obtained from the supernatants by liquid-liquid extraction and reconstitution in acetonitrile, achieving recoveries of 94.4 ± 9.6% for HHL and 99.2 ± 26.6% for OHHL for all processed samples . FurtheY. enterocolitica for the first time by monitoring produced OHHL. We further demonstrated that even single cell detection and reliable quantification is possible. Although the detection of small cell number requires several hours of co-cultivation, the presented method is considerably faster than classic microbiological cultivation techniques. The assay set up was successfully optimized, resulting in a stable blank signal throughout relevant duration of cultivation, facilitating the determination of a threshold value that defines the minimal luminescence signal, confirming a positive detection. With this approach, we achieved high sensitivity for detection of one initial cell in a sample after 15.3 h of co-cultivation. Hence, monitoring the luminescence signal and the time to exceed threshold also enables quantitative estimation of the initial cell concentration on the basis of the calibration curve plotted in 600 and cells per milliliter. The quantitative detection range is limited by high cell numbers, where autoinducer concentration is already high enough to induce expression of the lux operon. In this case, expression will start instantly, and strong luminescence signal increase can be observed within few minutes, as can be concluded from kinetics in Y. enterocolitica, further optimization has to be done, especially excluding luminescence signals induced by AIs from other bacteria. It is known that OHHL and similar AHLs that also might stimulate the lux expression cassette in the pMA biosensor are produced by a huge number of Enterobacteria. Therefore, a short pre-enrichment incubation step  in CIN selection media might be beneficial to reduce the number of other bacteria and to mainly enrich Y. enterocolitica. However, CIN medium is not 100% selective to Y. enterocolitica, and therefore additional steps such as, e.g., an alkali treatment, could be necessary to increase selectivity [Salmonella, Campylobacter, or Legionella. Exchanging the luminescence gene by fluorescence cassettes expressing different fluorophores might additionally enable multiplex detection of different bacteria in a single sample.To date, several approaches have been developed using bacterial biosensors to detect and quantitatively estimate different kinds of bacteria on the basis of their secreted autoinducers . Howeverectivity ,47. Henc


The formed deferoxamine-loaded hybrid materialswere characterized with several analytical techniques. Their adsorptiveproperties toward Fe(III) ions in aqueous samples, including pH-dependence,isothermal, kinetic, and thermodynamic experiments, were investigated.The materials were described with high values of maximal adsorptioncapacity qm, which varied between 87.41and 140.65 mg g–1, indicating the high adsorptivepotential of the DFO-functionalized materials. The adsorption processeswere also described as intense, endothermic, and spontaneous. Moreover,an exemplary magnetically active deferoxamine-modified material hasbeen proven for competitive in vitro binding of ferricions from the biological complex protoporphyrin IX–Fe(III),which may lead to a further examination of the materials’ biologicalor medical applicability.Deferoxamine (DFO)is one of the most potent iron ion complexingagent belonging to a class of trihydroxamic acids. The extremely highstability constant of the DFO–Fe complex (log β = 30.6)prompts the use of deferoxamine as a targeted receptor for scavengingFe(III) ions. The following study aimed at deferoxamine immobilizationon three different supports: poly(methyl vinyl ether- Several health disordersmay lead to an increased level of nontransferrin bound iron (NTBI),which might accumulate in healthy tissues, causing several dysfunctions,such as cardiac, hepatic, or pancreatic diseases. Burst release ofiron also occurs during subarachnoid hemorrhage (a devastating subtypeof stroke), which leads to hemoglobin breakdown, causing serious oxidativeinjuries and neuronal death. Moreover, iron excess promotes the formationof reactive oxygen species (ROS), which may oxidize various cell componentssuch as lipid membranes, nucleic acids, or protein. Iron overloadmay also trigger a more rapid proliferation of iron-demanding cancercells.Streptomyces pilosus. Deferoxamine as a hexadentate molecule coordinates iron ions ina ratio of 1:1 with an extremely high stability constant β ata level of 4.0 × 1030. Its complexes with other metalions are formed with much lower stability constants. This propertyhas prompted deferoxamine-mediated ion overload treatment for manyyears of clinical therapy.7 Moreover, free deferoxamine exhibits beneficial therapeuticeffects, such as antifibrotic effects, protection against acetaminophen-inducedliver injuries, or inhibition of neurodegenerative Alzheimer’sand Huntington’s diseases.11Among many classes of domains responsible foreffective chelationof metal ions, siderophores are the ones that bind iron selectivelyor exhibit extremely high binding constants. Deferoxamine (DFO) isa siderophore belonging to a class of trihydroxamic acids and is naturallysecreted by bacterium species in vivo absorptionto the gut, rapid renal excretion causing short plasma half-time,and sunlight hypersensitivity, which leads to enhanced productionof ROS.12 Thus, several immobilization and functionalizationapproaches to incorporate different DFO formulations for analyticaland biochemical applications have been investigated. Good pharmacokineticparameters and improved bioapplicability were proven for deferoxamineconjugates with various adamantane derivatives,13 reverse emulsion nanogels containing DFO and glycine,14 a poly membranemodified with DFO,16 or a synthesized c(RGDfK)–DFO–89Zr system.18Deferoxamine is being usedin clinical treatment; however, itsapplication is limited due to its poor 20 Sepharose gel,21 or filtration paper Whatman,22 leadingto biocompatible materials for direct Fe(III) sensing in aqueous orbiological samples, using classic analytical techniques. Interestingly,a few reports aimed at application of a new approach for quantificationof the amount of ferric ions chelated by DFO-functionalized materials,which involved a detection of Fe–O band signal intensitiesin FT-IR or surface-enhanced Raman scattering (SERS) spectra.25DFO properties has alsoprompted a design of functional materialsdedicated to adsorption or sensing of Fe(III) ions. The implementedDFO-functionalized materials were based on, e.g., mesoporous silicaMCM-41,alt-maleic anhydride) (PMVEAMA), silica microparticles,and magnetite nanoparticles, which were implemented as ferric ionscavengers. The characterized materials were subjected to studiesof their adsorptive properties toward Fe(III) ions, including a sequenceof pH-dependence, isothermal, kinetic, and thermodynamic studies.The comprehensive studies led to several parameters describing thematerials’ applicability for the metal binding, such as themost effective adsorption environment, materials’ adsorptioncapacities, rates of the adsorbate binding, or thermal coefficients.Moreover, the exemplary magnetite-based material was considered forcompetitive chelation of ferric ions from a biological complex ofprotoporphyrin IX (PPIX) and Fe(III) ions, which corresponds to anaturally occurring complex – hemin. The description of anadsorptive potential of the materials and characterization of their in vitro application toward competitive chelation of ferricions may lead to a new class of eco-friendly and biocompatible adsorbentsfinding application in biomedical science.The following research aimed to synthesize a series of deferoxamine-functionalizedhybrid materials based on three different supports: poly ions from aqueoussolutions to establish an influence of the support used on the adsorptiveproperties of the materials. Predominantly, the matrices’ sizeand functionalization potential would have had the most impact onthe materials’ adsorption efficiency, which has been investigatedin the following article.2.1alt-maleic anhydride) (PMVEAMA), commerciallyavailable amorphous silica microparticles functionalized with surfaceisocyanate and maleimide groups, and synthesized Fe3O4 nanoparticles encapsulated within the silica matrix, whichunderwent functionalization with deferoxamine via isocyanate–and maleimide–silyl linkers. The functionalization strategywas based on a reaction between a terminal free amine group of deferoxaminewith reactive pendant groups on the supports’ surface. Functionalizationof PMVEAMA was afforded by maleic anhydride ring opening at elevatedtemperature under nucleophilic attack of the deferoxamine amine group.For both silica and Fe3O4, the attachment of deferoxamine was performed either by amine groupaddition to a highly electrophilic carbon atom of pendant isocyanateor by Michael addition of the amine group to carbon–carbondouble bond of the maleimide ring. Accordingly, five hybrid materialswere obtained, which structures are collected in Figure S1.The designed adsorbents consisted of three differentsupports, which were biocompatible polymeric chains of poly(methylvinyl ether-3O4 nanoparticles wereobtained bycoprecipitation from an aqueous solution containing Fe(III):Fe(II)salts in a ratio of 2:1 under alkaline conditions (pH ∼10).26 The synthesized magnetite nanoparticles weresubsequently covered with a SiO2 layer, which was achievedby condensation of tetraethyl orthosilicate (TEOS) under alkalineconditions in a water/ethanol mixture. The obtained Fe3O4/SiO2 particles were then treated as a startingmaterial for obtaining the Fe3O4-based hybridmaterials. An introduction of the deferoxamine domain onto Fe3O4/SiO2 platform was achieved throughtwo different linkers: isocyanate– and maleimide–silyllinkers. The isocyanate linker was reacted with a solution of deferoxaminein DMF under a N2 atmosphere, and the resulting silyl derivativeof deferoxamine was incorporated into the silica matrix of Fe3O4/SiO2 material . The pre-synthesized maleimide linker was anchored to a magnetite-based support, prior to reaction withdeferoxamine, obtaining material 3b. In the case of PMVEAMA–deferoxamine preparation, a suspension of PMVEAMA intoluene was added to a solution of deferoxamine in DMF at temperatureof ∼110 °C, which led to a full opening of maleic ringsin the polymer chain. Moreover, silica-based materials were synthesizedby adding isocyanate- or maleimide-functionalized silica particlesto deferoxamine solution in DMF at room temperature, yielding materials 2a and 2b, respectively.The Fe2.2–1 (ν1), which is related to N–OH stretching, specific for DFOstructure.27 Such a signal is visible onlyin the spectrum of 1, which is due to its overlappingby a broad band originating from Si–O–Si stretchingof the silica matrix in each of the other materials. Nevertheless,signals at approximately 1570 and 1640 cm–1  andC=O(amide), respectively) undoubtedly prove theformation and incorporation of amide bonds, and thus the presenceof deferoxamine domains. Moreover, two bands at around 2855 and 2930cm–1  are related to C–H stretching of methylenegroups present in the deferoxamine structure. A signal at approximately1705 cm–1 (ν4) on the spectra of 1, 2b, and 3b may be attributedto the remaining unmodified domains, such as C=O stretchingof maleic anhydride of PMVEAMA or C=C stretching of the maleimidering in materials 2b and 3b. Each of thematerials was also characterized using thermogravimetric measurements. Nevertheless, a signal at around 35.5° appears to benarrower in the 3a and 3b spectra than forbare Fe3O4, implying the material size increase.Signals at approximately 30.2° are slightly wider for the spectraof the functionalized hybrid materials, which is caused by the overlappingof Fe3O4 and SiO2 reflexes, bothappearing at around 30.2°. Moreover, the hybrid materials’spectra show a tiny reflex at around 36.4°, indicating the neworgano-derivative XRD signal. On the basis of the positions of signalsand their full width at half-maximum (FWHM) values, the mean sizeof the characterized materials hklD wascalculated using the Scherrer equation, which mathematical expressionis given below, where k is the Scherrer constant[−], λ is the wavelength of X-ray irradiation [nm], B is the FWHM value [rad], and 2θ is the signal position[°]:3O4 nanoparticles was 15.39 nm, while themean sizes of materials 3a and 3b were 18.31and 18.92 nm, respectively, which indicates a proper silyl–deferoxaminegrafting, leading to the particles’ size increase.The resultsof XRD analysis of magnetite nanoparticle-based materials 2-based materials, the size of the particles isapproximately 50 μm, which is connected with the polymeric characterof the material’s 1 support, as well as with thesize of bare silica particles used for the preparation of materials 2a and 2b, which was between 40 and 63 μm.Therefore, the size of the silica particles after functionalizationwith DFO residues might have insignificantly increased. The obtainedmaterials, which are not based on the magnetite core, were also characterizedwith EDX–SEM after their treatment with Fe(III) ions. The Fe-mappingis presented in 3O4-based hybrid materials,which show the nanometric size of round-shaped particles of the deferoxamine-modifiedmaterials. The synthesized Fe3O4 nanoparticlesof 15.39 nm as a magnetically active support did not significantlyincrease after encapsulationwithin the silica matrix and DFO conjugation, which is an importantissue for nanomaterials applied as adsorbents.Allthe obtained deferoxamine-loaded hybrid materials were alsosubjected to visualization using the SEM technique 3A–E. For3a, as an exampleof the synthesized hybrid material. The used methods may highlightthe differences between the porous features of the deferoxamine-loadedmaterial before and after Fe adsorption. The molecular receptor chelatesthe cations by wrapping around them; therefore, it may influence thematerials’ pore sizes and surface area. The porous propertiesof both material 3a and Fe-loaded material 3a were determined using N2 adsorption–desorptionanalysis, which is presented in Figure S3. The shapes of the isotherms for both bare and iron-loaded material 3a can be classified as type IV, which postulates capillarycondensation of the adsorbed gas in small pores at pressures belowthe saturation pressure of the gas. Therefore, based on the isotherms’shapes, the material meets the criteria of mesoporosity.28 The material’s mesoporosity was alsoproven by BJH calculation during adsorption and desorption of nitrogen,which responded in the pore sizes of 3a and 3a–Fe(III) of 11.370 and 11.407 nm, respectively, calculated based on the adsorptioncurves, and 13.186 and 13.174 nm, respectively, for the desorptioncurves. Moreover, the volume of pores was established with mean valuesof 0.234 cm3 g–1 for material 3a and 0.239 cm3 g–1 for Fe-loaded material 3a. The surface areas of both materials were established usingthe BJH method, which gave 68.3 m2 g–1 for material 3a and 66.7 m2 g–1 for material 3a with chelated Fe(III) ions. All theparameters calculated for the two types of materials are very similar,with no drastic differences, which indicates that the formed deferoxamine–Fe(III)complex on the material’s surface has no significant impacton the porosity. Moreover, the second synthesized magnetite-basedmaterial  was characterized for its porousfeatures using the same analytical methods. The material was describedwith its pore sizes of 11.402 and 13.079 nm calculated from the adsorptionand desorption curves, respectively. Also, the surface area was calculatedto be 68.3 m2 g–1 and the mean pore volumewas calculated to be 0.279 cm3 g–1, whichjointly indicate the similarity of both obtained materials containingthe Fe3O4 core. Additionally, the pore sizedistributions established for the pristine magnetic materials andthe one complexed with Fe(III) ions are presented in Figure S4. The distribution profiles obtained for materials 3a and 3b are almost overlapped, while the poresize distribution of the material 3a–Fe(III) complexexhibits only insignificant change, which is consistent with the presentedBET analysis.The chelation of Fe(III) ions by deferoxamine domains may alsoinfluence the surface and the size of the pores of the materials.Thus, the most widely used techniques, which allow for assessmentof structural features of solids – the BET  isotherm and the BJH  method– were implemented for material m/z 561.5), but only the signals correspondingto the components of the buffer used; therefore, the materials’stability can be concluded.All the materials were tested for their stabilityin paraphysiologicalconditions of phosphate-buffered saline (PBS). After the incubation,the solutes were analyzed using ESI–MS in order to investigatewhether deferoxamine dissociates from the materials under the conditionsmimicking the biological environment. The spectra of the solutes showedno signals referring to the dissociated deferoxamine residue  or Fe(II) ions are presented in Figure S5b,c, respectively. Two signals corresponding to mono-and diprotonated complexes are visible at 614.4 and 307.7 m/z, respectively. Moreover, the signalpresent at 561.5 m/z related tofree deferoxamine can be a result of electrospray ionization mode,which leads to easier fragmentation. Interestingly, the signal at561.5 m/z is significantly lowerfor Fe(III)-complex than for Fe(II)-complex, which highlights thehigher affinity of ferric ions toward the formation of DFO–ironcomplexes. The choice of different supports for anchoring deferoxamine can also lead to conclusionson their influence on the final adsorptive properties. To fully characterizethe materials’ sorptive nature, several experiments were carriedout, including isothermal, kinetic, and thermodynamic studies.The synthesized materials weredesigned as chelating systems dedicated to Fe(III) ions since surface-introduceddeferoxamine exhibits high binding efficiency. The formation of thedeferoxamine–Fe complex was proven by ESI–MS measurements,which spectra are presented in 2.3.13 in more basic conditions for 5 mM solution,as well as in 5 mM solution in distilled water, which was characterizedto be of pH 2.45 ,Fe is the ferric ion, and H is the proton.29 Therefore, the adsorption may be limited only due to repulsive interactionsbetween excessive H+ and Fe3+ ions visible atpH 1. At the most acidic environment studied, the adsorption ratesreached 38–62% of the maximal adsorption capacity under thegiven conditions. The adsorption rates increased with increasing pH,reaching maxima at pH 2.45, which corresponds to Fe(III) solutionin pure distilled water. Then, qeq valuesslightly decreased, primarily due to the use of sodium salts as buffers’ingredients, leading to the competitive binding of Na+ ions.Nevertheless, the decrease is not drastic, ranging between 21.7 and30.8%.2.3.230 Graphical representations of the Langmuir and the Freundlichisotherms are presented in Figure S6, respectively,while the calculated parameters for both isothermal models are collectedin Theexperimental data obtained for adsorption isotherms were fittedto the Langmuir and the Freundlich models. The first model assumesa formation of the adsorbate monolayer on the adsorbent surface, whichis due to the equal binding efficiency of all the binding sites andneglecting the interactions between adsorbed molecules, while thelatter is mostly based on the assumption that adsorbate moleculesmay interact with each other via electrostatic, hydrogen, or π–πinteractions, forming the adsorbate multilayer.R2 value higher (≥0.997) and χ2 values lower (≤0.120) than those for the Freundlichmodel. This is consistent with the chemical nature of ferric ions,which hinders the intermolecular interactions, leading to the formationof the adsorbate monolayer. On the basis of the Langmuir fitting,the values of maximal adsorption capacity of the materials towardFe(III) were established. The qmax valuesvaried between 87.41 and 140.65 mg g–1, reachingthe highest values for SiO2 and Fe3O4/SiO2 particles conjugated with deferoxamine through themaleimide linker. Such a phenomenon might be a result of additionaliron ion trapping within a cyclic domain of maleimide. Nevertheless,satisfactory results were obtained for the other materials based onPMVEAMA and silica or SPIONs functionalized through the isocyanatelinker. Although the experimental data are not described preferablywith the Freundlich model, the values of 1/n constants connected with the intensity of the adsorptionprocess and heterogeneity of the adsorbent’s surface were calculated.The lower the 1/n value, the moreintense the adsorption process. For all the materials, the valuesranged between 0.56 and 0.79, indicating the efficiency of adsorptionprocesses.Undoubtedly, adsorption of Fe(III) ions on each hybrid materialfollows the Langmuir model, which is proven by the calculated 2.3.3R2 values for the pseudo–second–order kineticmodel , it is easily shown that the adsorption experiment follows the pseudo–second–orderkinetic model. This result implies that the formation of various interactionsbetween the adsorbent and analytes  is the adsorptionrate-limiting step.31 Accordingly, theadsorption of Fe(III) ions on the hybrid materials is limited by theircoordination by deferoxamine residues on the materials’ surface.The modeling allowed for the calculation of the initial metal ionadsorption rate, which appeared to be the highest for magnetite-basedparticles and the lowest for silica-based particles. Thus, the timeneeded for half-adsorption should be opposite to ik values, which is proven by the lowest t1/2 values for materials 3a and 3b, while the highest for materials 2a and 2b. In order to verify the physical nature of the rate-limitingstep, the intraparticle diffusion model was implemented to fit theexperimental data, in which the plot tq vs ln t may form a multilinear plot, as shown in 32 The presented plots show two separate phases;thus, two factors limit the adsorption rate within the adsorptionprogress. The first one is connected with an initial surface diffusion,while the second one is based on gradual adsorption limited by eitherintraparticle or pore diffusion. Moreover, the experimental kineticdata were fitted to the Elovich model  in order to demonstrate whether adsorption of ferric ionson the hybrid materials may include the heterogeneous diffusion processas the rate-limiting step. However, due to the relatively low R2 values calculated for this model (Table S1), such findings would be deniable.The obtained experimental data for the kineticstudies of Fe(III)adsorption on the synthesized hybrid materials were fitted to pseudo–first–orderand pseudo–second–order kinetics, intraparticle diffusiontheory, and the Elovich model. The highest linear correlation of theexperimental data was achieved for the pseudo–second–orderkinetic model 3, which icmodel 3 andthe2.3.4Kd vs 1/T are presentedin H°) and entropy (ΔS°) andGibbs free energy values (ΔG°) for experimentsconducted under the given thermal conditions. The parameters are collectedin Thermodynamic studies involved measurements ofthe amounts of metalions absorbed on the deferoxamine-functionalized particles in equilibriumstates (after 24 h incubation) in three different temperatures: 298,313, and 328 K. The obtained experimental data were fitted to thevan’t Hoff equation, which linear plots lnR2, which values were higher than 0.9870, andextremely low χ2 coefficients, indicating the goodlinearity of the plots. Adsorption of Fe(III) ions on the deferoxamine-loadedmaterials was found to be an endoenergetic process , which is more intense withthe temperature increase – values of Gibbs free energies forhigher temperature are more negative, and thus, the process is intensified.Interestingly, the established parameters are directly connected withthe ones obtained during kinetic studies. The silica-based materials 2a and 2b can be described as the least influencedby the temperature increase and the ones that exhibit the lowest adsorptionrate constants k2 and ik and the highest values of time needed for adsorption ofhalf-equilibrium adsorbate (t1/2). Moreover,for all the materials, entropy values are relatively high, which suggestthat an increased randomness mostly drives the metal ion adsorptionat the solution–adsorbent interface related to solvation effects.Such conclusions are rather evident, considering that Fe(III) adsorptionis based only on the formation of non-covalent coordination bondsbetween the receptor and analyte.All the plots werecharacterized with high correlation coefficients 2.3.52a and 2b. The materials wereincubated in three two- or three-component mixtures containing Fe(III),Al(III), and/or Cr(III) ions. The percentages of the metals adsorbedon the hybrid materials were determined using X-ray fluorescence (XRF)measurements  of the material–ioncomplexes, which are collected in The influence of an additionaltrivalent ion presence on the adsorptionselectivity toward ferric ions has been assessed using exemplary silica-basedmaterials 2.43a was investigatedfor competitive complexation of Fe(III) ions from the protoporphyrinIX–Fe(III) complex named hemin. PPIX–Fe complex formationand its interaction with Fe3O4–SiO2–NCO–DFO were monitored using electrospray-ionizationmass spectrometry (ESI–MS) analysis in positive mode, and thecorresponding spectra are presented in m/z appearing in the spectrum in positive mode, whichcorresponds to the protonated [(PPIX + Fe–2H + 2Na + H2O) + H]+ form and its sodium adduct, respectively,as well as their bicharged forms at 340.4 and 351.4 m/z. Also, the signal at 359.4 m/z is related to the bicharged protonated sodiumadduct of the [(PPIX + Fe + Cl–2H + 2Na) + H + Na]2+ form. The similar trend of the signals distances as for 340.4, 351.4,and 359.4 m/z is visible for thesignals at 453.5, 464.5, and 472.5 m/z, indicating PPIX–Fe complex adducts. Although the spectrumexhibits mono- and bicharged signals corresponding to free porphyrindomains not complexing Fe(III) ions , theirintensity is relatively low, which proves high Fe(III)-complexingefficiency. The obtained PPIX–Fe complex treated with an excessof deferoxamine led to a complete transfer of Fe(III) ions to theDFO domain, which is proven by both the appearance of a signal at614.4 m/z [DFO–2H + Fe]+ and the disappearance of signals corresponding to the PPIX+ Fe complex in m/z and a signalof free deferoxamine at 561.5 m/z caused by its excessive usage in the experiment, which proves thecompetitive extraction of iron ions by the studied siderophore. Theseresults have prompted examining the hybrid materials’ potentialfor competitive chelation of ferric ions, which was conducted usingmaterial 3a as an exemplary scavenger. After a very shortincubation of the material in the PPIX–Fe complex solution,the aqueous phase was analyzed, the spectrum of which is given in m/z correspondsto DFO–Fe complex, which is a consequence of a nanosized characterof the material used, not fully separable within the short time ofmagnetic separation, and thus getting to the ionization source. Nevertheless,it confirmed a very efficient competitive binding of Fe(III) ionswithin the deferoxamine-loaded material’s matrix, leaving PPIXuncomplexed. Similar results were observed for the samples containingmaterial 3a incubated in a series of buffer solutions,which were citric acid/sodium hydrogen phosphate buffers of pH valuesranging between 3 and 8, and phosphate-buffered saline (PBS) of pH7.4. The choice of such conditions was triggered by the differentpH of fluids in human organisms. The corresponding ESI–MS spectrapresented the signals originating from the ingredients of buffersand the signal referring to the complex of DFO and Fe(III) ions, asa pending organic domain on the not fully separated adsorptive nanoparticles.Therefore, the exemplary material 3a was proven for efficientferric ion transfer from its PPIX complex in a wide range of aqueousenvironments.The synthesized hybrid materials contain biologicallycompatible platforms and exhibit very promising Fe-adsorptive properties;they can find application in the treatment of diseases caused by long-termor sudden burst release of iron ions in human organisms. The exemplarydeferoxamine-loaded hybrid material 3O4 nanoparticles, which have already been proven as valuabletargeted drug- or energy-transporting platforms. One of the reasonsfor their high in vitro and/or in vivo applicability is their nanosize allowing for an enhanced circulationwithin the bloodstream. Also, the paramagnetic character of Fe3O4 nanoparticles allows for targeted transportbased on the three-dimensional concentration of the particles usingan external point or rotating magnetic field.36 Not only such features lead toa directed transport of drugs to target sites , improving the selectivity of the therapeutic effect,but also Fe3O4-based systems exhibit efficiencyin hyperthermia treatment, thanks to the possibility of heat generationat the specific organism site.37 The paramagneticfeatures of magnetite-based materials functionalized with deferoxaminewere examined using materials 3a and 3b. Figure S9 presents the concentration of the particlesusing a neodymium magnet in two different media , which afford physiological or paraphysiologicalconditions. The particles are easily concentrated with the magneteven at a distance between the magnet and the sample of 5 cm, whichindicates the easy directing of the particles using the external magneticfield. Therefore, the magnetite-based materials can find applicationas iron ion scavengers at targeted organism sites.Very satisfactory results of direct or competitivecomplexing offerric ions may lead to the materials’ further biological applications,especially the materials based on magnetically active Fe3–1, which correspond to 1.56to 2.52 mmol g–1. The highest values were obtainedfor magnetite-based materials 3a and 3b,which might be related to the use of a nanosized support. Moreover,material 3a, as a magnetically active exemplary material,was studied for competitive binding of Fe(III) ions from their complexwith PPIX, corresponding to hemin – an iron-containing porphyrinfound in human blood. The material exhibited high effectiveness, which,jointly with its ability for being magnetically directed, may leadto further biological application in the treatment of hematologicdiseases.The recent study presents the synthesis of deferoxamine-loadedhybrid materials using three different supports. The obtained materialswere characterized with several analytical techniques, involving thecharacterization of raw hybrid materials and their complexes withFe(III) ions. The materials were subjected to various adsorption studies,which responded in the comprehensive characterization of the materials’adsorptive potential toward ferric ions. All the materials showedhigh effectiveness in the adsorbate binding, reaching adsorption capacitiesof 87.41 to 140.65 mg g44.1alt-maleic anhydride) of average Mw ∼216,000Da and Mn ∼80,000 Da; tetraethylorthosilicate ≥99.0%, 3-(triethoxysilyl)propyl isocyanate 95%;(3-aminopropyl)triethoxysilane 97%; maleic anhydride 99%; 1,1,1,3,3,3-hexamethyldisilazanefor synthesis ≥98%; ZnCl2 ≥98%, anhydrous;Fe(ClO4)3·xH2O, low chloride; protoporphyrin IX (PPIX) ≥95%; human serumfrom human male AB plasma, USA origin, sterile-filtered). Silica modifiedwith surface isocyanate and maleimide groups was obtained from SiliCycleInc.  and characterized as follows: SiO2–maleimide  and SiO2–isocyanate .The other substances were of purity grade p.a. and obtained from POCH ,Stanlab  , and EUROCHEM  .Moreover, (NH4)2Fe(SO4)2·6H2O was supplied by Aktyn ,and DMSO was purchased from Merck , which wasdried over molecular sieves 4 Å prior to its use.The majority of the reagentswere obtained from Sigma Aldrich   operating between 20 and 1000 °Cwith a heating rate set for 5 °C min–1. Thesamples were heated in an airstream. The quantities of C, H, and Ncontents in the hybrid materials were calculated based on the elementalanalysis performed in an Elementar Vario EL III analyzer . The obtained DFO-loaded materials were also visualized usingan FEI Quanta FEG 250  scanning electron microscope(SEM) operating in a high vacuum condition of 70 Pa. The instrumentused an accelerating voltage of 10 kV and a working distance varyingbetween 9.9 and 10.4 mm. The SEM images were obtained with magnitudesof 2000× for materials 1, 2a, and 2b and of 100,000× for Fe3O4-basedmaterials 3a and 3b. Moreover, for non-magnetite-basedmaterials, energy dispersive X-ray spectroscopic (EDX) imaging wasperformed. The magnetite, magnetite–silica, and magnetite-basedhybrid materials were also characterized using a Bruker D8 Advance powder diffractometer (XRD). The apparatus usedCu Kα1 X-ray energy of wavelength λ of 1.5406 Å (Johanssontype) and worked in a high-angle mode in the 2θ range between6 and 60°. Also, magnetite-based materials 3a and 3b and the exemplary complex of material 3a withadsorbed Fe(III) ions were characterized with the Brunauer–Emmet–Teller(BET) isotherm performed using a Quantachrome Autosorb iQ . The samples were outgassed for 12 h at 100 °C priorto their analysis for nitrogen adsorption and desorption at a temperatureof 77.35 K. According to the experimental data, the samples’surface areas were established using the BET and Barrett–Joyner–Halenda(BJH) methods. The range of relative pressure p/0p used for the measurements was from 0.0 to1.0, where the applicability of the methods is from 0.05 to 0.3 andfrom 0.1 to 1.0 for the BET and BJH methods, respectively.Characterization of materialsinvolved using several analytical techniques. The FT-IR spectra ofdeferoxamine-loaded hybrid materials were recorded on a Bruker IFS66v/S  spectroscope operating in the wavelength rangebetween 400 and 4000 cm–1 with a resolution of 1 cm–1. The samples were placed in a poly(methyl methacrylate)(PMMA) cuvette , and the spectra wererecorded in triplicate in order to avoid any disturbances. The competitivenessin trivalent ion binding by materials 2a and 2b was monitored using a MiniPal2 X-ray spectrofluorometer (XRF) suppliedby Malvern PANalytical B.V.  equipped with arhodium vacuum tube as a source of X-rays. The analyses were performedfor 200 s with an X-ray tube voltage of 13 kV and automatically adoptedcurrent, which varied between 18 and 30 μA. Moreover, the measurementswere conducted using a no element-excluding filter.Theprogress of Fe(III) adsorption on the DFO-loaded hybrid materialswas monitored by UV–Vis assays using an Agilent 8453 spectrophotometer, operating in the range of wavelengths between200 and 1000 cm3a from a preparedPPIX–Fe (hemin) complex were monitored using an amaZon SL iontrap Bruker  mass spectrometer with an electrosprayionization source (ESI–MS). The samples were injected intothe ionization source at a flow rate of 10 μL min–1 by a syringe pump. The spectrometer worked in a so-called “enhancedresolution mode” and a detection range between 100 and 1000 m/z. The desolvating gas (N2) flowed at a rate of 800 L h–1, while the conegas (He) flowed at a rate of 50 L h–1. The voltageswere set at −4.5 and −0.5 kV for the capillary and theendplate offset, respectively.The materials’stability and the competitive binding ofFe(III) ions by the chosen material 4.34.3.12) and heatedto 110 °C. Under a nitrogen atmosphere, a solution of PMVEAMA(0.4 g) in 30 mL of toluene:DMF mixture  was added dropwisethrough a dropping funnel, with subsequent heating and mixing for16 h. Afterward, a cooled solution was treated with Et2O to obtain the brown precipitate. The crude product was dissolvedin EtOH and recrystallized with Et2O, yielding 1.16 g (93.5%)of material 1.A solutionof deferoxamine mesylate  in 30 mL of DMF was placedin a three-neck round-bottom flask and charged with a reflux condenser.The solution was purged with inert gas (N1: FT-IR (KBr)cm–1: 3399 , 2930 (ν C–H asym stretch), 2859 (ν C–Hsym stretch), 1769 (ν C=O anhydride ring stretch), 1700(ν C=O carboxylic acid stretch), 1654 (ν C=Oamide stretch), 1558 (ν N–H amide bend), 1438 (νO–H bend), 1193 (ν C–O stretch), 1051 (νN–OH stretch), 785 (ν N–H bend).Material 4.3.22a or material 2b, respectively. Afterward, the warm mixture was filteredoff, and the solid was washed with DMF (30 mL) and DCM (25 mL), obtainingwhite material 2a and yellow material 2b.Theanchoring of deferoxamine on the silica modified with either isocyanateor maleimide groups was based on the same synthetic protocol. A solutionof deferoxamine mesylate  in 40 mL of DMF was heatedto ∼75 °C and purged with nitrogen. Then, 2 g of isocyanate-or maleimide-modified was added in a few portions. Mixing under aninert atmosphere at elevated temperature was continued for 2 h forisocyanate-functionalized silica particles or 5 h for maleimide-functionalizedsilica particles, leading to material 2a: FT-IR (KBr) cm–1: 3427 , 2939 (νC–H asym stretch), 2891 (ν C–H sym stretch), 1647(ν C=O amide stretch), 1572 (ν N–H amidebend), 1092 , 805(ν Si–O–Si asym stretch), 467 (ν Si–O–Sibend).Material 2b: FT-IR (KBr) cm–1: 3447, 2926 (ν C–Hasym stretch), 2854 (ν C–H sym stretch), 1709 , 1655 (ν C=O amide stretch),1414 (ν O–H bend), 1094 , 958 (ν Si–OH bend), 803 (ν Si–O–Siasym stretch), 698 , 467 (νSi–O–Si bend).Material 4.3.34.3.3.13·6H2O and (NH4)2Fe(SO4)2·6H2O  in 400 mL of distilled water in a three-neckround-bottom flask was purged with N2. During mixing andconstant purging with the inert gas, a solution of 30 mL of NH4OH in 20 mL of distilled water was added dropwise with immediateprecipitation of magnetite. After ammonia was added, the mixture wasstirred for 1 h. The precipitate was collected using an external neodymiummagnet, then washed two times with distilled water (50 mL) and twotimes with ethanol (50 mL), and dried under vacuum at 50 °C for8 h, yielding magnetic nanoparticles . The obtainedFe3O4 nanoparticles were further encapsulatedwithin the silica matrix using the standard Stöber method.The dried magnetite nanoparticles were dispersed in 300 mL of H2O:EtOH mixture  on an ultrasound bath at room temperature.Next, 30 mL of ammonia was added. During continuous stirring, a solutionof TEOS  in 20 mL of EtOH was added dropwise.The mixture stayed in the ultrasound bath for 3 h, obtaining darkbrown particles. Afterward, the solid was separated using the magnet,washed two times with distilled water (50 mL) and two times with ethanol(50 mL), and dried under vacuum at 50 °C for 8 h, yielding Fe3O4–SiO2 platform.Magneticiron oxide was obtained using the standard coprecipitation method. An aqueoussolution of FeCl4.3.3.23O4–SiO2 particles was added in a few portions. Thesilyl-derivative incorporation into the silica matrix covering themagnetite core was carried out for 16 h at room temperature underan inert gas atmosphere (N2). The resulting deferoxamine-modifiedmagnetite-based hybrid material was separated with a magnet, washedtwo times with EtOH (20 mL) and two times with DCM (20 mL), and thendried under vacuum at 50 °C for 8 h. The resulting dark brownparticles were assigned as material 3a.Toa solution of deferoxamine mesylate  in 50 mL ofanhydrous DMSO placed in the ultrasound bath, (3-isocyanatopropyl)triethoxysilane was added. After mixture stirring for 2 hunder an inert gas atmosphere, 2.4 g of Fe3a: FT-IR (KBr) cm–1: 3384 , 2918 (ν C–H asym stretch),2855 (ν C–H sym stretch), 1634 (ν C=O amidestretch), 1435 (ν O–H bend), 1018 (ν Si–O–Sisym stretch), 953 (ν Si–OH bend), 795 (ν Si–O–Siasym stretch), 582 (ν Fe–O stretch).Material 4.3.3.33O4–SiO2 surface, which then underwent functionalization with deferoxamine.The maleimide-derivative was obtained in a three-step process: (1)To a solution of maleic anhydride  in 60 mL of anhydrousDMSO, 3-aminopropyltriethoxysilane  was added.The ring-opening process was handled under continuous stirring for2 h at room temperature. (2) Then, the mixture was heated to 80 °Cin an oil bath, and a solution of hexamethyldisilazane  in a DMSO:toluene mixture  and ZnCl2  were added. The reaction mixture was furtherstirred for 5 h at 80 °C. (3) Afterward, the mixture was cooledto room temperature, and then the unreacted reagents were extractedwith cold Et2O (3 × 75 mL). The obtained DMSO solutionof the silane maleimide-derivative was poured to Fe3O4–SiO2 (2.4 g) suspension dispersed in 40mL of anhydrous DMSO. The silane binding to the surface silica matrixwas continued for 16 h under a nitrogen atmosphere at room temperature.The obtained Fe3O4–SiO2–maleimideparticles were separated, washed one time with DMSO and three timeswith EtOH, and then dried under vacuum at 50 °C. The dried particleswere then poured into a solution of deferoxamine mesylate  in 50 mL of anhydrous DMSO. The reaction of deferoxaminefunctionalization in the ultrasound bath was carried out for 24 hat room temperature, under a nitrogen atmosphere. The resulting darkbrown particles were separated, washed with fresh solvents , and dried under vacuum (50 °C),obtaining material 3b.Synthesis of deferoxamine-functionalizedmagnetic particles throughthe maleimide linker was based on a synthesis of 3-maleimide-propyltriethoxysilane,with its further anchoring to Fe3b: FT-IR (KBr) cm–1: 3396 , 2930 (ν C–H asym stretch), 2855(ν C–H sym stretch), 1700 , 1630 (ν C=O amide stretch), 1577 (ν N–Hamide bend), 1404 (ν O–H bend), 1050 (ν Si–O–Sisym stretch), 797 (ν Si–O–Si asym stretch), 582(ν Fe–O stretch).Material 4.4The materials’stability in biological conditions was investigatedby incubation of 15 mg samples of each material in 20 mL of prepreparedphosphate-buffered saline (PBS), which affords paraphysiological conditions.The incubation was handled at 37 °C for 24 h with constant shaking.Afterward, the solids were centrifuged, and the solutes were injectedfor ESI–MS analysis.4.54.5.14)3·6H2O buffered in prepared solutions of pH 1 and 2 (hydrochloricacid/potassium chloride buffer), pH 3, 4, and 5 (citric acid/disodiumhydrogen phosphate), and pure distilled water. Each experiment involvedusing 10 mg of sample of each hybrid material, which was poured into10 mL of Fe(III) ion solution buffered in a particular medium. Eachsample was shaken for 24 h at room temperature. Afterward, the solidswere separated by filtration using a Schott funnel, centrifugation,or using a magnet, depending on the type of material used. The solutionswere then investigated for the amount of remaining metal ions, establishedusing UV–Vis spectrophotometric measurements (λmax = 297 nm). The amount of Fe(III) absorbed qeq on the hybrid material was calculated using the below equation,where c0 and ceq are the initial and the equilibrium concentrations of the metalused, respectively [mM], V is the volume of the solutionused [mL], m is the sample mass [mg], and M is the molar mass of the adsorbate [g mol–1].Adsorptionprocesses in different aqueous environments were performedusing 5 mM solutions of Fen1/].Isothermalstudies involved adsorption of Fe(III) ions from their perchloratesalt aqueous solution in distilled water at different concentrations:0.1, 0.5, 1, 2, 5, 10, and 20 mM. The experimental protocol and thequantification of the metal adsorbed on the materials were similarto those described in 4.5.3tq at time t, using the below equation, where tc is the concentration of the metal at time t [mM]:qe is thecalculated equilibrium amount of metal absorbed [mg g–1], k1 is the pseudo-first-order kineticsconstant [h–1], k2 isthe pseudo-second-order kinetics constant [g mg–1 h–1], kid is the intraparticlediffusion constant [mg g–1 h–1/2], Cid is the intraparticle diffusionplot intercept [mg g–1], α is the Elovichconstant [mg g–1 min–1], and βis the Elovich exponent [mg g–1]. Moreover, basedon the pseudo-first- and pseudo-second-order kinetic fitting, initialadsorption rate constants ik [mg g–1 h–1] were calculated, respectivelyt1/2 [h], which is equal to the time needed for adsorption of the halfamount of analyte adsorbed in equilibrium:The performingof kinetic studies was based on the quantification of the metal uptakefrom its aqueous solution depending on the contact time. Thus, 15mg of sample of each hybrid material was added to 20 mL of 5 mM solutionof Fe(III) ions in distilled water. The solute was collected in presettime intervals , in orderto calculate the collective amount of metal absorbed 4.5.4Kd is thedistribution coefficient [L g–1] calculated as qeq/ceq, R is the ideal gas constant (8.314 J mol–1 K–1), and ΔH° andΔS° are the standard enthalpy [J mol–1] and entropy [J mol–1 K–1], respectively, of Fe(III) adsorption on the particular hybrid material.Moreover, values of Gibbs free energies ΔG°[J mol–1 K–1] in particular conditionswere calculated:Thermodynamicstudies were based on the reaching Fe(III) adsorption equilibriumstate at three different incubation temperatures: 298, 313, and 328K. To 5 mL of ferric perchlorate aqueous solution in distilled waterof concentration 5 mM, 10 mg of sample of the hybrid material wasintroduced. Each mixture was incubated in 298, 313, or 328 K for 24h. Afterward, the amount of metal adsorbed on the hybrid materialwas calculated using the same protocol as given in 4.62a and 2b were incubated for 24 h with 10 mL samples ofthree different Fe-containing mixtures of trivalent ions . The mixtures, which contained Fe(ClO4)3, Cr(ClO4)3, and/or Al(ClO4)3 at their final concentration of 5 mM, were preparedusing distilled water as a solvent. After the incubation time, thesolids were centrifuged, the solutes were separated, and the material–ioncomplexes were dried under vacuum in the desiccator at room temperature.The dried samples were subjected to XRF analysis.A series of 50 mg samples of silica-based materials 4.73a was studied using a complex of protoporphyrin IX (PPIX) and Fe(III)ions. The complex was obtained by mixing 5 mL of a 0.1 mM solutionof PPIX in methanol with 5 mL of a 0.1 mM solution of Fe(III) ionsin distilled water for 24 h at room temperature. Afterward, 2.5 mLof the obtained PPIX–Fe complex was diluted with 2.5 mL ofH2O:MeOH (1:1) mixture, leading to the final complex concentrationof 0.025 mM, and 10 mg of 3a was purged into the solution.The mixture was shaken for 1 min, and then the material was separatedusing an external magnetic field. The formation of PPIX–Feand the progress of Fe-binding within the adsorptive material weremonitored using ESI–MS analysis. Moreover, the competitivenessstudies were also performed using pre-prepared citric acid/sodiumhydrogen phosphate buffers of pH 3, 4, 5, 6, 7, and 8 and phosphate-bufferedsaline (PBS). Briefly, 0.5 mL of a 0.5 mM solution of PPIX–Fein a mixture of H2O:MeOH (1:1) was added to 9.5 mL of thebuffers, and then 10 mg samples of 3a were added. Theincubation and analysis conditions were the same as described above.In order to investigate the competitiveness of thematerials in binding Fe(III) ions, the exemplary material