Functional upcycling of waste PET plastic to the hybrid magnetic microparticles adsorbent for cesium removal.

Accumulation of mismanaged plastic in the environment and the appearance of emerging plastic-derived pollutants such as microplastics strongly demand technologies for waste plastic utilization. In this study, polyethylene terephthalate (PET) from waste plastic bottles was directly utilized to prepare a matrix of an adsorbent for cesium (Cs+) removal. The organic matrix of PET-derived oligomers obtained by aminolysis depolymerization was impregnated with bentonite clay and magnetite nanoparticles (Fe3O4 NPs), playing the roles as a major adsorptive medium for Cs+ removal and as a functional component to primarily provide efficient separation of the hybrid adsorbent from aqueous system, respectively. The obtained hybrid composite microparticles were next tested as an adsorbent for the removal of Cs+ cation from aqueous solutions. The adsorption process was characterized by fast kinetics reaching ca. 60% of the equilibrium adsorption capacity within 5 min and the maximum adsorption capacity toward Cs+ was found to be 26.8 mg/g. The adsorption process was primarily dominated by the cationic exchange in bentonite, which was not significantly affected by the admixture of the competing mono- and divalent cations (Na+, K+, and Mg2+). The proposed approach here exploits the sustainable utilization scenario of plastic waste-derived material to template complex multifunctional nanocomposites that can find applications for pollution cleaning and environmental remediation.

Multi-color polymer carbon dots synthesized from waste polyolefins through phenylenediamine-assisted hydrothermal processing.

The large accumulation and low recycling rates of polyolefin waste have posed a threat to the environment and human health. The shortage of chemical recycling methods for polyolefins strongly demands the development of new and sustainable treatment technologies for hydrocarbon plastics to improve their waste management. In this study, polyethylene (PE) and polypropylene (PP) were utilized for the preparation of multi-color polymer carbon dots (PCDs) via a two-step hydrothermal (HT) synthesis involving (i) thermo-oxidative degradation of polyolefins to precursors containing plentiful oxygen-based functional groups, and (ii) modification with phenylenediamine (PDA). The fluorescence of PCDs depends on the structure of isomeric PDA and PCDs modified by ortho-, meta-, and para-PDA emit blue, green, and yellow color fluorescence, respectively. The formation mechanism of PCDs, involving dehydrative condensation and amination of PE or PP-derived precursors by PDA, was proposed. The obtained PCDs were utilized for the detection and quantification of Fe3+ ions at ppm concentrations. The proposed strategy here aims to broaden the scope of the chemical recycling methods for polyolefin plastic waste as well as to develop a conversion route of polyolefin to value-added materials.

Copper Ion Removal Using a Waste-Plastic-Derived Hydrogel Adsorbent Prepared via Microwave-Assisted PET Aminolysis.

Despite the tremendous progress in the development of functional materials from plastic waste to promote its recycling, only a few examples of hydrogel materials from plastic waste were reported. In this study, microwave-assisted depolymerization of waste PET plastic using polyamine was performed to prepare short aminophthalamide oligomers followed by chemically cross-linking into a hydrogel material. Catalyst-free microwave-assisted aminolysis of PET was completed within 30-40 s, demonstrating high efficiency of the depolymerization reaction. Subsequent epoxy cross-linking of the oligomers yielded a hydrogel with a swelling degree of ca. 92.1 times in pure water. The application of the obtained hydrogel for the removal of copper ions (Cu2+) from water was demonstrated. Efficient complexation of NH2 groups of the hydrogel with Cu2+ resulted in high adsorption capacities of the hydrogel material toward Cu2+ removal, which were the highest at neutral pHs and reached ca. 213 mg/g. The proposed type of environmental material is beneficial owing to its waste-derived nature and functionality that can be applied for the high-efficiency removal of a broad scope of known environmental pollutants.

Templating of catalytic gold and silver nanoparticles by waste plastic PET-derived hydrogel playing a dual role of a reductant and a matrix.

The progressive accumulation of discarded plastic in the environment demands further development of waste management of plastic waste and conversion technologies of such waste to value-added materials. Recently, the conversion of plastic waste to functional materials via chemical recycling has attracted considerable attention. In this report, plastic waste (PET) was utilized for the preparation of a hydrogel-based catalyst via a cross-linking reaction of PET-derived oligo(terephthalamide)s followed by the electroless metallization. The polymeric matrix of PET-derived hydrogel plays multiple roles of (i) an adsorption media for noble metal ions such as Au3+ and Ag+, (ii) a reducing agent of Au3+ and Ag+ ions to Au0 and Ag0, and (iii) a matrix for the controlled growth of Au and Ag nanoparticles (AuNPs and AgNPs). The obtained hybrid hydrogels after metallization contained well-dispersed AuNPs and AgNPs of 6.1 ± 3.7 nm or 6.1 ± 1.4 nm size, respectively. The catalytic activities of the hybrid hydrogels with metal nanoparticles were studied in a model system of p-nitrophenol reduction in an aqueous solution. The hybrid materials of both Au@hydrogel and Ag@hydrogel were catalytically active for the reduction of p-nitrophenol, obeying the first-order kinetics. Importantly, the AuNPs or AgNPs in the hydrogel matrix preserved the original catalytic activity after multiple p-nitrophenol reduction reactions, showing a promising reusability of the catalysts. The proposed here approach aims to broaden the scope of conversion routes of plastic waste to value-added materials as well as to develop new types of polymeric matrices for templating and growth of metal nanoparticles for catalytic applications.

Reconstituted TAD-size chromatin fibers feature heterogeneous nucleosome clusters.

Large topologically associated domains (TADs) contain irregularly spaced nucleosome clutches, and interactions between such clutches are thought to aid the compaction of these domains. Here, we reconstituted TAD-sized chromatin fibers containing hundreds of nucleosomes on native source human and lambda-phage DNA and compared their mechanical properties at the single-molecule level with shorter '601' arrays with various nucleosome repeat lengths. Fluorescent imaging showed increased compaction upon saturation of the DNA with histones and increasing magnesium concentration. Nucleosome clusters and their structural fluctuations were visualized in confined nanochannels. Force spectroscopy revealed not only similar mechanical properties of the TAD-sized fibers as shorter fibers but also large rupture events, consistent with breaking the interactions between distant clutches of nucleosomes. Though the arrays of native human DNA, lambda-phage and '601' DNA featured minor differences in reconstitution yield and nucleosome stability, the fibers' global structural and mechanical properties were similar, including the interactions between nucleosome clutches. These single-molecule experiments quantify the mechanical forces that stabilize large TAD-sized chromatin domains consisting of disordered, dynamically interacting nucleosome clutches and their effect on the condensation of large chromatin domains.

Fluorescent Nanoparticles Synthesized from DNA, RNA, and Nucleotides.

Ubiquitous on Earth, DNA and other nucleic acids are being increasingly considered as promising biomass resources. Due to their unique chemical structure, which is different from that of more common carbohydrate biomass polymers, materials based on nucleic acids may exhibit new, attractive characteristics. In this study, fluorescent nanoparticles (biodots) were prepared by a hydrothermal (HT) method from various nucleic acids (DNA, RNA, nucleotides, and nucleosides) to establish the relationship between the structure of precursors and fluorescent properties of biodots and to optimize conditions for preparation of the most fluorescent product. HT treatment of nucleic acids results in decomposition of sugar moieties and depurination/depyrimidation of nucleobases, while their consequent condensation and polymerization gives fluorescent nanoparticles. Fluorescent properties of DNA and RNA biodots are drastically different from biodots synthesized from individual nucleotides. In particular, biodots synthesized from purine-containing nucleotides or nucleosides show up to 50-fold higher fluorescence compared to analogous pyrimidine-derived biodots. The polymeric nature of a precursor disfavors formation of a bright fluorescent product. The reported effect of the structure of the nucleic acid precursor on the fluorescence properties of biodots should help designing and synthesizing brighter fluorescent nanomaterials with broader specification for bioimaging, sensing, and other applications.

Metal Ions Sensing by Biodots Prepared from DNA, RNA, and Nucleotides.

Nucleic acids that exhibit a high affinity toward noble and transition metal ions have attracted growing attention in the fields of metal ion sensing, toxic metal ion removal, and the construction of functional metal nanostructures. In this study, fluorescent nanoparticles (biodots) were synthesized from DNA, RNA, and RNA nucleotides (AMP, GMP, UMP, and CMP) using a hydrothermal (HT) method, in order to study their metal ion sensing characteristics. The fluorescent properties of biodots differ markedly between those prepared from purine and pyrimidine nucleobases. All biodots demonstrate a high sensitivity to the presence of mercury cations (Hg2+), while biodots prepared from DNA, RNA, and guanosine monophosphate (GMP) are also sensitive to Ag+ and Cu2+ ions, but to a lesser extent. The obtained results show that biodots inherit the metal ion recognition properties of nucleobases, while the nucleobase composition of biodot precursors affects metal ion sensitivity and selectivity. A linear response of biodot fluorescence to Hg2+ concentration in solution was observed for AMP and GMP biodots in the range 0-250 µM, which can be used for the analytic detection of mercury ion concentration. A facile paper strip test was also developed that allows visual detection of mercury ions in solutions.

Adsorptive Removal of Heavy Metal Ions, Organic Dyes, and Pharmaceuticals by DNA-Chitosan Hydrogels.

DNA-chitosan (DNA-CS) hydrogel was prepared by in situ complexation between oppositely charged DNA and chitosan polyelectrolytes via electrostatic cross-linking to study its adsorption characteristics. The DNA-chitosan hydrogel matrix contains (i) cationic (NH3+) and anionic (PO4-) sites for electrostatic binding with ionic species, (ii) -OH and -NH2 groups and heteroaromatic DNA nucleobases for chelation of heavy metal ions, and (iii) DNA double-helix for recognition and binding to small organic molecules of various structures and polarities. DNA-CS hydrogels efficiently bind with Hg2+, Pb2+, Cd2+, and Cu2+ metal cations of significant environmental concern. Adsorption capacities of DNA-CS hydrogels for studied metal ions depend on hydrogel composition and pH of solution and reach ca. 50 mg/g at neutral pHs. Hydrogels with higher DNA contents show better adsorption characteristics and notably higher adsorption capacity to Hg2+ ions. Because of the co-existence of cationic and anionic macromolecules in the DNA-CS hydrogel, it demonstrates an affinity to both anionic (Congo Red) and cationic (Methylene Blue) dyes with moderate adsorption capacities of 12.6 mg/g and 29.0 mg/g, respectively. DNA-CS hydrogel can also be used for adsorptive removal of pharmaceuticals on conditions that their molecules are sufficiently hydrophobic and have ionogenic group(s). Facile preparation and multitarget adsorption characteristics of DNA-CS hydrogel coupled with sustainable and environmentally friendly characteristics render this system promising for environmental cleaning applications.

DNA-Chitosan Hydrogels: Formation, Properties, and Functionalization with Catalytic Nanoparticles.

DNA-chitosan (DNA-CS) hydrogels were prepared on the basis of interpolyelectrolyte complexes (IPEC) in a co-assembled regime by in situ charging of the polysaccharide in a DNA solution. In contrast to poorly controlled coacervates formed upon mixing of DNA and CS solutions, stable DNA-CS IPEC hydrogels are formed at near-stoichiometric ratios of DNA and chitosan ionogenic groups. Structure, stability, and ion absorption properties of such hydrogels depended strongly on the ratio between cationic (CS) and anionic (DNA) counterparts in hydrogels. Abundant amino- and nitrogen-containing aromatic groups of co-assembled DNA and CS make their hydrogel an efficient, multitarget absorbent toward metal ions. Such strong affinity of both DNA and CS to Au3+ cation was used to entrap Au3+ ions into the DNA-CS hydrogels. Subsequent reduction of Au3+ ion inside hydrogels resulted in the formation of ∼2-3 nm size Au nanoparticles on DNA-CS scaffolds. Metallized hydrogels demonstrated catalytic activity in reduction of various nitroaromatics that depended on the ratio between CS and DNA in the hydrogel.

Internal Motion of Chromatin Fibers Is Governed by Dynamics of Uncompressed Linker Strands.

Chromatin compaction and internal motion are fundamental aspects of gene expression regulation. Here, we have investigated chromatin fibers comprising recombinant histone octamers reconstituted with double-stranded bacteriophage T4-DNA. The size of the fibers approaches the typical size of genomic topologically associated domains. Atomic force and fluorescence (correlation) microscopy have been used to assess the structural organization, histone-induced compaction, and internal motion. In particular, the fibers are stretched on arrays of nanochannels, each channel with a diameter of 60 or 125 nm. Major intrafiber segregation and fast internal fluctuations are observed. Full compaction was only achieved by triggering an attractive nucleosome interaction through the addition of magnesium cations. Besides compaction, histone complexation results in a dramatic decrease in the fiber's relaxation time. The relaxation times are similar to those of naked DNA with a comparable stretch, which indicates that internal motion is governed by the dynamics of uncompressed linker strands. Furthermore, the main reorganization process is association-dissociation of individually compacted regions. We surmise that the modulation of chromatin's internal motion by histone complexation might have implications for transcriptional bursting.

Compaction and self-association of megabase-sized chromatin are induced by anionic protein crowding.

Highly compacted chromatin, a complex of DNA with cationic histone proteins, is found in the nucleus of eukaryotic cells in an environment with a high concentration of macromolecular species, many of which possess a negative charge. In the majority of previous studies, however, these crowding conditions were experimentally modelled using neutral synthetic macromolecules such as polyethylene glycol (PEG). Despite the importance of the crowding agent charge in the condensation process of chromatin, to the best of our knowledge, the behavior of chromatin under conditions of anionic protein crowding has not been studied. Here, compaction of nearly megabase-long chromatin in the presence of the anionic globular protein BSA was investigated by single-molecule fluorescent microscopy (FM). We demonstrate different effects of anionic macromolecular crowders (MMCs) on DNA and chromatin, compared to neutral MMCs. While DNA molecules undergo gradual compaction into a globular form in the presence of ca. 20% w/v of BSA, chromatin fibres complete coil to globule transition at a much lower concentration of BSA (ca. 5% w/v). Furthermore, at higher concentrations of BSA in solution (>5% w/v), chromatin fibres self-associate and form large spherical or fibrillar supramolecular microstructures characterized by a high colloidal stability and dynamic intermolecular fluctuations. Formation of such self-organized colloids from chromatin is universal and characteristic of chromatin fibres of various lengths. Our results highlight the hitherto underappreciated effect of anionic MMC environment on chromatin higher-order structures that may play an important role in self-organization of chromatin in vivo.

Encapsulation of Long Genomic DNA into a Confinement of a Polyelectrolyte Microcapsule: A Single-Molecule Insight.

Encapsulation of nucleic acids is an important technology in gene delivery, construction of "artificial cells", genome protection, and other fields. However, although there have been a number of protocols reported for encapsulation of short or oligomeric DNAs, encapsulation of genome-sized DNA containing hundreds of kilobase pairs is challenging because the length of such DNA is much longer compared to the size of a typical microcapsule. Here, we report a protocol for encapsulation of a ca. 60 µm contour length DNA into several micrometer-sized polyelectrolyte capsules. The encapsulation was carried out by (1) compaction of T4 DNA with multivalent cations, (2) entrapment of DNA condensates into micrometer-sized CaCO3 beads, (3) assembly of polyelectrolyte multilayers on a bead surface, and (4) dissolution of beads resulting in DNA unfolding and release. Fluorescence microscopy was used to monitor the process of long DNA encapsulation at the level of single-DNA molecules. The differences between long and short DNA encapsulation processes and morphologies of products are discussed.

Amino Acid Sequence of Oligopeptide Causes Marked Difference in DNA Compaction and Transcription.

Compaction of T4 phage DNA (166 kbp) by short oligopeptide octamers composed of two types of amino acids, four cationic lysine (K), and four polar nonionic serine (S) having different sequence order was studied by single-molecule fluorescent microscopy. We found that efficient DNA compaction by oligopeptide octamers depends on the geometrical match between phosphate groups of DNA and cationic amines. The amino acid sequence order in octamers dramatically affects the mechanism of DNA compaction, which changes from a discrete all-or-nothing coil-globule transition induced by a less efficient (K4S4) octamer to a continuous compaction transition induced by a (KS)4 octamer with a stronger DNA-binding character. This difference in the DNA compaction mechanism dramatically changes the packaging density, and the morphology of T4 DNA condensates: DNA is folded into ordered toroidal or rod morphologies during all-or-nothing compaction, whereas disordered DNA condensates are formed as a result of the continuous DNA compaction. Furthermore, the difference in DNA compaction mechanism has a certain effect on the inhibition scenario of the DNA transcription activity, which is gradual for the continuous DNA compaction and abrupt for the all-or-nothing DNA collapse.

Hybrid porous magnetic bentonite-chitosan beads for selective removal of radioactive cesium in water.

Easy-to-obtain magnetic bentonite-chitosan hybrid beads (Bn-CTS) were prepared by immobilizing bentonite within a porous structure of chitosan beads to achieve a hybrid adsorption effect for the removal of cesium ion (Cs+) from water. The hybrid adsorbent, which had a porous structure and abundant binding sites contributed by both chitosan and bentonite, ensured superb adsorption characteristics. The paramagnetic character of the beads enabled their facile separation for recycling. The chitosan/bentonite ratio, pH and contact time were optimized to achieve the optimal Cs+ efficiency, and the adsorption kinetics and isotherms were thoroughly discussed. The adsorption kinetics obeyed the pseudo-second-order model, and the best fitted equation for equilibrium data was the Langmuir isotherm model. The maximum adsorption capacity of the bentonite-chitosan beads was 57.1 mg g-1. The adsorbent had excellent selectivity towards Cs+ adsorption in the presence of abundant cations (Li+, Na+, K+ and Mg2+). The adsorbent was able to be recycled by treating the beads with 0.1 mol L-1 of MgCl2 to quantitatively desorb Cs+ from the beads. Overall, the magnetic bentonite-chitosan beads can be used as a highly efficient adsorbent for radioactive waste disposal and management.

Efficient prevention of nanomaterials transport in the porous media by treatment with polyelectrolytes.

Contamination of soil by engineered nanomaterials (ENM) is an emergent environmental problem that urges the development of robust treatment protocols to prevent ENM transport through soil. We developed a method for efficient entrapment and retention of ENM in solid porous media of quartz sand with grain size of 300-500 µm used as a simple model of soil and studied the transport properties of multi-walled carbon nanotubes, fullerenes, silica and gold nanoparticles through the sand-packed column by UV-vis and fluorescent spectroscopy. The treatment of ENM-contaminated porous media with a mixture of oppositely charged polyelectrolytes, cationic poly(diallyldimethylammonium chloride) and anionic poly(acrylic acid) sodium salt, dissolved in NaCl solution followed by dilution in the column results in strong electrostatic interaction between the polyelectrolytes and a formation of inter-polyelectrolyte complexes (IPEC) that induce flocculation of ENM and adsorption to the surface of sand. The method demonstrates excellent ENM entrapment efficiency (>90%) and high capacity of several grams of ENM per 1 g of polyelectrolytes. The IPEC network formed after the treatment also serves as an efficient protection barrier for newly added ENM contaminants. The method is universal for various types of ENM (carbon ENM, metal and oxide nanoparticles) and equally efficient for distilled water, tap water, or lake water eluents.

Compaction of Single-Molecule Megabase-Long Chromatin under the Influence of Macromolecular Crowding.

The megabase-sized length of chromatin is highly relevant to the state of chromatin in vivo, where it is subject to a highly crowded environment and is organized in topologically associating domains of similar dimension. We developed an in vitro experimental chromatin model system reconstituted from T4 DNA (approximately 166 kbp) and histone octamers and studied the monomolecular compaction of this megabase-sized chromatin fiber under the influence of macromolecular crowding. We used single-molecule fluorescence microscopy and observed compaction in aqueous solutions containing poly(ethylene glycol) in the presence of monovalent (Na+ and K+) and divalent (Mg2+) cations. Both DNA and chromatin demonstrated compaction under comparable conditions in the presence of poly(ethylene glycol) and Na+ or Mg2+ salt. However, the mechanism of the compaction changed from a first-order phase transition for DNA to a continuous folding for megabase-sized chromatin fibers. A more efficient and pronounced chromatin compaction was observed in the presence of Na+ compared to K+. A flow-stretching technique to unfold DNA and chromatin coils was used to gain further insight into the morphology of partially folded chromatin fibers. The results revealed a distribution of partially folded chromatin fibers. This variability is likely the result of the heterogeneous distribution of nucleosomes on the DNA chain. The packaging of DNA in the form of chromatin in the crowded nuclear environment appears essential to ensure gradual conformational changes of DNA.

Single-molecule compaction of megabase-long chromatin molecules by multivalent cations.

To gain insight into the conformational properties and compaction of megabase-long chromatin molecules, we reconstituted chromatin from T4 phage DNA (165 kb) and recombinant human histone octamers (HO). The unimolecular compaction, induced by divalent Mg2+ or tetravalent spermine4+ cations, studied by single-molecule fluorescence microscopy (FM) and dynamic light scattering (DLS) techniques, resulted in the formation of 250-400 nm chromatin condensates. The compaction on this scale of DNA size is comparable to that of chromatin topologically associated domains (TAD) in vivo. Variation of HO loading revealed a number of unique features related to the efficiency of chromatin compaction by multivalent cations, the mechanism of compaction, and the character of partly compact chromatin structures. The observations may be relevant for how DNA accessibility in chromatin is maintained. Compaction of saturated chromatin, in turn, is accompanied by an intra-chain segregation at the level of single chromatin molecules, suggesting an intriguing scenario of selective activation/deactivation of DNA as a result of chromatin fiber heterogeneity due to the nucleosome positioning. We suggest that this chromatin, reconstituted on megabase-long DNA because of its large size, is a useful model of eukaryotic chromatin.

Novel highly porous magnetic hydrogel beads composed of chitosan and sodium citrate: an effective adsorbent for the removal of heavy metals from aqueous solutions.

This research focuses on the removal of heavy metal ions from aqueous solutions using magnetic chitosan hydrogel beads as a potential sorbent. Highly porous magnetic chitosan hydrogel (PMCH) beads were prepared by a combination of in situ co-precipitation and sodium citrate cross-linking. Fourier transform infrared spectroscopy indicated that the high sorption efficiency of metal cations is attributable to the hydroxyl, amino, and carboxyl groups in PMCH beads. Thermogravimetric analysis demonstrated that introducing Fe3O4 nanoparticles increases the thermal stability of the adsorbent. Laser confocal microscopy revealed highly uniform porous structure of the resultant PMCH beads, which contained a high moisture content (93%). Transmission electron microscopy micrographs showed that the Fe3O4 nanoparticles, with a mean diameter of 5 ± 2 nm, were well dispersed inside the chitosan beads. Batch adsorption experiments and adsorption kinetic analysis revealed that the adsorption process obeys a pseudo-second-order model. Isotherm data were satisfactorily described by the Langmuir equation, and the maximum adsorption capacity of the adsorbent was 84.02 mg/g. Energy-dispersive X-ray spectroscopy and X-ray photoelectron spectra analyses were performed to confirm the adsorption of Pb2+ and to identify the adsorption mechanism.

Photoinduced Fusion of Lipid Bilayer Membranes.

We have developed a novel system for photocontrol of the fusion of lipid vesicles through the use of a photosensitive surfactant containing an azobenzene moiety (AzoTAB). Real-time microscopic observations clarified a change in both the surface area and internal volume of vesicles during fusion. We also determined the optimal cholesterol concentrations and temperature for inducing fusion. The mechanism of fusion can be attributed to a change in membrane tension, which is caused by the solubilization of lipids through the isomerization of AzoTAB. We used a micropipet technique to estimate membrane tension and discuss the mechanism of fusion in terms of membrane elastic energy. The obtained results regarding this novel photoinduced fusion could lead to a better understanding of the mechanism of membrane fusion in living cells and may also see wider applications, such as in drug delivery and biomimetic material design.

Divalent cation shrinks DNA but inhibits its compaction with trivalent cation.

Our observation reveals the effects of divalent and trivalent cations on the higher-order structure of giant DNA (T4 DNA 166 kbp) by fluorescence microscopy. It was found that divalent cations, Mg(2+) and Ca(2+), inhibit DNA compaction induced by a trivalent cation, spermidine (SPD(3+)). On the other hand, in the absence of SPD(3+), divalent cations cause the shrinkage of DNA. As the control experiment, we have confirmed the minimum effect of monovalent cation, Na(+) on the DNA higher-order structure. We interpret the competition between 2+ and 3+ cations in terms of the change in the translational entropy of the counterions. For the compaction with SPD(3+), we consider the increase in translational entropy due to the ion-exchange of the intrinsic monovalent cations condensing on a highly charged polyelectrolyte, double-stranded DNA, by the 3+ cations. In contrast, the presence of 2+ cation decreases the gain of entropy contribution by the ion-exchange between monovalent and 3+ ions.