Advanced protein-embedded bimetallic nanocomposite optimized for in vivo fluorescence and magnetic resonance bimodal imaging.

HYPOTHESIS: The development of bimodal imaging probes represents a hot topic of current research. Herein, we deal with developing an innovative bimodal contrast agent enabling fluorescence imaging (FI)/magnetic resonance imaging (MRI) and, simultaneously, consisting of biocompatible nanostructures. Optimized synthesis of advanced protein-embedded bimetallic (APEBM) nanocomposite containing luminescent gold nanoclusters (AuNC) and superparamagnetic iron oxide nanoparticles (SPION), suitable for in vivo dual-modal FI/MR imaging is reported. EXPERIMENTS: The APEBM nanocomposite was prepared by a specific sequential one-pot green synthetic approach that is optimized to increase metals (Au, Fe) content and, consequently, the imaging ability of the resulting nanostructures. The protein matrix, represented by serum albumin, was intentionally chosen, and used since it creates an efficient protein corona for both types of optically/magnetically-susceptible nanostructures (AuNC, SPION) and ensures biocompatibility of the resulting APEBM nanocomposite although it contains elevated metal concentrations (approx. 1 mg·mL-1 of Au, around 0.3 mg·mL-1 of Fe). In vitro and in vivo imaging was performed. FINDINGS: Successful in vivo FI and MRI recorded in healthy mice corroborated the applicability of the APEBM nanocomposite and, simultaneously, served as a proof of concept concerning the potential future exploitation of this new FI/MRI bimodal contrast agent in preclinical and clinical practice.

Facile One-Pot Green Synthesis of Magneto-Luminescent Bimetallic Nanocomposites with Potential as Dual Imaging Agent.

Nanocomposites serving as dual (bimodal) probes have great potential in the field of bio-imaging. Here, we developed a simple one-pot synthesis for the reproducible generation of new luminescent and magnetically active bimetallic nanocomposites. The developed one-pot synthesis was performed in a sequential manner and obeys the principles of green chemistry. Briefly, bovine serum albumin (BSA) was exploited to uptake Au (III) and Fe (II)/Fe (III) ions simultaneously. Then, Au (III) ions were transformed to luminescent Au nanoclusters embedded in BSA (AuNCs-BSA) and majority of Fe ions were bio-embedded into superparamagnetic iron oxide nanoparticles (SPIONs) by the alkalization of the reaction medium. The resulting nanocomposites, AuNCs-BSA-SPIONs, represent a bimodal nanoprobe. Scanning transmission electron microscopy (STEM) imaging visualized nanostructures with sizes in units of nanometres that were arranged into aggregates. Mössbauer spectroscopy gave direct evidence regarding SPION presence. The potential applicability of these bimodal nanoprobes was verified by the measurement of their luminescent features as well as magnetic resonance (MR) imaging and relaxometry. It appears that these magneto-luminescent nanocomposites were able to compete with commercial MRI contrast agents as MR displays the beneficial property of bright luminescence of around 656 nm (fluorescence quantum yield of 6.2 ± 0.2%). The biocompatibility of the AuNCs-BSA-SPIONs nanocomposite has been tested and its long-term stability validated.

Evidence of Au(II) and Au(0) States in Bovine Serum Albumin-Au Nanoclusters Revealed by CW-EPR/LEPR and Peculiarities in HR-TEM/STEM Imaging.

Bovine serum albumin-embedded Au nanoclusters (BSA-AuNCs) are thoroughly probed by continuous wave electron paramagnetic resonance (CW-EPR), light-induced EPR (LEPR), and sequences of microscopic investigations performed via high-resolution transmission electron microscopy (HR-TEM), scanning transmission electron microscopy (STEM), and energy dispersive X-ray analysis (EDS). To the best of our knowledge, this is the first report analyzing the BSA-AuNCs by CW-EPR/LEPR technique. Besides the presence of Au(0) and Au(I) oxidation states in BSA-AuNCs, the authors observe a significant amount of Au(II), which may result from a disproportionation event occurring within NCs: 2Au(I) → Au(II) + Au(0). Based on the LEPR experiments, and by comparing the behavior of BSA versus BSA-AuNCs under UV light irradiation (at 325 nm) during light off-on-off cycles, any energy and/or charge transfer event occurring between BSA and AuNCs during photoexcitation can be excluded. According to CW-EPR results, the Au nano assemblies within BSA-AuNCs are estimated to contain 6-8 Au units per fluorescent cluster. Direct observation of BSA-AuNCs by STEM and HR-TEM techniques confirms the presence of such diameters of gold nanoclusters in BSA-AuNCs. Moreover, in situ formation and migration of Au nanostructures are observed and evidenced after application of either a focused electron beam from HR-TEM, or an X-ray from EDS experiments.

Distinctly Different Morphologies of Bimetallic Au-Ag Nanostructures and Their Application in Submicromolar SERS-Detection of Free Base Porphyrin.

Core-shell Au-Ag nanostructures (Au-AgNSs) are prepared by a seed-meditated growth, i.e., by a two-step process. The synthetic parameters greatly influence the morphologies of the final bimetallic Au-AgNSs, their stability and application potential as surface-enhanced Raman scattering (SERS) substrates. Direct comparison of several types of Au NPs possessing different surface species and serving as seeds in Au-AgNSs synthesis is the main objective of this paper. Borohydride-reduced (with varying stages of borohydride hydrolysis) and citrate-reduced Au NPs were prepared and used as seeds in Au-AgNSs generation. The order of reactants in seed-mediated growth procedure represents another key factor influencing the final Au-AgNSs characteristics. Electronic absorption spectra, dynamic light scattering, zeta potential measurements, energy dispersive spectroscopy and transmission electron microscopy were employed for Au-AgNSs characterization. Subsequently, possibilities and limitations of SERS-detection of unperturbed cationic porphyrin, 5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H,23H-porphine (TMPyP), were investigated by using these Au-AgNSs. Only the free base (unperturbed) SERS spectral form of TMPyP is detected in all types of Au-AgNSs. It reports about a well-developed envelope of organic molecules around each Au-AgNSs which prevents metalation from occuring. TMPyP, attached via ionic interaction, was successfully detected in 10 nM concentration due to Au-AgNSs.

The Effect of Fatty Acids and BSA Purity on Synthesis and Properties of Fluorescent Gold Nanoclusters.

Fluorescent gold nanoclusters (AuNCs) are envisaged as a novel type of fluorophores. This work reports on the first comparative study investigating the effect of presence/absence/abundance of fatty acids (namely palmitic acid, PA) or other substances (like glycoproteins and globulins) in the protein (bovine serum albumin, BSA) on synthesis and properties of the final AuNCs. The most popular template (BSA) and microwave (MW)-assisted synthesis of AuNCs have been intentionally chosen. Our results clearly demonstrate that the fluorescent characteristics (i.e., fluorescence lifetime and quantum yield) are affected by the fatty acids and/or other substances. Importantly, the as-prepared AuNCs are biocompatible, as determined by Alamar Blue assay performed on Hep G2 cell line.

Transformations of ferrates(iv,v,vi) in liquids: Mössbauer spectroscopy of frozen solutions.

The kinetics and mechanism of ferrate(iv), (v) and (vi) transformations in water and in polar organic solvents (namely ethanol and tetrahydrofuran) have been investigated by the method of 57Fe Mössbauer spectroscopy of frozen solutions. Ethanol with a very limited amount of water under an inert atmosphere, significantly slows down the transformation reactions of ferrates(iv and v) and provides direct proof of the existence of intermediate states. Simultaneously, ethanol is oxidized to caboxylates in the close vicinity of the surface of ferrate crystallites as proven by X-ray photoelectron spectroscopy. On the contrary, any transformation of ferrate(vi) in pure ethanol (with a very limited amount of water) was not observed. Mössbauer spectroscopy of frozen solutions enabled us to experimentally identify and quantify intermediates of ferrate(iv) and ferrate(v) transformations for the first time. Sodium ferrate(iv) in its tetrahedral form, Na4FeO4, undergoes a two-step charge disproportionation to Fe(iii) and Fe(vi) via a Fe(v) intermediate without any evolution of oxygen in polar protic and aprotic solvents, specifically 2Fe(iv) → Fe(iii) + Fe(v), and Fe(iv) + Fe(v) → Fe(iii) + Fe(vi), i.e. in sum 3Fe(iv) → 2Fe(iii) + Fe(vi). Ferrate(v) (K3FeO4) transforms to Fe(iii) and Fe(vi) without any indication of the Fe(iv) intermediate within the detection limit of the method. In addition to a charge disproportionation reaction proceeding in polar liquids, 3Fe(v) → Fe(iii) + 2Fe(vi), a competitive reduction of Fe(v) directly to Fe(iii) accompanied by oxygen evolution takes place in water. Oxygen evolution was also measured for ferrate(iv and vi) transformations in water, but to a higher and a smaller extent compared to ferrate(v), respectively. The thermodynamics of the suggested ferrate(iv) and ferrate(v) transformation pathways was examined by DFT calculations.

Impact of inorganic buffering ions on the stability of Fe(vi) in aqueous solution: role of the carbonate ion.

An iron compound of +6 oxidation state (Fe(VI)O4(2-), Fe(vi)) is a green molecule for various applications (water oxidation catalyst, organic transformation for synthesis, and water remediation agent). However, its use is hindered because of its inherent decay in aqueous solution. This study presents a systematic kinetics investigation of the decay of ferrate(vi) in the presence of inorganic buffering ions (borate, phosphate, and carbonate) at a pH range from 6.0 to 9.0. When the heterogeneous decay of Fe(vi) on ferric products was inhibited by phosphate, detailed kinetic analysis revealed that the carbonate anion enhanced the Fe(vi) decay rate, compared to phosphate and borate ions. The order of the Fe(vi) decay rate under neutral solution conditions was carbonate > phosphate ≥ borate. In alkaline solution, the decay rates of Fe(vi) were similar for the studied buffering ions. The decay of Fe(vi) in the presence of the carbonate ion was described by mixed first- and second-order kinetics and the first-order rate constant (k1') had a linear relationship with the concentration of the carbonate ion at a neutral pH (k1' = 0.023 + 3.54 × [carbonate] L mol(-1) s(-1)). The analysis of the Fe(vi) decay intermediates/products (˙O2(-), H2O2, and O2) suggests similar decay pathways in the presence of different buffering anions. The impact of carbonate ions on the size of the nanoparticles of the Fe(iii) precipitate, the final reduced form of Fe(vi), was studied using transmission electron microscopy, (57)Fe Mössbauer spectroscopy, and magnetization measurements. The results indicated that carbonate ions induce the formation of ultrasmall iron(iii) oxyhydroxide nanoparticles (<5 nm), which apparently contribute to increased decay of Fe(vi) due to their larger specific surface area. The described homogeneous reaction of carbonate with Fe(vi) has important implications in the efficiency of environmental Fe(vi) applications. On the other hand, the observed low reactivity of borate with Fe(vi) demonstrates that borate is the least reactive buffer in studies of Fe(vi) reactivity in neutral solutions.

Enhanced formation of silver nanoparticles in Ag+-NOM-iron(II, III) systems and antibacterial activity studies.

This work reports the role of iron redox pair (Fe(3+)/Fe(2+)) in the formation of naturally occurring silver nanoparticles (AgNPs) in the aquatic environment. The results showed that Fe(3+) or Fe(2+) ions in the mixtures of Ag(+) and natural organic matter enhanced the formation of AgNPs. The formation of AgNPs depended on pH and types of organic matter. Increase in pH enhanced the formation of AgNPs, and humic acids as ligands showed higher formation of AgNPs compared to fulvic acids. The observed results were described by considering the potentials of redox pairs of silver and iron species and the possible species involved in reducing silver ions to AgNPs. Dynamic light scattering and transmission electron microscopy measurements of AgNPs revealed mostly bimodal size distribution with decrease in size of AgNPs due to iron species in the reaction mixture. Minimum inhibitory concentration of AgNPs needed to inhibit the growth of various bacterial species suggested the role of surfaces of tested Gram-positive and Gram-negative bacteria. Stability study of AgNPs, formed in Ag(+)-humic acid/fulvic acids-Fe(3+) mixtures over a period of several months showed high stability of the particles with significant increase in surface plasmon resonance peak. The environmental implications of the results in terms of fate, transport, and ecotoxicity of organic-coated AgNPs are briefly presented.

Organic-coated silver nanoparticles in biological and environmental conditions: fate, stability and toxicity.

This review paper presents the overview of processes involved in transformation of organic-coated silver nanoparticles (AgNPs) in biological systems and in the aquatic environment. The coating on AgNPs greatly influences the fate, stability, and toxicity of AgNPs in aqueous solutions, biological systems, and the environment. Several organic-coated AgNP systems are discussed to understand their stability and toxicity in biological media and natural water. Examples are presented to demonstrate how a transformation of organic-coated AgNPs in an aqueous solution is affected by the type of coating, pH, kind of electrolyte (mono- or divalent), ionic strength, organic ligands (inorganic and organic), organic matter (fulvic and humic acids), redox conditions (oxic and anoxic), and light. Results of cytotoxicity, genotoxicity, and ecotoxicity of coated AgNPs to food chain members (plants, bacteria, and aquatic and terrestrial organisms) are reviewed. Key factors contributing to toxicity are the size, shape, surface coating, surface charge, and conditions of silver ion release. AgNPs may directly damage the cell membranes, disrupt ATP production and DNA replication, alternate gene expressions, release toxic Ag(+) ion, and produce reactive oxygen species to oxidize biological components of the cell. A progress made on understanding the mechanism of organic-coated AgNP toxicity using different analytical techniques is presented.

Mixtures of L-amino acids as reaction medium for formation of iron nanoparticles: the order of addition into a ferrous salt solution matters.

Owing to Mössbauer spectroscopy, an advanced characterization technique for iron-containing materials, the present study reveals previously unknown possibilities using l-amino acids for the generation of magnetic particles. Based on our results, a simple choice of the order of l-amino acids addition into a reaction mixture containing ferrous ions leads to either superparamagnetic ferric oxide/oxyhydroxide particles, or magnetically strong Fe0-Fe2O3/FeOOH core-shell particles after chemical reduction. Conversely, when ferric salts are employed with the addition of selected l-amino acids, only Fe0-Fe2O3/FeOOH core-shell particles are observed, regardless of the addition order. We explain this phenomenon by a specific transient/intermediate complex formation between Fe2+ and l-glutamic acid. This type of complexation prevents ferrous ions from spontaneous oxidation in solutions with full air access. Moreover, due to surface-enhanced Raman scattering spectroscopy we show that the functional groups of l-amino acids are not destroyed during the borohydride-induced reduction. These functionalities can be further exploited for (i) attachment of l-amino acids to the as-prepared magnetic particles, and (ii) for targeted bio- and/or environmental applications where the surface chemistry needs to be tailored and directed toward biocompatible species.

Air stable magnetic bimetallic Fe-Ag nanoparticles for advanced antimicrobial treatment and phosphorus removal.

We report on new magnetic bimetallic Fe-Ag nanoparticles (NPs) which exhibit significant antibacterial and antifungal activities against a variety of microorganisms including disease causing pathogens, as well as prolonged action and high efficiency of phosphorus removal. The preparation of these multifunctional hybrids, based on direct reduction of silver ions by commercially available zerovalent iron nanoparticles (nZVI) is fast, simple, feasible in a large scale with a controllable silver NP content and size. The microscopic observations (transmission electron microscopy, scanning electron microscopy/electron diffraction spectroscopy) and phase analyses (X-ray diffraction, Mössbauer spectroscopy) reveal the formation of Fe3O4/γ-FeOOH double shell on a "redox" active nZVI surface. This shell is probably responsible for high stability of magnetic bimetallic Fe-Ag NPs during storage in air. Silver NPs, ranging between 10 and 30 nm depending on the initial concentration of AgNO3, are firmly bound to Fe NPs, which prevents their release even during a long-term sonication. Taking into account the possibility of easy magnetic separation of the novel bimetallic Fe-Ag NPs, they represent a highly promising material for advanced antimicrobial and reductive water treatment technologies.

Interactions of aqueous Ag+ with fulvic acids: mechanisms of silver nanoparticle formation and investigation of stability.

This study investigated the possible natural formation of silver nanoparticles (AgNPs) in Ag(+)-fulvic acid (FA) solutions under various environmentally relevant conditions (temperature, pH, and UV light). Increase in temperature (24-90 °C) and pH (6.1-9.0) of Ag(+)-Suwannee River fulvic acid (SRFA) solutions accelerated the appearance of the characteristic surface plasmon resonance (SPR) of AgNPs. The rate of AgNP formation via reduction of Ag(+) in the presence of different FAs (SRFA, Pahokee Peat fulvic acid, PPFA, Nordic lake fulvic acid, NLFA) and Suwannee River humic acid (SRHA) followed the order NLFA > SRHA > PPFA > SRFA. This order was found to be related to the free radical content of the acids, which was consistent with the proposed mechanism. The same order of AgNP growth was seen upon UV light illumination of Ag(+)-FA and Ag(+)-HA mixtures in moderately hard reconstituted water (MHRW). Stability studies of AgNPs, formed from the interactions of Ag(+)-SRFA, over a period of several months showed that these AgNPs were highly stable with SPR peak reductions of only ~15%. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements revealed bimodal particle size distributions of aged AgNPs. The stable AgNPs formed through the reduction of Ag(+) by fulvic and humic acid fractions of natural organic matter in the environment may be transported over significant distances and might also influence the overall bioavailability and ecotoxicity of AgNPs.

Mechanisms and efficiency of the simultaneous removal of metals and cyanides by using ferrate(VI): crucial roles of nanocrystalline iron(III) oxyhydroxides and metal carbonates.

The reaction of potassium ferrate(VI), K(2)FeO(4), with weak-acid dissociable cyanides--namely, K(2)[Zn(CN)(4)], K(2)[Cd(CN)(4)], K(2)[Ni(CN)(4)], and K(3)[Cu(CN)(4)]--results in the formation of iron(III) oxyhydroxide nanoparticles that differ in size, crystal structure, and surface area. During cyanide oxidation and the simultaneous reduction of iron(VI), zinc(II), copper(II), and cadmium(II), metallic ions are almost completely removed from solution due to their coprecipitation with the iron(III) oxyhydroxides including 2-line ferrihydrite, 7-line ferrihydrite, and/or goethite. Based on the results of XRD, Mössbauer and IR spectroscopies, as well as TEM, X-ray photoelectron emission spectroscopy, and Brunauer-Emmett-Teller measurements, we suggest three scavenging mechanisms for the removal of metals including their incorporation into the ferrihydrite crystal structure, the formation of a separate phase, and their adsorption onto the precipitate surface. Zn and Cu are preferentially and almost completely incorporated into the crystal structure of the iron(III) oxyhydroxides; the formation of the Cd-bearing, X-ray amorphous phase, together with Cd carbonate is the principal mechanism of Cd removal. Interestingly, Ni remains predominantly in solution due to the key role of nickel(II) carbonate, which exhibits a solubility product constant several orders of magnitude higher than the carbonates of the other metals. Traces of Ni, identified in the iron(III) precipitate, are exclusively adsorbed onto the large surface area of nanoparticles. We discuss the relationship between the crystal structure of iron(III) oxyhydroxides and the mechanism of metal removal, as well as the linear relationship observed between the rate constant and the surface area of precipitates.

Non-chemical approach toward 2D self-assemblies of Ag nanoparticles via cold plasma treatment of substrates.

The nano-modification of selected substrates by means of atmospheric cold plasma treatment was exploited for the two-dimensional (2D) self-assembling of silver nanoparticles (Ag NPs). Such a useful combination of the cold plasma treatment of substrate surface and an immediate easy deposition of Ag NPs creating the 2D self-assemblies on the substrates is published for the first time, to the best of our knowledge. Except for the cold plasma treatment, mainly the following parameters influenced the resulting NP assemblies: the choice of solvent mixture, concentration of Ag NP dispersions, and the deposition technique. The 2D self-assemblies of Ag NPs, providing the same work function as a Ag electrode, were formed on the cold plasma-treated substrates when a drop-casting technique was employed. The possibility of an easy preparation of the Ag NP 2D self-assemblies on substrates without using any chemical agents and/or evaporating chamber could be exploited, e.g. in photovoltaic and light-emitting diode devices.

SERS-activating effect of chlorides on borate-stabilized silver nanoparticles: formation of new reduced adsorption sites and induced nanoparticle fusion.

Changes in morphology, surface reactivity and surface-enhancement of Raman scattering induced by modification of borate-stabilized Ag nanoparticles by adsorbed chlorides have been explored using TEM, EDX analysis and SERS spectra of probing adsorbate 2,2'-bipyridine (bpy) excited at 514.5 nm and evaluated by factor analysis. At fractional coverages of the parent Ag nanoparticles by adsorbed chlorides <0.6, the Ag colloid/Cl(-)/bpy systems were found to be constituted by fractal aggregates of Ag nanoparticles fairly uniform in size (10 +/- 2 nm) and SERS spectra of Ag(+)-bpy surface species were detected. The latter result was interpreted in terms of the presence of oxidized Ag(+) and/or Ag(n)(+) adsorption sites, which have been encountered also in systems with the chemically untreated Ag nanoparticles. At chloride coverages >0.6, a fusion of fractal aggregates into the compact aggregates of touching and/or interpenetrating Ag nanoparticles has been observed and found to be accompanied by the formation of another surface species, Ag-bpy, as well as by the increase of the overall SERS enhancement of bpy by factor of 40. The same Ag-bpy surface species has been detected under the strongly reducing conditions of reduction of silver nitrate by sodium borohydride in the presence of bpy. The formation of Ag-bpy is thus interpreted in terms of the stabilization of reduced Ag(0) adsorption sites by adsorbed bpy. The formation of reduced adsorption sites on Ag nanoparticle surfaces at chloride coverages >0.6 is discussed in terms of local changes in the work function of Ag. Finally, the SERS spectral detection of Ag-bpy species is proposed as a tool for probing the presence of reduced Ag(0) adsorption sites in systems with chemically modified Ag nanoparticles.

Characterization and surface-enhanced Raman spectral probing of silver hydrosols prepared by two-wavelength laser ablation and fragmentation.

A four step Ag foil laser ablation-Ag nanoparticle fragmentation procedure in ultrapure water was carried out both under argon and in air. Pulses of a high power Nd/YAG laser were used for laser ablation (1064 nm) and for the three step Ag hydrosol treatment in the absence of Ag foil in the sequence 1064-532-1064 nm. Transmission electron microscopy (TEM) and surface plasmon (SP) extinction spectra provide evidence of Ag nanoparticle fragmentation in the second and third step of the procedure carried out under argon. While polydispersity of Ag hydrosol increases in the second step, both the polydispersity and the mean size of the nanoparticles are reduced in the third step. Qualitative and quantitative surface-enhanced Raman scattering (SERS)/surface-enhanced resonance Raman scattering (SERRS) spectral probing of systems with Ag hydrosols and the selected adsorbates at 514.5 nm excitation shows that Ag hydrosols obtained in the second step of the preparation procedure carried out in air are the most suitable substrates for SERS/SERRS experiments performed at this excitation wavelength.