Immobilization of controlled Pu:Am ratio on actinide-specific affinity monolith support developed in capillary and coupled to inductively coupled plasma mass spectrometry.

The objective of this work was to develop an actinide-specific monolithic support in capillary designed to immobilize precise Pu:Am ratios and its coupling to inductively coupled plasma mass spectrometry (ICP-MS) for immobilized metal affinity chromatography applications. This format offers many advantages, such as reducing the sample amount and waste production, which are of prime importance when dealing with highly active radioelements. Four organic phosphorylated-based monoliths were synthesized in situ through UV photo-polymerization in capillary and characterized. The capillary coupling to ICP-MS was set up in conventional laboratory using Th and Sm as chemical analogues of Pu and Am. A dedicated method was developed to quantify online Th and Sm amounts immobilized on the monolithic capillaries, allowing to select the best monolith candidate poly(BMEP-co-EDMA)adp. By precisely adjusting the elemental composition in the loading solutions and applying the developed quantification method, the controlled immobilization of several Th:Sm molar ratios onto the monolith was successful. Finally, the capillary ICP-MS coupling was transposed in a glove box and by applying the strategy developed to design the monolithic support using Th and Sm, the immobilization of a 10.5 ± 0.2 (RSD = 2.3%, n = 3) Pu:Am molar ratio reflecting Pu ageing over 48 years was achieved in a controlled manner on poly(BMEP-co-EDMA)adp. Hence, the new affinity capillary monolithic support was validated, with only hundred nanograms or less of engaged radioelements and can be further exploited to precisely determine differential interactions of Pu and Am with targeted biomolecules in order to better anticipate the effect of Am on Pu biodistribution.

Determining the selectivity of a tetra-phosphorylated biomimetic peptide towards uranium in the presence of competing cations through the simultaneous coupling of HILIC to ESI-MS and ICP-MS.

A cyclic tetra-phosphorylated biomimetic peptide (pS1368) has been proposed as a promising starting structure to design a decorporating agent of uranyl (UO22+) due to its affinity being similar to that of osteopontin (OPN), a target UO22+ protein in vivo. The determination of this peptide's selectivity towards UO22+ in the presence of competing endogenous elements is also crucial to validate this hypothesis. In this context, the selectivity of pS1368 towards UO22+ in the presence of Ca2+, Cu2+ and Zn2+ was determined by applying the simultaneous coupling of hydrophilic interaction chromatography (HILIC) to electrospray ionization (ESI-MS) and inductively coupled plasma (ICP-MS) mass spectrometry. Sr2+ was used as Ca2+ simulant, providing less challenging ICP-MS measurements. The separation of the complexes by HILIC was first set up. The selectivity of pS1368 towards UO22+ was determined in the presence of Sr2+, by adding several proportions of the latter to UO2(pS1368). UO22+ was not displaced from UO2(pS1368) even in the presence of a ten-fold excess of Sr2+. The same approach has been undertaken to demonstrate the selectivity of pS1368 towards UO22+ in the presence of Cu2+, Zn2+ and Sr2+ as competing endogenous cations. Hence, we showed that pS1368 was selective towards UO22+ in the presence of Sr2+, but also in the presence of Cu2+ and Zn2+. This study highlights the performance of HILIC-ESI-MS/ICP-MS simultaneous coupling to assess the potential of molecules as decorporating agents of UO22+.

How Does Iron Storage Protein Ferritin Interact with Plutonium (and Thorium)?

The impact of the contamination of living organisms by actinide elements has been a constant subject of attention since the 1950s. But to date still little is understood. Ferritin is the major storage and regulation protein of iron in many organisms, it consists of a protein ring and a ferrihydric core at the center. This work sheds light on the interactions of early actinides (Th, Pu) at oxidation state +IV with ferritin and its ability to store those elements at physiological pH compared to Fe. The ferritin-thorium load curve suggests that ThIV saturates the protein (2840 Th atoms per ferritin) in a similar way that Fe does on the protein ring. Complementary spectroscopic techniques (spectrophotometry, infrared spectroscopy, and X-ray absorption spectroscopy) were combined with molecular dynamics to provide a structural model of the interaction of ThIV and PuIV with ferritin. Comparison of spectroscopic data together with MD calculations suggests that ThIV and PuIV are complexed mainly on the protein ring and not on the ferrihydric core. Indeed from XAS data, there is no evidence of Fe neighbors in the Th and Pu environments. On the other hand, carboxylates from amino acids of the protein ring and a possible additional carbonate anion are shaping the cation coordination spheres. This thorough description from a molecular view point of ThIV and PuIV interaction with ferritin, an essential iron storage protein, is a cornerstone in comprehensive nuclear toxicology.

Capping of Mn-Doped ZnS Quantum Dots with DHLA for Their Stabilization in Aqueous Media: Determination of the Nanoparticle Number Concentration and Surface Ligand Density.

Colloidal Mn2+-doped ZnS quantum dots (QDs) were synthesized, surface modified, and thoroughly characterized using a pool of complementary techniques. Cap exchange of the native l-cysteine coating of the QDs with dihydrolipoic acid (DHLA) ligands is proposed as a strategy to produce nanocrystals with a strong phosphorescent-type emission and improved aqueous stability. Moreover, such a stable DHLA coating can facilitate further bioconjugation of these QDs to biomolecules using established reagents such as cross-linker molecules. First, a structural and morphological characterization of the l-cysteine QD core was performed by resorting to complementary techniques, including X-ray powder diffraction (XRD) and microscopy tools. XRD patterns provided information about the local structure of ions within the nanocrystal structure and the number of metal atoms constituting the core of a QD. The judicious combination of the data obtained from these complementary characterization tools with the analysis of the QDs using inductively coupled plasma-mass spectrometry (ICP-MS) allowed us to assess the number concentration of nanoparticles in an aqueous sample, a key parameter when such materials are going to be used in bioanalytical or toxicological studies. Asymmetric flow field-flow fractionation (AF4) coupled online to ICP-MS detection proved to be an invaluable tool to compute the number of DHLA molecules attached to the surface of a single QD, a key feature that is difficult to estimate in nanoparticles and that critically affects the behavior of nanoparticles when entering the biological media (e.g., cellular uptake, biodistribution, or protein corona formation). This hybrid technique also allowed us to demonstrate that the elemental composition of the nanoparticle core remains unaffected after the ligand exchange process. Finally, the photostability and robustness of the DHLA-capped QDs, critical parameters for bioanalytical applications, were assessed by molecular luminescence spectroscopy.

Determination of the affinity of biomimetic peptides for uranium through the simultaneous coupling of HILIC to ESI-MS and ICP-MS.

Several proteins have been identified in the past decades as targets of uranyl (UO22+) in vivo. However, the molecular interactions responsible for this affinity are still poorly known which requires the identification of the UO22+ coordination sites in these proteins. Biomimetic peptides are efficient chemical tools to characterize these sites. In this work, we developed a dedicated analytical method to determine the affinity of biomimetic, synthetic, multi-phosphorylated peptides for UO22+ and evaluate the effect of several structural parameters of these peptides on this affinity at physiological pH. The analytical strategy was based on the implementation of the simultaneous coupling of hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). An essential step had been devoted to the definition of the best separation conditions of UO22+ complexes formed with di-phosphorylated peptide isomers and also with peptides of different structure and degrees of phosphorylation. We performed the first separations of several sets of UO22+ complexes by HILIC ever reported in the literature. A dedicated method had then been developed for identifying the separated peptide complexes online by ESI-MS and simultaneously quantifying them by ICP-MS, based on uranium quantification using external calibration. Thus, the affinity of the peptides for UO22+ was determined and made it possible to demonstrate that (i) the increasing number of phosphorylated residues (pSer) promotes the affinity of the peptides for UO22+, (ii) the position of the pSer in the peptide backbone has very low impact on this affinity (iii) and finally the cyclic structure of the peptide favors the UO22+ complexation in comparison with the linear structure. These results are in agreement with those previously obtained by spectroscopic techniques, which allowed to validate the method. Through this approach, we obtained essential information to better understand the mechanisms of toxicity of UO22+ at the molecular level and to further develop selective decorporating agents by chelation.

Functionalized phosphorescent nanoparticles in (bio)chemical sensing and imaging - A review.

Inorganic nanoparticles are a fascinating class of materials which promise great potential in numerous fields, including optical (bio)sensing. Many different kinds of such nanoparticles have been widely used for fluorescent sensing and imaging due to the different merits of fluorescent nanoparticles compared to molecular fluorophores. Progress made in the rational design of nanomaterials also allowed the synthesis of hybrid phosphorescent nanoparticles, that finds growing applications in sensing due to the combination of the interesting size- and shape-dependent properties of nanomaterials with a phosphorescence-type emission. In this review, we intend to highlight some of progress made in this active research area and update the database of various phosphorescent nanoparticles-based sensors on the basis of different sensing targets of interest in environmental, industrial and biomedical areas. Following an introduction and a discussion of merits of the synergy between nanomaterials and phosphorescence detection as compared to molecular luminophores the article assesses the kinds and specific features of nanomaterials often used in phosphorescence sensing. Specific examples on the use of phosphorescence nanoparticles in chemical sensing and bioimaging are given next. A final section intends to provide an overview of the prospects of such type of nanomaterials in the design of future devices for analytical chemistry.

Sensitive prostate specific antigen quantification using dihydrolipoic acid surface-functionalized phosphorescent quantum dots.

Herein, high-quality Mn-doped ZnS quantum dots (QDs) have been synthesized using a facile approach directly in aqueous media. The surface of the obtained QDs was further modified by cap-exchange of the native cysteine shell with dihydrolipoic acid (DHLA) ligands resulting in nanocrystals with high water-stability having an intense phosphorescent signal. Covalent bioconjugation of the DHLA-coated nanoparticles with an anti-IgG antibody was then carried out. Interestingly the QD immunoprobe (QD-labelled antibodies) maintained an intense phosphorescence emission, without any significant spectral-shift (as compared to the free QDs). Coupling of an asymmetric flow field flow fractionation technique to an elemental mass spectrometry detection enabled the accurate determination of the efficiency of the bioconjugation reaction. The obtained nanoparticle-antibody bioconjugate was then applied to develop a quantitative sandwich-type phosphorescent immunoassay for Prostate Specific Antigen (PSA), and a limit of detection (LOD) of 17 pg mL-1 of PSA was achieved and allow to quantify such biomarker in samples within clinically relevant levels. Finally, the assay was validated for the quantification of PSA in the cellular media of prostate cancer cells. Obtained results proved the robustness of the proposed immunoassay based on long-lived phosphorescence measurements against eventual photoluminescent interferences significantly affecting the conventional short-lived fluorescence detection.

Assessment of the removal of side nanoparticulated populations generated during one-pot synthesis by asymmetric flow field-flow fractionation coupled to elemental mass spectrometry.

Coupling of asymmetric flow field-flow fractionation (AF4) to an on-line elemental detection (inductively coupled plasma-mass spectrometry, ICP-MS) has been recently proposed as a powerful diagnostic tool for characterization of the bioconjugation of CdSe/ZnS core-shell Quantum Dots (QDs) to antibodies. Such approach has been used herein to demonstrate that cap exchange of the native hydrophobic shell of core/shell QDs with the bidentate dihydrolipoic acid ligands directly removes completely the eventual side nanoparticulated populations generated during simple one-pot synthesis, which can ruin the subsequent final bioapplication. The critical assessment of the chemical and physical purity of the surface-modified QDs achieved allows to explain the transmission electron microscopy findings obtained for the different nanoparticle surface modification assayed.

Highly sensitive nanoparticle-based immunoassays with elemental detection: Application to Prostate-Specific Antigen quantification.

One of the major challenges in developing novel assay methods for the detection of biomolecules is achieving high sensitivity, because of the ultralow concentrations typically in clinical samples. Here, a Mn-doped ZnS quantum dots-based immunoassay platform is presented for highly sensitive detection of cancer biomarkers. Ultrahigh sensitivity is achieved through gold deposition on the surface of the nanoparticle tags acting as catalytic seeds, thus effectively amplifying the size of the metallic nanoparticles after the immunoassay and before the tag detection. Elemental mass spectrometry measurement of the gold content allowed detection of Prostate-Specific Antigen (PSA) at the low attog mL(-1) level. Moreover, the developed method showed not only an extremely high sensitivity for PSA detection but also a broad dynamic range, higher than 8 orders of magnitude, particularly useful for clinical studies involving quantitative detection of diverse biomarkers at their very different relevant concentration levels. Its applicability to discriminate small differences in PSA concentrations at low levels (few pgmL(-1)) in real serum samples was successfully evaluated.

Sensitive targeted multiple protein quantification based on elemental detection of quantum dots.

A generic strategy based on the use of CdSe/ZnS Quantum Dots (QDs) as elemental labels for protein quantification, using immunoassays with elemental mass spectrometry (ICP-MS), detection is presented. In this strategy, streptavidin modified QDs (QDs-SA) are bioconjugated to a biotinylated secondary antibody (b-Ab2). After a multi-technique characterization of the synthesized generic platform (QDs-SA-b-Ab2) it was applied to the sequential quantification of five proteins (transferrin, complement C3, apolipoprotein A1, transthyretin and apolipoprotein A4) at different concentration levels in human serum samples. It is shown how this generic strategy does only require the appropriate unlabeled primary antibody for each protein to be detected. Therefore, it introduces a way out to the need for the cumbersome and specific bioconjugation of the QDs to the corresponding specific recognition antibody for every target analyte (protein). Results obtained were validated with those obtained using UV-vis spectrophotometry and commercial ELISA Kits. As expected, ICP-MS offered one order of magnitude lower DL (0.23 fmol absolute for transferrin) than the classical spectrophotometric detection (3.2 fmol absolute). ICP-MS precision and detection limits, however turned out to be compromised by procedural blanks. The full analytical performance of the ICP-MS-based immunoassay proposed was assessed for detection of transferrin (Tf), present at the low ng mL(-1) range in a complex "model" synthetic matrix, where the total protein concentration was 100 µg mL(-1). Finally, ICP-MS detection allowed the quantitative control of all the steps of the proposed immunoassay, by computing mass balances obtained, and the development of a faster indirect immunoassay format where the plate wells were directly coated with the whole protein mixture sample.