Changes in growth, lanthanide binding, and gene expression in Pseudomonas alloputida KT2440 in response to light and heavy lanthanides.

Pseudomonas alloputida KT2440 is a ubiquitous, soil-dwelling bacterium that metabolizes recalcitrant and volatile carbon sources. The latter is utilized by two redundant, Ca- and lanthanide (Ln)-dependent, pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ ADH), PedE and PedH, whose expression is regulated by Ln availability. P. alloputida KT2440 is the best-studied non-methylotroph in the context of Ln-utilization. Combined with microfluidic cultivation and single-cell elemental analysis, we studied the impact of light and heavy Ln on transcriptome-wide gene expression when growing P. alloputida KT2440 with 2-phenylethanol as the carbon and energy source. Light Ln (La, Ce, and Nd) and a mixture of light and heavy Ln (La, Ce, Nd, Dy, Ho, Er, and Yb) had a positive effect on growth, whereas supplementation with heavy Ln (Dy, Ho, Er, and Yb) exerted fitness costs. These were likely a consequence of mismetallation and non-utilizable Ln interfering with Ln sensing and signaling. The measured amounts of cell-associated Ln varied between elements. Gene expression analysis suggested that the Ln sensing and signaling machinery, the two-component system PedS2R2 and PedH, responds differently to (non-)utilizable Ln. We expanded our understanding of the lanthanide (Ln) switch in P. alloputida KT2440, demonstrating that it adjusts the levels of pedE and pedH transcripts based on the availability of Ln. We propose that the usability of Ln influences the bacterium's response to different Ln elements.IMPORTANCEThe Ln switch, the inverse regulation of Ca- and Ln-dependent PQQ ADH in response to Ln availability in organisms featuring both, is central to our understanding of Ln utilization. Although the preference of bacteria for light Ln is well known, the effect of different Ln, light and heavy, on growth and gene expression has rarely been studied. We provide evidence for a fine-tuning mechanism of Ca- and Ln-dependent PQQ ADH in P. alloputida KT2440 on the transcriptome level. The response to (non-)utilizable Ln differs depending on the element. Ln commonly co-occur in nature. Our findings underline that Ln-utilizing microbes must be able to discriminate between Ln to use them effectively. Considering the prevalence of Ln-dependent proteins in many microbial taxa, more work addressing Ln sensing and signaling is needed. Ln availability likely necessitates different adaptations regarding Ln utilization.

Exploring phosphate impact on arsenate uptake and distribution in freshwater phytoplankton: Insights from single-cell ICP-MS.

In this study, we investigated the interaction between arsenate (AsV) and phosphate (PO43-) in freshwater phytoplankton using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). This study aimed to elucidate the influence of varying PO43- concentrations on arsenic (As) uptake and distribution at the single-cell level, providing insights into intraspecies diversity. Two species of freshwater phytoplanktons, Scenedesmus acutus and Pediastrum duplex, were cultured under different concentrations of PO43- and AsV in a controlled laboratory environment. Scenedesmus acutus, a species with strong salt tolerance, and Pediastrum duplex, known for its weak salt tolerance, were selected based on their contrasting behaviors in previous studies. SC-ICP-MS revealed non-uniform uptake of As by individual phytoplankton cells, with distinct variations in response to PO43- availability. Arsenic uptake by both species declined with a high PO43- level after 7 days of exposure. However, after 14 days, As uptake increased in S. acutus with higher PO43- concentrations, but decreased in P. duplex. Moreover, our findings revealed differences in cell morphology and membrane integrity between the two species in response to AsV and various PO43- concentrations. S. acutus maintained cell integrity under all experimental culture conditions, whereas P. duplex experienced cell lysis at elevated AsV and PO43- concentrations. This study highlights the varying responses of freshwater phytoplankton to changes in AsV and PO43- levels and underscores the advantages of SC-ICP-MS over conventional ICP-MS in providing detailed, cellular level insights. These findings are crucial for understanding and managing As pollution in aquatic ecosystems.

Single-Cell Analysis of Elemental Contents by Laser Ablation-Inductively Coupled Plasma Mass Spectrometry.

Elemental analysis at the single-cell level is an emerging technique in the field of inductively coupled plasma mass spectrometry (ICP-MS). In comparison to the analysis of cell suspensions by fast time-resolved analysis, single-cell sampling by laser ablation (LA) allows the discriminatory analysis of single cells according to their size and morphology. In this study, we evaluated the changes in elemental contents in a single cell through differentiation of rat adrenal pheochromocytoma into neuron-like cells by LA-ICP-MS. The contents of seven essential minerals were increased about 2-3 times after the differentiation. In addition, we evaluated the degree of differentiation at the single-cell level by means of imaging cytometry after immunofluorescence staining of microtubule-associated protein 2 (Map2), a neuron-specific protein. The fluorescence intensities of Alexa Fluor 488-conjugated secondary antibody against the anti-Map2 primary antibody showed large variations among the cells after the onset of differentiation. Although the average fluorescence intensity was increased through the differentiation, there were still less-matured neuron-like cells that exhibited a lower fluorescence intensity after 5 days of differentiation. Since a positive correlation between the fluorescence intensity and the cell size in area was found, we separately measured the elemental contents in the less-matured smaller cells and well-matured larger cells by LA-ICP-MS. The larger cells had higher elemental contents than the smaller cells, indicating that the essential minerals are highly required at a later stage of differentiation.

Bioconcentration of Inorganic and Methyl Mercury by Algae Revealed Using Dual-Mass Single-Cell ICP-MS with Double Isotope Tracers.

Algae are an entry point for mercury (Hg) into the food web. Bioconcentration of Hg by algae is crucial for its biogeochemical cycling and environmental risk. Herein, considering the cell heterogeneity, we investigated the bioconcentration of coexisting isotope-labeled inorganic (199IHg) and methyl Hg (201MeHg) by six typical freshwater and marine algae using dual-mass single-cell inductively coupled plasma mass spectrometry (scICP-MS). First, a universal pretreatment procedure for the scICP-MS analysis of algae was developed. Using the proposed method, the intra- and interspecies heterogeneities and the kinetics of Hg bioconcentration by algae were revealed at the single-cell level. The heterogeneity in the cellular Hg contents is largely related to cell size. The bioconcentration process reached a dynamic equilibrium involving influx/adsorption and efflux/desorption within hours. Algal density is a key factor affecting the distribution of Hg between algae and ambient water. Cellular Hg contents were negatively correlated with algal density, whereas the volume concentration factors almost remained constant. Accordingly, we developed a model based on single-cell analysis that well describes the density-driven effects of Hg bioconcentration by algae. From a novel single-cell perspective, the findings improve our understanding of algal bioconcentration governed by various biological and environmental factors.

Study of Legionella pneumophila treatment with copper in drinking water by single cell-ICP-MS.

Legionella pneumophila is a persistent opportunistic pathogen that poses a significant threat to domestic water systems. Previous studies suggest that copper (Cu) is an effective antimicrobial in water systems. A rapid and sensitive quantification method is desired to optimize the conditions of L. pneumophila treatment by Cu and to better understand the interaction mechanisms between Cu and cells. In this study, we developed a highly sensitive single cell (SC)-ICP-MS method to monitor L. pneumophila cell concentration and track their uptake of Cu. The SC-ICP-MS method showed excellent sensitivity (with a cell concentration detection limit of 1000 cells/mL), accuracy (good agreement with conventional hemocytometry method), and precision (relative standard deviation < 5%) in drinking water matrix. The cupric ions (Cu2+) treatment results indicated that the total L. pneumophila cell concentration, Cu mass per cell, colony-forming unit counting, and Cu concentration in supernatant all exhibited a dose-dependent trend, with 800-1200 µg/L reaching high disinfection rates in drinking water. The investigation of percentages of viable and culturable, viable but nonculturable (VBNC), and lysed cells suggested there always were VBNC present at any Cu concentration. Experimental results of different Cu2+ treatment times further suggested that L. pneumophila cells developed an antimicrobial resistant mechanism with the prolonged Cu exposure. This is the first quantification study on the interactions of Cu and L. pneumophila in drinking water using SC-ICP-MS.

Single-cell ICP-MS for evaluating the Se-protective effect against MeHg+-induced neurotoxicity in human neuroblastoma cell line (SH-SY5Y).

The protective effect of selenium (Se) against Hg-induced neurotoxicity has been widely investigated; however, the mechanisms behind this interaction have not been fully elucidated yet. In the current work, the role of Se against MeHg+-induced cytotoxicity in the human neuroblastoma cell line (SH-SY5Y) is reported for the first time by tracking Hg uptake and accumulation at the single-cell level by inductively coupled plasma-mass spectrometry in single-cell mode (SC-ICP-MS). The influence of different Se species (SeMet, SeMeSeCys, citrate-SeNPs, and chitosan-SeNPs) on MeHg+ cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. SeMet and SeMeSeCys exhibited protective effects against MeHg+-induced cell death, particularly at high MeHg+ concentrations (LC50). In addition, chitosan-SeNPs showed greater protection compared to citrate-SeNPs when co-exposed with MeHg+. Interestingly, SC-ICP-MS unveiled the heterogeneous distribution of Hg uptake by SH-SY5Y cells. Co-exposure of SeMet and SeMeSeCys with MeHg+ led to a reduction of the amount of Hg accumulated per individual cell, which decreased the maximum level of Hg per cell by half (from 60 fg Hg cell-1 to 30 fg Hg cell-1) when SeMet was present, along with a decrease in the percentage of cells that accumulated the highest quantity of MeHg+. All these data corroborate the protective role of Se against Hg toxicity at the cellular level.

Dynamic elementomics of single-cell ICP-MS-derived signals in normal and calcium pump PMCA4-deficient mouse epididymal sperm during capacitation.

Currently, clinical analysis of male infertility mainly relies on parameters of semen and sperm cells. However, the high diagnostic failure rates indicate that the current assessment methods are not sufficient and a new approach to evaluating sperm function still needs to be developed. Here we explored the feasibility of single-cell inductively coupled plasma mass spectrometry (sc-ICP-MS)-derived profiles to determine the elemental characteristics in viable capacitated sperm under normal and deficient conditions. To validate the measurements, we used male sterile Pmca4-knockout (KO) mice with impaired calcium clearance, known to be dysregulated due to loss of calcium efflux capacity during sperm capacitation. Consistently, we observed significantly increased calcium intensities in Pmca4-KO sperm upon capacitation stimulation compared with control sperm from the caudaepididymides of wild-type control (WT) mice. More importantly, we explored that the characteristic signatures of calcium intensities in individual spikes derived from sc-ICP-MS was consistent with the dynamics of relative calcium levels in single sperm reported in the literature. Prominent alterations were also observed in the dynamic signatures of sc-ICP-MS-derived profiles of essential elements, particularly the redox-labile elements including copper, iron, manganese, selenium, and zinc in Pmca4-KO sperm compared to WT controls. Therefore, our study demonstrates that elementomics of sc-ICP-MS-derived signals can reveal ionic dysregulation in plasma membrane Ca2+-ATPase isoform 4 protein deficient sperm, and that sc-ICP-MS assay can be applied for functional analysis of viable sperm in functional activities, such as capacitation stimulation. We propose that cell elementomics can be used as an alternative approach to assessing sperm quality and male fertility at the single-cell level.

Investigation of the Impact of L-Phenylalanine and L-Tyrosine Pre-Treatment on the Uptake of 4-Borono-L-Phenylalanine in Cancerous and Normal Cells Using an Analytical Approach Based on SC-ICP-MS.

Boron has gained significant attention in medical research due to its B-10 isotope's high cross section for the reaction with thermal neutrons, generating ionizing particles that can eliminate cancer cells, propelling the development of boron neutron capture therapy (BNCT) for cancer treatment. The compound 4-borono-L-phenylalanine (BPA) has exhibited potential in BNCT clinical trials. Enhancing BPA uptake in cells involves proposing L-amino acid preloading. This study introduces a novel analytical strategy utilizing ICP-MS and single cell ICP-MS (SC-ICP-MS) to assess the effectiveness of L-tyrosine and L-phenylalanine preloading on human non-small cell lung carcinoma (A549) and normal Chinese hamster lung fibroblast (V79-4) models, an unexplored context. ICP-MS outcomes indicated that L-tyrosine and L-phenylalanine pre-treatment increased BPA uptake in V79-4 cells by 2.04 ± 0.74-fold (p = 0.000066) and 1.46 ± 0.06-fold (p = 0.000016), respectively. Conversely, A549 cells manifested heightened BPA uptake solely with L-tyrosine preloading, with a factor of 1.24 ± 0.47 (p = 0.028). BPA uptake remained higher in A549 compared to V79-4 regardless of preloading. SC-ICP-MS measurements showcased noteworthy boron content heterogeneity within A549 cells, signifying diverse responses to BPA exposure, including a subset with notably high BPA uptake. This study underscores SC-ICP-MS's utility in precise cellular boron quantification, validating cellular BPA uptake's heterogeneity.

Cytotoxicity, uptake and accumulation of selenium nanoparticles and other selenium species in neuroblastoma cell lines related to Alzheimer's disease by using cytotoxicity assays, TEM and single cell-ICP-MS.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, representing 80% of the total dementia cases. The "amyloid cascade hypothesis" stablishes that the aggregation of the beta-amyloid protein (Aß42) is the first event that subsequently triggers AD development. Selenium nanoparticles stabilized with chitosan (Ch-SeNPs) have demonstrated excellent anti-amyloidogenic properties in previous works, leading to an improvement of AD aetiology. Here, the in vitro effect of selenium species in AD model cell line has been study to obtain a better assessment of their effects in AD treatment. For this purpose, mouse neuroblastoma (Neuro-2a) and human neuroblastoma (SH-SY5Y) cell lines were used. Cytotoxicity of selenium species, such as selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys) and Ch-SeNPs, has been determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry methods. Intracellular localisation of Ch-SeNPs, and their pathway through SH-SY5Y cell line, have been evaluated by transmission electron microscopy (TEM). The uptake and accumulation of selenium species by both neuroblastoma cell lines have been quantified at single cell level by single cell- Inductively Coupled Plasma with Mass Spectrometry detection (SC-ICP-MS), with a previous optimisation of transport efficiency using gold nanoparticles (AuNPs) ((69 ± 3) %) and 2.5 mm calibration beads ((92 ± 8) %). Results showed that Ch-SeNPs would be more readily accumulated by both cell lines than organic species being accumulation ranges between 1.2 and 89.5 fg Se cell-1 for Neuro-2a and 3.1-129.8 fg Se cell-1 for SH-SY5Y exposed to 250 µM Ch-SeNPs. Data obtained were statistically treated using chemometric tools. These results provide an important insight into the interaction of Ch-SeNPs with neuronal cells, which could support their potential use in AD treatment.

Anticancer Screening of Ru(II) Photoredox Catalysts at Single Cancer Cell Level.

The rapid efflux of Pt-based chemotherapeutics by cancer cells is one of the major causes of drug resistance in clinically available drugs. Therefore, both the high cellular uptake as well as adequate retention efficiency of an anticancer agent are important factors to overcome drug resistance. Unfortunately, rapid and efficient quantification of metallic drug concentration in individual cancer cells still remains a tricky problem. Herein, with the help of newly developed single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), we have found that the well-known Ru(II)-based complex, Ru3, displayed remarkable intracellular uptake and retention efficiency in every single cancer cell with high photocatalytic therapeutic activity to overcome cisplatin resistance. Moreover, Ru3 has shown sensational photocatalytic anticancer properties with excellent in-vitro and in-vivo biocompatibility under light exposure.

Investigating the Cellular Uptake of Model Nanoplastics by Single-Cell ICP-MS.

A synthetic route to producing gold-doped environmentally relevant nanoplastics and a method for the rapid and high-throughput qualitative investigation of their cellular interactions have been developed. Polyethylene (PE) and polyvinyl chloride (PVC) nanoparticles, doped with ultrasmall gold nanoparticles, were synthesized via an oil-in-water emulsion technique as models for floating and sedimenting nanoplastics, respectively. Gold nanoparticles were chosen as a dopant as they are considered to be chemically stable, relatively easy to obtain, interference-free for elemental analysis, and suitable for bio-applications. The suitability of the doped particles for quick detection via inductively coupled plasma mass spectrometry (ICP-MS), operating in single-cell mode (scICP-MS), was demonstrated. Specifically, the method was applied to the analysis of nanoplastics in sizes ranging from 50 to 350 nm, taking advantage of the low limit of detection of single-cell ICP-MS for gold nanoparticles. As an initial proof of concept, gold-doped PVC and PE nanoplastics were employed to quantify the interaction and uptake of nanoplastics by the RAW 264.7 mouse macrophage cell line, using scICP-MS and electron microscopy. Macrophages were chosen because their natural biological functions would make them likely to internalize nanoplastics and, thus, would produce samples to verify the test methodology. Finally, the method was applied to assess the uptake by CaCo-2 human intestinal cells, this being a more relevant model for humanexposure to those nanoplastics that are potentially available in the food chain. For both case studies, two concentrations of nanoplastics were employed to simulate both standard environmental conditions and exceptional circumstances, such as pollution hotspot areas.

Exploring the performance of quadrupole, time-of-flight, and multi-collector ICP-MS for dual-isotope detection on single nanoparticles and cells.

To meet the demand for multi-element/isotope analysis at the single nanoparticle (NP) or cell level, different types of inductively coupled plasma mass spectroscopy (ICP-MS) have been used to simultaneously monitor multiple mass-to-charge ratios in single-particle/cell ICP-MS (SP/SC-ICP-MS) analysis. Systematic evaluation and comparison of the performance of these techniques are urgently required. Herein, three ICP-quadrupole (Q)-MS, two ICP-time of flight (TOF)-MS, and one multi-collector (MC)-ICP-MS instruments were employed to simultaneously detect 107Ag and 109Ag on single Ag NPs and Ag-exposed cyanobacteria cells. The evaluation was conducted by comparing the measured event-specific 109Ag:107Ag ratios with the natural ratio. Duration of NP or cell events and time resolution in the peak hopping mode were the main factors affecting the performance of ICP-Q-MS. Under the optimal condition (100 µs for both dwell time and settling time), less than 45% of the NP or cell events had a 109Ag:107Ag ratio deviating <30% from the natural ratio. Most events obtained via ICP-TOF-MS were paired events with both isotopes detected. For large-size NPs and cells with high exposure levels, nearly 80% of the events had a ratio deviation within ±30%. MC-ICP-MS performed particularly well in isotope determination with all the events having a ratio deviation within ±5%. For ICP-TOF-MS and MC-ICP-MS, the signal intensity of the events was the main factor affecting the accuracy of the measured 109Ag:107Ag ratios due to the counting statistics. The established methods and results provide insight on the analyses of two elements/isotopes or more on single NPs or cells. Based on the comparison of the advantages and limitations of these instruments, this study provides a critical reference for future multi-element/isotope SP/SC-ICP-MS analyses.

Towards single cell ICP-MS normalized quantitative experiments using certified selenized yeast.

In the search for a normalized procedure to replicate and compare single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) experiments, SELM-1, a certified reference material containing selenium enriched yeast cells has been used. Selenium concentrations (both, intra- and extracellular) have been measured using either sequential or simultaneous procedures. Regarding quantitative results, the sequential procedure involving cell washing followed by freeze drying of the washed material and intracellular Se quantification using SC-ICP-MS provided best results. In this case, intracellular Se accounted for 1304 ± 48 mg kg-1 (corresponding to 64% of the certified Se content). The average mass of Se per yeast cell was 41.6 fg Se with a dispersion of 1.6-279 fg Se/cell. In the isolated extracellular Se fraction, the Se concentration accounted for 412 ± 48 mg kg-1 (about 21% of the total Se). Thus, the sequential procedure provided a total Se recovery of about 85% with respect to the certified value. The direct dilution and simultaneous measurement of intra- and extracellular Se by SC-ICP-MS provided results of 1024 ± 42 mg kg-1 for intracellular and 316 ± 30 mg kg-1 for extracellular Se representing a total recovery of about 66%. In both cases, an initial thorough characterization of the cell density per solid weighed material was conducted by flow cytometry and the cell integrity ensured using confocal microscopy. These results clearly demonstrated that with appropriate sample preparation, SC-ICP-MS is a unique tool, which is capable of providing quantitative information about intracellular and extracellular Se. In addition, SELM-1 seems the ideal tool to enable data normalization at the single cell level to replicate, benchmark, and improve new SC-ICP-MS studies by using the same material for data validation.

Single-cell and bulk ICP-MS investigation of accumulation patterns of Pt-based metallodrugs in cisplatin-sensitive and -resistant cell models.

In research enabling preclinical development and attaining a deeper understanding of the behavior of metallodrugs in cancer cells with acquired resistance, intracellular Pt accumulation could be considered an important biomarker and analytical focus. In this work, Pt accumulation patterns in terms of the number of cells and Pt mass in single cells were precisely defined by using inductively coupled plasma-mass spectrometry (ICP-MS) operating in a fast time-resolved analysis mode. This technique is otherwise known as single-cell (SC)-ICP-MS. By applying the nascent and validated SC-ICP-MS technique, comparisons across three Pt drugs (cisplatin, carboplatin, and oxaliplatin) in the A2780 and A2780cis ovarian cancer cell models could be made. Additional roles of transporters on top of passive diffusion and the drugs' bioactivity could be postulated. The SC-ICP-MS-based observations also served as a cross-validation point to augment preexisting research findings on Pt-resistance mechanisms. Conjectures regarding S and Fe metabolism were also derived based on an additional and direct ICP-MS analysis of endogenous elements. Overall, our work not only confirms the utility of SC-ICP-MS in chemotherapeutic research, but also provided insights into further ICP-MS-based analytical capacities to be developed.

Review about Powerful Combinations of Advanced and Hyphenated Sample Introduction Techniques with Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for Elucidating Trace Element Species in Pathologic Conditions on a Molecular Level.

Element analysis in clinical or biological samples is important due to the essential role in clinical diagnostics, drug development, and drug-effect monitoring. Particularly, the specific forms of element binding, actual redox state, or their spatial distribution in tissue or in single cells are of interest in medical research. This review summarized exciting combinations of sophisticated sample delivery systems hyphenated to inductively coupled plasma-mass spectrometry (ICP-MS), enabling a broadening of information beyond the well-established outstanding detection capability. Deeper insights into pathological disease processes or intracellular distribution of active substances were provided, enabling a better understanding of biological processes and their dynamics. Examples were presented from spatial elemental mapping in tissue, cells, or spheroids, also considering elemental tagging. The use of natural or artificial tags for drug monitoring was shown. In the context of oxidative stress and ferroptosis iron, redox speciation gained importance. Quantification methods for Fe2+, Fe3+, and ferritin-bound iron were introduced. In Wilson's disease, free and exchangeable copper play decisive roles; the respective paragraph provided information about hyphenated Cu speciation techniques, which provide their fast and reliable quantification. Finally, single cell ICP-MS provides highly valuable information on cell-to-cell variance, insights into uptake of metal-containing drugs, and their accumulation and release on the single-cell level.

Absolute Quantification of Nanoparticle Interactions with Individual Human B Cells by Single Cell Mass Spectrometry.

We report on the absolute quantification of nanoparticle interactions with individual human B cells using quadrupole-based inductively coupled plasma mass spectrometry (ICP-MS). This method enables the quantification of nanoparticle-cell interactions at single nanoparticle and single cell levels. We demonstrate the efficient and accurate detection of individually suspended B cells and found an ∼100-fold higher association of colloidally stable positively charged nanoparticles with single B cells than neutrally charged nanoparticles. We confirmed that these nanoparticles were internalized by individual B cells and determined that the internalization occurred via energy-dependent pathways consistent with endocytosis. Using dual analyte ICP-MS, we determined that >80% of single B cells were positive for nanoparticles. Our study demonstrates an ICP-MS workflow for the absolute quantification of nanoparticle-cell interactions with single cell and single nanoparticle resolution. This unique workflow could inform the rational design of various nanomaterials for controlling cellular interactions, including immune cell-nanoparticle interactions.

Gold nanoclusters as elemental label for the sequential quantification of apolipoprotein E and metallothionein 2A in individual human cells of the retinal pigment epithelium using single cell-ICP-MS.

Gold nanoclusters (AuNCs) with a diameter of 1.99 nm on average were synthesized and applied as labels in immunoprobes for the determination of cytosolic proteins in individual human retinal pigment epithelium (HRPEsv) cells by single cell - inductively coupled plasma - mass spectrometry (sc-ICP-MS). For quantitative purposes, the number of gold atoms per immunoprobe (i.e., the amplification factor) was determined; 466 gold atoms on average were obtained. Human metallothioneins (MT), including the 2A isoform (MT2A), and apolipoprotein E (APOE) play an important role under inflammation and oxidation processes in the RPE. The new single biomarker strategy introduced was applied to the sequential determination of MT2A and APOE in HRPEsv cells under pro-inflammatory and control conditions through the development of immunoassays with the corresponding AuNCs immunoprobes and the measurement of the 197Au+ signal by sc-ICP-MS. In addition, 56Fe+ signal was measured as constituent element of HRPEsv cells in order to check the integrity of the cells after the immunoassay and to confirm the number of cell events detected when monitoring the protein label (197Au+). Optimisation of parameters related with the sample preparation for the analysis of cytosolic proteins in intact HRPEsv cells was carried out. The method was successfully applied to the determination of both proteins in control cells and cells treated with the recombinant human interleukin-1α. Quantitative results obtained per cell for the average protein amounts of APOE and MT2A using the sc-ICP-MS procedure were corroborated with commercial ELISA kits.

Evaluation of copper uptake in individual spores of Streptomyces coelicolor and endogenic nanoparticles formation to modulate the secondary metabolism.

Copper modulates secondary metabolism in Streptomyces. Although the cytosolic copper concentration is controlled by several chaperones and transporters, the formation of copper nanoparticles (NPs) and its relation to the antibiotic production has never been established in the model Streptomyces coelicolor. In this work, state-of-the-art analytical tools are used to evaluate the incorporation of copper in individual spores of S. coelicolor at different exposure concentrations (40, 80, and 160 µM Cu). Among them, the use of single cell-inductively coupled plasma-mass spectrometry revealed incorporation levels in the range of 2 to 2.5 fg/spore (median) increasing up to 4.75 fg/spore at the upper exposure concentrations. The copper storage within the spores in the form of NPs was evaluated using a combination of single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The obtained data confirmed the presence of NPs in the range of 8 to 40 (mean size 21 nm) inside S. coelicolor spores. The presence of the NPs was correlated with the actinorhodin production in liquid non-sporulating cultures amended with up to 80 µM Cu. However, further increase to 160 µM Cu, yielded to a significant decrease in antibiotic production. Secondary metabolism is activated under stressful conditions and cytosolic copper seems to be one of the signals triggering antibiotic production. Particularly, NP formation might contribute to modulate the secondary metabolism and prevent for copper toxicity. This work describes, for first time, the formation of endogenous copper NPs in S. coelicolor and reveals their correlation with the secondary metabolism.

Single-Cell Quantification of a Highly Biocompatible Dinuclear Iridium(III) Complex for Photocatalytic Cancer Therapy.

Quantifying the content of metal-based anticancer drugs within single cancer cells remains a challenge. Here, we used single-cell inductively coupled plasma mass spectrometry to study the uptake and retention of mononuclear (Ir1) and dinuclear (Ir2) IrIII photoredox catalysts. This method allowed rapid and precise quantification of the drug in individual cancer cells. Importantly, Ir2 showed a significant synergism but not an additive effect for NAD(P)H photocatalytic oxidation. The lysosome-targeting Ir2 showed low dark toxicity in vitro and in vivo. Ir2 exhibited high photocatalytic therapeutic efficiency at 525 nm with an excellent photo-index in vitro and in tumor-bearing mice model. Interestingly, the photocatalytic anticancer profile of the dinuclear Ir2 was much better than the mononuclear Ir1, indicating for the first time that dinuclear metal-based photocatalysts can be applied for photocatalytic anticancer treatment.

Cell population behavior of the unicellular red alga Galdieria sulphuraria during precious metal biosorption.

This study investigates the biosorption mechanism, including cell population behavior, of trace amounts of precious metals (gold, palladium, and platinum) in a unicellular red alga, Galdieria sulphuraria. Single-cell inductively coupled plasma mass spectrometry showed that the number of adsorbing cells and the concentration of adsorbed metal per cell varied depending on solution acidity and metal species. The X-ray absorption fine structure in 5 mM HCl solution indicated that the adsorbed Au formed inner-sphere complexes with S, whereas the adsorbed Pd and Pt formed an inner-sphere complexes with N and/or S. In 500 mM HCl solution, the adsorbed Au and Pd formed inner-sphere complexes only with S, and the Au formed a structure similar to Au2S. At higher acidity, Au and Pd were recovered by interacting with residues that formed more stable complexes, which was accompanied by changes in the behavior of cell populations adsorbing the metals. This is the first study to demonstrate the relationship between changes in the behavior of cell populations and chemical interactions that occur between substrate elements and biomaterial residues during biosorption. The findings of this study provide deeper insights into the biosorption mechanism and a background for the design of an environmentally friendly biosorbent.