Development of a single‐cell X‐ray fluorescence flow cytometer

An X‐ray fluorescence flow cytometer that can determine the total metal content of single cells has been developed. Capillary action or pressure was used to load cells into hydrophilic or hydrophobic capillaries, respectively. Once loaded, the cells were transported at a fixed vertical velocity past a focused X‐ray beam. X‐ray fluorescence was then used to determine the mass of metal in each cell. By making single‐cell measurements, the population heterogeneity for metals in the µM to mM concentration range on fL sample volumes can be directly measured, a measurement that is difficult using most analytical methods. This approach has been used to determine the metal composition of 936 individual bovine red blood cells (bRBC), 31 individual 3T3 mouse fibroblasts (NIH3T3) and 18 Saccharomyces cerevisiae (yeast) cells with an average measurement frequency of ～4 cells min^?1. These data show evidence for surprisingly broad metal distributions. Details of the device design, data analysis and opportunities for further sensitivity improvement are described.     

Poly‐L‐Arginine Grafted Silica Mesoporous Nanoparticles for Enhanced Cellular Uptake and their Application in DNA Delivery and Controlled Drug Release

Mesoporous silica nanoparticles (MSNs), that are capable of delivering gene and drugs to organisms in an effective and selective way have attracted much attention lately for its potential in the treatment of cancer. However, the successful application of MSNs for delivery of plasmid DNA or drugs requires surface modification of the silica with positively charged functional groups so that it binds to the negatively charged nucleic acids and also helps it penetrate through the cell membrane. We report for the first time the synthesis of a hybrid MSN where the cell penetrating cationic polypeptide poly‐L‐arginine synthesized by NCA polymerization is grafted onto the external surface of MSN using click chemistry. These poly‐L‐arginine grafted MSNs show low cytotoxity (85% cell viability at 100 μg/mL MSN concentration) and high cellular uptake by both HeLa and A549 (>90%). The poly‐L‐arginine grafted MSNs were used effectively to deliver mCherry DNA plasmid into cells leading to expression of the protein mCherry inside the cells (transfection efficiency 60%). In contrast, poly‐L‐arginine grafted non‐porous silica nanoparticles were unable to express the protein mCherry inside the cells although their uptake into the cells was as efficient as with poly‐L‐arginine grafted MSNs. We also show preliminary results to demonstrate that these hybrid MSNs can be used as a delivery vehicle for the anticancer drug Doxorubicin towards cancerous cells HeLa and A549. The biocompatibility of poly‐L‐arginine and its cell penetrating ability are expected to make these MSN conjugates very useful carriers for the delivery of genes and drugs into cancer cells.     

Cell Uptake of Lipid‐Coated Diamond

Fluorescent nanodiamonds (FNDs) can be used as nanoscale magnetic resonance sensors and stable optical labels. As a first step for using FNDs as nanosensors inside cells, they have to be ingested. Several techniques that improve particle uptake have been used. A simple approach based on commercially available liposomes is used to improve uptake. Uptake into colon cancer cells (HT‐29 cells) is demonstrated. Additionally, it is shown for the first time that one can facilitate diamond uptake into yeast cells by removing the cell wall and creating a so‐called spheroplast. Finally, the characteristics of FNDs coated with lipids and their behavior inside the cells are evaluated.     

Microfluidic chip-inductively coupled plasma mass spectrometry for trace elements and their species analysis in cells

Abstract

Accurate identification and quantification of trace elements and their species in cells is an important prerequisite for the exploration of their physiological function and related mechanisms of process involving trace elements/species in human body. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful analytical instrument for trace elements detection, while it still suffers from insufficient limits of detection, interference from complex cell matrix, and incompatible sample consumption in cells analysis. Microfluidic chips which possess advantages of low sample/reagent consumption, rapid analysis speed and high spatial resolution provide perfect miniaturized and integrated platforms for cell analysis. In this article, microfluidic chip-ICP-MS techniques for trace elements and their species analysis in cells were reviewed. Both chip-based pretreatment techniques (e.g., magnetic solid phase microextraction (MSPME), monolithic capillary microextraction (MCME), liquid phase microextraction (LPME)) including chip-based array microextraction techniques for trace elements and their species analysis and droplet chip for single cell analysis were introduced. The newly developed methods of microfluidic chips in combination with ICP-MS for trace elements and their species analysis in small numbers of cells and even single cell were critically discussed, including chip-based MSPME/MCME/LPME-(electrothermal vaporization-ICP-)MS, on-line chip-based array MSPME/MCME-ICP-MS, on-line chip-based array MSPME-high performance liquid chromatography-ICP-MS and online droplet chip-time-resolved ICP-MS. These methodologies were demonstrated with high sensitivity, high throughput, good matrix resistance and low sample/reagent consumption, contributing to the quantification of trace elements/species in cells and even single cells. Relevant 20 references are included herein, and the development trend of microfluidic chip-ICP-MS techniques for cells analysis is prospected.     

Trace element mapping of a single cell using a hard x‐ray nanobeam focused by a Kirkpatrick‐Baez mirror system

To visualize the distributions of trace elements in biological samples such as tissues and cells at high spatial resolution, we developed a scanning x‐ray fluorescence microscope (SXFM) at SPring‐8, using a Kirkpatrick‐Baez mirror optics that enables achromatic and highly efficient focusing. To evaluate performance regarding its application to biological samples, the SXFM was used at x‐ray energy of 15 keV to observe NIH/3T3 cells in which adenosine triphosphate (ATP) synthase β (specifically localized at the mitochondria) were labeled with gold colloidal particles. Various elemental distributions were visualized at the single‐cell level, including those for P, S, Cl, Ca, Fe, Cu, Zn and Au, and we obtained high‐resolution elemental distribution maps by magnifying the labeled single mitochondrion. Maximum spatial resolution achieved in the experiments was sub‐100 nm. Copyright © 2008 John Wiley & Sons, Ltd.     

Nuclear Uptake of Gold Nanoparticles Deduced Using Dual‐Angle X‐Ray Fluorescence Mapping

Studies into the cell nucleus' incorporation of gold nanoparticles (AuNPs) are often limited by ambiguities arising from conventional imaging techniques. Indeed, it is suggested that to date there is no unambiguous imaging evidence for such uptake in whole cells, particularly at the single nanoparticle level. This shortcoming in understanding exists despite the nucleus being the most important subcellular compartment in eukaryotes and gold being the most commonly used metal nanoparticle in medical applications. Here, dual‐angle X‐ray flouresence is used to show individually resolved nanoparticles within the cell nucleus, finding them to be well separated and 79% of the intranuclear population to be monodispersed. These findings have important implications for nanomedicine, illustrated here through a specific exemplar of the predicted enhancement of radiation effects arising from the observed AuNPs, finding intranuclear dose enhancements spanning nearly five orders of magnitude.     

Non‐invasive time‐course imaging of apoptotic cells by confocal Raman micro‐spectroscopy

Confocal Raman micro‐spectroscopy (CRMS) was used to measure time‐course spectral images of live cells undergoing apoptosis without using molecular labels or other invasive procedures. Human breast cancer cells (MDA‐MB‐231) were exposed to 300 µM etoposide to induce apoptosis, and Raman spectral images were acquired from the same cells at 2‐h intervals over a period of 6 h. The purpose‐built inverted confocal Raman micro‐spectrometer integrated an environmental enclosure and wide‐field fluorescence imaging. These key instrumental elements allowed the cells to be maintained under sterile physiological conditions (37 °C, 5% CO₂) and enabled viability and apoptosis assays to be carried out on the cells at the end of CRMS measurements. The time‐course spectral images corresponding to DNA Raman bands indicated an increase in signal intensity in apoptotic cells, which was attributed to DNA condensation. The Raman spectral images of lipids indicated a high accumulation of membrane phospholipids and highly unsaturated non‐membrane lipids in apoptotic cells. This study demonstrates the potential of CRMS for label‐free time‐course imaging of individual live cells. This technique may become a useful tool for in vitro toxicological studies and testing of new pharmaceuticals, as well as other time‐dependent cellular processes, such as cell differentiation, cell cycle and cell–cell interactions. Copyright © 2010 John Wiley & Sons, Ltd.     

Single‐cell resolution in high‐resolution synchrotron X‐ray CT imaging with gold nanoparticles

Gold nanoparticles are excellent intracellular markers in X‐ray imaging. Having shown previously the suitability of gold nanoparticles to detect small groups of cells with the synchrotron‐based computed tomography (CT) technique both ex vivo and in vivo, it is now demonstrated that even single‐cell resolution can be obtained in the brain at least ex vivo. Working in a small animal model of malignant brain tumour, the image quality obtained with different imaging modalities was compared. To generate the brain tumour, 1 × 10⁵ C6 glioma cells were loaded with gold nanoparticles and implanted in the right cerebral hemisphere of an adult rat. Raw data were acquired with absorption X‐ray CT followed by a local tomography technique based on synchrotron X‐ray absorption yielding single‐cell resolution. The reconstructed synchrotron X‐ray images were compared with images obtained by small animal magnetic resonance imaging. The presence of gold nanoparticles in the tumour tissue was verified in histological sections.     

The Scattering Problem in PT‐Symmetric Periodic Structures of 1D Two‐Material Waveguide Networks

A PT‐symmetric periodic structure with two‐material waveguide networks is constructed. In this study, how changing the number of cells affects the transmission properties is investigated. The results show that the PT‐unbroken (broken) region of the system is only determined by the cell structure, regardless of the number of unit cells. This means that any system has the same exceptional points (EPs), regardless of the number of cells and as long as the cell structure is consistent. In addition, it is confirmed that the coherent perfect absorbers and lasers (CPA lasers) occur in our model. The transfer matrix method is used to derive a sufficient condition for achieving the CPA laser point. A simple, effective formula for predicting the CPA laser state in an N unit cell system is derived.     

Inelastic electron lifetimes in simple metals: Influence of spin–orbit coupling

Inelastic single‐particle lifetimes due to electron–electron Coulomb interaction are computed ab‐initio for aluminium, silver, gold and copper using an all‐electron density‐functional calculation and a parameter‐free evaluation of the dielectric function. The novel feature is the inclusion of spin– orbit coupling in the wave functions. We show that, even in light metals, spin–orbit interaction is important for the calculation of inelastic lifetimes because it influences the scattering matrix elements. The importance of spin mixing on the lifetimes in aluminium is examined. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Radioisotope‐induced EDXRF investigation of elemental uptake in cauliflower grown at MSW‐contaminated site

A ¹⁰⁹Cd radioisotope‐induced energy dispersive X‐ray fluorescence (EDXRF) study has been performed on samples of cauliflower consisting of the flower, the leaves and the associated root soil. The cauliflowers are collected from farms near the main dumping site of municipal solid waste (MSW) in the city of Kolkata, India, and also from uncontaminated farms about 50 km away from the city. The systematic investigation is primarily aimed at achieving two correlated objectives. Firstly, a unified calibration approach is undertaken for the study tool viz., EDXRF spectrometer, through the use of same instrumental scattering constants for quantification in widely differing matrices like soil and plant. Quality control was done by quantitative reproduction of National Institute of Standards and Technology–Standard Reference Materials (NIST–SRMs). Subsequently, the second objective is to comparatively study elemental uptake in the cauliflower samples from contaminated and uncontaminated farms using the same calibration. This study suggests that the elemental concentrations in the root soils and leaves of the samples vary from farm to farm, whereby the concentrations of Cu, Zn and Pb in root soils of MSW‐contaminated farms are higher by almost an order of magnitude compared to uncontaminated farms. But the most notable feature of this study is the strikingly similar elemental concentrations in the edible flower part of all samples irrespective of the soil type. Plots of the ratio of concentrations of elements in leaf to soil and in flower to leaf, observed from the present EDXRF study suggests that a preferential uptake of elements takes place at different stages. Copyright © 2010 John Wiley & Sons, Ltd.     

Oxygen Electroreduction by Single PtPd Nanocubes Encaged in Hollow Carbon Nanospheres: Improved Durability and Strong Effect of Carbon‐Shell Thickness

As a cathode reaction in fuel cells, oxygen reduction reaction (ORR) plays a critical role in determining the overall performance of a fuel cell. It is still a big challenge to find effective ways to improve the catalytic activity, efficiency, and especially stability of ORR electrocatalysts. In the present study, single nanoparticle electrocatalysis for ORR is realized for the first time by encaging PtPd nanocube in hollow and porous carbon nanosphere (PtPd@HCS). Through tuning the carbon‐shell thickness by carbonization temperature, the effects of carbon‐shell thickness on ORR catalytic performance of PtPd@HCS are systematically investigated. The PtPd@HCS calcinated at 800 °C (PtPd@HCS‐800) with the thinnest carbon shell (3.52 nm) and rich pore structure exhibits enhanced ORR catalytic activity and stability. The strategy mentioned here is expected to provide a new method to design single nanoparticle electrocatalysts for fuel cells with high catalytic performance and reduced loading of precious metals.     

Structure and properties of the intercalation compound Cu<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript>TiSe<Subscript>2</Subscript>

Compounds in the pseudobinary Cu-TiSe₂ intercalation system are directly synthesized from elements. The phase diagram of the system is investigated, the solubility limit of copper is measured, and the structure of the material is determined. In the copper concentration range up to 60 mol %, single crystals are grown and the temperature dependence of the electrical resistance is measured. It is demonstrated that, in the concentration range under investigation, the intercalation of the system with copper gives rise to a set of phenomena observed upon intercalation of alkali metals.     

Detection of bacteria aided by immuno‐nanoparticles

Magnetic immuno‐nanorice particles were used for the capture and detection of Escherichia coli (E. coli) bacteria. The selectivity of the method was attained by attaching a specific anti‐E. coli antibody on the surface of the nanorice, binding exclusively to E. coli. The antibody attachment to the nanorice (60% sorption efficiency) took place through protein‐A molecules (82% uptake). Once E. coli was captured, the immuno‐nanorice‐bacteria complex was separated from the solution using the magnetic property of the nanorice. The detection of bacteria sorbed onto the immuno‐nanorice was accomplished using the ultra‐violet resonance Raman (UVRR) method, detecting single bacterial cells. Specific information concerning the aromatic residues tyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp) was derived. The discriminant function and cluster hierarchical analysis confirmed the specific and reliable bacteria‐detection capabilities. Copyright © 2007 John Wiley & Sons, Ltd.     

Comparison of standard‐based and standardless methods of quantification used in X‐ray fluorescence analysis: Application to the exoskeleton of clams

In this work, we aim at achieving the most accurate quantitative determination of the composition of exoskeletons of bivalves from Tagus estuary with Energy Dispersive X‐ray Fluorescence. Samples from the shells of clams ranging from the Bronze Ages to the 16th century A.D . belonging to the Museu Arqueológico de Almada, and also from the shells of clams collected recently in the same region, were analyzed for comparison of the trace element composition and detection of heavy metals. The analysis was performed with 2 Energy Dispersive X‐ray Fluorescence setups, one with triaxial geometry and another with a conventional geometry and vacuum capabilities. Samples were pressed as pellets, and the spectra collected with both setups were evaluated using standardless fundamental parameter based software's implemented in each setup, and by comparing with standard reference materials of similar matrix. The comparison of the results obtained with different methods lead to the conclusion that the most realistic results were obtained with calibration curves obtained with external standards and correction the fluorescent intensities with the Compton scattering peak. When comparing the obtained concentrations for all the analyzed periods, results showed a decrease of Fe in the 12th Century. Regarding the environmental current state of the Tagus estuary, there were no heavy metals detected above the safety regulations.     

Generation of experimental L x‐ray fluorescence cross‐sections for inter‐elements at inter‐energies EL1 ≤ E ≤ EK (computer program IGELCS)

The experimental values of L x‐ray fluorescence (XRF) cross‐sections are not available for all the elements in the range La–U, at all the photon energies E in the range EL1 ≤ E ≤ EK. To generate L XRF cross‐sections, where experimental measurements are not available, two empirical relations have been developed, one between the L XRF cross‐sections and photon energy and the other between the L XRF cross‐sections and atomic number. For the measured data on L XRF cross‐sections at incident energies between Ll and K edges of an element and the data on L XRF cross‐sections for elements in the range 57 ≤ Z ≤ 92 at an energy value, polynomial fits have been derived. The L XRF cross‐section values generated with the derived empirical relations were found to be in agreement with the experimental values within their experimental uncertainties. Subsequently, a software code IGELCS was developed to interpolate and to generate the cross‐sections at inter‐energies and for inter‐elements in a single computer run. The running of the software requires minimum input data on five elements at five common energies. Copyright © 2003 John Wiley & Sons, Ltd.     

Prominin‐1‐Specific Binding Peptide‐Modified Apoferritin Nanoparticle Carrying Irinotecan as a Novel Radiosensitizer for Colorectal Cancer Stem‐Like Cells

Resistance of cancer stem cells to radiotherapy remains a major obstacle to successful cancer management. Prominin‐1 (PROM1) is a cancer stem cell marker. Nanoparticle (NP) chemotherapeutics preferentially accumulate in tumors and are able to target cancer and cancer stem‐like cells through cancer cell‐specific ligands, making them uniquely suited as radiosensitizers for chemoradiation therapy. Using a biocompatible apoferritin NP, a PROM1‐targeted NP carrying irinotecan (PROM1‐NP) is engineered. The synergistic effect of the NP and irradiation is evaluated in PROM1‐overexpressing HCT‐116 colorectal cancer cell lines in vitro and in vivo. PROM1‐NP has a size of 17.2 ± 0.2 nm and surface charge of ?13.5 ± 0.2 mV. It demonstrates higher intracellular uptake than nontargeted NP or irinotecan alone. Treatment with PROM1‐NPs decreases HCT‐116 cell proliferation in a dose‐ and time‐dependent manner. In vitro radiosensitization reveals that PROM1‐NP is significantly more effective as a radiosensitizer than nontargeted NP or irinotecan. HCT‐116 tumor xenograft growth is markedly slower following treatment with PROM1‐NP plus irradiation, suggesting that PROM1‐NP is more effective as a radiosensitizer than irinotecan and nontargeted NP in vivo. This study provides the first preclinical evidence of the effectiveness of PROM1‐targeted NP formulation of irinotecan as a radiosensitizer.     

Determination of residual metal concentration in metallurgical slag after acid extraction using total reflection X‐ray fluorescence spectrometry

Total reflection X‐ray fluorescence spectrometry (TXRF) was used to determine residual metal content in metallurgical slag after sulfuric acid extraction. A slag sample provided by a copper smelter industry and submitted to a recycling process with the aim of extracting valuable metals was analyzed. Slurry sampling was evaluated as a simple sample preparation procedure for the determination of major (Si, Fe, K, Ca) and trace elements (Ti, Ni, Cu, Zn, As, Pb, Sr, Rb, Mn) in two certified reference materials (lake and river sediments) to evaluate the accuracy of the procedure. Precision was evaluated as relative standard deviation (%) of three replicates, being usually lower than 10%. Detection limits were in the range of 1.1–1079 µg g^?1. Additionally, it was evaluated to use Si – a major component of the slag – as intrinsic reference to determine residual elements in the slag residues. A two‐element internal standard (Ga + P) was employed to determine all the elements. Ga was used as internal standard for the determination of elements from K to Pb, and P was required for the quantification of Si. The optimal procedure of slurry sampling was applied for the analysis of the original metallurgical slag and the solid residues. It was found that As and Pb are accumulated in the slag, whereas Cu, Zn, and Fe are extracted to a percentage >90% from the slag. This was confirmed by analysis of the sulfuric acid extracts of the metallurgical slag. Copyright © 2014 John Wiley & Sons, Ltd.     

Assessment of chemical elements in cosmetics' eyeshadows by X‐ray fluorescence and International Nomenclature of Cosmetic Ingredients characterization

Energy dispersive X‐ray fluorescence (EDXRF) technique, as a qualitative and quantitative analysis, was used for inorganic chemical elements determination (K, S, Cl, Al, Si, Ca, Ti, Mn, Fe, Pb, Zn, Rb, and Bi) in eyeshadows for safe human use. International Nomenclature of Cosmetic Ingredients standardized nomenclature was used for labels investigation to obtain data on legal regularity. Data of 23 samples were clustered by similarity, measuring relative concentrations of detected chemical elements. Calculating the correlation among such values, a similarity matrix was used to generate a dendrogram. Pb was found in samples silver color S12E and copper color S22I above permissible limits (20 μg/g). Same composition was reported for the pink (S01A), black (S02A), and brown (S03A) samples, but the same chemical elements were not detected by EDXRF in them. The best correlation was found between samples S08D and S23 J (0.961). The least correlation was 0.0012 between S01A and S12E. The clustering analysis showed 7 groups of similar samples according to EDXRF data. Relations among 6 eyeshadows' colors and chemical compositions were discovered by using decision trees, where the most determinant elements were Mn, S, Cl, Ca, and Fe, in this order. Commercial regularization and International Nomenclature of Cosmetic Ingredients standardization of eyeshadows in Brazil are not fully complied by the manufacturers of the investigated brands.     

Accurate standard‐based quantification of X‐ray fluorescence data using metal‐contaminated plant tissue

Quantitative X‐ray fluorescence (XRF) measurements have been conducted on naturally lead‐contaminated samples. The calibration procedure using the ratio of fluorescence to Compton scattered radiation was investigated using Monte Carlo simulation. Experimental results with low‐energy photons (14 keV) and simulations show a very good linearity of the fluorescence to Compton ratio as a function of metal concentration. Lead (Pb), iron (Fe) and zinc (Zn) are measured in samples of Phaseolus vulgaris (bean seeds) that have been grown using a nutritive solution with different Pb dopings. Naturally contaminated samples are thus obtained. The calibration must be done for fixed conditions of X‐ray energy and scattering angle, while X‐ray beam intensity and detector to sample distance can change from one sample to another. Simulation allows to evaluate the matrix effect on the calibration curve, and shows that linearity is preserved even in the presence of other heavy elements in the fluorescence spectrum. However, calibration must be done using samples with similar matrix as it affects the slope of the curve. Copyright © 2010 John Wiley & Sons, Ltd.