Highly sensitive quantitative detection of glycans on exosomes in renal disease serums using fluorescence signal amplification strategies.

Exosomal glycoproteins play a significant role in many physiological and pathological processes. However, the detection of exosome surface glycans is currently challenged by the complexity of biological samples or the sensitivity of the methods. Herein, we prepared a novel fluorescent probe of biotin-functionalized nanocrystals (denoted as CdTe@cys-biotin) and applied it for the first time for the detection of the expression of exosomal surface glycans using a fluorescence amplification strategy. First, the dual affinity of TiO2 and CD63 aptamers of Fe3O4@TiO2-CD63 was utilized to rapidly and efficiently capture exosomes within 25 min. In this design, interference from other vesicles and soluble impurities can be avoided due to the dual recognition strategy. The chemical oxidation of NaIO4 oxidized the hydroxyl sites of exosomal surface glycans to aldehydes, which were then labeled with aniline-catalyzed biotin hydrazide. Using the high affinity between streptavidin and biotin, streptavidin-FITC and probes were successively anchored to the glycans on the exosomes. The fluorescent probe achieved the dual function of specific recognition and fluorescent labeling by modifying biotin on the surface of nanocrystals. This method showed excellent specificity and sensitivity for exosomes at concentrations ranging from 3.30 × 102 to 3.30 × 106 particles/mL, with a detection limit of 121.48 particles/mL. The fluorescent probe not only quantified exosomal surface glycans but also distinguished with high accuracy between serum exosomes from normal individuals and patients with kidney disease. In general, this method provides a powerful platform for sensitive detection of exosomes in cancer diagnosis.

Triple-sensitivity high-spatial-resolution X-ray computed tomography using a cadmium-telluride detector and its beam-hardening effect.

To observe blood vessels at high contrasts, we constructed a first-generation triple-sensitivity X-ray computed tomography (TS-CT) scanner using a cadmium-telluride (CdTe) detector and a triple-amplifying system. X-ray photons are absorbed by the CdTe crystal, and the electric charges produced by photons are converted into voltages using a current-to-voltage (I-V) amplifier, and the I-V output is amplified by a voltage-to-voltage (V-V) amplifier. The V-V output 1 is sent to a dual V-V amplifier through a 5.0-m-length coaxial cable and amplified to two-different outputs of 2 and 3. The three outputs 1-3 are sent to a personal computer through an analog-to-digital converter to reconstruct three tomograms simultaneously. In the TS-CT, the scattering photons from the object are extremely reduced using a 0.5-mm-diameter lead pinhole behind the object. The translation and rotation steps were 0.1 mm and 0.5°, respectively, and the spatial resolutions were 0.25 × 0.25 mm2. The scanning time was 19.6 min, and blood vessels were visible using gadolinium contrast media. In particular, the effective photon energy increased with increasing amplification factor of the amplifier caused by beam hardening of the object.

Indirect determination of mercury(II) by using magnetic nanoparticles, CdS quantum dots and mercury(II)-binding aptamers, and quantitation of released CdS by graphite furnace AAS.

This work describes an aptamer based method for highly sensitive determination of Hg(II). A Hg(II)-binding ssDNA aptamer was linked to silica-coated magnetic nanoparticles (magNPs). Then, a conjugate composed of graphene and CdS quantum dots (Gr-CdS) was linked to the complementary ssDNA. On mixing the two components, a duplex of type magNP-dsNNA-Gr/CdS is generated. If Hg(II) is added, it wills capturing the aptamer, and this leads to the release of Gr/CdS because of the formation of a stable thymine-Hg2+-thymine link. External magnetic force is used to remove the remaining complex. The released graphene-CdS is decomposed by HNO3 and injected into a graphite furnace AAS. The detectable amount of Cd is proportional to the concentration of Hg(II) in the sample. Under the optimal conditions, the method has a linear response in the 2.50 aM to 0.25 nM Hg(II) concentration range, and the detection limit is as low as 7.6 aM (at S/N = 3). It has high selectivity for Hg(II) over other metal ions. Graphical abstract.

Capabilities of asymmetrical flow field - Flow fractionation on-line coupled to different detectors for characterization of water-stabilized quantum dots bioconjugated to biomolecules.

The asymmetric flow field-flow fractionation (AF4) coupled on-line with elemental (inductively coupled plasma-mass spectrometry, ICP-MS) and molecular (fluorescence and UV) detection has been investigated as a powerful tool for the characterization of bioinorganic nano-conjugates. In this study, we described methods for the characterization of biotin-antibody complexes bioconjugated with streptavidin quantum dots (QDs-SA-b-Ab). Operating parameters of AF4 separation technique were optimized and two procedures are proposed using a channel thickness of 350 µm and 500 µm. The use of a 500 µm spacer allowed to achieve an efficient AF4 separation of the QDs-SA-b-Ab complexes from the excess of individual species used in the bioconjugation that was required for a proper characterization of the bioconjugates. Optimization of the AF4 allowed a separation resolution good enough to isolate the QDs-SA-b-Ab bioconjugates from the free excess of b-Ab and QD-SA. The efficiency of the bioconjugation process could be then calculated, obtaining a value of 86% for a 1 QDs-SA: 5 b-Ab bioconjugation ratio. In addition, sample recovery around 90% was achieved.

A novel cadmium-containing wastewater treatment method: Bio-immobilization by microalgae cell and their mechanism.

Heavy metal cadmium (Cd) has drawn tremendous comcern due to its rigorous environmental and health hazards. Herein, we have presented an efficient and economical strategy for the removal and recycling of hazardous Cd ions using microalgae cells as the bioreactors. Remarkably, the green bio-platform for the bioproduction of CdSe nanoparticles (NPs) was developed depending on their orderly regulated and sustainable cellular environment. The biofabricated CdSe NPs manifested favorable photoluminescence properties, and presented well monodispersed spherical morphology and certain crystallinity structure with mean size of smaller than 7 nm. Especially, the fluorescence "turn off" sensing system based on the CdSe NPs was established to detect Hg2+. The nanosensor enables the quantitative analyses of Hg2+ with a linear range of 0-2.0 µM and a detection limit of 0.021 µM. Furthermore, it was preliminarily speculated that the reducing biomolecules in the algae cells could be involved in the formation of CdSe NPs. This work not only provides new insights into the removal and recycling of hazardous Cd ions, but also brings a promising route for biosynthesis of CdSe NPs.

Triple-Labeling of Polymer-Coated Quantum Dots and Adsorbed Proteins for Tracing their Fate in Cell Cultures.

Colloidal CdSe/ZnS quantum dots were water solubilized by overcoating with an amphiphilic polymer. Human serum albumin (HSA) as a model protein was either adsorbed or chemically linked to the surface of the polymer-coated quantum dots. As the quantum dots are intrinsically fluorescent, and as the polymer coating and the HSA were fluorescent labeled, the final nanoparticle had three differently fluorescent components: the quantum dot core, the polymer shell, and the human serum albumin corona. Cells were incubated with these hybrid nanoparticles, and after removal of non-internalized nanoparticles, exocytosis of the three components of the nanoparticles was observed individually by flow cytometry and confocal microscopy. The data indicate that HSA is partly transported with the underlying polymer-coated quantum dots into cells. Upon desorption of proteins, those initially adsorbed to the quantum dots remain longer inside cells compared to free proteins. Part of the polymer shell is released from the quantum dots by enzymatic degradation, which is on a slower time scale than protein desorption. Data are quantitatively analyzed, and experimental pitfalls, such as the impact of cell proliferation and fluorescence quenching, are discussed.

Quantitative Assessment of Individual Populations Present in Nanoparticle-Antibody Conjugate Mixtures Using AF4-ICP-MS/MS.

A current remaining challenge in nanotechnology is the fast and reliable determination of the ratios between engineered nanoparticles and the species attached to their surface after chemical functionalization. The approach proposed herein based on the online coupling of asymmetric flow field-flow fractionation (AF4) with inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) allows for the first time the direct determination of such ratios in CdSe/ZnS core-shell quantum dot:rat monoclonal IgG2a antibody (QD:Ab) conjugate mixtures in a single run without any previous sample preparation (i.e., derivatization). AF4 provides full recovery and adequate resolution of the resulting bioconjugate from the excess of nanoparticles and proteins used in the different bioconjugation mixtures (1:1, 2:1, and 3:1 QD:Ab molar ratios were assessed). The point-by-point determination by ICP-MS/MS of the metal to sulfur ratios along the bioconjugate fractographic peak allowed disclosing the mixture of the different species in the bioconjugated sample, providing not only the limits of the range of QD:Ab ratios in the different bioconjugate species resulting after functionalization but also a good estimation of their individual relative abundance in the mixture. Interestingly, a wide variety of compositions were observed for the different bioconjugate mixtures studied (QD:Ab molar ratios ranging from 0.27 to 4.6). The resulting weighted QD:Ab ratio computed in this way for each bioconjugate peak matches well with both the global (average) QD:Ab ratio experimentally obtained by the simpler peak area ratio computation and the theoretical QD:Ab molar ratios assayed, which internally validates the procedure developed.

Cytotoxicity studies of quantum dots with the electroporation method.

In this study, the cytotoxicity of CdTe quantum dots (QDs) of various dimensions was examined using the electroporation method. The influence of the size of QDs on normal and tumour cell viability after 24 h of incubation with nanomaterials was examined. The three human cell lines were chosen for the tests: A549 (a tumour cell line derived from the lung), MRC-5 (normal fibroblasts from the lung) and HaCaT (normal keratinocytes from the skin). Accordingly, we modelled the effect of nanocrystals on various human tissues because nanoparticles can be introduced into an organism through different routes. We were also able to study which cells are more sensitive to nanoparticles: normal or tumour cells. The nanoparticles were introduced into cells through pores in the cell membranes that were generated by electrical pulses. The effectiveness of introducing nanocrystals into cells was determined as a function of the nanocrystal dimensions and accumulation locations. Moreover, the cytotoxicity of quantum dots was tested, and cell viability after electroporation was evaluated. We also investigated whether the introduced nanocrystals released cadmium ions.

Study of CdTe detection efficiency for medical applications using Geant4-based stochastic simulations.

The determination of detection efficiency and peak-to-total ratios has been performed for rectangular CdTe detectors for various x-ray and low-energy γ-ray source configurations including parallel beams, point, and cylindrical sources. The dependence of efficiency values on axial and off-axial distances, detector thickness and area, and source dimensions has been studied. The detector model developed in this work has been validated by comparing the Monte Carlo simulated values of detector efficiency for a parallel incident beam with the available published data and good agreement has been found with discrepancies remaining within 2% throughout the energy range. Geant4 simulations show nearly 100% photopeak and total efficiency with peak-to-total ratios approaching a maximum value of 1.0 for photons in the 4-70 keV energy range. Similar high values of detection efficiency have been obtained for brachytherapy I-125 seed sources having cylindrical geometries which indicates the suitability of CdTe detectors for the calibration of sources used in therapy. The logistic power curve was found excellent for empirically fitting the photopeak efficiency variations with axial displacement of the I-125 brachy source in the horizontal configuration. Geant4 simulations clearly show that small thicknesses, of the order of 0.5 mm, of CdTe material are sufficient for attaining almost 100% detection efficiency for low-energy photons having energies up to 100 keV.

Self-mediated pH changes in culture medium affecting biosorption and biomineralization of Cd2+ by Bacillus cereus Cd01.

Biomineralization is an interesting naturally occurring process of forming minerals by microorganisms, which offers an efficient way to sequester heavy metal ions within relatively stable solid phases. In this study, Bacillus cereus Cd01 was selected to investigate effects of self-mediated pH on biosorption and biomineralization of Cd2+ in whole 72h cultivation period. Results revealed that strain Cd01-mediated pH decrease of the cultivation medium from 7.0 to 6.1 inhibited biosorption of Cd2+ on Cd01 cells at the initial cultivation period, while an increased pH from 6.1 to 7.4 facilitated biosorption of Cd2+ on Cd01 cells at the middle and late cultivation period. The reasons were mainly that self-mediated pH altered cell surface hydrophobicity and cell membrane fluidity of strain Cd01. Moreover, biosorption and bioaccumulation of Cd2+ on Cd01 cells in the period of increased pH promoted biomineralization of Cd2+ observed by the transmission and scanning electron microscopes. The analyses of energy dispersive spectroscopy, X-ray photoelectron spectroscopy and select area electron diffraction demonstrated that Cd2+ loaded on Cd01 cells was biomineralized into polycrystalline and/ or amorphous cadmium sulfide and cadmium phosphate. These results suggest that strain Cd01 may play a potential role in biomineralization remediation of heavy metal contaminated soils.

Short-term assessment of cadmium toxicity and uptake from different types of Cd-based Quantum Dots in the model plant Allium cepa L.

We report on the toxicity and bioaccumulation of three different types of Cd-based quantum dots (QDs), dispersed in aqueous medium, for a model plant Allium cepa L. It is believed that encapsulation of nanoparticles should reduce their toxicity and increase their stability in different environments; in this work we studied how QD encapsulation affects their phytotoxicity. Core, core/shell, and core/shell/shell QDs (CdTe, CdTe/ZnS, and CdTe/CdS/ZnS QDs capped by 2-mercaptopropionic acid) were tested and CdCl2 was used as a positive control. After 24-h and 72-h exposure, total Cd content (MCd) and bioaccumulation factors (BAFs) were determined in all parts of A. cepa plants (roots, bulb, shoot), and the total length of the root system was monitored as a toxicity end-point. Measurements of total Cd content versus free Cd2+ content (with Differential Pulse Voltammetry, DPV) in exposure media showed differences in chemical stability of the three QD types. Correspondingly, selected QDs showed different toxicity for A. cepa and different Cd bioaccumulation patterns. CdTe QDs were the most toxic; their effect was similar to CdCl2 due to the release of free Cd2+, which was confirmed by the DPV measurements. Plants exposed to CdTe QDs also bioaccumulated the most Cd among all QD exposure groups. CdTe/ZnS QDs showed no toxicity and very low bioaccumulation of Cd in A. cepa; the main source of measured Cd in the plants were QDs adsorbed on their roots, which was confirmed by fluorescence microscopy. On the contrary, CdTe/CdS/ZnS QD toxicity and bioaccumulation patterns were similar to those of CdTe QDs and pointed to unstable CdS/ZnS shells.

Retention of CdS/ZnS Quantum Dots (QDs) on the Root Epidermis of Woody Plant and Its Implications by Benzo[a]pyrene: Evidence from the in Situ Synchronous Nanosecond Time-Resolved Fluorescence Spectra Method.

The retention of CdS/ZnS QDs on the epidermis has been confirmed to be one of the core procedures during the root uptake process. However, the retention mechanisms of QDs on the epidermis of woody plant were poorly understood for lacking of an appropriate QD quantitative method. In this study, a novel method for in situ determination of CdS/ZnS QDs retained on the root epidermis was established using synchronous nanosecond time-resolved fluorescence spectroscopy. No correlations between Kf values of oleylamine-CdS/ZnS QDs retained on the epidermal tissues and the surface/bulk composition of mangrove root were observed (p > 0.05) due to the existence of endocytosis mechanisms during the QD uptake processes. Moreover, the difference of the CdS/ZnS QDs in water and further translocated to xylem/phloem of root rather than the combination with cell wall/membranes was the predominant reason that caused the Kf values to follow the sequence of PEG-COOH-CdS/ZnS QDs < PEG-NH2-CdS/ZnS QDs ≪ oleylamine-CdS/ZnS QDs.

Chip-based magnetic solid phase microextraction coupled with ICP-MS for the determination of Cd and Se in HepG2 cells incubated with CdSe quantum dots.

The quantification of trace Cd and Se in cells incubated with CdSe quantum dots (QDs) is critical to investigate the cytotoxicity of CdSe QDs. In this work, a miniaturized platform, namely chip-based magnetic solid phase microextraction (MSPME) packing with sulfhydryl group functionalized magnetic nanoparticles, was fabricated and combined with inductively coupled plasma mass spectrometry (ICP-MS) for the determination of trace Cd and Se in cells. Under the optimized conditions, the limits of detection (LOD) of the developed chip-based MSPME-ICP-MS system are 2.2 and 21ngL-1 for Cd and Se, respectively. The proposed method is applied successfully to the analysis of total and released small molecular fraction of Cd and Se in Human hepatocellular carcinoma cells (HepG2 cells) incubated with CdSe QDs, and the recoveries for the spiked samples are in the range of 86.0-109%. This method shows great promise to analyze cell samples and the obtained results are instructive to explore the cytotoxicity mechanism of CdSe QDs in cells.

Effects of Cd(II) on the stability of humic acid-coated nano-TiO2 particles in aquatic environments.

The stability of nanoparticles (NPs) in aquatic environments is important to evaluate their adverse effects on aquatic ecosystems and human health. Nanoparticle stability is known to be influenced by coexisting ions and dissolved organic matter. This study was designed to investigate the effects of coexisting low-level Cd(II) on the stability of humic acid-coated nano-TiO2 (HA-TiO2) particles in aquatic environments by measuring their aggregation kinetics through time-resolved dynamic light scattering (DLS) and monitoring suspended HA-TiO2 concentrations via optical absorbance changes over time. The particles exhibited aggregation behavior consistent with the classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The results showed that Cd(II) concentration, pH, and ionic strength had various effects on the aggregation kinetics of the HA-TiO2 NPs. The HA-TiO2 particles aggregated faster as the Cd(II) concentration increased whereas the stability of the nanoparticles increased as the solution pH increased or ionic strength decreased regardless of the Cd(II) concentration. At the fixed pH and ionic strength conditions, the addition of Cd(II) promoted aggregation of nanoparticles, leading to higher attachment efficiencies. The enhanced aggregation of the HA-TiO2 NPs in the presence of coexisting cadmium ions in aqueous solutions indicated that the fate and transport of nanoparticles could be greatly affected by heavy metals in aquatic environments.

The role of intracellular trafficking of CdSe/ZnS QDs on their consequent toxicity profile.

BACKGROUND: Nanoparticle interactions with cellular membranes and the kinetics of their transport and localization are important determinants of their functionality and their biological consequences. Understanding these phenomena is fundamental for the translation of such NPs from in vitro to in vivo systems for bioimaging and medical applications. Two CdSe/ZnS quantum dots (QD) with differing surface functionality (NH2 or COOH moieties) were used here for investigating the intracellular uptake and transport kinetics of these QDs. RESULTS: In water, the COOH- and NH2-QDs were negatively and positively charged, respectively, while in serum-containing medium the NH2-QDs were agglomerated, whereas the COOH-QDs remained dispersed. Though intracellular levels of NH2- and COOH-QDs were very similar after 24 h exposure, COOH-QDs appeared to be continuously internalised and transported by endosomes and lysosomes, while NH2-QDs mainly remained in the lysosomes. The results of (intra)cellular QD trafficking were correlated to their toxicity profiles investigating levels of reactive oxygen species (ROS), mitochondrial ROS, autophagy, changes to cellular morphology and alterations in genes involved in cellular stress, toxicity and cytoskeletal integrity. The continuous flux of COOH-QDs perhaps explains their higher toxicity compared to the NH2-QDs, mainly resulting in mitochondrial ROS and cytoskeletal remodelling which are phenomena that occur early during cellular exposure. CONCLUSIONS: Together, these data reveal that although cellular QD levels were similar after 24 h, differences in the nature and extent of their cellular trafficking resulted in differences in consequent gene alterations and toxicological effects.

Enantioselective cellular uptake of chiral semiconductor nanocrystals.

The influence of the chirality of semiconductor nanocrystals, CdSe/ZnS quantum dots (QDs) capped with L- and D-cysteine, on the efficiency of their uptake by living Ehrlich Ascite carcinoma cells is studied by spectral- and time-resolved fluorescence microspectroscopy. We report an evident enantioselective process where cellular uptake of the L-Cys QDs is almost twice as effective as that of the D-Cys QDs. This finding paves the way for the creation of novel approaches to control the biological properties and behavior of nanomaterials in living cells.

Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay.

We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365 nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31 V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20 ngmL(-1) and a detection limit (DL) of 2 ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP).

Bioaccumulation and in-vivo dissolution of CdSe/ZnS with three different surface coatings by Daphnia magna.

Daphnia magna were exposed to nano-sized CdSe/ZnS quantum dots (QDs) having three different surface coatings. QDs were investigated for their aqueous stability in the test media (hard reconstituted laboratory water) and for their uptake, elimination, and in-vivo dissolution. Positively charged QDs (QEI) and negatively charged QDs (QSH) were electrostatically stable, whereas neutrally charged QDs (QSA) showed aggregation and sedimentation over 48-h. After 24h of exposure to QDs (100µg/L as total Cd), the D. magna whole body Cd concentration significantly increased with no mortality for all QDs. Uptake patterns differed among the three coatings and Cd concentration reached 1460±50, 1014±99, and 584±81µg Cd/g dry wt for QEI, QSH, and QSA, respectively. Significant amounts of QEI and QSA (40% and 43%, respectively) remained in the D. magna after 24h of depuration, while 89% QSH were readily excreted within the initial 1h of the depuration stage. Soluble Cd was released from QDs during both the uptake and depuration. Release of Cd was higher in QEI and QSA than QSH, possibly resulting from the longer retention of QEI and QSA in the D. magna than QSH. These results imply that the surface charge of QDs plays a significant role in both the exposure to organisms and the in-vivo dissolution of nanoparticles.

[Development of ICP-OES, ICP-MS and GF-AAS Methods for Simultaneous Quantification of Lead, Total Arsenic and Cadmium in Soft Drinks].

In this study, we developed methods to quantify lead, total arsenic and cadmium contained in various kinds of soft drinks, and we evaluated their performance. The samples were digested by common methods to prepare solutions for measurement by ICP-OES, ICP-MS and graphite furnace atomic absorption spectrometry (GF-AAS). After digestion, internal standard was added to the digestion solutions for measurements by ICP-OES and ICP-MS. For measurement by GF-AAS, additional purification of the digestion solution was conducted by back-extraction of the three metals into nitric acid solution after extraction into an organic solvent with ammonium pyrrolidine dithiocarbamate. Performance of the developed methods were evaluated for eight kinds of soft drinks.

In vivo study of immunogenicity and kinetic characteristics of a quantum dot-labelled baculovirus.

Nanomaterials conjugated with biomacromolecules, including viruses, have great potential for in vivo applications. Therefore, it is important to evaluate the safety of nanoparticle-conjugated macromolecule biomaterials (Nano-mbio). Although a number of studies have assessed the risks of nanoparticles and macromolecule biomaterials in living bodies, only a few of them investigated Nano-mbios. Here we evaluated the in vivo safety profile of a quantum dot-conjugated baculovirus (Bq), a promising new Nano-mbio, in mice. Each animal was injected twice intraperitoneally with 50 µg virus protein labelled with around 3*10(-5)nmol conjugated qds. Control animals were injected with PBS, quantum dots, baculovirus, or a mixture of quantum dots and baculovirus. Blood, tissues and body weight were analysed at a series of time points following both the first and the second injections. It turned out that the appearance and behaviour of the mice injected with Bq were similar to those injected with baculovirus alone. However, combination of baculovirus and quantum dot (conjugated or simply mixed) significantly induced stronger adaptive immune responses, and lead to a faster accumulation and longer existence of Cd in the kidneys. Thus, despite the fact that both quantum dot and baculovirus have been claimed to be safe in vivo, applications of Bq in vivo should be cautious. To our knowledge, this is the first study examining the interaction between a nanoparticle-conjugated virus and a living body from a safety perspective, providing a basis for in vivo application of other Nano-mbios.