Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.

BACKGROUND: A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses. METHODS: EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 × 107 cells were stained with AlexaFluor 488-conjugated specific antibodies or corresponding isotype controls. Amount and size of particles in normal scattering light mode (N mode) versus fluorescence mode (F mode, laser wavelength 488 nm) was measured using ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Cryo electron microscopy (EM) was used to verify the presence of membrane bilayer surrounded nanoparticles. RESULTS: All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit. CONCLUSION: Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.

Microwave-assisted synthesis of water-soluble, fluorescent gold nanoclusters capped with small organic molecules and a revealing fluorescence and X-ray absorption study.

Colourless solutions of blue light-emitting, water-soluble gold nanoclusters (AuNC) were synthesized from gold colloids under microwave irradiation using small organic molecules as ligands. Stabilized by 1,3,5-triaza-7-phosphaadamantane (TPA) or L-glutamine (GLU), fluorescence quantum yields up to 5% were obtained. AuNC are considered to be very promising for biological labelling, optoelectronic devices and light-emitting materials but the structure-property relationships have still not been fully clarified. To expand the knowledge about the AuNC apart from their fluorescent properties they were studied by X-ray absorption spectroscopy elucidating the oxidation state of the nanoclusters' gold atoms. Based on curve fitting of the XANES spectra in comparison to several gold references, optically transparent fluorescent AuNC are predicted to be ligand-stabilized Au5(+) species. Additionally, their near edge structure compared with analogous results of polynuclear clusters known from the literature discloses an increasing intensity of the feature close to the absorption edge with decreasing cluster size. As a result, a linear relationship between the cluster size and the X-ray absorption coefficient can be established for the first time.

Quantum dot loaded liposomes as fluorescent labels for immunoassay.

Liposomes loaded with water-soluble and water-insoluble quantum dots (QD) were for the first time applied as labels in different heterogeneous immunoassays for the determination of food contaminants, using mycotoxin zearalenone (ZEN) as a model. A great deal of work was devoted to the optimal choice of phospholipids for the liposomes preparation and to the factors which are important for the stability and size of obtained liposomes. Thin-film hydration and reverse-phase evaporation techniques were evaluated in terms of stability of the obtained liposomes and their efficiency for QD loading. Conjugation of liposomes with proteins and the influence of cross-linkers to the nonspecific interaction of the obtained liposomes with the surface of microtiter plates and cartridges were investigated and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester was found as the optimal cross-linker. The limits of detection (LOD) for ZEN of fluorescence-labeled immunosorbent assays were 0.6 µg kg(-1), 0.08 µg kg(-1), and 0.02 µg kg(-1), using QD, liposomes loaded with water-soluble QD, and water-insoluble QD, respectively. Similarly, the developed qualitative on-site tests using the different QD labels and taking into account the EU maximum residues level for ZEN in unprocessed cereals showed cutoff levels of 100, 50, and 20 µg kg(-1).

Process analysis of biofilms by photoacoustic spectroscopy.

Biofilms are aggregates of microorganisms and biopolymers which occur at aqueous interfaces. Biofilms play an important role in the degradation of pollutants in natural water systems as well as in wastewater treatment plants. In this communication, the use of photoacoustic spectroscopy (PAS) as a new biofilm monitoring technique is presented. PAS combines features of optical spectroscopy and ultrasonic tomography and allows a depth-resolved analysis of optically and acoustically inhomogeneous media. For the first time, both biofilm and bulk liquid were monitored by photoacoustic sensor heads. In this way, sorption of suspended iron(III) oxide particles on the outer and inner surfaces of the biofilm could be observed on-line and in situ. Colloids can act as carriers of pollutants and influence stability and degradation efficiency of biofilms.

On-line monitoring of opaque liquids by photoacoustic spectroscopy.

A new photoacoustic sensor system for on-line monitoring of highly concentrated and optical opaque liquid samples is presented. The dyeing of textiles is performed with highly concentrated dye solutions with concentrations ranging from 50 mg L(-1) up to 40 g L(-1). For process optimization and control of the wastewater, an on-line monitoring of the dye concentration is needed. Optical transmission measurements allow the determination of the dye concentration in a relatively small range. Samples with concentrations in the upper mg L(-1) and g L(-1) range have to be diluted before the measurement due to their optical opacity. Additionally, light-scattering particles have a strong effect on the transmitted light intensity. By photoacoustic spectroscopy, concentrations in condensed matter can be determined over several orders of magnitude. Furthermore, scattering particles do not generate any photoacoustic signal.

Characterization and cloning of the gene encoding the vacuolar membrane protein EXP-2 from Plasmodium falciparum.

As a contribution to the characterization of the parasitophorous vacuolar membrane from Plasmodium falciparum we have begun the identification of vacuolar membrane proteins. Exported protein-2 (EXP-2) is a vacuolar membrane protein exposed into the vacuolar space. To further characterize EXP-2, it was purified, and the 45 N-terminal amino acids were determined by micro-sequencing. Based on this information, partial cDNA and genomic fragments were amplified by PCR and used as probes for the isolation of complete cDNA and genomic DNA clones. The single copy gene is located on chromosome 14, and is transcribed during the ring stage of parasite development. The open reading frame encodes an N-terminal signal sequence which is cleaved from the mature protein. The amino acid composition of EXP-2 is characterized by charged amino acids, with a high abundance of aspartate residues in the C-terminal portion of the protein. In contrast to EXP-1, an integral protein of the vacuolar membrane, EXP-2 lacks a typical hydrophobic transmembrane domain. We suggest that EXP-2 may associate with the vacuolar membrane via an amphipathic helix located in the N-terminal half of the protein.