Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection.

Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-µL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (n = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with r2 value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 × 107 exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h.

Double click: dual functionalized polymeric micelles with antibodies and peptides.

Polymeric nanoparticle micelles provide a possible platform for theranostic delivery, combining the role of therapeutics and diagnostics in one vehicle. To explore dual-functional micelles, the amphiphilic copolymer of poly(d,l-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol)-X (P(LA-co-TMCC)-g-PEG-X) was self-assembled to form micelles, with X representing either azide or furan. Micelles of P(LA-co-TMCC)-g-PEG-azide and P(LA-co-TMCC)-g-PEG-furan terminal functional groups were used to conjugate dibenzylcyclooctyne and maleimide-modified probes, respectively, taking advantage of orthogonal coupling chemistry. To verify the utility of the dual-functional micelles, trastuzumab-maleimide antibodies and FLAG-dibenzylcyclooctyne peptides were covalently bound by sequential click chemistry reactions. SKOV-3luc cells that were treated with the dual-functionalized micelles showed colocalization of the antibodies and peptides by confocal imaging, demonstrating the promise of this approach.

Structure-activity examination of poly(glycoamidoguanidine)s: glycopolycations containing guanidine units for nucleic acid delivery.

In this study we synthesized a new series of polymers known as poly(glycoamidoguanidine)s (PGAGs). These new polymer structures were synthesized by copolymerizing a carbohydrate monomer (diester; galatarate or tartarate) with a diamine incorporating guanidine or methylguanidine as a charge center to create a polyamide backbone. These materials were strategically designed and compared to our previously studied DNA delivery vehicles, poly(glycoamidoamine)s (PGAAs), which contain secondary amines as the charge groups along the polymer backbone to examine the effect of charge center type on the cellular delivery efficiency of plasmid DNA (pDNA). The guanidine moieties within the PGAGs facilitate electrostatic binding with the negatively charged phosphate backbone of plasmid DNA (pDNA). Stable polymer-pDNA complexes (polyplexes) with sizes in the range of 60-200 nm are formed at polymer/pDNA charge ratios (N/P) of 5 and above. When the PGAGs are complexed with Cy5-labeled pDNA (Cy5-pDNA) at N/P ratios of 10 and 25, between 80 and 95% of HeLa cells were positive for Cy5 fluorescence, indicating effective cellular internalization of the polyplexes. The toxicity of both PGAA and PGAG polyplexes was studied via MTT assays, and over 95% cell survival was observed at N/P ratios of 5, 10, 15, 20, 25, and 30 in HeLa cells. Transgene expression was examined via luciferase assays at various N/P ratios in the absence and presence of serum. In the absence of serum, the PGAG polyplexes revealed similar transgene expression when compared to polyplexes formed with their analogous PGAA structures. In the presence of serum, one analog (Gg) consisting of galactarate copolymerized with the guanidine monomer yielded gene expression similar to the positive control, Glycofect Transfection Reagent. This new series of guanidine-containing oligomers are promising as a new design strategy to incorporate an alternative charge center type within the backbone of glycopolymer-based nucleic acid delivery vehicles.

Engineering Electromagnetic Hot-Spots in Nanoparticle Cluster Arrays on Reflective Substrates for Highly Sensitive Detection of (Bio)molecular Analytes.

Intense electromagnetic (EM) hot-spots arising at the junctions or gaps in plasmonic nanoparticle assemblies can drive ultrahigh sensitivity in molecular detection by surface-enhanced spectroscopies. Harnessing this potential however requires access to the confined physical space at the EM hot-spots, which is a challenge for larger analytes such as biomolecules. Here, we demonstrate self-assembly derived gold nanoparticle cluster arrays (NCAs) on gold substrates exhibiting controlled interparticle (<1 nm wide) and intercluster (<10 nm wide) hot-spots as highly promising in this direction. Sensitivity of the NCAs toward detection of small (<1 nm) or large (protein-receptor interactions) analytes in surface-enhanced Raman and metal-enhanced fluorescence assays is found to be strongly impacted by the size of the cluster and the presence of reflective substrates. Experiments supported by numerical simulations attribute the higher sensitivity to higher EM field enhancements at the hot-spots, as well as greater analyte leverage over EM hot-spots. The best-performing arrays could push the sensitivity down to picomolar detection limits for sub-nanometric organic analytes as well as large protein analytes. The investigation paves the way for rational design of plasmonic biosensors and highlights the unique capabilities of a molecular self-assembly approach toward catering to this objective.

Cylindrical illumination confocal spectroscopy: rectifying the limitations of confocal single molecule spectroscopy through one-dimensional beam shaping.

Cylindrical illumination confocal spectroscopy (CICS) is a new implementation of single molecule detection that can be generically incorporated into any microfluidic system and allows highly quantitative and accurate analysis of single fluorescent molecules. Through theoretical modeling of confocal optics and Monte Carlo simulations, one-dimensional beam shaping is used to create a highly uniform sheet-like observation volume that enables the detection of digital fluorescence bursts while retaining single fluorophore sensitivity. First, we theoretically show that when used to detect single molecules in a microchannel, CICS can be optimized to obtain near 100% mass detection efficiency, <10% relative SD in burst heights, and a high signal/noise ratio. As a result, CICS is far less sensitive to thresholding artifacts than traditional single molecule detection and significantly more accurate at determining both burst rate and burst parameters. CICS is then experimentally implemented, optically characterized, and integrated into separate two microfluidic devices for the analysis of fluorescently stained plasmid DNA and single Cy5 labeled oligonucleotides. CICS rectifies the limitations of traditional confocal spectroscopy-based single molecule detection without the significant operational complications of competing technologies.

Quantitative Assessment of Nanoparticle Biodistribution by Fluorescence Imaging, Revisited.

Fluorescence-based whole-body imaging is widely used in the evaluation of nanoparticles (NPs) in small animals, often combined with quantitative analysis to indicate their spatiotemporal distribution following systemic administration. An underlying assumption is that the fluorescence label represents NPs and the intensity increases with the amount of NPs and/or the labeling dyes accumulated in the region of interest. We prepare DiR-loaded poly(lactic- co-glycolic acid) (PLGA) NPs with different surface layers (polyethylene glycol with and without folate terminus) and compare the distribution of fluorescence signals in a mouse model of folate-receptor-expressing tumors by near-infrared fluorescence whole-body imaging. Unexpectedly, we observe that fluorescence distribution patterns differ far more dramatically with DiR loading than with the surface ligand, reaching opposite conclusions with the same type of NPs (tumor-specific delivery vs predominant liver accumulation). Analysis of DiR-loaded PLGA NPs reveals that fluorescence quenching, dequenching, and signal saturation, which occur with the increasing dye content and local NP concentration, are responsible for the conflicting interpretations. This study highlights the critical need for validating fluorescence labeling of NPs in the quantitative analysis of whole-body imaging. In light of our observation, we make suggestions for future whole-body fluorescence imaging in the in vivo evaluation of NP behaviors.

Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials.

The aim of this work was to study, in vitro, cell colonization of two biomaterials currently used for bone and cartilage repair, this step being important to understand the function of engineered tissues. Current methods that use histological approaches are not always suited to tissue-engineering analysis. We, therefore, set up a protocol to assess cell distribution, utilizing noninvasive confocal microscopy and fluorescent labels with a far red emission wavelength to optimize scaffold transparency and minimize light scattering. Hard (ceramic substitute) and soft (collagen sponge) biomaterials were seeded respectively, on one side of the scaffold, with human fibroblasts and bovine chondrocytes labelled with carbocyanine dyes (DiD and DiR). The mean penetration depth for DiR labelled fibroblasts and chondrocytes in the two scaffolds, around 270 m, was greater than for DiD (136-218 microm) labelled cells. These depths were independent of cell origin but were influenced by the nature of the scaffolds. Collagen sponge is transparent in contrast to ceramic substitutes where measurements could only be made in opened macropores. Besides the limits of the equipment, the limits of the supports were diffusion for collagen sponges and transmission for ceramic substitutes. Confocal microscopy techniques could thus be used to address the question of cell colonization of porous biomaterials in a noninvasive manner.

FISH and Calcofluor staining techniques to detect in situ filamentous fungal biofilms in water.

Filamentous fungi are a ubiquitous and diverse group of eukaryotic organisms and may contribute, along with bacteria, yeasts, protozoa and viruses, to the formation of biofilms in water distribution systems. However, fungal involvement in biofilms has not been demonstrated unambiguously. Furthermore, these fungi may be responsible for the production of tastes, odours and mycotoxins in drinking water making their early detection important. The detection of fme these problems a combination of two fluorescent techniques for direct detection was tested: (a) Fluorescence In Situ Hybridization (FISH) employing the universal rRNA probe EUK516, labelled with the red Cy3, followed by (b) staining with Calcofluor White MR2 fluorescent dye which stains fungal cell walls blue. Pure cultures of Penicillium brevicompactum were used to establish the methods followed by separate experiments with real water biofilm samples in PVC-C and cast iron coupons. FISH demonstrated eukaryotic microrganisms after approximately 5 h while the calcofluor method revealed chitinous filamentous structures in less than one hour. When the two methods were combined, additional resolution was obtained from the images of filamentous walls (blue) with intact protoplasm (red). In conclusion, FISH and Calcofluor staining provide rapid, direct and unambiguous information on the involvement of ff in biofilms which form in water.

Design and fabrication of a silica on silicon integrated optical biochip as a fluorescence microarray platform.

Previous research into the use of Flame Hydrolysis Deposition (FHD) of glasses in integrated optics has focused on the successful commercial exploitation of low cost optical devices within the field of telecommunications and optoelectronics. Recently we have sought to apply these fabrication technologies to the development of optical biochips, utilising their ability to be integrated with microfluidics as a 'Lab-on-a-chip' platform. In this paper, we carry this development forward by seeking to create a microarray of integrated optical sensing elements, addressed using a glass-polymer hybrid technology in which poly(dimethylsiloxane), PDMS, is used as an elastomeric packaging over-layer. In particular, we describe the wide range of modelling and microfabrication processes required for the successful manufacture, integration and packaging of such arrays. The integration of both optical and fluidic circuits in this device avoids precise alignment requirements and results in a compact, robust and reliable device. Finally, in this paper, we describe the implementation of a pumping system for delivering small amounts of fluid across the array together with an optical signal treatment.

The behavior of the fluorescent probe diS-C3-(5) in membrane and aqueous media.

Based on an analysis of back fluorometric titration data a partition coefficient, Kp = (5.70 +/- 0.95) x 10(4), and partition constant, K = (2.37 +/- 0.43) x 10(6), were found for the probe diS-C3-(5) in egg lecithin vesicle suspension. The relative probe quantity in an aqueous medium and in liposomes was calculated using these parameters. The number of chromophore states in this system was computer-analysed and it was shown that the probe fluorescence could be described by two fluorescing dye forms, aqueous and membrane monomers. The dependence of fluorescence intensity on the probe concentration was studied in various salt media, and a dimerization (association) constant Ka = 5 x 10(4) mol -1 . l in the buffer, and Ka = (8.1 +/- 1.5) x 10(4) mol-1 . l in 0.1 or 0.2 mol/l salt medium (KCl or NaCl) was found. From the fluorescence and absorption data critical concentrations of the onset of large probe aggregate formation were calculated for various aqueous media. The concentration dependence of the probe fluorescence in the membrane phase was calculated. The critical concentration of interaction characterizing the efficiency of the fluorescence concentration quenching processes (CCI) was found to be approx. 5-6 mol probe per 1000 mol lipid. The top probe concentration in a membrane (the "saturation" concentration) was estimated from the slope of the initial linear parts of the back fluorometric titration curves, and was found to be equal to (59 +/- 13) mol probe per 1000 mol lipid.

DNA amplification via polymerase chain reaction inside miniemulsion droplets with subsequent poly(n-butylcyanoacrylate) shell formation and delivery of polymeric capsules into mammalian cells.

There is a growing interest in the development of stable nanocapsules that could deliver the bioactive compounds within the living organism, and to release them without causing any toxic effects. Here the miniemulsion droplets were first used as "nanoreactors" for the amplification of single-molecule dsDNA template (476 and 790 base pairs) through PCR. Afterwards, each droplet was surrounded with a biodegradable PBCA shell by interfacial anionic polymerization, enabling therefore to deliver the PCR products into the cells. The size of the initial miniemulsion droplets and the final polymeric capsules was in the range of 250 and 320 nm, mainly depending on the type of the continuous phase and presence of dsDNA template molecules. The formation of PCR products was resolved with gel electrophoresis and detected with fluorescence spectroscopy in the presence of DNA specific dye (SYBRGreen). TEM studies were performed to prove the formation of the polymeric shell. The shell thickness was measured to be within 5-15 nm and the average molecular weight of the formed PBCA polymer was around 75000 g · mol(-1) . For the cell uptake experiments, the obtained nanocapsules were transferred from the organic phase into aqueous medium containing a water-soluble surfactant. The effect of the surfactant type (anionic, cationic or non-ionic) on the HeLa cell viability and nanocapsule uptake behavior was studied by CLSM and FACS. Confocal analysis demonstrated that nanocapsules stabilized with cationic (CTMA-Cl) and non-ionic (Lutensol AT50) surfactants show almost the same uptake, whereas capsules redispersed in anionic (SDS) surfactant possess a 30% higher uptake. The release of the encapsulated material within the cell was studied on the example of Cy5-labeled oligonucleotides showing the colocalization with mitochondria of MSCs cells.

Identification of single fluorescent labels using spectroscopic microscopy.

Detection of single, fluorescently labeled biomolecules is providing a powerful approach to measuring molecular transport, biomolecular interactions, and localization in biological systems. Because the biological molecules of interest rarely exhibit sufficient intrinsic fluorescence to allow observation of individual molecules, they are usually labeled with fluorescent dye molecules, fluorescent proteins, semiconductor nanocrystals or quantum dots, or fluorescently doped silica or polymer nanospheres to allow their detection. Differences in the photophysical and spectral properties of different labels allow one to identify individual molecules by distinguishing their corresponding labels. A simple approach to measuring fluorescence spectra of individual fluorescent labels can be implemented in a standard wide-field fluorescence microscope, where a grating or prism is incorporated into the path from the microscope to an imaging detector to disperse the emission spectrum. In this work, principal components and cluster analysis are applied to the identification of fluorescence spectra from single fluorescent labels, with statistical tests of the classification results. Spectra are determined from diffracted images of fluorescent nanospheres labels, where emission maxima are separated by less than 20 nm, and of single dye-molecule labels with 30 nm separation. Clusters of points in an eigenvector representation of the spectra correctly classify known labels (both nanospheres and single molecules) and unambiguously identify unknown labels in mixtures.

Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR.

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.

Single-molecule near-field optical energy transfer microscopy with dielectric tips.

The fluorescence lifetime and the fluorescence rate of single molecules are recorded as a function of the position of a Si3N4 atomic force microscopy tip with respect to the molecule. We observe a decrease of the excited state lifetime and the fluorescence rate when the tip apex is in close proximity to the molecule. These effects are attributed to the fact that the dielectric tip converts non-propagating near-fields to propagating fields within the dielectric tip effectively quenching the fluorescence. The spatial extension of the quenching area is of subwavelength dimensions. The results are discussed in terms of molecular fluorescence in a system of stratified media. The experiment provides surprising new insights into the interactions between a fluorescent molecule and a dielectric tip. The methodology holds promise for applications in ultra high-resolution near-field optical imaging at the level of single fluorophores.