Femtomolar detection of Tau proteins in undiluted plasma using surface plasmon resonance

The ability to directly detect Tau protein and other neurodegenerative biomarkers in human plasma at clinically relevant concentrations continues to be a significant hurdle for the establishment of diagnostic tests for Alzheimer’s disease (AD). In this article, we introduce a new DNA aptamer/antibody sandwich assay pairing and apply it for the detection of human Tau 381 in undiluted plasma at concentrations as low as 10 fM. This was achieved on a multichannel surface plasmon resonance (SPR) platform with the challenge of working in plasma overcome through the development of a tailored mixed monolayer surface chemistry. In addition, a robust methodology was developed involving various same chip control measurements on reference channels to which the detection signal was normalized. Comparative measurements in plasma between SPR and enzyme-linked immunosorbent assay (ELISA) measurements were also performed to highlight both the 1000-fold performance enhancement of SPR and the ability to measure both spiked and native concentrations that are not achievable with ELISA

Gel electrophoretic analysis of differently shaped interacting and non-interacting bioconjugated nanoparticles

The use of a simple gel electrophoretic method to study mixtures of differently shaped biofunctionalized nanoparticles (NP's) that undergo bioaffinity interactions is demonstrated. Both gold nanorods (NR's) and quasi-spherical nanoparticles (qNS's) were functionalized with an interacting antigen and antibody pairing (alpha-1 antitrypsin (AAT) protein and antiAAT) or non-interacting antibody controls (antiBNP). Gel-based measurements were accompanied with transmission electron microscopy (TEM) and UV-vis spectroscopy analysis before and after separation. Initial measurements of NR and qNS bioconjugates suspended individually were applied to optimize the gel separation conditions and it was demonstrated that higher particle uniformities could be obtained relative to the initial stock solutions. A series of NR and qNS mixtures prepared at various stoichiometric ratios were then compared for both interacting (antiAAT–AAT) and non-interacting (antiAAT–antiBNP) particle conjugates. Both gel images and extinction measurements were utilized to demonstrate reduced NP concentrations transported along the gel due to bioaffinity-induced NP assembly. This confirmed that gel electrophoresis can be extended to identifying particle aggregation associated with protein bioaffinity interactions as well as being an established tool for separating particles based on size, shape and surface chemistry

Real-time assessment of nanoparticle-mediated antigen delivery and cell response

Nanomaterials are increasingly being developed for applications in biotechnology, including the delivery of therapeutic drugs and of vaccine antigens. However, there is a lack of screening systems that can rapidly assess the dynamics of nanoparticle uptake and their consequential effects on cells. Established in vitro approaches are often carried out on a single time point, rely on time-consuming bulk measurements and are based primarily on populations of cell lines. As such, these procedures provide averaged results, do not guarantee precise control over the delivery of nanoparticles to cells and cannot easily generate information about the dynamics of nanoparticle-cell interactions and/or nanoparticle-mediated compound delivery. Combining microfluidics and nanotechnology with imaging techniques, we present a microfluidic platform to monitor nanoparticle uptake and intracellular processing in real-time and at the single-cell level. As proof-of-concept application, the potential of such a system for understanding nanovaccine delivery and processing was investigated and we demonstrate controlled delivery of ovalbumin-conjugated gold nanorods to primary dendritic cells. Using time-lapse microscopy, our approach allowed monitoring of uptake and processing of nanoparticles across a range of concentrations over several hours on hundreds of single-cells. This system represents a novel application of single-cell microfluidics for nanomaterial screening, providing a general platform for studying the dynamics of cell-nanomaterial interactions and representing a cost-saving and time-effective screening tool for many nanomaterial formulations and cell types

Phenotypic analysis of extracellular vesicles:a review on the applications of fluorescence

Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed

An ex vivo animal model to study the effect of transverse mechanical loading on skeletal muscle

In many populations like wheelchair and prosthetic users, the soft tissue is subject to excessive or repetitive loading, making it prone to Deep Tissue Injury (DTI). To study the skeletal muscle response to physical stress, numerous in vitro and in vivo models exist. Yet, accuracy, variability, and ethical considerations pose significant trade-offs. Here, we present an ex vivo approach to address these limitations and offer additional quantitative information on cellular damage. In this study, skeletal muscle tissue from Sprague Dawley rats was isolated and transversely loaded. Histological analysis and fluorescence staining demonstrated that the setup was suitable to keep the tissue alive throughout the experimental procedure. Mechanically induced cell damage was readily distinguishable through morphological changes and uptake of a membrane impermeable dye. Our comparably simple experimental setup can be adapted to different loading conditions and tissues to assess the cell response to mechanical loading in future studies

Detection and quantification of warfarin in pharmaceutical dosage form and in spiked human plasma using surface enhanced Raman scattering

Analytical approaches for the quantitation of warfarin in plasma are high in demand. In this study, a novel surface enhanced Raman scattering (SERS) technique for the quantification of the widely used anticoagulant warfarin sodium in pharmaceutical dosage form and in spiked human plasma was developed. The colloidal-based SERS measurements were carefully optimized considering the laser wavelength, the type of metal nanoparticles, their surface functionalization and concentration as well as the time required for warfarin to associate with the metal surface. Poly(diallyldimethylammonium chloride) coated silver nanoparticles (PDDA-AgNPs) were established as a substrate which greatly enhanced the weak warfarin Raman signal with high reproducibility. The limit of detection was calculated in both water and human plasma to be 0.56 nM (0.17 ngmL-1) and 0.25 nM (0.08 ngmL-1) respectively, with a high degree of accuracy and reproducibility. The proposed method is simple, economical, and easily applied for routine application requiring only small plasma samples and also could be potentially useful for pharmacokinetic research on warfarin

Gold suprashells : enhanced photothermal nanoheaters with multiple LSPR for broadband SERS

In this manuscript we report on a new type of self-assembled plasmonic nanostructure called gold suprashells, which are assembled around superparamagnetic iron oxide nanoparticle (SPION) cores. Gold suprashells have multiple surface plasmon resonances over a broad vis-NIR wavelength range, which makes them useful in applications where broadband absorption is required. For example, suprashells are efficient substrates that enhance SERS signals across multiple excitation wavelengths. This unique multi-resonant character is afforded by the suprashell structure, which comprises anisotropic assemblies of nanoparticles of tunable length. Furthermore, gold suprashells generate more heat when excited with a laser compared to the nanoparticle building blocks, therefore making them promising materials for photothermal applications. The suprashells can potentially be assembled onto any negatively charged core, which opens up multiple possibilities for the development of multifunctional core/suprashell nanoparticle designs. Here, we assemble gold suprashells around dextran-coated SPIONs in order to obtain plasmonic and magnetic nanoparticles. Cells that have internalized the multifunctional nanoparticles can be accumulated with a magnet and killed with a laser through the generation of plasmonic heat. This approach shows promise for the development of therapies aimed at killing circulating tumor cells (CTCs) utilizing the proposed magnetic and plasmonic nanoparticles

Covalent co-assembly between resilin-like polypeptide and peptide amphiphile into hydrogels with controlled nanostructure and improved mechanical properties

Covalent co-assembly holds great promise for the fabrication of hydrogels with controllable nanostructure, versatile chemical composition, and enhanced mechanical properties given its relative simplicity, high efficiency, and bond stability. This report describes our approach to designing functional multicomponent hydrogels based on photo-induced chemical interactions between an acrylamide-functionalized resilin-like polypeptide (RLP) and a peptide amphiphile (PA). Circular dichroism (CD) spectroscopy, electron microscopy, and amplitude sweep rheology were used to demonstrate that the co-assembled hydrogel systems acquired distinct structural conformations, tunable nanostructures, and enhanced elasticity in a PA concentration-dependent manner. We envisage the use of these materials in numerous biomedical applications such as controlled drug release systems, microfluidic devices, and scaffolds for tissue engineering

Tandem femto- and nanomolar analysis of two protein biomarkers in plasma on a single mixed antibody monolayer surface using surface plasmon resonance

The multiplexed detection of protein biomarkers in plasma present over a range of clinically relevant concentrations continues to be difficult for surface-based bioaffinity detection platforms such as surface plasmon resonance (SPR). As well as nonspecific adsorption, challenges include quantitative comparison between targets whose concentrations differ by orders of magnitude, regenerating SPR chips after plasma exposure, and the two- or four-channel limitation of many commercial SPR instruments limiting sample throughput. In this article, we explore an approach where two protein biomarkers alpha-1 antitrypsin (AAT) and Tau 381 are detected in tandem within a single SPR channel at micromolar and femtomolar concentrations, respectively. This was achieved by creating a mixed antibody (antiAAT and antiTau) monolayer on the chip surface. After the adsorption of AAT and/or Tau, further specificity was obtained via the adsorption of a DNA aptamer specific to each target. The detection range for each target was controlled via the relative surface density ratio of each antibody type as well as each aptamer concentration. Calibration measurements were performed in both buffer and spiked plasma with the detection of native concentrations of ∼39 fM (Tau) and ∼65 μM (AAT) in a human plasma sample. Finally, tandem measurements of both targets within the same SPR signal channel were demonstrated at these very different concentrations

Stability-indicating micellar enhanced spectro-fluorometric determination of Daclatasvir in its tablet and spiked human plasma

A fast, simple and sensitive micellar enhanced spectrofluorimetric method is performed for the determination of Daclatasvir dihydrochloride (DAC) in its pharmaceutical dosage form and in spiked human plasma. The fluorescence intensity (FI) was measured at 367 nm after excitation at 300 nm. In aqueous solution, the FI of DAC was greatly enhanced by >110% in the presence of sodium dodecyl sulphate (SDS). The detection method was linear over the range of 12.93 to 161.60 ng/mL, with a limit of detection of 1.75 ng/mL. The proposed method was successfully applied to the determination of DAC in its pharmaceutical dosage form and the mean % recovery of DAC in spiked human plasma was 95.42 ± 2.52. The developed methodology was also extended to stress studies of DAC after exposure to different forced degradation conditions including acidic, alkaline, photolytic, thermal and oxidative environments