Optimized polydopamine coating and DNA conjugation onto gold nanorods for single nanoparticle bioaffinity measurements.

Gold nanorods (NRs) have attracted a great deal of interest for a variety of biomedical and sensing applications. However, developing robust methods for biofunctionalizing NRs has continued to be challenging, especially for NR-DNA conjugates. This is due to the presence of cetyltrimethylammonium bromide (CTAB), which plays an essential role in controlling the anisotropic particle growth. In this article, we systematically explore the growth of a polydopamine (PDA) layer on a range of NR surfaces, comparing different polyelectrolyte and alkanethiol coatings as well as direct CTAB displacement. This revealed that the PDA layer thickness and growth rate is strongly dependent on the underlying nanorod functionalization chemistry and allowed us to establish a preferred route for the creation of stable, non-aggregated suspensions of PDA-coated NRs. The utility of this platform was then demonstrated by self-assembling packed monolayers of single-stranded DNA on the outer surface. Both the surface attachment and bioactivity of the resulting NR-DNA conjugates was then demonstrated by performing bulk solution and single nanoparticle imaging fluorescence measurements.

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.

Light-Triggered Inactivation of Enzymes with Photothermal Nanoheaters.

A universal method for inactivating enzymes on demand is introduced, which involves irradiating nanorod-bound enzymes with near-infrared light. The subsequent generation of plasmonic heat denatures the enzymes selectively without damaging other proteins or cell membranes present in the same solution.

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.

Dual nanoparticle amplified surface plasmon resonance detection of thrombin at subattomolar concentrations.

A novel dual nanoparticle amplification approach is introduced for the enhanced surface plasmon resonance (SPR) detection of a target protein at subattomolar concentrations. Thrombin was used as a model target protein as part of a sandwich assay involving an antithrombin (anti-Th) modified SPR chip surface and a thrombin specific DNA aptamer (Th-aptamer) whose sequence also includes a polyadenine (A30) tail. Dual nanoparticle (NP) enhancement was achieved with the controlled hybridization adsorption of first polythymine-NP conjugates (T20-NPs) followed by polyadenine-NPs (A30-NPs). Two different nanoparticle shapes (nanorod and quasi-spherical) were explored resulting in four different NP pair combinations being directly compared. It was found that both the order and NP shape were important in optimizing the assay performance. The use of real-time SPR measurements to detect target concentrations as low as 0.1 aM is a 10-fold improvement compared to single NP-enhanced SPR detection methods.

Ultrasensitive and ultrawide range detection of a cardiac biomarker on a surface plasmon resonance platform.

One of the main challenges in the development of new analytical platforms for ultrasensitive bioaffinity detection is jointly achieving a wide dynamic range in target analyte concentration, especially for approaches that rely on multistep processes as a part of the signal amplification mechanism. In this paper, a new surface-based sandwich assay is introduced for the direct detection of B-type natriuretic peptide (BNP), an important biomarker for cardiac failure, at concentrations ranging from 1 aM to 500 nM. This was achieved using nanoparticle-enhanced surface plasmon resonance (SPR) where a DNA aptamer is immobilized on a chemically modified gold surface in conjunction with the specific adsorption of antiBNP coated gold nanocubes in the presence of the biomarker target. A concentration detection range greater than eleven orders of magnitude was achieved through dynamic control of only the secondary nanoparticle probe concentration. Furthermore, detection at low attomolar concentrations was also achieved in undiluted human serum.

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.

Stabilized gold nanorod-dye conjugates with controlled resonance coupling create bright surface-enhanced resonance Raman nanotags.

The preparation and characterization of stable and non-aggregated colloidal suspensions of gold nanorod-molecular dye complexes which exhibit very bright surface-enhanced resonance Raman scattering (SERRS) signals is described. A systematic study was performed where both the localized surface plasmon resonance (LSPR) of the nanorod and the molecular resonance of dyes adsorbed onto the rod surface were selectively tuned with respect to the laser excitation wavelengths. Resonance coupling was found to be a significant factor in the overall SERRS enhancement. The polymer stabilized nanorod-dye conjugates were prepared without the added complexity of nanoparticle aggregation as well as having good control over the surface coverage and orientation of the dye molecules. Furthermore, we demonstrate that this new class of Raman nanotags greatly outperforms an approach based on quasi-spherical gold nanoparticles.

Nanoparticle-enhanced surface plasmon resonance detection of proteins at attomolar concentrations: comparing different nanoparticle shapes and sizes.

The application of biofunctionalized nanoparticles possessing various shapes and sizes for the enhanced surface plasmon resonance (SPR) detection of a protein biomarker at attomolar concentrations is described. Three different gold nanoparticle shapes (cubic cages, rods and quasi-spherical) with each possessing at least one dimension in the 40-50 nm range were systematically compared. Each nanoparticle (NP) was covalently functionalized with an antibody (anti-thrombin) and used as part of a sandwich assay in conjunction with a Au SPR chip modified with a DNA-aptamer probe specific to thrombin. The concentration of each NP-antibody conjugate solution was first optimized prior to establishing that the quasi-spherical nanoparticles resulted in the greatest enhancement in sensitivity with the detection of thrombin at concentrations as low as 1 aM. When nanorod and nanocage antibody conjugates were instead used, the minimum target concentrations detected were 10 aM (rods) and 1 fM (cages). This is a significant improvement (>10(3)) on previous NP-enhanced SPR studies utilizing smaller (~15 nm) gold NP conjugates and is attributed to the functionalization of both the NP and chip surfaces resulting in low nonspecific adsorption as well as a combination of density increases and plasmonic coupling inducing large shifts in the local refractive index at the chip surface upon nanoparticle adsorption.

Highly sensitive electrochemical detection of proteins using aptamer-coated gold nanoparticles and surface enzyme reactions.

A novel electrochemical detection methodology is described for the femtomolar detection of proteins which utilizes both DNA aptamer-functionalized nanoparticles and a surface enzymatic reaction. Immunoglobulin E (IgE) was used as a model protein biomarker, which possesses two distinct epitopes for antibody (anti-IgE) and DNA aptamer binding. A surface sandwich assay format was utilized involving the specific adsorption of IgE onto a gold electrode surface that was pre-modified with a monolayer of aptamer-nanoparticle conjugates followed by the specific interaction of alkaline phosphatase (ALP) conjugated anti-IgE. To clearly demonstrate the signal enhancement associated with nanoparticle use, anodic current measurements of the ALP catalyzed oxidation of the enzyme substrate 4-aminophenylphosphate (APP) were also compared with electrode surfaces upon which the aptamer was directly attached. The detection of an unlabelled protein at concentrations as low as 5 fM is a significant improvement compared to conventional electrochemical-based immunoassay approaches and provides a foundation for the practical use and incorporation of nanoparticle-enhanced detection into electrochemical biosensing technologies.

Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug-cell interactions.

Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800-3050 cm-1) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug-cell interactions at the subcellular scale.

A universal polymer shell-isolated nanoparticle (SHIN) design for single particle spectro-electrochemical SERS sensing using different core shapes.

Shell-isolated nanoparticles (SHINs) have attracted increasing interest for non-interfering plasmonic enhanced sensing in fields such as materials science, biosensing, and in various electrochemical systems. The metallic core of these nanoparticles is isolated from the surrounding environment preventing direct contact or chemical interaction with the metal surface, while still being close enough to enable localized surface plasmon enhancement of the Raman scattering signal from the analyte. This concept forms the basis of the shell isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique. To date, the vast majority of SHIN designs have focused on SiO2 shells around spherical nanoparticle cores and there has been very limited published research considering alternatives. In this article, we introduce a new polymer-based approach which provides excellent control over the layer thickness and can be applied to plasmonic metal nanoparticles of various shapes and sizes without compromising the overall nanoparticle morphology. The SHIN layers are shown to exhibit excellent passivation properties and robustness in the case of gold nanosphere (AuNP) and anisotropic gold nanostar (AuNS) core shapes. In addition, in situ SHINERS spectro-electrochemistry measurements performed on both SHIN and bare Au nanoparticles demonstrate the utility of the SHIN coatings. Correlated confocal Raman and SEM mapping was achieved to clearly establish single nanoparticle SERS sensitivity. Finally, confocal in situ SERS mapping enabled visualisation of the redox related molecular structure changes occurring on an electrode surface in the vicinity of individual SHIN-coated nanoparticles.

De Novo Design of Functional Coassembling Organic-Inorganic Hydrogels for Hierarchical Mineralization and Neovascularization.

Synthetic nanostructured materials incorporating both organic and inorganic components offer a unique, powerful, and versatile class of materials for widespread applications due to the distinct, yet complementary, nature of the intrinsic properties of the different constituents. We report a supramolecular system based on synthetic nanoclay (Laponite, Lap) and peptide amphiphiles (PAs, PAH3) rationally designed to coassemble into nanostructured hydrogels with high structural integrity and a spectrum of bioactivities. Spectroscopic and scattering techniques and molecular dynamic simulation approaches were harnessed to confirm that PAH3 nanofibers electrostatically adsorbed and conformed to the surface of Lap nanodisks. Electron and atomic force microscopies also confirmed an increase in diameter and surface area of PAH3 nanofibers after coassembly with Lap. Dynamic oscillatory rheology revealed that the coassembled PAH3-Lap hydrogels displayed high stiffness and robust self-healing behavior while gas adsorption analysis confirmed a hierarchical and heterogeneous porosity. Furthermore, this distinctive structure within the three-dimensional (3D) matrix provided spatial confinement for the nucleation and hierarchical organization of high-aspect ratio hydroxyapatite nanorods into well-defined spherical clusters within the 3D matrix. Applicability of the organic-inorganic PAH3-Lap hydrogels was assessed in vitro using human bone marrow-derived stromal cells (hBMSCs) and ex vivo using a chick chorioallantoic membrane (CAM) assay. The results demonstrated that the organic-inorganic PAH3-Lap hydrogels promote human skeletal cell proliferation and, upon mineralization, integrate with the CAM, are infiltrated by blood vessels, stimulate extracellular matrix production, and facilitate extensive mineral deposition relative to the controls.

Attomolar detection of protein biomarkers using biofunctionalized gold nanorods with surface plasmon resonance.

This paper describes an ultrasensitive surface plasmon resonance (SPR) detection method using biofunctionalized gold nanorods for the direct detection of protein biomarkers. Immunoglobulin E (IgE), which has separate antibody and DNA aptamer binding sites, was chosen as a model protein for which a sandwich assay platform was designed. Detection was achieved via the specific adsorption of unlabelled IgE proteins onto the surface immobilized aptamer followed by the specific adsorption of anti-IgE coated gold nanorods (Au-NRs). Using the biofunctionalized nanorods in conjunction with SPR, we were able to directly measure IgE proteins at attomolar concentrations. This is a remarkable 10(8) enhancement compared to conventional SPR measurements of the same surface sandwich assay format 'anti-IgE/IgE/surface bound IgE-aptamer' in the absence of gold nanorod signal amplification.

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.

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.

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.

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 ±â€¯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.

3D bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing.

The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated 3D bioprinting of thicker biofilms (>4 mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 d in culture. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had denser biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E. coli biofilms had greater resistance to tetracycline than thinner constructs over 7 d of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.

Enhanced bioaffinity sensing using surface plasmons, surface enzyme reactions, nanoparticles and diffraction gratings.

This paper introduces a novel approach to surface bioaffinity sensing based on the adsorption of nanoparticles onto a gold diffraction grating that supports the excitation of planar surface plasmons. A surface enzymatic amplification reaction is also incorporated into the detection scheme to enhance the sensitivity and utility of the nanoparticle-enhanced diffraction grating (NEDG) sensors. As a demonstration, the detection of microRNA is described where a combination of a surface polymerase reaction and DNA-modified nanoparticles is used to detect the bioaffinity adsorption of the target onto the probe-functionalized gold grating surface. The enzymatically-amplified NEDG sensors possess a great potential for a wide range of applications including the detection of biosecurity agents, DNA and RNA viruses, biomarkers, and proteins.