Plasmonic enhancement of fluorescence for biomedical diagnostics

The enhancement of fluorescence that can result from the proximity of fluorophores to metallic nanoparticles (NP’s) is investigated. This plasmonic enhancement, which is a result of the localized surface plasmon resonance (LSPR) at the metal surface, can be exploited in order to improve the signal obtained from optical biochips and thereby lower the limits of detection. The scale of the enhancement depends on many parameters such as NP size and shape, metal type and NP-fluorophore separation. Throughout the work, theoretical calculations were carried out, and, where relevant, theoretical predictions were compared with experimental measurements. Characterisation techniques used include TEM, AFM as well as optical fluorescence and absorption. The first section deals with the production of ordered arrays of nanostructures, of varying size and composition, on glass substrates using a nanosphere lithography technique. The ability to tune the peak wavelength of LSPR was demonstrated. Fluorescent dyes were then pin-printed onto the NP layer and the fluorescence enhancement was measured. The second body of work involved characterising the enhanced fluorescence from dyes attached to free NPs in solution. NPs of sizes ranging from 5 to 50nm radius and with different gold/silver alloy compositions were prepared by wet chemistry. The NPs were coated with silica shells to control the dye-NP separation and to minimise quenching. The dependence of the enhancement on NP size was found to agree well with theoretical calculations based on the Mie theory. The final body of work focused on the development of strategies applicable to polymer biochips. This included the development of techniques for immobilising NPs on plastic substrates. A range of dyes and a range of NP shapes were investigated. Dye-NP separation was controlled to nanometer precision by layer-by-layer deposition of polyelectrolytes. In this configuration, both dye quenching and enhancement effects were observed and characterised. The key result to emerge from this work was that it is possible to design an optical biochip enhancement platform where the NP shape, size and composition are optimised for the selected dye label and where the average dye-NP separation is designed to achieve maximum enhancement

Propagating and localized surface plasmon resonance sensing — A critical comparison based on measurements and theory

With its potential for ultrasensitive, label-free detection of molecular interactions, sensing methods based on the surface plasmon resonance (SPR) effect fully meet the requirements for modern analytical techniques. Already established by using propagating SPR in thin gold layers, the last years witnessed the emergence of another related technique utilizing extremely miniaturized noble metal sensor structures, based on a localized SPR. This paper provides a critical comparison of these kinds of SPR sensing, reviews the foundation of both general approaches, presents experimental data on exactly the same molecular model system using both techniques, as well as theoretical considerations in order to allow reasonable comparison. It highlights the specific features and effects, in order to provide guidance in choosing the right technique for given bioanalytical tasks. The study demonstrated the capabilities of LSPR for sensing of molecular layers even in the lower nanometer dimension. For the detection of small (bio)molecules, smaller particle diameters are favored regarding highest sensitivity. It also presents an approach to obtain refractive index and the thickness of a molecular film by analyzing the signal response of plasmonic sensors with metal nanoparticles. Moreover, an additional method for the improvement of the parameters' determination is introduced

A new strategy for silver deposition on Au nanoparticles with the use of peroxidase-mimicking DNAzyme monitored by Localized Surface Plasmon Resonance technique

Peroxidase-mimicking DNAzyme was applied as a catalyst of silver deposition on gold nanoparticles. This DNAzyme is formed when hemin binds to the G-quadruplex-forming DNA sequence. Such a system is able to catalyze a redox reaction with a one- or two-electron transfer. The process of silver deposition was monitored via a localized surface plasmon resonance technique (LSPR), which allows one to record scattering spectrum of a single nanoparticle. Our study showed that DNAzyme is able to catalyze silver deposition. The AFM experiments proved that DNAzyme induced the deposition of silver shells of approximately 20 nm thickness on Au nanoparticles (AuNPs). Such an effect is not observed when hemin is absent in the system. However, we noticed non-specific binding of hemin to the capture oligonucleotides on a gold NP probe that also induced some silver deposition, even though the capture probe was unable to form G-quadruplex. Analysis of SEM images indicated that the surface morphology of the silver layer deposited by DNAzyme is different from that obtained for hemin alone. The proposed strategy of silver layer synthesis on gold nanoparticles catalyzed by DNAzyme is an innovative approach and can be applied in bioanalysis (LSPR, electrochemistry) as well as in material sciences

2-LED-μspectrophotometer for rapid on-site detection of pathogens using noble-metal nanoparticle-based colorimetric assays

Novel point-of-care compatible methods such as colorimetric assays have become increasingly important in the field of early pathogen detection. A simple and hand-held prototype device for carrying out DNA-amplification assay based on plasmonic nanoparticles in the colorimetric detection is presented. The low-cost device with two channels (sample and reference) consists of two spectrally different light emitting diodes (LEDs) for detection of the plasmon shift. The color change of the gold-nanoparticle-DNA conjugates caused by a salt-induced aggregation test is examined in particular. A specific and sensitive detection of the waterborne human pathogen Legionella pneumophila is demonstrated. This colorimetric assay, with a simple assay design and simple readout device requirements, can be monitored in real-time on-site. © 2020 by the authors

Improving colloidal stability of silica nanoparticles when stored in responsive gel: application and toxicity study

When silica nanoparticles (SiNP) are stored in aqueous solution, even for few hours, they have a tendency to form agglomerates and therefore adapt inhomogeneous structures. Here we present a very practical method to store SiNP in responsive hydrogel. We have confirmed that SiNP kept in the responsive hydrogel do not undergo through undesirable morphological changes and while in storage they maintain their excellent colloidal stability. The effect of SiNP hollowing (i.e. dissolution of the core of the particles that leaves empty cavity inside) was significantly inhibited in the hydrogel, which is a critical feature for any nano-medical applications (e.g. controlled drug release). To demonstrate the applicability of the hydrogel-storing concept within a biologically relevant context, in this work we have evaluated the toxicological effects of the responsive SiNP-gel formulation in a model in vitro (human cell line U87GM and hemocompatibility using red blood cells) and ex ovo (hen’s egg test) experiments. Particles stored in the gel as well as the pure gel did not affect the hemocompatibility (hemolysis and erythrocyte aggregation) up to a concentration of 100?g/mL. Furthermore, systemic injections into the blood circulation of the chick area vasculosa confirmed the biocompatibility in a more complex biological environment. All evaluated toxicological values (haemorrhage, thrombosis, vascular lysis, and lethality) were comparable with the negative control and no differences in toxicological response could be observed between the SiNP stored in hydrogel and the control nanoparticles stored in the solution

Stabilizing silica nanoparticles in hydrogels: impact on storage and polydispersity

For successful nanomedicine, it is important that the unique, size-dependent physico-chemical properties of the nanomaterial remain predictably constant during both the storage and the manipulation of the material. Here a novel approach to preserve the colloidal stability and degradation of NPs is described. The concept is simple: (a) a solution of monodisperse particles is formulated into a responsive water- or PBS-based hydrogel; (b) the gel can be reversibly turned into a solution after long term storage by shaking it by hand; (c) the NP can be diluted and used in any desired application without the need for excessive manipulation. The differences between the physico-chemical properties of NPs stored in solution and in gel are compared. Two types of NPs were involved in this study: silica NPs of similar100 nm and Au-NPs of 30 and 80 nm in diameter. The key findings are: the fibrous matrix of the hydrogel limits the NP mobility{,} significantly reduces NP aggregation and conserves the NP morphology; both the hydrogelator and the NPs show negligible toxicity towards the model U937 human hematopoietic cell line; undesired leaching of cargo material loaded inside the particles is reduced{,} which could be an important feature for drug delivery systems

Microfluidic System for Cell Mixing and Particle Focusing Using Dean Flow Fractionation

Recent developments in the field of additive manufacturing processes have led to tremendous technological progress and opened directions for the field of microfluidics. For instance, new flexible materials for 3D printing allow the substitution of polydimethylsiloxane (PDMS) in microfluidic prototype development. Three-dimensional-printed microfluidic components open new horizons, in particular for the automated handling of biological cells (e.g., eukaryotic cells or bacteria). Here, we demonstrate how passive mixing and passive separation processes of biological cells can be realized using 3D printing concepts for rapid prototyping. This technique facilitates low-cost experimental setups that are easy to modify and adopt for specific detection and diagnostic purposes. In particular, printing technologies based on fused deposition modeling and stereolithography are used and their realization is discussed. Additive technologies enable the fabrication of multiplication mixers, which overcome shortcomings of current pillar or curve-based techniques and enable efficient mixing, also of biological cells without affecting viability. Using standard microfluidic components and state-of-the art 3D printing technologies, we realize a separation system based on Dean flow fragmentation without the use of PDMS. In particular, we describe the use of a 3D-printed helix for winding a capillary for particle flow and a new chip design for particle separation at the outlet. We demonstrate the functionality of the system by successful isolation of ~12 µm-sized particles from a particle mixture containing large (~12 µm, typical size of eukaryotic cells) and small (~2 µm, typical size of bacteria or small yeasts) particles. Using this setup to separate eukaryotic cells from bacteria, we could prove that cell viability is not affected by passage through the microfluidic systems

Propagating and localized surface plasmon resonance sensing — A critical comparison based on measurements and theory

With its potential for ultrasensitive, label-free detection of molecular interactions, sensing methods based on the surface plasmon resonance (SPR) effect fully meet the requirements for modern analytical techniques. Already established by using propagating SPR in thin gold layers, the last years witnessed the emergence of another related technique utilizing extremely miniaturized noble metal sensor structures, based on a localized SPR.This paper provides a critical comparison of these kinds of SPR sensing, reviews the foundation of both general approaches, presents experimental data on exactly the same molecular model system using both techniques, as well as theoretical considerations in order to allow reasonable comparison. It highlights the specific features and effects, in order to provide guidance in choosing the right technique for given bioanalytical tasks.The study demonstrated the capabilities of LSPR for sensing of molecular layers even in the lower nanometer dimension. For the detection of small (bio)molecules, smaller particle diameters are favored regarding highest sensitivity. It also presents an approach to obtain refractive index and the thickness of a molecular film by analyzing the signal response of plasmonic sensors with metal nanoparticles. Moreover, an additional method for the improvement of the parameters' determination is introduced. Keywords: Plasmon, Sensing, Nanoparticles, Metallic layers, Spectroscopy, thin-layer adsorptio

2-LED-µSpectrophotometer for Rapid On-Site Detection of Pathogens Using Noble-Metal Nanoparticle-Based Colorimetric Assays

Novel point-of-care compatible methods such as colorimetric assays have become increasingly important in the field of early pathogen detection. A simple and hand-held prototype device for carrying out DNA-amplification assay based on plasmonic nanoparticles in the colorimetric detection is presented. The low-cost device with two channels (sample and reference) consists of two spectrally different light emitting diodes (LEDs) for detection of the plasmon shift. The color change of the gold-nanoparticle-DNA conjugates caused by a salt-induced aggregation test is examined in particular. A specific and sensitive detection of the waterborne human pathogen Legionella pneumophila is demonstrated. This colorimetric assay, with a simple assay design and simple readout device requirements, can be monitored in real-time on-site