Nanoscale Photoluminescence Manipulation in Monolithic Porous Silicon Oxide Microcavity Coated with Rhodamine-Labeled Polyelectrolyte via Electrostatic Nanoassembling

Porous silicon (PSi) is a promising material for future integrated nanophotonics when coupled with guest emitters, still facing challenges in terms of homogenous distribution and nanometric thickness of the emitter coating within the silicon nanostructure. Herein, it is shown that the nanopore surface of a porous silicon oxide (PSiO2) microcavity (MC) can be conformally coated with a uniform nm-thick layer of a cationic light-emitting polyelectrolyte, e.g., poly(allylamine hydrochloride) labeled with Rhodamine B (PAH-RhoB), leveraging the self-tuned electrostatic interaction of the positively-charged PAH-RhoB polymer and negatively-charged PSiO2 surface. It is found that the emission of PAH-RhoB in the PSiO2 MC is enhanced (≈2.5×) and narrowed (≈30×) at the resonant wavelength, compared with that of PAH-RhoB in a non-resonant PSiO2 reference structure. The time-resolved photoluminescence analysis highlights a shortening (≈20%) of the PAH-RhoB emission lifetime in the PSiO2 MC at the resonance versus off-resonance wavelengths, and with respect to the reference structure, thereby proving a significant variation of the radiative decay rate. Remarkably, an experimental Purcell factor Fp = 2.82 is achieved. This is further confirmed by the enhancement of the photoluminescence quantum yield of the PAH-RhoB in the PSiO2 MC with respect to the reference structure. Application of the electrostatic nanoassembling approach to other emitting dyes, nanomaterials, and nanophotonic systems is envisaged

Biofunctionalized nanoporous aluminum oxide culture chips : for capture and growth of bacteria

Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis. After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2. Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated. In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface. With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system. In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide. In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment. </p

Stability of (Bio)Functionalized Porous Aluminum Oxide

Porous aluminum oxide (PAO), a nanostructured support for, among others, culturing microorganisms, was chemically modified in order to attach biomolecules that can selectively interact with target bacteria. We present the first comprehensive study of monolayer-modified PAO using conditions that are relevant to microbial growth with a range of functional groups (carboxylic acid, a-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane). Their stability was initially assessed in phosphate-buffered saline (pH 7.0) at room temperature. The most stable combination (PAO with phosphonic acids) was further studied over a range of physiological pHs (4–8) and temperatures (up to 80 °C). Varying the pH had no significant effect on the stability, but it gradually decreased with increasing temperature. The stability of phosphonic acid-modified PAO surfaces was shown to depend strongly on the other terminal group of the monolayer structure: in general, hydrophilic monolayers were less stable than hydrophobic monolayers. Finally, an alkyne-terminated PAO surface was reacted with an azide-linked mannose derivative. The resulting mannose-presenting PAO surface showed the clearly increased adherence of a mannose-binding bacterium, Lactobacillus plantarum, and also allowed for bacterial outgrowth

Carbohydrate [Presenting Self ]Assembled Monolayers: Preparation, Analysis, and Applications in Microbiology

This chapter commences with an overview of the different methods most commonly used to prepare glycosurfaces. The discussion on these methods is divided over three sections, each reflecting one of the three distinct approaches used to create glycosurfaces: direct formation of carbohydrate-containing self-assembled monolayers (SAMs), use of secondary (or tertiary) reactions to install a carbohydrate on a preformed SAM, and noncovalent immobilization of carbohydrates on a surface. Next, the chapter focuses on several key surface analysis techniques that can be used to characterize a prepared glycosurface and the type of information that can be obtained from each technique. Finally, it discusses several other applications of glycosurfaces in microbiology, focusing on binding, capture, and sensing of bacteria and bacterial toxins and on the multivalency effects that exert a large influence on the interaction between carbohydrates and proteins in biological systems and on fabricated glycosurfaces

Decoration of Porous Silicon with Gold Nanoparticles via Layer-by-Layer Nanoassembly for Interferometric and Hybrid Photonic/Plasmonic (Bio)sensing

Gold nanoparticle layers (AuNPLs) enable the coupling of morphological, optical, and electrical properties of gold nanoparticles (AuNPs) with tailored and specific surface topography, making them exploitable in many bioapplications (e.g., biosensing, drug delivery, and photothermal therapy). Herein, we report the formation of AuNPLs on porous silicon (PSi) interferometers and distributed Bragg reflectors (DBRs) for (bio)sensing applications via layer-by-layer (LbL) nanoassembling of a positively charged polyelectrolyte, namely, poly(allylamine hydrochloride) (PAH), and negatively charged citrate-capped AuNPs. Decoration of PSi interferometers with AuNPLs enhances the Fabry-Pérot fringe contrast due to increased surface reflectivity, resulting in an augmented sensitivity for both bulk and surface refractive index sensing, namely, about 4.5-fold using NaCl aqueous solutions to infiltrate the pores and 2.6-fold for unspecific bovine serum albumin (BSA) adsorption on the pore surface, respectively. Sensitivity enhancing, about 2.5-fold, is also confirmed for affinity and selective biosensing of streptavidin using a biotinylated polymer, namely, negatively charged poly(methacrylic acid) (b-PMAA). Further, decoration of PSi DBR with AuNPLs envisages building up a hybrid photonic/plasmonic optical sensing platform. Both photonic (DBR stop-band) and plasmonic (localized surface plasmon resonance, LSPR) peaks of the hybrid structure are sensitive to changes of bulk (using glucose aqueous solutions) and surface (due to BSA unspecific adsorption) refractive index. To the best of our knowledge, this is the first report about the formation of AuNPLs via LbL nanoassembly on PSi for (i) the enhancing of the interferometric performance in (bio)sensing applications and (ii) the building up of hybrid photonic/plasmonic platforms for sensing and perspective biosensing applications

Nanoscale Photoluminescence Manipulation in Monolithic Porous Silicon Oxide Microcavity Coated with Rhodamine-Labeled Polyelectrolyte via Electrostatic Nanoassembling

Porous silicon (PSi) is a promising material for future integrated nanophotonics when coupled with guest emitters, still facing challenges in terms of homogenous distribution and nanometric thickness of the emitter coating within the silicon nanostructure. Herein, it is shown that the nanopore surface of a porous silicon oxide (PSiO2) microcavity (MC) can be conformally coated with a uniform nm-thick layer of a cationic light-emitting polyelectrolyte, e.g., poly(allylamine hydrochloride) labeled with Rhodamine B (PAH-RhoB), leveraging the self-tuned electrostatic interaction of the positively-charged PAH-RhoB polymer and negatively-charged PSiO2 surface. It is found that the emission of PAH-RhoB in the PSiO2 MC is enhanced (≈2.5×) and narrowed (≈30×) at the resonant wavelength, compared with that of PAH-RhoB in a non-resonant PSiO2 reference structure. The time-resolved photoluminescence analysis highlights a shortening (≈20%) of the PAH-RhoB emission lifetime in the PSiO2 MC at the resonance versus off-resonance wavelengths, and with respect to the reference structure, thereby proving a significant variation of the radiative decay rate. Remarkably, an experimental Purcell factor Fp&nbsp;= 2.82 is achieved. This is further confirmed by the enhancement of the photoluminescence quantum yield of the PAH-RhoB in the PSiO2 MC with respect to the reference structure. Application of the electrostatic nanoassembling approach to other emitting dyes, nanomaterials, and nanophotonic systems is envisaged