Capillary filling dynamics in nanoporous anodic alumina for biosensing applications
- Autores: Chris Eckstein
- Directores de la Tesis: Lluís F. Marsal Garví (dir. tes.), Josep Ferré Borrull (dir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2019
- Idioma: español
- Tribunal Calificador de la Tesis: Josep Pallarès Marzal (presid.), Francisco J. Meseguer (secret.), Abel Santos Alejandro (voc.)
- Programa de doctorado: Programa de Doctorado en Tecnologías para Nanosistemas, Bioingeniería y Energía por la Universidad Rovira i Virgili
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
Intro Generic: Nanotechnology research has gained much interest in the last decades. Fabrication of materials at nano-scale levels has brought to light many new unique physico–chemical characteristics of materials which are otherwise not present in their bulk counterpart.
Harvesting these new properties has led to many unique applications in biotechnology, medicine and environmental sciences in recent years. Even though many exciting innovations have already been presented, a wealth of possibilities still lies ahead further uniting the fields of biology, physics, chemistry and material science.
Many of the fundamental biochemical reactions making life possible take place at the nanoscale level and being able to fabricate and modify materials at such level brings about benefits of understanding and controlling biological interactions of living cells, complex tissues or entire organisms with novel materials. Medical applications are bound to be made in the fields of drug delivery, tissue regeneration and wound healing. Environmental applications will further advance the detection and remediation of pollutants. Filtering gas, ions or other small molecules or harvesting and directing electromagnet waves are current applications in the fields of chemistry and physics.
Interdisciplinary techniques finding application across biology, chemistry and physics can be found in the form of sensing systems. The detection of substances or monitoring events at the nanoscale level can be found in cancer diagnostics, detection of environmental pollutants or the detection and monitoring of gaseous substances and light intensities.
The advantages of sensors based on nanomaterials lie in the application of ultra-small sample volumes, which is especially important in the field of biology where tissue samples are often minute or biological reagents are very expensive. Furthermore the downscaling of the reactions has led to new technologies such as lab-on-a-chip (LOC) or point-of-care (POC) devices which have the great potential of being small and portable and can be applied in situ, for faster and cheaper retrieval of data.
Nano-optofluidics is a recently introduced term which typically describes applications interfacing liquids carrying an analyte of interest with a solid as the matrix producing an optical signal detected by a measurement system. Nano-optofluidics possesses great potential for the development of novel LOC and POC devices which is what this PhD dissertation is aiming at.Intro Technical: Efforts put into exploiting optical properties of thin films for the development of optical biosensors date back until the 1970s where the vast majority of research output focused on surface plasmon resonance (SPR). The advent of nanostructures made of noble metals has since then spawned new research on localized SPR (LSPR) and waveguides.
A huge leap forward in research of optical sensing with Nanoporous Anodic Alumina (NAA) was seen when reflectometric interference spectroscopy (RIfS) was naturally adapted to NAA from porous silicon and other materials. Conventionally, RIfS is based on the detection of effective refractive index shifts in single layered NAA upon infiltrating with analytes presented as wellresolved peaks generated by the Fabry-Pérot effect. More recently Bragg reflectors (multi-layered structures of a nanoporous material with periodic refractive index) were presented as a promising alternative as sensing matrix which allows monitoring of optical transmittance dip position and intensity.
The term optofluidics was introduced in 2004 and represents a fairly young discipline which combines the fields of optics and fluidics at the micro- and nanoscale. Monitoring the infiltration of liquid into nanoporous structures via laser interferometry has very recently been demonstrated as a novel nano-characterisation tool in nanometrology.
Main Objectives: The main objective of this PhD dissertation lies in working towards the development of a novel type of nano-optofluidic biosensor based on nanopore radius reduction by advancing a recently introduced nano-metrological technique monitoring a laser reflected of the top and bottom surface of a nanoporous membrane while a liquid is traveling through the pores. This technique was termed fluid imbibition-coupled laser interferometry (FICLI) and will be referred to as such throughout this PhD dissertation.
It was at first necessary to learn different fabrication strategies of nanoporous anodic alumina (NAA) leading to a variety of nanostructured architectures which served as the basic porous matrix for the liquid to pass through for subsequent pore radius estimations using FICLI. Additionally, a range of chemical and biological surface modification strategies had to be controlled for the strategic immobilization of target analytes to the inside pore walls of NAA. For a better comparison of the FICLI technique it was necessary to apply further traditional optical NAA sample characterization techniques, such as scanning electron microscopy (SEM), confocal microscopy, UV/Vis, FTIR and photoluminescence spectroscopy.
A first milestone of this PhD dissertation was set to assess the sensitivity, accuracy and reproducibility of FICLI towards the estimation of pore radius modifications across different liquid-surface interfaces by using different liquids travelling through the pores of a set of consecutively modified NAA structures fabricated with different anodization electrolyte solutions.
A second milestone aimed at assessing complex chemical surface functionalizations and target specific immobilization of biological analytes onto the NAA pore walls and the effects on the estimations of pore radius changes via FICLI.
Results: In chapter 3 we addressed the fabrication of NAA membranes and how we modified and functionalized the surfaces of the pore walls. The FICLI set up and basic foundation of monitoring fluid movement within nanopores in real time and the subsequent pore radius estimation procedure were presented. In an experimental approach we discussed limitations and possibilities of FICLI and its advantages over alternative methods. With the considerations made in this chapter we established a FICLI measurement protocol for the following experiments. FICLI pore radius estimates are robust and reproducible and ready to be applied in large experiments in the following chapters In chapter 4 we addressed the reproducibility and accuracy of fluid imbibition coupled laser interferometry (FICLI) as a pore radius estimation technique under a set of changes to the physico-chemical parameters of the liquid-surface interface. We validated the technique by comparing it to results obtained by environmental scanning electron microscopy (ESEM).
The results showed good reproducibility and accuracy of FICLI when carefully adjusting the physico-chemical parameters of the liquids and carrier matrix in use and we obtained a linear relationship between pore widening times and increasing pore radius estimates in accordance to the literature for these materials.
However, FICLI and ESEM both present limiting bottlenecks towards the estimation of a pore radius with sub-nanometer accuracy. FICLI pore radius estimations are dependent on the use of accurate physico-chemical parameters of the liquid-surface interface during the analysis of the time-resolved interferograms. Herein lies the challenge, as certain parameters are still under ongoing discussion in the scientific community such as the dynamic contact angle within the nanopores and the changes of fluid parameters (i.e. viscosity, surface tension) due to the nano-confinement. In contrast, insufficient resolution of the ESEM images are the main limiting factor during radius estimations via image analysis as obscure boarders between pore and pore wall with low resolution lead to a highly subjective selection of measurement alignments.
Finally, we demonstrated that, in contrast to the purely topological nature of ESEM, FICLI is capable to characterize NAA beyond the surface and allows getting an insight into the internal geometry by giving pore radius estimates of NAA double and triple layer structures.
In chapter 5, it was demonstrated that FICLI is a highly sensitive method to detect and quantify changes in the pore surface properties and dimensions of NAA after the attachment of different biological and chemical analytes. The accuracy of FICLI for the characterization of pore size changes of NAA produced in oxalic and sulfuric acids has been shown. The radius increase due to wet chemical pore widening was detected irrespective of the electrolyte used to prepare the NAA, and the results are in good agreement with traditional NAA characterization methods such as ESEM and the literature. The electrostatic immobilization of biomolecules to the pore walls is a common and fast NAA modification technique. In this study, it was shown that the pore radius reduction of the NAA due to the immobilization of BSA is accurately estimated by FICLI. The radius reduction estimates fall into the expected size dimensions of the protein, and the radius reductions are reproducible irrespective of the NAA material and initial pore radius. Only slight differences are observed for NAA obtained with different electrolytes, which is an indication that the surface contact angle for such structures depends slightly of the electrolyte. The pore radius reduction due to the functionalization of NAA pore walls with APTESGTA was characterized. The observed radius reduction value suggests the deposition of an APTES multilayer due to reaction conditions. The subsequent covalent immobilization of streptavidin via imine bonds was further detected by a radius reduction. The dimensions of the protein were slightly overestimated and also suggest a multilayer deposition of the protein. The immobilization of IgGs via immune complexation with PA and secondary IgGs was detected by the characterization of NAA pore radius reductions and changing capillary filling dynamics. It was shown that a chain of immobilization events starting with the electrostatic immobilization of PA, followed by specific immune complexations of IgG and secondary IgG can be demonstrated by the size reduction of NAA pores. These findings provide great potential for the future development of target-specific biosensing systems based on the radius reduction of NAA and the changes in nanopore filling dynamics. Further research on real-time monitoring of filling dynamics variations can open the way to new sensing devices with a view to miniaturization.
Summary: In summary, the results presented in this PhD dissertation demonstrated the applicability of time-resolved interferometric monitoring of fluid dynamics as a powerful tool for the characterisation of nanopores. The accurate estimation of pore radii across a variety of different liquid surface interfaces showed the capability of this system to resolve changes in the nanopore pore dimension of below 2nm. Monitoring binding events of polymers and biomolecules on the pore walls immobilised via different strategies showed the potential for target-specific biosensing based on radius reduction in NAA and may well have opened the door for the development of novel lab-on-chip and point-of-care sensing devices.
In conclusion FICLI is a beneficial addition to nanometrology and relevant for applications where the characterisation of pores with a resolution on the nanometer level is important.
Other nano-characterisation and sensing applications might benefit from FICLI’s ease of use and sensitivity. For example the fabrication of carbon- or polymer-based nanotubes via NAA scaffolds might be a useful future application for FICLI to characterise the growth rate or total thickness of fabricated tubes before etching off the alumina matrix. Polyelectrolytes can be stimulated to swell or shrink by changing the local environment. Estimating the degree of swelling of such a functionalisation might give relevant insight for the development and characterisation of drug loading and releasing of drug delivery systems.
Determining the liquid contact angle within nanopores is currently still openly discussed and monitoring the pore filling dynamics in combination with high resolution electron microscopy producing more accurate pore radius estimates via image analysis, FICLI might contribute producing more accurate data on the dynamic contact angle within the nano-confinement.
