Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection

[EN] The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 mu M. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents.This work was financially supported by the E.U. FEDER, the Spanish Ministry of Science and Innovation (ADBIHOL-PID2019-110713RB-I00/AEI/10.13039/501100011033) and Generalitat Valenciana (PROMETEO/2020/094). M. I. Lucío acknowledges her Juan de la Cierva-Incorporación grant (IJC 2018-035355-I) funded by MCIN/AEI/10.13039/501100011033. P. Zezza acknowledges Generalitat Valenciana for her Grisolia fellowship grant.Zezza, P.; Lucío, MI.; Fernández, E.; Maquieira Catala, A.; Bañuls Polo, M. (2023). Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection. Biosensors. 13(3). https://doi.org/10.3390/bios1303031213

Cell Microarray Technologies for High-Throughput Cell-Based Biosensors

Due to the recent demand for high-throughput cellular assays, a lot of efforts have been made on miniaturization of cell-based biosensors by preparing cell microarrays. Various microfabrication technologies have been used to generate cell microarrays, where cells of different phenotypes are immobilized either on a flat substrate (positional array) or on particles (solution or suspension array) to achieve multiplexed and high-throughput cell-based biosensing. After introducing the fabrication methods for preparation of the positional and suspension cell microarrays, this review discusses the applications of the cell microarray including toxicology, drug discovery and detection of toxic agents.ope

Nanofabrication

We face many challenges in the 21st century, such as sustainably meeting the world's growing demand for energy and consumer goods. I believe that new developments in science and technology will help solve many of these problems. Nanofabrication is one of the keys to the development of novel materials, devices and systems. Precise control of nanomaterials, nanostructures, nanodevices and their performances is essential for future innovations in technology. The book "Nanofabrication" provides the latest research developments in nanofabrication of organic and inorganic materials, biomaterials and hybrid materials. I hope that "Nanofabrication" will contribute to creating a brighter future for the next generation

Glassy Materials Based Microdevices

Microtechnology has changed our world since the last century, when silicon microelectronics revolutionized sensor, control and communication areas, with applications extending from domotics to automotive, and from security to biomedicine. The present century, however, is also seeing an accelerating pace of innovation in glassy materials; as an example, glass-ceramics, which successfully combine the properties of an amorphous matrix with those of micro- or nano-crystals, offer a very high flexibility of design to chemists, physicists and engineers, who can conceive and implement advanced microdevices. In a very similar way, the synthesis of glassy polymers in a very wide range of chemical structures offers unprecedented potential of applications. The contemporary availability of microfabrication technologies, such as direct laser writing or 3D printing, which add to the most common processes (deposition, lithography and etching), facilitates the development of novel or advanced microdevices based on glassy materials. Biochemical and biomedical sensors, especially with the lab-on-a-chip target, are one of the most evident proofs of the success of this material platform. Other applications have also emerged in environment, food, and chemical industries. The present Special Issue of Micromachines aims at reviewing the current state-of-the-art and presenting perspectives of further development. Contributions related to the technologies, glassy materials, design and fabrication processes, characterization, and, eventually, applications are welcome

Biodetection and biointerface based on Nanostructured Aluminum Oxide (NAO): From fluorescence enhancement to MS effect on single neural cells

Anodic aluminum oxide (AAO) has been investigated and utilized in numerous products for almost a century. But the rapidly increasing interest in nanoscale materials and their outstanding properties has propelled nanoporous AAO to be used as a substrate for sensors and biosensors. Fluorescence-based biosensors are one type of optical biosensors which are very popular for detecting a variety of targets (DNA, RNA, glucose, enzyme, bacteria, etc.). The recent discovery of AAO fluorescence enhancement effect makes AAO more attractive since it has the great potential to be used in biosensing field to improve the sensor sensitivity. However, the mechanism of the AAO fluorescence enhancement effect has not be understood thoroughly. Based on the experimental and modeling results, it has been found that the main contributing factor to the fluorescence enhancement is probably the plasmonic Al nanoparticles (NPs) embedded in the film, while the nanopore dimensions have a limited contribution. Based on its fluorescence enhancement effect, a new class of molecular beacon biosensors is developed to detect specific hairpin DNA sequence. The sensor demonstrates excellent specificity and selectivity, indicating the great promise of this type of sensor for diagnostic applications. Furthermore, another optical biosensor has been developed based on AAO. TGF-β1 which is one type of growth factor secreted by pancreatic stellate cell (ITAF) has successfully detected by this sensor. It has been found that 10 ng/ml of purified transforming growth factor β1 (TGF-β1) can be readily detected in buffer with high specificity. TGF-β1 in a conditioned cell medium has also been detected successfully. By comparing with the reference data of purified TGF-β1, concentration of TGF-β1 secreted in the conditioned cell medium has been reasonable estimated. Finally, Transcranial magnetic stimulation (MS) effects on single neuron cell (N27) have been studied on both glass and AAO substrate. It has been found that MS not only has a negligible cytotoxic effect on N27 cells but also can speed up the N27 cell proliferation and regeneration

Engineering patterned and dynamic surfaces for the spatio-temporal control of cell behaviour

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Recent advancements in microfabrication technologies in combination with the use of light-responsive materials, allow to manipulate the shape of living cells in real-time in a non-invasive Spatio-temporal controlled way to introduce additional geometrically defined adhesion sites and to study relative cell behaviour. Here, the confocal laser technique is exploited for dynamically evaluate the variation over time of the tensional and morphological cell state. This method allows the precise control of specific actin structures that regulate cell architecture. Actin filament bundles, initially randomly organized in circular-shaped cells, are induced to align and distribute to form a rectangular-shaped cell in response to specific dynamic changes in the cell adhesion pattern. The changes in morphology also reflect dramatic changes in FAs distribution, cell mechanics, nuclear morphology, and chromatin conformation. The reported strategy is convenient to explore the cell-substrate interface and the mechanisms through which cell geometry regulates cell signalling in a facile and cost-effective manner and it open new routes to understand how the field of dynamic platforms should potentially contribute to unveil complex biological events such as the modulation of cell shape

Design of nanoscale responsive polymer film for sensor application

The concept of fabricating sensing or responsive surfaces at the nanoscale has been the subject of extensive investigations since they can function as nanoscale device as well as next-generation miniaturized sensor. Polymers offer particular insights into the developments of novel responsive materials, which can be integrated with sensory systems to alter structure and properties in response to environmental stimuli. These responsive polymer nanolayers change surface composition and properties modulated by external triggers such as temperature, pH, solvent exposure, and light. This study explores possibilities of designing such polymer surfaces that possess versatile sensing mechanisms via controlled reorganization of chain and functional groups in multi-component and composite surface and then applying this into actual sensory systems of critical relevance. To achieve this, we designed novel nano-structured materials based on the employment of polymer methodologies, chemical functionality, and provide new capabilities by adding nanoparticles/nanowires to alter responsive polymer systems. We selected polymer with very different chemistry, switchable properties, and the surface composition which can be precisely controlled and divided into four groups: (1) switchable adaptive polymer nanolayer on planar silicon substrates, (2) compliant polymer or nanocomposite nanolayer enhancing/improving the sensitivity of current cantilever based sensors, (3) polymer nanolayer on ZnO nanobelt with enhanced photosensitivity (4) assembled ultra thin film with optical grating 3D structures. Furthermore, we designed and fabricated compliant polymer or nanocomposite films for thermal, chemical and light sensitive systems which can greatly enhance the sensitivity of corresponding micro/nanodevices

Tungsten oxysulfide nanoparticles interspersed nylon based e-textile as a low cost, wearable multifunctional platform for ultra-sensitive tactile sensing and breath sensing applications

In this work, we report the hydrothermal synthesis of Tungsten Oxysulfide (WS2|O) nanoparticles interspersed onto porous, lightweight Nylon fabric as a clean-room-free, multifunctional sensor for detection of human breath, strain, and pressure. Morphological characterizations reveal the uniform dispersion of the conductive WS2|O nanosheets across the ultra-thin fibers of nylon. The fabricated WS2|O@nylon-based device as a breath sensor exhibits excellent sensitivity towards different breath patterns. The optimization studies resulted in a 4-layered high-performance piezoresistive wearable pressure sensor. It exhibited a sensitivity of 1.5 kPa−1, response time of 0.2 sec over a dynamic range of 50 Pa to 350 Pa. A Gauge factor of 24.2 and good mechanical stability across 10,000 cycles of compressive strain exhibited by the strain sensor makes it suitable for gesture recognition. The exceptional sensitivity, stability with good flexibility prove it as a promising device platform for the development of various wearable multifunctional sensor applications

Azobenzene-based Biomaterials as Dynamic Cell Culture Systems

The aim of this Thesis is to fabricate dynamic light-switchable biomaterials as scaffolds to study cell behavior in a more complex environment than the one generated by the use of static systems. We take advantage of compelling properties of azobenzenes to engineer photoresponsive 2D and semi-3D platforms to investigate different biological processes from adhesion up to differentiation. In vivo, in addition to the chemical and mechanical properties, the topographic cues play a main role to guide cell response. The ECM is filled with nano- to micro-meter scale landscapes (e.g., ridges, pores and fibers) in continuous remodeling. However, the topography of the most widely implemented in vitro platforms is static and difficulty mimics the dynamicity of ECM. Thus, we propose platforms, which can dynamically tune on demand their topographic properties upon external stimulation. In particular, azobenzene-containing systems can tune their properties under light illumination, recapitulating the spatial-temporal changes of the physiological cell environment. In Chapter 1, we will discuss about important properties of azobenzene molecules and present different applications of a variety of materials containing azobenzenes from amorphous materials to highly organize liquid crystal polymers. In particular, we will focus on the recent use of azopolymers as dynamic cell instructive materials. As of now, there is a lack of knowledge on the role of dynamic topography and, even more on its effect on stem cell differentiation. In the light of this, in Chapter 2 we will present a technique to photo patterning azopolymer thin films in situ by means of a laser-based confocal microscopy. Further, we will analyze the human mesenchymal stem cell (hMSC) response after the spatial-temporal dynamic topographic changes. In more details, a mass migration phenomenon of azopolymers elicited under light irradiation allows to emboss a variety of patterns on cell-populated azopolymer films. We will investigate the stem cell response on a switchable topography from a linear pattern to a grid both in term of cell cytoskeletal re-organization and cell differentiation. Our aim is to investigate the impact of dynamic remodeling of cell environment on hMSCs gene expression profile, in comparison to static surfaces. In order to achieve our goal, we will investigate the cell behavior over time, changing the topographic aspects of the substrate and analyzing the effect of dynamic cues in modulating cell morphology and osteogenic gene expression profile. In particular, we will investigate whether epigenetic effect induced by changes in the biophysical properties of the substrate over time would redirect the expression of lineage specific markers. In Chapter 3, we will discuss about an athermal photofluidization process that can directly reshape an azopolymer pillar array in the presence of cells to investigate the dynamic reassemble of F-Actin on deformed pillars. We will show that pillar arrays can be reshaped along the direction of laser polarization, resulting in elongated structures with controllable eccentricity. This light-driven phenomenon, permits to usesuch type of systems as platforms to analyze cell membrane curvature remodeling in respond to dynamic pillar reshaping. The plasma membrane wraps around the pillars, which generate local curvatures on cell membrane and trigger the F-actin accumulation. Human bone osteosarcoma epithelial cells (U2OS) will be used to investigate the reorganization of F-Actin during the platform transition from pillar to ellipsoidal-shape structures over time. In Chapter 4, we will focus on designing semi-3D hydrogel platforms containing azobenzene to engineer and manipulate culture systems in order to develop photoactuable cell confining systems. Acrylamide-modified gelatin containing azobenzene-based cross linkers will be used to microfabricate well-defined semi-3D photo-responsive structures by means of two-photon lithography (2PP). As proof of concept, we will show an example of an array of squared structures, where cells are physically confined between the adjacent gelatin blocks, which can be remotely stimulated. The light irradiation can be converted in a local mechanical stimulation able to deform the nucleus at a single-cell level. In Conclusion and Future Perspectives, a summary of the main results achieved in this thesis is presented and future applications are proposed

Switchable surfaces for biomedical applications

Switchable oligopeptides, able to expose of conceal biomolecules on a surface, upon the application of an electrical potential, represent a versatile tool for the development of novel devices, presenting potential biomedical applications. Recently, several studies have demonstrated the applicability of smart devices for the control of protein binding and cellular response. In this work; a detailed analysis of the steric requirements necessary to develop a mixed oligopeptide Self-Assembled Monolayer (SAM) presenting an optimum switching ability will be described. The influence of both the SAM components surface ratio and the switching unit length on the mixed SAMs switching performance will be investigated. The findings of this investigation will be used to develop, for the first time, a platform, based on electrically switchable oligopeptides, able to control the interaction between an antigen and its relative antibody. The influence of the biological medium on the oligopeptide switching ability will also be investigated. Finally, an orthogonal functionalisation strategy, will be investigated in detail, together with a new platform able to promote human sperm cells adhesion. The results of this research thesis will also represent the first building blocks towards the development of glass-gold rnicropattemed surfaces able to control the calcium signalling in human sperm cells, presenting potential applications in the improvement of in-vitro fertilisation (NF) treatments success rates