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Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles. The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system's classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts. This dissertation makes the following four major contributions in the wide-field common-path interferometric microscopy field: (1) formulating vectorial-optics based linear forward model that describes interferometric light scattering near planar interfaces in the quasi-static limit, (2) developing computationally efficient image reconstruction methods from defocus images to detect a single 25 nm dielectric nanoparticle, (3) developing asymmetric illumination based computational microscopy methods to achieve direct morphological visualization of nanoparticles at 150 nm, and (4) developing bond-selective interferometric microscopy to enable multispectral chemical imaging of sub-wavelength nanoparticles in the vibrational fingerprint region. Collectively, through these research projects, we demonstrate significant advancement in the wide-field common-path interferometric microscopy field to achieve high-resolution and accurate visualization and chemical characterization of a broad size range of individual biological nanoparticles with high sensitivity.

Diseases are often diagnosed by detection of disease-specific biomarkers in fluid samples. However, many state-of-the-art detection methods require a lab with complex machinery, trained operators, and/or lengthy analysis time. In contrast, point-of-care (POC) devices are brought to the patient's location, they are easy to use, and results are obtained almost immediately. Many current POC devices are too difficult to be used without a skilled assistant, and although many are able to detect analytes above a threshold value, they give little or no quantitative information. This work presents the development of polymer-based microfluidic devices capable of sensing and quantifying biomarkers in fluid samples in a straightforward manner using a novel biomarker assay termed "flow valve diagnostics". In this assay, an antibody-modified polydimethylsiloxane (PDMS) microchannel constricts due to the binding force between antibodies and antigens, stopping fluid flow. The flow distance is measured and correlated to antigen concentration. This detection method is an improvement over other methods because it is an innovative, non-instrumented, label-free, easy-to-use approach. These devices are small, portable, disposable, inexpensive, and thus ideal for use in POC testing. I have successfully fabricated flow valve devices with standard micromachining techniques, including photolithography, replica molding with PDMS, and plasma oxidation. Following fabrication, I compared two methods for attaching receptor biomolecules (e.g., antibodies) to the microchannel surfaces: non-specific adsorption and silanization with 3-glycidoxytrimethoxypropylsilane (GOPS). I used laser-induced fluorescence to determine that silanization with GOPS was the better method for biomolecule attachment. Finally, I tested antibody-modified flow valve devices with target antigens to determine if the antibody/antigen binding force was strong enough to cause channel pinching and flow stoppage. By modifying the device design and using higher antigen concentrations, I was able to show that flow valve devices can detect antigens in a concentration-dependent manner. Future work to improve the device design and to modify and test these devices with different receptor/target pairs will bring flow valve diagnostics closer to becoming a valuable asset in biomarker detection and POC testing.

Label-free imaging modalities offer numerous advantages, such as the ability to avoid the time-consuming and potentially disruptive process of physical staining. However, one challenge that arises in label-free imaging is the limited ability to extract specific structural or molecular information from the acquired images. To overcome this limitation, a novel approach known as digital staining or digital labeling has emerged. Digital staining leverages the power of deep learning algorithms to virtually introduce labels or stains into label-free images, thereby enabling the extraction of detailed information that would typically require physical staining. The integration of digital staining with label-free imaging holds great promise in expanding the capabilities of imaging techniques, facilitating improved analysis, and advancing our understanding of biological systems at both the cellular and tissue level. In this thesis, I explore supervised and semi-supervised methodologies of digital staining and the applications in augmenting label-free imaging modalities, particularly in the context of cell imaging and brain imaging. In the first part of the thesis, I demonstrate the novel integration of multi-contrast dark-field reflectance microscopy and supervised deep learning to enable subcellular immunofluorescence labeling and cell cytometry from label-free imaging. By leveraging the rich structural information and sensitivity of reflectance microscopy, this method accurately predicts subcellular features without the need for physical staining. As a result of the use of a novel multi-contrast modality, the digital labeling approach demonstrates significant improvements over the state-of-the-art techniques, achieving up to 3× prediction accuracy. In addition to fluorescence prediction, the method successfully reproduces single-cell level structural phenotypes related to cell cycles. The multiplexed readouts obtained through digital labeling enable accurate multi-parametric single-cell profiling across a large cell population. In the second part, I investigated a novel digital staining optical coherence tomography (DS-OCT) modality combining advantages of serial sectioning OCT and semi-supervised deep learning and demonstrated several advantages for the application of 3D histological brain imaging. The DS model is trained using a semi-supervised learning framework that incorporates unpaired translation, a biophysical model, and cross-modality image registration, which manifests broad applicability to other weakly-paired bioimaging modalities. The DS model enables the translation of S-OCT images to Gallyas silver staining, providing consistent staining quality across different samples. I further show that DS enhances contrast across cortical layer boundaries and enables reliable cortical layer differentiation. Additionally, DS-OCT preserves 3D-geometry on centimeter-scale brain tissue blocks. My pilot study demonstrates promising results on other anatomical regions acquired from different S-OCT systems, highlighting its potential for generalization in various imaging contexts. Overall, I investigate the problems of augmenting label-free imaging modalities with deep learning generated digital stains. I explored both supervised and semi-supervised methods for building novel DS frameworks. My work showcased two important applications in the field of immunofluorescence cell imaging and 3D histological brain imaging. On the one hand, the integration of DS techniques with multi-contrast microscopy has the potential to enhance the throughput of single-cell imaging cytometry, and phenotyping. On the other hand, integrating DS techniques with S-OCT holds great potential for high-throughput human brain imaging, enabling comprehensive studies on the structure and function of the brain. Through the exploration, I aim to shed light on the impact of digital staining in the field of computational imaging and its implications for various scientific disciplines.

Tesis por compendio / [ES] La tesis doctoral se centra en el desarrollo de un hidrogel sensible a analitos, funcionalizado con sondas de ADN, con estructura difractiva como transductor óptico para aplicaciones de diagnóstico in vitro. El primer capítulo incluye una visión general de los diferentes conceptos relacionados con el biosensado, los desarrollos recientes en el mercado del diagnóstico in vitro y, en particular, los biosensores de ADN. Además, se presenta la síntesis y caracterización de hidrogeles, su papel como matriz de soporte en biosensado y las estrategias de inmovilización. Por último, se explican los conceptos básicos de la holografía como nueva estrategia de detección y el papel de las diferentes redes de difracción en la biosensación. A continuación, en el Capítulo 2, se discuten los objetivos de este proyecto. El objetivo de esta investigación es desarrollar hidrogeles que incorporen sondas de ADN y dotarlas de una estructura difractiva para que actúen como transductores ópticos sin etiquetas. Se consideran dos tipos de estructuras difractivas: redes holográficas de relieve superficial (SRG) y redes de transmisión de volumen (VTG). La fase inicial de este trabajo se centró en la optimización de hidrogeles, ajustando su composición para que actuaran como biosensores holográficos. Se seleccionaron acrilamida y bisacrilamida para la preparación del hidrogel mediante reacción de polimerización por radicales libres. Además, para introducir la respuesta del analito en la red de hidrogeles 3D, hubo que investigar y poner a punto diferentes estrategias de inmovilización de biorreceptores. En el capítulo 3, la estrategia optimizada consiste en incorporar directamente sondas de ADN modificadas con acridita mediante copolimerización con monómeros de acrilamida durante la formación del hidrogel. Los hidrogeles funcionalizados con ADN se caracterizaron mediante imágenes de fluorescencia y se exploró su versatilidad mediante la fabricación de microarrays. Por último, el hidrogel optimizado sensible a los analitos se utilizó como plataforma para la preparación de SRG. El capítulo 4 describe otro enfoque adoptado para la funcionalización del hidrogel con sondas de ADN. Se añadió un comonómero de acrilato de propargilo al hidrogel de acrilamida, con el fin de introducir la presencia de residuos alcínicos y facilitar una mayor incorporación de las sondas de ADN. Las sondas de ADN utilizadas tenían grupos terminales tiol y se incorporaron mediante química de clic tiol-eno/tiol-yo, debido a la presencia de enlaces C-C dobles y triples. Con esta estrategia, se demostraron dos enfoques de inmovilización de sondas de ADN: durante y después de la síntesis del hidrogel. Los resultados preliminares mostraron que los SRGs tienen potencial para detectar directamente la hibridación de oligonucleótidos en un formato libre de etiquetas. En el capítulo 5, se optimizó el proceso de grabación de VTGs no inclinados en capas de hidrogel para mejorar el rendimiento del transductor. Tras una cuidadosa evaluación de los parámetros de grabación holográfica, las composiciones de las soluciones de incubación y los tiempos de incubación, las estructuras VTG se grabaron con una buena reproducibilidad, logrando una excelente eficiencia de difracción. Además, se estudió su estabilidad en agua para bioensayos. Por último, se observó que los VTG, modificados con oligonucleótidos, respondían selectivamente hibridándose sólo con la diana complementaria, a la vez que conservaban sus propiedades de difracción. El trabajo de investigación demostró la viabilidad de utilizar redes difractivas en capas de hidrogel como biosensores libres de etiquetas, capaces de detectar sondas de ADN, complementarias a la secuencia inmovilizada, en un medio acuoso. Por último, en el capítulo 6, se analizan comparativamente el rendimiento y la aplicabilidad de los distintos enfoques estudiados y se discuten las perspectivas futuras de los hidrogeles de ácidos nucleicos para la detección holográfica. / [CA] La tesi doctoral se centra en el desenvolupament d'un hidrogel sensible a anàlits, funcionalitzat amb sondes d'ADN, amb estructura difractiva com a transductor òptic per a aplicacions de diagnòstic in vitro. El primer capítol inclou una visió general dels diferents conceptes relacionats amb el biosensado, els desenvolupaments recents en el mercat del diagnòstic in vitro i, en particular, els biosensores d'ADN. A més, es presenta la síntesi i caracterització d'hidrogels, el seu paper com a matriu de suport en biosensado i les estratègies d'immobilització. Finalment, s'expliquen els conceptes bàsics de l'holografia com a nova estratègia de detecció i el paper de les diferents xarxes de difracció en la biosensación. A continuació, en el Capítol 2, es discuteixen els objectius d'este projecte. L'objectiu d'esta investigació és desenvolupar hidrogels que incorporen sondes d'ADN i dotar-les d'una estructura difractiva perquè actuen com a transductors òptics sense etiquetes. Es consideren dos tipus d'estructures difractivas: xarxes hologràfiques de relleu superficial (SRG) i xarxes de transmissió de volum (VTG). La fase inicial d'este treball es va centrar en l'optimització d'hidrogels, ajustant la seua composició perquè actuaren com biosensores hologràfics. Es van seleccionar acrilamida I bisacrilamida per a la preparació de l'hidrogel mitjançant reacció de polimerització per radicals lliures. A més, per a introduir la resposta de l'anàlit en la xarxa d'hidrogels 3D, va caldre investigar i posar a punt diferents estratègies d'immobilització de biorreceptores. En el capítol 3, l'estratègia optimitzada consisteix a incorporar directament sondes d'ADN modificades amb acridita mitjançant copolimerización amb monòmers d'acrilamida durant la formació de l'hidrogel. Els hidrogels funcionalitzats amb ADN es van caracteritzar mitjançant imatges de fluorescència i es va explorar la seua versatilitat mitjançant la fabricació de bioxips. Finalment, l'hidrogel optimitzat sensible als anàlits es va utilitzar com a plataforma per a la preparació de SRG. El capítol 4 descriu un altre enfocament adoptat per a la funcionalització de l'hidrogel amb sondes d'ADN. Es va afegir un comonómero de acrilato de propargilo a l'hidrogel d'acrilamida, amb la finalitat d'introduir la presència de residus alcínicos i facilitar una major incorporació de les sondes d'ADN. Les sondes d'ADN utilitzades tenien grups terminals tiol i es van incorporar mitjançant química de clic tiol-eno/tiol-ino, a causa de la presència d'enllaços C-C dobles i triples. Amb esta estratègia, es van demostrar dos enfocaments d'immobilització de sondes d'ADN: durant i després de la síntesi de l'hidrogel. Els resultats preliminars van mostrar que els SRGs tenen potencial per a detectar directament la hibridació de oligonucleótidos en un format lliure d'etiquetes. En el capítol 5, es va optimitzar el procés de gravació de VTGs no inclinats en capes d'hidrogel per a millorar el rendiment del transductor. Després d'una acurada avaluació dels paràmetres de gravació hologràfica, les composicions de les solucions d'incubació i els temps d'incubació, les estructures VTG es van gravar amb una bona reproducibilidad, aconseguint una excel·lent eficiència de difracció. A més, es va estudiar la seua estabilitat en aigua per a bioensayos. Finalment, es va observar que els VTG, modificats amb oligonucleótidos, responien selectivament hibridant-se només amb la diana complementària, alhora que conservaven les seues propietats de difracció. El treball de recerca va demostrar la viabilitat d'utilitzar xarxes difractivas en capes d'hidrogel com biosensores lliures d'etiquetes, capaces de detectar sondes d'ADN, complementàries a la seqüència immobilitzada, en un medi aquós. Finalment, en el capítol 6, s'analitzen comparativament el rendiment i l'aplicabilitat dels diferents enfocaments estudiats i es discuteixen les perspectives futures dels hidrogels d'àcids nucleics per a la detecció hologràfica. / [EN] The PhD thesis focuses on the development of an analyte-sensitive hydrogel, functionalised with DNA probes, with a diffractive structure as an optical transducer for in vitro diagnostic applications. The first chapter includes an overview of the different concepts related to biosensing, recent developments in the in vitro diagnostics market and, in particular, DNA biosensors. Furthermore, the synthesis and characterisation of hydrogels, their role as a support matrix in biosensing and immobilisation strategies are presented. Finally, the basic concepts of holography as a new detection strategy and the role of different diffraction gratings in biosensing are explained. Then, in Chapter 2, the objectives of this project are discussed. The aim of this research is to develop hydrogels that incorporate DNA probes and provide them with a diffractive structure to act as label-free optical transducers. Two types of diffractive structures are considered: surface-relief holographic gratings (SRGs) and volume transmission gratings (VTGs). The initial phase of this work focused on the optimisation of hydrogels, adjusting their composition to act as holographic biosensors. Acrylamide and bisacrylamide were selected for hydrogel preparation by free radical polymerisation reaction. Furthermore, in order to introduce the analyte response into the 3D hydrogel network, different bioreceptor immobilisation strategies had to be investigated and fine-tuned. In chapter 3, the optimised strategy is to directly incorporate acridite-modified DNA probes by copolymerisation with acrylamide monomers during hydrogel formation. The DNA-functionalised hydrogels were characterised by fluorescence imaging and their versatility was explored by microarray fabrication. Finally, the optimised analyte-responsive hydrogel was used as a platform for SRG preparation. Chapter 4 describes another approach adopted for functionalisation of the hydrogel with DNA probes. A propargyl acrylate comonomer was added to the acrylamide hydrogel in order to introduce the presence of alkyl residues and facilitate further incorporation of the DNA probes. The DNA probes used had thiol end-groups and were incorporated by thiol-ene/thiol-yo click chemistry, due to the presence of double and triple C-C bonds. With this strategy, two approaches to DNA probe immobilisation were demonstrated: during and after hydrogel synthesis. Preliminary results showed that SRGs have the potential to directly detect oligonucleotide hybridisation in a label-free format. In chapter 5, the recording process of unslanted VTGs in hydrogel layers was optimised to improve transducer performance. After careful evaluation of holographic recording parameters, incubation solution compositions and incubation times, the VTG structures were recorded with good reproducibility, achieving excellent diffraction efficiency. In addition, their stability in water for bioassays was studied. Finally, oligonucleotide-modified VTGs were found to respond selectively by hybridising only to the complementary target, while retaining their diffraction properties. The research work demonstrated the feasibility of using diffractive networks in hydrogel layers as label-free biosensors, capable of detecting DNA probes, complementary to the immobilised sequence, in an aqueous medium. Finally, in chapter 6, the performance and applicability of the different approaches studied are comparatively analysed and future prospects of nucleic acid hydrogels for holographic detection are discussed. / I would like to acknowledge the government of Valencia to for the PhD fellowship “Santiago Grisolia” and the BEFPI/2022 grant for a 4-months doctoral stay and also the Spanish Ministry of Economy and Competitiveness MINECO (ADBIHOL national project) for their financial support. / Zezza, P. (2023). Holographic biosensors made of DNA-functionalised hydrogels for in vitro diagnostic [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202597 / Compendio

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Yes / It has long been known that Tissue Factor (TF) plays a role in blood coagulation and has a direct thrombotic action that is closely related to cardiovascular risk, but it is becoming increasingly clear that it has a much wider range of biological functions that range from inflammation to immunity. It is also involved in maintaining heart haemostasis and structure, and the observation that it is down-regulated in the myocardium of patients with dilated cardiomyopathy suggests that it influences cell-to-cell contact stability and contractility, and thus contributes to cardiac dysfunction. However, the molecular mechanisms underlying these coagulation-independent functions have not yet been fully elucidated. In order to analyse the influence of TF on the cardiomyocitic proteome, we used functional biochemical approaches incorporating label-free quantitative proteomics and gene silencing, and found that this provided a powerful means of identifying a new role for TF in regulating splicing machinery together with the expression of several proteins of the spliceosome, and mRNA metabolism with a considerable impact on cell viability.

Yes / Rapid point of care tests for bacterial infection diagnosis are of great importance to reduce the misuse of antibiotics and burden of antimicrobial resistance. Here, we have successfully combined a new class of non-biological binder molecules with electrochemical impedance spectroscopy (EIS)-based sensor detection for direct, label-free detection of Gram-positive bacteria making use of the specific coil-to-globule conformation change of the vancomycin-modified highly branched polymers immobilized on the surface of gold screen-printed electrodes upon binding to Gram-positive bacteria. Staphylococcus carnosus was detected after just 20 min incubation of the sample solution with the polymer-functionalized electrodes. The polymer conformation change was quantified with two simple 1 min EIS tests before and after incubation with the sample. Tests revealed a concentration dependent signal change within an OD600 range of Staphylococcus carnosus from 0.002 to 0.1 and a clear discrimination between Gram-positive Staphylococcus carnosus and Gram-negative Escherichia coli bacteria. This exhibits a clear advancement in terms of simplified test complexity compared to existing bacteria detection tests. In addition, the polymer-functionalized electrodes showed good storage and operational stability.

Doctor of Philosophy / Department of Chemical Engineering / Vikas Berry / Graphene - a single atom thick two dimensional sheet of sp[superscript]2 bonded carbon atoms arranged in a honeycomb lattice - has shown great promise for both fundamental research & applications because of its unique electrical, optical, thermal, mechanical and chemical properties. Derivatization of graphene unlocks a plethora of novel properties unavailable to their pristine parent “graphene”. In this dissertation we have synthesized various structural and chemical derivatives of graphene; characterized them in detail; and leveraged their exotic properties for diverse applications. We have synthesized protein/DNA/ethylenediamine functionalized derivatives of graphene via a HATU catalyzed amide reaction of primary-amine-containing moieties with graphene oxide (GO) – an oxyfunctional graphene derivative. In contrast to non-specificity of graphene, this functionalization of GO has enabled highly specific interactions with analytes. Devices fabricated from the protein (concanavalin – A) and DNA functionalized graphene derivatives were demonstrated to enable label-free, specific detection of bacteria and DNA molecules, respectively, with single quanta sensitivity. Room temperature electrical characterization of the sensors showed a generation of ~ 1400 charge carriers for single bacterium attachment and an increase of 5.6 X 10[superscript]12 charge carriers / cm[superscript]2 for attachment of a single complementary strand of DNA. This work has shown for the first time the viability of graphene for bio-electronics and sensing at single quanta level. Taking the bio-interfacing of graphene to the next level, we demonstrate the instantaneous swaddling of a single live bacterium (Bacillus subtilis) with several hundred sq. micron (~ 600 µm[superscript]2) areal protein-functionalized graphene sheets. The atomic impermeability and high yield strength of graphene resulted in hermetic compartmentalization of bacteria. This enabled preservation of the dimensional and topological characteristics of the bacterium against the degrading effects of harsh environments such as the ultrahigh vacuum (~ 10[superscript]-5 Torr) and high intensity electron beam (~ 150 A/cm[superscript]2) in a transmission electron microscope (TEM) column. While an unwrapped bacterium shrank by ~ 76 % and displayed significant charge buildup in the TEM column; a wrapped bacterium remained uncontracted and undamaged owing to the graphenic wraps. This work has shown for the first time an impermeable graphenic encasement of bacteria and its application in high vacuum TEM imaging without using any lengthy traditional biological TEM sample preparation techniques. In an inch-scale, we fabricated robust free-standing paper composed of TWEEN/Graphene composite which exhibited excellent chemical stability and mechanical strength. This paper displayed excellent biocompatibility towards three mammalian cell lines while inhibiting the non-specific binding of bacteria (Bacillus cereus). We predict this composite and its derivatives to have excellent applications in biomedical engineering for transplant devices, invasive instrument coatings and implants. We also demonstrate a novel, ultra-fast and high yield process for reducing GO to reduced graphene oxide (RGO) using a facile hydride-based chemistry. The RGO sheets thus-produced exhibited high carrier mobilities (~ 100-600 cm[superscript]2/V•s) and reinstatement of the ambipolar characteristic of graphene. Raman spectra and UV-Vis spectroscopy on the RGO sheets displayed a high degree of restoration of the crystalline sp2 lattice with relatively low defects. We fabricated graphene nanoribbons (GNRs) – 1D structural derivatives of graphene – using a nano-scale cutting process from highly oriented pyrolytic graphite (HOPG) blocks, with widths pre-determinable between 5 nm to 600 nm. The as-produced GNRs had very high aspect ratio in the longitudinal direction (~ 0.01); exhibited predominantly mono-layered structure (< 10 % bilayer); and smooth edges (Raman I[subscript]D/G ~ 0.25 -0.28). Low temperature electrical transport measurements on back-gated thin film GNR devices were performed and a carrier mobility of ~ 20 ± 4 cm[superscript]2/V•s with sheet resistances of 2.2-5.1 MΩ / □ was extracted. Despite the ~ 50 nm thicknesses of the films, a clear bandgap scaling was observed with transport via variable range hopping (VRH) in 2 and 3 dimensions. This work demonstrates the first fully functional narrow pristine GNR thin-film field effect transistors (FETs). In addition we fabricated graphene quantum dots (GQDs) – 0D derivatives of graphene with dimensions < 100 nm – using a slight variation of our nano-scale cutting strategy, where the cleavage process is carried out in two dimensions. A high degree of control on the dimensions (Std. Dev. of ~ 5 nm for 50 X 50 nm square GQDs) and shape (pre-determinable between square, rectangle, triangle and trapezoid) of the as-synthesized GQDs is demonstrated. The optical properties of the GQDs such as the UV-Vis absorbance and photoluminescence were studied and their facile tunability was demonstrated depending on their dimensions. This work demonstrates for the first time the high throughput fabrication of GQDs with tunable dimensions and shape.