Raman-spectroscopy based cell identification on a microhole array chip

Circulating tumor cells (CTCs) from blood of cancer patients are valuable prognostic markers and enable monitoring responses to therapy. The extremely low number of CTCs makes their isolation and characterization a major technological challenge. For label-free cell identification a novel combination of Raman spectroscopy with a microhole array platform is described that is expected to support high-throughput and multiplex analyses. Raman spectra were registered from regularly arranged cells on the chip with low background noise from the silicon nitride chip membrane. A classification model was trained to distinguish leukocytes from myeloblasts (OCI-AML3) and breast cancer cells (MCF-7 and BT-20). The model was validated by Raman spectra of a mixed cell population. The high spectral quality, low destructivity and high classification accuracy suggests that this approach is promising for Raman activated cell sorting

Towards clinical translation of raman spectroscopy for tumor cell identification

In the modern world, cancer is one of the leading causes of death, and its early diagnostics remains one of the big challenges. Since cancer starts as a malfunction on the cellular level, the diagnostic techniques have to deal with single cells. Detection of circulating tumor cells (CTCs), which are present in the patient's blood, holds promise for the future theranostic applications, as CTCs represent the actual state of the primary tumor. Raman spectroscopy is a label-free technique capable of non-destructive and chemically-specific characterization of individual cells. In contrast to marker-based methods, the CTCs detected by Raman can be reused for more specific single-cells biochemical analysis methods. This thesis focuses on technological developments for Raman-based CTC identification, and encompasses the whole chain of involved methods and processes, including instrumentation and microfluidic cell handling, automation of spectra acquisition and storage, and chemometric data analysis. It starts with a design of custom application-specific instruments that we used to evaluate and optimize different experimental parameters. A major part is software development for automated acquisition and organized storage of spectral data in a database. With the automated measurement systems and the database in place, we were able to collect about 40.000 Raman spectra of more than 15 incubated cancer cell lines, healthy donor leukocytes, as well as samples originating from clinical patients. Additionally, the thesis gives an overview of data analysis methods and provides an insight into the underlying trends of the dataset. Although the cell identification models could not reliably differentiate between individual cancer cell lines, they were able to recognize tumor cells among healthy leukocytes with prediction accuracy of more than 95%. This work demonstrated an increase in the throughput of Raman-based CTC detection, and provides a basis for its clinical translation

Femtosecond Laser Micromachining of Advanced Fiber Optic Sensors and Devices

Research and development in photonic micro/nano structures functioned as sensors and devices have experienced significant growth in recent years, fueled by their broad applications in the fields of physical, chemical and biological quantities. Compared with conventional sensors with bulky assemblies, recent process in femtosecond (fs) laser three-dimensional (3D) micro- and even nano-scale micromachining technique has been proven an effective and flexible way for one-step fabrication of assembly-free micro devices and structures in various transparent materials, such as fused silica and single crystal sapphire materials. When used for fabrication, fs laser has many unique characteristics, such as negligible cracks, minimal heat-affected-zone, low recast, high precision, and the capability of embedded 3D fabrication, compared with conventional long pulse lasers. The merits of this advanced manufacturing technique enable the unique opportunity to fabricate integrated sensors with improved robustness, enriched functionality, enhanced intelligence, and unprecedented performance. Recently, fiber optic sensors have been widely used for energy, defense, environmental, biomedical and industry sensing applications. In addition to the well-known advantages of miniaturized in size, high sensitivity, simple to fabricate, immunity to electromagnetic interference (EMI) and resistance to corrosion, all-optical fiber sensors are becoming more and more desirable when designed with characteristics of assembly free and operation in the reflection configuration. In particular, all-optical fiber sensor is a good candidate to address the monitoring needs within extreme environment conditions, such as high temperature, high pressure, toxic/corrosive/erosive atmosphere, and large strain/stress. In addition, assembly-free, advanced fiber optic sensors and devices are also needed in optofluidic systems for chemical/biomedical sensing applications and polarization manipulation in optical systems. Different fs laser micromachining techniques were investigated for different purposes, such as fs laser direct ablating, fs laser irradiation with chemical etching (FLICE) and laser induced stresses. A series of high performance assembly-free, all-optical fiber sensor probes operated in a reflection configuration were proposed and fabricated. Meanwhile, several significant sensing measurements (e.g., high temperature, high pressure, refractive index variation, and molecule identification) of the proposed sensors were demonstrated in this dissertation as well. In addition to the probe based fiber optic sensors, stress induced birefringence was also created in the commercial optical fibers using fs laser induced stresses technique, resulting in several advanced polarization dependent devices, including a fiber inline quarter waveplate and a fiber inline polarizer based on the long period fiber grating (LPFG) structure

Electrons dynamics control by shaping femtosecond laser pulses in micro/nanofabrication: modeling, method, measurement and application

During femtosecond laser fabrication, photons are mainly absorbed by electrons, and the subsequent energy transfer from electrons to ions is of picosecond order. Hence, lattice motion is negligible within the femtosecond pulse duration, whereas femtosecond photon-electron interactions dominate the entire fabrication process. Therefore, femtosecond laser fabrication must be improved by controlling localized transient electron dynamics, which poses a challenge for measuring and controlling at the electron level during fabrication processes. Pump-probe spectroscopy presents a viable solution, which can be used to observe electron dynamics during a chemical reaction. In fact, femtosecond pulse durations are shorter than many physical/chemical characteristic times, which permits manipulating, adjusting, or interfering with electron dynamics. Hence, we proposed to control localized transient electron dynamics by temporally or spatially shaping femtosecond pulses, and further to modify localized transient materials properties, and then to adjust material phase change, and eventually to implement a novel fabrication method. This review covers our progresses over the past decade regarding electrons dynamics control (EDC) by shaping femtosecond laser pulses in micro/nanomanufacturing: (1) Theoretical models were developed to prove EDC feasibility and reveal its mechanisms; (2) on the basis of the theoretical predictions, many experiments are conducted to validate our EDC-based femtosecond laser fabrication method. Seven examples are reported, which proves that the proposed method can significantly improve fabrication precision, quality, throughput and repeatability and effectively control micro/nanoscale structures; (3) a multiscale measurement system was proposed and developed to study the fundamentals of EDC from the femtosecond scale to the nanosecond scale and to the millisecond scale; and (4) As an example of practical applications, our method was employed to fabricate some key structures in one of the 16 Chinese National S&T Major Projects, for which electron dynamics were measured using our multiscale measurement system

Trends of biosensing: plasmonics through miniaturization and quantum sensing

Despite being extremely old concepts, plasmonics and surface plasmon resonance-based biosensors have been increasingly popular in the recent two decades due to the growing interest in nanooptics and are now of relevant significance in regards to applications associated with human health. Plasmonics integration into point-of-care devices for health surveillance has enabled significant levels of sensitivity and limit of detection to be achieved and has encouraged the expansion of the fields of study and market niches devoted to the creation of quick and incredibly sensitive label-free detection. The trend reflects in wearable plasmonic sensor development as well as point-of-care applications for widespread applications, demonstrating the potential impact of the new generation of plasmonic biosensors on human well-being through the concepts of personalized medicine and global health. In this context, the aim here is to discuss the potential, limitations, and opportunities for improvement that have arisen as a result of the integration of plasmonics into microsystems and lab-on-chip over the past five years. Recent applications of plasmonic biosensors in microsystems and sensor performance are analyzed. The final analysis focuses on the integration of microfluidics and lab-on-a-chip with quantum plasmonics technology prospecting it as a promising solution for chemical and biological sensing. Here it is underlined how the research in the field of quantum plasmonic sensing for biological applications has flourished over the past decade with the aim to overcome the limits given by quantum fluctuations and noise. The significant advances in nanophotonics, plasmonics and microsystems used to create increasingly effective biosensors would continue to benefit this field if harnessed properly

Développement d’outils analytiques pour la détection de biomolécules directement dans des fluides sanguins

Cette thèse porte sur le développement de biocapteurs basés sur la technique de résonance des plasmons de surface (SPR) pour effectuer des analyses directement dans un fluide sanguin n’ayant subi aucune purification ou dilution. L’ensemble des biocapteurs discutés exploiteront un instrument SPR portable développé dans le groupe du professeur Masson. Le premier volet de la thèse portera sur le processus d’interférence lié à l’adsorption non spécifique du sérum à la surface du capteur. L’analyse des biomolécules adsorbées sera effectuée en combinant la SPR à la spectrométrie de masse. Les informations obtenues seront exploitées pour la construction de biocapteurs adaptés à l’analyse en milieu sanguin. Un premier biocapteur développé ciblera la protéine antigène prostatique spécifique (APS) contenue dans le sérum servant de biomarqueur pour dépister le cancer de la prostate. Pour détecter les faibles concentrations de cette protéine directement dans le sérum, un matériel plasmonique microstructuré sera utilisé pour amplifier les signaux obtenus et sera recouvert d’une monocouche peptidique minimisant l’adsorption non spécifique du sérum. L’instrument SPR aura été adapté pour permettre également la détection simultanée de fluorescence. Un test ELISA sera ainsi effectué en parallèle du test SPR. Chacune des techniques fournira un contrôle pour la deuxième, tout en permettant de détecter le biomarqueur au niveau requis pour dépister la maladie. La combinaison des deux méthodes permettra aussi d’élargir la gamme dynamique du test de dépistage. Pour terminer, l’instrument SPR portable sera utilisé dans le cadre de détection de petites biomolécules ayant un potentiel thérapeutique directement dans un échantillon de sang. Des peptides ayant une activité anti-athérosclérotique pourront ainsi être détectés à même un échantillon de sang ni purifié ni dilué, et ce à des concentrations de l’ordre du micromolaire. Une modification de la microfluidique via l’introduction d’une membrane poreuse au cœur de celle-ci sera la clé permettant d’effectuer de telles analyses. La présente thèse met de l’avant de nouvelles stratégies et des modifications instrumentales permettant d’analyser des protéines et des petites molécules directement dans un échantillon non purifié de sérum ou de sang. Les modifications apportées au système fluidique, à l’instrument SPR et au niveau du biocapteur employé permettront d’effectuer des biodétections dans des matrices aussi complexes que les fluides sanguins. Les présents travaux mettent en lumière la capacité d’un instrument SPR/fluorescence portable à faire en 12 minutes la biodétection d’un marqueur du cancer de la prostate directement dans un échantillon de sérum. Finalement, on rapporte ici un des premiers articles où un biocapteur SPR est utilisé à même un échantillon de sang non-purifié pour faire des biodétections.This thesis discusses the development of surface plasmon resonnance (SPR) biosensors to perform detection directly on unpurified and undiluted blood based fluids such as serum or blood. Every biosensor discussed in the following chapters rely on a home-built portable SPR device developed in Professor Masson’s research laboratories. Non-specific adsorption, which greatly hinders biosensing in crude fluids, will be the first topic of the thesis. Serum adsorption was performed on the SPR sensor surface and then characterized by SPR and mass spectrometry. This study provided useful information for biosensing directly in blood-based fluids. It also provided a better fundamental understanding of the nonspecific adsorption process on surfaces. The first biosensor was developed to detect prostate specific antigen (PSA), a protein normally contained in serum, which is a known biomarker for prostate cancer. In order to detect low concentrations of this protein directly in serum, a microstructured gold film was used to amplify the signal generated by the binding event on the biosensor. A peptide monolayer covered the metallic surface of the sensor to reduce non-specific protein adsorption. The SPR portable instrument was modified to simultaneous detect fluorescence in order to perform a SPR and ELISA test in a single instrumental platform. Each technique provided a control for the other for detection of the prostate cancer biomarker at concentration levels required for the screening of the disease. The SPR and ELISA combination also extended the dynamic range of the biosensing assay. Finally, the portable SPR device was used to detect small biomolecules with potential therapeutic activity directly in a sample of blood. Peptides with an anti-atherosclerotic activity were thus detected in an unpurified and undiluted blood sample at micromolar concentration. The addition of a porous membrane to the microfluidic used for the biosensing assay facilitated the successful detection of these molecules in whole blood. The present thesis describes novel strategies and instrumental modifications to unlock the possibility of performing biosensing directly on unpurified and undiluted blood-based fluids. Modifications of the fluidic system, the SPR instrument and biosensor used will allow detection in fluids with high complexity such as serum or blood. The work described herein reports a prostate cancer screening assay performed in 12 minutes directly in serum using a portable SPR/fluorescence instrument. Finally, this thesis reports one of the first scientific papers where a SPR biosensor is used to perform analysis directly in blood

Superfocusing, Biosensing and Modulation in Plasmonics

Plasmonics could bridge the gap between photonics and electronics at the nanoscale, by allowing the realization of surface-plasmon-based circuits and plasmonic chips in the future. To build up such devices, elementary components are required, such as a passive plasmonic lens to focus free-space light to nanometre area and an active plasmonic modulator or switch to control an optical response with an external signal (optical, thermal or electrical). This thesis partially focuses on designing novel passive and active plasmonic devices, with a specific emphasis on the understanding of the physical principles lying behind these nanoscale optical phenomena. Three passive plasmonic devices, designed by conformal transformation optics, are numerically studied, including nanocrescents, kissing and overlapping nanowire dimers. Contrary to conventional metal nanoparticles with just a few resonances, these devices with structural singularities are able to harvest light over a broadband spectrum and focus it into well-defined positions, with potential applications in high efficiency solar cells and nanowire-based photodetectors and nanolasers. Moreover, thermo-optical and electrooptical modulation of plasmon resonances are realized in metallic nanostructures integrated with either a temperature-controlled phase transition material (vanadium dioxide, VO2), or ferroelectric thin films. Taking advantage of the high sensitivity of particle plasmon resonances to the change of its surrounding environment, we develop a plasmon resonance nanospectroscopy technique to study the effects of sizes and defects in the metal-insulator phase transition of VO2 at the single-particle level, and even single-domain level. Finally, we propose and examine the use of two-dimensional metallic nanohole arrays as a refractive index sensing platform for future label-free biosensors with good surface sensitivity and high-throughput detection ability. The designed plasmonic devices have great potential implications for constructing nextgeneration optical computers and chip-scale biosensors. The developed plasmon resonance nanospectroscopy has the potential to probe the interfacial or domain boundary scattering in polycrystalline and epitaxial thin films

Microstructuring of materials with laser technologies for biomedical applications

This thesis presents the use of laser technologies for structuring different materials for applications in biomedicine. One of the aims of this work is the fabrication of fluidic chips for their employment as preclinical devices. By direct or indirect laser techniques, materials like soda-lime glass, titanium or tantalum are structured. Dimensions from microns to millimetres are achieved, depending on the final application of the chip. In particular, a device that imitates a coronary bifurcation is fabricated by laser technologies and soft-lithography methods. It is validated by culturing endothelial cells in their inner walls that withstand flow conditions. Other structures, like microchannels, a circulating tumour cells capturing chip or patterns over titanium and tantalum are manufactured

Surface Engineering Of Gold Nanoparticles And Their Applications

Gold nanoparticles (AuNPs) with their unique sizes, shapes, and properties have generated much enthusiasm over the last two decades, and have been explored for many potential applications. The successful application of AuNPs depends critically on the ability to modify and functionalize their surface to provide stability, compatibility, and special chemical functionality. This dissertation is aimed at exploring the chemical synthesis and surface modification of AuNPs with the effort to (1) control the number of functional groups on the particle surface, and to (2) increase the colloidal stability at the physiological conditions. To control the functionality on the particle surface, a solid phase place exchange reaction strategy was developed to synthesize the 2 nm AuNPs with a single carboxylic acid group attached on the particle surface. Such monofunctional AuNPs can be treated and used as molecular nanobuilding blocks to form more complex nanomaterials with controllable structures. A necklace -like AuNP/polymer assembly was obtained by conjugating covalently the monofunctional AuNPs with polylysine template, and exhibited an enhanced optical limiting property due to strong electromagnetic interaction between the nanoparticles in close proximity. To improve the colloidal stability in the psychological condition, biocompatible polymers, polyacrylic acid (PAA), and polyethylene glycol (PEG) were used to surface modify the 30 nm citrate-stabilized AuNPs. These polymer-modified AuNPs are able to disperse individually in the high ionic strength solution, and offer as the promising optical probes for bioassay applications. The Prostate specific antigen (PSA) and target DNA can be detected in the low pM range by taking advantages of the large scattering cross section of AuNPs and the high sensitivity of dynamic light scattering (DLS) measurement. In addition to the large scattering cross section, AuNPs can absorb strongly the photon energy at the surface plasmon resonance wavelength and then transform efficiently to the heat energy. The efficient photon-thermal energy conversion property of AuNPs has been used to thermal ablate the Aβ peptide aggregates under laser irradiation toward Alzheimer\u27s disease therapy

Fabrication of 2D colloidal crystals over large areas for biosensing

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física Aplicada. Fecha de lectura: 23-06-2017Los dispositivos basados en la Resonancia de Plasmón Superficial se aplican extensamente como biosensores para identificar distintas biomoléculas de manera sensible, rápida, en tiempo real, libre de marcadores y/o con la posibilidad de multidetección. Actualmente, los desafíos al desarrollo de este tipo de dispositivos se encuentran en los altos costes de la instrumentación empleada, la limitada capacidad de conectar otras técnicas analíticas y una carencia de sensibilidad cuando se aplican a la detección de analitos muy diluidos o con pequeño peso molecular. Con el fin de vencer estos desafíos, se ha dirigido una extensa investigación al desarrollo de plataformas SPR avanzadas, basadas en materiales innovadores y métodos de fabricación que permiten una producción económica y masiva de grandes superficies nanoestructuradas, así como de dispositivos miniaturizados y de bajo coste. En este trabajo de tesis se ha llevado a cabo la optimización de un proceso para la producción de superficies plasmónicas sobre grandes áreas. La técnica de nanofabricación propuesta combina litografía coloidal y procesos plasma. En particular, hemos estudiado en detalle la formación de monocapas coloidales según el método Langmuir-Blodgett, sirviendo de máscaras para crear nanoestructuras en sustratos sólidos. El proceso implica un ataque plasma, la deposición de oro y la eliminación del residuo coloidal para obtener una superficie que consiste en cristales plasmónicos bidimensionales. Ajustando los parámetros del proceso es posible diseñar estructuras en materiales diferentes, controlando con precisión la relación de aspecto de la estructura final y, por consiguiente, la posición espectral de la respuesta óptica de acuerdo con el sistema de adquisición apropiado. La versatilidad de este método de fabricación ofrece gran potencial para un eficiente, fácil y masivo desarrollo de cavidades de oro con forma, diámetro y periodicidad controlables. Finalmente, se ha demostrado que estas superficies trabajan como plataformas eficaces en experimentos de SPR y SERS para una detección rápida, de tiempo real y sensible de distintos analitos protéicos (como la proteína pentraxin PTX3) y genómicos (como el gen del tumor de Wilms).Surface plasmon resonance (SPR)-devices are widely applied as biosensing platfoms to perform a potentially sensitive, rapid, real time, label free and/or multiplexed detection. As a drawback, this technology is often challenged by high instrumentation costs, poor interfacing capabilities with other analytical techniques and a lack of sensitivity when applied to direct detection of highly diluted targets or analytes of small molecular weight. In order to overcome these challenges, an extensive research has addressed the development of advanced SPR platforms based on innovative nanomaterials and fabrication methods, enabling a low cost and massive production of large area nanostructured surfaces, as well as affordable miniaturized detection devices. In this thesis we report the optimization of a protocol for the production of large area plasmonic surfaces. The proposed nanofabrication technique combines colloidal lithography and plasma processes. In particular, we deeply studied the Langmuir-Blodgett formation of colloidal monolayers, acting as efficient etching masks when transferred on solid substrates. The sequential etching process, gold deposition and particle lift off allowed obtaining a surface made of 2D plasmonic crystals. By adjusting the process parameters it is possible to nanostructure different materials, leading to a fine tuning of the final structure aspect ratio and, consequently, of the spectral position of the optical response according to a proper acquisition setup. The versatility of this fabrication method shows great potential for easy and massive parallel fabrication of gold cavity arrays with a tailorable shape, diameter and periodicity. These surfaces have been proved to work as sensitive platforms in SPR and SERS experiments for a fast, real time and multiplexing detection of proteomic (long pentraxin PTX3) and genomic (Wilms tumor gene) biomarkers