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

Unravelling Nonspecific Adsorption of Complex Protein Mixture on Surfaces with SPR and MS

Characterization of protein adsorption to surfaces has implications from biosensing to protective biocoatings. While research studies have principally focused on determining the magnitude of protein adsorption to surfaces, the proteins involved in the process remains only broadly identified and has not been investigated on several surfaces. To further elucidate the nonspecific adsorption process of serum to surfaces, surface plasmon resonance (SPR) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) were used in combination to obtain quantitative and qualitative information about the process of protein adsorption to surfaces. To validate the technique, crude serum was nonspecifically adsorbed on four self-assembled monolayer (SAM) on gold: 16-mercaptohexadecanoic acid (16-MHA), 11-mercaptoundecane­(ethylene glycol)₃-COOH (PEG), 3-MPA-LHDLHD-OH, and 3-MPA-HHHDD-OH. Direct MS analysis of the nonspecifically adsorbed proteins suggested the presence of a variety of protein (BSA, IgG, and apolipoprotein A-1). Performing a trypsin digestion of the nonspecifically adsorbed proteins confirmed the presence of BSA and apolipoprotein A-1 and further revealed the complexity of the process by detecting the presence of complement C3, SHC-transforming protein 1, and kininogen 2. The level of nonspecific adsorption on different surfaces measured by SPR sensing directly correlated with the intensity of the serum protein and indirectly with the tryptic peptides measured by MS. Detailed analysis of the BSA peptides digested on 16-MHA and for BSA digested in solution was used to investigate the orientation of BSA on this surface. The combination of SPR and MS allows the quantitative and qualitative understanding of protein adsorption processes to surfaces

Surface Plasmon Resonance Imaging-MALDI-TOF Imaging Mass Spectrometry of Thin Tissue Sections

Identification and quantification of proteins in imaging of biological samples are a challenge in today’s science. Here, we demonstrate a novel surface plasmon resonance imaging-matrix assisted laser desorption ionization imaging mass spectrometry (SPRi-MALDI IMS) coupled technique competent for the acquisition of multiparametric information by creating a tissue section imprint on an SPRi sensor surface. Correlated images were acquired in SPRi and in MALDI IMS for abundant proteins from a single mouse kidney tissue. The spatial organization of the transferred proteins from the tissue to the SPRi surface was preserved and imaged by SPR and MALDI MS. Surface chemistry was selected to nonspecifically adsorb and retain high concentrations of proteins on the SPRi surface. The diffusion kinetics were controlled to ensure fast transfer of proteins from the tissue sections with minimal lateral diffusion to achieve high spatial fidelity transfer. Lastly, the SPRi instrument was modified to insert a tissue sample in the fluidics chamber to facilitate the real-time measurement of the transfer process. The MALDI IMS experimental conditions, such as matrix deposition and the interface between the SPRi prism and the MALDI IMS instrument, were also optimized. The results show quantitative and regioselective SPRi images correlating to MALDI IMS images of different proteins transferred from a single tissue section

Plasmonic sensing in crude biofluids with microhole arrays

présenté par Jean-François MassonInternational audienc

Plasmonic sensing in crude biofluids with microhole arrays

présenté par Jean-François MassonInternational audienc

Silver-Assisted Laser Desorption Ionization For High Spatial Resolution Imaging Mass Spectrometry of Olefins from Thin Tissue Sections

Silver has been demonstrated to be a powerful cationization agent in mass spectrometry (MS) for various olefinic species such as cholesterol and fatty acids. This work explores the utility of metallic silver sputtering on tissue sections for high resolution imaging mass spectrometry (IMS) of olefins by laser desorption ionization (LDI). For this purpose, sputtered silver coating thickness was optimized on an assorted selection of mouse and rat tissues including brain, kidney, liver, and testis. For mouse brain tissue section, the thickness was adjusted to 23 ± 2 nm of silver to prevent ion suppression effects associated with a higher cholesterol and lipid content. On all other tissues, a thickness of at 16 ± 2 nm provided the best desorption/ionization efficiency. Characterization of the species by MS/MS showed a wide variety of olefinic compounds allowing the IMS of different lipid classes including cholesterol, arachidonic acid, docosahexaenoic acid, and triacylglyceride 52:3. A range of spatial resolutions for IMS were investigated from 150 μm down to the high resolution cellular range at 5 μm. The applicability of direct on-tissue silver sputtering to LDI-IMS of cholesterol and other olefinic compounds presents a novel approach to improve the amount of information that can be obtained from tissue sections. This IMS strategy is thus of interest for providing new biological insights on the role of cholesterol and other olefins in physiological pathways or disease

Enhanced SPR Biosensing with microhole arrays and peptide monolayers

présenté par JF MassonInternational audienc

Plasmonic Nanopipette Biosensor

Integrating a SERS immunoassay on a plasmonic “patch clamp” nanopipette enabled nanobiosensing for the detection of IgG. A SERS response was obtained using a sandwich assay benefiting from plasmon coupling between a capture Au nanoparticle (AuNP) on a nanotip and a second AuNP modified with a Raman active reporter and an antibody selective for IgG. The impact of nanoparticle shape and surface coverage was investigated alongside the choice of Raman active reporter, deposition pH, and plasmonic coupling, in an attempt to fully understand the plasmonic properties of nanopipettes and to optimize the nanobiosensor for the detection of IgG. These probes will find applications in various fields due to their nanoscale size leading to the possibility of spatially and temporally addressing their location near cells to monitor secretion of biomolecules

Tracking Silent Hypersensitivity Reactions to Asparaginase during Leukemia Therapy Using Single-Chip Indirect Plasmonic and Fluorescence Immunosensing

Microbial asparaginase is an essential component of chemotherapy for the treatment of childhood acute lymphoblastic leukemia (cALL). Silent hypersensitivity reactions to this microbial enzyme need to be monitored accurately during treatment to avoid adverse effects of the drug and its silent inactivation. Here, we present a dual-response anti-asparaginase sensor that combines indirect SPR and fluorescence on a single chip to perform ELISA-type immunosensing, and correlate measurements with classical ELISA. Analysis of serum samples from children undergoing cALL therapy revealed a clear correlation between single-chip indirect SPR/fluorescence immunosensing and ELISA used in clinical settings (<i>R</i>² > 0.9). We also report that the portable SPR/fluorescence system had a better sensitivity than classical ELISA to detect antibodies in clinical samples with low antigenicity. This work demonstrates the reliability of dual sensing for monitoring clinically relevant antibody titers in clinical serum samples

Development of Escherichia coli Asparaginase II for Immunosensing: A Trade-Off between Receptor Density and Sensing Efficiency

The clinical success of Escherichia coli l-asparaginase II (EcAII) as a front line chemotherapeutic agent for acute lymphoblastic leukemia (ALL) is often compromised because of its silent inactivation by neutralizing antibodies. Timely detection of silent immune response can rely on immobilizing EcAII, to capture and detect anti-EcAII antibodies. Having recently reported the use of a portable surface plasmon resonance (SPR) sensing device to detect anti-EcAII antibodies in undiluted serum from children undergoing therapy for ALL (Aubé et al., <i>ACS Sensors</i> <b>2016</b>, <i>1</i> (11), 1358–1365), here we investigate the impact of the quaternary structure and the mode of immobilization of EcAII onto low-fouling SPR sensor chips on the sensitivity and reproducibility of immunosensing. We show that the native tetrameric structure of EcAII, while being essential for activity, is not required for antibody recognition because monomeric EcAII is equally antigenic. By modulating the mode of immobilization, we observed that low-density surface coverage obtained upon covalent immobilization allowed each tetrameric EcAII to bind up to two antibody molecules, whereas high-density surface coverage arising from metal chelation by N- or C-terminal histidine-tag reduced the sensing efficiency to less than one antibody molecule per tetramer. Nonetheless, immobilization of EcAII by metal chelation procured up to 10-fold greater surface coverage, thus resulting in increased SPR sensitivity and allowing reliable detection of lower analyte concentrations. Importantly, only metal chelation achieved highly reproducible immobilization of EcAII, providing the sensing reproducibility that is required for plasmonic sensing in clinical samples. This report sheds light on the impact of multiple factors that need to be considered to optimize the practical applications of plasmonic sensors