10 research outputs found
DĂ©veloppement dâoutils analytiques pour la dĂ©tection de biomolĂ©cules directement dans des fluides sanguins
Cette thĂšse porte sur le dĂ©veloppement de biocapteurs basĂ©s sur la technique de rĂ©sonance des plasmons de surface (SPR) pour effectuer des analyses directement dans un fluide sanguin nâayant subi aucune purification ou dilution. Lâensemble des biocapteurs discutĂ©s exploiteront un instrument SPR portable dĂ©veloppĂ© dans le groupe du professeur Masson. Le premier volet de la thĂšse portera sur le processus dâinterfĂ©rence liĂ© Ă lâadsorption non spĂ©cifique du sĂ©rum Ă la surface du capteur. Lâanalyse des biomolĂ©cules adsorbĂ©es sera effectuĂ©e en combinant la SPR Ă la spectromĂ©trie de masse. Les informations obtenues seront exploitĂ©es pour la construction de biocapteurs adaptĂ©s Ă lâanalyse en milieu sanguin. Un premier biocapteur dĂ©veloppĂ© ciblera la protĂ©ine antigĂšne prostatique spĂ©cifique (APS) contenue dans le sĂ©rum servant de biomarqueur pour dĂ©pister le cancer de la prostate. Pour dĂ©tecter les faibles concentrations de cette protĂ©ine directement dans le sĂ©rum, un matĂ©riel plasmonique microstructurĂ© sera utilisĂ© pour amplifier les signaux obtenus et sera recouvert dâune monocouche peptidique minimisant lâadsorption non spĂ©cifique du sĂ©rum. Lâinstrument SPR aura Ă©tĂ© adaptĂ© pour permettre Ă©galement la dĂ©tection simultanĂ©e de fluorescence. Un test ELISA sera ainsi effectuĂ© en parallĂšle du test SPR. Chacune des techniques fournira un contrĂŽle pour la deuxiĂšme, tout en permettant de dĂ©tecter le biomarqueur au niveau requis pour dĂ©pister la maladie. La combinaison des deux mĂ©thodes permettra aussi dâĂ©largir la gamme dynamique du test de dĂ©pistage.
Pour terminer, lâinstrument SPR portable sera utilisĂ© dans le cadre de dĂ©tection de petites biomolĂ©cules ayant un potentiel thĂ©rapeutique directement dans un Ă©chantillon de sang. Des peptides ayant une activitĂ© anti-athĂ©rosclĂ©rotique pourront ainsi ĂȘtre dĂ©tectĂ©s Ă mĂȘme un Ă©chantillon de sang ni purifiĂ© ni diluĂ©, et ce Ă des concentrations de lâordre du micromolaire. Une modification de la microfluidique via lâintroduction dâune membrane poreuse au cĆur de celle-ci sera la clĂ© permettant dâeffectuer de telles analyses.
La prĂ©sente thĂšse met de lâavant de nouvelles stratĂ©gies et des modifications instrumentales permettant dâanalyser des protĂ©ines et des petites molĂ©cules directement dans un Ă©chantillon non purifiĂ© de sĂ©rum ou de sang. Les modifications apportĂ©es au systĂšme fluidique, Ă lâinstrument SPR et au niveau du biocapteur employĂ© permettront dâeffectuer des biodĂ©tections dans des matrices aussi complexes que les fluides sanguins. Les prĂ©sents travaux mettent en lumiĂšre la capacitĂ© dâun instrument SPR/fluorescence portable Ă faire en 12 minutes la biodĂ©tection dâun marqueur du cancer de la prostate directement dans un Ă©chantillon de sĂ©rum. Finalement, on rapporte ici un des premiers articles oĂč un biocapteur SPR est utilisĂ© Ă mĂȘme un Ă©chantillon de sang non-purifiĂ© pour faire des biodĂ©tections.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 Massonâs 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-mercaptoundecaneÂ(ethylene
glycol)<sub>3</sub>-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 todayâs 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 ÎŒm down to the high resolution cellular range at 5 ÎŒm.
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 âpatch clampâ
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><sup>2</sup> > 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 (AubeÌ et al., <i>ACS Sensors</i> <b>2016</b>, <i>1</i> (11), 1358â1365), 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