Selective target protein detection using a decorated nanopore into a microfluidic device.

Solid-state nanopores provide a powerful tool to electrically analyze nanoparticles and biomolecules at single-molecule resolution. These biosensors need to have a controlled surface to provide information about the analyte. Specific detection remains limited due to nonspecific interactions between the molecules and the nanopore. Here, a polymer surface modification to passivate the membrane is performed. This functionalization improves nanopore stability and ionic conduction. Moreover, one can control the nanopore diameter and the specific interactions between protein and pore surface. The effect of ionic strength and pH are probed. Which enables control of the electroosmotic driving force and dynamics. Furthermore, a study of polymer chain structure and permeability in the pore are carried out. The nanopore chip is integrated into a microfluidic device to ease its handling. Finally, a discussion of an ionic conductance model through a permeable crown along the nanopore surface is elucidated. The proof of concept is demonstrated by the capture of free streptavidin by the biotins grafted into the nanopore. In the future, this approach could be used for virus diagnostic, nanoparticle or biomarker sensing.

Surface Plasmon Resonance Imaging-Mass Spectrometry Coupling on Antibody Array Biochip: Multiplex Monitoring of Biomolecular Interactions and On-Chip Identification of Captured Antigen.

The coupling of surface plasmon resonance imaging (SPRi) with mass spectrometry (MS) offers a very promising multidimensional analysis. This system takes advantage of the two well-established techniques: SPR, which allows for the analysis of biomolecular interactions through the determination of kinetic and thermodynamic constants, and MS, which can characterize biological structures from mass measurements and fragmentation experiments. Here, a protocol for the coupling of SPRi with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described using a biochip grafted by antibodies in an array format. Interaction between ß-lactoglobulin antibodies and the protein antigen is detected and analyzed by SPRi. Then, the arrayed biochip which fitted a commercially MALDI target was inserted in a MALDI source, and mass spectra were recorded directly from the biochip surface from each antibody spot, showing protein ions attributed to the corresponding specific protein antigens.

Solid-State Nanopore Easy Chip Integration in a Cheap and Reusable Microfluidic Device for Ion Transport and Polymer Conformation Sensing.

Solid-state nanopores have a huge potential in upcoming societal challenging applications in biotechnologies, environment, health, and energy. Nowadays, these sensors are often used within bulky fluidic devices that can cause cross-contaminations and risky nanopore chips manipulations, leading to a short experimental lifetime. We describe the easy, fast, and cheap innovative 3D-printer-helped protocol to manufacture a microfluidic device permitting the reversible integration of a silicon based chip containing a single nanopore. We show the relevance of the shape of the obtained channels thanks to finite elements simulations. We use this device to thoroughly investigate the ionic transport through the solid-state nanopore as a function of applied voltage, salt nature, and concentration. Furthermore, its reliability is proved through the characterization of a polymer-based model of protein-urea interactions on the nanometric scale thanks to a hairy nanopore.

Versatile cyclodextrin nanotube synthesis with functional anchors for efficient ion channel formation: design, characterization and ion conductance.

Biomimetic ion channels with different materials have been extensively designed to study the dynamics in a confined medium. These channels allow the development of several applications, such as ultra-fast sequencing and biomarker detection. When considering their synthesis, the use of cheap, non-cytotoxic and readily available materials is an increasing priority. Cyclodextrins, in supramolecular architectures, are widely utilized for pharmaceutical and biotechnological applications. Recent work has shown that short nanotubes (NTs) based on alpha-cyclodextrin (α-CD) assemble transient ion channels into membranes without cytotoxicity. In this study, we probe the influence of new cyclodextrin NT structural parameters and chemical modifications on channel formation, stability and electrical conductance. We report the successful synthesis of ß- and γ-cyclodextrin nanotubes (ß-CDNTs and γ-CDNTs), as evidenced by mass-spectrometry and high-resolution transmission electron microscopy. CDNTs were characterized by their length, diameter and number of CDs. Two hydrophobic groups, silylated or vinylated, were attached along the γ-CDNTs, improving the insertion time into the membrane. All NTs synthesized form spontaneous biomimetic ion channels. The hydrophobic NTs exhibit higher stability in membranes. Electrophysiological measurements show that ion transport is the main contribution of NT conductance and that the ion energy penalty for the entry into these NTs is similar to that of biological channels.

Biomimetic ion channels formation by emulsion based on chemically modified cyclodextrin nanotubes.

Biomimetic ion channels can be made to display the high sensitivity of natural protein nanopores and to develop new properties as a function of the material used. How to design the best future biomimetic channels? The main challenges are to control their sensitivity, as well as their syntheses, chemical modifications, insertion and lifetime in a lipid membrane. To address these challenges, we have recently designed short cyclodextrin nanotubes characterized by mass spectrometry and high-resolution transmission electron microscopy. They form non-permanent ion channels in lipid bilayers. Here we show how to improve the nanotube insertion in order to limit multiple insertions, how to stabilize biomimetic channels into the membrane, and how to understand the ion dynamics in confined medium scale.

Functionalized Solid-State Nanopore Integrated in a Reusable Microfluidic Device for a Better Stability and Nanoparticle Detection.

Electrical detection based on single nanopores is an efficient tool to detect biomolecules, particles and study their morphology. Nevertheless the surface of the solid-state membrane supporting the nanopore should be better controlled. Moreover, nanopore should be integrated within microfluidic architecture to facilitate control fluid exchanges. We built a reusable microfluidic system integrating a decorated membran, rendering the drain and refill of analytes and buffers easier. This process enhances strongly ionic conductance of the nanopore and its lifetime. We highlight the reliability of this device by detecting gold nanorods and spherical proteins.

Optimized Synthesis According to One-Step Process of a Biobased Thermoplastic Polyacetal Derived from Isosorbide.

This paper describes both the synthesis and characterization of a biobased and non-aromatic polyacetal produced from the reaction between isosorbide and methylene chloride. The reaction was conducted in an aprotic dipolar and harmless solvent using a one-step, fast and economical procedure. The chemical composition of this polymer was investigated using Nuclear Magnetic Resonance and Fourier Transform Infra-Red spectroscopies. The molecular weights were examined by size exclusion chromatography and MALDI-TOF spectrometry. The synthesis conditions (concentration, mixing speed, solvent nature, stoichiometry, addition mode of one reactan) were found to strongly influence both polymer architecture and reaction yield. Under moderated stirring conditions, the polyacetal was characterized by a larger amount of macro-cycles. Inversely, under higher intensity mixing and with an excess of methylene chloride, it was mainly composed of linear chains. In this latter case, the polymeric material presented an amorphous morphology with a glass transition temperature (Tg) close to 55 °C. Its degradation temperature was evaluated to be close to 215 °C using thermogravimetry according to multi-ramp methodology. The chemical approach and the physicochemical properties are valuable in comparison with that characteristic of other isosorbide-based polyacetals.

Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications.

Biomimetic membrane channels offer a great potential for fundamental studies and applications. Here, we report the fabrication and characterization of short cyclodextrin nanotubes, their insertion into membranes, and cytotoxicity assay. Mass spectrometry and high-resolution transmission electron microscopy were used to confirm the synthesis pathway leading to the formation of short nanotubes and to describe their structural parameters in terms of length, diameter, and number of cyclodextrins. Our results show the control of the number of cyclodextrins threaded on the polyrotaxane leading to nanotube synthesis. Structural parameters obtained by electron microscopy are consistent with the distribution of the number of cyclodextrins evaluated by mass spectrometry from the initial polymer distribution. An electrophysiological study at single molecule level demonstrates the ion channel formation into lipid bilayers, and the energy penalty for the entry of ions into the confined nanotube. In the presence of nanotubes, the cell physiology is not altered.

Biomarkers probed in saliva by surface plasmon resonance imaging coupled to matrix-assisted laser desorption/ionization mass spectrometry in array format.

Detection of protein biomarkers is of major interest in proteomics. This work reports the analysis of protein biomarkers directly from a biological fluid, human saliva, by surface plasmon resonance imaging coupled to mass spectrometry (SPRi-MS), using a functionalized biochip in an array format enabling multiplex SPR-MS analysis. The SPR biochip presented a gold surface functionalized by a self-assembled monolayer of short poly(ethylene oxide) chains carrying an N-hydroxysuccinimide end-group for the immobilization of antibodies. The experiments were accomplished without any sample pre-purification or spiking with the targeted biomarkers. SPRi monitoring of the interactions, immune capture from the biochip surface, and finally on-chip matrix-assisted laser desorption/ionization-MS structural identification of two protein biomarkers, salivary α-amylase and lysozyme, were successively achieved directly from saliva at the femtomole level. For lysozyme, the on-chip MS identification was completed by a proteomic analysis based on an on-chip proteolysis procedure and a peptide mass fingerprint.

Further Insight into the Detailed Characterization of a Polydisperse Cyclodextrin-Based Polyrotaxane Sample by Electrospray Ionization Mass Spectrometry.

An easy and fast approach based on electrospray mass spectrometry (ESI-MS) was developed to provide a detailed characterization of a mixture containing polydisperse cyclodextrin-based polyrotaxane (CD-based PR). Here, method gave access to usual data such the weight-average molecular weight, the number-average molecular weight and the polydispersity index, but also to more specific features as the average number of CDs threaded and the average threading degree. Moreover, the nature and the average number of groups grafted per CD, such as sulfate or silyl groups, can be accurately determinate. This ESI-MS approach advantageously complements the widely used NMR and SEC methods and, thereby, constituting a milestone in the actual MS bottleneck regarding the analysis of polydisperse supramolecular assemblies.

Analysis of a polydisperse polyrotaxane based on poly(ethylene oxide) and α-cyclodextrins using nanoelectrospray and LTQ-Orbitrap.

Polyrotaxanes (PR) are among the most studied interlocked molecules in the field of supramolecular chemistry. Cyclodextrin based polyrotaxanes (CD based PRs) are well-known to be difficult to analyze by mass spectrometry (MS). Nanoelectrospray (nanoESI) employed during mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments turns out to be particularly useful to analyze these noncovalent assemblies. While ESI/nanoESI based spectra usually contain multicharged species which greatly complicate the interpretation, particularly for such complex mixtures analysis, the hyphenation with a high resolution analyzer such as Orbitrap could overcome this limitation. This Article reports efforts to achieve a detailed structural deciphering by nanoESI-MS and nanoESI-MS/MS of CD based PRs constituted of αCDs, unmodified or surrounded by 1 or 2 sulfation(s), which were threaded along polydisperse poly(ethylene oxide) α,ω-dipyrenyl chains. The described method is more sensitive and less sample consuming than a typical NMR experiment and in good agreement with size-exclusion chromatography (SEC) results. Moreover, as compared to MALDI-TOF MS analysis, all populations were presumably elucidated without discrimination effect. Therefore, this MS development allowed us to estimate the PR sample content with 16 to 35 ethylene oxide units, 1 to 5 αCDs threaded, and 0 to 10 sulfo groups grafted on the overall CDs. Finally, the method afforded the possibility to unambiguously attribute supramolecular architectures from 2276.0278 to 7767.8342 g·mol(-1) corresponding to poly[2]- to poly[6]rotaxanes.

Two independent ways of preparing hypercharged hydrolyzable polyaminorotaxane.

The aim of this work is to synthesize new PEO-based polyrotaxanes from modified cyclodextrins. Two strategies are discussed and compared. In the first, a pseudopolyrotaxane was formed between alpha,omega- PEO dimethacrylate and alpha-cyclodextrin. A coupling reaction between 1-pyrenebutyric acid N-hydroxysuccinimide ester was carried out to block the cyclic molecules onto the PEO. Cyclodextrins of the supramolecular assemblies were then oxidized using sodium periodate and reacted with spermine to form a potentially highly charged polyrotaxane. In the second strategy, cyclodextrins were first modified, and used to form the polyrotaxane through the pseudopolyrotaxane synthesis followed by the blocking reaction. Acidic titration allowed quantifying the number of amine functions borne by the supramolecular assemblies through two variables: the number of rings per polymer chain and the number of spermine groups per cyclic molecule. The supramolecules obtained by both strategies are discussed.

Amphiphilic poly[(propylene glycol)-block-(2-methyl-2-oxazoline)] copolymers for gene transfer in skeletal muscle.

Amphiphilic triblock copolymers such as poly(ethylene glycol-b-propylene glycol-b-ethylene glycol) PE6400 (PEG(13)-PPG(30)-PEG(13)) have been recently shown to promote gene transfer in muscle. Herein we investigated the effect of a chemical change of the PEG moiety on the transfection activity of these compounds. We synthesized new amphiphilic copolymers in which the PEG end blocks are replaced by more hydrophilic poly(2-methyl-2-oxazoline) (PMeOxz) chains of various lengths. The resulting triblock PMeOxz-PPG-PMeOxz compounds were characterized by NMR, SEC, TGA, and DSC techniques and assayed for in vivo muscle gene transfer. The results confirm both the block structure and the monomer unit composition (DP(PG)/DP(MeOxz)) of the new PPG(34)-PMeOxz(41) and PPG(34)-PMeOxz(21) triblock copolymers. Furthermore, in vivo experiments show that these copolymers are able to significantly increase DNA transfection efficiency, despite the fact that their chemical nature and hydrophilic character are different from the poloxamers. Overall, these results show that the capacity to enhance DNA transfection in skeletal muscle is not restricted to PEG-PPG-PEG arrangements.

Shape-tunable polymer nodules grown from liposomes via ring-opening metathesis polymerization.

A new inisurf (acting as surfactant and initiator) molecule for ring-opening metathesis polymerization (ROMP) was synthesized and used in aqueous solution in order to control the size and shape of polymer nodules grown from liposomes. Nodules were observed to grow in size with conversion of monomer, and depending on the monomer used, they adopted either a spherical or comet-like shape. Here, we investigate polymer production from a liposome surface. We use a hydrophobic derivative of the Grubbs catalyst positioned at the liposome surface to allow for ROMP of monomers dissolved in the aqueous outer phase. We obtain nodules of polymer that can grow up to tens of micrometers, unveiling new efficient possibilities of polymerization from a membrane in an aqueous solution.