Poly(N-isopropylacrylamide) | EPFL Graph Search

Poly(N-isopropylacrylamide) (variously abbreviated PNIPA, PNIPAM, PNIPAAm, NIPA, PNIPAA or PNIPAm) is a temperature-responsive polymer that was first synthesized in the 1950s. It can be synthesized from N-isopropylacrylamide which is commercially available. It is synthesized via free-radical polymerization and is readily functionalized making it useful in a variety of applications. PNIPA dissolves in water, however, when these solutions are heated in above their cloud point temperature, they undergo a reversible lower critical solution temperature (LCST) phase transition from a soluble hydrated state to an insoluble dehydrated state. Although it is widely believed that this phase transition occurs at , the actual temperatures may differ 5 to 10 °C (or even more) depending on the polymer concentration, molar mass of polymer chains, polymer dispersity as well as terminal moieties. Furthermore, other molecules in the polymer solution,

Bioconjugates for bioseparation by affinity precipitation

Matteo Costioli

Recent advances in the fields of biotechnology and medicine have intensified the necessity for pharmaceutical-grade biomacromolecules purification (e.g. enzymes, antibodies, plasmid DNA). One of the key factors for a successful downstream process is the possibility to isolate and concentrate the target biomolecules in one step. Affinity precipitation is a promising technique for the purification of genetically engineered molecules. This method combines the ability of the affinity techniques to specifically capture the target molecule from a diluted feed with the concentration effect typical of a precipitation technique. Affinity precipitation is an interesting alternative to established (chromatographic) technologies. This method uses stimulus-responsive affinity macroligands (AML) to capture and then coprecipitate the target molecule. A typical AML consists of a "smart polymer" to which an affinity ligand is linked. The polymer mediates the response to an external stimulus (e.g. a change in temperature, pH or salt content) and the affinity tag mediates the selective binding of the target molecule. Upon stimulation, the entire affinity complex is precipitated. In this thesis the design and characterization of new AML to be used in affinity precipitation were studied. Moreover, their applications in the purification of protein or plasmid DNA were also described. Free radical chain transfer polymerization of N-isopropylacrylamide (NIPAAm) was used to synthesize the AML precursor, i.e., a semitelechelic thermoresponsive telomer carrying a functional group. This latter has then been reacted, using different bioconjugation techniques, to synthesis the final AML. Telomerization kinetic studies were performed for the determination of the best polymerization conditions. In a first approach, the AML was constructed using the well known avidin/biotin system. A carbodiimid-coupling strategy was used to conjugate an avidin molecule to a poly-N-isopropylacrylamide polymer (PNIPAAm) which carries a carboxylic acid end group. Then, a single stranded poly-CTT biotinylated oligonucleotide was reacted with the avidin/polymer complex to create the final AML (BiotTF-AML). This AML was found to bind tightly and with high specificity to a specific plasmid DNA via a triple helix interaction. Triple helix affinity precipitation (THAP) process was then used for the purification of plasmid DNA from a clear lysate (recovery yield > 70%, no detectable protein contamination). Another AML synthesis strategy (to be used in the THAP process) was proposed. It was based on the chemical ligation between a PNIPAAm carrying a hydrazide moiety and an aldehyde group on the poly-CTT (coupling efficiency 80%). Moreover, it was also shown that the AML employed for the plasmid DNA purification can be used in the transfection of mammalian cells with no loss in protein expression. Finally, a direct telomerization approach using a modified FLAG-peptide as telogen, was shown to be a useful strategy for a one pot synthesis of an AML employed in the affinity precipitation of antibody.

EPFL2004

Cellulose Nanocrystals with Tethered Polymer Chains: Chemically Patchy versus Uniform Decoration

Alix Vaimiti Marie Dupire, Alke Fink, Harm-Anton Klok, Théo Gaston Gérard Lachat, Justin Orazio Zoppe

The site-specific surface modification of colloidal substrates, yielding "patchy" nanoparticles, is a rapidly expanding area of research as a result of the new complex structural hierarchies that are becoming accessible to chemists and materials scientists through colloidal self-assembly. The inherent directionality of cellulose chains, which feature a nonreducing and a reducing end, within individual cellulose nanocrystals (CNCs) renders them an interesting experimental platform for the synthesis of asymmetric nanorods with end-tethered polymer chains. Here, we present water-tolerant reaction pathways toward patchy and uniformly modified CNC hybrids based on atom transfer radical polymerization (ATRP) and initiators that were linked to the CNCs with carbodiimide-mediated coupling and Fischer esterification, respectively. Various monomers, including N-isopropylacrylamide (NIPAM), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), and sodium 4-vinylbenzenesulfonate (4-SS), were polymerized from both types of initiator-modified CNCs, yielding chemically patchy and uniform CNC hybrids, via surface-initiated ATRP (SI-ATRP). Interestingly, the stereochemistry of tethered PNIPAM was affected by the precise location of ATRP initiating sites, as evidenced by H-1 NMR and circular dichroism (CD) spectroscopy. This effect may be related to the inherent right-handed chirality of CNCs. CNC/PMETAC hybrids were labeled with gold nanoparticles (AuNPs) in order to visualize the precise location of polymer tethers via cryo-electron microscopy. In some instances, the AuNPs were indeed concentrated at the end groups of the patchy CNC hybrids.

2017

Screening of a Human Antibody Phage Display Library Against a Peptide Antigen Using Stimuli-Responsive Bioconjugates

Dorothée Dumoulin, Caroline Vandevyver

A synthetic antibody phage library, (ETH-2) was screened against the MUC-1 peptide. Two standard methods were used, namely screening on immunotubes coated with the peptide antigen and on streptavidin-coated paramagnetic particles together with a biotinylated MUC-1 peptide. The results were compared with those obtained using a novel approach based on a stimuli-responsive bioconjugate of avidin and poly-(N-isopropylacrylamide), also in combination with the biotinylated peptide. The poly-(N-isopropylacrylamide)-based bioconjugate is soluble below a temperature of 30 degrees C in the aqueous solutions pertinent to this study, but precipitates rapidly once this temperature is surpassed. The process is reversible, and the bioconjugate redissolves once the temperature is lowered again. The stimuli-responsive bioconjugate was used to isolate specific phage from the library in several rounds of panning, consisting of repeated thermoprecipitation/redissolution cycles in a procedure known as "affinity precipitation." As Jar as we know, this is the first time a compound as big as a viral particle has been specifically captured by a stimuli-responsive bioconjugate. Compared to the two other investigated screening techniques, affinity precipitation of the phage led to a greater variety of genetically, different specific phage, while the process was easy to handle and required no dedicated equipment safe for a centrifuge and a thermostated water bath.

2008

Bioconjugates for bioseparation by affinity precipitation

Matteo Costioli

EPFL2004