Application of molecularly imprinted polymer nanoparticles for degradation of the bacterial autoinducer N-hexanoyl homoserine lactone.

A novel bacterial quorum quenching system is presented. For the first time the degradation of N-l-hexanoyl homoserine lactone (C6-AHL), a Gram-negative quorum sensing autoinducer, has been enhanced using molecularly imprinted nanoparticles (MIP NPs) which were prepared using transition state analogue of the γ-lactone ring hydrolysis as template.

A novel thermal detection method based on molecularly imprinted nanoparticles as recognition elements.

Molecularly Imprinted Polymers (MIPs) are synthetic receptors that are able to selectively bind their target molecule and, for this reason, they are currently employed as recognition elements in sensors. In this work, MIP nanoparticles (nanoMIPs) are produced by solid-phase synthesis for a range of templates with different sizes, including a small molecule (biotin), two peptides (one derived from the epithelial growth factor receptor and vancomycin) and a protein (trypsin). NanoMIPs are then dipcoated on the surface of thermocouples that measure the temperature inside a liquid flow cell. Binding of the template to the MIP layer on the sensitive area of the thermocouple tip blocks the heat-flow from the sensor to the liquid, thereby lowering the overall temperature measured by the thermocouple. This is subsequently correlated to the concentration of the template, enabling measurement of target molecules in the low nanomolar regime. The significant improvement in the limit of detection (a magnitude of three orders compared to previously used MIP microparticles) can be attributed to their high affinity, enhanced conductivity and increased surface-to-volume ratio. It is the first time that these nanosized recognition elements are used in combination with thermal detection, and it is the first report on MIP-based thermal sensors for determining protein levels. The developed thermal sensors have a high selectivity, fast measurement time (<5 min), and data analysis is straightforward, which makes it possible to monitor biomolecules in real-time. The set of biomolecules discussed in this manuscript show that it is possible to cover a range of template molecules regardless of their size, demonstrating the general applicability of the biosensor platform. In addition, with its high commercial potential and biocompatibility of the MIP receptor layer, this is an important step towards sensing assays for diagnostic applications that can be used in vivo.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.