Formation of protein molecular imprints within Langmuir monolayers: A quartz crystal microbalance study

Abstract

Protein imprinting leading to enhanced rebinding of ferritin to ternary lipid monolayers is demonstrated using a quartz crystal microbalance. Monolayers consisting of cationic dioctadecyldimethylammonium bromide, non-ionic methyl stearate, and poly(ethylene glycol) bearing phospholipids were imprinted with ferritin at the air/water interface of a Langmuir-Blodgett trough and transferred hydrated to hydrophobic substrates for study. This immobilization was shown by fluorescence correlation spectroscopy to significantly hinder any further diffusion of lipids, while rebinding studies demonstrated up to a six-fold increase in ferritin adsorption to imprinted versus control monolayers. A diminished rebinding of ferritin to its imprint was observed through pH reduction to below the protein isoelectric point, demonstrating the electrostatic nature of the interaction. Rebinding to films where imprint pockets remained occupied by the template protein was also minimal. Studies with a smaller acidic protein revealed the importance of the steric influence of poly(ethylene glycol) in forming the protein binding pockets, as albumin-imprinted monolayers showed low binding of ferritin, while ferritin-imprinted monolayers readily accommodated albumin. The controllable structure-function relationship and limitations of this system are discussed with respect to the application of protein imprinting in sensor development as well as fundamental studies of proteins at dynamic interfaces.

The rational development of molecularly imprinted polymer-based sensors for protein detection

“…These include the capture of protein complexes at existing surfaces, as in the example shown in Figure 5, 94 and the organisation of surfactant-like molecules in Langmuir monolayers. 123 While these 2-D imprints can show some selectivity, it is unlikely to ever be a suitable method for sensing applications, as non-specific binding is always likely to be high with such materials.…”

Electrosynthesized molecularly imprinted polymers for protein recognition

“…This approach restricts the formation of imprinted binding sites to the surface of a polymer or to a very thin polymer layer the thickness of which is comparable to the size of the protein template. In extreme cases proteins are captured by 2D monomolecular layers of suitable functional monomers anchored to an existing surface [25,26]. To maximize the binding capacity of the respective MIPs, generation of polymeric nanostructures with high area/volume ratio comes as a natural necessity to take full advantage of the surface imprinted sites.…”

“…Furthermore, the applied potential can be pulsed in order to create films with enhanced adhesion to the electrode surface [26]. The application of pulsed potential aids also the replenishment of the depleted solution layer adjacent to the electrode by the low diffusivity macromolecular template [79], [83], and thus to enhance the incorporation of the protein template into the growing polymer.…”

Biosensing Applications of Molecularly Imprinted Nanomaterials

“…Piletsky et al [148] prepared a self-assembled monolayer (SAM) of hexadecyl mercaptan on a gold electrode in the presence of a cholesterol template and used the imprinted SAM to measure the cholesterol concentration using potassium ferricyanide as the electrochemical probe. The use of an organic SAM to create molecular recognition sites on gold electrodes for developing various chemical sensors was reported by several laboratories [149][150][151][152][153][154]. Using nanoparticles as scaffolds, Boal and Rotello [155] assembled multivalent binding sites for flavin on gold colloids, where diaminopyridine-and pyrene-functionalized thiols were chosen as building blocks.…”

“…Photo-induced electron transfer is a very popular sensing mode for fluorescent molecular recognition [131,154,175,176]. The electron-transfer mechanism is also applicable for the fluorescence detection of nonfluorescent analyte.…”