Designing Molecularly Imprinted Polymers for sensing proteins is still a somewhat empirical process due to the inherent complexity of protein imprinting. Based on Bovine Serum Albumin as a model analyte, we explored the influence of a range of experimental parameters on the final sensor responses. The optimized polymer contains 70% cross linker. Lower amounts lead to higher sensitivity, but also sensor response times substantially increase (to up to 10 h) at constant imprinting effect (signal ratio MIP/NIP on quartz crystal microbalance—QCM). However, by shifting the polymer properties to more hydrophilic by replacing methacrylic acid by acrylic acid, part of the decreased sensitivity can be recovered leading to appreciable sensor responses. Changing polymer morphology by bulk imprinting and nanoparticle approaches has much lower influence on sensitivity.
This work describes the development of molecularly imprinted polymer (MIP) thin films by using reassembled S-layer protein arrays as templates. Crystalline bacterial cell surface layer (S-layer) proteins are among the most abundant biopolymers on earth and form the outermost cell envelope component in a broad range of bacteria and archaea. The unique feature of S-layer based imprints is the crystalline character of the reassembled S-layer protein lattice leading to a precisely controllable periodicity of surface functional groups and topographical features. By determining the Young (elastic) modulus of the S-layer protein with respect to that of the polymer at its gel point, the feasibility of the S-layer based biomolecular imprinting was confirmed. After imprinting the polymer with an S-layer coated silicon stamp, the sensitivity of the imprints and their selectivity in relation to various other proteins were investigated by quartz crystal microbalance (QCM) studies. Furtheron, polycationic ferritin (PCF) was bound in a dense packing on the S-layer and subsequently used for stamping. Successful rebinding of PCF proved that the S-layer lattice can be used as a template for making imprints of densely packed and, probably, perfectly oriented biologically functional molecules, a concept that can in principle be extended to a wide range of other biomolecules (e.g. antibodies).
Herein we report novel approaches to the molecular imprinting of proteins utilizing templates sizing around 10 nm and some 100 nm. The first step comprised synthesizing nanoparticles of molecularly imprinted polymers (MIP) towards bovine serum albumin (BSA) and characterizing them according to size and binding capacity. In a second step, they were utilized as templates. Quartz crystal microbalances (QCM) coated with MIP thin films based on BSA MIP nanoparticles lead to a two-fold increase in sensor responses, compared with the case of directly using the protein as the template. This also established that individual BSA molecules exhibit different “epitopes” for molecular imprinting on their outer surfaces. In light of this knowledge, a possible MIP-based biomimetic assay format was tested by exposing QCM coated with BSA MIP thin films to mixtures of BSA and imprinted and non-imprinted polymer (NIP) nanoparticles. At high protein concentrations (1000 ppm) measurements revealed aggregation behavior, i.e., BSA binding MIP NP onto the MIP surface. This increased sensor responses by more than 30% during proof of concept measurements. At lower a BSA concentration (500 ppm), thin films and particles revealed competitive behavior.
In this paper, we study the effects of the structure, defects, and surface morphology of zinc oxide thin films on the enhancement of the Raman signal of organic molecules. The SERS substrates of Ag/ZnO thin film with low cost, stability, and high sensitivity are prepared to detect the rhodamine 6G (R6G) reagent and metronidazole (MNZ) standard. Zinc oxide (ZnO) thin films are produced by a simple and low-cost sol−gel dip-coating method. The films are dip-coated and heated at 250 °C for 45 min. The deposition process is repeated from 1 to 6 times to obtain ZnO films with various thicknesses. Following that, ZnO films are annealed at 500 °C for different duration from 30 to 150 min to obtain a good structure and porous surface which enhance the incident light scattering for SERS. The effects of different film thicknesses and annealing times on the surface morphology, structure, defects, and optical properties of ZnO thin films are investigated by field-emission scanning electron microscopy, Raman spectroscopy, ultraviolet−visible spectroscopy, and reflectance spectroscopy. The results indicate that the films with 6 layers and annealed in 120 min show the best behavior in crystallinity and surface roughness. Finally, silver nanoparticles (Ag NPs) are deposited onto the ZnO films with different sputtering times by the DC magnetron sputtering method to form the active SERS substrates. The optimal SERS sample of Ag (10 s)/ZnO (6 layers, 500 °C, 120 min) is highly sensitive, stable, and has spectral reproducibility. The SERS substrate detects R6G solution at a limit of detection (LOD) of 10 −14 M, an enhancement factor (EF) of 2.62 × 10 13 , and detects MNZ solution with a LOD of 0.01 ppm and EF of 3.30 × 10 6 . It is very stable for at least 6 months as stored away from moisture and light and has a high spectral reproducibility with the RSD of the method in three Ag/ZnO samples of 10.70, 7.34, and 10.44%.
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