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.
Food standards and quality control are important means to ensure public health. In the last decade, melamine has become a rather notorious example of food adulteration: Spiking products with low-cost melamine in order to feign high amino acid content exploits the lack in specificity of the established Kjeldahl method for determining organic nitrogen. This work discusses the responses of a sensor based on quartz crystal microbalances (QCM) coated with molecularly imprinted polymers (MIP) to detect melamine in real life matrices both in a selective and a sensitive manner. Experiments in pure milk revealed no significant sensor responses. However, sensor response increased to a frequency change of −30Hz after diluting the matrix ten times. Systematic evaluation of this effect by experiments in melamine solutions containing bovine serum albumin (BSA) and casein revealed that proteins noticeably influence sensor results. The signal of melamine in water (1600 mg/L) decreases to half of its initial value, if either 1% BSA or casein are present. Higher protein concentrations decrease sensor responses even further. This suggests significant interaction between the analyte and proteins in general. Follow-up experiments revealed that centrifugation of tagged serum samples results in a significant loss of sensor response, thereby further confirming the suspected interaction between protein and melamine.
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.
A molecular imprinting strategy was combined with mass-sensitive transducers to generate robust and reliable biomimetic sensor systems for the detection of bioparticles. The patterning of polymers with bioanalytes enabled us to detect Escherichia coli (E. coli) bacteria with quartz crystal microbalance (QCM). The QCM sensor results were compared with direct Atomic Force Microscopy (AFM) measurements—bacteria cells adhering to the sensor coatings were counted. The recognition sites generated by Bacillus subtilis (B. subtilis) spores could successfully and reversibly recognize the template analyte and ensured rapid sensing. Cross sensitive measurements clearly showed the advantage of the molecular imprinting strategy, by which spores of Bacillus species (subtilis and thuringiensis) could easily be differentiated and selectively detected. The growth of B. subtilis from its spores was observed at 42 °C in appropriate nutrient solution of glucose and ammonium sulfate over a period of 15 h. Moreover, the growth of B. subtilis bacteria from its respective spores was studied by increasing the glucose concentration until saturation effect of the sensor. The polymeric sensor coatings were patterned to fix the B. subtilis in order to investigate osmotic effects according to a frequency response of 400 Hz by altering the ionic strength of 0.1 M.
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