The Role of the Initiator System in the Synthesis of Acidic Multifunctional Nanoparticles Designed for Molecular Imprinting of Proteins

Multifunctional nanoparticles have been shown earlier to bind certain proteins with high affinity and the binding affinity could be enhanced by molecular imprinting of the target protein. In this work different initiator systems were used and compared during the synthesis of poly (N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) nanoparticles with respect to their future applicability in molecular imprinting of lysozyme. The decomposition of ammonium persulfate initiator was initiated either thermally at 60 °C or by using redox activators, namely tetramethylethylenediamine or sodium bisulfite at low temperatures. Morphology differences in the resulting nanoparticles have been revealed using scanning electron microscopy and dynamic light scattering. During polymerization the conversion of each monomer was followed in time. Striking differences were demonstrated in the incorporation rate of acrylic acid between the tetramethylethylenediamine catalyzed initiation and the other systems. This led to a completely different nanoparticle microstructure the consequence of which was the distinctly lower lysozyme binding affinity. On the contrary, the use of sodium bisulfite activation resulted in similar nanoparticle structural homogeneity and protein binding affinity as the thermal initiation

Electrosynthesized molecularly imprinted polyscopoletin nanofilms for human serum albumin detection

Molecularly imprinted polymers (MIPs) rendered selective solely by the imprinting with protein templates lacking of distinctive properties to facilitate strong target-MIP interaction are likely to exhibit medium to low template binding affinities. While this prohibits the use of such MIPs for applications requiring the assessment of very low template concentrations, their implementation for the quantification of high-abundance proteins seems to have a clear niche in the analytical practice. We investigated this opportunity by developing a polyscopoletin-based MIP nanofilm for the electrochemical determination of elevated human serum albumin (HSA) in urine. As reference for a low abundance protein ferritin-MIPs were also prepared by the same procedure. Under optimal conditions, the imprinted sensors gave a linear response to HSA in the concentration range of 20–100 mg/dm3, and to ferritin in the range of 120–360 mg/dm3. While as expected the obtained limit of detection was not sufficient to determine endogenous ferritin in plasma, the HSA-sensor was successfully employed to analyse urine samples of patients with albuminuria. The results suggest that MIP-based sensors may be applicable for quantifying high abundance proteins in a clinical setting

Electrosynthesized molecularly imprinted polymers for protein recognition

Molecularly imprinted polymers (MIPs) for the recognition of proteins are expected to possess high affinity through the establishment of multiple interactions between the polymer matrix and the large number of functional groups of the target. However, while highly affine recognition sites need building blocks rich in complementary functionalities to their target, such units are likely to generate high levels of non-specific binding. This paradox, that nature solved by evolution for biological receptors, needs to be addressed by the implementation of new concepts in molecular imprinting of proteins. Additionally, the structural variability, large size and incompatibility with a range of monomers made the development of protein MIPs to take a slow start. While the majority of MIP preparation methods are variants of chemical polymerization, the polymerization of electroactive functional monomers emerged as a particularly advantageous approach for chemical sensing application. Electropolymerization can be performed from aqueous solutions to preserve the natural conformation of the protein templates, with high spatial resolution and electrochemical control of the polymerization process. This review compiles the latest results, identifying major trends and providing an outlook on the perspectives of electrosynthesised protein-imprinted MIPs for chemical sensing

MIPs and Aptamers for Recognition of Proteins in Biomimetic Sensing

Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either “evolution in the test tube” of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the “biological” degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application

Electrosynthesized MIPs for Transferrin: Plastibodies or Nano-Filters?

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered

Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples

Highly selective multifunctional magnetic nanoparticles containing a thermoresponsive polymer shell were developed and used in the sample pretreatment of urine for the assessment of lysozymuria in leukemia patients. Crosslinked poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) was grown onto silica-coated magnetic nanoparticles by reversible addition fragmentation chain transfer (RAFT) polymerization. The lysozyme binding property of the nanoparticles was investigated as a function of time, protein concentration, pH, ionic strength and temperature and their selectivity was assessed against other proteins. High-abundant proteins, like human serum albumin and γ-globulins did not interfere with the binding of lysozyme even at elevated concentrations characteristic of proteinuria. A sample cleanup procedure for urine samples has been developed utilizing the thermocontrollable protein binding ability of the nanoparticles. Method validation was carried out according to current bioanalytical method validation guidelines. The method was highly selective, and the calibration was linear in the 25 to 1000 µg/mL concentration range, relevant in the diagnosis of monocytic and myelomonocytic leukemia. Intra- and inter-day precision values ranged from 2.24 to 8.20% and 1.08 to 5.04%, respectively. Intra-day accuracies were between 89.9 and 117.6%, while inter-day accuracies were in the 88.8 to 111.0% range. The average recovery was 94.1 ± 8.1%. Analysis of unknown urine samples in comparison with a well-established reference method revealed very good correlation between the results, indicating that the new nanoparticle-based method has high potential in the diagnosis of lysozymuria

Enzymatic digestion as a tool for removing proteinaceous templates from molecularly imprinted polymers

A proteolytic digestion procedure including pre-treatment steps is proposed for the efficient removal of protein templates from molecularly imprinted polymers.</p

MIPs and Aptamers for Recognition of Proteins in Biomimetic Sensing

Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either “evolution in the test tube” of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the “biological” degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application