Molecularly Imprinted Polymers for Compound-Specific Isotope Analysis of Polar Organic Micropollutants in Aquatic Environments

Compound-specific isotope analysis (CSIA) of polar organic micropollutants in environmental waters requires a processing of large sample volumes to obtain the required analyte masses for analysis by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). However, the accumulation of organic matter of unknown isotopic composition in standard enrichment procedures currently compromises the accurate determination of isotope ratios. We explored the use of molecularly imprinted polymers (MIPs) for selective analyte enrichment for ¹³C/¹²C and ¹⁵N/¹⁴N ratio measurements by GC/IRMS using 1<i>H</i>-benzotriazole, a typical corrosion inhibitor in dishwashing detergents, as example of a widely detected polar organic micropollutant. We developed procedures for the treatment of >10 L of water samples, in which custom-made MIPs enabled the selective cleanup of enriched analytes in organic solvents obtained through conventional solid-phase extractions. Hydrogen bonding interactions between the triazole moiety of 1<i>H</i>-benzotriazole, and the MIP were responsible for selective interactions through an assessment of interaction enthalpies and ¹⁵N isotope effects. The procedure was applied successfully without causing isotope fractionation to river water samples, as well as in- and effluents of wastewater treatment plants containing μg/L concentrations of 1<i>H</i>-benzotriazole and dissolved organic carbon (DOC) loads of up to 28 mg C/L. MIP-based treatments offer new perspectives for CSIA of organic micropollutants through the reduction of the DOC-to-micropollutant ratios

Thin Walled Imprinted Polymer Beads Featuring Both Uniform and Accessible Binding Sites

A novel approach addressing the classical deficiencies of molecularly imprinted polymers (MIPs), that is, low binding capacity and nonuniform binding sites, is reported. The thin walled beads were produced in two steps by first grafting thin MIP films, under controlled (RAFT) or noncontrolled conditions, from porous silica beads following previously reported procedures. The resulting composites were compared in terms of film thickness, the grafted layer homogeneity, the effect of different support morphologies, and for their ability to recognize the template in chromatographic or static binding tests. Thus, using l-Phenylalanine anilide (L-PA) as template to imprint poly­(MAA-co-EDMA) in such a way led to nanometer thick films where the resulting composite were able to selectively retain the template in relation to the thickness of the grafted film. In the second step, removing the silica supports from the above composites by etching, led to nanometer thin walled beads with structure, morphology and recognition properties strongly depending on grafting chemistry (RAFT or non-RAFT), monomer dilution and on the film thickness of the original composite. Thus whereas the thicker walled materials retained their mesoporous morphology and displayed enhanced enantioselectivity, load capacity, and higher surface areas compared to their composite precursors, the thin walled beads showed lower surface areas indicating network collapse. The thin walled beads prepared under dilute conditions in absence of RAFT displayed a perfectly uniform binding site distribution and a saturation capacity exceeding that of a conventional monolithic MIP. The beads prepared by RAFT control showed a further enhanced saturation capacity significantly exceeding that of the reference material. Finally, the reduced hydrophobic character of the thin walled materials indicated the existence of two separate pore systems with different pore wettabilities

Antibody-Free Biomarker Determination: Exploring Molecularly Imprinted Polymers for Pro-Gastrin Releasing Peptide

Biomarker mass spectrometry assays are in high demand, and analysis of pro-gastrin releasing peptide (ProGRP) as a small cell lung cancer marker has been recently investigated by mass spectrometry after immunoextraction. In this article, we introduce an assay based on molecularly imprinted polymers (MIPs) targeting the proteotypic peptide of ProGRP as a possible alternative to current immuno-based assay. The MIPs were prepared by surface-initiated reversible addition–fragmentation chain transfer polymerization and were introduced as sorbents for the cleanup and enrichment of a ProGRP signature peptide from tryptically treated serum samples. The use of an appropriate solid-phase extraction protocol allowed specific extraction of the target peptide while depleting other peptides that arose from the sample digestion, hence resulting in reduced background. The selective extraction of a ProGRP signature peptide, after digestion of serum samples, translates into a time- and cost-effective method suited for bottom-up analysis wherever targeted peptide extraction from complex matrices is required

Reversible Self-Assembled Monolayers (rSAMs): Adaptable Surfaces for Enhanced Multivalent Interactions and Ultrasensitive Virus Detection

We report on the design of pH-switchable monolayers allowing a reversible and ordered introduction of affinity reagents on sensor surfaces. The principal layer building blocks consist of α-(4-amidinophenoxy)­alkanes decorated at the ω-position with affinity ligands. These spontaneously self-assemble on top of carboxylic acid terminated SAMs to form reversible homo or mixed monolayers (rSAMs) that are tunable with respect to the nature of the head group, layer order and stability while featuring pH responsiveness and the dynamic nature of noncovalent build assemblies. We show that this results in a range of unique biosensor features. As a first example a sialic acid rSAM featuring strong lectin affinity is here used to sense hemagglutinin and influenza virus (H5N1) at the pM and fM level by in situ ellipsometry in a fully reversible fashion. We believe that the rSAM concept will find widespread use in surface chemistry and overall for boosting sensitivity in affinity biosensors

Theoretical Studies of 17-β-Estradiol-Imprinted Prepolymerization Mixtures: Insights Concerning the Roles of Cross-Linking and Functional Monomers in Template Complexation and Polymerization

In this study, computational methods were employed in efforts to elucidate physical mechanisms underlying the ligand selectivity of polymeric sorbents produced through the molecular imprinting of 17-β-estradiol. Previous computational and experimental studies had identified candidate systems applicable to the development of synthetic polymeric receptors for the detection and possible removal of pollutants with endocrine-disrupting properties. Here we present a series of comprehensive molecular dynamics studies of candidate molecular imprinting prepolymerization systems. The results from the studies highlight the role of the cross-linker and the importance of the interplay between functionalities of the various monomers employed in template complexation. The significance of these results for future studies is discussed

Catalytic Formation of Disulfide Bonds in Peptides by Molecularly Imprinted Microgels at Oil/Water Interfaces

This work describes the preparation and investigation of molecularly imprinted polymer (MIP) microgel (MG) stabilized Pickering emulsions (PEs) for their ability to catalyze the formation of disulfide bonds in peptides at the O/W interface. The MIP MGs were synthesized via precipitation polymerization and a programmed initiator change strategy. The MIP MGs were characterized using DLS analysis, SEM measurement, and optical microscopy analysis. The dry and wet MIP MGs showed a hydrodynamic diameter of 100 and 280 nm, respectively. A template rebinding experiment showed that the MIP MGs bound over two times more template (24 mg g^–1) compared to the uptake displayed by a nonimprinted reference polymer (NIP) MG (10 mg g^–1) at saturation. Using the MIP MGs as stabilizers, catalytic oxidation systems were prepared by emulsifying the oil phase and water phase in the presence of different oxidizing agents. During the cyclization, the isolation of the thiol precursors and the oxidizing reagents nonselectively decreased the formation of the byproducts, while the imprinted cavities on the MIP MGs selectively promoted the intramolecular cyclization of peptides. When I₂ was used as the oxidizing agent, the MIP-PE-I₂ system showed a product yield of 50%, corresponding to a nearly 2-fold increase compared to that of the nonimprinted polymer NIP-PE-I₂ system (26%). We believe the interfacial catalysis system presented in this work may offer significant benefits in synthetic peptide chemistry by raising productivity while suppressing the formation of byproducts

Methyleneation of Peptides by <i>N</i>,<i>N</i>,<i>N</i>,<i>N</i>‑Tetramethylethylenediamine (TEMED) under Conditions Used for Free Radical Polymerization: A Mechanistic Study

Free radical polymerization is often used to prepare protein and peptide-loaded hydrogels for the design of controlled release systems and molecular imprinting materials. Peroxodisulfates (ammonium peroxodisulfates (APS) or potassium peroxodisulfates (KPS)) with <i>N</i>,<i>N</i>,<i>N</i>,<i>N</i>-tetramethylethylenediamine (TEMED) are frequently used as initiator and catalyst. However, exposure to these free radical polymerization reagents may lead to modification of the protein and peptide. In this work, we show the modification of lysine residues by ammonium peroxodisulfate (APS)/TEMED of the immunostimulant thymopentin (TP5). Parallel studies on a decapeptide and a library of 15 dipeptides were performed to reveal the mechanism of modification. LC–MS of APS/TEMED-exposed TP5 revealed a major reaction product with an increased mass (+12 Da) with respect to TP5. LC–MS² and LC–MS³ were performed to obtain structural information on the modified peptide and localize the actual modification site. Interpretation of the obtained data demonstrates the formation of a methylene bridge between the lysine and arginine residue in the presence of TEMED, while replacing TEMED with a sodium bisulfite catalyst did not show this modification. Studies with the other peptides showed that the TEMED radical can induce methyleneation on peptides when lysine is next to arginine, proline, cysteine, aspargine, glutamine, histidine, tyrosine, tryptophan, and aspartic acid residues. Stability of peptides and protein needs to be considered when using APS/TEMED in <i>in situ</i> polymerization systems. The use of an alternative catalyst such as sodium bisulfite may preserve the chemical integrity of peptides during in situ polymerization

Reversible Self-Assembled Monolayers (rSAMs) as Robust and Fluidic Lipid Bilayer Mimics

Lipid bilayers, forming the outer barrier of cells, display a wide array of proteins and carbohydrates for modulating interfacial biological interactions. Formed by the spontaneous self-assembly of lipid molecules, these bilayers feature liquid crystalline order, while retaining a high degree of lateral mobility. Studies of these dynamic phenomena have been hampered by the fragility and instability of corresponding biomimetic cell membrane models. Here, we present the construct of a series of oligoethylene glycol-terminated reversible self-assembled monolayers (rSAMs) featuring lipid-bilayer-like fluidity, while retaining air and protein stability and resistance. These robust and ordered layers were prepared by simply immersing a carboxylic acid-terminated self-assembled monolayer into 5–50 μM aqueous ω-(4-ethylene glycol-phenoxy)-α-(4-amidinophenoxy)­decane solutions. It is anticipated that this new class of robust and fluidic two-dimensional biomimetic surfaces will impact the design of rugged cell surface mimics and high-performance biosensors

Molecularly Imprinted Porous Monolithic Materials from Melamine–Formaldehyde for Selective Trapping of Phosphopeptides

Thirty-five melamine–formaldehyde (MF) monolithic materials with bimodal pore distributions were synthesized in fused silica capillaries by catalyst-free polycondensation, starting with an aqueous MF precondensate, using acetonitrile as the macroporogen and a variety of aliphatic polyethers and triblock copolymeric surfactants as porogens and mesoporogens, respectively. By varying the prepolymer composition and the type and molecular weight of the polymeric porogen components, a library of porous monolithic materials was produced, covering a range of meso- and macroporous properties. A multivariate evaluation revealed that the amount of surfactant was the strongest contributor to specific surface area and pore volume and to the inversely related mesopore size, whereas the macropore dimensions were controlled mainly by the amount of aliphatic polyether porogen. One of these capillary monoliths, chosen based on the combination of meso- and macropores providing optimal percolative flow and accessible surface area, was synthesized in the presence of <i>N</i>-Fmoc and <i>O</i>-Et protected phosphoserine and phosphotyrosine to prepare molecularly imprinted monoliths with surface layers selective for phosphopeptides. These imprinted monoliths were characterized alongside nonimprinted monoliths by a variety of techniques and finally evaluated by liquid chromatography–mass spectrometry in the capillary format to assess their abilities to trap and release phosphorylated amino acids and peptides from partly aqueous media. Selective enrichment of phosphorylated targets was demonstrated, suggesting that these materials could be useful as trapping media in affinity-based phosphoproteomics

Phosphotyrosine Biased Enrichment of Tryptic Peptides from Cancer Cells by Combining pY-MIP and TiO₂ Affinity Resins

Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific, and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY-imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP- and TiO₂-based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO₂ combination as compared to TiO₂ alone. pY constituted up to 8% of the pY-MIP-TiO₂-enriched phosphopeptide fractions. The pY-MIP-TiO₂ and the TiO₂ protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842, respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO₂-enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic, and almost exclusively monophosphorylated, as compared to TiO₂ alone. These unique properties render the pY-MIP-based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research