Molecularly Imprinted Materials Research Articles

Sensitive and specific detection of Listeria monocytogenes in food samples using imprinted upconversion fluorescence probe prepared by emulsion polymerization method

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen with high morbidity and mortality rates, necessitating rapid detection methods. Current techniques, while reliable, are labor-intensive and not amenable to on-site testing. We report the design and synthesis of a novel imprinted upconversion fluorescence probe through Pickering emulsion polymerization for the specific detection of L. monocytogenes. The probe employs trimethylolpropane trimethacrylate and divinylbenzene as cross-linkers, acryloyl-modified chitosan as a functional monomer, and the bacterium itself as the template. The developed probe demonstrated high specificity and sensitivity in detecting L. monocytogenes, with a limit of detection of 72 CFU/mL. It effectively identified the pathogen in contaminated salmon and chicken samples, with minimal background interference. The integration of molecular imprinting and upconversion fluorescence materials presents a potent and reliable approach for the rapid and specific detection of L. monocytogenes, offering considerable potential for on-site food safety testing.

Study on the Selectivity of Molecular Imprinting Materials Determined through Hydrogen Bonding on Template Molecular Structures of Flavonoids.

Molecular imprinting technology is widely used for the specific identification of compounds, but the selective recognition mechanisms of the same compounds still need to be further studied. Based on differences in hydrogen bond size and orientation, molecularly imprinted polymers (MIPs) were designed to adsorb flavonols with the same parent core and different hydroxyl groups. A surface-imprinted material was designed with silicon dioxide as the carrier, myricetin as the template molecule, and methacrylic acid (MAA) as the functional monomer. Scanning electron microscopy (SEM), Brunauer-Emmett-Teller surface area (BET) analyses, Fourier-transform infrared spectroscopy (FT-IR), and other characterization experiments were carried out. The intrinsic mechanism of the MIPs was also explored. The MIPs showed good adsorption of myricetin and other flavonoids through hydrogen bonding and steric hindrance. The adsorption capacity was 3.12-9.04 mg/g, and the imprinting factor was 1.78-3.37. Flavonoids with different hydroxyl groups in different numbers and directions had different hydrogen bond strengths with functional monomers. R2, R4, and R1 on 2-phenylchromogenone had stronger electronegativity, and the hydroxyl group was also more likely to form and have stronger hydrogen bonds. The hydroxyl negativity and the degree of steric hindrance of flavonoids played a major role in the recognition of molecularly imprinted materials. This study is of great significance for the synthesis of and selection of templates for analogous molecular imprinting materials.

A More Efficient Method for Preparing a MIP-CQDs/ZnO1-x Photodegradant with Highly Selective Adsorption and Photocatalytic Properties.

The application of semiconductor photocatalysts in wastewater treatment always has a drawback, which is the lack of selectivity for pollutants, but molecular imprinting technology (MIT) is a remarkable method for preparing highly selective adsorbents for low concentration target pollutants. Up to now, the research of molecular imprinting materials has mainly focused on organic polymers, and there has been little research on inorganic molecular imprinting materials. In the present work, we introduced carbon quantum dots (CQDs) into the flower-like hierarchical ZnO to prepare photocatalysts CQDs/ZnO. Further, with ciprofloxacin (CIP) as the template molecule, a molecular imprinting material MIP-CQDs/ZnO1-x was prepared by introducing both oxygen vacancies and imprinted cavities into CQDs/ZnO by the hydrothermal calcination method. It can not only increase the concentration of oxygen vacancies and broaden the light absorption range of zinc oxide without changing the crystal form of ZnO but also make it have the characteristics of preferential adsorption and degradation of CIP during the degradation process. Under the synergistic effect of CQDs, oxygen vacancies, and molecularly imprinted cavities, the molecularly imprinted material exhibits excellent photocatalytic and selective adsorption performance.

Fenpicoxamid-Imprinted Surface Plasmon Resonance (SPR) Sensor Based on Sulfur-Doped Graphitic Carbon Nitride and Its Application to Rice Samples.

This research attempt involved the development and utilization of a newly designed surface plasmon resonance (SPR) sensor which incorporated sulfur-doped graphitic carbon nitride (S-g-C3N4) as the molecular imprinting material. The primary objective was to employ this sensor for the quantitative analysis of Fenpicoxamid (FEN) in rice samples. The synthesis of S-g-C3N4 with excellent purity was achieved using the thermal poly-condensation approach, which adheres to the principles of green chemistry. Afterwards, UV polymerization was utilized to fabricate a surface plasmon resonance (SPR) chip imprinted with FEN, employing S-g-C3N4 as the substrate material. This process involved the inclusion of N,N'-azobisisobutyronitrile (AIBN) as the initiator, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, methacryloylamidoglutamic acid (MAGA) as the monomer, and FEN as the analyte. After successful structural analysis investigations on a surface plasmon resonance (SPR) chip utilizing S-g-C3N4, which was imprinted with FEN, a comprehensive investigation was conducted using spectroscopic, microscopic, and electrochemical techniques. Subsequently, the kinetic analysis applications, namely the determination of the limit of quantification (LOQ) and the limit of detection (LOD), were carried out. For analytical results, the linearity of the FEN-imprinted SPR chip based on S-g-C3N4 was determined as 1.0-10.0 ng L-1 FEN, and LOQ and LOD values were obtained as 1.0 ng L-1 and 0.30 ng L-1, respectively. Finally, the prepared SPR sensor's high selectivity, repeatability, reproducibility, and stability will ensure safe food consumption worldwide.

Preparation of bifunctional monomer molecularly imprinted polymer filled solid-phase extraction for sensitivity improvement of quantitative analysis of sulfonamide in milk

To solve the problems of unstable adsorption performance and high organic solvent consumption of traditional molecular imprinting materials, we developed a water-resistant and highly adsorptive molecularly imprinted sulfamethazine polymer (MIP) through a novel synthetic strategy consisting of the application of mixed functional monomers combined with hydrophobic crosslinkers. The results showed that the imprinting factor of the prepared MIP was increased from 0.93 to 4.38, and that the relative selection coefficient k' was 5.59. With the application of the material to be developed as a solid-phase extraction (SPE) filler for sulfonamides (SAs) in milk combined with UPLC-MS/MS, the validated method showed a sensitive quantification limit (0.89 µg/kg), a steady recovery (95.55%-97.97%) and an excellent precision (0.08%-2.92% RSD). Moreover, after 5 times usage, the recovery rate of MIP-SPE was still above 90%. Therefore, the prepared materials showed high performance of molecular adsorption and were water-resistant, which was also considered an excellent filler of SPE for SAs extraction in food or other fields.

Sol-gel polymerization of silylated amino acids around a protein template yields selective biomimetic imprints

The development of artificial receptors able to selectively recognize a target protein is of particular interest in separation, diagnostics, and therapeutics fields. Herein, we disclose a method to prepare biomimetic and functionalized protein imprints in biocompatible conditions avoiding any protein denaturation. For that purpose, a set of different hybrid silylated amino acid derivatives were synthesized and used without tetraethyl orthosilicate to prepare our molecularly imprinted polymers, allowing to reduce to a minimum of the silicon amount, in order to obtain imprints made almost entirely of amino acids to mimic paratope surfaces of antibodies. Such functional building blocks were polymerized on the surface of magnetic silica nanoparticles at pH 8.5 in ultrapure water in the presence of two globular proteins: cytochrome C or lysozyme. The resulting imprinted hybrid materials were evaluated for their adsorption capacity, specificity, and selectivity by quartz-crystal microbalance with dissipation and magnetic enzyme-linked immunosorbent assay (ELISA) assays. High imprinting factors of 8.7 were measured for these biomimetic hybrid materials (corresponding to approximately 4000 and 450 ng of protein per cm2 immobilized on molecularly imprinted polymers and non-imprinted polymer nanoparticles, respectively), representing a significant breakthrough in sol-gel-based molecular imprinting materials. Moreover, competition experiments performed by magnetic ELISA (mELISA) show very good specificity of our imprints at the usual concentrations of ELISA measurements.

Construction and Properties of New-Type Photo-Responsive Molecular Imprinting Materials

Construction and Properties of New-Type Photo-Responsive Molecular Imprinting Materials

Fast exhaustive enrichment and electrochemical quantitative detection of anthocyanins from natural products by using dual responsive and dummy molecularly imprinted polymers

In this work, a new adsorbent was synthesized using dummy-template molecular imprinting technology, magnetic separation technology and pH-responsive technology, which was used to quickly enrich anthocyanins in fruit and detect with electrochemical methods. On the surface of magnetic particles, the molecularly-imprinted materials were synthesized by using quercetin as the dummy-template, 4-vinyl phenylboronic acid and methacrylic acid as the functional monomers. The microstructure was characterized by scanning electron microscopy, transmission electron microscope, Fourier transform infrared spectroscopy, N2 adsorption–desorption, thermal gravimetric analysis, vibrating sample magnetometer and X-ray photoelectron spectroscopy. Compared with structural analogue, dummy-template magnetic and pH-responsive molecularly imprinted polymers (DMMIPs) had shorter kinetic equilibrium time, higher selectivity and better adsorption capacity toward anthocyanins. The binding capacity and imprinting factor of the dummy molecularly imprinted polymer could reach 59.22 mg/g and 1.56, respectively. The anthocyanins’ content of blueberry and grape peel crude extracts were consistent with the reference reports.

Improvement of selective catalytic oxidation capacity of phthalates from surface molecular-imprinted catalysis materials: Design, mechanism, and application

Phthalates (PAEs) are widely distributed environmental pollutants. As an effective method, advanced oxidation usually has low efficiency and excessive cost. In this study, surface molecular imprinting was used to enhance the targeted catalytic degradation ability of PAEs in the advanced oxidation process. Molecular simulation results show that more stable hydrogen bonds can be formed between carboxyl components and phthalates. With acrylic acid as a functional monomer, the adsorption capacity and catalytic capacity for diethyl phthalate on molecular imprinting material (C-MIL-100-MIP) were improved significantly reached to 19.1 mg g−1 and 12.05 mg g−1. Although amino groups can also provide hydrogen bonds, the catalyst synthesized using amino as functional monomer is not well. This result is consistent with the calculation result. The actual wastewater treatment results showed that the removal capacity of DMP and DEP on C-MIL-100-MIP was 44.4% and 64.7% higher than that of MIL-100. Moreover, in the PS catalysis process, C-MIL-100-MIP could adsorb PS and generate more free radicals within 30 min than that of carbonized MIL-100 and MIL-100. This feature can significantly improve the efficiency of PS usage. The results show that molecular computation can provide an efficient method for the design of catalytic materials with specific recognition ability and using molecular imprint method to improve the catalysis efficiency has a certain application prospect in the industrial wastewater treatment.

Specific adsorption of tetracycline from milk by using biocompatible magnetic molecular imprinting material and evaluation by ECD

Biocompatible magnetic molecularly imprinted polymers (BMMIPs) were prepared with Zein for the first time, and were used to enrich tetracycline compounds selectively. Innovative combination of BMMIPs and electrochemistry to obtain lower detection line to satisfy industrial detection demands. Using Zein as the crosslinking agent, the polymers were synthesized on the surface of Fe3O4 particles. The scanning electron microscope, transmission electron microscope and X-ray diffraction technologies were used to characterize BMMIPs. Through optimization, BMMIPs attained large adsorption capacity (236.40 mg/g) with fast kinetics (40 min) and followed the Langmuir isotherm and pseudo-second-order kinetic models. BMMIPs had good recognition ability, the selective factors of oxytetracycline, chlortetracycline, doxycycline were 4.78, 4.23, and 3.39, respectively. Excellent linearity was attained in the range of 0.025–500 μg/mL, with low detection limits and low quantitation limits of 0.025 and 0.083 μg/mL. According to our exploring, BMMIPs was ideal materials for enrichment of tetracycline in complex biological samples.

Molecularly Imprinted Materials for Selective Biological Recognition.

Molecular imprinting is an approach of generating imprinting cavities in polymer structures that are compatible with the target molecules. The cavities have memory for shape and chemical recognition, similar to the recognition mechanism of antigen-antibody in organisms. Their structures are also called biomimetic receptors or synthetic receptors. Owing to the excellent selectivity and unique structural predictability of molecularly imprinted materials (MIMs), practical MIMs have become a rapidly evolving research area providing key factors for understanding separation, recognition, and regenerative properties toward biological small molecules to biomacromolecules, even cell and microorganism. In this review, the characteristics, morphologies, and applicability of currently popular carrier materials for molecular imprinting, especially the fundamental role of hydrogels, porous materials, hierarchical nanoparticles, and 2D materials in the separation and recognition of biological templates are discussed. Moreover, through a series of case studies, emphasis is given on introducing imprinting strategies for biological templates with different molecular scales. In particular, the differences and connections between small molecular imprinting (bulk imprinting, "dummy" template imprinting, etc.), large molecular imprinting (surface imprinting, interfacial imprinting, etc.), and cell imprinting strategies are demonstrated in detail. Finally, future research directions are provided.

Progress of stimuli-responsive molecular imprinted materials for capture/release of proteins

To date, molecular imprinting (MIP) materials towards proteins have been utilized in a wide range of applications encompassing the fields of samples purification, proteomics, life science, biosensor, pharmaceutical research and drug delivery. Although tremendous progress has been made in MIP technique field, the large size of macromolecules would lead to low transmission and diffusion efficiency during MIP processes, and further cause difficult in adsorption and desorption of target proteins. Therefore, great challenges still remain to be addressed, especially in the design of unique and advanced MIP materials towards macromolecules. More recently, stimuli-responsive MIP materials have attracted considerable interest because they can respond to external stimuli while modulating their affinities for the target proteins and endowing switchable capacities of the binding/releasing processes. In this review, an overview of different stimuli-responsive MIP materials towards proteins developed from 1998 to now, especially the synthetic methods, monomer species, intelligent modulating principle and application has been provided. Moreover, the future of intelligent stimuli-responsive MIP materials for target proteins is expounded. In the first section, the classical MIP fabrication process and mechanism of MIP materials have been described: template incubating with the function monomers, polymerization and template removing. Then the fundamental properties of classical MIP materials including high selectivity, stability and excellent reusability without loss of activity have been discussed. However, the MIP materials are mainly utilized as adsorbents for extraction of the small molecules. When it comes to template with large molecules, such as proteins, the MIP materials suffered from the lower diffusion efficiency and needed harsh elution conditions. Therefore, to approach challenges, new materials-the stimuli-responsive MIP materials should be explored. In the second section, several methods for synthesis of stimuli-responsive MIP materials have been introduced in detail, including bulk polymerization, emulsion polymerization, precipitation polymerization, suspension polymerization and surface imprinting method. Moreover, the advantages and shortcomings of each method and its application have been summarized. Combination of MIP materials with stimuli-responsive properties has attracted significant research interest. In the third section, a general summary of stimuli-responsive MIP technology has been made, including the design of thermo/pH sensitive MIP materials and its application in proteins analysis. Finally, this review performs prospect for the future development of stimuli-responsive MIP technology including the limitation in theoretical study, synthesis method and commercial products. The stimuli-responsive MIP materials have displayed great potential anyhow in several applications including drug delivery and environmental protection owing to their versatility properties.

Modification of mesoporous silica with molecular imprinting technology: A facile strategy for achieving rapid and specific adsorption

In order to improve the diffusion kinetics of molecularly imprinted materials (MIMs), applying imprinting technology to mesoporous materials is a promising strategy. In the present study, an imprinting approach based on the combination of mesoporous silica materials and molecular imprinting technology is reported. Molecularly imprinted material (MIM) for 2,4-dichlorophenoxyacetic acid (2,4-D) was prepared by using 2,4-D as the template molecule, alkyne-modified β-cyclodextrin and propargyl amine as the combinatorial functional monomers and SBA-15 as the supporter. The functional monomers were anchored to the azide-modified SBA-15 by azide–alkyne Click reaction. The synthesized MIM was characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), thermal gravimetric analysis (TGA), low-angle X-ray diffraction (XRD) and N2 adsorption–desorption analysis. The interactions between template and functional monomers were studied by proton NMR analysis and UV–vis experiments. The results of the equilibrium binding experiments and selective tests showed that the prepared MIM has binding affinity and specificity for a group of analytes which have similar size and shape to those of template. Binding kinetic experiments demonstrated that the present imprinting approach can effectively enhance the mass transfer rate. The solid phase extraction of 2,4-D using MIM as the adsorbent was investigated. The extraction conditions for the processes of loading, washing and eluting were optimized. The recoveries of the molecularly imprinted solid phase extraction (MISPE) column for 2,4-D were 76.3–88.9% with relative standard deviations (RSD) of 3.48–7.64%.

Magnetic nanoparticles combining teamed boronate affinity and surface imprinting for efficient selective recognition of glycoproteins under physiological pH

Boronate-functionalized surface imprinted nanoparticles that function under physiological pH would be highly desirable for specific enrichment of target glycoproteins. In this work, magnetic molecularly imprinted polymers integrated with low pKa teamed boronate affinity (Fe3O4@PGMA-TBA/MIPs) were fabricated for selective separation of glycoprotein at pH = 7.4. The teamed boronate affinity (TBA) was formed by boron-nitrogen (B-N) coordination between 1,6-hexamethylenediamine and 3-aminophenylboronic acid, and then was fixed on the surface of magnetic poly(glycidyl methacrylate) (Fe3O4@PGMA) through ring-opening reaction. After immobilizing the template glycoproteins (ovalbumin, OVA), surface imprinting layer was deposited onto Fe3O4@PGMA-TBA surface via redox polymerization of aniline, and Fe3O4@PGMA-TBA/MIPs with obvious core–shell-shell structure were prepared by removing template. Fe3O4@PGMA-TBA/MIPs were demonstrated with an imprinted polymer film (10–20 nm) and exhibited superparamagnetic property (Ms = 32 emu g−1) and magnetic stability after multiple regenerations. Besides, the results of 11B MAS NMR spectrum and zeta potentials confirmed the presence of TBA resulting from B-N coordination. Additionally, taking advantage of surface imprinting and TBA, as-prepared Fe3O4@PGMA-TBA/MIPs posed high binding capacity (190.7 mg g−1) and fast capture kinetics (50 min) toward OVA under physiological pH. More importantly, because of the TBA was electroneutral at pH = 7.4, Fe3O4@PGMA-TBA/MIPs not only exhibited superior specific recognition toward OVA (imprinting factor IF = 7.51), but also maintained the activity of OVA from practical samples analysis. Therefore, this work opened up a universal route for developing intelligent controllability molecular imprinting materials for the specific separation of glycoproteins in biomedical filed under physiological pH.

Molecular imprinting of 5-fluorouracil in particles with surface RAFT grafted functional brushes

Molecularly imprinted particles with surface grafted functional polymer brushes were synthesized and evaluated aiming at the targeting of 5-fluorouracil (5-FU). A molecular imprinting material for 5-FU was prepared through precipitation crosslinking polymerization, in the presence of a RAFT agent, and choosing either methacrylic acid (MAA) or 2-hydroxyethyl methacrylate (HEMA) as functional monomers. In a later step, different kinds of functional polymer brushes, namely poly(methacrylic acid), poly(2-hydroxyethyl methacrylate) and poly(N-isopropylacrylamide) were grafted in the particles through a “grafting from” process starting on the RAFT groups present in their surface. The accomplishment of 5-FU imprinting was proven through comparative drug uptake measurements with imprinted/non-imprinted materials. Enhancement of 5-FU release in alkaline conditions comparatively to acidic environments was shown for particles with different combinations of molecular imprinting and grafted functional brushes. The MAA imprinting system with PMAA grafted functional brushes shows the highest improvement in 5-FU release triggered by a pH change from 2 to 10. Furthermore, for particles with PNIPA grafted brushes, a boost in drug release was also shown at T=20°C as compared to 40°C. The novel achievements reported here put into evidence the combined benefits of molecularly imprinted particles with surface grafted functional polymer brushes concerning molecular recognition and stimulated 5-FU release.

Molecular Dynamics Simulation Study of the Selectivity of a Silica Polymer for Ibuprofen.

In the past few years, the sol-gel polycondensation technique has been increasingly employed with great success as an alternative approach to the preparation of molecularly imprinted materials (MIMs). The main aim of this study was to study, through a series of molecular dynamics (MD) simulations, the selectivity of an imprinted silica xerogel towards a new template—the (±)-2-(P-Isobutylphenyl) propionic acid (Ibuprofen, IBU). We have previously demonstrated the affinity of this silica xerogel toward a similar molecule. In the present study, we simulated the imprinting process occurring in a sol-gel mixture using the Optimized Potentials for Liquid Simulations-All Atom (OPLS-AA) force field, in order to evaluate the selectivity of this xerogel for a template molecule. In addition, for the first time, we have developed and verified a new parameterisation for the Ibuprofen® based on the OPLS-AA framework. To evaluate the selectivity of the polymer, we have employed both the radial distribution functions, interaction energies and cluster analyses.

Construction of imprint sites in mesopores of SBA-15 via thiol-ene click reaction

This paper reports a new strategy for preparation of 2D molecularly imprinted materials (MIM) through the exploitation of thiol-ene click chemistry. 2D MIM for cholesterol was prepared using cholesterol as the template, acrolyl-modified β-cyclodextrin (acrolyl-β-CD) as the functional monomer, and thiol-modified SBA-15 (thiol-SBA-15) as the supporter. In this method, acrolyl-β-CD molecules were assembled around the templates by formation of template–monomer inclusion compounds. Then the acrolyl-β-CD molecules were anchored to the walls of the mesopores of SBA-15 via the thiol-ene click chemistry. After removal of the template molecules, the resulting recognition sites were formed in the mesopores of SBA-15. TEM, XRD, and N2 adsorption–desorption analysis results demonstrated that the synthesized MIM had a highly ordered mesoporous structure. Results from FT-IR revealed that acrolyl-β-CDs have been successfully conjugated to SBA-15. The density of β-CDs attached on the MIM is determined by EA and TGA. The equilibrium binding amounts of MIM and non-imprinted materials (NIM) for cholesterol are 53.50 and 25.24 μmol/g, respectively. The 2D MIM exhibited binding affinity and specificity for a group of analytes which have similar size and shape to those of template. The application of the prepared materials as stationary phases in thin layer chromatography was investigated preliminarily. The imprinted materials had higher retention ability for the template than the NIM. The retention factors (Rf) of cholesterol on MIM and NIM are 0.62 and 0.81, respectively. The solid phase extraction of cholesterol using MIM as the adsorbent was further investigated. The recoveries of the molecularly imprinted solid phase extraction column for cholesterol were 77.1–94.7 % with relative standard deviations (RSD) of 2.25–6.78 %.

Selective adsorption of elastase by surface molecular imprinting materials prepared with novel monomer

A new functional monomer with diol groups was synthesized and applied to fabricate surface molecular imprinting polymers (SMIPs) microspheres for selective adsorption of elastase.

Molecular dynamics simulations of molecularly imprinted polymer approaches to the preparation of selective materials to remove norfloxacin

ABSTRACTA novel molecularly imprinted polymer (MIP) designed by molecular dynamics (MD) simulations was successfully prepared with norfloxacin as a template molecule, methyl acrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a crosslinker. According to the theoretical prediction and experimental preparation methods, three kinds of molecular imprinting materials were designed and synthesized with MD simulations and molecular imprinting technology. The best ratio of the template to the functional monomer to the crosslinker was 1:8:40 in these studies. The experimental results illustrate that the MD simulations were credible in compounding the components of the MIPs. The structure of the prepared polymers were characterized with various methods. To analyze the adsorption performances, many kinds of static adsorption tests, including kinetic, isotherm, and selectivity tests, were used. The results indicate that the novel adsorbents conformed to the pseudo–second‐order kinetic equation and followed the Langmuir isotherm model. The adsorption amounts of MIP2 at a ratio of 1:8:40 were about 29.35 mg/g at 298 K. The selective adsorption and reusable performance of norfloxacin were excellent. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 132, 42817.

Methyleneation of peptides by N,N,N,N-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 N,N,N,N-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-MS(2) and LC-MS(3) 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 in situ polymerization systems. The use of an alternative catalyst such as sodium bisulfite may preserve the chemical integrity of peptides during in situ polymerization.