Non-imprinted Control Polymer Research Articles

Synthesis and characterization of core–shell magnetic molecularly imprinted polymer nanocomposites for the detection of interleukin-6

Interleukin-6 (IL-6) belongs to the cytokine family and plays a vital role in regulating immune response, bone maintenance, body temperature adjustment, and cell growth. The overexpression of IL-6 can indicate various health complications, such as anastomotic leakage, cancer, and chronic diseases. Therefore, the availability of highly sensitive and specific biosensing platforms for IL-6 detection is critical. In this study, for the first time, epitope-mediated IL-6-specific magnetic molecularly imprinted core–shell structures with fluorescent properties were synthesized using a three-step protocol, namely, magnetic nanoparticle functionalization, polymerization, and template removal following thorough optimization studies. The magnetic molecularly imprinted polymers (MMIPs) were characterized using dynamic and electrophoretic light scattering (DLS and ELS), revealing a hydrodynamic size of 169.9 nm and zeta potential of +17.1 mV, while Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy techniques showed characteristic peaks of the polymer and fluorescent tag, respectively. Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM) investigations confirmed the successful encapsulation of the magnetic core within the ca. 5-nm-thick polymeric shell. The MMIP-based electrochemical sensing platform achieved a limit of detection of 0.38 pM within a linear detection range of 0.38–380 pM, indicating high affinity (dissociation constant KD = 1.6 pM) for IL-6 protein in 50% diluted serum samples. Moreover, comparative investigations with the non-imprinted control polymer demonstrated an imprinting factor of 4, confirming high selectivity. With multifunctional features, including fluorescence, magnetic properties, and target responsiveness, the synthesized MMIPs hold significant potential for application in various sensor techniques as well as imaging.

Solid-phase extraction of organophosphates from polluted waters on a matrix-imprinted sorbent

Abstract The study aims to synthesize a selective matrix imprinted sorbent for the extraction of parathion and malathion. The structural unit of the polymeric framework was 2-methylpropanoic acid, the intermolecular crosslinker was ethylene glycol dimethacrylate, the polymerization initiator was azobisisobutyronitrile, the porogen was xylene, and the analyte was parathion. The synthesis was carried out under conditions of heating the reaction mixture to 65 °C, after which the matrix was washed with methanol to remove the analyte. For comparison purposes, non-imprinted control polymer was used as a negative control, which was prepared similarly, but without the addition of the analyte. The identification and quantification of organophosphates were performed by gas chromatography, and the morphological characteristics of the sorbents were evaluated by scanning electron microscopy. The optimal buffer for the purification of organophosphates was acetate buffer with a pH of 4; the optimal organic eluent was methanol. The limit of detection for para- and malathion was 0.1 μg/ml; the limit of quantification was 0.3 μg/ml. Linearity in the extraction conditions was observed in the range of 0.1–1 μg/ml for parathion and 0.1–2 μg/ml for malathion. The developed method will enable quick, selective, and cost-effective extraction of organophosphates from various substrates.

A rapid synthesis of molecularly imprinted polymer nanoparticles for the extraction of performance enhancing drugs (PIEDs).

It is becoming increasingly more significant to detect and separate hormones from water sources, with the development of synthetic recognition materials becoming an emerging field. The delicate nature of biological recognition materials such as the antibodies means the generation of robust viable synthetic alternatives has become a necessity. Molecularly imprinted nanoparticles (NanoMIPs) are an exciting class that has shown promise due the generation of high-affinity and specific materials. While nanoMIPs offer high affinity, robustness and reusability, their production can be tricky and laborious. Here we have developed a simple and rapid microwaveable suspension polymerisation technique to produce nanoMIPs for two related classes of drug targets, Selective Androgen Receptor Modulators (SARMs) and steroids. These nanoMIPs were produced using one-pot microwave synthesis with methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as a suitable cross-linker, producing particles of an approximate range of 120-140 nm. With the SARMs-based nanoMIPs being able to rebind 94.08 and 94.46% of their target molecules (andarine, and RAD-140, respectively), while the steroidal-based nanoMIPs were able to rebind 96.62 and 96.80% of their target molecules (estradiol and testosterone, respectively). The affinity of nanoMIPs were investigated using Scatchard analysis, with Ka values of 6.60 × 106, 1.51 × 107, 1.04 × 107 and 1.51 × 107 M-1, for the binding of andarine, RAD-140, estradiol and testosterone, respectively. While the non-imprinted control polymer (NIP) shows a decrease in affinity with Ka values of 3.40 × 104, 1.01 × 104, 1.83 × 104, and 4.00 × 104 M-1, respectively. The nanoMIPs also demonstrated good selectivity and specificity of binding the targets from a complex matrix of river water, showing these functional materials offer multiple uses for trace compound analysis and/or sample clean-up.

Plastic Antibodies Mimicking the ACE2 Receptor for Selective Binding of SARS-CoV-2 Spike.

Molecular imprinting has proven to be a versatile and simple strategy to obtain selective materials also termed “plastic antibodies” for a wide variety of species, i.e., from ions to macromolecules and viruses. However, to the best of the authors’ knowledge, the development of epitope‐imprinted polymers for selective binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is not reported to date. An epitope from the SARS‐CoV‐2 spike protein comprising 17 amino acids is used as a template during the imprinting process. The interactions between the epitope template and organosilane monomers used for the polymer synthesis are predicted via molecular docking simulations. The molecularly imprinted polymer presents a 1.8‐fold higher selectivity against the target epitope compared to non‐imprinted control polymers. Rebinding studies with pseudoviruses containing SARS‐CoV‐2 spike protein demonstrate the superior selectivity of the molecularly imprinted matrices, which mimic the interactions of angiotensin‐converting enzyme 2 receptors from human cells. The obtained results highlight the potential of SARS‐CoV‐2 molecularly imprinted polymers for a variety of applications including chem/biosensing and antiviral delivery.

Investigation of polyacrylamide hydrogel-based molecularly imprinted polymers using protein gel electrophoresis.

In conjunction with polyacrylamide gel electrophoresis (PAGE), molecular imprinting methods have been applied to produce a multilayer mini-slab in order to evaluate how selectively and specifically a hydrogel-based molecularly imprinted polymer (MIP) binds bovine haemoglobin (BHb, ~64.5 kDa). A three-layer mini-slab comprising an upper and lower layer and a MIP, or a non-imprinted control polymer dispersion middle layer has been investigated. The discriminating MIP layer, also based on polyacrylamide, was able to specifically bind BHb molecules in preference to a protein similar in molecular weight such as bovine serum albumin (BSA, ~66 kDa). Protein staining allowed us to visualise the protein retention strength of the MIP layer under the influence of an electric field. This method could be applied to other proteins with implications in effective protein capture, disease diagnostics, and protein analysis.

Facilitating serum determination of neuron specific enolase at clinically relevant levels by coupling on-line molecularly imprinted solid-phase extraction to LC-MS/MS

The identification and quantification of biomarkers is essential for the diagnosis, treatment, and long-term monitoring of many human diseases. In the present work, macromolecular synthetic receptors with pre-determined affinity and selectivity for the signature peptide of a prognostically significant small cell lung cancer (SCLC) biomarker - neuron-specific enolase (NSE) – were prepared in a porous polymer microsphere format using a template-directed synthesis strategy performed under precipitation polymerization conditions. The polymer microspheres were packed into short trap columns and then exploited as molecularly selective sorbents in a fully automated, on-line molecularly imprinted solid-phase extraction (MISPE) protocol. The on-line MISPE protocol was optimised with respect to the composition of the loading mobile phase, the flow rate, and the extraction time. The molecularly imprinted polymers (MIPs) showed high affinity and useful selectivity for the peptide target - the hexapeptide ELPLYR - compared to non-imprinted control polymers. The MIPs were able to retain the biomarker on-column for extraction times of up to 20 min, and the on-line MISPE method enabled complete recovery of the biomarker over the linear range 10–100 ng mL−1 when the biomarker was present in spiked ammonium bicarbonate solution (R2 = 0.994). For extractions of ELPLYR from very complex biological matrices, the recoveries of ELPLYR from reversed-phase SPE (RP-SPE)-treated and untreated digested human serum were 100.8 ± 6.2% and 61.6 ± 1.9%, respectively. Extractions of ELPLYR from spiked untreated digested serum were linear in the range of 7.5–375 ng mL−1 (R2 = 0.99). The limit of detection (LOD) and limit of quantification (LOQ) for the biomarker in digested serum were estimated to be 1.8 ng mL−1 and 6.0 ng mL−1, respectively, which is below the median reference level of NSE in humans (8.6 ng mL−1). This work sets in place the basis for a new diagnostic tool for SCLC that is sensitive, robust, automated, and antibody-free, and which works very well with complex human plasma samples.

The molecular imprinting effect of propranolol and dibenzylamine as model templates: Binding strength and selectivity

Recent studies have shown anomalies with the most studied non-covalent molecularly imprinted polymer, the propranolol imprinted one. This imprinted polymer, like many others, binds more template than the non-imprinted control polymer, but its selectivity in template adsorption is only slightly or not at all improved by imprinting, depending on the compound compared. The reasons for this anomaly are discovered here. Simple experiments show that acid homoassociation in the prepolymerisation complex is the likely cause of the anomaly. The specific conductivity of prepolymerization mixtures at different functional monomer to template ratios follows a pattern observed in homoassociating systems. Analysis of the optimal prepolymerization mixture shows that on average two molecules of the functional monomer are complexed to the basic template, even if the template lacks any other hydrogen bonding functional group than the amino group. Molecular modeling calculations provide the structure and stability of the homoassociated prepolymerization complexes. These results lead to a plausible interpretation of the anomaly, which may not be unique for the propranolol imprinted polymer, but may affect all imprinted polymers made for basic templates by using acidic functional monomers. The analytical applications of the new imprinting model are demonstrated.

Advances in the development of molecularly imprinted polymers for the separation and analysis of proteins with liquid chromatography.

This review documents recent advances in the design, synthesis, characterization, and application of molecularly imprinted polymers in the form of monoliths and particles/beads for the use in the separation and analysis of proteins with solid-phase extraction or liquid chromatography. The merits of three-dimensional molecular imprinting, whereby the molecular template is randomly embedded in the polymer, and two-dimensional imprinting, in which the template is confined to the surface, are described. Target protein binding can be achieved by either using the entire protein as a template or by using a protein substructure as template, that is, a peptide, as in the "epitope" approach. The intended approach and strategy then determine the choice of polymerization method. A synopsis has been provided on methods used for the physical, chemical, and functional characterizations and associated performance evaluations of molecularly imprinted and nonimprinted control polymers, involving a diverse range of analytical techniques commonly used for low and high molecular mass analytes. Examples of recent applications demonstrate that, due to the versatility of imprinting methods, molecularly imprinted monoliths or particles/beads can be adapted to protein extraction/depletion and separation procedures relevant to, for example, protein biomarker detection and quantification in biomedical diagnostics and targeted proteomics.

New Methods to Study the Behavior of Molecularly Imprinted Polymers in Aprotic Solvents.

This work presents three new experimental methods for studying molecular imprinting. The electric conductivity measurements of the pre-polymerization mixture of amine templates in an aprotic solvent provide evidence of ionic dissociation of the pre-polymerization complexes. The displacement measurement of the template propranolol from its molecularly imprinted polymer (MIP) using a quaternary ammonium ion in toluene, shows that this MIP behaves as an ion exchanger even in a non-polar solvent. The same experiment also shows that template binding to the MIP from toluene involves ionic interaction. The third experimental method introduced here serves to study the models of template binding on MIPs. To this end the binding isotherm of propranolol (PR) has been measured on a polymer mixture consisting of non-imprinted control polymer (NIP) and a stronger binding acidic polymer, respectively. All three methods are suitable for studying several other imprinting systems.

The Selectivity of Polymers Imprinted with Amines.

One of the main reasons for making molecularly imprinted polymers (MIPs) has been that MIPs interact selectively with a specific target compound. This claim is investigated here with the example of a widely used type of noncovalent MIP, the MIP for the beta blocker propranolol. Adsorption isotherms of this MIP and of a nonimprinted control polymer (NIP), respectively, have been measured with a series of compounds in the porogen solvent acetonitrile. The results, visualized as “selectivity ladders”, show that the MIP binds propranolol and many other amines better than the NIP does, but the selectivity of the MIP is actually inferior to that of the NIP. The selectivity of either polymer for propranolol is modest against many amines, but is remarkable with respect to other compounds. The contribution of imprinting towards selectivity can be better appreciated when three MIPs, made with different amine templates, are compared among themselves. Each MIP is seen to bind its own template slightly better than the other two MIPs do. In media different from the porogen, the selectivity patterns may change substantially. Propranolol seems to have properties that make it stand high on the selectivity scale in different solvents, albeit for different reasons.

Biomimetic recognition and peptidase activities of transition state analogue imprinted chymotrypsin mimics

The first peptidolysis reaction utilizing transition state analogue imprinted polymer was demonstrated utterly in the viewpoint of size and shape-selective substrate recognition. The enzyme mimic polymer was synthesized from the amino acid triad histidine, aspartic acid and serine in presence of phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate transition state analogue (TSA) by molecular imprinting technique. The polymer catalyst synthesized exhibited high selectivity and performed as a reliable tool for peptidolytic reactions. The peptidase activity of the enzyme mimic polymer catalyst was investigated by following the hydrolysis of dipeptides spectrophotometrically at 207nm and the kinetic parameters, rate acceleration kaccand imprinting efficiencykim, were evaluated. The imprinted peptidase displayed a rate acceleration of 1.67×103 contrasted with the uncatalyzed peptidolysis and an imprinting efficiency of 45 over the non-imprinted control polymer. The artificial peptidase amazingly promoted the hydrolysis of dipeptides having Phe/Tyr amino acid as the C-terminal residues discriminating chymotrypsin specific and non-specific substrates. The mimic exhibited higher rate acceleration and substrate specificity towards peptides compared to amino acid p-nitroanilides. Despite the fact that natural enzyme is much superior to the MIP catalyst in hydrolase activities, the mimic portrays high thermal stability, prolonged life span and unrivaled recyclability and reusability.

Geometrical effect of 3D-memory cavity on the imprinting efficiency of transition-state analogue-built artificial hydrolases

Highlighting biomimetic recognition and shape-selective binding, highly crosslinked transition-state analogue-imprinted artificial hydrolases are synthesized from amino acid monomers. Two different transition-state analogues—TSAs—are used for the preparation of the enzyme mimic polymer catalysts. The catalytic hydrolysis of amino acid p-nitroanilides is found to be dependent on the geometry of the TSA imprints on the polymer matrix. The imprinted TSA facilitates tetrahedral complementarity to the transition-state intermediate of hydrolysis. The geometry of the 3D-memory cavity fabricated by the print molecule along with the catalytic entities is accountable for the higher catalytic competence of the imprinted enzyme mimics over the non-imprinted control polymers. The super crosslinked macroporous polymer matrix, in which the catalytic functions are suitably oriented in a ‘3D pocket’ for selective binding of the substrate through H-bonding, is accountable for the high imprinting efficiency of the imprinted polymer catalysts. The imprinted mimics are found to be exhibiting cross-selectivity in their catalytic properties. Even though the mimics could not compete with native enzyme, they exhibit higher thermal stability, increased shelf-life and superior reusability.

FIB and MIP: understanding nanoscale porosity in molecularly imprinted polymers via 3D FIB/SEM tomography.

We present combined focused ion beam/scanning electron beam (FIB/SEM) tomography as innovative method for differentiating and visualizing the distribution and connectivity of pores within molecularly imprinted polymers (MIPs) and non-imprinted control polymers (NIPs). FIB/SEM tomography is used in cell biology for elucidating three-dimensional structures such as organelles, but has not yet been extensively applied for visualizing the heterogeneity of nanoscopic pore networks, interconnectivity, and tortuosity in polymers. To our best knowledge, the present study is the first application of this strategy for analyzing the nanoscale porosity of MIPs. MIPs imprinted for propranolol - and the corresponding NIPs - were investigated establishing FIB/SEM tomography as a viable future strategy complementing conventional isotherm studies. For visualizing and understanding the properties of pore networks in detail, polymer particles were stained with osmium tetroxide (OsO4) vapor, and embedded in epoxy resin. Staining with OsO4 provides excellent contrast during high-resolution SEM imaging. After optimizing the threshold to discriminate between the stained polymer matrix, and pores filled with epoxy resin, a 3D model of the sampled volume may be established for deriving not only the pore volume and pore surface area, but also to visualize the interconnectivity and tortuosity of the pores within the sampled polymer volume. Detailed studies using different types of cross-linkers and the effect of hydrolysis on the resulting polymer properties have been investigated. In comparison of MIP and NIP, it could be unambiguously shown that the interconnectivity of the visualized pores in MIPs is significantly higher vs. the non-imprinted polymer, and that the pore volume and pore area is 34% and approx. 35% higher within the MIP matrix. This confirms that the templating process not only induces selective binding sites, but indeed also affects the physical properties of such polymers down to the nanoscale, and that additional chemical modification, e.g., via hydrolysis clearly affects that nature of the polymer.

Selective solid-phase extraction using molecularly imprinted polymers for analysis of venlafaxine, O-desmethylvenlafaxine, and N-desmethylvenlafaxine in plasma samples by liquid chromatography–tandem mass spectrometry

This paper focuses on the development of a novel miniaturized molecularly imprinted solid-phase extraction (MISPE) and ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method to determine venlafaxine (VEN), O-desmethylvenlafaxine (ODV), and N-desmethylvenlafaxine (NDV) in plasma samples. The molecularly imprinted polymer (MIP) was prepared by the precipitation polymerization approach; VEN, metacrylic acid, ethylene glycol dimethacrylate, 2,2-azobisisobutyronitrile, and toluene were used as template, monomer, crosslinker, initiator, and porogen solvent, respectively. MIP and of the non-imprinted control polymer (NIP) sorbents were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. MIP phase presented higher extraction efficiency (MISPE, using plasma samples spiked with VEN) than the NIP phase (84 and 49% recovery rates, respectively). Analysis of other antidepressants with different chemical structures by MISPE-UHPLC–MS/MS attested to the selectivity of the developed MIP. The developed method presented precision assays with coefficients of variation (CV) smaller than 15%; accuracy assays with relative standard error (RSE%) values ranging from −12 to 16%, and linear ranges from 3 to 700ngmL−1 for VEN, from 5 to 700ngmL−1 for ODV, and from 3 to 500ngmL−1 for NDV. The coefficients of determination (r2) were higher than 0.995. The lack-of-fit test also attested to the linearity of this method. This method was successfully applied to determine VEN, NDV, and ODV in plasma samples from depressed patients undergoing therapy with VEN.

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging.

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core-shell particles specifically recognizing glucuronic acid (GlcA) or N-acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP-GlcA and red QDs conjugated with MIP-NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non-imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.

Dopaminergic receptor–ligand binding assays based on molecularly imprinted polymers on quartz crystal microbalance sensors

Molecularly imprinted polymers (MIPs) have been successfully applied as selective materials for assessing the binding activity of agonist and antagonist of dopamine D1 receptor (D1R) by using quartz crystal microbalance (QCM). In this study, D1R derived from rat hypothalamus was used as a template and thus self-organized on stamps. Those were pressed into an oligomer film consisting of acrylic acid: N-vinylpyrrolidone: N,N′-(1,2-dihydroxyethylene) bis-acrylamide in a ratio of 2:3:12 spin coated onto a dual electrode QCM. Such we obtained one D1R-MIP-QCM electrode, whereas the other electrode carried the non-imprinted control polymer (NIP) that had remained untreated. Successful imprinting of D1R was confirmed by AFM. The polymer can re-incorporate D1R leading to frequency responses of 100–1200Hz in a concentration range of 5.9–47.2µM. In a further step such frequency changes proved inherently useful for examining the binding properties of test ligands to D1R. The resulting mass-sensitive measurements revealed Kd of dopamine∙HCl, haloperidol, and (+)-SCH23390 at 0.874, 25.6, and 0.004nM, respectively. These results correlate well with the values determined in radio ligand binding assays. Our experiments revealed that D1R-MIP sensors are useful for estimating the strength of ligand binding to the active single site. Therefore, we have developed a biomimetic surface imprinting strategy for QCM studies of D1R-ligand binding and presented a new method to ligand binding assay for D1R.

Molecularly imprinted polymers for protein capture, sequestration, and delivery

Event Abstract Back to Event Molecularly imprinted polymers for protein capture, sequestration, and delivery John Clegg1, 2, Justin Zhong3, Heidi R. Culver1, 2 and Nicholas A. Peppas1, 2, 3, 4 1 University of Texas at Austin, Department of Biomedical Engineering, United States 2 University of Texas at Austin, Institute for Biomaterials, Drug Delivery, and Regenerative Medicine, United States 3 University of Texas at Austin, McKetta Department of Chemical Engineering, United States 4 University of Texas at Austin, College of Pharmacy, United States Introduction: Molecularly imprinted polymers (MIPs) for protein detection have been limited to a few model protein templates[1]. MIPs entrap protein within their pores during polymerization, requiring denaturing or even destructive purification techniques to purify and extract templates[2]. The cost of many therapeutic proteins is therefore prohibitive to their use as imprinting templates. Herein, we hypothesized that rationally-selected templates, possessing similar geometry, molecular weight and isoelectric point to disease-relevant biomarkers could be used as low-cost alternatives. We demonstrate a MIP system, using Trypsin as a low-cost template, to capture and sequester low molecular weight, positively charged biomolecules while excluding larger, negative proteins. The ability to displace and release a model therapeutic protein through template binding is also investigated. Materials and Methods: Trypsin was allowed to pre-assemble with methacrylate monomers possessing hydrophilic, positive, or negative moieties, as well as hydrophobic core nanoparticles and crosslinker in phosphate buffer under a nitrogen atmosphere. Following polymerization, entrapped trypsin and unreacted monomers were extracted with 10 washes in 10% acetic acid (Fig. 1). Non-imprinted control polymers (NIPs) were prepared in the same manner, excluding Trypsin. Hydrogel microparticles were subjected to binding assays, with proteins of varying charge and molecular weight (Fig. 2A). MIP sequestration and delivery of cytochrome c was demonstrated through loading cytochrome c within MIPs and NIPs, and monitoring payload release over 24 hours in the presence and absence of trypsin. Results and Discussion: MIPs bound more trypsin than NIPs (Fig. 2B) and similar quantities of cytochrome c and lysozyme, while binding small quantities of hemoglobin (Fig. 2C). Normalization of equilibrium rebinding for MIPs to NIPs (Imprinting Factor) revealed no difference in relative protein binding between trypsin, lysozyme, cytochrome c, which were all significantly greater than bovine hemoglobin (Fig 2D). All imprinting factors were significantly different from 1, (p< .05), demonstrating a measurable impact of molecular imprinting on protein binding. MIPs exhibited 84.9% loading efficiency of cytochrome c, as compared to 52.3% for NIPs. MIPs were capable of sequestering cytochrome c in 0.1x PBS, releasing only 5.7% of payload released in 24h, while subsequently releasing 41.5% during 24h incubation with trypsin. Conclusion: Trypsin imprinting impacts cytochrome c, lysozyme, trypsin, and hemoglobin binding through exclusion on the basis of size and electric potential. Cytochrome c and lysozyme bind in similar capacity to Trypsin, likely as their similar charge and smaller size permit entry and sequestration into trypsin-MIP pores. These proteins can be efficiently loaded, excluded, sequestered, and delivered. Future studies, using rationally-selected imprinting templates with structural similarity to clinical biomarkers will determine if this fully-synthetic, low cost system can be employed in medical diagnostics and drug delivery. NSF Graduate Research Fellowship; Pratt Foundation; UT-Portugal Collaborative Research Program

Development of a molecularly imprinted solid-phase extraction sorbent for the selective extraction of telmisartan from human urine.

A novel molecularly imprinted solid-phase extraction with spectrofluorimetry method has been developed for the selective extraction of telmisartan from human urine. Molecularly imprinted polymers were prepared by a noncovalent imprinting approach through UV-radical polymerization using telmisartan as a template molecule, 2-dimethylamino ethyl methacrylate as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, N,N-azobisisobutyronitrile as an initiator, chloroform as a porogen. Molecularly imprinted polymers and nonimprinted control polymer sorbents were dry-packed into solid-phase extraction cartridges, and eluates from cartridges were analyzed using a spectrofluorimeter. Limit of detection and limit of quantitation values were 11.0 and 36.0 ng/mL, respectively. A very high imprinting factor (16.1) was achieved and recovery values for the telmisartan spiked in human urine were in the range of 76.1-79.1%. In addition, relatively low within-day (0.14-1.6%) and between-day (0.11-1.31%) precision values were obtained. Valsartan was used to evaluate the selectivity of sorbent as well. As a result, a sensitive, selective, and simple molecularly imprinted solid-phase extraction with spectrofluorimetry method has been developed and successfully applied to the direct determination telmisartan in human urine.

Spectroscopic and quartz crystal microbalance (QCM) characterisation of protein-based MIPs

We have studied acrylamide-based polymers of varying hydrophobicity (acrylamide, AA; N-hydroxymethylacrylamide, NHMA; N-isopropylacrylamide, NiPAm) for their capability of imprinting protein. Rebinding capacities (Q) from spectroscopic studies were highest for bovine haemoglobin (BHb) MIPs based on AA, Q=4.8±0.21<NHMA, Q=4.3±0.32<NiPAm, Q=3.6±0.45, while also demonstrating low selectivities for non-template proteins (<30±5%), with the exception of bovine serum albumin (BSA, >76±0.5%). When applied to the QCM sensor as thin-film MIPs, NHMA MIPs were found to exhibit best discrimination between MIP and non-imprinted control polymer (NIP) in the order of NiPAm<AA<NHMA. The extent of template removal and rebinding, using both crystal impedance and frequency measurements, demonstrated that 10% (w/v):10% (v/v) sodium dodecyl sulphate:acetic acid (pH 2.8) was efficient at eluting template BHb (with 80±10% removal). Selectivity studies of NHMA BHb-MIPs revealed higher adsorption and selective recognition properties to BHb (64.5kDa) when compared to non-cognate BSA (66kDa), myoglobin (Mb, 17.5kDa), lysozyme (Lyz, 14.7kDa) thaumatin (Thau, 22kDa) and trypsin (Tryp, 22.3kDa). The QCM gave frequency shifts of ∼1500±50Hz for template BHb rebinding in both AA and NHMA MIPs, whereas AA-based MIPs exhibited an interference signal of ∼2200±50Hz for non-cognate BSA in comparison to a ∼500±50Hz shift with NHMA MIPs. Our results show that NHMA-based hydrogel MIP are superior to AA and NIPAM.

Pending templates imprinted polymers—hypothesis, synthesis, adsorption, and chromatographic properties

This is the first time when protein-imprinted polymers are prepared with "pending templates." The polymers were synthesized in the presence of a real sample (chicken egg white), rather than any known commercial proteins. Compared with a simultaneously synthesized nonimprinted control polymer, the polymers show higher adsorption capacity for abundant components (as "pending templates") in the original sample. Chromatography experiments indicated that the columns made of the imprinted polymers could retain abundant species (imprinted) and separate them from those not imprinted. Thus, the sample could be split into dimidiate subfractions with reduced complexities. "Pending template imprinting" suggests a new way to investigate molecular imprinting, especially to dissect, simplify, and analyze complicated samples through a series of polymers just imprinted by the samples per se.