Enhanced selective capture of phosphomonoester lipids enabling highly sensitive detection of sphingosine 1-phosphate.

Sphingolipids play crucial roles in cellular membranes, myelin stability, and signalling responses to physiological cues and stress. Among them, sphingosine 1-phosphate (S1P) has been recognized as a relevant biomarker for neurodegenerative diseases, and its analogue FTY-720 has been approved by the FDA for the treatment of relapsing-remitting multiple sclerosis. Focusing on these targets, we here report three novel polymeric capture phases for the selective extraction of the natural biomarker and its analogue drug. To enhance analytical performance, we employed different synthetic approaches using a cationic monomer and a hydrophobic copolymer of styrene-DVB. Results have demonstrated high affinity of the sorbents towards S1P and fingolimod phosphate (FTY-720-P, FP). This evidence proved that lipids containing phosphate diester moiety in their structures did not constitute obstacles for the interaction of phosphate monoester lipids when loaded into an SPE cartridge. Our suggested approach offers a valuable tool for developing efficient analytical procedures.

Urea-Based Imprinted Polymer Hosts with Switchable Anion Preference.

The design of artificial oxyanion receptors with switchable ion preference is a challenging goal in host-guest chemistry. We here report on molecularly imprinted polymers (MIPs) with an external phospho-sulpho switch driven by small molecule modifiers. The polymers were prepared by hydrogen bond-mediated imprinting of the mono- or dianions of phenyl phosphonic acid (PPA), phenyl sulfonic acid (PSA), and benzoic acid (BA) using N-3,5-bis-(trifluoromethyl)-phenyl-N-4-vinylphenyl urea (1) as the functional host monomer. The interaction mode between the functional monomer and the monoanions was elucidated by 1H NMR titrations and 1H-1H NMR NOESY supported by molecular dynamic simulation, which confirmed the presence of high-order complexes. PPA imprinted polymers bound PPA with an equilibrium constant Keq = 1.8 × 105 M-1 in acetonitrile (0.1% 1,2,2,6,6-pentamethylpiperidine) and inorganic HPO42- and SO42- with Keq = 2.9 × 103 M-1 and 4.5 × 103 M-1, respectively, in aqueous buffer. Moreover, the chromatographic retentivity of phosphonate versus sulfonate was shown to be completely switched on this polymer when changing from a basic to an acidic modifier. Mechanistic insights into this system were obtained from kinetic investigations and DSC-, MALDI-TOF-MS-, 1H NMR-studies of linear polymers prepared in the presence of template. The results suggest the formation of template induced 1-1 diad repeats in the polymer main chain shedding unique light on the relative contributions of configurational and conformational imprinting.

Highly Efficient Synthesis and Assay of Protein-Imprinted Nanogels by Using Magnetic Templates.

We report an approach integrating the synthesis of protein-imprinted nanogels ("plastic antibodies") with a highly sensitive assay employing templates attached to magnetic carriers. The enzymes trypsin and pepsin were immobilized on amino-functionalized solgel-coated magnetic nanoparticles (magNPs). Lightly crosslinked fluorescently doped polyacrylamide nanogels were subsequently produced by high-dilution polymerization of monomers in the presence of the magNPs. The nanogels were characterised by a novel competitive fluorescence assay employing identical protein-conjugated nanoparticles as ligands to reversibly immobilize the corresponding nanogels. Both nanogels exhibited Kd <10 pM for their respective target protein and low cross-reactivity with five reference proteins. This agrees with affinities reported for solid-phase-synthesized nanogels prepared using low-surface-area glass-bead supports. This approach simplifies the development and production of plastic antibodies and offers direct access to a practical bioassay.

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.

Ratiometric Fluorescence Detection of Phosphorylated Amino Acids Through Excited-State Proton Transfer by Using Molecularly Imprinted Polymer (MIP) Recognition Nanolayers.

A 2,3-diaminophenazine bis-urea fluorescent probe monomer (1) was developed. It responds to phenylphosphate and phosphorylated amino acids in a ratiometric fashion with enhanced fluorescence accompanied by the development of a redshifted emission band arising from an excited-state proton transfer (ESPT) process in the hydrogen-bonded probe/analyte complex. The two urea groups of 1 form a cleft-like binding pocket (Kb >1010  L2 mol-2 for 1:2 complex). Imprinting of 1 in presence of ethyl ester- and fluorenylmethyloxycarbonyl (Fmoc)-protected phosphorylated tyrosine (Fmoc-pTyr-OEt) as the template, methacrylamide as co-monomer, and ethyleneglycol dimethacrylate as cross-linker gave few-nanometer-thick molecularly imprinted polymer (MIP) shells on silica core microparticles with excellent selectivity for the template in a buffered biphasic assay. The supramolecular recognition features were established by spectroscopic and NMR studies. Rational screening of co-monomers and cross-linkers allowed to single out the best performing MIP components, giving significant imprinting factors (IF>3.5) while retaining ESPT emission and the ratiometric response in the thin polymer shell. Combination of the bead-based detection scheme with the phase-transfer assay dramatically improved the IF to 15.9, allowing sensitive determination of the analyte directly in aqueous media.

Ultratrace Detection of Histamine Using a Molecularly-Imprinted Polymer-Based Voltammetric Sensor.

Rapid and cost-effective analysis of histamine, in food, environmental, and diagnostics research has been of interest recently. However, for certain applications, the already-existing biological receptor-based sensing methods have usage limits in terms of stability and costs. As a result, robust and cost-effective imprinted polymeric receptors can be the best alternative. In the present work, molecularly-imprinted polymers (MIPs) for histamine were synthesized using methacrylic acid in chloroform and acetonitrile as two different porogens. The binding affinity of the MIPs with histamine was evaluated in aqueous media. MIPs synthesized in chloroform displayed better imprinting properties for histamine. We demonstrate here histamine MIPs incorporated into a carbon paste (CP) electrode as a MIP-CP electrode sensor platforms for detection of histamine. This simple sensor format allows accurate determination of histamine in the sub-nanomolar range using an electrochemical method. The sensor exhibited two distinct linear response ranges of 1 × 10-10-7 × 10-9 M and 7 × 10-9-4 × 10-7 M. The detection limit of the sensor was calculated equal to 7.4 × 10-11 M. The specificity of the proposed electrode for histamine is demonstrated by using the analogous molecules and other neurotransmitters such as serotonin, dopamine, etc. The MIP sensor was investigated with success on spiked serum samples. The easy preparation, simple procedure, and low production cost make the MIP sensor attractive for selective and sensitive detection of analytes, even in less-equipped laboratories with minimal training.

An Epitope-Imprinted Biointerface with Dynamic Bioactivity for Modulating Cell-Biomaterial Interactions.

In this study, an epitope-imprinting strategy was employed for the dynamic display of bioactive ligands on a material interface. An imprinted surface was initially designed to exhibit specific affinity towards a short peptide (i.e., the epitope). This surface was subsequently used to anchor an epitope-tagged cell-adhesive peptide ligand (RGD: Arg-Gly-Asp). Owing to reversible epitope-binding affinity, ligand presentation and thereby cell adhesion could be controlled. As compared to current strategies for the fabrication of dynamic biointerfaces, for example, through reversible covalent or host-guest interactions, such a molecularly tunable dynamic system based on a surface-imprinting process may unlock new applications in in situ cell biology, diagnostics, and regenerative medicine.

Different expression levels of glycans on leukemic cells-a novel screening method with molecularly imprinted polymers (MIP) targeting sialic acid.

Sialic acid (SA) is normally expressed on the cell membranes and is located at the terminal position of the sugar chains. SA plays an important role for regulation of the innate immunity, function as markers of the cells and can be recognized by a variety of receptors. Interestingly, the level of SA expression is increased on metastatic cancer cells. The availability of specific antibodies against SA is limited and, therefore, biomarker tools for detection of SA are lacking. We have recently presented a novel method for specific fluorescence labeling of SA molecular imprinted polymers (MIP). Here, we have performed an extended screening of SA expression by using SA-MIP and included four different chronic lymphocytic leukemia (CLL) cell lines, conveniently analyzed by flow cytometry and fluorescence microscopy. SA expression was detected in four cell lines at different levels, and the SA expression were verified with lectin-FITC. These results show that SA-MIP can be used as a plastic antibody for detection of SA using both flow cytometry and fluorescence microscopy. We suggest that SA-MIP can be used for screening of different tumor cells of various stages, including CLL cells.

Sialic Acid-Imprinted Fluorescent Core-Shell Particles for Selective Labeling of Cell Surface Glycans.

The expression of cell surface glycans terminating with sialic acid (SA) residues has been found to correlate with various disease states there among cancer. We here report a novel strategy for specific fluorescence labeling of such motifs. This is based on sialic acid-imprinted core-shell nanoparticles equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups allowing environmentally sensitive fluorescence detection at convenient excitation and emission wavelengths. Imprinting was achieved exploiting a hybrid approach combining reversible boronate ester formation between p-vinylphenylboronic acid and SA, the introduction of cationic amine functionalities, and the use of an NBD-appended urea-monomer as a binary hydrogen-bond donor targeting the SA carboxylic acid and OH functionalities. The monomers were grafted from 200 nm RAFT-modified silica core particles using ethylene glycol dimethacrylate (EGDMA) as cross-linker resulting in a shell thickness of ca. 10 nm. The particles displayed strong affinity for SA in methanol/water mixtures (K = 6.6 × 10(5) M(-1) in 2% water, 5.9 × 10(3) M(-1) in 98% water, B(max) ≈ 10 µmol g(-1)), whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 × 10(3) M(-1) in 98% water). In cell imaging experiments, the particles selectively stained different cell lines in correlation with the SA expression level. This was further verified by enzymatic cleavage of SA and by staining using a FITC labeled SA selective lectin.

Lipidomic "deep profiling": an enhanced workflow to reveal new molecular species of signaling lipids.

Current mass spectrometry-based lipidomics aims to comprehensively cover wide ranges of lipid classes. We introduce a strategy to capture phospho-monoester lipids and improve the detection of long-chain base phosphates (LCB-Ps, e.g., sphingosine-1-phosphate). Ten novel LCB-Ps (d18:2, t20:1, odd carbon forms) were discovered and characterized in tissues from human and mouse, as well in D. melanogaster and S. cerevisiae. These findings have immediate relevance for our understanding of sphingosine-1-phosphate biosynthesis, signaling, and degradation.

Microcontact-Imprinted Optical Sensors for Virulence Factors of Periodontal Disease.

Periodontitis (gum disease) is a common biofilm-mediated oral condition, with around 7% of the adult population suffering from severe disease with risk for tooth loss. Moreover, periodontitis virulence markers have been found in atherosclerotic plaque and brain tissue, suggesting a link to cardiovascular and Alzheimer's diseases. The lack of accurate, fast, and sensitive clinical methods to identify patients at risk leads, on the one hand, to patients being undiagnosed until the onset of severe disease and, on the other hand, to overtreatment of individuals with mild disease, diverting resources from those patients most in need. The periodontitis-associated bacterium, Porphyromonas gingivalis, secrete gingipains which are highly active proteases recognized as key virulence factors during disease progression. This makes them interesting candidates as predictive biomarkers, but currently, there are no methods in clinical use for monitoring them. Quantifying the levels or proteolytic activity of gingipains in the periodontal pocket surrounding the teeth could enable early-stage disease diagnosis. Here, we report on a monitoring approach based on high-affinity microcontact imprinted polymer-based receptors for the Arg and Lys specific gingipains Rgp and Kgp and their combination with surface plasmon resonance (SPR)-based biosensor technology for quantifying gingipain levels in biofluids and patient samples. Therefore, Rgp and Kgp were immobilized on glass coverslips followed by microcontact imprinting of poly-acrylamide based films anchored to gold sensor chips. The monomers selected were N-isopropyl acrylamide (NIPAM), N-hydroxyethyl acrylamide (HEAA) and N-methacryloyl-4-aminobenzamidine hydrochloride (BAM), with N,N'-methylene bis(acrylamide) (BIS) as the crosslinker. This resulted in imprinted surfaces exhibiting selectivity towards their templates high affinity and selectivity for the templated proteins with dissociation constants (K d) of 159 and 299 nM for the Rgp- and Kgp-imprinted, surfaces respectively. The former surface displayed even higher affinity (K d = 71 nM) when tested in dilute cell culture supernatants. Calculated limits of detection for the sensors were 110 and 90 nM corresponding to levels below clinically relevant concentrations.

Glucuronide directed molecularly imprinted solid-phase extraction: isolation of testosterone glucuronide from its parent drug in urine.

Two molecularly imprinted polymers (MIPs) that we recently described to be class-selective for glucuronides have been successfully exploited for the molecularly imprinted solid-phase extraction (MISPE) of testosterone glucuronide (TG) from its parent drug (T) in urine. Both sorbents targeted the glucuronate fragment but feature different functional groups for binding the carboxylate anion, MIP1, a neutral 1,3-diarylurea group, and MIP2, a cationic imidazolium functionality. MISPE-HPLC-UV methods developed using both sorbents allowed the extraction of TG from its parent compound in urine samples spiked at 150, 300 or 600 ng mL(-1) for TG and at 50 ng mL(-1) for T. By comparing the performance of the two sorbents it came out that MIP1 is a more suitable SPE packing than MIP2, since it isolated the glucuronide with a higher precision (RSD 2-5%, n = 3) and with an enhanced enrichment factor (EF = 4.2). On the basis of these results, the imprinted receptor MIP1 can be applied for the direct extraction of TG in doping and clinical analysis and to selectively capture any other relevant glucuronated metabolite avoiding tedious deconjugation steps prior to quantification.

Oxoanion Imprinting Combining Cationic and Urea Binding Groups: A Potent Glyphosate Adsorber.

The use of polymerizable hosts in anion imprinting has led to powerful receptors with high oxyanion affinity and specificity in both aqueous and non-aqueous environments. As demonstrated in previous reports, a carefully tuned combination of orthogonally interacting binding groups, for example, positively charged and neutral hydrogen bonding monomers, allows receptors to be constructed for use in either organic or aqueous environments, in spite of the polymer being prepared in non-competitive solvent systems. We here report on a detailed experimental design of phenylphosphonic and benzoic acid-imprinted polymer libraries prepared using either urea- or thiourea-based host monomers in the presence or absence of cationic comonomers for charge-assisted anion recognition. A comparison of hydrophobic and hydrophilic crosslinking monomers allowed optimum conditions to be identified for oxyanion binding in non-aqueous, fully aqueous, or high-salt media. This showed that recognition improved with the water content for thiourea-based molecularly imprinted polymers (MIPs) based on hydrophobic EGDMA with an opposite behavior shown by the polymers prepared using the more hydrophilic crosslinker PETA. While the affinity of thiourea-based MIPs increased with the water content, the opposite was observed for the oxourea counterparts. Binding to the latter could however be enhanced by raising the pH or by the introduction of cationic amine- or Na+-complexing crown ether-based comonomers. Use of high-salt media as expected suppressed the amine-based charge assistance, whereas it enhanced the effect of the crown ether function. Use of the optimized receptors for removing the ubiquitous pesticide glyphosate from urine finally demonstrated their practical utility.

Ultratrace enrichment of tyrosine phosphorylated peptides on an imprinted polymer.

Novel molecularly imprinted polymers (MIPs) designed to bind the side chain of phosphotyrosine can be used as artificial receptors for affinity-based enrichment of proteolytic peptides. In comparison with general enrichment methods for phosphorylated peptides such as TiO(2)-based methods, the pTyr-imprinted polymers offered high selectivity for pTyr-containing peptides down to the low fmol level. This suggests MIPs as a new tool for affinity-based proteomics.

Combinatorial Design of a Sialic Acid-Imprinted Binding Site.

Aberrant glycosylation has been proven to correlate with various diseases including cancer. An important alteration in cancer progression is an increased level of sialylation, making sialic acid one of the key constituents in tumor-specific glycans and an interesting biomarker for a diversity of cancer types. Developing molecularly imprinted polymers (MIPs) with high affinity toward sialic acids is an important task that can help in early cancer diagnosis. In this work, the glycospecific MIPs are produced using cooperative covalent/noncovalent imprinting. We report here on the fundamental investigation of this termolecular imprinting approach. This comprises studies of the relative contribution of orthogonally interacting functional monomers and their synergetic behavior and the choice of different counterions on the molecular recognition properties for the sialylated targets. Combining three functional monomers targeting different functionalities on the template led to enhanced imprinting factors (IFs) and selectivities. This apparent cooperative effect was supported by 1H NMR and fluorescence titrations of monomers with templates or template analogs. Moreover, highlighting the role of the template counterion use of tetrabutylammonium (TBA) salt of sialic acid resulted in better imprinting than that of sodium salts supported by both in solution interaction studies and in MIP rebinding experiments. The glycospecific MIPs display high affinity for sialylated targets, with an overall low binding of other nontarget saccharides.

Tailored Polymer-Based Selective Extraction of Lipid Mediators from Biological Samples.

Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

Molecularly Imprinted Polymers for the Removal of Antidepressants from Contaminated Wastewater.

Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants regularly detected in the environment. This indicates that the existing wastewater treatment techniques are not successfully removing them beforehand. This study investigated the potential of molecularly imprinted polymers (MIPs) to serve as sorbents for removal of SSRIs in water treatment. Sertraline was chosen as the template for imprinting. We optimized the composition of MIPs in order to obtain materials with highest capacity, affinity, and selectivity for sertraline. We report the maximum capacity of MIP for sertraline in water at 72.6 mg g-1, and the maximum imprinting factor at 3.7. The MIPs were cross-reactive towards other SSRIs and the metabolite norsertraline. They showed a stable performance in wastewater-relevant pH range between 6 and 8, and were reusable after a short washing cycle. Despite having a smaller surface area between 27.4 and 193.8 m2·g-1, as compared to that of the activated carbon at 1400 m2·g-1, their sorption capabilities in wastewaters were generally superior. The MIPs with higher surface area and pore volume that formed more non-specific interactions with the targets considerably contributed to the overall removal efficiency, which made them better suited for use in wastewater treatment.

Selective detection of phospholipids using molecularly imprinted fluorescent sensory core-shell particles.

Sphingosine-1-phosphate (S1P) is a bioactive sphingo-lipid with a broad range of activities coupled to its role in G-protein coupled receptor signalling. Monitoring of both intra and extra cellular levels of this lipid is challenging due to its low abundance and lack of robust affinity assays or sensors. We here report on fluorescent sensory core-shell molecularly imprinted polymer (MIP) particles responsive to near physiologically relevant levels of S1P and the S1P receptor modulator fingolimod phosphate (FP) in spiked human serum samples. Imprinting was achieved using the tetrabutylammonium (TBA) salt of FP or phosphatidic acid (DPPA·Na) as templates in combination with a polymerizable nitrobenzoxadiazole (NBD)-urea monomer with the dual role of capturing the phospho-anion and signalling its presence. The monomers were grafted from ca 300 nm RAFT-modified silica core particles using ethyleneglycol dimethacrylate (EGDMA) as crosslinker resulting in 10-20 nm thick shells displaying selective fluorescence response to the targeted lipids S1P and DPPA in aqueous buffered media. Potential use of the sensory particles for monitoring S1P in serum was demonstrated on spiked serum samples, proving a linear range of 18-60 µM and a detection limit of 5.6 µM, a value in the same range as the plasma concentration of the biomarker.

Phospholipid imprinted polymers as selective endotoxin scavengers.

Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of polymerizable bis-imidazolium and urea hosts were evaluated as cationic and neutral hosts for phosphates and phosphonates, the latter used as mimics of the phospholipid head groups. The bis-imidazolium hosts interacted with the guests in a cooperative manner leading to the presence of tight and well defined 1:2 ternary complexes. Optimized monomer combinations were subsequently used for imprinting of phosphatidic acid as an endotoxin dummy template. Presence of the aforementioned ternary complexes during polymerization resulted in imprinting of lipid dimers - the latter believed to crudely mimic the endotoxin Lipid A motif. The polymers were characterized with respect to template rebinding, binding affinity, capacity and common structural properties, leading to the identification of polymers which were thereafter subjected to an industrially validated endotoxin removal test. Two of the polymers were capable of removing endotoxin down to levels well below the accepted threshold (0.005 EU/mg API) in pharmaceutical production.