Sudhirkumar Shinde

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.

Multiplexed MALDI-MS arrays for screening of MIP solid phase extraction materials.

Technology that facilitates rapid investigation of solid phase extraction protocols using very small amounts of sorbent can save both time and money. The microfabricated ISET (Integrated Selective Enrichment Target) interfaced with MALDI mass spectrometry is able to provide an efficient, economic and generic optimization process for SPE sample preparation. The SPE is performed in a rapid and parallel fashion, with a processing time off only 2h per ISET with 96 samples. Each of the 96 wells on the ISET can hold 600nL of SPE sorbent. The ability to work with small amounts of sorbent and samples in the ISET platform provides a big advantage when developing affinity sorbents, such as molecularly imprinted polymers (MIPs). Here it is demonstrated that an amount of 25mg phosphoserine imprinted MIP (pS-MIP) sorbent can allow for analysis of more than 500 ISET nanovials using a multitude of different conditions. In the presented case, the multiplexed experiments allowed for early discovery of unspecific interactions and subsequent minimization of these, resulting in a protocol that provided improved enrichment of phosphopeptides.

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

Abstract 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.

Validation of molecularly imprinted polymers for side chain selective phosphopeptide enrichment

Selective enrichment techniques are essential for mapping of protein posttranslational modifications (PTMs). Phosphorylation is one of the PTMs which continues to be associated with significant analytical challenges. Particularly problematic are tyrosine-phosphorylated peptides (pY-peptides) resulting from tryptic digestion which commonly escape current chemo- or immuno- affinity enrichments and hence remain undetected. We here report on significant improvements in this regard using pY selective molecularly imprinted polymers (pY-MIPs). The pY-MIP was compared with titanium dioxide (TiO2) affinity based enrichment and immunoprecipitation (IP) with respect to selective enrichment from a mixture of 13 standard peptides at different sample loads. At a low sample load (1pmol of each peptide), IP resulted in enrichment of only a triply phosphorylated peptide whereas TiO2 enriched phosphopeptides irrespective of the amino acid side chain. However, with increased sample complexity, TiO2 failed to enrich the doubly phosphorylated peptides. This contrasted with the pY-MIP showing enrichment of all four tyrosine phosphorylated peptides at 1pmol sample load of each peptide with a few other peptides binding unselectively. At an increased sample complexity consisting of the standard peptides spiked into mouse brain digest, the MIP showed clear enrichment of all four pY- peptides.

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.

Reflux precipitation polymerization: a new synthetic insight in molecular imprinting at high temperature

The synthesis of uniform molecularly imprinted polymer (MIP) microspheres (MSs) using distillation precipitation polymerization (DPP) at high temperature has attracted great interest in the field of molecular imprinting. However, there are still some shortcomings in this method. In this work, to create uniform MIP MSs in a short time and to demonstrate the effects of high temperature on imprinting performance, a new precipitation polymerization method (reflux precipitation polymerization, RPP) was used for the first time to fabricate MIP MSs in this study. The SEM images of the polymeric MSs indicate the presence of template molecules could improve the particle morphology and size uniformity. The specific molecular recognition of the monodispersed MIP MSs was confirmed by fluorescence measurement and HPLC-UV analysis. The binding behavior of the MIP MSs was simulated using the heterogeneous Freundlich isotherm, which shows that the MIP MSs produced by the RPP possess compatible selectivity in comparison with those produced by traditional PP method. It is noted that, for the first time, we demonstrated that molecular imprinting at high temperature was only successful when electrostatic interactions played important roles in the imprinting process.

Mesoporous polymeric microspheres with high affinity for phosphorylated biomolecules

Phosphate plays a central role in the environment and in biology. This has led to the demand for a specific phosphate receptor that can be used to sense, enrich or separate phosphate and phosphorylated molecules from complex mixtures. Here, we report an approach for the production of phospho-affinity porous beads with controllable size and pore structure leading to significantly improved chromatographic properties. The beads were prepared by polymerization of bis-imidazolium based host monomer and crosslinker inside the pores of macroporous silica beads that post-etching resulted in mesoporous polymer replicas. The silica precursor, silica–polymer composite and porous polymer replica were characterized by using SEM, IR spectroscopy, optical microscopy and thermogravimetric analysis. Bis-imidazolium functionalized materials were capable of binding and enriching the signaling lipid sphingosine-1-phosphate from plasma samples. Moreover, these engineered materials combined with artificial receptors can recognize phosphorylated amino acids/peptides and are applicable to selective recognition of a broad class of phosphorylated biomolecules.

Ratiometric fluorescence detection of phosphorylated amino acids through excited‐state proton transfer 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.

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.