An Effective Polymer Cross-Linking Strategy To Obtain Stable Dispersions of Upconverting NaYF₄ Nanoparticles in Buffers and Biological Growth Media for Biolabeling Applications

Ligands on the nanoparticle surface provide steric stabilization, resulting in good dispersion stability. However, because of their highly dynamic nature, they can be replaced irreversibly in buffers and biological medium, leading to poor colloidal stability. To overcome this, we report a simple and effective cross-linking methodology to transfer oleate-stabilized upconverting NaYF₄ core/shell nanoparticles (UCNPs) from hydrophobic to aqueous phase, with long-term dispersion stability in buffers and biological medium. Amphiphilic poly­(maleic anhydride-<i>alt</i>-1-octadecene) (PMAO) modified with and without poly­(ethylene glycol) (PEG) was used to intercalate with the surface oleates, enabling the transfer of the UCNPs to water. The PMAO units on the phase transferred UCNPs were then successfully cross-linked using bis­(hexamethylene)­triamine (BHMT). The primary advantage of cross-linking of PMAO by BHMT is that it improves the stability of the UCNPs in water, physiological saline buffers, and biological growth media and in a wide range of pH values when compared to un-cross-linked PMAO. The cross-linked PMAO–BHMT coated UCNPs were found to be stable in water for more than 2 months and in physiological saline buffers for weeks, substantiating the effectiveness of cross-linking in providing high dispersion stability. The PMAO–BHMT cross-linked UCNPs were extensively characterized using various techniques providing supporting evidence for the cross-linking process. These UCNPs were found to be stable in serum supplemented growth medium (37 °C) for more than 2 days. Utilizing this, we demonstrate the uptake of cross-linked UCNPs by LNCaP cells (human prostate cancer cell line), showing their utility as biolabels

Primary sequence alignment of <i>T</i>. <i>congolense</i> calflagin with those of <i>T</i>. <i>cruzi</i> FCaBP and <i>T</i>. <i>brucei</i> Tb24.

<p>The secondary structural elements (α-helices and β-strands) are depicted as cones and arrows, respectively, and were derived from the I-TASSER based structure prediction for <i>T</i>. <i>congolense</i> calflagin, the x-ray crystal structure of <i>T</i>. <i>cruzi</i> FCaBP (3CS1) and the NMR data for <i>T</i>. <i>brucei</i> Tb24 (2LVV). The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted in green, salmon, cyan, and yellow, respectively. Residues in the 12-residue Ca²⁺ binding loops at position 1, 3, 5 and 12 are underlined. Invariant basic residues on the protein surface that are associated with membrane binding are colored blue. Non-conserved surface-exposed residues are highlighted using bold print.</p

Multiple sequence alignment of the <i>T</i>. <i>congolense</i> calflagins.

<p>The positions of the two MS-identified tryptic peptides identified by mass spectrometry are highlighted in yellow and red boxes. The lower case, white highlighted v represent amino acid (valine-isoleucine) differences in two of the ORFs.</p

Structural characterization and surface analysis of <i>T</i>. <i>congolense</i> calflagin Modeled <i>T</i>. <i>congolense</i> calflagin (middle panel) was compared to the crystal structure of <i>T</i>. <i>cruzi</i> FCaBP (left vertical column) and to the NMR structure of <i>T</i>. <i>brucei</i> Tb24 (right vertical column).

<p>(A) the predicted model of <i>T</i>. <i>congolense</i> calflagin (middle vertical column) exhibits four EF-hands motifs. The EF-hands (EF1, EF2, EF3 and EF4) are colored green, salmon, cyan, yellow, accordingly. Left panel: structural alignment of <i>T</i>. <i>congolense</i> (grey) over <i>T</i>. <i>cruzi</i> FCaBP (green). Right panel: <i>T</i>. <i>congolense</i> calflagin structure aligned over <i>T</i>. <i>brucei</i> Tb24 (magenta). (B) Surface representation of the three calflagin models overlapping panel A, showing exposed hydrophobic (gray), basic (blue) and acidic (red) respectively. (C) 180° rotation of B in the <i>y-axis</i>. Black arrows pointing towards putative epitopes and numbered according to their respective α-helix location.</p

MALDI-TOF mass spectrum of the trypsin-digested ~26 kDa gel band recognized by mAb Tc6/42.6.4.

<p>The peak at 1227.72 m/z corresponds to the peptide VLQMHELTTR and the peak at 1457.51 m/z corresponds to the peptide LSFNEVCSGCER (with two carbamidomethylations).</p

Calflagin-based serodiagnosis of trypanosome infections in Ugandan cattle.

<p>(A) ELISA signal intensities resulting from bovine IgG and IgM antibodies binding to solid-phase adsorbed recombiant <i>T</i>. <i>congolense</i> calflagin. All values were normalized against signals elicited from plasma of laboratory raised, trypanosome negative calves. (B) ROC curves generated from the serodiagnostic ELISA results. Areas under curve for IgG and IgM were 0.623 and 0.709 respectively. For reference, an area under the curve of 1.00 equates to a test with 100% sensitivity and specificity, whereas an area under the curve of 0.500 indicates that a test has no value in differentiating between the binary population.</p

Confocal immunofluorescence microscopy showing localization of <i>T</i>. <i>congolense</i> calflagin in the presence and absence of calcium.

<p>Maximum intensity projections of fixed <i>T</i>. <i>congolense</i> PCF were probed with mAb Tc6/42.6.4 and anti-para-flagellar rod protein in the presence (A, B, and C) and absence (D, E, and F) of calcium. Green: calflagin; Red: para-flagellar rod protein; Blue: DAPI/DNA.</p

Immunoblot detection of antigen in the four major life cycle stages of <i>T</i>. <i>congolense</i> IL3000 using mAb Tc6/42.6.4.

<p>BSF: bloodstream forms; PCF: procyclic culture forms; EMF: epimastigote forms; MCF: metacyclic forms. Modified with permission from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004510#pntd.0004510.ref001" target="_blank">1</a>].</p

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation.

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.</p