Surface-enhanced Raman spectroscopic-encoded beads for multiplex immunoassay

A new type of encoded bead, which uses surface-enhanced Raman scattering (SERS), is described for multiplex immunoassays. Silver nanoparticles were embedded in sulfonated polystyrene (PS) beads via a polyol method, and they were used as SERS-active substrates. Raman-label organic compounds such as 4-methylbenzenethiol (4-MT), 2-naphthalenethiol (2-NT), and benzenethiol (BT) were then adsorbed onto the silver nanoparticles in the sulfonated PS bead. Although only three kinds of encoding have been demonstrated here, various combinations of these Raman-label organic compounds have the potential to give a large number of tags. The Raman-label-incorporated particles were then coated with a silica shell using tetraethoxyorthosilicate (TEOS) for chemical stability and biocompatibility. The resulting beads showed unique and intense Raman signals for the labeled organic compounds. We demonstrated that SERS-encoded beads could be used for multiplex detection with a model using streptavidin and p53. In our system, the binding event of target molecules and the type of ligand can be simultaneously recognized by Raman spectroscopy using a single laser-line excitation (514.5 nm).     

Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.     

Mild,Selective Oxidation of Aromatic Alcohols Using β-Cyclodextrin-Functionalized Glass Microparticles: Characterization,Stability, and Application

The surface of glass microparticle (GMP) was functionalized with β-cyclodextrin (→ GMP-β-CD) and was characterized by x-ray photoelectron spectroscopy (XPS). GMP-β-CD was used to catalyze oxidation of alcohols into aldehydes and katones with excellent yield (86–92%). The modified surface of GMP-β-CD showed no change or degradation after repeated use as confirmed from XPS analysis after 10 cycles.

[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]     

Liposome solubilization induced by complexation with dimeric β-cyclodextrin

Dimeric β-cyclodextrins (β-CD) were prepared from the reaction of native β-CD with epichlorohydrin under basic conditions, and the effects on the diacetylene (DA) and polydiacetylene (PDA) liposomes have been investigated. Vesicular DA was solubilized in the presence of dimeric β-CD with the consequent inhibition of polymerization. The result is attributed to the formation of a complex between dimeric β-CD and DA liposomes, and it is clearly differentiated from that of monomeric β-CD. Furthermore, the ordered supramolecular structure of PDA was perturbed by the dimeric β-CD, which was detected from the visible color change. Finally, the morphological characteristics and size of PDA in the absence and presence of dimeric β-CD were examined using transmission electron microscopy and dynamic light scattering The results show fused structure of size more than 200 nm along with the deformation of the vesicles, and they represent a novel phenomenon of liposome structure induced by complexation with dimeric β-CD. The evaluated physicochemical characteristics can be applied to the development of carbohydrate-based detergents.     

Magnetic field induced aggregation of nanoparticles for sensitive molecular detection

A molecular detection method utilizing the magnetically induced aggregation of silver nanoparticle (NP)-embedded silica NPs for SERS activation is described. Here, silver embedded magnetic NPs (Ag-M-dots) composed of a magnetic core and silica shells, on whose surface silver NPs were formed, were used. Because the magnetic field induced aggregated Ag-M-dots exhibit a strong SERS signal compared to the dispersed Ag-M-dots, the system allows for the detection of adsorbed Raman label compound even at the 100 fM level. Adenine was tested as a model biocompound and its Raman spectrum could be observed at concentrations as low as 1 pM. The experimental results were supported by the theoretical calculations.     

Encoding peptide sequences with surface-enhanced Raman spectroscopic nanoparticles

Peptides synthesized on microbeads were encoded by chemically and physically adsorbing surface-enhanced Raman spectroscopic nanoparticles (SERS dots) on the microbead surface during the synthesis, which could be easily and rapidly decoded by Raman spectroscopy.     

Fluorescence-based multiplex protein detection using optically encoded microbeads

Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.     

Synthesis of photolabile o-nitroveratryloxycarbonyl (NVOC) protected peptide nucleic acid monomers

The chemical synthesis of peptide nucleic acid (PNA) monomers was accomplished using various combinations of the o-nitroveratryloxycarbonyl (NVOC) group (N-aminoethylglycine backbone) and base labile acyl-type nucleobase protecting groups (anisoyl for adenine and cytosine; isobutyryl for guanine), thus offering a photolithographic solid-phase PNA synthetic strategy compatible with photolithographic oligonucleotide synthesis conditions and allowing the in situ synthesis of PNA microarrays in an essentially neutral medium, by avoiding the use of the commonly used deprotection reagents such as trifluoroacetic acid or piperidine. Convenient methods were also explored to prepare 1-(carboxymethyl)-4-N-(4-methoxybenzoyl)cytosine and 9-(carboxymethyl)-2-N-(isobutyryl)guanine with good yields.     

STEP signaling pathway mediates psychomotor stimulation and morphine withdrawal symptoms,but not for reward,analgesia and tolerance

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP^C230X−/− mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP^C230X−/− mice displayed reduced hyperlocomotion in response to intrastriatal injection of the μ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.     

Preparation of polydiacetylene immobilized optically encoded beads

Polydiacetylene (PDA), which can change the chromic and fluorescence properties by inducing environmental perturbations, is immobilized on planar solid supports for many biological applications. In this work, we immobilize PDA onto optically encoded spherical beads (PDA-SERS beads). The prepared PDA immobilized beads (36 μm) exhibit a blue color without fluorescence. By inducing stress, their color and fluorescence properties are changed to red with fluorescence. The SERS spectra of the PDA-SERS beads can be recognized over the PDA background. Moreover, our PDA immobilization methods are successfully applied to silica-surface SERS-encoded beads (5 μm) and proven to also be useful in fluorescence encoding systems.