Novel Glycolipid Chips with a Double Layer of Au Nanoparticles for Biological Toxin Detection.

Glycolipid chips having a double layer of Au nanoparticles are proposed for detection of biological toxins. The sugar-modified chips constitute an under and an upper layer of Au nanoparticles of 20-80 nm diameter on glass plates, and Au nanoparticles of each layer are linked with 1,8-octanedithiol by a self-assembled monolayer (SAM) technique. A tris-sialo glycosphingolipid, ganglioside GT1b, having lipoic amide at the sphingosine part was immobilized on the Au outside surface of the upper layer, and botulinum toxin (type A heavy chain) was detected by localized surface plasmon resonance (LSPR). The GT1b-Cer-coated chip having a double layer of Au nanoparticles enhanced the toxin detection by LSPR more than those with single monolayers. The LSPR response changed according to the sizes of Au nanoparticles in each under and upper layer. The combination of 60 and 40 nm Au nanoparticles in the under and upper layer, respectively, gave the best result, which enabled the toxin detection at concentrations below 5 ng/mL with the portable LSPR device.

1H-NMR Karplus Analysis of Molecular Conformations of Glycerol under Different Solvent Conditions: A Consistent Rotational Isomerism in the Backbone Governed by Glycerol/Water Interactions.

Glycerol is a symmetrical, small biomolecule with high flexibility in molecular conformations. Using a 1H-NMR spectroscopic Karplus analysis in our way, we analyzed a rotational isomerism in the glycero backbone which generates three kinds of staggered conformers, namely gt (gauche-trans), gg (gauche-gauche), and tg (trans-gauche), at each of sn-1,2 and sn-2,3 positions. The Karplus analysis has disclosed that the three rotamers are consistently equilibrated in water keeping the relation of 'gt:gg:tg = 50:30:20 (%)' at a wide range of concentrations (5 mM~540 mM). The observed relation means that glycerol in water favors those symmetric conformers placing 1,2,3-triol groups in a gauche/gauche geometry. We have found also that the rotational isomerism is remarkably changed when the solvent is replaced with DMSO-d6 or dimethylformamide (DMF-d7). In these solvents, glycerol gives a relation of 'gt:gg:tg = 40:30:30 (%)', which means that a remarkable shift occurs in the equilibrium between gt and tg conformers. By this shift, glycerol turns to also take non-symmetric conformers orienting one of the two vicinal diols in an antiperiplanar geometry.

Assembly of Glycochips with Mammalian GSLs Mimetics toward the On-site Detection of Biological Toxins.

According to our previously proposed scheme, each of three kinds of glycosphingolipid (GSL) derivatives, that is, lactosyl ceramide [Lac-Cer (1)] and gangliosides [GM1-Cer (2) and GT1b-Cer (3)], was installed onto the glass surface modified with Au nanoparticles. In the present study, we tried to apply microwave irradiation to promote their installing reactions. Otherwise, this procedure takes a lot of time as long as a conventional self-assembled monolayer (SAM) technique is applied. Using an advanced microwave reactor capable of adjusting ambient temperatures within a desired range, various GSL glycochips were prepared from the derivatives (1)-(3) under different microwave irradiation conditions. The overall assembling process was programed with an IC controller to finish in 1 h, and the derived GSL glycochips were evaluated in the analysis of three kinds of biological toxins [a Ricinus agglutinin (RCA120), botulinum toxin (BTX), and cholera toxin (CTX)] using a localized surface plasmon resonance (LSPR) biosensor. In the LSPR analysis, most of the irradiated GSL chips showed an enhanced response to the targeting toxin when they were irradiated under optimal temperature conditions. Lac-Cer chips showed the highest response to RCA120 (an agglutinin with ß-D-Gal specificity) when the microwave irradiation was conducted at 30-35 °C. Compared to our former Lac-Cer glycochips with the conventional SAM condition, their response was enhanced by 3.6 times. Analogously, GT1b chips gained an approximately 4.1 times enhancement in their response to botulinum type C toxin (BTX/C) when the irradiation was conducted around at 45-60 °C. In the LSPR evaluation of the GM1-Cer glycochips using CTX, an optimal condition also appeared at around 30-35 °C. On the other hand, the microwave irradiation did not lead to a notable increase compared to the former GM1-Cer chips derived with the SAM technique. Judging from these experimental results, the microwave irradiation effectively promotes the installing process for all the three kinds of the GSL derivatives, while the optimal thermal condition becomes different from each other. Many bacterial and botanic proteinous toxins are composed of such carbohydrate binding domains or subunits that can discriminate both the key epitope structure and the dimension of glycoconjugates on the host cell surface. It is assumed that the optimal irradiation and thermal conditions are required to array these semi-synthetic GSL derivatives on the Au nanoparticles in a proper density and geometry for tight adhesion with each of the biological toxins.

Silicon nitride sugar chips for detection of Ricinus communis proteins and Escherichia coli O157 Shiga toxins.

Lactosides having either an amino-triethylene glycol or an azido-triethylene glycol were designed and synthesized, and the two derivatives were immobilized onto silicon nitride (SiN) surfaces. When a click reaction was applied for the immobilization of the azido-sugar, a Ricinus communis lectin (RCA120) was detected with a higher response by reflectometric interference spectroscopy (RIfS). When an N-hydroxysuccinimide (NHS) method was applied for the sugar immobilization, the response was less than that of the click one. The response of bovine serum albumin (BSA) as the negative control was negligible, but the lactose-SiN chip prepared by the click method suppressed nonspecific binding more effectively than did the chip from the NHS method. Next, we examined an antibody-immobilized SiN chip prepared by the click reaction. The detection response was, however, lower than that of the lactose-SiN chip, meaning that the sugar-chip by the click reaction was superior to the antibody-chip. Finally, to detect Shiga toxins from Escherichia coli O157:H7, globotrisaccharide (Gb3) with an azido-triethylene glycol was synthesized and immobilized onto the SiN chip by the click reaction. The Gb3-SiN chips enabled us to detect the toxins at concentrations less than 100 ng/mL. RCA120, horse gram, gorse lectins and BSA showed no response to the Gb3-SiN chip, showing a high specificity for the toxin.

Remarkable functions of sn-3 hydroxy and phosphocholine groups in 1,2-diacyl-sn-glycerolipids to induce clockwise (+)-helicity around the 1,2-diacyl moiety: Evidence from conformation analysis by 1H NMR spectroscopy.

Cell-membrane glycerolipids exhibit a common structural backbone of asymmetric 1,2-diacyl-sn-glycerol bearing polar head groups in the sn-3 position. In this study, the possible effects of sn-3 head groups on the helical conformational property around the 1,2-diacyl moiety in the solution state were examined. 1H NMR Karplus relation studies were carried out using a series of 1,2-dipalmitoyl-sn-glycerols bearing different sn-3 substituents (namely palmitoyl, benzyl, hydrogen, and phosphates). The 1H NMR analysis indicated that the helical property around the 1,2-diacyl moiety is considerably affected by these sn-3 substituents. The sn-3 hydroxy group induced a unique helical property, which was considerably dependent on the solvents used. In CDCl3 solution, three staggered conformers, namely gt(+), gg(-) and tg, were randomized, while in more polar solvents, the gt(+) conformer with (+)-helicity was amplified at the expense of gg(-) and tg conformers. The sn-3 phosphocholine in phosphatidylcholine exhibited a greater effect on the gt(+) conformer, which was independent of the solvents used. From the 1H NMR analysis, the helical conformational properties around the 1,2-diacyl moiety conformed to a simple empirical rule, which permitted the proposal of a conformational diagram for 1,2-dipalmitoyl-sn-glycerols in the solution states.

Thermophilic Talaromyces emersonii Flavin Adenine Dinucleotide-Dependent Glucose Dehydrogenase Bioanode for Biosensor and Biofuel Cell Applications.

Flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) was identified and cloned from thermophilic filamentous fungi Talaromyces emersonii using the homology cloning method. A direct electron transfer bioanode composed of T. emersonii FAD-GDH and a single-walled carbon nanotube was produced. Enzymes from thermophilic microorganisms generally have low activity at ambient temperature; however, the T. emersonii FAD-GDH bioanode exhibits a large anodic current due to the enzymatic reaction (1 mA cm-2) at ambient temperature. Furthermore, the T. emersonii FAD-GDH bioanode worked at 70 °C for 12 h. This is the first report of a bioanode with a glucose-catalyzing enzyme from a thermophilic microorganism that has potential for biosensor and biofuel cell applications. In addition, we demonstrate how the glycoforms of T. emersonii FAD-GDHs expressed by various hosts influence the electrochemical properties of the bioanode.

Bis(ß-lactosyl)-[60]fullerene as novel class of glycolipids useful for the detection and the decontamination of biological toxins of the Ricinus communis family.

Glycosyl-[60]fullerenes were first used as decontaminants against ricin, a lactose recognition proteotoxin in the Ricinus communis family. A fullerene glycoconjugate carrying two lactose units was synthesized by a [3 + 2] cycloaddition reaction between C60 and the azide group in 6-azidohexyl ß-lactoside per-O-acetate. A colloidal aqueous solution with brown color was prepared from deprotected bis(lactosyl)-C60 and was found stable for more than 6 months keeping its red color. Upon mixing with an aqueous solution of Ricinus communis agglutinin (RCA120), the colloidal solution soon caused precipitations, while becoming colorless and transparent. In contrast, a solution of concanavalin A (Con A) caused no apparent change, indicating that the precipitation was caused specifically by carbohydrate-protein interactions. This notable phenomenon was quantified by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results were discussed in terms of detection and decontamination of the deadly biological toxin in the Ricinus communis family.

Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers.

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.

Localized surface plasmon resonance detection of biological toxins using cell surface oligosaccharides on glyco chips.

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (ß-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm ß-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.

Glycotechnology for decontamination of biological agents: a model study using ricin and biotin-tagged synthetic glycopolymers.

Two types of biotin-tagged glycopolymers carrying lactose or glucose in clusters along the polyacrylamide backbone were prepared and subjected to decontamination analyses with the plant toxin ricin. A buffer solution containing the toxin was treated with one glycopolymer followed by streptavidin-magnetic particles. Supernatant solutions were analyzed with surface plasmon resonance and capillary electrophoresis, and revealed that the lactose glycopolymer "captured" this toxin more effectively than the glucose polymer. Free toxin was not detectable in the supernatant after treatment with the glycopolymer and magnetic particles; >99% decontamination was achieved for this potentially fatal biological toxin.

Molecular design, synthesis and bioactivity of glycosyl hydrazine and hydrazone derivatives: notable effects of the sugar moiety.

Assuming that the water solubility of our previous hydrazone derivatives would improve after modification with sugars while keeping or modulating their notable biological activities, we designed and synthesized some glycosyl hydrazine and hydrazone derivatives. Bioassay results indicated that the antitumor activity of our previously prepared hydrazones reduced or disappeared after modification with sugars. On the contrary, some glycosyl derivatives displayed much better antifungal activity against selected fungi. Obviously, a small sugar can change the biological activity of hydrazones significantly.

Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column.

Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin. Lactose, which was immobilized on monolithic silica, was used as a capture ligand for ricin extraction from the sample solution, and the silica was supported in a disk-packed spin column. Recovery of ricin was more than 40%. After extraction, the extract was digested with trypsin and analyzed by LC/MS. The accurate masses of molecular ions and MS/MS spectra of the separated peptide peaks were measured by Fourier transform-MS and linear iontrap-MS, respectively. Six peptides, which were derived from the ricin A-(m/z 537.8, 448.8 and 586.8) and B-chains (m/z 701.3, 647.8 and 616.8), were chosen as marker peptides for the identification of ricin. Among these marker peptides, two peptides were ricin-specific. This method was applied to the determination of ricin from crude samples. The monolithic silica extraction removed most contaminant peaks from the total ion chromatogram of the sample, and the six marker peptides were clearly detected by LC/MS. It takes about 5 h for detection and identification of more than 8 ng/ml of ricin through the whole handling, and this procedure will be able to deal with the terrorism using chemical weapon.

Preparation and evaluation of lactose-modified monoliths for the adsorption and decontamination of plant toxins and lectins.

A series of sugar-modified porous silica monoliths with different sugar ligands (ß-lactoside, ß-N-acetyllactosaminide, ß-d-galactoside, ß-d-N-acetylgalactosaminide and ß-d-glucoside) and linkers were prepared and evaluated using plant toxins and lectins including ricin and a Ricinus communis agglutinin (RCA(120)). Among these sugar monoliths, a lactose monolith carrying a triethylene glycol spacer adsorbed ricin and RCA(120) with the highest efficiency. The monolith showed no binding with albumin, globulin, and lectins from Jack beans, Osage orange, Amur maackia and wheat germ. All these data support the utility of the lactose-modified monolith as a tool for adsorption and decontamination of plant toxins.

Use of lactose against the deadly biological toxin ricin.

Developing a technology for detecting and decontaminating biological toxins is needed. Ricin from Ricinus communis is a highly poisonous toxin; it was formerly used for an assassination in London and in postal attacks in the United States. Ricin is readily available from castor beans and could be used as a biological agent. We propose using glycotechnology against the illegal use of ricin. Lactose (a natural ligand of this toxin) was incorporated into polyacrylamide-based glycopolymers at variable sugar densities (18-100%) and evaluated with surface plasmon resonance (SPR) spectroscopy and the real agent, ricin. Glycopolymers (18-65% lactose densities) effectively interfered with the toxin-lactoside adhesion event (>99% efficiency within 20 min). This supported the notion of using the mammary sugar lactose against a deadly biological toxin.

Library assembly of mono-, di- and tri-O-sulfated beta-D-xylopyranosides; effect of O-sulfation on pyranose ring conformation.

With molluscan sulfatase-catalyzed de-O-sulfation reactions, a series of mono-, di- and tri-O-sulfated p-nitrophenyl beta-D-xylopyranosides were assembled and applied to a 1H NMR study to examine the effect of O-sulfate groups on the equilibration between pyranose 4C1 and 1C4 conformations.

Multivalent galacto-trehaloses: design, synthesis, and biological evaluation under the concept of carbohydrate modules.

Galacto-trehalose (GT) is a novel class of 1,1'-linked nonreducing disaccharide having an α-galactoside epitope. In this study, a pair of α,α- and α,ß-GT isomers were prepared in one pot with our α-glycosylation method, converted into vinyl monomers and then subjected to radical copolymerization with a second sugar (4-acrylamidophemyl ß-Glc or ß-GlcNAc) in the presence of acrylamide. The derived glycopolymers were assayed with α-galactoside-specific proteins (BSI-B(4) lectin and Shiga toxin-1) to show the results that both α,α- and α,ß-isomers are recognized by these carbohydrate-binding proteins more strongly in forms of the GT polymers. Moreover, the glycopolymer carrying both α,α-GT and ß-GlcNAc along the polymer chain showed an integrated detoxifying activity to the E. coli toxin as the result of a "module effect" of the second sugar.

[Highly sensitive detection technology for biological toxins applying sugar epitopes].

The Shiga toxin is a highly poisonous protein produced by enterohemorrhagic Escherichia coli O157. This bacterial toxin causes the hemolytic uremic syndrome. Another plant toxin from castor beans, ricin, is also highly toxic. The toxin was used for assassination in London. Recently, there were several cases of postal matter containing ricin. Both toxins are categorized as biological warfare agents by the Centers of Disease Control and Prevention. Conventional detection methods based on the antigen-antibody reaction, PCR and other cell-free assays have been proposed. However, those approaches have drawbacks in terms of sensitivity, analytical time, or stability of the detection reagents. Therefore, development of a facile and sensitive detection method is essential. Here we describe new detection methods applying carbohydrate epitopes as the toxin ligands, which is based on the fact that the toxins bind cell-surface oligosaccharides. Namely, the Shiga toxin has an affinity for globobiosyl (Gb(2)) disaccharide, and ricin binds the beta-D-galactose residue. For Shiga toxin detection, surface plasmon resonance (SPR) was applied. A polyanionic Gb(2)-glycopolymer was designed for this purpose, and it was used for the assembly of Gb(2)-chips using alternating layer-by-layer technology. The method allowed us to detect the toxin at a low concentration of LD(50). A synthetic carbohydrate ligand for ricin was designed and immobilized on the chips. SPR analysis with the chips allows us to detect ricin in a highly sensitive and facile manner (10 pg/ml, 5 min). Our present approaches provide a highly effective way to counter bioterrorism.

A novel sugar-probe biosensor for the deadly plant proteinous toxin, ricin.

Because of the illegal use of highly toxic ricin from the castor-oil plant, Ricinus communis, in bioterrorism and suspected white powder cases, anti-terrorism measures for the toxin are urgently required. Here we demonstrate a facile and sensitive detection method using synthetic analogues of beta-lactosyl- and beta-d-galactosyl ceramides as the ligands based on the fact that ricin binds cell-surface oligosaccharides. Sugar-probes having lipoic acids as anchor functions were synthesized via either a chemical or chemoenzymatic way and were immobilized on the sensor chips by a self-assembled monolayer technique. Surface plasmon resonance (SPR) analysis using these carbohydrate probes allowed us to detect the toxin in a highly sensitive and facile manner (10 pg/mL, 5 min), being the best benchmark as a method for detecting the toxin. In addition, a visual monitoring method was developed, in which sugar-coated Au nanoparticles were utilized for discriminating ricin from other proteins in a facile manner, taking 10-30 min for judgment.

Glycochips from polyanionic glycopolymers as tools for detecting Shiga toxins.

An alternating layer-by-layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb(2)), beta-lactoside (beta-Lac), or alpha-D-mannoside (alpha-Man) residues were prepared, and used for the detection of Shiga toxins, Stx-1 and Stx-2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx-1 and Stx-2 show binding specificity for the Gb(2) glycochip as well as a weak affinity for the beta-Lac glycochip. The affinity constants of these toxins depended strongly on the sugar content of the Gb(2) polymer used to prepare the glycochip. Greater affinity was observed for chips with a higher sugar content (up to 43 %) in the Gb(2) glycopolymer. The maximal affinity constants of Stx-1 and Stx-2 (K(a)=10(8)-10(9) M(-1)) enabled highly sensitive and facile analysis (10 ng mL(-1), 30 min). When Gb(2) glycopolymers were used as competitors, Stx-1 and Stx-2 behaved differently from one another in terms of their SPR response; this allowed us to perform discriminative analysis between the two toxins.

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction.

When alpha-D-GlcNAc-OC(6)H(4)NO(2) -p and beta-D-(6-sulfo)-GlcNAc-OC(6)H(4)NO(2)-p (2) were used as substrates, beta-N-acetylhexosaminidase from Aspergillus oryzae transferred the beta-D-(6-sulfo)-GlcNAc(unit from 2 to alpha-D-GlcNAc-OC(6)H(4)NO(2) -p to afford beta-D-(6-sulfo)-GlcNAc-(1-->4)-alpha-D-GlcNAc-OC(6)H(4)NO(2)-p (3) in a yield of 94% based on the amount of donor, 2, added. beta-D-(6-sulfo)-GlcNAc-(1-->4)-alpha-D-Glc-OC(6)H(4)NO(2)-p (4) was obtained with alpha-D-Glc-OC(6)H(4)NO(2) -p as acceptor in a similar manner. With a reaction mixture of 2 and beta-D-GlcNAc-OC(6)H(4)NO(2)-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of beta-D-GlcNAc from 1 to 2, affording disaccharide beta-D-GlcNAc-(1-->4)-beta-(6-sulfo)-D-GlcNAc-OC(6)H(4)NO(2)-p (5) in a yield of 13% based on the amount of 1 added.