Advances in endotoxin analysis.

The outer membrane of gram-negative bacteria is primarily composed of lipopolysaccharide (LPS). In addition to protection, LPS defines the distinct serogroups used to identify bacteria specifically. Furthermore, LPS also act as highly potent stimulators of innate immune cells, a phenomenon essential to understanding pathogen invasion in the body. The complex multi-step process of LPS binding to cells involves several binding partners, including LPS binding protein (LBP), CD14 in both membrane-bound and soluble forms, membrane protein MD-2, and toll-like receptor 4 (TLR4). Once these pathways are activated, pro-inflammatory cytokines are eventually expressed. These binding events are also affected by the presence of monomeric or aggregated LPS. Traditional techniques to detect LPS include the rabbit pyrogen test, the monocyte activation test and Limulus-based tests. Modern approaches are based on protein, antibodies or aptamer binding. Recently, novel techniques including electrochemical methods, HPLC, quartz crystal microbalance (QCM), and molecular imprinting have been developed. These approaches often use nanomaterials such as gold nanoparticles, quantum dots, nanotubes, and magnetic nanoparticles. This chapter reviews current developments in endotoxin detection with a focus on modern novel techniques that use various sensing components, ranging from natural biomolecules to synthetic materials. Highly integrated and miniaturized commercial endotoxin detection devices offer a variety of options as the scientific and technologic revolution proceeds.

Chemical Synthesis of Human Milk Oligosaccharides: para-Lacto-N-hexaose and para-Lacto-N-neohexaose.

Human milk oligosaccharides (HMO) have emerged as a very active area of research in glycoscience and nutrition. HMO are involved in the early development of infants and may help to prevent certain diseases. The development of chemical methods for obtaining individual HMO aids the global effort dedicated to understanding the roles of these biomolecules. Reported herein is the chemical synthesis of two common core hexasaccharides found in human milk, i. e. para-lacto-N-hexaose (pLNH) and para-lacto-N-neohexaose (pLNnH). After screening multiple leaving groups and temporary protecting group combinations, a 3+3 convergent coupling strategy was found to work best for obtaining these linear glycans.

Atomic force microscopy study of the complexation of sterols and the glycoalkaloid α-tomatine in Langmuir-Blodgett monolayers.

Glycoalkaloids are secondary metabolites produced by plants that aid in their protection from pathogens and pests. They are known to form 1:1 complexes with 3ß-hydroxysterols such as cholesterol causing membrane disruption. So far, the visual evidence showcasing the complexes formed between glycoalkaloids and sterols in monolayers has been mainly restricted to some earlier studies using Brewster angle microscopy which were of low resolution showing the formation of floating aggregates of these complexes. This study is aimed at using atomic force microscopy (AFM) for topographic and morphological analysis of the aggregates of these sterol-glycoalkaloid complexes. Langmuir-Blodgett (LB) transfer of mixed monolayers of the glycoalkaloid α-tomatine, sterols, and lipids in varying molar ratios onto mica followed by AFM examination was performed. The AFM method allowed visualization of the aggregation of sterol-glycoalkaloid complexes at nanometer resolution. While aggregation was observed in mixed monolayers of α-tomatine with cholesterol and in mixed monolayers with coprostanol, no sign of complexation was observed for the mixed monolayers of epicholesterol and α-tomatine, confirming their lack of interaction found in prior monolayer studies. Aggregates were observed in transferred monolayers of ternary mixtures of α-tomatine with cholesterol and the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or egg sphingomyelin (egg SM). The formation of aggregates was found to be less prevalent for mixed monolayers of DMPC and cholesterol containing α-tomatine than it was for mixed monolayers containing egg SM and cholesterol with α-tomatine. The observed aggregates were generally elongated structures, of a width ranging from about 40-70 nm.

Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold.

The potential to generate variable pore sizes, simplistic surface modification, and a breadth of commercial uses in the fields of biosensors, actuators, drug loading and release, and the development of catalysts have unquestionably accelerated the usage of nanoporous gold (NPG)-based nanomaterials in research and development. This article describes the process of the generation of hierarchical bimodal nanoporous gold (hb-NPG) by employing a step-wise procedure involving electrochemical alloying, chemical dealloying techniques, and annealing to create both macro- and mesopores. This is done to improve the utility of NPG by creating a bicontinuous solid/void morphology. The area available for surface modification is enhanced by smaller pores, while molecular transport benefits from the network of larger pores. The bimodal architecture, which is the result of a series of fabrication steps, is visualized using scanning electron microscopy (SEM) as a network of pores that are less than 100 nm in size and connected by ligaments to larger pores that are several hundred nanometers in size. The electrochemically active surface area of the hb-NPG is assessed using cyclic voltammetry (CV), with a focus on the critical roles that both dealloying and annealing play in creating the necessary structure. The adsorption of different proteins is measured by solution depletion technique, revealing the better performance of hb-NPG in terms of protein loading. By changing the surface area to volume ratio, the created hb-NPG electrode offers tremendous potential for biosensor development. The manuscript discusses a scalable method to create hb-NPG surface structures, as they offer a large surface area for the immobilization of small molecules and improved transport pathways for faster reactions.

Facile fabrication of hierarchically nanostructured gold electrode for bio-electrochemical applications.

Nanoporous gold (NPG) is one of the most extensively investigated nanomaterials owing to its tunable pore size, ease of surface modification, and range of applications from catalysis, actuation, and molecular release to the development of electrochemical sensors. In an effort to improve the usefulness of NPG, a simple and robust method for the fabrication of hierarchical and bimodal nanoporous gold electrodes (hb-NPG) containing both macro-and mesopores is reported using electrochemical alloying and dealloying processes to engineer a bicontinuous solid/void morphology. Scanning electron microscopy (color SEM) images depict the hierarchical pore structure created after the multistep synthesis with an ensemble of tiny pores below 100 nm in size located in ligaments spanning larger pores of several hundred nanometers. Smaller-sized pores are exploited for surface modification, and the network of larger pores aids in molecular transport. Cyclic voltammetry (CV) was used to compare the electrochemically active surface area of the hierarchical bimodal structure with that of the regular unimodal NPG with an emphasis on the critical role of both dealloying and annealing in creating the desired structure. The adsorption of different proteins was followed using UV-vis absorbance measurements of solution depletion revealing the high loading capacity of hb-NPG. The surface coverage of lipoic acid on the hb-NPG was analyzed using thermogravimetric analysis (TGA) and reductive desorption. The roughness factor determinations suggest that the fabricated hb-NPG electrode has tremendous potential for biosensor development by changing the scaling relations between volume and surface area which may lead to improved analytical performance. We have chosen to take advantage of the surface architectures of hb-NPG due to the presence of a large specific surface area for functionalization and rapid transport pathways for faster response. It is shown that the hb-NPG electrode has a higher sensitivity for the amperometric detection of glucose than does an NPG electrode of the same geometric surface area.

N-Alkylated Analogues of Indolylthio Glycosides as Glycosyl Donors with Enhanced Activation Profile.

While studying indolylthio glycosides, previously we determined their activation profile that required large excess of activators. This drawback was partially addressed in the present study of N-alkylated SInR derivatives. The activation process was studied by NMR and the increased understanding of the mechanism led to a discovery of different activation pathways taking place with SIn versus SInR derivatives. Also investigated was orthogonality of the SInR leaving groups versus thioglycosides and selective activation of thioimidates over SInR glycosides.

Expanding the scope of stereoselective α-galactosylation using glycosyl chlorides.

Recently, we reported that silver(I) oxide mediated Koenigs-Knorr glycosylation reaction can be dramatically accelerated in the presence of catalytic acid additives. We have also investigated how well this reaction works in application to differentially protected galactosyl bromides. Reported herein is the stereoselective synthesis of α-galactosides with galactosyl chlorides as glycosyl donors. Chlorides are easily accessible, stable, and can be efficiently activated for glycosylation. In this application, the most favorable reactions conditions comprised cooperative Ag2SO4 and Bi(OTf)3 promoter system.

HPLC-Based Automated Synthesis of Glycans in Solution.

As the 21st century unfolds with rapid changes, new challenges in research and development emerge. These new challenges prompted us to repurpose our HPLC-A platform that was previously used in solid phase glycan synthesis to a solution phase batch synthesis described herein. The modular character of HPLC allows for implementing new attachments. To enable sequential synthesis of multiple oligosaccharides with the single press of a button, we supplemented our system with a four-way split valve and an automated fraction collector. This enabled the operator to load all reagents and all reactants in the autosampler, press the button to start the repetitive automation sequence, leave the lab, and upon return find products of multiple reactions ready for purification, analysis, and subsequent application.

Streamlined access to carbohydrate building blocks: Methyl 2,4,6-tri-O-benzyl-α-d-glucopyranoside.

Presented herein is an improved synthesis of a common 3-OH glycosyl acceptor. This compound is a building block that is routinely synthesized by many research groups to be used in glycosylation refinement studies. The only known direct synthesis by Koto lacks regioselectivity and relies on chromatography separation using hazardous solvents. Our improved synthetic approach relies on Koto's selective benzylation protocol, but it is followed by acylation-purification-deacylation sequence. Although this approach involves additional manipulations, it provides consistent results and is superior to other indirect strategies. Also obtained, albeit in minor quantities, is 4-OH acceptor, another common building block.

Nanoporous Gold Monolith for High Loading of Unmodified Doxorubicin and Sustained Co-Release of Doxorubicin-Rapamycin.

Nanoparticles (NPs) have been widely explored for delivering doxorubicin (DOX), an anticancer drug, to minimize cardiotoxicity. However, their efficiency is marred by a necessity to chemically modify DOX, NPs, or both and low deposition of the administered NPs on tumors. Therefore, alternative strategies should be developed to improve therapeutic efficacy and decrease toxicity. Here we report the possibility of employing a monolithic nanoporous gold (np-Au) rod as an implant for delivering DOX. The np-Au has very high DOX encapsulation efficiency (>98%) with maximum loading of 93.4 mg cm-3 without any chemical modification required of DOX or np-Au. We provide a plausible mechanism for the high loading of DOX in np-Au. The DOX sustained release for 26 days from np-Au in different pH conditions at 37 °C, which was monitored using UV-Vis spectroscopy. Additionally, we encased the DOX-loaded np-Au with rapamycin (RAPA)-trapped poly(D,L-lactide-co-glycolide) (PLGA) to fabricate an np-Au@PLGA/RAPA implant and optimized the combinatorial release of DOX and RAPA. Further exploiting the effect of the protein corona around np-Au and np-Au@PLGA/RAPA showed zero-order release kinetics of DOX. This work proves that the np-Au-based implant has the potential to be used as a DOX carrier of potential use in cancer treatment.

A Streamlined Regenerative Glycosylation Reaction: Direct, Acid-Free Activation of Thioglycosides.

Our group has previously reported that 3,3-difluoroxindole (HOFox) is able to mediate glycosylations via intermediacy of OFox imidates. Thioglycoside precursors were first converted into the corresponding glycosyl bromides that were then converted into the OFox imidates in the presence of Ag2 O followed by the activation with catalytic Lewis acid in a regenerative fashion. Reported herein is a direct conversion of thioglycosides via the regenerative approach that bypasses the intermediacy of bromides and eliminates the need for heavy-metal-based promoters. The direct regenerative activation of thioglycosides is achieved under neutral reaction conditions using only 1 equiv. NIS and catalytic HOFox without the acidic additives.

Recent Advances in the Application of Glycan-Modified Self-Assembled Monolayers.

Glycans have many important roles in human health and disease in processes such as infection, fertilization, cellular development, cellular adhesion, cancer metastasis and immune system response. The presentation of glycan structures on surfaces for screening of their interaction with protein binding partners, interactions with individual cells, and development of bioassays is an actively developing field. Self-assembled monolayers (SAMs) of glycan terminated alkanethiols on gold have found application in many of these areas. Additionally, more complex structures such as glycan modified polymers on gold surfaces have provided new routes for multivalent glycan presentation. Glycans have also been conjugated to monolayers formed on other useful substrates such as glass or silicon wafers. SAMs have been formed both by direct immobilization of glycan terminated alkanethiols and by conjugation of glycans to pre-formed SAMs with reactive terminal groups. The structure of the SAMs has been characterized using a range of methods including surface spectroscopy, scanning probe microscopy, and electrochemical methods. The binding of proteins to these SAMs has been followed using methods including surface plasmon resonance and electrochemical techniques such as impedance spectroscopy. In this chapter, we will seek to review the recent literature concerning SAMs containing terminal glycans, with a focus on their biomolecular interactions. The applications of these glycan-modified SAMs to the screening and study of protein and cellular binding and to biosensor and assay development will be reviewed.

A pH sensitive thiolated ß-cyclodextrin-modified nanoporous gold for controlled release of doxorubicin.

This article reports a novel thiolated ß-cyclodextrin (HS-ß-CD) modified nanoporous gold (NPG) wire for pH sensitive delivery of doxorubicin (DOX) in controlled manner. Nanoporous gold is a versatile material because of its three-dimensional nanoscale network of pores, facile surface functionalization, biocompatibility, and high capacity for the DOX payload. HS-ß-CD can form supramolecular inclusion complexes with DOX affording the possibility of altering the controlled release behavior. DOX is one of the most potent anti-tumor drugs used in the treatment of different cancers. The binding of HS-ß-CD and DOX was examined using UV-Vis spectroscopy. The prepared NPG structure exhibited excellent properties for controlled drug release outlining the potential of a pH sensitive drug implant for biomedical applications. This delivery system could improve local targeting of the drug as well as alter the rate of release of DOX near tumors.

The development of a dedicated polymer support for the solid-phase oligosaccharide synthesis.

Reported herein is the development of a novel polystyrene-based resin that we named PanzaGel. The resin was equipped with diethylene glycol-derived cross-linker with the dedicated application to polymer supported glycan synthesis in mind. After investigating its swelling properties and obtaining encouraging data for its chemical and thermal stability we accessed the amenability of PanzaGel to the HPLC-based platform for the automated synthesis. Comparable glycosylation results to those with traditional supports have been obtained in the synthesis of glycans up to pentasaccharide that was obtained in 30% overall yield. The automated synthesis set-up implemented a common analytical autosampler for delivering all reagents for all steps of the glycan synthesis and cleavage.

Adhesion layer-free attachment of gold on silicon wafer and its application in localized surface plasmon resonance-based biosensing.

The use of a metallic adhesion layer between plasmonic-active nanostructures and a solid supported is known to dampen the plasmonic response. To overcome this problem, organic adhesion layers have been introduced, which in turn can undermine the stability of the film. Moreover, both types of layers limit the regeneration of the nanostructures for multiple uses. Here we report a quick and simple approach to prepare intermediate adhesion layer-free binding of nanostructured films of gold on silicon wafers. The approach involves scratching and etching of the silicon wafer before sputter coating with a thin layer of Au. The plasmonic-active nanostructures were then prepared on this thin Au film using electrochemical deposition. As-prepared plasmonic-active nanostructured thin films of gold (PANTF-Au) are easy to handle, physically robust, and can be regenerated. The bulk refractive index sensitivity of PANTF-Au is 150 nm/RIU with the figure of merit 1.4, and with a plasmonic field-decay length of 27 nm. We further used these thin films to study interactions between lectin and glycoprotein inside a flow cell as well as on a microplate made of PANTF-Au. The PANTF-Au can be easily integrated with electrochemical devices and microfluidics, which can help to pave the way toward the development of ideal optical-electrochemical point-of-care biosensors.

Chemical synthesis of human milk oligosaccharides: lacto-N-neohexaose (Galß1 → 4GlcNAcß1→)2 3,6Galß1 → 4Glc.

The first chemical synthesis of lacto-N-neohexaose (LNnH) has been completed using a convergent synthetic strategy. The reaction conditions and donor-acceptor combinations have been carefully refined to minimize side reactions and achieve high yields in all glycosylation steps. Lacto-N-neotetraose, another common human milk oligosaccharide, was also synthesized en route to the target LNnH.

HPLC-assisted automated oligosaccharide synthesis: the implementation of the two-way split valve as a mode of complete automation.

Reported herein is the development of a user-friendly platform for simple and transformative automation based on standard HPLC equipment. We showcase how the improved platform works in application to the completely automated, a "press of the button," synthesis of various glycan sequences.

Nanomaterials for Biosensing Lipopolysaccharide.

Lipopolysaccharides (LPS) are endotoxins, hazardous and toxic inflammatory stimulators released from the outer membrane of Gram-negative bacteria, and are the major cause of septic shock giving rise to millions of fatal illnesses worldwide. There is an urgent need to identify and detect these molecules selectively and rapidly. Pathogen detection has been done by traditional as well as biosensor-based methods. Nanomaterial based biosensors can assist in achieving these goals and have tremendous potential. The biosensing techniques developed are low-cost, easy to operate, and give a fast response. Due to extremely small size, large surface area, and scope for surface modification, nanomaterials have been used to target various biomolecules, including LPS. The sensing mechanism can be quite complex and involves the transformation of chemical interactions into amplified physical signals. Many different sorts of nanomaterials such as metal nanomaterials, magnetic nanomaterials, quantum dots, and others have been used for biosensing of LPS and have shown attractive results. This review considers the recent developments in the application of nanomaterials in sensing of LPS with emphasis given mainly to electrochemical and optical sensing.

Chemical Synthesis of Human Milk Oligosaccharides: Lacto-N-hexaose Galß1→3GlcNAcß1→3 [Galß1→4GlcNAcß1→6] Galß1→4Glc.

The first synthesis of lacto-N-hexaose (LNH) has been completed using a convergent strategy. The donor-acceptor protecting-leaving group combinations were found to be of paramount significance for achieving successful glycosylations and minimizing side reactions. Lacto-N-tetraose, another common human milk oligosaccharide, was also obtained en route to the target LNH.

The chemical synthesis of human milk oligosaccharides: Lacto-N-tetraose (Galß1→3GlcNAcß1→3Galß1→4Glc).

The total chemical synthesis of lacto-N-tetraose (LNT) has been completed using both convergent and linear strategies. Similarly to that of our previous HMO syntheses, the donor-acceptor protecting-leaving group combinations were found to be of paramount significance to achieving successful glycosylations and minimizing side reactions.