The authors thank the Thin Film Technology Division, Instrument Technology Research Center (ITRC) and National Applied Research Laboratories, Hsinchu Science Park, Taiwan. This work was supported by the National Science Council (grant no. MOST 105-0210-01-13-01) and Academia Sinica.

<ol>
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The authors have nothing to disclose.

A class of HIV-1 bNAbs including PG9, PG16, and PGTs 128, 141 - 145 were reported to be highly potent in neutralizing 70 - 80% of circulating HIV-1 isolates. The epitopes of these bNAbs are highly conserved among the variants of the entire HIV-1 group M, therefore they may guide the effective immunogen design for an HIV vaccine to elicit neutralizing antibodies23,24,25. As a part of our ongoing efforts to identify the glycan epi...

N-glycans on glycoproteins are covalently linked to the asparagine (Asn) residue of the consensus Asn-Xxx-Ser/Thr sequon, which affect several biological processes such as protein conformation, antigenicity, solubility, and lectin recognition1,2. The chemical synthesis of N-linked oligosaccharides represents a significant synthetic challenge because of their huge structural micro heterogeneity and highly branched architecture. Careful selection of protecting groups to tune reactivity of building blocks, achieving selectivity at anomeric centers, and proper use of promoter/activator(s) are key elements in synthesis of complex oligosaccharides. To solve this problem of complexity, a great amount of work to advance N-glycan synthesis was reported recently3,4. In spite of these robust approaches, finding an effective method for the preparation of a wide range of N-glycans (~20,000) remains a major challenge.
The rapid mutation rate of HIV-1 to achieve the extensive genetic diversity and its ability to escape from neutralizing antibody response, is among the greatest challenges to develop a safe and prophylactic vaccine against HIV-15,6,7. One effective tactic that HIV uses to avoid the host immune response is the post-translational glycosylation of envelope glycoprotein gp120 with a diverse N-linked glycans derived from the host glycosylation machinery8,9. A recent report regarding the precise analysis of recombinant monomeric HIV-1 gp120 glycosylation from human embryonic kidney (HEK) 293T cells suggests the occurrence of structural microheterogeneity with a characteristic cell-specific pattern10,11,12. Therefore, understanding the glycan specificities of HIV-1 bNAbs requires well characterized gp120 related N-glycan structures in a quantity sufficient for analysis.
The discovery of glycan microarray technology provided high throughput-based exploration of specificities of a diverse range of carbohydrate-binding proteins, viruses/bacterial adhesins, toxins, antibodies, and lectines13,14. The systematic glycans arrangement in an arrayed chip-based format could determine problematic low affinity protein-glycan interactions through multivalent presentation15,16,17,18. This chip-based glycan arrangement conveniently appears to effectively mimic cell-cell interfaces. To enrich the technology and overcome the uneven issue associated with conventional array formats, our group recently developed a glycan array on an aluminum oxide-coated glass (ACG) slide using phosphonic acid-ended glycans to enhance the signal intensity, homogeneity, and sensitivity19,20.
To improve the current understanding about glycan epitopes of newly isolated HIV-1 broadly neutralizing antibodies (bNAbs), we have developed a highly efficient modular strategy for the preparation of a broad array of N-linked glycans21,22 to be printed on an ACG slide (see Figure 1). Specificity profiling studies of HIV-1 bNAbs on an ACG array offered the unusual detection of hetero-glycan binding behavior of highly potent bNAb PG9 that was isolated from HIV infected individuals23,24,25.

<table width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">64197</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetonitrile</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75058</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic anhydride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108247</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Anhydrous magnesium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7487889</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Boron trifluoride ethyl etherate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">109637</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Bovine serum albumin</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">9048468</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bio-Gel P2 polyacrylamide</td>
			<td style="width:123px;">Bio-Rad</td>
			<td style="width:119px;">1504118</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bis(cyclopentadienyl)hafnium(IV) dichloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12116664</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">&#946;-1, 4 Galactosyl transferases from bovine milk</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">48279</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="width:168px;height:84px;">BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)</td>
			<td style="width:123px;">Arrayit</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium molybdate</td>
			<td style="width:123px;">TCI</td>
			<td style="width:119px;">C1794</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium nitrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">16774213</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Clean glass slide&#160;</td>
			<td style="width:123px;">Schott&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Cytidine-5&#8242;-monophospho-N-acetylneuraminic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">3063716</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Deuterated chloroform</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">865496</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Donkey Anti-Human IgG (Alexa Fluor647 conjugated</td>
			<td style="width:123px;">Jackson Immuno Research, USA</td>
			<td style="width:119px;">709605098</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dichloromethane</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75092</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Diethylaminosulfur trifluoride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">38078090</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dimethylformamide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">68122</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethyl acetate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">141786</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethylene glycol</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">107211</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">FAST frame slide incubation chambers</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Guanosine 5&#39;-diphospho-b-L-fucose disodium salt&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">15839700</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab tracer 2.0 software&#160;</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:164px;">Section 4 of the Protocol</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GenePix Pro 4300A reader (microarray image analysis)</td>
			<td style="width:123px;">moleculardevices</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">www.moleculardevices.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GraphPad Prism Software (Image processing )</td>
			<td style="width:123px;">GraphPad Software, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">http://www.graphpad.com/guides/prism/6/user-guide/</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Lithium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">1310652</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Manganese chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7773015</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Methanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">67561</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">N-butanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">71363</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Oxalic acid</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">144627</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Palladium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12135227</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Phosphate Buffered Saline</td>
			<td style="width:123px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">10010023</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Pyridine</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">110861</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">P-Toluene sulfonic acid monohydrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">773476</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Silver triflate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">2923286</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium bicarbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7647145</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Sodium hydrogen carbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium methoxide&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">124414</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7757826</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Toluene&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108883</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tris buffer&#160;</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">N/A</td>
			<td style="width:164px;">Ultra-pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tween-20</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">9005645</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Uridine diphosphate galactose (UDP-galactose)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">137868521</td>
			<td style="width:164px;"></td>
		</tr>
	</tbody>
</table>

chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

A modular chemo-enzymatic strategy for the synthesis of a wide array of N-glycans is presented in Figure 1. The strategy is based on the fact that diversity can be created at beginning by chemo-enzymatic synthesis of the three important modules, followed by the &#945;-specific mannosylation at the 3-O and/or 6-O position of the mannose residue of the common core trisaccharide of N-glycans. Considering the structural diversi...

Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

chemo-enzymatic-synthesis-n-glycans-for-array-development-hiv

Research

JoVE Journal

Chemistry

8.6K Views.  Academia Sinica. A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

Video: Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...