We thank Phil Griffin and Tera Lavoie of the Soft Matter Characterization Facility and Advanced Electron Microscopy Facility, respectively, at The University of Chicago. We also thank Xiaobing Zuo and Soenke Seifert of the Advanced Photon Source at Argonne National Laboratory and NIST Center for Hierarchical Materials Design (CHiMaD) for support. We thank Jeff Ting and Michael Lueckheide for their contributions to this work.

The authors have nothing to disclose.

As mentioned above, the protocols presented here are written with a focus on oligonucleotides as the polyanion component and pLys-PEG as the cationic-neutral block copolymer, but we have tested them with a variety of polymers, such as poly(acrylic acid), polyglutamate, and PEG-poly(vinylbenzyl trimethylammonium), and believe they will be generally applicable for most polyelectrolyte pairs. One parameter that may need to be optimized is the salt concentration used for annealing, because it should be high enough that PCMs ...

When oppositely charged polyelectrolytes are mixed in aqueous solution, entropy gain from release of their counterions causes demixing of the solution into a polymer-rich condensed phase and a polymer-depleted supernatant1,2,3,4,5, a phenomenon known as polyelectrolyte complexation. If a neutral hydrophilic block is conjugated to one or both of the polyelectrolytes, nanoscale phase separation occurs instead (Figure 1A). The resulting self-assembled core-shell nanoparticles are variously referred to as polyelectrolyte complex micelles (PCMs), polyion complex micelles, block ionomer complexes, or coacervate-core micelles by analogy to surfactant micellization, even though all components of the system are hydrophilic6,7. A PCM&#39;s ability to encapsulate hydrophilic molecules such as proteins and nucleic acids, as well as the extensive tunability offered by the block copolymer carrier architecture makes them attractive candidates for delivering therapeutic molecules in vivo8,9,10,11,12,13.
Delivering therapeutic nucleic acids to cellular targets is a particularly important challenge, and one for which PCMs offer several advantages. Therapeutic nucleic acids (genetic DNA, mRNA, and oligonucleotides such as siRNA) have immense potential for improving human health, but must overcome numerous biological and physical barriers to realize that potential14,15,16. Bare nucleic acids are degraded by serum and cellular nucleases, are quickly cleared from circulation, and their strong negative charge makes it difficult for them to penetrate cell membranes without assistance. Current approaches for overcoming these barriers include costly chemical modifications to prevent damage from nucleases and/or encapsulation into various lipid nanoparticles assembled via hydrophobic interactions15,17,18. While these methods have proven effective for local injections and liver targeting, systemic use presents significant limitations of toxicity, immunogenicity, and limited biodistribution16. By contrast, PCMs use the negative charge of nucleic acids to condense them within the phase-separated core, while the neutral corona provides a steric barrier against degradation as well as a platform for incorporating ligands to enhance targeting or internalization11,19. In vitro and animal studies have shown that PCMs can effectively deliver various nucleic acid payloads20,21,22,23,24, but weaknesses in our ability to predict PCM properties such as size, shape, and stability from the properties of the constituent polymers have hindered their wider adoption.
Recent work by our group and others in the field has begun to address this problem by developing structure-property, and in some cases structure-property-function relationships for PCMs formed from nucleic acids and various cationic-neutral polymers7,25,26,27. Two consistent themes that have emerged from these studies are the importance of developing well-controlled, repeatable protocols for PCM assembly and the benefit of using multiple techniques to characterize the resulting nanoparticles. Polyelectrolytes, particularly those with high charge density like nucleic acids, interact with each other very strongly, and appear to readily become kinetically trapped upon mixing, resulting in PCM preparations that are highly sensitive to small variations in procedure and display high polydispersity and poor repeatability from batch to batch. PCMs have also been shown to adopt a wide range of shapes and sizes depending on the atomic-level configurations of their components, and capturing this diversity with any individual characterization technique is very difficult, particularly since some common techniques such as dynamic light scattering (DLS) require assumptions about particle shape for their interpretation.
In this article, we discuss material design and selection for PCMs, with a focus on oligonucleotides and cationic-neutral diblock copolymers. We then describe a salt annealing protocol that uses high salt concentrations followed by slow dialysis to avoid kinetic trapping during PCM assembly. The polyelectrolytes are mixed in high salt conditions where electrostatic attractions are screened, then the salt concentration is slowly lowered to allow the polyelectrolytes to settle into their most energetically favorable configurations, analogous to the slow cooling process of thermal annealing. Using this protocol, we are regularly able to achieve exceptionally low polydispersity and high repeatability for oligonucleotide PCMs7,26. Finally, we describe how four separate measurement techniques can be used to characterize PCMs over a very wide range of length scales, from external morphology to internal structure: DLS, multi-angle light scattering (MALS), small angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). We hope that these protocols will enable more researchers to effectively explore the capabilities of these interesting nanoparticles.
Polymer Selection and Preparation 
PCM properties are strongly influenced by the physical and chemical characteristics of the constituent polymers, making polymer selection a critical step in the design process. The most well-characterized block copolymers for nucleic acid PCMs are linear diblocks such as poly(lysine)-poly(ethylene glycol) (pLys-PEG), but PCMs can be formed between polyelectrolytes and a variety of hydrophilic neutral-charged polymers, which can be generated in a high throughput manner28. The choice of charged group strongly affects the stability of ion pairing and shape of micelles26, and PCM size has been shown to increase with the length of the charged block5,7,26 (Figure 2), thus allowing PCM properties to be tuned for the requirements of a desired application. For linear diblocks, we have found that the charged block should have at least 10 charges and be strongly charged at the desired pH. Longer charged blocks may promote PCM formation with oligonucleotides such as siRNA, which are difficult to complex with shorter blocks21. We have successfully observed PCM formation with block lengths up to 200, and the literature describes longer polymers. More flexibility is available in the choice of neutral blocks24, but experience has shown that very short neutral blocks lead to aggregation rather than nanoparticle formation, and that the minimum neutral length increases with charged block length. For pLys-PEG, a PEG MW of at least 3,000&#8211;5,000 is required for pLys lengths below ~50, and longer lengths are required as the charged block is increased further. Increased neutral block length results in increased PCM size, particularly shell thickness, due to steric crowding of the neutral polymers.
This manuscript presents a protocol for preparing PCMs from lyophilized high-purity pLys-PEG and oligonucleotides of known quantity, but should be readily adaptable to other systems as well. We have tested it successfully with several charged polypeptides, including polyarginine and polyglutamic acid, as well as several synthetic polyelectrolytes, such as polyacrylic acid and poly(vinylbenzyl trimethylammonium). We also describe preparing PCMs with a stoichiometric ratio of polyelectrolyte charges, but this is easily modified. We find it easiest to work in charge concentration units (c.c.), which also naturally accommodates polymers that are not fully charged. If either polymer is not well-characterized, care should be taken to accurately determine the polymer lengths/masses and ensure that excess salt is not present beyond that needed for charge neutralization by dialysis, for example. The presence of any retained water should also be accounted for when concentrations are calculated. Nucleic acid concentration can be conveniently quantified by absorbance at 260 nm, and the presence or absence of terminal phosphates should be considered when calculating the c.c.
When using oligonucleotides as polyanions, the hybridization state and chemical structure help determine the propensity for self-assembly and the characteristics of the resulting PCM5,7,26. Optimizing these, within the requirements for biological efficacy if using PCMs for delivery, will increase the likelihood of forming the desired structures. Helpful tools for analyzing hybridization include MATLAB functions for nucleic acids, NUPACK29, and IDT OligoAnalyzer. We recommend analyzing a candidate sequence to understand the strength of binding to 1) itself in a hairpin formation; 2) another copy of the same sequence (self-dimer); and 3) to other oligonucleotides present in the system. DNA and RNA melting temperatures for a specific sequence can also be calculated using the nearest-neighbor method30,31. Thermal annealing of nucleic acids (step 2.3) denatures any residual secondary structure in the individual nucleotides and promotes equilibrium folding.
PCM Characterization and Analysis 
A wide range of techniques are available for characterizing nanoparticles, including static and dynamic light scattering, small angle scattering of electrons or neutrons, and electron microscopy. In this article, we provide protocols for two light scattering techniques, small angle X-ray scattering, and two electron microscopy techniques.
DLS measures the autocorrelation of temporal fluctuations in scattering intensity at one angle from Brownian motion of the sample. Fitting this data can provide hydrodynamic radius and polydispersity for spherical micelles (Figure 3). Multiple angle light scattering (MALS) measures the static scattering intensity at many angles. This angular dependence describes the shape of the nanoparticle but is limited to length scales longer than ~50 nm for visible light, which limits its effectiveness for smaller nanoparticles. Both techniques are based on refractive index mismatch and primarily describe the outside dimensions of the nanoparticle.
Small angle X-ray scattering (SAXS) uses X-rays as the scattering probe, and their shorter wavelength allows measurements over a range of ~0.1&#8211;100 nm. Fitting the observed scattering intensity vs. angle (conventionally expressed as momentum transfer q) provides information on PCM morphology (i.e., size and shape) and also internal structure. If an absolute intensity calibration is available, and if the scattering intensity can be extrapolated to zero angle, PCM mass and aggregation number can also be estimated32, making SAXS an extremely versatile and valuable method. Small angle neutron scattering (SANS) is sensitive over a similar range of length scales but is only available at specialized facilities and will not be explicitly discussed in this article33,34,35.
Recent years have seen the advent of benchtop SAXS instruments, but we find that synchrotron sources are better suited for PCM characterization, as their higher intensity allows data to be collected much faster for these low-contrast samples. We provide a brief protocol for acquiring PCM SAXS data at Beamline 12-ID-B at the Advanced Photon Source (Argonne National Laboratory, USA) from a user perspective. This protocol should be applicable to most synchrotron sources, but consulting with local staff before proposing an experiment is highly recommended. We also provide a data reduction and analysis protocol using Irena36, a free set of macros written for Igor Pro. Irena includes a versatile set of form factors for modeling SAXS data and allows for construction of multicomponent models that are capable of describing the complex scattering profile of PCMs (see Representative Results, Figure 4). Irena also has comprehensive documentation and tutorials available online. Before attempting the procedures below, we recommend familiarization with these, particularly the tutorial &#34;Modeling of SAXS data with two main scatterer populations&#34;.
Radiation damage is a concern for X-ray scattering, but several measures can be employed to minimize it. In particular, we recommend using a flow cell setup with a syringe pump and PCM sample flowing during data acquisition, rather than a sealed capillary. This also greatly simplifies background subtraction. We also suggest taking multiple exposures of the flowing sample rather than one longer one in order to limit the flux that any single volume of sample sees and to allow for comparison of the exposure data to identify any damage.
In contrast to the scattering techniques, which generally require fitting to interpret, transmission electron microscopy (TEM) provides a real space visual image of the nanoparticles by passing an electron beam through the sample and projecting an image on a scintillation screen (Figure 5). We present protocols for two TEM techniques in this article. Cryo TEM freezes micelle samples into a thin layer of vitreous ice, preserving structural conformation with minimal foreign substances, optimal for micelles &#8804;~10-100 nm in radius. Negative stain TEM uses a heavy metal salt (e.g., uranium) to surround the sample after it has been dried on the surface of a grid. The dense stain will scatter more electrons than the sample, adding contrast and producing a negative image of the sample. Cryo TEM is recommended for high-quality images. However, it is more costly, time consuming, and may not provide sufficient contrast. When this is a concern, negative stained samples should be used. Examples of each are shown in Figure 5.
Each of these techniques reports on slightly different aspects of the nanoparticles, with different strengths and limitations. Light scattering is readily available, and is often the fastest approach, but has substantial limitations in size and shape resolution. SAXS can provide information over a large range of length scales at reasonably high throughput, but requires specialized equipment to acquire the data, as well as modeling to interpret it. TEM images are straightforward to interpret but can be limited in contrast and are inherently low throughput. Our experience has shown that using multiple techniques for characterization greatly increases the information that can be obtained about PCM properties and simplifies interpretation of data sets obtained from each one alone. For example, SAXS and TEM primarily examine a PCM&#39;s dense core, while light scattering reports on the overall dimensions of the nanoparticle. Thus, combining them allows measurement of both core and corona size. TEM&#39;s ability to acquire real space images can provide ground truth data to enable selection of appropriate form factors for modeling SAXS data that might otherwise be ambiguous. This article describes protocols for all four techniques, and an example process for using them to characterize an unknown sample is given in the Discussion section.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70 mm circle filter paper</td>
			<td>Whatman</td>
			<td>1001-070</td>
			<td>Filter paper for wicking during grid prep</td>
		</tr>
		<tr>
			<td>Carbon Film TEM grid</td>
			<td>Electron Microscopy Sciences</td>
			<td>CF200-Cu</td>
			<td>TEM grid</td>
		</tr>
		<tr>
			<td>DAWN</td>
			<td>Wyatt Technology</td>
			<td>DAWN</td>
			<td>MALS instrument</td>
		</tr>
		<tr>
			<td>DNA oligonucleotide</td>
			<td>Integrated DNA Nanotechnologies Inc</td>
			<td></td>
			<td>Custom oligonucleotide</td>
		</tr>
		<tr>
			<td>Lacey Carbon TEM grid</td>
			<td>Electron Microscopy Sciences</td>
			<td>LC200-Cu</td>
			<td>TEM grid</td>
		</tr>
		<tr>
			<td>Methoxy-poly(ethylene glycol)-block-poly(l-lysine hydrochloride) PEG5k - PLKC50</td>
			<td>Alamanda Polymers Inc</td>
			<td>mPEG5K-b-PLKC50</td>
			<td>Example block copolymer</td>
		</tr>
		<tr>
			<td>Milli-Q</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td>Ultrapure water</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>For measuring nucleic acid concentration</td>
		</tr>
		<tr>
			<td>negative-action tweezers</td>
			<td>Dumont</td>
			<td>N7</td>
			<td>Tweezers for grid preparation</td>
		</tr>
		<tr>
			<td>Parafilm &#34;M&#34;</td>
			<td>Bemis Company Inc</td>
			<td>PM996</td>
			<td>Laboratory film</td>
		</tr>
		<tr>
			<td>Quantifoil Holey Carbon TEM grid</td>
			<td>Electron Microscopy Sciences</td>
			<td>Q210CR1.3</td>
			<td>TEM grid</td>
		</tr>
		<tr>
			<td>Research Goniometer and Laser Light Scattering System</td>
			<td>Brookhaven Instruments</td>
			<td>BI-200SM</td>
			<td>DLS/MALS instrument</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer G2 2K 0.5 mL</td>
			<td>Thermo Scientific Pierce Protein Biology</td>
			<td>87723</td>
			<td>Dialysis cartridge</td>
		</tr>
		<tr>
			<td>small volume cuvette</td>
			<td>Brookhaven Instruments</td>
			<td>BI-SVC</td>
			<td>Cuvette for DLS/MALS</td>
		</tr>
		<tr>
			<td>Solarus 950 Advanced Plasma System</td>
			<td>Gatan</td>
			<td>Solarus 950</td>
			<td>Plasma system for TEM grids</td>
		</tr>
		<tr>
			<td>Talos TEM</td>
			<td>FEI</td>
			<td>Talos</td>
			<td>TEM used for cryo samples</td>
		</tr>
		<tr>
			<td>Tecnai Spirit TEM</td>
			<td>FEI</td>
			<td>Spirit</td>
			<td>TEM used for dry samples</td>
		</tr>
		<tr>
			<td>Uranyl Formate</td>
			<td>SPI-Chem</td>
			<td>16984-59-1</td>
			<td>For negative staining samples for TEM</td>
		</tr>
		<tr>
			<td>Vitrobot</td>
			<td>FEI</td>
			<td>Vitrobot</td>
			<td>Vitrification robot for cryo grid preparation</td>
		</tr>
	</tbody>
</table>

assembly and characterization of polyelectrolyte complex micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

In order to illustrate the characterization methods described above, we show typical results for PCMs assembled from oligonucleotides and block copolymers of various lengths and chemistries (Figure 1). Figure 2 provides an example of how PCM core size (as determined from SAXS and TEM, Figure 4 and Figure 5) varied with charged block length. Figure 3 shows DLS data and fitti...

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Assembly and Characterization of Polyelectrolyte Complex Micelles

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

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10.6K Views.  The University of Chicago. We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Video: Assembly and Characterization of Polyelectrolyte Complex Micelles

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

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 ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...