1Organic Chemistry Institute and CeNTech, Westfälische Wilhelms-Universität Münster, 2Laboratory of Macromolecular and Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, 3Laboratory of Materials and Interface Chemistry and Soft Matter Research Unit, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology
The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.
We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.
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.
We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.
Here are some highlights from the November 2011 Issue of Journal of Visualized Experiments (JoVE).
Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy
We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.
1Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, 2Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, 3Presently at the Interdisciplinary Center for Wide Band-gap Semiconductors, University Of California Santa Barbara
Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.
We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.
1Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 2Carolina Center for Nanotechnology Excellence, University of North Carolina
This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.
We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.
Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry
1Biomedical Engineering Department, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, 3Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Institute for Nanobiotechnology, Johns Hopkins University School of Medicine
A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.
Crystal structure of protein–DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.
We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.
1Department of Chemistry, City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies, 2Department of Chemical Engineering, City College of New York
Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.
We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.
Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.
RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus
In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.
Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.
1Department of Physics and Astronomy, The University of Texas at San Antonio, 2Centro de Investigaciones en Optica A. C., 3Department of Biology and Neurosciences Institute, The University of Texas at San Antonio
We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.
Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups
We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering
This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.
Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.
Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography
A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.
We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.
Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.
1Fischell Department of Bioengineering, University of Maryland, 2Institute for Bioscience and Biotechnology Research, University of Maryland, 3Department of Materials Science and Engineering, University of Maryland
This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.
The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.
Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting
1Institute of Microengineering (IMT), Photovoltaics and Thin Film Electronics Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), 2Department of Electrical Engineering and Computer Sciences, University of California, Berkeley
We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.
In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.
Here are some highlights from the October 2011 Issue of Journal of Visualized Experiments (JoVE).
Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets
A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.
SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.
Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.
Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.
Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry
Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).
We present methods for fabrication of patterned microstructures of vertically aligned carbon nanotubes (CNTs), and their use as master molds for production of polymer microstructures with organized nanoscale surface texture. The CNT forests are densified by condensation of solvent onto the substrate, which significantly increases their packing density and enables self-directed formation of 3D shapes.
The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here's a look at what's coming up in the February 2013 Issue of Journal of Visualized Experiments (JoVE).
Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.
A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.
Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.
1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University Medical Center, 2Department of Pharmacology, Center for Stem Cell Biology, Vanderbilt University Medical Center, 3Vanderbilt University Medical Center
Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, 3Department of Neurology, University of California, Los Angeles
Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.
Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.
As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.
Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.
The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.
The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.