Name: dna-magnetic particle binding analysis by dynamic and electrophoretic light scattering
Uploaded: 2017-11-09T15:00:00
Description: Watch this Scientific Journal Video about DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering at JoVE.com

The financial support by Czech Science Foundation (project GA CR 17-12816S) and CEITEC 2020 (LQ1601) is greatly acknowledged.

1. Magnetic Nanoparticles Synthesis
<ol>
	<li>Synthesis of magnetic nanoparticles stabilized with citrate (MNPs)
	<ol>
		<li>Add 20 mmol of FeCl3 &#8729; 6H2O (5.406 g) and 10 mmol of FeCl2 &#8729; 4H2O (1.988 g) in 20 mL of deoxygenated double distilled water (ddd water). Stir obtained solution vigorously in beaker under N2 atmosphere using a mechanical stirrer until a transparent solution is obtained.</li>
		<li>Under rapid mechanical stirring, a...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+commercial+DNA+extraction+kits+for+isolation+and+purification+of+bacterial+and+eukaryotic+DNA+from+PAH-contaminated+soils.">Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.</a> Can J Microbiol. 57 (8), 623-628 (2011).</li><li>Rachmayanti, Y., Leinemann, L., Gailing, O., Finkeldey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+from+processed+and+unprocessed+wood:+Factors+influencing+the+isolation+success.">DNA from processed and unprocessed wood: Factors influencing the isolation success.</a> Forensic Sci Int Genet. 3 (3), 185-192 (2009).</li><li>Munir, M. T., Umar, S., Shahzad, K. A., Shah, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+Magnetic+Nanoparticles+for+Hepatitis+B+Virus+Detection.">Potential of Magnetic Nanoparticles for Hepatitis B Virus Detection.</a> J Nanosci Nanotechnol. 16 (12), 12112-12123 (2016).</li><li>Ulbrich, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+drug+delivery+with+polymers+and+magnetic+nanoparticles:+covalent+and+noncovalent+approaches,+release+control,+and+clinical+studies.">Targeted drug delivery with polymers and magnetic nanoparticles: covalent and noncovalent approaches, release control, and clinical studies.</a> Chem Rev. 116 (9), 5338-5431 (2016).</li><li>Pershina, A. G., Sazonov, A. E., Filimonov, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticles-DNA+interactions:+design+and+applications+of+nanobiohybrid+systems.">Magnetic nanoparticles-DNA interactions: design and applications of nanobiohybrid systems.</a> Rus Chem Rev. 83 (4), 299 (2014).</li><li>Xu, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+nanoparticles+characterization:+Sizing+and+zeta+potential+measurement.">Progress in nanoparticles characterization: Sizing and zeta potential measurement.</a> Particuol. 6 (2), 112-115 (2008).</li><li>Krickl, S., Touraud, D., Kunz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+ethanolamine+stabilized+natural+rubber+latex+from+Taraxacum+kok-saghyz+and+from+Hevea+brasiliensis+using+zeta-potential+and+dynamic+light+scattering+measurements.">Investigation of ethanolamine stabilized natural rubber latex from Taraxacum kok-saghyz and from Hevea brasiliensis using zeta-potential and dynamic light scattering measurements.</a> Ind Crops Prod. 103, 169-174 (2017).</li><li>Haddad, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Isolation+of+DNA+by+Polycharged+Magnetic+Particles:+An+Analysis+of+the+Interaction+by+Zeta+Potential+and+Particle+Size.">The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.</a> Int J Mol Sci. 17 (4), (2016).</li><li>Tenorio-Neto, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicron+magnetic+core+conducting+polypyrrole+polymer+shell:+Preparation+and+characterization.">Submicron magnetic core conducting polypyrrole polymer shell: Preparation and characterization.</a> Mater Sci Eng C Mater Biol Appl. 61, 688-694 (2016).</li><li>Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+ferromagnetic+iron+oxide+particles+by+polyester+resin.">Encapsulation of ferromagnetic iron oxide particles by polyester resin.</a> e-Polym. 8 (1), 1-9 (2008).</li><li>Ghorbani, Z., Baharvand, H., Nezhati, M. N., Panahi, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+polymer+particles+modified+with+beta-cyclodextrin.">Magnetic polymer particles modified with beta-cyclodextrin.</a> J Polym Res. 20 (7), (2013).</li><li>Heger, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramagnetic+Nanoparticles+as+a+Platform+for+FRET-Based+Sarcosine+Picomolar+Detection.">Paramagnetic Nanoparticles as a Platform for FRET-Based Sarcosine Picomolar Detection.</a> Sci Rep. 5, (2015).</li><li>Navarro, E., Serrano-Heras, G., Casta&#241;o, M. J., Solera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+detection+chemistry.">Real-time PCR detection chemistry.</a> Clin Chim Acta. 439, 231-250 (2015).</li></ol>

In this protocol, the theoretical principles that explain DNA binding to magnetic particles via zeta potential were under question. The protocol describes the synthesis and modification of magnetic nanoparticles and microparticles. Method for preparation of DNA control and binding solution are also described. Two strategies are shown here for screening of DNA-particle interactions: quantitative PCR and DLS approaches. DLS provides three indicators of physicochemical changes in particles: particle size, polydispersity ind...

DNA isolation is one of the most essential steps in molecular biology. The development of nucleic acid extraction methods has great impact on the emerging fields of genomics, metagenomics, epigenetics, and transcriptomics. There is a wide range of biotechnological applications for DNA isolation including medical (forensic/diagnostic tools and prognostic biomarkers), and environmental applications (metagenomic biodiversity, pathogen prevalence, and surveillance). There has been increasing demand to purify and isolate DNA from different materials and in different scales such as blood, urine, soil, wood, and other kinds of samples.1,2,3,4
Nano- and micro-sized particles are suitable for DNA isolation due to their high surface area and particularly when they can be immobilized by a magnetic field. Physicochemical properties of particles, such as size or charge, can greatly influence their ability to bind target biomolecules.5 To further enhance binding of biomolecules and to stabilize particles, different chemical modifications (surface coatings) can be utilized. The many different strategies for binding are classified according to covalent and non-covalent interactions.6 The size of particles directly affects their magnetization properties, whereas particle composition can be tailored by incorporation of metallic, alloy or other materials that can influence its density, porosity, and surface.7 There is no reliable way to measure surface charge of small particles. Instead, electric potential at the slipping plane (some distance away from nanoparticle surface) can be measured.8 This value is called zeta potential and it is a potent tool that is usually used for evaluation of nano- and microparticle stability via DLS.9 Since its value is highly dependent not only on the pH and ionic strength of the dispersive environment, but also on the surface characteristics of the particles, it can also prove the changes in this surface caused by the interaction between the particles and molecule of interest.10
On the other hand, DNA structure in dehydrated conditions (A-DNA form) exhibits compacted conformations that facilitate its precipitation (aggregation) when compared to commonly occurring B-DNA form. Electrostatic (ionic and H-bond) are the major forces controlling the binding of DNA to other materials due to their sterically accessible phosphate and nitrogen bases (particularly guanine).7,10
In this work, three representative chemical modifications of magnetic nanoparticles and microparticles are analyzed (Figure 1A). The method of synthesis and chemical modification of nanoparticles and microparticles is described. A binding solution, that accords to theoretical principles of DNA precipitation (pH, ionic strength, and dehydration), is used to evaluate DNA binding and elution. Quantitative PCR is used to evaluate the elution efficiency of DNA from the representative nanoparticles and microparticles (Figure 1B). Particle size, polydispersity index, and zeta potential are important parameters that are used to visualize the physicochemical changes that occur on particle surface (Figure 1C). It is important to emphasize on the chemical characterization of magnetic particle surface. While this step was beyond the scope of this protocol, several modern techniques can be applied to investigate the efficiency of chemical modifications.11,12,13,14 Fourier transform infrared spectroscopy (FTIR) can be used to evaluate the infrared spectrum of particle surface and compare it to the spectrum of free chemical modifiers. X-ray photoelectron spectroscopy (XPS) is another technique that can be used to identify the elemental composition of material surface. Other electrochemical, microscopic and spectroscopic methods can be used to shed the light on the quality of particle synthesis. This work highlights a new perspective to analyzing DNA-magnetic particles interactions via DLS.

<table>
	<tbody>
		<tr>
			<td>Iron(III) chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>207926</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Iron(II) chloride tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>380024</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Iron(II) sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>F8263</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Penta</td>
			<td>10060-11000</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>W302600</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Tetraethyl orthosilicate</td>
			<td>Sigma-Aldrich</td>
			<td>131903</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched, average Mw ~25,000</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>221228-M&#160;</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Penta</td>
			<td>71250-11000</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Potassium nitrate</td>
			<td>Sigma-Aldrich</td>
			<td>P6083</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>1.05012</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>ow-molecular-weight cut-off membrane (Mw=1 kDa)</td>
			<td>Spectrum labs</td>
			<td>G235063</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Overhead Stirrer</td>
			<td>witeg Labortechnik GmbH</td>
			<td>DH.WOS01035</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Memmert GmbH + Co.</td>
			<td>84198998</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Bandelin</td>
			<td>795</td>
			<td>Magnetic particle synthesis</td>
		</tr>
		<tr>
			<td>BRAND UV cuvette micro</td>
			<td>Sigma-Aldrich</td>
			<td>BR759200-100EA</td>
			<td>Cuvette for size measurement</td>
		</tr>
		<tr>
			<td>BRAND cap for UV-cuvette micro</td>
			<td>Sigma-Aldrich</td>
			<td>BR759240-100EA</td>
			<td>Cuvette caps for size measurement</td>
		</tr>
		<tr>
			<td>Folded Capillary Zeta Cell</td>
			<td>Malvern</td>
			<td>DTS1070</td>
			<td>Cuvette for zeta potential measurement</td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td>ZEN3600</td>
			<td>Device for measurement of size and zeta potential</td>
		</tr>
		<tr>
			<td>Infinite 200 PRO 
			NanoQuant instrument</td>
			<td>Tecan</td>
			<td>396 227 V1.0, 04-2010</td>
			<td>device for measurement of DNA concentration</td>
		</tr>
		<tr>
			<td>SYBR Green Quantitative RT-PCR Kit</td>
			<td>Sigma-Aldrich</td>
			<td>QR0100</td>
			<td>PCR kit</td>
		</tr>
		<tr>
			<td>Mastercycler pro S instrument</td>
			<td>Eppendorf</td>
			<td>6325 000.013</td>
			<td>Thermocycler</td>
		</tr>
		<tr>
			<td>MinElute kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S7670</td>
			<td>DNA binding</td>
		</tr>
	</tbody>
</table>

dna-magnetic particle binding analysis by dynamic and electrophoretic light scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

Using the protocol described here for chemical synthesis and modification of magnetic particles, six magnetic particles were synthesized and analyzed for DNA binding. A summary of the analysis is shown in Table 1. By comparing the particle size in water and in binding solution, it is clear that all particles aggregated in binding solution by 2 - 22 folds. Some particles further aggregated to more folds in the presence of DNA; however this was not directly correlated with ...

Watch this Scientific Journal Video about DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering at JoVE.com

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

The overall goal of this experiment is to analyze binding of DNA and Magnetic Particles via Dynamic and Electrophoretic Light Scattering.This method can help answer key questions in the Biotechnology field such as, how fast and strong is the binding between nucleic acid and magnetic particles.The main advantage of this technique is the visualization of physical-chemical changes on the particles such as size, polydispersity or zeta potential.The implications of this technique extend towards molecular diagnosis because of increasing demand for fast and sensitive DNA isolation.In this procedure, add 20 millimoles of Ferric Chloride and ten millimoles of Ferrous Chloride in 20 milliliters of Deoxygenated Double Distilled Water.Next, stir the obtained solution vigorously in beaker under Nitrogen gas atmosphere using a mechanical stirrer until a transparent solution is obtained.Then, under rapid mechanical stirring, add 150 milliliters of one molar of aqueous ammonia drop by drop using dropping funnel over an hour.Afterward, transfer the solution into a screwable vessel and heat the solution at 75

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Research

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Chemistry

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. ...

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on how to prepare the SERS-active silver nanoaggregate, record a video of certain blinking spots in the microscopic image, and analyze the blinking statistics. In this analysis, a power law reproduces the probability distributions for bright events relative to their duration. The probability distributions for dark events are fitted by a power law with an exponential function. The parameters of the power law represent molecular behavior in both bright and dark states. The random walk model and the speed of the molecule across the entire silver surface can be estimated. It is difficult to estimate even when using averages, autocorrelation functions, and super-resolution SERS imaging. In the future, power law analyses should be combined with spectral imaging, because the origins of blinking cannot be confirmed by this analysis method alone.

This protocol describes the analysis of blinking surface-enhanced Raman scattering due to the random walk of a single molecule on a silver surface using power laws.

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the &#34;A-form&#34; conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...