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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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. 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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 ...

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