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, add 150 mL of 1 M NH4OH, drop by drop using dropping funnel (take 1 h or more if needed). Transfer the solution into a screwable vessel after all NH4OH is added. Screw the vessel and heat solution at 75 &#176;C for 30 min. 
		CAUTION: NH4OH is classified as corrosive and dangerous for environment. 
		NOTE: Prepare NH4OH solution from high grade 28% NH4OH in water using deoxygenated water.</li>
		<li>Collect the black precipitate using magnet and wash it using 100 mL of ddd water. Repeat 3 times. Add 50 mL of 0.5 M tri-sodium citrate dihydrate to precipitate. Screw the vessel. 
		NOTE: Use deoxygenated solution of tri-sodium citrate dihydrate.</li>
		<li>Re-disperse the precipitate via sonication (35 kHz, amplitude of 100%) and heat vessel at 80 &#176;C for 1 h. 
		NOTE: A sonicator of sufficient power is needed.</li>
		<li>Let the solution cool to room temperature. Collect precipitate using a magnet and remove supernatant. Re-disperse black mud in 50 mL of ddd water using sonicator (35 kHz, amplitude of 100%).</li>
		<li>Transfer aliquots (~25 mL) into 50 mL centrifuge tubes and add 20 mL of acetone. Centrifuge at 2400 x g for 10 min and remove supernatants.</li>
		<li>Re-disperse precipitate and dialyze solution against water using low molecular weight cut-off dialysis device (500 - 1,000 Da) of suitable volume for 24 h. 
		NOTE: Prepare the dialysis device according to manufacturer&#39;s requirements.</li>
	</ol>
	</li>
	<li>Synthesis of magnetic nanoparticles modified with branched polyethylenimine (MNPs_PEI)
	<ol>
		<li>Add 2 mL of obtained MNPs solution (~0.1 g/mL) into a glass beaker. Add 38 mL of ddd water and sonicate (35 kHz, amplitude of 100%) the suspension (10 min).</li>
		<li>Under rapid mechanical stirring, add 10 mL of 10% branched polyethylenimine (average 25 kDa). Stir solution for subsequent 3 h.</li>
		<li>Collect precipitate using magnet and remove supernatant. Re-disperse precipitate in 20 mL ddd water. Repeat 5 times then sonicate the final suspension (35 kHz, amplitude of 100%).</li>
	</ol>
	</li>
	<li>Synthesis of magnetic nanoparticles particles modified with silica (MNPs_TEOS)
	<ol>
		<li>Add 10 mL of obtained MNPs solution (~0.1 g/mL) into a glass beaker. Add 60 mL of water and 130 mL of ethanol. Sonicate solution and add 6 mL of 28% NH4OH.</li>
		<li>Under rapid mechanical stirring, add 1 mL of tetraethyl orthosilicate. Stir solution for subsequent 12 h.</li>
		<li>Collect black precipitate using magnet and wash it using 50 mL of ethanol (3 times) and 50 mL of water (3 times). Re-disperse particles in 10 mL of ddd water and sonicate the suspension (35 kHz, amplitude of 100%).</li>
	</ol>
	</li>
	<li>Synthesis of magnetic nanoparticles modified with amino groups (MNPs_APTES)
	<ol>
		<li>Add 5 mL of obtained MNPs_TEOS solution (~0.1 g/mL) into a glass beaker. Add 45 mL of water and 50 mL of ethanol. Sonicate solution (35 kHz, amplitude of 100%).</li>
		<li>Under rapid mechanical stirring, add 0.5 mL of (3-aminopropyl)triethoxysilane. Stir solution for subsequent 3 h.</li>
		<li>Collect black precipitate using magnet and wash it using 50 mL of ethanol (3 times) and 50 mL of water (3 times). Re-disperse particles in 5 mL of ddd water and sonicate the suspension (35 kHz, amplitude of 100%).</li>
	</ol>
	</li>
</ol>
2. Magnetic Microparticles Synthesis
<ol>
	<li>Synthesis of magnetic microparticles stabilized with citrate (MMPs)
	<ol>
		<li>Add 25 mL of 2 M KNO3, 12.5 mL of 1 M KOH and 205.875 mL of ddd water into a screwable vessel. Add 6.675 mL of 1 M FeSO4 during magnetic stirring. Immediately transfer vessel to a preheated water bath (90 &#176;C) for 2 h. 
		NOTE: Prepare KNO3 and KOH solutions in ddd water.</li>
		<li>Let the solution cool to room temperature. Collect black precipitate using magnet and wash it using 50 mL of ddd water (5 times). Add 50 mL of 0.5 M tri-sodium citrate dihydrate to precipitate. Screw the vessel.</li>
		<li>Redisperse the precipitate via sonication (35 kHz, amplitude of 100%) and heat vessel at 80 &#176;C for 1 h. Collect black precipitate using magnet and wash it using 50 mL of ddd water (10 times).</li>
		<li>Modify MMPs with branched polyethylenimine (MMPs_PEI), silica (MMPs_TEOS) and amino groups (MMPs_APTES) similar to MNPs protocol. Refer to sections 2.2, 2.3 and 2.4, respectively for more details.</li>
	</ol>
	</li>
	<li>Synthesis of magnetic microparticles modified with branched polyethylenimine (MMPs_PEI)
	<ol>
		<li>Add 5 mL of MMPs solution (~0.1 g/mL) into a glass beaker. Add 38 mL of ddd water and sonicate the solution (35 kHz, amplitude of 100%, 10 min). Under rapid mechanical stirring add 10 mL of 10% branched polyethylenimine (average 25 kDa). Stir solution for subsequent 3 h.</li>
		<li>Collect precipitate using a magnet and remove supernatant. Redisperse precipitate in 20 mL of double distilled water. Repeat 5 times then sonicate the final solution (35 kHz, amplitude of 100%).</li>
	</ol>
	</li>
	<li>Synthesis of magnetic microparticles modified with silica (MMPs_TEOS)
	<ol>
		<li>Add 5 mL of MMPs solution (~0.1 g/mL) into a glass beaker. Add 60 mL of water and 130 mL of ethanol. Sonicate suspension (35 kHz, amplitude of 100%) and add 6 mL of 28% NH4OH.</li>
		<li>Under rapid mechanical stirring, add 1 mL of tetraethyl orthosilicate. Stir solution for subsequent 12 h.</li>
		<li>Collect black precipitate using a magnet and wash it using 50 mL of ethanol (3 times) and 50 mL of water (3 times). Redisperse particles in 5 mL of ddd water and sonicate the suspension (35 kHz, amplitude of 100%).</li>
	</ol>
	</li>
	<li>Synthesis of magnetic microparticles modified with amino groups (MMPs_APTES)
	<ol>
		<li>Add 3 mL of MMPs_TEOS solution (~0.1 g/mL) into a glass beaker. Add 45 mL of water and 50 mL of ethanol. Sonicate the suspension (35 kHz, amplitude of 100%).</li>
		<li>Under rapid mechanical stirring, add 0.5 mL of (3-Aminopropyl) triethoxysilane. Stir solution for subsequent 3 h.</li>
		<li>Collect black precipitate using magnet and wash it using 50 mL of ethanol (3 times) and 50 mL of water (3 time). Redisperse particles in 3 mL of ddd water and sonicate the suspension (35 kHz, amplitude of 100%).</li>
	</ol>
	</li>
</ol>
3. Magnetic Particle Size Analysis Using DLS
<ol>
	<li>Particle size in water
	<ol>
		<li>Use a DLS instrument that applies a back scattering detector angle (173&#176;) and a beam wavelength of 633 nm. 
		NOTE: Back scattering DLS is suitable for concentrated samples where multiple scattering can be avoided. Side scattering (detector angle 90&#176;) and forward scattering (detector angle 15&#176;) DLS are suitable for smaller particles and mixed particles, respectively.</li>
		<li>Prior to measurement in water, sonicate particles for 3 min. 
		NOTE: Use diluted solution of the particles. For this purpose, mix 9.6 &#181;L of particles with 96 &#181;L of water.</li>
		<li>Carefully pipette 50 &#181;L of particle solution into polystyrene latex cuvettes and avoid formation of bubbles. 
		NOTE: For each sample, use disposable cuvette (See table of materials).</li>
		<li>Note the clarity of the cuvette and the direction of light beam. Insert the cell in the sample holder and close the chamber.</li>
		<li>Use the following parameters for measurement: temperature: 25 &#176;C; refractive index of dispersive phase (Iron): 2.344; refractive index of dispersive environment (water): 1.333 
		NOTE: Perform measurement of the refractive index using a refractometer for more accurate results, particularly when developing novel or hybrid materials.</li>
	</ol>
	</li>
</ol>
4. Preparing DNA Stock, Magnetic Particles and Binding Buffer
<ol>
	<li>DNA stock
	<ol>
		<li>Use 10 mM Tris-HCL buffer (pH = 8) for dilution and storage of DNA. Use a standard DNA sample that can be amplified specifically with a direct PCR protocol in the laboratory (Refer to section 5.2 for reaction settings and conditions). To amplify 498 base pair (bp) of the xis gene from bacteriophage &#955; control, use the following primers. Forward primer: 5&#39;-CCTGCTCTGCCGCTTCACGC-3&#39;; Reverse primer: 5&#39;-TCCGGATAAAAACGTCGATGACATTTGC-3&#39;</li>
		<li>Purify PCR product using commercial DNA purification kit. 
		NOTE: Commercial kits using silica column-based procedure yield &#62; 100 ng/&#181;L purified DNA.</li>
		<li>Measure concentration of DNA using spectrophotometric method (absorbance at 260 nm). Dilute the DNA stock of 1011 copies/&#181;L as instructed above to working solutions of 1010, 109, 108, and 107 copies/&#181;L. 
		NOTE: The number of copies of PCR product can be calculated according to the following equation, assuming average molecular weight is 650 Da for each bp: 
		DNA copies/&#181;L = (quantity in ng/&#181;L * 6.022x1023 copies/mol) / (498 bp * 650 g/mol of bp * 1x109 ng/g)</li>
	</ol>
	</li>
	<li>Magnetic particles
	<ol>
		<li>Allow particles to sediment without the magnet and remove supernatant. Mix 1: 20 v/v ratio of magnetic particles with ddd water (particles: water). 
		NOTE: Volume-based approach is used to describe quantities of magnetic particles since equal masses of MNPs and MMPs were readily difficult to compare.</li>
	</ol>
	</li>
	<li>Binding buffer
	<ol>
		<li>For rapid preparation of 0.75 M sodium acetate-HCL solution (pH = 6), mix 1 mL of 3 M sodium acetate with 3 mL of 1 mM HCL. 
		NOTE: All solutions used in binding can be stored at 4 &#176;C. Note that low temperature significantly increases binding particularly in the case of ethanol solution. Carefully control the temperature of solutions for better reproducibility and accuracy of screening.</li>
	</ol>
	</li>
</ol>
5. Testing Efficiency of DNA Isolation Using Quantitative PCR
<ol>
	<li>DNA isolation by modified MMPs or MNPs
	<ol>
		<li>Use a 96-well plate and a magnet customized for 96-well plate for small volume experiments.</li>
		<li>Use the following binding solution mixture: 8 &#181;L modified MMPs or MNPs, 10 &#181;L sodium acetate-HCl (0.75 M, pH = 6), 60 &#181;L of 85% ethanol, 10 &#181;L DNA (1010 copies solution).</li>
		<li>Mix well by pipetting and place the plate on the magnet for 3 min. While the plate is on the magnet, remove the solution and add 100 &#181;L of 85% ethanol for washing.</li>
		<li>Remove ethanol. 
		NOTE: Allow the ethanol to dry for few seconds but do not let the particles over-dry or show cracks. If the particles over-dry they will be difficult to dissolve with elution solution.</li>
		<li>Remove the plate from the magnet and add 40 &#181;L of elution solution. Mix well by pipetting. Place the plate back on the magnet and collect eluted DNA (~37 &#181;L). Store eluent at -20 &#176;C until further analysis.</li>
	</ol>
	</li>
	<li>Quantitative PCR
	<ol>
		<li>Use the primers that were used to prepare stock DNA in step 4.1.1 to quantify copies of the xis gene, according to the protocol by Haddad et al.10</li>
		<li>Prepare master mix solution according to the amount of samples to be tested, then distribute the mixture into PCR tubes or a white background 96-well plate.</li>
		<li>Use the following quantities for a complete volume of 20 &#181;L PCR mixture: 10 &#181;L polymerase / intercalating dye mixture (see table of materials), 2 &#181;L DNA sample, 2 &#181;L forward primer (10 &#181;M), 2 &#181;L reverse primer (10 &#181;M)</li>
		<li>Set up the thermocycler according to the following program: 95 &#176;C for 120 s, 40 cycles (95 &#176;C for 15 s, 64 &#176;C for 15 s, 68 &#176;C for 45 s), 68 &#176;C for 5 min.</li>
		<li>Using triplicates for standards and samples, calculate the cycle threshold and construct a standard curve to estimate the number of copies of DNA retrieved in each sample. The instrument used here performs this automatically.</li>
		<li>Here, the number of copies of DNA is estimated for total elution volume (~40 &#181;L). Percentage of DNA retrieved corresponding to original amount added to binding solution (totally 1011 copies) can vary beyond the 0 to 100 interval.</li>
	</ol>
	</li>
</ol>
6. DNA-magnetic Particles Binding Analysis Using DLS
<ol>
	<li>Binding of DNA
	<ol>
		<li>For large volume experiments (in 1.5 mL tube), use the following binding solution mixture: 96 &#181;L modified MMPs or MNPs, 120 &#181;L sodium acetate-HCl (0.75 M, pH = 6), 720 &#181;L of 85% ethanol, 120 &#181;L DNA (1010 copies solution) or 120 &#181;L Tris-HCL buffer (10 mM, pH = 8) for control.</li>
	</ol>
	</li>
	<li>Zeta potential analysis
	<ol>
		<li>Prepare two binding solutions, one with DNA and another with Tris-HCL buffer for control as instructed in step 6.1.1. To avoid formation of bubbles, carefully pipette 800 &#181;L of the binding mixture into a disposable cell. 
		NOTE: Use disposable cuvette for measurement of surface zeta potential (See table of materials).</li>
	</ol>
	</li>
	<li>Use the following parameters for measurement on DLS instrument: Smoluchowski model with an F(ka) of 1.5, Temperature: 25 &#176;C, refractive index of dispersive phase (Iron): 2.344, refractive index of dispersive environment (Water): 1.333. 
	NOTE: The rate of DNA-magnetic particle interaction directly affects the measurement of replicates. A single measurement by the instrument takes 60-70 s. Here, the instrument was instructed to take 10 measurements with 1 min delay intervals. For simplicity, the results will be shown at time = 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 min from the first reading.</li>
</ol>

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The authors have nothing to disclose.

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 index and zeta potential. Particle size changes directly indicate aggregation or disintegration. Polydispersity index value describes the distribution of particle species in the system ranging from monodispersed (index &#60; 0.05) to highly polydispersed (index &#62;0.7). Zeta potential can classify particles according to surface charge where highly positive particles show values greater than 30 mV and highly negative particles show values lower than -30 mV. As predicted, the zeta potential of polycharged magnetic particles in binding solution relatively decreased when the particles were exposed to DNA. The exception to this rule is MNPs_TEOS. These particles only differ in that they possess a unique highly negative surface charge (Figure 2). This suggests that ionic strength played an important role in holding the particles, DNA and positive ions together. On the other hand, the changes in particle size can indicate the role of precipitation/aggregation of DNA-particles during the binding process, particularly in presence of longer DNA strands.
During synthesis of magnetic particles and chemical modifications, there are several critical steps which require attention: firstly, the reproducibility of the method of synthesis depends greatly on accurate quantities of chemical precursors used in synthesis as well as good stirring and timely controlled addition of NH4OH. To acquire a homogenized population of particles (of similar size, shape, density and composition), it is important to follow sonication and stirring steps thoroughly when instructed. Secondly, in many realistic cases the chemical modifications do not predict the reality of what is on the surface of magnetic particles. A comprehensive characterization step is required to confirm the chemical composition of the particle surface, as described in the introduction section. Among the commonly used techniques, researchers rely on FTIR, XPS, electrochemistry, spectroscopy as well as DLS.
In addition, several critical steps are required during the DLS measurements and analysis: firstly, the choice of binding solution affects various steps including chemical compatibility with particles and DNA and also spectral properties. Choice of refractive indices of binding solution and particle material can be based on previous studies or evaluated by refractometry. Secondly, interactions between the particles and the electrodes in the cuvettes should be monitored carefully as oxidation/reduction process can affect measurement. Thirdly, as shown in Figure 3, the rate of binding can be a function of time. It is critical to observe the stability of interaction, and this is a major advantage for application of DLS in such studies. Here, the observer is monitoring particle aggregation and physiochemical changes in real-time. Fourthly, polydispersity of the system can set limitations to the scope of analysis. Polydispersity index value below 0.05 describes a monodispersed system while values above 0.7 describe a polydispersed system of various sizes of particles. The size of particles and distribution of various particle species can be studied using various types of DLS, including back-scattered, side-scattered and forward scattered, as mentioned in protocol section 3.1. Finally, sonication of particles prior to testing can obviously affect their physical properties (e.g. size and surface area) and if done excessively it can cause release of some chemical modifications.
In this protocol, information acquired via DLS is compared to data acquired by quantitative PCR, also known as real-time PCR. The latter is the gold standard for detection and quantitation of DNA.15 PCR technique is widely used in laboratories, hospitals and universities. However, when testing DNA exposure to novel and synthesized materials it is important to take the chemistry of PCR into consideration. Polymerase enzyme, the key element of polymerase chain reaction, is sensitive to certain chemical inhibitors and enhancers. Addition of internal controls, testing of the synthetic materials, and good laboratory practice can help reduce biases in PCR results. Mechanisms of action for inhibitors and enhancers vary between chemicals that directly bind to polymerase and others that chelate ions from the Mg2+-dependent polymerase in solution. Some insights on DNA isolation are difficult to get using PCR screening. DLS can show cases when DNA binds to particles but is rather washed or does not elute (DNA not detected by PCR). For example, both MNPs_APTES and MMPs_APTES showed significant decrease in zeta potential upon DNA exposure (Table 1) but the DNA detected in elution did not correlate with these results. It is possible to modify the binding, washing, elution solutions to control the release of DNA from particles. DLS is a relatively faster method for evaluation of DNA-particle interactions. The stability and rate of interaction can also be observed by this method (Figure 3), which can impact the time used in magnetic particles- based DNA isolation protocols.
The challenges for using DLS in analysis are straight forward. This technique requires a relatively large sample volume for analysis (~1 mL) when compared to PCR. Some parameters like refractive indices require careful evaluation when using new solutions in the samples. Furthermore, it is very common to observe oxidation/reduction reactions on the electrodes of cuvettes when testing novel material. Careful assessment and knowledge of the chemical composition of the sample can help evaluate the feasibility of re-using the same cuvette. It is worth mentioning that DLS analysis is limited to the specificity and sensitivity provided by PCR, yet it provides different kind of insight to the DNA-particle binding process.
Like all spectroscopic techniques, modifications and troubleshooting of DLS aim for development of more accurate and sensitive measurements. This can be achieved by providing better understanding of the nature of the material tested physically and chemically. In other words, optical properties and details of chemical composition are the key to gain accurate and sensitive data from DLS. Optical properties can be acquired by refractometry, while chemical composition of particles / particles&#39; surface can be studied by various techniques such as FTIR, XPS, electrochemistry and other spectroscopic methods. In this protocol, binding solution can be improved by using various ionic strengths, other dehydrating agents (e.g. isopropanol), various pH and low temperatures. On the other hand, the chemical modifications of particle surface are left to the chemists&#39; imagination.
The development of materials for isolation of DNA will continue to rise in the next decades with more focus on specificity, purity and limit of detection. The quality of extraction methods plays important role in studies of single cell scale, metagenomic scales and samples of unusual origins. At the present time, it is important to perform DLS analysis of nanoparticles and microparticles modified with various kinds of materials. This step is required before establishing a strong theoretical model that describes the behavior of DNA-particle interactions.

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&amp;nbsp;</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&lt;br/&gt; 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 DNA retrieval detected by quantitative PCR (Table 1). Comparison of zeta potential (10 measurements in ~2 min intervals) of particles in controls versus with DNA showed sharp drop in three particles; namely: MNPs_APTES, MMPs_TEOS and MMPs_APTES (Figure 2). Trends in zeta potential over time were also observed, showing variations in the first three readings before they are stabilized in most cases (Figure 3). Variation after exposure to DNA was mostly observed in MMPs (Figure 2). Lower standard deviation was observed by omitting the first three readings representing first 5 min after exposure of particles to DNA (Table 1).
Overall view of the analysis in Table 1 shows the different characteristics of DNA-particle binding as attributed to magnetic particle size and chemical modifications. Briefly, MNPs_PEI nanoparticles increased in size after binding DNA by four folds with no significant change in polydispersity index or zeta potential. However, DNA was not retrievable in this procedure. MNPs_TEOS nanoparticles increased in size and decreased in polydispersity index with unnoticeable change in zeta potential after DNA exposure, yet they were the best particles in their retrieval rates at 20.3%. MNPs_APTES nanoparticles decreased slightly in size and noticeably in zeta potential upon DNA exposure. MMPs_PEI microparticles did not show change in size upon DNA exposure, however they increased in polydispersity index and decreased slightly in zeta potential. MMPs_TEOS microparticles showed alternative size characteristics to their nanoparticle counterparts i.e. MNPs_TEOS. Surprisingly, they decreased noticeably in zeta potential but not beyond the range displayed by MNPs_TEOS. MMPs_APTES increased in particle size upon DNA exposure and also decreased in zeta potential. It is important to note that both MNPs_APTES and MMPs_APTES retained similar sizes after chemical modification despite the fact that they were made from different sized precursors. They have also displayed similar binding properties. MNPs_TEOS showed no significant change in zeta potential by their values were in the lowest extreme detected in this study.
The follow up over time for zeta potential showed stabilizing values after 5 - 10 min, particularly in the case of MNPs (Figure 3A, 3C and 3E). A continuous trend in zeta potential was found in some MMPs readings (Figure 3B, 3D and 3F). In some cases, variations in zeta potential corresponded to variations in polydispersity index and particle size but not with DNA retrieval by elution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56815/56815fig1.jpg" /> 
Figure 1: Protocol scheme. (A) Chemical modifications of magnetic particles including branched polyethyleneimine, silica and amino groups. (B) DNA isolation steps using binding buffer and magnetic particles immobilized on magnet, followed by quantitative PCR. (C) DNA-particle binding analysis using DLS. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56815/56815fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56815/56815fig2.jpg" /> 
Figure 2: Zeta potential of representative magnetic particles in presence and absence of DNA. Exposure to DNA caused visible decrease of zeta potential in MNPs_APTES, MMPs_TEOS and MMPs_APTES. Error bars represent standard deviation (N = 10). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56815/56815fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56815/56815fig3.jpg" /> 
Figure 3: Zeta potential of representative magnetic particles over time in presence and absence of DNA. Zeta potential of particles in binding solution was measured at time = 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 min. Gap between the zeta potential curves of control vs. with DNA are obvious in cases of MMPs_TEOS, MNPs_APTES and MMPs_APTES. (A) Zeta potential of MNPs_PEI is highly positive indicating no interactions. (B) Zeta potential of MMPs_PEI decreased over time suggesting a very slow interaction between particles and DNA. (C) Zeta potential of MNPs_TEOS was very negative in absence and presence of DNA. (D) Zeta potential of MMPs_TEOS shows lower values in presence of DNA when compared to controls. (E) Zeta potential of MNPs_APTES shows lower values in presence of DNA when compared to controls. (F) Zeta potential of MMPs_APTES shows lower values in presence of DNA when compared to controls. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56815/56815fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Characteristics</td>
			<td>MNPs_PEI</td>
			<td>MNPs_TEOS</td>
			<td>MNPs_APTES</td>
			<td>MMPs_PEI</td>
			<td>MMPs_TEOS</td>
			<td>MMPs_APTES</td>
		</tr>
		<tr>
			<td>Particle Size (nm)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>in water</td>
			<td>141.8</td>
			<td>91.3</td>
			<td>1106.4</td>
			<td>825.0</td>
			<td>1281.3</td>
			<td>1281.3</td>
		</tr>
		<tr>
			<td>in binding solution</td>
			<td>531.2</td>
			<td>1990.1</td>
			<td>3091.0</td>
			<td>2669.0</td>
			<td>2669.0</td>
			<td>1990.1</td>
		</tr>
		<tr>
			<td>in binding solution with DNA</td>
			<td>1990.1</td>
			<td>3091.0</td>
			<td>2669.0</td>
			<td>2669.0</td>
			<td>1990.1</td>
			<td>4145.4</td>
		</tr>
		<tr>
			<td>observed change</td>
			<td>increased</td>
			<td>increased</td>
			<td></td>
			<td></td>
			<td></td>
			<td>increased</td>
		</tr>
		<tr>
			<td>Polydispersity index</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>in water</td>
			<td>0.237</td>
			<td>0.240</td>
			<td>0.219</td>
			<td>0.410</td>
			<td>0.281</td>
			<td>0.206</td>
		</tr>
		<tr>
			<td>in binding solution</td>
			<td>0.225</td>
			<td>0.773</td>
			<td>0.090</td>
			<td>0.107</td>
			<td>0.374</td>
			<td>0.397</td>
		</tr>
		<tr>
			<td>in binding solution with DNA</td>
			<td>0.240</td>
			<td>0.298</td>
			<td>0.282</td>
			<td>0.301</td>
			<td>0.387</td>
			<td>0.319</td>
		</tr>
		<tr>
			<td>observed change</td>
			<td></td>
			<td>decreased</td>
			<td>increased</td>
			<td>increased</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeta potential (mV)*</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>in binding solution</td>
			<td>42.29</td>
			<td>-27.83</td>
			<td>33.23</td>
			<td>30.80</td>
			<td>-6.86</td>
			<td>-0.66</td>
		</tr>
		<tr>
			<td>(&#177;SD)</td>
			<td>1.15</td>
			<td>1.17</td>
			<td>2.89</td>
			<td>8.99</td>
			<td>7.99</td>
			<td>5.42</td>
		</tr>
		<tr>
			<td>in binding solution with DNA&#160;</td>
			<td>41.81</td>
			<td>-25.97</td>
			<td>12.28</td>
			<td>27.81</td>
			<td>-23.67</td>
			<td>-15.22</td>
		</tr>
		<tr>
			<td>(&#177;SD)</td>
			<td>2.02</td>
			<td>0.80</td>
			<td>2.21</td>
			<td>4.33</td>
			<td>0.95</td>
			<td>4.28</td>
		</tr>
		<tr>
			<td>observed change</td>
			<td></td>
			<td></td>
			<td>decreased</td>
			<td></td>
			<td>decreased</td>
			<td>decreased</td>
		</tr>
		<tr>
			<td>Quantitative PCR retrieval**</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Copy number retrieved&#160;</td>
			<td>1.22E+03</td>
			<td>1.01E+09</td>
			<td>3.18E+06</td>
			<td>1.44E+06</td>
			<td>3.76E+08</td>
			<td>6.88E+06</td>
		</tr>
		<tr>
			<td>(&#177;SD)</td>
			<td>8.05E+02</td>
			<td>7.55E+07</td>
			<td>2.83E+06</td>
			<td>1.98E+06</td>
			<td>1.08E+08</td>
			<td>3.33E+06</td>
		</tr>
		<tr>
			<td>DNA retrieval percentage</td>
			<td>0.0</td>
			<td>20.3</td>
			<td>0.1</td>
			<td>0.0</td>
			<td>7.5</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>(&#177;SD)</td>
			<td>0.0</td>
			<td>1.5</td>
			<td>0.1</td>
			<td>0.0</td>
			<td>2.2</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td colspan="7">* Only stable measurements after 5 minutes of binding are shown (N=7).</td>
		</tr>
		<tr>
			<td colspan="7">** R&#178; = 0.940 for triplicates of standards.</td>
		</tr>
	</tbody>
</table>
Table 1: Characterization of representative magnetic particles and their abilities to bind and retrieve DNA. Upon exposure to binding solution, all particles showed aggregation (increase in particle size). When DNA was present in binding solution, particle size increase was visible in MNPs_PEI, MNPs_TEOS and MMPs_APTES. Particles were polydisperse systems in binding solution, particularly MNPs_TEOS which became less polydispersed upon binding of DNA. MNPs_APTES showed increase in polydispersity index and decrease in zeta potential upon exposure to DNA. MMPs_TEOS and MMPs_APTES showed large decrease in zeta potential upon exposure to DNA. MNPs_TEOS and MMPs_TEOS showed the highest DNA retrieval rates of 20.3% and 7.5%, respectively, as shown by quantitative PCR. MNPs_APTES and MMPs_APTES showed 0.1% DNA retrieval rates.

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 ...

dna-magnetic-particle-binding-analysis-dynamic-electrophoretic-light

Nanyang Technological University

Research

JoVE Journal

Chemistry

12.0K Views.  Mendel University in Brno. 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.

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

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Biochemistry

Frequency Mixing Magnetic Detection Scanner for Imaging Magnetic Particles in Planar Samples

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. It consists of two magnetic measurement heads on both sides of the sample mounted on the legs of a u-shaped support. The sample is locally exposed to a magnetic excitation field consisting of two distinct frequencies, a stronger component at about 77 kHz and a weaker field at 61 Hz. The nonlinear magnetization characteristics of superparamagnetic particles give rise to the generation of intermodulation products. A selected sum-frequency component of the high and low frequency magnetic field incident on the magnetically nonlinear particles is recorded by a demodulation electronics. In contrast to a conventional MPI scanner, p-FMMD does not require the application of a strong magnetic field to the whole sample because mixing of the two frequencies occurs locally. Thus, the lateral dimensions of the sample are just limited by the scanning range and the supports. However, the sample height determines the spatial resolution. In the current setup it is limited to 2 mm. As examples, we present two 20 mm &#215; 25 mm p-FMMD images acquired from samples with 1 &#181;m diameter maghemite particles in silanol matrix and with 50 nm magnetite particles in aminosilane matrix. The results show that the novel MPI scanner can be applied for analysis of thin biological samples and for medical diagnostic purposes.

A scanner for imaging magnetic particles in planar samples was developed using the planar frequency mixing magnetic detection technique. The magnetic intermodulation product response from the nonlinear nonhysteretic magnetization of the particles is recorded upon a two-frequency excitation. It can be used to take 2D images of thin biological samples.

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. ...

Engineering

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

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 ...

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Imaging the Motion of Fully Reconstituted Cdc45/Mcm2-7/GINS (CMG)

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Biology

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...