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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Magnetic Nanoparticles Synthesis

1. Synthesis of magnetic nanoparticles stabilized with citrate (MNPs)
   1. Add 20 mmol of FeCl₃ ∙ 6H₂O (5.406 g) and 10 mmol of FeCl₂ ∙ 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.
   2. 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 °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.
   3. 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.
   4. Re-disperse the precipitate via sonication (35 kHz, amplitude of 100%) and heat vessel at 80 °C for 1 h.
      NOTE: A sonicator of sufficient power is needed.
   5. 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%).
   6. 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.
   7. 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's requirements.
2. Synthesis of magnetic nanoparticles modified with branched polyethylenimine (MNPs_PEI)
   1. 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).
   2. Under rapid mechanical stirring, add 10 mL of 10% branched polyethylenimine (average 25 kDa). Stir solution for subsequent 3 h.
   3. 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%).
3. Synthesis of magnetic nanoparticles particles modified with silica (MNPs_TEOS)
   1. 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.
   2. Under rapid mechanical stirring, add 1 mL of tetraethyl orthosilicate. Stir solution for subsequent 12 h.
   3. 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%).
4. Synthesis of magnetic nanoparticles modified with amino groups (MNPs_APTES)
   1. 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%).
   2. Under rapid mechanical stirring, add 0.5 mL of (3-aminopropyl)triethoxysilane. Stir solution for subsequent 3 h.
   3. 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%).

2. Magnetic Microparticles Synthesis

1. Synthesis of magnetic microparticles stabilized with citrate (MMPs)
   1. 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 °C) for 2 h.
      NOTE: Prepare KNO₃ and KOH solutions in ddd water.
   2. 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.
   3. Redisperse the precipitate via sonication (35 kHz, amplitude of 100%) and heat vessel at 80 °C for 1 h. Collect black precipitate using magnet and wash it using 50 mL of ddd water (10 times).
   4. 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.
2. Synthesis of magnetic microparticles modified with branched polyethylenimine (MMPs_PEI)
   1. 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.
   2. 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%).
3. Synthesis of magnetic microparticles modified with silica (MMPs_TEOS)
   1. 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.
   2. Under rapid mechanical stirring, add 1 mL of tetraethyl orthosilicate. Stir solution for subsequent 12 h.
   3. 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%).
4. Synthesis of magnetic microparticles modified with amino groups (MMPs_APTES)
   1. 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%).
   2. Under rapid mechanical stirring, add 0.5 mL of (3-Aminopropyl) triethoxysilane. Stir solution for subsequent 3 h.
   3. 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%).

3. Magnetic Particle Size Analysis Using DLS

1. Particle size in water
   1. Use a DLS instrument that applies a back scattering detector angle (173°) 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°) and forward scattering (detector angle 15°) DLS are suitable for smaller particles and mixed particles, respectively.
   2. Prior to measurement in water, sonicate particles for 3 min.
      NOTE: Use diluted solution of the particles. For this purpose, mix 9.6 µL of particles with 96 µL of water.
   3. Carefully pipette 50 µL of particle solution into polystyrene latex cuvettes and avoid formation of bubbles.
      NOTE: For each sample, use disposable cuvette (See table of materials).
   4. Note the clarity of the cuvette and the direction of light beam. Insert the cell in the sample holder and close the chamber.
   5. Use the following parameters for measurement: temperature: 25 °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.

4. Preparing DNA Stock, Magnetic Particles and Binding Buffer

1. DNA stock
   1. 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 λ control, use the following primers. Forward primer: 5'-CCTGCTCTGCCGCTTCACGC-3'; Reverse primer: 5'-TCCGGATAAAAACGTCGATGACATTTGC-3'
   2. Purify PCR product using commercial DNA purification kit.
      NOTE: Commercial kits using silica column-based procedure yield > 100 ng/µL purified DNA.
   3. Measure concentration of DNA using spectrophotometric method (absorbance at 260 nm). Dilute the DNA stock of 10¹¹ copies/µL as instructed above to working solutions of 10¹⁰, 10⁹, 10⁸, and 10⁷ copies/µL.
      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/µL = (quantity in ng/µL * 6.022x10²³ copies/mol) / (498 bp * 650 g/mol of bp * 1x10⁹ ng/g)
2. Magnetic particles
   1. 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.
3. Binding buffer
   1. 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 °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.

5. Testing Efficiency of DNA Isolation Using Quantitative PCR

1. DNA isolation by modified MMPs or MNPs
   1. Use a 96-well plate and a magnet customized for 96-well plate for small volume experiments.
   2. Use the following binding solution mixture: 8 µL modified MMPs or MNPs, 10 µL sodium acetate-HCl (0.75 M, pH = 6), 60 µL of 85% ethanol, 10 µL DNA (10¹⁰ copies solution).
   3. 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 µL of 85% ethanol for washing.
   4. 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.
   5. Remove the plate from the magnet and add 40 µL of elution solution. Mix well by pipetting. Place the plate back on the magnet and collect eluted DNA (~37 µL). Store eluent at -20 °C until further analysis.
2. Quantitative PCR
   1. 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
   2. 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.
   3. Use the following quantities for a complete volume of 20 µL PCR mixture: 10 µL polymerase / intercalating dye mixture (see table of materials), 2 µL DNA sample, 2 µL forward primer (10 µM), 2 µL reverse primer (10 µM)
   4. Set up the thermocycler according to the following program: 95 °C for 120 s, 40 cycles (95 °C for 15 s, 64 °C for 15 s, 68 °C for 45 s), 68 °C for 5 min.
   5. 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.
   6. Here, the number of copies of DNA is estimated for total elution volume (~40 µL). Percentage of DNA retrieved corresponding to original amount added to binding solution (totally 10¹¹ copies) can vary beyond the 0 to 100 interval.

6. DNA-magnetic Particles Binding Analysis Using DLS

1. Binding of DNA
   1. For large volume experiments (in 1.5 mL tube), use the following binding solution mixture: 96 µL modified MMPs or MNPs, 120 µL sodium acetate-HCl (0.75 M, pH = 6), 720 µL of 85% ethanol, 120 µL DNA (10¹⁰ copies solution) or 120 µL Tris-HCL buffer (10 mM, pH = 8) for control.
2. Zeta potential analysis
   1. 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 µL of the binding mixture into a disposable cell.
      NOTE: Use disposable cuvette for measurement of surface zeta potential (See table of materials).
3. Use the following parameters for measurement on DLS instrument: Smoluchowski model with an F(ka) of 1.5, Temperature: 25 °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.

Results

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 ...

Discussion

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...

Materials

List of materials used in this article

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