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<!doctype html>
<html>
<head>
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.11.1/jquery.min.js"></script>
<link rel="stylesheet" type="text/css" href="/styles/style.css">
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<body>
<h1 class="h1">Final Protocols</h1>
<ul>
<li><a href="#solid_silica_prep">dSiO<sub>2</sub> Particles Preparation</a></li>
<li><a href="#adsorb_dna_solid">Adsorb DNA on these particles → Detection of this adsorption</a></li>
<li><a href="#adsorb_protein_solid">Adsorb Protein on these particles → Detection of this adsorption</a></li>
<li><a href="#adsorb_dna_protein_solid">Adsorb DNA+Protein simultaneously on these particles → Detection of this adsorption</a></li>
<li><a href="#hmsn_prep">Make Shell (i.e. Hollow MSN) → Detection whether DNA and Protein is still inside</a></li>
<li><a href="#activity_enzyme">Activity Check of the Enzyme which is entrapped inside</a></li>
<li><a href="#attach_dna_hmsn">Attach DNA on outer surface → Detection</a></li>
</ul>
<h2>Synthesis of Hollow MSN → AT A GLANCE</h2>
<p>Hollow Mesoporous silica nanoparticles are produced first by depositing a mixture of CTAC and TEOS on a hard sphere of dSiO<sub>2</sub>. Then the hard central dSiO<sub>2</sub> sphere was removed via a Na<sub>2</sub>CO<sub>3</sub>based etching process. Characterization is done via SEM/TEM and pore size analysis via BET and BJH methods.Modulation of the cavity size can be done by changing the size of dSiO<sub>2</sub> hard sphere. For changing the pore size, the only possible option seems to be that of changing the ratio CTAC vs TEOS in the reaction mixture, although the same needs to be confirmed via proper experimentation.</p>
<div id="main">
<h2 id="solid_silica_prep">dSiO<sub>2</sub> Particles Preparation</h2>
<ul>
<li>
<h3>Materials</h3>
<ol>
<li>Ethanol</li>
<li>Ammonia</li>
<li>TEOS</li>
</ol>
</li>
<li>
<h3>Procedure</h3>
<ol>
<li>In a typical synthesis, 35.7 mL of absolute ethanol was mixed with 5 mL water and 0.8 mL of ammonia and stirred for 5-10 minutes at room temperature.</li>
<li>Then 1 mL of TEOS was added and the mixture was allowed to react at room temperature for 1 hour.</li>
<li>Afterward, dSiO2 nanoparticles were washed with water and ethanol and suspended in 20 mL of water.</li>
</ol>
</li>
</ul>
<h2 id="adsorb_dna_solid">Adsorb DNA on these particles → Detection of this adsorption</h2>
<ul>
<li>
<h3>Materials</h3>
<ol>
<li>Silica Nanoparticles (dSiO<sub>2</sub>).</li>
<li>Plasmid DNA</li>
<li>Guanidine hydrochloride (Guanidine-HCl) 2M at pH 5.2 :: binding buffer</li>
<li>Deionized water</li>
<li>1M NaOH</li>
<li>1M HCl</li>
</ol>
</li>
<li>
<h3>AT A GLANCE</h3>
<p>Adsorption of DNA on dSiO<sub>2</sub> nanoparticles is based on three driving forces:
<ul>
<li>Electrostatic Screening effect</li>
<li>Dehydration Effect</li>
<li>Generation of intermolecular hydrogen bonds</li>
</ul>
DNA being negative charged can't come in direct contact with these nanoparticles, hence methods employed aim at reducing the repulsion due to negative charges. Guanidine-HCl salt provide the cations used to reduce these electrostatic effects.The sorbent and the adsorbent can be hindered due to water. Guanidine-HCl is a chaotropic salt and captures water molecules thereby dehydrating DNA and reduce water bound to the sorbent and adsorbent surfaces. This step increases adsorption capacity of the adsorbent.DNA adsorbs on silica by the means of intermolecular H-bonding between the phosphate groups of the DNA backbone and the silanol groups of silica surface. Low pH is required for protonation of silanol and phosphate. pH is important for intermolecular H-bonding.These are negatively charged particles.</p>
</li>
<li>
<h3>Procedure</h3>
<ol>
<li>250 µg/mL DNA is taken in deionized water.</li>
<li>10 µL of DNA solution is added into 0.5 mL centrifuge tube with 0.2 mg of nanoparticles and 10 µL deionized water and 20 µL binding buffer.</li>
<li>The mixture was well dispersed by vortex for 30s and then continuously shook at 25°C for 20 hours.</li>
</ol>
</li>
<li>
<h3>Characterization</h3>
<p>The DNA concentration was determined by using a NANODROP 1000 spectrophotometer(Thermo Scientific, USA). The concentration was an average of duplicate measurements.</p>
</li>
<li>
<h3>Detection</h3>
<p>The three techniques given below may be used for detection:</p>
<ul>
<li>Gel Electrophoresis: DNA containing nanoparticles will give a bright band near the well.</li>
<li>Zetasizer - For measuring Zeta Potential. A large change in zeta potential occurs due to DNA adsorption.</li>
<li>DLS - For measuring Hydrodynamic Diameter. It increases due to DNA adsorption.</li>
</ul>
</li>
</ul>
<h2 id="adsorb_protein_solid">Adsorb Protein on these particles → Detection of this adsorption</h2>
<ul>
<li>
<h3>Materials</h3>
<ol>
<li>Solid Silica Nanoparticles</li>
<li>T7 RNA Polymerase</li>
<li>DNA sequence with T7 promoter and terminator sequences(to test the activity once absorbed)</li>
<li>Buffer Solution for Pol.:</li>
<ul>
<li>20 mM Tris-HCl</li>
<li>100 mM NaCl</li>
<li>0.1 mM EDTA</li>
<li>1 mM DTT</li>
<li>50% Glycerol</li>
</ul>
<li>Buffer solutions of different pHs to be studied.</li>
</ol>
</li>
<li>
<h3>Overview</h3>
<p>The adsorbtion of protein onto the silica nanoparticle surface depends on a number of factors, primarily pH, Ion strength of incubation solution and temperature. The pH mainly affects the adsorbtion process. It affects the surface charge of the protein. The pH of the solution must lie between the point of zero charge of the silica and the isoelectric point of the protein causing the two to have opposite charges. So, the following method will be adopted to check the conditions of maximum adsorbtion onto the silica nanoparticle.</p>
</li>
<li>
<h3>Procedure</h3>
<ol>
<li>Three solutions containing different conditions of pH were prepared. One will be a pH that lies between the pzc of the silica nanoparticle and isoelectric point of the protein (pH ~ 4.5-55). One will be the standard buffer conditions of storage of the T7 RNA Pol(20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol, pH 7.5 @ 25°C) . Also, one other pH of 6 was selected to study the effect of pH in more detail.</li>
<li>Then, the adsorbtion was measured at different points of time at intervals of 1 minute for the first 15 minutes as the process of adsorbtion when the charges are opposite is expected to be instantaneous. However, to elucidate the adsorbtion process the adsorbtion was measured at intervals of one hour after that for a period of 5 hours and further if necessary.The adsorbtion will be studied by the technique mentioned in the following section of this document.</li>
<li>Another important study will be the study of the activity of the enzyme once adsorbed. The adsorbtion conditions may affect the protein’s activity positively or negatively meaning that even if the adsorbtion may be less at some conditions, its activity at some condition may be more, resulting in a higher yield of the reaction product(in this case RNA). The activity studies will also be carried out as per procedure mentioned in this document.</li></li>
<li>Once these studies are made the conditions resulting in the most significant amount of RNA production will be selected as the optimum conditions after ensuring that there is no error in experimental techniques and analysis.</li>
</ol>
</li>
<li>
<h3>Detection</h3>
<ul>
<li>Non-denaturing SDS Page can be done to detect the presence of proteins in the nanoparticles sample.</li>
</ul>
</li>
</ul>
<h2 id="adsorb_dna_protein_solid">Adsorb DNA+Protein simultaneously on these particles → Detection of this adsorption</h2>
<p>We will use above steps to first adsorb DNA, then protein and then detecting their presence. </p>
<h2 id="hmsn_prep">Make Shell (i.e. Hollow MSN) → Detection whether DNA and Protein is still inside</h2>
<ul>
<li>
<h3>Preparation of Mesoporous Silica Nanoparticle Layer over dSiO<sub>2</sub></h3>
<ul>
<li>
<h4>Materials</h4>
<ol>
<li>CTAC</li>
<li>TEOS</li>
<li>TEA</li>
</ol>
</li>
<li>
<h4>Procedure</h4>
<ol>
<li>CTAC (2 g) and TEA (20 mg) were dissolved in 20 mL of high Q water and stirred at room temperature for 1 h.</li>
<li>Then, 10 mL of dSiO2 water solution was added and stirred at room temperature for 1 h before addition of 0.15 mL of TEOS.</li>
<li>The mixture was stirred for 1 h at 80°C in a water bath to form MSN layer over dSiO<sub>2</sub>.</li>
</ol>
</li>
</ul>
</li>
<li>
<h3>Etching of dSiO<sub>2</sub> to form Hollow MSN</h3>
<ul>
<li>
<h4>Materials</h4>
<ol>
<li>CTAC</li>
<li>Sodium Carbonate</li>
<li>Sodium Chloride</li>
<li>Methanol</li>
</ol>
</li>
<li>
<h4>Procedure</h4>
<ol>
<li>Both mixture and water bath were cooled down to 50°C followed by addition of 636 mg of sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>), which was under constant stirring for 30 min to form HMSN.</li>
<li>To remove the CTAC, the product was extracted for 24 h with a 1 wt% solution of NaCl in methanol at room temperature.</li>
<li>This process was carried out for at least 3 times to ensure complete removal of CTAC.</li>
</ol>
</li>
<li>
<h4>Characterization</h4>
<ol>
<li>SEM and TEM imaging.</li>
<li>Surface area measurement done via Brunauer–Emmett–Teller (BET) method using nitrogen adsorption and desorption isotherms.</li>
<li>The pore size distribution plot will be obtained by the Barrett–Joyner–Halenda (BJH) method.</li>
<li>The zeta potentials can also be measured, to check for coagulation.</li>
<li>The pH of the solutions is to be recorded with pH meter.</li>
</ol>
</li>
</ul>
</li>
</ul>
<h2 id="activity_enzyme">Activity Check of the Enzyme which is entrapped inside</h2>
<p>We will put dNTPs in the solution. The nanoparticles now cantains enzyme entrapped which should start producing RNA which would come out from the nanoparticles in the solution which could be detected by Spectrophotometry. </p>
<h2 id="attach_dna_hmsn">Attach DNA on outer surface → Detection</h2>
<p>Pre- synthesised MSN are functionalised with (isocyanate) NCO groups. DNA is modified with (amino) NH2 groups and attached to the MSN according to the protocol given below.</p>
<ul>
<li>
<h3>Materials</h3>
<ol>
<li>Anhydrous toluene</li>
<li>TSPI</li>
<li>Amino modified DNA</li>
<li>Anhydrous Acetonitrile</li>
<li>Binding buffer (consisting of 10 mM 3-(N-morpholino)- propanesulfonic acid (MOPS))</li>
<li>50 mM NaNO3</li>
<li>0.05% (by volume) Triton X-100 (pH = 7.0))</li>
</ol>
</li>
<li>
<h3>Procedure</h3>
<ol>
<li>500 mg of prepared MSN was refluxed(boil such as to collect back the vapours) in 80 mL of anhydrous toluene with 0.25 mL of 3-(triethoxysilyl)propyl isocyanate(TSPI) for 20 h to immobilize the isocyanate group on the surface. </li>
<li>The isocyanate-functionalized MSN (MSNNCO) was collected and washed with anhydrous toluene three times and then dried in an oven at 70°C to evaporate the toluene.</li>
<li>The amino modified DNA was suspended in anhydrous acetonitrile.</li>
<li>24 µL of 1 mM amino modified DNA solution was added to 1 mg of MSN-NCO in 1.5 mL centrifuge tubes, followed by adjustment of the total volume to 30 µL with anhydrous acetonitrile.</li>
<li>The solution was agitated at room temperature for 30 min; then, binding buffer was added to adjust the overall volume to 200 µL.</li>
<li>The mixture was shaken for 1 more hour at room temperature (1000 rpm).</li>
</ol>
</li>
<li>
<h2>The binding efficiency of DNA, which was in the range of 86 to 91%</h2>
</li>
</ul>
</div>
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