Name: synthesis and characterization of amphiphilic gold nanoparticles
Uploaded: 2019-07-02T13:00:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of Amphiphilic Gold Nanoparticles at JoVE.com

Z.P.G. and F.S. thank the Swiss National Science Foundation and, specifically, NCCR &#39;Molecular Systems Engineering&#39;. Z.L. and F.S. thank the support of the Swiss National Science Foundation Division II grant. All authors thank Quy Ong for fruitful discussions and for proofreading the manuscript.

1. Synthesis of 11-mercapto-1-undecanesulfonate (MUS)
NOTE: This protocol can be used at any scale desired. Here, a 10 g scale-of-product is described.
<ol>
	<li>Sodium undec-10-enesulfonate 
	<ol>
		<li>Add 11-bromo-1-undecene (25 mL, 111.975 mmol), sodium sulfite (28.75 g, 227.92 mmol), and benzyltriethylammonium bromide (10 mg) to a mixture of 200 mL of methanol (MeOH) and 450 mL of deionized (DI) water (4:9 v/v MeOH:H2O ratio) in a 1 L round-bottom flask.</li>...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+tail+protrusions+mediate+the+insertion+of+nanoparticles+into+model+cell+membranes.">Lipid tail protrusions mediate the insertion of nanoparticles into model cell membranes.</a> Nature Communications. 5, 4482-4493 (2014).</li><li>Cagno, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+non-toxic+antiviral+nanoparticles+with+a+virucidal+inhibition+mechanism.">Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism.</a> Nature Materials. 17 (2), 195-203 (2018).</li><li>Uzun, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-soluble+amphiphilic+gold+nanoparticles+with+structured+ligand+shells.">Water-soluble amphiphilic gold nanoparticles with structured ligand shells.</a> Chemical Communications. 2 (2), 196-198 (2008).</li><li>Huang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+stability+of+self-assembled+monolayer-coated+gold+nanoparticles:+The+effects+of+surface+compositional+and+structural+heterogeneity.">Colloidal stability of self-assembled monolayer-coated gold nanoparticles: The effects of surface compositional and structural heterogeneity.</a> Langmuir. 29 (37), 11560-11566 (2013).</li><li>Carney, R. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+energy+change+for+insertion+of+charged,+monolayer-protected+nanoparticles+into+lipid+bilayers.">Free energy change for insertion of charged, monolayer-protected nanoparticles into lipid bilayers.</a> Soft Matter. 10 (4), 648-658 (2014).</li><li>Ong, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Ligand+Shell+for+Mixed-Ligand+Coated+Gold+Nanoparticles.">Characterization of Ligand Shell for Mixed-Ligand Coated Gold Nanoparticles.</a> Accounts of Chemical Research. 50 (8), 1911-1919 (2017).</li><li>Marbella, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+techniques+for+noble+metal+nanoparticles.">NMR techniques for noble metal nanoparticles.</a> Chemistry of Materials. 27 (8), 2721-2739 (2015).</li><li>Templeton, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivity+of+Monolayer-Protected+Gold+Cluster+Molecules+Steric+Effects.">Reactivity of Monolayer-Protected Gold Cluster Molecules Steric Effects.</a> Journal of American Chemical Society. 120 (8), 1906-1911 (1998).</li><li>Harkness, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+mass+spectrometry+strategy+for+the+relative+quantitation+of+ligands+on+mixed+monolayer-protected+gold+nanoparticles.">A structural mass spectrometry strategy for the relative quantitation of ligands on mixed monolayer-protected gold nanoparticles.</a> Analytical Chemistry. 82 (22), 9268-9274 (2010).</li></ol>

This protocol describes first the synthesis of MUS ligand and, then, the synthesis and characterization of amphiphilic MUS:OT gold nanoparticles. Synthesizing MUS with minimal salt content enables a better reliability of the stoichiometric ratio between the ligands during the nanoparticle synthesis, which is a key factor for the reproducible synthesis of MUS:OT nanoparticles with a target hydrophobic content (Figure 8). The use of methanol as a common solvent for MUS and OT, along with the s...

The ligand shell of gold nanoparticles can be engineered to exhibit several different properties that can be applied to address challenges in biomedicine1,2,3,4. Such versatility allows for the control of the intermolecular interactions between nanoparticles and biomolecules5,6,7. Hydrophobicity and charge play a decisive role, as well as other surface parameters that affect how nanoparticles interact with biomolecules5,8,9. To tune the nanoparticles&#39; surface properties, the choice of thiolate molecules that make up the ligand shell offers a myriad of possibilities, according to the characteristics sought. For example, a mixture of ligand molecules with hydrophobic and hydrophilic (e.g.,&#160;charged) end groups are often used to generate amphiphilic nanoparticles10,11.
One prominent example of this type of nanoparticles is protected by a mixture of OT and MUS (hereafter called MUS:OT nanoparticles) that has been shown to possess many relevant properties12,13,14. First, with a ligand shell composition of 66% MUS (hereafter 66:34 MUS:OT), the colloidal stability of the nanoparticles is high, reaching up to 33% in weight in deionized water, as well as in phosphate-buffered saline (1x, 4 mM phosphate, 150 mM NaCl)15. Moreover, these particles do not precipitate at relatively low pH values: for example, at pH 2.3 and with salt concentrations of 1 M NaCl15, these nanoparticles remain colloidally stable for months.The stoichiometric ratio between the two molecules on the ligand shell is important because it dictates the colloidal stability in solutions with a high ionic strength16.
These particles have been shown to traverse the cell membrane without porating it, via an energy-independent pathway1,12. The spontaneous fusion between these particles and lipid bilayers underlies their diffusivity through cell membranes17. The mechanism behind this interaction is the minimization of contact between a hydrophobic solvent-accessible surface area and water molecules upon fusion with lipid bilayers18. Compared to all-MUS nanoparticles (nanoparticles having only the MUS ligand on their shell), the higher hydrophobicity on mixed MUS:OT nanoparticles (for example, at a 66:34 MUS:OT composition) increases the span of the core diameter that can fuse with lipid bilayers18.Different self-assembly organizations of the ligand shell correlate to distinct binding modes of 66:34 MUS:OT nanoparticles with various proteins, such as albumin and ubiquitin, when compared to all-MUS particles19.Recently, it has been reported that 66:34 MUS:OT nanoparticles can be utilized as a broad-spectrum antiviral agent that irreversibly destroys the viruses because of multivalent electrostatic bindings of MUS ligands and nonlocal couplings of OT ligands to capsid proteins14.In all these cases, it has been found that the hydrophobic content, as well as the core size of the nanoparticles, determines how these bio-nano interactions take place. These diverse properties of MUS:OT nanoparticles have prompted many computer simulation studies that aimed to clarify the mechanisms underpinning the interactions between MUS:OT particles and various biological structures such as lipid bilayers20.
The preparation of MUS:OT-protected Au nanoparticles poses a few challenges. First, the charged ligand (MUS) and the hydrophobic ligand (OT) are immiscible. Thus, the solubility of the nanoparticles and of the ligands needs to be taken into account throughout the synthesis, as well as during characterization. Additionally, the purity of the MUS ligand molecules&#8212;specifically, the content of inorganic salts in the starting material&#8212;influences the quality, reproducibility, as well as the short- and long-term colloidal stability of the nanoparticles.
Here, a detailed synthesis and characterization of this class of amphiphilic gold nanoparticles protected by a mixture of MUS and OT are outlined. A protocol for the synthesis of the negatively charged MUS ligand is reported to ensure the purity and, hence, the reproducibility of different nanoparticle syntheses. Then, the procedure to generate these nanoparticles, based on a common one-phase synthesis, followed by thorough purification, is reported in detail. Various necessary characterization techniques21, such as TEM, UV-Vis, TGA, and NMR, have been combined to obtain all the necessary parameters for any further biological experiments.

<table>
	<tbody>
		<tr>
			<td>11-bromo-1-undecene</td>
			<td>Sigma Aldrich</td>
			<td>467642-25 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Sulfite</td>
			<td>Sigma Aldrich</td>
			<td>S0505-250 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyltriethyl-ammonium bromide</td>
			<td>Sigma Aldrich</td>
			<td>147125-25 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>BDH1135-2.5 LP</td>
			<td></td>
		</tr>
		<tr>
			<td>DI water</td>
			<td>Millipore</td>
			<td>ZRXQ003WW</td>
			<td>Deionized water</td>
		</tr>
		<tr>
			<td>1 L round bottom flask</td>
			<td>DURAN</td>
			<td>24 170 56</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>1.00930 EMD Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirring bar</td>
			<td>Sigma Aldrich</td>
			<td>Z329207,</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning High Vacuum Grease</td>
			<td>Sigma Aldrich</td>
			<td>Z273554 ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtering flask</td>
			<td>DURAN</td>
			<td>20 201 63</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtering Buchner Funnel</td>
			<td>FisherSci</td>
			<td>11707335</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol &#62;99.8%, ACS, Reagent</td>
			<td>VWR</td>
			<td>2081.321DP</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium dioxide</td>
			<td>Sigma Aldrich</td>
			<td>151882 ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioacetic acid 96%</td>
			<td>Sigma Aldrich</td>
			<td>T30805 ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon black</td>
			<td>Sigma Aldrich</td>
			<td>05105-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite</td>
			<td>Sigma Aldrich</td>
			<td>D3877 SIGMA-ALDRICH</td>
			<td>Filtration medium</td>
		</tr>
		<tr>
			<td>Condenser</td>
			<td>Sigma Aldrich</td>
			<td>Z531154</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, ACS reagent 37%</td>
			<td>Sigma Aldrich</td>
			<td>320331 SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, BioXtra, pellets (anhydrous)</td>
			<td>Sigma Aldrich</td>
			<td>S8045 SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>VWR</td>
			<td>525-0155P</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL round bottom flask</td>
			<td>DURAN</td>
			<td>24 170 37</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL round bottom flask</td>
			<td>DURAN</td>
			<td>24 170 46</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid, fACS reagent 70%</td>
			<td>Sigma Aldrich</td>
			<td>438073 SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold(III) chloride trihydrate &#62;99.9% trace metal basis</td>
			<td>Sigma Aldrich</td>
			<td>520918 ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>1-octanethiol &#62;98.5%</td>
			<td>Sigma Aldrich</td>
			<td>471836 ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Borohydride purum p.a.&#62;96%</td>
			<td>Sigma Aldrich</td>
			<td>71320 ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>Separatory funnel</td>
			<td>SIgma Aldrich</td>
			<td>Z330655 SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Funnel</td>
			<td>DURAN</td>
			<td>21 351 46</td>
			<td></td>
		</tr>
		<tr>
			<td>2V folded filtering papers</td>
			<td>Whatman</td>
			<td>1202-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon filters</td>
			<td>Merck</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine, ACS reagent, &#62;99.8%, solid</td>
			<td>Sigma Aldrich</td>
			<td>207772 SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm NMR-Tubes, Type 5HP (high precision)</td>
			<td>Armar</td>
			<td>32210.503</td>
			<td>Length 178 mm</td>
		</tr>
		<tr>
			<td>Methanol-d4 99.8 atom%D</td>
			<td>Armar</td>
			<td>16400.2035</td>
			<td></td>
		</tr>
		<tr>
			<td>TGA crucible</td>
			<td>Thepro</td>
			<td>9095-9270.45</td>
			<td></td>
		</tr>
		<tr>
			<td>400 mesh carbon supported copper grid</td>
			<td>Electron Microscopy Science</td>
			<td>CF400-Cu</td>
			<td></td>
		</tr>
		<tr>
			<td>quartz cuvette</td>
			<td>Hellma Analytics</td>
			<td>100-1-40</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis and characterization of amphiphilic gold nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

The reaction steps to synthesize MUS are shown in Figure 1. The 1H NMR spectra of the product of each step are represented in Figure 2. The synthesis workflow of the binary MUS:OT amphiphilic gold nanoparticles is described in Figure 3. Following the synthesis, the workup of the nanoparticles consisted of washing the particles several times with ethanol and DI water. Prior to any character...

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Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

This protocol addresses the batch-to-batch reproducibility of sulfonate and amphiphilic gold nanoparticles that our laboratory uses in experiments with cells, viruses, and proteins. This technique addresses the inorganic contaminants common to this type of synthesis. It also introduces checks and balances after each step to ensure the reproducibility of amphiphilic gold nanoparticles.This technique is laborious and requires patience. The level of difficulty depends on scale, so start at a small scale, and familiarize yourself with each instrument and step before scaling up. First, add 11-bromo-1-undecene, sodium sulfite, and benzyltriethylammonium bromide to 200 milliliters of methanol and 450 milliliters of deionized water, in a one liter round bottom flask.Reflux the reaction mixture at 102 degrees Celsius for 48 hours until the solution becomes colorless. After workup, suspend the isolated white powder in 400 milliliters of methanol in a round bottom flask. Using a filtering flask and borosilicate filter, filter the solution to remove the methanol insoluble inorganic byproducts.Next, dissolve approximately 30 grams of sodium undec-10-enesulfonate in 500 milliliters of methanol, in a one-liter round bottom flask. Add 2.6 times excess of thioacetic acid to the solution, and stir it in front of a 250 watt UV lamp overnight. Once the reaction is complete, evaporate the methanol completely.Dry the powder under vacuum, and then disperse it in diethyl ether. Filter the mixture and wash the solid product with diethyl ether to remove any excess thioacetic acid until no more colored substances appear in the diethyl ether supernatant. After drying the solid under high vacuum, dissolve it in methanol, yielding a yellow to orange solution.Next, add three grams of carbon black to the solution and mix vigorously. Then, filter the mixture through Celite, covering 2/3 of a fluted filter paper. To synthesize MUS, combine approximately 400 milliliters of one molar hydrochloric acid and 35 grams of sodium 11-acetylthio-undecanesulfonate in a one liter round bottom flask.Reflux the mixture at 102 degrees Celsius for 12 hours to cleave the thioacetate group and obtain a thiol. On the following day, transfer the product to a two liter round bottom flask. To keep the solution acidic and prevent crystallization of inorganic salts, add 200 milliliters of one molar sodium hydroxide, and 400 milliliters of deionized water to the flask to give a final volume of one liter.Store the clear solution at four degrees Celsius overnight to crystallize the product as fine solids that are viscous when wet. On the following day, decant the clear supernatant. Then, transfer the product to 50 milliliter centrifuge tubes and centrifuge for five minutes at 4000 times g.Following centrifugation, decant the supernatant into another round bottom flask. Transfer the white pellets to centrifuge tubes, and dry under high vacuum to obtain methanol-soluble MUS in about 30%yield. Weigh 177.2 milligrams of gold(III)chloride trihydrate in a small glass vial.Following this, dissolve 87 milligrams of MUS in 10 milliliters of methanol, in a 20 milliliter glass vial. Sonicate the solution in an ultrasonic bath until no solid material is visible, to ensure complete dissolution. Add 26 microliters of 1-Octanethiol to the methanol solution, and agitate it to mix the ligands.Add 500 milligrams of sodium borohydride to 100 milliliters of ethanol in a 250 milliliter round bottom flask. Stir vigorously until the solution is clear. Dissolve the gold salt in 100 milliliters of ethanol in a 500 milliliter round bottom flask and stir at 800 RPM until the gold salt dissolves completely.Next, add the ligand solution to the reaction mixture. Wait 15 minutes for the formation of the gold-thiolate complex, which is indicated by a color change from translucent yellow to turbid yellow. Add the previously prepared sodium borohydride solution drop-wise to the reaction mixture, using a separatory funnel.Adjust the interval time of the drops so that the addition of sodium borohydride takes about an hour. Once the sodium borohydride addition is complete, remove the addition funnel and allow the reaction mixture to stir for another hour. Then, remove the magnetic stir bar using a magnet placed on the outside of the round bottom flask.Store the reaction mixture at four degrees Celsius overnight to precipitate the nanoparticles. After decanting the supernatant ethanol, transfer the remaining precipitant to 50 milliliter centrifuge tubes and centrifuge for three minutes at 4000 times g. After centrifugation, decant the supernatant.Disperse the nanoparticles again with ethanol by vortexing. Then, centrifuge the samples again. Dry the nanoparticles under vacuum to remove the residual ethanol.To clean the nanoparticles from free hydrophilic ligands, dissolve the precipitates in 15 milliliters of deionized water, and transfer the solutions to centrifuge tubes with filtration membranes of 30 kilodalton cutoff molecular weight. Concentrate the nanoparticle solutions by centrifugation for five minutes at 4000 times g. Following centrifugation, add 15 milliliters of deionized water, and centrifuge to concentrate again.To turn the nanoparticles into a manageable powder, freeze-dry the remaining aqueous solution. To characterize the nanoparticles by ligand ratio, prepare a 150 milligram per milliliter methanol-d4 solution of iodine. Add 600 microliters of the solution to approximately five milligrams of nanoparticles in a glass vial to etch the nanoparticles.Wrap the cap of the vial with paraffin film and sonicate it in an ultrasonic bath for 20 minutes. Then, transfer the solution to an NMR tube and acquire a proton NMR spectrum with 32 scans. The MUS synthesis is shown here.The proton NMR spectra of the product of each step are represented here. The synthesis workflow of the binary MUS Octanethiol amphiphilic gold nanoparticles is described here. Prior to characterization, the cleanliness of the nanoparticles from unbound free ligands was monitored by proton NMR.The size distribution of the nanoparticles was characterized by TEM, which shows that the average diameter is 2.4 nanometers pointing to approximately 18.08 nanometers squared of surface area, and 7.23 nanometers cubed of volume per particle. Localized surface plasma and resonance absorption was measured by acquiring UV-vis spectra. Representative proton NMR spectra with peak assignments and integration for determining the ligand ratio in the iodine-etched nanoparticles are shown here.The NMR spectrum of the nanoparticles showed that the ratio of MUS to Octanethiol is 85 to 15. The surface coverage of the nanoparticles was examined by TGA. Based on TGA data, ligand density can be estimated to be 4.8 ligands per nanometer squared.The stoichiometric ratios versus the NMR ratios of Octanethiol resulting from various syntheses are compared here. The most important things to remember in this procedure are to, on one side, the removal of inorganic impurities while preparing MUS ligands, and on the other side, the workup of the nanoparticles. This procedure is amenable to various combinations of ligands, but make sure to always characterize each batch individually.One question this procedure can answer, for example, is to what extent the ligand ratio corresponds to the stoichiometry found on the surface of the nanoparticles. Scaling up the production of the ligand and proving the batch-to-batch reproducibility of the nanoparticles, has allowed us to tackle important questions related to biology and medicine. For example, we have established the virucidal properties of these nanoparticles.Please, work carefully when using the UV lamp, and always wear protective gloves when handling liquid nitrogen. Always follow chemical safety rules.

Research

JoVE Journal

Chemistry

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