This work is supported by Ministry of Science and Technology, Taiwan, Republic of China, under grant number MOST 105-2221-E-011-008, and also by Advanced-Connectek Inc., Taipei, Taiwan, ROC under contract number RD Ref. No. 6749 and Dept. Ref. No. 011 through the Graduated Institute of Electro-Optical Engineering, National Taiwan University of Science and technology.

Caution: Use latex gloves, protective eyeglasses, and a laboratory coat for performing the experiment. Whenever using the biosafety cabinet, turn on the cabinet fan and keep the cabinet door half-closed.
1. Preparation of Glass Beads
<ol>
	<li>Place 100 glass beads of 3 mm diameter in a 100-mL laboratory bottle, and then cap it tightly.</li>
	<li>Autoclave the contents at 120 &#176;C for 10 min.</li>
	<li>Leave the bottle to cool down to room temperature, then place it in the biosafety cabinet.</li>
</ol>
2. Preparation of Lysogeny Broth (LB)
<ol>
	<li>Dissolve 8 g powder of LB-Lennox broth in a 500-mL laboratory bottle with 400 mL of water.</li>
	<li>Stir the contents with a PTFE magnetic stirring bar for 20 min, and then cap it tightly.</li>
	<li>Autoclave the contents at 120 &#176;C for 10 min.</li>
	<li>Leave the solution to cool down to room temperature and place it in the biosafety cabinet.</li>
	<li>Using a pipette, aliquot the broth into eight 15-mL centrifuge tubes in the biosafety cabinet (12.5 mL each).</li>
	<li>Aliquot the remaining broth into three 100-mL laboratory bottles in the biosafety cabinet (100 mL each). Cap the three bottles tightly. Keep them in the biosafety cabinet.</li>
</ol>
3. Culture of Shewanella algae
<ol>
	<li>Use the deep-frozen cryopreserved strain.</li>
	<li>In the biosafety cabinet, pick out 1 mL of the frozen material from the frozen tube with a stainless-steel spatula, and place it into a centrifuge tube prepared in step 3.5.</li>
	<li>Incubate the cultures for 24 h in a 37 &#176;C incubator.</li>
</ol>
4. Preparation of LB-Lennox (Broth with Agar) Petri Dishes
<ol>
	<li>Dissolve two tablets of LB-Lennox (broth with agar) into a 100-mL laboratory bottle with 100 mL of water.</li>
	<li>Stir the contents with a PTFE magnetic stirring bar for 20 min and then cap it tightly.</li>
	<li>Autoclave the contents at 120 &#176;C for 10 min.</li>
	<li>In the biosafety cabinet, aliquot by hand 100 mL of solution into 4 Petri dishes, ensuring each receive ~25 mL. Leave the solution to cool down to room temperature.</li>
</ol>
5. Preparation of Monoclonal Bacteria
<ol>
	<li>In the biosafety cabinet, label the three bottles prepared in step 2.6, #1, #2 and #3, respectively.</li>
	<li>Pipette 0.1 mL of the resultant bacterial suspension in step 3.3 into bottle #1. Cap the bottle and swing it by hand for 1 min to get a homogenous solution.</li>
	<li>Pipette 0.1 mL of the resultant bacterial liquid in step 5.2 into bottle #2. Cap the bottle and swing it by hand for 1 min to get a homogenous solution.</li>
	<li>Pipette 0.1 mL of the resultant bacterial liquid in step 5.3 into bottle #3. Cap the bottle and shake it by hand for 1 min to get a homogenous solution.</li>
	<li>Pipette the liquid in bottle #3 into the 4 Petri dishes prepared in step 4.4, using a volume of 0.02 mL each.</li>
	<li>Put the glass beads prepared in step 1.3 into the 4 Petri dishes used, 4 beads in each dish.</li>
	<li>Close the lids of the Petri dishes and shake them by hand for 1 min.</li>
	<li>Turn the Petri dishes upside down and incubate in a 37 &#176;C incubator for 24 h.</li>
</ol>
6. Multiplication of Monoclonal Bacteria
<ol>
	<li>Fetch 7 tubes prepared in step 2.5.</li>
	<li>Pick out the resultant monoclonal bacteria from the 4 Petri dishes prepared in step 5.8 with a stainless steel-spatula, and put them into 7 tubes separately.</li>
	<li>Leave the 7 tubes in a 37 &#176;C incubator for 24 h.</li>
	<li>Pick out the one with the largest light scattering using the visual colorimetric method.</li>
</ol>
7. Preparation of LB-Lennox Broth with Glucose and Salt
<ol>
	<li>Put 10 g of LB-Lennox broth, 10 g of NaCl, and 10 g of glucose into a 500-mL laboratory bottle. Add water until the volume reaches 450 mL.</li>
	<li>Stir the contents with a PTFE magnetic stirring bar for 20 min.</li>
	<li>Autoclave the contents at 120 &#176;C for 10 min.</li>
</ol>
8. Preparation of Sodium Tungstate
<ol>
	<li>Put 16.5 g of Sodium Tungstate Na2WO4&#65294;2H2O into a 100-mL laboratory bottle with a stainless-steel spatula. Add water until the volume reaches 50 mL.</li>
	<li>Stir the contents with a PTFE magnetic stirring bar for 20 min.</li>
	<li>Autoclave the contents at 120 &#176;C for 10 min.</li>
	<li>In the biosafety cabinet, get filtrate via a vacuum fiberglass filter with pores of 1 &#181;m.</li>
</ol>
9. Preparation of LB with Glucose, Salt, and Sodium Tungstate
<ol>
	<li>In the biosafety cabinet, pour the filtrate gained in step 8.4 by hand into the solution with glucose and salt prepared in step 7.3.</li>
	<li>In the biosafety cabinet, aliquot with a pipette the 500 mL resultant solution in step 9.1 into 10 x 50 mL centrifuge tubes.</li>
</ol>
10. Culture of Bacteria
<ol>
	<li>In the biosafety cabinet, fetch the liquid prepared in step 6.4 and aliquot it with a pipette into the 10 test tubes prepared in step 9.2, with each tube receiving 0.05 mL.</li>
	<li>Incubate the 10 tubes in a 37 &#176;C incubator for 120 h.</li>
</ol>
11. Harvest of BME minerals
<ol>
	<li>Ultrasonicate each of the 10 tubes in step 9.2 at 20 KHz with 150 W for 1 h.</li>
	<li>Centrifuge the tubes at 2,025 x g for 1 h.</li>
	<li>Remove the clear liquid in the tubes with a pipette, add water, and then repeat steps 11.1 and 11.2 one more time.</li>
	<li>Remove the clear liquid in the tubes with a pipette, add alcohol, and then ultrasonicate them at 20 KHz with 150 W for 1 h.</li>
	<li>Centrifuge the tubes at 2,025 x g for 1 h.</li>
	<li>Repeat steps 11.4 and 11.5 one more time</li>
	<li>Harvest BME minerals by removing the clear liquid in the tubes with a pipette; afterwards, immediately cap the tubes without running any drying process.</li>
</ol>
12. Oscillating Temperature with Pandoraea sp. and Molybdate
<ol>
	<li>Culture Pandoraea sp. in the same way as in steps 2, 3, 4, 5, and 6 for Shewanella algae. The result of this step corresponds to that of step 6.4.</li>
	<li>Make LB broth with both glucose and salt in the same way as in steps 7, 8, and 9, except that the 16.5 g of sodium tungstate in step 7.1 is replaced with 12 g of sodium molybdate, Na2MoO4 &#183; 2H2O. The result of this step corresponds to that of step 9.2.</li>
	<li>In the biosafety cabinet, fetch the liquid prepared in step 12.1, and aliquot it with a pipette into the 10 tubes prepared in step 12.2, with each tube receiving 0.05 mL.</li>
	<li>Incubate the 10 tubes in step 12.3 under oscillating temperatures for 120 h in a reciprocal shaking bath, oscillating the temperature 5 times between 25 &#176;C and 37 &#176;C, with each temperature lasting for 12 h.</li>
</ol>

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The authors have nothing to disclose.

Concerning the self-consistency of the experimental results, the preparation and multiplication of monoclonal bacteria are critical. This experiment, different from the template synthesis experiments15,16, employed bioactive gram-negative bacteria. To get a single wall, we chose prokaryotic bacteria instead of eukaryotic bacteria like yeast15. To achieve a spherical shape with a length-to-diameter ratio of 1:1, instead of a larger length-to-diameter ratio16, we fed bacteria with a much higher concentration of oxyanions to manipulate them to shrink into a spherical shape, making microcapsules with a single, round, and thin wall (&#60;30 nm).
Since the BME mainly relies on adjusting the concentration of oxyanions to control the metabolism of bacteria, it features two limitations. Firstly, the concentration of oxyanions is limited by the solubility, though the concentration should be as high as possible. Second, most bacterial metabolisms will stop at a temperature over 45 &#176;C or under 5 &#176;C, respectively the upper and lower bounds of our experiment.
Despite these two limitations, the BME has great potential for making metal oxide materials of practical interest. To substantiate this claim, we are going to try this method to make zirconium microcapsules and iron microcapsules&#8212;the former being a good candidate material for artificial bones, and the latter for drug delivery.

Metal oxide nanoparticles are exploited for drug delivery1, construction artificial bones2, heterogeneous catalysis3, field emission4,5, solar cells6, gas sensors7, and lithium batteries8. For practical applications, the mechanical strength of both nanocrystals and their microstructure are crucial. Among the microstructures, hollow shell structures can be used to create lightweight, mechanically robust materials9. Among hollow shell structures, a spherical shape is known to be more rigid than an ellipsoidal shape; the latter has a larger length-to-diameter ratio than the former10,11. This work describes a protocol for synthesizing spherical microcapsules via bacteria with a non-toxic method under an ambient condition, which contrasts with the alternative methods, including the template synthesis method12, ultrasonic-spray-assisted synthesis method13 and hydrothermal method14. Some of the alternative methods require templates12, some a temperature as high as 500 &#176;C13, and some a high pressure14. As for the resulting structure, the template synthesis method utilizing the yeast template brings about a core-shell structure15, instead of one with a single wall, and the one utilizing the E. coli template produces a structure with length-to-diameter ratio of 1.7:0.8, and is not spherical.16.
In this work, we have made metal oxide microcapsules with a single wall and of spherical shape under an ambient condition by exploiting bacterial metabolism. In bacterial glycolysis, a chemical process that metabolizes carbon sources, like glucose and lactose, carbon sources are considered to be the origin of the reducing power generated therein. We manipulated bacterial metabolism by adjusting the concentration of carbon sources to achieve desired ends. This method is environment-friendly, using non-toxic agents and consuming much less electricity power. Lastly, this method allows the mass production of microcapsules simply by increasing the volume of broth.
Prior to the method, there have been another two methods utilizing bacterial metabolism to make minerals: biologically induced mineralization (BIM)17 and biologically controlled mineralization (BCM)18. Neither BIM nor BCM can be used for making sodium tungstate and molybdate tungstate microcapsules like our process, which is designated as the bacterial mineral excretion (BME)19. In this experiment, the shape of microcapsules can be controlled to have a length-to-diameter ratio from 10:1 to 1:1, and the size of nanoparticle grains that form the shells can be adjusted ranging from 15 nm to 110 nm.

<table><tbody><tr><td>LB(Lennox)broth with agar tablets</td><td>Sigma-Aldrich</td><td>L7075</td><td>1 tablet for 50 mL broth with agar</td></tr><tr><td>LB (Lennox) broth</td><td>Sigma-Aldrich</td><td>L3022-1KG</td><td>LB (Lennox) powder 1 kg</td></tr><tr><td>Dextrose anhydrous</td><td>Nihon Shiyaku Reagent</td><td>PL 78695</td><td>glucose</td></tr><tr><td>Sodium Tungstate</td><td>Nihon Shiyaku Reagent</td><td>PL 76050</td><td>Na&lt;sub&gt;2&lt;/sub&gt;WO&lt;sub&gt;4&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sodium Molybdate</td><td>Nihon Shiyaku Reagent</td><td>PL103564</td><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sodium Chloride</td><td>Nihon Shiyaku Reagent</td><td>PL 68131</td><td>NaCl</td></tr><tr><td>Ethanol 99.5%</td><td>Acros organics</td><td>AC615090040</td><td>CH&lt;sub&gt;3&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;OH</td></tr><tr><td>Water</td><td>Made in our university</td><td></td><td>de-ionlized water</td></tr><tr><td>Autoclave</td><td>Tomin Medical Equipmenco, Ltd., Taipei City, Taiwan, ROC</td><td>TM-329</td><td>heat to 120 &amp;deg;C for 10 min</td></tr><tr><td>Centrifuge</td><td>Digit System Laboratory System, New Taipei City, Taiwan, ROC</td><td>DSC302SD</td><td>centrifuge at 2025 x g</td></tr><tr><td>-80 &amp;deg;C Refrigerator</td><td>Panasonic</td><td>MDF-U3386S</td><td>Use to deep-freeze cryopreserve strain</td></tr><tr><td>Ultrasonic Homogenizer Sonicator Processor Cell Disruptor</td><td>Lenox</td><td>UPS-150</td><td>frequency 20 KHz power 150 W</td></tr><tr><td>Incubator</td><td>Customer made</td><td>custom made</td><td>heat to 40 &amp;deg;C or cool to 18 &amp;deg;C with time cotrol</td></tr><tr><td>Reciprocal shaking baths</td><td>Kingtech Scientific Co., Ltd</td><td>WBS-L</td><td></td></tr><tr><td>Digital Stirring Hot Plate</td><td>Corning</td><td>#6797-620D</td><td>use with PTFE magnetic stirring bar</td></tr><tr><td>Biosafety cabinet</td><td>Zong Yen co., LTD</td><td>ZYBH-420</td><td>All bacteria related process are done here</td></tr><tr><td>Scanning electron microscope</td><td>JEOL</td><td>JSM-6500F</td><td>SEM Images</td></tr><tr><td>50 mL centrifudge tube</td><td>Falcon</td><td>14-432-22</td><td></td></tr><tr><td>15 mL centrifudge tube</td><td>Falcon</td><td>14-959-53A</td><td></td></tr><tr><td>Laboratory bottle 100 mL</td><td>Duran</td><td>21 801 24 5</td><td></td></tr><tr><td>Laboratory bottle 500 mL</td><td>Duran</td><td>21 801 44 5</td><td></td></tr><tr><td>Stainless steel spatula</td><td>Chemglass</td><td>CG-1981-10</td><td></td></tr><tr><td>PTFE Disposable Stir Bars</td><td>Fisher</td><td>S68066</td><td></td></tr><tr><td>Plastic Petri Dishes</td><td>Fisher</td><td>S33580A</td><td></td></tr><tr><td>Shewanella algae</td><td>Courtesy of author #3</td><td>Courtesy of author #3</td><td></td></tr><tr><td>Pandoraea sp.</td><td>Courtesy of author #3</td><td>Courtesy of author #3</td><td></td></tr></tbody></table>

synthesizing sodium tungstate and sodium molybdate microcapsules via bacterial mineral excretion

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the two metal oxides&#39; corresponding nanoparticles&#8212;the former being as small as 22 nm and the latter 15 nm. We fed two strains of bacteria, Shewanella algae and Pandoraea sp., with various concentrations of tungstate or molybdate ions. The concentrations of tungstate and molybdate were adjusted to make microcapsules of different length-to-diameter ratios. We found that the higher the concentration the smaller the nanoparticles were. The nanoparticles came in with three length-to-diameter ratios: 10:1, 3:1 and 1:1, which were achieved by feeding the bacteria respectively with a low concentration, a medium concentration, and a high concentration. The images of the hollow microcapsules were taken via the scanning electron microsphere (SEM). Their crystal structures were verified by X-ray diffraction (XRD)&#8212;the crystal structure of molybdate microcapsules is Na2MoO4 and that of tungstate microcapsules is Na2WO4 with Na2W2O7. These syntheses all were accomplished under a near ambient condition.

Figure 1 shows genuine spherical microcapsules. Both the two strains of bacterium, Shewanella algae and Pandoraea sp., originally have a length-to-diameter ratio of 3:1. For achieving the length-to-diameter ratio of 1:1, a high concentration (&#62;100 mM) of metal oxyanions is required. A low concentration (&#60;5 mM) of oxyanions can result in a length to diameter ratio of 10:1, as that in Figure 2, which may result from the influx of the oxyanions, blocking the binary fission of bacteria. Lastly, for achieving a length-to-diameter ratio of 3:1, like that in Figure 3, a medium concentration (~20 mM) of oxyanions is needed. The formation of spherical shells, with a length-to-diameter ratio of 1:1, may be brought about by bacterial drives that make themselves shrink their surface area to balance the intake of oxyanions while diffusing oxyanions through the cell membrane. The three figures together indicate the length-to-diameter ratio may be tuned to from 10:1 to 1:1 simply by adjusting the concentration of oxyanions.
Figure 4 and Figure 5 show the nanoparticle grains of sodium molybdate in different sizes: the smaller one being 15 nm, and the larger one 110 nm. Note that in Figure 5, on the non-shattered shells, particles of 110 nm can still be chained up to each other, forming porous shells. The larger one was gained through oscillating the temperature of the culturing broth 5 times between 25 &#176;C and 37 &#176;C, with each temperature lasting for 12 h. During the temperature oscillation, grains of different sizes can not only be produced but also maintain the micro-spherical structure, which means we can make microcapsules with different grain sizes, from 15 nm to 110 nm, just by controlling the broth temperature.
Figure 6 shows the broken wall with larger grains staying next to the opening of the wall. The wall thickness is about 22 nm and the larger grain is about 40-60 nm. The difference in size may result from different metabolic processes, which are not yet identified.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57022/57022fig1.jpg" /> 
Figure 1: The SEM image of hollow spherical shells with a length-to-diameter ratio of 1:1. This structure was made of sodium tungstate excreted by Shewanella algae with glucose as the carbon source. Reprinted with permission from ECS J. of Solid State Sci. and Tech., 6(3), N3113 (2017). Copyright 2017, The Electrochemical Society. <a href="https://cloudflare.jove.com/files/ftp_upload/57022/57022fig1large.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/57022/57022fig2.jpg" /> 
Figure 2: The SEM image of hollow long filament shells with a length-to-diameter ratio of 10:1. This structure was made of sodium molybdate excreted by Pandoraea sp. with glucose as the carbon source. Reprinted with permission from ECS J. of Solid State Sci. and Tech., 6(3), N3113 (2017). Copyright 2017, The Electrochemical Society. <a href="https://cloudflare.jove.com/files/ftp_upload/57022/57022fig2large.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/57022/57022fig3.jpg" /> 
Figure 3: The SEM image of broken hollow rod-shaped shells with a length-to-diameter ratio of 3:1. This structure was made of sodium tungstate excreted by Shewanella algae with glucose as the carbon source. Reprinted with permission from ECS J. of Solid State Sci. and Tech., 6(3), N3113 (2017). Copyright 2017, The Electrochemical Society. <a href="https://cloudflare.jove.com/files/ftp_upload/57022/57022fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57022/57022fig4.jpg" /> 
Figure 4: The SEM image of shattered sodium molybdate shells with a grain particle size of 15 nm. <a href="https://cloudflare.jove.com/files/ftp_upload/57022/57022fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57022/57022fig5.jpg" /> 
Figure 5: The SEM image of shattered and non-shattered sodium molybdate shells with a grain particle size of 110 nm. <a href="https://cloudflare.jove.com/files/ftp_upload/57022/57022fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57022/57022fig6.jpg" /> 
Figure 6: The SEM image of broken hollow shells with a length-to-diameter ratio of 1:1. This structure was made of sodium tungstate excreted by Shewanella algae with glucose as the carbon source. Granules with a size about 40-60 nm hang outside the shell right next to a big hole, while the shell itself is made of granules with a size about 22 nm. Reprinted with permission from ECS J. of Solid State Sci. and Tech., 6(3), N3113 (2017). Copyright 2017, The Electrochemical Society. <a href="https://cloudflare.jove.com/files/ftp_upload/57022/57022fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion at JoVE.com

Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion

This work presents a protocol for manufacture sodium tungstate and sodium molybdate microcapsules via bacteria and their corresponding nanoparticles.

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the ...

synthesizing-sodium-tungstate-sodium-molybdate-microcapsules-via

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.5K Views.  National Taiwan University of Science and Technology. This work presents a protocol for manufacture sodium tungstate and sodium molybdate microcapsules via bacteria and their corresponding nanoparticles.

Video: Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion

Reverse Microemulsion-mediated Synthesis of Monometallic and Bimetallic Early Transition Metal Carbide and Nitride Nanoparticles

A reverse microemulsion is used to encapsulate monometallic or bimetallic early transition metal oxide nanoparticles in microporous silica shells. The silica-encapsulated metal oxide nanoparticles are then carburized in a methane/hydrogen atmosphere at temperatures over 800 &#176;C to form silica-encapsulated early transition metal carbide nanoparticles. During the carburization process, the silica shells prevent the sintering of adjacent carbide nanoparticles while also preventing the deposition of excess surface carbon. Alternatively, the silica-encapsulated metal oxide nanoparticles can be nitridized in an ammonia atmosphere at temperatures over 800 &#176;C to form silica-encapsulated early transition metal nitride nanoparticles. By adjusting the reverse microemulsion parameters, the thickness of the silica shells, and the carburization/nitridation conditions, the transition metal carbide or nitride nanoparticles can be tuned to various sizes, compositions, and crystal phases. After carburization or nitridation, the silica shells are then removed using either a room-temperature aqueous ammonium bifluoride solution or a 0.1 to 0.5 M NaOH solution at 40-60 &#176;C. While the silica shells are dissolving, a high surface area support, such as carbon black, can be added to these solutions to obtain supported early transition metal carbide or nitride nanoparticles. If no high surface area support is added, then the nanoparticles can be stored as a nanodispersion or centrifuged to obtain a nanopowder.

A &#8220;removable ceramic coating method&#8221; is presented in visual format for the synthesis of non-sintered and metal-terminated monometallic and bimetallic early transition metal carbide and nitride nanoparticles with tunable sizes and crystal structures.

A reverse microemulsion is used to encapsulate monometallic or bimetallic early transition metal oxide nanoparticles in microporous silica shells. The ...

Synthesis and Reaction Chemistry of Nanosize Monosodium Titanate

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the material with Au(III) ions. The synthesis method was derived from a sol-gel process used to produce micron-sized monosodium titanate (MST), with several key modifications, including altering reagent concentrations, omitting a particle seed step, and introducing a non-ionic surfactant to facilitate control of particle formation and growth. The resultant nMST material exhibits spherical-shaped particle morphology with a monodisperse distribution of particle diameters in the range from 100 to 150&#160;nm. The nMST material was found to have a Brunauer-Emmett-Teller (BET) surface area of 285 m2g-1, which is more than an order of magnitude higher than the micron-sized MST. The isoelectric point of the nMST measured 3.34 pH units, which is a pH unit lower than that measured for the micron-size MST. The nMST material was found to serve as an effective ion exchanger under weakly acidic conditions for the preparation of an Au(III)-exchange nanotitanate. In addition, the formation of the corresponding peroxotitanate was demonstrated by reaction of the nMST with hydrogen peroxide.

The surfactant mediated sol-gel synthesis of nanosized monosodium titanate is described, along with preparation of the corresponding peroxide modified material. An ion-exchange reaction with Au(III) is also presented.

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the ...

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature solvents. These coordinating ligands help solubilize and stabilize the nanoparticles while in solution, and can influence the resulting size and reactivity of the nanoparticles during their formation. Despite the ubiquity of using ligands during synthesis, the presence of aliphatic ligands on the nanoparticle surface can result in a number of problems during the end use of the nanoparticles, necessitating further ligand stripping or ligand exchange procedures. We have developed a way to synthesize cadmium sulfide (CdS) nanoparticles using a unique sulfur copolymer. This sulfur copolymer is primarily composed of elemental sulfur, which is a cheap and abundant material. The sulfur copolymer has the advantages of operating both as a high temperature solvent and as a sulfur source, which can react with a cadmium precursor during nanoparticle synthesis, resulting in the generation of ligand free CdS. During the reaction, only some of the copolymer is consumed to produce CdS, while the rest remains in the polymeric state, thereby producing a nanocomposite material. Once the reaction is finished, the copolymer stabilizes the nanoparticles within a solid polymeric matrix. The copolymer can then be removed before the nanoparticles are used, which produces nanoparticles that do not have organic coordinating ligands. This nascent synthesis technique presents a method to produce metal-sulfide nanoparticles for a wide variety of applications where the presence of organic ligands is not desired.

Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction.

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature ...

Nanosponge Tunability in Size and Crosslinking Density

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with controlled dimensions. This approach begins with synthesis of a functionalized lactone which is key to the pendant functionalization of the resulting polymer. Valerolactone (VL) and allyl-valerolactone (AVL) are then copolymerized using ring-opening polymerization. Post-polymerization modification is then used to install an epoxide moiety on some or all of the pendant allyl groups. Epoxy-amine chemistry is employed to form nanoparticles in a dilute solution of both polymer and small molecule diamine crosslinker based on the desired nanosponge size and crosslinking density. Nanosponge sizes can be characterized by transmission electron microscopy (TEM) imaging to determine the dimension and distribution. This method provides a pathway by which highly tunable polyesters can create tunable nanoparticles, which can be used for small molecule drug encapsulation. Due to the nature of the backbone, these particles are hydrolytically and enzymatically degradable for a controlled release of a wide range of hydrophobic small molecules.

This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Molten-Salt Synthesis of Complex Metal Oxide Nanoparticles

The development of feasible synthesis methods is critical for the successful exploration of novel properties and potential applications of nanomaterials. Here, we introduce the molten-salt synthesis (MSS) method for making metal oxide nanomaterials. Advantages over other methods include its simplicity, greenness, reliability, scalability, and generalizability. Using pyrochlore lanthanum hafnium oxide (La2Hf2O7) as a representative, we describe the MSS protocol for the successful synthesis of complex metal oxide nanoparticles (NPs). Furthermore, this method has the unique ability to produce NPs with different material features by changing various synthesis parameters such as pH, temperature, duration, and post-annealing. By fine-tuning these parameters, we are able to synthesize highly uniform, non-agglomerated, and highly crystalline NPs. As a specific example, we vary the particle size of the La2Hf2O7&#160;NPs by changing the concentration of the ammonium hydroxide solution used in the MSS process, which allows us to further explore the effect of particle size on various properties. It is expected that the MSS method will become a more popular synthesis method for nanomaterials and more widely employed in the nanoscience and nanotechnology community in the upcoming years.

Here, we demonstrate a unique, relatively low-temperature, molten-salt synthesis method for preparing uniform complex metal oxide lanthanum hafnate nanoparticles.

The development of feasible synthesis methods is critical for the successful exploration of novel properties and potential applications of nanomaterials. ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Bioengineering

Triplet Fusion Upconversion Nanocapsule Synthesis

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has significant implications for producing high energy light beyond a material's surface. However, the deployment of UC materials has been stymied due to poor material solubility, high concentration requirements, and oxygen sensitivity, ultimately resulting in reduced light output. Toward this end, nanoencapsulation has been a popular motif to circumvent these challenges, but durability has remained elusive in organic solvents. Recently, a nanoencapsulation technique was engineered to tackle each of these challenges, whereupon an oleic acid nanodroplet containing upconversion materials was encapsulated with a silica shell. Ultimately, these nanocapsules (NCs) were durable enough to enable triplet fusion upconversion-facilitated volumetric three-dimensional (3D) printing. By encapsulating upconversion materials with silica and dispersing them in a 3D printing resin, photopatterning beyond the surface of the printing vat was made possible. Here, video protocols for the synthesis of upconversion NCs are presented for both small-scale and large-scale batches. The outlined protocols serve as a starting point for adapting this encapsulation scheme to multiple upconversion schemes for use in volumetric 3D printing applications.

This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has ...

Engineering

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in magnetic resonance imaging (MRI). While iron oxide nanoparticles provide constant negative contrast on MRI over typical experimental timeframes, MnO generates switchable positive contrast on MRI through dissolution of MnO to Mn2+ at low pH within cell endosomes to &#8216;turn ON&#8217; MRI contrast. This protocol describes a one-pot synthesis of MnO nanoparticles formed by thermal decomposition of manganese(II) acetylacetonate in oleylamine and dibenzyl ether. Although running the synthesis of MnO nanoparticles is simple, the initial experimental setup can be difficult to reproduce if detailed instructions are not provided. Thus, the glassware and tubing assembly is first thoroughly described to allow other investigators to easily reproduce the setup. The synthesis method incorporates a temperature controller to achieve automated and precise manipulation of the desired temperature profile, which will impact resulting nanoparticle size and chemistry. The thermal decomposition protocol can be readily adapted to generate other metal oxide nanoparticles (e.g., iron oxide) and to include alternative organic solvents and stabilizers (e.g., oleic acid). In addition, the ratio of organic solvent to stabilizer can be changed to further impact nanoparticle properties, which is shown herein. Synthesized MnO nanoparticles are characterized for morphology, size, bulk composition, and surface composition through transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. The MnO nanoparticles synthesized by this method will be hydrophobic and must be further manipulated through ligand exchange, polymeric encapsulation, or lipid capping to incorporate hydrophilic groups for interaction with biological fluids and tissues.

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of ManganeseII Acetylacetonate

This protocol details a facile, one-pot synthesis of manganese oxide (MnO) nanoparticles by thermal decomposition of manganese(II) acetylacetonate in the presence of oleylamine and dibenzyl ether. MnO nanoparticles have been utilized in diverse applications including magnetic resonance imaging, biosensing, catalysis, batteries, and waste water treatment.

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in ...