Name: synthesis of functionalized magnetic nanoparticles, their conjugation with the siderophore feroxamine and its evaluation for bacteria detection
Uploaded: 2020-06-16T14:30:00
Description: Watch this Scientific Journal Video about Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection at JoVE.com

The authors gratefully acknowledge Professor Klaus Hantke (University of T&#252;bingen, Germany) for kindly supply the Yersinia enterocolitica strains used in this work. This work was supported by grants AGL2015-63740-C2-1/2-R and RTI2018-093634-B-C21/C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union. Work in University of Santiago de Compostela and University of A Coru&#241;a was also supported by grants GRC2018/018, GRC2018/039, and ED431E 2018/03 (CICA-INIBIC strategic group) from Xunta de Galicia. Finally, we want to thank to Nuria Calvo for her great collaboration doing the voice-off this video protocol.

NOTE: For the reactions performed under inert atmosphere conditions, all the glassware was previously dried in an oven at 65 &#176;C, sealed with a rubber septum and purged with argon three times.
1. Synthesis of magnetic nanoparticles conjugated with feroxamine
<ol>
	<li>Synthesis of Fe3O4 magnetic nanoparticles (MNPs)
	<ol>
		<li>Add 0.5 g of Fe(acac)3 in a 20 mL glass vial and then mix with 10 mL of benzyl alcohol.</li>
		<li>Sonicate this mixture for 2 mi...

<ol>
	<li>Pan, Y., Du, X., Zhao, F., Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticles+for+the+manipulation+of+proteins+and+cells.">Magnetic nanoparticles for the manipulation of proteins and cells.</a> Chemical Society Reviews. 41 (7), 2912-2942 (2012).</li><li>Zheng, T., Nolan, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Siderophore-based+detection+of+Fe(III)+and+microbial+pathogens.">Siderophore-based detection of Fe(III) and microbial pathogens.</a> Metallomics. 4, 866-880 (2012).</li><li>Hider, R. C., Kong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+biology+of+siderophores.">Chemistry and biology of siderophores.</a> Natural Product Reports. 27 (5), 637-657 (2010).</li><li>Sandy, M., Butler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Iron+Acquisition:+Marine+and+Terrestrial+Siderophores.">Microbial Iron Acquisition: Marine and Terrestrial Siderophores.</a> Chemical Reviews. 109 (10), 4580-4595 (2010).</li><li>Pinna, N., Grancharov, S., Beato, P., Bonville, P., Antonietti, M., Niederberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetite+Nanocrystals+:+Nonaqueous+Synthesis,+Characterization.">Magnetite Nanocrystals : Nonaqueous Synthesis, Characterization.</a> Chemistry of Materials. 17 (15), 3044-3049 (2005).</li><li>Li, Y. S., Church, J. S., Woodhead, A. L., Moussa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+silica+coated+iron+oxide+magnetic+nano-particles.">Preparation and characterization of silica coated iron oxide magnetic nano-particles.</a> Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy. 76 (5), 484-489 (2010).</li><li>Chen, J. P., Yang, P. C., Ma, Y. H., Tu, S. J., Lu, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+tissue+plasminogen+activator+by+binding+to+silica-coated+magnetic+nanoparticle.">Targeted delivery of tissue plasminogen activator by binding to silica-coated magnetic nanoparticle.</a> International Journal of Nanomedicine. 7, 5137-5149 (2012).</li><li>El-Boubbou, K., Gruden, C., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+glyco-nanoparticles:+a+unique+tool+for+rapid+pathogen+detection,+decontamination,+and+strain+differentiation.">Magnetic glyco-nanoparticles: a unique tool for rapid pathogen detection, decontamination, and strain differentiation.</a> Journal of the American Chemical Society. 129 (44), 13392-13393 (2007).</li><li>Mart&#237;nez-Matamoros, D., 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=Preparation+of+functionalized+magnetic+nanoparticles+conjugated+with+feroxamine+and+their+evaluation+for+pathogen+detection.">Preparation of functionalized magnetic nanoparticles conjugated with feroxamine and their evaluation for pathogen detection.</a> RSC Advances. 9 (24), 13533-13542 (2019).</li><li>Cozar, 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=Raman+and+surface-enhanced+Raman+study+of+desferrioxamine+B+and+its+Fe(III)+complex,+ferrioxamine+B.">Raman and surface-enhanced Raman study of desferrioxamine B and its Fe(III) complex, ferrioxamine B.</a> Journal of Molecular Structure. 788 (1-3), 1-6 (2006).</li><li>Shebanova, O. N., Lazor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a-C:H+and+oxygen-containing+Si:C:H+films+by+Raman+spectroscopy+and+XPS.">Characterisation of a-C:H and oxygen-containing Si:C:H films by Raman spectroscopy and XPS.</a> Journal of Solid State Chemistry. 174 (4), 424-430 (2003).</li><li>Gonz&#225;lez, P., Serra, J., Liste, S., Chiussi, S., Le&#243;n, B., P&#233;rez-Amor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+spectroscopic+study+of+bioactive+silica+based+glasses.">Raman spectroscopic study of bioactive silica based glasses.</a> Journal of Non-Crystalline Solids. 320 (12), 92-99 (2003).</li><li>Veres, 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=Characterisation+of+a-C:H+and+oxygen-containing+Si:C:H+films+by+Raman+spectroscopy+and+XPS.">Characterisation of a-C:H and oxygen-containing Si:C:H films by Raman spectroscopy and XPS.</a> Diamond and Related Materials. 14 (3-7), 1051-1056 (2005).</li><li>You, Y., 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=Visualization+and+investigation+of+Si-C+covalent+bonding+of+single+carbon+nanotube+grown+on+silicon+substrate.">Visualization and investigation of Si-C covalent bonding of single carbon nanotube grown on silicon substrate.</a> Applied Physics Letters. 93 (10), 103111-103113 (2008).</li><li>Graf, N., 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=XPS+and+NEXAFS+studies+of+aliphatic+and+aromatic+amine+species+on+functionalized+surfaces.">XPS and NEXAFS studies of aliphatic and aromatic amine species on functionalized surfaces.</a> Surface Science. 603 (18), 2849-2860 (2009).</li><li>Michaeli, W., Blomfield, C. J., Short, R. D., Jones, F. R., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+HMDSO/O2+plasma+deposits+using+a+high-sensitivity+and+-energy+resolution+XPS+instrument:+curve+fitting+of+the+Si+2p+core+level.">A study of HMDSO/O2 plasma deposits using a high-sensitivity and -energy resolution XPS instrument: curve fitting of the Si 2p core level.</a> Applied Surface Science. 137 (1-4), 179-183 (2002).</li><li>Liana, A. E., Marquis, C. P., Gunawan, C., Gooding, J. J., Amal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T4+bacteriophage+conjugated+magnetic+particles+for+E.+coli+capturing:+Influence+of+bacteriophage+loading,+temperature+and+tryptone.">T4 bacteriophage conjugated magnetic particles for E. coli capturing: Influence of bacteriophage loading, temperature and tryptone.</a> Colloids and Surfaces B: Biointerfaces. 151, 47-57 (2017).</li><li>Fang, W., Han, C., Zhang, H., Wei, W., Liu, R., Shen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+amino-functionalized+magnetic+nanoparticles+for+enhancement+of+bacterial+capture+efficiency.">Preparation of amino-functionalized magnetic nanoparticles for enhancement of bacterial capture efficiency.</a> RSC Advances. 6, 67875-67882 (2016).</li><li>Zhan, S., 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=Efficient+removal+of+pathogenic+bacteria+and+viruses+by+multifunctional+amine-modified+magnetic+nanoparticles.">Efficient removal of pathogenic bacteria and viruses by multifunctional amine-modified magnetic nanoparticles.</a> Journal of Hazardous Materials. 274, 115-123 (2014).</li></ol>

This protocol describes the synthesis of a conjugate between magnetic nanoparticles and the siderophore feroxamine by covalent bonding. The synthesis of magnetite was carried out using the protocol reported by Pinna et al.5 followed by silica coating to protect the magnetic core of corrosion in aqueous systems, to minimize the aggregation and to provide a suitable surface for functionalization6. The silica coating process was modified. Instead of carrying out three coatings...

The bacteria detection methods using MNP are based on the molecular recognition of antibodies, aptamers, bioprotein, carbohydrates conjugated to MNP by the pathogenic bacteria1. Taking into account that siderophores are recognized by specific receptors on the outer membrane of bacteria, they could also linked to MNP to increase their specificity2. Siderophores are small organic molecules involved in the Fe3+ uptake by bacteria3,4. The preparation of conjugates between siderophores and MNP along with their evaluation for the capture and isolation of bacteria has not yet been reported.
One of the crucial steps in the synthesis of conjugates of magnetic nanoparticles with small molecules is the selection of the type of bond or interaction between them to ensure that the small molecule is attached to the surface of the MNP. For this reason, the procedure to prepare the conjugate between magnetic nanoparticles and feroxamine&#8212;the siderophore recognized by Yersinia enterocolitica&#8212;was focused at the generation of a modifiable surface of the MNP to allow linking it covalently to the siderophore by carbodiimide chemistry. In order to get an uniform magnetite nanoparticles (MNP) and to improve nucleation and size control, a solvolysis reaction with benzyl alcohol was carried in a thermal block without shaking5. Then, a silica coating was generated by St&#246;ber method to confer protection and improve the stability of the nanoparticles suspension in aqueous media6. Taking into account the structure of the feroxamine, the introduction of amine groups is necessary to produce suitable nanoparticles (MNP@SiO2@NH2) to be conjugated with the siderophore. This was achieved by condensation of (3-aminopropyl)triethoxysilane (APTES) with the alcohol groups present on the surface of the silica modified nanoparticles (MNP@SiO2) using a sol-gel method7.
In parallel, the feroxamine iron(III) complex was prepared by complexation of the commercially available deferoxamine with iron acetyl acetonate in aqueous solution. N-succinylferoxamine, bearing succinyl groups that will act as linkers, was obtained by the reaction of feroxamine with succinic anhydride.
The conjugation between MNP@SiO2@NH2 and N-succinylferoxamine to give MNP@SiO2@NH@Fa was carried out through carbodiimide chemistry using as coupling reagents benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 1-hydroxybenzotriazole (HOBt) in a soft basic media to activate the terminal acid group in N-succinylferoxamine8.
Once the MNPs were characterized, we evaluated the capabilities of bare and functionalized magnetic nanoparticles to capture wild type (WC-A) and a mutant of Y. enterocolitica lacking feroxamine receptor FoxA (FoxA WC-A 12-8). Plain MNPs, functionalized MNPs and the conjugate MNP@SiO2@NH@Fa were allowed to interact with each Y. enterocolitica strain. The bacteria-conjugate aggregates were separated from the bacteria suspension by the application of a magnetic field. The separated aggregates were rinsed twice with phosphate buffered saline (PBS), re-suspended in PBS to prepare serial dilutions and then, they were plated for colony counting. This protocol demonstrates each step of the synthesis of MNP@SiO2@NH@Fa, the structural characterization of all the intermediates and the conjugate, and a bacterium capture assay as an easy way to evaluate the specificity of the conjugate in relation to the intermediates.9

<table>
	<tbody>
		<tr>
			<td>1-Hydroxybenzotriazole hydrate 
			HOBT</td>
			<td>Acros</td>
			<td>300561000</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2&#8242;-Bipyridyl</td>
			<td>Sigma Aldrich</td>
			<td>D216305</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Aminopropyltriethoxysilane 99%</td>
			<td>Acros</td>
			<td>151081000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution 28% NH3</td>
			<td>Sigma Aldrich</td>
			<td>338818</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate BOP Reagent</td>
			<td>Acros</td>
			<td>209800050</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>822259</td>
			<td></td>
		</tr>
		<tr>
			<td>Deferoxamine mesylate salt &#62;92,5% (TLC)</td>
			<td>Sigma Aldrich</td>
			<td>D9533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous, 96%</td>
			<td>Panreac</td>
			<td>131085</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate, Extra Pure, SLR, Fisher Chemical</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) acetylacetonate 97%</td>
			<td>Sigma Aldrich</td>
			<td>F300</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth (Lennox)</td>
			<td>Sigma Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine, 99.5+%, AcroSeal</td>
			<td>Acros</td>
			<td>459591000</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide, 99.8%, Extra Dry, AcroSeal</td>
			<td>Acros</td>
			<td>326871000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine, 99.5%, Extra Dry, AcroSeal</td>
			<td>Acros</td>
			<td>339421000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex LH-20</td>
			<td>Sigma Aldrich</td>
			<td>LH20100</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic anhydride &#62;99%</td>
			<td>Sigma Aldrich</td>
			<td>239690</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetraethyl orthosolicate &#62;99,0%</td>
			<td>Sigma Aldrich</td>
			<td>86578</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of functionalized magnetic nanoparticles, their conjugation with the siderophore feroxamine and its evaluation for bacteria detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

An exhaustive structural characterization is carried out in order to determine the morphology and the properties of each intermediate and the final conjugate. For this purpose, the techniques XRD, FT-IR, Raman spectroscopy, TGA, TEM, EDX mapping and XPS are used in order to demonstrate the formation of the conjugate. The oxidation states of the atoms at the surface of the nanoparticles acquired by X-ray photoelectron spectroscopy (XPS) are the most relevant data to confirm the formation of covalent bonds between the nano...

Watch this Scientific Journal Video about Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection at JoVE.com

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Magnetic nanoparticles are ideal tools for analytical science and nano medicine due to their strong magnetic properties and the easy functionalization of their surface. We report here the first instance of a conjugate between amino functionalized silica magnetic particles and the siderophore feroxamine along with evaluation for the capture of photogenic material. Siderophores are small, organic molecules which play a key role in the iron partake mechanism, and they are recognized by specific outer membrane receptors of bacteria.This video displays, step-by-step, an efficient protocol to prepare magnetic iron nanoparticles. Then, they were coated with silica to protect dispersion and to protect the magnetic nucleus. The resulting reactor surface was functionalized with amine groups.Synthesis of magnetic nanoparticles conjugated with feroxamine. Add 0.5 gram of Fe(acac)3 in a 20 mL glass vial. Then, mix with a 10 mL of benzyl alcohol.Sonicate this mixture for 2 minutes. Then, transfer to a heating block and heat at 180 degrees Centigrades for 72 hours. Rinse the nanoparticles with 96%ethanol.And centrifuge for 30 minutes. Separate the nanoparticles from the supernatant by magnetic attraction, using a neodymium magnet. Discard the residual solvent.Discard the supernatant, alternating with sonication, until the solvent looks clear. Prepare a suspension of 2 grams of MNP in 80 milliliters of isopropanol, add 4 milliliters of 21%ammonia, 7.5 milliliters of distilled water, and 0.56 milliliters of TEOS. Heat the mixture at 40 degrees Centigrades for 2 hours, with continuous stirring.Then, sonicate for 1 hour. Separate the MNP with a magnet, discard the supernatant, disperse it in 30 milliliters of isopropanol, add ammonia, distilled water, and TEOS, in the same order as described before. Heat the mixture at 40 degrees Centigrades for two hours with continuous stirring.Then, sonicate for one hour. Remove and wash conveniently the magnetic stir bar, to recover all the material. Discard the supernatant and rinse the nanoparticles with 96%ethanol three times, alternating with sonication.Dry the nanoparticles under vacuum at room temperature for 12 hours. Rinse 500 milligrams of the MNP@SIO2 obtained from previous step with dimethylformamide. Sonicate them and discard.Re-suspend the particles in a round-bottom flask, stir with a magnetic bar and add 9 milliliters of APTES. Stir the mixture at 60 degrees Centigrades for 12 hours. Discard the supernatant and rinse the nanoparticles with 96%ethanol three times, alternating with sonication.Dissolve a 100 mg of deferoxamine, mesylate salt and 53.0 milligrams of iron acetylacetonate in 5 milliliters of distilled water. Stir the mixture overnight at room temperature. Wash the resulting product three times with 20 milliliters of ethyl acetate in a separation funnel.Remove the organic solvent under vacuum. Freeze-dry the aqueous phase to afford feroxamine and a red solid. Add 350 milligrams of succinic anhydride to a solution of 100 mg of feroxamine in 5 milliliters of pyridine in a 50 milliliters round-bottom flask.Stir the resulting mixture, at room temperature, for 16 hours. Remove the excess of pyridine under reduced pressure. Dissolve the reaction crude in 3 milliliters of methanol.Transfer the methanolic solution into a LH-20 column and elute at 0.5 mL/minute. Collect the red fraction and remove the methanol under vacuum. Rinse 30 mg of dry amine functionalized nanoparticles twice with DMF and sonicate the nanoparticles in a 100 milliliter Erlenmeyer flask for 30 minutes.Prepare a solution of 200 milligrams N-succinylferoxamine, 173 milligrams Benzotriazole 1-yl-oxy-tris-phosphonium hexafluorophosphate BOP, 46 mg 1-hydroxybenzotriazole HOBt and 128.8 milligrams N, N-diisopropylethylamine DIPEA, in 10 mL of DMF, in a 50 milliliter round-bottom flask. Suspend the previously rinsed MNP@SiO2@NH2 in a 3 milliliter of DMF, under sonication in dry and oxygen free conditions. Use argon atmosphere.Add, dropwise, mix A to mix B.Shake the final mixture, using an orbital shaker, at room temperature overnight. Separate the resulting conjugate from the suspension using a magnet. Bacterial assay with Y.enterocolitica strains to quantify the isolation and capture of pathogenic bacteria with nanoparticles.Prepare a suspension of all the intermediate nanoparticles and the final conjugate in PBS at 1 mg/mL in sterile tubes. Prepare a culture of Y.entorocolitica in 5 mL of Luria Bertani, LB, broth overnight. Prepare a 5 mL of iron deficient Trypcase Soy Broth, TSB, by the addition of 50 microliters of 10 mM 2, 2-bipyridyl solution.Inoculate the 5 milliliter of iron deficient TSB broth with 50 microliters of the overnight culture of Y.enterocolitica. Incubate at 37 degrees Centigrades with agitation until an OD600 of 0.5 to 0.8 is reached. Take a 100 microliters of the overnight culture and dilute it in a tube containing 900 microliters of PBS to obtain the first 1/10 dilution.Then, prepare a 1/100 dilution from the first dilution using the same procedure to get a concentration of bacterial cells at 10 to the power of 6 Colony Forming Units per milliliter approximately. Add a 100 microliters of nanoparticles suspension and 1 mg/mL to 1 milliliter of the 1/100 bacteria dilution. Separate the MNP bacteria aggregates, by using a magnet, discard, carefully, the supernatant.Rinse the separated nanoparticles twice with 1 millimeter PBS using a vortex. Prepare four successive 1/10 dilutions from the former suspension, until a 10 to the power of minus 4 dilution. Plate 10 microliters of each dilution onto TS agar plates.Photograph the plate with a gel digitalizer in epi white mode. Process the image, using the convenient software, to amplify a spot to count the number of individual colonies. To confirm the formation of the covalent bond between the nanoparticles and the siderophore, we employ FT-IR Raman spectroscopy to monitoring the synthesis, observing how new bands appear in agreement with a new functional groups in each step.TEM and SEM microscopy were used to observe the morphology and particle size. Thermogravimetric analysis to measure the weight loss due to the addition of organic material. And XPS allow us to analyze the different oxidation states of the atoms in the surface and confirming the formation of covalent bonds.Finally, we used all the intermediates on the final conjugate to test its ability to capture bacteria, in order to establish their properties as biosensor. This protocol was designed to take advantage of the high biocompatibility of magnetite nanoparticles. The coating with silica improves the dispersion and protect the magnetic nucleus from degradation in aqueous media.And also give a reactive surface to functionalize with amine groups which are necessary to covalently bind an acid modified siderophore by carbodiimide chemistry. In this case, N-succynilferoxamine. The conjugate bacteria capture capability was tested and as a result, a low number of colonies was found, without significant difference with the intermediates due to a specific interactions on bulky effects presented in the PBS bacterial suspension.

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Chemistry

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to the preparation of new organic dyes. Although numerous strategies have been reported to access naphthalene scaffolds, many procedures still present limitations in terms of incorporating functionality, which in turn narrows the range of available substrates. The development of versatile methods for direct access to substituted naphthalenes is therefore highly desirable.
The Diels-Alder (DA) cycloaddition reaction is a powerful and attractive method for the formation of saturated and unsaturated ring systems from readily available starting materials. A new microwave-assisted intramolecular dehydrogenative DA reaction of styrenyl derivatives described herein generates a variety of functionalized cyclopenta[b]naphthalenes that could not be prepared using existing synthetic methods. When compared to conventional heating, microwave irradiation accelerates reaction rates, enhances yields, and limits the formation of undesired byproducts.
The utility of this protocol is further demonstrated by the conversion of a DA cycloadduct into a novel solvatochromic fluorescent dye via a Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. Fluorescence spectroscopy, as an informative and sensitive analytical technique, plays a key role in research fields including environmental science, medicine, pharmacology, and cellular biology. Access to a variety of new organic fluorophores provided by the microwave-assisted dehydrogenative DA reaction allows for further advancement in these fields.

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is 1H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4&#8217;-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This ...

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.

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 ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...