Editing Magnetic beads Preparation Methods (section)

== Polydopamine Modified Glutathione disulfide Superparamagnetic IronOxide Nanoparticles ==

[https://www.ncbi.nlm.nih.gov/pubmed/30678236 https://www.ncbi.nlm.nih.gov/pubmed/30678236]&nbsp;[https://www.mdpi.com/2079-4991/9/2/138 https://www.mdpi.com/2079-4991/9/2/138]



[[File:Polydopamine Modified Superparamagnetic Iron Oxide2.png|500px|Polydopamine Modified Superparamagnetic Iron Oxide2.png]]

Iron oxide nanoparticles (Fe3O4, IONPs) were prepared by a facile chemical co-precipitationmethod [21] where FeCl3·6H2O (34.6 g) and FeCl2·4H2O (12.7 g), in the molar ratio of 2:1,were dissolved in 1.5 L of water. Then, Fe3O4 nanoparticles were precipitated quickly by addingammonia (120 mL, 28%) in the solution at 22 ◦C. The obtained black suspension was stirred for 2–3 min.The obtained black precipitate was then magnetically decanted and washed thoroughly with deionized

(DI) water until the solution reached pH 8. The precipitate was then kept in nitric acid solution (1 mM,pH 3) and dialyzed against nitric acid solution (10 mM) for 48 h. Dialyzed nanoparticles were thencentrifuged for 15 min at 20,000 G with deceleration to obtain stable IONPs. The supernatant was thenused for further characterizations and surface modification Synthesis of Polydopamine-modified IONPs: pDA-IONPsA quantity of 100 mg of Fe3O4 (10 mg mL−1) was dispersed under continuous stirring in 25 mL of10 mM DA solution (PBS, 10 mM, pH 9.0) for 24 h at 22 ◦C. After, polydopamine-modified nanoparticles(pDA-IONPs) were magnetically decanted, washed thoroughly 5 times with ultrapure water using ultrafiltration to remove the non-reacted DA (ensured using UV), and further dispersed in water.



Preparation of GSSG-modified Nanoparticles

GSSG-modified nanoparticles were prepared using 20 mg of pDA-IONPs, which were dispersed in 1 mg mL􀀀1 GSSG solution (10mMPBS, pH 9.4) for 24 h under continuous magnetic agitation (700 rpm) at 22 �C. The GSSG-modified nanoparticles (GSSG-pDA-IONPs) were magnetically separated using magnetic syringe [22], washed with PBS buffer and checked with DTT to ensure the complete removal of unreacted GSSG. DTT reduces the unreacted disulfide, giving a yellow color solution indicating the presence of disulfide group. The resultant nanoparticles were then washed and re-dispersed in PBS. The above nanoparticles were then reduced to glutathione to get free –SH. Briefly, GSSG modified nanoparticles (800 �L of the suspension) were first mixed with Tris buffer (0.2 M, pH 9), after which, DTT (0.01 M) was added and the reduction of disulfide was allowed to proceed for 1 h at 22 �C. To ensure the reduction, DTNB (0.01 M) in acetate buffer (0.2 M, pH 5) was reacted with the sample and the absorbance was measured at 408 nm. S-nitrosation was then performed using NaNO2 (0.4 mM). The reaction took place when GSH-pDA-IONPs were successively added in NaNO2 solution at 37 �C and pH 2.5 for 30 min in the dark. UV-vis spectra were recorded to see the formation of S-nitrosation and the resultant nanoparticles were finally washed with PBS buffer and re-dispersed in PBS.