If you can use an ultracentrifuge, I suggest you to try it even if it take long time (more than an hour), If you have suspension in lower layer, it will go down to the bottom if the thensity of the nanoparticles is higher than the solvent's. Of course in many times you can not resuspend aggregated nanoparticles,

But if you can't make pellet you can remove the upper solvent and refill the tube in several times.

Trying different solvent to make pellets is also a way, but cell will die.

If you have other structures like cells, or something large, you will centrifuge the cells too, means you can not separate them. There is a method called density gradient centrifugation that maybe can help you.

DOI: 10.1002/anie.200805047

Hi Ewa,

I think the easiest and most succesfull method is making the particles to flocculate by changing the pH of the fluid, I mean, looking for the Isoelectric Point of the particles.

But, this would change the isoomosticity of your fluid. I'm not sure if you want it to happen. The changes in the media are reversible, so if you do not have anything that could damage because of different pH, I think this method could help you.

If it doesn't help you, good luck and never give up!

David

Hi Ewa Stepien

a few days ago when I review a paper I read : The reaction mixtures prepared from the seeds were kept for 7 days at room temperature for stabilization and subsequently they were centrifuged at 2000 rpm for 1 hour and redispersed in distilled water. The procedure was repeated three times and remnant pellets were dried and powdered for SEM and XRD.

you cam use it .

Thanks Jahari for your guidance, but... I do not need to extract nanoparticles to have pure NP-powder. My target is to obtain NP-free solution. So repeated ultracentrifugation may be a good method, however time consuming. I will try to apply the second step of ultracentrifugation.

Ewa

Thanks David (Bake). Of course Im aware about suspended cells. To remove cells I usually preform pre-centrifugation at 150-500 g (depends on cells) or if I use human plasma I remove platelets and micoplatelets at 3000g. Gradient ultracentrifugation is an excellent method for separation of nanoparticles and others (lipoproteins) for further investigation. However in my experiment I need to have “unmodified” NP-free supernatant.

Ewa

Good idea by David (Lago-Cachón). Changing the pH is reversible, but can influence not only isoomosticity of solvent, but can also influence solubility of my NPs. I want to avoid it because I need to know their solubility in medium.

Summarizing, repeated ultracentrifugation, but I still do not have any idea about time and speed, to have a time effective procedure.

As far as Abbas Azhar suggestions, I nor sure if 2000 rpm for 1 hour is good for my preparation. RPM means nothing if we do not know about a rotor radius and angle. The question is about NPs sedimentation coefficient. If they are small they need more acceleration to centrifuge.

Can anyone guide me on how to proceed for Reactive oxygen Species formation and their detection formed by magnetite iron oxide nanoparticle coated with protein ?

Ewa , you are right . in mentioned paper author's mean is you should stabilized first and then sediment their.

For less force or time to remove nanoparticles with centrifugation you can also try concentrate the sample before centrifugation. it is always help me but I working with tens of milliliters so it is easy for me.

You can also use polymers as a surfactant. using it in small concentration it makes links between nanoparticles helping to aggregate (in larger concentration - general use - the effect is opposite). If you have negatively charged surface you have to use positively charged polymer.

150,000g seems high to still be getting poor sedimentation. How homogeneous are your particles? Does the culture media contain added serum? You will probably remove a number of serum components at this speed. Additionally, if serum is present your particles will most likely possess a serum corona which will make the effective size of your particles larger. I have been separating highly charged qdots (12-20nm) at 20,0000g 30mins in the abscence of serum, the pellet is loose but I just avoid disturbing it.

I would try molecular sieving chromatography, e.g. Sepharose 6b. Would let supernatant go through while retaining nanoparticles. An other option would be ultra-filtration, a technique used to get rid of viral particles from plasmas for transfusion (comparable size with your nanoP. Hope that helps. PP

Kez, Thank you for your answer. I use serum free medium for incubation of cells with NPs. They are regular in size between 22 and 40 nm. Problem is that I have very high concentrations (1 mg/mL). In these concentrations the pellet is loose and a suspension above is visible. Philippe, ultra-filtration is a method of choice in lower concentrations. In case of my media, filters become clogged immediately. I will try repetitive centrifugation in lower speed.

Hi Ewa, you could dilute your suspension before centrifugation and achieve the higher conc when you re-suspend if you think this would help. Also make sure the deceleration of the centrifuge is set to the lowest value as this will help maintain the pellet. Alternatively, dialysis and rotary evaporation might work.