Dear Arathi, Though I do not work with Silver nano particles, but if I correctly understand your problem, may I suggest you that when you prepare your Ag nanoparticles, is it possible for you to do the dialysis of your nano suspension to get rid of Ag ions so that once you have dialyzed nano preparation, you can safely use them for your down stream experiments, without any possibility of precipitation. I am not sure how far this will be helpful. Good luck.

Thanks for the suggestion Rajesh.But I have no problem making nanoparticles . Although its not dialysis,  we employ filtration too. My concern is after adding to biological media (especially silver nitrate). Nanoparticles tend to release ions over time and same when they come in contact with biological substances. As far as I know, there is no simple way to distinguish which form of silver it is. Thats why people use silver nitrate as ionic control (which was not working with mine, so looking for alternatives)

Hi Arathi, 

I guess this paper can provide you some answers. Please have a look.

http://pubs.rsc.org/en/content/articlepdf/2012/ra/c2ra20684f

Dear Arathi, if I understood correctly you want your AgNPs not to release any ions, is that true? In that case I would try to stabilize NPs with something (citrates? cysteine?). Still, I am not sure if it is possible to stop release of ions completly. 

Other thing is that your biological media themselves can react with silver, e.g. DMEM contains glucose and glutamine which will react with silver for sure. 

As to suggestion of Rajesh - dialysis will be better way to get rid of silver ions than filtration.

 Hi Arathi,

Your problem is a genuine one. Although, I have not published in the field of particularly, silver nanoparticles, but acco. to the literature i have read and some present work experience, I can say that this is the inherent property of silver nanoparticles to loose the Ag+ ions in the media. And these Ag+ ions react with Cl- ions in the media and forms the AgCl ppt. You cant rule out the exact role of Ag NPs or Ag+ ions.

Generally, people do not bother that after adding in the media, what would be the fate of AgNPs. Infact, It can be a good study to show that how exactly, the fate of AgNPs will alter while adding into the media.

By the way, what you can do is, one time you can perform an experiment and take the UV measurements of media with nanoparticles (without cells), at different time periods at 400 nm or so. By this way, you will have an idea, that how the nanoparticles concentration is decreasing in your systems and how they are changing in the ionic form with time. So, you can put in supporting information that the result is not because of purely Ag NPs.  

Besides, ICP-MS, there are are other techniques like fluorescence (if your NPs fluoresce) and XPS etc. But UV spectrophotometer is good and easily available instrument which you can use.

Good luck.

Hello Arathi,

I have used AgNo3 as Ag+ ions effect control in chemical reactivity studies for nano particles.The reason for using silver nitrate is that it is one of the silver salts with high water solubility. I think you can probably use other higly soluble silver salts like AgF or Ag acetate. The AgF is very soluble, but , in your case, there may be biological effects from F- ions. The silver acetate is not as soluble (9 g/L, I think). The main problem is, no change in anion is going to solve your precipitation problem which is caused by Ag+ itsef, in combination with the anions in your medium. And you can not replace the Ag+ ions as control, because the formation of Ag+ ions is what you want to account for.

I do not think you can fix this.

My question is: why are you using such high concentrations to begin with? They are unrealistic for any exposure scenario to nano particles and I am sure even Ag+ can cause biological effects at such concentrations.

The other issue you should be aware of is that if you are using 30-500 ppm of Ag+, you are also using 30-500 ppm pf nano Ag and adding this to your culture medium. In my experience (and according to data in the literature), in the presence of solutions with high ionic strangth nanoAg particles quickly agglomerate and leave solution. You need to mesure the real concentration of nanoAg in the medium you are using, not base your calculations on the amount of nanoAg you are adding to it.

Otherwise you conclusions will be difficult to defend at publication.

Just my to cents.

Whatever form of Ag+ you use, the Ag+ will react with the Cl- in your media to form AgCl, since the Ksp is on the order of 10^-10. 

Others pointing out colloidal stability of your AgNPs is very important.  If electrostatically stabilized colloids are added to cell culture media with no proteins present, aggregation ensues (MacCuspie).  Even the order of addition (AgNPs + FBS + media) is critically important (Zook et al.)

Single Particle ICP-MS is a great approach to look at dissolution of the AgNPs...you'll get the size distribution change, particle concentration change, and dissolved fraction concentration change all in one series of measurements, assuming you optimize your measurement conditions correctly (see challenges with gold NP spICP-MS in Liu et al.).

Article Stable nanoparticle aggregates/agglomerates of different siz...

Article Colloidal stability of silver nanoparticles in biologically ...

Article Capabilities of Single Particle Inductively Coupled Plasma M...

Here is a paper about the absorbance method mentioned above:

Article Measuring silver nanoparticle dissolution in complex biologi...

It has been reported by many researchers that the antibacterial activity of AgNPs is due to the release of Ag+ ions. The antibacterial activity of AgNPs increases with the decrease of their size because smaller AgNPs tend to release more Ag+ ions in solution. There are also some reports indicating that the activity of AgNPs is prevented in the absence of oxygen since these NPs are not oxidized to Ag+. I, therefore, think that AgNO3 and AgNPs both release Ag+ but since the nitrate releases more silver cations it is stronger antibacterial agent than AgNPs with the same concentration.You can't get read of Ag+ by adding Cl- because the produced AgCl precipitate also releases Ag+ in solution.Washing the prepared AgNPs thoroughly with deionized water can almost remove any trapped Ag+ ions in these NPs.

@ All Thanks for the suggestions and papers provided!

@ Anca-Maria Tugulea 

Thanks for the alternatives. I know I cant avoid Ag+ and ppts but am wondering having ppts, if its okay to use that as a control since I dont see such ppts with nano!

The reason for using such a high concentrations are to make sure we have LC50 covered and have concentrations above it-- from our previous results

As far as  AgNP and media, yeah I accounted that and yes you are right --majority goes to media so what actually goes to cells is very less; this is what we want for our next step anyway.. so sure point noted there!

@ Robert

yeah s-ICPMS will tell me more about nano form and so I don't have to use AgNO3 as a control anymore. I just don't have access to it so trying for alternatives for ionic silver controls. Thanks though