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Dear all,
I’m also having some problems with the washing steps. Right after synthesis, the MNPs are quickly attracted to the magnet rendering the supernatant completely clear. Subsequent washing steps however, give rise to an increasingly dark colored solution.
I have tried degassing the water with N2 (with a homemade contraption) and also boiling it to remove most of the dissolved oxygen. For practical reasons, I did let the water cool down first in a Schott bottle and after opening it seemed to have been an airtight seal. Would it make sense to do the washing steps with 80°C water?
Is it possible that I introduce too much oxygen into the system when I dissolve the magnetic pellet by shaking too vigorously? Or are there other potential pitfalls I should pay attention to?
Thank you very much in advance!
Kind regards, Jonas
Dear Jonas,
Sorry to hear about your troubles with synthesis. We also occasionally had troubles like you are experiencing. There are a few things that are important here.
1) The correct ratio of Fe2+ to Fe3+ (otherwise proper MNPs have no chance to grow properly).
2) It can also be that the chemicals (iron salts) are old. The Fe2/3 solution should be translucent and without precipitates. Either buy fresh salts, or filter the solution. Otherwise, the existing precipitates could influence the MNP formation
3) From our experience, preheating the alkali solution is mostly sufficient to get a good synthesis. Degassing can help a bit too.
4) pH of the washing solution should not be too acidic. Otherwise you simply dissolve the uncoated MNPs.
I hope this helps,
T
Dear Jonas,
Sorry to hear about your troubles with synthesis. We also occasionally had troubles like you are experiencing. There are a few things that are important here.
1) The correct ratio of Fe2+ to Fe3+ (otherwise proper MNPs have no chance to grow properly).
2) It can also be that the chemicals (iron salts) are old. The Fe2/3 solution should be translucent and without precipitates. Either buy fresh salts, or filter the solution. Otherwise, the existing precipitates could influence the MNP formation
3) From our experience, preheating the alkali solution is mostly sufficient to get a good synthesis. Degassing can help a bit too.
4) pH of the washing solution should not be too acidic. Otherwise you simply dissolve the uncoated MNPs.
I hope this helps,
T
Dear Tomek,
Thank you for your fast reply! And don’t get me wrong, I’m very happy that you and your team have made this protocol available to the research community. It would be a really great way to stretch our budget.
1) If the ratio would be wrong would I still get a magnetic precipitate before washing? Or does the faulty ratio cause MNPs that easily break down?
2) I will definitely filter the solution next time first. I did not see any real precipitate, but it wasn’t translucent either, it had more of a (slightly) cloudy appearance.
3) I’ll include these steps again in my next trial.
4) I’ll check the pH of the water (UPDATE: it turned out to be around 5.8) and if necessary I’ll use Tris buffer maybe (pH8?), is the use of Tris expected to interfere with the silica coating protocol?
Is it also important how quickly the iron salt solution is added to the sodium hydroxide? I don’t have a burette in the lab so I use a Pasteur pipette and I try to add the solution in a dropwise fashion.
Thank you again for the help and the beautifully documented protocols!
Kind regards, Jonas
Dear Jonas,
To answer your questions,
1) Yes, from my experience if the ratio is not optimal, one gets precipitate which is also magnetic (not as strong thou) which then in washing gives brownish “color”
2) The solution should be clear and not cloudy. Not sure what exactly the “precipitate” suspension is, but removing them with filtration helps the synthesis
4) We don’t add Tris or any buffer, the only thing what I would do is eventually to add a drop of NaOH to pure water that you use for washing if the pH is too low. Normally, the MNPs should not desolve in H2O during wash and normally we don’t addjust the pH of the water. But, in case your H2O is very acidic, one could consider adjusting it.
Regarding the speed of addition, I don’t think it matters too much here. We add it dropwise, but I also made MNPs by adding the whole iron solution at once. It does change the MNPs size and magnetic properties slightly, but they also work.
I’m glad that you like the protocols, let me know how they worked for you and I’m happy to advise shall you have any more troubles.
Cheers,
T.
Dear Tomek,
Thank you for taking the time to exhaustively answer my questions.
I will try the protocol again when I have some free time in the lab (might take some time though) and I’ll report back here.
Thank you again!
Kind regards, Jonas
Hi everyone,
I’ve just produced silica-coated MNPs. In my first attempt I’ve tried to do it in the same scale as in the protocol but got poor results – the MNPs lost their magnetic properties during washes. I’ve scaled down the procedure 4x and it worked very good. I’ve coated approx. 50% of the beads with TEOS with overnight incubation (the procedure was scaled down by 10x as compared to the original one). I’ve tested the beads in plasmid isolation and genomic DNA isolation from tissues – in both cases the MNPs worked very well (I’ve checked the DNA in electrophoresis and PCR, respectively). Here are some of my thoughts and tips that you may find useful:
– I think that the first attempt of the synthesis failed because I was not able to control the temperature of the NaOH solution. The heating plate of my magnetic stirrer was not efficient enough.
– I’ve filtered the FeCl2/FeCl3 solution, but it seems that the salts themselves do not have to be the highest quality – I’ve decided to use cheap chemicals from a local company and they worked very well
-I’ve degassed the NaOH solution using ultrasound bath instead of vacuum pump.
– The ethanol used in TEOS-coating indeed does not have to be 99%. We have used 96% technical grade ethanol. Tomek, thanks for the tip.
The BOMB project is an awesome idea and I’m very grateful that you decided to share your protocols with the rest of the world. Great work!
Cheers – Gosia
Hey Gosia,
Thanks a lot for the thorough feedback! Findings like these are excactly what we need!
Cheers
PhilHow do you safely store a 300kg pulling steady magnet?
Ferrous surfaces are everywhere, particularly near a fume hood. Would an electromagnet be suitable for this purpose? Any recommendations?
Indeed, one needs to be very careful with these magnets. We usually store them in a plastic box in a drawer and only take them out for the respective application. In our fume hoods we have the possibility to place the magnet next to the MNP container without any ferrous surfaces in the near environment (<50 cm), which is sufficient in our experience. However, I cannot emphasise enough to be very careful and never use two magnets simultaneously!
I would assume an electromagnet should be suitable as well, if you have one. We never tried one, but it should work as well.
Dear all,
After a long hiatus, I tried the protocol again, paying attention to the different critical points. I filtered my iron salts solution to have a perfectly clear solution to start with, degassed the solutions using sonification and also adjusted the pH of the water.
But again, I’m having the same problem: after a few washes the solution gets increasingly dark.
I took some of the bead solutions and checked them with the light microscope (400x magnified) and I can see a lot of irregular large pieces. They do seem to react to the magnetic field, but their presence must indicate I did something wrong during synthesis right? I have included 2 external links for pictures here:
Thank you for your help.
Kind regards, Jonas
Dear Jonas,
Sorry to hear that it still didn’t work for you. One thing that comes to my mind is that the iron salts could be old. Buying fresh good quality chemicals solved a similar issue for another user. And then the synthesis worked like a charm. Not sure if this is the trouble here, but small amounts of fresh salts one can buy relatively cheaply and if this indeed solves the issue would be great.
Cheers,
T
Dear Tomek,
Thank you for your kind reply, maybe the age/quality is indeed the issue because I have the impression that the washing steps are going even worse than before…
I think I’ll just have to bite the bullet now and order pre-made paramagnetic beads which I can then use with the different protocols your team has developed. If that all works out, we can still try to synthesize beads ourselves at a later time.
Thank you for your help!
Kind regards, Jonas
Dear all,
I work with magnetic beads in serology, specifically carboxyl coated magnetic beads from Luminex. I couple antigens in those beads and then perform antibody detection.
I was wondering if it is possible to use particles produced with the “protocol #1.1 magnetic core nanoparticles synthesis” the same way I use the beads I currently use.
Does anybody knows if it is possible?
If you need more information, please let me know.
My best regards,
Guilherme
Dear Guilherme,
Thank you for the question. The protocol 1.1 magnetic core particles are not stable long term. They have to be coated either with silica (protocols 2.1) or carboxyl (protocol 3.1) for full functionality. I guess if you were using COOH beads before, then you could use the #3.1 beads for coupling. Another chemistry would have to be used for silica beads.
Hope this helps,
Best regards,
Tomek
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