I am trying to measure the mean size of J774A.1 exosomes. According to previous literature, the mean size should be 100 ± 20 nm. When I run the dynamic light scatter on my 100 nm latex bead control in the same solvent as my exosome sample (1x PBS, 0.14 M NaCl), my results are perfect. However, when I run the same protocol on my sample, I get these horrible results with means 3x the size the exosomes are supposed to be, high polydispersity index, as well as baseline errors as high as 44%. Does anyone have any suggestions on how I can correct this? I've tried gentle sonication to break up the aggregated particles, chilling them to 4 C before running the sample in the DLS, filtering with a 0.22 um filter.
Which technique was used in the previous study, DLS as well?
Dynamic light scattering is very sensitive to small amounts of aggregates, because larger particles scatter exponentially more light than smaller particles. Assuming that your exosome sample is cleaned up, you could try filtration through a larger poresize (0.45 micrometer) or gentle centrifugation with a table-top centrifuge. If the scattering intensity is quite low, can the sample concentration be increased further? It does not sound like a problem with too much signal, as that rarely shows baseline errors. Without looking at the real data, this is difficult to diagnose. If the problem persists, please post the derived countrate, the intercept of the correlation function, mean size and PDI. You could also attempt to make DLS measurements at 4C directly.
First thing you can try is to perform a UV-Vis absorption of your samples. if your sample show high absorption at the same wavelength of UV-Vis laser, then you are experiencing large error. The other test you can do to get sure the result you get are viable, is to dilute the samples by a factor of for example 5, 50, and 500 and carry out the DLS again. Usually each photo should be scattered by one particles, the reason why you need to have very diluted samples.
Sometimes centrifugation may help as well. Light scattering is very sensitive to even very few larger particles/agglomerates/ dust. [ http://www.materials-talks.com/blog/2014/11/17/tips-tricks-for-nanoparticle-characterization/#exosomes ]
It may also be a good idea to look into nanoparticle tracking analysis NTA. See "NTA or DLS - which is better suited for nanoparticle size analysis?" at http://www.materials-talks.com/blog/2016/09/15/nta-or-dls/
Can anybody please tell about the minimum quantity of exosomes required for DLS analysis? Thank you in advance.
- Badges
- Science topic
- Similar topics
- Philosophy
- Esthetics
- Interpretation