I think your sample is not stable in the solvent you prepared. They tend to aggregate and form clusters of different sizes. One thing you can test is the zeta potential. When the zeta potential is in the range -5 ~+5 mV, the sample could not form stable colloid. In this case you might need to find another solvent to disperse your sample and get the accurate size distribution.

Please try to do few things;

Sonicate your sample as much time as possible (if sonication will not effect to your self assembled structures),

Optimeize the sample concentration of DLS measiumeuerement by Diluting and re measuring your sample as many times as possible,

Please try to make sure that you are using suitable parameters in the software program (i.e., absorbance, refractive index, solvent type, cell type etc.)

Thanks for your answers.

Xiaojing Xia : the zeta potential is about 24

Naveen Kumar Reddy Bogireddy : I sonicated them for 10 min, upon dilution the sizes getting larger (for example for 0.1 mg/ml size was larger than 0.5 mg/ml), and thank you for the last one that you advice me, I will check them

You should calculate the hydropathicity of your peptide. This may just be its natural behavior. If your peptide is amphiphillic you will need to use a solvent that dissolves hydrophobic and hydrophillic susbstances, or make a mixed solution ( for example buffer and acetonitrile or buffer and an alcohol (50:50 or 80:20, depends).

Saeedeh Khazaei thanks

Welcome