Diffusion Of Ca²⁺ Ions During The Rupturing Process Of MWCNTs Processed Aloe Protoplast In Hypotonic Solution

Carborn nanotubes?CNTs?are widely applied in the fields such as biomedical engineering,electronics and environment science with its unique structure and superior physical and chemical properties.Along with its rapid application,the bio-safety of CNTs becomes the hot spot.Calcium ions are widely existed in cells,also they play important role as the second messenger.Investigating the effects of CNTs processing on the Ca²⁺ level in cells can improve our understanding on the toxical mechanism of CNTs.This thesis mainly focused on the Ca²⁺ diffusion during the rupturing process of a single Aloe cell protoplast processed with multi-wall carborn nanotubes?MWCNTs?,and its relations to the damage of cell membrane.Aloe cell protoplasts were prepared with enzymolysis method.There viability was confirmed by FDA.Purified MWCNTs was ultrasound dispersed in Ca²⁺-free culture medium to obtain 35 mg/L suspensions.Aloe cell protoplasts were equally divided into two parts,one was mixed with 30 mL MWCNTs suspensions?test group?,the other was mixed with blank Ca²⁺-free culture medium?control group?.Both the test and the control were cultured in the same condition for13 h before Ca²⁺ testing.Ca²⁺ testing was carried out with calcium ion selective micro-electrode.The tip size of the electrode was 2³ ?m,the average response was better than 27mV/pCa,the detection limit was lower than1.52×10^-6mol/L,the response time was 0.6 s.The results showed that when the protoplast ruptured,Ca²⁺ concentration exhibited sharp rise and a slow descending.It is very interesting that the peak value of the Ca²⁺ concentration was much higher than that in normal cells cytoplasm.This may be related to the destruction of Ca²⁺ pool.The transient of Ca²⁺ concentration was calculated theoretically by solving the diffusion equation.After fitting the experimental data,the apparent diffusion coefficient was obtained.The apparent diffusion coefficients of both the test(2.14×10^-5cm²/s)and the control(2.64×10^-5cm²/s)were lower than the free-diffusion coefficient(5.3×10^-6cm²/s)of Ca²⁺.The reason may be that in hypotonic culture medium,the pressure within the protoplast increased,this high pressure accelerated the diffusion of Ca²⁺.We also noticed that the apparent diffusion coefficient of control was lower than that of test,indicating a relative low pressure in the protoplast of test compared with that in the protoplast of the control.This meant a decrease of the strength of the cell membrane caused by MWCNTs processing.