| Objective:To construct fluorescent probes and magnetic separation probe by FA-PAMAM targeted system combined with biotin-streptavidin amplification system for ovarian cancer SKOV3 cells separation and detection.Methods:Streptavidin?SA?was covalently conjugated to QDs and MNPs through the active ester method,and analyzed their physical properties through agarose gel electrophoresis and Melvin laser granulomere,to verify success of the coupling.Biotinylated FA was synthesized through the carrier PAMAM using the same method,FTIR spectrometer was used to detect new chemical bond formation.Through the nature of SA-QDs specific binding with biotinylated FA,FA targeted SA-QDs fluorescent probe could specific target to SKOV3 cells.The fluorescence of FA-PAMAM targeted system combined with BAS signal amplification system were observed under fluorescent inverted microscope,and the FR lower expression A549 cells were regarded as control group,to verify the target specificity of the probe.FA-PAMAM targeted system combined with biotin-streptavidin magnetic signal amplification system for the capture model of CTCs was constructed.By two-step incubation,first joined biotinylated FA incubating with SKOV3 cell for a certain period,then put SA-MNPs in.SKOV3 cells were separated under the action of 1.0 T magnetic field.In order to obtain high capture rate,the separation conditions were optimized.Detected the toxic effect of the capture system through MTT test,and observed the culture of capture SKOV3 cells to prove that the isolated cells by capture system can be used for further research.Results:1.Melvin laser granulomere results showed that the particle size distributions of QDs?17.86 nm?and MNPs?24.48 nm?were relatively concentrated,with excellent monodispersity,the sizes of SA-QDs?29.47 nm?and SA-MNPs?30.33 nm?were increased after coupled with SA;Zeta potential determination results found that the surfaces of QDs and MNPs showed negative potential in ultrapure water,-?58.10±4.56?mV and-?41.60±3.26?mV respectively,the negative potential of SA-QDs and SA-MNPs were reduced after coupled with SA,-?26.30±2.06?mV and-?27.00±2.11?mV respectively.QDs,SA-QDs,MNPs and SA-MNPs were all moving from the cathode to positive pole in agarose gel.But after coupled with SA,the moving speed of SA-QDs and SA-MNPs were slower than that of MNPs and QDs,respectively.And when excited by ultraviolet light,QDs and SA-QDs transmitted strong red fluorescence.FTIR spectra showed that the stretching vibration absorption peak of C=C key on the benzene ring can be seen in 1550 cm-11650 cm-1,while the stretching vibration absorption peak C-H key on the benzene ring can be seen in 3200 cm-13350 cm-1,these characteristic peaks were from FA,confirmed that the FA had successfully coupled to the PAMAM.At the same time the stretching vibration absorption peak of biotin can be seen in 1650 cm-11750cm-1,indirect proof of FA-PAMAM-biotin coupling success.2.The FA targeted SA-QDs fluorescence probes could specifically recognize the FR positive SKOV3 cells,the average fluorescence intensity up to 114.92±2.87 a.u.In contrast,the fluorescence intensity reduced when the cells were pretreated with excess FA?57.86±7.58 a.u?,and the fluorescence intensity of low FR expressing lung adenocarcinoma A549 cells were weaken?14.94±0.83 a.u?.Simultaneously,we found that FA targeted SA-QDs fluorescence probes was distinguished from QDs probes without PAMAM?47.81±1.35 a.u?or BAS?77.50±2.43 a.u?mediated by a stronger fluorescent intensity.3.The capture model of CTCs was constructed,capture condition optimization results showed that FA-PAMAM-biotin imputed amounts to 2 pmol,FA-PAMAM-biotin reacted with SKOV3 cells for 60 min,then 70?g SA-MNPs incubation for 30 min,and magnetic separation 30 min were optimal capture conditions,capture efficiency up to a maximum of 83.41%.4.MTT colorimetric method showed that the materials used in the experiment were non-toxic to cells,and the cells could still maintain good activity after magnetic separation.Conclusion:1.SA-QDs and SA-MNPs were successfully coupled with stable and good biocompatibility,and the FA-PAMAM-biotin was successfully synthesized.2.SA modified QDs combined biotinylated FA can targeting mark SKOV3 cell,the dyeing method has obvious quantum dot fluorescent signal amplification effect.3.Combination of FA-PAMAM targeted system and BAS signal amplification system mediated magnetic signal amplification,can specifically and efficiently recognize FR expression on SKOV3 cells capture,and the cells captured have good biological activity.The capture system has a certain application prospect in terms of early detection of CTCs. |
PDF Full Text Request