Biotin-streptavidin Based Immunomagnetic Signal Amplification System For Ovarian Cancer Circulating Tumor Cells Separation From Human Whole Blood

Objective:1.To stably synthesize the streptavidin（SA）conjugated magnetic nanoparticles（SA-MNPs）and verified its synthesis and physical characteristics.2.Folic acid and biotin were stably conjugated through the carrier of bovine serum albumin forming biotinylated folate,and its features were tested.3.Present a simple and efficient immunomagnetic separation method for capturing cancer cells using SKOV3 cells as a model using FA-conjugated MNPs based on the biotin～SA coupling system to explore a new method for early detection of ovarian cancer（OC）.4.Verifying the effect of biotin～SA based immunomagnetic nanoparticles system in separation of circulating tumor cells from OC patient peripheral blood samples to show its potential application in early stage diagnosis of clinical cases.Methods:SA was covalently coupled with MNPs through the method of EDC/NHSS,the physical characteristics of the prepared MNPs-SA were verified by Transmission electron microscope（TEM）,dynamic light scattering（DLS）,and agarose gel electrophoresis.Biotinylated folate was synthesized through the carrier bovine serum albumin using the same method,which was tested by Fourier transform infrared spectrometer（FTIR）.The conjugates MNPs-biotin and MNPs-PEG-FA were prepared using the same method as the MNPs-SA conjugates,all with a molar ratio of biotin-BSA or FA-PEG-NH2 to MNPs at 20:1.4 test samples were prepared by spiking 450 cells of Hoechst.33342-stained SKOV3 cells into 1 m L human blood（1:10⁹/ml）.An appropriate of biotin-FA was added to the cell suspension in a 1.5 m L centrifuge tube while none was added to three other tubes containing the cells.After incubation in a vertical mixing device at37^oC for 1 h（the optimal conditions）,50μL of the obtained MNPs-SA conjugates/MNPs-biotin conjugates/MNPs-PEG-FA conjugates were added to the mixed cell suspension and incubated with mixing for 1 h.The cells was captured on the wall of the tube in the presence of a high magnetic field（1.0 T）in 2 h.The captured cells were re-suspended in 50μL PBS.The capture efficiency（CE）was measured by imaging and counting the pre-stained SKOV3 cells under a fluorescence microscope.By comparing the CE,the targeting specificity of folic acid to SKOV3cells were tested,and a stronger magnetic signal of biotin-SA based immunomagnetic nanoparticles separation system were validated.Since rapidity of the biotin～SA based immunomagnetic separation system for CTCs isolation were crucial factors for its practical utility,attempts were made initially to optimize the magnetic signal amplification conditions and meticulous selection of other magnetic separation parameters.In this study,different coupling ratios and molar ratios of MNPs-SA conjugates and biotin-FA were optimized,and the specificity of the MNPs-SA～biotin-FA complexes to capture tumor cells was investigated.And we investigated the efficiency to capture SKOV3 cells（ca.450cells per m L）in whole blood for different durations of incubation and different separation time.The capability of the MNPs-SA～biotin-FA complexes to capture rare SKOV3 cells were also investigated in different medium.The viability of the captured CTCs is very important for further investigation of their malignant nature and to assess the invasive potential of individual CTCs in clinical studies.Thus,the MNPs-SA～biotin-FA captured cells were cultured on a plate to examine their bioactivity.After the CTCs were captured from matrix using the MNPs-SA～biotin-FA system developed above,a commonly used ICC method was applied to identify and enumerate CTCs from non-specifically trapped blood cells,including goat polyclonal antibody anti-HE4,a marker for SKOV3 cells and QSH550-labeled rabbit anti-goat Ig G as well as Hoechst.33342 nuclear staining.Having proved that the tumor cells could be captured with MNPs-SA～biotin-FA complexes efficiently and specifically,we applied this system to detect OC cells in whole blood samples from 20 OC patients and 100 healthy females to judge its value in practical application.The isolated cells were also identified with ICC,and cells positive for HE4-QDs and Hoechst were enumerated as CTCs.Results:1.The TEM images revealed that the SA-MNPs were well dispersed without aggregation with a uniform size distribution without significant change in morphology after conjugation.The size distribution of SA-MNPs conjugates was from 18.06 to 85.9 nm with an average size of 28.53 nm.Compared with the TEM results,the average hydrodynamic size was bigger than that of the unconjugated MNPs with 25 nm.The zeta potential of SA-MNPs went-0.44 m V while the MNPs on its own was at-26.86 m V.The changes in the MNPs before/after conjugation with SA were demonstrated in the gel electrophoresis.mobility in 1%（w/v）agar gel at 90V over 30 min showed faster mobility of the unconjugated MNPs compared with the SA-MNPs.2.FTIR shows that biotin was covalently attached to the amine-functional groups on the surface of BSA forming the bands that are unique to conjugated biotin at 1540 and～1660 cm^-1that are indicative of amide II（N-H bend+C-N stretch）and amide I（C=O stretch）vibrational modes,respectively.These bands indicated the amide bonds formed between biotin and BSA.Besides,the characteristic peak(1606cm^-1)of cyclobenzene loops which are present in FA were shown in biotinylated folate.3.In this study,conjugates of SA-MNPs,biotin-MNPs,FA-PEG-MNPs and FA-biotin～SA-MNPs were prepared to capture OC SKOV3 cells.The results showed that the conjugates of MNPs-PEG-FA（42%）and FA-biotin～SA-MNPs complexes exhibited a higher CE than that of MNPs-SA and MNPs-biotin,while the biotin-FA with SA-MNPs was the highest with a CE of 73.1±9.5%（mean±SD）,which showed that FA could specifically target to SKOV3 cells,and the biotin～SA based immunomagnetic nanoparticles system demonstrated a stronger magnetic singal.4.The optimized parameters for the biotin～SA mediated immunomagnetic nanoparticle separation process showed that MNPs to SA at 1:10 to form MNPs-SA and biotin to BSA-PEG-FA（biotin-FA as biotin-BSA-PEG-FA,where FA is folic acid or folate）ratio of 40:1 to form biotin-FA provided the best conditions for conjugation.During SKOV3 capture,4 MNPs-SA conjugates complexed with one（1）biotin-FA and resulted in the capture of>91.4±4.7%SKOV3 cells using 1 h incubation with biotin-FA,1 h incubation with MNPs-SA and 2 h exposure time to a magnetic separator.In contrast,only 20.5±3.5%folate receptor negative A549 cells were captured.Moreover,the isolated cells were used directly for culture and immunocytochemistry identification for specificity without pretreatment.The immunomagnetic nanoparticle separation was slightly affected by the matrices as shown by 98±2%,83.5±6.1%,80.0±7.8%capture efficiency of SKOV3 cells in PBS,lysed blood,and whole blood,respectively,but still allowed for extraction of the CTCs even in untreated thick whole blood.This indicated that the MNPs-SA～biotin-BSA-PEG-FA system was successfully used in whole blood with relatively high capture efficiency.5.There were no obvious green fluorescence in 100 healthy females whole blood,and 16 out of 20 OC patients showed strong green fluorescence,which showed a consistency of 80%to clinical diagnostic criteria.Conclusion:1.MNPs-SA and biotinylated folate were successfully synthesized.2.Present a simple and efficient immunomagnetic separation method for capturing cancer cells using SKOV3 cells as a model using FA-conjugated MNPs based on the biotin～SA coupling system by the specific combination of FA and FR.3.The biotin～SA based immunomagnetic nanoparticle isolation system can separate OC SKOV3 cells from female whole bood without destructive,which showed a promising tool for CTC enrichment and detection of early stage OC.