Construction Of Multi-Layer SiO₂ Nanoparticles Scaffolds Lined Into Capillary And Its Circulating Tumor Cell Capturewith High Load

Circulating tumor cells(CTC)are free tumor cells in patients'blood,which can be used as biomarkers for diagnosis and prognosis.Although many CTC detection methods have been developed,the capture efficiency and detection sensitivity still need to be improved.The purpose of this study is to construct multi-layer SiO₂nanoparticles lined scaffolds in quartz glass capillary(200 mm(L)×1.0 mm(D))and modified aptamers to study its ability to capture CTC specifically.Firstly,based on the amino colorimetric method of silk surface,the static colorimetric reaction device,one-way flow washing device and static hydrolysis reaction device were assembled to obtain the amino colorimetric method suitable for the capillary inner surface to characterize the amino content,which was used as a quality control method for the construction process of multi-layer nanoparticles lined scaffolds in the capillary.Then,the capillary with 1st-layer nanoparticles lined scaffolds(1-LNLS)was optimized in the circulating flow reaction system.In the first step,the average amino density on the inner surface of capillary increased from 0.0193 nmol/mm²to 0.1180 nmol/mm²when the optimal amino reaction time of 10h and the concentration of amino reagent APTES was 5%,based on the maximum amino density on the capillary inner surface.In the second step,the amino SiO₂nanoparticles(Asi NP)with hydration particle size of 101.89 nm and zeta potential increased from-42.65 m V to-15.68 m V were prepared.In the third step,1-LNLS was constructed by the activation rate and grafting concentration of PAA was 10%and 4.0 mg/ml,respectively,and the loading concentration of Asi NP was 3.2 mg/ml.Then,PAA and Asi NP were grafted onto 1-LNLS to construct 2nd-layer(2-LNLS)and3rd-layer nanoparticle lined scaffolds(3-LNLS)in capillary.Based on the maximum amino density on the capillary inner surface,2-LNLS was prepared with the concentration of PAA and Asi NP were 2.0 mg/ml and 1.5 mg/ml,respectively,and 3-LNLS was prepared with the concentration of PAA and Asi NP were 0.8 mg/ml and 0.5 mg/ml,respectively.1-LNLS,2-LNLS and 3-LNLS were characterized by SEM and AFM.According to the SEM positive characterization results,the nanoparticles were immobilized on the inner surface of the capillary,and the nanoparticles still had good morphology after immobilization.,and the imbedded nanoparticles on the inner surface increased gradually from 1-LNLS,2-LNLS to 3-LNLS.The SEM side characterization results showed that the stack height of nanoparticles increased in turn.AFM characterization showed that compared with the inner surface of blank capillary,the roughness of aminated capillary,1-LNLS,2-LNLS and 3-LNLS increased in turn.The root mean square roughness of the inner surface of those capillary was 3.68,8.97,20.3,35.1 and 49.5 nm,respectively,indicating that 1-LNLS,2-LNLS and 3-LNLS were successfully prepared.1-LNLS-PAA,2-LNLS-PAA and 3-LNLS-PAA were prepared by grafting PAA onto multi-layer nanoparticles lined scaffolds,and then,1-LNLS-Apt 2-LNLS-Apt and 3-LNLS-Apt were prepared by grafting aptamers.A549 cells were used to simulate CTC for high-density cell suspension capture experiment(500000 cells/ml),the number of specific captured cells in each1-LNLS-Apt,2-LNLS-apt and 3-LNLS-Apt was 6999,11102 and 21037,respectively,and the number of non-specific captured cells of per 1-LNLS-PAA,2-LNLS-PAA and 3-LNLS-PAA without fixation of aptamers was 3857,3692 and 3428,respectively.In conclusion,the non-specific capture was not related to the number of scaffold layers,and the specific capture increased with the increase of the number of scaffold layers,which indicating that the increase of scaffold roughness was conducive to the increase of the cell loading in capillary,and the maximum cell loading of 3-LNLS-Apt reached 3346.7 cells/cm².In extremely low density cell suspension,namely 5000 cells/ml and 100 cells/ml,the capture efficiency of 3-LNLS-Apt was71.14%and 92.38%,respectively.Compared with the same type of capture capillary,the capture efficiency of the literature was 88%at the density of 100 cells/ml,and the cell density of3-LNLS-Apt was 9.4 cells/cm²,which was higher than that of 6.8 cells/cm²in literature.Under whole blood interference,1260 target cells could still be captured by 3-LNLS-APT.In conclusion,the multi-layer nanoparticles lined scaffolds constructed in the capillary in this paper can further realize the high-load and high-sensitive capture of CTC.