A Microfluidic Chip-Based System For The Isolation And Detection Of Drug-Resistant Cells In Acute Myeloid Leukemia

Acute myeloid leukemia（AML）is a common malignant disease with a high mortality rate,because the treatment of this disease is mainly based on drug chemotherapy,which can easily lead to drug resistance and relapse.Several studies show that CXC chemokine receptor4（CXCR4）overexpression is highly correlated with poor prognosis and drug resistance in AML patients.Therefore,isolation and detection of AML cells with CXCR4 overexpression will be crucial for the treatment of AML.Currently,the mainstream cell sorting methods are flow cytometry and magnetic activated cell sorting,but flow cytometry requires high instrumentation requirements,while magnetic activated cell sorting requires large sample volumes and low separation efficiency,both of which have their shortcomings.In response to the current status of drug resistance and relapse in AML and the limitations of current cell sorting methods,this work designed a microfluidic chip-based method for the isolation and detection of drug-resistant cells in AML,combining magnetic nanoprobes with microfluidic chips,which could achieve rapid isolation and detection of drug-resistant cells in AML and provide an effective means of prognosis detection for AML patients.Firstly,Fe₃O₄@OA nanoparticles of 10 nm and 18 nm size were successfully prepared by high-temperature thermal decomposition method,and the saturation magnetization intensities of both were 80.69 emu/g[Fe] and 86.81 emu/g[Fe],respectively.Subsequently,the Fe₃O₄@OA nanoparticles with a higher magnetization intensity of 18 nm size were selected for the experiments,and two types of magnetic fluorescent nanoprobes were successfully prepared.The first one was a magnetic fluorescent nanoprobe based on antibody 12G5（Fe₃O₄@PEG@12G5-F647）and the second one was a magnetic fluorescent nanoprobe based on small molecule peptides（Fe₃O₄@PEG-F647/BKT140）,and the hydrodynamic size,surface zeta potential,and fluorescence intensity of the probes were characterized.The targeting effects of these two probes were investigated by using confocal microscopy,fluorescence microscopy,and flow cytometry,and the results showed that the antibody12G5-based magnetic fluorescent nanoprobe had a higher targeting rate.Therefore,the highly targeted antibody-based magnetic fluorescent nanoprobe was selected for the next experiments.Then the microfluidic chip was designed and the rationality of its design was verified by simulation results.After that,the initial screening of viscoelastic sheath fluid was performed by rotational rheometry and used for the separation of erythrocytes and HL-60 cells in microfluidic chips.According to the separation efficiency of different sheath solutions at different flow rates,the sheath solution was selected as PEO solution with a mass fraction of0.2 wt% and the flow rate was set to 200 μL/h,which had a high cell separation efficiency.The conditions of probe targeting cells were further explored.When the probe was used at 30μg,the volume of the probe and cell solution mixing system was 250 μL,the incubation time was 20 min,and the flow parameters applicable to the cell separation were selected,the magnetic capture efficiency of the U-shaped array capture zone of the microfluidic chip was82.92% ± 7.03% for the target cells under the action of external magnetic field.Finally,a microfluidic chip-based fluorescence quantitative detection system for drug-resistant AML cells was designed and constructed.The application of the detection scheme of the system includes the following four processes,in order of sample processing,probe labeling,microfluidic separation and capture,and fluorescence signal detection.The system was further applied for the quantitative fluorescence analysis of HL-60 cell samples,and the accuracy and linear range of the assay were obtained.The results showed that 1380 ～20000 target cells could be detected out of 4 × 105 cells.The accuracy results were within10%,proving the high accuracy of the present assay system.In addition,comparing this system with a flow cytometry-based system for achieving fluorescence quantification of drug-resistant cells,the consistency analysis again verified the high confidence of this solution.