Microfluidics Enabling Liquid Biopsy And Single-cell Sequencing

Liquid biopsy allows noninvasive detection of circulating tumor cells(CTCs),exosomes,and circulating tumor DNAs in biofluids,providing real-time,dynamic,and comprehensive physiological and pathological information,holding great value for tumor diagnosis,prognosis assessment,efficacy monitoring,recurrence prediction,tumor metastasis and drug resistance research.Celluar heterogeneity commonly exists in cell populations,which is masked by traditional bulk cell analysis.Single-cell transcriptome analysis dissects cell heterogeneity by profiling the gene expression of single-cells.Recently,microfluidic technology has been widely used in the field of liquid biopsy and single-cell analysis owing to the advantages of low consumption of samples and reagents,rapid reaction,high detection sensitivity,automation,miniaturization,integration,high throughput,accurate fluid control,as well as good biocompatibility.However,due to the scarcity and heterogeneity of CTCs,and the interference by background cells,current microfluidics-based CTC detection methods still have same limitations,such as insufficient enrichment efficiency,purity,release efficiency as well as viability.Additionally,current single-cell analysis methods based on microfluidics are not suitable for efficient and parallel single-CTC transcriptome analysis due to the potential CTC loss in the processes of cell transfer and single-cell capture.To solve the abovementioned issues,this thesis developed a series of innovative microfluidic strategies for efficient capture and controlled release of CTCs,as well as for efficient capture and parallel transcriptome analysis of single CTCs.The main work is as follows.(1)Stimuli-responsive microfluidic interface for noninvasive myeloma evaluationA stimuli-responsive ligand decorated microfluidic chip(MRD-Chip)was developed for efficient capture,controlled release and cytogenetic analysis of circulating myeloma cells(CMCs).By decorating the herringbone chips with CD 138 antibody targeting plasma cells and introducing a disulfide bond linker between the microfluidic interface and CD138 antibody,the MRD-Chip can caprure circulating myeloma cells with high efficicncy and release the captured CMCs via a thiol-exchange reaction.Immunofluorescence assay was performed to identify the cell type of captured cells,and the CMC counts were used to monitor the therapeutic efficacy of multiple myeloma patients.Furthermore,the chromosome abnormality of released CMCs was checked by fluorescence in-situ hybridization(FISH)technology.This method has the advantages of low sampling invasiveness,high sample throughput,high capture efficiency and controllable release of CMCs,as well as compatibility of released cells with cytogenetic analysis,paving a new way for noninvasive myeloma evaluation.(2)Biomimetic multivalent aptamer "nanoclimber" functionalized microfluidic chip for highly sensitive minimal residual disease detectionA multivalent aptamer functionalized microfluidic chip was developed for highly sensitive minimal residual disease detection.Inspired by the climbing plants,we synthesized the multivalent aptamer "nanoclimber"(MANC)through a free radical copolymerization reaction.Due to the flexible skeleton and multivalent aptamer configuration,MANC exhibts higher affinity towards residual leukemia cells than individual aptamer in complex biofluids.Furthermore,we coupled the MANC with a microfluidic chip based on the principle of "deterministic lateral displacement" for efficiently capture residual leukemia cells in the peripheral blood from acute T lymphoblastic leukemia patients.Nuclease was chosen to degrade the MANC for efficient and nondestructive release of residual leukemia cells.This method possesses the advantages of low sampling invasiveness,high capture efficiency,high purity,and high release efficiency of residual leukemia cells,providing new opportunities for noninvasive and highly sensitive minimal residual disease detection.(3)DNA-programmed ordered multivalent aptamer microfluidic interface for liquid biopsyA cubic DNA nanostructure(CDN)-programmed orientation-ordered multivalent aptamer(CDN-Apt)functionalized microfluidic interface(CDN-Apt-Chip)was developed for efficient capture and release of residual leukemia cells.The CDN-Apt is prepared by DNA self-assembly,and shows higher affinity tawords residual leukemia cells than individual aptamer due to multiple aptamer configuration.Benefiting from the ordered orientation and multivalent binding effect,the CDN-Apt-Chip can capture residual leukemia cells with high efficiency.Additionally,efficient and nondestructive release of residual leukemia cells was achieved by using nuclease to degrade the CDNApt.The released cells were subjected to T cell receptor sequencing to reveal the T cell clonal dynamics related to treatment response.This method shows the advantages of low sampling invassiveness,high sample throughput,high capture efficiency and release efficiency of residual leukemia cells,high utilization of aptamer,as well as compatibility of released cells with transcriptome analysis,providing a new paradigm for microfluidic interface regulation and promoting the clinical application of aptamerbased liquid biopsy.(4)CTC-Paired-Chip:Efficient single circulating tumor cell capture and parallel transcriptome sequencingA microfluidic strategy(CTC-Paired-Chip)based on the principle of differential flow resistance" was proposed for efficient and paired capture of single-CTCs/singleDNA barcoded beads,and parallel transcriptome analysis of single-CTCs(CTC-Pairedseq).According to the principle of "differential flow resistance",single DNA barcoded beads and single CTCs were captured in paired microfluidic chambers one by one.By integrating and accurately controlling the microvalve and micropump on CTC-PairedChip,rapid droplet generation and mixing,fast cell lysis,efficient mRNA caprure and bead washing,as well as efficient reverse transcription and enzyme digestion reaction sequentially took palce on a single chip.After bead retrieval,cDNA amplification,library construction and high-throughput sequencing,we realized parallel single-CTC transcriptome sequencing.This method has the advantages of high utilization of CTCs and DNA barcoded beads,high detection sensitivity,low contamination of cell-free mRNA,and capable of parallel single-cell analysis,providing an effective tool for the heterogeneity study of CTCs.