Biomembrane-Si Nanopore Array Based Optical Biosensor And Drug Screening System Construction

Bio-membrane system has the function of blocking exogenous injury and maintaining a stable, orderly life activity. As an outer structure of organism, bio-membrane system provide necessary energy, information and materials exchange to keep life activities. Capability of selective permeability across bio-membrane systems provides function of exchange, as well as protection. For those drugs targeting to physiological processing within cells, it’s necessary and vital to have the ability of transporting through bio-membrane into intracellular environment. While, the blockage effect of bio-membrane largely limits the bioavailability of intracellular targeting drugs due to blockage effect. Therefore, bio-membrane permeability assays play an importakt role in drug screening.During the last three decades, the study of bio-membrane system function and drug permeability developed gradually from the complicated in vivo research to in vitro experiment. In vitro models included computer simulation, molecular-based model, artificial membrane based model, cell-based model and 3D-tissue-based model. Efficient method to study membrane permeability was limited. At present, the mostly used methods were based on fluorescent label or electric detection. However, fluorescent probe might have some influence on cell function and interrupt the natural structure of drugs. Chip design and device based on electric signal detection were always complicated and easy to be polluted, resulting in the poor reproducibility. To overcome the shortcomings of existing methods, we are aiming to establish a label-free optical detection platform for the study of bio-membrane function and drug permeability. This thesis constructed a microfluidic Si nanopore array supported artificial bio-membrane platform. Based on the micro-structure and optical signal of Si nanopore array, the pore blockage effect of bio-membrane on nanopore array was studied. On this optical biosensor platform, the selective ion permeability of ion channel protein, kinetic cell membrane translocation behavior of peptides and drug permeability through blood-brain barrier (BBB) were studied by FT-RIS. These works laid the foundation for the use of this label-free optical biosensor on drug screening.The main contents of this thesis are as follows:(1) A Si nanopore array-hydrogel supported artificial cell membrane system was established. With the signal amplification of pH sensitive hydrogel, sensitive pH sensing ability of the pSi-hydrogel sensor was studied by real-time FT-RIS. Si nanopore array supported phospholipid bilayers (PLBs) were constructed integrating with microfluidic chip. The pore blockage effect of PLBs on H+ permeability was verified by FT-RIS. After ion channel protein inserted, part of the ion permeability of PLBs was verified to recover. On this basis, blockage effect of ion channel inhibitor on ion channel protein was studied to establish a promising system for ion channel inhibitor drug screening.(2) A pSi nanopore array supported artificial cell membrane system was established to study the peptides permeability. The integrity and mobility of cell membrane was verified by FT-RIS, FRAP and steady-state current measurement. Based on the pore blockage effect of artificial cell membrane, the kinetic transport processing of peptides through cell membrane was monitored by real-time FT-RIS. Three types peptides with different action mode on cell membrane were studied to establish a model to discriminate cargo peptides from antimicrobial peptides (AMPs). The discrimination performance was consistent with cell-based and microbial culture-based experiment. Finally the platform for cargo peptides and AMPs drug screening was established.(3) A pSi nanopore array supported in-vitro BBB model was established. Co-cultured with extracellular matrix Matrigel, hBMECs were successfully cultured into monolayer with tight junction structure. The integrity of in-vitro BBB model was verified by FT-RIS and immunofluorescence. Based on the pore blockage effect of BBB, an innovation label-free optical detection to study in-vitro BBB model was demonstrated. Permeability decreasing of BBB was observed during 5 days culture. Acetylcholine and fluorescein sodium were used as model molecules to test the in-vitro BBB model. On this model, concentration based permeability of three antibiotics (chloromycetin, ciprofloxacin and eythromycin) was studied to establish a platform for antibiotics drug screening targeting to brain injury.(4) A pSi nanopore array supported immobilized bacteria system was established based on label-free immunoassay. This system was especially suitable to bacteria and cell analysis. Bacteria were immobilized on the nanopore array by immunoaffinity between bacteria and antibody. Based on the pore blockage effect of bacteria, in-direct FT-RIS method by detecting optical probe BSA was established to study the component and density of artificial bacterial biofilm. This system provides potential application in low drug-resistance antibiotics screening.(5) In the last section, the author introduced her research during one-year exchange program in Stanford University, USA. A colorimetric sensor array was constructed for diagnosis of inflammatory disease by distinguishingly responding to urine samples from patients with different diseases. High-throughput imaging and computational graph analysis were combined to analysis the cross response pattern to body fluid samples with similar disease. Statistic algorithm was introduced to obtain the best sensor array. This sensing array allowed the identification of 94% of Kawasaki disease subjects (ROC AUC:0.981) in the training set and 94% of KD subjects (ROC AUC:0.873) in the testing set from other febrile illnesses. Literature mining was done by statistic method to discuss the probable sensing mechanism.