Fluorescence Nanopore Arrays And Its Applications In Single-Molecule Biosensing

Since Kasianowicz et al.demonstrated the first single-stranded nucleic acid molecule sensing by staphylococcus aureus alpha hemolysin(?-HL)in 1996,nanopore technology has gained wide attention due to its advantages of high sensitivity and its prospect of single-molecule sequencing.It has gradually developed into a systematic single molecule biophysics method,which plays an important role in basic chemistry,life science,clinical diagnosis and environmental detection.In 2014,Oxford Nanopore Technologies released the first commercial nanopore sequencer,which pushed the development of nanopore field to a climax.Subsequently,nanopore sequencing technology rapidly became the third generation sequencing technology by virtue of its long length,non-amplification and its unique advantages in epigenetic sequencing and genome sequencing of new species,opening the“One thousand-dollar genome”era.With the rapid development of big data and cloud computing,genome,transcriptome,epigenome and structural genome have become research trends,thus people have an increasing urgent demand for high-throughput nanopore sequencing technology.However,due to the limitations of existing microelectronic and microelectrode fabrication techniques,the conventional electrophysiological nanopore technology relying on patch clamp cannot significantly increase its throughput without increasing the cost and device size.Compared with electrical analysis,optical imaging technology has incomparable advantages in throughput.Inspired by the optical patch clamp technology,Bayley et al.proposed a fluorescence nanopore technology,which monitoring the single-molecule behavior inside a single nanopore by probing the calcium ion flux.This technology increases the theoretical throughput to 10⁴/mm²,while the capability of single-molecular sensing and low detection cost are retained,which has wide industrial applications.This paper focuses on solving the existing problems of fluorescence nanopore technology,reducing technical barriers,establishing the finite element model to provide theoretical support,and finally constructing fluorescent nanopore chip with simple operation,stable performance,low cost and high throughput for clinical detection and industrial application.The main contents of this paper are as follows:1.Motion-type modulation and immobilization of biological nanopores for optical nanopore recordingsBiological nanopores are a kind of natural channel proteins,the lateral motion of which on the lipid bilayer is not conducive to the collection of fluorescence signals and data analysis.The motion of individual biological nanopores on the lipid bilayer could be monitored by probing the calcium ion flux through the biological nanopores.By simply adjusting the osmotic pressure across a lipid bilayer,the nanopore motion could be controllably restricted.This method has a wide range of generality that could be applicable to the commonly used staphylococcus aureus alpha hemolysin(?-HL)and mycobacterium smegmatis porin A(MspA)nanopores.The fluorescence recovery after photobleaching(FRAP)experiment proved that adjusting osmotic pressure could indirectly change the interaction between the lipid bilayer and the agarose hydrogel substrate,thus effectively regulating the lipid bilayer movement and ultimately affecting the movement of nanopores.The optical single-channel recordings(oSCRs)showed that compared with the moving nanopores,immobilized nanopores had a high signal to noise ratio(SNR)of fluorescence readout,while the capability of single-molecule sensing was remained.Further finite element simulation(FEM)provided theoretical supporting for relevant experimental results.This simple,efficient and label-free immobilization strategy ensures long-time and stable fluorescence readout and promotes the realization of high-throughput nanopore arrays.2.Electrode-free nanopore sensing by diffusioptophysiologyInspired by natural transmembrane transports,we proposed the first electrode-free fluorescence nanopores technique without any external electronic components,and named it Diffusioptophysiology(DOP).Through FEM and single-molecule cyclodextrin sensing,the feasibility of DOP was demonstrated theoretically and experimentally.By introducing an osmotic pressure,increasing calcium ion concentration and using large diameter biological nanopores,the sensing performance of DOP is further optimized with improved the capture efficiency and signal to background ratio(SBR).Single-molecule sensing of small molecules,macromolecules,and biomacromolecules was successfully demonstrated.A fingertip-sized microdroplet array was also constructed for low-cost and high-throughput nanopore detection,which providing a new concept for clinical diagnosis with a disposable nanopore sensor.3.Calcium ion flux regulated single-molecule nucleic acid sensing using nanopores In the study of fluorescence nanopore technology,the duration time of nucleic acids translocating through an?-HL nanopore was significantly prolonged.This effectively compensated the limited time resolution of the fluorescence detection system,but the mechanism of this phenomenon has not been studied in depth.Through experiments under the KCl/CaCl₂ buffer combination,we found that the calcium ion flux acting as the fluorescent signal source in the hydrogel is the key factor to slow nucleic acids translocation.We further proved that this phenomenon is applicable to nucleic acids with different sequences and different nanopores.We inferred that the calcium ion flux can bind to the phosphate groups on the nucleic acid backbone,shielding the negative charge of the nucleic acid and causing the deceleration of the nucleic acid.In the presence of a calcium ion flux,we directly discriminated three RNA homopolymers,poly(rA)₂₃,poly(rC)₂₃ and poly(rU)₂₃.We found that the presence of a calcium ion flux can stabilize the structure of nucleic acids and provide richer details when passing through the pore,which wolud be helpful for the structure detection of complex nucleic acid molecules.4.Machine learning assisted RNA tertiary structure profiling using nanoporesThe flexibility of RNA makes it difficult to analyze its structure using classical structural biology methods.The presence of a calcium ion flux can stabilize the RNA structure,which provides an opportunity for RNA tertiary structure profiling using biological nanopores.In previous studies,since biological nanopores usually had a narrow opening,highly folded RNA could not access to the lumen of the nanopore.Inspired by DNA sequencing,we utilized the wide nano-cavity of Msp A as a nano-trap to partially or completely capture RNA molecules,and realized RNA tertiary structure detection at the single-molecule level.This demonstrated the great potential and advantages to detect macromolecular structures using biological nanopores.In the presence of a calcium ion flux,we directly observed the tertiary structure information and unfolding process of miRNA,si RNA and tRNA.In order to optimize feature recognition,we introduced a machine learning algorithm for automatically signal truncation and recognition,which providing a powerful bioinformatics tool for nanopore data processing.Finally,combined with MspA nanopores and machine learning,we directly detected tRNA in biological samples,which would provide a new label-free and transcription-free method for evaluating tRNA expression levels in basic research and rapid diagnosis.