Functionalized Quartz-Based Nanopore Biosensors For Single Cell Analysis

With the continuous development of the new technological revolution,people have rapidly entered the information age.The first problem we face in the process of using information is how to quickly obtain accurate and reliable information?Sensors are the main way to obtain information and detection means in the field of nature and production.The sensor is a detection device that can sense the signal measured by the outside world and convert it into an electrical signal or other desired form of signal output according to certain rules.The current electrochemical biosensor is a new branch developed in the field of sensor technology,which combines biological and electrochemical measurement technology,is an interdisciplinary comprehensive detection technology,in food detection,chemical industry,environmental monitoring,disease diagnosis,drug screening,gene sequencing and clinical aspects have been widely used.Nanopore detection technology presents superior performance that is not available in general materials with its unique physical and chemical properties such as surface effect,macroscopic quantum tunneling effect and size effect,etc.and the electrochemical nanobiosensors constructed with nanopores are an emerging sensor detection tool developed in the 21st century by combining the two leading technologies of nanotechnology and biology.Solid-state quartz conical nanopore sensor is a kind of electrochemical biosensor,because of the outstanding advantages and properties such as easy preparation,good mechanical properties,easy surface modification,good stability,etc.,with resistance pulse and ion rectification as the output signal have been successfully used in single molecule detection,single cell research,protein detection,small molecule detection,metal ion detection,nucleic acid detection and ion device development and other related fields.It has great research significance for the development of life sciences and life medicine.In this context,based on the combination of existing nanopore detection technology,novel quartz nanopores and electrochemical detection technology and nanopositioning equipment,the paper develops a cone-shaped functionalized quartz nanopore biosensor with good specificity,high sensitivity,low detection limit and label-free characteristics.The dynamic content of small molecules and protein kinases in single cells and the tumor biomarkers secreted by single cells in a limited space were monitored and analyzed in real time by a single-cell nanochemical assay and an inverted microscope.The details are as follows:In Chapter 2,we have developed a conical nanopore sensor with enzymatic function,immobilizes glutathione transferase(GST)on the surface of quartz nanopores with a series of chemical modifications,and utilizes the specific binding of GST to glutathione(GSH)to achieve the nanopore sensor by the change of the charged nature of GST and GSH on the inner surface of nanopore in neutral electrolyte solution at pH=7.20～7.40 with ionic current as the output signal.The label-free glutathione small molecule detection by the nanopore sensor showed linear correlation in the range of 0.10-5.00 mM,and the lower limit of detection reached 62μM.GSH is the most abundant non-protein thiol in cells,and GSH is present in almost every cell in the body,contributing significantly to the maintenance of normal activities of the body.The nanosensor constructed by GST has the advantage of negligible damage to the cells due to its small size,which enables real-time detection of intracellular GSH.It provides a new method for the study of nanopores in single cells.In Chapter 3,we have developed a dual nanopore sensor based on the morphology of single nanopore,the morphological configuration of the dual nanopore sensor can effectively eliminate the interference in the current circuit of living cells due to the presence of membrane impedance in the cell membrane,the dual nanopore biosensor constructed by screening the medium affinity peptide probe combined with protein kinase A(PKA),so that the effective pore size reduction of the nanopore is achieved by using the ion current as the output signal to realize the reversible,sensitive and labelless detection of PKA catalytic subunit activity,and its detection limit reaches 0.04 nM,which showed a linear correlation in the range of 0.12-3.75 nM,and the sensor surface was sealed by thiol-methoxypoly(ethylene glycol)(SH-mPEG)to avoid non-specific adsorption in complex biological environments.PKA is a key molecule responsible for the normal functioning of all cellular processes and plays a critical role in many cellular regulations.It transfers phosphate groups from adenosine triphosphate(ATP)to serine or threonine residues of specific proteins for phosphorylation.The phosphorylated protein by protein kinase regulates the activity of target proteins,which further affects the expression of related genes,thus altering the function of the target protein.Abnormalities of PKA in cells can cause disruption of intracellular regulatory mechanisms and cellular pathway functioning.The development of a peptide-modified dual nanopore sensor based on this enables direct monitoring of PKA catalytic subunit activity in individual living cells.It provides an example of the application of dual nanopores for single-cell intracellular protein quantification and a promising assay tool for single-cell analysis.In Chapter 4,based on the development of peptide-modified dual nanopore sensor in Chapter 3 to achieve real-time current monitoring of PKA activity changes in different cells,as the intracellular active PKA kinase responds to the peptide on the dual nanopore causing the current to decrease in real time until saturation,combined with the working curve of PKA concentration to quantitative analysis of PKA activity content of different cells.Moreover,the dynamic changes of PKA activity in different cells induced by different stimuli were distinguished.We also verified the feasibility of dual nanopore biosensing for the detection of changes in PKA activity using the genetically encoded fluorescent sensor,and the experimental results were almost identical between nanopore biosensing and biosensing for the detection of changes in PKA activity.This illustrates that the feasibility of constructing a dual nanopore sensor with peptide probes for the spatiotemporally resolved detection of dynamic PKA activity in living cells.Provides a simple and effective approach to cancer diagnosis and drug screening.It helps nanopore sensors to develop in the direction of in vivo detection.In Chapter 5,A dual nanopore sensor constructed with CD63 aptamer was developed,The modified CD63 aptamer can specifically bind to the quadruple transmembrane CD63 protein of exosomes surface,and the effective pore size of the nanopore sensor is reduced by binding to exosomes,and a highly sensitive exosome detection is achieved with an ionic current as the output response signal with a lower limit of detection up to 3.4 × 106 Particle/mL.exosomes,as biomarkers of cancer,carry cargo of genetic or signaling alterations that reflect the origin of cancer cells and can provide diagnostic or prognostic potential in cancer detection.Because exosomes are stably existed in most body fluids and their content is similar to that of parental cells,exosomes are expected to serve as a liquid biopsy tool for various diseases,making their detection critical.the CD63 aptamer-modified dual nanopore sensor combined with a microarray-on-a-chip device enables real-time monitoring of exosome secretion from single cells over time.This provides a promising detection tool for disease diagnosis and early detection of cancer.It is also a further extension of the application of functionalized dual nanopore sensors.