Accurate Measurement And Identification Based On Glass Conical Nanopipettes

With the development of nanotechnology and molecular biology,nanopore biosensing has received close attentions.In recent years,glass conical nanopipettes have been widely used in bionics,biosensing and single-cell analysis due to their simple preparation methods,excellent chemical and physical stability.Currently,nanopipette biosensors have been comprehensively developed,including the construction of p H-,temperature-,polarity-sensitive channels,and biomolecules recognition.However,there are still many deficiencies.On one hand,the single structure and simple identification principle for most materials made the interface difficult in flexible controlling,which may affect the sensitivity and selectivity of sensing.On the other hand,nanoamp currents are susceptible to the interference of non-specific adsorption on the nanopipettes,which affected the accuracy of molecular measurements.Therefore,the innovation of materials and identification principles,and the development of measurement methods are of great significance to achieve accurate measurement of biomolecules and improve the analytical performance.Therefore,this paper focuses on the “accurate measurement and identification of glass conical nanopipettes”.Combined with the innovative of interface materials and the support of measurement technologies,this research improves the sensitivity and accuracy of nanopipettes in biosensing.Besides,the design of functional molecules can be used to achieve the precise position of target molecule at the subcellular level,which expands the application of nanopipettes in single-cell analysis field.This work provides new strategies and platforms for the accurate measurement of biomolecules,and facilitates the rapid development of nanopipettes in the field of measurement and identification.Specifically,this paper includes the following four parts.Chapter 1.Overview This chapter mainly introduces the research background of glass conical nanopipettes and their applications in biosensing fields,focusing on the development and application of sensing measurement methods.Besides,the mechanisms of intermolecular interactions within the confined space of nanopipette are also described in detail.And the application of functional molecules and materials in molecular sensing are reviewed.Finally,the research purpose and significance,the main research contents and innovation points of this paper are expounded.Chapter 2.A highly selective ATP-responsive biomimetic nanochannel based on smart copolymer ATP-sensitive potassium(KATP)channels couple intracellular metabolism to the electrical activity by regulating K+ flux across the plasma membrane,thus playing an important role in both normal and pathophysiology.KATP channel is a heteromultimer of two subunits(potassium channel subunit(Kir6.x)and sulfonylurea receptor(SUR))and exhibits dynamic functions with adjustability and reversibility.The SUR drives the opening and closing of Kir6.x in an ATP-responsive manner to regulate K+ transport.However,the construction of most biomolecular nanopipette sensors simply relied on the effect of surface charge on ion transport,ignoring the structural features and the cooperative regulation of protein channels in organisms.In this work,inspired by the protein structure and recognition mechanism,a novel smart polymer is designed and synthesized to realize the functional modification of glass conical nanopipettes.Among them,N-isopropylacrylamide(PNIPAAm)is used as the conformational driving unit,and phenylthiourea(CPT)and phenylboronic acid(PBA)are used as the functional recognition unit of ATP molecule.By combining with the recognition unit,ATP changes the hydrogen bond environment around the PNIPAAm chain,and converts the recognition of ATP into its dynamic conformational transition,showing excellent on-off regulation ability.Based on the accurate grasp of protein structure and recognition mechanism,the nanopipette sensor shows excellent stability and reversibility.Moreover,the synergistic effect between the polymer functional units improves the detection sensitivity,and the biomimetic channel has good selectivity in the complex organism environment.Chapter 3.Rational design of a self-assembled surfactant film in nanopipettes: combined fluorescence and electrochemical sensing Ion rectification is the main sensing measurement method of glass conical nanopipettes.However,nanopipette electrochemistry is easily disturbed by non-specific adsorption on the surface.To further optimize the analytical performance of nanopipette sensing,we develop a novel nanopipette surface functionalization method.In this work,the self-assembled approach based on the hydrophobic interaction is able to effectively immobilize the function molecules onto nanopipette surface,offering an adjustable surface structure with tailor-made selectivity and sensitivity.In addition,the self-assembled approach is readily generalizable to organic fluorescent probe molecules and applicable for synergizing the ion current and fluorescent analysis of nanopipettes.Using arginine(Arg)as a model target,an amphiphilic fluorophore molecule(DIl AQ)is first designed and synthesized in our work.The introduction of fluorophore provids an additional signal supplement for nanopipette electrochemistry,which further improves the detection accuracy.In addition,the self-assembled surfactant film can sensitively tune the immobilized functional units on the surface by simply changing the ratio of surfactant to functional units,optimizing the interface properties.Finally,the sensor is successfully used for Arg metabolism level assessment,demonstrating that Arg metabolism shows an age-related difference in Alzheimer's disease.Since amphiphilic functional molecules can be varied to meet any specific detection purpose,this work opens up newopportunities for the construction of nanopipette measurement platforms.Chapter 4.Precise analysis of protein phosphorylation under oxidative stress in single cells based on reactive near-infrared fluorescent probe and polymer-functionalized glass conical nanopipettes The supplementation of the fluorescent signal can solve the interference of non-specific adsorption of nanopipettes and improve the accuracy of measurement,but it is difficult to obtain molecular structural information and lack of spatial resolution.In this work,we develop a reactive fluorescent probe molecule(NIR-HP)to achieve precise localization of proteins under oxidative stress at the subcellular level.At the same time,the nanopipette is connected with the mass spectrometry test platform to realize the in-situ analysis of the phosphorylation modification of proteins under oxidative stress in a single living cell with high spatial resolution.In order to realize the enrichment of phosphorylated peptides in nanopipette,we synthesize a polymer that can selectively bind phosphate groups for the modification of glass conical nanopipettes.Further,functionalized glass conical nanopipettes are used to achieve in situ capture of labeled proteins and selective enrichment of phosphorylated peptides with confocal microscopy and micromanipulators.Finally,nanopipette is used as electrospray ion source to construct a mass spectrometry system to analyze protein phosphorylation modification by proteomics.This work not only enables the in-situ capture of labeled proteins at the subcellular level,but also provides a possibility for the analysis of protein phosphorylation modification sites under oxidative stress in combination with mass spectrometry.This work is of great significance for the research on glass conical nanpipettes in basic fields such as cell metabolism and neurodegenerative diseases.