New Methods For Photoelectrochemical Biosensing And Single-Cell Electrochemical Analysis

Biosensor uses bioactive materials and signal conversion elements to realize the measurement of analytes.The physiological activities of life and physiological diseases usually associate with specific biomarkers,the detection of which is of great significance for understanding the process of physiological activities and the diagnosis,treatment and prevention of diseases.Cells are the basic units of life,and many diseases are caused by pathological changes of cells.Therefore,the study of the cellular structure and function and investigation of their behavioral activities,such as cell division,morphological changes and senescence,are of great significance to explore the law of life activities and the nature of life processes as well as the origin of specific diseases.At present,cell analysis is gradually moving from group behavior research to single-cell level.Single-cell analysis requires high sensitivity,good selectivity,as well as and high spatial and temporal resolution.Therefore,the development of method for single-cell analysis is an important challenge for analysts.The research work of this paper includes two parts.The first part is photoelectrochemical（PEC）biosensor.The semiconducting polymer dots（Pdots）was used as photoactive material to construct PEC DNA sensor,p H sensor and enzymatic sensor.The second part is single-cell biosensor.Nucleic acid aptamer,deoxyribonuclease（DNAzyme）and other nucleic acid probes were modified in nanopipette to detect different metal ions in a single cell using ionic current rectication and other effects.The main contents are as follows:1.Energy transfer between semiconducting Pdots and gold nanoparticles in a PEC system:a case application for DNA analysisIn this work,the phenomenon of energy transfer（ET）between Pdots and Au NPs is explored for the first time.Firstly,the carboxyl group modified polymer dots（Pdots）were prepared by coprecipitation method.Then it was assembled on the indium tin oxide（ITO）electrode.Afterwards,the DNA probe（p DNA）was immobilized on the surface of the electrode.After the DNA hybridization with the Au NP-tethered complementary ss DNA（Au-t DNA）,the Au NPs were brought in the close proximity of Pdots.Upon light stimulation,the ET between Pdots and Au NPs was generated,the photocurrent intensity evidently decreased.The ET was applied in the PEC biosensor for the sensitive detection of DNA.The results exhibited that the prepared Pdots has excellent photoelectrical properties.The DNA sensor based on Pdots has high sensitivity and good stability.2.A polymer dots-based PEC p H sensorIn this work,Pdots was explored for PEC p H sensor for the first time.Firstly,the carboxyl group modified Pdots was prepared by coprecipitation method.With the decrease of p H value,the configuration of Pdots will convert from the stretched one to coiled one.In addition,the higher the concentration of the proton,the higher the reduction potential.Upon illumination,the photogenerated electron-hole pairs were separated with more electrons transferred from Pdots to the protons and thus generated high photocurrent.With the increase of p H value,the concentration of the proton will decrease.The photogenerated electrons were captured by dissolved oxygen,the potential of the oxygen reduction was decreased with the increased p H value.The fewer photogenerated electrons transferred from Pdots to the oxygen,and thus low photocurrent.The results exhibited that the proposed p H sensor has a wide detection range with high selectivity,good reversibility and stability,which had a potential application in the field of p H sensing.3.3D semiconducting polymer/graphene networks:toward PEC enzymatic bioanalysisIn this work,a highly sensitive photocathodic enzymatic bioanalysis was constructed based on three-dimensional（3D）graphene structure for the first time.Firstly,porous 3D graphene was synthesized by hydrothermal and freeze-dry processes,and then mixed with semiconducting polymer to obtain 3D semiconducting polymer/graphene（SP/G）networks with unique porosity and large surface area.Afterwards,the as-prepared hybrid was immobilized onto the indium tin oxide（ITO）.Exemplified by sarcosine oxidase（SOx）as a model biocatalyst,an innovative 3D SP/G-based photocathodic bioanalysis was achieved.The results exhibited that the 3D SP/G-based photocathodic enzymatic bioanalysis is of high sensitivity,good stability,reproducibility and selectivity.4.Electrochemical analysis of single cell Mg²⁺based on nano-capillariesThe Au-decorated nanopipette was prepared sequentially using laser-based puller and sputtering method.Then the Mg²⁺recognized DNAzyme（Mg DH）was modified onto the inner wall of Au-decorated nanopipette,which was of high specificity and biocompatibility.The recognition reaction of Mg²⁺to Mg DH will lead to the cleavage of Mg DH,which will reduce the charge density of the inner wall of the nanotip and thus change the detection signal,realizing the analysis of intracellular Mg²⁺in a single cell.Afterward,the Mg²⁺in cancer cells and normal cells were analyzed.Finally,α1-adrenoceptor was used to stimulate the cells,resulting the outflow of intracellular Mg²⁺.The intracellular Mg²⁺is analyzed before and after stimulation by the as-prepared nanosensor.The results indicated that the prepared nanosensor can be used for sensitive detection of the intracellular Mg²⁺.5.Electrochemical analysis of single cell K⁺based on nano-capillariesBased on the successful detection of Mg²⁺in a single cell,another nanosensor for detecting K⁺in a single cell was prepared.The key of this method is to prepare the ultra-small tip with a pore diameter of 20 nm.The G-rich K⁺aptamers were modified on the inner wall of the nanotip by silanization and amidation reactions.Initially,the aptamer was immobilized on the inner surface of the nanotip in the shape of the random single-stranded structure.The K⁺will combine with the aptamer through diffusion effect.At this time,the aptamer exhibited a four stranded G-quadruplex structure,resulting in a decrease of the effective pore size.After the binding of K⁺with[2.2.2]-crytand,the conformation of the aptamer was recovered to the random single-stranded structure,indicating the good reversibility of the nanosensor.Through the corresponding relationship between the ion current signal and the K⁺concentration,the concentration of K⁺in a single cell can be estimated.In the early stage of apoptosis,the concentration of K⁺decreased significantly coupled with the decrease of cell volume.According to this phenomenon,the intracellular K⁺can be measured after the induction by drugs for the same time,and then the effect of the different drugs was evaluated.6.Electrochemical analysis of single Cell Na⁺/K⁺（sodium to potassium ratio）based on nanocapillariesOn the basis of the above studies,a new method for electrochemical detection of intracellular Na⁺/K⁺was designed to fully understand the synergistic effect of intracellular metal ions and its effect on pathological process.Initially,K⁺recognized DNA triple-helix molecules（DTHMS）and Na⁺recognized DNAzyme（Na A43SE）were respectively modified onto the Au-decorated nanopipette.The presence of target metal ions will result in the change of the charge density of the inner wall of the nanotip and thus change the detection signal.Then the modified nanopipettes were used to detect single-cell Na⁺/K⁺in normal cells and cancer cells.Thereafter,the cells were induced with different drugs,and the corresponding effects were statistically evaluated using 50 cells.