Research Of Electrochemical Cytosensor Based On Functional Nanomaterials

Cytosensor have become a hot research topic based on the important role of cells in life sciences and human health.Notably,electrochemical cytosensor plays a more significant part in analysis and detection of target cells due to inherent advantages,such as miniaturization,easy operation,high sensitivity,satisfactory selectivity,rapid response,affordability,real-time,non-destructive analysis and so on.With the development of nanotechnology,nanomaterials have been gradually applied in biosensors.The significant roles of nanomaterials in cytosensing lie in their ability to address some key issues,including design of the cell compatible interface,facilitation of the electron transfer in electrochemical reactions,achievement of efficient transduction of the bio-recognition event and improvement of response times.In general,nanomaterials were used as nanocarriers to immobilize aptamers,antibodies,enzymes and other biological molecules as well as some electronic mediators.In this paper,besides using nanomaterials as nanocarriers,a strategy of non-enzymatic catalysis signal amplification was established based on the catalytic effect of nanomaterials mimicking enzymes.Compared with traditional enzymatic signal amplification strategies,non-enzymatic catalysis nanomaterials have the advantages of good stability,low cost,easy preparation and modification.The aim of this paper is to construct electrochemical cytosensors with high sensitivity,selectivity,stability and wide application by preparing nanomaterials with excellent properties for the sensitive detection of cell concentration,type,apoptosis,internal/external molecules and metabolic substrates.The main research contents include the following four parts,which are summarized as follows:?1?A simple and sensitive non-enzymatic electrochemical biosensor was developed for the determination of hydrogen peroxide?H₂O₂?and real-time monitoring the release of H₂O₂ from living cells based on the covalent bonding of dendrimer-encapsulated Pt nanoclusters and carbon nanotubes to form nanocomposites?Pt-DENs/CNTs?.The nanocomposites were equipped with good biocompatibility,large specific surface area and excellent electron transport properties.In addition,based on the synergistic effect of Pt-DENs and CNTs,the prepared biosensors exhibited excellent catalytic performance for the reduction of H₂O₂.Ultrasensitive detection of H₂O₂ was achieved with a linear range concentration of3-400?M with a detection limit of 0.8?M.In addition,the obtained biosensor could be applied to detect H₂O₂ released from living cells,and the amount of H₂O₂ released per cell was about 0.158 fmol.The biosensor showed good stability,reproducibility and selectivity.?2?A non-enzymatic sandwich-structured amperometric cytosensor was constructed for the ultrasensitive detection of circulating tumor cells?CTCs?and simultaneously evaluated the dynamic expression of N-glycans on the cell surface.The cytosensor was constructed by using a lectin-functionalized palladium-gold nanoparticle?NPs??Pd@Au-Con A?as a nanoprobe,and the Fe₃O₄@SiO₂ NPs as a magnetic nanocarrier.The Pd@Au-Con A nanoprobe not only specifically recognized glycans on the surface of CTC,but also amplified current signals because Pd@Au NPs can effectively catalyzed the reduction of H₂O₂.Fe₃O₄@SiO₂ NPs have excellent magnetic separation ability,large specific surface area and easy bio-functionalization,which used to immobilize SYL3C aptamer for capturing CTC.In this paper,human breast cancer cells?MCF-7?were used as model cells,and the constructed cytosensor showed a lower detection limit(30 cell mL^-1)and a wider linear detection range(1×10²-1×10⁶ cell mL^-1).The method sensitively realized the detection of the quantity of cells as well as exhibited high sensitivity,excellent stability and reproducibility.In addition,the developed cytosensor could effectively monitor the variation of cell surface glycans interfered by the external treatment.According to these excellent new properties of our cytosensor,it may have a great potential for the early diagnosis of cancer as well as contributing to reveal biological processes and diseases associated with N-glycan.?3?An ultrasensitive amperometric cytosensor was constructed in the basis of signal amplified method via applying PGO-Au nanocomposite as matrix and Cu₂O@PtPd nanocomposite as signal label,which used to quantitatively detect early apoptotic cells and evaluate the therapeutic effect of anticancer drugs.PGO-Au nanocomposite possessed excellent electrical conductivity,good biocompatibility and large ratio of volume/surface,which effectively enhanced the immobilization of annexin V to capture the early apoptotic cells.More importantly,Cu₂O@PtPd nanocomposite was utilized as signal amplified label to improve the catalytic efficiency of the cytosensor toward the reduction of H₂O₂.Human non-small cell lung cancer?A549?cells were selected as model cells and cisplatin as apoptotic inducer.The established cytosensor exhibited good stability,selectivity and reproducibility.The linear detection range of early apoptotic cells was 50-5×10⁷ cell mL^-1,and the detection limit was down to 20 cell mL^-1.It might push the envelope alternative to detect the early stage of apoptosis and evaluate therapeutic effect of anticancer drugs.?4?A sensitive electrochemical aptamer cytosensor was constructed based on double recognition aptamer and hybrid signal amplification probe for the detection of human hepatoma cells?HepG2?.Toluidine blue?Tb?and hemin/G-quadruplex-labeled Fe₃O₄@Au nanocomposites were used as labels for amplified signals to obtain sensitive current signal responses.Au NPs electrodeposited on glassy carbon electrodes were used as substrate solids to immobilize TLS11a aptamer which specifically captured HepG2 cells.The proposed cytosensor had high sensitivity,wide linear range,satisfactory repeatability and better selectivity,and could be used for the detection of human blood samples.The linear detection range of HepG2 cells was1×10²-1×10⁷ cells mL^-1,and the detection limit was 15 cells mL^-1.In view of these advantages,the constructed electrochemical aptamer cytosensor provides an important diagnostic tool for liver cancer cell detection,and has great potential in early diagnosis and clinical analysis of cancer.