Novel Biosensors For Enzyme Based On The Multi-functional Interface

Enzyme is one of important substances in creatures.There are more than 5,000 enzymes working in various types of biochemical reactions.The abnormality of enzyme activity is often closely related to the occurrence of the disease.Studies have found that abnormality in Furin activity was associated with neuron-degenerative diseases such as Alzheimer's disease and Parkinson's disease;overexpression in human epidermal growth factor erb2(HER2),which exhibits protein kinase activity on the cell surface,indicated many sorts of cancer,especially the breast cancer.Therefore,the detection of relevant enzymes and their activities in cells are of great significance for understanding the corresponding pathological or physiological processes.In this dissertation,electroanalytical chemistry and surface-enhanced Raman spectroscopy were utilized to develop highly selective,sensitive and anti-interference enzyme sensor for analysis of intracellular enzyme activity(concentration).The details are as follows:(a)a highly sensitive and selective ratiometric electrochemical sensor for the detection of intracellular furin activity was developed.Based on the principle that Furin can selectively cleave peptides containing Arg-X-Lys/Arg-Arg?-X sequences,we have designed a multi-signal peptide(P3)containing this sequence.The results showed that the detection sensitivity of the multiple signal site P3 to furin was amplified by 2.3 times compared to the single produced by peptide P1 with single recognition site.Second,a highly conductive multi-branched structure of gold bell flower nanomaterials was prepared,which significantly increased the specific surface area of the electrode and further realized signal amplification.In addition,the internal reference molecule MB was co-assembled at the gold nano bellflowers and in this way ratiometric electrochemical sensor was constructed.This dual signal output mode can effectively avoids the interference of the real sample system and significantly improve the accuracy of enzyme assay.The sensor has a good electrochemical response and a wide linear range(1 U/L~50 U/L)for furin.Finally,using this novel sensor,we successfully achieved the analysis of Furin activity in U251 cells and MDA-MB-468 cells.(b)The integrated EC-SERS smart platform was prepared for cell capture and insitu detection of intracellular HER2 protein based on the disordered mesoporous gold film and electrochemically controlled RGD peptide.First,organic molecules TMTMS that can react with RGD peptide for cell adhesion under potential,as well as specific peptide H2 responding to HER2,and organic molecule Fc-ATPs were successfully synthesized and characterized.Next,disordered mesoporous gold films with high EF values and anti-fouling was prepared.TMTMS molecules are modified onto gold films via Au-S bond and they are activated by potential and thus react with RGD to form cyclic RGD peptide which can efficiently and selectively fulfill the adhesion of cell.Another peptide H2 responding to the HER2 is anchored onto the RGD peptide with the help of EDC/NHS to form amide.As a result,Fc-ATP can be inserted into H2 under the phosphorylation of HER2.The RGD-H2+RGD/TMTMS/MesoGF/Au/ITO interface constructed from the above materials and molecules can achieve highly selective and efficient cell capture,and the number of adhered cells was about 380 per unit area(HeLa cell as the model).Notably,the captured cells were in good and original shape and exhibited high biological activity.In addition,the interface can achieve the renewal of interface at-0.2 V.By optimizing the concentration of Fc-ATP probe to 200 ?g/mL,5 to 100 ng/mL HER2 was achieved based on the SERS technology.Besides,the sensory interface has good selectivity for anions,cations,amino acids,and other enzymes or proteins that act similarly to HER2 in cells.Finally,based on the above analytical performance,concentration of HER2 in HeLa cells and SK-BR-3 cells was successfully determined by SERS imaging and efficiency of anti-HER2 drugs,such as trastuzumab(Herceptin)was evaluated as well.