Study Of Tumor Biology With Electrochemical Biosensing Techniques

Tumor has endangered the lives of human beings a lot.The study of tumor biology is helpful to reveal the rule of development of tumors,which will further benefit the prevention,diagnosis and treatment of tumors.Due to the high sensitivity and high selectivity,electrochemical biosensing techniques have now been widely used to study electron transfer in biological system at the molecular or cellular level.Herein we have fabricated several biosensors for the study of tumor biology.The main work is described as follows:1.Electrochemical study of the acetylation of p53 C-terminal domainp53 is an important tumor suppressor and transcription factor.The acetylation of p53 C-terminal domain(CTD)plays an essential role on its function.In this chapter,an electrochemical method to evaluate the effect of CTD acetylation on the interaction between CTD and DNA has been developed.In this study,peptides derived from the CTD of p53 with single or multiple acetylations are immobilized on electrode.Since the DNA binding ability of the peptide is correlative to the extent of acetylations,ferrocene-tagged dsDNA is used as the molecule beacon.Therefore,the determination of acetylation can be achieved with ferrocene as the electrochemical readout.The result shows that the DNA binding ability is enhanced with an increase in the extent of acetylation.The method is very simple and provides a novel way to study protein modificaitons.2.Assay of matrix metalloproteinases-2(MMP-2)based on the assembly of collagen-DNA complex on electrode surfaceMatrix metalloproteinases-2(MMP-2)are essential for the breakdown of extracellular matrix in the metastasis of tumors.In this chapter,the activity of MMP-2 is assayed electrochemically by preparing collagen-DNA complex on the surface of an electrode.The complex is fabricated on the electrode due to the similarity of collagen triple helix and DNA double helix in topology.Collagen in the complex is the substate of MMP-2.So when collagen in the complex is digested by MMP-2,[Fe(CN)6]3-/4-can get close to the electrode,which results in a higher electrochemical signal.Thus a new kind of electrochemical biosensor for the detection of MMP-2 is developed with a linear detection range from 0.1 to 1 pg/mL.Moreover,protein entrapment can also be achieved by blocking target proteins in the complex,thus further analysis can be conducted on the target proteins.3.Study of CUB-domain-containing protein mediated cell adhesionCell adhesion to the extracellular matrix is an important function in tumor metastasis.In this chapter,10%polymethyl methacrylate(PMMA)is fabricated on the surface of an electrode to culture cells by electrospinning.The electrospun membrane possesses excellent biological compatibility and can be used for three-dimensional(3D)cell culture.Overexpression of CUB-domain-containing protein(CDCP1)leads to cell rounding and a loss of adhesion phenotype.The gap junction between cells may act as channels through the cell membrane.So,when cells are electrically stimulated,gap junctions of the cells effectively connect electrically to the cells.As a result,the sensitivity of cells by externally applying electrical field increases and cells may act as a conductor for a short time by considerable biochemical and biophysical changes.However,after the immunoreactions between CDCP1 and its antibody,cells adhere onto the electrode surface,forming the cell layer which interferes in the electron transfer.Therefore,the role of CDCP1 in cell adhesion is revealed by electrochemical technique.The method is simple and provides a new platform to study the effect of molecules on cell adhesion.4.Peptide-based electrochemical approach for apoptosis evaluationThe inhibition of apoptosis leads to the proliferation of tumors.This chapter reports a strategy to assemble apoptotic cells on a solid surface using a peptide as the recognition element.The peptide modified on an electrode is designed to contain the sequence that can recognize externalized phosphatidylserine on apoptotic cells,and can then capture the cells onto the electrode surface.In the electrochemical system,the immobilized cells can not only provide significant steric hindrance for electron transfer,but also shield the positive charges of the peptide that can attract negatively charged electrochemical probes.Therefore,the obtained electrochemical signals drop significantly after the incubation of apoptotic cells,which can be used to reveal the apoptosis level.The experiment results of this approach are in accord with other standard methods such as fluorescent and colorimetric assays.Moreover,this electrochemical method is simple,cost-effective,convenient,sensitive,and holds great potential toward apoptosis evaluation,therapeutic effect assessment and deeper cellular biological studies.5.iRGD based electrochemical study of doxorubicin inside a cellChemotherapy is now one of the main treatments for tumors.The killing effect of drugs on tumor cells relies on the intracellular drug concentration.This chapter reports a method to electrochemically study doxorubicin inside a cell with iRGD peptide as the trasnfection reagent.Due to the enhancement of the cell penetration caused by iRGD peptide,DNA molecules,previously modified on a gold electrode surface,can be easily transfected into the cells.At the same time,doxorubicin,an anticancer drug,can also be transfected into cells with high penetration.Consequently,doxorubicin binds to DNA chains through electrostatic interaction,and the redox reaction is transferred out of the cell across the cell membrane.As a result,electrical communication between the inner part of cells and an electrode is achieved.The method is more gentle than the previous reports and may provide a novel way to get information inside of cells.6.The measurement of intracellular pH changes based on DNA-templated capsid protein nanotubesIntracellular pH(pHi)is a fundamental modulator of cell function.Minute changes in pHi may cause great effects in many cellular activities such as metabolism and signal transduction.In this chapter,we report an electrochemical pHi sensor based on viral coat proteins-DNA nanotubes modified gold electrode.The sensor is pH sensitive due to the pH dependent electrochemical property of methylene blue and cell permeable owing to the polyarginine domain of the cowpea chlorotic mottle virus coat protein(CCMV CPs).Moreover,since the pH sensor can be translocated into cells without any further operations,the measurement of pHi changes can be greatly simplified.The pH sensor has a broad pH spectrum in the pH range from 4.0 to 9.0 and sensitively responses to the pH changes of cells,so it might be useful to study pH dependent biological and pathological processes in the future.