Electrochemical And Photoelectrochemical Sensing For Apoptosis And Drug Theropy Of Tumor Cells

In the decades,cancer is always one of the main reasons to limit the life span of human beings,especially in the developed modern society with advanced medical level.The diagnosis and treatment of cancers,the study of cancer drugs,the monitoring of drug effect are always the leading fields for science.Traditional techniques for cancer diagnosis include spectroscopy,chromatography,etc.These techniques are often accompanied by false-positive results,leading to negative effects for diagnosis.Electrochemical sensing is a kind of electrochemical technique,combining electrochemical response with biological molecular recognition.Photoelectrochemical sensing is another electrochemical and optical technique,based on the combination between photoelectrochemical response and biological molecular recognition.Both of them could be applied in the fields of cancer diagnosis and drug effect monitoring,with simple devices,low cost,high sensitivity,weak interferences,simple miniaturization and integration.The results can be complementary with traditional diagnostic techniques,more authentic and persuasive.Apoptosis,a programmed cell death,is important and meaningful in cell metabolism,cell proliferation and death,especially for cancer cells.Thus,apoptosis is often used to evaluate the cancer therapy and drug effect.At present,the traditional methods for apoptotic detection usually depend on optics and chromatographic techniques,which are easily disturbed by false-positive results,reducing the accuracy,as well as tumor diagnosis.Electrochemical and photoelectrochemical sensing,with high sensitivity and low interference,can obviously reduce false-positive results,improving the evaluation of cancer theropy and drug effect.Based on biotechnologies and nanotechnologies,this dissertation builds several electrochemical and photoelectrochemical sensors with high sensitivity and low interference,to recognize and detect apoptotic signal molecules,realizing diagnosis of apoptosis and evaluation of drug effect.The main results were summarized as follows:1.Graphene-CdS Nanocomposites:Facile One-Step Synthesis and Enhanced Photoelectrochemical CytosensingGraphene-CdS(GR-CdS)nanocomposites were prepared in a one-step synthesis in aqueous solution.The synthetic approach was simple and fast,and it may be extended for the synthesis of other GR-metal-sulfide nanocomposites.The asprepared GR-CdS nanocomposite films inherited excellent electrontransport properties of GR.In addition,heteronanostructure of the GR-CdS nanocomposites facilitated the spatial separation of the charge carriers,thus resulting in enhanced photocurrent intensity,which makes it a promising candidate for photoelectrochemical applications.This strategy was used for the fabrication of an advanced photoelectrochemical cytosensor,based on these GR-CdS nanocomposites,by using a layer-by-layer assembly process.This photoelectrochemical cytosensor showed a good photoelectronic effect and cell-capture ability,and had a wide linear range and low detection limit for Hela cells.The as-synthesized GR-CdS nanocomposites exhibited obviously enhanced photovoltaic properties,which could be an efficient platform for many other high-performance photovoltaic devices.2.Chronic Myeloid Leukemia Drug Evaluation Using a Multisignal Amplified Photoelectrochemical Sensing PlatformChronic myeloid leukemia(CML)is a malignant clone disease of hematopoietic stem cells.At present,the most effective therapy for CML is bone marrow transplantation,but this procedure is expensive,and it is often difficult tofind appropriately matched bone marrow donors.As an alternative to marrow transplantation,a more effective anticancer drug should be developed to cure the disease;in addition,an effective system to evaluate the activity of the drug needs to be developed.Herein,we present a novel antileukemia drug evaluation method based on a multisignal amplified photoelectrochemical sensing platform that monitors the activity of caspase-3,a known marker of cell apoptosis.Manganese-doped CdS@ZnS core-shell nanoparticles(Mn:CdS@ZnS)were synthesized via a simple wet chemical method,which provided a stable photocurrent signal.A DEVD-biotin peptide and streptavidin-labeled alkaline phosphatise(SA-ALP)were immobilized successively at these nanoparticles through amide bonding and through specific interaction between biotin and streptavidin,respectively.The photocurrent of this sensing platform improved as the ALP hydrolyzed the substrate 2-phospho-lascorbic acid(AAP)to ascorbic acid(AA),a more efficient electron donor.The activity of caspase-3 was detected using this sensing platform,and thus,the efficacy of nilotinib for targeting K562 CML cells could be evaluated.The results indicate that nilotinib can effectively induce apoptosis of the K562 cells.This sensing platform exhibited sensitive,reproductive,and stable performance in studying the nilotinib-induced apoptosis of K562 CML cells,and the platform could be utilized to evaluate other anticancer drugs.3.CdSeTe@CdS@ZnS Quantum Dots Sensitized Macroporous TiO2 Film:A Multisignal Amplified Photoelectrochemical PlatformA macroporous TiO2 film(M-TiO2),which was prepared by burning off the polystyrene microsphere(PS)template from a PS/TiO2 composite film,can provide a large active surface,improve electron-transport performance,and increase the photocurrent.Furthermore,core-shell-shell CdSeTe@CdS@ZnS quantum dots(QDs)were introduced to sensitize the M-TiO2 film,which can efficiently broaden the absorption spectra range,separate and transfer charge carriers,reduce recombination loss,and improve photovoltaic response,with a sensitization shell of CdS and a passivation shell of ZnS.A multisignal amplified photoelectrochemical platform was fabricated by further modifying this film with a combination of biotin-DEVD-peptide(Biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Gly-Cys)(which is specifically cleaved by caspase-3)and streptavidin-labeled alkaline phosphatase(SA-ALP).Under the enzymatic catalysis of ALP with the substrate 2-phospho-l-ascorbic acid trisodium salt(AAP),ascorbic acid(AA)was generated as a better electron donor,leading to increased photocurrent output.The activity of caspase-3,which depends on the amount of residual peptide on the electrode,was inversely proportional to the amount of AA.By monitoring the variation of photocurrent caused by AA,caspase-3 activity and the therapeutic effect of nilotinib(a special medicine of chronic myeloid leukemia,CML)were indirectly detected and evaluated.The photoelectrochemical platform can be used as a potential evaluation system for monitoring caspase-3 activity and drug effects.4.Toward Therapeutic Effects Evaluation of Chronic Myeloid Leukemia Drug:Electrochemical Platform for Caspase-3 Activity SensingIn recent decades,advanced therapies and novel scientific drug evaluation systems for chronic myeloid leukemia(CML)treatment are very urgent due to its increasing morbidity.The combination of dasatinib with tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL)was supposed to be effective for leukemia therapy.Taking full advantage of novel nano-biotechnology,we have developed a robust electrochemical cytosensing approach to profile the therapeutic effects of dasatinib and TRAIL by probing the activity of caspase-3 from apoptotic CML cells.The sensor was on a base of a glassy carbon electrode(GCE)modified with nano-materials composed of Au nanoparticles(AuNPs),poly(dimethyldiallyl ammonium chloride)(PDDA),and carbon nanotubes(CNTs).Then the platform immobilized the biotinylated DEVD-peptide(biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Gly-Cys)via the strong bonding between AuNPs and the thiol group(Au-S bond).In particular,the sensor was then constructed with the environmentally friendly alkaline phosphatase(ALP)via the specific interaction between the biotin and streptavidin,and could retest detection indirectly for caspase-3 sensing by detecting the differential pulse voltammetry(DPV)signal of enzymatic catalysis product,ascorbic acid(AA).The results indicated that either dasatinib or TRAIL could successfully induce the apoptosis of CML cells,while the combination of dasatinib and TRAIL resulted in an improved therapeutic effect,suggesting a novel optimized strategy for CML therapy.This novel electrochemical sensing strategy exhibits attractive advantages of environmental benignity,simple performance,high stability,and may be readily expanded to evaluate other cancer therapeutic effects.5.Simultaneous Detection of Tumor Cell Apoptosis Regulators Bcl-2 and Bax through a Dual-Signal-Marked Electrochemical ImmunosensorB-cell lymphoma 2(Bcl-2)and Bcl-2-associated X protein(Bax)are often used to monitor the apoptosis of tumor cells and evaluate cancer drug effect.In this work,a novel sandwich-type dual-signal-marked electrochemical biosensor was fabricated for simultaneous detection of Bcl-2 and Bax proteins.Reduced graphene oxide(RGO)layers were used as substrate to immobilize Bcl-2 and Bax antibodies for further capturing target antigens.CdSeTe@CdS quantum dots(QDs)and Ag nanoclusters(NCs)with antibody modification and mesoporous silica amplification were used as signal probes,which were proportional to the amount of Bcl-2 and Bax antigens.Mesoporous SiO2 can provide a larger surface area,more effectively charged by ethylene imine polymer or poly(diallyldimethylammonium chloride)to adsorb more probes.The Bcl-2 and Bax proteins were determined indirectly by the detection of oxidation peak currents of Cd and Ag using anodic stripping voltammetry,showing a good linear relationship in the protein concentration range from 1 ng/mL to 250 ng/mL.The detection limit of trace protein level was 0.5 fmol.The biosensor was further introduced to investigate Bcl-2 and Bax expressions from nilotinib-treated chronic myeloid leukemia K562 cells.With the increase of drug dosage and incubation time,the up-regulation for Bax and down-regulation for Bcl-2 were observed,which indicated that the apoptosis level of K562 cells could be regulated by Bcl-2 family.The ratio of Bax/Bcl-2 was further calculated for evaluation of its drug effect and apoptosis level.The limited cell amount for detection reached less than 1×103 cells,much lower than traditional methods.Furthermore,completely independent detection step and stable acid solutions containing Ag+and Cd2+for long-time storage contribute to reducing the error from the sample differences and avoiding the potential errors from the photodegradation offluorescent probes,enzymolysis of DNA,or inactivation of enzyme during an excess experimental period.