Simultaneous Detection of Tumor Cell Apoptosis Regulators Bcl-2 and Bax through a Dual-Signal-Marked Electrochemical Immunosensor.

B-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 × 10(3) cells, much lower than traditional methods. Furthermore, completely independent detection step and stable acid solutions containing Ag(+) and Cd(2+) for long-time storage contribute to reducing the error from the sample differences and avoiding the potential errors from the photodegradation of fluorescent probes, enzymolysis of DNA, or inactivation of enzyme during an excess experimental period.

[1]  A. Strasser,et al.  The BCL-2 protein family: opposing activities that mediate cell death , 2008, Nature Reviews Molecular Cell Biology.

[2]  J. Griffin,et al.  Comparing nilotinib with dasatinib as second-line therapies in patients with chronic myelogenous leukemia resistant or intolerant to imatinib – a retrospective chart review analysis , 2013, Current medical research and opinion.

[3]  Jun‐Jie Zhu,et al.  Electrochemiluminescence energy transfer-promoted ultrasensitive immunoassay using near-infrared-emitting CdSeTe/CdS/ZnS quantum dots and gold nanorods , 2013, Scientific Reports.

[4]  M. Minden,et al.  Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. , 1998, Blood.

[5]  Linting Li,et al.  Toward therapeutic effects evaluation of chronic myeloid leukemia drug: electrochemical platform for caspase-3 activity sensing. , 2014, Biosensors & bioelectronics.

[6]  Taeghwan Hyeon,et al.  Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a mesoporous silica shell for magnetic resonance and fluorescence imaging and for drug delivery. , 2008, Angewandte Chemie.

[7]  S. Woolf,et al.  An evidence-based analysis of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone marrow transplantation in treating the chronic phase of chronic myeloid leukemia: developed for the American Society of Hematology. , 1999, Blood.

[8]  S. Manon,et al.  Bax: Addressed to kill. , 2011, Biochimie.

[9]  S. Chandna,et al.  Putative partners in Bax mediated cytochrome-c release: ANT, CypD, VDAC or none of them? , 2009, Mitochondrion.

[10]  Chun Li,et al.  Flexible graphene films via the filtration of water-soluble noncovalent functionalized graphene sheets. , 2008, Journal of the American Chemical Society.

[11]  G. Wallace,et al.  Processable aqueous dispersions of graphene nanosheets. , 2008, Nature nanotechnology.

[12]  Jun‐Jie Zhu,et al.  Photochemical synthesis of fluorescent Ag nanoclusters and enhanced fluorescence by ionic liquid , 2013 .

[13]  Qingming Shen,et al.  Chronic myeloid leukemia drug evaluation using a multisignal amplified photoelectrochemical sensing platform. , 2014, Analytical chemistry.

[14]  T. Tsuruo,et al.  5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins. , 1998, British Journal of Cancer.

[15]  Gwyn T. Williams,et al.  Molecular regulation of apoptosis: Genetic controls on cell death , 1993, Cell.

[16]  Jian Zhang,et al.  Mono-(2-ethylhexyl) phthalate induces apoptosis through miR-16 in human first trimester placental cell line HTR-8/SVneo. , 2016, Toxicology in vitro : an international journal published in association with BIBRA.

[17]  Liang Tan,et al.  Quantification of Bax protein on tumor cells based on electrochemical immunoassay , 2013 .

[18]  Joseph Wang,et al.  Nanoparticle‐Based Electrochemical Bioassays of Proteins , 2007 .

[19]  John Calvin Reed,et al.  Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance. , 1996, Oncogene.

[20]  A. Yakovlev,et al.  Caspase-dependent apoptotic pathways in CNS injury , 2001, Molecular Neurobiology.

[21]  R. Budd,et al.  Activation-induced cell death. , 2001, Current opinion in immunology.

[22]  F. Tas,et al.  Serum bcl-2 and survivin levels in melanoma , 2004, Melanoma research.

[23]  Neeta Singh,et al.  Apoptotic signaling induced by Tiazofurin-an in vitro study , 2006 .

[24]  J. Goldman,et al.  Molecular heterogeneity in complete cytogenetic responders after interferon-α therapy for chronic myelogenous leukemia: low levels of minimal residual disease are associated with continuing remission , 2000 .

[25]  Guodong Liu,et al.  Electrochemical coding for multiplexed immunoassays of proteins. , 2004, Analytical chemistry.

[26]  G. Dewson,et al.  Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis , 2013, Cell Death and Differentiation.

[27]  Tapash Chakraborty,et al.  Properties of graphene: a theoretical perspective , 2010, 1003.0391.

[28]  A. W. Boersma,et al.  Bax upregulation is an early event in cisplatin-induced apoptosis in human testicular germ-cell tumor cell line NT2, as quantitated by flow cytometry. , 1997, Cytometry.

[29]  X. Xia,et al.  A green approach to the synthesis of graphene nanosheets. , 2009, ACS nano.

[30]  Chunxiang Xu,et al.  Quantum-dot-based biosensor for simultaneous detection of biomarker and therapeutic drug: first steps toward an assay for quantitative pharmacology. , 2012, The Analyst.

[31]  S. Cory,et al.  The Bcl-2 protein family: arbiters of cell survival. , 1998, Science.

[32]  A. Gewirtz.  Oligonucleotide therapeutics: clothing the emperor. , 1999, Current opinion in molecular therapeutics.

[33]  Gbenga Anthony Adefolaju,et al.  Effects of HIV protease, nucleoside/non-nucleoside reverse transcriptase inhibitors on Bax, Bcl-2 and apoptosis in two cervical cell lines. , 2014, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[34]  Jun‐Jie Zhu,et al.  Graphene quantum dots as fluorescence probes for turn-off sensing of melamine in the presence of Hg(2+). , 2014, ACS applied materials & interfaces.

[35]  S. Cory,et al.  The Bcl2 family: regulators of the cellular life-or-death switch , 2002, Nature Reviews Cancer.

[36]  A. Barrett,et al.  The role of stem cell transplantation for chronic myelogenous leukemia in the 21st century. , 2015, Blood.

[37]  S. Korsmeyer,et al.  Cell Death Critical Control Points , 2004, Cell.

[38]  J C Reed,et al.  Mitochondria and apoptosis. , 1998, Science.

[39]  Yu Xiaowen,et al.  Electrochemical Biosensing Based on Graphene Modified Electrodes , 2014 .

[40]  S. Marcé,et al.  A thirty-five nucleotides BCR-ABL1 insertion mutation of controversial significance confers resistance to imatinib in a patient with chronic myeloid leukemia (CML). , 2015, Experimental and molecular pathology.

[41]  Xuecheng Chen.  Core/shell structured silica spheres with controllable thickness of mesoporous shell and its adsorption, drug storage and release properties , 2013 .

[42]  S. Korsmeyer,et al.  Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death , 1993, Cell.

[43]  S. Satija,et al.  Using Self-Assembly To Control the Structure of DNA Monolayers on Gold: A Neutron Reflectivity Study , 1998 .

[44]  W. S. Hummers,et al.  Preparation of Graphitic Oxide , 1958 .

[45]  Yun Xiang,et al.  Quantum-dot/aptamer-based ultrasensitive multi-analyte electrochemical biosensor. , 2006, Journal of the American Chemical Society.

[46]  S. Kaufmann,et al.  Emerging understanding of Bcl-2 biology: Implications for neoplastic progression and treatment. , 2015, Biochimica et biophysica acta.

[47]  Joseph Wang,et al.  Simultaneous electrochemical measurement of metal and organic propellant constituents of gunshot residues. , 2012, The Analyst.

[48]  C. Sawyers,et al.  Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. , 2001, The New England journal of medicine.

[49]  Jun-Jie Zhu,et al.  Ultrasensitive multi-analyte electrochemical immunoassay based on GNR-modified heated screen-printed carbon electrodes and PS@PDA-metal labels for rapid detection of MMP-9 and IL-6. , 2014, Biosensors & bioelectronics.

[50]  J. Rowley.  A New Consistent Chromosomal Abnormality in Chronic Myelogenous Leukaemia identified by Quinacrine Fluorescence and Giemsa Staining , 1973, Nature.

[51]  P. Nowell,et al.  A minute chromosome in human chronic granulocytic leukemia , 1960 .