Hydrogen peroxide sensors for cellular imaging based on horse radish peroxidase reconstituted on polymer-functionalized TiO₂ nanorods.

We describe the reconstitution of apo-horse radish peroxidase (apo-HRP) onto TiO(2) nanorods functionalized with a multifunctional polymer. After functionalization, the horse radish peroxidase (HRP) functionalized TiO(2) nanorods were well dispersible in aqueous solution, catalytically active and biocompatible, and they could be used to quantify and image H(2)O(2) which is a harmful secondary product of cellular metabolism. The shape, size and structure of TiO(2) nanorods (anatase) were analyzed by transmission electron microscopy (TEM), high resolution TEM (HRTEM), electron diffraction (ED) and X-ray diffraction (XRD). The surface functionalization, HRP reconstitution and catalytic activity were confirmed by UV-Vis, FT-IR, CLSM and atomic force microscopy (AFM). Biocompatibility and cellular internalization of active HRP reconstituted TiO(2) nanorods were confirmed by a classical MTT cytotoxicity assay and confocal laser scanning microscopy (CLSM) imaging, respectively. The intracellular localization allowed H(2)O(2) detection, imaging and quantification in HeLa cells. The polymer functionalized hybrid system creates a complete sensor including a "cell positioning system" in each single particle. The flexible synthetic concept with functionalization by post-polymerization modification allows introduction of various dyes for sensitisation at different wavelengths and introduction of various anchor groups for anchoring on different particles.

[1]  M. Toledano,et al.  ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis , 2007, Nature Reviews Molecular Cell Biology.

[2]  Karthikeyan Subramani,et al.  Magnetic resonance imaging tracking of stem cells in vivo using iron oxide nanoparticles as a tool for the advancement of clinical regenerative medicine. , 2011, Chemical reviews.

[3]  Itamar Willner,et al.  Reconstitution of apo-glucose dehydrogenase on pyrroloquinoline quinone-functionalized au nanoparticles yields an electrically contacted biocatalyst. , 2005, Journal of the American Chemical Society.

[4]  J. Luong,et al.  Cellulose nanocrystal/gold nanoparticle composite as a matrix for enzyme immobilization. , 2009, ACS applied materials & interfaces.

[5]  J. Cooney.  Chromatographic Gel Media for Large Scale Protein Purification , 1984, Bio/Technology.

[6]  Prakash D Nallathamby,et al.  Photostable single-molecule nanoparticle optical biosensors for real-time sensing of single cytokine molecules and their binding reactions. , 2008, Journal of the American Chemical Society.

[7]  Patrik Schmuki,et al.  TiO2‐Nanoröhren: Synthese und Anwendungen , 2011 .

[8]  M. Beal,et al.  Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases , 2006, Nature.

[9]  Zhilian Zhou,et al.  The pursuit of a scalable nanofabrication platform for use in material and life science applications. , 2008, Accounts of chemical research.

[10]  M. Jaroniec,et al.  Fabrication and enhanced visible-light photocatalytic activity of carbon self-doped TiO2 sheets with exposed {001} facets , 2011 .

[11]  Jeffrey N. Anker,et al.  Biosensing with plasmonic nanosensors. , 2008, Nature materials.

[12]  Felix Kratz,et al.  Polymer therapeutics: concepts and applications. , 2006, Angewandte Chemie.

[13]  Colin L. Masters,et al.  Neurodegenerative diseases and oxidative stress , 2004, Nature Reviews Drug Discovery.

[14]  Victor S-Y Lin,et al.  Mesoporous silica nanoparticle based controlled release, drug delivery, and biosensor systems. , 2007, Chemical communications.

[15]  Xiaoling Yang,et al.  Immobilization of horseradish peroxidase in three-dimensional macroporous TiO2 matrices for biosensor applications , 2009 .

[16]  W. Tremel,et al.  Biomolecular conjugation inside synthetic polymer nanopores via glycoprotein-lectin interactions. , 2011, Nanoscale.

[17]  D. Balding,et al.  HLA Sequence Polymorphism and the Origin of Humans , 2006 .

[18]  D. Astruc,et al.  Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. , 2004, Chemical reviews.

[19]  Wolfgang Tremel,et al.  Synthesis and bio-functionalization of magnetic nanoparticles for medical diagnosis and treatment. , 2011, Dalton transactions.

[20]  Jörg Maser,et al.  Biology of TiO2–oligonucleotide nanocomposites , 2003, Nature materials.

[21]  Patrick Theato,et al.  Synthesis of well‐defined polymeric activated esters , 2008 .

[22]  S. Nie,et al.  Luminescent quantum dots for multiplexed biological detection and imaging. , 2002, Current opinion in biotechnology.

[23]  C. Larabell,et al.  Quantum dots as cellular probes. , 2005, Annual review of biomedical engineering.

[24]  W. Tremel,et al.  V2O5 Nanowires with an Intrinsic Peroxidase‐Like Activity , 2011 .

[25]  Philippe Rostaing,et al.  Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking , 2003, Science.

[26]  W. Tremel,et al.  Double-stranded RNA polyinosinic-polycytidylic acid immobilized onto gamma-Fe2O3 nanoparticles by using a multifunctional polymeric linker. , 2007, Small.

[27]  Manuel Serrano,et al.  The common biology of cancer and ageing , 2007, Nature.

[28]  H. Goesmann,et al.  Nanoparticulate functional materials. , 2010, Angewandte Chemie.

[29]  Shuguang Zhang,et al.  Emerging biological materials through molecular self-assembly. , 2002, Biotechnology advances.

[30]  R. Zentel,et al.  Synthesis of pentafluorophenyl(meth)acrylate polymers: New precursor polymers for the synthesis of multifunctional materials , 2005 .

[31]  Monty Liong,et al.  Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs. , 2007, Small.

[32]  C. Niemeyer,et al.  Kovalente Hämin‐DNA‐Addukte zur Herstellung einer neuartigen Klasse artifizieller Häm‐Enzyme , 2005 .

[33]  M. Grätzel,et al.  Facile preparation of large aspect ratio ellipsoidal anatase TiO2 nanoparticles and their application to dye-sensitized solar cell , 2009 .

[34]  M. Terrones,et al.  Covalent 2D and 3D networks from 1D nanostructures: designing new materials. , 2007, Nano letters.

[35]  George M Whitesides,et al.  Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. , 1998, Angewandte Chemie.

[36]  H. Dai,et al.  In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. , 2020, Nature nanotechnology.

[37]  H. A. Therese,et al.  From single molecules to nanoscopically structured functional materials: Au nanocrystal growth on TiO2 nanowires controlled by surface-bound silicatein. , 2006, Angewandte Chemie.

[38]  C. Niemeyer,et al.  Kinetic analysis of semisynthetic peroxidase enzymes containing a covalent DNA-heme adduct as the cofactor. , 2006, Chemistry.

[39]  C. Niemeyer,et al.  DNA-directed immobilization of horseradish peroxidase-DNA conjugates on microelectrode arrays: towards electrochemical screening of enzyme libraries. , 2007, Chemistry.

[40]  M. Hecht,et al.  Peroxidase activity of de novo heme proteins immobilized on electrodes. , 2007, Journal of inorganic biochemistry.

[41]  Dongsheng Xu,et al.  Controllable Synthesis of TiO2 Single Crystals with Tunable Shapes Using Ammonium-Exchanged Titanate Nanowires as Precursors , 2010 .

[42]  J. Matthew Mauro,et al.  Long-term multiple color imaging of live cells using quantum dot bioconjugates , 2003, Nature Biotechnology.

[43]  W. Tremel,et al.  Reactive polymers: a versatile toolbox for the immobilization of functional molecules on TiO2 nanoparticles. , 2006, Angewandte Chemie.

[44]  Evan W. Miller,et al.  Molecular imaging of hydrogen peroxide produced for cell signaling , 2007 .

[45]  W. R. Taylor,et al.  In vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles. , 2007, Nature materials.

[46]  S. Nie,et al.  Quantum dot bioconjugates for ultrasensitive nonisotopic detection. , 1998, Science.

[47]  E. Giannelis,et al.  Activity increase of horseradish peroxidase in the presence of magnetic particles. , 2008, Journal of the American Chemical Society.

[48]  B. Dickinson,et al.  A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells. , 2008, Journal of the American Chemical Society.

[49]  Robert Langer,et al.  Quantum dot-aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer. , 2007, Nano letters.

[50]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[51]  Kenji Yamamoto,et al.  Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker. , 2003, Biochemical and biophysical research communications.

[52]  J. Boilot,et al.  Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells. , 2009, Nature nanotechnology.

[53]  Aicheng Chen,et al.  Coadsorption of horseradish peroxidase with thionine on TiO2 nanotubes for biosensing. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[54]  Adam Heller,et al.  Electrical Wiring of Glucose Oxidase by Reconstitution of FAD-Modified Monolayers Assembled onto Au-Electrodes , 1996 .

[55]  É. Boisselier,et al.  Dendrimers designed for functions: from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine. , 2010, Chemical reviews.

[56]  P. Théato,et al.  RAFT Polymerization of Pentafluorophenyl Methacrylate: Preparation of Reactive Linear Diblock Copolymers , 2005 .

[57]  E. Schenborn,et al.  Reporter gene vectors and assays , 1999, Molecular biotechnology.

[58]  Erkki Ruoslahti,et al.  Nanocrystal targeting in vivo , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[59]  Eugenii Katz,et al.  Improving enzyme–electrode contacts by redox modification of cofactors , 1995, Nature.

[60]  Katz,et al.  Integration of Layered Redox Proteins and Conductive Supports for Bioelectronic Applications. , 2000, Angewandte Chemie.

[61]  M. Bruchez,et al.  Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots , 2003, Nature Biotechnology.

[62]  Vincent Noireaux,et al.  In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles , 2002, Science.

[63]  Peter Greil,et al.  Hydroxyapatite growth on anodic TiO2 nanotubes. , 2006, Journal of biomedical materials research. Part A.

[64]  H. A. Therese,et al.  dsRNA-functionalized multifunctional gamma-Fe2O3 nanocrystals: a tool for targeting cell surface receptors. , 2008, Angewandte Chemie.

[65]  Ho-Joong Kim,et al.  Responsive nanostructures from aqueous assembly of rigid-flexible block molecules. , 2011, Accounts of chemical research.

[66]  E. Torres,et al.  Apoenzyme reconstitution as a chemical tool for structural enzymology and biotechnology. , 2009, Angewandte Chemie.

[67]  Suntharampillai Thevuthasan,et al.  PEGylated inorganic nanoparticles. , 2011, Angewandte Chemie.

[68]  Itamar Willner,et al.  Design of Amperometric Biosensors and Biofuel Cells by the Reconstitution of Electrically Contacted Enzyme Electrodes , 2008 .

[69]  Christine M. Micheel,et al.  Cell Motility and Metastatic Potential Studies Based on Quantum Dot Imaging of Phagokinetic Tracks , 2002 .

[70]  S. Mecking,et al.  Nanoparticles of conjugated polymers. , 2010, Chemical reviews.

[71]  J. Burnell.  Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase - Pi-dependent pyrophosphorylase from bacteria , 2010, BMC Biochemistry.

[72]  Scott E. Fraser,et al.  In vivo visualization of gene expression using magnetic resonance imaging , 2000, Nature Biotechnology.