A new route to the considerable enhancement of glucose oxidase (GOx) activity: the simple assembly of a complex from CdTe quantum dots and GOx, and its glucose sensing.

A new complex consisting of CdTe quantum dots (QDs) and glucose oxidase (GOx) has been facilely assembled to achieve considerably enhanced enzymatic activity and a wide active temperature range of GOx; these characteristics are attributed to the conformational changes of GOx during assembly. The obtained complex can be simultaneously used as a nanosensor for the detection of glucose with high sensitivity. A mechanism is put forward based on the fluorescence quenching of CdTe QDs, which is caused by the hydrogen peroxide (H2O2) that is produced from the GOx-catalyzed oxidation of glucose. When H2O2 gets to the surface of the CdTe QDs, the electron-transfer reaction happens immediately and H2O2 is reduced to O2, which lies in electron hole traps on CdTe QDs and can be used as a good acceptor, thus forming the nonfluorescent CdTe QDs anion. The produced O2 can further participate in the catalyzed reaction of GOx, forming a cyclic electron-transfer mechanism of glucose oxidation, which is favorable for the whole reaction system. The value of the Michaelis-Menton constant of GOx is estimated to be 0.45 mM L(-1), which shows the considerably enhanced enzymatic activity measured by far. In addition, the GOx enzyme conjugated on the CdTe QDs possesses better thermal stability at 20-80 degrees C and keeps the maximum activity in the wide range of 40-50 degrees C. Moreover, the simply assembled complex as a nanosensor can sensitively determine glucose in the wide concentration range from micro- to millimolar with the detection limit of 0.10 microM, which could be used for the direct detection of low levels of glucose in biological systems. Therefore, the established method could provide an approach for the assembly of CdTe QDs with other redox enzymes, to realize enhanced enzymatic activity, and to further the design of novel nanosensors applied in biological systems in the future.

[1]  S. Genuth,et al.  The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. , 1993, The New England journal of medicine.

[2]  Igor L. Medintz,et al.  Self-assembled nanoscale biosensors based on quantum dot FRET donors , 2003, Nature materials.

[3]  V. Rotello,et al.  Integrated magnetic bionanocomposites through nanoparticle-mediated assembly of ferritin. , 2007, Journal of the American Chemical Society.

[4]  A Heller,et al.  "Wired" enzyme electrodes for amperometric determination of glucose or lactate in the presence of interfering substances. , 1994, Analytical chemistry.

[5]  Zeev Rosenzweig,et al.  Synthesis and application of quantum dots FRET-based protease sensors. , 2006, Journal of the American Chemical Society.

[6]  Guo-Li Shen,et al.  Amperometric glucose biosensor based on a surface treated nanoporous ZrO2/Chitosan composite film as immobilization matrix , 2004 .

[7]  Igor L. Medintz,et al.  Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates , 2006, Nature materials.

[8]  Samir Kumar Pal,et al.  Structural and Functional Characterization of Enzyme-Quantum Dot Conjugates: Covalent Attachment of CdS Nanocrystal to α-Chymotrypsin , 2007 .

[9]  B. D. Malhotra,et al.  Immobilization of glucose oxidase onto Langmuir–Blodgett films of poly-3-hexylthiophene , 2003 .

[10]  C. Soeller,et al.  DNA hybridization detection with blue luminescent quantum dots and dye-labeled single-stranded DNA. , 2007, Journal of the American Chemical Society.

[11]  Bo Tang,et al.  A new nanobiosensor for glucose with high sensitivity and selectivity in serum based on fluorescence resonance Energy transfer (FRET) between CdTe quantum dots and Au nanoparticles. , 2008, Chemistry.

[12]  A. Heeger,et al.  Beyond superquenching: Hyper-efficient energy transfer from conjugated polymers to gold nanoparticles , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Hui Jiang,et al.  Enzyme-quantum dots architecture for highly sensitive electrochemiluminescence biosensing of oxidase substrates. , 2007, Chemical communications.

[14]  J. Irudayaraj,et al.  Examination of Cholesterol oxidase attachment to magnetic nanoparticles , 2005, Journal of nanobiotechnology.

[15]  Itamar Willner,et al.  Optical detection of glucose and acetylcholine esterase inhibitors by H2O2-sensitive CdSe/ZnS quantum dots. , 2008, Angewandte Chemie.

[16]  Junbai Li,et al.  Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability. , 2007, Biochemical and biophysical research communications.

[17]  G. Bayramoglu,et al.  Congo Red attached monosize poly(HEMA-co-MMA) microspheres for use in reversible enzyme immobilisation , 2002 .

[18]  Albena Ivanisevic,et al.  Enzymatic clipping of DNA wires coated with magnetic nanoparticles. , 2005, Journal of the American Chemical Society.

[19]  Joseph Irudayaraj,et al.  Activity of glucose oxidase functionalized onto magnetic nanoparticles , 2005, Biomagnetic research and technology.

[20]  G. S. Wilson,et al.  Rotating ring-disk enzyme electrode for biocatalysis kinetic studies and characterization of the immobilized enzyme layer , 1980 .

[21]  Bansi D Malhotra,et al.  Application of thiolated gold nanoparticles for the enhancement of glucose oxidase activity. , 2007, Langmuir : the ACS journal of surfaces and colloids.

[22]  T. Godjevargova Behavior of glucose oxidase immobilized on ultrafiltration membranes obtained by copolymerizing acrylonitrile and N-vinylimidazol , 2000 .

[23]  Igor L. Medintz,et al.  Quantum dot bioconjugates for imaging, labelling and sensing , 2005, Nature materials.

[24]  Zeev Rosenzweig,et al.  Glucose oxidase–magnetite nanoparticle bioconjugate for glucose sensing , 2004, Analytical and bioanalytical chemistry.

[25]  Dale M. Willard,et al.  CdSe−ZnS Quantum Dots as Resonance Energy Transfer Donors in a Model Protein−Protein Binding Assay , 2001 .

[26]  Lars Baltzer,et al.  Substrate modulation of the activity of an artificial nanoesterase made of peptide-functionalized gold nanoparticles. , 2007, Angewandte Chemie.

[27]  H. Mattoussi,et al.  Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays. , 2002, Analytical chemistry.

[28]  L. Pollegioni,et al.  Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum. , 1999, European journal of biochemistry.

[29]  Itamar Willner,et al.  Lighting-up the dynamics of telomerization and DNA replication by CdSe-ZnS quantum dots. , 2003, Journal of the American Chemical Society.

[30]  Yuehe Lin,et al.  Glucose Biosensors Based on Carbon Nanotube Nanoelectrode Ensembles , 2004 .

[31]  J. Castillo,et al.  Determination of Glucose in Blood Based on the Intrinsic Fluorescence of Glucose Oxidase , 1997 .

[32]  Itamar Willner,et al.  Probing biocatalytic transformations with CdSe-ZnS QDs. , 2006, Journal of the American Chemical Society.

[33]  V. V. Mozhaev,et al.  Strategy for Stabilizing Enzymes Part One: Increasing Stability of Enzymes via their Multi-Point Interaction with a Support , 1990 .

[34]  R. Kopelman,et al.  Analytical properties and sensor size effects of a micrometer-sized optical fiber glucose biosensor. , 1996, Analytical chemistry.

[35]  Sudhakar R. Sainkar,et al.  PEPSIN-GOLD COLLOID CONJUGATES: PREPARATION, CHARACTERIZATION, AND ENZYMATIC ACTIVITY , 2001 .

[36]  Min-Hung Liao,et al.  Direct Binding and Characterization of Lipase onto Magnetic Nanoparticles , 2003, Biotechnology progress.

[37]  E. Anslyn,et al.  Using an indicator displacement assay to monitor glucose oxidase activity in blood serum. , 2007, Organic letters.

[38]  A. Bard,et al.  Electrochemistry and electrogenerated chemiluminescence of CdTe nanoparticles , 2004 .

[39]  V. Rotello,et al.  Regulation of α-chymotrypsin activity on the surface of substrate-functionalized gold nanoparticles , 2006 .

[40]  V. Rotello,et al.  Effect of ionic strength on the binding of alpha-chymotrypsin to nanoparticle receptors. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[41]  P Guyot-Sionnest,et al.  Electrochromic nanocrystal quantum dots. , 2001, Science.

[42]  H. Jia,et al.  Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility , 2003, Biotechnology and bioengineering.

[43]  David E Benson,et al.  A modular nanoparticle-based system for reagentless small molecule biosensing. , 2005, Journal of the American Chemical Society.

[44]  Eunkeu Oh,et al.  Inhibition assay of biomolecules based on fluorescence resonance energy transfer (FRET) between quantum dots and gold nanoparticles. , 2005, Journal of the American Chemical Society.

[45]  Alexander Eychmüller,et al.  Strongly Photoluminescent CdTe Nanocrystals by Proper Surface Modification , 1998 .