Multiplex detection of nucleases by a graphene-based platform

In this article, we present a new method for the multiplex detection of nucleases by using graphene oxide (GO) as a platform. We introduce a Y-shaped DNA (Y-DNA) as the multiplex probe. The 5′ termini of the Y-DNA are labeled with carboxy fluorescein (FAM), 6-carboxy-X-rhodamine (ROX) and cyanine 5 (Cy5) and they include three nuclease cleavage sites corresponding to PvuII, EcoRV and HaeIII, respectively. Upon the addition of nucleases, the nucleases cleave the corresponding sites in Y-DNA. Then, short dsDNA fragments containing fluorophores were released from the Y-DNA. These dsDNA fragments were unstable and easy to unwind into two short ssDNAs. They were then adsorbed onto the GO surface. Because of the excellent electronic transference of GO, the fluorescence intensity of the fluorophores can be quenched efficiently. Therefore, by monitoring the fluorophores’ fluorescence change before and after the addition of the nucleases, it is easy to establish a platform of a Y-DNA/GO complex for the simultaneous multiplex detection of nucleases.

[1]  Min Zhang,et al.  Interaction of peptides with graphene oxide and its application for real-time monitoring of protease activity. , 2011, Chemical communications.

[2]  G. Eda,et al.  Graphene oxide as a chemically tunable platform for optical applications. , 2010, Nature chemistry.

[3]  Huang-Hao Yang,et al.  Amplified aptamer-based assay through catalytic recycling of the analyte. , 2010, Angewandte Chemie.

[4]  Hongje Jang,et al.  A Graphene‐Based Platform for the Assay of Duplex‐DNA Unwinding by Helicase† , 2010, Angewandte Chemie.

[5]  Yuehe Lin,et al.  Aptamer/graphene oxide nanocomplex for in situ molecular probing in living cells. , 2010, Journal of the American Chemical Society.

[6]  H. Ju,et al.  Fluorescence resonance energy transfer between quantum dots and graphene oxide for sensing biomolecules. , 2010, Analytical chemistry.

[7]  Filip Braet,et al.  Carbon nanomaterials in biosensors: should you use nanotubes or graphene? , 2010, Angewandte Chemie.

[8]  Chunhai Fan,et al.  A Graphene Nanoprobe for Rapid, Sensitive, and Multicolor Fluorescent DNA Analysis , 2010 .

[9]  Xin Lu,et al.  Fast and Facile Preparation of Graphene Oxide and Reduced Graphene Oxide Nanoplatelets , 2009 .

[10]  Daoben Zhu,et al.  Fluorescence logic-signal-based multiplex detection of nucleases with the assembly of a cationic conjugated polymer and branched DNA. , 2009, Angewandte Chemie.

[11]  Huang-Hao Yang,et al.  A graphene platform for sensing biomolecules. , 2009, Angewandte Chemie.

[12]  A. Govindaraj,et al.  Binding of DNA nucleobases and nucleosides with graphene. , 2009, Chemphyschem : a European journal of chemical physics and physical chemistry.

[13]  Guoliang Zhang,et al.  Deoxygenation of Exfoliated Graphite Oxide under Alkaline Conditions: A Green Route to Graphene Preparation , 2008 .

[14]  Zhuang Liu,et al.  PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. , 2008, Journal of the American Chemical Society.

[15]  Jun Li,et al.  Hairpin fluorescence DNA probe for real-time monitoring of DNA methylation. , 2007, Analytical chemistry.

[16]  Dan Luo,et al.  Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes , 2005, Nature Biotechnology.

[17]  O. Fleck,et al.  DNA repair nucleases , 2004, Cellular and Molecular Life Sciences CMLS.

[18]  N. Grindley,et al.  Mechanisms of site-specific recombination. , 2003, Annual review of biochemistry.

[19]  A. Jeltsch,et al.  A fluorimetric assay for on-line detection of DNA cleavage by restriction endonucleases. , 2002, Journal of biotechnology.

[20]  E. Rangarajan,et al.  Sugar non-specific endonucleases. , 2001, FEMS microbiology reviews.

[21]  A. Jeltsch,et al.  Structure and function of type II restriction endonucleases. , 2001, Nucleic acids research.

[22]  J. Thorson,et al.  A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Ma.  Genes, greens, and vaccines , 2000, Nature Biotechnology.

[24]  W. Tan,et al.  Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA. , 2000, Nucleic acids research.

[25]  K. Kasai,et al.  Assay of trypsin activity by capillary isoelectric focusing with laser‐induced fluorescence detection , 1998, Electrophoresis.

[26]  M. Kawakita,et al.  Development of a sensitive and accurate enzyme-linked immunosorbent assay (ELISA) system that can replace HPLC analysis for the determination of N1,N12-diacetylspermine in human urine. , 1998, Journal of biochemistry.

[27]  M. Eigen,et al.  Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[28]  M. Lieber.  The FEN‐1 family of structure‐specific nucleases in eukaryotic dna replication, recombination and repair , 1997, BioEssays : news and reviews in molecular, cellular and developmental biology.

[29]  F. Eckstein,et al.  Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides. , 1988, Nucleic acids research.

[30]  P. McEuen,et al.  Controlled assembly of dendrimer-like DNA , 2004, Nature materials.

[31]  C. Stein,et al.  Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences , 2000, Nature Biotechnology.

[32]  Lee Sp,et al.  Fluorescence assays for DNA cleavage. , 1997 .