Immobilization of self-quenched DNA hairpin probe with a heterobifunctional reagent on a glass surface for sensitive detection of oligonucleotides Arvind Misra * , Mohammad Shahid Nucleic Acids Research Laboratory, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India article info Article history: Received 19 May 2009 Revised 3 July 2009 Accepted 7 July 2009 Available online 24 July 2009 Keywords: Hairpin probe Quenching Guanosine abstract A new sensitive method for the detection of nucleic acids on a glass surface has been described. The self- quenched DNA hairpin probe is immobilized on a glass surface utilizing heterobifunctional reagent, N-(3- triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC). In the closed state fluorescence intensity was quenched due to the presence of guanosine residues in close vicinity of fluo- rophore while on hybridization with perfectly matched complementary target strand fluorescence was restored. Efficiency and specificity of immobilization as well as thermal stability at variable temperature and pH conditions have been discussed in detail. The method employed has potential for the detection of single nucleotide variations and other diagnostic studies. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Nucleic acids based detection and quantification methods play an important role in the field of DNA-diagnostics and drug discov- ery. The labeled oligonucleotides with organic fluorophores instead of radioisotopes are routinely being used to recognize the DNA se- quences in homogeneous as well as in heterogeneous hybridiza- tion assays. 1,2 Oligonucleotides having hairpin like structure— hairpin probes, are labeled with a fluorophore at one end and a quencher molecule (conventional/natural) at the other terminal. Recently, such kind of single labeled hairpin probe has emerged as an effective diagnostic tool in molecular biology studies similar to the conventional molecular beacons. 3,4 The molecular beacons comprise stem–loop like structure (ideally, 4–7 nucleobase pairs in stem region and 15–25 nucleobase pairs in loop region) and are usually dual labeled with a fluorophore (donor dye) at 5 0 -end and with an acceptor (as a quencher), which may or may not be fluorescent, at the 3 0 -end. In the absence of perfectly matched com- plementary target strand fluorophore and quencher molecule re- main in proximity and as a result of dipole–dipole interaction between them, the fluorescence intensity of the probe quenches efficiently by the phenomenon of fluorescence resonance energy transfer (FRET). 5 In the case of molecular beacons, the strategy requires labeling at both ends of a single stranded oligonucleotide with a specific fluorescent dye which generally limits the yield of synthesized oli- gonucleotide probe and is also an expensive approach. To circum- vent the limitations possessed by the molecular beacons, hairpin probes have been designed and developed involving a single fluo- rescent molecule at one end and an acceptor molecule, like nucle- obase or amino acid, on the other end, to quench the fluorescence intensity of the fluorophore in the closed state conformation. For this purpose, a number of unique non-fluorescent quenchers in- stead of traditional ones, ranging from DNA nucleotides (guano- sine, deazaguanosine) to gold nanoparticles have already been introduced successfully. 6–10 Additionally, instead of utilizing inter- actions between the two extrinsic probes, interaction of fluoro- phores with DNA nucleobases or with amino acids has potentially used for the specific detection of DNA or RNA se- quences and antibodies at the single molecular level. 11–13 The fluorescence quenching by mono-labeled hairpin probe is based on the phenomenon of photoinduced electron transfer (PET), in between the first excited singlet state of fluorophore and ground state of a quenching molecule when present in proxim- ity, in which electron donating moiety like guanosine oxidized to form radical ion pair (G +Å ) and return to ground state via radiation- less charge recombination. 14,15 Both the tools are successfully ap- plied in diagnostics to detect the presence of the specific target nucleic acid sequences, after hybridization with matched comple- mentary target strands, by strong increase in the fluorescence intensity signal. Therefore, makes the homogeneous assay more reliable, sensitive and versatile. For high-throughput screening of biomolecules microarray technology (biochip), in last decades, has become a potent tool with diversified applications in genetic analysis, molecular diag- nostics, and drug discovery. 16–26 Of two well-established method- 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.07.015 * Corresponding author. Tel.: +91 0542 2307321x104; fax: +91 0542 2368127/ 175. E-mail address: arvindmisra2003@yahoo.com (A. Misra). Bioorganic & Medicinal Chemistry 17 (2009) 5826–5833 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc