Conformationally Gated Electrochemical Gene Detection Chad E. Immoos, [b] Stephen J. Lee, [c] and Mark W. Grinstaff* [a] Introduction Identifying specific nucleic acid sequences of viral or bacterial pathogens, hereditary diseases, or genetic abnormalities is of widespread interest in the areas of medicine, biotechnology, and homeland security. Current methods for DNA detection include both sequencing techniques and hybridization assays. The advantages of hybridization assays over sequencing tech- niques include rapid detection of the analyte and the elimina- tion of sample amplification and purification steps. Electro- chemical detection schemes possess several advantages in- cluding sensitive electrochemical transducers, single amplifica- tion, low cost, rapid detection, minimal power requirements, and compatibility with microfabrication techniques. [1±3] Several methods for sequence-specific DNA detection based on elec- trochemical methods have been reported. The groups of Barton and Thorp describe the detection of DNA using an elec- trocatalytic scheme for signal amplification. [4±6] Electrochemilu- minescence assays have also been reported for the detection of specific DNA sequences. [7] Another common assay design is a sandwich-type assay possessing three components: a cap- ture strand, a target strand, and a probe strand containing an electroactive reporter group. [8±10] Very recently, the electro- chemical detection of DNA by using immobilized hairpins [11,12] and single-stranded DNA [13] has been reported. Such electro- chemical DNA sensors, in which electron-transfer dynamics are altered as a consequence of a structural rearrangement in- duced by hybridization, are of interest. Herein, we report the synthesis and characterization of ferrocene-labeled DNA hair- pins, and the use of a solid-phase hybridization assay on a gold-ball electrode to electrochemically detect DNA. The design criteria for this conformationally gated DNA- detection device are: 1) an easily addressable redox probe and 2) a large structural/conformational change upon hybridization of the target DNA strand. Ferrocene was chosen as the electro- active probe in these studies because of its proven utility in biological diagnostics, [9,14,15] its stability during DNA synthe- sis, [16,17] and its accessible redox potential under physiological conditions. To afford significant structural rearrangement of the electroactive DNA, we selected a DNA stem-loop sequence, with the loop DNA sequence complementary to a specific DNA target. Binding of target DNA induces a hairpin-to-duplex tran- sition. This structural rearrangement has been previously char- acterized by using fluorescence resonance energy transfer (FRET) techniques with fluorophore-labeled hairpins. [18] Such fluorophore-labeled hairpins, termed ™molecular beacons∫, are of interest for diagnostic applications. [19] For our studies, when the DNA hairpin is immobilized on the electrode, the five 3’- and 5’-terminal nucleobases form a stem that places the 5’-ter- minal redox probe in close proximity to the electrode surface, thereby making it electrochemically accessible. Upon binding of the complementary DNA strand to the exposed loop por- tion of the immobilized sequence, the hairpin will open to the extended-duplex form; this increases the distance between the 5’-terminal ferrocene and the electrode surface. The increased distance between the redox probe and the electrode is mani- fested as a loss of the electrochemical signal (Scheme 1). Results and Discussion The bifunctionalized 26-mer oligodeoxynucleotide was synthe- sized by using a 3’-hexylthiol-modified controlled-pore glass (CPG) resin and a ferrocene phosphoramidite on an ABI 392 solid-phase DNA synthesizer. [17] The probe sequence contained within this hairpin motif is complementary to a sequence char- [a] Dr. M. W. Grinstaff Departments of Chemistry and Biomedical Engineering, Boston University 590 Commonwealth Avenue, Boston, MA 02215 (USA) Fax:(+ 1)617-353-6466 E-mail:mgrin@chem.bu.edu [b] Dr.C.E.Immoos Department of Chemistry, Duke University Durham, NC 27708 (USA) [c] Dr.S.J.Lee Army Research Office Research Triangle Park, NC 27709 (USA) The synthesis and characterization of a 26-base DNA hairpin con- taining both a redox-active reporter (ferrocene) and terminal thiol functionality for electrochemical gene detection is described. This electrochemical DNA sensor exploits electron-transfer dy- namics that alter as a consequence of a large structural rear- rangement (hairpin-to-duplex) induced by hybridization of the target DNA sequence. Melting temperature and circular dichroism studies confirm that the 26-mer DNA forms a hairpin structure in the absence of target DNA. The loop region of the DNA hairpin is shown to form a stable duplex in the presence of complementary single-stranded DNA. Atomic force microscopy and ellipsometry experiments of immobilized self-assembled DNA monolayers sug- gest that hybridization with complementary DNA affords a con- formational change that alters the electrochemical response. 1100 ¹ 2004 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim DOI: 10.1002/cbic.200400045 ChemBioChem 2004,5,1100±1103