Dendritic amplification of DNA analysis by oligonucleotide-functionalized Au-nanoparticles Fernando Patolsky, Koodali T. Ranjit, Amir Lichtenstein and Itamar Willner* Institute of Chemistry, The Farkas Center for Light-Induced Processes, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. E-mail: willnea@vms.huji.ac.il Received (in Cambridge, UK) 20th March 2000, Accepted 3rd May 2000 Published on the Web 25th May 2000 Dendritic amplification of DNA analysis is accomplished by the application of 5A- and 3A-terminated oligonucleotide- functionalized Au-colloids complementary to the analyte DNA. The development of DNA sensors attracts recent research efforts directed to gene analysis, identification of genetic disorders, tissue matching, and forensic applications. 1,2 Pho- tonic detection of DNA was accomplished by the use of fluorescence-labeled oligonucleotides, 3 the use of surface plasmon resonance, SPR, 4 and through the application of oligonucleotide-modified Au-nanoparticles which exhibit plas- mon absorbance. 5,6 Electronic transduction of oligonucleotide– DNA recognition events, and the amplified transduction of DNA sensing, are major challenges in DNA-based bio- electronics. 7 Electrostatic attraction of transition metal com- plexes 8 or dyes 9 was used for the voltammetric probing of the formation of double-stranded assemblies. Amplified electronic transduction of DNA sensing events was accomplished by the application of enzyme labels that bind to the double-stranded (ds) assembly, 10 the use of protein labels, 11 or the use of branched oligonucleotides. 12 Other amplification routes of DNA sensing events included the application of enzymes that biocatalyze the precipitation of an insoluble product on the transducer, 13 or the use of liposomes that bind to the ds-DNA assembly. 14,15 Electrochemical 12–15 or microgravimetric, quartz-crystal-microbalance, 11,16 methods have been applied as electronic transduction signals for the sensing events. Here, we report on a novel method to amplify DNA sensing events by the application of oligonucleotide-functionalized Au-nanoparticles for the amplified sensing of DNA by a dendritic-type amplifica- tion route. Microgravimetric, quartz-crystal-microbalance (QCM) experiments are used to follow the amplified DNA- sensing processes. Scheme 1(a) shows the method of the amplified sensing of a target DNA. A primer thiol-functionalized oligonucleotide 1 is assembled on the Au-electrode, and the target analyte DNA 2, hybridizes with the sensing interface. The primary amplification of the sensing process is performed by the interaction of the surface with the 3-functionalized Au-particles. The secondary, dendritic-type amplification is performed by the interaction of the resulting interface with the analyte sample 2 that is pre- treated with the 1-functionalized Au-nanoparticle. The mass associated with the Au-nanoparticles linked to the crystal in the primary hybridization step, and the dendritic structure formed in the secondary hybridization process, amplify the sensing of 2. Note that the amplifying Au-nanoparticles that are used in the two modification steps have different modification layers, and are functionalized with the 3A-terminated and the 5A-terminated thiolated oligonucleotides, 3 and 1, respectively. Both oligonu- cleotides, 3 and 1, are complementary to the two ends of the analyte 2. The 1- and 3-functionalized Au-nanoparticles were prepared according to the literature 6 by the reaction of citrate- stabilized Au-nanoparticles (12 ± 1 nm, 5 mL) with the thiolated oligonucleotides 1 or 3 for 20 h. Fig. 1(A), curve (a), shows the frequency changes of the Au- quartz crystal (AT-cut, 9 MHz) functionalized with 1 (surface coverage 1.4 3 10 211 mol cm 22 ) as a result of interaction with the analyte 2 (2 3 10 28 M). A frequency decrease of ca. 9 Hz is observed. Treatment of the resulting interface with the 3-functionalized Au-nanoparticles results in an additional frequency change of ca. 60 Hz [Fig. 1(A), curve (b)], indicating that the binding of the Au-nanoparticles indeed amplified the primary sensing of 2. The amplified sensing of 2 is specific and the interaction of the sensing interface with the non-com- plementary DNA 2a, at a high concentration (5 3 10 26 M), does not yield any change in the crystal frequency (ca. 21 Hz) even after its interaction with the 3-functionalized Au-nano- Scheme 1 (a) Dendritic amplified DNA-sensing using oligonucleotide-functionalized Au-nanoparticles. (b) Immobilization of the thiolated oligonucleotide 1 on a glass support. This journal is © The Royal Society of Chemistry 2000 DOI: 10.1039/b002221g Chem. Commun., 2000, 1025–1026 1025