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Immobilization and Hybridization of DNA on an Aluminum(III) Alkanebisphosphonate Thin Film with Electrogenerated Chemiluminescent Detection
- Source :
- Journal of the American Chemical Society. 117:2627-2631
- Publication Year :
- 1995
- Publisher :
- American Chemical Society (ACS), 1995.
-
Abstract
- Immobilization of single-stranded (ss) DNA on an electrode covered with an aluminum alkanebisphosphonate film by immersion in an ss-DNA solution was demonstrated with ss-DNA labeled with R~(bpy)3~+. This was detected by observing the electrogenerated chemiluminescence (ECL) produced upon oxidation in a solutior containing trin-propylamine. Surface hybridization was shown by immobilization of unlabeled ss-DNA, followed by exposure to a complementary labeled strand of DNA to produce R~(bpy)3~+-labeled double-stranded (ds) DNA on the surface. The extent of DNA hybridization was determined by ECL. Hybridization of unlabeled DNA, e.g., poly(dA) and poly(dT), could be detected by treatment with R~(phen)3~+ and ECL of this species associated with the ds-DNA. In a similar way, an eight-base single strand of DNA was determined by immobilizing it on the film followed by hybridization of the eight-base complementary strand. The surface-immobilized eight-base pair ds-DNA was also detected by observing the ECL of intercalated Ru(phen)3*+. The immobilization and hybridization of DNA on a selfassembled thin film is of interest both in studies of molecular recognition of DNA and in various application^.'-^ Apart from providing a predictable surface that binds a complementary strand of DNA on substrates for studying DNA double-helix formation and its interactions with proteins and other molecules: a film with single-stranded (ss) DNA can form the basis of a DNA biosensor, e.g., by serving as a designed surface modified electrode. DNA diagnostics has become an important area in molecular biology and biotechnology studies, with applications to the determination of disease-causing and food-contaminating organisms and in forensic and environmental investigations. However, traditional methods for DNA sequencing have several disadvantages, e.g., they are labor intensive, expensive, time consuming, and difficult to a u t ~ m a t e . ~ . ~ The development of new DNA biosensors has involved the application of optical methods (e.g., luminescence, ellipsometry, and pseudo-Brewster angle reflectometry), piezoelectric devices (e.g., surface acoustic wave, quartz crystal microbalance), and electrochemical techniques (e.g., cyclic voltammetry and square wave voltammeElectrogenerated chemiluminescence (ECL) has also been applied to DNA analysis, e.g., of DNA produced by the polymerase chain reaction by capturing R~(bpy)3~+-labeled DNA on magnetic beads, transporting the beads to an electrochemical cell, and detecting emission under electrochemical exc i t a t i~n .~ ,~ 'Abstract published in Advance ACS Abstracts, February 15, 1995. (1) Furuta, H.; Magda, D.; Sessler, J. L. J. Am. Chem. SOC. 1991, 113, 978. In a previous study, we demonstrated that an aluminum(1II) alkanebisphosphonate film can be used to immobilize calf thymus double-stranded (ds) DNA based on the interaction of the film metal center (A13+) with the phosphate group of DNA and that the immobilized DNA could be determined by ECL detection upon oxidation of a solution of R~(phen)3~+ intercalated with DNA and tri-n-propylamine.10 The technique for the fabrication of the aluminum alkanebisphosphonate, AL2(CdBP), film by sequential adsorptionheaction steps has been described previously.lo,'l We report here that the film can also be used to immobilize ss-DNA (e.g., poly(dA), poly(dT), poly(dC), and eight-base and 30-base synthesized DNA chains). After the immobilization, the film can be employed to recognize a complementary strand of DNA in a solution followed by ECL detection. The strategy employed is shown in Figure 1. Experimental Section Apparatus. The Plexiglas cell designed for ECL and electrochemical studies of the film immobilized on the gold surface supported on the silicon wafer was described previously. A saturated calomel reference electrode (SCE) and a platinum wire counterelectrode were used for all measurements. The electrochemical measurements coupled with ECL experiments were camed out with a Model 175 universal programmer, a Model 173 potentiostat (Princeton Applied Research, PAR, Princeton, NJ), and an Omnigraphic 2000 x-y recorder (Houston Instruments, Houston, TX). The ECL emission was detected with a Model C1230 single-photon-counting system (Hamamatsu Corp., Bridgewater, NJ) utilizing a Hamamatsu R928P PMT, cooled to -20
Details
- ISSN :
- 15205126 and 00027863
- Volume :
- 117
- Database :
- OpenAIRE
- Journal :
- Journal of the American Chemical Society
- Accession number :
- edsair.doi...........99a141edaf48caa76ce3c764cce63454