Full Paper Electrochemical Genoassay Design for Allele-Specific Detection of Toll-Like Receptor-2 Gene Polymorphism Pinar Kara, a Seda Cavdar, a Afig Berdeli, b Mehmet Ozsoz b * a Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova, Izmir, Turkey *e-mail: mehmet.ozsoz@ege.edu.tr b Laboratory of Molecular Medicine, Department of Pediatrics, Faculty of Medicine, Ege University, 35100 Bornova, Izmir, Turkey Received: May 29, 2007 Accepted: June 19, 2007 Abstract An allele-specific voltammetric genoassay for the detection of allele-specific toll-like receptor-2 gene arg753gln polymorphism (TLR-2) from polymerase chain reaction (PCR) amplified real samples was described in this study. Meldola blue (MDB), an intercalator molecule, was used as hybridization label. The wild-type and mutant type oligonucleotideprobeswereimmobilizedontodisposablegraphiteelectrodesurfacesbycovalentattachmentmethod. The extent of hybridization between probe and target sequences was determined by using differential pulse voltammetry (DPV). As a result of the interaction between MDB and DNA at electrode surface, the MDB signal observed from probe sequence before hybridization and after hybridization with MM sequence is lower than that observed after hybridization with complementary sequence. The differences between the MDB reduction peaks obtained from probe modified, hybrid modified and MM modified electrode were used to detect TLR-2 from PCR amplifiedrealsamples.Thediscriminationofhomozygousandheterozygousalleleswasalsoestablishedbycomparing the peak currents of MDB reduction signals. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity. Keywords: Electrochemical genosensor,Intercalation, Toll-like receptors, Polymorphism, Polymerase chain reaction DOI: 10.1002/elan.200703944 1. Introduction Determinationofspecificnucleicacidsequencesinbiolog- ical and environmental samples can lead to early diagnosis of inherited human diseases also identification and detec- tionofpathogens[1].Detectionofinfectiousdiseasespecies and genetic mutation at the molecular level provides the possibilityofperformingreliablediagnosisevenbeforeany symptoms of the disease appear [2]. Molecular diagnostics basedonanalysisofthegenomicsequenceshaveoffereda highlysensitiveandquantitativemethodforthedetectionof infectiousdiseasepathogensandpolymorphisms[3–4].As new genes are discovered due to the developing of the Human Genome Project and seem to be important in primarypatientandprenataldiagnosis,moleculardiagnosis laboratories have been challenged to develop rapid and cheap detection of specific DNA sequences and their mutationsinindividuals. Conventional methods for the determination of specific DNA sequences are generally based on electrophoresis separationswherebyDNAisseparatedaccordingtolength and stained with ethidium bromide. Sequence specificity can be achieved by transferring the separated DNA to a membrane and hybridizing with a radioactively labeled probe. This method is very sensitive but complex handling with hazardous radioactive labels are necessary. The other methods which are achieved by labeling the probe with biotin[5–6]digoxigenin[7]orfluorescentdyes[8]inorder toavoidtheuseofhazardousradioactivelabelsarealsonot suitable for the routine analysis because of the long, expensive and complicated steps of these procedures. In comparison with these conventional methods; a new, as sensitive as these methods and sequence specific detection ofnucleicacidhybridizationbyusingelectrochemicalDNA biosensorshasrecentlybeenreported[9–14]. There have been intensive studies to apply modern voltammetric methods in nucleic acid research and DNA analysis. DNA biosensor technologies are currently under intenseinvestigationowingtotheirgreatpromiseforrapid and low-cost detection of specific DNA sequences in human,viralandbacterialnucleicacids[15–18]. ElectrochemicalDNAbiosensors,basedonelectrochem- icaltransductionofhybridizationevents,havegreatpromise for the task of pharmaceutical, clinical, environmental and forensicapplications.Suchdevicescouplethehighspecific- ityofDNAhybridizationreactionswiththehighsensitivity, lowcostandportabilityofelectrochemicaltransducers.The electrochemical biosensors can be assembled to a mini- aturizedarraywhichistermedDNAchips[19–21]. Recently there has been a considerable interest in developing a DNA electrochemical biosensor for safe and rapid applications in genetic analysis as alternative to conventionalmethods.Thedevelopmentofelectrochemical genosensorsholdsgreatpromisefordetectinginheritedand 1875 Electroanalysis 19, 2007,No.18,1875–1882 # 2007 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim