title: Electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere label for detection of Vibrio cholerae.
abstract: Vibrio cholerae is a Gram-negative bacterium that causes cholera, a diarrheal disease. Cholera is widespread in poor, under-developed or disaster-hit countries that have poor water sanitation. Hence, a rapid detection method for V. cholerae in the field under these resource-limited settings is required. In this paper, we describe the development of an electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere (AuNPs-PSA) reporter label. The reporter label mixture was prepared by lyophilization of AuNPs-PSA-avidin conjugate with different types of stabilizers. The best stabilizer was 5% sorbitol, which was able to preserve the dried conjugate for up to 30 days. Three methods of DNA hybridization were compared and the one-step sandwich hybridization method was chosen as it was fastest and highly specific. The performance of the assay using the lyophilized reagents was comparable to the wet form for detection of 1aM to 1fM of linear target DNA. The assay was highly specific for V. cholerae, with a detection limit of 1fM of PCR products. The ability of the sensor is to detect LAMP products as low as 50ngµl(-1). The novel lyophilized AuNPs-PSA-avidin reporter label with electrochemical genosensor detection could facilitate the rapid on-site detection of V. cholerae.
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Semeling, Kedah, Malaysia.
authors.forename: Pei Sheng
authors.initials: PS
authors.lastname: Liew
authors.affiliation: Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkhuntien-chaitalay Road, Thakam, Bangkok 10150, Thailand. Electronic address: l_benjap@yahoo.com.
authors.forename: Benchaporn
authors.initials: B
authors.lastname: Lertanantawong
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Semeling, Kedah, Malaysia.
authors.forename: Su Yin
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Semeling, Kedah, Malaysia.
authors.forename: Ravichandran
authors.initials: R
authors.lastname: Manickam
authors.affiliation: School of Chemical Sciences and Food Technology, Faculty of Science and Technology, University Kebangsaan Malaysia, Bangi, Selangor 43600, Malaysia.
authors.forename: Yook Heng
authors.initials: YH
authors.lastname: Lee
authors.affiliation: Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkhuntien-chaitalay Road, Thakam, Bangkok 10150, Thailand; School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkhuntien-chaitalay Road, Thakam, Bangkok 10150, Thailand.
authors.forename: Werasak
authors.initials: W
authors.lastname: Surareungchai
mesh_terms: D001681:Biological Assay; D015374:Biosensing Techniques / Q000379:methods*; D002771:Cholera / Q000175:diagnosis* / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis*; D055664:Electrochemical Techniques / Q000379:methods*; D005612:Freeze Drying; D006046:Gold / Q000737:chemistry*; D006801:Humans; D007840:Latex; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry*; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D014734:Vibrio cholerae / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D007840:Latex; D006046:Gold
keywords: Cholera; Genosensor; Gold nanoparticles; LAMP; Layer-by-layer modified latex; Lyophilization; PCR products; Vibrio cholerae