title: An impedance immunosensor based on low-cost microelectrodes and specific monoclonal antibodies for rapid detection of avian influenza virus H5N1 in chicken swabs.
abstract: Early screening of suspected cases is the key to control the spread of avian influenza (AI) H5N1. In our previous studies, an impedance biosensor with an interdigitated array microelectrode based biochip was developed and validated with pure AI H5 virus, but had limitations in cost and reliability of the biochip, specificity of the antibody against Asian in-field H5N1 virus and detection of H5N1 virus in real samples. The purpose of this study is to develop a low-cost impedance immunosensor for rapid detection of Asian in-field AI H5N1 virus in chicken swabs within 1h and validate it with the H5N1 virus. Specific monoclonal antibodies against AI H5N1 virus were developed by fusion of mouse myeloma cells with spleen cells isolated from an H5N1-virus-immunized mouse. Dot-ELISA analysis demonstrated that the developed antibodies had good affinity and specificity with the H5N1 virus. The microelectrodes were redesigned with compact size, fabricated using an improved wet-etching micro-fabrication process with a higher qualified production rate of 70-80%, and modified with the antibodies by the Protein A method. Equivalent circuit analysis indicated that electron transfer resistor was effective with the increase in impedance after capturing of the H5N1 viruses. Linear relationship between impedance change and logarithmic value of H5N1 virus at the concentrations from 2(-1) to 2(4) HAU/50 μl was found and the lower limit of detection was 2(-1) HAU/50 μl. No obvious interferences from non-target viruses such as H6N2, H9N2, Newcastle disease virus, and infectious bronchitis virus were found. Chicken swab tests showed that the impedance immunosensor had a comparable accuracy with real-time RT-PCR compared to viral isolation.
authors.affiliation: MOA Key Laboratory of Agricultural Information Acquisition Technology (Beijing), China Agricultural University, Beijing 100083,China. Electronic address: jianhan@cau.edu.cn.
authors.forename: Jianhan
authors.initials: J
authors.lastname: Lin
authors.affiliation: Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Ronghui
authors.initials: R
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, China.
authors.forename: Peirong
authors.initials: P
authors.lastname: Jiao
authors.affiliation: State Key Lab of Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Science, Beijing 100083, China.
authors.forename: Yuntao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liao
authors.affiliation: State Key Lab of Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Science, Beijing 100083, China.
authors.forename: Yude
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Modern Precision Agriculture System Integration Research Key Laboratory of Ministry of Education, China Agricultural University, Beijing 100083,China.
authors.forename: Maohua
authors.initials: M
authors.lastname: Wang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology; D015374:Biosensing Techniques; D002645:Chickens / Q000276:immunology; D017097:Electric Impedance; D004797:Enzyme-Linked Immunosorbent Assay; D053124:Influenza A Virus, H5N1 Subtype / Q000302:isolation & purification* / Q000472:pathogenicity; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology*; D051379:Mice; D008839:Microelectrodes
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral
keywords: Asian in-field H5N1 virus; Avian influenza detection; Impedance immunosensor; Interdigitated array microelectrode