title: Ultrasensitive non enzymatic multiple immunosensor for tumor markers detection by coupling DNA hybridization chain reaction with intercalated molecules.
abstract: In this study, we tried coupling the small signal molecules that could intercalate into DNA double helix with hybridization chain reaction (HCR) technique to fabricate a multiple immunosensor. Doxorubicin hydrochloride (DXH) and methylene blue (MB) were used as signal molecules and alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were selected as model biomarkers. The immunosensor mainly consists of three parts as follows: First, two different primary antibodies (Ab1) immobilized on the surface of gold nanoparticles (Au NPs); Second, secondary antibodies (Ab2) conjugated with DNA primer; Third, long DNA concatemers from HCR were used as a carrier to intercalate amounts of signal molecules (DXH or MB). A sandwich immunocomplex was formed among primary antibodies, target biomarkers and secondary antibodies conjugated with DNA primer via specific recognition reaction. Afterwards, DNA concatemers intercalating amounts of DXH or MB were linked to DNA primer via DNA hybridization. Square wave voltammetry (SWV) was employed to record the response signals from electroactive molecules DXH and MB, and two distinguishable signals were obtained, which peak potentials were at about -0.30V (corresponding to MB) and -0.70V (corresponding to DXH, both vs SCE), respectively. The signal intensities of MB and DXH were linearly related to the logarithm of biomarkers concentration in the range of 0.05pgmL-1-25ngmL-1, and the limit of detection were 0.03pgmL-1for CEA and 0.02pgmL-1for AFP (at S/N=3), respectively. Furthermore, the immunosensor exhibited a sensitive electrochemical response to biomarkers in human serum samples and the results obtained were in accordance with reference method, indicating the immunosensor can be applied to real sample analysis in clinic diagnosis.
authors.affiliation: College of Chemistry and Materials Science, the Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chem-Biosensing, Anhui Normal University, Wuhu 241000, People's Republic of China.
authors.forename: Jinjin
authors.initials: J
authors.lastname: Guo
authors.affiliation: College of Chemistry and Materials Science, the Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chem-Biosensing, Anhui Normal University, Wuhu 241000, People's Republic of China.
authors.forename: Junchun
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Chemistry and Materials Science, the Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chem-Biosensing, Anhui Normal University, Wuhu 241000, People's Republic of China.
authors.forename: Junjun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: College of Chemistry and Materials Science, the Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chem-Biosensing, Anhui Normal University, Wuhu 241000, People's Republic of China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Zhang
authors.affiliation: College of Chemistry and Materials Science, the Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chem-Biosensing, Anhui Normal University, Wuhu 241000, People's Republic of China. Electronic address: zhyz65@mail.ahnu.edu.cn.
authors.forename: Yuzhong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000032:analysis / Q000097:blood; D015374:Biosensing Techniques / Q000379:methods*; D002272:Carcinoembryonic Antigen / Q000032:analysis / Q000097:blood*; D017931:DNA Primers / Q000737:chemistry; D004317:Doxorubicin / Q000737:chemistry; D055664:Electrochemical Techniques / Q000379:methods; D006046:Gold / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007364:Intercalating Agents / Q000737:chemistry*; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D008751:Methylene Blue / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D000509:alpha-Fetoproteins / Q000032:analysis*
chemical_list: D055910:Antibodies, Immobilized; D014408:Biomarkers, Tumor; D002272:Carcinoembryonic Antigen; D017931:DNA Primers; D007364:Intercalating Agents; D000509:alpha-Fetoproteins; D006046:Gold; D004317:Doxorubicin; D008751:Methylene Blue
keywords: Doxorubicin hydrochloride; Hybridization chain reaction; Methylene blue; Multiple immunosensor; Square wave voltammetry