title: Amperometric sensing of HIF1α expressed in cancer cells and the effect of hypoxic mimicking agents.
abstract: Hypoxia inducible factor 1 alpha (HIF1α) overexpression was detected in cancerous cells using an amperometric immunosensor with a nano-bioconjugate. The sensor probe was fabricated by covalently immobilizing the antibody (anti-HIF1α) onto a composite layer of functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA) formed on a layer of gold nanoparticles (AuNPs). A nano-bioconjugate with hydrazine and a secondary antibody of HIF1α (sec-Ab2) attached on AuNPs reveals the immunoreaction at the sensor probe through the catalytic reduction of H2O2 by hydrazine at -0.35V vs. Ag/AgCl. Morphology and performance of the sensor probe were characterized using FE-SEM, XPS, EIS, and cyclic voltammetry. The calibration plot at optimized experimental conditions shows a dynamic range of 25-350pM/mL with a detection limit of 5.35±0.02pM/mL. The reliability of the sensor was evaluated using non-cancerous Vero and cancerous MCF-7 cell lysates, where the HIF1α expression was compared with three cancerous cell lines MCF-7, PC-3, and A549. Furthermore, the sensor probe confirms the stable expression of HIF1α in the A549 lung cancer cells when exposing them to hypoxic mimicking agents Co, Ni, and Mn ions. Of these, Co ions show the highest stabilization effect on HIF1α followed by Ni and Mn ions, respectively.
authors.affiliation: Department of Chemistry and Institute of BioPhysio Sensor Technology (IBST), Pusan National University, Busan 46241, South Korea.
authors.forename: Khalil K
authors.initials: KK
authors.lastname: Hussain
authors.affiliation: Department of Chemistry and Institute of BioPhysio Sensor Technology (IBST), Pusan National University, Busan 46241, South Korea.
authors.forename: N G
authors.initials: NG
authors.lastname: Gurudatt
authors.affiliation: Department of Chemistry and Institute of BioPhysio Sensor Technology (IBST), Pusan National University, Busan 46241, South Korea.
authors.forename: Tanveer Ahmad
authors.initials: TA
authors.lastname: Mir
authors.affiliation: Department of Chemistry and Institute of BioPhysio Sensor Technology (IBST), Pusan National University, Busan 46241, South Korea. Electronic address: ybshim@pusan.ac.kr.
authors.forename: Yoon-Bo
authors.initials: YB
authors.lastname: Shim
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry*; D001565:Benzoates / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods; D045744:Cell Line, Tumor; D055664:Electrochemical Techniques / Q000379:methods*; D006046:Gold / Q000737:chemistry; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis*; D007118:Immunoassay / Q000379:methods; D053768:Metal Nanoparticles / Q000737:chemistry; D009369:Neoplasms / Q000473:pathology*; D011108:Polymers / Q000737:chemistry*; D015203:Reproducibility of Results; D013876:Thiophenes / Q000737:chemistry*
chemical_list: D055910:Antibodies, Immobilized; D001565:Benzoates; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011108:Polymers; D013876:Thiophenes; C066730:polythiophene; D006046:Gold
keywords: Cancer cells; Conductive polymer; HIF1α; Hypoxia; Immunosensor