title: Cell-Free Protein Synthesis Approach to Biosensing hTRβ-Specific Endocrine Disruptors.
abstract: Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor β in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.
authors.affiliation: Department of Chemical Engineering, Brigham Young University , Provo, Utah 84602, United States.
authors.forename: Amin S M
authors.identifier: 0000-0002-6550-8783
authors.initials: AS
authors.lastname: Salehi
authors.affiliation: Department of Chemical and Biomolecular Engineering, Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Miriam J
authors.initials: MJ
authors.lastname: Shakalli Tang
authors.affiliation: Department of Chemical Engineering, Brigham Young University , Provo, Utah 84602, United States.
authors.forename: Mark T
authors.initials: MT
authors.lastname: Smith
authors.affiliation: Department of Chemical Engineering, Brigham Young University , Provo, Utah 84602, United States.
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Hunt
authors.affiliation: Department of Chemical and Biomolecular Engineering, Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Law
authors.affiliation: Department of Chemical and Biomolecular Engineering, Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: David W
authors.identifier: 0000-0003-0810-8836
authors.initials: DW
authors.lastname: Wood
authors.affiliation: Department of Chemical Engineering, Brigham Young University , Provo, Utah 84602, United States.
authors.forename: Bradley C
authors.identifier: 0000-0003-4438-183X
authors.initials: BC
authors.lastname: Bundy
mesh_terms: D015374:Biosensing Techniques; D004353:Drug Evaluation, Preclinical; D052244:Endocrine Disruptors / Q000032:analysis*; D006801:Humans; D008024:Ligands; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D037042:Thyroid Hormone Receptors beta / Q000737:chemistry*
chemical_list: D052244:Endocrine Disruptors; D008024:Ligands; D011993:Recombinant Fusion Proteins; D037042:Thyroid Hormone Receptors beta