title: Protein quantification using controlled DNA melting transitions in bivalent probe assemblies.
abstract: Homogenous protein assays, despite the potential for mix-and-read workflows, have eluded widespread acceptance due to interferences in biological matrices and limited multiplexability. Here, we employ standard qPCR instrumentation for thermofluorimetric analysis of bivalent probe (TFAB) assemblies, allowing protein levels to be quantitatively translated into multiplexable DNA melting transitions within 30 min. As protein-bound bivalent probes are thermodynamically more stable than unbound probes, differential thermal analysis can remove background analytically, without physical separation. Using either antibody-oligonucleotides or aptamers as probes, TFAB is validated for protein quantification in buffer, human serum, and human plasma and for assaying hormone secretions from endocrine cells. The direct optical method exhibits superior scalability, allowing detection of only 1 amol of protein in microfluidic channels of 100 pL volume. Overall, we demonstrate TFAB as a robust and generalizable homogeneous protein assay with superior performance in biological matrices.
authors.affiliation: Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University , Auburn, Alabama 36849, United States.
authors.forename: Joonyul
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University , Auburn, Alabama 36849, United States.
authors.forename: Juan
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University , Auburn, Alabama 36849, United States.
authors.forename: Andresa B
authors.initials: AB
authors.lastname: Bezerra
authors.affiliation: Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University , Auburn, Alabama 36849, United States.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Holtan
authors.affiliation: Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University , Auburn, Alabama 36849, United States.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Brooks
authors.affiliation: Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University , Auburn, Alabama 36849, United States.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Easley
mesh_terms: D004247:DNA / Q000737:chemistry*; D055098:Endocrine Cells / Q000166:cytology; D005470:Fluorometry; D006801:Humans; D046210:Microfluidic Analytical Techniques; D015335:Molecular Probes / Q000737:chemistry*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D044366:Transition Temperature
chemical_list: D015335:Molecular Probes; D011506:Proteins; D004247:DNA