title: Upconverting nanophosphors as reporters in a highly sensitive heterogeneous immunoassay for cardiac troponin I.
abstract: Photon upconverting nanophosphors (UCNPs) have a unique capability to produce anti-Stokes emission at visible wavelengths via sequential multiphoton absorption upon infrared excitation. Since the anti-Stokes emission can be easily spectrally resolved from the Stokes' shifted autofluorescence, the upconversion luminescence (UCL) is a highly attractive reporter technology for optical biosensors and biomolecular binding assays - potentially enabling unprecedented sensitivity in separation-based solid-phase immunoassays. UCL technology has not previously been applied in sensitive detection of cardiac troponin I (cTnI), which requires highly sensitive detection to enable accurate and timely diagnosis of myocardial infarction. We have developed an UCL-based immunoassay for cTnI using NaYF4: Yb(3+), Er(3+) UCNPs as reporters. Biotinylated anti-cTnI monoclonal antibody (Mab) and Fab fragment immobilized to streptavidin-coated wells were used to capture cTnI. Captured cTnI was detected from dry well surface after a 15 min incubation with poly(acrylic acid) coated UCNPs conjugated to second anti-cTnI Mab. UCL was measured with a dedicated UCL microplate reader. The UCL-based immunoassay allowed sensitive detection of cTnI. The limit of detection was 3.14 ng L(-1). The calibration curve was linear up to cTnI concentration 50,000 ng L(-1). Plasma recoveries of added cTnI were 92-117%. Obtained cTnI concentrations from five normal plasma samples were 4.13-10.7 ng L(-1) (median 5.06 ng L(-1)). There is yet significant potential for even further improved limit of detection by reducing non-specifically bound fraction of the Mab-conjugated UCNPs. The assay background with zero calibrator was over 40-fold compared to the background obtained from wells where the reporter conjugate had been excluded.
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6 A, 20520 Turku, Finland. Electronic address: nkelon@utu.fi.
authors.forename: Nina
authors.initials: N
authors.lastname: Sirkka
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6 A, 20520 Turku, Finland.
authors.forename: Annika
authors.initials: A
authors.lastname: Lyytikäinen
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6 A, 20520 Turku, Finland.
authors.forename: Tanja
authors.initials: T
authors.lastname: Savukoski
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6 A, 20520 Turku, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D015374:Biosensing Techniques; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D009203:Myocardial Infarction / Q000175:diagnosis; D036103:Nanotechnology; D012680:Sensitivity and Specificity; D019210:Troponin I / Q000097:blood*
chemical_list: D019210:Troponin I
keywords: Cardiac troponin I; Immunoassay; Upconversion luminescence; Upconverting nanophosphors