Upconverting nanophosphors as reporters in a highly sensitive heterogeneous immunoassay for cardiac troponin I

• Detection of attomole analyte quantity in microwell using upconversion luminescence.
• Upconverting nanophosphors enable detection of femtomolar analyte concentration.
• Excellent analyte recovery from plasma in upconversion luminescence based immunoassay.

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: Yb3+, Er3+ 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.

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