Terbium and Rhodamine as Labels in a Homogeneous Time-resolved Fluorometric Energy Transfer Assay of the β Subunit of Human Chorionic Gonadotropin in Serum

Abstract

Background: Fluorescence resonance energy transfer (FRET) is a powerful tool in analytical chemistry. The aim of the present work was to use FRET to design a homogeneous immunoassay. Methods: We used a highly fluorescent terbium (Tb3+) chelate (donor) and the organic fluorochrome rhodamine (acceptor) combined with time-resolved detection of the acceptor emission in homogeneous assay format for the measurement of the β subunit of human chorionic gonadotropin (βhCG) in serum. We used two antibodies labeled with Tb3+ and rhodamine, respectively, recognizing different epitopes on βhCG. The close proximity between the labels in the immunocomplex permitted energy transfer between the pulse-excited Tb3+ donor (decay time >1 ms) and the acceptor rhodamine (decay time of 3.0 ns). The prolonged emission of donor-excited acceptor (energy transfer) was measured after the short-lived background and acceptor emissions had decayed. The emission of donor-excited rhodamine was measured at a wavelength of where the emission of unbound donor is minimal. Results: The energy transfer signal was directly proportional to the βhCG concentration in the sample. The limit of detection was 0.43 μg/L, and the assay was linear up to 200 μg/L. Total assay imprecision in the range 10–185 μg/L was between 7.5% and 2.8%. Conclusions: Although less sensitive than heterogeneous, dissociation-enhanced europium-based separation assays, the presented assay format has advantages such as speed and simplicity, which make the assay format ideal for assays requiring a high throughput.

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

“…Alternatively, two antibodies with selectivity for different epitopes on the protein surface can be used in sandwich-type fluorescence immunoassay. Labeling of both the antibodies with either a FRET donor or acceptor would result in FRET fluorescence upon binding of both antibodies to the protein [ 71 ]. However, the labeling of biopolymers, e.g., proteins and antibodies, with either a FRET donor or acceptor can be cumbersome and expensive and could induce conformational and functional changes of the targets, resulting in aberrancy.…”

A Protein L -Based Immunodiagnostic Approach Utilizing Time-Resolved Förster Resonance Energy Transfer

“…Time-resolved FRET (TR-FRET) unites the properties of TRF and FRET, which is especially advantageous when analyzing biological samples. As TR-FRET -based methods induce relatively low background fluorescence, this technique has been widely applied in medical research and diagnostics [2] – [10] . Overall, TR-FRET -based applications offer a viable alternative for the conventional multistep diagnostic tests, such as enzyme-linked immunosorbent assay (ELISA).…”

Photon Upconversion in Homogeneous Fluorescence‐based Bioanalytical Assays

“…The light absorption can be avoided by using fluorescent probes with both excitation and emission wavelengths in the near‐infrared window (650–1100 nm), where biological materials are most transparent to light, but autofluorescence and scattered excitation light will still limit the assay sensitivity 68,69 . Efficient solution for the autofluorescence and scattered excitation light is provided by the time‐resolved fluorometry 70,71 and especially the application of long‐lifetime fluorescent lanthanide labels as donors in time‐resolved FRET (TR‐FRET) assays 66,72,73 . The need for UV excitation, however, has limited the use of TR‐FRET assays with strongly colored samples, such as whole blood, which efficiently absorbs both UV and visible light 74 .…”