Luminescent lanthanides – Evolution of eporter systems for immunoassays

Tero Soukka

Lanthanides among other rare earths are considered strategic materials due to their indispensable role in many high-tech electronic, optical and magnetic applications (e.g. green technologies) and strong domination of China over their supply. In biotechnology the trivalent lanthanide ions have been famous for their unique narrow-banded, long-lifetime luminescence characteristics, which have enabled sensitive bioanalytical assays utilizing various lanthanide reporters and time-gated fluorescence detection already for four decades [1]. The full potential of luminescent lanthanides as reporters, however, goes nowadays far beyond – with the ultimate dream to reach of the holy grail of luminescence, the molecular upconversion in aqueous environment [2].

The use of microsecond time-resolution and pulsed excitation in fluorometric measurement of long-lifetime lanthanide luminescence dates back to late 1970’s [3] and the development of dissociation enhanced lanthanide fluoroimmunoassay technology [4]. Thereafter, the development of intrinsically luminescent lanthanide chelates and highly stable cryptates facilitated the use of time-gated detection in versatile fluorescence-based bioanalytical applications, such as fluorescence resonance energy-transfer with additional advantages. The quest for ever lower limit of detection resulted also adaptation of pure inorganic lanthanide nanocrystals as well as polystyrene nanoparticles dyed with tens of thousands of lanthanides complexes as reporters to demonstrate assays, where the detectability of the reporters can be excluded from the sensitivity limiting factors [5, 6]. The practical sensitivity limits for the applications, however, are defined by the requirement of high-intensity ultraviolet excitation together with time-gated detection, the non-specifically bound fraction of the nanoparticle bioconjugates, and the detectable long-lifetime background photoluminescence from many non-optimized solid-phase materials. 

Lanthanide-based photon upconversion is an extraordinary class of photoluminescence that was first introduced to the bioanalytical assays in the late 1990’s [7]. The lanthanide-doped inorganic upconverting nanoparticles (UCNPs) are able to produce visible upconversion luminescence, i.e. anti-Stokes emission under infrared excitation by sequential absorption of two or more photons as a result of the ladder-like long-lived intermediate energy-states. The photon upconversion is an extremely rare - practically nonexistent - phenomenon in the nature and, due to the large intrinsic anti-Stokes shift, it allows total elimination of the matrix and solid-phase auto-fluorescence and scattered excitation by simple spectral separation – without temporal resolution. The availability of low-cost, high-power infrared laser diode light sources developed for fiber-optic telecommunication also renders this technology immediately applicable [8]. Thus, the UCNPs are an attractive reporter technology for highly sensitive in vitro diagnostic assays with no limitations due to solid-phase material background photoluminescence.

The high detectability of the reporter is a necessity for ultrasensitive immunoassays, but in real applications the other factors, such as the specificity and binding affinity the antibodies, the compatibility of the assay with clinically relevant matrices, the usability and expenses of the test platform and total assay time achievable, are equally important. Even the UCNPs as reporters can provide a significant advantage on the detectability, as a downside their larger dimensions compared to molecular reporters affect the binding kinetics and the total assay time required, bring challenges with steric hindrance in molecular recognition, and enhance the combined effect of the weak interactions causing non-specific binding. These drawbacks, however, can be largely – but not quite completely – circumvented with optimized test designs and surface chemistry of the nanoparticles. Optimization has resulted demonstration of a supersensitive upconversion luminescence immunoassay for cardiac Troponin I on a conventional microtitration plate platform [9].

References:
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[9] Raiko, K. et al. (2021) Supersensitive photon upconversion based immunoassay for detection of cardiac troponin I in human plasma. Clin Chim Acta 523: 380-385. doi: 10.1016/j.cca.2021.10.023.