Fluorescence polarization assays: Another mainstream in detection of biohazards

Abstract

Infectious pathogens such as virus and drug-resistant bacteria are widespread, incurring high incidence, mortality rates, and significant socioeconomic burdens. Standard diagnostic methods such as bacterial culture, nucleic acid testing (NAT), and immunoassays have been used for containment. These methods, however, face technical challenges. Procedures are complicated, time/labor-intensive, and depend on bulky, expensive instruments. NAT systems based on quantitative polymerase chain reaction (qPCR) and clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) use fluorescence readouts, relying on costly fluorophore (F)-quencher (Q) reporter probes. In addition, the fluorescence readout is susceptible to external noises, affecting the sensitivity and reliability of the analysis.

Attributed to an underlying principle in signal readout, a fluorescence polarization (FP) system outperforms the fluorescent ones. It interrogates the polarization of emitted light from Fs upon excitation with linearly polarized light. Fs’ rotational behaviors that mainly rely on their molecular weights govern the degree of polarization of emitted light, resulting in corresponding FP signal change. Thus, FP system works with a simpler, cost-effective Q-free reporter probe. In addition, it provides robust signals with ratiometric nature of the readout.

This review discusses diagnostic systems for biohazards that leverage FP readout and novel molecular/immuno-assays. We will explore the assay designs, principles, and practical features of these systems, while addressing current challenges. A critical discussion will suggest future directions for further advancements, aiming to make FP-based diagnostic systems a standard tool for clinical care.