FRET-Based Aptasensor for the Selective and Sensitive Detection of Lysozyme

Abstract

Lysozyme is a conserved antimicrobial enzyme and has been cited for its role in immune modulation. Increase in lysozyme concentration in body fluids is also regarded as an early warning of some diseases such as Alzheimer’s, sarcoidosis, Crohn’s disease, and breast cancer. Therefore, a method for a sensitive and selective detection of lysozyme can benefit many different areas of research. In this regard, several aptamers that are specific to lysozyme have been developed, but there is still a lack of a detection method that is sensitive, specific, and quantitative. In this work, we demonstrated a single-molecule fluorescence resonance energy transfer (smFRET)-based detection of lysozyme using an aptamer sensor (also called aptasensor) in which the binding of lysozyme triggers its conformational switch from a low-FRET to high-FRET state. Using this strategy, we demonstrated that the aptasensor is sensitive down to 2.3 picomoles (30 nM) of lysozyme with a dynamic range extending to ~2 µM and has little to no interference from similar biomolecules. The smFRET approach used here requires a dramatically small amount of aptasensor (~3000-fold less as compared to typical bulk fluorescence methods), and it is cost effective compared to enzymatic and antibody-based approaches. Additionally, the aptasensor can be readily regenerated in situ via a process called toehold mediated strand displacement (TMSD). The FRET-based aptasensing of lysozyme that we developed here could be implemented to detect other protein biomarkers by incorporating protein-specific aptamers without the need for changing fluorophore-labeled DNA strands.

Wireless Network of Silver Film Lysozyme Aptasensor Based on Fractal Measurement

“…Based on a nonradiative energy transfer phenomenon between two fluorophores in short distances (<10 nm), i.e., FRET effect, the FRET aptasensor uses aptamers as probes of high affinity and specificity in the detection of lysozyme. Because of the advantages of fast response, noncontamination, and high specificity, FRET aptasensor has been extensively exploited in lysozyme detection studies [9][10][11][12][13][14]. To achieve sensitivity improvement, FRET sensors based on various materials, structures, and probes have been reported [15][16][17][18][19].…”

Recent Advances in Aptamer Sensors

“…The aptamers themselves can be modified with a fluorophore and quencher materials at both ends without affecting their binding affinity toward the targets, so a FRET aptasensor strategy can be easily constructed in the presence of targets inducing fluorescence quenching [ 156 , 157 , 158 ]. Kang et al provided a dual-mode readout DNA biosensor combining both an aptamer and a DNAzyme to assay ATP with two different mechanisms, using fluorometric and colorimetric signals ( Figure 8 D), respectively.…”

Aptamer-Based Fluorescent Biosensor for the Rapid and Sensitive Detection of Allergens in Food Matrices

“…Lys, as an allergen in egg and a biomarker of many diseases, its detection and quantification are of great significance in clinical diagnosis [167]. Sapkota et al developed an aptamer sensor based on single-molecule FRET (smFRET) for the detection of Lys (Figure 5a) [168]. One of the arms has a blocking chain (B1), which is extended by 15 nucleotides to partially hybridize to the aptamer.…”

“…aptamer sensor based on single-molecule FRET (smFRET) for the detection of Lys (Figure 5a) [168]. One of the arms has a blocking chain (B1), which is extended by 15 nucleotides to partially hybridize to the aptamer.…”