A surface plasmon field-enhanced fluorescence reversible split aptamer biosensor

Abstract

Surface plasmon field-enhanced fluorescence is reported for the readout of a heterogeneous assay that utilizes low affinity split aptamer ligands. Weak affinity ligands that reversibly interact with target analytes hold potential for facile implementation in continuous monitoring biosensor systems. This functionality is not possible without the regeneration of more commonly used assays relying on high affinity ligands and end-point measurement. In fluorescence-based sensors, the use of low affinity ligands allows avoiding this step but it imposes a challenge associated with the weak optical response to the specific capture of the target analyte which is also often masked by a strong background. The coupling of fluorophore labels with a confined field of surface plasmons is reported for strong amplification of the fluorescence signal emitted from the sensor surface and its efficient discrimination from the background. This optical scheme is demonstrated for time-resolved analysis of chosen model analytes - adenoside and adenosine triphosphate - with a split aptamer that exhibits an equilibrium affinity binding constant between 0.73 and 1.35 mM. The developed biosensor enables rapid and specific discrimination of target analyte concentration changes from low μM to mM in buffer as well as in 10% serum.

Polyclonal Aptamers for Specific Fluorescence Labeling and Quantification of the Health Relevant Human Gut Bacterium Parabacteroides distasonis

“…Thus, bioaffinity per se has a pivotal role as a key construction and functionality element in the development of biosensors, which is mainly still realized by the use antibodies or antibody derivatives [15]. However, this role of affinity molecules can be taken over with success by aptamers [38,39].…”

ATP‐Responsive and ATP‐Fueled Self‐Assembling Systems and Materials

“…Here, the ATP-binding aptamer is split into two fragments, which can be joined together upon binding with ATP ( Figure 4d). The split aptamer was reported to have a K d of 350 × 10 −6 m, [70] which is about two orders of magnitude higher than those for the affinity interaction with the native (not split) aptamer in the bulk solution, K d ≈ 6 × 10 −6 m. Although the split aptamer has a weaker binding affinity with ATP, it critically complements conventional ATP-binding DNA-aptamer-based self-assembly systems. In this direction, Jayawickramarajah and co-workers for instance developed a synthetic transducer based on self-assembling DNA-small molecule chimeras that were able to convert a chosen biological input, ATP in this case, into the displacement of a protein inhibitor bound within cucurbit [7]uril (CB7; Figure 5c).…”

“…Here, the ATP‐binding aptamer is split into two fragments, which can be joined together upon binding with ATP (Figure 4d). The split aptamer was reported to have a K d of 350 × 10 −6 m , [ 70 ] which is about two orders of magnitude higher than those for the affinity interaction with the native (not split) aptamer in the bulk solution, K d ≈ 6 × 10 −6 m . Although the split aptamer has a weaker binding affinity with ATP, it critically complements conventional ATP‐binding DNA‐aptamer‐based self‐assembly systems.…”

Dual-Valve and Counter-Flow Surface Plasmon Resonance

“…Surface plasmon resonance (SPR) has been demonstrated as a technique of choice for real-time determinations of binding affinity and kinetics between biologically important molecules. − SPR has gained additional attention in the pharmaceutical industry, because it is viable for detecting binding of small molecules to purified proteins or receptors at cell membranes . Besides, SPR detection does not require the addition of an external or exogenous label to small molecules, which could significantly change the binding behavior.…”