Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection

Abstract

Aptamer-based lateral flow assays (LFAs) are an emerging field of aptamer applications due to numerous potential applications. When compared to antibodies, potential advantages like cost effectiveness or lower batch to batch variations are evident. The development of LFAs for small molecules, however, is still challenging due to several reasons, primarily linked to target size and accessible interaction sites. In small molecule analysis, however, aptamers in many cases are preferable since immunogenicity is not required and they may exhibit even higher target selectivity. We report the first cross-recognition of a small molecule (ampicillin) and a protein (C-reactive protein), predicted by in-silico analysis, then experimentally confirmed - using two different aptamers. These features can be exploited for developing an aptamer-based LFA for label-free ampicillin detection, functioning also for analysis in milk extract. Most importantly, the principal setup denotes a novel, transferable and versatile general approach for detection of small molecules using competitive LFAs, unlikely to be generally realized by aptamer-DNA-binding otherwise.

Worldwide aflatoxin contamination of agricultural products and foods: From occurrence to control

“…When a sample is dropped on the sample pad, it moves across the strip via capillary force to react with the immune‐reagents immobilized on the membrane and forms a readable complex (Koczula & Gallotta, 2016). There are direct and indirect LFIAs formats, however, the indirect format is the approach used for the detection of small molecules like aflatoxins (Kaiser et al., 2018).…”

Multiplex Lateral Flow Immunoassay: An Overview of Strategies towards High-throughput Point-of-Need Testing

“…Aptamers are produced in vitro, thus avoiding the use and sacrifice of animal hosts, lowering batch to batch variations, and allowing for the selection of aptamers in non-physiological conditions (that simulate conditions in which the aptamer will be applied). Immunogenicity is not required and the toxicity of the target compound does not hamper the obtainment of aptamers [37,38].…”

Development of nucleic acid aptamer-based lateral flow assays: A robust platform for cost-effective point-of-care diagnosis

“…This is mainly because of the difficulty in developing high-affinity and specific aptamers for small molecule recognition. Despite novel strategies such as the crosslink mediated reporter aggregation (Figure 11 ) and the cross-recognition aptamer-based detection 58 have shed light on this area, further investigation is still imperative for efficient small molecule detection. This is especially true given the growing interest in the small endogenous metabolites-based disease diagnosis.…”

“…Even so, immobilization of small molecules on membranes may lead to the conformational change of the molecule and results in the failure of aptamer-target recognition 57 . As a result, the binding capacity of the detection aptamer to the immobilized target molecules must be experimentally tested 58 . To solve this problem, in a pioneer study, Lars Kaiser and colleagues developed a cross-recognition aptamer-based competitive LFA for small molecule - ampicillin - detection 58 .…”

“…As a result, the binding capacity of the detection aptamer to the immobilized target molecules must be experimentally tested 58 . To solve this problem, in a pioneer study, Lars Kaiser and colleagues developed a cross-recognition aptamer-based competitive LFA for small molecule - ampicillin - detection 58 . Via an in silico analysis of the sequence homologies between ampicillin and C-reactive protein aptamers, the authors first identified an aptamer sequence displaying affinity to both ampicillin and C-reactive protein.…”