Background

The overuse of antibiotics in aquaculture is a global phenomenon. In the 1950s, it was discovered that low doses of antibiotics could significantly increase meat production, and this unexpected bonus was quickly industrialized, making antibiotics an official feed additive.

Antibiotics enter the human body through farm-raised livestock and are absorbed via the food chain. Long-term consumption of antibiotic-contaminated foods may lead to health issues such as allergies, kidney toxicity, and hearing impairment^[1][2]. Furthermore, these medications disrupt the balance of gut microbiota, significantly increasing risks of intestinal disorders including pseudomembranous colitis and colorectal cancer^[3].

Adaptor molecules exhibit advantages such as high affinity, specificity, easy chemical modification, and low immunogenicity. Adaptor-based biosensors demonstrate rapid and sensitive characteristics in small molecule detection^[4]. However, research on antibiotic-specific adaptor screening and sensor development remains limited. Existing reported sensors face issues like insufficient sensitivity or reliance on complex signal amplification strategies.

Due to the small molecular size and subtle structural differences of antibiotics, the sequence space capable of forming high-affinity binders with antibiotics is narrow. As a result, the number of effective aptamer sequences is significantly fewer compared to those for toxins and protein targets. Moreover, in complex biological matrices, lipids, proteins, and multivalent cations can non-specifically adsorb aptamers, leading to false positives/negatives. The signal-to-noise ratio of aptamer-fluorescence systems in real samples is often lower than 3, necessitating the introduction of additional signal amplification techniques. This complicates the operation and undermines the advantage of rapid detection^[5].

Purpose

Our team, HunanU, was committed to the research and development of a new type of antibiotic biosensor. Through the combination of identification module and signal amplification module, we could realize the detection of antibiotics in colleges and universities, aiming to provide a sensitive and feasible technical solution for antibiotic monitoring.

Principles and Methods

In this project, we built a fluorescent sensor based on aptamer to realize the rapid, sensitive and highly specific detection of antibiotics, and designed a simple and feasible signal amplification technology route.

The designed antibiotic detection system consists of a target recognition module and a signal amplification module. The recognition system employs aptamer-based biomolecular probes called ligand-binding oligo (LBO). It binds to antibiotic molecules in the solution system, causing structural changes and releasing a short strand called short release oligo (SRO)^[5].

In the signal amplification module, we devised two amplification strategies to accommodate different application scenarios: T7 in vitro transcription–based amplification and CRISPR-Cas12a–mediated amplification.

In the T7 system, the released short strand hybridizes with a downstream T7 promoter, initiating transcription. The transcription product is an aptamer RNA that enhances the fluorescence of a specific dye, enabling the quantification of antibiotic residues through fluorescence intensity. In the CRISPR-Cas12a system, the short strand hybridizes with a downstream crRNA, activating the trans-cleavage activity of the Cas12a protein. This activity cleaves reporter molecules in the system, thereby amplifying the detection signal.

In the project, we selected ampicillin for the preliminary experiments, while vancomycin was used as the target in the formal experimental group. Ampicillin is widely used in animal husbandry to treat bacterial infections including those of the respiratory and intestinal tracts. Vancomycin is considered a last-resort antibiotic for humans. However, its illegal use in animal breeding could give rise to more serious antibiotic resistance among consumers^[6].

Beyond focusing on our specific target, we also aimed to develop a modular and tunable platform capable of detecting multiple antibiotics through the replacement of aptamers.

Point of View

One significant advantage of our system is its high flexibility and tunability. By adjusting the number of complementary nucleotides, we can modulate the ease of short-strand release, thereby controlling the system's sensitivity and enabling the detection of antibiotics across a wide concentration range.

Moreover, our design is highly modular: by simply replacing the aptamer sequence in LBO in the future, the platform can be readily adapted to detect a variety of targets beyond antibiotics, including metal ions, toxins, and biomacromolecules.

We have already realized target recognition and signal amplification via a two-step cascade reaction. Looking ahead, we are focused on enhancing detection convenience by developing an artificial cell that autonomously performs recognition and amplification upon sample addition, eliminating manual intervention. This system will be applied in detection kits to extend the technology’s reach beyond the lab. Together with public, community, and scientific partners, we aim to employ synthetic biology to safeguard food security—this is our vision for the future.