A novel fluorescent biosensor based on affinity-enhanced aptamer-peptide conjugate for sensitive detection of lead(II) in aquatic products.

Lead contamination is a major concern in food safety and, as such, many lead detection methods have been developed, especially aptamer-based biosensors. However, the sensitivity and environmental tolerance of these sensors require improvement. A combination of different types of recognition elements is an effective way to improve the detection sensitivity and environmental tolerance of biosensors. Here, we provide a novel recognition element, an aptamer-peptide conjugate (APC), to achieve enhanced affinity of Pb2+. The APC was synthesized from Pb2+ aptamers and peptides through clicking chemistry. The binding performance and environmental tolerance of APC with Pb2+ was studied through isothermal titration calorimetry (ITC); the binding constant (Ka) was 1.76*106 M-1, indicating that the APC's affinity was increased by 62.96% and 802.56% compared with the aptamers and peptides, respectively. Besides, APC demonstrated better anti-interference (K+) than aptamer and peptide. Through the molecular dynamics (MD) simulation, we found that more binding sites and stronger binding energy between APC with Pb2+are the reasons for higher affinity between APC with Pb2+. Finally, a carboxyfluorescein (FAM)-labeled APC fluorescent probe was synthesized and a fluorescent detection method for Pb2+ was established. The limit of detection of the FAM-APC probe was calculated to be 12.45 nM. This detection method was also applied to the swimming crab and showed great potential in real food matrix detection.

A Novel Aptamer-Imprinted Polymer-Based Electrochemical Biosensor for the Detection of Lead in Aquatic Products.

Lead contamination in aquatic products is one of the main hazard factors. The aptasensor is a promising detection method for lead ion (Pb(II)) because of its selectivity, but it is easily affected by pH. The combination of ion-imprinted polymers(IIP) with aptamers may improve their stability in different pH conditions. This paper developed a novel electrochemical biosensor for Pb(II) detection by using aptamer-imprinted polymer as a recognition element. The glassy carbon electrode was modified with gold nanoparticles and aptamers. After the aptamer was induced by Pb(II) to form a G-quadruplex conformation, a chitosan-graphene oxide was electrodeposited and cross-linked with glutaraldehyde to form an imprint layer, improving the stability of the biosensor. Under the optimal experimental conditions, the current signal change (∆I) showed a linear correlation of the content of Pb(II) in the range of 0.1-2.0 µg/mL with a detection limit of 0.0796 µg/mL (S/N = 3). The biosensor also exhibited high selectivity for the determination of Pb(II) in the presence of other interfering metal ion. At the same time, the stability of the imprinted layer made the sensor applicable to the detection environment with a pH of 6.4-8.0. Moreover, the sensor was successfully applied to the detection of Pb(II) in mantis shrimp.

Detection of Cadmium(II) in Aquatic Products Using a Rolling-Circle Amplification-Coupled Ratio Fluorescent Probe Based on an Aptamer-Peptide Conjugate.

The existing aptamers for cadmium (Cd2+), the common toxic heavy metal contaminant in food, cannot meet the requirements for detecting Cd2+ in rapid detection methods. In previous work, we found that coupling aptamer-peptide conjugates (APCs) with peptides and aptamers can provide a less disruptive method with a significantly improved affinity. Moreover, we found that the spatial conformation of aptamers and peptides is crucial for obtaining proper affinity in APC. Therefore, we describe a simple design strategy to obtain a series of APCs with different affinities by designing peptide orientations (N-terminal, C-terminal). The best affinity was found for APC(C1-N) with a binding constant (Ka) of 2.23 × 106 M-1, indicating that the APC(C1-N) affinity was significantly increased by 829.17% over aptamer. Finally, a rolling-circle amplification (RCA)-coupled ratio fluorescence-based biosensor for Cd2+ detection was established with a detection limit of 0.0036 nM, which has great potential for practical aquatic product detection.

Fluorescence Resonance Energy Transfer Aptasensor of Ochratoxin A Constructed Based on Gold Nanorods and DNA Tetrahedrons.

Ochratoxin A (OTA) contamination of corn has received significant attention due to the wide distribution and high toxicity of OTA. The maximum residue limit standard of OTA in corn has been established by the Chinese Government and other unions. Nanoparticle-based fluorescence resonance energy transfer (FRET) assays are promising methods for the sensitive and fast detection of OTA. However, satisfactory detection sensitivity is commonly achieved with complicated signal amplification processes or specific nanoparticle morphologies, which means that these assays are not conducive to fast detection. This study proposes a simple and novel strategy to improve the sensitivity of FRET aptasensors. In this strategy, a DNA tetrahedron was first used in gold nanorod-based FRET aptasensors. DNA tetrahedron-modified gold nanorods are used as fluorescent acceptors, and Cy5-modified complementary sequences of the OTA aptamer are used as fluorescent donors. The aptamers of OTA are embedded in the DNA tetrahedrons, and FRET occurs when the aptamers hybridize with the Cy5-modified complementary sequences. The aptamer-integrated DNA tetrahedron modified on the surface of gold nanorods acts as an anchor, thus avoiding the crowding and entanglement of aptamers. Due to the competitive combination between the OTA aptamers and complementary sequences, the greater the amount of OTA, the less the amount of Cy5-modified complementary sequences that bind with the aptamers and the less the amount of Cy5 that is quenched. Thus, the fluorescence intensity is positively related to the OTA concentration. In this study, in the concentration range of 0.01-10 ng/mL, the fluorescence intensity was found to be linearly related to the logarithmic concentration of OTA. The limit of detection was calculated to be 0.005 ng/mL. The specificity of the developed biosensor was demonstrated to be efficient. The accuracy and stability of the developed aptasensor were also tested, and the method exhibited good performance in real samples.