Novel sandwich-type electrochemiluminescence aptasensor based on luminol functionalized aptamer as signal probe for kanamycin detection.

A novel sandwich electrochemiluminescence (ECL) aptasensor was developed for highly sensitive detection of kanamycin using luminol-functionalized aptamer as a signal probe. The aptasensor used polyethyleneimine (PAMAM), molybdenum disulfide, and multi-walled carbon nanotubes as the substrate, which provided enough binding sites for aptamer1 (the aptamer which modified NH2) coupling. We found that kanamycin could be detected using the aptamer1 containing the same base sequence as aptamer2 (the aptamer which modified SH) on the electrode self-assembly. In addition, PAMAM nanocomposites can be used to effectively improve the ECL intensity by loading a high volume of luminol molecules and silver nanoparticles. In the presence of kanamycin, the sandwiched aptasensor was formed between aptamer1 and the probe of aptamer2 connecting silver nanoparticles, luminol, and PAMAM, resulting in a proportional increase of ECL intensity. Since the significantly enhanced loading of luminol by PAMAM accelerated the electron transfer, the sensitive aptasensor exhibited a wide linear range of detection from 1 × 10-3 to 1 × 103 ng/mL and a low detection limit of 0.21 pg/mL (S/N) for kanamycin. The fabricated aptasensor was successfully applied in quantitative analysis of kanamycin in milk samples.

Non-immobilized GO-SELEX of aptamers for label-free detection of thiamethoxam in vegetables.

Due to the massive use of thiamethoxam (TMX) pesticide and the accumulated potential hazards exposure, the detection of TMX is of great significance to food and ecological safety. In this study, aptamers with affinity for TMX were obtained through graphene oxide assisted systematic evolution of ligands by exponential enrichment (GO-SELEX). After 9 rounds of positive and counter selection, 5 candidate sequences were obtained, among which seq.20 had the highest affinity for TMX, and its dissociation constant (Kd) was 210.47 ± 79.37 nM. Then, the aptamer was further truncated based on structural analysis. The truncated aptamers (seq.20-1, seq.20-2) exhibited higher affinity (Kd = 118.34 ± 13.85 nM, Kd = 123.35 ± 29.80 nM), which seq.20-2 had only 37 bases. Furthermore, circular dichroism spectroscopy showed that TMX induced the conformation of aptamer from B-form structure to hairpin structure, and then formed a stable TMX-ssDNA complex. Finally, the truncated aptamer (seq.20-2) and the original aptamer (seq.20) were used as recognition elements to construct colorimetric aptasensors based on gold nanoparticles for the detection of TMX. It was found that the sensitivity of the former (LOD = 1.67 ± 0.12 nM, S/N = 3) was better than that of the latter (LOD = 3.33 ± 0.23 nM, S/N = 3). Feasibility of truncated aptamer as recognition element in the detection of TMX in vegetable samples was preliminarily verified.

Broad-specificity time-resolved fluorescent immunochromatographic strip for simultaneous detection of various organophosphorus pesticides based on indirect probe strategy.

The massive use of organophosphorus pesticides (OPs) poses a great threat to food safety, human health and environmental protection. As there are many kinds of pesticides, their detection is facing a severe challenge. The simultaneous detection of multiple organophosphorus pesticides in one test is a problem to be solved at present. In this paper, a time-resolved fluorescent immunochromatographic (TRFIA) strip is prepared by using broad-specificity antibodies (Abs) of OPs as the recognition element. Abs were connected to europium oxide latex microspheres using sheep anti-mouse antibodies (SaMIgG) to form an indirect probe. This strategy could effectively realize signal amplification, and could save the amount and protect the activity of Abs. After the detection, the color change of the test line (T-line) was observed to make qualitative judgment under UV-light (365 nm). Then, the images of the positive sample were analyzed by using ImageJ to complete the quantitative detection. Under optimal construction and operating conditions, the limit of detection of the strip could reach 0.53 ng g-1. And the TRFIA strip performed well in the additive test of vegetable samples. It is inexpensive to prepare, convenient to carry, and easy to operate. More importantly, it improves the detection efficiency and meets the needs of rapid field testing of a large number of samples.