Evolution of Interferon-Gamma Aptamer with Good Affinity and Analytical Utility by a Rational In Silico Base Mutagenesis Post-SELEX Strategy.

The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is conventionally an effective method to identify aptamers, which are oligonucleotide sequences with desired properties to recognize targets specifically and sensitively. However, there are some inherent limitations, e.g., the loss of potential high-affinity sequences during biased iterative PCR enrichment processes and the limited structural diversity of the initial library, which seriously restrict their real-world applications. To overcome these limitations, the in silico base mutagenesis post-SELEX strategy based on the low Gibbs free energy (ΔG) and genetic algorithm was developed for the optimization of the interferon-gamma aptamer (B1-4). In the process of evolution, new sequences were created and the aptamer candidates with low ΔG values and advanced structures were produced. After five rounds of selection, systematic studies revealed that the affinity of the newly developed evolutionary aptamer (M5-5) was roughly 10-fold higher than that of the parent aptamer (B1-4), and an aptasensor detection system with a limit-of-detection (LOD) value of 3.17 nM was established based on the evolutionary aptamer. The proposed approach provided an efficient strategy to improve the aptamer with low energy and a high binding ability, and the good analytical utility thereof.

High-Throughput Aptamer Microarrays for Fluorescent Detection of Multiple Organophosphorus Pesticides in Food.

A novel high-throughput aptamer microarray fluorescent method based on thioflavin T (ThT) was established for the sensitive detection of phoxim, parathion, fensulfothion, and isocarbophos. In this work, the aptamers in binding buffer tended to have the antiparallel G-quadruplex structure, which can bind ThT and release its potential fluorescence signal. However, when the organophosphorus pesticides (OPs) were present, partial aptamers preferred to bind them, forcing the displacement of ThT from the G-quadruplex and resulting in the significant decrease in fluorescence signal. Under optimal experimental conditions (12T spacer, 300 nM aptamer, and 80 µM ThT), the OP aptamer microarray has low limits of detection of 25.4 ng/mL for phoxim, 12.0 ng/mL for parathion, 7.7 ng/mL for fensulfothion, and 9.9 ng/mL for isocarbophos. The accuracy and reliability of the method is further verified by testing the recovery rate of OPs spiked in two different complicated sample matrices (pears and radishes). It is worth mentioning that not only the developed aptamer microarray technology has low sensitivity and a broad spectrum, but it also allows for high-throughput and rapid analysis of a variety OPs, which overcomes some of the shortcomings of other OP detection methods.

Simultaneous determination of zearalenone and ochratoxin A based on microscale thermophoresis assay with a bifunctional aptamer.

Nowadays, contamination of various mycotoxins in crops and their products exposes increasing risks to human health. Efficient determination methods are urgently needed. Herein, a bifunctional aptamer and a simple aptasensor based on microscale thermophoresis assay (MST) were constructed for the first time for simultaneous determination of two mycotoxins, i.e. zearalenone (ZEN) and ochratoxin A (OTA). The bifunctional aptamer was engineered by splicing a ZEN aptamer and an OTA aptamer with a linker according to the structure analysis of aptamers. The binding mechanism of the bifunctional aptamer to ZEN and OTA were revealed basing on the molecular docking studies. The MST assay proved that the bifunctional aptamer showed high affinity and specificity towards ZEN and OTA. Furthermore, a bifunctional aptamer-based MST-aptasensor was developed for simultaneous detection of ZEN and OTA in corn oil sample. The MST-aptasensor provided a limit of detection (LOD) of 0.12 nM, with satisfactory recoveries of 93.31-104.19% and excellent selectivity, indicating that the bifunctional aptamer and MST-aptasensor had great potential in practical applications.

Development of a lateral flow aptamer assay strip for facile identification of theranostic exosomes isolated from human lung carcinoma cells.

Exosomes are Extracellular Vesicles (EV) that own unique structural features and functions and have gradually become the hot research spot in recent years. The tumor-derived exosomes contain various types of useful biological information, and medical identification of exosomes relied on the specific characterization of membrane surface proteins. In this study, in order to rapidly identify non-small cell lung cancer (NSCLC)-derived exosomes, based on an aptamer against CD63 protein on exosome membrane, a low cost lateral flow aptamer assay (LFAA) test strip using nanogold particles as visualization probes was successfully developed for facile identification of A549 exosomes isolated from human lung carcinoma cells diluted from 6.4 × 109 particles/mL herein.