Probing high-affinity aptamer binding region and development of aptasensor platform for the detection of cylindrospermopsin.

Cylindrospermopsin (CYN) is one of the most concerning cyanotoxins due to its potential toxicity and spreading to various environments including drinking water. CYN has potential interferences with human and animal metabolic pathways, which influence the functions of organs including liver, kidneys, lungs, etc. CYN is involved in the inhibition of protein synthesis and detachment of ribosomes from the endoplasmic reticulum membrane. It also interacts with soluble proteins, which are associated with protein translations. It is believed that cytochrome 450 is responsible for the rapid toxicity of CYN. Researchers are urged to develop a high-throughput screening method for the detection of CYN in water. Construction of low cost, rapid, and sensitive analytical methods for the detection of CYN is challenging. Here, we used graphene oxide (GO) as the fluorescence sensing platform for probing the high affinity of the short aptamer derived from the wild-type long aptamer-CYN sensing. The biosensor construction involved two steps: first, quenching the fluorescence of fluorescent-labelled truncated aptamer using GO as a quencher and, second, fluorescence recovery in the presence of CYN by competitive binding between the target and GO. One of the truncate aptamers has a 12-fold higher affinity and enhances sensitivity compared to the long aptamer sequence. The limit of detection of the high affinity truncated aptamer is 17 pM which is 6-fold lower than the long aptamer (100 pM). The sensor specifically detects CYN in the presence of other potential interfering toxins. The performance of the sensor was validated using CYN spiked tap water with very good recovery percentage. A rapid and highly sensitive detection of CYN from water resources has been achieved using this method.

An aptamer based fluorometric microcystin-LR assay using DNA strand-based competitive displacement.

The high-affinity region of a truncated aptamer was applied to the development of a sensitive method for the determination of microcystin-LR (MC-LR) using competitive displacement and molecular beacons. In this assay, the fluorophore and quencher labelled complementary sequences of the aptamer are hybridized with the truncated aptamer to form a fluorophore-quencher pair. In the presence of MC-LR, the aptamer duplex dissociates, and the fluorophore-quencher pair is separated. This turn leads to an increase in the yellow fluorescence which is best measured at excitation/emission wavelengths of 555/580 nm. One of the truncated aptamers showed a 50-fold increase in the affinity (0.93 nM) compared to the wild type aptamer (50 nM). The truncated sequence shows considerable cross-reactivity with L congeners but none with other congeners. The assay works in 0.5 to 200 nM MC-LR concentration range. It was applied to spiked tap water samples and gave recoveries around 95 ± 5%. Graphical abstract Schematic representation of a method for determination of microcystin-LR via fluorescence that is induced by competitive displacement of complementary DNA strands in a truncated dsDNA aptamer.

Fluorometric determination of okadaic acid using a truncated aptamer.

Okadaic acid (OKA), a marine toxin produced by dinoflagellates, is responsible for most human diarrhetic shellfish poisoning-associated health disorders. A competitive displacement assay for OKA is described here. An OKA-binding aptamer was truncated with two sequences, one labeled with 6-carboxyfluorescein (FAM), and one with a quencher. On addition of OKA, it will bind to the aptamer and green fluorescence pops up because label and quencher become spatially separated. One of the truncated aptamers exhibis an excellent binding capability (Kd 2.77 nM) for OKA compared to its full-length aptamer (526 nM). The selectivity of the assay was proven by the successful fluorometric determination of OKA in the presence of common diarrhoetic toxins and in shellfish extracts. The detection limit is as low as 39 pg·mL-1. Graphical abstract Schematic representation of the competitive displacement assay for okadaic acid (OKA). The OKA-binding aptamer is truncated with two parts, one labeled with 6-carboxyfluorescein (FAM), and one with a quencher. On addition of OKA, green fluorescence pops up because label and quencher become spatially separated.

Aptamer selection and aptasensor construction for bone density biomarkers.

Osteoporosis (OP) is a bone disease involved in dysregulation of one of the bone metabolism arms, formation, or desorption cause a porous bone. Osteocalcin (OC) and beta-crosslap (BC), are the well-known markers for OP, which are connected to bone formation and desorption, respectively. In addition to the OP biomarker, BC is also used as an estrogen replacement therapeutic monitoring. ELISA and other antibody-based detection methods are routinely used for measuring OC and BC. These methods have limitations that include thermostability, sensitivity, sacrificing animals, and cost of production. However, aptamer-based-assays are of interest to overcome these drawbacks and achieve the most specific and robust application. Herein, specific aptamers for OC and BC were selected by the systematic evolution of ligands by exponential enrichment (SELEX) method from the pool of ssDNA library with 60 random sequences. The binding affinity (Kd) of the selected aptamers were evaluated against the respective biomarkers. The high-affinity aptamers of OC and BC showed the Kd values of 59 and 55 nM respectively. A graphene oxide-based aptasensors were fabricated from the high-affinity aptamers, and the detection limits of OC and BC were found to be 0.4 pg/ml and 0.21 pg/ml, respectively. These aptasensors have been tested with OC and BC spiked buffer samples and validated using serum samples collected from osteoporotic rats.

Mapping the binding region of aptamer targeting small molecule: Dabigatran etexilate, an anti-coagulant.

Aptamers are single-stranded DNA or RNA, which have attracted considerable scientific interest due to their characteristic of specific and selective binding to target molecules. They are evolved from the in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). This paper reports a simple experimental approach to elucidate the binding region of small targets binding aptamers. A previously isolated 60-mer aptamer for the anti-coagulant dabigatran etexilate (DBG) was used for this investigation. Complimentary sequences labelled with a fluorophore and a quencher were used for testing the binding region by change in the fluorescence signal. The full-length aptamer was truncated to multiple shorter copies including a 38 nucleotides sequence that showed 47 fold high affinity compared to the original aptamer. Circular dichroism spectroscopy (CD) measurements indicate that the 38-mer is remarkably more sensitive than the parent aptamer. The truncated 38-mer sequence was used to construct a turn-on fluorescence sensor with the detection limit of 1 nM. The performance of the sensor was examined in blood serum samples and showed excellent recovery percentages exceeding 98%. The reported screening protocol could be applied to the growing small targets aptasensors that require efficient binding aptamer sequences coupled with optimum signal transduction methods.

Aptameric biosensor for the sensitive detection of major shrimp allergen, tropomyosin.

The development of a sensitive and rapid detection approach for allergens in various food matrices is essential to assist patients in managing their allergies. The most common methods used for allergen detection are based on immunoassays, PCR and mass spectrometry. However, all of them are very complex and time-consuming. Herein, an aptamer biosensor for the detection of the major shrimp allergen tropomyosin (TM) was developed. Graphene oxide (GO) was used as a platform for screening of the minimal-length aptamer sequence required for high-affinity target binding. A fluorescein dye labeled GO quenches the truncated aptamer by π-stacking interactions. After the addition of TM, the fluorescence was restored due to the competitive binding of the aptamer to GO. One of the truncated aptamers was found to bind to TM with four-fold higher affinity (30 nM) compared to the full-length aptamer (124 nM), with a limit of detection (LOD) of 2 nM. The aptamer-based sensor demonstrates the sensitive, selective, and specific detection of TM in 30 min. The performance of the sensor was confirmed using TM spiked chicken soup, resulting in a high percentage recovery (~97 ± 10%). The association of GO and labelled aptamer sensor platform has shown the rapid detection of TM in food, which is compared to other methods very sensitive, specific and performs in high throughput application.