Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay

Abstract

Detection of the small molecule 25-hydroxyvitamin D (25(OH)D) as the most relevant marker for vitamin D supply suffers from a high variability of results using the current detection methods, such as high-performance liquid chromatography (HPLC) and immunoassays. A new detection approach using a highly specific aptamer directed against 25(OH)D was established in this study based on the target-induced dissociation (TID) sensing approach. In this work, the aptamer was investigated regarding its structural properties as well as its binding affinity by using microscale thermophoresis (MST). Moreover, complementary oligonucleotides were designed based on the aptamer structure and were evaluated in MST experiments. Binding experiments of immobilized aptamers were conducted in microarray experiments. It could be shown that the aptamer exhibited the usual B-DNA structure and did not form any G-quadruplexes. The design of complementary oligonucleotides for the TID assay identified a putative 25(OH)D binding site within the aptamer. The limit of detection of the established competitive assay was determined to be 5.4 nM, which sets the stage for the development of a biosensor system.

Aptasensors for Point-of-Care Detection of Small Molecules

“…Lee et al demonstrated the ability of a TID-based AU-NP aptasensor to monitor the small molecule hydroyvitamin D3 (25(OH)D 3 ) in the micromolar range [ 33 ]. These findings could be verified and further extended by the introduction of Cy5-labelled complementary oligonucleotides [ 34 ].…”

All-Optical Planar Polymer Waveguide-Based Biosensor Chip Designed for Smartphone-Assisted Detection of Vitamin D

“…To demonstrate the capability of the sensor system for biosensing applications, we demonstrate the detection of 25-hydroxyvitamin D (25OHD) as a model analyte from diluted serum samples. Since 25OHD is a relatively small molecule and thus only induces a relatively small refractive index change above the SPR sensor surface, a competitive aptamer-based assay recently developed by our team [18], was applied. In this assay, a DNA aptamer [19] directed against 25OHD is used in combination with an oligonucleotide that is complementary to a section of the aptamer which is involved in binding of the aptamer target.…”

“…In the absence of the target, the oligonucleotide forms a duplex with the aptamer, while in the presence of 25OHD, the aptamer binds to its target instead. Through this process, the binding of the small molecule 25OHD, which is hard to detect by SPR in general, is translated into the dissociation of the larger oligonucleotide [18]. To further increase the 25OHD-induced changes of the refractive index above the sensor surface, gold nanoparticles (AuNPs) were used for signal enhancement, as described recently for aptamer-based detection of CRP [17].…”

“…Concentrations of 0 nM, 25 nM, 50 nM, and 100 nM 25-hydroxyvitamin D were prepared in BB supplemented with 10% Ethanol, 500 mM NaCl, and 10% 25-hydroxyvitamin D-depleted human serum. All solutions additionally contained 10 nM complementary oligonucleotide (sequence: AAA-GTC-ACA-C, [18]) with a 5 terminal biotin modification. The solutions were incubated on the sensor for 30 min to allow binding of 25-hydroxyvitamin D to the aptamer as well as hybridization of the oligonucleotide to those aptamer molecules that have not bound 25-hydroxyvitamin D under the given conditions.…”

Recent Advancements in Aptamer-Based Surface Plasmon Resonance Biosensing Strategies

“…Consequently, by incorporating these nucleic acid aptamers into various sensing platforms, it is possible to subject these probes to repeated use without loss of functionality, thereby allowing a device to be potentially recyclable. Numerous aptamer-based approaches have been reported for chemical and biological detection, such as surface-enhanced Raman spectroscopy [40,41], SPR [42,43], fluorescence [44,45], colorimetry [46,47], and electrochemistry [48,49].…”