Impact of the Coverage of Aptamers on a Nanoparticle on the Binding Equilibrium and Kinetics between Aptamer and Protein

Abstract

Knowledge of the interaction between aptamer and protein is integral to the design and development of aptamer-based biosensors. Nanoparticles functionalized with aptamers are commonly used in these kinds of sensors. As such, studies into how the number of aptamers on the nanoparticle surface influence both kinetics and thermodynamics of the binding interaction are required. In this study, aptamers specific for interferon gamma (IFN-γ) were immobilized on the surface of gold nanoparticles (AuNPs), and the effect of surface coverage of aptamer on the binding interaction with its target was investigated using fluorescence spectroscopy. The number of aptamers were adjusted from an average of 9.6 to 258 per particle. The binding isotherm between AuNPs–aptamer conjugate and protein was modeled with the Hill-Langmuir equation, and the determined equilibrium dissociation constant (K′D) decreased 10-fold when increasing the coverage of aptamer. The kinetics of the reaction as a function of coverage of aptamer were also investigated, including the association rate constant (k on) and the dissociation rate constant (k off). The AuNPs–aptamer conjugate with 258 aptamers per particle had the highest k on, while the k off was similar for AuNPs–aptamer conjugates with different surface coverages. Therefore, the surface coverage of aptamers on AuNPs affects both the thermodynamics and the kinetics of the binding. The AuNPs–aptamer conjugate with the highest surface coverage is the most favorable in biosensors considering the limit of detection, sensitivity, and response time of the assay. These findings deepen our understanding of the interaction between aptamer and target protein on the particle surface, which is important to both improve the scientific design and increase the application of aptamer–nanoparticle based biosensor.

Influence of Gold/Silver Ratio in Ablative Nanoparticles on Their Interaction with Aptamers and Functionality of the Obtained Conjugates

“…For future works, the addition of a poly-thymine spacer between the nanoparticle binding thiol group and the folding sequence will help to facilitate the formation of a functional conjugate as thymine holds the lowest affinity of all four nucleobases. Furthermore, using fluorophore labels in the aptamer sequence could allow a more sensitive surface coverage readout compared to the pure UV/vis measurement done in this study by using the well-established fluorescence assay . This approach would also allow the utilization of replacement assays with biotin.…”

“…Furthermore, using fluorophore labels in the aptamer sequence could allow a more sensitive surface coverage readout compared to the pure UV/vis measurement done in this study by using the well-established fluorescence assay. 82 This approach would also allow the utilization of replacement assays with biotin.…”

Molecular Engineering of Aptamer Self-Assemblies Increases in Vivo Stability and Targeted Recognition

“…Nucleic acid (NA) aptamers are short, single-stranded oligonucleotides generated by an in vitro technique termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). − Aptamers have a unique tertiary structure with high affinity and specificity for targets, such as small molecules, ions, proteins, cells, bacteria, and viruses. − With these merits, aptamers are commonly referred to as chemical antibodies (Abs). , However, compared to Abs, aptamers have a wider range of targets and can be selected from toxins, nonimmunogenic substances, and other small molecules. ,, In addition, aptamers have several superior advantages, including a small molecular weight, low immunogenicity, ease of synthesis and modification, and high batch-to-batch stability . Such advantages afford aptamers with broad application prospects in metal ion detection, biosensing, − bioimaging, drug delivery, and cancer therapy. , They can, moreover, act as signal mediators, agonists, or antagonists to regulate the growth and behavior of target cells, as well as form aptamer–drug conjugates through drug modification. − …”

A Lab-in-a-Syringe Device Integrated with a Smartphone Platform: Colorimetric and Fluorescent Dual-Mode Signals for On-Site Detection of Organophosphorus Pesticides

“…CTAB (2 mL, 10 mM) and 15 mL of HAuCl 4 (1 mM) were added into the glassware treated with aqua regia and stirred for 15 min along with the color from yellow to orange. Then, 2 mL of freshly prepared NaBH 4 (100 mM) was added into the above solution under ultrasonic conditions with a quick color change from orange to wine-red and stirred for another 2 h. The obtained CTAB-Au NP solution was left to rest overnight and stored at 4 °C for further use, and the concentration was calculated through the Beer–Lambert law …”

“…Then, 2 mL of freshly prepared NaBH 4 (100 mM) was added into the above solution under ultrasonic conditions with a quick color change from orange to wine-red and stirred for another 2 h. The obtained CTAB-Au NP solution was left to rest overnight and stored at 4 °C for further use, and the concentration was calculated through the Beer−Lambert law. 30 Dual-Mode Detection of AchE Activity. To verify the feasibility of detecting OPs, AchE detection was required.…”