Colorimetric Aptasensor of Vitamin D3: A Novel Approach to Eliminate Residual Adhesion between Aptamers and Gold Nanoparticles

Abstract

Colorimetric aptasensors based on gold nanoparticles (AuNPs) commonly feature ssDNA probes nonspecifically adsorbed to surface gold particles. A major limitation of this versatile method is the incomplete dissociation of the adsorbed nontarget binding segments of the aptamer sequence upon target binding. This results in weak or nonexistent sensor performance by preventing the particles from aggregating when the optimized salt concentration is added. Rather than removing the nonbinding nucleotides flanking the binding region of the aptamer, proposed herein is an alternative strategy, simply introducing a centrifugation and resuspension step after target recognition that eliminates residual binding between the aptamer and the surface of the particles. The performance of two different vitamin D3 (VTD3) aptamers were tested. The method enhanced the performance of the sensor that used the higher detection limit (1 µM) aptamer by fourfold. The superiority of the proposed method became apparent in a nonworking colorimetric sensor became a highly sensitive sensor with a one nanomolar detection level and excellent discrimination against potential interfering molecules including VTD2 when the centrifugation and resuspension process was implemented. The level of VTD3 in human blood was determined colorimetrically after extraction with n-hexane. The results were in agreement with those obtained by HPLC. The proposed method could be applied to aptamers targeting small molecules with no need to reprocess the SELEX-isolated sequence by knowing the binding region and removing the flanking primers.

Label-Free Fluorescent Aptasensor for Small Targets via Displacement of Groove Bound Curcumin Molecules

“…The observed specificity is achieved as a result of the non-disrupted 3D structure of aptamer upon binding with curcumin established above, as well as the intrinsic specificity of this aptamer sequence. The specificity results reported here are in agreement with those reported previously, when the same aptamer sequence and interfering molecules were examined using a colorimetric gold aggregation sensor [44]. Additionally, the excellent specificity found by this study is broadly consistent with the study of Lee et al when other interfering molecules were examined [51].…”

“…The mixture was allowed to react for 15 min, then fluorescence measurements were conducted as noted above. For the detection of VTD3 from blood samples, the previously published extraction protocol by our group [44] was flowed as well as all the additional assessment steps of VTD3 concentrations using HPLC. After n-hexane purification, extraction, and drying steps, the samples were re-dissolved in 0.1 mM NaCl salted water containing 5% ethanol and spiked with the desired VTD3 concentration.…”

Aptamers used for biosensors and targeted therapy

“…5 C) [ 107 ]. Structure-switching designs were also employed for the detection of other targets, such as vitamin D3 [ 108 ], abscisic acid [ 109 ], cancer cells [ 110 ], interleukin-6 [ 111 ], and chloramphenicol [ 112 ]. These methods based on AuNPs are simple and cost-effective because the color change can be conveniently assayed by the naked eye.…”

Biomedical Application of Electroactive Polymers in Electrochemical Sensors: A Review

“…Counter, or negative, selection steps can be utilized to select against nucleic acid strands that may have an affinity toward molecularly similar interfering species (control cells in Figure 20). Aptamer-based sensors, also known as aptasensors, have been used for the electrochemical detection of immunoglobulin E [99], fluorescence-based detection of Hepatitis B virus e antigen [100], and colorimetric detection of Vitamin D3 [101]. Examples of CP-based aptasensors are given below.…”