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.

Figure 1

AuNP colorimetric aggregation-based sensing method. The Figure illustrates the proposed suppression effect of nonbinding flanking nucleotide on target binding signals, adhering to the particles after target detection, which prevents aggregation, and the role of the proposed method (centrifugation and resuspension) to eliminate the residual adhesion of these nonbinding sequences.

Figure 2

(A) Salt tolerance experiments with bare AuNPs, AuNP-Lee aptamer (100 nM), and AuNP-Lee aptamer + VTD3 (1 µM) when using vortexing and centrifugation sensing methods. The optimal salt concentration is indicated by the black arrow. (B) UV-visible spectra of the sensor performance after independent incubation with control (buffer only) and increasing VTD3 concentrations and application of centrifugation and resuspension step. (C) Colorimetric aptasensor response towards a range of VTD3 concentrations using the AuNP-Lee aptamer (vortexed and centrifuged) compared with control experiments using AuNP-70-mer random ssDNA exposed to the same experimental steps. Top panels show photos of the sensor response in both cases, i.e., vortexed and centrifuged conditions. The sensor response to a range of VTD2 concentrations is also shown. Error bars indicate standard deviation of the mean of three independent experiments starting from particle functionalization.

Figure 3

(A) Salt tolerance experiments of bare AuNPs, AuNP-Bruno aptamer (100 nM), and AuNP-Bruno aptamer + VTD3 (1 µM and 20 µM, respectively) when using vortexing and centrifugation sensing methods. Optimal salt concentration is indicated by the black arrow. (B) Colorimetric aptasensor response towards a range of VTD3 concentrations (µM range) using the AuNP−Bruno aptamer (vortexed and centrifuged) compared with control experiments using AuNP-70-mer random ssDNA exposed to the same experimental steps. Top panels show photos of the sensor response in vortexed and centrifuged conditions for both cases. Raw UV-visible spectra are provided in the Supporting Information. Error bars indicate standard deviation of the mean of three independent experiments starting from particle functionalization.

Figure 4

Selectivity examinations of interfering molecules at 600 nM and 10 µM using the AuNP-Lee aptamer and AuNP-Bruno aptamer, respectively. Error bars indicate standard deviation of the mean of three independent experiments starting from particle functionalization. Top panels show photos of the sensor responses to the interfering molecules. Note that the order of the different samples is the same order presented in the figure.

Figure 5

(A) Colorimetric aptasensor response with Lee aptamer towards a range of VTD3 concentrations spiked into and extracted from human blood using n-hexane, with redissolution in detection buffer under the centrifugation condition compared with control experiments using AuNP-70-mer random ssDNA exposed to the same experimental steps. Top panels show photos of the sensor response towards spiked VTD3 concentrations and its response to the native VTD3 concentration in the blood. Error bars indicate the standard deviation of the mean of three independent experiments starting from particle functionalization. (B) HPLC analysis of spiked VTD3 concentration in blood samples. Red arrows indicate the signal response for native VTD3 concentration extracted from blood. Raw UV-visible spectra and HPLC chromatograms are provided in the Supporting Information.