Experimental and theoretical approaches for the selective detection of thymine in real samples using gold nanoparticles as a biochemical sensor

Abstract

We report a simple, selective and cost effective method for the qualitative and quantitative determination of thymine in a DNA standard and urine samples using gold nanoparticles (AuNPs) as a label-free colorimetric biochemical sensor. The mechanism for the detection of thymine is demonstrated via the color change of the AuNPs from pink to blue, followed by the shift of the localized surface plasmon resonance (LSPR) absorption band to a higher wavelength with the introduction of an analyte. The selective detection of thymine was experimentally verified by performing a control experiment with nucleobases, other biomolecules, metal ions and anions. In addition, the computation density functional theory (DFT) and time dependent density functional theory (TD-DFT) using the Gaussian (C.01) program highlighted that the electrostatic potential behavior of the thymine molecule facilitated a non-covalent interaction toward gold for the selective detection of analytes, and the computation was also used to calculate a UV-Vis absorption band as well. The calculated absorption band of the AuNPs with thymine, obtained using TD-DFT, was found to be very close to the experimental data. The omnicapped truncated tetrahedral (ν ₃-tetrahedral) Au₂₀ cluster structure was considered as the model for the AuNP optimization. The linear range obtained for the quantitative determination of thymine was found to be 10-1200 ng mL^-1 with a limit of detection of 3 ng mL^-1. The advantages of using the AuNPs as a biochemical sensor are that they provide a facile and low cost method and are selective for the qualitative and quantitative determination of thymine in a DNA standard and in urine samples in comparison to chromatographic and electrochemical methods.

Fig. 1. A glass vial containing (a) the AuNPs and (b) the AuNPs with thymine, along with their respective UV-Vis spectra. TEM images of (c) the AuNPs and (d) the AuNPs with thymine. (e) The enlarged view of the AuNPs with HR-TEM and (f) the lattice structure of the AuNPs.

Fig. 2. DLS measurements of (a) the AuNPs and (b) the AuNPs with thymine showing the percentage distribution of the particles before and after the addition of thymine. FTIR spectra of (c) pure DBS and AuNPs/DBS and (d) pure thymine and the AuNPs with thymine.

Fig. 3. Images of glass vials containing the AuNPs with different biomolecules (500 ng mL⁻¹): (a) the AuNPs, (b) adenine, (c) cytosine, (d) guanine, (e) glucose, (f) haemoglobin, (g) thymine, (h) urea and (i) bilirubin, along with their UV-Vis spectral data using the AuNPs as a biochemical sensor at pH 7.0 for 5 min of reaction time at room temperature.

Fig. 4. UV-Vis spectra of (a) the AuNPs, and the AuNPs with a fixed concentration (500 ng mL⁻¹) of nucleobase (b) adenine, (c) guanine, (d) thymine and (e) cytosine in the absence of the AuNPs, as well as of (f) adenine + AuNPs, (g) guanine + AuNPs, (h) cytosine + AuNPs, (i) thymine + AuNPs and (j) all nucleobases + AuNPs.

Fig. 5. (a) The optimized labeled structure of thymine, (b) thymine showing charge distribution at each atom, (c) the interaction of thymine with Au-ESP and (d) the HOMO–LUMO of thymine and gold.

Fig. 6. Images of glass vials containing the DBS–AuNPs and different concentrations of thymine (10, 50, 100, 200, 400, 800 and 1200 ng mL⁻¹) using the DBS–AuNPs as a colorimetric probe at pH 7.0 for 10 min of reaction time at room temperature, along with the respective UV-Vis spectra.

Fig. 7. Effect of the absorbance ratio (at 650/525 nm) of biomolecules, metal ions and anions in the presence and absence of the AuNPs for selective determination of thymine. The concentrations of the diverse substances such as bilirubin, glucose, uric acid, and creatine (600 mg L⁻¹), haemoglobin, guanine, adenine, and cytosine (450 mg L⁻¹), Na⁺, K⁺, Fe³⁺, Ca²⁺, Cl⁻, and PO₄³⁻ (850 mg L⁻¹) and Zn²⁺ and Cu²⁺ (700 mg L⁻¹) were spiked into separate glass vials containing the AuNPs, and the pH of the sample solution (pH 7.0) and the reaction time (5 min) were maintained.