A colorimetric nanoprobe based on enzyme-immobilized silver nanoparticles for the efficient detection of cholesterol

Abstract

A large number of cardiovascular diseases have recently become of serious concern throughout the world. Herein, we developed a colorimetric probe based on functionalized silver nanoparticles (AgNPs) for the efficient sensing of cholesterol, an important cardiovascular risk marker. A simple sodium borohydride reduction method was employed to synthesize the AgNPs. The cholesterol oxidase (ChOx)-immobilized AgNPs interact with free cholesterol to produce H₂O₂ in proportion to the concentration of cholesterol, resulting in decreased AgNP absorbance (turn-off) at 400 nm due to electron transfer between the AgNPs and H₂O₂. The response of the sensor can also be observed visually. The absorption intensity of the AgNPs is recovered (turn-on) upon the addition of sodium dodecyl sulfate due to the inhibition of ChOx. This on-off mechanism was effectively applied to detect cholesterol within the concentration range 10-250 nM with a low detection limit of approximately 0.014 nM. Moreover, the selectivity of the sensor toward cholesterol was analyzed in the presence of a range of interfering organic substances such as glucose, urea, and sucrose. Finally, the potential of the proposed sensor was evaluated using real samples.

Fig. 1. Mechanism of the production of H₂O₂ by AgNPs immobilized with ChOx in the presence of cholesterol and the optical response of the nanoprobe.

Fig. 2. (A) UV-visible spectra of (a) AgNPs, (b) AgNPs conjugated with EDC/NHS and ChOx, and (c) AgNPs and ChOx in the presence of cholesterol. (B) FTIR spectra of trisodium citrate and citrate-capped AgNPs. (C) FTIR spectra of AgNPs (a) conjugated with EDC/NHS, (b) in the presence of H₂O₂, and (c) in the presence of cholesterol. TEM images of (D) AgNPs, (E) AgNPs conjugated with EDC/NHS and immobilized with ChOx, and (F) AgNPs conjugated with EDC/NHS and immobilized with ChOx in the presence of cholesterol.

Fig. 3. Activation of functional groups on the surfaces of AgNPs using EDC/NHS and conjugation with ChOx.

Fig. 4. UV-visible spectra of AgNPs (A) in the presence of different concentrations of H₂O₂ (10–150 μM) and (B) at different time intervals (0–40 min) in the presence of 150 μM H₂O₂. The inset of (A) shows the change in absorbance as a function of H₂O₂ concentration. (C) Plot of absorbance vs. time in the presence 150 μM H₂O₂. UV-visible spectra of (D) AgNPs conjugated with EDC/NHS and immobilized with ChOx in presence of various concentrations of cholesterol (1–50 nM) and (E) AgNPs conjugated with EDC/NHS immobilized with ChOx in presence of 250 nM cholesterol. The inset of (D) shows the change in absorbance as a function of the cholesterol concentration. (F) Plot of absorbance vs. time for AgNPs immobilized with ChOx in the presence of 250 nM cholesterol. The insets of (C) and (F) show zoomed in images of linear areas of the plots.

Fig. 5. (A) UV-visible spectra of AgNP-EDC/NHS + ChOx + cholesterol with different concentrations of SDS. (B) The linear calibration curve between absorbance and SDS concentration. (C) Inhibition percentages of ChOx in the presence of different concentrations of SDS.

Fig. 6. Logic gate implementation: (A) truth table for the applied logic gate; (B) absorbance for different input signals; and (C) a representation of the logic gate relating to the applied truth table [NOT, OR].

Fig. 7. The Lineweaver–Burk plot for the determination of the enzymatic activity of functionalized AgNPs immobilized with ChOx.

Fig. 8. UV-visible spectra of (A) AgNO₃ with various concentrations of NaBH₄ and (B) AgNPs with various concentrations of ChOx in the presence of cholesterol. Relative enzymatic activity of ChOx as a function of (C) temperature and (D) pH.

Fig. 9. AgNP absorbance in the presence of various potentially interfering biomolecules during cholesterol sensing.