Reagent-Free Colorimetric Cholesterol Test Strip Based on Self Color-Changing Property of Nanoceria

Abstract

Paper-based test strip consisting of cerium oxide nanoparticles (nanoceria) as hydrogen peroxide (H₂O₂)-dependent color-changing nanozymes and cholesterol oxidase (ChOx) has been developed for convenient colorimetric determination of cholesterol without the need for chromogenic substrate. The construction of the cholesterol strip begins with physical adsorption of nanoceria on the paper surface, followed by covalent immobilization of ChOx via silanization, chitosan-mediated activation, and glutaraldehyde treatment of the nanoceria-embedded paper matrices. In the presence of cholesterol, ChOx catalyzes its oxidation to produce H₂O₂, which forms peroxide complex on the nanoceria surface and induces visual color change of the nanoceria-embedded paper from white/light yellow into intense yellow/orange, which was conveniently quantified with an image acquired by a conventional smartphone with the ImageJ software. Using this strategy, target cholesterol was specifically determined down to 40 μM with a dynamic linear concentration range of 0.1-1.5 mM under neutral pH condition, which is suitable to measure the serum cholesterol, with excellent stability during 20 days and reusability by recovering its original color-changing activity for 4 consecutive cycles. Furthermore, the practical utility of this strategy was successfully demonstrated by reliably determining cholesterol in human blood serum samples. This study demonstrates the potential of self color-changing nanozymes for developing colorimetric paper strip sensor, which is particularly useful in instrumentation-free point-of-care environments.

Keywords: cholesterol determination; cholesterol oxidase; nanoceria; paper strip; reagent-free colorimetric assay.

Figure 1

Schematic illustration of the paper strip incorporating nanoceria and ChOx for the smartphone-mediated reagent-free colorimetric determination of cholesterol.

Figure 2

SEM images of (A) bare paper strip, (B) paper strip incorporating both nanoceria and ChOx, and the corresponding EDS maps of (C) Ce, (D) N, and (E) C elements.

Figure 3

(A) Real images and the corresponding dose-response curve for H₂O₂ determination using nanoceria-embedded paper strip and (B) linear calibration plot. The error bars represent the standard deviation of three independent measurements.

Figure 4

(A) Real images and the corresponding color intensities of the selective colorimetric detection of cholesterol using paper strips incorporating nanoceria and ChOx. A 10 mM concentration of cholesterol was used, while 100 mM of other substrates was used in the experiments. (B) Real images, dose-response curve, and the corresponding linear calibration plots for cholesterol determination using paper strips incorporating nanoceria and ChOx. The error bars represent the standard deviation of three independent measurements.

Figure 5

(A) Long-term storage stability and (B) reusability of paper strips for cholesterol detection.