Development of a Novel Colorimetric pH Biosensor Based on A-Motif Structures for Rapid Food Freshness Monitoring and Spoilage Detection

Abstract

Accurate methods for assessing food freshness through colorimetric pH response play a critical role in determining food spoilage and ensuring food quality standards. This study introduces a novel unlabeled DNA sequence, poly-dA₂₀, designed to exploit the colorimetric properties of both the single strand and the fold-back A-motif structure in conjunction with gold nanoparticles (AuNPs) under varying pH conditions. When exposed to storage temperatures of 4 °C and 25 °C, the color variations in the AuNP solution, influenced by pH level changes in mutton and sea bass samples' different storage periods, are easily discernible to the naked eye within a minute. The ratio of UV absorption values at 527 nm and 700 nm (A₅₂₇/A₇₀₀) demonstrates a strong linear correlation with both the storage duration and pH of the food samples. Furthermore, a comprehensive analysis combining the total volatile basic nitrogen (TVB-N) value with the A₅₂₇/A₇₀₀ ratio is employed for precise assessment of food freshness. The innovative pH-responsive sensing strategy not only provides a new approach for on-site food freshness and spoilage detection systems but also serves as a valuable tool for pH-related biological detection in clinical diagnostic applications.

Keywords: A-motif; colorimetric detection strategy; food safety; pH-responsive DNA nanostructures; smart biosensor.

Figure 1

(A) Schematic diagram of a short oligonucleotide sequence composed of homopolymeric deoxyadenosines, where structural units consisting of phosphate groups, deoxyribose, and adenine are linked via phosphodiester bonds. (B) shown the folding structure of A-motif caused by the base pairing scheme in AH⁺ -H⁺A base pairs comprising protonated adenosines. Red 1 represents the N1 protonated site on adenines. (C) Schematic diagram of colorimetric biosensing strategy based on A-motif structure.

Figure 2

(A) Schematic diagram of the transformation of the poly-dA₂₀ strand from a random coil state to two distinct A-motif structures at varying pH conditions (pH < 4 and 4 ≤ pH < 7). (B) Circular dichroism results of the poly-dA₂₀ probes under different pH conditions. The red dotted arrows represent the trend of the CD spectrum as pH decreases from 7 to 4. (C) Fluorescence spectra of Cy3-DNA probes at a concentration of 100 nM under varying pH conditions (pH = 4, 7, and 8). The insert diagram illustrates the structure of the Cy3-poly-dA₂₀ strand. (D) Fluorescence spectra of Cy3-A₂₀ probes labeled with quenched (Q) groups, BHQ2, at a concentration of 100 nM in different pH environments (pH = 4, 7, and 8). The insert diagram illustrates the structural transformation of the F-A₂₀-Q probes in response to acidic and alkaline environments.

Figure 3

(A) Visual images before and after the introduction of poly-dA₂₀ chains into AuNPs solutions under varying pH conditions. (B) UV–vis absorption spectra of poly-dA₂₀/AuNPs mixed solutions at different pH levels. (C) UV–vis absorption spectra of AuNPs at various final concentrations of NaCl. Insert: Visual images of AuNPs at various final NaCl concentrations. (D) Visual color variations in poly-dA₂₀/AuNPs system at different pH values after introducing a final concentration of 0.15 M NaCl. (E) UV–vis absorption spectra of poly-dA₂₀/AuNPs system at different pH values after introducing a final concentration of 0.15 M NaCl. (F) Plot of the absorption ratio (A₅₂₇/A₇₀₀) at different pH values. Insert: a locally linear relationship between absorbance ratio and pH value from pH 6.0 to 8.0. The error bars represent the standard deviation of three measurements.

Figure 4

(A) Schematic diagram of sample handling process for mutton and sea bass. (B) The time–pH curve of mutton during storage at 4 °C and 25 °C. (C) Visual images of AuNP colorimetric pH sensor based on A-motif to mutton samples stored at 4 °C for different days. The images depict the progression of 0, 1, 2, 3, 4, 5, 6, and 7 days from left to right. (D) UV–vis absorption spectra of mutton samples stored for different days at 4 °C using A-motif colorimetric pH sensing system. (E) The linear correlation between the storage time of mutton samples at 4 °C and the A₅₂₇/A₇₀₀ ratio using the colorimetric sensing system. (F) Visual images of AuNPs colorimetric pH sensor based on A-motif to mutton samples stored at 25 °C for different hours. The images depict the progression of 0, 8, 16, 24, 48, and 72 h from left to right. (G) UV–vis absorption spectra of mutton samples stored for different hours at 25 °C using A-motif colorimetric pH sensing system. (H) The linear correlation between the storage time of mutton samples at 25 °C and the A₅₂₇/A₇₀₀ ratio using the colorimetric sensing system. (I) The time–pH curve of sea bass during storage at 4 °C and 25 °C. (J) Visual images of AuNP colorimetric pH sensor based on A-motif to sea bass samples stored at 4 °C for different days. The images depict the progression of 0, 1, 2, 3, 4, 5, 6, and 7 days from left to right. (K) The linear correlation between the storage time of sea bass samples at 4 °C and the A₅₂₇/A₇₀₀ ratio using the colorimetric sensing system. (L) Visual images of AuNPs colorimetric pH sensor based on A-motif to sea bass samples stored at 25 °C for different hours. The images depict the progression of 0, 8, 16, 24, and 48 h from left to right. (M) The linear correlation between the storage time of sea bass samples at 25 °C and the A₅₂₇/A₇₀₀ ratio using the colorimetric sensing system.

Figure 5

(A) Variations in TVB-N values and A₅₂₇/A₇₀₀ ratios in mutton samples over preservation time and different pH values during storage at 4 °C. (B) Variations in TVB-N values and A₅₂₇/A₇₀₀ ratios in mutton samples over preservation time and different pH values during storage at 25 °C. (C) Variations in TVB-N values and A₅₂₇/A₇₀₀ ratios in sea bass samples over preservation time and different pH values during storage at 4 °C. (D) Variations in TVB-N values and A₅₂₇/A₇₀₀ ratios in sea bass samples over preservation time and different pH values during storage at 25 °C. The red dashed lines correspond to the threshold TVB-N values, set at 15 mg/100 g for mutton samples and 20 mg/100 g for sea bass samples. The blue area signifies the fresh stage of the food sample, while the yellow area indicates the spoilage stage of the food sample.