Monitoring Changes in the Concentration of Orange microRNAs During Storage Using a Multiplexed Optical Biosensor Based on Förster Resonance Energy Transfer

Introduction
Agricultural products undergo morphological, physiological, and biochemical changes during transportation and storage. Measuring these changes can help assess mechanical damage and storage quality, although many indicators are not always specific to particular treatments or conditions. In recent years, researchers have explored the role of microRNAs as biomarkers for evaluating postharvest quality in fruit such as tomatoes, bananas, pears, and peaches. microRNAs are small non-coding RNA molecules (17–23 nucleotides) that regulate gene expression and play a key role in fruit development and quality. Specific microRNAs, including miRNA397 and miRNA828, are particularly important in determining the storage quality of oranges.
Förster resonance energy transfer (FRET) is a widely used technique in microRNA biosensing due to its simplicity and high sensitivity. It involves energy transfer between two light-sensitive molecules. This study aims to develop an optical FRET-based biosensor capable of simultaneously detecting miRNA397 and miRNA828 in orange fruit tissue. The innovation lies in applying this method to fruit tissues and evaluating its performance for monitoring fruit quality during cold storage of orange fruit.
Materials and Methods
For biosensor construction, HPLC-purified oligonucleotide sequences, the nucleic acid dye TOTO, and fluorescently labeled oligonucleotides (Cy3 and Cy5) were obtained from Sangon Biotech (China). Complementary oligonucleotides (probes) and fluorescent-labeled sequences were designed to hybridize with the target microRNAs, forming stable nucleic acid duplexes. In this system, hybridization between the target microRNA and complementary probes produces double-stranded nucleic acids that enable a fluorescence-based detection mechanism. The intercalating dye TOTO binds to these duplexes and, when excited at 440 nm, emits a broad fluorescence spectrum between 500 and 700 nm. This emission acts as an energy donor that excites Cy3 and Cy5 fluorophores, leading to FRET. As microRNA concentration increases, hybridization efficiency and fluorescence intensity also increase, enabling quantitative analysis. The system allows simultaneous detection of two target microRNAs because Cy3 and Cy5 have distinct excitation and emission spectra. Cy3 and Cy5 have absorption peaks at 550 and 650 nm, and emission peaks at 570 and 670 nm, respectively.
A total of 63 fresh, uniform oranges were collected from a farm near Gorgan (Golestan Province) and transported to the laboratory. The fruit were stored under three temperature conditions (2, 4, and 6 °C) and seven storage periods (0 to 30 days), following a completely randomized design with three replications. Total RNA, including microRNAs, was extracted from fruit tissues using the TRIzol method, involving phase separation with phenol–chloroform and RNA precipitation with isopropanol. Hybridization reactions were performed in PBS buffer, followed by incubation and addition of TOTO dye. Fluorescence signals were measured using a portable spectrometer at 440 nm excitation. Statistical analysis was performed using two-way ANOVA and Duncan’s test (5% significance level) in MATLAB 2018b, with P < 0.05 considered significant, enabling evaluation of storage temperature and time effects on microRNA expression.
Results and Discussion
The biosensor response, in the form of relative fluorescence intensity, increased with rising microRNA concentration. A wide linear detection range was obtained from 0 to 9 pM, indicating strong analytical performance. The system showed acceptable reproducibility, with low standard deviation values across measurements, demonstrating stable and reproducible biosensor behavior. Recovery experiments were performed by spiking known amounts of microRNA (20–200 fM) into total RNA extracted from orange samples. The biosensor achieved recovery rates mainly between 95% and 110%, indicating acceptable accuracy. At lower concentrations (20 fM), higher variability was observed, reflecting reduced accuracy near the detection limit. At higher concentrations, recovery became more stable (approximately 97–106%), confirming improved reliability in moderate and high concentration ranges.
Biological results showed that both miRNA397 and miRNA828 levels decreased over the 30-day storage period, suggesting gradual changes in gene expression during postharvest storage. The decline was most pronounced during the first 10 days, likely due to rapid physiological changes, followed by a slower decrease. Lower storage temperatures (2 and 4 °C) helped better preserve microRNA levels compared to 6 °C, although differences were not extreme. From a biological perspective, miRNA397 is associated with lignin biosynthesis by regulating laccase genes; its reduction may increase lignification and fruit firmness, contributing to quality loss. miRNA828 regulates MYB transcription factors involved in flavonoid and anthocyanin pathways, so its decline may affect phenolic composition and antioxidant capacity. Overall, the biosensor effectively captured both analytical performance and biologically meaningful changes during fruit storage, demonstrating its potential for postharvest quality monitoring.
Conclusion
Conventional microRNA measurement methods often suffer from low sensitivity, limited linear range, and insufficient accuracy, leading to increased interest in biosensor technologies. Biosensors offer advantages such as high sensitivity, rapid response, reproducibility, and cost-effectiveness, making them strong alternatives. In this study, a FRET-based optical biosensor was developed to simultaneously detect two key microRNAs (miRNA397 and miRNA828) in postharvest oranges. Using a single excitation wavelength, the system enabled selective and accurate detection through probe hybridization. Results showed that microRNA levels significantly decreased over 30 days of cold storage, indicating fruit quality deterioration and confirming the biosensor’s effectiveness for rapid quality monitoring.