The scientific problem the team set out to solve is a familiar one. Seafood is among the most perishable foods on the market. Its high water activity, abundant endogenous autolytic enzymes, near-neutral pH, and porous tissue structure create ideal conditions for microbial growth and biochemical breakdown. Freshness therefore determines flavor, texture, smell, and appearance, and assessing it quickly matters enormously for food safety and consumer protection. Scientists typically track several markers of spoilage, including total volatile basic nitrogen, the K-value reflecting ATP degradation, total viable counts of bacteria, and hypoxanthine. Of these, hypoxanthine has attracted growing attention because it behaves in a particularly useful way: while early ATP metabolites such as inosine monophosphate fluctuate sharply within the first twenty-four hours, hypoxanthine begins accumulating at the initial storage stage and keeps rising steadily over a longer period, making it a stable and reliable indicator of progressive deterioration under refrigerated or ambient conditions.

The concentration ranges involved define what any practical sensor must achieve. In fresh seafood, hypoxanthine levels typically sit below 10 micromolar, whereas spoiled products exceed 50 micromolar, with accumulation kinetics that vary between species during refrigerated storage. Conventional detection methods, including high-performance liquid chromatography, gas chromatography–mass spectrometry, and electrochemical techniques, can measure these levels accurately, but they demand costly equipment, trained personnel, labor-intensive sample preparation, and large volumes of organic solvents. Electrochemical approaches are simpler but often suffer from poor stability in complex food matrices and require frequent electrode modification and calibration. Fluorescence sensing offers a compelling alternative because it is fast, sensitive, easy to operate, and consumes minimal sample. Among fluorescence strategies, ratiometric sensing, which measures the ratio of two emission signals rather than a single intensity, provides built-in self-calibration against variations in probe concentration, excitation intensity, and environmental factors, significantly improving accuracy and reliability.

The heart of the new platform is a fluorescent nanomaterial synthesized in a single hydrothermal step from citric acid, urea, and boric acid. Carbon quantum dots have drawn tremendous interest because of their tunable fluorescence, excellent photostability, low toxicity, biocompatibility, and easy synthesis from abundant precursors. Unlike conventional semiconductor quantum dots such as CdSe or PbS, they are metal-free and environmentally friendly, making them especially suitable for food safety applications. Doping the carbon framework with heteroatoms such as nitrogen and boron further enhances performance. In this work, the co-doping proved decisive: the N, B-CDs achieved a fluorescence quantum yield of 12.89 percent, compared with just 0.17 percent for boron-only dots, 1.36 percent for nitrogen-only dots, and 0.35 percent for undoped dots. Transmission electron microscopy showed uniformly dispersed spherical particles roughly two nanometers in diameter, while X-ray photoelectron spectroscopy and infrared spectroscopy confirmed the successful incorporation of carbon–nitrogen, carbon–boron, nitrogen–boron, and related bonds into the carbon skeleton.

The dots also proved remarkably robust, a critical property for real-world food analysis. Their fluorescence remained stable across neutral and alkaline pH conditions, and even in strongly acidic environments the decrease was modest. Salt tolerance was excellent: after adding sodium chloride at concentrations up to 800 millimolar, fluorescence remained above 80 percent of its initial level, an important feature because real food samples often contain large amounts of salts and electrolytes. The dots withstood temperatures from 4 to 70 degrees Celsius, two hours of continuous ultraviolet irradiation without photobleaching, repeated cycles of UV light and darkness, and thirty days of refrigerated storage with essentially no loss of signal. This combination of brightness and resilience underpins the sensor’s suitability for practical monitoring outside the laboratory.

The detection chemistry is an elegant enzymatic cascade. Xanthine oxidase selectively oxidizes hypoxanthine to uric acid, generating hydrogen peroxide as a byproduct. Horseradish peroxidase then uses that hydrogen peroxide to oxidize o-phenylenediamine, producing 2,3-diaminophenazine, a compound that absorbs strongly near the blue emission of the carbon dots and glows orange at 570 nanometers. As hypoxanthine concentration rises, the dots’ blue emission at 440 nanometers is progressively suppressed while the orange peak grows, and the ratio of the two signals tracks the analyte directly. Mechanistic experiments revealed that the quenching arises mainly from static quenching, through formation of a ground-state complex between the dots and the phenazine product, with a secondary contribution from the inner filter effect. Temperature-dependent Stern–Volmer plots, an unchanged fluorescence lifetime of about 6.5 nanoseconds upon addition of the product, and zeta potential measurements all ruled out energy transfer and confirmed the proposed pathway. Control experiments verified that no color change occurs unless hypoxanthine is present, and the enzyme-driven recognition conferred outstanding selectivity against twenty-two common ions, six amino acids, and small molecules such as urea, glutathione, and ascorbic acid.

Analytical performance was strong in both solution and paper formats. In solution, the ratio of the two emissions responded linearly across 0.1 to 125 micromolar, with a detection limit of 0.106 micromolar, far below the 10 micromolar freshness threshold. A smartphone-based color analysis of photographed solutions performed comparably, reaching a detection limit of 0.072 micromolar. The reaction reached completion within five minutes at pH 6, a compromise condition where protonation of the phenazine product enhances the inner filter effect enough to offset slight losses in enzyme activity and dot fluorescence. When applied to extracts from fish, shrimp, and clams purchased at a supermarket in Wuhan, the solution method returned hypoxanthine concentrations closely matching those measured by high-performance liquid chromatography, with recoveries between 99.00 and 111.10 percent and relative standard deviations below 1.10 percent. A small positive bias in fish samples was traced to matrix effects and corrected with matrix-matched calibration, without affecting freshness classification.

The paper-based version is where the technology becomes genuinely portable. Filter paper strips were activated with alkali, treated with glutaraldehyde, and then soaked in the carbon dot solution so that the dots were covalently grafted to the cellulose via Schiff base bonds, a strategy that overcomes the limited stability and reproducibility of physically adsorbed sensors. Reagents are dropped onto the strip in sequence, and after five minutes the strip is photographed under a 365-nanometer UV lamp inside a dark box using fixed camera settings. The color shifts from bright blue through green to orange-yellow as hypoxanthine rises, and the red-to-blue channel ratio extracted from the images yielded a detection limit of 0.128 micromolar across a working range of 0.1 to 225 micromolar. Because different smartphones process color differently, the team introduced a simple normalization step using a blank reference strip, which cut the variation between an Honor, an iPhone, and a Huawei device from 9.2 percent to 3.1 percent. Ten independently prepared strips gave nearly identical signals, confirming excellent batch reproducibility.

Most importantly for practical use, the researchers established and validated visual freshness thresholds that require no computation at all. A red-to-blue ratio below 1.17, corresponding to hypoxanthine below 10 micromolar, indicates fresh seafood; ratios between 1.17 and 3.61 indicate sub-fresh product; and ratios above 3.61, with hypoxanthine above 50 micromolar, signal spoilage. When 90 independent seafood samples stored at room temperature for up to sixty hours were classified by the paper sensor and checked against chromatographic reference measurements, the overall accuracy was 91.1 percent, and crucially no fresh sample was ever mistaken for spoiled, nor any spoiled sample for fresh. Time-course monitoring revealed that fish spoiled markedly faster than shrimp and clams under identical conditions, demonstrating the platform’s ability to capture species-dependent spoilage kinetics. With its covalent grafting retaining more than 95 percent of signal after thirty days, a five-minute response, dual solution and paper formats, and validation across three seafood types, the platform offers a low-cost, user-friendly route to real-time quality assessment at points of sale, in resource-limited settings, and potentially in the hands of consumers themselves.

Subject of Research: A ratiometric fluorescence sensor using nitrogen and boron co-doped carbon dots for smartphone-assisted detection of hypoxanthine to monitor seafood freshness

Article Title: Smartphone-assisted ratiometric fluorescence sensing of hypoxanthine using N, B-co-doped carbon dots for on-site seafood freshness monitoring

Article References: Zhong, Y., Shao, K., Zou, Y., Li, D., Guo, Y., & Wang, D. (2026). Smartphone-assisted ratiometric fluorescence sensing of hypoxanthine using N, B-co-doped carbon dots for on-site seafood freshness monitoring. Food Chemistry: X, 39, Article 104526. https://doi.org/10.1016/j.fochx.2026.104526

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104526

Keywords: carbon quantum dots, hypoxanthine, seafood freshness, ratiometric fluorescence, paper-based sensor, smartphone detection, food safety, nitrogen boron co-doping, xanthine oxidase, spoilage monitoring, fluorescence quenching, point-of-care testing