Recent advances in ratiometric electrochemical sensors for food analysis

Ratiometric electrochemical sensors are renowned for their dual-signal processing capabilities, enabling automatic correction of background noise and interferences through built-in calibration, thus providing more accurate and reproducible measurements. This characteristic makes them highly promising for food analysis. This review comprehensively summarizes and discusses the latest advancements in ratiometric electrochemical sensors and their applications in food analysis, emphasizing their design strategies, detection capabilities, and practical uses. Initially, we explore the construction and design strategies of these sensors. We then review the detection of various food-related analytes, including nutrients, additives, metal ions, pharmaceutical and pesticide residues, biotoxins, and pathogens. The review also briefly explores the challenges faced by ratiometric electrochemical sensors in food testing and potential future directions for development. It aims to provide researchers with a clear introduction and serve as a reference for the design and application of new, efficient ratiometric electrochemical sensors in food analysis.

Electrochemical methods are especially effective in the field of food analysis due to their unique advantages (Gu et al., 2022;Xu, Yang, Zhang, Lu, & Bai, 2023;Zhang et al., 2021;Zhou et al., 2023).These methods are renowned for their high sensitivity, rapid response times, and minimal sample preparation requirements.Additionally, compared to traditional analytical techniques such as chromatography and mass spectrometry, electrochemical methods are generally more costeffective and portable.Ratiometric electrochemical sensors mark a significant advancement in electrochemical analysis technologies (Kodr et al., 2021;Liu, Dong, Zhang, & Tian, 2017;Luo et al., 2022;Qin, Liu, Meng, Liu, & You, 2024;Zhang et al., 2022).Unlike traditional sensors that rely on a single signal output, ratiometric sensors employ two electrochemical signals and use the ratio of these signals as the output.This dual-signal approach provides inherent error correction capabilities, which are absent in traditional single-signal sensors, thereby greatly enhancing accuracy, sensitivity, and reliability.The primary advantage of ratiometric sensors in food analysis is their ability to correct for background noise and potential interferences that could affect measurements.This feature is particularly valuable in complex food matrices, where various substances may interfere with the detection of specific analytes.Additionally, ratiometric sensors often incorporate built-in correction features, which enhance their sensitivity and allow for more precise quantification of target analytes.
To date, a few review articles have summarized and analyzed ratiometric electrochemical sensors (Cui, Li, Zou, & Zhang, 2018;Jin, Sun, Sun, & Gui, 2021;Spring, Goggins, & Frost, 2021;Wei et al., 2022;Xu, Wang, & Liu, 2022;Yang et al., 2018;Zhang, Wen, Wang, Yang, & Sun, 2022;Zhu, Wang, Yang, Chen, & Yu, 2023), contributing significantly to the field.However, reviews specifically discussing the application of ratiometric electrochemical sensors in food analysis are still relatively rare.Given the importance of food analysis and the advantages of ratiometric electrochemical sensors, this review paper will systematically and comprehensively outline the application of ratiometric electrochemical sensors in food analysis.The paper will first introduce the types of ratiometric electrochemical sensors (internal reference type and dual-signal response type) and the construction principles of those used in food analysis.We will then detail the detection of various targets in food analysis, including the construction, detection principles, and analytical performance of the sensors.Finally, this review will provide insights into the current state and future development of ratiometric electrochemical sensors in food analysis.We believe this review will offer researchers in the field a clear introduction to the application of ratiometric electrochemical sensors in food analysis and guide the design and application of new, efficient ratiometric electrochemical sensors (Fig. 1).(See Table 1.)

Design strategies for ratiometric electrochemical sensors
Ratiometric electrochemical sensors utilize the ratio of signals from multiple redox-active molecules to provide signal output.These advanced devices are distinguished by their ability to correct for background noise, which offers more reliable and sensitive measurements than those provided by traditional electrochemical sensors.It is important to note that ratiometric electrochemical sensors differ from conventional dual-signal electrochemical biosensors.Conventional dual-signal biosensors use two independent signal sources for qualitative and quantitative analysis of the target, enhancing detection reliability.In contrast, ratiometric electrochemical sensors generally use the ratio of two signals to correct background noise and interference, thereby improving detection accuracy and repeatability.A primary advantage of ratiometric sensors is their "built-in calibration" feature, typically arising from the use of two electrochemically active probes that respond to environmental changes under similar conditions.This setup helps counteract variable external factors, ensuring more accurate and consistent readings.The characteristics and corresponding design approaches of ratiometric electrochemical sensors mainly fall into two categories (Fig. 1).

Internal reference type
In this design, a fixed internal reference signal (I) is established, which remains unchanged upon the introduction of the target analyte.Another signal (II) that varies with the concentration of the target analyte is produced.The target is analyzed accurately using the response ratio (S I /S II ).The electroactive probes generating signals are diverse, including electroactive small molecules (such as ferrocene, methylene blue, toluidine blue, thionine, Nile blue), metal-organic frameworks (MOFs), metallic nanoclusters, oxide nanomaterials, etc.The generation of the electrochemical signal (II) can be of two types: direct electrochemical signals produced by the oxidation/reduction of the target analyte itself, and indirect electrochemical signals often achieved through antigen-antibody recognition, nucleic acid aptamer binding, followed by the introduction of additional signal probes.

Dual-signal response type
This strategy employs two different electrochemical probes, each responsive to the target analyte, but with distinct mechanisms of action.The essence of this approach lies in the detailed analysis provided by comparing the electrochemical signal responses of each probe to changes in analyte concentration.Such comparative analysis can be achieved by strategically adjusting the proximity of each probe to the electrode or by modulating their electrochemical responses through the introduction or removal of probes from the electrode surface in response Fig. 1.Schematic illustration of the construction of ratiometric electrochemical sensors and their application in food analysis.

Applications of ratiometric electrochemical sensors in food analysis
Ratiometric electrochemical sensors exhibit outstanding analytical performance and have promising prospects in food analysis.Based on the detection targets of ratiometric electrochemical sensors, they can be mainly divided into categories for detecting nutrients, additives, metal ions, pharmaceutical and pesticide residues, biotoxins, and pathogens.The following discussion will categorize and elaborate on these applications, underscoring the unique advantages and potential uses of ratiometric electrochemical sensors in ensuring food safety and quality.

Nutrients
Nutrients in food refer to essential and beneficial components that are crucial for assessing the quality of food products and medicinal ingredients (Shao, Hou, Jia, & Zheng, 2022;Similä, Ovaskainen, Virtanen, & Valsta, 2006).Accurate measurement of specific nutrient content is vital for ensuring the quality and safety of food items, as well as for understanding their nutritional value and health benefits.Ratiometric electrochemical sensors have proven particularly useful in the detection of specific types of nutrients such as quercetin and caffeic acid.
Quercetin (Qu) is emphasized as an important flavonoid due to its widespread occurrence in various plants and its significance in human diets (Kandemir, Tomas, McClements, & Capanoglu, 2022;Lai & Wong, 2022;Oh, Ambigaipalan, & Shahidi, 2021).Gui and Wang et al. have reported a direct ratiometric electrochemical method for detecting quercetin (Yu, Jin, Gui, Lv, & Wang, 2017).This assay employed thionine (TH) as the reference electroactive substance to analyze Qu.TH and Qu displayed distinct oxidation peaks at different potentials (− 0.22 V for TH and 0.18 V for Qu), suitable for ratiometric measurements.Detection is based on differential pulse voltammetry (DPV), which measures the oxidation peak intensities of quercetin and thionine at specific voltages.The ratiometric method calculates the ratio of these peak intensities (I Qu /I TH ), which linearly correlates with the concentration of quercetin in the solution.The sensor demonstrates high sensitivity and selectivity in detecting Qu, effectively overcoming potential interferences from real food samples.
Polyphenolic compounds are well-known for their excellent antioxidant properties, among which caffeic acid (CAE) is a key polyphenol, particularly abundant in chrysanthemum tea (Matsui, Tanaka, & Iwahashi, 2017;Naveed et al., 2018).Due to its stability, caffeic acid is often used as a benchmark for assessing the total content of polyphenolic compounds.Wang and Xie et al. have engineered a sophisticated ratiometric electrochemical sensor utilizing poly(methylene blue) (PMB) electropolymerized onto flower-like nickel-based metal-organic frameworks (Ni-TPA MOFs) for the precise detection of CAE (Yin, Zhuang, Xiao, Wang, & Xie, 2021).This sensor is built around a GCE that has been modified with a nanocomposite comprising PMB and Ni-TPA MOFs.Here, PMB acts as the internal reference probe, while Ni-TPA MOFs significantly boosts the electrochemical signal strength The sensor's functionality is based on a ratiometric electrochemical sensing strategy that measures the ratio of dual current peak intensities for CAE and PMB across different redox potentials.The sensor responds linearly across a CAE concentration range of 0.25-15.0mM with a detection limit of 0.2 mM, and its accuracy has been corroborated against the traditional Folin-Ciocalteu method.

Additives
Food additives are substances added intentionally to food during various stages like processing, manufacturing, or storage to enhance appearance, texture, or preservation.Detecting food additives is crucial for ensuring food safety, compliance with regulatory standards, protecting consumer rights, enhancing public trust, and fostering the development of the food industry.Currently, ratiometric electrochemical sensors are widely used for the detection of various food additives (Fig. 2).These include preservatives such as propyl gallate, nitrites, and malachite green; antioxidants like vanillin, hydroquinone, kojic acid (KA), and glutathione; color additives; as well as substances that may remain in food from processing, such as bisphenol A and hydrogen peroxide (H 2 O 2 ).

Preservatives
Wang et al. developed a dual-ratiometric electrochemical sensor for detecting the food preservative propyl gallate (PG) (Yin, Wang, & Zhuang, 2021).The construction of the sensor involves anchoring anthraquinone (AQ) and multi-walled carbon nanotube (MWCNT) onto the GCE surface to leverage their excellent electrochemical activity and conductivity, enhancing the sensitivity for PG detection.As internal reference electroactive probes, AQ and MWCNT facilitate the display of three distinct current peaks corresponding to the electro-oxidation of PG, AQ, and MWCNT upon PG detection.The sensor quantifies PG through two ratiometric signals (I PG /I AQ and I PG /I MWCNT ), which linearly correlate with PG concentration ranging from 1.0 to 30.0 μM, with detection limits estimated at 0.67 μM and 0.76 μM, respectively.The sensor demonstrates satisfactory results in detecting PG in edible oil samples, showing high sensitivity and selectivity.
Malachite Green (MG), a dye and medicinal agent used in aquaculture, possesses antibacterial, antifungal, and parasiticidal properties but is a toxic triphenylmethane molecule associated with carcinogenic and teratogenic risks in humans.Zhou and Wang et al. developed a ratiometric electrochemical sensor using three-dimensional conductive metal-organic frameworks (CMOFs) to detect MG in fish samples (Li et al., 2023).The CMOFs, Cu 3 (HHTP) 2 , was synthesized through a onepot process combining the ligand 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) with copper ions, with N,N-dimethylformamide (DMF) added as a limiting agent to control nanorod growth and morphology.Cu 3 (HHTP) 2 functioned as an internal reference and displayed exceptional electrocatalytic activity and charge transfer efficiency, significantly enhancing the sensor's sensitivity to MG. Demonstrations showed that this electrochemical sensor offered a broad detection range (5-1000 nM) and a low detection limit of 1.34 nM, exhibiting excellent selectivity and interference resistance.Additionally, the sensor's application in actual fish samples validated its potential for food safety assurance.

Antioxidants
Tert-butylhydroquinone (TBHQ) is a synthetic antioxidant used primarily in edible oils to extend shelf life, but its excessive use can degrade food quality and pose health risks, leading to regulated maximum permissible levels.Wang et al. described the development of a ratiometric electrochemical sensor for detecting TBHQ (Cao, Wang, Zhuang, Wang, & Ni, 2019).The sensor utilizes a GCE modified with electrochemically deposited MnO 2 and reduced graphene oxide (ERGO), forming the working electrode MnO 2 /ERGO/GCE.This method involves using two distinct signals at different potentials: one from TBHQ and the other from the internal MnO 2 reference probe.The sensor exhibited two linear response ranges: 1.0-50.0μM and 100.0-300.0μM, with a detection limit of 0.8 μM.Ma et al. developed a ratiometric electrochemical sensor based on the self-assembly of Co NC/CNT and methylene blue for effective detection of TBHQ (Fig. 3) (Zhang et al., 2024).The sensor uses carbon nanotube-encapsulated Co/nitrogen-doped carbon (Co NC/CNT) and methylene blue (MB) as internal reference signals to enhance accuracy.The construction process includes self-assembling ZIF-67/CNT materials, which are then pyrolyzed in an argon atmosphere to form Co NC/CNT.This is followed by electrostatic adsorption to combine with MB, forming a Co NC/CNT/MB composite.Under optimized conditions, the ratio of the net peak currents of TBHQ to MB shows a linear relationship with TBHQ concentration.The sensor demonstrates excellent selectivity, repeatability, reproducibility, and stability, making it suitable for detecting TBHQ in real edible oil samples.
Vanillin is a widely used additive in food, cosmetics, and pharmaceuticals, primarily for its distinctive sweet aroma and antioxidant properties (Moradi, 2022).Jin and Gui et al. developed a novel ratiometric electrochemical aptasensor for the detection of vanillin that enhances sensitivity and selectivity through the use of advanced composite materials and biomolecular recognition elements (Sun, Jiang, Jin, & Gui, 2019).The sensor integrates Ketjen black and ferrocene dual-doped Metal-Organic Frameworks (ZIF-8, zeolite imidazole frameworks), combined with gold nanoparticles (AuNPs).A GCE is modified with these materials and a DNA aptamer that specifically binds to vanillin, creating a robust sensing platform.The method utilizes dual signal outputs from the target molecule vanillin and an internal reference (ferrocene incorporated into ZIF-8).The sensor operates within a vanillin concentration range of 10 nM to 0.2 mM, with a detection limit of 3 nM.The sensor has been successfully tested vanillin in real food samples (chocolate and nougat) e.Recently, Lee et al. developed a new sensor to detect the antioxidant kojic acid, structurally similar to vanillin, using an electrode modified with MXene/PB/AuNPs nanocomposites (Karuppaiah, Koyappayil, Go, & Lee, 2023).
Glutathione (GSH), a widely utilized food additive, is renowned for its potent antioxidant and detoxification properties (Tsiasioti, Zotou, & Tzanavaras, 2021).Liu et al. developed a highly sensitive and selective dual-signal, intrinsic self-calibrating electrochemical sensor for detecting GSH, leveraging the enhanced electrochemical properties of in situ electrodeposited silver nanoparticles on a nickel-iron Prussian blue analog (Ag/NiFe PBA) (Xu et al., 2022).Two distinct oxidation peaks attributed to Ag and Fe species appear on the Ag/NiFe PBA-modified electrode.In the presence of chloride ions, Ag is oxidized to form silver chloride (AgCl), and subsequent addition of GSH triggers a specific interaction that significantly reduces the peak current of AgCl, while the peak current of NiFe PBA remains unchanged.The ratio of these peak currents (I AgCl /I Fe ) serves as the signal output.The sensor exhibits an extraordinarily low detection limit of 0.12 nM.Successfully applied to various vegetable and serum samples, this sensor demonstrates its effectiveness and potential for broad applications in food safety and clinical diagnostics.

Colorants
Food colorants, also referred to as food additives, are primarily utilized to enhance food colors, attracting consumers and improving product appearances.Sunset Yellow (SY), a synthetic azo dye commonly used in beverages, candies, desserts, and processed foods, has raised health concerns due to its potential allergenic and carcinogenic risks (Barciela, Perez-Vazquez, & Prieto, 2023).Sun and Xu et al. developed a ratiometric electrochemical sensor for SY using polydopamine (PDA) and nickel sulfide on hollow carbon spheres (NiS@HCS) as primary materials (Chen et al., 2022).The NiS@HCS was synthesized via a hydrothermal method to enhance its catalytic activity and conductivity.Polydopamine served as an internal reference signal, forming a film on the GCE through electropolymerization, enhancing the sensor's sensitivity and selectivity toward SY.The sensor quantifies SY based on the ratio of electrochemical signals from SY and PDA, where PDA serves as an internal reference.The sensor was tested in real food samples such as rice vinegar, cooking wine, and carbonated drinks, showing good recovery rates and consistency with UV-Vis spectroscopy.Subsequently, Wu, Zhang, and Ma et al. developed a gold nanoparticle-based ratiometric molecularly imprinted electrochemical sensor for SY (Wu et al., 2023).Compared to traditional sensors, molecularly imprinted biosensors offer higher specificity and sensitivity due to the specific recognition of template molecules by molecularly imprinted polymers (MIPs).This sensor is not affected by other structurally similar interferents and performs well in the analysis of real samples.

Others
Other substances primarily include residuals from food processing or storage, such as bisphenol A (BPA) and hydrogen peroxide (H 2 O 2 ).Bisphenol A (BPA), used in polycarbonate plastics and epoxy resins, is an environmental endocrine disruptor that mimics estrogen and can cause reproductive, developmental, and other health issues (Beitollahi et al., 2022).Wang et al. developed a ratiometric electrochemical sensor for detecting BPA (Sun, Xiao, Tang, Zhuang, & Wang, 2021).This sensor uses a GCE modified with a composite of polythionine (PTH) and gold nanoparticles (AuNPs).The ratiometric strategy leverages the ratio of oxidation peak currents between BPA and the internal reference PTH for self-calibration, enhancing the reliability of the detection.The sensor also exhibited high sensitivity, stability, and selectivity, demonstrating its potential for practical application in real water samples.Pang et al. described a ratiometric electrochemical sensor for simultaneously detecting BPA and bisphenol S (BPS), using a carbon cloth electrode modified with a covalent organic framework (COF-LZU1) and AgNPs (Pang, Wang, Shen, & Qiao, 2022).
Hydrogen peroxide (H 2 O 2 ) is used in the food industry as a disinfectant and bleaching agent to control microorganisms and maintain food color and texture, but its residues must be monitored due to potential health hazards like irritation and oxidative stress that can cause cellular damage (Abdelshafy, Hu, Luo, Ban, & Li, 2024;Xing et al., 2022).Kan et al. developed a ratiometric electrochemical sensor for the quantitative detection of H 2 O 2 in food samples (Luo & Kan, 2022).The sensor is based on the specific dissociation reaction of 4-aminophenylboronic acid ester (ABAPE) triggered by H 2 O 2 to generate electroactive 4-aminophenol (4-AP), providing high selectivity.The sensor uses polythiophene (TH) as the reference probe and is modified with Ketjen black (KB) and gold nanoparticles (AuNPs) on the electrode surface, enhancing the electrocatalytic activity toward 4-AP.The sensor measures the ratio of 4-AP to TH currents, which correlates well with H 2 O 2 concentrations.Moreover, the ratiometric strategy sensor demonstrates good accuracy, reliability, and stability, successfully detecting H 2 O 2 in milk, beer, and juice samples with satisfactory results.Following that, Li et al. described a ratiometric electrochemical sensor based on a ZIF-8/ COOH-MWCNTs composite specifically designed for the detection of H 2 O 2 (Zhao et al., 2023).The oxidation peak current of MB serves as the internal reference signal, while the response electrochemical signal is generated from the oxidation peaks of 4-AP, produced by the reaction of H 2 O 2 with ABAPE.

Metal ions
Monitoring heavy metals is an essential task in food testing, and ratiometric electrochemical sensors have also been used for monitoring Cd 2+ and Pb 2+ .Zhang et al. developed a dual-signal ratiometric electrochemical sensor based on semi-complementary aptamers for detecting cadmium in mussels (Chen, Liu, Su, Zhang, & Zou, 2021).This sensor utilizes a homemade multi-functional screen-printed electrode (SPE) that enables both primary ratiometric sensing and auxiliary on-chip pH monitoring.The preparation process of the sensor involves electrodepositing AuNPs onto the SPE.The Cd 2+ -specific aptamers are then attached to the AuNPs through Au-S bonds to form Apt/AuNPs/SPE. Next, ssDNA@PTH-Au (reference probe) is dropped onto the electrode, where it hybridizes with the Cd 2+ aptamer to produce ssDNA@PTh-Au/ Apt/AuNPs/SPE.The binding of Cd 2+ to its specific aptamer changes the aptamer's configuration and expels the previously hybridized ssDNA@PTH-Au, resulting in a reduction in the reference signal.Meanwhile, the signal for Cd 2+ increases.This change forms a "signal on/off" type of ratiometric detection system where the target signal (I Cd ) and the reference signal (I PTH-Au ) constitute a ratiometric sensing system.The sensor demonstrates a detection range from 2 × 10 − 3 to 8 × 10 − 1 mg•L − 1 , with a detection limit of 7 × 10 − 1 mg•L − 1 .This sensor has been successfully applied to the determination of cadmium in actual mussel samples, showing satisfactory recovery results, confirming its practicality and accuracy.
Zhang et al. developed a sensor employing a composite electrode composed of ZrO 2 , Ni/Co-MOFs, and AuNPs, which was modified with hairpin-structured nucleic acid chains tagged with anthraquinone-2carboxylic acid (AQ) (Wang et al., 2023).The sensor uses two targetspecific aptamer strands, each marked with an electrochemical probe ferrocene (Fc) for Cd 2+ and MB for S. aureus, enabling simultaneous detection.This ratiometric sensor operates by measuring the ratio of the MB and AQ signals to the Fc signal, effectively leveraging the redox potential differences between these markers to achieve precise and specific detection of both targets.Zou et al. developed a ratiometric electrochemical sensor based on the synergistic action of semicomplementary aptamer pairs and Ag nanowires@zeolitic imidazolate framework-8 (AgNWs@ZIF-8) for detecting Pb 2+ in fish (Han et al., 2023).Dai et al. developed a sensor composed of curcumin (p-CCM), MWCNTs, and layered double hydroxides (LDHs), forming a novel ratiometric electrochemical sensing interface for Cd 2+ and Pb 2+ (Duan et al., 2024).

Pharmaceutical and pesticide residues
Pharmaceutical and pesticide residues are chemicals that can remain in food products due to agricultural activities and pharmaceutical applications.These substances can have profound health implications, including fostering antibiotic resistance, disrupting hormonal functions, and causing other toxicological impacts.Therefore, it is essential to detect and quantify these residues to ensure consumer safety and meet regulatory standards.Ratiometric electrochemical sensors have proven to be highly effective for this purpose.Such sensors have already been applied to detect a range of pharmaceutical residues (like tetracycline, kanamycin, trimethoprim, and carbendazim) and pesticide residues (such as imidacloprid, carbaryl, parathion-methyl, phoxim, malathion, profenofos, and glyphosate) (Fig. 4).

Fig. 4.
Schematic illustrations of the molecular structures of various pharmaceuticals and pesticides detected by currently reported ratiometric electrochemical sensors in food products.

Pharmaceutical residues
Tetracycline (TET) is a broad-spectrum antibiotic widely used in animal husbandry, which can leave residues in animal-derived food products, potentially causing health issues such as allergic reactions and antimicrobial resistance (Khezerlou et al., 2023;Wang, Yin, & Gunasekaran, 2023).Guo and Zhang et al. developed a separable ratiometric electrochemical sensor designed effectively for detecting TET residues (Xu et al., 2017).This innovative sensor comprises two distinct aptasensors: Aptasensor 1 utilizes screen-printed carbon electrodes (SPCEs) that are electrodeposited with AuNPs to create a foundational layer for the attachment of TET-specific aptamers via Au-S bonds; Aptasensor 2 similarly incorporates carbon nanofibers (CNFs) combined with AuNPs on SPCEs, where TET-specific aptamers are also attached.Upon exposure to TET, the aptamers bound to TET undergo a spatial conformation change, affecting the electrochemical characteristics of the nanocomposites.This alteration leads to measurable changes in the electrical current at each aptasensor.The ratiometric measurement (ΔI CNFs /ΔI Fc , where ΔI CNFs and ΔI Fc represent the current changes in Aptasensor 2 and Aptasensor 1, respectively) ensures precision.The sensor exhibits a detection range for TET from 10 − 8 -10 − 3 g•L − 1 , with a detection limit of 3.3 × 10 − 7 g•L − 1 , and has been successfully validated in real milk samples.Kanamycin (KAN) is an aminoglycoside antibiotic widely used in animal husbandry, and its residues can pose health risks such as antibiotic resistance (Tang & Tao, 2023).Li and Xu et al. developed a ratiometric electrochemical aptasensor for detecting KAN using a Fclabeled primer and a graphdiyne-methylene blue (GDY-MB) nanocomposite (Gao et al., 2022).The sensor operates by forming a hairpin structure when KAN binds to the aptamer, enhancing the ferrocene signal near the electrode surface.Exonuclease I is used to increase the signal through cyclic amplification.This aptasensor demonstrates high stability and specificity, effectively detecting KAN in milk samples.Subsequently, Jin and colleagues developed an enzyme-free ratiometric electrochemical aptasensor for detecting KAN in food (Jin et al., 2024).Utilizing an entropy-driven strand displacement reaction, the sensor demonstrated high specificity, reproducibility, and remarkable sensitivity.Deng et al. developed a molecularly imprinted ratiometric electrochemical sensor for sensitive and selective detection of trimethoprim (TMP), another broad-spectrum antibiotic (Deng et al., 2024).The sensor utilizes a 3D -1D hetero-nanoflower structure composed of MoS 2 and carbon nanotubes as the substrate, which enhances electrical conductivity and provides a larger surface area to optimize sensor performance.The sensor employs a molecularly imprinted polymer for specific recognition and quantification of TMP, using ferrocene as a reference for ratiometric measurement.
Carbendazim (CBZ) is a broad-spectrum fungicide extensively used in agriculture, known to cause potential chromosomal abnormalities and environmental hazards (Wang, Shi, Liu, & Laborda, 2020;Zhou, Guo, Wang, Wang, & Zhang, 2023).Gao and Lu et al. developed a ratiometric electrochemical sensor for detecting CBZ, which is constructed from a composite of MXene@Ag nanoclusters and aminofunctionalized multi-walled carbon nanotubes (NH 2 -MWCNTs) (Zhong et al., 2021).This combination enhances the sensor's electrocatalytic capabilities, and the presence of Ag nanoclusters provides a stable reference signal essential for the ratiometric detection method.The addition of NH 2 -MWCNTs improves the electrochemical signals of CBZ and Ag, leading to signal amplification and increased sensitivity.The sensor demonstrates a good linear relationship between the ratio of CBZ to Ag signal intensities (I CBZ /I AgNCs ) and the concentration of CBZ, ranging from 0.3 nM to 10 μM, with a detection limit of 0.1 nM.

Pesticide residues
Current advancements in ratiometric electrochemical sensors are predominantly focused on detecting organophosphorus pesticides (OPs), such as phoxim, parathion-methyl, malathion, and profenofos.These pesticides are known for their broad-spectrum insecticidal effects and relatively rapid action, primarily inhibiting acetylcholinesterase (AChE), which can be potentially toxic to both the environment and nontarget organisms, including humans (Sarlak et al., 2021;Wan, Liu, Pi, & Wang, 2023).
Zhang et al. have developed a ratiometric electrochemical immunosensor utilizing DNA tetrahedral nanostructures (DTNS) for detecting phoxim (PHO) in vegetables (Fig. 5) (Su et al., 2022).The sensor is constructed by modifying a GCE with AuNPs and anchoring DTNS via gold-sulfur (Au -S) bonds.MB, absorbed onto the DTNS, forms the MB/ DTNS/AuNPs/GCE substrate.The DTNS adheres spontaneously to the modified electrode, creating a stable three-dimensional structure that offers numerous binding sites for the internal reference signal probe, MB.A monoclonal antibody (m-Ab) is vertically linked to the apex of the DTNS, selectively responding to the antigenic phoxim and enhancing the target signal.This configuration establishes a ratiometric index, I Phoxim / I MB , through the DPV technique.The sensor exhibits a robust linear response in the range of 0.1-30 μg•L − 1 and a detection limit of 3 ng•L − 1 .
Then, the Zhang group developed a novel ratiometric electrochemical immunosensor for the detection of parathion-methyl (PTM) (Su et al., 2022).Initially, AuNPs were electrodeposited onto a SPE, which was then modified with antibodies targeting PTM.MXene-Au nanocomposites were synthesized via a hydrothermal method, and MB, serving as the reference signal probe, was adsorbed onto MXene-Au through electrostatic adsorption.Subsequently, MXene-Au-MB was conjugated with the PTM antigen (ATG) to form a competitive signal probe complex, MXene-Au-MB-ATG.During detection, PTM competes with MXene-Au-MB-ATG for binding to the SPE.The presence of PTM causes partial dissociation of MXene-Au-MB-ATG from the antibodies, altering the sensor's current signal.The sensor utilizes a dual-electricfield mode, enhanced by DC-biased sinusoidal excitations, to amplify the immunoreaction.
Guo and Liu et al. introduced a dual-ratiometric electrochemical aptasensor for the simultaneous detection of two organophosphorus pesticides, malathion (MAL) and profenofos (PRO) (Li et al., 2023).This sensor integrates an innovative hairpin tetrahedral DNA nanostructure (HP-TDN) with metal ions and nanocomposites, building a complex sensory framework that enhances signal amplification.The sensor's architecture utilizes AuNPs embedded in a zeolitic imidazolate framework (ZIF-8) to form an Au@ZIF-8 nanocomposite, significantly boosting conductivity and biocompatibility.The HP-TDN structure, tagged with TH, provides specific binding sites for the Pb 2+ and Cd 2+ tags linked to the MAL and PRO aptamers.When MAL and PRO are present, their corresponding aptamers bind to these analytes and dissociate from HP-TDN's complementary strands, leading to reduced oxidation currents for Pb and Cd.Thionine's oxidation current remains stable and serves as a reliable reference, enabling ratiometric measurements.The ratios of the oxidation currents (I Pb /I TH and I Cd /I TH ) are used to quantify the concentrations of MAL and PRO.
Ratiometric electrochemical sensors are also employed to detect various other types of pesticides, including the neonicotinoid imidacloprid (Liu et al., 2021;Zhang et al., 2020), the carbamate carbaryl (Zhang, Zhang, et al., 2020), and the herbicide glyphosate (Zhao, Guo, Lan, & Liu, 2024).Zou et al. developed a novel signal on-off ratiometric electrochemical sensor to detect imidacloprid (IMI), enhancing selectivity and stability through molecularly imprinted polymer (MIP) technology (Zhang et al., 2020).The sensor uses 6-(Ferrocenyl)hexanethiol (FcHT) as an internal reference signal and creates an MIP film on a GCE through electropolymerization.This film specifically recognizes IMI by its shape and chemical functionalities.Initially, AuNPs are electrodeposited on the GCE to expand the area available for FcHT and MIP attachment.Subsequently, FcHT is self-assembled in a solution to form an FcHT/AuNPs layer.An MIP layer is then formed over this layer in the presence of IMI template molecules by electropolymerizing o-phenylenediamine, followed by acid treatment to remove the template molecules, thus creating a functional MIP/FcHT/AuNPs/GCE electrode.Under optimal conditions, the sensor exhibits a linear response range from 0.5 to 100 μM and a detection limit of 47 nM.Later, the same X.Hu et al. research group developed an IMI ratiometric electrochemical sensor based on a flexibly fabricated vibratory electrode module (Liu et al., 2021).This construction utilizes a composite modification of AuNPs, Prussian Blue (PB), and β-cyclodextrin (β-CD), with PB serving as the internal reference signal and β-CD as the molecular recognizer.

Biotoxins
Biotoxins are harmful substances produced by microorganisms, plants, or animals that can accumulate in food and water, posing significant health risks to humans.Monitoring these toxins is essential for food safety and public health because they can cause a variety of health issues including acute and chronic poisoning, neurological damage, immune suppression, and cancer.Ratiometric electrochemical sensors have been effectively employed to detect various biotoxins (Fig. 6).These include mycotoxins such as aflatoxins, ochratoxin A, zearalenone, patulin, deoxynivalenol, and citrinin; marine toxins like saxitoxin; bacterial toxins such as streptomycin; algal toxins including microcystin; and plant toxins like the Cry1Ab protein.

Mycotoxins
Aflatoxin B1 (AFB1), produced by molds, is a highly toxic chemical Fig. 5. Schematic illustration of the construction and detection principle of a ratiometric electrochemical immunosensor for phoxim (Su, Wang, et al., 2022).
Fig. 6.Molecular structures of various biotoxins detected by currently reported ratiometric electrochemical sensors in food products.
Upon specific binding with its aptamer, the conformational change in the aptamer causes the MB-labeled DNA strand to detach from the electrode surface, enhancing the signal from the Fc label while diminishing the MB signal.This "signal-on/off" mode simplifies the sensor's signal reading process and enhances the accuracy and sensitivity of detection.In the ECL system, CdTe/CdS/ZnS quantum dots and luminol serve as the signal sources, with horseradish peroxidase-modified gold nanorods acting as the quencher/enhancer.In the absence of AFB1, cDNA hybridizes with the aptamer, maintaining both ECL signals.When AFB1 is present, the aptamer binds with the target and releases the cDNA sequence, leading to an increase in the ECL signal from the quantum dots and a decrease from the luminol.
Using dual-detection modalities to construct a ratiometric sensing system is a highly effective strategy.Fang et al. developed an innovative ratiometric sensing approach based on ECL and electrochemical methods for detecting AFB1 (Lv et al., 2022).This strategy incorporates a newly synthesized ECL emitter, BPYHBF, integrated with a composite material of multi-walled carbon nanotubes (MWCNTs) within a ferrocene-modified phenolic resin metal-organic framework (Fc-MOF).The sensor employs a competitive immunoassay format, where BPYHBF serves as a luminescent tag competing with the target antigen.This configuration provides robust ECL signals in the presence of tri-npropylamine.Additionally, the MWCNTs/Fc-MOF is used as the sensing base to enhance the internal reference electrochemical signal, thus allowing for self-calibration to enhance sensitivity and reliability.Recently, Feng and Cao et al. developed a dual-mode electrochemical/ photoelectrochemical (EC/PEC) aptasensor for the highly sensitive detection of AFB1 (Fig. 7) (Zhang et al., 2024).The sensor was designed using a novel composite material, Au NPs/PC ZIF-8 -ZnO, synthesized from ZIF-8 precursors integrated with ultrasmall gold nanoparticles (Au NPs) to enhance the sensing substrate.Thiolated MB-labeled single-stranded DNA (ssDNA-MB) was anchored to the composite via Au-S bonds.This ssDNA-MB hybridized with a Fc-labeled aptamer (Apt-Fc) to form double-stranded DNA (dsDNA).In the presence of target AFB1, the dsDNA dissociates due to the specific binding of AFB1 to Apt-Fc, leading to the detachment of the Fc label from the electrode surface and the reformation of the hairpin structure of ssDNA-MB.This causes a decrease in the Fc oxidation peak current and an increase in the MB oxidation peak current in the EC mode, facilitating ratiometric electrochemical detection.Additionally, the sensitization effect of MB enhances the photoelectrochemical current response of the electrode, allowing for a "signal off-on" PEC detection mode.Liu and You et al. utilized a hybridization chain reaction (HCR) to amplify the detection signal, using MB and Fc as electroactive indicators to generate response and reference signals, respectively, constructing a label-free, homogeneous electrochemical aptasensor for detecting AFB1 in grains (Zhu, Liu, Li, Chen, & You, 2022).Additionally, other nucleic acid technologies such as exonuclease I (Exo I) and a DNAzyme-driven tripedal DNA walker were also employed to build ultra-sensitive ratiometric electrochemical sensors for AFB1 (Cui et al., 2022;Liang et al., 2024;Lv et al., 2023;Wang, Qian, An, Lu, & Huang, 2019).
Ochratoxin A (OTA), produced by fungi like Aspergillus and Penicillium and found in grains and various foods, is monitored for its kidney Fig. 7. Schematic illustration of the construction and detection principle of a ratiometric electrochemical and photoelectrochemical dual-mode aptasensor for AFB1 (Zhang, Li, et al., 2024).
damage, immunosuppression, and potential carcinogenic effects to ensure public health safety (Schrenk et al., 2020).In 2018, Wang and Lin et al. developed a highly reproducible ratiometric aptasensor that combines ECL and electrochemical signals to detect OTA (Lin et al., 2018).This sensor design utilized two probes: a Ru(phen) 3 2+ labeled working probe (Ru(phen) 3

2+
-wp) generating the ECL signal, and a MB labeled reference probe (MB-rp) generating the electrochemical signal.Liu and You et al. reported a ratiometric electrochemical aptasensor for detecting OTA, employing dual signal amplification through MB and Fctagged DNA.The specific binding of OTA induces changes in DNA structure, altering the electrochemical signals on the sensor, enabling highly sensitive and precise detection of OTA (Zhu et al., 2020).Utilizing DNA strands labeled with MB and Fc, as well as nucleic acid hybridization and strand displacement reactions, other highly sensitive ratiometric electrochemical sensors have also been developed for the detection of OTA (Liang et al., 2021;Liu et al., 2023).
Recently, novel nanostructured materials have been successfully employed to develop electrochemical sensors for detecting OTA.Yang and Hu et al. developed a sensor based on DNA tetrahedral nanomaterial (NTH), combined with zirconium metal-organic framework (UiO-66) as a signal tag for the detection of OTA (Li et al., 2022).In this sensor, UiO-66 and an electrolyte solution of [Fe(CN) 6 ] 3− /4− are utilized as the signal probe and the internal reference probe, respectively.Wu and Hu et al. reported a novel dual-signal ratiometric electrochemical aptasensor based on cobalt metal-organic frameworks (Co-MOFs) for the detection of OTA in foods (Guan et al., 2023).This biosensor utilizes Co-MOFs as the signal probe and MB as the internal reference probe, demonstrating high sensitivity and reproducibility.The sensor design incorporates DNA walker technology along with a cyclic nucleic acid enzymatic cleavage process to amplify the detection signal for OTA.Recently, Wang et al. developed a novel high-sensitivity dual-signal ratiometric electrochemical aptasensor for OTA, utilizing nanoporous gold (NPG) (Wang et al., 2024).The high surface area and conductivity of NPG facilitate the attachment of a greater number of aptamers, significantly amplifying the signals.Furthermore, Suo and Wei et al. developed a ratiometric electrochemical aptasensor for detecting ZEN in corn flour, using MB and Ag + as dual-signal probes, enhanced with Au@Pt/Fe-N-C nanocomposites (Suo et al., 2022).This design significantly improves the sensor's sensitivity due to the large surface area and high conductivity of the nanocomposites.
Antigen-antibody recognition and molecular imprinting technologies have also given rise to other ratiometric electrochemical sensors for detecting OTA.Xu and Liu et al. developed a versatile immunosensor capable of detecting OTA, AFB1, and zearalenone (ZEN) (Qileng et al., 2020).This sensor combines PEC and electrochemical signal responses, utilizing the PEC signals generated by cadmium sulfide nanoparticles under light-activated conditions and signals produced by CuS electrochemical reactions for dual validation and ratiometric detection of target analytes.Additionally, the same research group introduced a novel ratiometric immunosensor for distinguishing different ochratoxins in mixtures (Qileng et al., 2020).Using the molecular imprinting technology, Zeng et al. developed another Ratiometric sensor with enhanced specificity for detecting OTA (Hu, Xia, Liu, Chen, & Zeng, 2022).
Ratiometric electrochemical sensors have been developed to detect other mycotoxins such as patulin, deoxynivalenol, and citrinin.Wang and Liu et al. have detailed a dual-mode aptasensor combining EC and PEC methods for the ratiometric detection of patulin (PAT) (Liu et al., 2023).This sensor utilizes CdTe quantum dots sensitized with gold nanorods (CdTe QDs/Au NRs) to generate photocurrent, while a ferrocene (Fc)-tagged aptamer produces redox current.The quantitative detection of PAT is achieved by the ratio of PEC to EC signals.Dai et al. developed a ratiometric electrochemical immunosensor based on ironbased metal-organic frameworks (Fe-MOF) and gold nanoparticles (AuNP) for the detection of deoxynivalenol (DON) in cereal products (Shu et al., 2024).The sensor uses Fe-MOF/AuNP as the signal probe combined with immunoglobulin G (IgG)-tagged, and [Fe(CN) 6 ] 3− /4− as the internal reference probe, forming a competitive capture and detection system for DON.Zeng et al. developed a novel ratiometric electrochemical sensor for selective detection of citrinin (Hu, Liu, Xia, Zhao, & Zeng, 2021).Poly(thionine) serves a dual role as both the MIP and the reference probe, while [Fe(CN) 6 ] 3− /4− acts as the indicator probe, whose signal decreases upon addition of citrinin, allowing for ratiometric measurement of citrinin levels.

Pathogens
Pathogens, including bacteria, viruses, fungi, and parasites, can contaminate food and water, leading to illnesses ranging from mild gastroenteritis to severe diseases like meningitis and sepsis.Due to their high sensitivity and specificity, ratiometric electrochemical sensors have been developed to detect pathogens, specifically targeting organisms like Salmonella and Staphylococcus aureus.
Salmonella is a common bacterium that can be transmitted through consuming contaminated food or water, causing symptoms such as diarrhea and fever, and requiring antibiotics in severe cases.Currently, there are several studies on ratiometric electrochemical sensors for detecting Salmonella, which specifically target biomarkers such as Salmonella esterase and characteristic nucleic acids.Huang et al. developed two ratiometric electrochemical probe molecules, named Sal-CAF and Sal-NBAF, based on ferrocene moieties specifically for detecting Salmonella esterase (Kumaravel, Jian, Huang, Huang, & Hong, 2022).The octyl esters in Sal-CAF can be cleaved by the Salmonella esterase, initiating a trimethyl lock mechanism that releases electron-rich aminoferrocene derivatives.This action shifts the DPV peak potential from 0.29 V to − 0.08 V (vs Ag/AgCl), facilitating a ratiometric response.Furthermore, the same research group enhanced the sensitivity for detecting Salmonella esterase by combining the highly selective probe Sal-CAF with an electrode modified with graphene quantum dots-gold nanoparticles, achieving a detection limit of 35.62 × 10 CFU•mL − 1 (Kumaragurubaran et al., 2023).Yang and Zhang et al. developed a novel ratiometric electrochemical biosensor designed for rapid and sensitive detection of Salmonella enterica serovar Typhimurium in food products (Yu, Yuan, Zhang, Guo, Lu, Yang, et al., 2022).This biosensor utilizes saltatory rolling circle amplification (SRCA) paired with a dualsignal electrochemical readout to improve both specificity and sensitivity.It features an electrode surface immobilized with mercaptomodified β-cyclodextrin and Au NPs, providing a robust platform for SRCA reactions.Subsequently, the same research group combined SRCA with the CRISPR/Cas12a system to develop a novel ratiometric electrochemical biosensor for the ultrasensitive and specific detection of Salmonella (Zheng et al., 2023).By effectively integrating rapid SRCA amplification and the trans-cleavage capabilities of Cas12a, signal amplification was achieved, thereby eliminating non-specific amplification.The sensor was able to detect Salmonella at concentrations as low as 2.08 fg•μL − 1 in pure cultures.
Staphylococcus aureus (S. aureus) is a common pathogen that can cause various infections, ranging from minor skin infections to lifethreatening diseases such as pneumonia, meningitis, and sepsis.Wu and Yang et al. developed a dual-mode ratiometric aptasensor based on DNAzyme activation recycling for the ultrasensitive and accurate detection of S. aureus (Shan, Kuang, Feng, Wu, & Yang, 2023).This sensor utilized ECL and electrochemical signals for dual-mode detection to achieve a ratiometric response to the target.Specifically, the probe DNA labeled with an ECL emitter (probe 2-Ru) contains a blocked DNAzyme and is partially hybridized with the aptamer, which is then captured by the probe DNA labeled with an EC indicator (probe 1-MB) on the electrode surface.When S. aureus is present, the conformational change of probe 2-Ru activates the blocked DNAzymes, leading to the recycling cleavage of probe 1-MB, bringing the ECL tag closer to the electrode surface.Due to the reverse change tendencies of the ECL and EC signals, the sensor achieves quantitative detection of S. aureus.Meanwhile, the same research group developed another highly sensitive ratiometric electrochemical sensor for detecting S. aureus based on dual DNA recycling amplification and Au NPs@ZIF-MOF-modified electrodes (Shan, Xie, Zhou, Wu, & Yang, 2023).

Conclusions and perspectives
Ratiometric electrochemical sensors represent an advanced electroanalytical method that effectively overcomes the background noise issues inherent in traditional electrochemical sensors by utilizing the ratio of signals from multiple electrochemically active substances, thus enhancing measurement accuracy and repeatability.This review comprehensively discusses the application of ratiometric electrochemical sensors in food analysis, assessing their unique advantages in food safety and quality detection.We have explored the two main types of ratiometric electrochemical sensors: the internal reference type and the dual-signal response type.Moreover, the review highlights emerging methods for enhancing sensor specificity using antibody recognition, aptamer binding, and molecularly imprinted polymers, as well as signal amplification techniques involving nucleic acid technologies and nanomaterials, opening new pathways to improve sensor sensitivity and selectivity.
Despite the widespread attention ratiometric electrochemical sensors have received in food analysis in recent years and the significant progress made, several challenges remain, such as the complexity of sensor design and manufacturing, the lack of effective in situ real-time analysis, and the limited range of detectable targets.Future developments could focus on several directions: (1) Developing electrode base composite functional materials with more controllable structure and performance that not only provide stable reference signals but also catalyze target reactions to generate response signals, thereby simplifying sensor construction and operation; (2) Constructing ratiometric electrochemical sensors for food analysis based on microelectrodes, utilizing the characteristic of in situ analysis to facilitate real-time monitoring of food; (3) Developing detection systems based on responsive organic electrochemical probes, where organic small molecules with precise structures and adjustable properties, coupled with different recognition groups, could allow the use of single probe molecules to conveniently detect various analytes.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 2 .
Fig. 2. Schematic illustrations of the molecular structures of various types of food additives detected by currently reported ratiometric electrochemical sensors.

Fig. 3 .
Fig. 3. Schematic illustration of the construction and detection principle of a ratiometric electrochemical sensor for TBHQ based on Co NC/CNT/MB composite(Zhang, Liu, et al., 2024).

Table 1
A summary of the ratiometric electrochemical sensors for food analysis.