Ratiometric Fluorescence Immunoassay Based on MnO₂ Nanoflakes for Sensitive and Accurate Detection of Tricaine

Abstract

Tricaine is a common anesthetic used in the long-distance transport of live fish. Recently, its negative impact on human health has aroused extensive concern. Thus, rapid and reliable techniques for tricaine residue analysis are essential to ensuring the quality of aquatic products. Herein, a specific anti-tricaine monoclonal antibody (Mab) was prepared. Then, a sensitive and robust ratiometric fluorescence ELISA (RF-ELISA) was constructed for detecting tricaine based on two MnO 2 nanoflakemediated (MnO 2 NFs) fluorogenic reactions. In the RF-ELISA protocol, MnO 2 NFs with oxidase-like activity can trigger the formation of fluorescent 2,3-diaminophenazine (oxOPD) with an emissive peak at 570 nm from non-fluorescent ophenylenediamine (OPD), while ascorbic acid (AA) can decompose MnO 2 NFs to lose their oxidase-mimicking activity, which is accompanied by the oxidation of AA into dehydroascorbic acid (DHAA). The subsequent reaction between the generated DHAA and OPD will result in the production of 3-(1,2-dihydroxy ethyl)furo [3,4-b]quinoxalin-1(3H)-on (DFQ), which has a potent emission peak at 445 nm. By virtue of the alkaline phosphatase (ALP) labeled on the antibody, which can catalyze the production of AA from ascorbic acid 2-phosphate (AAP), the concentration of tricaine can be linked to the variation of the RF signal (F 445 /F 570 ) via a competitive immunoreaction. After optimization, RF-ELISA displayed a detection limit (LOD) of 0.28 ng/mL toward tricaine (in buffer solution), which was 376-fold lower than that of the traditional colorimetric ELISA. For practical application, the LODs of RF-ELISA for tricaine detection in shrimp and tilapia samples were determined to be 2.8 and 5.6 ng/g, respectively. Recoveries for spiked shrimp and tilapia samples, as well as the validation data from LC−MS/MS, showed that RF-ELISA exhibited good accuracy, precision, and reliability. This RF-ELISA protocol opened up new ways for tricaine and other-target analyses in food safety detection.

Development of broad-spectrum immunoassay with monoclonal antibody to detect five eugenols and study of their molecular recognition mechanism

“…The performance of antibodies in binding to small molecules is widely recognized as being heavily dependent on the structure of the immunizing hapten ( Luo et al, 2017 ). Typically, an ideal hapten is considered to be one that retains as much of the target molecule's structure as possible after being conjugated to a carrier protein ( Liang et al, 2023 ). Previously, Shen et al designed and synthesized eugenol-containing hapten by introducing a four-carbon-length carboxylic spacer arm at the hydroxyl group site of eugenol, and a broad-spectrum mAb selective for four EUGs was obtained ( Shen et al, 2019 ).…”

“…Live fish is preferred by consumers due to their appealing taste and high nutritional content; however, the transportation and handling of live fish often subject them to stress, resulting in an elevated risk of disease and mortality, and a decline in their edible quality ( Liang et al, 2023 ). Anesthetics can induce a sedative effect in fish, reducing their mobility, thereby lowering their vulnerability to infection and improving their survival rate ( Fadillah et al, 2020 ).…”

Development of an Open Droplet Microchannel-Based Magnetosensor for Immunofluorometric Assay of Trimethoprim in Chicken and Pork Samples with a Wide Linear Range

“…32,33 Because fluorescence signal readout immunoassay technology has inherent advantages over colorimetric and absorbance-based analysis, including nondestructive mode of operation, rapid signal generation, high sensitivity, and selectivity, fluorescence immunosensors have become the most widely used and widely applied immunosensors. 34,35 Nevertheless, the majority of fluorescent immunosensors still have various limitations, including the requirement for complex signal response procedures, biotoxicity, strong fluorescent backgrounds, high light signal susceptibility, and inadequate homogeneity. 36 Therefore, the conversion of the fluorescent signal to a grayscale output may further improve the detection performance, such as the sensor's detection range and the signal output's stability, by reducing the sensitivity of the signal.…”

“…However, microfluidic immunosensors that have been constructed still need to address certain significant issues, namely, the accessibility of signal inputs, the type of data collection, and the complexity of signal processing. According to the signal output mechanism, colorimetric, fluorescent, and electrochemical signal systems are the most commonly reported readout devices for immunoassay techniques. , Because fluorescence signal readout immunoassay technology has inherent advantages over colorimetric and absorbance-based analysis, including nondestructive mode of operation, rapid signal generation, high sensitivity, and selectivity, fluorescence immunosensors have become the most widely used and widely applied immunosensors. , Nevertheless, the majority of fluorescent immunosensors still have various limitations, including the requirement for complex signal response procedures, biotoxicity, strong fluorescent backgrounds, high light signal susceptibility, and inadequate homogeneity . Therefore, the conversion of the fluorescent signal to a grayscale output may further improve the detection performance, such as the sensor’s detection range and the signal output’s stability, by reducing the sensitivity of the signal. − In addition, the coupled magnetic bead mode improves and broadens the linear range, while simplifying immunoassay procedures and effectively avoiding background interference.…”