Abstract: Rhamnolipids have hydrophilic groups and are composed of 1～2 molecules of rhamnose. They are a type of lipid substance containing rhamnose structure. As a biological surfactant, it has the characteristics of high surface activity, low toxicity, and natural degradation, and has great potential application value in the fields of petroleum industry, food, medicine, cosmetics, green agriculture, and ecological environment. This study used phenol-sulfuric acid method, anthrone-sulfuric acid method, and 3, 5-dinitrosalicylic acid method, replacing traditional spectrophotometers with enzyme-linked immunosorbent assay, to determine the mass concentration of rhamnolipids. Subsequently, a micro rapid method for detecting the mass concentration of rhamnolipids was developed, aiming to achieve rapid, accurate, and sensitive determination of rhamnolipids. The phenol-sulfuric acid method was chosen as the optimal detection method for the mass concentration of rhamnolipids, we successfully established a phenol-sulfuric acid micro detection system, and optimized the detection conditions. Using an enzyme-linked immunosorbent assay reader, it was found that the absorbance value at 479 nm and the slope of the standard curve were both high. This allows for rapid detection of the mass concentration of rhamnolipids, saving time, hazardous chemicals, reducing the generation of waste gases, and improving detection efficiency. With the successful application of the phenol-sulfuric acid micro detection system, we can achieve rapid screening of a large number of samples, with simple operation, reduced time and labor costs, and reduced dependence on expensive equipment. The system can quickly detect the mass concentration of rhamnolipids in the transformation solution, laying the foundation for screening high transformation strains.
Key words: rhamnolipids; phenol-sulfuric acid method; anthrone-sulfuric method; 3, 5-dinitrosalicylic acid method; high-throughput
（Acta Laser Biology Sinica, 2025, 34(5): 466-472）