Visual authenticating hazardous adulterant phenolphthalein in slimming foods: Target-mimicking hapten epitope improved immunoassay

Screening for the hazardous adulterant phenolphthalein (PTH) in slimming foods is necessary. Herein, the linkage of the PTH target epitope with various spacer arms was proposed for hapten design, aiming to produce highly sensitive and specific antibodies targeting PTH. To understand the influence of spacer arms on epitope, comprehensive evaluations were conducted using computer-aided chemistry and animal immunization. The resulting antibody exhibited maximal half-inhibitory concentration (IC50) of 0.25 ng/mL. Then, a lateral flow immunoassay (LFIA) was established with detection capability for screening (CCβ) of less than 140, 240, and 25 ng/g for PTH in tea, instant coffee, and oral liquid, respectively. Furthermore, blind sample results agreed well with LFIA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Therefore, this work not only provides a robust tool for detecting PTH adulteration but also suggests that the careful pairing of spacer arms with hapten epitope is a key factor in advancing rational hapten design.