Abstract: In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC–BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC–BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC–BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075–0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%–103.7% with a relative standard deviation of 2.5%–3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC–BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.

一种新型莱克多巴胺-蛋白质载体结合技术在侧向流试纸条检测动物饲料中莱克多巴胺含量中的应用

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[41]List of electronic supplementary materials

[42]Fig. S1 Results of MALDI-TOF-MS analysis

[43]Fig. S2 Lateral flow strip test for RAC detection using the RAC–BSA⁽¹⁾ prepared via the Mannich reaction and the RAC–BSA⁽²⁾ prepared using previous method for the immobilization on test line

[44]Fig. S3 TEM image of AuNPs

[45]Fig. S4 UV-vis spectra of AuNPs and monoclonal antibody against RAC–AuNPs conjugated

[46]Fig. S5 Cross reactivity test by loading running buffer, RAC, SAL, CLB, TER, NE, and PHE

[47]Fig. S6 Storage stability after keeping the strip tests for 0 to 5 months after loading running buffer as negative control and 1 ng/mL of RAC as positive control

[48]Fig. S7 Effect of the volume ratio of methanol and running buffer of 0:100, 5:95, 10:90, 20:80, and 40:60 toward the lateral flow strip test

[49]Fig. S8 Relationship between the ∆gray intensity and the concentrations of the non-spiked (0) and spiked RAC in animal feed at 0.075–1.000 ng/g