Detection of aflatoxin B₁ by immunochromatographic test strip based on CuS nanoflowers and nanobody

Aflatoxin B₁(AFB₁) is a toxic metabolite typically produced by Aspergillus flavus and parasitic fungi, not only contaminates crops but also readily enters the human body through the food chain, affecting human health. Given the widespread distribution, severe contamination, significant hazards to humans and animals, as well as the difficulty in removal of AFB₁, there is an urgent need for a convenient and sensitive detection method. In contrast to instrumental analysis methods, immunochromatographic test strip (ICTS) offers advantages such as short detection times, high efficiency, and no requirement for specialized personnel. In view of the wide applications of ICTS and the excessive variety and quantity of crops tested, it is necessary to reduce the cost of ICTS. Nanobody G8 was fused with an anti-digoxin antibody to construct the expression vector pET25b-G8-DIG, which was then transformed into E. coli BL21 for prokaryotic expression. Following nickel column purification, the synthesized G8-DIG fusion protein was confirmed to have a molecular weight of approximately 30 kDa through SDS-PAGE gel electrophoresis. The multifunctional fusion protein G8-DIG was able to bind to both AFB₁ antigen and digoxin (DIG) antigen. Copper sulfide nanoflowers (CuS Nanoflowers, CuS NFs) were synthesized by the reaction of copper nitrate trihydrate and thiourea, and the nanomaterial was confirmed to possess a floral structure with dimensions around 300 nm through TEM and SEM analysis. Optimization of the ICTS including the optimal mass concentration of CuS NFs solution, the amount of G8-DIG added, the mass concentration of the T-line, and the probe quantity revealed that the detection limit (LOD) of this method was 2.4 ng/mL, with a detection range of 6.87-96.21 ng/mL. The spiked recovery rate ranged from 86% to 110%, and the coefficient of variation (CV) was less than 10.9%. When tested on corn quality control samples, the recovery rate was 111.8%. Furthermore, the specificity of the method was assessed by detecting OTA, T-2, DON, and ZEN as interfering samples on the test strip, demonstrating excellent specificity of the developed method. Utilizing DIG-BSA in place of the secondary antibody traditionally used for the control line on test strip improved the stability and cost-effectiveness of the test strip. The selected CuS NFs and G8-DIG, serving as signal probes, not only enable the detection of AFB₁ but also reduce the cost of the test strip.