Hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide nanocomposite as a substitute of enzyme-labeled second antibody for ultrasensitive detection of antibodies.

Enzyme-labeled secondary antibody is often used to amplify the output signal in the process of antibody detection. However, its preparation process is complex and time-consuming. Herein, we fabricated an innovative hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide (HRBGO) nanocomposite, used as a substitute of enzyme-labeled second antibody. The synthetic HRBGO was loaded with generous rhodamine B and modified with boronic acid. Therefore, the HRBGO could selectively label the carbohydrate chains of Fc fragment of primary antibody through specific boronate affinity recognition, and then perform signal output and amplification by releasing rhodamine B. To verify the practicability of HRBGO, trastuzumab as a humanized monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2) was selected as model antibody. A glycosylation site-blocked / HER2-immobilized magnetic nanoparticles (GHMN) was also prepared for selectively capturing trastuzumab from complex samples via specific immunoaffinity. Because the glycosylation sites of HER2 can also be labeled with the HRBGO by boronate affinity recognition, these sites were blocked by a masking agent to minimize the background signal. For specific and ultrasensitive detection of trastuzumab, the integration of GHMN and HRBGO was proposed and optimized in detail. Trastuzumab detection based on HRBGO consisted of three steps: specific capture, selective labeling, and output signal. The proposed strategy provided ultrahigh sensitivity with limit of detection of 0.35 fg mL-1 and was successfully applied in the detection of trastuzumab in spiked serum sample with recovery and relative standard deviation in the range of 98.7-103.8% and 3.8-6.0%, respectively. To assess universal applicability, the HRBGO was also successfully used for the determination of anti-SARS-COV2 RBD antibody in human serum sample.