Unique Fluorescent Protein-Encoded Microbeads with Supramaximal Encoding Capacity and High Sensitivity for Multiplex Bioassays

Fluorescence-encoded microbeads (FEBs) have been widely used as a critical component in multiplexed biomolecular assays. Here, we propose a simple, sustainable, low-cost, and safe strategy for preparing FEBs by assembling fluorescent proteins (FPs) onto magnetic microbeads (MBs) via chemical coupling. Combining the type of FP, the concentration of FP, and the size of the magnetic microbeads as encoding elements, an ultralarge encoding capacity with 506 barcodes was obtained. We demonstrate that the FP-based FEBs have good stability during long-term storage and tolerate the use of an organic solution. Multiplex detection of femtomolar ssDNA molecules was achieved via flow cytometry, and the detection procedure is simple and fast because it does not require amplification and washing strategies. The advantages of this advanced method for multiplex detections including high sensitivity, specificity, accuracy, repeatability, rapidity, and cost-effectiveness show a broad application prospect in basic and applied research fields such as disease diagnosis, food safety, environmental protection, proteomics, genomics, and drug screening.