Development of Multiplexed Biosensors Using Beads Coated with Gold Nanoclusters and Carbon Dots

A multiplexed sensor using fluorescent materials is a good strategy to detect multiple antigens simultaneously to accurately diagnose a single disease with multiple biomarkers or to distinguish diseases with similar symptoms. To quantify each concentration of analytes, the fluorescent intensity of dyes labeling each antigen should be measured, and the intensity peak of dyes needed to be separated. Nowadays, bead-based microfluidic platforms have been investigated to make the detection easier and enable the multiplexing sensor. Because in these platforms, sandwich ELISA (enzyme-linked immunosorbent assay) took place in a plate with microwells and only one bead could enter the microwell, the concentration of each antigen could be easily quantified by counting the number of each bead captured in the microwell. However, conventional fluorescent dyes utilized encoded beads often had short lifespans, biocompatibility issues, and complex conjugation methods. Furthermore, the narrow Stoke’s shift of the dyes causes interactions between the dyes, which makes multiplexing difficult. Thus, the detection area or time has been divided to enable multiplexed sensors.
In these regards, gold nanoclusters or carbon dots were good candidates because they have high biocompatibility, photostability, easy synthesis methods, and long lifespan. Moreover, since gold nanoclusters have a large Stoke’s shift, using carbon dots together could minimize the interaction between fluorophores to create an efficient multiplexed biosensor. It can be allowed to fabricate multiplexed biosensors that could measure each antigen simultaneously under the same excitation wavelength. In this study, magnetic particles coated with fluorescent gold nanoclusters or blue carbon dots were introduced to the microwell for the development of multiplexed fluorescent biosensors. These platforms lead to an easier read-out of the information of multiple antigens simultaneously and higher sensitivity. Magnetic beads were coated with TEOS (Tetraethyl orthosilicate) containing carbon dots and gold nanoclusters to put an adequate amount of fluorophore in the shell for sufficient fluorescent intensity. Synthesis methods of magnetic particles coated with gold nanoclusters or carbon dots were optimized while controlling pH and solvents, and we characterized the stability and size distribution to ensure sensor reproducibility. Our multiplexed biosensor is a sensor that can accurately determine the type of disease with multiple biomarkers. If different types of antibodies are immobilized in the same space, the ratio of antibodies can be changed, and it can reduce the reproducibility of biosensors. Thus, the microwell will be coated with one type of antibody which can detect epitopes held by two analytes. Then encoded beads reacted with antigens will be flown to the microwell. In the detection line, the ratio of fluorescent intensity can be attained, and the amount of each antigen can be quantified by each fluorescent intensity. The measurement of each antigen simultaneously will be compared to the measurement of each one separately. By comparing fluorescent intensity and LOD (Limit of Detection), the accuracy of our multiplexed sensor platform can be checked. In our platform, the background noise which occurs in the interaction between the fluorophores can be efficiently reduced and can detect two antigens more sensitively.