Magnetic Lab-On-A-Bead Fluorescence Assay For Detection Of Proteins In Low Concentration

Abstract

In modern world, the fate of toxic proteins in the environment needs a subject of great concern due to their potential detrimental effects on living organisms. These proteins can have significant impacts on ecosystems and human health. Immunoassays, which are diagnostic tests based on antibody-antigen interactions, offer a quantitative method for detecting various diseases. Given the extensive selection of antibodies, these assays have become a cornerstone of modern diagnostics. This technique involves creating a sandwich structure using two antibodies binding to a single specific antigen. This dual-antibody approach enhances the specificity and sensitivity of disease detection in immunoassays. Several technologies have been proposed for protein quantification, including ELISA, PCR, FTIR, SERS and flow cytometry. However, these methods still have limitations such as non-specificity, intricacy, and high expenses. As a result, an optical system has been developed to monitor toxic proteins in real-time, combining the advantages of IMS and fluorescence in a dual-mode assay for accurate protein quantification. We present a novel imaging technique that utilizes commonly used fluorescent complexes and incorporates digital analysis to detect analytes. We investigate a magnetic-recovery lab-on-a- bead protein detection method using 1 um Ser-mag carboxylate modified microparticles as both substrate and sensor. By employing microparticles, we harness the available spatial information to create a ratiometric signal that remains unaffected by variations in microparticle number and volume during the binding process. This eliminates the primary source of uncertainties typically encountered in traditional ensemble assays. The technique involves coating microbeads with capture molecules that specifically bind to the target protein, immersing them in the liquid sample, performing magnetic recovery, and then staining with a fluorescent dye. The bead-protein complexes are subsequently analysed using Laser Confocal Scanning Microscopy at the single bead level. Furthermore, we investigate the influence of various parameters on the measurement process and their impact on the results. This approach offers simple assay protocols, short incubation times, minimal reagent consumption, and eliminates the need for enzymatic signal amplification. To achieve direct counting and imaging of single molecules, we implemented a co-registration process where widefield and fluorescence images are combined. A mask is created, and particle counting is performed by blindly selecting particles in the widefield image to exclude any undesired fluorescence background. By estimating a threshold value, we enhance the fluorescent signal, thereby increasing the measurement sensitivity. Various metrics were employed to develop signal detection. We verified that the fraction of fluorescent beads scales with the concentration of the target protein in the liquid samples, reaching a limit as low as Zeptomolar (100 zM) for model assay systems. The practical benefits of the dual mode IMS/Fluorescence assay are demonstrated through the detection of bacterial proteins in environmental samples and clinical biomarkers in human serum.