Abstract

As a kind of biosensor which is easy to produce and use, lateral flow immunoassay (LFIA) has been favored by people for a long time and applied in various fields such as disease diagnosis, drug testing and environment monitoring. However, due to its low sensitivity, it doesn't quite meet the requirements of some important biomarkers with a very low cutoff value, which limits its application. In this study, multi-layer surface functionalized nanospheres were designed to improve the sensitivity of LFIA. The BSA layer significantly increased the number of sites available for biotinylation. The streptavidin layer bound the inner and outer layers and was loaded with abundant Alexa Fluor 647 (a stable and efficient fluorescent dye). Biotinylated antibodies efficiently and firmly bound to streptavidin and formed the outermost layer. This method could achieve high loading efficiency of antibodies and fluorescent molecules, thus realizing detection signal amplification. The probes were applied to LFIA for the detection of cardiac troponin I (cTnI, one of the most representative cardiac markers) and the results showed high sensitivity (0.049 ng mL−1). The working range of this assay (the concentration of cTnI) was 0.049 to 50 ng mL−1. Besides, the performance in linearity (R2 > 0.99), accuracy (R2 > 0.975), repeatability (CV < 10%) and thermal acceleration stability also met clinical needs well. Therefore, this method provided a promising probe preparation scheme for fast and sensitive in vitro diagnosis.