Sandwich assay based deployable devices for fluorescent biosensing of cytokines

Abstract

Cytokines are a large, diverse family of small soluble proteins (4-60 kDa) expressed by immune and nonimmune cells. They play significant roles in cellular signalling, activation, growth and differentiation, resulting in a wealth of information related to health and disease. Defects in the regulatory networks may cause fluctuation of cytokine levels and accompany in inflammation or diverse diseases, indicating that cytokine measurement could be an effective approach for characterizing the function of immune system, diagnosing and predicting disease, and evaluating the effects of treatment. However, detection of cytokines is challenging because of their low biological concentration (pg/mL), complicated interacting networks, and inhomogeneous distribution. The current methods for detection of cytokines are mainly based on immunoassay techniques. Among them, enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) have become the most common cytokine quantification tools due to the simple protocols and relatively high sensitivity (pg/mL). However, all these approaches were limited on multiplex monitoring of in vivo cytokines with ultralow concentration (fg/mL) and superb spatial resolution. In this project, four novel biosensors have developed for sensitive, multiplex and in vivo monitoring of cytokines, which include three published papers and two revised manuscripts. Optical fibre and stainless steel were applied as the substrates for the development of the deployable biosensors with the capability of in vivo application. Sandwich structure was designed with capture module (antibody) immobilized on the surface of substrates and detection module (antibody or aptamer) conjugated with fluorescent reporter for signal amplification and readout. We first tested the sandwich-based biosensors in in vitro experiments by assessing and optimising their performance in PBS buffer, serum, plasma, saliva, perspiration, and whole blood. After validation of in vitro work, we applied the biosensors for in vivo monitoring of cytokines in rat brain or spine cord for the study of chronic pain or the development of diseases. The findings indicated that the designed sensing devices have the potential to be used for sensitive and multiplex monitoring of analytes (such as cytokines) in clinical practice and help with the better understanding of various disease conditions.