Biphasic electrochemical immunoassay based on tyramine-DNA cascade signal amplification for detection of interleukin-8 in human saliva samples

Oral cancer poses a significant global public health challenge. The sensitive detection of salivary biomarkers, such as interleukin-8 (IL-8), holds promise for the early diagnosis of oral cancer. In this work, we developed a novel biphasic electrochemical immunoassay (BEIA) method for highly sensitive IL-8 detection in human saliva samples by integrating the conventional immunoassay method with homogeneous electrochemical sensing model. The recognition of IL-8 is achieved through a sandwich immunoreaction on the surface of magnetic beads (MBs). Subsequently, tyramide signal amplification (TSA) is performed to capture a large number of trigger DNA probes on MBs. These trigger DNA probes then initiate an exonuclease III-assisted signal amplification (EASA) reaction, resulting in the hydrolysis of numerous methylene blue (MB)-labeled signal DNA strands and release of MB into solution phase. The electrochemical signal of MB in solution phase is then monitored on an indium tin oxide (ITO) electrode using square wave voltammetry (SWV). With the introduction of TSA-EASA cascade signal amplification, IL-8 can be detected with high sensitivity. The developed method demonstrates a good linear relationship with IL-8 in the concentration range of 1.0 pg mL⁻¹ to 1000 pg mL⁻¹ and achieves a detection limit of 0.28 pg mL⁻¹ (S/N = 3). Additionally, the developed method exhibits high selectivity and can accurately determine IL-8 levels in clinical salivary samples, offering a promising alternative for point-of-care testing and early-stage diagnosis of oral cancer. Furthermore, the method can be easily adapted for the detection of other targets through the simple substitution of antibody pairs.