Label-free and ultra-sensitive H1N1 influenza virus immunosensor based on a triple-enhanced LIG/PB/PEDOT/Au microelectrode

Laser-induced 3D porous graphene enables scalable, low-cost biosensing. • PB/PEDOT/Au composite synergistically enhances stability, antifouling, and electroactivity. • The immunosensor achieves ultrasensitive detection of H1N1 and a wide dynamic range. • Label-free and pretreatment-free detection is successfully demonstrated in complex biological matrices. As a major cause of seasonal influenza, H1N1 virus requires advanced monitoring. Custom-patterned conductive graphene microelectrodes are essential for building relevant sensing platforms. In this study, a label-free H1N1 electrochemical immunosensor was developed based on a sequentially engineered laser-induced graphene (LIG)/Prussian blue (PB)/poly3,4-ethylenedioxythiophene (PEDOT)/Au nanocomposite microelectrode. Unlike previous studies that focused on single-component modifications, our approach integrates multiple functional layers to synergistically enhance signal stability, sensitivity, and antifouling performance. The LIG microelectrode was fabricated using a low-cost intelligent semiconductor laser, which directly patterned a three-electrode system on a polyimide (PI) film. Subsequently, PB, PEDOT, and gold nanoparticles (Au NPs) were sequentially electrodeposited onto the LIG electrode. PB served as an ultrasensitive electrochemical signal reporter, while the encapsulating PEDOT polymer layer not only stabilized the electrical signal of PB in neutral environments but also significantly enhanced the electrode's antifouling properties. To construct the label-free immunosensor, Au NPs were introduced to improve conductivity and were functionalized via Au–S–COOH bonding for the immobilization of H1N1 antibodies. Under optimal conditions, the LIG/PB/PEDOT/Au composite electrode exhibited excellent electrochemical activity and conductivity. The fabricated immunosensor demonstrated ultrahigh sensitivity with a wide concentration range from 0.1 fg mL −1 to 100 ng mL −1 , and an extremely low detection limit of 0.063 fg mL −1 . The results confirmed that the immunosensor possesses excellent selectivity, precision, and stability, and is thus suitable for the analysis of complex biological samples without the need for labeled probes and sample pretreatment. Therefore, this environmentally friendly and label-free immunosensing platform represents a significant advancement in point-of-care biosensing and demonstrates strong potential for rapid on-site screening and prevention of infectious disease outbreaks. A label-free immunosensor based on laser-induced graphene microelectrode modified with PB/PEDOT/Au nanocomposite enables ultra-sensitive detection of H1N1 virus in complex biological samples without pretreatment. Download: Download high-res image (120KB) Download: Download full-size image