A novel ultrasensitive and simple amplified immunosensing strategy is designed based on a surface-enhanced fluorescence (SEF) nanohybrid made from covalently conjugated thionine–gold nanoparticles (GNP–Th), as a novel amplified fluorescence label, and magnetic nanoparticles (MNPs), as a biological carrier, used for hepatitis B virus surface antigen (HBsAg) detection. This immunosensing strategy operates on the basis of the capture and then release of the amplified fluorescence label. Capturing of the antiHBs-antibody (Ab)-modified GNP–thionine hybrid (GNP–Th-Ab) is carried out through the formation of a two-dimensional (sandwich) probe between this amplified label and antiHBs-antibody-modified magnetic nanoparticles (MNP-Ab), in the presence of a target antigen and using an external magnetic force. Afterward, releasing of the captured fluorescence label is performed using a protease enzyme (pepsin) by a digestion mechanism of grafted antibodies on the GNP–thionine hybrid. As a result of antibody digestion, the amplified fluorescent hybrids (labels) are released into the solution. To understand the mechanism of enhanced fluorescence, the nature of the interaction between thionine and gold nanoparticles is studied using the B3LYP density functional method. In such a methodology, several new mechanisms and structures are used simultaneously, including a SEF-based metal nanoparticle–organic dye hybrid, dual signal amplification in a two-dimensional probe between the GNP–thionine hybrid and MNPs, and a novel releasing method using protease enzymes. These factors improve the sensitivity and speed, along with the simplicity of the procedure. Under optimal conditions, the fluorescence signal increases with the increment of HBs antigen concentration in the linear dynamic range of 4.6 × 10–9 to 0.012 ng/mL with a detection limit (LOD) of 4.6 × 10–9 ng/mL. The proposed immunosensor has great potential in developing ultrasensitive and rapid diagnostic platforms.

Graphdiyne's (GDY's) outstanding features have made it a novel 2D nanomaterial and a great candidate for electronic gadgets and optoelectronic devices, and it has opened new opportunities for the development of highly sensitive electronic and optical detection methods as well. Here, we testified a non-covalent grafting strategy in which GDY serves as a charge carrier layer and a bioaffinity substrate to immobilize biological receptors on GDY-based field-effect transistor (FET) devices. Firm non-covalent anchoring of biological molecules via pyrene groups and electrostatic interactions in addition to preserved electrical properties of GDY endows it with features of an ultrasensitive and stable detection mechanism. With emerging new forms and extending the subtypes of the already existing fatal diseases, genetic and biological knowledge demands more details. In this regard, we constructed simple yet efficient platforms using GDY-based FET devices in order to detect different kinds of biological molecules that threaten human health. The resulted data showed that the proposed non-covalent bioaffinity assays in GDY-based FET devices could be considered reliable strategies for novel label-free biosensing platforms, which still reach a high on/off ratio of over 10 4 . The limits of detection of the FET devices to detect DNA strands, the CA19-9 antigen, microRNA-155, the CA15-3 antigen, and the COVID-19 antigen were 0.2 aM, 0.04 pU mL −1 , 0.11 aM, 0.043 pU mL −1 , and 0.003 fg mL −1 , respectively, in the linear ranges of 1 aM to 1 pM, 1 pU mL −1 to 0.1 μU mL −1 , 1 aM to 1 pM, 1 pU mL −1 to 10 μU mL −1 , and 1 fg mL −1 to 10 ng mL −1 , respectively. Finally, the extraordinary performance of these label-free FET biosensors with low detection limits, high sensitivity and selectivity, capable of being miniaturized, and implantability for in vivo analysis makes them a great candidate in disease diagnostics and pointof-care testing.

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