The ability to detect low concentrations of analytes and in particular low‐abundance biomarkers is of fundamental importance, e.g., for early‐stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single‐molecule bioaffinity assays. While many review articles have highlighted the potentials of single‐molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real‐world applications as one should expect. This Review provides a theoretical background on single‐molecule—or better digital—assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single‐molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.

No abstract

Enzyme immunoassays are widely used for detection of analytes within various samples. However, enzymes as labels suffer several disadvantages such as high production cost and limited stability. Catalytic nanoparticles (nanozymes) can be used as an alternative label in immunoassays overcoming the inherent disadvantages of enzymes. Prussian blue nanoparticles (PBNPs) are nanozymes composed of the Fe[Fe(CN)]-based coordination polymer. They reveal peroxidase-like activity and are capable of catalyzing the oxidation of colorless 3,3',5,5'-tetramethylbenzidine in the presence of HO to form intensely blue product. Here, we introduce the method for conjugation of PBNPs with antibodies and their application in nanozyme-linked immunosorbent assay (NLISA). Sandwich NLISA for detection of human serum albumin in urine was developed with limit of detection (LOD) of 1.2 ng·mL and working range up to 1 μg·mL. Furthermore, the microbial contamination of Salmonella Typhimurium in powdered milk was detected with LOD of 6 × 10 colony-forming units (cfu)·mL and working range up to 10 cfu·mL. In both cases, a critical comparison with the same immunoassay but using native peroxidase as label was realized. The achieved results confirmed the suitability of PBNPs for universal and robust replacement of enzyme labels.