The growing need to optimize immunoassay performance driven by interest in analyzing individual cells has resulted in a decrease in the amount of sample required. Miniaturized immunoassays that use ultra-small femtoliter to attoliter sample volumes, a range known as the extended nanospace, can satisfy this analytical need; however, capturing every targeted molecule without loss in extended nanochannels for subsequent detection remains challenging. This is the first report of a successful extended nanofluidics-based quantitative immunochemical reaction capable of high capture efficiency using a femtoliter-scale sample volume. A novel patterning method using a photolithographic technique with vacuum ultraviolet light and low-temperature (100 °C) bonding enables patterning of functional groups for antibody immobilization before bonding, resulting in an immunochemical reaction space of only 86 fL. Reaction rate analyses indicate a decrease in the required sample volume to 810 fL and improvement in the limit of detection to 3 zmol, 5-6 orders of magnitude better than possible with the microfluidic immunoassay format. Highly efficient (near 100%) immunochemical reactions on a seconds time scale are possible due to the nm-scale diffusion length, which should be advantageous for the analysis of ultra-low-volume samples.

Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

Single molecule analysis is desired in many areas that require the analysis of ultra-small volume and/or extremely low concentration samples (e.g., single-cell biology, medicine diagnosis, virus detection, etc.). Due to the ultra-small volume or concentration, the sample contains only single or countable analyte molecules. Thus, specific single molecules should be precisely processed and detected for analysis. However, except nucleic acids, most molecules are difficult to amplify, and a new analytical methodology for specific single molecules is thus essential. For this, efficient chemical processing and detection, which are important analytical elements, should be developed. Here, we report a single-molecule ELISA (enzyme-linked immunosorbent assay) device utilizing micro/nanofluidic technology. Both chemical processing and detection were integrated into an ultra-small space (10 nm in size), and the integration allowed precise processing (∼100% capture) and detection of a specific single molecule (protein) for the first time. This new concept and enabling technology represent a significant innovation in analytical chemistry and will have a large impact on general biology and medicine.

Understanding nanoconfinement phenomena is necessary to develop nanofluidic technology platforms. One example of nanoconfinement phenomena is shifts in reaction equilibria toward reaction products in nanoconfined systems, which have been predicted theoretically and observed experimentally in DNA hybridization. Here we demonstrate a convection-limited nanofluidic immunoassay that achieves total capture of a target analyte and an apparent shift in the antibody-antigen reaction equilibrium due to nanoconfinement. The system exhibits wavefronts of the target analyte that propagate along the length of the nanochannel at a velocity much slower than that of the carrier fluid. We apply an analytical model describing the propagation of these wavefronts to determine the density of capture antibody binding sites in the enclosed nanochannel for a known concentration of the target analyte. We then use this binding site density to estimate the concentration of solutions with 5× and 10× less analyte. Our analysis suggests that nanoconfinement results in a preference toward binding of the target analyte with the surface-grafted capture antibody, as evidenced by an apparent reduction in the equilibrium dissociation constant. Our findings motivate the advancement of new biomedical and chemical synthesis technologies by leveraging nanoconfinement effects, and demonstrate a useful platform for studying the effect of nanoconfinement on chemical systems.