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.

Digital enzyme-linked immunosorbent assay (ELISA) is a powerful analytical method for highly sensitive protein biomarker detection. The current protocol of digital ELISA requires multiple washing steps and signal amplification using an enzyme, which could be the potential drawback in in vitro diagnosis. In this study, we propose a digital immunoassay method, which we call “Digital HoNon-ELISA” (digital homogeneous non-enzymatic immunosorbent assay) for highly sensitive detection without washing and signal amplification. Target antigen molecules react with antibody-coated magnetic nanoparticles, which are then magnetically pulled into femtoliter-sized reactors. The antigens on the particles are captured by antibodies anchored on the bottom surface of the reactor via molecular tethers. Magnetic force enhances the efficiency of particle encapsulation in the reactors. Subsequent physical compartmentalization of the particles enhances the binding efficiency of antigen-carrying particles to the antibodies. The tethered particles show characteristic Brownian motion within a limited space by the molecular tethering, which is distinct from free diffusion or nonspecific binding of antigen-free particles. The number of tethered particles directly correlates with the concentration of the target antigen. Digital HoNon-ELISA was used with a prostate-specific antigen to achieve a detection of 0.093 pg/mL, which is over 9.0-fold the sensitivity of commercialized highly sensitive ELISA (0.84 pg/mL) and comparable to digital ELISA (0.055 pg/mL). This digital immunoassay strategy has sensitivity similar to digital ELISA with simplicity similar to homogeneous assay. Such similarity allows for potential application in rapid and simple digital diagnostic tests without the need for washing and enzymatic amplification.

Piezoelectric shear strain was measured for c-axis oriented epitaxial Pb(Zr,Ti)O 3 (PZT) thin films. The PZT films, with a composition near the morphotropic phase boundary (MPB), were epitaxially grown on (001) MgO substrates and then microfabricated into a rectangular shape by wet etching of the films. Lateral electrodes were deposited on both sides of the PZT films, to apply an external electric field perpendicular to the polarization. A sinusoidal input voltage of 100 kHz was applied between the lateral electrodes, and in-plane shear vibration was measured by a laser Doppler vibrometer. In-plane displacement due to shear mode piezoelectric vibration was clearly observed and increased proportionally with the voltage. Finite element method (FEM) analysis was conducted to determine the horizontal electric field in the PZT film, and the piezoelectric coefficient d 15 was calculated to be 440 Â 10 À12 m/V. The d 15 of the PZT film represents the intrinsic shear piezoelectric effect, which is slightly smaller than that of bulk PZT, due to the absence of extrinsic effects such as longitudinal and transverse piezoelectric strain or domain rotation.

Homogeneous digital immunoassay is a powerful analytical method for highly sensitive biomarker detection with a simple protocol. By using this method, we demonstrated the simultaneous multiple protein detection.

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. On the other hand, we have developed a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In this study, we present a method that enables the multiplex detection of proteins based on Droplet‐free Digital ELISA. First, two types of paramagnetic beads immobilized with antibodies specific to different target proteins were incubated with a sample solution, and target proteins were captured on their specific beads. Then, these beads were labeled with horseradish peroxidase (HRP), and tyramide substrate reacted with HRP on beads, resulting in products of this reaction deposited on those beads. This signal amplification on beads makes it possible to count the number of labeled beads digitally. We used this approach to simultaneously detect IL‐6 and HBs Ag with single molecule resolution. The obtained limit of detections were 0.1 pg/mL and 0.013 IU/mL, respectively. Our method has potential applications in simple in vitro diagnostic systems for simultaneous and high‐sensitive detection of protein biomarkers.

The orientation dependence of shear mode piezoelectric properties has been investigated for epitaxial Pb(Zr,Ti)O 3 (PZT) thin films with composition near the morphotropic phase boundary. (101)-and (111)-oriented PZT films were epitaxially grown on SrTiO 3 (STO) substrates by rf magnetron sputtering and microfabricated into rectangular-shaped specimens to apply a horizontal electric field using lateral electrodes. The application of a sinusoidal input voltage of 100 kHz generated in-plane shear vibration, which was measured using a laser Doppler vibrometer. Inplane displacement proportionally increased with applied voltage for each PZT film. When a horizontal electric field was applied to (101)PZT along the directions parallel and perpendicular to [1 1 10]STO, the shear piezoelectric coefficient d 15 was calculated to be 110 and 305 pm/V, respectively. On the other hand, d 15 of (111)PZT under the electric field parallel to [1 1 10]STO was calculated to be 160 pm/V. These results indicate that the shear mode piezoelectric coefficients of the epitaxial PZT films not only show large values compatible with bulk ceramics, but also strongly depend on the crystallographic orientation and the direction of the electric field. #

Digital homogeneous non-enzymatic immunosorbent assay (digital Ho-Non ELISA) is a new class of digital immunoassay. In this paper, we developed a multiparameter single-particle motion analysis method for a highly sensitive digital Ho-Non ELISA.

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