Wash- and Amplification-Free Digital Immunoassay Based on Single-Particle Motion Analysis

Abstract

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.

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

“…Akama et al [138] combined tethered particle monitoring with an immunoassay in femtoliter arrays.I nt he "digital homogeneous non-enzymatic immunosorbent assay" (HoNon-ELISA), antibody-decorated magnetic particles were used to separate the analyte from the sample matrix. Then, they were magnetically pulled into the microreactors of af emtoliter array chip for the confinement of the antibodyantigen reaction.…”

Fortschritte in der optischen Einzelmoleküldetektion: Auf dem Weg zu höchstempfindlichen Bioaffinitätsassays

“…Silver et al [137] entwickelten einen Sandwich-Immunassay zum Nachweis von PSA auf der Basis von TPM. Akama et al [138] kombinierten TPM mit einem Immunassay in Femtoliter-Arrays.I m" digitalen homogenen nicht enzymatischen Immunosorbent Assay" (HoNon-ELISA) wurden mit Antikçrper überzogene magnetische Mikrosphären verwendet, um den Analyten von der Probenmatrix zu trennen. Daraufhin wurden die Mikrosphären magnetisch in die Mikroreaktoren eines Femtoliter-Array-Chips gezogen, um die Antikçrper-Antigen-Reaktion einzuschränken.…”

A digital protein microarray for COVID-19 cytokine storm monitoring

“…16 Despite its full automation and ultra-high sensitivity, the high assay and instrumentation costs and large physical footprint prevent its application for timely near-bedside diagnosis. Several groups invented microfluidic platforms for lab-on-a-chip operation of digital assays [17][18][19][20] and notably, Yelleswarapu et al 21 demonstrated a mobile-phone-based, droplet microfluidic digital immunoassay for point-of-care (POC) settings. More recently, a few previous studies 22, 23 have implemented a digital assay platform applicable to the clinical treatment of a COVID-19-induced cytokine storm.…”