Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus

Shifting the paradigm in RNA virus detection: integrating nucleic acid testing and immunoassays through single-molecule digital ELISA

“…These advancements are evident in several aspects of immunological assays, including reaction carriers, recognition elements, signal amplification, and reading methods. Of particular importance is the fusion of single-molecule detection technology and immunological assays, resulting in single-molecule counting immunity, which is also known as digital ELISA ( 38 , 39 ). This revolutionary technique has overcome the sensitivity limitations of traditional immunological assays, thus offering an extraordinary level of sensitivity and high resolution that macroscopic measurements have failed to achieve.…”

“…This strategic deviation circumvents the inaccuracies introduced by macro-level signal fluctuations, thereby providing an overwhelming advantage for detecting target molecules at low concentrations. In fact, for the same target molecule, the detection limit of digital assays is generally at least 1/1000 that of conventional assays ( 39 , 40 ).…”

Improvement of Sensitivity and Speed of Virus Sensing Technologies Using nm- and μm-Scale Components

“…After the first report, the research group of Quanterix Corporation continued to develop digital ELISA-related technologies, including the dynamic range broadening of assays by using the average enzyme number per bead measured with digital and analogue hybrid methods [ 97 ], polymer-based microwell sensing plate with fluidic channel for automation of assays [ 98 ], and the theoretical analysis of digital assays [ 99 ], and commercialized the automated digital ELISA systems such as the Simoa series [ 100 ]. Simoa series has been widely used in viral protein detection, which includes the detection of HIV (LOD = 2.5 fg/mL in serum) [ 101 ], influenza virus (LOD = 0.59 fmol/L and 0.99 fmol/L for nucleoprotein and hemagglutinin in buffer solution) [ 102 ], and SARS-CoV-2 (20 fg/mL for N protein in nasopharyngeal swab) [ 103 , 104 , 105 ]. The sensitivity of dELISA is approximately 1000-fold higher than analogue ELISA.…”

“…As mentioned above, dELISA for virus detection [ 101 , 102 , 103 , 104 , 105 ] achieves high sensitivity by combining the principle of digital detection and the efficient capture, concentration, and washing of targets with the help of magnetic particles. However, to increase the resolution of detection, it is preferable that only one magnetic particle is accommodated in a well.…”

“…Figure 8 shows an outline of the detection principle of MCDIA. MCDIA uses digital detection technology, similar to the dELISA [ 101 , 102 , 103 , 104 , 105 ] which has been described above. The well array used consists of pL-sized holes which can accommodate many magnetic particles per well.…”