Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology

Abstract

Abstract. Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product developmentfeedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

Enhancement of Molecular Transport into Film Stacked Structures for Micro-Immunoassay by Unsteady Rotation

“…For subsequent reaction steps, the 3D-Stack can be easily moved to the adjacent well containing the reagents; finally, to quantify the enzyme reaction, the 3D-Stack is removed, and the remaining reaction solution in the wells can be read using a conventional plate reader. In our previous studies, the detection sensitivity of the 3D-Stack was higher than that of conventional methods when measurements were performed with the same reaction time as conventional methods [18,19]. Suzuki focused on the flow between films and suggested that there is a trade-off between surface area increase and flow rate increase, which affects the sensitivity of ELISA [20].…”

Direct immunoassay on a polyester microwell plate for colorimetric detection of the spike protein in swab and saliva samples

“…Developing new substrates for immunoassays can contribute to creating affordable and accurate biosensors that may allow quick diagnosis. 4,7,8,[19][20][21][22] Colorimetric immunoassays on inexpensive platforms have been demonstrated to be promising for POC detection of SARS-CoV-2. A recent example showing the development of wax-printed ELISA paper plate assay was successfully demonstrated to detect the S protein in saliva samples.…”

Design of 3D Structure Membrane for the Increased Sensitivity in Enzyme Linked Immunosorbent Assay (mELISA)

“…The ability of ELISA explains the use of an enhanced molecule absorption area for a greater bonding area between the solid support and antibody. The 3D printed fabricated membrane provided enhanced molecule detection sensitivity as compared to that of the conventional ELISA [9]. In addition, gold nanoparticles (AuNPs) were used as probes of the antibody-conjugated with HRP (horseradish peroxidase) in order to enhance the detection of the area of breast cancer biomarker [10].…”