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Beads-on-a-Tip testing for ultrasensitive antigen detection across a large dynamic range
Ziwei Wu1, Yangjian Cai1, Yitong Zhao1,2
1Institute for Biomedical Materials and Devices (IBMD), Faculty of Science University of Technology Sydney Sydney New South Wales Australia.
Insights
This study introduces a novel Beads-on-a-Tip design for ultra-sensitive antigen detection, significantly improving lateral flow immunoassay (LFIA) sensitivity for point-of-care testing (POCT). The new method achieves a lower limit of detection than existing LFIAs.
Area of Science:
- Biomedical Engineering
- Analytical Chemistry
- Point-of-Care Diagnostics
Background:
- Lateral flow immunoassays (LFIAs) are widely used for point-of-care testing (POCT) due to their low cost and ease of use.
- Current LFIAs often lack quantitative capabilities and suffer from limited sensitivity (nanogram range) due to passive fluidics and non-specific binding.
- Nitrocellulose membranes in traditional LFIAs contribute to high background noise and reduced assay performance.
Purpose of the Study:
- To develop an enhanced LFIA platform with improved sensitivity and quantitative capabilities for antigen detection.
- To overcome the limitations of passive capillary fluidics and non-specific binding in conventional LFIAs.
- To establish a novel diagnostic system for ultra-sensitive detection of biomarkers, using SARS-CoV-2 spike antigen as a model.
Main Methods:
- A novel 'Beads-on-a-Tip' design was developed, replacing nitrocellulose membranes with magnetic beads in a pipette tip.
- Capture antibodies were conjugated to magnetic beads for sandwich immunoassay, enabling active washing and enrichment of low-abundant antigens.
- Upconversion nanoparticles (UCNPs) were utilized as luminescent reporters to enhance detection sensitivity.
Main Results:
- The Beads-on-a-Tip design achieved a significantly reduced limit of detection (LOD) of 706 fg/mL for SARS-CoV-2 spike antigen.
- The assay demonstrated a broad dynamic range spanning over 7 orders of magnitude (500 fg/mL to 10 μg/mL).
- Optimization of incubation time and UCNP-to-bead ratio was crucial for enhancing immunocomplex formation and signal-to-noise ratio.
Conclusions:
- The developed Beads-on-a-Tip LFIA system offers a substantial improvement in sensitivity and quantitative accuracy for POCT.
- This novel approach effectively mitigates non-specific binding and background noise, leading to superior assay performance.
- The system presents a promising solution for ultra-sensitive, quantitative diagnostics, particularly for low-abundant biomarkers.
Abstract:
Lateral flow immunoassays (LFIAs) are low-cost, rapid, and easy to use for point-of-care testing (POCT), but the majority of the available LFIA tests are indicative, rather than quantitative, and their sensitivity in antigen tests are usually limited at the nanogram range, which is primarily due to the passive capillary fluidics through nitrocellulose membranes, often associated with non-specific bindings and high background noise. To overcome this challenge, we report a Beads-on-a-Tip design by replacing nitrocellulose membranes with a pipette tip loaded with magnetic beads. The beads are pre-conjugated with capture antibodies that support a typical sandwich immunoassay. This design enriches the low-abundant antigen proteins and allows an active washing process to significantly reduce non-specific bindings. To further improve the detection sensitivity, we employed upconversion nanoparticles (UCNPs) as luminescent reporters and SARS-CoV-2 spike (S) antigen as a model analyte to benchmark the performance of this design against our previously reported methods. We found that the key to enhance the immunocomplex formation and signal-to-noise ratio lay in optimizing incubation time and the UCNP-to-bead ratio. We therefore successfully demonstrated that the new method can achieve a very large dynamic range from 500 fg/mL to 10 μg/mL, across over 7 digits, and a limit of detection of 706 fg/mL, nearly another order of magnitude lower than the best reported LFIA using UCNPs in COVID-19 spike antigen detection. Our system offers a promising solution for ultra-sensitive and quantitative POCT diagnostics.
