(C-102) Streamlined Proximity Extension Assay for Highly Sensitive Serum Protein Detection using Antifouling POEGMA Polymer-Coated Magnetic Beads

Introduction:: Magnetic bead-based proximity extension assay (magPEA) is a highly sensitive technique that involves the use of beads to capture proteins, along with a pair of antibody-oligo conjugated probes to detect proteins via PCR amplification [1]. Despite its high sensitivity, the performance of magPEA can be compromised by biofouling, a phenomenon in which biomolecules non-specifically adsorb onto the bead surface. To mitigate this issue, blocking reagents are often employed to treat the beads prior to use, and extensive washing steps are required to minimize nonspecific adsorption. These methods, however, introduce complexity into assay manipulation, prolong the total assay time, and impede high-throughput analysis. Here we describe a concise approach to minimize biofouling by coating magnetic beads with POEGMA polymer. Using the antifouling magnetic beads, we established a streamlined PEA assay and demonstrated sub-femtomolar detection sensitivity without beads blocking and washing.


Materials and Methods:: We generated the POEGMA polymer brush on amine-terminated Dynabeads via enzymatically degassed surface-initiated atom transfer radical polymerization (SI-ATRP) [2, 3]. To activate the beads, a bromo-initiator was first attached onto the surface, and we then utilized a glucose oxidase-assisted (oxygen consuming agent) SI-ATRP scheme to grow POEGMA polymer brushes in ambient environment for 2 hours. The beads were washed with 50% Tetrahydrofuran in PBS-Br buffer to remove residual POEGMA and stored in PBS before use. The capture antibody was immobilized onto the beads by mixing them with antibody solution and drying them under vacuum at a pressure of 100kPa for at least 8 hours, where the thick polymer brush allowed for the physical adsorption of the antibody without covalent coupling. We performed the magPEA assay without beads blocking, whereby for each reaction, we mixed 5×10^5 beads with 100 µL IL-8 standard protein spiked in 20% Fetal bovine serum (FBS) and incubated at room temperature for 15 min on an end-over-end rotator. The tube was then put onto a magnetic rack to pellet the beads and remove the supernatant. We added 20 µL PEA probe buffer to resuspend the beads and incubated at room temperature for another 15 min. Finally, we removed the PEA probe buffer and resuspended the beads in 10 µL PCR buffer and ran a one-pot extension-PCR amplification (Figure 1A).


Results, Conclusions, and Discussions:: In consistent with previous reports [2, 3], the POEGMA polymer brush significantly reduced nonspecific binding on magnetic beads compared to bare magnetic beads. Moreover, POEGMA brush simplifies capture antibody coating and eliminates the requirement of multiple steps of chemical activation and conjugation. Using FITC-labelled BSA as a proof-of-example, we observed a significantly greater immobilization capacity for BSA on POEGMA beads compared with the bare beads, suggesting that the protein can be effectively attached to the beads through physical adsorption under vacuum (Figure 1B). We then evaluated the antifouling effect of POEGMA beads by incubating them with FITC-BSA. The lower fluorescence intensity observed on the POEGMA beads in contrast to the bare beads also indicated the antifouling capability of polymer brush (Figure 1C). To assess the performance of our POEGMA beads in conducting PEA assays, we tested various IL-8 protein concentrations spiked in 20% FBS ranging from 10 ng/mL to 10 fg/mL. The Ct values obtained after PCR amplification were used to build the standard curve with a four-parameter logistic (4PL) fit. The limit of detection (LOD) was calculated by using the mean Ct value of negative control minus 3-fold standard deviations as the threshold. We achieved a LOD of 20.3 fg/mL for IL-8 protein, which is comparable to our previous magPEA assay, but with significantly simplified and shortened workflow. Figure 1D).

In conclusion, we developed an antifouling magnetic bead-based PEA assay for streamlined protein detection with high sensitivity. The simplified workflow makes our method a promising tool for automatic and high-throughput analysis, with the added benefit of being adaptable to portable devices for point-of-care protein detection [4].


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[1] Zhang et al. Analytical Chemistry 94.36 (2022): 12481-12489.

[2] Navarro et al. Journal of the American Chemical Society 141.7 (2019): 3100-3109.

[3] Van Andel et al. ACS applied materials & interfaces 9.44 (2017): 38211-38221.

[4] Zhang et al. Biosensors and Bioelectronics 195 (2022): 113656.