# Enzymatic Protein Immobilization for Nanobody Array

## TL;DR

This paper introduces a new method for attaching proteins to surfaces using an enzyme, which improves detection accuracy and efficiency in biomolecule arrays.

## Contribution

A site-specific protein immobilization strategy using OaAEP1 for nanobody arrays is introduced, offering higher efficiency and specificity.

## Key findings

- The OaAEP1-based method enables site-specific immobilization of nanobodies on surfaces.
- The method was validated using a nanobody-targeting eGFP model system.
- The approach shows potential for rapid biomolecule detection and diagnostic applications.

## Abstract

Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody’s anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing OaAEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields.