One-step gold nanoparticle signal amplification in a 3D paper-based device for rapid detection of anti-interferon-γ autoantibodies.

The diagnosis of adult-onset immunodeficiency (AOID) associated with neutralizing anti-interferon-γ autoantibodies (anti-IFN-γ Abs) remains highly challenging due to the lack of specific clinical manifestations and routine laboratory detection methods. To address this issue, we developed a three-dimensional paper-based analytical device featuring one-step signal amplification (3D-osPAD) for the rapid and highly sensitive detection of anti-IFN-γ Abs. This device incorporates an in situ gold signal amplification strategy within the paper matrix, wherein Au ions from HAuCl4 are reduced to Au by 2-(N-morpholino)ethanesulfonic acid (MES buffer) and deposited onto gold nanoparticles (AuNPs) to enhance the colorimetric signal. To achieve one-step assay, the probe (AuNPs@IFN-γ) and all reagents required for the gold deposition reaction are pre-dried and stored in separate cellulose layers of the device. By leveraging the design of vertical microfluidic channels and hydrophilic-hydrophobic patterned paper zones, all reactions are automatically initiated, proceed sequentially, and complete upon a single sample loading step. This method effectively reduces the detection limit of anti-IFN-γ Abs from 0.1 μg mL to 0.01 μg mL, achieving a 10-fold increase in sensitivity, while the entire detection and signal amplification process can be completed within 10 min. Clinical validation demonstrated that this platform exhibited 100 % sensitivity and specificity in distinguishing samples from patients with AOID from healthy controls. The automated 3D-osPAD provides a simple means to monitor anti-IFN-γ antibody titers over time, thereby supporting clinical management and improving patient care.