Decentralized quantification of neutrophils in whole blood using a rapid gold nanoparticle-based paper biosensor

Abstract

Here, we introduce a minimal-equipment method to rapidly quantify blood neutrophils using a gold nanoparticle (AuNP)-based origami paper biosensor (oPB). The oPB consists of two main components: AuNPs confined within a polystyrene sulfonate (PSS)-coated paper reservoir and an origami paper strip optimized for both cell capture and nanoprobe deposition. The AuNPs were functionalized with specific antibodies targeting a granulocytic membrane marker and validated for neutrophil enumeration in a sample. The integration of blood cells into the proposed AuNP-based oPB and their subsequent analysis were achieved by identifying a paper substrate that enables both cell flow-through and nanoprobe transfer across the origami layers, while ensuring stable cell capture under standard paper-based immunoassay protocols. The biosensor was calibrated using mock blood samples prepared with real specimens, ensuring that pre-processing steps mimicked those expected to be applied to actual clinical samples. The calibration demonstrated robust linearity across the evaluated range of cell densities, from pathologically low to normal neutrophil levels, and achieved both high sensitivity (limit of detection: 9.2 neutrophils·μL⁻¹) and high specificity, with no interference from the blood matrix or other leukocyte subsets. Finally, when tested with clinical blood samples, the biosensor accurately stratified patients exhibiting normal neutrophil levels, mild/moderate neutropenia and severe neutropenia, in less than 20 min, without requiring specialized laboratory instrument for either sample pre-processing or result readout. The results shown here pave the way for using the proposed AuNP-based oPB to rapidly analyze blood cells at any point of care, enabling early diagnosis and timely clinical intervention.