Enhanced diffusometric immunosensing with grafted gold nanoparticles for detection of diabetic retinopathy biomarker tumor necrosis factor-α

Diffusometry is sensitive to geometric changes of particles. Target antigens can be detected through diffusivity changes resulting from their immunoreactions by functionalizing particle surface with a specific antibody. Considering that Brownian motion is a self-driven phenomenon, diffusometric immunosensing features several characteristics, such as no-washing steps, rapid detection, high flexibility, and high sensitivity. Until recently, this technique has been applied to many biomedical fields, such as monitoring of microorganism motility and diagnosis of diseases with biomarkers. Despite the abovementioned advantages, diffusivity changes in conventional diffusometry can be compromised at low-abundance antigens because proteins are much smaller than capture particles. To overcome such restriction, we present an improved diffusometric immunosensing technique by grafting additional gold nanoparticles (AuNPs) to capture particles to enhance size changes. A diabetic retinopathy (DR) biomarker, tumor necrosis factor-α was selected to evaluate the proposed immunosensing technique. Spherical AuNPs showed better enhancement than rod-like AuNPs during measurement. Limit of detection was improved by at least 100-fold down to 10 pg/mL. A dichotomous method was also developed to enable rapid detection and avoid tedious calibration. The relationship of concentrations between the two solutions used can be explicitly determined by comparing diffusivity of an unknown concentration of target molecules with that of a reference solution. Minimum discernible concentration reached as low as twofold higher or lower than basal concentration. Tear samples were collected from four volunteers, including three healthy subjects and one proliferative DR patient to prove the concept in diagnosis of the disease. All data showed good agreement with preset conditions. The technique eventually provides an insight into rapid diagnoses of diseases in the early stage.