EGaIn-Modified ePADs for Simultaneous Detection of Homocysteine and C-Reactive Protein in Saliva toward Early Diagnosis of Cardiovascular Disease

Homocysteine (Hcy) and C-reactive protein (CRP) are critical biomarkers for numerous chronic diseases, with cardiovascular disease (CVD) being the most prevalent. The ability to simultaneously detect both biomarkers in point-of-care settings is in high demand for CVD early diagnosis and prevention. Herein, we prepared the eutectic gallium indium (EGaIn) nanoparticles decorated with p-phenylenediamine (PPD) on the surface to facilitate the subsequent attachment of gold nanoparticles (AuNPs) to achieve EGaIn-PPD@Au, which was modified on the screen-printed electrochemical paper-based analytical devices (ePADs). Aptamers that are specific to Hcy and CRP were then immobilized on the EGaIn-PPD@Au surface to achieve the sensing interface on ePADs. The presence of EGaIn-PPD@Au significantly enhanced the electrical conductivity, leading to amplified electrochemical signals. This aptasensor demonstrated high specificity, capable of detecting Hcy in a range of 1-50 μM with a detection limit of 0.22 μM, and the detection range for CRP was 1-100 ng/mL with a detection limit of 0.039 ng/mL. The aptasensor also effectively detected Hcy and CRP in clinical saliva samples, yielding an area under the curve (AUC) of about 0.80 when the individual biomarker was considered and 0.93 when both biomarkers were taken into account. The positive correlation observed between salivary and blood concentrations of Hcy and CRP, coupled with their association with cardiovascular disease (CVD), suggested the potential of this methodology as a noninvasive point-of-care strategy for the early diagnosis of CVD.