A Portable Dual-Mode Aptasensor for Rapid Detection of Influenza A Hemagglutinin

We report a highly portable dual modality aptasensing platform for the detection of surface glycoprotein, hemagglutinin (HA), of the influenza A virus. The sensing device consists of a colorimetric detection component composed of a microfluidic paper-based analytical device (μPAD) having four analyte testing areas, as well as a flexible, miniaturized three-electrode system. Both sensing components are brought together and encapsulated within a microfluidic holder, which increases the integrity of the device, mediates sample flow, and allows for ease of translation to point-of-care (POC) use. Colorimetric detection using the μPADs resulted in a detection limit of 0.62 μg/mL in buffer over a linear range of 0.75-50 μg/mL HA (R² = 0.9905), with stability over 30 days (up to 97.3% signal retention). The electrochemical component uses impedance spectroscopy to measure changes in charge transfer resistance (Rct) upon aptamer-target recognition using hexaamineruthenium(III) chloride as a redox probe. Through the addition of the electrochemical component, the detection limit was improved to 8.5 ng/mL in buffer over a linear range of 0.015-25 μg/mL HA (R² = 0.9730), with stability over 7 days (113.1% signal retention). When using the device with a modeled real sample medium of artificial human saliva, colorimetric analysis resulted in detection of HA down to 1.0 μg/mL, while the electrochemical component resulted in a detection limit of 14.9 ng/mL. Additionally, while carrying out tests in dilute human saliva composed of a fifty-fifty mixture of pooled human saliva and TE buffer, detection limits using the μPAD nearly matched the artificial saliva's calculated limit at 1.04 μg/mL and the electrochemical platform reached a comparable but even lower limit of detection at 4.11 ng/mL HA, displaying promise for real world applicability. Both sensing platforms exhibited excellent selectivity when used against other surface proteins of common viral pathogens. While using the combined device, there are five target-testing zones (4 colorimetric, 1 electrochemical), decreasing the likelihood of false-negative/false-positive outcomes.