(449f) Integrated Microfluidic-Electrochemical Sensor Prototype for Detection of Prion Biomarkers

Animal diseases pose significant economic, health, and security threats, potentially devastating livestock populations and jeopardizing food industries. Rapid and accurate diagnosis is critical for effective containment. Prion diseases, including Bovine Spongiform Encephalopathy (BSE), present particular challenges due to their infectious and fatal nature. Current detection methods, such as real-time quaking-induced conversion (RT-QuIC) and Western blotting, are laborious and unreliable. Addressing this pressing need, we present a comprehensive approach to animal disease detection by developing a modular, point-of-care (POC) microfluidic bio-detection system. This electrochemical sensing method utilizes a flow-through Nanoporous Capacitive Electrode microfluidic chip, offering a compact and efficient platform for on-site testing. The chip integrates carbon-based transducer material (CBTM) functionalized with aptamers specific to PrP, ensuring high sensitivity and selectivity in detecting the target biomolecule. The concentration of the target analyte in the sample can be determined by measuring the electrochemical response of the microfluidic chip before and after exposure to the target analyte. For this purpose, we have used electrochemical impedance spectroscopy in combination with equivalent circuit modeling. The variation in the charge transfer resistance due to the binding of the target analyte to the CBTM results in a reduction in the active electrode surface area, which is used as a quantifier for the target analyte concentration.

Using the automated ESSENCE detection system, the α-PrP^SC apta­mer functionalized CNT CBTM was run against decreasing concentrations of Pr^P protein in PBS buffer solution. While we aim to detect actual PrP^SC prions and degenerated Pr^P proteins, we have instead used PrP protein (precursor to the prion) as a surrogate. Through automated fluid control and Electrochemical Impedance Spectroscopy (EIS) data acquisition, our system provides a user-friendly, field-deployable solution for animal disease surveillance. Integrating single-walled carbon nanotubes (SWCNTs) as the CBTM further enhances the sensor's performance, offering rapid and reliable detection of PrP. The 45-mer Aptamer-functionalized SWCNTs demonstrate remarkable selectivity for PrP, enabling the accurate identification of BSE-related biomarkers. Our detection experiments show promising results, with a detection limit in the low ng/mL range, approximately five picomolar (pM), highlighting system sensitivity. A calibration curve generated from experimental data exhibits a linear relationship (R² ~ 1), demonstrating the system's quantitative capabilities.

The preliminary results underscore the feasibility of employing aptamer-functionalized SWCNTs in a microfluidic electrochemical sensor for BSE detection. The development of this system represents a significant advancement in animal disease surveillance, offering a rapid, sensitive, and reliable tool for on-site testing.

We incorporated a custom algorithm to identify optimal measurement conditions to determine signal stability during measurement. A stable EIS response is detected consistently by studying the signal drift using the variation in the root mean squared error of a particular time index signal to the baseline signal. A stable EIS response is consistently detected by analyzing signal drift using variation in the root mean squared error (RMSE) of time-indexed signals compared to a baseline. In this approach, we tested multiple flow rates (1.5 µL/min, 3 µL/min, and 5 µL/min) under both flow and no-flow conditions. Results suggest stabilization time increases with flow rate: ~30 minutes at 1.5 µL/min and ~15 minutes at 3 µL/min. After the flow is stopped, the signal stabilizes within 20-30 minutes, regardless of the rate.

As expected, the flow rate and presence/absence of flow affect specific electrochemical parameters of the system, primarily the electrochemical double-layer. Since multiple concomitant factors influence the system's behavior, identifying the electrochemical component of interest is crucial to achieving enhanced sensing.

Since multiple factors influence system behavior, identifying the key electrochemical component is crucial for enhanced sensing. A single-stranded DNA(ssDNA)-based sensor was employed to identify key protocol elements. The CBTM was functionalized with a 25-mer ssDNA probe, and complementary and non-complementary sequences were used as target and control, respectively.

The optimized protocol developed through this system improves detection capabilities, including detection limit and sensitivity. Our approach also lays the groundwork for future integration with sample preparation systems, enabling comprehensive surveillance and early detection of emerging threats. This study presents a novel strategy for animal disease detection, leveraging cutting-edge technology to address the growing challenges of infectious pathogens. We offer a versatile platform capable of transforming veterinary diagnostics by combining microfluidics, electrochemical sensing, and aptamer-based molecular recognition.