Precision Agriculture: MXene-Based 3D Biosensors for Early Detection of Subclinical Mastitis in Dairy Cows
Authors: Ataei Kachouei Matin , Corl Benjamin A , Ali Azahar 
Published: August 2024

INTRODUCTION

Mastitis, inflammation of mammary glands, is caused by pathogens like Streptococcus and Staphylococci, posing an economic challenge in the US dairy industry. Subclinical mastitis (SCM), asymptomatic and hard to detect, leads to prolonged milk yield declines. SCM costs the US over $2 billion annually. Current tests include somatic cell count, California Test, and Wisconsin Mastitis Test, but they offer periodic, qualitative measurements with moderate sensitivities (less than 90%) and specificities (85%). Consequently, there is a pressing need for reliable, early, continuous, and on-site detection of SCM to enhance milk yield, control the spread of infection, and health management, and improve the well-being of dairy cows.
Herein, we developed a 3D-printed MXene sensor for the detection of mastitis, providing a high sensitivity and a ≤1 min response time. This tool can aid US livestock farmers in dairy herd management.

SENSOR FABRICATION

Device manufacturing comprises three steps. First, the CAD design of the sensor (Figure 2) guides printing using a 25 μm resolution extrusion-based 3D printer. A UV-curable epoxy resin forms the sensor base, with unique microstructures enhancing surface area (Figure 3). The sensor geometry incorporates a ~280 μm periodic lateral pattern for increased surface area and a 3D geometric diffusion interface. Uncontrollably, a random microfold pattern emerges due to the epoxy resin's photo-sensitivity. These microstructures, acting as microelectrodes, amplify the sensor's surface area, boosting analytical sensitivity.
Next, a 100nm gold layer is applied to the sensor base using an e-beam evaporator with a shadow mask. Sensing materials are then drop-casted onto each electrode. The NAGase sensing electrode is coated with Ti3C2 nanosheets (MXene) decorated with anti-NAGase. The pH sensing electrode modified with poly(3-octyl-thiophene) and is covered by a hydrogen ion-selective membrane (H-ISM). The reference electrode (RE) is constructed by applying Ag/AgCl paste onto grooves around each working electrode (Figure 2).

RESULTS AND DISCUSSION

First, surface morphologies and electrochemical characterizations were conducted. FE-SEM images depict the lateral pattern and micro-folds of the sensor base (Figure 3). EDS and mapping analyses confirm uniform MXene distribution on the gold surface. Moreover, cyclic voltammetry (CV) explores electrode electrochemical behavior in different media. CV of MXene with ferro/ferricyanide shows distinctive peaks for mediator oxidation and reduction. CV without a mediator reveals characteristic MXene oxidation peaks, highlighting the sensor's mediator-free capability for direct applications in dairy farms without need for mediator solution.
For pH sensing, open circuit potentiometry (OCP) was used. Figure 4 shows OCP response rising with increasing pH values. The calibration graph in Figure 4 establishes a linear correlation between pH values and OCP, aligning with the Nernstian equation for ion-selective electrodes. The pH sensor successfully measures milk sample pH, demonstrating remarkable selectivity in the complex milk matrix.
The NAGase biosensor employs two sensing modalities for laboratory and field applications. Electrochemical impedance spectroscopy with a mediator generates Nyquist plots for calculating charge transfer resistance (Rct) in the presence of NAGase. Figure 5 displays a calibration graph with a linear correlation between Rct and the logarithmic concentration of NAGase. The NAGase-anti-NAGase interaction forms immunocomplexes on the MXene surface, increasing resistance to the mediator's redox reaction. This feature serves as the sensing mechanism for NAGase, a potential biomarker in mastitis.
Due to MXene's self-oxidation tendency without a mediator, CV technique used as a mediator-free alternative for practical field applications. In Figure 6, voltammograms show decreasing oxidative and reductive currents with increasing NAGase concentration. Oxidative peak currents used as the signal, exhibiting a negative correlation with NAGase concentration. The immunocomplexes formation acts as an insulative shield, impeding redox reactions. This innovative sensor aims to reduce time and effort in detecting mastitis in dairy cows, controlling pathogen spread.

Figure 1