Accurate and real-time monitoring of biomarker proteins, such as Tumor Necrosis Factor (TNF) alpha, plays a vital role in early disease diagnosis, effective treatment design, and personalized health management strategies. However, existing detection methods, including enzyme-linked immunosorbent assay (ELISA), radioimmune assays (RIA), and polymerase chain reaction (PCR), have significant drawbacks regarding sensitivity, cost, time, and labor efficiency, emphasizing the urgent need for alternative biosensing techniques. Here, we present a mass-based biosensing approach utilizing aptamers for the real-time detection of proteins, using TNF-alpha as the model analyte. The recognition process is based on the selective binding of the target molecule to the aptamer's unique three-dimensional structure. By utilizing a quartz crystal microbalance (QCM) as the transducing element, real-time detection of target binding is translated into a linear decrease in resonant frequency due to the change in mass upon target binding. The developed aptasensor enabled real-time quantification of TNF-alpha with high reliability, sensitivity, and specificity. The sensitivity of the sensor ranged from 14.5 nM to 115.6 nM, in which a linear correlation between target concentration and frequency decrease rate was found. Successful sensor regeneration demonstrated potential for continuous measurements in solution. By directly monitoring the change in mass during sensor fabrication and upon analyte binding, this platform provides key mechanistic insights in the surface functionalization process during sensor fabrication and analyte binding kinetics during sensor operation. In the future, incorporation of alternative target receptors, by simply changing the aptamer sequence, can broaden the analyte spectrum, making this platform highly versatile. We hereby demonstrate a technology that can be utilized for various biosensing platforms upon minimal modifications, including electrochemical and optical systems, for a wide range of macromolecular analytes.

Endotoxin is a deadly pyrogen, rendering it crucial to monitor with high accuracy and efficiency. However, current endotoxin detection relies on multistep processes that are labor-intensive, time-consuming, and unsustainable. Here, we report an aptamer-based biosensor for the real-time optical detection of endotoxin. The endotoxin sensor exploits the distance-dependent scattering of gold nanoparticles (AuNPs) coupled to a gold nanofilm. This is enabled by the conformational changes of an endotoxin-specific aptamer upon target binding. The sensor can be used in an ensemble mode and single-particle mode under dark-field illumination. In the ensemble mode, the sensor is coupled with a microspectrometer and exhibits high specificity, reliability (i.e., linear concentration to signal profile in logarithmic scale), and reusability for repeated endotoxin measurements. Individual endotoxins can be detected by monitoring the color of single AuNPs via a color camera, achieving single-molecule resolution. This platform can potentially advance endotoxin detection to safeguard medical, food, and pharmaceutical products.