Real-Time Detection of Streptavidin with a Novel Optical Cavity-Based Biosensor

Introduction: Recent advancements in optical cavity-based biosensors, especially those with two reflective surfaces, have significantly improved label-free detection technologies. Utilizing microfluidic and micro/nanofabrication technologies, these innovations have enabled the miniaturization of biosensors and the ability to detect multiple analytes from small sample volumes. The strong light confinement within optical cavity resonators makes them highly sensitive to changes in the surrounding medium. This sensitivity, combined with the ability to operate without labeling, makes these biosensors ideal for point-of-care diagnostics, offering rapid and accurate detection crucial for timely medical intervention.
In our research, we have developed a simple optical cavity-based biosensor using two reflective surfaces to dynamically measure biomarker binding. Utilizing differential detection methods, we have successfully identified biomolecular interactions, including organic molecule-protein and protein-protein interactions with enhanced sensitivity [1]. Additionally, we constructed a prototype of a cost-effective point-of-care biosensing system using economical optical components [2]. Building upon these achievements, we further enhanced the limit of detection (LOD) of our system by fine-tuning the reflectance of the surfaces and optimizing the differential detection approach [3]. Here, we employed an improved 3-aminopropyltriethoxysilane (APTES) functionalization process and demonstrated a real-time detection of streptavidin (STV) using biotin as the receptor molecule with an improved sensitivity compared to what we reported previously. The simplicity of optical design, combined with advanced detection methods, offers broad applicability across a range of biosensing applications, such as healthcare diagnostics and environmental monitoring.

Materials and

Methods: The silver layer (Ag) was deposited on a glass substrate and patterned using a photolithography process. Spin-on-Glass (SOG) was then spin-coated and cured. An SU8-2010 layer was patterned using a photolithography process to define the microfluidic channel. Finally, both substrates were bonded together using UV glue and cured under UV light. The microfluidic channel has a length of 20 mm, an average height of 12.65 µm, and a width of 1 mm. To immobilize the bioreceptor (sNHS-biotin), the surface was treated with a 0.095% solution of APTES in methanol for 20 minutes, then rinsed with methanol and baked at 110°C for 30 minutes. The silanized sensing region was then incubated in 10 µL of a 5 mg/mL sNHS-biotin solution for 1 hour and subsequently rinsed with deionized (DI) water. Areas outside the sensing region were blocked with 1% BSA for 30 minutes, rinsed with DI water, and dried under a gentle stream of N2 gas.

Results, Conclusions, and Discussions: Within the functionalized sensing region, a circular area with a diameter of 187 μm was used for data processing, calculating average intensities and the differential value (η). The setup consisted of two 10 mA laser diodes at wavelengths of 808 nm and 880 nm, a beam splitter, neutral filters, a mirror, and a camera (Figures 1a and 1b). The sample was positioned between the camera and the incoming laser light. Initially, the channel was filled with DI water to establish baseline data. Subsequently, solutions of different STV concentrations were injected into the inlet port of the channel, followed by rinsing with DI water. The intensity variations before, during, and after the injection of STV were recorded in real-time by the camera, and the last 2 minutes of data after the DI water rinse was used to report the results.
The experimental results from our optical cavity-based biosensor demonstrate its capability to detect real-time changes in differential value across STV concentrations ranging from 78 ng/ml to 2.5 µg/ml (Figure 1d). At the higher concentrations of 1.25 µg/ml and 2.5 µg/ml, significant signal persistence was observed, with average intensities of 0.0923 and 0.0910, respectively. This persistence likely results from robust STV-biotin binding, nonspecific bindings, and surface saturation, where more STV molecules remain bound to the biosensor’s surface even post-rinse. These factors contribute to the signal increase after rinsing, indicating the sensor's sensitivity and potential for detecting high analyte levels. Conversely, the lowest concentration (78 ng/ml) showed a significantly lower average signal intensity of 0.0095. This indicates more effective rinsing with minimal nonspecific interactions and fewer STV molecules bound to the biotin. The minimal residual signal at this concentration underscores the biosensor's ability to accurately detect low analyte concentrations. The differential response across concentration levels suggests it is critical to improve surface treatment to reduce nonspecific binding and ensure the complete removal of unbound molecules to improve the biosensor’s performance.

Acknowledgements (Optional): This work was supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under Award R15GM146233.

References
1. Rho, D.; Kim, S. Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein. Biosensors 2021, 11, 4.
2. Rho, D.; Breaux, C.; Kim, S. Demonstration of a Low-Cost and Portable Optical Cavity-Based Sensor through Refractive Index Measurements. Sensors 2019, 19, 2193.
3. Sypabekova, M.; Hagemann, A.; Kleiss, J.; Morlan, C.; Kim, S. Optimizing an Optical Cavity-Based Biosensor for Enhanced Sensitivity. IEEE Sensors Journal 2023, 23, 25911-25918, doi:10.1109/JSEN.2023.3317678.