Resonant Acoustic Profiling
Helge R. Schnerr, Ph.D., senior researcher, TTP LabTech, described applications of RAP as embodied in the company’s RAPid 4 system for label-free bioassays. RAP measures the oscillation of a functionalized resonating quartz crystal, which decreases as molecules bind to its surface.
Target molecules are attached to the sensor surface through direct linkage or capture, then samples containing potential binding partners are applied to the sensor surface. Frequency changes in oscillation of quartz crystal resonators, proportional to the mass of molecules bound to the surface, are measured over time to characterize the binding of molecules onto the surface, providing information about the specificity, affinity, kinetics, and concentration of molecular binding interactions in real-time.
Unlike optical biosensors, RAP is said to be unaffected by solvents, eliminating the need to run calibration to normalize for the effects of organic solvents such as DMSO, or components of crude cell lysates, culture medium, or serum samples.
In describing the advantages of RAP-based technologies, Dr. Schnerr contrasted it to optical label-free biosensor methods, explaining that optical label-free biosensor methods ultimately detect and measure changes in dielectric constant or refractive index of a solution in close proximity to the surface of the sensor substrate.
“The advantages of powerful techniques under extremely well-controlled conditions are often minimized when trying to apply these methods in routine analytical procedures,” she explained. As optical methods rely on proximity-based detection, any analyte that is within the evanescent sensing field (typically 300 nm for most SPR devices) is detected as bound. This is the case whether it is physically bound to the receptor or simply in close proximity to the surface of the sensor.
In contrast, RAP measures only those materials that are acoustically coupled to the sensor surface, that is, binding-based detection rather than proximity-based detection. The process of measuring refractive index changes with optical methods to infer mass changes, imparts a number of other intrinsic limitations—in particular, the masking of binding events that occurs in sample environments that have variant refractive indices.
The RAPid 4 system analyzes up to four samples or combinations of samples and control materials in parallel, processing an average of 350 samples per day. According to Dr. Schnerr, RAPid 4 development has incorporated significant innovation, including the stress-free mounted crystal holding two resonating centers that replaces the conventional O-ring design used in other QCM devices. The new design is said to improve baseline stability and control referencing, enabling the study of slower off-rates, plus it creates a smaller flow cell volume for improved kinetics with higher sensitivity.
Dr. Schnerr noted that the RAPid 4 biosensor is well-suited to the development of biotherapeutics because it allows direct measurements in crude and complex samples, thereby eliminating expensive time-consuming purification of often limited material while delivering high-content information.
Efficiency is further refined through automation and the elimination of analyte labelling. In addition, the real-time nature of acoustic detection allows prompt decisions to be made associated with biotherapeutic pharmacology, clone selection, culture conditions, and purification efficiency even in the early stages of development.
The collected data can also be used to accurately determine the concentration of target molecules across a broad 3-log dynamic range. Detection of kinetic data can be performed in real time over a range of samples and concentrations.