Are you ready to go label - free?

With real-time detection and the ability to handle complex mixtures - acoustic biosensor technology is ideal for label free bio-analysis

SELECTIVE molecular interaction is a fundamental process essential for life, being the basis for enzyme-substrate, antibody–antigen and ligand-receptor activity. Radioactive or fluorescent molecular labels have enabled significant research on such proteins but they can have drawbacks, especially where their addition alters the binding kinetics. In response, many companies have developed label-free, real-time biosensors for biotechnology applications to complement or even displace other detection technologies. The continuous evaluation of the formation of biocomplexes provides more detailed information compared to label-based techniques, which measure the amount of the bound label only at the end of the binding process.

The widespread adoption of biosensors started in the early 1990s when the first optical platforms based on surface plasmon resonance (SPR) entered the market.  Although mostly advanced optical systems are utilised, piezoelectric and acoustic devices represent similar but significantly less expensive alternatives.

The number of publications that involve the use of quartz crystal microbalance (QCM) technology has increased rapidly since the mid 1980s and in particular since the 1990s. Acoustic biosensors have been employed in the label-free detection of an incredibly broad range of analytes - from interfacial chemistries and lipid membranes to small molecules and whole cells (Marx, 2003; Skládal, 2003; Cooper and Singleton, 2007). However, most of the data have been generated using “home built” QCM systems, often with insufficient attention to surface chemistry and assay reproducibility.

Any biosensor that utilises acoustics must possess a variety of integrated technical controls to facilitate the highest level of sensitivity, accuracy and precision. Previous attempts to exploit this detection method for biomolecular analysis have been limited by poor sample delivery mechanics, inadequate thermal controls and the lack of a multi-sensor analysis platform. To address the need of robust acoustic detection system that can generate reproducible data for a huge variety of analytes, instruments based on piezoelectric acoustic sensors have been developed more recently offering complimentary biosensor capability.

One such instrument is the RAPid 4 acoustic biosensor system which exploits resonant acoustic profiling (RAP). The instrument combines five key design components namely a novel acoustic biosensor, microfluidics, sensor cassettes, industrial liquid handling and intuitive control and analysis software. Its development involved significant innovation. For instance, a stress-free crystal mount replaces the conventional O-ring design used in other QCM devices. The new design markedly improves baseline stability enabling the study of slower off-rates, plus creates a smaller flow cell for improved kinetics with higher sensitivity. Secondly, novel algorithms have been implemented for more accurate determination of the resonant frequency change to observe the molecular interactions.

The RAPid 4 is a flow-based analysis system which performs label-free

“Acoustic biosensors have been employed in the label-free detection of an incredibly broad range of analytes - from interfacial chemistries and lipid membranes to small molecules and whole cells”

interaction analysis in real-time. By directly detecting the association and dissociation of molecules on the surface of a quartz crystal, the acoustic system enables the determination and quantification of protein interactions in buffered solutions and in complex mixtures that may contain solvents, serum, growth media or other impurities. In addition, the initial binding rate can be used to accurately determine the concentration of target molecules across a broad dynamic range. Currently, few technologies offer life scientists the ability to detect real-time kinetic data of this quality across such a broad range of samples, sample purities and concentrations.  RAPid 4 is designed to analyse up to 4 samples or combinations of samples and control materials in parallel, typically processing an average of 350 samples per day.  As a fully automated platform, it requires minimal user intervention and runs unattended for days.

The analytical capabilities of acoustic biosensors are ideally suited to the development of biotherapeutics, a rapidly expanding area of drug research. Direct measurement in crude and complex samples eliminates 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.

Use of acoustic biosensors extends from research, through drug development and into preclinical studies, and includes several key applications:

• optimisation of recombinant protein and antibody affinities as biopharmaceuticals
• assessment of the concentration and integrity of proteins 
• immunogenicity studies
• improvement of protein expression, extraction and purification processes
• quality control of production processes in manufacturing

The application of acoustics is expanding the scope of label-free interactions analysis by enabling methods complementary to optical technologies such as surface plasmon resonance. The further introduction of robotics and refinement of analytical software routines suggests that the mainstream use of acoustic biosensors in biopharmaceutical research and production is just around the corner.