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Mar 15, 2009 (Vol. 29, No. 6)

Advances in Label-Free Assay Screening

Advantages and Limitations of New Tools Such as RAP, Optical Biosensors, and SPR

  • 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.

  • Microparticle-Based GC-SPR Assays

    Click Image To Enlarge +
    The prototype Toshiba system viewed in close-up

    Carl Norman, Ph.D., principle research scientist at Toshiba’s Cambridge Research Lab, described Toshiba’s bioassay platform incorporating microparticles and label-free GC-SPR (grating-coupled surface plasmon resonance) detection. The prototype-stage assay platform is being developed in collaboration with the Institute of Biotechnology at the University of Cambridge.

    In conventional and microarray-based GC-SPR, an analyte of interest is flowed across specific receptors (e.g., antibodies or other proteins) that have been spotted and immobilized onto a gold-coated sensor chip. The chip surface is illuminated with polarized monochromatic light that can be made to couple to electrons in the gold surface to form a surface plasmon. Efficient coupling occurs only at a specific angle of incidence, termed the GC-SPR angle, thereby reducing the intensity of reflected light. 

    Shifts in the GC-SPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this type of assay system can assess multiple interactions between analyte and receptor such as antigen-antibody interactions on a single chip. Modifications of the basic ELISA immunoassay using GC-SPR for label-free real-time variant of this solid-phase immunoassay have been developed allowing multiple assessments antibody-antigen interactions on the same sensor chip.

    GC-SPR has been applied to cell-based assays allowing, for example, antigen expression by detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.

    But, according to Dr. Norman, use of spotted microarrays on single surfaces has some significant limitations. Spots may be subject to drying problems, irregular analyte distribution, or spot overlap—particularly if a low surface tension solvent is used. More fundamentally, the use of a single surface limits the assay to just one set of preparation (e.g., incubation time/temperature) or hydration conditions per assay. As such, it is extremely cumbersome, not to say impossible, to use more than one surface chemistry on a single-surface 2-D microarray.

    Dr. Norman explained that Toshiba’s platform combines the joint advantages of discretely functionalized code-bearing microparticles with multiplexed GC-SPR-based detection and analysis. Shape-encoded, free-standing carrier particles are produced from a silicon master mold via a soluble substrate technology that is both inexpensive and scalable. A gold-coated optical grating on the surface of each particle allows GC-SPR measurements. An automated reader system determines the code of the particles and measures their SPR signals in a multiplexed format.

    The major advantage in using particles, each coded batch functionalized in isolation from the others, is that a wide range of surface preparation conditions and different surface chemistries can be combined in one single assay. For example, a 10% amino-terminated self-assembled monolayer (SAM) could be used to immobilize molecule A, while a 5% carboxyl-terminated SAM could be used to immobilize molecule B, but both immobilized molecules could then be combined in the same assay. Using standard microarrays, this would require two different assays to be performed—one for each molecule.

    “The time and cost saving of the Toshiba system becomes significant when an even higher number of different molecule types need to be tested,” Dr. Norman continued.   “It should also prove to be a useful process-optimization tool, as all possible process conditions could be combined into a single assay, and then directly compared in just one test.”

    Dr. Norman added that Toshiba’s system, now at the working prototype stage, has a throughput of about 50 particles per assay, which he says is expected to increase “dramatically” in the commercial prototype. The instrument could become available within the next two years, initially aimed at lab-based research in the general biotech and drug discovery arenas.


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