March 15, 2009 (Vol. 29, No. 6)
Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications
Advantages and Limitations of New Tools Such as RAP, Optical Biosensors, and SPR
Currently, most assays for detection of biologically relevant binding events use radioactive or fluorescent dyes to tag one or more molecules. The need for multiple reagents limits label-based assay flexibility and speed, particularly in HTS formats.
In cell-based assays, label dependence may produce artifacts because of manipulation of cells either through label-induced toxicity or other effects and requirements for over-expression of targets or reporter proteins. Label-based assays may also necessitate several ligand immobilization and detection methods and serial experiments to yield sufficient information, contributing considerably to the cost of drug discovery and development.
Recently, academic and industry scientists presented new techniques to facilitate label-free screening methods as assay tools for multiple applications at Select Biosciences’ “Screening, MedChem, and ADMET Europe” conference, held in Berlin last month.
According to label-free assay developers, label-based assays restrict scientists to investigating one signaling pathway per ligand-receptor complex, and do not incorporate multiple-pathway readouts likely to be involved in a single ligand binding interaction. Cell-based label-free technologies, such as optical biosensors that leverage characteristics of cells that change in response to signal transduction may facilitate fast and accurate real-time readout capabilities for cell-based and other assays.
Advantages and limitations of label-free technologies, including resonant acoustic profiling (RAP), optical biosensors, and microparticle-based surface plasmon resonance (SPR), were described at the ADMET meeting as well as progress in their use for drug discovery applications.
Julio Martin, Ph.D., manager of ultra-HTS, molecular discovery research at GlaxoSmithKline, believes that label-free sensors hold promise for filling significant gaps in the drug discovery process. Dr. Martin commented that, while “the explosion of genomics in the 90s was expected to generate an abundant supply of new targets amenable to high-throughput screening assays, the delivery has not lived up to the high expectations.”
Dr. Martin imagines a simple technological platform that enables testing any target for hit identification in a similar generic fashion, for instance, through direct binding of compounds to protein. At the same time, the platform should be testing those same compounds in a more complex biological setting that closely resembles the native system, for example in a primary cell line. “This is what label-free can offer,” said Dr. Martin.
Optical resonance grating sensors in particular, according to Dr. Martin, have proven to be versatile platforms that can detect simple binding events between small compounds and biomolecular targets to complex phenotypic changes in nonengineered cells.
Optical index of refraction platforms offer a fundamental advantage because they do not require a direct electrical connection between the excitation source, the detection transducer, and the transducer surface (i.e., the sensor). “Optical biosensors and corresponding detectors can be designed to allow measurement of images with resolution of a few microns, that is, an area as small as 1 cm2 can be used to perform several hundred parallel determinations,” he explained.
Dr. Martin added that “optical biosensors fulfill all the requirements for adoption as a label-free, universal drug discovery platform because they are amenable to high throughput that is rapid, robust, and relatively inexpensive and that can assess large numbers of compounds in a single independent experiment.”
Serial G-protein coupled receptor activation, Dr. Martin said, is particularly suited to optical label-free assay because G-proteins constitute the most abundant target class for current drug therapeutics and attract a significant part of the drug discovery efforts in pharma. Both native cells expressing endogenous GPCRs and cells overexpressing one particular receptor have been monitored by optical grating sensors in response.
“Distinct temporal fingerprints are exhibited,” he continued “that seem to be characteristic of the particular receptor coupling pathways triggered by each ligand on a determined cellular background. Optical label-free assays allow the possibility of studying agonist trafficking in any cellular background, including native primary cells, without additional experimental complications other than cell handling. The key is having a strategy and tools to deconvolute the combination of signatures. Deciphering the signature encrypted in a temporal response may be the Rosetta stone of cellular label-free.”
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
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.
GPCR Activation Response
John Gatfield, Ph.D., senior lab head of molecular biology at Actelion Pharmaceuticals, described the use of his company’s technology to characterize coupling pathways of a prototypic GPCR activation response in recombinant and nonrecombinant cells. GPCRs comprise one of the largest classes of drug targets; about 50% of all marketed drugs act directly or indirectly on this receptor family. These receptors are the focus of multiple drug discovery efforts currently under way in pharmaceutical companies.
Dr. Gatfield described cell-based assays that use impedance measurements to analyze GPCR activation response patterns in recombinant and nonrecombinant cells. Cellular parameters such as attachment, spreading growth, death, and morphology cause impedance change in response to ligand binding and receptor pathway activation.
Acea Biosciences’ xCELLigence System, developed with Roche, consisting of a microelectronic biosensor array incorporated into each well of 16 and 96 well microplates, enables cell activity measurements in cell monolayers without labels or reporters. The system measures activity in real time, providing instantaneous readings of experimental results and improving the ability to identify cellular changes that reflect response of the cells to certain stimuli (added drugs, hormones, etc.).
Measurements analyze the interaction of living cells with the microelectronic sensor array. The system detects and quantitates changes in electrical impedance as the living cells interact with and electrically insulate the biocompatible microelectrode surface in the microplate well. The degree of electrical insulation is then converted by an algorithm to a specific parameter, the Cell Index, which can be used to characterize cellular activities including cell proliferation, cytotoxicity, adhesion, receptor tyrosine kinase activity, and G-coupled protein pathway responses. The cell index is directly proportional to the cell layer induced electrical impedance.
The system can be applied to functional monitoring of GPCR linked to different activation pathways; the sensitivity of the assay parallels those of standard assays such as calcium and cAMP measurements. As a label-free technology it theoretically allows for the simultaneous analysis of multiple pathways in one experimental well.
Indeed, one especially interesting use of the technology is the dissection of GPCR signaling in recombinant and nonrecombinant cells in response to natural ligands or synthetic compounds. As an illustration, Dr. Gatfield presented the response pattern of a Gq- and Gs-coupled GPCR to its natural ligand as being biphasic. By using specific pathway inhibitors of the Ca2+ pathway or the cAMP pathway he could attribute the two response phases to the two different coupling pathways.
“As one potential application in the pharmaceutical industry, this multiplexing of several GPCR signaling pathways in one assay format would allow a rapid characterization of full agonists versus biased agonists after a screening campaign,” Dr. Gatfield added.
Patricia F. Dimond, Ph.D. ([email protected]), is a life science consultant.