There is no doubt that sequencing the human genome was an enormous technical achievement. But it didn't lead to the expected quantum leap in understanding protein function or the disease process.
Instead, we began to appreciate the importance of alternative mRNA splicing, differential tissue expression, and post-translational modification in generating the components of biochemical pathways, tissue modeling, and normal physiological functioning.
Proteomics has allowed us to identify and quantify proteins expressed in normal and disease states. However, proteomics does not ascribe function so additional methods, such as biomolecular interaction analysis (BIA), are imperative to unravel the myriad interconnected pathways and systems whose balanced function is essential for health.
"Molecules in the body and cells work by interacting with each otherthat is why this field is so important," says Hans-H Trutnau, managing director of biosensor service company Kinomics (www. kinomics.com). "BIA enables you to measure any pair of interactions you can imagine involving proteins, DNA, RNA, antibodies, or ligands."
Biosensors rely on the exquisite binding properties of cells and biomolecules for their functional partners. When partners come together, changes in different physical parameters, including mass or enthalpy, are measured by transducers producing an electrical signal proportional to the interaction. Data analysis can extend the information content to quantify the interaction's kinetics, specificity, and strength as well as any conformational changes.
Of the various types of biosensors, those that measure the affinity of one molecule for another are currently making the biggest contribution to analyzing biomolecular interactions.
SPR Leads the Market
According to Gerry Ronan, CEO of Farfield Sensors (www.farfield-sensors.co.uk), "The BIA market is dominated by Biacore (www.biacore.com) whose surface plasmon resonance (SPR) systems use optical sensors to measure the rate of mass addition and removal from which kinetic and affinity parameters can be calculated."
Stefan Lfs, vp and CSO at Biacore, claims, "We established the market for label-free, real-time investigation of protein interactions. There are now over 4,000 peer-reviewed publications describing work based on Biacore's systems, and these are being added to at the rate of 700800 every year."
Central to an SPR biosensor is a glass-surfaced chip that supports a thin layer of gold, providing both the means to immobilize or covalently link the test protein and to generate the physical conditions for SPR.
Surface plasmons are collective oscillations of free electrons constrained in the metal film. Polarized light from a prism excites the electrons resonantly.
As molecules are immobilized on a sensor surface, the refractive index at the interface between the surface and a solution flowing over the surface changes, altering the angle at which the reduced-intensity light is reflected from the glass. The change in angle, caused by binding or dissociation of molecules from the sensor surface, is proportional to the mass of bound material.
"SPR has no lower resolution limit. We can detect down to 200 daltons, maybe lower, depending on the density of target molecules on the chip surface," Lfs maintains. "Low sample concentration can be a problem, but with a sufficiently high binding affinity, even this can be managed. Sample protein preparation and processing remain as the key issues curtailing broader technology adoption."
"In the early years, Biacore's customers within the pharmaceutical and biotechnology industries used their instruments in the same way as academics, and later on for target identification and validation.
"As different systems and applications have developed, it has become possible to address later stages in the development process including lead optimization and secondary screening.
"Our instruments are also suitable for process control, in particular of biopharmaceutical drugs like antibodies and therapeutic proteins and also immunogenic studies. The newly launched Biacore T100 system now meets the regulatory demands made when working in the GLP and GMP environment."
Biacore acknowledges the contribution made by customers in developing new applications. "The majority of these involve proteins, although a significant number are now DNA-protein," Lfs comments.
"Some customers investigate more fundamental surface interactions to develop new surfaces minimizing nonspecific binding for different types of medical devices. Compared with five years ago, we can now measure the direct binding of small molecule drugs with target proteins."
Kinomic's Trutnau identifies two potential bottlenecks to the increased usage of biosensorssoftware and hardware. "Data analysis to produce a kinetic profile still takes time. Kinetic analysis is important, for example, when selecting a therapeutic antibody to treat a particular cancer.
"Rapid binding may not be so critical as knowing that an antibody sticks to its targeted tumor cell long enough to do the job. Our software produces results two- to tenfold faster than other systems. A single button click sets off the automated data analysis. Users love the on-screen traffic light system that shows whether measurements are consistent."
On the hardware side, most industry observers agree there is a need to increase the throughput capability of commercially available biosensors. Biacore is currently rolling out an instrument that, it says, will produce five to ten times more data per unit time. To complement this, early in the year, Biacore acquired FLEXChip from HTS Biosystems (www.htsbiosystems.com).
Cambridge Consultants (CCL; www.cambridgeconsultants.com) modified the SPR technique to enable an array of binding sites to be measured simultaneously. Direct imaging of the binding surface is achieved with interferometric phase imaging.
A single wavelength, ultra-low divergence beam illuminates the binding area. The phase of the reflected light varies linearly in response to small changes in refractive index at the surface, producing a measurable image.
"Apart from microfluidics, the system doesn't need any moving parts," explained Martin French, CCL's clinical diagnostics business leader. "Our proprietary interferometer can be of monolithic construction, lending itself to multiplexing. We estimate that the system is capable of examining over 1,000 individual binding sites within a high density protein array of 10 mm2."
With development partner Orla Protein Technologies (www. orlaproteins.com), CCL demonstrated proof-of-principle for the multiplexed analysis of antibody binding to specific proteins at the recent "37th Annual Oak Ridge Conference".
Looking to the future, French said, "The high-value diagnostics market is where we'd like to go. This type of technology can be miniaturized, which means there is great potential for screening patients at the point of care."
Help is at Hand
Biosensors are sophisticated instruments and therefore are not an insignificant investment. Suppliers provide after-sales support and publication of application protocols to expand fields of use and ensure quality, reproducible measurements.
Biaffin (www.biaffin.com) provides bioanalytical services, specializing in BIA using Biacore's SPR instrumentation. Oliver Diekman, BIA project manager, explains, "We work for many pharmaceutical companies, even those that already have SPR capability, establishing and validating assays."
"Buying an instrument doesn't guarantee a good result. Users have to be aware of the problems of mass transport in a flow-through system. If molecules enter a system slower than they bind to the sensor, you measure the flow rate, not the binding rate.
"Also, if a chip has an excess of binding sites, molecules can move from one site to the next without being detected, introducing measurement uncertainty. Customers use Biaffin because of our experience in eliminating these artifacts, speed, and reliability."
"SPR systems that measure mass addition or removal from a surface, tend to run out of steam when the interaction molecule is small, as is the case with drug candidates," comments Ronan.
"In addition, since all proteins tend to be sticky, measurements are plagued by nonspecific binding. Farfield's take, an optical method called Dual Polarization Interferometry (DPI), is different. (See Application Note on page 34).
"Using our molecular microscope, we measure structural changes in the protein as a result of its binding to another molecule. Conformation can be determined by quantifying the immobilized protein's size. Density gives us information about protein folding. Nonspecific binding does not induce a structural change so the measurement is clean."
Farfield isn't just about detecting interactions, it wants to understand them too. A month ago, the company announced the award of a competitive Small Business Research Initiative grant from the U.K.'s Biotechnology and Biological Sciences Research Council to examine the structure and behavior of glycosylated biomolecules.
The same technology found in quartz watches or mobile phones is finding new applications in the life sciences. Allan Marchington, CEO of Akubio (www.akubio. com), explains. "Each crystal vibrates at a set frequency that changes as mass is added to the surface.
"Our instrument incorporates a quartz crystal resonator onto which a molecular target is coupled, usually an antibody or a protein. A potential binding partner is passed over the surface. Changes in vibrational frequency indicate changes in mass and therefore biomolecular interactions."
"With Akubio's system, the interaction is measured directly. This is solid-state technology, so it is miniaturizable, potentially to a hand-held, point-of-care diagnostic device, it is available in multiple formats, and throughput can easily be increased.
"Quartz crystal technology doesn't suffer from mass transport limitations in which a still layer over the sensor surface causes sample molecules to travel the last 300 nm by diffusion.
SPR systems measure refractive index to within about 300 nm of the surface. Binding molecules frequently come off and re-bind, and if they don't travel out of the 300-nm range, their movements are not registered. With quartz crystal technology, it only needs a few water molecules to appear between the bound and unbound molecules for the system to register therefore kinetics measurements are more accurate."
One of the biggest advantages is that Akubio's technology is not reliant on refractive index, therefore it is less dependent on sample solvent, and crude samples can be used. Akubio's first instrument is now in beta testing with a launch planned for 2006.
"Ultimately, all screening will become real-time and label-free. Of the main approaches, including optical and acoustic techniques, the winner will be the most versatile technology with the broadest application range," Marchington concludes.