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Jun 15, 2006 (Vol. 26, No. 12)

Going a Label-Free Route with Biosensors

New Technologies for Calorimetry and SPR Aim to Transcend the Drawbacks to Labels

  • Molecular labels have enabled many scientific advances that would not otherwise have been possible. However, labels have drawbacks. They disrupt the accurate measurement of kinetic constants, particularly binding equilibria, and problems, such as antibody cross reaction or impure solutions, can occur. Many companies are developing label-free biosensors for biotechnology applications.

    Calorimetry is one of the more robust methods for label-free sensing. There are two main approaches to the science of calorimetry. In differential scanning calorimetry (DSC), the energy of a thermal transition is measured while the temperature in the reaction chamber is slowly raised. In isothermal titration calorimetry, the instrument passively measures the heat produced during a chemical reaction.

    The Nano DSC III is the most recent DSC to be released by Calorimetry Sciences (www.calorimetrysciences.com). Its strongest feature is its sensitivity. The instrument can detect changes in heat down to the nanowatt or microwatt range, generated by as little as 10 micrograms of protein. The technique is nondestructive when used on proteins that denature reversibly. "We do have problems. Sometimes proteins as they unfold expose hydrophobic domains. Those domains will clump, and precipitate out of solution. A lot of times that is irreversible. We have the advantage of the capillary cell. It attenuates problems with aggregation of protein," says Rusty Russell, vp science. The Nano DSC III is also capable of measuring volumetric properties of solutions.

    MicroCal (www.microcalorimetry.com) also manufactures DSC and ITC systems for research. "We sell ultrasensitive calorimeters that give you information that complements SPR," says Steve Spotts, vp of sales and marketing. The company’s premier product is the AutoITC, a fully automated version of isothermal titration calorimetery with an autosampler front end. The instrument can achieve a throughput of 100 samples per week unattended, according to MicroCal.

    Spotts explains how the instrument can be used in combination with surface plasmon resonance. "SPR will give you Kon and Koff rates. Calorimetry provides a complete thermodynamic profile and can elucidate the mechanism of that binding. Not only can you tell whether it’s binding, but how, for example, specific or nonspecific binding. ITC gives you more information so you can go back and modify the molecule to bind more tightly, or less tightly."

    Thermometric (www.thermormetric.com) developed a multichannel, multifunctional ITC with applications in whole cells or viruses, or monitoring processes that have a long duration. The reaction chamber holds two to three millimeters, and there are up to 48 channels (one chamber each) per instrument.

    "We have high sensitivity, down to nanowatt quantities. We have 48 channels. When experiments take a long time, then we have the best instrument for long-time measurements. If you only do short times, it’s more labor loading our instrument. When you look at these blood cultures for microorganisms, it takes a couple of hours to do the measurement, and you need a high sensitivity," says managing director Jaak Suurkuusk, Ph.D.

    Microplate Differential Calorimetry by Vivactis (www.vivactis.com) is a unique variation on the traditional calorimeter that performs calorimetric measurements in 96-well plate format. The system is fully automated from its robotic sample-handling front end to data acquisition and data handling at the back end. The reaction is mixed directly on the sensor, so as to capture the full heat of reaction.

    According to Peter van Gerwen, Ph.D., vp of research and operations, "In a conventional calorimeter, you have a measurement cell and a reference cell, the reference cell being clean water, and it’s used to measure the amount of heat you dissipate. In our system, you’re not measuring heat in absolute terms, but only the difference in heat between two wells. This means that this system is less sensitive, for example, to mixing heat. In conventional calorimetry, the mixing heat would interfere with the reaction heat. In our system, which is a true differential system, you can inject in both reference well and measurement well, thereby compensating for mixing heat, so that you see only the heat of binding. It also means you can run more complex assays than a conventional calorimeter. You can play with it."

  • Surface Plasmon Resonance

    SPR is based on the properties of reflection and refraction of light. It measures the shadow created at a certain refraction index when surface plasmons are excited to become photons. Typically, a gold film is used as the interface material, and the light beam targeted at the opposite side of the interface from the covalently bound surface ligands.

    Biacore (www.biacore.com) specializes in systems for analyzing protein interactions to determine specificity, association and dissociation constants, affinity, and concentration of interactant. Biacore offers sensor chips with surface chemistries for general and specialized applications for use with SPR sensors. "What is critical with our systems is the sensor surface," says marketing director, Helena Nilshans. "Our wide range of sensor surfaces allows users to attach many different molecules."

    Improvements over many successive generations of technology have resulted in enhanced sensitivity and ease of use. "We easily detect interactions involving small molecules such as pharmaceutical drug candidates in several of our systems. This level of sensitivity is a limitation of many other SPR-based systems." Interactions that can be monitored by Biacore systems include protein-protein or protein-peptide interactions and others involving small molecules, membrane proteins, nucleic acids, viruses, and carbohydrates.

    Another physical property that can be exploited for biological sensing is sound. Sound waves can be generated by the vibration of a quartz crystal. Binding events trigger detection through a change in mass.

    Akubio (www.akubio.com) uses a sensor based on a quartz crystal that is coated with gold. Under electrical current, it oscillates at a specific frequency. Binding events change this frequency in proportion to the amount of mass bound to the surface. The customer immobilizes a protein of choice to the surface, creating a regenerable custom biosensor for a specific protein interaction. "You can regenerate the surface, and use it multiple times. We’ve used them over a hundred times, while retaining activity," says John Watkins, Ph.D., applications specialist at Akubio. Applications for this type of technology include protein-protein interactions, affinities, antibody binding, and biotech applications. "It’s a label-free technology that’s going to help people perform measurements they weren’t able to do previously and save time working in crude samples."

    Accurion Scientific Instruments (Nanofilm Technologie; www.nanofilm.de) utilizes the vibrations of a quartz crystal as well, but as a wave propagated along the surface of the crystal. According to Martin Laging, applications manager, surface accoustic wave technology (SAW) is similar to surface plasmon resonance. "This technology is first of all very much related to so-called SPR studies, and also very well recognized in the field of molecular biology. There’s one significant difference. With SPR, you detect mass changes on the surface. With a QCM-based method, like SAW, you can detect mass changes and changes in viscosity on the surface. It can be interesting, because if you have conformational changes, like a protein layer, sometimes proteins change conformation, and this changes viscosity as well. These are two effects you can monitor."

    Protiveris (www.protiveris.com) is one of a small number of companies pioneering cantilever technology for biosensors. A cantilever is a small piece of silicon, approximately five hundred microns long, and one micron thick. By attaching a capture molecule to the end of the cantilever, one can detect binding events. "If the cantilever were a mile long, instead of five hundred microns, we could monitor down to a few inches at the end," says Robert Cain, Ph.D., director of research and development at Protiveris.

    Cantilever bending is related to the change in surface stress, and the change in resonant frequency is related to the change in mass. An array of lasers detects these changes in the cantilevers. According to Dr. Cain, the detection of dual properties is an advantage of cantilever technology. "The other label-free technologies are all a function more or less of a change in mass. The change in surface stress is something cantilevers can do that other techniques can’t do. The change in surface stress is related to surface energy. If you’re measuring change in surface stress and change in resonant frequency, you’re getting change in energy per binding event."

  • Fiber Optics

    Luna Innovations(www.lunainnovations.com) uses fiber optics to detect binding events at microcantilevers. %Fiber optics improve the robustness of the sensor. You can basically put them anywhere. All you need is a very small area to position the fiber optic above or below. You can run a fiber for miles. The lasers have to be appropriately aligned. With an electrical current system, the electronics can fail. Other systems are not as robust, not as rugged as fiber optics-based systems,% says Rik Obiso, Ph.D., the director of life sciences at Luna.

    Luna’s cantilever system is still early in development, but they have experimented with putting antibodies on the cantilevers to test for various infectious diseases, and also the use of polymers. Luna is looking at markets in universities, government, and biodefense. Some of their earliest projects, however, are with NASA.

    %We’re working on an application with NASA for recycled water supplies, spacecraft, and mold issues. So if an astronaut goes into space with a sickness, it can be detected quickly and screened in recycled air. The microenvironment created by spacecraft is one we targeted initially.%

    Corning (www.corning.com) introduced optical fiber in the 1970s and has been making microplates since the early days of microplates. They have brought these two lines of development together in an optically based label-free sensing system based on a 384-well microtiter plate, the Corning Epic system. The technology resembles SPR in that it measures the change in a reflected wavelength by perturbation of a field above the surface of the well bottom.

    %It’s unique for us to be putting this in a microplate,% says Tom Lynch, Epic business manager for Corning. %Corning has been working with resonant wave guides for a long time. We’ve been able to put this in a 384-well microplate, which allows high-throughput screening to occur. Some of the label-free technology might have stopped at 96-wells, but we have 384.% Epic’s throughput is estimated to be around 40,000 wells per eight-hour shift.

  • Ion Channel Chemistry

    There are some unique technologies in the field of label-free biosensors, as well. Ambri (www.ambri.com) makes use of the chemistry of ion channels to create a label-free sensing system. Functioning sodium channels are embedded in a lipid bilayer near a gold electrode. When sodium molecules pass through the channels, a signal is registered at the electrode. By attaching antibodies to the outside of the channel surface, you can create a sensor that will return a reduced signal when the target molecule is bound to the antibody, blocking sodium from entering.

    The system is being developed as a medical device, to be used, for example, in a clinic setting with blood samples. "We have indeed operated with whole blood," explains Bruce Cornell, Ph.D., chief scientist at Ambri. "The fact that it is an electrical signal means you’re not faced with more conventional optical interferences when putting colored, opaque, or fluorescing samples in it. Although we have interferences in our world as well, they’re quite different. We feel we’re better off measuring electrical current. You don’t have to have an optical-to-electronic conversion. Ultimately, if you have an optical system you have to come up with some kind of photodiode."

    Label-free sensing technologies have a lot of potential advantages, but also a great deal of competition. Nanogen(www.nanogen.com) developed an electronic addressing system for assembling DNA microchips. They experimented with electronic sensing, but ultimately settled on a fluorescence label-based detection system for their product.

    "I have not seen any biosensor that is label-free that has achieved the same performance and sensitivity as the fluorescent systems. Nanogen did have a project before my time on cantilever biosensors and had some significant IP. We’ve done direct electronic detection. None have delivered the performance or sensitivity we needed. The evolution of technology takes a lot of time. When something else is so well established, it has to find its own niche first," says Graham Lidgard, Ph.D., senior vp of R&D for Nanogen.

    Axela Biosensors (www.axelbiosensors) introduced its new dotLab system, which is scheduled to launch in the fall. The open assay platform reportedly enables real-time monitoring of protein interactions.

    "There is a strong need for a platform with the capabilities of the dotLab System that can address the large, underserved market for protein detection," says Rocky Ganske, president and CEO of Axela Biosensors. "In addition, the translational research studies that health researchers will perform using the dotLab System are a natural precursor to Axela’s planned move into the larger clinical diagnostics market."

    The core of the dotLab System is Axela’s Diffractive Optics Technology. This biosensor technology permits researchers "to rapidly identify and quantify therapeutic and clinically relevant protein biomarkers without the need for fluorescent tags," continues Ganske. "The dotLab System will also allow scientists in academia and pharmaceutical research to detect and understand molecular interactions at their bench in real-time. Initial applications for the dotLab System will focus on immunoassays and their development and include rapid reagent characterization, antibody pairing, and cross reactivity studies."

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