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."