Temperature Control Inside the Capillary

KCE on its own offers an alternative to spectroscopic methods KCE-based measurements are based on the dependence of one of the binding parameters (kon, ko , or Kd) on temperature. First, a calibration curve of binding parameter versus temperature is built for an affinity pair of choice, which can, for example, be an aptamer and its protein target. Advantageously, the calibration curve does not need to be built with a CE instrument in question. It can be built with another (verified) CE instrument or with a non-CE technique such as SPR. Second, the same binding parameter is measured in the CE instrument in question and the calibration curve is used to find the temperature inside the capillary. In addition to solving the chicken and the egg problem, the KCE approach also measures an effective temperature in the total volume of the capillary, which is a more relevant value than that measured at the detection point. 

Measurement of Temperature Inside the Capillary The use of NECEEM for measuring temperature inside the capillary has been demonstrated experimentally by measuring koff as a function of temperature for the interaction between SSB protein and fluorescently labeled ssDNA (Berezovski and Krylov, 2004). The calibration curve was built with a verified Beckman MDQ CE instrument, which uses a liquid heat exchanger to remove excess heat efficiently from the capillary. This calibration curve was then used to measure the temperature inside the capillary in a custom-built instrument with the capillary exposed to the ambient atmosphere at 20°C. It was found that the temperature inside the capillary was 15°C higher than the ambient temperature. As affinity interactions are very sensitive to temperature, the unjustified assumption that the capillary temperature 

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