22 September 2009

Math helps take more precise temperatures

Checking the temperature on some things is simple, however measuring the temperatures in microfluidic systems—tiny, channel-lined devices used in medical diagnostics, DNA forensics, and “lab-on-a-chip” chemical analyzers—is not very easy, as their current “thermometer” can precisely calibrate to one reference temperature.

There is now a proposed a mathematical solution that enables researchers to calibrate the “thermometer” for microfluidic systems that would allow users to measure all temperatures, said scientists at the National Institute of Standards and Technology (NIST).

Reactions taking place in microfluidic systems often require heating, meaning users must accurately monitor temperature changes in fluid volumes ranging from a few microliters (a droplet approximately 1 millimeter in diameter) to sub-nanoliters (a droplet approximately 1/10 of millimeter in diameter). A common DNA analysis technique, for example, depends heavily on precise temperature cycling. Ordinary, thermometers or other temperature probes are useless at such tiny dimensions, so some groups have turned to temperature-sensitive fluorescent dyes, particularly rhodamine B. The intensity of the dye’s fluorescence decreases with increasing temperature. The idea is the dye can be a noninvasive way to map the range of temperatures occurring within a microfluidic system during heating and, in turn, provide a means of calibrating that system for experiments.

However, the technique currently requires the user to base all readings on the fluorescence at a single reference temperature. Previous groups have developed “calibration curves” that relate temperature to rhodamine B fluorescent intensity based on a reference temperature of about 23°C (a technique first proposed by NIST researchers David Ross, Michael Gaitan and Laurie Locascio in 2001). But it turns out, the curves are only good for that one temperature.

Now, the NIST team of Jayna J. Shah, Michael Gaitan, and Jon Geist said changing the reference point, such as the higher temperature when a microfluidic system first heats up, introduces errors when a dye intensity-to-temperature calculation occurs using current methods.

“Our analysis shows that a simple linear correction for a 40°C reference temperature identified errors between minus 3 to 8°C for three previously published sets of calibration equations derived at approximately 23°C,” Shah said.

To address the problem, the NIST team developed mathematical methods to correct for the shift experienced when the reference temperature changes. This allowed the researchers to create generalized calibration equations that can apply to any reference temperature.

Microfluidic DNA amplification (production of numerous copies of DNA from a tiny sample) by the polymerase chain reaction (PCR) is one procedure that could benefit from the new NIST calculations, Shah said. “PCR requires a microfluidic device to be cycled through temperatures at three different zones starting around 65°C, so a useful dye intensity-to-temperature ratio would have to be based on that temperature and not a reference point of 23°C,” she said.

For related information, go to www.isa.org/instruments.