Ultrasensitive Detection of Nucleic Acids in a Microchip

We have combined a laser-based confocal fluorescence detector with a state-of-the-art glass microchip to develop an electrophoresis apparatus much more sensitive than conventional systems.

This instrument is designed to utilize laser induced fluorescence as a means of detection (schematic). A continuous wave argon ion laser (514nm) is employed to excite the molecules migrating in the microchip's separation channel. The fluorescent photons are collected by a confocal objective (40 x 0.65NA) and detected by a photomultiplier tube. The detected signal is amplified, discriminated, and sent to the computer to be analyzed. A labVIEW program is used for acquiring data and for controlling the high voltage relay circuit which, in turn, controls the applied electric field in accordance with the microchip's injectional/separational design.

In order to attain a high degree of sensitivity, and high resolution, the various experimental parameters are optimized. These parameters include the intensity of the laser, loading/running voltages, loading/running times, and sampling interval size. The laser intensity, about 10 mW, is adjusted so that only a small percentage of molecules are photodestructed. The loading/running voltages are set such that the maximum separation occurs in the shortest time period, but does not cause overheating of the microchip, which, in turn, causes bubble formation in the channels. Optimal loading/running times are set according to the electrophoretic velocities involved. Enough time is allowed for the analyte to reach the intersection, but not to saturate it. Lastly, the sampling interval size is optimized according to running time.

Other external factors needed to be considered for optimal sensitivity are: alignment of the laser with the microchip and optics, choosing the proper objective and filters, maintaining proper fluid levels and cleanliness in the microchip's reservoirs and channels, and elimination of unwanted background noise.

Recent experimental results show that microchip electrophoresis is an effective process for analyzing chemical and biochemical components at very high speeds and low concentrations. Typically, this has been the domain of gel and capillary fluorescence-based electrophoresis. The microchip technology has many benefits over those more conventional methods of analysis. For example, the chip can analyze a mixture in seconds where it would take capillary electrophoresis at least 20 minutes and gel electrophoresis 1 hour to do the same analysis. The microchip can detect sample concentration in the range of 100 picomolar which is at least two orders of magnitude greater than conventional CE. Furthermore, gel and microchip electrophoresis are comparable in price, but capillary electrophoresis is tens of thousands of dollars more than either. In short, microchip electrophoresis is quicker, more sensitive, and cheaper than conventional techniques used to electrophoretically analyze chemical mixtures.

Future improvements will include the incorporation of time-gating electronics to increase the limit of detection even more, and the use of infrared excitation to eliminate unwanted background noise.