Capillary Electrophoresis

Recent Publications

On-chip CE with Single Molecule Sensitivity

Capillary electrophoresis (CE) is an analytical technique in which molecules with different electrophoretic mobilities are separated by applying an external electric field. CE has applications to drug discovery and screening, clinical analysis, and pathogen detection. Recent advances in microfabrication technology have enabled CE analysis on microchips. The main advantages of miniaturization include high separation electric fields, reduced sample reagent, fast assays, and highly sensitive detection. On-chip CE can be performed using electrokinetic injection using a cross-like microchip geometry (see movie below):





Electrophoretic Seperation

Figure 1. Movie of the electrokinetic injection and separation of two simple fluorophores (bodipy and fluorescein) in a microfabricated capillary electrophoresis system. The channels shown are 50 m wide and 20 m deep. The fluorescence images are 20 ms exposures and consecutive images are separated by 250 ms.

We are developing an on-chip CE system with single-molecule detection sensitivity. Such a system has applications towards single cell assays and other sparse sample assays. The system is shown in Figure 2 and consists primarily of an argon-ion laser, an inverted microscope, a simple optical train, a computer-controlled eight-channel high voltage supply, and an intensified CCD camera. Preliminary experiments (Figure 3) using this setup show that a mixture of fluorescein and bodipy dyes at initial concentration of 400 pM and 800 pM, respectively, can be separated with an SNR of 61.3 (based on fluorescein peak). We are currently optimizing the system to achieve single-molecule sensitivity.

We are optimizing the performance of state-of-the-art on-chip electrophoresis devices by designing optimal geometries [1], flow control strategies [2,3], and signal integration methods [4]. One important area we are emphasizing is novel methods for field amplified sample stacking (FASS). FASS is a robust sample preconcentration technique which can be easily integrated with on-chip CE [5]. In FASS, gradients in electrical conductivity are used to generate gradients in sample ion electrophoretic velocity. The sample ions exiting a region of high electric field "stack" on entering the low electric field region. We have developed an experimentally validated dispersion model to study the dynamics of the FASS process. We are currently using the model to investigating the parameter space for optimizing on-chip FASS [6]. Also, we have developed a novel FASS-CE chip that uses a photoinitiated porous polymer structure to facilitate sample injection and flow control for high gradient FASS [4]. Using this chip system we have demonstrated more than 1000-fold electropherogram signal increase. We have also initiated development of nanometer-scale channels in an attempt to develop novel separation modalities.

Figure 2. Schematic of experimental setup. The imaging system consists of an inverted, epifluorescent microscope (Olympus IX70, NY) fitted with a 60X water immersion objective (NA 0.9), an Intensified CCD camera (Roper Scientific I-PentaMAX, NJ), and an argon-ion laser (Lexel Model 95, CA). The mechanical shutter (Uniblitz LS3ZM2, NY) and ICCD are triggered by TTL signals from a pulse generator (BNC Model 555, CA). A concave lens (f = -100 mm) is used to tailor the in-channel laser spot size. An eight-channel computer-controlled high voltage supply (Micralyne uTk, Alberta, Canada) controls on-chip electrokinetic flows.

Figure 3. Electropherogram of an on-chip separation bodipy and fluorescein. The distance between detector and injector is 2 mm. The images were collected at the rate of 25 Hz with 5 ms shutter exposure time. Detection spot size (integrated on the CCD chip) was 100 m2. The first peak represents bodipy and is followed by fluorescein peak with an SNR of 61.3.

References

  1. Molho, J.I., A.E. Herr, B.P. Mosier, J.G. Santiago, T.W. Kenny, R.A. Brennen, G.B. Gordon, B. Mohammadi, "Optimization of Turn Geometries for On-Chip Electrophoresis," Analytical Chemistry, Vol. 73, No. 6, pp. 1350-1360, 2001.
  2. Bharadwaj, R., J.G. Santiago, and B. Mohammadi, "Design and Optimization of On-Chip Capillary Electrophoresis," Electrophoresis, Vol. 23, pp. 2729-2744, 2002.
  3. Chen, C.-H., H. Lin, J.G. Santiago, and S. Lele, "Analysis of Convective Electrokinetic Flow Instabilities," to be submitted to Journal of Fluid Mechanics, 2003.
  4. Jung, B., R. Bharadwaj, and J.G. Santiago, "Thousand-Fold Signal Increase using Field Amplified Sample Stacking for On-Chip Electrophoresis," Vol. 24, No. 21, Electrophoresis, 2003.
  5. Bharadwaj R. and J.G. Santiago, "On-chip field amplified sample stacking under suppressed electrosomotic flow conditions," to be presented at the 7th International Conference on Miniaturized Chemical and BioChemical Analysis Systems, Squaw Valley, US.
  6. Bharadwaj, R. and J.G. Santiago, "The Dynamics and Efficiency of Field Amplified Sample Stacking," to be submitted, 2003.