Chemical cytometry

Pang, Z., Liu, X., Al-Mahrouki, A., Berezovski, M., Krylov, S. N. (2006). Selection of surfactants for cell lysis in chemical cytometry to study protein-DNA interactions. 

Dovichi NJ, Hu S. Chemical cytometry. CurrOpin Chem Biol 2003 7 603-8. 

Arkhipov, S.N. et al.. Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells. Cytometry A, 63, 41, 2005. 

Chemical Cytometry Capillary Electrophoresis Analysis at the Level of the Single Cell... 

Chemical Cytometry of Proteins, Biogenic Amines, and Metabolic... 

Chemical Cytometry of Biogenic Amines and Proteins Using One-Dimensional... 

In contrast, chemical cytometry employs powerful ultrasensitive analytical methods to characterize the composition of single cells. In principle, chemical cytometry can resolve hundreds of components from a single cell. 

We instead employ fluorogenic reagents, such as 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ), in our experiments. These compounds are nonfluorescent until they react with a primary amine in the presence of a nucleophile. Fluorogenic reagents have very few fluorescent impurities and generate a very low background signal that does not interfere in the chemical cytometry experiment. 

The use of chemical cytometry for the study of GFP fusion proteins deals with these issues. Electrophoretic conditions can be manipulated to minimize overlap of GFP and autofluorescent components, so that GFP can be detected on a very low background, which improves detection limits. The migration time of the fusion will reflect any post-translational modifications to the protein, including proteolysis of the target protein. 

FIGURE 21.6 Chemical cytometry of a single yeast cell expressing the GFP-Gal4 fusion protein. The autofluorescence signal is similar in amplitude to the signal due to the fluorescent protein without electrophoresis, accurate quantiflcation of fluorescence due to the fusion protein would be difficult. 

FIGURE 21.7 Chemical cytometry of a single D. radiodurans cell expressing GFP. The set of peaks appears to be associated with proteolytic fragments of GFP. 

We have developed two-dimensional CE systems for the characterization of proteins and biogenic amines. The use of this technology for chemical cytometry is similar to the use of onedimensional electrophoresis a cell is aspirated into the column, lysed, and its components labeled with FQ. For two-dimensional electrophoresis, components are separated based on CSE in the first-dimension capillary. Fractions are then transferred across an interface to a second capillary, where they undergo additional separation based on micellar electrokinetic chromatography (MECC) before detection by fluorescence. The voltage drop across the first capillary is set to zero during the second dimension separation, holding components stationary. In a typical experiment, 300 fractions are transferred between capillaries under computer control. 

This research group coined the terms chemical cytometry and metabolic cytometry in 1999. Metabolic cytometry is a form of chemical cytometry that monitors biosynthetic and biodegradation enzymatic cascades in a single cell. 

FIGURE 21.13 Metabolic cytometry. A substrate (top) is fluorescently labeled (star). This substrate can undergo biosynthesis (left) or biodegradation (right). As long as the fluorescent tag remains intact, the metabohc products can be monitored by chemical cytometry. 

Instrumentation for chemical cytometry consists of three parts. The first is an injection module that facilitates aspiration of a cell into the capillary. The second is instrumentation to perform one- or two-dimensional CE. The third is a high sensitivity laser-induced fluorescence detector. 

Both one- and two-dimensional CE have been used for chemical cytometry. One-dimensional electrophoresis is similar to conventional experiments, albeit with the issues of sample loading, lysis, and on-column labeling. 

FIGURE 21.20 Typical timing diagram used in two-dimensional CE for chemical cytometry. The distal end of capillary 2 is held in the sheath-flow cuvette, which is at ground potential. 

The field of chemical cytometry is in its infancy, and it is clear that there is much work to be done before the technology is widely used. First, instrumentation throughput must be increased. Flow cytometry today can process a 100,000 cells/s. While chemical cytometry will not produce similar throughput, it is reasonable to expect the technology to process 10,000 cells/day in onedimensional electrophoresis and perhaps 1000 cells/day in two-dimensional electrophoresis. Such instrumentation will likely resemble the multiple-CE systems that have become ubiquitous in DNA sequencing. - ... 

Wu, H., Wheeler, A. and Zare, R. N. Chemical cytometry on a picoUter-scale integrated microfinidic chip. Pmc. Natl Acad. Sci. USA 2004 101 12809-12813. 

Hu, K., Ahmadzadeh, H. and Krylov, S.N. Asymmetry between sister cells in a cancer cell line revealed by chemical cytometry. Anal. Chem. 2004 76 3864—3866. 

Wu, H.K, Wheeler, A., andZare, R.N. Chemical cytometry on a picoliter-scale integrated microfluidic clap, Proceedings of the National Academy of Sciences 2004, 101351, 12809-12813. 

---