Continued progress in understanding cellular physiology requires new strategies for biochemical measurements in solitary cells, multiple cells, and subcompartments of cells. Large spatial gradients in the concentrations of molecules and presumably the activities of enzymes can occur in cells. Consequently, there is a critical need for measurement techniques for mammalian cells with control over the numbers or regions of cells interrogated. In the present work, we developed a strategy to rapidly load the cytoplasmic contents of either multiple cells or a subregion of a single cell into a capillary. A single, focused pulse from a laser created a mechanical shock wave which disrupted a group of cells or a portion of a cell in the path of the shock wave. Simultaneously, the cytoplasm was loaded into a capillary for electrophoretic separation. The size of the region of cellular disruption (and therefore the volume of cytoplasm collected) was controlled by the amount of energy in the laser pulse. Higher energies could be used to sample groups of cells while much lower energies could be utilized to selectively sample the tip of a neuronal process. The feasibility of performing measurements on subcellular compartments was also demonstrated by targeting reporter molecules to these compartments. A reporter localized to the nucleus was detected on the electropherogram following laser-mediated disruption of the cell and the nucleus. Finally, we demonstrate that this method terminated cellular reactions with sufficient rapidity that cellular membrane repair mechanisms were not activated during cytoplasmic collection. The combined ability to preselect a spatial region of a cell or cells and to rapidly load that region into a capillary will greatly enhance the utility of CE in the biochemical analysis of cells.

With the impressive growth in gene sequence data that has become available, recombinant proteins represent an increasingly vast source of molecular components, with unique functional and structural properties, for use in biotechnological applications and devices. To facilitate the use, manipulation, and integration of such molecules into devices, a controllable method for their chemical modification was developed. In this approach, a trifunctional labeling reagent first recognizes and binds a His-tag on the target protein's surface. After binding, a photoreactive group on the trifunctional molecule is triggered to create a covalent linkage between the reagent and the target protein. The third moiety on the labeling reagent can be varied to bring unique chemical functionality to the target protein. This approach provides: (1) specificity in that only His-tagged targets are modified, (2) regio-specific control in that the target is modified proximal to the His-tag, the position of which can be varied, and (3) stoichiometric control in that the number modifications is limited by the binding capacity of the His-tag. Two such labeling reagents were designed, synthesized, and used to modify both N- and C-terminally His-tagged versions of the enzyme murine dihydrofolate reductase (mDHFR). The first reagent biotinylated the enzyme,while the second served to attach an oligonucleotide to yield a protein-DNA conjugate. In all cases, modification in this manner brings new functionality to the protein while leaving the enzymatic activity intact. The protein-DNA conjugate was used to specifically immobilize the active enzyme through DNA hybridization onto polystyrene microspheres, a step toward creating a functional protein microarray.

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