Instrument development for the analysis of low abundance analytes in single cells and small volume samples

摘要

Understanding the cell-to-cell differences between cells is important for both fundamental biology and in identifying normal and pathological functioning. Biogenic amines, which include catecholamines and indolamines, are of particular interest due to their presence throughout the central and peripheral nervous systems in many species, as well as their association with a wide variety of higher order behaviors such as sleep, memory formation, feeding, and mood; however, they are low abundance analytes since they are present in localized regions of the nervous system in femtomole to attomole quantities. Also, when sampling from the nervous system, the amines are often present within a complex matrix of proteins, salts, lipids, and other common biological compounds, which can complicate the detection and identification of trace levels of amines. This combination prompts the use of technologies that enable single cell measurements. Single cell measurements also provide insight into cell-specific metabolism, as different cell types are both quantitatively and qualitatively unique. Cell-specific metabolism distinguishes a cell that is morphologically similar to its neighbors but has a different molecular complement, which may result in a different function or indicate a difference in cell status. Differences in metabolism could also indicate potentially pathological behavior. The goal has been the design, construction, and validation of analytical instruments to enable single cell characterization. The high sensitivity and low sample consumption of capillary electrophoresis (CE) combined with the selectivity and sensitivity of laser-induced native fluorescence detection (LINF) makes CE-LINF well suited to study single cells and even subcellular organelles; however, the isolation and loading of such small samples into the CE system is challenging. This issue is addressed by designing, constructing, and interfacing a single beam optical trap with a laboratory-built CE system that uses multi-channel LINF detection, which has been optimized for single cell analyses. The optical trap is formed by tightly focusing the output of a Nd:YAG laser with a high numerical aperture objective. Once the cell is localized within the trap, the capillary inlet is moved adjacent to the trapped cell using a combination of a computer-controlled micromanipulator and a microscope stage. The cell is then released from the trap and pressure injected into the capillary. Cell lysis occurs within the capillary and the cellular constituents are subsequently separated and detected. Detection takes place using multi-channel LINF, which has been optimized for selective excitation and detection of biogenic amines. Briefly, a 224 nm HeAg hollow cathode ion laser is used in combination with a sheath-flow cuvette; the fluorescence emission is collected and measured using three channel detection with each photomultiplier tube having its own wavelength range selected with the appropriate dichroic mirror. This instrument allows unambiguous identification of a variety of catecholamines and indolamines based on differences in both their fluorescence emission profiles and migration times. This system, both as a hyphenated instrument and as individual components, has been used for several neurochemical applications, including detecting trace levels of indolamines in microdialysis samples and in single pinealocytes, the indolamine-containing cells of the pineal gland. These analyses highlight the ability of the system to isolate and manipulate single cells and perform injections and separate and detect low abundance analytes in samples with high concentrations of salts.