1. Field of the Invention
The invention relates to the field of analytical chemistry in the areas of cellular biochemical and biomedical analysis and in particular to a laser microsurgery apparatus and method performed in less than one second for controllably lysing a single cell or selected cells and then collecting all or a selected portion of the cellular contents for immediate chemical analysis.
2. Description of the Prior Art
Dramatic progress in our understanding of biological processes has been made possible by studies of single cells. Research directed at the individual cells of an organism has utilized recent technological advances in optical and chemical methods. Laser-based techniques for manipulation of single cells and subcellular structures have enabled the performance of surgery on single cells. Ultrasensitive chemical analysis methods have now been used in biochemical studies of single cells. New knowledge gained from such single-cell studies is already finding medical and commercial applications. It can be expected that technology for the manipulation and analysis of single cells will play an important role in such areas as biomedical research, drug discovery, diagnosis of disease, and medical treatment.
In the past decade the tools of the analytic chemist have been applied to biochemical studies of living cells. For example, single neuronal cells from nonmammalian species have been analyzed through the use of capillary electrophoresis. By virtue of the large size of these neuronal ganglion cells subcellular measurements have been possible. Typically these cells are approximately 0.1 to 0.2 millimeters in diameter. In the prior art technique, the end lead of a capillary is etched to a fine point and used to sample cytoplasm from a cell or disrupted cellular fragment. Cytoplasmic contents are separated within the capillary and either detected on or off line.
For smaller mammalian cells in the range of 10 to 15 microns in diameter, the entire cell is loaded into the capillary and then the cell is lysed with a hypotonic or detergent-containing buffer. The cellular contents are separated in the capillary and detected by a variety of methods such as laser induced fluorescence or eletrochemical detection. However, cellular lysis is not easily controllable either as to the time at which it takes place or over the duration during which it occurs. For measurements on mammalian cells, the temporal resolution of the measurement techniques and the effects of perturbing the cell prior to complete lysis are important issues to consider. Lysis is defined for the purposes of this specification as the disruption of at least a portion of the plasma membrane with the release of at least a portion of the cellular contents. The manner in which the cell is lysed will govern the time which is required to terminate the biochemical reactions in progress, that is the manner of lysis will govern the temporal resolution of the biological measurement. Also, the manner of lysis will influence cellular processes occurring during the period of sampling. Many biological events take place on time scales of seconds or less. The enzymes typically have turnover numbers on the order of one to ten thousand per second. Metabolite concentrations can change greater than ten-fold in one second. Accurate measurement of such cellular properties by analytical techniques requires then that complete cell lysis occurs rapidly in comparison to the rate of change of the measured parameter. If disruption of the cell membrane occurs at a rate which is too slow, significant changes in the parameter can occur during the very lysis of the cell resulting in an inaccurate view of the actual physiological state of the cell.
In addition, membrane permeabilization during lysis results in the influx of extracellular ions such as calcium (Ca.sup.2+), activating many enzymes including kinases, phosphatases, proteases and nucleases. Even after disruption of the plasma membrane, biochemical processes will proceed until reactions are terminated, that is by separation of the reactants or denaturation of the molecules. Therefore, in order to accurately measure many cellular processes, complete cell lysis must be performed within milliseconds or less. The prior art chemical lysis is incapable of providing this speed in any manner which is controllable so that effective cellular analysis is possible.
Another important consideration in making whole cell measurements is the effect caused by manipulation of the cell prior to sampling. In studies of nonadherent or free floating cells using capillary electrophoresis the use of electroosmotic flow to move the cell into the capillary inlet may impact the cell and hence the process which is to be measured. A large body of literature exists on the biological effects of electrical fields. Unfortunately, most of the literature addresses AC rather than static fields so that the effects of low DC electric field strengths on cellular physiology have not been well characterized. However, there is no doubt that there is some effect. Application of an electric field with a gradient of the order of 1-2 kV/cm, can induce permeabilization of the cell membranes, a phenomenon known as electroporation. The potential gradients below 1-2 kV/cm, which are generally used for capillary electrophoresis, namely on the order of 400 V/cm, have important effects on cellular physiology. At an electric field strength of 167 V/cm for a five millisecond duration, there were localized increases in cellular permeability with concomitant influx of calcium ions (Ca.sup.2+) has been demonstrated. Such calcium ion influx can activate numerous cellular processes that may affect biochemical measurements.
In order to move a cell into a capillary, investigators have induced electroosmotic flow using potential gradients varying from 10 V/cm to 300 V/cm. While potential gradients in the range of 10-20 V/cm are unlikely to perturb cellular physiology, higher field strengths most certainly will.
Adherent cells or cells that adhere to a substrate, such as a glass slide or pipette are not amenable to manipulation by electroosmotic flow or hydrodynamic flow without first removing them from their attaching substrate. However, the mechanical stress induced by such removal can trigger a variety of cellular responses. Nearly all cells express abundant adhesion proteins at their surfaces. Structures known as focal adhesions provide a structural link between the cytoskeleton and the extracellular matrix. Focal adhesions consist of integrins and other proteins which are linked to a variety of intracellular signal transduction pathways. Mechanical stresses act through these membrane components to activate numerous enzymes including tyrosine kinases, serine/threonine kinases, G-proteins, proteases and others. Activations of these pathways trigger immediate and long term changes in the cellular physiology. For this reason mechanical manipulation, especially with adherent cells, prior to the time of sampling may interfere with measurement of the cellular biochemistry.
What is needed is a method to completely or at least partially lyse a single cell in milliseconds or less in a manner which is controlled both as to time and place and which does not affect the cellular physiology prior to lysis followed by loading of the cellular contents into a device for analysis such as a capillary for analyte separation and detection.
USPTO Backgrounds
USPTO Backgrounds