Optical fiberless sensors

Details Optical fiberless sensors Optical fiberless sensors

2000-09-20

2002-04-30

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S004000, C435S029000, C435S164000, C435S173300, C435S805000, C422S082050, C422S087000

Reexamination Certificate

active

06379955

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to optical fiberless sensors, method of fiberless sensor fabrication and uses of such sensors in cells.
BACKGROUND
The ability of cells, tissues, an organ system and an entire organism to rapidly respond and adapt to exogenous stimuli is a requirement for the maintenance of life. Exposure of a single cell to such stimuli can manifest itself in a variety of ways, including a flux of essential intracellular ions (i.e. Na+, K+, Ca++, Cl−, H+), as well as changing oxygen and glucose levels. These changes can trigger additional signaling cascades, ultimately resulting in the recruitment of the appropriate cellular machinery for a response to the stimuli.
Of course, some stimuli are pathogenic to cells. Such stimuli cause a combination of linked and cascading biochemical events leading up to disease and/or cell death. For example, exposure to bacteria, viruses, toxins and toxicants may result in a myriad of intra/extracellular responses, depending on the pathogen or pathogenic agent in question and the route of exposure. The determination and understanding of which of the “downstream” biochemical signals elicited are indicators of physical, chemical or mechanical injury are fundamental to the development of countermeasures and therapy.
Classical biochemical investigations of the toxicologic effects of chemicals on organs and tissues were typically performed on homogenates. This approach reduced complex arrays of cells to a uniform blend. While providing important new information on fundamental mechanisms of toxicology/pharmacology, these studies are limited in their ability to discriminate between cells which are passively or actively involved.
More recent molecular and imaging techniques have improved cellular resolution. However, these newer imaging techniques frequently provide only static “snapshots” of dynamic cellular processes. Other approaches, while more dynamic, suffer from the fact that the approach alters the cells under study. For example, commercially available fluorescent probes used in the detection of calcium fluxes, chemically bind the moiety in question and potentially alter its homeostasis in situ.
Clearly, the most extensive work done intracellularly focused on the direct injection of dyes into the cell. While this method has provided researchers with a simple technique to study cellular processes, it has also proven problematic. For instance, the dye may itself be toxic, or otherwise interfere with the cell chemistry.
Another problem is that there is no way to position the dye once it is introduced into the cell. Often, the dye is selectively trapped in some organelles, rather than dispersed evenly throughout the cell.
An additional, critical limitation with the dye injection approach is that the technology is currently limited in selectivity to a small number of analytes. For instance, while there are good dyes for calcium ion detection, there are none for potassium, sodium or chloride.
Fiber optic probes, or optodes, with a polymer sensing element, solve the above problems of dye injection. See W. Tan et al., “Submicrometer Intracellular Chemical Optical Fiber Sensors,” Science 258:778 (1992). These micro-fiberoptic sensors (100-1000 nm) are based on optical grade silica fibers pulled to submicron size. The pulled fiber tips are much less fragile than those of the electrochemical microsensors, which are made from pulled micropipettes. Attached to the tip is a dye-polymer matrix, which is very durable and smooth and runs tightly bound to the tip, even during penetration of biological tissues. The matrix on the end of the fiber often includes several components, such as a chromoionophore, an ionophore, and appropriate ionic additives, all trapped inside a polymer layer, so that no chemicals are free to diffuse throughout the cell. The effects of toxicity of the dyes are thus minimized. Also, the probe can be carefully positioned in the cell, allowing any specific area to be imaged or monitored.
Nonetheless, the fiber optic probes have the significant drawback of being unable to easily monitor more than one location in the cell. For monitoring more than one location, multiple probes are needed. Due to size constraints, it can prove difficult to position several fibers inside a single cell. Moreover, even the insertion of single fiber sensor can easily damage a cell or short out the cross membrane electrical potential and having several fibers compounds this problem.
Thus, improved methods for studying cells and intracellular analytes are needed. Such improved methods should be amenable to monitoring the cell at more than one location and should have minimal toxicity.
SUMMARY OF THE INVENTION
The invention relates generally to optical fiberless sensors, method of fiberless sensor fabrication and uses of such sensors in cells. The sensors of the present invention are: (1) small enough to enter a single mammalian cell relatively non-invasively, (2) fast and sensitive enough to catch even minor alterations in the movement of essential ions and (3) mechanically stable enough to withstand the manipulation of the sensor to specific locations within the cell.
Importantly, the fiberless sensors of the present invention are non-toxic and permit the simultaneous monitoring of several cellular processes. In one embodiment, the present invention contemplates the use of such fiberless sensors to monitor a single cell exposed to a variety of noxious or trophic stimuli.
The fiberless sensors of the present invention are particularly useful for the direct, real-time, non-invasive, intracellular studies of chemical insults and in elucidation of subcellular mechanisms of action induced by pathogens and related toxins. These sensors are immensely smaller, faster and more sensitive than fiber-optic sensors currently used. The spatially and temporarily highly resolved and highly detailed chemical information gained from using these sensors, greatly speeds up current protocols of research and also leads to new and improved methodologies.
In one embodiment, the present invention contemplates a method comprising: a) providing i) one or more cells, ii) a plurality of fiberless optical sensors, and iii) a means for detecting said sensors; b) introducing said plurality of sensors into said one or more cells; and c) detecting said sensors in said cells with said detecting means.
In another embodiment, the present invention contemplates a method comprising: a) providing i) one or more cells, ii) a plurality of fiberless optical sensors, iii) an exogenous cellular stimulus, and iv) a means for detecting said sensors; b) introducing said plurality of sensors into said one or more cells; c) stimulating said one or more cells with said exogenous cellular stimulus, and d) detecting said sensors in said cells with said detecting means.
In one embodiment, the present invention contemplates a method comprising: a) providing i) first and second preparations of cells, ii) a plurality of fiberless optical sensors, iii) an exogenous cellular stimulus, and iv) a means for detecting said sensors; b) introducing said plurality of sensors into said first and second preparations of cells; c) stimulating said first preparation of cells with said exogenous stimulus, d) detecting said sensors in said cells with said detecting means, and e) comparing the sensors in said first preparation of cells with the sensors in said second preparation of cells.
It is not intended that the present invention be limited by the nature of the cells. Both prokaryotic and eukaryotic cells can be monitored using the sensors of the present invention. Among eukaryotic cells, it is specifically contemplated that the sensors of the present invention are introduced into mammalian cells. All types of mammalian cells are contemplated (e.g. oocytes, epithelial cells, etc.). In some embodiments, cells such as neurons and astrocytes in primary culture are contemplated. Thus, the present invention contemplates generally compositions comprising mammalian cel

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