A pH micro-probe, a temperature micro-probe, and an immuno-based micro-probe each include a shaft for transmuting an input light signal and a tip for inserting into a cell or other substance for measuring pH, temperature, and/or antigens. The pH micro-probe and the temperature micro-probe each include a luminescent material positioned on the tip of the micro-probe. The light signal excites the luminescent material so that the luminescent material emits a luminescent light signal. The luminescent light signal has a property value dependent on the pH or temperature being measured and reflects back through the shaft for being measured by a light signal measuring device. The immuno-based micro-probe includes a reflective material that has an effective refractive index dependent on the number of antigen-antibody bonds present on the reflective material.

BACKGROUND

Probes are often used for measuring acidity level (pH), temperature, and antigens of substances and test samples. Conventional probes typically are too large to make localized measurements, thus making pH, temperature, and antigen measurements in micro scale such as in a single cell, in a specific local spot, and micro-scale gradient change difficult. This particularly prevents intracellular measurements from being taken. Some conventional probes can make intracellular measurements but not without destroying the cell being probed.

SUMMARY

The present invention solves the above-described problems and provides a distinct advance in the art of pH, temperature, and immuno-based probes.

A pH micro-probe constructed in accordance with an embodiment of the present invention comprises a shaft, an ultra-thin mirror coating applied to at least part of the shaft, a tip, and a luminescent dye-doped coating and an ultra-thin mirror coating applied to the tip. The tip of the pH micro-probe may be inserted into a biological cell or similar substance whose pH is being measured. An input light signal is transmitted into the shaft and reflected off of the mirror coating so as to continue traveling into the tip. The input light signal reflects off of the mirror coating applied to the tip so as to excite the luminescent dye-doped coating. The luminescent dye-doped coating emits a pH dependent luminescent light signal. The luminescent light signal travels back through the shaft for being measured by a light signal measuring device.

A temperature micro-probe constructed in accordance with another embodiment of the present invention comprises a shaft and mirror coating similar to the pH micro-probe and a tip that includes a microcavity extending into the tip and a luminescent material positioned on or in the microcavity. The tip of the temperature micro-probe is inserted into a substance whose temperature is being measured. An input light signal is transmitted through the shaft so as to excite the luminescent material. The luminescent material emits a temperature dependent luminescent light signal. The luminescent light signal travels back through the shaft for being measured by a light signal measuring device.

An immuno-based micro-probe constructed in accordance with yet another embodiment of the present invention comprises a shaft and mirror coating similar to the pH micro-probe and temperature micro-probe, a tip, and a thin film coated on the tip. The thin film includes a number of nano holes extending therethrough. The tip of the immuno-based micro-probe is inserted into a sample being measured. A number of antibodies will then be immobilized on the thin film. Antigens in the sample will begin to bond to the antibodies. An input light signal is transmitted through the shaft so that some of the input light signal reflects off of the thin film and some of the input light signal passes through the nano holes. The amount of light reflecting off of the thin film is dependent on the number of antigens or ratio of antigen-antibody bonds to total number of antibodies.

A pH micro-probe constructed in accordance with another embodiment of the present invention comprises a shaft and a tip. The shaft includes a central fiber and a plurality of surrounding fibers. The shaft is connected to the tip at the shaft's distal end and is tapered from its proximal end to its distal end.

The central fiber is connected to the tip at the shaft's distal end (i.e., a distal end of the central fiber is connected to the tip) and allows an input light signal from an input light source to travel therethrough towards and into the tip. The central fiber includes a mirror coating surrounding the central fiber and configured to guide the input light signal through the central fiber. The mirror coating may be applied to a portion or all of an outer surface of the central fiber and may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material. The central fiber may be an elongated transparent member formed of glass or other suitable transparent material.

The surrounding fibers are connected to the tip at the shaft's distal end (i.e., distal ends of the surrounding fibers are connected to the tip) and allow a luminescent light signal to travel into and through the surrounding fibers from the tip. Each surrounding fiber includes a mirror coating surrounding the surrounding fiber and configured to reflect a luminescent light signal traveling through the surrounding fibers so as to guide the luminescent light signal through the surrounding fibers. Each mirror coating may be applied to a portion or all of an outer surface of one of the surrounding fibers and may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material. The surrounding fibers are twisted around the central fiber and may be elongated transparent members formed of glass or other suitable transparent material. In one embodiment, the surrounding fibers are adjacent to each other and may include six surrounding fibers.

The tip is configured to contact or be inserted into a cellular substance and has a luminescent dye doped coating and a mirror coating. The tip is connected to the central fiber and the surrounding fibers at the shaft's distal end and has a diameter of between 3 and 5 micrometers. In one embodiment, the tip widens from the distal end of the shaft and has a rounded shape. Importantly, the tip affords optical communication of the luminescent signal arising from the input light signal received from the central fiber to propagate back through the surrounding fibers.

The luminescent dye doped coating interacts with the light signal to generate a pH dependent or other characteristic dependent luminescent light signal. The luminescent dye-doped coating may be a thin film at least partially formed of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) or similar material, and may include an aerogel or similar material, organically modified silicate (ORMOSIL) or similar material, or any other suitable material. The luminescent dye-doped coating may be applied to a portion or all of an outer surface of the tip and may have a thickness on the nano scale or micro scale. The luminescent dye-doped coating may be approximately 100 nm to approximately 2 μm thick. The luminescent dye-doped coating may overlap the shaft's distal end.

The mirror coating at least partially reflects the luminescent light signal within the tip and may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material applied over the luminescent dye doped coating. The mirror coating may overlap the shaft's distal end.

In use, the tip of the pH micro-probe may be inserted into a biological cell or similar substance whose pH is being measured. The micro-probe can be connected at the shaft's proximal end to a light source such as a light ray generator for generating an input light signal. The input light signal is transmitted through the central fiber towards the shaft's distal end into the tip and the luminescent light signal is reflected back into the surrounding fibers at the shaft's distal end and through the surrounding fibers towards the shaft's proximal end. The micro-probe can also be connected to a light signal measuring device near the shaft's proximal end for receiving the luminescent light signal from the surrounding fibers so that a property value can be measured for determining the pH or other characteristic of the cellular substance.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Turning now to the drawing figures, and particularlyFIG. 1, a pH micro-probe10constructed in accordance with an embodiment of the invention is illustrated. The micro-probe10broadly includes a shaft12, a mirror coating14applied to at least part of the shaft12, a tip16, and a luminescent dye-doped coating18and a mirror coating20applied to at least part of the tip16.

The shaft12allows a light signal to be transmitted therethrough and one embodiment of the shaft12is an elongated transparent member formed of glass or other suitable transparent material. The shaft12may be tapered so that a distal end of the shaft12is narrower or smaller than its proximal end. The shaft12also allows an output luminescent light signal to travel from the tip16and through the shaft12, as described below.

The mirror coating20reflects the light signal traveling through the shaft12so as to guide the light signal through the shaft12. The mirror coating20may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material and may be applied to a portion or all of an outer surface of the shaft12.

The tip16allows the luminescent dye-doped coating18to interact with the substance being tested and is positioned at the distal end of the shaft12. The tip16may be the distal end of the shaft12itself or may be an extension or attachment connected to the shaft12. The tip16may be bulb shaped (elongated, egg shaped, or spherical) and may be wider or larger than the distal end of the shaft12.

The luminescent dye doped coating18interacts with the light signal to generate a pH dependent luminescent light signal. The luminescent dye-doped coating may be a thin film at least partially formed of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) or similar material, and may include an aerogel or similar material, organically modified silicate (ORMOSIL) or similar material, or any other suitable material. The luminescent dye-doped coating18may be applied to a portion or all of an outer surface of the tip16and may have a thickness on the nano scale or micro scale. In one embodiment the luminescent dye-doped coating18may be approximately 100 nm to approximately 2 μm thick.

The mirror coating20at least partially reflects the light signal within the tip16and may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material applied over the luminescent dye doped coating18.

In one embodiment, a protective film22such as a platinum belt may be applied between the luminescent dye doped coating18and the mirror coating20, as shown inFIG. 2. The protective film22protects the luminescent dye doped coating18and the tip16from damage.

Use of the pH micro-probe10will now be described in more detail. The tip16of the pH micro-probe10may be inserted into a micro-volume solution or the intracellular or intercellular substance (cytoplasm, etc.) of a cell. An input light signal is then transmitted from a light signal generator into the proximal end of the shaft12. The light signal will reflect off of the mirror coating20so as to continue traveling through the shaft12to the distal end of the shaft12. The light signal will then continue into the tip16and will reflect off of the mirror coating20so as to bounce around inside the tip16. The light signal will hit and be at least partially absorbed by the luminescent dye doped coating18. The dye doped coating18will emit a luminescent light signal such as a fluorescent light signal. The luminescent light signal will reflect inside of the tip16and eventually exit the tip16into the shaft12. The luminescent light signal will reflect off of the mirror coating14so as to continue traveling from the distal end of the shaft12to the proximal end of the shaft12. The luminescent light signal will then exit the proximal end of the shaft12and continue traveling through fiber optic components to a light signal measuring device.

The luminescent light signal has a property value indicative of the pH of the measured substance. For example, the intensity of the luminescent light signal may be dependent on the pH of the substance. A fluorescence ratio (the ratio of the fluorescence of the luminescent dye doped coating18when excited at a first wavelength compared to a second wavelength (e.g. 560 nm compared to 640 nm) may also be measurable against the pH of the substance.

The above-described pH micro-probe10provides several advantages over conventional pH measuring methods. For example, the pH micro-probe10allows the pH of the cellular substance of a single cell or a very small sample to be measured. The pH micro-probe10confines the pH measurement to the cellular or other substance in contact with the tip16so that the pH of adjacent cells or adjacent material does not affect the measurement. This is particularly useful in heterogeneous environments such as biological cells. The pH micro-probe10also allows for the measurement of pH via fluorescence ratios, which eliminates the need to account for certain factors such as how much dye doped coating20is used.

In another embodiment of the invention, a pH micro-probe100similar to the above-described micro-probe10includes a shaft102having a central fiber104and one or more surrounding fibers106, as shown inFIG. 3.

The central fiber104allows an input light source to travel therethrough and includes a core108similar to the shaft12and a mirror coating110similar to the mirror coating20described above.

The surrounding fibers106allow the luminescent light signal to travel therethrough and each include a core and mirror coating similar to the core108and mirror coating110of the central fiber104. The surrounding fibers106may be twisted around the central fiber104or may maintain an initial orientation in relation to the central fiber104. In one embodiment there are six surrounding fibers106forming a hexagonal pattern around the central fiber104.

The pH micro-probe100is used in substantially the same manner as the pH micro-probe10described above except that the light signal is transmitted through the central fiber104and the luminescent light signal is reflected back through the surrounding fibers106and optionally the central fiber104.

Turning now toFIG. 4, a temperature micro-probe200constructed in accordance with another embodiment of the invention is illustrated. The temperature micro-probe200broadly includes a shaft202, a tip204, at least one microcavity206extending into the tip204, and a luminescent material208positioned in the microcavity206.

The shaft202allows a light signal to be transmitted therethrough and may be substantially similar to the shaft12described above.

The tip204allows the light signal to interact with the luminescent material208and may be substantially similar to the tip16described above. In one embodiment, the tip204is the distal end of the shaft202.

The microcavity206allows the luminescent material208to adhere to the tip204and interact with the light signal and may be formed in the tip204near the distal end of the shaft202. The microcavity206may be formed by chemical etching via hydrofluoric acid (such as a 20% hydrofluoric acid solution) and may extend less than 30 μm along the tip204. In one embodiment, the microcavity206has a volume of 5 μm by 5 μm by 5 μm or less.

The luminescent material208interacts with the light signal to generate a temperature dependent luminescent light signal and may be a thin film or coating of fluorescent material or a number of quantum dots. In one embodiment, the luminescent material208is a Rhodamine dye such as R6G. In another embodiment, the luminescent material208is a number of quantum dots in a liquid phase or solid phase coating on the surface. In yet another embodiment, the luminescent material208is a phosphor or phosphorescent material

Use of the temperature micro-probe200will now be described n more detail. The tip204of the temperature micro-probe200is inserted into the substance whose temperature is to be measured. An input light signal is then transmitted into the proximal end of the shaft202from a light signal generator. The light signal travels to the distal end of the shaft202until it reaches the microcavity206. The light signal will then be at least partially absorbed by the luminescent material208. The luminescent material208will emit a luminescent light signal such as a fluorescent light signal. The luminescent light signal will travel to the proximal end of the shaft202, exit the proximal end of the shaft202, and continue traveling through fiber optic components to a light signal measuring device.

The luminescent light signal has a property value indicative of the temperature of the substance. For example, the wavelength of the luminescent light signal may be dependent on the temperature of the substance. In one embodiment, the full width of half of the maximum value of the wavelength (FWHM) may be measured. As another example, the intensity or peak intensity of the luminescent light signal at a specified wavelength may be dependent on the temperature of the substance. In another embodiment, the luminescent decay time may be measured to determine the temperature of the substance. As an example, the luminescent decay time of phosphor may be dependent on temperature.

The above-described temperature micro-probe200provides several advantages over conventional temperature measuring instruments. For example, the temperature micro-probe200allows the temperature of very small amounts of substances (including intracellular and intercellular substances) to be measured. The temperature micro-probe200allows for localized measurements to be made without influence of nearby temperatures. This is particularly useful in heterogeneous environments such as biological cells.

Turning now toFIGS. 5 and 6, an immuno-based micro-probe300constructed in accordance with another embodiment of the invention is illustrated. The immuno-based micro-probe300broadly includes a shaft302, a tip304, a thin film306, and a number of nano holes308.

The shaft302allows a light signal to be transmitted therethrough and may be substantially similar to the shafts12,202described above.

The tip304allows the light signal to interact with the thin film306and may be substantially similar to the tips16,204described above. In one embodiment, the tip304is the distal end of the shaft302.

The thin film306allows half antibodies to be immobilized thereon and may be applied to or coated on at least a portion of the tip304. The thin film306may be formed of gold or any other suitable material.

The nano holes308allow at least a portion of the light signal to pass through the thin film306and extend through the thin film306so that the thin film306exhibits a porous texture.

Use of the immuno-based micro-probe300will now be described in more detail. The tip304of the immuno-based micro-probe300is inserted into a sample to be measured. An input light signal is then transmitted into the proximal end of the shaft302from a light signal generator. The light signal will travel to the distal end of the shaft302and will continue into the tip304. Some of the input light signal will reflect off of the thin film306and some of the input light signal will pass through the nano holes308Antigens in the sample will bind to a layer of half antibodies immobilized on the thin film306. As more antigens bind to the antibodies, the reflection intensity at spectral wavelength will increase. That is, more of the input light signal will reflect back into the shaft302for being detected by a light signal measuring device. As such, the number of antigen-antibody bonds or the ratio of antigen-antibody bonds to non-bonds can be measured as a function of the measured reflection intensity of the reflected light signal.

The above-described immuno-based micro-probe300provides several advantages over conventional immuno-based detection methods. For example, the immuno-based micro-probe300allows the antigens of a very small sample, such as a single cell, to be measured. The immuno-based micro-probe300allows for localized measurements to be made. This is particularly useful in heterogeneous environments.

Turning now toFIGS. 7 and 8, a pH micro-probe400constructed in accordance with another embodiment of the invention is illustrated. The micro-probe400broadly comprises a shaft402and a tip404.

The shaft402includes a central fiber406and a plurality of surrounding fibers408. The shaft402is connected to the tip404at the shaft's distal end412and is tapered from its proximal end414to its distal end412.

The central fiber406is connected to the tip404at the shaft's distal end412(i.e., a distal end of the central fiber406is connected to the tip404) and allows an input light signal from an input light source to travel therethrough towards and into the tip404. The central fiber406includes a mirror coating410asurrounding the central fiber406and configured to guide the input light signal through the central fiber406. The mirror coating410amay be applied to a portion or all of an outer surface of the central fiber406and may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material. The central fiber406may be an elongated transparent member formed of glass or other suitable transparent material.

The surrounding fibers408are connected to the tip404at the shaft's distal end412(i.e., distal ends of the surrounding fibers408are connected to the tip404) and allow a luminescent light signal to travel into and through the surrounding fibers408from the tip404. Each surrounding fiber408includes a mirror coating410bsurrounding the surrounding fiber408and configured to reflect a luminescent light signal traveling through the surrounding fibers408so as to guide the luminescent light signal through the surrounding fibers408. Each mirror coating410bmay be applied to a portion or all of an outer surface of one of the surrounding fibers408and may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material. The surrounding fibers408are twisted around the central fiber406, as best seen inFIG. 8, and may be elongated transparent members formed of glass or other suitable transparent material. In one embodiment, the surrounding fibers408are adjacent to each other and may include six surrounding fibers, as shown inFIG. 8.

The tip404is configured to contact or be inserted into a cellular substance and has a luminescent dye doped coating416and a mirror coating418. The tip404is connected to the central fiber406and the surrounding fibers408at the shaft's distal end412and has a diameter of between 3 and 5 micrometers. In one embodiment, the tip404widens from the shaft's distal end412and has a rounded shape. Importantly, the tip404affords optical communication of the luminescent signal arising from the input light signal received from the central fiber406to propagate back through the surrounding fibers408.

The luminescent dye doped coating416interacts with the light signal to generate a pH dependent or other characteristic dependent luminescent light signal. The luminescent dye-doped coating416may be a thin film at least partially formed of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) or similar material, and may include an aerogel or similar material, organically modified silicate (ORMOSIL) or similar material, or any other suitable material. The luminescent dye-doped coating416may be applied to a portion or all of an outer surface of the tip404and may have a thickness on the nano scale or micro scale. The luminescent dye-doped coating416may be approximately 100 nm to approximately 2 μm thick. The luminescent dye-doped coating416may overlap the shaft's distal end412, as shown inFIG. 7.

The mirror coating418at least partially reflects the luminescent light signal within the tip404and may be a thin film at least partially formed of silver, aluminum, gold, or other reflective material applied over the luminescent dye doped coating416. The mirror coating418may overlap the shaft's distal end412, as shown inFIG. 7.

Use of the micro-probe400will now be described in detail. The tip404of the pH micro-probe400may be inserted into a biological cell or similar substance whose pH is being measured. The micro-probe400can be connected at the shafts proximal end414to a light source such as a light ray generator for generating an input light signal. The input light signal is transmitted through the central fiber406towards the shaft's distal end412into the tip404and the luminescent light signal is reflected back into the surrounding fibers408at the shaft's distal end412and through the surrounding fibers408towards the shafts proximal end414. The micro-probe400can also be connected to a light signal measuring device near the shaft's proximal end414for receiving the luminescent light signal from the surrounding fibers408so that a property value can be measured for determining the pH or other characteristic of the cellular substance.