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Description  |
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The invention relates to optical means for remotely monitoring pH and,
particularly, for invasive, or direct, monitoring of blood pH.
In medicine, invasive, or direct, monitoring of blood acid-base parameters
and other selected ions is desirable, and in many cases necessary, in the
management of critically ill patients or those undergoing complex surgical
procedures. In particular, blood pH is regulated within very narrow bounds
in normal individuals, varying no more than several hundredths of a pH
unit from an average of 7.40. The pH is directly dependent on bicarbonate
and dissolved CO.sub.2 concentrations in the blood. As a consequence,
several anesthetic agents and diseases affect blood pH, either directly or
indirectly. In particular, diabetic acidosis, which arises from depletion
of serum bicarbonate, and pulmonary disorders and anesthetic agents, which
affect respiration, can cause rapidly increased blood pCO.sub.2, which in
turn can produce striking alterations in blood pH. Either of these events
are life threatening. Thus, there is an important medical need for
directly monitoring blood pH.
Currently, the most widespread methods for direct blood pH measurement, or
direct blood electrolyte monitoring, involve the use of ion-selective
electrodes. While such electrodes can provide rapid and accurate
measurements, there are several disadvantages to their use. The familiar
glass pH electrode does not readily lend itself to the construction of
invasive devices. Although miniature glass electrodes have been mounted on
flexible catheters, small glass electrodes are inherently fragile and
therefore present serious risks to the patient. Indeed, most investigators
of in vivo blood pH have not employed invasive electrodes, but rather have
adopted the somewhat more cumbersome technique in which an arterial-venous
shunt is constructed to allow blood flow past a rigidly mounted,
mechanically protected glass electrode.
Electrical interference is a major problem with high-resistance
microelectrodes such as glass electrodes. Low-resistance miniature
electrodes are available, and can give satisfactory measurements in the
presence of other electrical equipment, but these require that the
amplifying and processing electronics be physically close to the
electrodes. Thus, the capability for remote measurements is lost. The most
common electrical interference occurs in the 50-60 Hz and radio frequency
ranges. While such interference can be reduced by special filtering
electronics, both forms of interference can cause DC shifts which are
easily overlooked.
Finally, the use of currently available electrodes can present direct
hazards to patient safety. Electronically based transducers can pose an
electrical hazard, especially when other such transducers are used at the
same time, and polyvinyl chloride-based electrodes widely used with
inophores, such as valinomycin, can be dissolved by many gaseous
anesthetics.
The electrical interference problem of electrodes is not limited to their
uses in medicine. In any environment where high sensitivity is critical,
electrical noise generated by extraneous fields will be a problem. Other
problems inherent to the use of electrodes include the susceptibility of
wire leads and couplings to deterioration under corrosive conditions, or
conditions of alternating temperatures.
Many of the above-mentioned difficulties with current information-gathering
technology can be overcome by using remote, in situ optical probes coupled
to a detector by optical waveguides, or fiber optics, e.g., Hirschfeld,
"Remote Fiber Fluorimetric Analysis," Energy and Technology Review, pgs.
17-21 (July 1980); Borman, "Optrodes," Anal. Chem., Vol. 53, pgs.
1616A-1618A (December 1981); and Peterson and Vurek, "Fiber Optic Sensors
for Biomedical Applications," Science, Vol. 224, pgs. 123-129 (Apr. 13,
1984). Fiber optics are durable, corrosion-resistant, heat-resistant,
impervious to electrical or magnetic interference, and are available in
very small diameters, which makes them amenable for use with miniature
probes.
Peterson, et al., in U.S. Pat. No. 4,200,110, issued Apr. 29, 1980,
disclose a pH sensing device which employs an optical transducer connected
to a detector by two fiber optics. The optical transducer is a
membranous-walled chamber which contains particles on which colorimetric
pH sensitive dyes are attached or impregnated. The dye is illuminated by
white light transmitted by one fiber optic, and the light scattered by the
dye-covered particles is collected by the other fiber optic. Use of more
than one fiber optic reduces sensitivity because precise alignment of the
illuminating and light-collecting fibers must be maintained, and because
illumination of dye molecules is less efficient when separate fibers are
used for illumination and collection than if a single fiber is used for
both collection and illumination. Moreover, the presence of the membrane
between the pH-sensitive dye and the fluid being monitored substantially
reduces the response time of the device to rapid changes in pH.
Numerous workers have used fluorescein or other dyes bound to solid
supports as quantitative pH indicators. Haaijman and Van Dalen,
"Quantification in Immunofluorescence Microscopy: A New Standard for
Fluorescein and Rhodamine Emission Measurement," Journal of Immunological
Methods, Vol. 5, pgs, 359-374 (1974), quantified the fluorescent response
of fluorescein bound to Sephadex supports to changes in pH. Hirschfeld, in
Borman, "Optrodes," cited above, reported binding fluorescein to porous
glass supports for the purpose of measuring pH, futher results being
reported in Hirschfeld et al., "Feasibility of Using Fiber Optics for
Monitoring Groundwater Contaminants," Optical Engineering, Vol. 22, pgs,
527-531 (October 1983). Saari and Seitz, in Analytical Chemistry, Vol. 54,
pgs. 821-823 (April 1982), disclose a pH sensor based on fluorescence
generated by a fluoresceinamine immobilized on a controlled pore glass
support. Immobilization was achieved by reacting controlled pore glass
derivatized with isothiocyanate groups with a saturated solution of
fluoresceinamine. Saari and Seitz report that their procedure for
immobilizing fluoresceinamine reduces the intensity of the fluorescent
signal by approximately three orders of magnitude from that of a
comparable amount of unbound fluoresceinamine. They also report an
extremely unfavorable signal-to-noise ratio of approximately 1:2 at pH 3.
The foregoing illustrates the limitations of the current pH-sensing
technology, especially in the area of medical applications. An alternative
to available pH sensing methods which overcame some of these limitations
would be highly advantageous for remote pH-sensing applications,
particularly in situ monitoring of blood pH.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide a pH-sensing
apparatus which generates an optical signal related to the magnitude of
the pH sensed.
Another object of the invention to provide a pH-sensing apparatus which
generates an optical signal related to the magnitude of the pH sensed and
which transmits said optical signal to a detector by a fiber optic.
Another object of the invention is to provide a low cost, low maintenance
pH sensor capable of remotely monitoring pH and compatible with a
multi-position monitoring system utilizing optical sensors.
Another object of the invention is to provide an optically based pH sensor
capable of monitoring the pH of physiological fluids, particularly blood.
These and other objects are attained in accordance with the present
invention wherein a support material is provided on which organic dye
molecules are covalently attached at a surface density falling within a
predetermined range. The invention is an application of the discovery that
the pH dependent fluorescent response of bound organic dye molecules
depends critically on (1) the surface density of organic dye molecules
bound to the support material and (2) the nature of the covalent linkage
between the organic dye molecules and the support material. The invention
is operated by contacting the support material on which the organic dye is
attached with the fluid whose pH is to be determined. When in contact, the
organic dye on the support material is illuminated so that it is caused to
fluoresce. The intensity of organic dye fluorescence is then related to
pH.
Preferably the invention comprises a fiber optic for transmitting an
illumination beam to the organic dye molecules and for collecting the
fluorescence thereof, and preferably the support material is in the form
of a particle, hereinafter referred to as a carrier particle. An
illumination beam is transmitted from a first end of the fiber optic to a
second end of the fiber optic, the illumination beam comprising light from
at least one associated light source. A carrier particle with covalently
bonded organic dye molecules is attached to the second end of the fiber
optic so that light from the illumination beam emanating from the second
end causes the covalently bound organic dye molecules to fluoresce. The
organic dye molecules are attached to the carrier particle at a surface
density within the predetermined range. A portion of the fluorescence from
the bound organic dye molecules is collected by the second end of the
fiber optic and transmitted to the first end of the fiber optic, the
transmitted portion of the fluorescence comprising a fluorescent signal.
At the first end of the fiber optic the fluorescent signal is separated
from the illumination beam and analyzed.
The present invention is addressed to problems associated with remote pH
monitoring in hostile or inaccessible regions. It advantageously overcomes
many of these problems by combining rugged, high quality fiber optics with
simple in situ transducers for generating fluorescent signals related to
ambient pH. For pH measurements in physiological fluids, the problem of
poor signal-to-noise ratio is overcome by adjusting the surface density of
organic dye molecules bound to a carrier particle so that it falls within
a predetermined range characterized by a strong fluorescent response in
the range of physiological pHs.
In addition, all particular embodiments of the invention are amenable for
use with a multi-position sensing system which comprises many sensors, all
of which feed signals to a single station for analysis. Such a
configuration can reduce costs by obviating the need for separate
analyzers for each sensor, and can increase reproducibility between
sensors by having all signals analyzed by the same instrument.
BRIEF DESCRIPTION OF THE DRAWINGS
Further objects of the invention, together with additional features
conbtributing thereto and advantages accruing therefrom will be apparent
from the following descriptions of preferred embodiments of the invention
which are shown in the accompanying drawings, which are incorporated in
and form a part of the specification. In the drawings:
FIG. 1 diagrammatically illustrates an optical configuration suitable for
use with the present invention.
FIG. 2 diagrammatically illustrates an optical configuration which includes
means for monitoring Raman backscatter from the fiber optic.
FIG. 3 is a curve describing the relationship between fluorescence
intensity and pH for an embodiment of the invention employing fluorescein
covalently linked to a controlled-pore glass carrier particle via
aminopropyl silane coupling agents.
FIG. 4 is a curve describing the relationship between fluorescence
intensity and pH for an embodiment of the invention employing fluorescein
covalently linked to a controlled-pore glass carrier particle via
aminoaryl silane coupling agents.
DETAILED DESCRIPTION OF THE INVENTION
Reference will now be made in detail to the present preferred embodiments
of the invention, an example of which is illustrated in the accompanying
drawings.
In accordance with the present invention an apparatus is provided for
measuring pH by means of an in situ fluorescent probe which generates a
fluorescent signal whose intensity varies with ambient pH. The fluorescent
probe comprises a support material onto which a plurality of organic dye
molecules are covalently attached so that there is a sufficient number of
non-dimerized organic dye molecules on the support material to generate a
fluorescent signal. A minimum number of organic molecules that do not form
dimers is at least 10.sup.3 attached molecules. A sufficient number of
these organic molecules that do not form dimers and generate a fluorescent
signal since they are illuminated by the illumination beam is at least
10.sup.3 attached molecules. The invention is operated by first placing
the fluorescent probe in contact with the fluid whose pH is to be
determined, i.e., an associated fluid. Next, the fluorescent probe is
caused to fluoresce by illuminating it with light from a suitable light
source. A portion of the resulting fluorescence is then collected, and its
intensity is related to pH.
Preferably the support material is in the form of a particle, hereinafter
referred to as a carrier particle, attached to an end of a fiber optic. In
accordance with this embodiment an illumination beam from an associated
light source is transmitted from a first end of the fiber optic to a
second end of the fiber optic, the carrier particle being attached to the
second end of the fiber optic so that light from the illumination beam
emanating from the second end illuminates the carrier particle causing the
attached organic dye molecules to fluoresce. A portion of the generated
fluorescence is collected by the second end of the same fiber optic and
transmitted to the first end of the fiber optic, the transmitted
fluorescence forming a fluorescent signal. At the first end of the fiber
optic the fluorescent signal is separated from the illumination beam and
analyzed.
Many different support materials and many different techniques for
covalently attaching organic dye molecules, such as fluorescein, are
available for use in accordance with the present invention. Such materials
and techniques are well known in the arts of affinity chromatography and
enzyme immobilization technology, e.g., Jakoby and Wilchek, editors
"Affinity Techniques," Methods in Enzymology, Vol. 34 (Academic Press, NY,
1974), Mobach, editor, "Immobilized Enzymes," Methods in Enzymology, Vol.
44 (Academic Press, NY, 1976), and Maugh II, Science, Vol. 223, pgs.
474-476 (Feb. 3, 1984). Among support materials, inorganic support
materials, such as controlled-pore glass, are preferred. Inorganic support
material suitable for use in accordance with the present invention are
described by Messing and Weetall, U.S. Pat. No. 3,519,538 issued July 7,
1970, entitled, "Chemically Coupled Enzymes," Weetall, U.S. Pat. No.
3,652,761 issued Mar. 28, 1972, entitled "Immunochemical Composites and
Antigen or Antibody Purification Therewith," and Weetall, "Covalent
Coupling Methods for Inorganic Support Materials," in Methods of
Enzymology, Vol. 44, pgs. 134-148 (Academic Press, NY, 1976). Accordingly,
these references are incorporated by reference for their descriptions of
inorganic support materials and means for covalently coupling organic
molecules thereto.
The preferred means of covalently bonding organic dye molecules to an
inorganic support material is by way of a silane coupling agent. Silane
coupling agents are silicon compounds which possess two different kinds of
reactivity: organofunctional and siliconfunctional. That is, silane
coupling agents have a silicon portion with an affinity for inorganic
materials such as glass or aluminum silicate, and they have an organic
portion which may be tailored to combine with a variety of other organics,
such as fluorescein, or some suitably derivatized version thereof, such as
fluorescein isothiocyanate. The most preferred support material is
controlled-pore glass. Controlled pore glass is commercially available in
a variety of forms manufactured according to the techniques of Hood et
al., U.S. Pat. No. 2,106,764, Chapman et al., U.S. Pat. No. 3,485,687, and
Haller, U.S. Pat. No. 3,549,524. Moreover, it is commercially available in
a variety of pore sizes and with a variety of different silane coupling
agents already attached (Pierce Chemical Company, Handbook and General
Catalog, Rockford, IL, 1983). Alternatively, silane coupling agents
suitable for use with the present invention can be prepared and attached
to control pore glass in accordance with the teaching of Weetall and
Filbert, "Porous Glass for Affinity Chromatography Applications," in
Methods of Enzymology, Vol. 34, pgs. 59-72 (Academic Press, NY, 1974).
Accordingly, this article is incorporated by reference.
Aminopropyl and aminoaryl silane coupling agents having formulas --O.sub.3
Si(CH.sub.2).sub.3 NH.sub.2 and
##STR1##
respectively, are preferred.
A crucial feature of the present invention is the requirement that the
organic dye molecules be attached to the support material such that a
sufficient number of the molecules remained non-dimerized for a
fluorescent signal to be generated. It has been discovered that when
certain organic dyes are attached to a support material at high surface
densities adjacent dye molecules interact, and in so doing reduce the
fluorescence response of the interacting molecules. It is believed that
this interaction involves the formation of dimers, akin to the
dimerization process described by Chambers et al., in "Effect of Dimer
Formation on the Electronic Absorption and Emission Spectra of Ionic Dyes,
Rhodamines and Other Common Dyes," J. Physical Chemistry, Vol. 78, pgs
380-387 (1974).
In accordance with the invention any organic dye whose fluorescence is
substantially reduced by dimerization is amenable for use in the
invention. In particular, this class of dyes includes sulforhodamine,
rhodamine, eosin B, eosin Y, acriflavine, proflavine acridine orange, and
fluorescein. Fluorescein is the preferred organic dye for measuring pH in
the physiological range.
In accordance with the invention the fluorescent probe comprising the
support material and bound organic dye molecules can be characterized
either by the number of non-dimerized covalently attached fluorescein
molecules present, or by the average density of bound organic dye
molecules (whether dimerized or not). In the latter case the approximate
length of the coupling agent must be known and the bound organic dye must
be uniformly and substantially randomly distributed on the surface of the
support material. The number of non-dimerized organic dye molecules
covalently attached to a support material is referred to as the first
plurality of organic dye molecules. The number of organic dye molecules
covalently attached to a support material uniformly and substantially
randomly distributed at a density within a preferred range is referred to
as the second plurality of organic dye molecules.
At least two factors determine the preferred range of densities for
particular support materials and coupling agents: the surface density of
the coupling agent on the support material, and the length and flexibility
of the coupling with an attached organic dye molecule. The surface density
of organic dye molecules covalently attached to a support material can be
expressed in any convenient units, e.g., molecules per square micrometer,
or the like. If attached dye molecules are distributed uniformly and
substantially randomly on the surface of the support material and if the
approximate length of the coupling agent is known, then the probability
that there will be an attached organic dye molecule within interaction
distance of any given attached dye molecule can be readily computed, e.g.,
Pielou, An Introduction to Mathematical Ecology (Wiley-Interscience, NY,
1969) pgs. 111-123, which pages are incorporated by reference. From this
probability the number of non-interacting attached organic dye molecules
can be computed, and an expected fluorometric response estimated. Thus,
for a given support material and coupling agent an upper limit on organic
dye molecule surface density can be computed. The lower limit of organic
dye molecule surface density is determined by detector sensitivity and
noise level. The upper and lower limits define an operable, or preferred,
range of surface densities. Within the operable range there is a most
preferred surface density where there is a maximum of non-interacting
organic dye molecules.
The nature of the surface of the support material is important in
determining the lower limit of preferred surface densities: the greater
amount of available surface area per illuminated region of support
material the lower the limit of operable densities. For example,
controlled-pore glass possesses a highly convoluted surface, and its
surface area per volume is exceedingly high. Thus, the lower limit of the
preferred density range for controlled-pore glass is substantially lower
than that of other support materials, such as cross-linked polymers, and
the like. In all cases, however, the density within the
illumination-collection region must be high enough so that there is a
sufficient number of organic dye molecules to generate a fluorescent
signal. By way of example, current detection apparatus can readily detect
a fluorescent signal from as few as 10.sup.3 fluorescein molecules,
Haaijman and Van Dalen, "Quantification in Immunofluorescence Microscopy:
A New Standard for Fluorescein and Rhodamine Emission Measurement,"
Journal of Immunological Methods, Vol. 5, pgs. 359-374 (1974).
To insure that the surface density of organic dye molecules lie within the
operable range, either the surface density of coupling agents can be
controlled, or the reaction between the coupling agents and the organic
dye (or suitable derivatives thereof) can be controlled. The latter
alternative is particularly applicable when commercially available
derivatized controlled-pore glass is used, as the surface density of the
coupling agents is quite high.
If the surface density of coupling agents is relatively sparse so that
interaction between organic dye molecules is precluded (or minimal), then
the coupling agents can be completely reacted with the organic dye,
saturating the coupling agents with organic dye molecules. If the surface
density of the coupling agents is relatively high, then procedures must be
chosen which prevent saturation of the coupling agents with organic dye
molecules. Such prevention can be accomplished using standard chemical
techniques such as disabling some of the coupling agents (for example by
including a reactant which competes with the organic dye for coupling
agent reaction cites, but which does not substantially affect the
fluorescence output of the organic dye), or adjusting reaction times,
reactant concentrations, and the like.
Non-specific binding or adsorption is a common problem in procedures for
covalently bonding molecules to support materials. That is, in many cases
the molecule to be covalently bonded also has an affinity for ionic or
hydrogen bonding with the support material. | | |
