Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
2000-04-27
2003-03-11
Warden, Jill (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S051000, C422S083000, C422S085000, C422S068100, C422S082080, C436S172000
Reexamination Certificate
active
06531097
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus and a method for determining the concentration of a substance. In one particular application, the invention relates to apparatus and a method for determining the concentration of oxygen within living tissue cells.
BACKGROUND TO THE INVENTION
For clarity, the term “assay substance” will be used in this specification to refer to the particular substance that an apparatus or method according to the invention is intended to detect.
It is well-known that a large number of fluorescent dyes measurably change their fluorescing characteristics in dependance upon the concentration of specific substances. For example, it is known that the fluorescence of the fluorophor tris(4,7-diphenyl-1,10-phenanthroline) ruthenium chloride is decreased in an inverse relationship to concentration of oxygen. It is recognised that both the intensity and the duration of light emitted by fluorescence of this substance are reduced in the presence of oxygen.
This phenomenon has been applied in measurement of oxygen tension in tumours, for example, as disclosed in the paper of W. K. Young, B. Vojnovic and P. Wardman: “Measurement of oxygen tension in tumours by time-resolved fluorescence” British Journal of Cancer (1996) 74 (Supl.XXVII) S256-S259. In this disclosure, apparatus for measuring oxygen tension comprises a sensor in which a fluorophor is localised in a polymer and coated on an end surface of an optical fibre. A pulsed laser is used to apply pumping light to the fibre, which light is transmitted to the fluorophor. Following the pulse, the fluorophor emits light, which travels back along the fibre to a detector. An assessment of the concentration of oxygen present is made on the basis of the time taken for the fluorescent emission to decay, the rate of decay increasing with the concentration. This sensor is advantageous in that it is relatively unreactive with biological tissue and will not affect living cells even after a prolonged contact with them, and in that it does not consume oxygen during detection.
The operation of such apparatus is, to a large extent, satisfactory. However, the apparatus does have a major disadvantage. In order to generate fluorescence of sufficient intensity to be measurable, a high-intensity light source is needed. In practice, it has been found that a laser source is required. While laser sources are readily available, they are relatively costly, such that they represent a significant portion of the total cost of a sensor.
SUMMARY OF THE INVENTION
It is an aim of the invention to provide a system for detecting the concentration of an assay substance which has all of the advantages of the above-described prior art system, but which does not require use of a laser light source.
According to a first of its aspects, the invention provides a sensor for detecting concentration of an assay substance comprising an optical fibre having an end surface on which is disposed a polymer body within which a multiplicity of particles is immobilised, on which particles is adsorbed a fluorophor, the polymer being such as to allow the assay substance to permeate into the body to come into contact with the fluorophor, and the fluorophor being selected as having a fluorescent activity which is measurably altered in the presence of the assay substance.
It has been found that the pattern of distribution of the fluorophor in a sensor embodying the invention is particularly effective in transmitting a high proportion of the light generated by the fluorophor back along the fibre for detection.
Preferably, the sensor comprises a single optical fibre which carries light from a pumping light source to the polymer body and which carries light emitted by the fluorophor from the polymer body to a detector. This further simplifies the construction of the sensor.
It has been found to be advantageous for the numeric aperture of the optical fibre to be greater than 0.3, and, in some embodiments, yet more advantageous to be greater than 0.4. In particularly preferred embodiments, a numerical aperture on the range 0.45 to 0.5 is selected. Numerical apertures in these ranges are particularly suited to collection of light from the fluorophor in the body of active material.
An optical fibre for use in a sensor embodying the invention is advantageously of less than 300 &mgr;m in diameter. A diameter of approximately 200 &mgr;m has been found to be particularly suitable.
The particles may suitably be silica gel particles. It is generally preferable for such particles to be of as small a size as possible, since this maximises the surface area on which dye can be adsorbed and also promotes cross-linking of the polymer matrix. At present, silica particles having an average diameter of 5 &mgr;m are readily available. It is preferable that the silica particles are of an average diameter of 5 &mgr;m or less.
In a particularly useful embodiment of the invention, the assay substance is oxygen. In such embodiments, a suitable polymer is a silicone rubber. A suitable fluorophor for use in such embodiments is a ruthenium complex dye, for example tris(4,7-diphenyl-1,10-phenanthroline). However, many other dyes could also be used, a prime desirable property being that the dye has a relatively long fluorescent lifetime; preferably in the order of several &mgr;s when unquenched by the assay substance.
In principle, a dye with a short fluorescent lifetime could be used. If this is the case, a correspondingly fast light source must be used to excite the fluorophor, and the light source must be controlled by suitably fast switching circuitry.
From another of its aspects, the invention provides a method of making a sensor for measuring the concentration of an assay substance in which a fluorescent dye is adsorbed onto a multiplicity of solid particles, subsequently dispersing the particles in a liquid polymer, applying the liquid polymer and the particles contained in it to an end surface of an optical fibre, and curing the liquid polymer to form a polymer body on the end surface of the optical fibre, the dye having been selected as to have a fluorescent activity which is measurably altered in the presence of the assay substance, and the polymer having been selected to be permeable to the assay surface when in its cured condition.
In such methods, the optical fibre typically comprises a core, a cladding, and a protective buffer coating which covers the cladding. In such cases, the method includes removal of the buffer coating from an end portion of the fibre prior to application of the liquid polymer, and subsequent to curing of the liquid polymer, a protective coating is applied to cover the said end portion and the polymer body. In such embodiments, the protective coating may be formed from the same liquid polymer as is applied to the end portion of the fibre. The polymer of the protective coating may be substantially pure or it may incorporate opaque particles such as carbon black. This latter arrangement isolates the fluorophor from ambient light, while the former arrangement may offer greater acceptability for use in contact with biological tissue.
Alternatively, the method may include insertion of the said end portion through a hollow member, such that its end face, on which the liquid polymer is applied, projects from the hollow member, and subsequent to curing of the polymer layer, a protective coating is applied to cover an end face of the tubular member and the polymer body. The hollow member may be a hollow needle made, for example, from steel or a ceramic material. The arrangement described in this paragraph has the advantage of having high mechanical durability.
In a third of its aspects, the invention provides a system for measuring the concentration of an assay substance comprising:
a sensor having a sensing body including fluorophor, which fluorophor has a fluorescent activity which is measurably altered in relation the concentration of the assay substance and light conveying means for conveying light to and from the fluorophor;
a pumping light sou
Vojnovic Borivoj
Wardman Peter
Young William K.
Burns Doane Swecker & Mathis L.L.P.
Cancer Research Campaign Technology Ltd.
Siefke Sam P.
Warden Jill
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