Assay apparatus and methods
First Claim
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1. Apparatus for use in assays of fluid suspensions, said apparatus comprising:
- means for delimiting a first volume of said fluid suspension;
means for introducing a pre-established quantity of a fluorescent material into said first volume, said fluorescent material being soluble in a unique fluid phase of said suspension and being capable when so dissolved of emitting fluorescent radiation when optically excited;
means for delimiting within said first volume a second volume limited to primarily a single phase of said suspension and throughout which fluorescence in said fluorescent material may be optically excited and observed.
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Abstract
An assay apparatus and methods employing total internal fluorescence to define a thin observed volume of a multi-phase suspension from which the suspended phase, because of its size and shape, is substantially excluded. Observations of the fluorescence of a known quantity of fluorescent material introduced into a known volume of the sample containing the observed volume permits quantitation of the volumes of the suspended and suspending phases.
108 Citations
10 Claims
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1. Apparatus for use in assays of fluid suspensions, said apparatus comprising:
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means for delimiting a first volume of said fluid suspension; means for introducing a pre-established quantity of a fluorescent material into said first volume, said fluorescent material being soluble in a unique fluid phase of said suspension and being capable when so dissolved of emitting fluorescent radiation when optically excited; means for delimiting within said first volume a second volume limited to primarily a single phase of said suspension and throughout which fluorescence in said fluorescent material may be optically excited and observed.
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2. Apparatus for use in immunoassays of a sample suspension containing an antigen-antibody complex incorporating a fluorescent tag capable of emitting fluorescent radiation over a first frequency band in response to a first predetermined band of exciting radiation, said apparatus comprising:
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means for delimiting a first volume of said sample suspension; means for introducing a pre-established quantity of a fluorescent material into said first volume, said fluorescent material being soluble in a unique fluid phase of said sample suspension and being capable when dissolved of emitting fluorescent radiation over a second frequency band when excited by a second predetermined band of said exciting radiation; and means for delimiting within said first volume a second volume limited to primarily a single phase of said suspension and throughout which fluorescence in said first and second bands may be excited and observed. - View Dependent Claims (3, 4)
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5. A method for assay of a fluid suspension, said method employing an optical element transmissive to excitation radiation which can excite fluorescence of a fluorescent material soluble in a unique fluid phase of said sample suspension, said optical element also being transmissive to fluorescence of said fluorescent material, said method comprising the steps of:
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immersing a known area of said optical element in a fluid sample to be assayed; controlling the immersion of said optical element so as to insure said known area is completely covered with a thin layer of said fluid sample of known thickness; introducing a pre-established quantity of said fluorescent material into the volume of said fluid sample established by said known area and said known thickness; incubating said optical element in said volume of fluid sample for a time in excess of the time required for diffusional processes to distribute said fluorescent material within said volume; illuminating said optical element with radiation so as to excite fluorescence, by an evanescent wave, of said fluorescent material dissolved in said unique fluid phase; and measuring the intensity of said fluorescence. - View Dependent Claims (6, 7)
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8. A method for immunoassay employing an optical element transmissive to excitation radiation which can excite fluorescence of a fluorescent tag included as a constituent of an antigen-antibody complex to be assayed in a sample suspension, said element being also transmissive to excitation radiation which can excite fluorescence of a second fluorescent material soluble in a unique fluid phase of said sample suspension, said element being also transmissive to fluorescence of said tag and of said second fluorescent material, said element having selected moieties of said antibody-antigen complex attached to a known area of its surface, said method comprising the steps of:
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immersing said optical element in a sample suspension to be assayed so as to form said complex; controlling the immersion of said optical element so as to insure said known area is completely covered with a thin layer of said sample of known thickness; introducing a pre-established quantity of said second fluorescent material into the volume of said sample suspension established by said known area and said known thickness; incubating said submerged optical element for a time in excess of the time required for diffusional processes to scavenge said volume; illuminating said optical element with said excitation radiation so as to excite fluorescence, by an evanescent wave, of said tag in said complex attached to said element and said second fluorescent material dissolved in said unique fluid phase; and measuring the relative intensities of said fluorescence. - View Dependent Claims (9, 10)
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Specification