GB2142156A - Fluorescent spectroscopy - Google Patents

Abstract

A method for conducting fluorescent spectroscopy which involves irradiating a test sample with light of a first wavelength which causes the test sample to fluoresce light of a second wavelength and measuring a portion of the fluorescent light; irradiating the test sample with light of the first wavelength at reduced intensity and measuring the light of the first wavelength transmitted through the test sample; and comparing the intensity of the fluorescent light to the transmitted light. <IMAGE>

Description

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GB 2 142 156 A

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SPECIFICATION

Fluorescence spectroscopy method

5 This invention relates to chemical analysis techniques, and more particularly to a method and apparatus for analyzing substances by exciting with a band of radiant energy and monitoring the fluorescence generated by such an excitation.

United States Patents 3,664,744; 3,748,044; 3,831,618; 3,811,780; 3,900,289; 3,833,304 and 3,817,425, describe in detail a bichromatic spectrophotometer and related apparatus. An Operation Manual entitled 10 Abbott VP Bichromatic Analyzer (1978) available through Abbott Laboratories Diagnostic Division, 1921 Hurd Drive, Irving, Texas 75062 further describes bichromatic analyzers and the details of their operation.

The present invention seeks to include a movable filter assembly which converts this type of bichromatic spectrophotometer into a fluorescent spectrophotometer with a sensitivity level sufficient to perform precise fluorescence immunoassay measurements on extremely diluted solutions.

15 In order to rapidly and accurately analyze the concentration of a particular substance present in a chemical specimen, such as blood, serum and urine, chemists have extensively relied on photometric measurements using filter photometers and monochromatic servomechanism spectrophotometer systems.

The increased need for sensitivity and specificity in optic detection and the advent of new immunotagging techniques shifted emphasis to fluorimetric measurements and instrumentation. Thus, dedicated filter 20 fluorimeters and spectrofluorimeters have been developed and are commercially available. However, all these prior instruments suffer from a serious deficiency-the inability to change from one mode of measurement, for example photometry, to another, for example fluorimetry, in the same instrument. This deficiency prevents the covering of a wide concentration range.

Since dedicated fluorimeters use primarily right angle illumination geometry and absorption spectropho-25 tometers use primarily straight through illumination geometry, converting from one mode of measurement to the other requires substantial hardware changes, such as the insertion of mirrors to divert the beam or the use of an auxiliary light source. Both modifications further require substantial cumbersome operator interaction and calibration.

As an example of such a prior, cumbersome multipurpose fluoro/spectrophotometric apparatus, in 30 Analytical Biochemistry 42,494-504,1971, Britton Chance, D. Mayer, and V. Legallais, there is described a dual-wavelength spectrophotometer and fluorimeter using interference filters which measure a fluoresc-ence/absorbance difference ratio using two light sources and three detectors. In U.S. Patent 3,811,777, there is described apparatus for measuring the fluorescence intensity of tissue material and correcting it or separately measuring reflectance measurements on the same sample. This technique is not applicable to 35 dilute solutions, as encountered in immunoassay measurements because of the small penetration of the exciting beam in the solution and the low reflectance signal level. Further, it is not convertible into an absorbance measurements mode as in the technique described in this specification. The present application describes techniques which alleviate these problems and enable either fluorescence or absorption to be measured on a dilute solution on the same light path, using the same light source and detector, thus 40 minimizing hardware complexity and operator intervention.

Another problem encountered in prior fluorescence intensity instruments is the dependence of the detected intensity on sample geometry and position. Thus, very accurate sample repositioning is very crucial for obtaining accurate correlation between fluorescence intensity and concentration. C.A. Parker, Photo-luminescence of Solutions, Elsevier, Amsterdam, 1968, p. 220-234, teaches that straight through, in-line 45 illumination is much less critically dependent on the exact position of the cuvette holding the specimen than frontal illumination, and is preferred to right angle excitation in precise measurement of fluorescence intensity of solutions contained in cylindrical cells or cuvettes with optically imperfect surfaces. It is desired, therefore, to make use of these advantages of in-line excitation geometry.

According to the present invention, a fluorescence spectrophotometer for investigating the fluorescent 50 characteristics of a test sample, comprises:-

a) a light source providing light of a first wavelength for excitation of the test sample thereby producing fluorescent light of a second wavelength from the test sample;

b) detector means for detecting light of the first and second wavelength;

c) a test sample holder transparent to light of the first and second wavelength;

55 d) a filter assembly comprising a frame having a plurality of filter members mounted therein, said filter assembly movable between a test position and a reference position, in the test position one filter member allowing the passage of light of the first wavelength from the light source to the test sample and one filter member allowing the passage of light of the second wavelength from the test sample to the detector means and in the reference position one filter member allowing the passage of light of the first wavelength from the 60 light source to the test sample and one filter member allowing the passage of light of the first wavelength transmitted through the test sample to the detector means;

e) means for directing light from the light source to the test sample and means for directing light from the test sample to the detector; and f) a means for moving the filter assembly between the test and reference positions operatively associated 65 with the filter assembly.

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GB2 142 156 A

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One disadvantage with the in-line, straight through, excitation geometry is that part of the exciting beam transmitted through the optics will cause a high measurement blank (C.A. Parker, 1968). We discovered one may use multilayered three-cavity filters, sharp cut-off filters, and neutral density filters to achieve high rejection of the excitation beam and achieve sensitivities equivalent to perpendicular excitation. As an 5 example, detectable concentrations of fluorescein of 1.48 x 10~8 M to as low as 7.4 x 10~9Mcanbe achieved using the techniques described.

While this high level of sensitivity may suffice for many fluorescence measurements, at least another order of magnitude increase in sensitivity is required for precise fluorescence immunoassay measurements. As an example, using straight through excitation geometry, the detected fluorescent light levels are extremely low, 10 requiring sensitivity levels for immunoassay measurements in the order of 10-10 M fluorescein. We have now further discovered that the sensitivity of the apparatus just referred to is limited due to the background transmitted light as well as due to the coupling of stray light between filter elements.

Accordingly the invention further provides a fluorescence spectrophotometer for investigating the fluorescent characteristic of a test solution comprising:

15 a) a light source providing light of a first wavelength for excitation of the test solution thereby producing fluorescent light of a second wavelength from the test solution;

b) detector means for detecting light of the first and second wavelength;

c) a test solution holder transparent to light ofthefirstand second wavelength;

d) a filter assembly comprising a frame having a plurality of filter members mounted therein, said filter 20 assembly movable between a test position and a reference position, in the test position one filter member allowing the passage of light of the first wavelength from the light source to the test solution and one filter member allowing the passage of light of the second wavelength from the test solution to the detector means and in the reference position one filter member allowing the passage of light of the first wavelength transmitted through the test solution to the detector means;

25 e) said filter assembly including light shielding means for preventing the coupling of stray light between said filter members;

f) means for directing light from the light source to the test solution and means for directing light from the test solution to the detector; and g) a means for moving the filter assembly between the test and reference positions operatively associated 30 with the filter assembly.

The invention also provides a filter assembly comprising a frame having a pair of test filters and a pair of reference filters mounted therein, members of the test filter pair lying on a circular line 180° apart, one member providing for the passage of light of a first wavelength for excitation of a test solution and one member providing for the passage of fluorescent light of a second wavelength, members of the reference 35 filter pair located on the circular line 180° apart between members of the test filter pair, each member of the reference filter pair providing for the passage of light of the first wavelength and light shielding means for preventing the coupling of stray light between said filter members.

In a preferred embodiment, the apparatus includes a filter frame movably mounted on a base and having a pair of test filters and a pair of reference filters mounted on the filter frame, with the frame movable between 40 a test position and a reference position. In an in-line or straight through optical geometry, and with the filter frame in the reference position, excitation light from a light source at a first wavelength may be passed through one of the reference filters, a test solution holder, and the other reference filter. With the filter frame in the test position, excitation light may be passed through one of the test filters and to the test solution holder, with the other test filter passing fluorescent light of a second wavelength from the test solution. A 45 pre-filter passing excitation light and blocking fluorescent light is inserted between the excitation light source and the filters. This minimizes background transmitted light and fluorescent light in undesired optical paths to reduce optical coupling between the filter elements. Light shielding or baffle means are provided to prevent the undesired coupling of stray light or reflected light between the filter elements, and thereby significantly increase the instrument's sensitivity.

50 First baffle members on the top filter frame surface form a separate walled compartment for each filter to prevent stray light from optically coupling between filters across the top of the filter frame. Second baffle members on the bottom filter frame surface prevent stray light optically coupling between filters across the filter frame bottom surface.

In a constructed embodiment of the invention, a detected sensitivity of 1.9 x 10~10 M fluorescein was 55 obtained.

Further according to the invention, there is provided a method for conducting fluorescence spectroscopy comprising:

irradiating a test sample with light of a first wavelength which causes the test sample to fluoresce light of a second wavelength and measuring a portion of the fluorescent light;

60 irradiating the test sample with light of the first wavelength at reduced intensity and measuring the light of the first wavelength transmitted through the test sample; and comparing the intensity of the fluorescent light to the transmitted light.

One constructional arrangement according to the invention will now be described byway of example with reference to the accompanying drawings, in which :-65 Figure / is a schematic view of an improved fluorescent spectrophotometer with a sectional view of a filter

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assembly having a pre-filter and light shielding or baffling means in accordance with the present invention;

Figure 2 is a bottom view of a filter wheel in the filter assembly;

Figure 5 is a top view of the filter wheel in the filter assembly;

Figure 4 is a top view of a carriage or base with the filter wheel shown in dashed lines.

5 Referring to the drawings, there is illustrated a light source 1 and a filter assembly 3 having a filter wheel 9 with filters 5,6,7 and 8 mounted therein. Filter wheel shaft 15 rotatably mounted in a carriage or base 16 is molded integrally into filter wheel 9 and is used to couple the filter wheel to a drive motor. A test solution holder 4 and a detector such as photomultiplier detector 13 feeds detector output information into a data processor 14.

10 A pre-filter 17 is mounted in a cavity 18 formed in carriage 16 to minimize background transmitted light. A series of continuous raised ridges 19 (see Figure 3) provided on the top surface of filter wheel 9 form walled compartments which respectively surround each of the filters 5,6,7,8 to block and prevent stray light from optically coupling between the filters at the filter wheel top surface. Also, respective annular ridges 20,21 project downwardly from the bottom surface of filter 9 (see Figures 1 and 2) to block stray light below the 15 filter wheel from optically coupling between filters. This stray light may consist, in part, of excitation light reflected from many surfaces below the filter wheel. Ridges 20,21 are mated with corresponding respective annular grooves 22,23 provided in carriage 16.

In operation, in the test position, light from light source 1 is directed by prism 2 to filters 17 and 5, each of which is a 500 nm narrow band interference filter which serves to pass light of an excitation wavelength to 20 prism 11 which directs the excitation light to the test sample 4. Fluorescing light from the test solution is directed by prism 12 to a 530 nm sharp cut-off filter 6 which passes light to the detector 13. Rotation of filter wheel assembly 9 through 180° places filters 7 and 8 (500 nm narrow band interference and neutral density filters) in the path of the light as it travels from the light source to the detector thereby producing a reference signal.

25 A cartridge containing filter assembly 3 and carriage 16 can be conveniently inserted into an Abbott VP™ bichromatic absorption spectrophotometer to convert it to a fluorescence spectrophotometer. One may utilize the principles of this invention to adapt other spectrophotometers to enable fluorescence measurements.

The signal obtained from a conventional bichromatic spectrophotometer converted to a fluorescence 30 spectrophotometer by the apparatus of the present invention is proportional to the logarithm of the ratio of the fluorescence intensity to the reference intensity. In the case of very dilute solutions, the signal is linear with concentrations over one order of magnitude in change in concentration, and in the case of larger concentration ranges the signal is proportional to the logarithm of the concentration over several orders of magnitude of concentration change.

35 The neutral density filters are selected to adjust the intensity of the excitation light transmitted through the test solution to the detector in the reference mode, and therefore, adjusts the sensitivity range of the measurements.

Those skilled in optics will recognize a variety of light sources, detectors, and filter combinations suitable for achieving the purposes of this invention. A variety of prisms, mirrors, lens, and collimators are suitable 40 means for directing light from the light source to the test sample and from the test sample to the detector. Similarly, a wide variety of data handling techniques are available for processing electrical signals resulting from the test (fluorescing light) and reference (excitation light transmitted through the test sample) beams.

The filters in filter assembly 3 for measuring fluorescing substances are listed in Table I, it being understood that filter 17 is the same as filter 5 in each instance. Thus, pre-filter 17 has optical transmittance 45 and blocking characteristics matching those of excitation filter 5, and therefore increases the blocking characteristics of the excitation filter at the fluorescence wavelength.

TABLE I

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6

7

8

Fluorescing Substance

490

nm

515

nm

490

nm

490

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Fluorescein

405

nm

450

nm

405

nm

405

nm

Umbelliferone

340

nm

460

nm

340

nm

340

nm a-naphthol, NADH

366

nm

470

nm

366

nm

366

nm

8-anilinonephthaiene

319

nm

445

nm

319

nm

319

nm

Homovanillic acid/H202

Those skilled in the optic arts will recognize the use of narrow band-pass filters, cut-off filters and the like for reference and fluorescence.

60 As an example for using this invention in fluorescence immunoassay measurements, a constructed embodiment of the invention was employed in the determination of theophylline in human serum samples. Filter assembly 3 included a pre-filter 17 at 405 nm, an excitation filter 5 at 405 nm, a fluorescence filter 6 at 460 nm, with a broad band extending from 440-470 nm, and reference filters 7,8 at 405 nm. The pre-filter, excitation and reference filters were narrow band interference filters. This filter assembly was placed in an 65 Abbott VP™ bichromatic analyzer available from Abbott Laboratories, Irving, Texas.

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Theophylline was determined using the following methods and reagents. The reagents were commercially available from Ames Division, Miles Laboratories, Elkhardt, Indiana 46515.

The theophylline in the solution was reacted with a reagent containing an antibody to theophylline and an enzyme, (3-galactosidase. A theophylline derivative labeled with a substrate for this enzyme, (3-galactosyl-umbeiliferone-theophylline conjugate was added to the mixture. This drug derivative is non-fluorescent under the conditions of the assay; however, hydrolysis catalyzed by p-galactosidase yields a fluorescent product. When antibody to theophylline reacts with the labeled theophylline, it protects it, making it virtually inactive as a substrate forthe p-galactosidase. Competitive binding reactions are set up with a constant amount of the theophylline labeled reagent, a limiting amount of antibody, and the clinical serum or plasma sample containing theophylline:

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labeled theophylline + antibody + theophylline it

(antibody/labeled theophylline) + {antibody/theo-phylline)

p-galactosidase fluorescense produced in proportion to the theophylline level in the serum sample

Standards and samples were prediluted 1:51 using a 1:20 diluted bicine buffer as specified by the kit manufacturer. 100 ul aliquotes of each prediluted standard and sample was placed in the sample cups in the multicuvette assembly of the Abbott VP™ bichromatic analyzer. The enzyme/antibody reagent provided by the manufacturer in a concentrated form was diluted 1:30 using bicine buffer and was loaded in the reagent reservoir. The fluoregenic drug reagent supplied by Ames was loaded in an auxiliary reagent reservoir. The Abbott VP™ bichromatic analyzer was set up with a dispense ratio of 1:26 (10 ul sample + 250 ul reagent). The auxiliary reagent dispenser was to 10.22 ul at station 21. Temperature in the incubator water bath was 30°C. The cuvette and the sample processing module were covered with black plastic covers. The instrument was set to run in an end point mode after priming the reagent and auxiliary manifolds.

Signal from the 10th revolution (27 minutes) incubation time were plotted against standard concentration of theophylline. Theophylline concentration in the solutions was determined from the plot. This data was correlated with enzymatic immunoassay data performed on a spectrophotometer in an absorbance mode using EMIT™ reagents from Syva Company. The date correlated well between the two methods.

It may be particularly noted that a significant advantage of this invention resides in that since both fluorescence and reference signals are seen by the same detector, prior problems in transients and fluctuations in the light source are eliminated.

Rather than the rotating filter wheel described herein, the principles of this invention may be applied to provide other moving filter assemblies, such as a sliding, vibrating or reciprocating filter assembly wherein the two test filters and the two reference filters may be sequentially and repetitively inserted in the optical path.

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Claims (1)

1. A method for conducting fluorescence spectroscopy comprising:

45 irradiating a test sample with light of a first wavelength which causes the test sample to fluoresce light of a second wavelength and measuring a portion of the fluorescent light;

irradiating the test sample with light of the first wavelength at reduced intensity and measuring the light of the first wavelength transmitted through the test sample; and comparing the intensity of the fluorescent light to the transmitted light.

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Printed in Ihe UK for HMSO, D8818935, 12/84, 7102.

Published by The Patent Office, 25 Southampton Buildings, London, WC2A 1AY, from which copies may be obtained.