WO1993010437A1

WO1993010437A1 - A method relating to the quantification of a substance with the aid of fluorescence

Info

Publication number: WO1993010437A1
Application number: PCT/SE1992/000792
Authority: WO
Inventors: Håkan DREVIN; Aija Mattila-Fjelner; Erik Ringberg
Original assignee: Kabi Pharmacia Ab
Priority date: 1991-11-20
Filing date: 1992-11-18
Publication date: 1993-05-27
Also published as: SE9103427D0; AU3053592A

Abstract

A method of measuring fluorescence emitted by a fluorophore when assaying a substance (analyte) in a sample, wherein a measured florescence signal is compared with a standard curve in which signal strenght (emission) is a function of the analyte concentration. The method is mainly characterized in that (i) the fluorescence values which correspond to the analyte concentration interval (c) of the standard curve are measured at the same emission wavelength (lambdaem) or at the same excitation wavelength (lambdaex) wherein the interval (c) is divided into a finite number of part-intervals C1,.. Ca,... Cn, where a^- is an integer 1«a«n and n^- is an integer »2 and (ii) that the signal (fluorescence)emitted from the sample is measured for the excitation wavelength and at the emission wavelength respectively which correspond to the wavelengths used when measuring corresponding values on the standard curve.

Description

A Method Relating to the Quantification of a

Substance with the Aid of Fluorescence

The invention pertains to a method of expanding the range within which the concentration of a substance (analyte) contained in a sample can be quantified by means of a fluorescence measuring process.

Fluorescence measuring methods are now widely used for the quantitative determination of low-concentration analytes. The methods involve relating measured fluorescence signals to the amount of analyte present (concentration, content, etc.), with the aid of a standard curve or with the aid of standard values, where fluorescence (emission, signal

strength) is a function of analyte concentration (the amount of analyte present). The analyte concerned may be the

fluorescent substance itself or some other substance which will inf)uence the ability of the fluorophore to fluoresce. The test system may be designed so that the strength of measured fluorescence will increase or decrease with increasing amounts of analyte.

One drawback with fluorescence-measuring processes is that the intrinsic absorbance of the fluorophore will affect the result at high measuring values. This places a limitation on the measuring range when high fluorescence values are to be used. In order to overcome this drawback, it is often

elected to use absorbance-measuring processes. Alternatively, the sample tested may be diluted. Both alternatives result in uncertainty factors. Absorbance-measuring processes are less sensitive. Dilution of the test sample implies an additional process step, and also introduces the risk of dilution error. We have now developed a method of circumventing these drawbacks, while enabling the sensitivity of the fluorescence-measuring process to be retained. The result is an expanded measuring range.

In our solution to the problem, there is used a standard curve which is obtained by measuring the emission values for different part-intervals I ₁, ....I_a, .....I_n of the curve, either for different excitation wavelengths or different emission wavelengths. The emission values of a part-interval (I_a) corresponding to lower emission in a conventional standard curve are obtained by measuring emission subsequent to excitation at a wavelength at which the fluorophore has higher extinction, compared with a part-interval (I_a+1) corresponding to higher emission in the conventional standard curve. Alternatively, the emission for the part-interval (I_a) is measured at a wavelength at which the emission of the fluorophore is higher than the emission at the

wavelength at which emission for the part-interval (I_a+1) is measured. In the first alternative, which is the preferred alternative, the different intervals are measured, or determined, preferably at a common emission wavelength. In the latter alternative, excitation for the different intervals preferably takes place at a common excitation wavelength.

Intervals having a lower index a correspond to fluorescence (emission) that has lower measured values in a conventional standard curve. The interval Ia. borders on the intervals

I_a-1 and I_a+1, preferably with a given overlap, n is an integer >/ = 2, although preferably 10 or lower, a is an integer from 1 to n. It is convenient in many instances to normalize the measured emission values to neighbouring intervals, so that the standard curve will be continuous.

Normalization is conveniently effected towards the part-interval which corresponds to the lowest measured fluorescence. See the experimental part. The measured fluorescence (emission) intervals of the standard curve are corresponded by a concentration range which can be divided into part-intervals C₁ . . . ... . .

C_a.......C_n, where a and n have the aforesaid significance and the boundaries of the part-intervals find direct correspondence in the boundaries of the part-interval I_a. When the increasing fluorescence corresponds to increasing

analyte concentration, the left and the right limits in I_a+1 are higher than the corresponding limits in I_a. When

increasing fluorescence corresponds to a decreasing analyte concentration, the left and the right limits in I_a+1 are lower than the corresponding limits in I_a.

The invention thus relates to a method of quantifying with the aid of fluorescence emitted from a fluorophore the presence of a substance (analyte) in a sample incorporated into an assay medium, said method comprising the steps of comparing measured fluorescence values with a standard curve where the signal strength (emission) is a function of the amount of analyte. The preferred characteristic features of the invention are

(i) that the fluorescence values which correspond to the

standard curve intervals of analyte concentration (C): a. are measured at the same emission wavelength

(lambda_em), but that excitation for a part-interval

(C_a) of the analyte concentration range and

excitation for a part-interval (C_a+1) of the concentration range has occurred at different excitation wavelengths (lambda_ex(a) and

lambda_ex(a+1) respectively); said lambda_ex(a) and lambda_ex(a+1) being selected so that the extinction of the fluorophore at lambda_ex(a) is greater than at lambda_ex(a+1), or b. are measured at the same excitation wavelength

(lambda_ex), but that the emission for a part- interval (C_a) of the analyte concentration range and for a part-interval (C_a+1) of the analyte concentration range has been measured at different emission wavelengths (lambda_em(a) and lambda_em(a+1) respectively); said lambda_em(a) and lambda_em(a+1) being selected so that the fluorophore emission at lambda_em(a) is greater than at lambda_em(a+1), and

(ii) that the signal (fluorescence) emitted by the sample, i.e. the assay medium is measured for the excitation wavelength and at the emission wavelength which

corresponds to the measured standard curve values.

By "assay medium" is meant the sample plus all constituents present in the medium on which the fluorescence measurement is performed.

The indexes a and a+1 for wavelength (lambda) refer to the part-interval I_a and C_a and I_a+1 and C_a+1, respectively.

Naturally, wavelengths shall be chosen so as to obtain with the emission an acceptable signal/noise ratio.

In the alternative according to (i:a), lambda is preferably chosen as the wavelength for emission maximum (= lambda_emmax).

The wavelength for excitation maximum (lambda_exmax) is

preferably smaller than or equal to lambda_ex(a), which is smaller than lambda_ex(a+1), i.e. lambda_exmax </ = lambda_ex(a)

< lambda_ex(a+1), but preferably lambda_exmax = lambda_ex(a)

< lambda_{ex(a+1 )}. This does not exclude the possibility that lambda_{ex( a+1 )} < lambda_{ex( a )} </ = lambda_exmax, with preference for lambda_{ex( a+1 )} < lambda_{ex( a )} = lambda_exmax. Lambda_ex(a) and lambda_ex(a+1) are preferably chosen in the same excitation peak, such that the quotient between the extinction at lambda_ex(a) and lambda_ex(a+1) will be > 2, preferably 10 or more.

In the alternative according to (i:b), lambda is

preferably chosen to be the wavelength for excitation

maximum ( = lambda_exmax). The wavelength for emission maximum

(lambda_emmax) is preferably smaller than or equal to

lambda_em(a) which is smaller than lambda_em(a+1), i.e.

lambdae_emmax is </ = lambda_em(a) < lambda_em(a+1), with

preference to lambda_emmax = lambda_em(a) < lambda_em(a+1).

This does not exclude the possibility that lambda_em(a+1)

< lambda_em(a) </ = lambda_emmax, with preference to

lambda_em(a+1) < lambda_em(a) = lambda_emmax

Lambda_em(a) and lambda_em(a+1) are preferably chosen in the same emission peak, so that the quotient between emission at lambda_em(a) and lambda_em(a+1) will be > 2, preferably 10 or more.

Figures 1 and 2 illustrate superimposed excitation and

emission relating to 4-methylumbelliferone. Figure 1 illustrate a preferred embodiment according to alternative i:a. Figure 2 illustrates a preferred embodiment according to alternative i:b. The significance of the various lambda will be evident from the Figures.

The fluorescent substance measuxed in the assay shall preferably exhibit an emission maximum at 300 nm or at a lower wavelength. Stokes shift should be greater than 10 nm, and preferably above 30 nm. The substance may be organic or inorganic. Those compounds of most interest include compounds that have an umbelliferone structure, a rhodamine structure, a fluorescein structure, etc., and fluorescent lanthanide chelates (primarily Eu³⁺. Tb³⁺, Sm³⁺ and Dy³⁺). These latter substances in particular have a large Stokes shift with emission maxima which are well-separated from their respective excitation maxima and from emission wavelengths of proteins and other substances present in biological samples. The fluorescence of lanthanide chelate is often long-lived, which renders the chelates more suited for assaying with time-resolved fluorescence spectroscopy.

The invention can be applied with several different types of fluorescence techniques.

The fluorescence signal is directly related to the amount of analyte present (concentration) in the sample in those

instances when the fluorophore used in the assay is the analyte itself.

Indirect coupling between fluorescence signal and analyte concentration has been applied, among other things, in

methodology which utilizes bio-affinity reactions between receptor-ligand. Depending on the methodology applied, an elevated fluorescence signal may, in this particular case, indicate either a higher or a lower analyte concentration.

One example in this regard is found when determining enzymatic activity with the aid of a fluorogenic substrate. Depending on whether or not the substrate" or the product formed is fluorescent, a reduction or increase in the fluorescence signal signifies a higher enzyme activity. By adding standardized quantities of reagent (cofactors, coenzyme, enzyme activity, substrate, cosubstrate, etc.) to biological

samples, it is possible to assay different components that take part in or are essential to the enzyme activity. Other popular bio-affinitive methods include ligand-receptor methods which utilize a bio-affinitive reactant which is labelled with a fluorophore group or with a group which can give rise to fluorescence, so as to form a receptor-ligand complex in an amount which is related to measured fluorescence and the amount of analyte present. Examples of methods which use bio-affinitive ligand-receptor-pairs are antigen/hapten and antibodies (immuno assays).

The fluorescence signal is normally a measure of the concentration of fluorophore in the assay medium. This applies to methods where the fluorophore is an analyte, enzymedetermining methods which utilize a fluorogenic substrate, heterogeneous receptor-ligand methods which utilize

fluorogenic labels, etc. exceptions are receptor-ligand methods, in which the fluorophore concentration when

determining fluorescence are independent of the analyte concentration in the assay medium, but with which the signal is modulated (amplified or reduced) because of direct or indirect interactions with the analyte (homogeneous

receptor-ligand assays, for instance homogeneous fluoroimmuno assays).

In the ideal case, the ratio of the extinction for lambda_em(a) to lambda_em(a+1) and the ratio of the emission at lambda_em(a) and the emission at lambda_em(a+1) is directly proportional to the "breadth" of the concentration of the composite

interval total range. Utilized reactions in which the

fluorescing substance takes part, are liable to set limits which render the range more narrow.

The primary advantages afforded by the invention are obtained when assaying substances which can be found in a broad

concentration range (two or more powers of ten) in the type of sample concerned. The invention is therewith particularly beneficial in respect of samples that have been taken from living material, such as whole blood, serum, plasma, urine, cerebro spinal fluid, etc. The method can also be applied to assaying environmental contaminants in air, water, soil and living material. With regard to receptor-ligand methods, the primary use of the invention is found in so-called heterogeneous variants, e.g. heterogeneous immunoassays of, e.g., "sandwich" or competitive (inhibition) types.

The invention will now be exemplified with reference to Examples, and is defined in the accompanying Claims which form part of this specification.

EXPERIMENTAL PART

Example 1: Determining 4-methyl-umbelliferone in aqueous solution. Excitation at different wavelengths. Fluorescence measurement at a common wavelength.

There was prepared a 1 Mm solution of 4-methyl-umbelliferone in 0.2 M glycine buffer, pH 10.6 with 0.1% TWEEN 20. The fluorescence-measuring process was carried out on a diluted series (dilution step 1/2: concentrations, see below).

Separate measurements were carried out with excitation at 365 nm and 412 nm respectively. Emission was measured at 450 nm. The fluorometer used was an LS 5 (Perkin Elmer, England). The measuring process was carried out with 1 cm quartz cuvettes, with an incident signa'l from one side of the cuvette.

Table 1

Blanking against water. The glycine buffer value is not subtracted. Conc.MUF

ex/ em ex/ em

m 365/450 412/450

μm FU

Glycine buffer 3.0 .62

0.008 9.2

0.015 15.8

0.031 27.1

0.06 48.3 1.1

0.12 92.4 1.3

0.24 168 2.3

0.49 301 3.4

0.98 615 6.3

1.95 1,150 (Max!! ) 12.1

3.9 24.3

7.8 49.8

15.6 102

31.3 199

62.5 359

125 670

250 1,150 (Max!! )

The measured values for ex₄₁₂ were multiplied by 32, in order to provide a continuous standard curve from the emission values (lambda₃₆₅ and lambda₄₁₂) obtained with excitation at 365 nm and 412 nm respectively.

The result: The normal measuring range of 4-methylumbelliferone is 0.008-0.98 Nm (120 times measuring range) with the cuvette concerned and without dilution. Combination measuring according to the invention extended the measuring range to 0.008-125 Nm (about 15,000 times). Example 2: Determining 4-methyl-umbelliferone in aqueous solution. Excitation at the same wavelength. Fluorescence measurement at different wavelengths.

Solutions having the concentration according to Table 2 were prepared analogously with the solutions recited in Example 1. Excitation was effected at 410 nm in two assay series. Fluorescence was measured at 455 nm and 500 nm respectively.

Table 2

Blanking against water.

Test ex/ em ex/ em

Conc.MUF 410/455 410/500

μM FU FU

0.0042 25.9 5.4

0.016 106 20

7.0 474 94.9

31 1,150 390

120 710 931

640 701 847

The measurement values obtained show that the measuring range cannot be extended to the'same extent when applying this embodiment of the invention. Example 3: Determining enzymatic activity when the enzyme is a label group in an enzyme immuno assay .

The test used was a commercially available test designated Pharmacia CAP IgE FEIA (Kabi Pharmacia Diagnostics AB, Uppsala, Sweden). The test protocol involved incubating a solid-phase-bound anti-IgE antibody with a serum sample, wherein IgE present in the sample binds to the solid phase (the matrix). The matrix was then washed and incubated with galactosidase-labelled anti-IgE antibody so as to form the complex

matrix anti-IgE IgE anti-IgE-beta-galactosidase.

The matrix was then washed and incubated with substrate (4-methyl-umbelliferyl-beta-galactoside (= MUFG)). The reaction was halted after ten minutes and liberated

4-methyl-umbelliferone was eluted from the matrix and assayed fluorometrically, in our case by excitation at 365 nm and 412 nm and measuring emission at 450 nm for respective excitation wavelengths. The test is designed so that the fluorescence measured (liberated 4-methylumbelliferone) becomes a function of the amount of IgE present in the sample. The matrix used had a sponge-like character and the matrix pores were able to accommodate the whole of the liquid volume in which the immune reaction was carried out.

In order to obtain a standard curve there was used a sample which comprised a dilution series having the IgE activities recited in the following Table. Table 3

Test / / / KU/1 ex em ex em ex ex

No. 365/450 412/450 365+412

1 7,360 162 7,360 10

2 13,552 247 13,552 30

3 23,200 326 23,200 100

4 55,152 682 55,152 300

5 130,880 1,650 132,000 1,000

6 283,840 4,063 325,000 3,000

7 571,888 11,696 935,640 10,000

8 584,832 12,732 1,018,720 30,000

9 579,696 12,036 962,840 100,000

Serum

The fluorescence-assaying process was carried out in an FC 96 Fluorocounter (Pharmacia Diagnostics AB, Uppsala, Sweden).

By dividing the values in columns 365/450 with corresponding values in the column 412/450, quotients were obtained which are constant for 4, 5 and 6 and equal to 80. The values in the column 412/450 have since been multiplied with 80. A combination of the values 1, 2, 3 and 4 in column 365/450 with multiplied values of 5, 6, 7, 8 and 9 in column 412/450 gives the column 365/412/ 450. Assay data shows that the measuring range is narrower for this system than for t"he system described in Example 1. The assaying range is limited by the solubility of the substrate (ca. 0.5 mM MUFG = 25 nmol MUFG/50 μl = ca 125,000 FU. Beta-galactosidase is able to work at a full enzyme rate until about 0.25 mM MUFG remains in the solution (= 60,000 FU). The upper measurement signal that can be used is then 60,000 FU, which is about three times more than what the instrument used can measure. The lower part of the assaying range is limited by the non-specific signal. When the non-specific signal contains 0.2% of the total activity, good precision is required in order to be able to distinguish between the lowest standard point (10 KU/l) and the blank value.

It is possible to increase the upper limit of 60,000 FU with the aid of other substrates or other enzymes, for instance 4-methyl-umbelliferyl phosphate (MUFF) in

combination with alkaline phosphatase.

Claims

1. A method of measuring the fluorescence deriving from a fluorophore when assaying a substance (analyte) in an assay medium in which method a measured fluorescence signal is compared with a standard curve in which the signal strength

(emission) is a function of analyte concentration,

ch ar a c t e r i z ed in that

(i) the fluorescence values which correspond to the analyte concentration interval (C) of the standard curve

a. are measured at the same emission wavelength

(lambda_em), but that excitation for a part-interval

(C_a) of the analyte concentration range and excitation for a part-interval (C_a+1) of the concentration range that has occurred at different excitation wavelengths (lambda_ex(a) and lambda_ex(a+1) respectively) so chosen that the extinction of the fluorophore at lambda_ex(a). will be greater than at lambda_ex(a+1), or

b. are measured at the same excitation wavelength

(lambdaex), but that the emission for a partinterval (C_a) of the analyte concentration range and for a part-interval (C_a+1) of the analyte concentration range has been measured at different emission wavelengths (lambda_em(a) and lambda_em(a+1) respectively) chosen so that the emission for the fluorophore at lambda_em(a) is greater than at lambda_em(a+1),

wherein the interval (C) is divided into a finite

number of part-intervals C ₁,...C_a,....C_n, where a is an integer 1 </ = a </ = n and n is an integer >/ = 2, and

(ii) that the signal (fluorescence) emitted from the

assay medium is measured for the excitation wavelength and at the emission wavelength respectively which

correspond to the wavelengths used when measuring

corresponding values on the standard curve.

2. A method according to Claim 1, c h a r a c t e r i z e d in that the fluorescence values corresponding to the signal strength range of the curve are measured according to method step (i:a).

3. A method according to any one of Claims 1-2, c h a r a c t e r i z e d in that lambdaem is the wavelength of emissionmax ( = lambda_emmax).

4. A method according to any one of Claims 1-3, c h a r a c t e r i z e d in that lambda_exmax </ = lambda_ex(a) < lambda_ex(a+1), where lambda_exmax is the wavelength of excitation max.

5. A method according to any one of Claims 1-4, c h a r a c t e r i z e d in that lambda_ex(a) =

lambdaexmax

6. A method according to Claims 1, c h a r a c t e r i z e d in that the fluorescence values corresponding to the signal-strength range on the standard curve are measured according to method step (i:b).

7. A method according to Claim 6, c h a r a c t e r i z e d in that lambda is the wavelength for excitation max (= lambda_exmax).

8. A method according to any one of Claims 1 and

Claims 6-7, c h a r a c t e r i z e d in that

lambdaemmax </ = lambda_em(a) < lambda_em(a+1), where lambdaemmax = the wavelength for emissionmax.

9. A method according to any one of Claims 1 and Claims

6-8, c h a r a c t e r i z e d in that lambda_em(a) = lambda_emmax.

10. A method according to any one of Claims 1-9,

cha r a c t e r i z e d in that the fluorophore used has a Stokes shift > 10 nm.

11. A method according to any one of Claims 1-10,

ch ar a c t e r i z e d in that the fluorophore used has an emissionmax that is greater than 300 nm.

12. A method according to any one of Claims 2-5 and Claims 10-11, c h a r a c t er i z e d in that the quotient between the extinction for lambda_em(a) and lambda_em(a+1) is greater than 2.

13. A method according to any one of Claims 6-11,

c h a r a c t e r i z e d in that the quotient between the emission for lambda_em(a) and lambda_em(a+1) is greater than

2.

14. A method according to any one of Claims 1-13,

cha r a c t e r i z e d in that the sample has a biological origin.

15. A method according any one of Claims 1-14,

char a c te r i z e d in that the method is a part of a method which utilizes the bio-affinity between receptor and ligand, preferably an immunological method.

16. A method according to any one of Claims 1-15,

char a ct e r i z e d in that the method is used to determine enzymatic activity.

17. A method according to any one of Claims 1-16,

cha r ac t e r i z e d in that n is 2.