FI96641C - Biospecific assay method - Google Patents

Description

96641 BIOSPECIFIC METHOD OF DETERMINATION - BIOSPECIFIK BESTÄMNINGSMETOD 5

Immunoassay is an established biospecific assay method and its various applications are widely used in routine diagnostics and research laboratories.

Another group of biospecific assays, although still under development, is the DNA and RNA hybridization assay. Biospecific assays generally use two biospecific reagents, a primary reagent and a secondary reagent (e.g., antibody, DNA, or RNA probe), each of which binds to specific determinants of the analyte molecule to form a complex of three molecules (one of these) and normally a reagent. Currently used labels include radioisotopes, enzymes, luminescent labels, and fluorescent labels.

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There is an ever-increasing need for multiparameter analytics in routine diagnostics. Unfortunately, the current methods do not allow the use of more than two or three labels to be measured simultaneously, because the spectrometric separation of the signals given by these different labels is not possible with sufficient accuracy. The emission spectra of different radioisotopes or fluorescent labels overlap considerably and, as a result, the separation of different analytes from the same sample is poor in the required concentration range.

It is an object of the present invention to provide an improved method for multiparametric biospecific assays.

The multiparameter assay of this invention is performed using artificially prepared microparticles representing different analytes as a solid support. The microparticles in each of the different classes 96641 are first coated with a specific primary reagent (antibody, DNA, RNA), i.e. the microparticles serve as a solid support for these reagents and the biospecific reaction. This invention is characterized in particular by the fact that microparticles representing different classes are prepared from a mixture of different monomer components or contain different indicators (D) so that they can be identified and distinguished from each other on the basis of Raman spectra I0. The determination of the analyte concentration is carried out on the basis of a biospecific reaction on the surface of the microparticles, the amount of label F bound to the surface of the microparticles being measured on the basis of the signal given by it.

15 In the following text, a substance used as a label for a biospecific reaction F is referred to as a fluorescent, phosphorescent or electroluminescent (= electrochemiluminescent), core-luminescent or bioluminescent camp.

The features of the invention appear from claim 1.

For the method according to the invention, there are 11 properties. a the following steps:

Microparticles are prepared in advance, divided into different classes, so that these microparticles of different classes 50 contain one or more different components Di, D2, D3, ... ettavissa .. which are identifiable by their l · aman-spect spectrum tr:: · and the presence or combination of which indicates the class of microparticles in question.

These microparticles of different classes are coated with the biospecific primary reagents that bind the analytes in question and then the microparticles are mixed together.

Biospecific secondary reagents that bind different analytes are labeled with photoluminescent Mole-5 channel F, and these reagents are mixed together.

- Add the sample to be determined and the biospecific f ister to the microparticles. mixing of secondary reagents, after which complexes of different analyte molecules and labeled biospecific reagents begin to bind to the reagent molecules on the surface of the micro-particles due to bioaffinity.

- The binding reaction is stopped by adding a diluent and at the same time diluting the concentration of labeled biospecific reagents not bound to the microparticles.

- The Raman emission of the components D contained in the microparticles is excited by a light beam and the

A class of 20 microparticles based on Raman spectral details.

The dye F on the surface of the microparticles is excited by a beam of light, by chemical or electrical activation, and its emission intensity is measured by a photon detector whose electrical signal intensity is proportional to that. analyytir. concentration.

It is essential for the sensitivity of biospecific K) determination methods based on the use of microparticles that the signal given by the indicator used to identify the class of microparticles does not interfere with the measurement of the signal given by the photo lumen label.

15 The principles of multiparametric biospecific assays have been published in the past. In most cases, however, the multiparametric method has been based on the use of solid reaction substrates 96641 4 in which biospecific reagents can be attached as separate and optically separable regions (e.g. PCT WO 84/01031).

The use of uniform microparticles as a solid support for a biospecific assay offers many advantages over the method described above. Uniform sized microparticles with a diameter of 1-200 μm can be prepared from a suitable polymer so that they fit well into an aqueous suspension. Biospecific primary reagents are bound to the surface of microparticles by physical adsorption or covalent bonding. In the method of the present invention, a sample, thus containing all the analytes in question, is incubated with a mixture of secondary reagents and microparticles in as little as possible; 1 to 100 microliters) in order to achieve the most complete reaction possible in a short time. Since the average distance between the analyte molecules and the labeled reagents in the reaction on the surface of the microparticles is very small, the reaction equilibrium can be achieved quickly and resulted in complexes of the reagent and the analyte 25 labeled with the reagents on the surface of the microparticles. Such a structure is commonly referred to in the literature as a layered structure. If the incubation times can be precisely adjusted, there is no need to drive the reaction to the endpoint. after incubation, the reaction solution is diluted sufficiently to reduce the concentration of free labeled 3 () secondary reagent and the amount of labeled complexes on the surface of the individual microparticles. with a hundred photon detector based on emission F. In this method, a one-to-two-fold dilution 55 is sufficient to distinguish the signals from the bound and free labeled reagent fraction sufficiently effective because the microphotometric detector used in this method is able to optically distinguish the light emitted by the microparticle signal at its 96641 measuring chamber focus.

A statistically sufficient number of microparticles are analyzed and the emission F signal strength of each particle is recorded on a computer.

Several multiparameter biospecific assay methods based on the use of artificially prepared microparticles have been published in the past: 1. A method in which the analyte class is based on the color of the microparticles and is detected by measuring the absorbance of 15 optically analyte particles (J. G. Streefkerk & et al .: Protides).

Biol. Fluids 24 (1976) 811-4).

2. A method in which the identification of an analyte class is based on the use of microparticles of different sizes and the identification is performed by measuring the diameter of the particle to be analyzed optically (T. M. McHugh & et al .: Journal of

Immunological Methods 95 (1986) 57-61).

3. A method in which microparticles are identified by fluorescent dyes mixed with them and the biospecific signal is measured by another fluorescent dye such as FITC (EP-A-126 450, GOIN 33/58) 50 4. A method using a short-lived a fluorescence (discharge time nanosecond) emitting dye for the identification of microparticles and a long-lived fluorescence emitting dye (discharge time 10 microseconds to 2 milliseconds) to measure analyte concentrations -45.

96641 5. A method using a short-lived fluorescent (discharge time nanosecond) dye to identify microparticles and a chemical or bioluminescent molecule (discharge time several seconds) to measure the concentrations of the analytes and to separate the fluorescence signal and luminescence signal and luminescence signal at different times (SF Patent No. 89837).

10 6. A method using a short-lived fluorescence (discharge time nanosecond) emitting dye to identify microparticles and a phosphorescence-generating molecule (discharge time 10 microseconds 15 to 2 milliseconds) to measure the concentration of analytes patent 90695).

7. A method using long-lived fluorescent dyes such as fluorescent chelates of lanthanide ions Tb, Dy, Eu and Sm to identify microparticles and measure the biospecific signal 25 (SF Application No. 931198).

In the method of the present invention, bio- or chemiluminescent labels (F) can be used to measure the biospecific reaction, and their measurement can be performed luminometrically with a photon counter. Electroluminescent or electro-chemiluminescent labels whose emission is activated can also be used. electrically and the signal F is measured with a photon counter.

Raman emission-based microparticle detection 55 requires a laser beam and a different measurement step in these methods, whereby the detection has no effect on the measurement sensitivity of the signal F.

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96641 7 In the method of the present invention, all fluorescent or phosphorescent labels (F) giving long-term photon emission can also be used to measure the biospecific reaction, and their measurement can be performed by time-resolved measurement. Fluorescence and phosphorescence excitation as well as Raman excitation can occur with the same or separate laser beam. This method has the advantage that the scattering of the light used to excite the label F and the Raman emission D and the background fluorescence caused by it do not interfere with the measurement of the label F, and thus a good measurement sensitivity is obtained.

Conventional organic fluorescent dyes can also be used as the photoluminescent label F15 of the biospecific reagent in the method of the present invention. Conventional organic fluorescent dyes have the advantage of high excitation light absorption and quantum fluorescence efficiency, but their use is hampered by the background signal from scattering and autofluorescence, which limits the measurement sensitivity. By using confocal optics and sufficiently good spectrometric separation to measure the signals of these photoluminescent labels, even good measurement sensitivities can be achieved (S. Nie & al.

25 Science 226 (1994) 1018-1021).

Essential to the method of the present invention, however, is the finding that the problem of scattering and autofluorescence can be eliminated if dual photon excitation is used in the excitation.

Dual photon excitation can occur when the targeting of intense excitation light makes the density of photons, per unit volume and per unit time, so high that the energy of the two photons is absorbed into the same chromophore. In this case, the energy absorbed is also the sum of the energy of two photons. Double photon excitation of fluorescent substances is a known technique e.g. in the field of spectroscopy and microscopy (U.S Pat.

96641 β 5,034,613). According to the concepts of probability calculus, the absorption of a single photon into a dye is an independent event, and the absorption of several photons is a series of individual, independent events.

5 The probability of absorption of a single photon can be described by a linear function as long as the excitable energy states are not saturated. The absorption of two photons is a nonlinear process. In double photon excitation, excitation of the dye molecule 10 occurs only when both photons are absorbed simultaneously. The absorption probability of two photons is the product of the absorption probabilities of individual photons. The emission caused by two photons is thus a quadratic process.

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The characteristics of an optical system for excitation of a microparticle can be described by the response of the system to a point light source. The point light source forms an intensity distribution characteristic of the optical system 20 at the focal point (point response) due to the effect of diffraction. Normalized, this intensity distribution is the probability distribution of how photons from a point light source arrive in the focus range. Probability distribution of the first photon in the dual photon technique; a is the normalized product of the intensity distributions of the second photon. The probability distribution thus obtained is clearly more limited in 3-dimensional space, especially in the depth direction, than the probability distribution of a single photon. 50 Dual photon excitation thus induces only the fluorescence generated in the clearly defined 3-u-ct vicinity of the focal point of the light beam.

t 55 When toner is excited by dual photon excitation, light scatter from the focus point environment and optical components is significantly reduced compared to normal excitation. Furthermore, double phyton excitation il ftS’i Itlii iiUAl, i i 96641 s reduces the generation of background fluorescence in the sample outside the target point, in the sample environment, and in the optics. Since the excited light beam must be focused to the smallest possible point, the dual photon excitation 5 is best suited for examining small sample volumes or structures, which is also the case in the method according to the present invention.

The advantage of dual photon excitation is also that, for example, visible or near-infrared (NIR) light can be used for excitation in the ultraviolet region or the blue region. Similarly, tuning in the visible area can be done with a light beam in the NIR area. Because the wavelength of the light source is significantly longer than the dye emission wavelength, scattering and possible autofluorescence at and around the light source can be effectively attenuated by using low-pass filters (at least 10 orders of magnitude) and 2 «to prevent access to the fluorescence emission detector.

In most cases, dual photon excitation requires the use of high-power ultrashort pulse laser devices.

However, it has been found that double photon excitation can also be detected with continuous laser light 25 (DE patent application P 44 14 0940.9-42 and P. Hänninen SaJ,

Proceedings for Three-Dimensional Microscopy, SPIE-The

International Society for Optical Engineering, 1994, Vol.

2184, 66-71). In our experiments, we have found that very good signal i-background ratio and sensitivity can be achieved with double photon excitations and short-lived fluorescent labels.

Examples of short-lived fluorescent dyes suitable for double photon excitation include coumarin and rhodamine derivatives.

The problem with long-lived (Tw = 1 millisecond) fluorescent and phosphorescent labels is the long measurement time they require, 96641 10 due to the saturation of the excitation states of the labels limiting the intensity of the vinyl light to such a low level that one microparticle must be measured for up to one second. 5 The measurement of signals from labels based on chemiluminescence, bioluminescence and electroluminescence also requires at least one second. Because short-lived (T. = i r.anosecond 1) 10 fluorescent labels can be used in a method based on double photon excitation, the intensity of the excitation light can be up to 106 times stronger than the excitation intensity of long-lived fluorescent labels.

Short-lived stamps provide a stronger signal and can be measured in a correspondingly shorter time. 15 This has the advantage that, in the time available, a larger number of microparticles can be measured for a single assay, the results of which can be averaged. This achieves a higher measurement accuracy and does not require such a high accuracy for the size distribution of the microparticles. The stronger signal also allows the fluorescence of the label bound to the microparticles to be measured from the fast moving flow.

25 The use of dual photon tuning was invented by n.nor. it is particularly advantageous in the process according to suggested that the Raman excitation used to identify the particle and the double photon excitation of the label indicating the amount of analyte F can be performed with the same laser beam having a wavelength of 50 in the red or NIR range. The use of red or NIR light is advantageous in Raman winch because in this wavelength range less background fluorescence interfering with Ramar. Measurement is generated.

35 Raman scattering is the inelastic scattering of light associated with molecular structures, in which the energy quanta corresponding to the characteristic vibrations of the molecular structures are mixed with the scattering-inducing light. The Raman spectrum appears:! * IM UV I * -M * '· · 11 96641 as the wavelength change of the scattered light on either side of the wavelength of the excitation light. The Raman spectrum contains similar molecular structure information as the infrared spectrum (IR). The Raman spectrum can also be obtained from very small targets such as microparticles (P. Dhamelincourt & et al. Spectroscopy Europe 5/2 (1993) 16-26). Therefore, the Raman spectrum can be used to identify and distinguish between different molecular structures used to make microparticles.

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Microparticles belonging to different classes can be prepared by known pclymerization methods in such a way that the monomer mixtures used for the syntheses of the different particle lucers differ from each other on the basis of their chemical composition. The monomer mixtures used for the preparation of the particle classes may differ not only in the grades or quantities of monomer they contain but also in the nature or quantity of the other additional indicator substances. Examples of monomers that are polymerizable together and have significantly different Raman spectra include styrene, deuterated styrene (= styrene-D6), methyl methacrylate, and acrylonitrile. Indicators to be added to the monomer mixture may be, for example, oligo- and polymers of the above-mentioned monomers or aliphatic and aromatic halogen compounds.

Compounds 30 used to identify microparticles can be added to batches of microparticles of different classes in various combinations to form microparticles having a Raman signal of various indicators indicating the class of the particle as a binary number or some other number system. 35 The binary number 0 is expressed, for example, as an indicator concentration 0 and the binary number 1 is expressed as a concentration other than zero.

96641 12

For example, with styrene being the main component of the monomer mixture used in the particle synthesis and the indicators used in the particle synthesis of the other three monomers mentioned above, 23 mice or 8 different classes of particles can be prepared from these monomers when delivered according to the binary number 5 system. The Raman spectra of different classes of polymer particles made from said monomers can be distinguished either by comparing the spectra of the H) particle classes as a whole by the method disclosed in U.S. Patent 5,313,406 or by comparing the intensities of the individual peaks in the spectra. Figures 1 show the Raman spectra of said compounds and their structural analogs, from which it can be seen that the Raman spectrum of each compound has characteristic peaks missing from the spectra of the other said compounds.

Figure 1 shows the Raman spectra of polystyrene (Ia), benzene (Ib) 2 O and deuterated d6-benzene (Ic). Comparing these spectra with each other, it is observed that by replacing the hydrogen atoms of the benzene ring with deuterium atoms, the Raman peak at wavelengths 3000-3100 shifts to wavelengths 2260-2300. At the same time, based on 25, the Raman spectrum of D6-styrene (or D6-polystyrene) differs from that of H6-styrene (or H6-polystyrene), and thus deuterated styrene (polystyrene) can be used as an indicator of particle class in an H6-styrene polymer.

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Figure 1 also shows the Raman spectrum of polymethyl methacrylate (Id). In this spectrum, the following is observed: a peak characteristic of the carbonyl group at wavenumbers of 1700-1750 l / cm. In this wavenumber range, there is no polystyrene 1 la 35 (la) and no other indicator compounds under consideration have peaks in their Raman spectrum. Thus, the methyl methacrylate polymer was suitable as an indicator determining the class of polystyrene particles 96641 is, together with the other indicators mentioned above.

The Raman spectrum of polyacrylonitrile has a peak due to the nitrile group at wavelengths of 2200-2250 1 / cm.

In this wavelength range, neither polystyrene nor the indicator compounds under consideration have peaks in their Raman spectra. Thus, the acrylic-nitrile polymer is suitable for use as an indicator of the polystyrene-10 particle class in combination with other indicator compounds under consideration.

Similar examples of other indicator compounds whose Raman spectra differ from the Raman spectra of polystyrene and other mentioned Li indicators can be shown in large numbers.

The identification of a class of microparticle based on the Raman spectrum it provides can be accomplished by a number of known methods. Raman emission is best excited by a laser beam. For example, a conventional lattice spectrometer with a CCD row detector can be used to record the Raman frequency spectrum, from which the entire spectrum is obtained simultaneously. The interferogram of Raman 25 (which is a Fourier transform of the frequency spectrum) can be recorded using, for example, a simple interferometer connected to a CCD line detector. In both cases, the frequency spectrum and the interferogram can be recorded in a very short time, since Raman indicators can account for up to 1% of the total mass of microparticles and the laser intensity used can be so high as to produce a strong signal in a very short measurement time of less than 1 millisecond. The identification of the microparticles 35 can take place on the basis of individual peaks of the frequency spectrum or by comparing the registered frequency spectrum or interferogram with the reference spectra corresponding to different classes. The comparison is made most quickly and most simply by the cross-correlation method known from U.S. Patent 5,313,406. Computation can be performed with modern microcomputer technology in as little as 1 millisecond.

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The measurement of the microparticles can be performed on a slide or other support on which the microparticles have been placed and attached chemically or physically, or in a liquid suspension 10 in a reaction chamber, a liquid channel or a separate measuring cuvette. The measurement may be based on scanning microscopy, or the measurement optics may be targeted at the microparticles in some other way, or it may be imaging. The measurement can also take place in 15 flow cuvettes into which the diluted microparticle suspension is introduced. The flow of the microparticle suspension can either be stopped or the flow can be running at the time of measurement. The measurement can also take place for flow cytometers in the usual way in a cuvette or in free air. The aperture of the measuring cuvette is selected according to the size of the microparticle used and the photoluminescence emission is measured with a photomultiplier tube or other sensitive photon detector or a suitable image detector. 25 microchannel image amplifiers and a solid state image detector (CCD) can also be used as detectors. A sufficient number of microparticles are identified and analyzed to obtain a statistically accurate result for each analyte. The measurement results are related to the 50 measurements performed on standard samples, on the basis of which the final results are calculated.

It is also characteristic of the present invention that the microphotometric measurement system disclosed herein is capable of optically distinguishing light emission from the surface 55 of the microparticles from light emission from a solution in the vicinity of the microparticles. This resolution is based on the fact that when the reaction solution is diluted considerably, only one microparticle fits into the focus range of the photon detector 96641 15 at a time and the background signal caused by the free labeled reagents in the solution is small.

When dual photon excitation is used, the excitation efficiency 5 is proportional to the square of the excitation light power. This property of dual photon excitation substantially enhances the separation of light emission from the surface of microparticles from the solution in the vicinity of the microparticles and the light emission from optical parts. Optical separation achieves sufficient resolution of the free and bound fractions without chemical or physical separation, but if unavoidable, separation can be enhanced by any known separation method such as washing, filtration or centrifugation.

The method according to the present invention and the equipment required for it can be implemented in practice in many different ways. However, it is essential in the invention that each individual microparticle representing the analyte class is identified by Raman spectrometry. The intensity of the biospecific reaction is measured simultaneously or separately and thus a multipararea biospecific assay can be performed on the same sample.

With a measurement arrangement based on time resolution and / or double photon excitation, the fluorescence emission F can be distinguished from the fluorescence and scattering.

It will be apparent to those skilled in the art that various embodiments of the invention may vary within the scope of the claims set forth below.

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Claims (6)

1. Biospecific multiparametric assay method, in which pre-fabricated, microparticles divided into different classes sensitive to different analytes are coated with biospecific reagents that bind different analytes, where 5. the different microparticles belonging to different classes are mixed and the sample is added to this mixture, - a mixture of with luminescent molecules labeled biospecific secondary reagents are added, initiating a reaction between analyte molecules, labeled secondary reagents, and immobilized reagents to the microparticles, dilute the reaction solution to reduce the concentration of the free labeled reagents, bound to each particle is obtained by measuring the electrical signal strength of the detector, characterized in that the belonging to different classes of microparticles contain such different molecular structures or different indicators on the basis of which they are identified and separated from each other by means of Raman spectra. 2. A method according to claim 1, characterized in that the photoluminescent marker is a fluorescent molecule and that its excitation occurs with double photon excitation. 96641 i9 3. A method according to claim 1, characterized in that the photoluminescent marker is a fluorescent or phosphorescent molecule and its excitation occurs as a single photon excitation. 4. A method according to claim 1, characterized in that the photoluminescent marker is a chemiluminescent, bioluminescent or electroluminescent molecule. 5. A method according to claim 2 or 3, characterized in that the Raman excitation of the microparticles and the excitation of the photoluminescent marker 15 are carried out with pure light beam. A method according to any one of claims 1 to 3, characterized in that the exciter and the detection of the photoluminescent marker are confocal. »