RU2362987C2 - Application of biphoton-excited fluorescence in field of clinical chemistry for analysis of components contained in clinical sample - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to field of analytic clinical chemistry. Analysed component is subjected to chemical reaction or reactions in one or several stages, or reactions in reaction sequence resulting in change of quantitatively determined property of compound or compounds. Said chemical reaction or reactions or reaction sequence results in formation of compound with biphoton fluorescence, or in change of characteristics of biphoton fluorescence in reaction system containing at least one compound with biphoton fluorescence, and analysed component is quantitatively evaluated by exciting said compound or compounds with biphoton fluorescence and measuring intensity of biphoton-escited fluorescence and comparing obtained values of measured fluorescence with method of data standardisation based on measurements obtained from standard material of said analysed component.

EFFECT: novel method of analysis of components contained in clinical sample.

17 cl, 7 dwg

Description

Field of Invention

The present invention relates to the field of clinical chemistry and to the use of two-photon-excited fluorescence as a principle for detecting the quantitative determination of components analyzed in a clinical sample.

BACKGROUND OF THE INVENTION

Publications and other materials used in the description to illustrate the prior art in this field, in particular additional information regarding practice, are incorporated into the text by reference.

Clinical Chemistry Assays

The components defined in a clinical sample, the authors mean in this context, metabolites or other components of the serum, which are usually measured in practice in the field of clinical chemistry. Examples of such analyzed components are the following components: glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, bilirubin, creatinine, total proteins, iron, magnesium, urea, uric acid, ASAT, ALAT, amylase, LDH (LDH), GT, alkaline phosphatase, lipase and creatine kinase. In this context, the detectable components contained in the clinical sample do not include those components that are measured by affinity-based methods such as quantitative immunoassays, competitive binding assays, antigenic bridge formation assays, or agglutination assays (The Immunoassay Handbook, 1994, David Wild, Editor, ISBN 0-333-51179-4).

The components most commonly determined in a clinical sample are analyzed by methods based on photometric determination. Analytical protocols include the addition of one or more reagents to the sample (e.g., blood, plasma, serum, urine or other body fluid, diluted or undiluted in an appropriate aqueous or non-aqueous diluent) and incubation under constant conditions in one or more cases after the addition of the reagent or in between reagent additions. And finally, the absorption of the analyzed mixture is measured at one or more wavelength ranges, followed by calculation of the concentration of the analyzed component using standard calibration curves. Most clinical assays are based on an enzyme-catalyzed reaction, where the enzyme acts either as an analyte component or as a specific reaction catalyst for an analyte component. As a result of the reaction catalyzed by the enzyme, a change in the structure of the substrate molecule occurs, which leads to a change in the absorption characteristics of the substrate. The change in the photometric readings at the appropriate wavelength is proportional to the concentration of the analyzed component in the sample. In the case when the analyzed component acts as a substrate and the enzyme serves as a reagent, the reaction may end, while the measurement is carried out at the end point, while when the enzyme acts as the analyzed component, the reaction proceeds under strictly controlled conditions and the measurement is carried out with using kinetic techniques. In this context, the term “kinetic measurement” means that the sample is quantified either at a given predetermined time point (or time points), or, alternatively, if the kinetics of the reaction are well characterized and mathematically modeled, the reaction can be evaluated at any specific time point (or time points) followed by calculation of the final enzymatic activity (concentration in units of activity) using kinetic equations calculated separately for this in advance th case. Analysis programs and methodologies commonly used in clinical practice are described in textbooks and literature (Tiez Texbook of Clinical Chemistry, Ed. C. Burtis and E. Ashwood, W.B. Saunders Company). Another type of clinical analysis is represented by a type that does not include enzymatic catalysis, but has a pure chemical base (non-enzymatic reactions). Such assay components include, for example, bilirubin analysis with a diazoreagent, whey protein analysis with a biuret reaction, and albumin analysis with a BCG reagent (bromcresol green) (Tiez Texbook of Clinical Chemistry, p.702). As a result of these reactions, a colored final product is formed, the concentration of which is measured by photometry in the corresponding wavelength region. The most typical form of photometric analyzer in clinical practice includes a single light beam and single photometric cuvettes. This means that any variation in the optical length of single cells or in the optical properties of the walls of the cells causes inaccuracy in the final analysis result. Because of these photometric properties and, on the other hand, because of the stringent requirements for the accuracy of clinical analyzes, the quality requirements of single-cell ditches are extremely necessary. For this reason, clinical analysis cuvettes are and remain costly. In the practice of clinical chemistry, the price of a cuvette is the basis of the cost of clinical analyzes performed using photometry.

Some of the analyzed components of the clinical analysis were also determined by methods based on the detection of fluorescence (single-photon-excited fluorescence). These analyzes were carried out by replacing the chromogenic substrate (or reagent) used in the photometric methods with a fluorogenic substrate (or reagent). As a result, the fluorescence signal emanating from the analyzed mixture is proportional to the concentration of the component. Examples of such assays are glucose analysis using glucose oxidase, peroxidase and Amplex Red ™ (fluorogenic substrate, trade name for molecular probes, Cat.No. A-12222) as reagents, and creatinine analysis using creatinase, creatinase, sarcosine oxidase, peroxidase and Amplex Red as a reagent. Another example is the analysis of amylase using fluorophore-labeled starch as a reagent (or another starch analog, such as amylopectin or an appropriate synthetic reagent, such as α-1,4-oligosaccharide), where labeled starch is a substrate for the enzyme, and hydrolysis of the substrate results to increase fluorescence as the enzyme digests the polymer reagent (or oligomer) (Fluorescence Microplate Assays, Molecular Probes, Seventh edition, 2002, p.51). Another example of the fluorometric methods of analysis is the analysis of alkaline phosphatase using fluorescein-labeled phosphate as a fluorogenic substrate (Handbook of fluorescent probes and research products , 9 ^{th Ed., P.420, Molecular Probes} , Eugene, OR, USA, 2002). This analysis is based on an increase in the fluorescence intensity upon hydrolysis of the fluorogenic substrate with the analyzed component. However, fluorescence-based assays are used mainly in research laboratories, while their use in clinical practice is very limited. In general, fluorescence-based methods give orders of magnitude with greater sensitivity and a wider dynamic range than photometric-based methods. In research laboratories, these qualities give a clear advantage, but in the field of clinical chemistry this advantage did not have a useful application, since the requirements for the sensitivity or dynamic range of analyzes in the field of clinical chemistry are often rather not high enough. Instead, the reliability, accuracy of the results and the cost of the reagent are usually considered as more important criteria for analyzes in the field of clinical chemistry. Using the above criteria as an example, it was shown that until now, fluorometry has not demonstrated clear advantages over photometry as a method of detection in clinical practice.

Applications of fluorescence in bioaffinity assays

Single-photon-excited fluorescence has found various applications in the field of bioanalytics. Applications such as immunological analysis, DNA hybridization analysis, and receptor binding analysis using fluorescence as a detection method have been introduced over the past three decades. In these analyzes, specific bio-affinity reactions are used to determine the analyte component in the sample. The amount of analyte component can be determined by monitoring the fluorescence signal, which depends on the amount of analyte component bound. These assays may also be based on monitoring changes in fluorescence properties during a specific binding reaction. The indicated change in the fluorescence property may include either a change in the fluorescence intensity, a change in the emission wavelength, a change in the pulse decay time, or in the polarization of the fluorescence.

Immunological tests have been widely used in clinical diagnosis to determine certain diseases or physiological conditions. Immunoassays can be divided into two different types of assays, competitive and non-competitive assays. In a competitive method, labeled antigen (second biospecific reagent) competes with the analyte for binding to a limited number of antibodies (first biospecific reagent). The concentration of the analyte component can be determined from the ratio of the labeled antigen bound to the antibody, or from the ratio of the free fraction of the labeled antigen. In a non-competitive method (immunometric method), the analyzed component binds to an excess of binding antibody (first biospecific reagent). Excess labeled antibody (second biospecific reagent) binds to another portion of the component being analyzed. The amount of analyte component can be determined based on the fraction of labeled antibody bound to the analyte component. The physical separation of bound and free fractions is a necessary technique before detection, if the registration principle does not make it possible to distinguish the signal of the bound fraction from the signal of the free fraction. Thus, analysis methods are divided into split analyzes and non-split analyzes, often also referred to as heterogeneous and homogeneous analyzes. [Miyai K., Principles and Ptactice of immunoassay, (ed. Price CP and Newman DJ) Stockton Press, New York 1991, 246 and Hemmilä IA, Applications of Fluorescence in Immunoassays, (ed. Winefordner JD) John Wiley & Sons, New York 1991]. The coated tube method is typically used for separation assays, and in such cases the free fraction is separated by repeated washing steps.

The sensitivity and dynamic range of fluorometry for single-photon excitation are higher and broader than those for photometry. However, sensitivity is sufficient only for a limited number of analyzes. Time-resolved fluorescence, chemiluminescence, or electrochemiluminescence fluorescence methods provide improved sensitivity compared to a conventional steady state (i.e., instantaneous) fluorescence. These methods are known both in the field of scientific research and in diagnostics, and they are considered along with the most sensitive analytical methods (Hemmila and Mukkala, Crit. Rev. Clin. Lab. Sci. 2001).

Two-photon excitation

In 1931, Maria Göppert-Mayer [Ann. Phys. 9 (1931) 273] showed that a molecule can simultaneously absorb two photons. This phenomenon remained for a long time without any practical application until sources of intense laser radiation became available. Two-photon excitation occurs when, when focusing an intense light source, the photon density per unit volume and per unit time becomes high enough so that two photons can be simultaneously absorbed by the same chromophore. The absorbed energy consists of the sum of the energies of two photons. The possibility of two-photon excitation will depend on the degree of density of the 2nd photon. Thus, the absorption of two photons is a second-order nonlinear process. The simultaneous absorption of two photons by one chromophore brings the chromophore into an excited state. Then, the aforementioned excited state is attenuated by spontaneous emission of a photon with a higher energy than the illumination photons. In this context, a process that involves two-photon excitation and subsequent relaxation accompanied by radiation is called two-photon-excited fluorescence (TPE). TPE typically has emission characteristics similar to those of single-photon-excited fluorescence for the same chromophore [Hu C. and Webb W.W., J. Opt. Soc. Am. B, 13 (1996) 481]. The excitation spectrum, however, sometimes expands and / or shifts hypsochromely when compared with the spectrum of single-photon excitation. Molecules that are excited by the simultaneous absorption of two photons and produce excited states and fluorescence radiation are referred to in this context as two-photon fluorescent dyes.

One of the key characteristics of two-photon excitation is that excitation takes place only in a clearly defined 3-dimensional space (3D) of the focal point. The consequence of this characteristic is a high 3D concentration in the space of the produced fluorescent radiation. Due to the nonlinear nature of the excitation, minimal background fluorescence is produced outside the focal volume, i.e., in the medium surrounding the sample and in the optical components. Another key characteristic of two-photon excitation is that illumination and radiation occur in significantly different wavelength regions. The result of this characteristic is that the leakage of scattered light in a channel that detects fluorescence excitation can be reduced using a low-pass filter (decrease by at least 10 orders of magnitude). Since the excitation volume is small (in the area of femtoliters, i.e. 10-15 liters), two-photon excitation is most suitable for observing small volumes of samples and structures.

Bioanalytical applications of fluorescence with two-photon excitation

One of the earliest reports regarding the analytical use of two-photon excitation was the publication of Sepaniak et al. [Anal. Chem. 49 (1977), 1554]. The authors discussed the possibility of using fluorescence with two-photon excitation for recording HPLC. The low background and simplicity of the system have been demonstrated. Researchers Lakowicz et al. [J. Biomolec. Screening 4 (1999) 355] reported the use of multiphoton excitation in screening with high throughput. They showed that two-photon-induced fluorescence of fluorescein can be measured with high reliability in multi-well plates with high density.

Most of the bioanalytical application of fluorescence with two-photon excitation, described in the literature, refers to two-photon microscopy [Denk W. et al. US patent 5034613, Denk W. et al., Science 248 (1990) 73]. The use of two-photon fluorescence excitation in laser scanning microscopy gives 3D spatial resolution without using the small holes needed in confocal microscopy. Together with a simple optical model, two-photon excitation microscopy provides 3D spatial resolution comparable to that of conventional single-photon confocal microscopy. The development also leads to the industrial production of two-photon laser scanning microscopic systems. An obstacle to the development of the method is the requirement of an expensive laser capable of generating strong ultrashort pulses with a high repetition rate.

The latest development of less expensive laser technology is very encouraging in connection with the application of fluorescence technology with two-photon excitation in routine bioanalytical methods [Hänninen P. et al., Nat. Biotechnol. 18 (2000) 548; Soini J.T. et al. Single Mol. 1 (2000) 203; Soini JT (2002) Crit. Rev. Sci. Instr., International poetications WO 98/25143 and WO 99/63344]. According to the international publication WO 98/25143 and the international publication WO 99/63344, instead of expensive lasers with a mode locking mode for two-photon excitation, microchip lasers pumped by passive Q-switched LEDs can be used. These lasers are monolithic, small, simple and low in cost. International publications WO 98/25143 and WO 99/63344 describe the use of two-photon-excited fluorescence in the detection of bioaffinity analysis data. This bioaffinity assay method uses microparticles as a bioaffinity binding solid phase to which the first biospecific reagent binds. This bioaffinity assay method uses a biospecific second reagent that is labeled with a two-photon fluorescent dye. According to the methods described in international publications WO 98/25143 and WO 99/63344, bioaffinity complexes are formed on the surface of microparticles, and the amount of bioaffinity complexes is quantified by measuring the intensity of two-photon-excited fluorescence emanating from individual microparticles. Thus, this method allows bioaffinity analysis without separation in microvolumes.

The labeled second bio-affinity reagent binds to the surface of the microparticles either through the molecule of the analyzed component to form three-component bio-affinity complexes (non-competitive, immunometric method), or it binds directly to the first bio-specific reagent to form two-component bio-affinity complexes (competitive binding method). The first and second biospecific reagents are biologically active molecules, such as haptens, biologically active ligands, drugs, peptides, polypeptides, proteins, antibodies or antibody fragments, nucleotides, oligonucleotides or nucleic acids. According to international publications WO 98/25143 and WO 99/63344, a laser with high two-photon excitation efficiency is focused on the reaction suspension, and two-photon-excited fluorescence emanating from single microparticles is measured when they move throughout the focal volume of the laser beam. Alternatively, microparticles can be "captured" during the period of registration of fluorescence by an optical trap, which is caused by a laser beam. The capture of microparticles to the focal point of the laser beam is based on the optical pressure that is produced on the microparticle by means of a laser beam. According to the international publication WO 98/25143, optical capture increases the duration of the particle in the focal volume of the laser beam and increases the working cycle of fluorescence detection. The optical arrangement of a typical two-photon excitation fluorometric device is shown in Figure 1 (ArcDia ™ TPX Plate Reader). The design of the device may vary depending on the individual use and application. The most important components that distinguish a device are:

- The laser beam is focused using the lenses of the microscope objective with a minimum of numerical aperture from 0.4 to 0.7.

- To capture the microparticles, a two-dimensional piezoelectric scanner is used, which is able to stop scanning instantly when the microparticle is detected near the focal volume.

- The near-infrared laser is a pulsed laser with a pulse duration shorter than 10 nanoseconds, a pulse repetition rate above 10 kHz and an average beam power in the sample of the order of 100 mW, with TEM 00 type of output of a polarized beam. A typical laser is a Nd: YAG or Nd: LBS microchip laser with passive Q switching [Danailov M.B. & ai., Appl. Phys. B 73, 1-6 (2001)]. An alternative laser may be a mode-locked Yb-promoted fiber laser [A. Grudinin & ai., Optics Letters (2003), Vol. 28 Issue 17 Page 1522].

The fluorescence signal in the visible region of the optical spectrum is determined using CPM photomultipliers using simple photon counting.

Current status of the issue in in vitro diagnostic testing

The main areas of in vitro diagnostic testing (IVD) are: clinical analyzes, bioaffinity tests, hematology and microbiology. Of these four areas, diagnosis is often required in clinical chemistry practice, however, the highest sales volume and growth rate are accounted for by bioaffinity analyzes. Other areas, microbiology and hematology, together account for 1/6 of IVD sales, but they are necessary for a complete initial diagnosis and treatment.

In the usual laboratory practice of small and medium-sized hospitals or clinics, usually three types of analyzers are required: 1) a device for counting blood cells in hematology, 2) an automatic analyzer for clinical chemistry and 3) an analyzer for immunological analyzes. Microbiological testing can be performed quickly using test strips, by visualizing microbial cultures, or by immunoassay, so specialized tools are not required to read the test results.

As discussed above, at least three core analyzers are required in the in vitro diagnostic laboratory today to perform some of the analyzes that are most often needed. Such a large number of analyzers is costly, and these costs are paid back only when the throughput of the samples in the laboratory is high enough so that the standby time of the analyzer is as low as possible. According to the current trend in industrialized countries, the healthcare system is undergoing critical analysis regarding cost-effectiveness. The government that pays compensation for expenses in the health system is in a difficult position to reduce costs. Based on this situation, both clinical laboratories and manufacturers of analyzers and test kits are forced to reduce the total cost of analysis operations and carry out IVD maintenance more efficiently. As a result, IVD is centralized to mechanically performed work in large laboratories. However, this trend contradicts the need for intensive care, emergency therapy and outpatient care, where IVD results of analysis of samples of one patient are required without delay. The trend towards centralization is not applicable in rural areas, where the distances from the doctor’s office to the central hospital are considerable. Thus, in addition to the trend towards centralization, there is an increased need for on-site health checks (ROS) and for the distribution of IVD analyzers.

The trend towards centralization IVD

The trend towards centralization leads to the fact that high-performance IVD analyzers are installed in central laboratories. Collection of samples from patients will occur either locally in remote doctor's offices, or in medical centers or in a reception center IVD laboratory. In the first case, samples taken from patients should be collected and transported from remote areas by courier to the central IVD laboratory, while in the latter case, transportation of the patients themselves is required. Obviously, the direct verification costs are minimal when the analyzes are performed in batches in a central laboratory using high-performance analyzers. However, health care structures usually focus on analyzes that require direct costs and are not able to calculate the indirect costs of IVD throughout the health system. Part of the money is spent on transportation of samples or patients to central laboratories and a small part is given for (i) patient waiting time, (ii) numerous visits by the patient, and (iii) free-standing samples in uncontrolled conditions. In a small number of cases, the overall cost structure of IVD services and logistics is taken into account when the IVD methodology and devices are chosen by the authorities. In addition, the variable waiting time of samples of an individual patient under uncontrolled conditions (temperature, exposure to daylight) inevitably causes changes in the composition of the sample, leading to erroneous analysis results and an erroneous diagnosis.

Tests performed at health care facilities

Platforms for medical care (ROS) are designed to test single samples and are characterized by a limited portfolio of orders. Typically, when testing single samples at ROS points, non-standard methods of analysis are used, they obtain high-quality or semi-quantitative results, work without the supervision of specialists in clinical chemistry and without resorting to a database, usually, ROS tests are based on the use of collected one-time sites for analysis. Some of them are based on immunochromatographic methods, which include a chromatographic medium and a test zone with an immobilized immune reagent. The determination can take place visually or, for example, using the photometric method, the fluorometric method, the resonance of a surface plasmon, or the electroluminescent method. ROS testing is effective. for costs with limited use - 1000-10000 tests per year. In large quantities exceeding 10,000 tests per year, ROS tests become less cost effective due to the high price of a one-time analysis of the component. ROS devices are practical only in cases where qualitative results are immediately required for samples of a single patient.

Decentralization of IVD Diagnostic Testing Tools

In contrast to the trend towards centralization, there is a clear increase in the need for the distribution of IVD services and analyzers. By decentralization of diagnostic IVD testing, the authors mean the practical implementation of IVD diagnostics using compact complex analyzers for IVD, which are distributed in rooms where samples are taken from patients, such as local doctors' offices or remote main medical institutions of the region. The analyzers are connected to the database of the central laboratory through a long-distance communication network. The analyzers function and their work is monitored by specialists in the field of clinical chemistry in a central laboratory, so the analyzers work like chemical sensors located close to the patient.

The technology for distributing IVD funds is still underdeveloped. Until now, there is no exemplary and suitable methodology and commercially available equipment that would make decentralized IVD diagnostics cost-effective. This is one of the main reasons for the current trend towards centralization of IVD in industrialized countries. An exemplary analyzer for decentralized IVD diagnostics should perform all of the most commonly required tests, including both clinical tests and sensitive immunoassays, with one detector. In order to carry out cost-effective analyzes, the analyzer for decentralized IVD diagnostics must perform all analyzes without separation, in other words, without washing steps. Such a separation-free analysis format should greatly simplify the handling of fluid technology, thus making the apparatus smaller and less expensive to manufacture. The physical size of the analyzer should be suitable for desktop use. The analyzer should allow operations in small volumes to be analyzed. This together with the format without separation (without washing stages) should save reagents and liquid consumables, thereby increasing the cost-effectiveness of the method. Work on the analyzer should not require personnel with the knowledge required in the clinical laboratory. The cost of the analyzer should not be higher than, for example, the cost of a small analyzer used in clinical chemistry, or an analyzer used in immunology. The device should include a specialized data processing program for applications in clinical chemistry and for the quantification of analyzes, and should communicate with a data network for observation by a specialist in a central laboratory. This means that analyzers should be distributed in remote locations where samples are taken, while clinical chemistry expertise, application of analysis results, quality and content control should be centralized. Currently, there are few commercially available analyzers and technologies that would satisfy these requirements. Small analyzers used in clinical chemistry and analyzers used in immunology are usually available as separate units. Some of the commercial analyzers used in clinical chemistry also carry out immunoassays in a non-split format. However, these immunoassays are usually based on the principles of either turbidimetry or nephelometry (agglutination analysis), which do not allow highly sensitive analyzes, but are limited to the analysis of components with a relatively high clinical initial concentration.

In order to cope with the analysis of components with a lower initial concentration, some manufacturers have recently developed analyzers that combine clinical assays with immunological assays using the principle of heterogeneous immunoassay. An example of such an analyzer is the Adaltis analyzer (Adaltis Italia S.p.A, Bologna, Italy), which performs clinical analysis by recording data using photometry and immunoassays by recording data using chemiluminescence. The principle on which heterogeneous immunoassay is based does not, however, allow for analysis without separation and therefore is not exemplary for decentralized IVD diagnostics. In conclusion, the IVD market is looking for a new tool technology that will meet the requirements discussed above.

One of the most important problems of the photometric methodology is the need for expensive test ditches. If the analyzer is equipped with reusable flow cells, the cost of the analysis cell is not a problem. However, reusable flow cells are not commonly used at present because of their sensitivity to the component being analyzed and the remaining reagent and contamination. Thus, for most commercial analyzers used in clinical chemistry, single photometric cuvettes are used. Due to the nature of photometric data recording, the result of the analysis depends on the optical quality of the tested cell and on the optical path length. These quality requirements force the creation of high industrial technologies, which makes the cell production expensive and the corresponding analyzes are prone to error due to changes from the cell to the cell. For this reason, cuvettes for analyzes used in clinical chemistry are and remain expensive. Indeed, the cuvette is the main component of the costs of analyzes in the field of clinical chemistry, which are based on photometric data recording.

An additional problem regarding the accuracy of photometric analyzes is related to interference from the sample matrix. Serum and plasma are typical samples where metabolites are analyzed. However, the sample may contain interfering matrix components, such as bilirubin, hemoglobin from hemolysed red blood cells, or turbidity due to particles, lipids. All of these substances can greatly interfere with photometric measurements if the sample is not diluted to a large extent before analysis.

The third problem of photometric data recording relates to the analyzed volumes. The analytical sensitivity of photometric recording depends on the length of the optical path. The longer the optical path, the higher the dilution factor can be used. This increases the tolerance range for interference from matrix components and the accuracy of registration. Typical optical lengths for commercial analyzers are 5 to 8 mm. Depending on the geometry of the cell, this length leads to an analyzed volume of 100-300 microliters. Such a cuvette is not optimal for immunological analyzes, since the cost of reagents for immunoassays is usually ten times higher than the cost of reagents for clinical chemistry. Therefore, analyzers used in clinical chemistry with photometric recording are relatively large in size, and to work with them, rather large volumes of reagents and diluents are required. This property increases the cost of immunoassays, and the advantage of small analyzed volumes cannot be taken.

Summarizing the above, the authors conclude that the market for IVD diagnostics does not achieve the desired results regarding the technology and analyzer, which should facilitate the implementation of routine clinical analyzes and highly sensitive immunological analyzes and which should perform all the most frequently required analyzes with a methodology without separation in microvolumes. In addition, the methodology should minimize or eliminate the need for liquid treatment other than dilution and distribution of the sample. The methodology should facilitate the analysis with high accuracy by using single-use ditches with low cost and without the need for pre-fabricated coated tubes or other expensive analysis component. Thus, the analyzer must combine the functions of two large clinical analyzers - an analyzer for clinical chemistry and an immunoanalyzer, and thus contribute to the implementation of cost-effective analyzes in microvolumes.

The purpose and essence of the invention

One objective of the present invention is to provide an improved method for the quantification of components in the field of clinical chemistry contained in a clinical sample.

Another objective of the present invention is the use of a device for improved quantification of components in the field of clinical chemistry contained in a clinical sample.

It is also an object of the present invention to provide an improved system for quantifying the components in clinical chemistry contained in a clinical sample.

Another objective of the present invention is to develop a program for a system for the quantitative evaluation of components in the field of clinical chemistry contained in a clinical sample.

Thus, the present invention relates to an in vitro diagnostic method for quantifying a component in the field of clinical chemistry contained in a clinical sample, where the analyzed component

a) undergoes a chemical reaction or reactions with a reagent or reagents, proceeding in one or non-slip steps, or a sequence of reactions, or

b) catalyzes a chemical reaction or reactions, or a reaction in a sequence of reactions, reagent or reagents, in one or more stages;

in a reaction system, wherein said reaction, or reactions, or a sequence of reactions leads to a change in the quantitatively determined property of a compound or compounds of said reaction, or reactions, or a sequence of reactions. A characteristic feature of the method is that

i) the specified chemical reaction, or reactions, or a sequence of reactions leads to

- the formation of compounds with two-photon fluorescence or

- a change in the properties of two-photon fluorescence of the reaction system, which includes at least one compound with two-photon fluorescence;

and

ii) said analyte is quantified by excitation of said compound or two-photon fluorescence compounds and measurement of two-photon-excited fluorescence intensity and correlation of said measured fluorescence intensity with a standardization method based on measurements obtained from a reference material of said analyte component.

The present invention also relates to the use of a two-photon fluorescence based fluorometric device for in vitro diagnostic quantification of an analyte component or components in a clinical sample or samples, wherein the method for said quantitative determination of one or more of these analyte components includes one or more chemical reactions leading to the formation of at least one two-photon fluorescence compound, or to a change the properties of two-photon fluorescence of a reaction system comprising at least one compound with two-photon fluorescence.

The present invention also relates to a system for in vitro diagnostic quantification of at least one analyte component in a clinical sample or samples. Characteristic of the system is that it includes

a) a fluorometric device that uses two-photon-excited fluorescence to quantify one or more components analyzed in clinical samples, and

b) a data processing unit with a program for specialized data processing of said quantitative assessment of said analyte component or components using said fluorometric device, wherein said quantitative assessment of one or more of said analyte components includes one or more chemical reactions leading to at least one compound with two-photon fluorescence or to a change in the properties of two-photon fluorescence of a reaction system containing it least one compound with two-photon fluorescence.

The present invention also relates to a software product for a system that is characterized in that the software product comprises means for controlling the data processing unit of the quantification system in order to carry out or control the quantification of the analyzed component by excitation of a compound or compounds with two-photon fluorescence, measuring the intensity of two-photon-excited fluorescence and correlation of the indicated measured fluorescence intensity with the standard method ation data based on measurements obtained from reference material of said analyte.

Brief Description of Drawings

Figure 1 is an example of an optical configuration of a fluorometric detector, which is based on two-photon excitation and is used as a measuring device according to the present invention.

Figure 2 is a graphical depiction of the effect of the degree of hemolysis on the restoration of the signal of single-photon-excited fluorescence and two-photon-excited fluorescence.

Figure 3 presents the response curve of the signal and the accuracy profile of the analysis of bilirubin.

Figure 4 presents the response curve of the signal in the analysis of bilirubin in a logarithmic scale.

5 shows standard glucose assay curves with three different incubation time periods.

6 shows a standard alkaline phosphatase assay curve.

7 shows a standard curve for the analysis of creatinine.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the development of technologies and methodologies that satisfy the requirements for a decentralized analyzer for IVD diagnostics described above. The invention relates to the use of two-photon-excited fluorescence (TPE) technology and equipment used for clinical analyzes in the field of clinical chemistry. The invention is based on the surprising discovery that TPE can be used not only for sensitive bio-affinity assays (which have already been described in the literature, as mentioned above), but also for the analyzed components contained in a clinical sample, and the invention allows these two classes of assays to be performed with cost-effectiveness through the same analyzer. The significance of the invention increases, given the surprising discovery that the TPE methodology is exceptionally acceptable to the interference created by the sample matrix. The matrix can adversely affect analyzes that are based on photometry and conventional single-photon excitation fluorescence. The TPE methodology allows for immunological and clinical analyzes without separation, in microvolumes, using single low-cost cuvettes (for example, standard microtiter plates). Ditches with the quality required for spectrophotometric determination are not required in this case. The invention significantly helps to reduce the overall cost of clinical tests, since a cell having spectrophotometric quality is the most expensive component of a typical clinical analysis. Thus, the TPE methodology allows highly sensitive bio-affinity analyzes and clinical analyzes using a single instrument with improved technical characteristics. The TRE methodology also allows you to reduce the analyzed volumes to 10% of the volumes used in ordinary practice. The TPE analyzer may be benchtop and does not include any liquid treatment other than sample dilution and distribution.

Terms

- Single-photon-excited fluorescence: A process that involves the linear absorption of a single photon by a fluorophore and the subsequent attenuation of an excited state, accompanied by radiation.

- Two-photon-excited fluorescence (TPE): A process in which a chromophore is excited by the simultaneous absorption of two photons, followed by attenuation of the excited state, accompanied by radiation.

- Two-photon fluorescence compound: A compound that can be excited by two-photon excitation, i.e. by simultaneously absorbing two photons, followed by attenuation of the excited state, accompanied by radiation.

- Bioaffinity assays: General name for all biological assays that are based on affinity binding reactions, i.e. on the reaction in which bioaffinity complexes are formed. These assays include, for example, immunoassays and nucleic acid hybridization assays. Assays that are based on enzyme-catalyzed chemical reactions are not affinity binding assays.

- Component analyzed by bio-affinity analysis: General name for the analyzed components, which are quantified in clinical practice by bio-affinity analyzes, using, for example, antibodies or DNA probes as a specific affinity binding probe.

- Clinical chemistry assays: Generic name for quantitative assays, which include a chemical reaction that is measured on a regular basis in clinical practice, excluding bioaffinity assays.

- Component analyzed in a clinical sample: General name for the analyzed components, which are quantitatively determined by means of “analyzes based on methods of clinical chemistry”.

- Fluorogenic substrate: A substrate that is characterized by either an increase or decrease in the fluorescence intensity, or a change in the radiation wavelength when it undergoes a reaction catalyzed by an enzyme.

- 3SD: Standard deviation multiplied by a factor of 3. The specified function is constantly used to determine the lower limit of data recording.

- Measurement of kinetic characteristics: The sample is quantified either at a specific given time point (or specific time points), or, alternatively, if the reaction kinetics are well characterized and mathematically modeled, the reaction can be measured at any specific time point (or time points) with the subsequent calculation of the final enzymatic activity (concentration in units of activity) using the kinetic equation established before work for this particular used i.

- Data standardization analysis: The quantification is based on the use of an appropriate reference material for calibrating the signal response received from the detector in the corresponding concentration component or activity units analyzed, and on the method of specific response information provided by the manufacturer along with the reference material.

- Chemical reaction. A reaction wherein chemical or biochemical compounds react to form a new compound or compounds, i.e. a reaction where the chemical primary structure of a compound changes, or a reaction where a change in chemical structure occurs at the level of covalent binding.

Preferred Embodiments

The present invention relates to an in vitro diagnostic assay method by which it is possible to quantitatively analyze components in clinical samples (e.g., blood, plasma, serum, urine or other body fluids) using the principle of two-photon-excited fluorescence as a method of recording data. The analysis is carried out without separation.

A typical method according to the present invention is an in vitro diagnostic method for quantifying the component analyzed in a clinical sample, wherein the component analyzed in the clinical sample

a) undergoes a chemical reaction or reactions with a reagent or reagents, or

b) catalyzes a chemical reaction or reactions of a reagent or reagents;

in a reaction system comprising a reaction or reactions in the sequence where said reaction, or reactions of said analyte component, or catalyzed reactions lead to a change in the measured characteristic of a compound or compounds of said reaction system. A characteristic of the method is that

i) said chemical reaction, or reactions of said analyte component, or catalytic reactions lead to

- the formation of a compound exhibiting two-photon fluorescence or

- a change in the properties of two-photon fluorescence of a reaction system comprising at least one compound exhibiting two-photon fluorescence; and

ii) said analyte component is quantified by excitation of said compound or compounds exhibiting two-photon fluorescence, and measuring the intensity of two-photon-excited fluorescence, and correlating said measured fluorescence intensity with a method for standardizing data not based on measurements obtained from a reference material of said analyte component .

A preferred method according to the present invention includes the steps of:

a) contacting the clinical sample containing the component to be analyzed with the reagent or reagents specific to the analysis;

b) the implementation in the reaction system of a chemical reaction of the analyzed component with the specified reagent or reagents or the implementation of the chemical reaction or reactions of the specified reagent or reagents catalyzed by the analyzed component;

c) possibly repeating steps a) and b) one or more times;

d) said reaction or reactions of a step or steps b) lead to the formation of a compound exhibiting two-photon fluorescence, or lead to a change in the characteristics of two-photon fluorescence of said reaction system comprising at least one compound exhibiting two-photon fluorescence; and

f) quantifying the analyte component by exciting said compound or compounds exhibiting two-photon fluorescence, measuring the two-photon-excited fluorescence intensity and correlating said measured fluorescence intensity with a data standardization method based on measurements obtained from the reference material of said analyte component.

Measurements of the intensity of the fluorescent signal emanating due to two-photon excitation of fluorescence were performed kinetically or by signal at the end point.

Quantification of an analyte component or components contained in a clinical sample can be carried out for several samples, as well as for several components analyzed in a sample, by repeating the method of the present invention for each sample and / or component being analyzed.

Typically, an analyte component includes, but is not limited to, albumin, total protein, hemoglobin, ammonia, carbonate, direct bilirubin, total bilirubin, calcium, chloride, iron, magnesium, phosphate, HDL cholesterol, LDL cholesterol, total cholesterol, creatinine, fructosamine , glucose, lactate, triglycerides, urea, uric acid, acid phosphatase, alkaline phosphatase, alanine aminotransferase, aspartate inotransferase, pancreatic amylase, total amylase, cholinesterase, creatine kinase, glutamate ltransferazoy, glutamate dehydrogenase, hydroxybutyrate, lactate dehydrogenase, and lipase.

A fluorometric device using the principle of fluorescence upon two-photon excitation can be used, according to the present invention, for the quantitative evaluation of one or more of the analyzed components contained in clinical samples. According to a preferred embodiment of the present invention, some components analyzed in a clinical sample, for example, those described above, can be quantified using the same fluorometric device that uses the principle of fluorescence upon two-photon excitation. Typically, at least 2, preferably 5, more preferably 10, even more preferably 20, and most preferably all of the components analyzed in the clinical sample described above can be sequentially quantified using the same device. The same fluorometric device can, of course, be used for the quantitative determination of the analyzed components by the bio-affinity method.

The device to be used according to the invention typically includes a pulsed laser for two-photon-excited fluorescence with a pulse duration of shorter than 10 nanoseconds, with a pulse repetition rate above 10 kHz, with TEM 00 type of output of a polarized beam, with an average power of radiation pulses in the sample from 20 to 200 mW, preferably from 85 to 120 mW, and most preferably about 100 mW.

According to the present invention, the concentration of the analyte component can be quantified using two-photon-excited fluorescence. Typically, a laser is directed to the reaction suspension through objective lenses, preferably a high numerical aperture, and two-photon-excited fluorescence is generated in the focal volume (or volumes), collected from the same volume (or volumes) and quantified in one or more wavelength channels.

According to the present invention, the fluorescence intensity is usually proportional to the concentration or activity of the analyte component. The response signal may come from the analyzed component as such, or it may come from the reagent or reaction product, the concentration of which either decreases (reagent) or increases (reaction product), as a function of the concentration of the analyzed component. The response signal may also come from the product of the reaction or the product of the reaction sequence or cascade of reactions, where the analyzed component acts as a reaction component or catalyzes at least one of the reactions from the reaction sequence or cascade of reactions.

Alternatively, the signal may come from a fluorescent reagent, the emission from which is absorbed (or quenched) by the reaction product, where the formation of said reaction product is proportional to the concentration or activity of the component being analyzed.

Some of the methods for analyzing components defined in a clinical sample that were originally developed for recording single-photon-excited fluorescence can be used as such or after a small modification in the form of registration of two-photon-excited fluorescence, while some other methods of analysis are not constructive, but require large modifications so that they can be used to register two-photon-excited fluorescence. Whether the method is applicable as such or requires modifications depends on the chemical properties of the fluorogenic component of the analysis method.

The invention also relates to a system and a software product for implementing a quantitative method. The system includes many elements from the previous known system, with the help of which a quantitative analysis is carried out, but the control unit of the system monitors the elements to ensure the implementation of the quantitative method according to the invention. The system control unit preferably includes a processor, a memory unit, and a measurement device. Signals from photomultipliers and possible other sensors are received and measured in a measuring device. Measurement parameters and received data are stored in a memory block. The program that provides the processor is also stored in memory. The storage software product includes processor control means for implementing the steps of the quantification method of the invention. A preferred software product may include methods for adjusting a data processing device included in a quantification system to perform or monitor any combination of one or more steps of the method of the invention.

Advantages of the Invention

The present invention describes the use of two-photon-excited fluorescence as a method of recording clinical analysis data, characterized by the following surprising advantages compared to the implemented methods:

Advantage 1:

The interference caused by the presence of matrix components in the sample, such as hemoglobin (hemolysed serum) or bilirubin (i.e. jaundice, bilirubinemia), has been shown to be significantly less with data recording by means of two-photon-excited fluorescence than with photometry. In addition, the same advantage is found when data recording by means of two-photon-excited fluorescence is compared with conventional single-photon-excited fluorometry. This advantage is illustrated by Example 1, which describes the study of the dependence of the fluorescence signal intensity on the degree of hemolysis of a sample. The results of this study are presented graphically in figure 2. The figure shows that an increase in the degree of hemolysis is accompanied by a decrease in the fluorescence signal due to absorption by hemoglobin. In the case of single-photon-excited fluorometry, an increase in the degree of hemolysis from 0 to 1.5 g / liter causes a significant decrease in the fluorescence signal, from 100% drops to 55%, while hemolysis practically does not exert the same degree of two-photon-excited fluorescence. influence. The degree of hemolysis, 1.5 g / liter, is rather moderate and is often found in the clinical laboratory. This example demonstrates that analyzes of clinical chemistry with registration using two-photon-excited fluorescence are extremely tolerant to matrix effects, therefore, registration using two-photon-excited fluorescence gives significantly improved accuracy compared to analyzes with registration using single-photon-excited fluorescence. In general, the analyzed components of the clinical sample are characterized by a rather narrow normalized range of values. This means that even a slight change in the concentration of the analyzed component can have a significant physiological effect or reflect a significant physiological disturbance. Such analyzes require high accuracy and accuracy. From the point of view of equipment, accuracy of 2-3% CV (coefficient of variation) can be easily achieved using various registration methods, such as phonometry or single-photon fluorometry, but maintaining accuracy in the same area is a major problem for modern technologies for recording analysis data in clinical chemistry. Indeed, many of the methods of analysis in clinical chemistry, which are based on photometry or single-photon-excited fluorometry, cannot provide satisfactory accuracy for samples containing a matrix component that interferes with determination. Registration by means of two-photon-excited fluorescence described in this invention eliminates the problem of the accuracy of the analysis, since it is extremely resistant to the influence of matrix components, such as serum hemolysis products.

Advantage 2

A second advantage of the present invention relates to analysis cuvettes. Due to the property of two-photon-excited fluorometry, the intensity of the fluorescence signal does not depend on the length of the optical path or on the total volume of the cell. Instead, TRE fluorescence is generated only in the focal volume of the beam with diffraction divergence, which is located several hundred micrometers from the back surface of the cell window. A fluorescent signal is collected from the same beam with diffraction divergence by means of the same objective lenses (epifluorescence recording). Thus, this excitation format eliminates the need for cuvettes with an exact optical length and encourages the use of single cuvettes for low cost analysis, such as standard microtiter plates. This advantage is illustrated in Example 2, which describes the study of the influence of the optical quality of a cuvette window for analysis on the accuracy of fluorescence measurements. The results (table 1) show that both microtiter tablets, one with a bottom window made of high-quality optical glass and the other with a bottom window made of untreated polymer film, show almost equal signal accuracy (<2% CV). This example indicates that two-photon-excited fluorescence promotes the use of standard low cost cuvettes without compromising analysis accuracy. Therefore, the TPE registration method eliminates the need for expensive cells of high optical quality and promotes more cost-effective analyzes in clinical chemistry than the prior art methods based on photometry or single-photon-excited fluorometry.

Advantage 3

A third advantage of the present invention relates to assay volumes. Since the TPE signal level does not depend on the analyzed volume, registration with the TPE allows to reduce the analyzed volumes without affecting the analysis efficiency. Typical assay volumes in modern clinical chemistry practice range from 100 to 200 μl. With registration by TPE, the analyzed volumes can be easily reduced to 10 μl or even lower. Such a decrease in the analyzed volume is usually accompanied by a decrease in reagent consumption and cost. This aspect enhances the cost-effectiveness of the TPE registration method.

Advantage 4

A fourth advantage of the present invention is a higher detection sensitivity by TPE compared to photometry. The lowest registration limit of the TPE microfluorometer (see Example 1) for a fluorophore in an aqueous buffer solution such as the ArcDia BF560 fluorophore in serum is about 100 rM (10 ^-10 mol / liter), while photometry (path length = 5- 8 mm) allows you to register the same compound (molar absorption coefficient of 80,000 M ^-1 · cm ^-1 ) at a concentration of 100 nM (10 ^-7 mol / liter). Thus, the difference in the registration limits of the two registration methods is three orders of magnitude. As for the analyzes in clinical chemistry, the difference in the limits of registration is probably less in favor of TPE, due to the limitations resulting from the kinetics of the reaction of the methods of analysis. Since components analyzed in a clinical sample usually exist at higher concentrations, the detection limit for photometry is usually very satisfactory. However, a lower limit is advantageous when the sample contains an interfering matrix component. With increasing sample dilution, the matrix component is less affected by the TPE registration method, so this method provides greater analysis accuracy.

Advantage 5

A fifth advantage of the present invention stems from the fact that analyzers used in clinical chemistry based on TPE detection make it possible to carry out highly sensitive immunological assays in a non-split format. A prototype of such an analyzer has now been constructed, and its use for highly sensitive bio-affinity assays has been demonstrated. Thus, the TPE detection method allows us to develop an analyzer that will satisfy all the requirements suitable for the successful functioning of an analyzer designed for decentralized IVD diagnostics, (i) by performing both analysis in clinical chemistry and sensitive ones: immunoassays with the same a recording device, (ii) by providing an analysis type without separation, (iii) by developing a simple design of a compact benchtop analyzer, (iv) allowing analysis with reduced reaction volumes, (v) by means of automated operations after “manual” loading of the sample (in this case, personnel with special experience in clinical chemistry are not required), (vi) by providing communication with the data transmission network for observation by an expert in the central laboratories, (vii) through analysis with increased profitability compared to the most modern methods.

Detailed description of figures

Figure 1

Figure 1 is an example of an optical configuration of a fluorometric detector, which is based on two-photon excitation and is used as a measuring device according to the present invention. Design may vary depending on individual use and application. Typically, the elements are characterized as follows.

- The cuvette is a disposable cuvette or microtiter plate including an optical bottom window. Various microfluidic chips (technology based on laboratory chips) can also be used.

- The lenses are the lenses of a microscope objective with a numerical aperture of at least 0.5.

- The scanner is a two-dimensional piezoelectric scanner that can stop scanning instantly if the microparticle is near the focal beam.

- The laser is a near-infrared pulsed laser with a radiation wavelength shorter than 10 nanoseconds, with a pulse repetition rate above 10 kHz and an average radiation pulse power of 10 to 100 mW, with TEM 00 type of polarized beam output. A typical laser is a Nd: YAG or Nd: LBS microchip laser with passive Q switching.

- BP is a filter for bandwidth.

- M1 and M2 are mirrors for reflecting the beam.

- BS is a beam splitting device.

- The pH is a hole.

- SD is a light scattering detector operating at the output laser wavelength.

- DM1-DM3 are dichroic mirrors.

- PD is a photodiode for monitoring a laser pulse.

- M3 is a mirror.

- F1-F2 are interference filters that are optimally tuned to the emission wavelength of the labeled reagent.

- CPM1-CPM3 are photomultipliers for single-photon counting.

- The control unit, including:

- a measuring device for receiving signals from sensors / photomultipliers,

- a processor for monitoring elements of the system for implementing the quantitative method according to the invention,

- a memory unit for storing measurement results and other analysis data and a program implemented programmatically for the processor.

Figure 2

Figure 2 shows the effect of the degree of hemolysis on the return of a single-photon-excited fluorescence signal (triangle) and two-photon-excited fluorescence (square).

Figure 3

Figure 3 shows the signal response curve (square) and the corresponding accuracy profile (triangle) of the bilirubin analysis method. Accuracy values are given in units of coefficient of variation (CV%).

Figure 4

Figure 4 presents the curve of the signal response of the analysis method of bilirubin in a logarithmic scale. A 3SD level of negative control is also presented (horizontal line).

Figure 5

Figure 5 shows the standard curves of a glucose analysis method with three different incubation time periods. The figure also shows the level of 3SD negative control samples.

6

Figure 6 presents the standard curve of the method of analysis of alkaline phosphatase. The ordinate (slope) is calculated based on the change in the fluorescence intensity as a function of time, and it corresponds to the enzymatic activity of the sample.

7

7 shows a standard curve for creatinine. The intersection of the standard curve and the line for 3SD negative control samples (horizontal dashed line) gives the detection limit for this analysis method.

EXAMPLES

The invention is illustrated by examples 1-15 as follows, however, applications in cases where the present invention has proven advantages are not limited to these examples.

Example 1

The effect of hemolysis on the signal intensity of single-photon-excited fluorescence and two-photon-excited fluorescence

Bovine serum albumin (BSA) was tagged with a fluorescent reagent, ArcDia BF560 (Arctic Diagnostic Oy, Turku, Finland) to obtain a label-BSA conjugate with a degree of substitution of 2, 7. The conjugate was diluted with a hemoglobin solution (variable concentration of hemoglobin, Tris 50 mM, NaCl 150 mm

NaN ₃ 10 mM, Tween 20 0.01%, BSA 0.5%, pH 8.0) to a concentration of 100 nM. This solution was dispensed into the wells of a microtiter plate of either a 384-well type (Greiner BIO-One GmbH, Code: 788096, µClear SV-plate, 2 ^nd Gen, with black walls and a bottom made of a polymer film), or a 96-well type (Greiner BIO -One GmbH, Code 655096) in quantities of 10 and 100 μl, respectively. Quantification with a 96-well plate was carried out using a conventional single-photon-excited fluorometer (Ascent, Thermo Electron Oy, Vantaa, Finland) using a 530 nm excitation filter and a 590 nm emission filter. Quantification with a 384-well plate was performed with an ArcDia ™ TPX Plate Reader (Arctic Diagnostic Oy, Turku, Finland), a prototype microfluorometer specifically designed to measure fluorescence from the surface of individual microparticles according to the ArcDia TPX bioaffinity assay principle [Hänninen et al. , Nat. Biotechnol. 18 (2000) 548]. The device used in this study is a modification of a system that has recently been described in detail by Soini et al. (Rev. Sci. Instr. 2002, 37, 7, 2680). Using a tablet reader, you can measure samples in standard microtiter tablets. It is equipped with an automatic focusing function for accurately positioning the laser beam in the sample located in the well of a microtiter plate, and a long working distance of the lens to facilitate reading from microwells of various bottom thicknesses. The optical configuration of the device is shown in figure 1. A passive Q-switched Nd: YAG microchip laser pumped by LASER LEDs (NanoPulse NP-07014-400, Nanolase, Meylan France) was used as a light source for fluorescence upon two-photon excitation. The laser generates pulses with a duration in nanoseconds at 1064 nm with a 17 kHz pulse repetition rate. The incident laser beam is focused through the bottom of the microwell of the tablet into the sample using microscope objective lenses (Lens, Leica C-Plan 63x / 0.75, Leica Microsystems, Bensheim, Germany). The average power of laser pulses from a focused laser beam is ~ 40 mW. The signal of two-photon-excited fluorescence is directed through a dichroic mirror DM1 and filtered using a dichroic mirror DM2 kg passband filter F1 and recorded using a CPM1 photomultiplier in the wavelength range of 535-600 nm. The fluorescence signals of three repeated samples were averaged and normalized, presented as a function of hemoglobin concentration in Fig. 2, which shows a significant effect of hemoglobin on a single-photon-excited fluorescence signal, while in the case of two-photon-excited fluorescence, only a weak effect was observed. The results obtained indicate that the TPE detection method gives significantly greater analysis accuracy than methods based on single-photon-excited fluorescence.

Example 2

The influence of the material of the cell on the accuracy of definitions between the cells

Mouse monoclonal antibodies were tagged with a ArcDia BF 560-SE fluorescent label (Arctic Diagnostics Oy, Turku, Finland) according to the standard protocol [MEWaris et al. Anal. Biochem. 309 (2002), 67-74] to give a conjugate with a degree of substitution of 3.2 tags per IgG. The conjugate was diluted with buffer (Tris 50, NaCl 150 mm, NaN ₃ 10 mM, BSA 0.5%, Tween 20 0.01%, pH 8.0) to a concentration of 50 nM and distributed into the wells of two different 384-well microplates. One of the microplates was a standard low-cost microplate with a polymer film bottom window (Greiner BIO-One GmbH, Code: 788096, µClear SV-plate, 2 ^nd Gen, with black walls and a polymer film bottom), while the other tablet was a special tablet with a bottom window made of high-quality optical glass (Greiner, SensoPlateTM 96 Well). Samples were measured using a two-photon-excited microfluorimeter (ArcDia TPX Plate Reader described in Example 1) in eight parallel samples using an integration time of 10 seconds per well. Changes between cuvettes were calculated in units of the coefficient of variation. The results obtained show (table 1) that two different tablets give comparable measurement accuracy between the cuvettes.

Table 1 Accuracy of TPE measurements using (i) cuvettes made of high-quality optical glass and (ii) cuvettes with an optical wall made of a polymer film Polymer film tablet Optical glass tablet Ditch No. Signal CV (%) Ditch No. Signal CV (%) one 63.0 0, 95 one 66.1 0.81 2 64.0 2 67.2 3 63.3 3 67.5 four 64,4 four 66.8 5 63,2 5 66.8 6 64.7 6 67.2 7 63.8 7 67.0 8 63.6 8 66.0

Example 3

Analysis of total bilirubin

The analysis of total bilirubin was carried out according to a modified protocol for the analysis of total bilirubin Sigma Diagnostics ^® (procedure 605), which is based on the reaction between bilirubin and diazonium salts. The 605 analysis procedure was performed except that no alkaline tartrate reagent was added. Bilirubin was dissolved in a mixture containing 1 part of dimethyl sulfoxide and 2 parts of sodium carbonate (100 mM, aq.), And then diluted with buffer (bovine serum albumin 40 g / l, Tris 100 mM, pH 7.4) to obtain bilirubin standards 2, 5, 10, 15 and 20 mg / dl. A standard sample of bilirubin in an amount of 2 μl was placed in a cuvette for analysis (384-well plate, Greiner BIO-ONE GmbH, Code: 788096), then a solution of caffeine (10 μl, caffeine 25 g / l, sodium benzoate 38 g / l, was added in a solution of NaAc, code 605-2, Sigma) and a solution of a diazoreagent (5 μl, sulfanilic acid 1.25 mm, NaNO ₂ 1 mm, Hcl 0.05 M). The mixture was well mixed, followed by the addition of a cysteine reagent (1 μl, code 605-6, Sigma, reconstructed with 10.5 ml of H ₂ O). The reaction mixture was incubated for 3 min at room temperature. Fluorescence emanating from the sample was collected at 630-660 nm using a two-photon excitation microfluorimeter (ArcDia TPX Plate Reader described in Example 1) using an integration time of 10 seconds. The fluorescent signal took place as a function of the bilirubin standard, as shown in FIGS. 3 and 4 (i.e., the standard curve).

Example 4

Glucose test

Fluorometric glucose analysis is based on two consecutive enzymatic reactions. First, glucose is oxidized by molecular oxygen in the presence of glucose oxidase. As a result of the reaction, gluconolactone and hydrogen peroxide are formed. In the next, second reaction, the fluorogenic reagent, Amplex Red ™, is oxidized with hydrogen peroxide in the presence of the horseradish peroxidase enzyme (HRP) to produce the resorufin fluorescent product. The reaction procedure was as follows: a standard glucose stock solution was prepared by dissolving 0.198 g of D- (+) glucose monohydrate in 10 ml of assay buffer (NaH ₂ PO ₄ 10 mM, NaCl 150 mM, pH 6.2). The stock solution was then diluted with buffer to give glucose standards of various concentrations. An assay reagent cocktail was prepared by mixing, in equal volume proportions, glucose oxidase (6 U / ml, Sigma G-7016), HRP (300 mU / ml, Sigma P8375) and Amplex Red (75 PM, Molecular Probe A-12222) . The reagent cocktail was used without delay. An assay reagent cocktail (75 μl) was dispensed into the assay wells (Greiner BIO-One GmbH 96-well plate, cat. No. GRE 655096) and a standard sample (75 μl) was added. Samples were incubated for various time periods (15 min, 30 min and 1 h) at 32 ° C and then measured at specific time periods using a two-photon excitation microfluorimeter (ArcDia TPX Plate Reader described in Example 1) using an integration time of 10 seconds. The signal response and analysis accuracy compared between the cuvettes are presented in figure 5 and table 2, respectively.

table 2 Glucose accuracy compared between cuvettes Glucose (μM) 15 minutes Incubation time 30 min 1 hour one four% four% 2% 3 3% 3% 3% 10 2% one% one% thirty one% one% 0.02% one hundred one%

Example 5

Amylase Analysis

For analysis of amylase, the α-1,4-oligosaccharide was subjected to fluorophore and quenching agent labeling with respect to the reducing end and non-reducing end, respectively. The resulting conjugate showed a decrease in fluorescence emission compared with the emission of free fluorophore due to the proximity of quenching by a quenching agent fragment. The conjugate functions as a substrate for the amylase enzyme and, thus, can be used to determine the activity of amylase in clinical samples. In the presence of amylase, the oligosaccharide conjugate is cleaved. Thus, the fluorophore and the quenching agent are split into parts, which leads to less efficient quenching and an increase in detectable fluorescence emission.

Example 6

Alkaline phosphatase assay

Analysis of alkaline phosphatase (ALP, EC 3.1.3.1) can be carried out using a fluorogenic substrate, hydrolysis of which by alkaline phosphatase enzyme leads to increased fluorescence. The increase in fluorescence as a function of time (= slope) is proportional to the activity of the analyzed component. In this example, analysis was performed using DDAO phosphate (Molecular Probes, D-6487) as a fluorogenic substrate. The analysis was performed as follows: an ALP stock solution was prepared by dissolving 0.54 mg of lyophilized alkaline phosphatase (P-5931, Sigma, 42 U / mg) in 0.54 ml of water. The ALP stock solution was further diluted with a 1.0 mM MgCl ₂ solution to give standard ALP solutions with various concentrations. An DDAO phosphate stock solution was prepared by dissolving 1.11 mg of DDAO phosphate in 0.525 ml of N, N-dimethylformamide. An assay reagent was prepared by diluting 66 μl of the DDAO phosphate stock solution with 7.36 ml of glycine buffer (100 mM glycine, 1.0 M MgCl ₂ , 1.0 mM ZnCl ₂ , pH 10.4). The assay reagent (75 μl) was dispensed into wells (96-well Greiner BIO-One GmbH cat. No. GRE-655096 plate), followed by the addition of a standard ALP sample (7.5 μl). The reaction mixture was incubated at 32 ° C and fluorescence was measured kinetically using a microfluorimeter with: two-photon excitation (ArcDia TPX Plate Reader) at intervals of 5 minutes with an integration time of 15 seconds.

Based on the kinetic data, the increase in fluorescence intensity as a function of time (= slope) for each standard sample was calculated, and the results are presented in Fig.6.

Example 7

Creatinine analysis

This method of creatinine analysis is based on the use of various enzymes and a two-step analysis protocol. In the first stage, creatine is metabolized through three sequential enzyme-catalyzed reactions (creatinase, sarcosine oxidase, catalase). The removal of creatine is followed by a second step, where creatinine is analyzed by four sequential enzyme-catalyzed reactions (creatininase, creatinase, sarcosine oxidase and horseradish peroxidase). The cascade of reactions leads to the formation of a fluorescent end product "resorufin".

A standard creatinine solution (10 microliters, in a buffer containing 10 mM NaH ₂ PO ₄ , 150 mM NaCl, titrated to pH 8) was dispensed into wells (384-well plate from Greiner BIO-ONE, cat. No. GRE 7810966) and further 5 μl of reagent 1 was added for analysis (containing sarcosine oxidase 80 kE / L, creatinase 140 kE / L, horseradish peroxidase 20 kE / L, catalase solution 200 kE / L). The reaction mixture was incubated at room temperature in the dark for 45 minutes. Then 5 μl of reagent 2 for analysis (containing creatininase 60 kE / L, Amplex Red 300 μM) was dispensed into the wells. The reaction mixture was incubated for 15 min and fluorescence was measured using an ArcDia TPX Plate Reader (Koskinen JO et al., 2004, Anal. Biochem. 328, 210-218) using a solution measurement mode and integration time of 10 sec. The results are presented in Fig.7.

Example 8

Creatine Kinase Assay

Creatine kinase catalyzes the reaction between creatine phosphate and ADP (ADP) to form creatine and ATP (ATP). The method of analysis of creatine kinase is based on the determination of hydrogen peroxide after the conversion of creatine with the addition of creatinase and sarcosine oxidase. The sequence of reactions leads to the release of hydrogen peroxide, which is measured by the standard method using fluorogenic substrate "Ampiex Red" and horseradish peroxidase as a catalyst.

Example 9

Alanylaminotransferase Assay (ALAT)

The method of analysis of alanine aminotransferase is based on the determination of pyruvate after the conversion of L-alanine and 2-oxoglutarate to pyruvate and L-glutamate. Further, pyruvate is reacted with phosphate using a pyruvate oxidase catalyst to form acetyl phosphate, hydrogen peroxide and carbon dioxide. Finally, hydrogen peroxide is measured by a standard method using an Amplex Red fluorogenic substrate and horseradish peroxidase as a catalyst.

Example 10

Aspartate aminotransferase assay (ASAT)

Aspartate aminotransferase catalyzes the conversion of L-aspartate and 2-oxoglutarate to oxaloacetate and L-glutamate. Further, oxaloacetate is converted to pyruvate by the addition of oxaloacetate decarboxylate. Further, pyruvate interacts with phosphate, the reaction catalyzes pyruvate oxidase to form acetyl phosphate, hydrogen peroxide and carbon dioxide. At the end of the reaction, hydrogen peroxide was measured by a standard method using an Amplex Red fluorogenic substrate and horseradish peroxidase as a catalyst.

Example 11

Calcium Analysis

Calcium was quantified using specific calcium chelating agents in a manner that is characterized by an increase in fluorescence efficiency or a change in the emission wavelength when complexed with calcium ions.

Example 12

Total cholesterol analysis

Initially, the cholesterol esters of the sample are converted to cholesterol and fatty acids with the addition of cholesterol esterase. Then, cholesterol is converted to cholest-4-en-3-one by a reaction catalyzed by cholesterol oxidase. This conversion is accompanied by the release of hydrogen peroxide, which is determined quantitatively by the standard method, using the Amplex Red fluorogenic reagent as a substrate, horseradish peroxidase as a catalyst, and detection using a two-photon excitation fluorometer.

Example 13

HDL Cholesterol Analysis

Fractions of lipoprotein other than HDL are blocked by the addition of blocking reagents (α-cyclodextrin and Mg ²⁺ ). The HDL cholesterol fraction is converted to cholesterol-4-en-3-one by adding the cholesterol oxidase enzyme with increased specificity to HDL cholesterol. This conversion is accompanied by the release of hydrogen peroxide, which is quantified by the standard method using Amplex Red fluorogenic reagent as a substrate, horseradish peroxidase as a catalyst, and detection is carried out using a two-photon excitation fluorometer.

Example 14

Triglyceride Analysis

Triglycerides are hydrolyzed by the addition of lipase to form fatty acids and glycerol. Then, glycerol is phosphorylated by treating ATP and glycerol kinase to form glycerol-3-phosphate and ADP. Finally, glycerol-3-phosphate is oxidized via O ₂ with the addition of glycerol phosphate oxidase to form dihydroxyacetone phosphate and hydrogen peroxide. Hydrogen peroxide is quantitatively determined by the standard method using Amplex Red fluorogenic reagent as a substrate, horseradish peroxidase as a catalyst, and detection using a two-photon excitation fluorometer.

Claims (17)

1. In vitro diagnostic method for the quantitative determination of the component contained in a clinical sample, used in the field of clinical chemistry, where the analyzed component
a) undergoes a chemical reaction or reactions with a reagent or reagents in one or more stages, or in a sequence of reactions, or
b) catalyzes a chemical reaction or reactions, or a reaction in a sequence of reactions of a reagent or reagents in one or more stages
in the reaction system, wherein said reaction or reactions, or reaction sequence, changes the measured property of a compound or compounds of said reaction or reactions or reaction sequence, characterized in that
i) the specified chemical reaction or reactions, or a sequence of reactions leads to
the formation of a compound with two-photon-excited fluorescence, or
a change in the properties of two-photon-excited fluorescence of a reaction system containing at least one two-photon fluorescence compound; and
ii) said analyte component is quantified by excitation of said compound or two-photon fluorescence compounds and measurement of two-photon-excited fluorescence and correlation of fluorescence measurement data with a standardization method based on measurements obtained from a reference material of said analyte component. 2. The method according to claim 1, characterized in that it includes stages
a) contacting the clinical sample containing the component to be analyzed with a specific reagent or reagents for analysis;
b) the implementation in the reaction system of a chemical reaction of the analyzed component with the specified reagent or reagents, or the implementation of the chemical reaction or reactions of the specified reagent or reagents catalyzed by the analyzed component;
c) possibly repeating steps a) and b) one or more times;
d) said reaction or reactions of step or steps b) leads to the formation of a compound exhibiting two-photon fluorescence, or leads to a change in the characteristics of two-photon fluorescence of said reaction system comprising at least one compound exhibiting two-photon fluorescence; and
e) quantifying said analyte component by exciting said compound or compounds exhibiting two-photon fluorescence, measuring two-photon-excited fluorescence and correlating said measured fluorescence values with a standardization method based on measurements obtained from a reference material of said analyte component. 3. The method according to claim 1 or 2, characterized in that the fluorescence resulting from two-photon excitation is measured kinetically. 4. The method according to claim 1 or 2, characterized in that the fluorescence resulting from two-photon excitation is measured as a signal at the end point. 5. The method according to claim 2, characterized in that the quantitative determination of the analyzed component contained in the clinical sample is carried out for several samples by repeating steps a) to e) according to claim 2 for each sample. 6. The method according to claim 2, characterized in that several of the analyzed components contained in the clinical sample are quantified by repeating steps a) to e) according to claim 2 for each analyzed component. 7. The method according to claim 1 or 2, characterized in that the component or components contained in the clinical sample is selected from the group consisting of albumin, total protein, hemoglobin, ammonia, carbonate, direct bilirubin, total bilirubin, calcium, chloride, iron, magnesium, phosphate, HDL cholesterol, LDL cholesterol, total cholesterol, creatinine, fructosamine, glucose, lactate, triglycerides, urea, uric acid, acid phosphatase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, pancreatic amyl cholinesterase, creatine kinase, glutamyl transferase, glutamate dehydrogenase, hydroxybutyrate dehydrogenase, lactate dehydrogenase and lipase. 8. The use of a fluorometric device operating on the principle of fluorescence during two-photon excitation for in vitro diagnostic quantification of the analyzed component or components contained in a clinical sample or samples, where the specified quantitative determination of one or more of these analyzed components includes one or more chemical reactions, leading to the formation of at least one two-photon fluorescence compound, or to a change in the properties of two-photon fluorescence fluorescence of the reaction system comprising at least one compound with two-photon fluorescence. 9. The application of claim 8, characterized in that the device is a pulsed laser for two-photon-excited fluorescence
a) with laser pulses shorter than 10 ns,
b) with a pulse repetition rate above 10 kHz,
c) with TEM 00 type of output of the polarized beam, and
d) with an average beam power in the sample of from 20 to 200 mW, preferably from 85 to 120 mW, and most preferably about 100 mW. 10. The use of claim 8 or 9, characterized in that the analyzed component or components contained in the clinical sample are selected from the group consisting of albumin, total protein, hemoglobin, ammonia, carbonate, direct bilirubin, total bilirubin, calcium, chloride , iron, magnesium, phosphate, HDL cholesterol, LDL cholesterol, total cholesterol, creatinine, fructosamine, glucose, lactate, triglycerides, urea, uric acid, acid phosphatase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, pancreatic ase total amylase, cholinesterase, creatine kinase, glutamyl transferase, glutamate dehydrogenase, hydroxybutyrate dehydrogenase, lactate dehydrogenase and lipase. 11. The use of claim 10, characterized in that the same fluorometric device is sequentially used to quantify at least 2, preferably 5, more preferably 10, even more preferably 20 and most preferably all components of the group analyzed in a clinical sample . 12. The use of claim 8 or 9, characterized in that the same fluorometric device is also used for the quantitative determination of components analyzed by the bio-affinity method. 13. System for in vitro diagnostic quantification of at least one analyte component contained in a clinical sample or samples, characterized in that the system includes
a) a fluorometric device operating on the principle of fluorescence upon two-photon excitation, for quantifying one or more components analyzed in a clinical sample, a control unit including a memory unit and a measuring device, and
b) a data processing unit with a program for specialized data processing for the specified quantitative determination of the specified analyzed component or components using the specified fluorometric device, where the specified quantitative determination of one or more of these analyzed components includes one or more chemical reactions leading to at least at least one compound with two-photon fluorescence, or to a change in the properties of two-photon fluorescence of the reaction system, won at least one compound with two-photon fluorescence. 14. The system according to item 13, characterized in that it includes shops for cuvettes intended for analysis, containing test tubes for samples and cuvettes. 15. The system according to item 13 or 14, characterized in that it includes a dispensing device for diluting the sample and for distributing it into cuvettes for analysis. 16. The system according to item 13 or 14, characterized in that it includes mechanisms for moving the cuvette for analysis and / or distribution head. 17. The system according to item 13 or 14, characterized in that it includes a control unit for automatic control of the system.

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