DE102008013997B4 - Method for the quantitative detection of analytes in liquid medium - Google Patents

Abstract

Method for the quantitative detection of an analyte in a liquid medium by adding probes which consist of detection molecules which are capable of binding to the analyte and the permanent magnetic or magnetizable nanoparticles which mark the detection molecules, the detection being carried out by means of a measurement method, the magnetic measurement of which is the mean of the volume-weighted size distribution of the aggregates and / or the scattering of the size distribution of the aggregates, with the following steps: a) providing a liquid sample which contains the analyte, b) adding the probe in at least one concentration series, wherein either the relative concentration of the analyte or the concentration of the probe is varied , c) Determination of the mean of the volume-weighted size distribution of the aggregates and / or the scatter of the size distribution of the aggregates as a measure of the agglutination for the concentration series produced under b) by d) determination of the extreme value of the magnetic measurand plotted over the concentration ratios of the concentration series and e) calculation of the analyte concentration from the position of the extreme value of the magnetic measurand by comparison with ...

Description

The invention relates to a method for the quantitative detection of analytes in a liquid medium, in which the analyte is detected by means of probes which consist of detection molecules specific for the analyte and marking permanent magnetic or magnetizable nanoparticles bound to the detection molecules.

The basic mode of action of such a detection consists in the specific binding between the molecule to be quantified (the analyte) and a so-called detection molecule (also detection reagent or detection molecule or detection reagent). Via the coupled marking particles, the reaction products between analyte and detection molecule should then be recognized and quantified.

Such detection methods are inter alia for the detection of biomolecules, such. As proteins, peptides, DNA segments or antigens, whole or parts of viruses or bacteria or other foreign macromolecules, very common. The fast and reliable detection of biomolecules is one of the major challenges in modern medicine. In addition to the detection of disease indicators (bacteria, certain recognition proteins) is another field of application for such detection methods, as the genus described above, the so-called "therapeutic drug monitoring" (TDM), in the blood drugs or the concentration of hormones or Enzyme can be detected.

As detection molecules for detection in medical assays serve z. As antibodies (in immunoassays) or receptors (in receptor assays). The detection molecules are thereby added to the analyte substance. In order to detect the detection molecules, they are previously labeled and thus molecular probes are produced. The marking takes place in the prior art z. B. with a fluorescent molecule, a radioactive atom or an enzyme which, for example, after addition of a substrate causes a color reaction (enzyme-linked immunosorbent assay (ELISA)).

The principle of immunoagglutination and immunoprecipitation is described in Lottspeich, F .; Zorbas, H. (ed.): Bioanalytics. Heidelberg: Academic Publisher GmbH, 1998, pp. 75-79, chapter 5.3. It discusses homogeneous and non-homogeneous test procedures using probes from labeled detection molecules. The marker for the detection molecule can either lead to precipitation or be detected in a homogeneous sample, for example spectroscopically. The detection molecule linked to the marker is an antibody. The characteristic behavior of such systems with excess of the probe or probe undershot, the so-called prozone effect, can be seen in the "Heidelberger Kurve". For a quantitative evaluation on this basis, it is essential that the equivalence of epi- and paratop is correctly indicated by the measurand.

Depending on the variety of applications, a wide range of immunoassay procedures (including receptor assays) are available. A distinction is made between so-called heterogeneous immunoassays, in which the analyte-probe complexes formed are separated from the unbound probes, z. B. by immobilization on slides or centrifugation and subsequent washing and rinsing steps, of the so-called homogeneous assays in which the signal of the probe is so modified by the coupling (modulation) that then the signals of the bound and the unbound probes are distinguished can. The separation step can be omitted in homogeneous assays. As a result, homogeneous assays are faster to carry out, require less labor and human resources and are sometimes more precise.

In addition to the complex and safety-related radioimmunoassays, most immunoassay variants use optical signal transmitters (fluorescence probes, ie fluorescence assays, [LISA]). If these fluorescence assays are designed as homogeneous assays in original media, background signal effects can be produced in a variety of ways. So z. For example, in serum the fluorescent light can be significantly attenuated by binding the probes to the ubiquitous albumin. In contrast, no special requirements are attached to the optical properties of the medium in the case of magnetic detection methods, and the magnetic field used in the detection method passes virtually uninfluenced by all magnetically or slightly magnetizable media. Thus, measurements in whole blood or other body fluids can be carried out without further treatment. In particular, these detection methods can also be used in vivo.

Magnetic Relaxation Immunoassay (MARIA) uses magnetic nanoparticles (MNPs) as markers for the probes. From the DE 195 03 664 C2 is a method for magnetorelaxometric quantitative detection of analytes in liquid and solid phases is known in which the relaxation of the magnetic moment of the sample after switching off an external magnetic field is measured. This is an integral measurement method, ie the measurement signal is the sum of the contributions of all MNPs. The method is based on the targeted exploitation of significantly different relaxation times for discrimination between bound and unbound superparamagnetic, ferrimagnetic or ferromagnetic colloidal particles, which is why the construction of a homogeneous assay with this method is possible. Absolute values are measured, ie the magnetic flux in the sample is related to the concentration of the analyte.

From the DE 195 08 772 A1 a method for the quantitative detection of analytes in liquid and solid phases is known in which ferrimagnetic or ferromagnetic colloidal particles of suitable magnetic coercive field strength and suitable magnetic moment labeled analytes used in an immobilized sample to be measured and the concentration of the analyte by the extent of the specific binding to the labeled detection particle dependent magnetic remanence is determined.

Enpuku, US Pat. No. 6,770,489 B3 describes the construction of an immunoassay or detection method for an analyte in which the detection reagent is labeled with magnetic nanoparticles and the quasi-static magnetic remanence is measured.

In the magnetic detection methods described above, two different measuring sequences have hitherto been implemented: (1) Captive antibodies were immobilized on a solid phase, to which antigens could then be coupled, after which the sample was incubated with the probes. (2) The analyte is in solution or suspension and is cross-linked by the addition of another binding agent. The resulting aggregates are then z. B. centrifugation and then detected with the probes. Using calibration curves, the analyte concentrations of unknown samples can now be determined. To determine the fraction of bound probes, a reference sample (100% immobilized probes) should be measured with the same conditions as for the measurement of the sample (eg equal distance between sample and detector).

The previous methods therefore require considerable equipment and personnel costs for sample preparation and measurement.

The invention has for its object to find a method in which the disadvantages are avoided in the prior art and the analyte can be measured directly in solution or suspension without Separations- or Immobilisierungsschritte and quantified with high reliability.

The object is achieved by a method according to claim 1.

The invention is based on the finding that the mean value of the volume-weighted size distribution of the aggregates and / or the scattering of the size distribution of the aggregates has an extreme value for a specific ratio of probes to analyte molecules. These maximum values are clearly pronounced and therefore analytically well evaluable. In addition, there is also a maximum in the number of probes organized in the aggregates and a minimum in the number of free probes. This applies at least to multivalent analytes and probes.

For the method according to the invention, it is necessary to use a measured variable which strictly correlates with, for example, the size distribution of the aggregates (see above). This has been verified so far for various magnetic measurement methods. In concrete terms, the magnetic measuring method is always suitable for the purposes of the invention if the measured variable depends on the Brownian relaxation in the carrier liquid, ie here in the medium or the matrix of the sample, since this corresponds to the volume (the hydrodynamic volume) of the measuring complex. d. H. the complex of analyte (s) and probe (s) is linked.

Fundamentally suitable as a magnetic measuring method in the context of this invention is therefore the measurement of the magnetic relaxation (as immunoassay MARIA) and the measurement of the magnetic susceptibility. Other measuring methods are suitable, provided that they give the detection curve described in more detail below, which the person skilled in the art can find out in the context of preliminary tests.

The US Pat. No. 6,825,655 B2 (Minchole) already describes a measuring method for the quantitative determination of an analyte in solution by means of magnetic susceptibility. The excitation here is an alternating magnetic field, which leads to a time-delayed and amplitude-modulated fluctuation of the magnetic moment of the sample. This behavior also depends on the bonding state of the magnetic nanoparticles. However, Minchole explicitly avoids agglomeration in order to arrive at safer results in the chosen evaluation method.

• (a) jeweils die Relaxation des magnetischen Momentes der Probe nach Abschalten eines externen Magnetfeldes,
• (b) jeweils die komplexe magnetische Suszeptibilität, insbesondere unter Variation der Frequenz und Bestimmung der frequenzabhängigen komplexen magnetischen Suszeptibilität,

gemessen.In preferred embodiments:

• (a) respectively the relaxation of the magnetic moment of the sample after switching off an external magnetic field,
• (b) in each case the complex magnetic susceptibility, in particular with variation of the frequency and determination of the frequency-dependent complex magnetic susceptibility,

measured.

The detection curve or measurement curve of the method according to the invention is the dependence of the selected measured variable, that is to say for example the magnetic relaxation, on the ratio of the analyte / probe concentration. This measurement curve must have a clearly evaluable extreme value. Otherwise, adjust the procedure by varying the probes or the probe concentration until the trace is the characteristic shape as shown to remove has. Preferably, therefore, the extreme value whose location is used to determine the concentration of the analyte is a highly developed extreme within a bell-like curve.

In this case, the agglutination of analyte and probe correlates with the selected measured variable in such a way that the mean value of the volume-weighted size distribution of the aggregates and / or the scattering of the size distribution of the aggregates has an extreme value for a certain number ratio of probes to analyte molecules. If this can be proven, a reliable quantification of the analyte with the new detection method can be assumed.

In a very preferred embodiment, the concentration ratio found is used to determine the absolute concentration of the analyte solely from the extreme value determining concentration ratio of analyte and probe via a calibration curve.

The calibration curve can be found by measurements on reference material with known analyte and probe concentrations or by model assumptions based on the number of binding sites of probes and analytes and size assumptions on probes and analytes.

Preferably, the calibration curve is recorded by measurements on reference material, and more preferably by at least one concentration series with variation of the analyte or the probe concentration.

In a preferred embodiment, the concentration series is prepared by a dilution series of the analyte and the concentration of the probes is kept constant within the series. Equivalently, the analyte could be concentrated, which would be technically far more complicated than the dilution series.

Alternatively, the concentration series can be made by varying the probe concentration and the concentration of the analyte within the series is kept the same. These possibilities apply both to the recording of the calibration curves and to the concentration series for the quantitative analytical measurements.

In both cases, it may be advantageous to record several concentration series, in particular with changed individual concentration values, in order to increase the accuracy or to reduce the statistical dispersion.

In contrast to the methods described in the prior art, the new method quantifies aggregates which are formed in a single reaction step by the agglutination of the analyte with the probes. Quantification here means that a parameter correlating with the size distribution of the aggregates, above the proportion of aggregated probes and, in turn, the concentration of the analyte is determined. This is done by measuring several samples with different concentration ratio between analyte and probes, as described above, and applying a suitable mathematical model to the measurement curve, e.g. B. a magnetic relaxation curve (eg., MARIA measurement) is adjusted. The dependence of the parameters describing the aggregates on the ratio of the concentrations of probes to analyte is the concentration determination curve. The key point of the new method is that the concentration determination curve has a strongly developed maximum with a certain number ratio of probes to analyte molecules and that this is associated with an agglutination maximum at a certain analyte-probe ratio. From the position of the maximum of the concentration determination curve, the analyte concentration can be inferred conversely with known probe concentration.

In the simplest case, the determination of the extreme value and the exact position of the extreme value with respect to the analyte-to-probe ratio (exact abscissa value) is done by interpolation of the measurement points / measurements, alternatively by another mathematical curve analysis of the measurement curve, by mathematical curve fitting the exact determination of the extreme value takes place.

The analyte concentration is preferably calculated by comparing the extreme value position of the sample measurement with the extreme value positions of at least one calibration curve. It is generally less advantageous to calculate the absolute value of the maximum, i. H. the maximum value of the measurand, for analysis.

In a preferred embodiment, the analyte is a biomolecule, in particular an antigen or an antibody. Such analytes are often multivalent, so that those desired for the invention Evidence curve can be easily obtained. In principle, however, the method can be carried out for any analyte which can be detected quantitatively with a detection molecule connected to a labeling nanoparticle.

The detection molecules for the preferred analytes "biomolecules" are preferably antibodies, specific antibody fragments, antigens, specific antigen fragments, receptor-binding agonists or antagonists, generally specific peptides, proteins, nucleotides, DNAs or RNAs. The liquid medium in which the analyte is located, d. H. the sample matrix is preferably an aqueous medium, especially for samples of biomolecules.

The nanoparticles which label the detection molecules are preferably monodisperse, ie. H. as uniform as possible (measured by mean volume or mean diameter). A certain size distribution can be accepted. The size of the particles is preferably between 1 and 400 nm (average diameter), more preferably between 1 and 200 nm. For certain measuring methods, certain particle sizes are advantageous, which the person skilled in the art can select accordingly.

The magnetic or magnetizable nanoparticles are preferably of superparamagnetic, ferromagnetic or ferrimagnetic substances, preferably of iron oxide, barium ferrite or strontium ferrite or cobalt ferrite.

The nanoparticles can be surface-treated or carry a surface coating.

The preparation of the nanoparticles as such and the probes of nanoparticles and detection molecules is known in the art and therefore need not be carried out separately here. It will include the above mentioned DE 195 03 664 C2 directed. Both nanoparticles for labeling as well as complexes of nanoparticles and various detection molecules are commercially available.

• 1. Es ist vergleichsweise einfach und schnell durchzuführen und dabei robust, d. h. wegen der erfindungsgemäßen Kurvenanpassung wenig empfindlich auf Schwankungen und Inhomogenitäten bei Einzelmessungen; das Verfahren ist daher unanfällig gegenüber Messartefakten und zumindest bei den magnetischen Messverfahren ist das intrinsische Untergrundsignal nur sehr gering und in der Regel vernachlässigbar;
• 2. In die Nachweisreaktion sind nur die Analyten und die Sonden involviert. Es sind weder eine vorherige Immobilisierung der Analyten noch andere Manipulationen der Probe notwendig;
• 3. Es ist nur eine Bindungsreaktion nötig; bei Auswertung rein magnetischer Signale ist die Probenmatrix i. a. gut transparent;
• 4. Die Auswertung von Absolutwerten der Messgröße ist nicht erforderlich, auf die Amplitude der Signale kommt es daher nicht an; aufgrund der starken Korrelation zwischen Aggregatgröße und Messgröße (z. B. magnetischer Relaxationszeit) kann auf eine Kalibrierung der Amplitude verzichtet werden. Das ist insbesondere für einen Einsatz der genannten magnetischen Messverfahren wichtig, wo die Sonden nur grob lokalisiert werden können. Eine Quantifizierung über die Signalamplituden würde zu stark erhöhten Analyseunsicherheiten führen, da das Signal der magnetischen Marker in der dritten Potenz vom Abstand Probemolekül/Detektor abhängt.

The method has numerous advantages over known analysis methods:

• 1. It is comparatively simple and quick to perform while robust, ie because of the curve adjustment according to the invention little sensitive to fluctuations and inhomogeneities in individual measurements; the method is therefore not susceptible to measurement artifacts and at least in the magnetic measurement method, the intrinsic background signal is only very small and usually negligible;
• 2. Only the analytes and the probes are involved in the detection reaction. Neither prior immobilization of the analytes nor other manipulations of the sample are necessary;
• 3. Only one binding reaction is necessary; when evaluating purely magnetic signals, the sample matrix is generally well transparent;
• 4. The evaluation of absolute values of the measured variable is not required, so the amplitude of the signals is not important; Due to the strong correlation between aggregate size and measured variable (eg magnetic relaxation time), calibration of the amplitude can be dispensed with. This is particularly important for use of the mentioned magnetic measuring methods, where the probes can only be roughly localized. A quantification via the signal amplitudes would lead to greatly increased analysis uncertainties, since the signal of the magnetic markers in the cube depends on the distance between the sample molecule / detector.

Provided the probes are reproducible, stable and have no non-specific binding, the location of the aggregation maximum should be determined only by the concentration of the analyte and its binding ability (valence). Routine measurements are then possible without constant calibration measurements. The location of the maximum of the concentration determination curve of a batch of probes would need to be only once at the beginning.

General implementation of the procedure:

To determine the concentration of the analyte, a calibration measurement is first performed to obtain the exact relationship between analyte concentration and measurement. Thereafter, the sample is measured with the analyte to be quantified. The analyte concentration is calculated from the measurement signal using the calibration curve. As an example for measuring the agglutination of the nanoparticulate probes in the presence of the analyte, the magnetorelaxometry is chosen here.

1. Calibration measurement

First, a concentration series of the analyte A with the known concentration c _{A is} produced. The N samples of the analyte present in the concentrations c _{A, i} = c _A / V _i where i = 1 to N, where V _{i are} the respective dilution factors, are now compared with the nanoparticulate probes (eg, antibodies, labeled with magnetic nanoparticles) with the fixed concentration c _s incubated. At approximately equal binding valences of analyte and probe, c _s should be within the concentration range of c _{A, i} . Otherwise, the Adjust concentration values of the concentration series accordingly. The incubation time should be selected so that the binding reaction reaches the equilibrium state, which can be determined by preliminary tests if necessary. As a choice for the V _i is z. B. a geometric series such. B. V _i = 3 '. After incubation, the relaxation curves are to be measured with a suitable relaxometer, and the relaxation times τ _i or other suitable parameters describing the agglutination, which are accessible by the measuring method, such as. B. the aggregate size to determine. Now, this parameter is plotted as a function of the concentration ratio κ _i = c _s / c _{A, i} . If there is agglutination, these points, eg. B. (κ _i , τ _i ) have a local maximum or minimum. The concentration ratio κ 'at which this extremum is located is to be determined. Optionally, a mathematical function that well describes the course of the points in the area of the line maximum can now be adapted to the measured values in the area of the line maximum. Thus, by interpolating the measurement points, the value of κ 'can be calculated with higher accuracy.

2. Measurement of the analyte of unknown concentration

Here are at least two different ways to perform the measurement:

2.1. Variation of the analyte concentration

The same procedure is used for this measurement as for the calibration curve, except that instead of the analyte of known concentration, the sample to be analyzed containing analyte A of unknown concentration c _A is used. From this sample, therefore, a dilution series is prepared, whereby now only the dilution factors V _{i are} known. The samples are incubated with the probes at the fixed concentration c _s , and the parameter ( _s) describing the agglutination are measured and plotted against the quantity ω _i = c _s / V _i . If no local maximum has been established, as already mentioned above, c _s should be changed so that its value is within the range of c _A / V _i to be estimated, provided the binding valence of probes and analytes is approximately equal. The value ω ', at which the agglutination is maximum, is to be determined as described above. With this value ω ', the analyte concentration can now be calculated using the calibration value κ' by means of c _A = ω '/ κ'.

2.2. Variation of probe concentration

Of the nanoparticulate probes, a concentration series c _{s, i} = c _s / V _{i is} to be prepared, the expected analyte concentration c _A having to be in this range if, as mentioned above, the binding valences of analyte and probes are approximately equal, ie c _{s, max} <c _A <c _{s, 1} . Now, the N aliquots of the analyte are to be incubated with the probes of the concentrations c _{s, i} . The minimum incubation time should be chosen so that the binding reaction of the sample with the lowest probe concentration reaches the equilibrium state, as expected, the reaction time is the longest. Thereafter, the relaxation curves are to be measured and, in turn, the relaxation times or another suitable and accessible parameter to be determined and applied as a function of the probe concentration c _{s, i} . As described above, the probe concentration c _s ', at which the extremum of the measured values lies, may be determined by interpolation. This calculates the unknown analyte concentration to c _A = c _s '/ κ'.

EXAMPLE

A magnetic relaxation measurement of biotin is carried out as an example analyte:
Measurement method: MRX measurement
Analyte: Biotin-BSA (BBSA)
Probes: MNP probes (fluidMagSA, chemicell GmbH)
Incubation time: 18 hours

A trace of the magnetic relaxation times (in milliseconds) of a concentration series of incubated samples with different ratios of MNP concentration to BBSA concentration was recorded. For the relaxation time, for the average aggregate diameter of the volume-weighted size distribution in nanometers and for the scattering parameter of the size distribution of the aggregates, a clear maximum in the form of a single peak of curves is consistently shown. The result is in shown.

The location and shape of the aggregation maximum is also determined by the structure of the probes. When using larger nanoparticles, which accordingly have a larger number of specifically binding detection molecules, the maximum aggregation occurs at a smaller probe-to-analyte ratio ( ).

By selecting the size of the probes, it is thus possible to influence an appropriate position of the measuring curve.

In the accompanying drawings show:

: Concentration Determination Curves: Relaxation time of the MRX traces measured 18 hours after the start of the incubation of biotin-BSA (BBSA) with MNP probes (fluidMagSA, chemicell GmbH), depending on the probe-to-analyte ratio. By fitting a relaxation model to the relaxation curves, the median and scattering parameters of the aggregate size distributions were determined.

: Relaxation times for the cross-linking of structurally different MNP probes with biotin-BSA. Incubation times were 18 h (fluidMagSA) and 72 h (μMACS * SA), respectively. The fluidMagSA MNP are single core particles with a mean total diameter of about 25 to 30 nm; According to the manufacturer, the diameter of the μMACS * SA multinuclear particles is about 50 nm. In a control experiment, the analyte solutions were incubated with probes which were previously saturated with the receptor molecule of the analyte (biotin) and thus can no longer bind specifically. It turns out that the probes do not bind nonspecifically.

Claims (13)

Method for the quantitative detection of an analyte in a liquid medium by addition of probes consisting of detection molecules capable of binding with the analyte and persistent or magnetizable nanoparticles which label the detection molecules, the detection being effected by a measuring method whose magnetic parameter is the mean of the volume-weighted size distribution of the aggregates and / or the dispersion of the size distribution of the aggregates, with the following steps: a) providing a liquid sample containing the analyte, b) addition of the probe in at least one concentration series, whereby either the relative concentration of the analyte or the concentration of the probe is varied, c) Determination of the mean value of the volume-weighted size distribution of the aggregates and / or the dispersion of the size distribution of the aggregates as a measure of the agglutination for the concentration series produced under b) d) Determination of the extreme value of the applied via the concentration ratios of the concentration series magnetic quantity and e) Calculation of the analyte concentration from the position of the extreme value of the magnetic measured variable by comparison with a calibration curve. A method according to claim 1, characterized in that the relaxation of the magnetic moment of the sample is measured after switching off an external magnetic field, wherein the magnetic parameter is the re-laxationszeit. A method according to claim 1, characterized in that as the measuring method in each case at least one measurement of the complex magnetic susceptibility is carried out, in particular with variation of the frequency and determination of the frequency-dependent complex magnetic susceptibility. Method according to one of claims 1 to 3, characterized in that the measuring method and / or the probes are selected so that the extreme value of the measured variable is a strongly developed extreme value, preferably a maximum. Method according to one of claims 1 to 4, characterized in that the calibration curve was found by measurements on reference material with known analyte and probe concentrations or by model assumptions on the basis of the number of binding sites of probes and analytes and size assumptions to probes and analytes. Method according to one of claims 1 to 5, characterized in that the concentration series is produced by a dilution series of the analyte and that the concentration of the probes within the row is kept constant. Method according to one of claims 1 to 5, characterized in that the concentration series is prepared by varying the probe concentration and that the concentration of the analyte within the series is kept the same. Method according to one of claims 6 or 7, characterized in that a plurality of concentration series, in particular with altered individual concentration values, are produced. Method according to one of claims 1 to 8, characterized in that the determination of the extreme value for the measurement curve of the sample to be examined is effected by interpolation or by mathematical curve analysis of the entire measurement curve. Method according to one of claims 1 to 9, characterized in that the analyte is a biomolecule, in particular an antigen or an antibody. Method according to one of claims 1 to 10, characterized in that the detection molecules are antibodies, specific antibody fragments, antigens, specific antigen fragments, receptor binding agonists or antagonists, generally specific peptides, proteins, nucleotides, DNAs or RNAs. Method according to one of claims 1 to 11, characterized in that the detection molecules of the marking magnetic or magnetizable nanoparticles particles of superparamagnetic, ferromagnetic or ferrimagnetic Substances, preferably of iron oxide, barium ferrite, strontium ferrite or cobalt ferrite are. Method according to one of claims 1 to 12, characterized in that the liquid medium is an aqueous medium.

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