SE520150C2 - Quantitative and qualitative analysis of chemical substances - Google Patents

Abstract

The analysis can be made directly on an analyte (A), which in itself has a sufficiently large magnetic permeability, so that a measurable local variation of the material constant occurs dependent upon the formation of a complex between the analyte and the recognition component (I) and which can be detected with a coil (S). The analyte contains a ferromagnetic material (M). The analysis can also be made on an analyte which has low magnetic permeability, whereby a marker (B) with a sufficiently large magnetic permeability is allowed to bind (covalent or non-covalent) to the analyser, either before or after the binding of the analyte to the recognition component, thus inducing measurable local variations of the material constant, dependent upon the formation of a complex between the analyte coupled to the marker and the recognition component. The analyte-binding marker contains partly a ferromagnetic material and partly a binding substance (C) which is directed towards an epitope on the analyte other than that towards which the recognition component is direct.

Description

520 Analyzes can be performed on analytes (A) which have low magnetic permeability (ulel) allowing an analyte binding marker (B) with a sufficiently large magnetic permeability (ur >> l). bind (covalently or non-covalently) to the analyte, antigen before or after the binding of the analyte to the recognition component (I), and thus induce measurable local changes in the material constant (ur) due to the formation of a complex between the analyte (linked to the marker) and the recognition component.

The analyte binding marker (B) contains both a ferromagnetic material (M) and a binding substance (C) which is directed towards a different epitope on the analyte than the recognition component (I) is directed towards. See figure lb.

~ Analyzes can be performed on analytes (A), which have low magnetic permeability (ur: 1) whereby an analyte-like marker (E) with a sufficiently large magnetic permeability (ur >> l) is allowed to in a process competitive with the analyte (A) bind to the recognition component (I) and thus induce measurable local changes in the material constant (ur) due to the formation of a complex (AI or B1) between the analyte (A) or the marker (B) and the recognition component (I). The analyte-like marker (B) contains both a ferromagnetic material (M) and a substance (D) which is analyte (A) or an analyte-like substance which can compete for binding to the recognition component (I). Se ñgur le.

The magnetic permeability of the material inside and near a coil or conductor affects its inductance. This means that inductance measurements enable determinations of magnetic permeability, which in wet cases leads to the content of analyte being quantifiable. This can be done by measuring the magnetic permeability of the recognition component (possibly including the matrix to which it is immobilized) directly. A more indirect approach involves measuring the decrease in magnetic peneability in the measurement solution due to a portion of the analyte or marker adhering due to the molecular recognition on the recognition component.

The inductance of a long coil is determined by the equation: L = (uA / l) N 2 (generally inside the coil) where u denotes the magnetic permeability, l denotes the length of the coil, A denotes the cross-sectional area of the coil and N is the number of wound turns on the coil. Thus, if A, 1 and N are kept constant, and if a controlled amount of material is inside the coil, the magnetic permeability of this material can be determined.

The inductance can be measured in several different ways: * By placing the coil in an electrical measuring bridge, e.g. a Maxwell jetty.

* By measuring the resonant frequency of an LC circuit in which the coil is included.

* By applying a potential pulse and measuring the current response.

* By applying a current pulse or a non-constant current (where the current change per unit time di / dt is kept constant) and the voltage response is studied.

* By inductive connection between two coils.

* By measuring the impedance of the coil. 520 150 Q gj, _ '; ', L,, _. . .

Magnetism has been used for specific insulation on an industrial scale, e.g. the s.k. The HGMS technology (High Gradient Magnetic Separator) which is used in connection with large processes, such as water purification, processing of kaolin etc., as well as cell / protein insulation with magnetically actuable particles (eg from the manufacturer Dynal). There are also inductive sensors on the market, which are based on a coil capturing a magnetic field change. These inductive sensors react to physical properties e.g. that a fl otter in a tank is provided with a magnet or a ferromagnetic material. The inductive sensor thus reacts to the liquid level of the tank.

Previously known are a large number of patents, which include measurements of magnetic fields or magnetic perineability for determining the content of magnetic material in a sample, such as e.g. magnetite in iron ore, iron oxide in slag, the catalytically active nickel content in hydrogenation of grease, hard metal alloy control, the iron content in oils, detection of paramagnetic ions, ferromagnetic materials in cement mixtures, and magnetic oxygen analyzers. In these determinations, there is no molecular recognition of the analyte, which contributes to an often low specificity and that the analysis is severely limited to ferromagnetic materials. Mention may also be made of the following patents: Magnetisla active reagent carriers [see patent US 667414]: These are useful for the transport of antibodies, enzymes or haptens, in e.g. immunoassayer. The document only relates to the use of the carrier as a means of isolation or branching and it is suggested that this may be combined with other chemical analysis techniques. However, measurements of the magnetic permeability of the carrier particles per se are not affected at all. The same applies to another patent specification [see patent JP 62118255 A], where magnetic particles are used in such a way for chemical analysis, whereby the measurement itself takes place by means of light scattering phenomena.

Measurement of gastric field changegr has been reported previously i.a. in combination with magnetotactic bacteria for the prediction of hypoxia in water [see patent US 7873031: This patent specification deals with magnetic field measurements on magnetically enriched layers of bacteria containing magnetic particles. The amount of such bacteria in the layer (which contributes to the strength of the magnetic field) correlates to the oxygen content in the surrounding environment.

Furthermore, a work has recently been published describing biomagnetic neurosensors4. This work is also based on measurements of magnetic fields, which in this case are due to ion migrations (which correspond to an electric current that gives rise to an induced magnetic field) in a nerve. thus, it is a question of a completely different type of detection, where it is not a question of measurements of a material constant, as in our case. It should also be pointed out that magnetic field measurements are sensitive to interference from the large amount of electronic noise that exists around us. Furthermore, only substances that affect the nerve or bacteria can be detected and the response can be very difficult to interpret and complex.

To practically eczema live the n a procedure proposed by us in this P. p. ~ Y. patent application, we report below results from preliminary studies.

An example of a useful marker and its determination: To exemplify with a possible marker, we chose to use special iron oxide-dextran particles, which exhibit a large magnetic permeability. However, the particles are so small (approximately 1-300 nm) that the solution is colloidal and the dextran sheath (in combination with the particles having a low magnetic retention) contributes to no aggregation of the particles. Measurements of the magnetic permeability of solutions containing different concentrations of such particles were performed.

The solutions were placed in a measuring coil whose inductance was affected and in turn affected either the resonant frequency (see Figure 2) of an LC circuit in which the coil was included or the balancing (see Figure 3), expressed as a voltage difference (response), by a Maxwell bridge in which the coil was included. From the two figures (2 and 3) it appears that the relationship between output signal (resonant frequency change in Hz or voltage difference (response) in mV) is in a linear relationship with the content of particles in the measurement solution (expressed in the form of the concentration even). This example illustrates that it is quite possible to use the described techniques, based on measurements of magnetic permeability, to determine the level of marker / analyte, which is useful if determinations of the marker content-lanalithium decrease in the measurement solution are to be determined.

Example of a chemical analysis of a model analyte which per se has a sufficiently large magnetic permeability: In this sub-experiment we chose to use the iron oxide-dextran particles as a model analyte for analytes which per se have a sufficiently large magnetic permeability for a determination, without markers , shall be possible.

Concanavalin A was selected as a recognition component (because it binds to dextran) and immobilized on a carrier (sepharose gel / Pharmacia). After incubation in measurement solutions containing different contents of model analyte, the Maxwellbrygge principle, described earlier, was used to analyze the magnetic penile stability of the complex formed. Figure 4 shows that the voltage difference (response) (upper curve) increases with the original content of model analyte in the measurement solution (expressed in the form of the concentration of iron). When the same experiment was performed on a carrier without Concanavalin A, no voltage differences were noted (lower curve). This confirms the importance of the molecular recognition for the chemical analysis.

Example of a chemical analysis of an analyte (glucose), which has low magnetic permeability (urr-1). by a competitive procedure between the analyte and a marker: In this sub-experiment we show that it is possible to quantitatively and qualitatively measure the content of an analyte, which has low magnetic perneability (ul-el). We chose as a model to use glucose as analyte, iron oxide-dextran particles (described above) as the competitive marker and Concanavalin-A as the recognition component. Concanavalin-A was immobilized on a vehicle (sepharose gel / Pharmacia). Because glucose and the marker (via their dextran component) both have affinity for Concanavalin-A, they competed for binding to it. At low levels or in the absence of glucose, only the marker binds to the recognition component. With increasing glucose content in the measurement solution, an ever smaller proportion of the two bound substances (glucose and marker respectively) to the recognition component of the marker is constituted. Thus, it is possible in this way to determine the glucose concentration in an unknown sample. By placing the recognition component Concanavalin-A inside the coil, the amount of bound marker could be detected for different glucose concentrations. The result is depicted in Figure 5.

From this calibration curve for glucose analysis it appears that the voltage difference of the Maxwell bridge, compensated for the maximum binding of the marker, (response change) is related to the glucose content in the measurement solution (upper curve).

The control experiment without recognition component confirms that the absence of molecular recognition means that this connection is missing (lower curve). We also managed to perform measurements on glucose with the recognition component immobilized inside the coil, thus demonstrating a chemical (bio) tendon for glucose. 520 2 LITERATURE REFEREINSEilš i 150 112 * 5, ¥ '.' --r _.-'- V ¿'I 1. E. Dj urle, Electricitetslära, Teknisk Högskolelitteratur i Stockholm AB, 1983 2. D. Kriz, Towards chemical sensors, Lund 1994 3. L. Grahm, H.-G. J ubrink and A. Lauber, Elektrisk mätteknik 1 & 2, Bokförlaget teknikinforxnation, Lund and Linköping 1992. 4. Christopher W. Babb, David R. Coon, Garry A. Rechnitz, Anal. Chem., 1995, 67, 763-769. '.i = ~ “c =' l '520150 gi: a' f 'y: ax fl ig;: *' .. 25 .it 3 F IGURTEXTER Figure 1. Three different variants (a, b and c) of a measurement procedure based on the combination of molecular recognition and measurement of magnetic pezmeability.

Figure 2. Resonance frequency measurements on measurement solutions containing different contents of iron oxide-dextran particles (marker).

Figure 3. Maxwell bridge measurements on measuring solutions containing different contents of iron oxide-dextran particles (marker).

Figure 4. Maxwell bridge measurements on concanawalin-A sepharose (recognition component carrier) that have been incubated in measurement solutions containing different levels of iron oxide-dextran particles (model analyte).

Figure 5. Maxwell bridge measurements on concanaxaline-A. sepharose (recognition component carrier) which has been incubated in measurement solutions containing different levels of glucose (model analyte) and constant levels of iron oxide-dextran particles (core), and where a competitive binding has taken place.

Claims (10)

10 l5 20 25 30 35 520 150 7 PATENT REQUIREMENTS 1. l. Method for quantitative and qualitative determination of chemical substances (analytes) characterized - characterized in that the principle of operation is partly based on measurement of magnetic permeability by means of a coil containing said substance and also on the use of one or more recognition components , which are responsible for the specific molecular identification of the analyte through various types of interactions both covalent but also non-covalent such as, for example, electrostatic, hydrophobic, hydrogen bonds and van der Waals. Method according to claim 1 and also characterized in that the analyte per se has sufficient (pr >> l) change of the material constant (pr) of the complex large magnetic permeability so that a measurable between the analyte and the recognition component takes place upon binding . Method according to claim 1 and also characterized in that the analyte has low magnetic permeability (pfßl) wherein an analyte-binding marker with a sufficiently large magnetic permeability (pr >> l) is allowed to bind (covalent or non-covalent) to the analyte, either before or after the binding of the analyte to the recognition component, and thus inducible measurable changes in the material constant (pr) of the complex between the analyte (coupled to the marker) and the recognition component. Method according to claim 1 and also characterized in that the analyte has low magnetic permeability (pfwl) wherein an analyte-like marker with a sufficiently large magnetic permeability (pr >> l) is allowed to bind in a process competitive with the analyte. to the recognition component and thus induce measurable changes in the material constant (pr) of the complex between the analyte or marker and the recognition component. lO 15 20 25 30 35 520 150 8 Method according to either of Claims 1 to 4 and also characterized in that measurable changes in the material constant (s) of the measurement solution itself, after incubation with the recognition component and subsequent separation thereof, were used for the qualitative and quantitative determination of the analyte. Method according to either of Claims 3 to 5 and also characterized in that the marker substances obtain a sufficiently large magnetic permeability (from >> 1) because they contain materials with a high magnetic permeability such as transition elements and phases thereof: iron, nickel, cobalt, gadolium and manganese, as well as chemical compounds or alloys or semiconductors, the contents of which are marked markers. Furthermore, in cases where they are analyte-binding, they contain the recognition component that binds (covalently or non-covalently) to an epitope on the analyte other than the recognition component described in claims 1-5, and in cases where they are analyte-like, they contains covalently or non-covalently bound analyte or other substance which in its form and location of functional groups mimics the analyte and thus can bind to the recognition component described in claims 1-5. Method according to either of Claims 1 to 6 and also characterized in that the measurements of the magnetic permeability take place by measuring the inductance of said coils with an electrical measuring bridge (eg Maxwell) or by means of the resonant frequency of an LC circuit. in which the coil is included or by means of potential / current pulses or by methods where the potential / current derivative is constant with respect to time or with impedance measurements or measurements based on inductive coupling. Device whose operating principle is based on either of claims 1-7 and which is also characterized in that the measurement of the magnetic permeability and the recognition component are physically integrated, the device being a chemical sensor or biosensor in in those cases the recognition component has a biological origin. A device whose operating principle is based on either of claims 1-7 and which is also characterized in that the measurement of the magnetic permeability and the recognition component are physically separated or integrated in a flow system where analyte is injected, the device being a chemical flow injection analysis system (FIA). Device whose principle of operation is based on either of claims 1 to 9 and which is also characterized by the fact that it is intended for clinical diagnostics (for eg proteins, viruses, HIV, bacteria, phages, fungi, hormones, antigens, antibodies , cancer markers and other disease markers, metabolites, glucose, toxins, performance enhancing agents and drugs) in various types of body fluids (such as whole blood, plasma, serum, saliva, urine, sweat, tears, tissue extracts and faeces extracts) or for industrial analysis or for applications in agricultural technology / veterinary medicine or for military defense purposes or as part of medical devices or for environmental monitoring applications or for applications in robotics.