DE102020212466B4 - Device and procedure for rapid test - Google Patents

Abstract

Device for analyzing an aqueous sample for the presence of at least one analyte, which has a sample recess (1), a mixing device (2), a concentrating device (3), an excitation device (4) and a measuring device (5) which are arranged around a common longitudinal axis (6), wherein a sample vessel (7) can be arranged in the sample recess (1), wherein the mixing device (2) is set up to generate a first controlled, time-varying magnetic gradient field in the region of the sample recess (1), wherein the concentrating device (3) is set up to generate a second controlled, time-varying magnetic gradient field in the region of the sample recess (1), the region of greater magnetic flux density of which is arranged in the region of the measuring device (5), wherein the excitation device (4) is arranged in the region of the sample recess (1) and is set up to generate an excitation field, wherein the measuring device (5) is set up to record values for a time-varying magnetic field and to transmit them as a measurement signal to a Evaluation unit and the evaluation unit is set up to generate an analysis signal from the measurement signal, characterized in that it has an excitation coil as excitation device (4) and that it has a measuring coil as measuring device (5) which is set up to record values for the time-varying voltage induced in the measuring coil as a measurement signal and that it has an arrangement of two coils as compensation device, of which the first coil is identical in construction to the excitation coil and the second coil is identical in construction to the measuring coil and is connected anti-series to it and is arranged coaxially within the first coil, and the compensation device is arranged at a distance from the common longitudinal axis (6).

Description

The present invention relates to a device and a method that can be carried out therewith for analyzing aqueous samples for the presence of an analyte.

In the process, an aqueous sample is mixed with magnetic nanoparticles and examined for the presence of an analyte by applying a magnetic field. The method has the advantage that it allows the detection of the analyte in one step and, in particular, without washing out the magnetic nanoparticles that are not bound to the analyte.

Due to its simple procedure, the method according to the invention has the advantage, particularly when using the device according to the invention to carry it out, that it can be carried out at the location where the sample is taken ("point-of-care analysis") and in particular does not require sending to a specialized analysis laboratory. The device according to the invention has the particular advantage that it is easy to operate and therefore no laboratory training is required for its use in the method according to the invention, but only a short instruction.

Due to its inventive steps of concentrating the magnetic nanoparticles and detecting their relaxation behavior, the method has the further advantage that the analyte can be detected more quickly and the analysis signals obtained with the method have a higher signal-to-noise ratio than can be achieved with conventional methods, in particular ELISA tests or PCR tests.

State of the art

The DE 42 18 638 C2 describes a device and method for determining particle size distributions using scattered light measurements.

US 2006 / 0 292 630 A1 describes a sensor coated with capture molecules for detecting biomolecules in a magnetic field. For this purpose, recesses with Hall elements underneath are arranged in the surface of the sensor. Above and below the sensor there is a coil of an electromagnet that generates a magnetic gradient field.

US 2008 / 0 309 323 A1 describes a method for detecting biomolecules in which magnetic nanoparticles coated with capture molecules are added to a solution containing the biomolecules in order to aggregate therein, the aggregates are placed on a sensor and immobilized there by means of magnetic fields, and the biomolecules are detected by detecting the magnetic stray field.

Object of the invention

The invention has the object of providing a device and a method that can be carried out with it, with which an aqueous sample can be tested for the presence of an analyte. In particular, the invention has the object of providing an alternative device that is suitable for the point-of-care analysis of an aqueous sample for the presence of an infectious disease and for the use of which no special technical training is required for the method according to the invention.

Description

The invention solves the problem with the features of the claims and in particular provides a device which is suitable for use as a measuring device for analyzing an aqueous sample for the presence of at least one analyte and has a sample recess, a mixing device, a concentrating device, an excitation device and a measuring device, all of which are arranged around a common longitudinal axis, wherein a sample vessel can be arranged in the sample recess, wherein the mixing device is set up to generate a first controlled, time-varying magnetic gradient field in the region of the sample recess, wherein the concentrating device is set up to generate a second controlled, time-varying magnetic gradient field in the region of the sample recess, the region of greater magnetic flux density of which is arranged in the region of the measuring device,
wherein the excitation device is arranged in the region of the sample recess preferably around the common longitudinal axis and is designed to generate an excitation field, wherein the measuring device is designed to record values for a temporally variable magnetic field and to transmit them as a measurement signal to an evaluation unit,
wherein a compensation device is optionally set up to record values for the time-varying magnetic field and to pass them on to the evaluation unit as a compensation signal and
the evaluation unit is set up to generate an analysis signal from the measurement signal and optionally the compensation signal.

1. a. Befüllen eines Probengefäßes, das für den Analyten spezifische magnetische Nanopartikel und einen Messbereich aufweist, mit der wässrigen Probe, und Versiegeln des Probengefäßes,
2. b. Mischen der wässrigen Probe durch Anlegen eines ersten gesteuerten magnetischen Gradientenfeldes im Bereich des Probengefäßes, um eine Probenmischung herzustellen,
3. c. Aufkonzentrieren der magnetischen Nanopartikel aus der Probenmischung im Messbereich des Probengefäßes durch Anlegen eines zweiten gesteuerten zeitlich veränderlichen magnetischen Gradientenfeldes im Bereich des Probengefäßes,
4. d. Detektieren des Analyten durch Anlegen eines magnetischen Anregungsfeldes im Messbereich des Probengefäßes, um die magnetischen Nanopartikel zu magnetisieren, Aufnehmen der Werte des zeitlich veränderlichen Magnetfeldes der magnetisierten magnetischen Nanopartikel als Messsignal und optional Aufnehmen der Werte für die induzierte elektrische Spannung des Anregungsfeldes als Kompensationssignal,
5. e. Erzeugen zumindest eines Analysesignals aus dem Messsignal und optional dem Kompensationssignal, Vergleichen des Analysesignals der wässrigen Probe mit einem Referenzsignal und bei einem Analysesignal, das vom Referenzsignal abweicht, Anzeigen des Vorhandenseins des Analyten in der wässrigen Probe,

umfasst.Furthermore, the invention relates to a method for analyzing an aqueous sample for the presence densein at least one analyte, which can be carried out in particular using the device according to the invention and comprises the steps

1. a. Filling a sample vessel containing magnetic nanoparticles specific for the analyte and a measuring range with the aqueous sample and sealing the sample vessel,
2. b. Mixing the aqueous sample by applying a first controlled magnetic gradient field in the region of the sample vessel to produce a sample mixture,
3. c. Concentrating the magnetic nanoparticles from the sample mixture in the measuring area of the sample vessel by applying a second controlled, time-varying magnetic gradient field in the area of the sample vessel,
4. d. Detecting the analyte by applying a magnetic excitation field in the measuring area of the sample vessel in order to magnetize the magnetic nanoparticles, recording the values of the time-varying magnetic field of the magnetized magnetic nanoparticles as a measurement signal and optionally recording the values for the induced electrical voltage of the excitation field as a compensation signal,
5. e. generating at least one analysis signal from the measurement signal and optionally the compensation signal, comparing the analysis signal of the aqueous sample with a reference signal and, in the case of an analysis signal that deviates from the reference signal, indicating the presence of the analyte in the aqueous sample,

includes.

The aqueous sample is filled into the sample vessel in step a. of the method. According to the invention, the sample vessel is sealed after being filled with the aqueous sample, so that the further process steps take place without opening the sample vessel and in particular without removing parts of the aqueous sample from the sample vessel. The method therefore has the advantage that the analysis for the presence of analyte is not influenced by decanting or washing the sample, in particular washing out the magnetic nanoparticles not bound to the analyte. The method has the further advantage that an infectious or toxic aqueous sample cannot escape from the sample vessel and the analysis therefore does not have to be carried out in a safety laboratory, but can be carried out, for example, at the location where the sample was taken (point-of-care analysis).

The aqueous sample may already contain the magnetic nanoparticles before step a. of the method. In particular, the aqueous sample may be a mouthwash solution containing magnetic nanoparticles, which may be filled into the sample vessel after rinsing the mouth. Alternatively, the aqueous sample may be mixed with the magnetic nanoparticles before step a. of the method and then filled into the sample vessel.

Optionally, the aqueous sample is derived from a human and is, for example, saliva, a tissue swab, a mouthwash, urine, CSF or blood. In this embodiment, the aqueous sample is preferably prepared by mixing a sample derived from a human with a predetermined amount of a buffer solution, wherein the buffer solution may comprise DNase inhibitor, RNase inhibitor and/or protease inhibitor. In this embodiment, the analyte is preferably a biomolecule whose presence indicates or excludes infection with a disease, e.g. an antibody against an infectious agent or a surface protein of a virus.

Alternatively, the aqueous sample can be an environmental sample, e.g. a sample from a body of water or a soil sample suspended in water.

The analyte is detected in the method according to the invention by specific binding of the analyte to magnetic nanoparticles. Magnetic nanoparticles are generally made of a ferrimagnetic, ferromagnetic or paramagnetic material such as iron oxide and can be influenced by a magnetic field, eg moved in one direction along the magnetic field. The magnetic nanoparticles are preferably spherical and have a diameter of at least 1 nm up to 1 µm, measured e.g. by dynamic light scattering, wherein preferably a suspension of the magnetic nanoparticles is illuminated by means of coherent light and a Fraunhofer diffraction pattern is imaged onto a light-sensitive multi-element detector of a recording device and converted into analog electrical signals and a computer calculates the particle size distribution from the digitized analog signals via the solution of a Fredholm integral equation, in particular in a particle size meter, e.g. available as Nanophox from Sympatec GmbH (Clausthal-Zellerfeld, Germany), wherein preferably the diameter of at least 80% of the magnetic nanoparticles deviates from an average selected diameter by at most 20%. Preferably the magnetic nanoparticles are selected according to the size of the analyte to be detected. In particular, larger nanoparticles can be selected for the detection of a larger analyte, e.g. a virus, and for the detection of a smaller analyte, e.g. a protein, smaller nanoparticles.

According to the invention, the magnetic nanoparticles are specific for the analyte, i.e. the magnetic nanoparticles are designed to specifically bind the analyte. The magnetic nanoparticles are preferably specific for the analyte in that they have a binder that can specifically bind to the analyte. In particular, the magnetic nanoparticles can be covalently bound to the binder or coated with the binder.

The binder may comprise or consist of amino acids and/or nucleotides, and may in particular be a binding protein, e.g. an antibody or antibody fragment or lectin or darpin, or may be an antigen, e.g. a surface protein or protein fragment of a pathogen, or may be an oligonucleotide which preferably binds specifically to a genetic sequence of DNA and/or RNA.

Preferably, the analyte is a biomolecule. In particular, the analyte preferably comprises or consists of nucleotides, at least one fatty acid, sugars, amino acids or a combination of at least two of these.

In the embodiment in which the binder is an antibody, the analyte is preferably an antigen, e.g. a surface protein or protein fragment of a viral or bacterial pathogen or a surface protein of an endogenous cell, and/or has a surface structure specifically bound by the binder. In this embodiment, the analyte can alternatively be a low molecular weight molecule, e.g. a hormone or a biomarker.

In the embodiment in which the binder is an antigen, the analyte is preferably an endogenous biomolecule that specifically binds to the binder, e.g. a B cell receptor, a T cell receptor or an antibody.

In the embodiment in which the binder is an oligonucleotide, the analyte preferably has a nucleotide sequence that is at least partially complementary to the sequence of the binder and is more preferably the genetic material of a pathogen, e.g. a virus, or the body's own genetic material.

The device has at least one sample recess in which a sample vessel can be arranged, preferably fixed therein. The sample recess is preferably designed as a depression with a base and an opening. The longitudinal axis of the sample recess spanned between the base and the opening is the common longitudinal axis of the device, around which the mixing device, the concentrating device, the excitation device, the measuring device and optionally the compensation device are preferably arranged.

Optionally, the sample recess has a region of larger cross-section and tapers to a region of smaller cross-section, preferably in the region of its base. In this embodiment, the excitation device and the measuring device are preferably arranged in the region of smaller cross-section around the sample recess.

A sample vessel can be arranged in the sample recess, which preferably has a shape that fits the sample recess precisely and a volume of at least 10 µL up to 10 mL, e.g. 500 µL or 1 mL. The sample vessel has a measuring area that is preferably arranged at one of its ends, more preferably at the end that is arranged in the sample recess towards the bottom.

In general, the sample vessel contains the magnetic nanoparticles specific for the analyte; in particular, the nanoparticles can already be placed in the sample vessel before step a. of the method, e.g. in a dilution solution, or can be filled into the sample vessel together with the aqueous sample in step a.

Optionally, the sample vessel is mounted in the device so as to be displaceable along the common longitudinal axis and/or rotatable about the common longitudinal axis, e.g. movable along a spiral movement in the device, in particular along a thread.

In the method, a reference sample is optionally analyzed before step a., wherein steps b. to e. of the method are carried out and the sample vessel contains a reference sample which has magnetic nanoparticles, optionally in a diluent solution, and no analyte. It is preferred that after the analysis of the reference sample, the method runs with the same sample vessel, i.e. that the aqueous sample is filled into the same sample vessel in which the reference sample is contained, the sample vessel is sealed and then steps b. to e. of the method run again. By analyzing the reference sample and then the aqueous sample in the same sample vessel, the method has the advantage that the aqueous sample can be analyzed with greater accuracy.

In general, the sample vessel has a cross-section that preferably tapers towards one of its ends to a region of smaller cross-section, in particular towards the end that is in the direction tion of the bottom of the sample recess. In the embodiment with a tapered sample vessel, the measuring area is preferably arranged in the area of smaller cross-section of the sample vessel or is formed by it. In this embodiment, the area of smaller cross-section of the sample vessel preferably has a volume of at most 50 µL, preferably at most 10 µL or at most 1 µL, in particular a volume of at most 1%, preferably at most 0.5% of the volume of the sample vessel. The method has the advantage that by concentrating the magnetic nanoparticles in the measuring area of the sample vessel, the measurement signal recorded by the measuring device is amplified in comparison to a non-concentrated aqueous sample.

The device has a mixing device that is set up to mix the aqueous sample in order to produce a sample mixture. The mixing device is generally set up to generate a first gradient field, i.e. a first magnetic field with a location-dependent flux density, in the region of the sample recess. Magnetic nanoparticles are moved in a magnetic gradient field in the direction of the magnetic flux density that is greater in magnitude. The first gradient field is preferably a controlled rotating magnetic gradient field and is more preferably time-dependent. The first gradient field is preferably set up so that the magnetic nanoparticles are moved at least partially perpendicular to the common longitudinal axis.

In a preferred embodiment, the device can have an arrangement of electromagnets, in particular of at least 2 electromagnets, preferably at least 3, at least 4 or at least 5 electromagnets as a mixing device, which are preferably arranged around the common longitudinal axis. In this embodiment, a control unit is set up to supply the electromagnets with current in a controlled manner one after the other or simultaneously in order to generate a preferably rotating first gradient field, and when the time-varying magnetic field is detected, the control unit no longer supplies the electromagnets with current. This gives the device and method the advantage that the measurement of the time-varying magnetic field is not disturbed by the first controlled magnetic gradient field.

In an alternative embodiment, the device can have an arrangement of permanent magnets, in particular of at least 1 permanent magnet, preferably at least 2, at least 3, at least 4 or at least 5 permanent magnets as a mixing device, which are preferably arranged around the common longitudinal axis, more preferably in a ring shape or spiral shape along the common longitudinal axis, and are optionally mounted so that they can move. Permanent magnets are generally designed to generate a magnetic gradient field without having to be supplied with current and can be made of, for example, aluminum, nickel, cobalt, neodymium, bismuth, manganese, iron, boron, samarium, plastic or combinations or alloys thereof. This gives the device and method the advantage that less current is required to carry out the method and the first gradient field generated is a strong magnetic field. In this embodiment, a control unit is configured to move the permanent magnets and the aqueous sample relative to one another and is optionally configured in particular to move the permanent magnets in a controlled manner towards or away from the common longitudinal axis and/or to move the permanent magnets in a radial movement around the common longitudinal axis and/or to move the permanent magnets along the common longitudinal axis and/or combinations thereof, e.g. to move the permanent magnets along a spiral path around the longitudinal axis, preferably in the direction of the measuring area, in order to generate a first controlled, time-varying magnetic gradient field. Alternatively, the permanent magnets can preferably be mounted fixedly in the device. The sample vessel is preferably arranged in the device so as to be longitudinally displaceable along the common longitudinal axis and/or rotatable about its longitudinal axis or the common longitudinal axis, and the control unit is configured to displace the sample vessel along the common longitudinal axis and/or rotate it about its axis in order to generate a first controlled, time-varying magnetic gradient field.

In a further alternative embodiment, the device can have a coil arrangement made up of at least one pair of identically constructed coils with a coil radius and a coil axis as a mixing device. The coils of the at least one pair of coils are preferably ring-shaped and are arranged parallel to one another, the distance between the coils of the at least one pair of coils preferably being equal to their coil radius and the coils of the pair of coils preferably being arranged at the same distance from the common longitudinal axis around the sample recess. The coils are arranged with their coil axis orthogonal or optionally parallel to the common longitudinal axis. In this embodiment, a control unit is set up to apply alternating current to the coils of the at least one pair of coils, preferably with alternating current in opposite directions, i.e. with a sinusoidal alternating current of the same frequency and opposite phase, i.e. shifted by 180°. It is optionally preferred that the coil arrangement voltage has a first and a second pair of coils, wherein the coils of a coil pair are identical in construction to one another and are arranged parallel to one another, wherein the coils of the first coil pair have a first coil radius and a first coil axis and the coils of the second coil pair have a second coil radius and a second coil axis, wherein the distance between the coils of a coil pair is equal to their coil radius and the coils of a coil pair are each arranged at the same distance from the common longitudinal axis and the first coil axis, second coil axis and the common longitudinal axis are each arranged orthogonally to one another and a control unit is set up to supply the coils of the first and second coil pairs with alternating current in a controlled manner, in particular the coils of the first coil pair with opposing alternating current of a first phase and the coils of the second coil pair with opposing alternating current of a second phase which is shifted by 90° to the first phase. The amplitude of the alternating current can be the same or different for each coil pair and/or each coil. Alternatively, one coil of a coil pair can be wound in reverse to the other coil and the coils of the coil pair can be supplied with alternating current in the same direction, ie alternating current of the same phase and frequency.

The first controlled magnetic gradient field preferably has a magnetic gradient field strength of at least 10 T/m, measurable by spaced apart magnetic flux density sensors.

In step b. of the method, the aqueous sample filled into the sample vessel is mixed by applying a first controlled magnetic gradient field in the region of the sample vessel in order to produce a sample mixture. In particular, the first controlled magnetic gradient field can be applied by moving a mixing device according to the invention by a control unit and/or applying alternating current to it.

It has been shown that by applying a first controlled, time-varying magnetic gradient field and the resulting movement of the magnetic nanoparticles through the aqueous sample, the analyte can be quickly bound to the magnetic nanoparticles without the need for additional mixing, e.g. by mechanical shaking of the sample vessel.

Optionally, the sample vessel has additional magnetic particles as mixing particles. Mixing particles are generally made of a ferromagnetic, ferrimagnetic, paramagnetic or diamagnetic material, e.g. iron oxide, and do not bind specifically to the analyte. Mixing particles are preferably spherical and have a diameter of at least 1 µm. It has been shown that when the first controlled magnetic gradient field is applied to the sample vessel, the mixing particles cause additional mixing of the aqueous sample by being moved by the magnetic field.

To concentrate the magnetic nanoparticles from the sample mixture, the device has a concentrating device which is set up to generate a second gradient field in the area of the sample recess, i.e. a second magnetic field with a location-dependent flux density, whereby according to the invention the area of greater magnetic flux density of the gradient field is generated in the area of the measuring device. Preferably, the second gradient field is a controlled, time-varying magnetic gradient field and is further preferably set up so that the magnetic nanoparticles are moved at least partially along the common longitudinal axis, more preferably in the direction of the measuring area of the sample vessel.

In a preferred embodiment, the device has a magnet as a concentrating device. The magnet can be an electromagnet and in particular have a core wound around a coil and can be supplied with current, or can be a permanent magnet, for example. The magnet preferably has a cylindrical shape and is more preferably tapered at one end. This gives the magnet the advantage that the magnetic nanoparticles are focused at one point and thus concentrated more effectively than with a non-tapered core. In this embodiment, it is further preferred that the magnet is arranged along the common longitudinal axis and with its tapered end pointing towards the bottom of the sample recess and the sample recess behind it. The magnet is preferably mounted in the device so that it can move along the common longitudinal axis. It has been shown that by moving the magnet towards the sample recess, the magnetic nanoparticles can be better concentrated and that by moving the magnet away from the sample recess, the values for the time-varying magnetic field in the measuring device can be determined more accurately.

In an alternative embodiment, the device has a coil arrangement comprising at least one pair of identical coils with a coil radius and a coil axis, which together men with a control unit form the concentrating device of the device. The coils of the at least one coil pair are preferably ring-shaped and are arranged parallel to one another, wherein the coils of the coil pair are each arranged at the same distance from the common longitudinal axis around the sample recess. The coils are arranged with their coil axes orthogonal or optionally parallel to the common longitudinal axis. In this embodiment, the control unit is set up to apply alternating current in opposite directions to the coils of the at least one coil pair. Alternatively, one coil of the coils of a coil pair can be wound in the opposite direction to the other coil and the coils of the coil pair can be applied with alternating current in the same direction.

Optionally, the concentrating device and the mixing device of the device can be formed at least partially by the same arrangement of coils or electromagnets or permanent magnets. In a preferred embodiment, the control unit is preferably set up to first apply current in a controlled manner to an arrangement of electromagnets arranged around the common longitudinal axis in order to generate a first controlled, time-varying magnetic gradient field, and then to no longer apply current to the arrangement of electromagnets and instead to apply current in a controlled manner to an electromagnet arranged along the common longitudinal axis in order to generate a second controlled, time-varying magnetic gradient field.

Generally preferably, the second controlled magnetic gradient field generated by the concentrating device has a field gradient of at least 10 T/m.

In step c. of the method, the magnetic nanoparticles are concentrated from the sample mixture in the measuring area of the sample vessel. The magnetic nanoparticles are preferably concentrated by moving them along the sample vessel in the direction of the measuring area by applying a second controlled, time-varying magnetic gradient field in the area of the sample vessel. By concentrating the magnetic nanoparticles, the method has the advantage that it enables a more sensitive determination of the analyte than with conventional methods, in particular ELISA methods or PCR methods.

In the embodiment in which the magnetic gradient field is generated by an electromagnet that can be moved along the common longitudinal axis, the electromagnet is preferably moved in step c. of the method from a first position with a greater distance from the measuring area to a second position with a smaller distance from the measuring area and is supplied with direct current in order to apply a second controlled, time-varying magnetic gradient field in the area of the sample vessel, and after step c. the electromagnet is no longer supplied with current and is moved from the second position to the first position.

For detecting the analyte, the device has an excitation device which is preferably arranged in the region of the sample recess around the common longitudinal axis and is designed to generate a time-varying magnetic excitation field in the region of the sample recess.

The excitation device is preferably a coil or a coil arrangement that is designed to generate a magnetic excitation field, in particular a homogeneous sinusoidal magnetic field. A coil (excitation coil) that can be supplied with current can be arranged as an excitation device in the region of the sample recess coaxially around the common longitudinal axis, preferably spaced radially from the sample recess and supplied with current in order to generate an excitation field. Alternatively, a Helmholtz coil arrangement that can be supplied with current can be arranged as an excitation device around the sample recess and supplied with alternating current in the same direction by a control unit in order to generate an excitation field.

The excitation device is preferably spaced radially from the sample recess, with a measuring device being arranged in the radial distance between the excitation device and the sample recess. It has been shown that the presence of analyte can be detected at large amplitudes of the excitation field, which reach the non-linear region of the magnetization curve of the magnetic nanoparticles, due to the harmonics of the excitation frequency.

The excitation field generated by the excitation device is preferably a pulsed magnetic field with a pulse length of 0.01 s, preferably at least 0.1 s up to 10 s, preferably up to 5 s and a magnetic flux density of at least 0.1 mT, preferably at least 10 mT up to 1,000 mT, measured by the time-dependent magnetization of the aqueous sample.

Alternatively, the excitation field generated by the excitation device can be a homogeneous sinusoidal magnetic field and have a field frequency of at least 0.1 Hz, preferably at least 1 Hz or 1 kHz, up to 2 MHz, preferably up to 25 kHz, and a magnetic flux density of at least 0.01 mT, preferably at least 0.1 mT up to 10 mT, measured by the frequency-dependent magnetization of the aqueous sample.

Optionally, the excitation device is configured to generate at least two sinusoidal magnetic fields with mutually different frequencies as an excitation field. In this embodiment, the measuring device can have a plurality of excitation coils, each of which is configured to generate a sinusoidal magnetic field with exactly one field frequency, wherein the field frequencies of the generated sinusoidal magnetic fields differ, or can have an excitation coil that is configured to generate an excitation field with a plurality of mutually different superimposed field frequencies.

A measuring device is arranged in the device, which is set up to record values for the time-varying magnetic field of the magnetic nanoparticles in the aqueous sample and to send them as a measurement signal to the evaluation unit. Values for the time-varying magnetic field can be, for example, values for the time-varying voltage induced in a coil, or values for the time-varying resistance in a magnetic field sensor.

Optionally, the measuring device is shielded against magnetic fields that are not the excitation field. In particular, the measuring device can be shielded against the first gradient field or the second gradient field.

In a preferred embodiment, the measuring device is designed as a coil (measuring coil) and is more preferably arranged around the sample recess, in particular around the area of smaller diameter. The measuring coil is preferably surrounded by an excitation coil as an excitation device, i.e. the excitation coil is arranged at a greater radial distance than the measuring coil around the sample recess, and the measuring coil is set up to record values for the time-varying induced voltage as values for the time-varying magnetic field.

Alternatively, the measuring device can be designed as a magnetic field sensor, e.g. as an AMR sensor, Hall sensor, TMR sensor, GMR sensor or fluxgate detector. The measuring device is preferably arranged so that it can record values for the time-varying magnetic field with little interference, e.g. an AMR sensor can be arranged as a measuring device along the common longitudinal axis immediately adjacent to the bottom of the sample recess in the device and can be set up to record values for the time-varying electrical resistance as values for the time-varying magnetic field.

The measurement signal can, for example, have or consist of the relaxation time, the phase angle between the excitation magnetic field and the measurement signal in the case of sinusoidal excitation and/or the ratio of the amplitudes of individual harmonics.

According to the invention, the device for compensating the measurement signal has a compensation device that is set up to record a compensation signal. In particular, the compensation device is set up to record values for a temporally variable magnetic field and to send them to the evaluation unit as a compensation signal. By calculating the measurement signal with the compensation signal, the method has the advantage that the sample can be tested more precisely for the presence of analyte than with conventional methods, since the analysis signal generated from this has less signal noise. A device with a compensation device has the advantage that it can be set up anywhere to analyze an aqueous sample for the presence of analyte, in particular without special shielding against other magnetic fields.

Preferably, the compensation device is arranged to record the compensation signal simultaneously with the recording of the measurement signal.

In a first embodiment, the device has an arrangement of two coils as compensation devices, of which the first coil is identical in construction to the excitation coil as excitation device and the second coil is identical in construction to the measuring coil as measuring device and is connected anti-serially to it and is arranged coaxially within the first coil. In this embodiment, the compensation device is arranged at a distance from the excitation device and the sample recess. The compensation device is set up to generate a compensation field that has the same field frequency, field amplitude and phase as the excitation field, and to record values for the time-varying compensation field as a compensation signal, which is preferably the inverted signal of the excitation field.

In a second embodiment with a measuring coil as a measuring device, the device has at least one coil as a compensation device, which is identical in construction to the measuring coil as a measuring device. In this embodiment, the compensation device and the measuring device are arranged parallel to one another in the same excitation device and the compensation device is anti-serial to the measuring device. In this embodiment, the excitation field is also the compensation field and the compensation device is set up to record values for the time-varying compensation field as a compensation signal, which is preferably the inverted signal of the excitation field. In this embodiment, the device has the advantage that the impedance of the excitation device is low and the device can be built in a space-saving manner. The compensation device optionally consists of two partial coils that are arranged in parallel on both sides of the measuring device. This gives the device the advantage that the measuring device can be arranged in the center of the excitation device and the measurement signal it records has fewer interference signals.

In the method, the analyte is detected in step d. For this purpose, a magnetic excitation field is generated in the area of the sample vessel, which is designed to periodically magnetize the magnetic nanoparticles in the aqueous sample. The magnetized magnetic nanoparticles generate time-varying dipolar magnetic fields in the magnetic excitation field, which are detected according to the invention by recording values for the time-varying magnetic field of the magnetized magnetic nanoparticle as a measurement signal and optionally values for the time-varying compensation field as a compensation signal. The measurement signal and optionally the compensation signal are then passed to an evaluation unit.

It has been shown that magnetic nanoparticles with bound analytes can be distinguished from magnetic nanoparticles without bound analytes by the fact that they have changed dynamic magnetic properties and, in particular, the time-varying dipolar magnetic field they generate has a changed amplitude of harmonic field frequencies and changed delays between magnetic moments or a changed phase shift to the excitation field. It is currently assumed that these effects are caused by an influence of the analyte binding on the relaxation behavior of the magnetic nanoparticles. This relaxation generates a magnetic flux, the temporal change of which generates, for example, an induced electrical voltage in a measuring coil. This induced electrical voltage is greater the greater the change per time in the magnetization of the magnetic nanoparticles.

To indicate the presence of analyte in the aqueous sample, the device has an evaluation unit that is set up to generate an analysis signal. The evaluation unit is optionally set up to calculate the measurement signal with the compensation signal in order to generate a compensated signal. Furthermore, the evaluation unit is preferably set up to apply a time derivative and/or a Fourier transformation or Laplace transformation, Hilbert transformation, correlation and/or a digital lock-in method to the measurement signal or to the compensated signal in order to generate an analysis signal. The analysis signal can in particular be derived from values for the voltage induced in a coil or, for example, the electrical resistance induced in a magnetic field sensor in the fundamental frequency of the excitation field and harmonics, i.e. integer multiples of the fundamental frequency of the excitation field that acts on the aqueous sample.

The evaluation unit is preferably designed to indicate the presence of analyte in the aqueous sample, and in particular to compare the analysis signal of the aqueous sample with a reference signal, i.e. the analysis signal of a reference sample or an aqueous sample without analyte, and to indicate the presence of the analyte in the aqueous sample in the case of an analysis signal that deviates from the reference signal, preferably deviates significantly. The reference signal can be obtained by analyzing a reference sample before, after or during the analysis of the aqueous sample, or can be obtained, for example, from a database for reference signals.

Optionally, the evaluation unit is configured to indicate the presence of the analyte only in the case of an analysis signal that deviates significantly from the reference signal, preferably by a value that is at least as large as the random signal noise, e.g. by at least 1% or at least 5% or at least 10%, particularly preferably with a signal-to-noise ratio of greater than 3, which corresponds to greater than 99% certainty, wherein the signal is the difference between the analysis signal and the reference signal and the noise is the standard deviation of the reference signal determined from a predetermined number of at least 3 measurements with a reference sample.

In the method, at least one analysis signal is generated from the measurement signal recorded in step d. and optionally the compensation signal in step e. For this purpose, the measurement signal of the aqueous sample is optionally calculated with the compensation signal, e.g. subtracted, in order to generate a compensated signal. The measurement signal or the compensated signal is then preferably converted into a time derivative and/or a Fourier transformation or Laplace transformation, Hilbert transformation, correlation and/or a digital Lock-in procedure to generate an analysis signal.

The analysis signal is preferably a spectrum that represents values for the magnetization intensity of the magnetic nanoparticles as a function of their vibration frequency or as a function of time.

In step e. of the method, the analysis signal of the aqueous sample is compared with a reference signal, i.e. the analysis signal of a reference sample, and if the analysis signal deviates from the reference signal, the presence of the analyte in the aqueous sample is indicated. The presence of the analyte is optionally only indicated if the analysis signal deviates significantly from the reference signal, preferably by a value that is at least as large as the random signal noise, e.g. by at least 1% or at least 5% or at least 10%, particularly preferably with a signal-to-noise ratio of greater than 3, which corresponds to greater than 99% certainty, where the signal is the difference between the analysis signal and the reference signal and the noise is the standard deviation of the reference signal determined from a predetermined number of at least 3 measurements with the reference sample. It has been shown that the specificity of the measurement method depends on the biological specificity.

Alternatively or additionally, in step e., a calibration curve or calibration line for the analysis signal as a function of the concentration of analyte in the aqueous sample can be recorded by measuring at least 2 aqueous samples with a previously known concentration of analyte and the measured analysis signal can be placed in the calibration curve or calibration line in order to determine the concentration of analyte in the aqueous sample.

Optionally, at least 2 sample vessels can be placed in the sample recess of the device at the same time. The process steps of mixing and concentrating the aqueous sample then run simultaneously for all aqueous samples filled into the sample vessels. It is preferred that the detection runs one after the other for each aqueous sample. It has been shown that the process can run faster by mixing and concentrating several aqueous samples at the same time.

Optionally, the sample vessel has specific magnetic nanoparticles with at least two different binders, each of which is specific for one analyte. It is generally preferred that each nanoparticle carries only one binder and is therefore only specific for one analyte. Furthermore, the magnetic nanoparticles are preferably designed so that they can be detected simultaneously, for example by nanoparticles that are specific for one analyte having different dynamic magnetic properties than nanoparticles that are specific for another analyte. In this embodiment, it is preferred that the aqueous sample comes from a human and only the presence of at least two different analytes indicates an infection with a disease.

In general, the method is suitable for the rapid detection of analyte in the aqueous sample. In particular, the method is optionally designed so that the steps of filling the sample vessel with the aqueous sample to indicating the presence of the analyte in the aqueous sample are carried out directly one after the other within a maximum of 30 minutes, preferably a maximum of 10 minutes or a maximum of 5 minutes.

Optionally, the sample recess, mixing device, concentrating device, excitation device and measuring device can be arranged in a fixed location in the device. This gives the device the advantage of being less susceptible to failure due to fewer moving parts. It has been shown that the analysis that can be carried out with the device allows a reliable analysis of aqueous samples even when the sample recess, mixing device, concentrating device, excitation device and measuring device are arranged in a fixed location.

• - 1 schematisch eine Ausführungsform des Geräts und
• - 2 schematisch eine weitere Ausführungsform des Geräts zeigen.

The invention will now be described in more detail with reference to the figures shown in

• - 1 schematically an embodiment of the device and
• - 2 schematically show another embodiment of the device.

In 1 the device is shown schematically, in which a sample recess 1, a mixing device 2, a concentrating device 3, an excitation device 4 and a measuring device 5 are arranged around a common longitudinal axis 6, wherein a sample vessel 7 can be arranged in the sample recess 1. The sample recess 1 and the sample vessel 7 taper towards their lower end as shown in the illustration to an area of smaller cross-section, which forms the measuring area 8. The mixing device 2 and the concentrating device 3 are formed by the same arrangement of electromagnets, wherein the core of each electromagnet is aligned with its north pole N in the direction of the sample vessel 7 and with its south pole S in the direction away from the sample vessel 7 and is wound by a coil that can be charged with alternating current. The mixing device 2 and concentrating device 3, controlled, time-varying magnetic gradient fields are generated in the region of the sample recess 1, with the region of greater magnetic flux density of the second controlled gradient field being arranged in the region of the measuring device 5. The sample vessel 7 is arranged in the device so as to be movable along the common longitudinal axis 6.
The excitation device 4 is designed as an excitation coil and is arranged in the area of the sample recess 1 at a radial distance around the common longitudinal axis 6. The excitation device 4 is set up to generate an excitation field in the measuring area 8 of the sample recess 1. The measuring device 5 is designed as a measuring coil and is arranged at the radial distance between the sample recess 1 and the excitation device 4. The measuring device 5 is set up to record values for a temporally variable magnetic field and to transmit them as a measurement signal to an evaluation unit.

2 shows the device with a sample recess 1, a mixing device 2, a concentrating device 3, an excitation device 4 and a measuring device 5, all of which are arranged around a common longitudinal axis 6, wherein a sample vessel 7 can be arranged in the sample recess 1. The sample recess 1 and the sample vessel 7 taper towards their lower end as shown in the illustration to an area of smaller cross-section, which forms the measuring area 8. The mixing device 2 and the concentrating device 3 of this embodiment are formed by the same arrangement of permanent magnets, each of which is aligned with its north pole N in the direction of the sample vessel 7 and with its south pole S in the direction away from the sample vessel 7 and can be moved by a control unit relative to the sample vessel 7 or to the sample recess 1 and to the common longitudinal axis 6. Otherwise, this embodiment corresponds to that in 1 shown embodiment.

Example 1: Analysis of an aqueous sample for the presence of a virus protein

A virus protein was detected as analyte in an aqueous sample using the method according to the invention.

Iron oxide nanoparticles with an average diameter of 80 nm and a protein A coating were used as magnetic nanoparticles. Due to their protein A coating, the magnetic nanoparticles used are designed to bind monoclonal antibodies specific to the virus protein and thus bind specifically to the virus protein. The concentration of the magnetic nanoparticles was 5 mg/mL with a molar particle concentration of 20 pmol/mL. As a reference sample, the magnetic nanoparticles without analyte were filled into the sample vessel of the analyzer and a reference measurement was carried out according to steps b. to e. of the procedure.

The virus protein was then filled into the sample vessel to a total volume of 1 mL, and the sample vessel was sealed. A first and second controlled magnetic gradient field was applied in the area of the sample vessel, generated by two NdFeB permanent magnets, each measuring 10×10×30 ^mm3, as a mixing device and a concentrating device, which were arranged around the sample vessel on opposite sides and with their magnetic north pole aligned towards the sample vessel, at a distance of 10 mm from the sample vessel. The aqueous sample was mixed by the first and second controlled magnetic gradient field by controlled rotation of the sample vessel around its longitudinal axis between the permanent magnets and the detection particles were then concentrated from the sample volume in the measuring area of the sample vessel within a few minutes. The gradient field had a field gradient of 80 T/m.

After the controlled removal of the first and second magnetic gradient fields from the area of the sample vessel, the sample vessel with the aqueous sample was transferred into the sample well of the device and the analyte was detected.

To detect the analyte, the reference sample and the aqueous sample were exposed to a sinusoidal excitation field of frequency f ₀ = 500 Hz and an excitation amplitude of 10 mT/µ ₀ and an averaging time of 1 s. The ratio of the third 3f ₀ and fifth 5f ₀ harmonics of the excitation frequency Sf ₀ /3f ₀ was determined as the analysis signal.

A change in the analysis signal of more than three times the standard deviation of the reference signal was defined as a threshold for statistically significant evidence.

To investigate the detection limit and the linearity of the dynamic range of the analyzer, different concentrations of the virus protein as analyte in the range from 0.5 nM to 5 nM were analyzed using the method. A calibration curve was generated from the analysis signals, which shows the change in the analysis signal depending on the analyte concentration. For a sufficiently high analyte concentration (above a quantification limit of five times the standard deviation In addition to qualitative detection, the concentration of the analyte in the aqueous sample could be quantified by means of the mean reference signal determination.

Claims (15)

Device for analyzing an aqueous sample for the presence of at least one analyte, which has a sample recess (1), a mixing device (2), a concentrating device (3), an excitation device (4) and a measuring device (5) which are arranged around a common longitudinal axis (6), wherein a sample vessel (7) can be arranged in the sample recess (1), wherein the mixing device (2) is set up to generate a first controlled, time-varying magnetic gradient field in the region of the sample recess (1), wherein the concentrating device (3) is set up to generate a second controlled, time-varying magnetic gradient field in the region of the sample recess (1), the region of greater magnetic flux density of which is arranged in the region of the measuring device (5), wherein the excitation device (4) is arranged in the region of the sample recess (1) and is set up to generate an excitation field, wherein the measuring device (5) is set up to record values for a time-varying magnetic field and to transmit them as a measurement signal to an evaluation unit. and the evaluation unit is set up to generate an analysis signal from the measurement signal, characterized in that it has an excitation coil as excitation device (4) and that it has a measuring coil as measuring device (5) which is set up to record values for the time-varying voltage induced in the measuring coil as a measurement signal and that it has an arrangement of two coils as compensation device, of which the first coil is identical in construction to the excitation coil and the second coil is identical in construction to the measuring coil and is connected anti-series to it and is arranged coaxially within the first coil, and the compensation device is arranged at a distance from the common longitudinal axis (6). Device for analyzing an aqueous sample for the presence of at least one analyte, which has a sample recess (1), a mixing device (2), a concentrating device (3), an excitation device (4) and a measuring device (5) which are arranged around a common longitudinal axis (6), wherein a sample vessel can be arranged in the sample recess (1), wherein the mixing device (2) is set up to generate a first controlled, time-varying magnetic gradient field in the region of the sample recess (1), wherein the concentrating device (3) is set up to generate a second controlled, time-varying magnetic gradient field in the region of the sample recess (1), the region of greater magnetic flux density of which is arranged in the region of the measuring device (5), wherein the excitation device (4) is arranged in the region of the sample recess (1) and is set up to generate an excitation field, wherein the measuring device (5) is set up to record values for a time-varying magnetic field and to pass them on to an evaluation unit as a measurement signal and the Evaluation unit is set up to generate an analysis signal from the measurement signal, characterized in that it has a measuring coil as a measuring device (5), which is set up to record values for the time-varying voltage induced in the measuring coil as a measurement signal and that it has at least one coil as a compensation device, which is identical in construction to the measuring coil, wherein the compensation device and the measuring coil are arranged parallel to one another in the same excitation device (4) and the compensation device is connected anti-serially to the measuring coil. Device according to one of the Claims 1 until 2 , characterized by an arrangement of electromagnets as a mixing device (2) and by a control unit which is arranged to supply the electromagnets with current in a controlled manner one after the other or simultaneously. Device according to one of the Claims 1 until 2 , characterized by an arrangement of permanent magnets as a mixing device (2) and by a control unit which is arranged to move the permanent magnets and the aqueous sample relative to one another. Device according to one of the preceding claims, characterized in that it has an electromagnet as a concentrating device (3) which can be energized and which is displaceably mounted in the device. Device according to one of the Claims 1 until 4 , characterized in that its concentrating device (3) has a coil arrangement comprising at least one coil pair of identically constructed coils with a coil radius and a coil axis, wherein the coils of the at least one coil pair are arranged parallel to one another, wherein the coils of the coil pair are arranged with their coil axis orthogonal or optionally parallel to the common longitudinal axis (6), and a control unit is set up to supply the coils of the at least one coil pair with alternating current. Device according to one of the preceding claims, characterized in that it has an excitation coil as excitation device (4), and the excitation field generated by the excitation coil is a pulsed magnetic field. Device according to one of the Claims 1 until 3 or 6 until 7 , characterized in that the sample recess (1), mixing device (2), concentrating device (3), excitation device (4) and measuring device (5) are arranged stationary in the device. Method for analyzing an aqueous sample for the presence of at least one analyte, in particular carryable using a device according to one of the preceding claims, with the steps a. filling a sample vessel (7), which has magnetic nanoparticles specific for the analyte and a measuring range, with the aqueous sample and sealing the sample vessel (7), b. mixing the aqueous sample by applying a first controlled, time-varying magnetic gradient field in the region of the sample vessel (7) to produce a sample mixture, c. concentrating the magnetic nanoparticles from the sample mixture in the measuring range of the sample vessel (7) by applying a second controlled, time-varying magnetic gradient field in the region of the sample vessel (7), d. detecting the analyte by applying a magnetic excitation field in the measuring range of the sample vessel (7) to magnetize the magnetic nanoparticles, recording the values of the time-varying magnetic field of the magnetized magnetic nanoparticles as a measurement signal, e. Generating at least one analysis signal from the measurement signal, comparing the analysis signal of the aqueous sample with a reference signal and, in the case of an analysis signal that deviates from the reference signal, indicating the presence of the analyte in the aqueous sample, characterized in that in step d. values for a time-varying compensation field are recorded and passed to an evaluation unit as a compensation signal, and that in step e. the analysis signal is generated from the measurement signal and the compensation signal. Procedure according to Claim 9 , characterized in that the aqueous sample is derived from a human and is mixed with a predetermined amount of a buffer solution comprising DNase inhibitor, RNase inhibitor and/or protease inhibitor prior to step a., and the presence of analyte in the aqueous sample indicates infection with a disease. Method according to one of the Claims 9 until 10 , characterized in that the sample vessel (7) has additional magnetic particles as mixed particles. Method according to one of the Claims 9 until 11 , characterized in that in step c. an electromagnet is moved from a first position at a greater distance from the measuring area to a second position at a smaller distance from the measuring area and is supplied with direct current in order to apply a second controlled time-varying magnetic gradient field in the region of the sample vessel (7), and after step c. the electromagnet is no longer supplied with current and is moved from the second position to the first position. Method according to one of the Claims 9 until 12 , characterized in that in step e. the analysis signal of the aqueous sample is compared with a reference signal and the presence of the analyte in the aqueous sample is only indicated if the analysis signal deviates significantly from the reference signal. Procedure according to Claim 13 , characterized in that before step a. a reference sample is analyzed which has magnetic nanoparticles and no analyte, wherein steps b. to e. of the method are carried out, and that after the analysis of the reference sample the aqueous sample in step a. is filled into the same sample vessel (7) in which the reference sample is contained, the sample vessel (7) is sealed and then steps b. to e. of the method are carried out again. Method according to one of the Claims 9 until 13 , characterized in that the steps of filling the sample vessel (7) with the aqueous sample until indicating the presence of the analyte in the aqueous sample are carried out directly one after the other within a maximum of 30 minutes.

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