Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
  • G01R33/00—Arrangements or instruments for measuring magnetic variables
  • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
  • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
  • G01R33/09—Magnetoresistive devices
  • G01R33/093—Magnetoresistive devices using multilayer structures, e.g. giant magnetoresistance sensors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
  • G01R33/00—Arrangements or instruments for measuring magnetic variables
  • G01R33/12—Measuring magnetic properties of articles or specimens of solids or fluids
  • G01R33/1269—Measuring magnetic properties of articles or specimens of solids or fluids of molecules labeled with magnetic beads

Definitions

• the invention relates to a method for sensing a magnetic stray field generated by a magnetizable object when magnetized.
• the invention further relates to a magnetic sensor which performs said sensing and the use of such a sensor in a biochip for e.g. molecular diagnostics, biological sample analysis or chemical sample analysis.
• micro-arrays or biochips are revolutionizing the analysis of samples for DNA (desoxyribonucleic acid), RNA (ribonucleic acid), nucleic acids, proteins, cells and cell fragments, tissue elements, etcetera.
• Applications are e.g. human genotyping (e.g. in hospitals or by individual doctors or nurses), medical screening, biological and pharmacological research, detection of drugs in saliva.
• the aim of a biochip is to detect and quantify the presence of a biological molecule in a sample, usually a solution.
• Biochips also called biosensors, biosensor chips, biological microchips, gene- chips or DNA chips, consist in their simplest form of a substrate on which a large number of different probe molecules are attached, on well defined regions on the biochip, to which molecules or molecule fragments that are to be analysed can bind if they are matched.
• substrate may include any underlying material or materials that may be used, or upon which a device, a circuit or an epitaxial layer may be formed.
• substrate may also include a semiconductor substrate such as e.g. a doped silicon, a gallium arsenide (GaAs), a gallium arsenide phosphide (GaAsP), an indium phosphide (InP), a germanium (Ge), or a silicon germanium (SiGe) substrate.
• the "substrate” may include, for example, an insulating layer such as a SiO 2 or an Si 3 N 4 layer in addition to a semiconductor substrate portion.
• substrate also includes glass, plastic, ceramic, silicon-on- glass, silicon-on-sapphire substrates.
• substrate is thus used to define generally the elements for layers that underlie a layer or portions of interest.
• substrate may be any other base on which a layer is formed, for example a glass or metal layer.
• a fragment of a DNA molecule binds to one unique complementary DNA (c-DNA) molecular fragment.
• the occurrence of a binding reaction can be detected, e.g. by using fluorescent markers that are coupled to the molecules to be analysed.
• fluorescent markers magnetizable objects can be used as magnetic markers that are coupled to the molecules to be analysed. It is the latter type of markers which the present invention is dealing with.
• said magnetizable objects are usually implemented by so called superparamagnetic beads. This provides the ability to analyse small amounts of a large number of different molecules or molecular fragments in parallel, in a short time.
• One biochip can hold assays for 10-1000 or more different molecular fragments.
• a biochip consisting of an array of sensors (e.g. 100) based on the detection of superparamagnetic beads may be used to simultaneously measure the concentration of a large number of different molecules (e.g. protein, DNA, drugs of abuse, hormones) in a sample fluid (e.g. a solution like blood or saliva).
• the sample fluid comprises a target molecule species or an antigen. Any biological molecule that can have a magnetic label (marker) can be of potential use.
• the measurement may be achieved by attaching a superparamagnetic bead to the target, magnetizing this bead with an applied magnetic field, and using (for instance) a Giant Magneto Resistance (GMR) sensor to detect the stray field of the magnetized beads.
• GMR Giant Magneto Resistance
• the magnetic field generator may comprise a current flowing in a wire which generates a magnetic field, thereby magnetizing a superparamagnetic bead.
• the stray field from the superparamagnetic bead introduces an in-plane magnetization component in the GMR, which results in a resistance change.
• Figure 2 shows an embodiment of a magnetic sensor MS on a substrate SBSTR.
• a single or a multiple of such (a) sensor(s) may be integrated on the same substrate SBSTR to form a biochip BCP as is schematically indicated in Figure 1.
• the magnetic sensor MS comprises a magnetic field generator which, in this example, is integrated in the substrate SBSTR e.g. by a first current conducting wire WR 1 . It may also comprise a second (or even more) current conducting wire WR 2 . Also other means instead of a current conducting wire may be applied to generate the magnetic field H.
• the magnetic field generator may also be located outside (external excitation) the substrate SBSTR.
• each magnetic sensor MS a magnetoresistive element, for example a giant magnetoresistive resistor GMR, is integrated in the substrate SBSTR to read out the information gathered by the biochip BCP, thus to read out the presence or absence of target particles TR via magnetizable objects thereby determining or estimating an areal density of the target particles TR.
• the magnetizable objects are preferably implemented by so called superparamagnetic beads SPB.
• Binding sites BS which are able to selectively bind a target TR are attached on a probe element PE.
• the probe element PE is attached on top of the substrate SBSTR.
• Each probe element PE is provided with binding sites BS of a certain type.
• Target sample TR is presented to or passed over the probe element PE, and if the binding sites BS and the target sample TR match, they bind to each other.
• the superparamagnetic beads SPB are directly or indirectly coupled to the target sample TR.
• the superparamagnetic beads SPB allow to read out the information gathered by the biochip BCP.
• Superparamagnetic particles are suspended in a (polymer) binder or a matrix of which at zero applied magnetic field the time-averaged magnetization is zero due to thermally induced magnetic moment reversals that are frequent on the time scale of the magnetization measurement. The average reversal frequency is given by
• KV (with K the magnetic anisotropy energy density and V the particle volume) is the energy barrier that has to be overcome, and V 0 is the reversal attempt frequency (typical value: 10 9 s "1 ), k is the Boltzmann constant, and T is the absolute temperature (in Kelvin).
• the magnetic field H magnetizes the superparamagnetic beads SPB which as a response generate a stray field SF which can be detected by the GMR.
• the GMR should preferably be positioned in a way that the parts of the magnetic field H which passes through the GMR is perpendicular to the sensitive direction of the layer of the GMR.
• a total external (that is to say not internal in the GMR) field for which the GMR is sensitive is indicated by H ext in Figure 2.
• the accuracy of the measurement may be too bad.
• the magnetic cross-talk is tenths of times higher than the magnetic stray field SF. This of course makes the measurement useless.
• the invention provides a magnetic sensor for sensing a magnetic stray field generated by a magnetizable object when magnetized and for generating an electrical object signal which depends on the sensed magnetic stray field, comprising a magnetic field generator for generating a magnetic main field for magnetizing the magnetizable object, and cross-talk reduction means for reducing the effect of a cross-talk signal component in the electrical object signal caused by magnetic cross-talk between the magnetic main field and the magnetic stray field, wherein the cross-talk reduction means is arranged for distinguishing a signal property between the cross-talk signal component and the remaining part of the electrical object signal and for generating an electrical output signal.
• the cross-talk By distinguishing a signal property between the cross-talk signal component and the remaining part of the electrical object signal the cross-talk can be isolated from the object signal and thus a cross-talk free output signal can be created.
• the signal property may for instance be the phase of a signal. Discrimination between the magnetic cross-talk, thus magnetic field coming directly from the magnetic field generator, and the magnetic stray field, thus magnetic field coming from the magnetic field generator via the paramagnetic beads, is possible by the fact that the magnetic stray field lags in time compared to the (direct) magnetic field.
• the cross-talk reduction means may comprise means for generating an orthogonal electrical signal which is orthogonal in relation to the cross-talk signal component, and a multiplier for multiplying the orthogonal electrical signal with the electrical object signal. The resulting signal after said multiplication forms a basis for the electrical output signal. The resulting signal may be directly used as the electrical output signal.
• the cross-talk reduction means comprises a low- pass filter for filtering said resulting signal after said multiplication.
• the low-pass filter then produces the electrical output signal.
• the frequency of the magnetic main field may be chosen such that the phase difference between the sensed magnetic stray field and the magnetic main field expressed in radians approximately equals ⁇ /2 +n ⁇ , in which n is an integer number. By doing so the gain of the sensor is maximized.
• Another signal property may for instance be the amplitude of a signal. This can for instance be applied if the magnetic main field comprises a first magnetic signal having a first frequency and a second magnetic signal having a second frequency which is substantially larger than the first frequency and which is larger than the magnetization cut-off frequency of the magnetizable object. For frequencies higher than the magnetization cut-off frequency the strenght of the produced magnetic stray field will ever decrease by increasing frequency.
• the first and second magnetic signals produce a first electrical signal component in the electrical object signal having the first frequency and having a first amplitude and producing a second electrical signal component in the electrical object signal having the second frequency and a second amplitude.
• the cross-talk reduction means comprises means for measuring the amplitudes of the first and the second electrical signal components and for mutually subtracting the measured amplitudes.
• the resulting signal after said subtraction forms a basis for the electrical output signal.
• the resulting signal may be directly used as the electrical output signal.
• the cross-talk reduction means comprises a low- pass filter for filtering said resulting signal after said multiplication. The low-pass filter then produces the electrical output signal.
• the first frequency is lower than said magnetization cut-off frequency and the second frequency is much higher than said magnetization cut-off frequency.
• the invention also provides a magnetic sensor for sensing a magnet stray field generated by a magnetizable object when magnetized and for generating an electrical object signal which depends on the sensed magnetic stray field, comprising a first external magnetic field generator for generating a first magnetic main field for magnetizing the magnetizable object, and cross-talk reduction means for reducing a crosstalk signal component in the electrical object signal caused by magnetic cross-talk between the first external magnetic main field and the magnetic stray field, the cross-talk reduction means comprising a second external magnetic field generator for generating a second magnetic main field for compensating for the magnetic cross-talk between the first external magnetic main field and the magnetic stray field SF.
• the first and second external magnetic field generators may for instance comprise first and second coils through which respectively first and second AC-currents, having the same frequency, flow during operation of the magnetic sensor.
• the cross-talk reduction means may comprise means for adapting the ratio of the amplitudes of the first and second AC-currents for minimizing the magnetic cross-talk. It may further comprise means for adapting the difference of the phases of the first and second AC-currents for further reducing the magnetic cross-talk. This is an easy way of adapting the mutual strength of the first and second magnetic fields and their mutual phases since no mechanical adaptations are necessary. Possibly the adaptation can be performed automatically, e.g. by applying generally known feedback techniques.
• the invention further provides a method for sensing a magnetic stray field generated by a magnetizable object when magnetized comprising the steps of: - in a first main step applying the steps of:
• Figure 1 shows a biochip comprising a substrate and a plurality of magnetic sensors
• Figure 2 shows an embodiment of a magnetic sensor with integrated magnetic field excitation
• Figure 3 shows the resistance of a GMR as a function of the magnetic field component in the direction in which the layer of the GMR is sensitive to magnetic fields
• Figure 4 shows an embodiment of the invention in which the phase difference of the cross-talk signal and the sensed stray field is used to discriminate between the crosstalk signal component in the object signal and the remainder of the object signal;
• Figure 5 shows a bode-diagram for further elucidating the inventive embodiment as shown in Figure 6,
• Figure 6 shows an embodiment of the invention in which the amplitude difference of the cross-talk signal and the sensed stray field is used to discriminate between the cross-talk signal component in the object signal and the remainder of the object signal;
• Figure 7 shows an embodiment of the invention in which an additional magnetic field is generated to compensate for the magnetic cross-talk field
• Figure 8 shows an inventive method in which before the actual (bio-) measurement, when no paramagnetic beads are present yet, the amplitude of the object signal is measured and stored, and during the actual measurements, when paramagnetic beads are present, the stored amplitude is subtracted from the amplitude of the current object signal, thereby delivering a cross-talk- free output signal.
• any other means which have a property (parameter) which depends on magnetic field such as certain types of resistors like a tunnel magnetoresistive (TMR) or an anisotropic magnetoresistive (AMR) can be applied.
• TMR tunnel magnetoresistive
• AMR anisotropic magnetoresistive
• GMR is the magnetoresistance for layered structures with conductor interlayers in between so-called switching magnetic layers
• TMR is the magneto-resistance for layered structures comprising magnetic metallic electrode layers and a dielectric interlayer.
• a first magnetic film is pinned, what means that its magnetic orientation is fixed, usually by holding it in close proximity to an exchange bias layer, a layer of antiferromagnetic material that fixes the first magnetic film's magnetic orientation.
• a second magnetic layer or free layer has a free, variable magnetic orientation. Changes in the magnetic field, in the present case originating from changes in the magnetization of the superparamagnetic particles SPB, cause a rotation of the free magnetic layer's magnetic orientation, which in turn, increases or decreases the resistance of the GMR structure. Low resistance generally occurs when the sensor and pinned layers are magnetically oriented in the same direction. Higher resistance occurs when the magnetic orientations of the sensor and pinned layers (films) oppose each other.
• TMRs are made of two ferromagnetic electrode layers separated by an isolating (tunnel) barrier.
• This barrier must be very thin, i.e., of the order of lnm. Only then, the electrons can tunnel through this barrier. This is a quantum-mechanical transport process.
• the magnetic alignment of one layer can be changed without affecting the other by making use of an exchange bias layer. Changes in the magnetic field, in the present case originating from changes in the magnetization of the superparamagnetic particles SPB, cause a rotation of the sensor film's magnetic orientation, which in turn, increases or decreases resistance of the TMR structure.
• FIG 4 shows an embodiment of the invention in which the phase difference of the cross-talk signal and the sensed stray field SF is used to discriminate between the cross-talk signal component in the object signal U OB and the remainder of the object signal.
• the magnetic sensor MS comprises a generator G for generating an AC- voltage/current having a radian frequency (O 1 . It generates an AC-current through a wire WR 1 which as a response generates a magnetic main field H (see Figure 2).
• the magnetic main field H magnetizes the superparamagnetic beads SPB which as a response generate a stray field SF (see also Figure 2).
• the GMR can sense the magnetic stray field SF since it comprises a large component in the sensitive (x-) direction of the GMR. (See the x- direction in Figure 2.)
• H x ⁇ the magnetic cross-talk
• the GMR is biased with a current i s delivered by a DC-current source IBIA S -
• the current i s generates an internal magnetic field in the GMR. Therefore by choosing the appropriate value for the current i s the curve shown in Figure 3 can be "moved” horizontally and a suitable sensitivity of the GMR can be chosen. In most cases the highest possible sensitivity ("highest" negative slope in the curve of Figure 3) will be chosen.
• the magnetic sensor MS further comprises an amplifier AMP, a phase shifter PHSFT, a multiplier MP, and a frequency low-pass filter LPF. An input of the amplifier AMP is coupled to receive a voltage change, caused by the stray field SF, across the GMR.
• This voltage change is buffered and preferably amplified by the amplifier AMP which delivers an object signal U OB at an output of the amplifier AMP.
• An input of the phase shifter PHSFT is coupled to receive an AC- signal which is in phase with the AC-current through the wire WR 1 , and thus also in phase with the main magnetic field H and the cross-talk field H x ⁇ .
• the phase shifter PHSFT delivers an orthogonal electrical signal U ORT which is 90 degrees delayed in phase compared to the AC-signal at the input of the phase shifter PHSFT.
• the magnetic cross-talk H x ⁇ is expressed by equation [I]:
• H b depends on the number of beads.
• the relaxation time constant ⁇ neel expresses the relaxation time when the excitation field is reduced to zero, (see for Neel relaxation: "Journal of Magnetism and Magnetic Materials 194 (1999) page 62 by R. K ⁇ tiz et al.). Due to the time it takes to magnetize the paramagnetic beads SPB, the stray field SF of the paramagnetic beads SPB will lag behind the applied magnetic main field H. This is in contrast with the cross-talk field H XT which have approximately the same phase as the phase of the magnetic main field H. At a radian frequency ⁇ « 1/ ⁇ eei the phase lag ⁇ is small.
• the phase of the cross talk field H XT which is essentially a component of the applied magnetic main field H, does not change. This still results in a phase difference between the magnetic cross talk field H XT and the stray field SF from the paramagnetic beads SPB in the magnetic sensor MS.
• the object signal U OB is demodulated with the orthogonal electrical signal This demodulation is performed by the multiplier MP which multiplies the object signal U OB with the orthogonal electrical signal U ORT thereby delivering a cross-talk free signal U MP at the output of the multiplier MP forming a basis for the electrical output signal U 0 which is a measure for the amount of target particles TR (see Figure 2).
• the object signal U OB is expressed by equation [3]:
• FIG. 6 shows an embodiment of the invention in which the amplitude difference of the cross-talk signal H x ⁇ and the sensed magnetic stray field SF is used to discriminate between the cross-talk signal components in the object signal U OB and the remainder of the object signal.
• the magnetic sensor MS comprises the AC-generator G, which now generates a first frequency ft and a second frequency f 2 , the wire WR 1 , the GMR, the DC-current source I BIAS , the amplifier AMP, and the frequency low-pass filter LPF.
• a first input of the multiplier MP 1 and a first input of the multiplier MP 2 are coupled to the output of the amplifier AMP.
• a second input of the first multiplier MP 1 is coupled via the gain adapter G v , to receive an AC-signal having the first frequency ft.
• a second input of the second multiplier MP 2 is coupled to receive an AC-signal having the second frequency f 2 .
• the subtracter DFF is coupled with a first input to the output of the first multiplier MP 1 for receiving inter alia a first DC-signal DC 1 , and with a second input to the output of the second multiplier MP 2 for receiving inter alia a second DC-signal DC 2 .
• An output of the subtracter DFF is coupled to an input of the low-pass filter LPF to deliver the resulting signal after the subtraction.
• An output of the low-pass filter LPF delivers the output signal U 0 .
• the summing means SM adds up the two AC-signals from the generator G and supplies an AC-current through the wire WR 1 having both the first and the second frequencies ft and f 2 (having equal amplitudes).
• the gain adapter G v may alternatively be put in between the generator G and the second input of the second multiplier MP 2 .
• Other alternative positions for the gain adapter G v are: in between the output of the amplifier AMP and the first input of the first multiplier MP 1 , in between the output of the amplifier AMP and the first input of the second multiplier MP 2 , in between the output of the first multiplier MP 1 and the first input of the subtracter DFF, and in between the output of the second multiplier MP 2 and the second input of the subtracter DFF.
• Figure 5 shows a bode-diagram for further elucidating the inventive embodiment as shown in Figure 6.
• the applied magnetic main field H (see Figure 2) consists of two frequencies: a relatively low frequency ft and a relatively high frequency f 2 .
• the magnetic cross talk H x ⁇ is not frequency dependent, so the amplitude of the resulting cross talk signal will be the same for both frequencies.
• the cross-talk signal H x ⁇ is expressed by equation [5]:
• H xJ , H 1 COSO)J + H 2 cos ⁇ 2 t [5]
• the amplitude of the signal at ft contains both cross talk and signal from the paramagnetic beads SPB, while the amplitude of the signal at f 2 is just cross talk since the stray field SF has practically reduced to zero at f 2 , as can be deducted from the bode-diagram of Figure 5.
• the stray field SF is expressed by equation [2].
• Figure 7 shows an embodiment of the invention in which an additional magnetic main field is generated to compensate for the magnetic cross-talk field. In the previous mentioned embodiments of the invention the effect of the magnetic cross-talk is reduced. It is however also possible to more directly eliminate the magnetic cross-talk.
• a first external magnetic field generator implemented by a first coil L A for generating a first magnetic main field H A for magnetizing the magnetizable object SPB
• cross-talk reduction means for reducing a cross-talk signal component in the electrical object signal caused by magnetic cross-talk between the first external magnetic field H A and the magnetic stray field SF (not indicated in Figure 7, but see Figure 2).
• the cross-talk reduction means comprises a second external magnetic field generator implemented by a second coil L B for generating a second magnetic main field H B for compensating for the magnetic cross-talk between the first external magnetic main field H A and the magnetic stray field SF.
• This method of using interfering fields H A , H B with the same phase and frequency to cancel the components in the x-direction can be used for sensors using an internally applied field (like in the previous embodiments) to magnetize the paramagnetic beads SPB or using an externally applied field to magnetize the paramagnetic beads SPB as is indicated in Figure 7.
• the second coil L B is used to cancel any magnetic cross talk resulting from a slight misalignment of the first coil L A from the applied first magnetic main field H A not being perfectly homogeneous or any other reason which causes cross-talk for instance reasons previously mentioned with the other embodiments of the invention.
• the first coil L A which is used to magnetize the paramagnetic beads SPB, is misaligned in such a way that the field H A it generates has a small component in the x-direction.
• this misalignment is indicated by the angle ⁇ not being equal to zero.
• first and second AC-currents having the same frequency, flow through respectively the first and second coils L A and L B .
• the second coil L B By using the second coil L B to generate the second magnetic main field H B with the same x-component amplitude, but opposite in direction, the x-component (which is cross-talk) can be cancelled.
• the resulting field H res has thus no x-component, i.e.
• Figure 8 shows an inventive method in which before the actual (bio-) measurement, when no paramagnetic beads SPB are present yet, the amplitude of the object signal U OB is measured and stored, and during the actual measurements, when paramagnetic beads are present, the stored amplitude is subtracted from the amplitude of the current object signal U OF , thereby delivering a cross-talk- free output signal.
• the magnetic sensor MS During a first main step the magnetic sensor MS generates a magnetic main field H in the absence of any magnetizable object e.g. in the absence of paramagnetic beads. Then the magnetic field H is sensed as if this field would have come from paramagnetic beads in case it would have been present. Thus an electrical object signal U OB is created in the absence of any paramagnetic beads. This means that the object signal U OB is basically 100 percent magnetic cross-talk. The electrical object signal U OB is filtered by the frequency low-pass LPF. The resulting output signal U 0 is a pure DC-signal. This signal is stored in a memory MM.
• the magnetic sensor MS During a second main step the magnetic sensor MS generates a magnetic main field H while the paramagnetic beads SPB are present. Then the magnetic field H is sensed and a further electrical object signal U FOB is generated. Because of the fact that now paramagnetic beads SPB are present the further electrical object signal U FOB is both determined by the sensed magnetic stray field SF, and thus comprises information with regard to the amount of targets TR (see Figure 2), and magnetic cross-talk. The further electrical object signal U OB is also filtered by the frequency low-pass LPF. The resulting further output signal U 0 F is also a pure DC-signal.
• the stored DC-signal during the first main step is then subtracted, by a subtracter SBSTR, from the currently generated DC-signal thereby generating a DC-output voltage U OUT which is a cross-talk free DC-signal representing the number of targets TR.
• a subtracter SBSTR subtracts the DC-output voltage from the currently generated DC-signal thereby generating a DC-output voltage U OUT which is a cross-talk free DC-signal representing the number of targets TR.
• said subtraction may also be reversed.

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