HK1033771B - Apparatus and methods for employing magnetic particles for quantitative measurement of target particles - Google Patents

Description

Apparatus and method for quantitatively detecting target particles using magnetic particles

The technical field to which the invention belongs

The present invention relates to detecting the presence of magnetic particles, and more particularly to a method for quantitatively detecting the accumulation of these magnetic particles by alternating current excitation and induction detection of the magnetic moment amplitude of the vibration of the particles caused by the excitation frequency.

Background

Considerable attention has been given to techniques for determining the presence and possible concentration levels of particulates in larger mixtures or solutions containing minute particulates. In certain circumstances, it is desirable to measure certain organic compounds with very low concentrations. For example, in medicine, it is useful to determine the concentration of a given type of molecule, which is usually in solution, either in a natural physiological fluid form (e.g., blood or urine), or directed to a living system (e.g., a drug or contaminant).

One widely used method for detecting the presence or absence of microparticles of a compound of interest (known as an assay) is an immunoassay. In this method, detection of a given molecular species (called a ligand) is achieved by using a second molecular species (called a counterligand or receptor) which must bind specifically to the first compound of interest. Detection of the presence or absence of the ligand is accomplished by detecting or inferring, directly or indirectly, the extent of binding between the ligand and the counterligand.

Several detection and measurement methods are discussed in U.S. patent 4,537,861(Elings et al.). This patent describes several methods for achieving homogeneous immunization in solution with a binding reaction between a ligand and an anti-ligand, typically an antigen and an antibody. The Elings document creates a spatial pattern formed by a spatial array of discrete regions of counter-ligand material and ligand-based material, which are distributed in a dispersed manner to interact with the spatial array of discrete regions of counter-ligand material to produce a binding reaction between the ligand and counter-ligand in the spatial pattern, the bound complex being labeled with a particular physical characteristic. When the labeled binding complexes accumulate in a spatial pattern, the desired immunoassay can be provided by a scanning device. The scanning device may be based on fluorescence, optical density, optical dispersion, color and reflectance, etc.

Labeled coupling complexes are accumulated on specially prepared surfaces or optically transparent catheters or containers by applying a local magnetic field to a solution of the coupled (bind) complexes with magnetic particles, according to Elings. The magnetic particles range in size from 0.01 to 50 microns. Once the coupling complex is magnetically aggregated in solution, the scanning techniques described previously can be employed.

Magnetite-formed magnetic particles and inert matrix materials have long been used in the field of biochemistry. Their diameter ranges from a few nanometers to a few micrometers, with magnetite ranging from 15% to 100%. They are generally described as superparamagnetic particles, and if the size range is large, are referred to as magnetic beads. The usual method is to coat the surface of these particles with some bioactive material that will cause them to bind tightly to the particular microscopic substance or particle of interest (e.g., protein, virus, cell, DNA fragments). These particles become "handles" that can move or immunize objects with magnetic fields, which are typically provided by strong permanent magnets. The Elings patent is an example of the use of magnetic particles as labels. Products specially manufactured with rare earth magnets or pole pieces to accomplish this goal are commercially available.

Although these magnetic particles are used in practice only for moving or immune-coupled objects, some experimental work has been done using particles as labels to detect the presence of coupled objects. Labels are typically coupled to an object of interest using radioactive, fluorescent, or phosphorescent molecules. Magnetic tagging is a very attractive technology if detectable in sufficiently small quantities, as other tagging technologies have various serious weaknesses. Radiological methods create health and handling problems and are relatively slow. Fluorescence or phosphorescence techniques have limitations in terms of quantification accuracy and dynamic range, as the emitted photons are absorbed by other materials in the sample. See Japanese patent publication 63-90765, published 4/21 (Fujiwara et al) 1988.

Because the signal from very small volumes of magnetic particles is very weak, researchers have naturally attempted to create detectors based on superconducting quantum interference devices (SQUIDs). Superconducting quantum interference device amplifiers are well known to be the most sensitive magnetic field sensing devices in many situations. However, there are some specific difficulties with this approach. Because the pick-up coils of superconducting quantum interference devices must be maintained at cryogenic temperatures, the sample must be cooled in order to obtain the maximum coupling with these coils. This process results in a tedious process where the detection becomes unacceptable. The complexity and cryogenics of the superconducting quantum interference device itself make it difficult to use as an inexpensive desktop instrument. Even designs based on "high-tech" superconductors do not completely overcome these obstacles and introduce some new difficulties (fuziwara et al).

There are also more conventional methods to detect and quantify magnetic particles. These methods involve some form of magnetometer, the sample being placed in a strong magnetic field and the sample being subjected to a force, typically a change in the apparent weight of the sample as the intensity changes. An example of this technique is shown in Rohr patents 5,445,970 and 5,445,971. A more sophisticated technique is to detect the effect of particles on the deflection or vibration of a tiny mechanical cantilever. (Baselt et al, "force microscopy based biosensor", naval research laboratory, J.Vac, Science Tech.B.Vol 14, No.2(5pp) (4 months 1996)). These methods are limited in that they rely on the conversion of inherent magnetic effects into mechanical responses. These responses must be distinguished from a large number of other types of mechanical effects such as vibration, viscosity, and buoyancy.

An inexpensive, room temperature, desktop instrument that can directly detect and quantify very small amounts of magnetic particles would have significant application.

Description

In general, the present invention provides a method and apparatus for directly detecting and measuring very small accumulated magnetic particles (such as magnetite) and related materials to which they are coupled.

The invention essentially consists of a device for quantitative detection of target particles using magnetic particles, the magnetic particles and target particles combining to form a magnetically coupled composite sample, said device comprising: a mobile base with samples stored in a defined pattern; applying a variable magnetic field to a magnetizer of the sample; a magnetic field sensing element having an output signal conductor; means for moving said sample to a magnetic field and for producing a resultant output signal in operative relationship with said sensing element; a signal processor including a processor and an analysis element, the signal processor converting the output signal from the sensing element to provide a signal indicative of the quantity of the sample in a pattern; and means for converting said quantity indicating signal into a form useful to a person.

Magnetic particles or beads are coupled to the target particles using known methods to provide a magnetic sample element or magnetic coupling complex. One well-defined mode of magnetic sample elements is to deposit them on a planar base and then apply a high amplitude high frequency magnetic field to excite the magnetic particles in the sample. This causes the magnetic particles to oscillate at the excitation frequency like localized magnetic poles. The magnetic field from the sample is tightly coupled to the inductive sensing coil, which is made into a gradiometer structure. This configuration makes the sensing coil insensitive to the large and uniform magnetic field used to excite the sample to the greatest extent possible. Furthermore, the geometry of the coil is designed to match the spatial pattern of the sample to provide a significantly larger response depending on the relative position of the sample and the coil. The induced voltage across the sensing coil is carefully amplified and processed by phase sensitive detection. The induced pickup from the driving magnetic field itself serves as a reference signal for the phase detector circuit. The output of the phase detector is further filtered and then digitized.

The signal amplitude is modulated by moving the sample relative to the sensing coil array. This allows one to reject the signal simply because of the imbalance of the coils, the inconsistency of the drive field, the crossing of the circuit, or any other source of signal that is not apparent from the sample itself. The digitally shaped curve of signal amplitude versus sample position is compared to a theoretical response curve obtained using a suitable curve fitting technique. This provides a very accurate estimate of the magnetic content of the sample based on the inherent instrument noise and offset.

Brief description of the drawings

The objects, advantages and features of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings. The drawings illustrate the following:

FIG. 1 is a perspective view of a desktop version of the present invention.

Fig. 2 is an enlarged plan view of the embodiment of the sensing coil of the present invention of fig. 1.

Fig. 3 is a mechanical schematic perspective view of the invention of fig. 1.

Fig. 4 is an electronic schematic block diagram of the invention of fig. 1.

Fig. 4A is an enlarged plan view of the base of fig. 1 with the sensing coil positioned.

Fig. 4B is a perspective view of the base connector metal housing.

Fig. 5 is an enlarged plan view of another embodiment of the sensing coil of the present invention of fig. 1.

Fig. 6 is a waveform of a sensing coil output signal when a material to be measured passes through the sensing coil.

PREFERRED EMBODIMENTS

Preferred embodiments of the present invention will now be described with reference to the accompanying drawings, and in particular figures 1 to 3.

Readout module

The readout module contains several different subsystems that are sample movement controls with a base on which resides a sample of the magnetic-coupled composite for detection, and which provide the necessary relative motion within the system; a magnetizer that applies an excitation signal to the sample; a sensing coil as a signal pickup device for picking up a signal generated by the sample; the drive circuit is used for providing drive current for the magnetizer coil; an amplifier/phase detector/digitizer coupled to the sensing coil for receiving and processing the output signal; and a microprocessor chip providing bidirectional communication between an external Personal Computer (PC) and the readout module.

A. Sample motion control

Magnetic particles are coupled to target particles by conventional methods to create magnetically coupled composite samples. The target particles may include atoms, single molecules, biological cells, and the like. The magnetically coupled composite sample accumulates several to several hundred particles and settles at a predetermined location 11 near a base or disk 12 (fig. 3). Methods of forming coupling compounds and attaching them to predetermined locations of the disk are well known and standard techniques may be used. The discs are mounted on a shaft 13 and extend down to a serrated wheel disc 14. A suitable rotation device, such as a stepper motor 16, has a shaft 17 extending at its distal end to the worm gear assembly 15. The motor tracking PC66 controls the rotational movement of the puck 12 via signals applied by line 18. Of course, wireless means may be employed to couple the PC and the system of the present invention, if desired.

In a preferred embodiment, as presently contemplated, the disk 12 is approximately 47 millimeters in diameter and approximately 0.25 millimeters thick. The disc may be made of glass, plastic, silicon, or the like. For practical purposes, the thickness ranges from about 0.1 mm to 1.0 mm. In this particular example, the wheel 14 is connected to the disc 12 by a shaft 13 and is rotated by a motor 16 through a 120-tooth worm reduction gear. Of course, a variety of different rotary drives may be employed.

The magnetic conductor 21 is moved linearly relative to the disc 12 by a rotating means, such as a stepper motor 22, having a lead screw 23 on its shaft 24 for 40 revolutions per cycle. The sleeve 25 is provided with an internally threaded bore, the threads of which cooperate with the threads of the helical lead screw. A control signal is applied from the microcomputer 65 to the motor 22 through line 26. Likewise, the specific case of the rotary drive described herein is merely an example, and other suitable elements having different characteristics may be employed.

B. Magnetic conductor

In the preferred embodiment, ferrite toroid core 31 (fig. 4) has a gap 32 approximately 1.5 mm wide. In the described embodiment the core diameter is about 30 mm. The driving coil 33 is wound in a single layer to cover 270 degrees of the ring-shaped magnetic core 31, and the coil is symmetrical with respect to the gap. The feedback loop 34 is wound around the ring-shaped core body at a position of about 180 degrees from the gap (opposite to the gap). The loop 34 may be external to the coil 33 or between the coil 33 and the toroidal core. It may consist of several or many turns, depending on the necessary and appropriate feedback function. The purpose of the feedback loop is to sense or express the magnetic field of the gap 32, enabling the signal processing or output circuitry to be self-correcting for temperature drift and the like. This is used to enhance accuracy and is not a necessary operation of the present system. The toroidal core magnetizer array is mounted in the insulator inner chamber 35, which may be formed of fiber glass. The inner chamber 35 has a slot 36 (fig. 4) corresponding to the location of the notch 32. The slot/notch is shaped and configured to selectively receive the edge of the rotatable disk 12 and provide space for a sensor coil base, as described in more detail below.

C. Sensing coil

Referring now specifically to fig. 2, 4, and 4A, insulator base 41 is fitted into slot 36 of inner chamber 35 and extends to notch 32. Coupling pads 40, 42 are provided at the proximal end, and a sensing coil 43 is mounted on the base adjacent the tip. The base is preferably made of quartz or silicon and the sensing element is a thin film thin copper coil. The base and sensing coils can be constructed using standard thin film fabrication techniques, with conductors entering and leaving each coil at two different levels. For example, the entry trace 49 may be placed on the base surface by standard photolithographic lithographic processing, a layer of diverging quartz is overlaid on the entry conductor, and then the coil 43 and output conductor 44 are similarly processed, with a protective quartz layer added on top. The layers can be connected using conventional methods.

The interfacing of the sensing coils in sequence forms a gradiometer structure and is connected to the coupling pads 40, 42 by conductive tracks 44, 49 and to the signal processing circuitry by twisted pair 45. The use of twisted pairs helps to reduce drift signals or interfering pick-up.

The width of the helical coil shown in fig. 2 is about 5 microns and the pitch between the helical traces is about 10 microns. The thickness of the sensing coil traces is typically around 1 micron. The diameter of each complete coil is about 0.25 mm.

The relatively long and narrow base 41 is made and the coupling pads 40, 42 are relatively far from the toroid gap, which helps to minimize the wander pick-up of the bond wires 45. A metal housing 46 (fig. 4B) is employed in the coupling region to further reduce drift signal or interference pickup. The nearest connector of the base is slid into slot 50 after the wires are connected. The metal housing is essentially a small section of a drum with a thicker enclosure, typically made of copper. The metal housing provides electromagnetic shielding to facilitate mechanical handling, but is not necessary to the system of the present invention.

Another embodiment of a sensing coil is shown in fig. 5. The planar configured coil 47 is elongated to be rectangular. The tracking range is about the same as the coil of fig. 2, and the combined coil width is also about 0.25 mm. The coil is approximately 1-2 mm in length and is connected to the coupling pads 52, 53 by wires 48, 51.

D. Driving circuit

The magnetic drive circuit, shown on the left in fig. 4, includes a pair of high current, high speed operational amplifiers 54, 55. The primary winding 56 of the transformer provides power and the amplifier provides a drive current in excess of one amp to magnetize or drive the coil 33 at a frequency of about 200 KHz. The drive circuitry is highly balanced to minimize common mode noise pickup in the sense loops or coils 43, 47. A smaller secondary winding 57 is coupled to loop 34 along the magnetizing coil to provide a feedback voltage to operate amplifiers 54, 55, oscillating at a regulated amplitude and frequency. The secondary winding 57 also provides an optimized reference signal for the phase detection circuit described below.

E. Amplifier/phase detector/digitizer

Low noise integrated device amplifiers are the basis of this circuit, although better noise performance is possible using discrete components. The amplifier 61 is a transformer coupled to the sensing coil to reduce common mode noise signals, facilitating the removal of the imbalance between the magnetizer and the sensing coil. The transformer coupling method is conventional and is located within the amplifier 61 but is not specifically shown in the figure. The phase sensitive detector 62 also employs a target specific integrated circuit design. The output of the phase detector is applied to a low pass filter 63 and then digitised in an a/D converter 64. The converter should have a high resolution, such as a 20-bit sigma-delta converter. The converter chip has excellent noise rejection at 60Hz and 50Hz frequencies, which is helpful to maximize the sensitivity of the provided instrument. It is available from a number of manufacturers.

F. Microcomputer with a memory for storing a program for executing a program

The microcomputer 65 includes a microprocessor chip such as a Motorola HCll and a built-in port to provide bi-directional serial communication to the PC66 through a serial port plugged into the PC. It also provides a dedicated component in communication with the serial a/D converter 64 and the stepper motors 16 and 22. A simple command language is written directly to the microcomputer 65 allowing the PC to send commands and accept responses and data.

G. Human-machine interface

The PC provides the operating commands for the system of the present invention. It runs the system through an RS232 interface, for example, from the RS232 interface of the microcomputer.

H. System operation

In a relatively straightforward and familiar arrangement, well-defined dots or patterns of magnetic particle complexes containing the sample are deposited at one or more locations 11 near the edge of the disk 12. Following a control signal from the PC, the stepper motor 22 is energized to rotate the lead screw 23 to move the array of magnetizers towards the sample disk 12. When the sample position 11 near the edge of the disk 12 is aligned with the sense coils 43, 47 in the middle of the toroid gap 32, the stepper motor 22 is stopped and a high amplitude (e.g., 1 amp) high frequency (200KHz) signal is applied to the toroid drive coil 33. The signal from the PC66 then drives the stepper motor 16 to rotate the disk to move the sample dots past the sensing coil. The high amplitude, high frequency magnetic field of the gap 32 excites the magnetic particle sample in the gap. The toroidal core is intentionally driven to saturation to achieve a notched magnetic field strength of about 1000 oersted. The magnetic particles will produce magnetic oscillations at the excitation frequency that behave like localized magnetic poles. Assuming that the physical location of the magnetic particles is in close proximity to the sensing coil, the magnetic field from the sample is closely coupled to the sensing coil of the inclinometer configuration. Because of the sensing coil of the slope gauge arrangement, the sensing coil is relatively large and the output of the uniform excitation magnetic field is substantially 0 or none. To obtain the maximum possible response, the geometry of the sensing coil is configured to match the spatial pattern of the sample. That is, the sample pattern points cannot be larger than about 0.25 mm. The response signal changes significantly depending on the relative position of the sample and the coil.

The signal of the sensing coil in an environment with a driving magnetic field and no sample is used as a reference signal of the signal processing part of the invention. As the sample moves through one sensing coil after another, the phase of the coil output signals is reversed by 180 degrees, as shown in fig. 6, thereby providing a very powerful detection technique. The induced voltage is amplified by an amplifier 61 and processed by a phase detector 62. The signal is filtered, digitized and passed through the microcomputer 65 to the PC to provide an output signal to the PC. Indicator 67 may be any type of useful device to provide information to the system operator. It may be a visual indicator, communicating information by digital or graphical means; it could also be some type of light system, or an audible indicator, or a combination of these or other indicators.

The output signal amplitude can be modulated by moving the sample relative to the sensing coil array. This allows rejection of the signal only because of system and external inputs, and not because of the sample itself. The digitally shaped curve of signal amplitude versus sample position was compared to a theoretical response curve stored in PC66 obtained using a suitable classical curve fitting technique. The result of this operation provides a very accurate estimate of the magnetic content of the sample, with the inherent instrument noise and offset excluded.

Although preferred embodiments of the present invention have been described above, some other alternative embodiments of the present invention should be mentioned. We have described two sensor coils above, but there are other possible configurations. The aforementioned magnetizer moves relative to the sample disk, but the disk and coupled stepper motor may be configured to move relative to the magnetic drive array if desired. The aforementioned toroidal core uses a rectangular area, but other shapes may be used. As regards the number of sample particles at the spot 11 of the disc 12, a 0.25 mm spot of the sample element may contain, for example, about 10 magnetic particles of 5 micron size. Or about 1200 particles of 1 micron size.

Claims (21)

1. An apparatus for quantitative detection of target particles using magnetic particles, the magnetic particles and target particles being bound to form a magnetically coupled composite sample, the apparatus comprising:

a movable base (12) on which the sample is deposited in a defined pattern;

a magnetizer (31, 32, 33) for applying an alternating magnetic field to the sample;

a magnetic field (43) sensing element having an output signal conductor (45);

means (22, 23, 24, 25 and 14, 15, 16, 17) for moving said sample into a magnetic field in operative relationship with said sensing element, said sensing element having a resulting output signal; and

a signal processor (62, 64, 65, 66) comprising a processor and an analyzing element for converting said output signal of said sensing element to provide a signal indicative of a pattern of said sample amount.

2. The apparatus of claim 1, wherein the sensing element is an inductive sensing coil.

3. The apparatus of claim 2, wherein the sensing elements are two separate sensing coils.

4. The apparatus of claim 3, wherein the sensing coils are connected in a inclinometer configuration.

5. The apparatus of claim 3, wherein the sensing coil is a circular spiral.

6. The apparatus of claim 3, wherein the sensing coil is rectangular in shape.

7. The device of claim 1, wherein the moving means provides relative motion in two dimensions between the sample and the magnetic field applying means.

8. The device of claim 7, wherein the mobile device comprises:

a motor (22) and screw means (23, 24, 25) for linearly moving the magnetic field applying means relative to the movable base; and

motor means (14, 15, 16, 17) for moving said moveable mount and sample past the magnetic field applying means in a predetermined manner.

9. The apparatus of claim 1, wherein the magnetizer includes:

a ring-shaped magnetic core (31) having a notch (32) at one side;

a drive coil (33) wound around the core but left empty at the gap; and

means for applying an alternating current power source to the drive coil.

10. The apparatus of claim 9, further comprising a feedback loop (34) coupled to the magnetic core and the drive coil, an output of the feedback loop being connected to the signal processor (62) to cause the signal processor to self-correct for external effects.

11. The device of claim 9, wherein the sensing element (43) is mounted in fixed relation to the sensor mount (41) and extends into the gap.

12. The apparatus of claim 11, wherein the sensing element is two separate sensing coils mounted on the sensor base and connected in a gradiometer configuration, the sensing coils being positioned at the gap.

13. The apparatus of claim 1, wherein the signal processor comprises:

an amplifier (61) coupled to an output of the sensing element;

a phase sensitive detector (62) connected to the amplifier to adjust output signal conditions;

an analog-to-digital converter (64) for converting the output signal to a digital form; and

computing means (65, 66, 67) for receiving said digital signal and converting it into a form usable by a person and providing control signals to said device.

14. The apparatus of claim 8, wherein:

the movable base is a disk on which a plurality of patterns of samples are applied; and

the motor is a stepping motor adapted to rotate the disc in accordance with a signal from the signal processor.

15. The apparatus of claim 12, wherein said sensor mount is elongated and has coupling pads (40, 42) at its proximal end connected to conductors (44, 49) for inputting and outputting signals from said sensing coil, said sensing coil being mounted at a distal end of said sensor mount, said sensor mount further comprising a conductor housing (46) surrounding said coupling pads and said proximal end of said sensing mount for reducing drift signals and interfering pickup.

16. A method of quantitatively measuring target particles coupled to magnetic particles to form a magnetically coupled composite sample, the method comprising the steps of:

applying at least one sample pattern (11) on a base (12);

establishing a magnetic field at a predetermined location proximate to the inductive sense coil;

moving the sample in a predetermined manner across the magnetic field, exciting the magnetic particles to distribute in a pattern and produce magnetic field oscillations;

coupling magnetic field oscillations generated by the magnetic particles to the inductive sense coil;

detecting a voltage generated by the induction sensing coil; and

the amplitude of the induced voltage is measured to determine the number of oscillating magnetic particles.

17. The method of claim 16, wherein the sensing step is accomplished by a pair of sense coils (43) connected in a gradiometer configuration.

18. The method of claim 16, wherein the base is a rotatable disk.

19. The method according to claim 18, wherein the magnetic field is established at a gap (32) on a toroidal core (31) having a drive coil (33) wound thereon.

20. The method of claim 19, further comprising the steps of:

applying a plurality of groups of sample models which are separated from each other on at least one area of the edge of the disc;

the edge of the movable disk enters a gap of the annular iron core; and

the disc is rotated so that the sample passes through the notch.

21. The method of claim 16, wherein the magnetic field is established in a toroidal core (31) around which the drive winding (33) is wound, the step of switching being performed by a signal processor, the method further comprising the steps of:

applying an alternating drive signal to the drive coil to create a magnetic field;

feeding back the ac drive signals of the drive coils to a signal processor (62, 64, 65, 66); and

the feedback signal is used to correct errors that occur in the signal processor due to external influences.