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WO2005111614A1 - Systeme de detection de particules magnetiques et technique d'execution d'une d'un dosage par liaison - Google Patents

Systeme de detection de particules magnetiques et technique d'execution d'une d'un dosage par liaison Download PDF

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Publication number
WO2005111614A1
WO2005111614A1 PCT/GB2005/001892 GB2005001892W WO2005111614A1 WO 2005111614 A1 WO2005111614 A1 WO 2005111614A1 GB 2005001892 W GB2005001892 W GB 2005001892W WO 2005111614 A1 WO2005111614 A1 WO 2005111614A1
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WIPO (PCT)
Prior art keywords
reaction site
magnetic particles
coil
target analyte
reaction
Prior art date
Application number
PCT/GB2005/001892
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English (en)
Inventor
Janice Kiely
Peter Hawkins
Richard Luxton
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Randox Laboratories Limited
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Filing date
Publication date
Application filed by Randox Laboratories Limited filed Critical Randox Laboratories Limited
Publication of WO2005111614A1 publication Critical patent/WO2005111614A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/015—Pretreatment specially adapted for magnetic separation by chemical treatment imparting magnetic properties to the material to be separated, e.g. roasting, reduction, oxidation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core

Definitions

  • MAGNETIC PARTICLE DETECTOR SYSTEM AND METHOD OF PERFORMING BINDING ASSAY
  • the invention relates to a magnetic particle detector system and a method for performing a binding assay such as an immunoassay.
  • the system is particularly useful in connection with assays such as immunoassays but can be used in other applications involving the binding of magnetic particles to a reaction surface.
  • Binding assays use complementary partners in such a way that one of the partners in a sample can be quantified.
  • Typical examples of binding partners include: any antibody and corresponding antigen; hormone and hormone receptor; hormone and hormone binding protein; drug and drug receptor; enzyme and cofactor; chelating or complex-forming agent and an ion.
  • One of the most common forms of binding assay is the immunoassay where the binding ⁇ artners are antibodies and the targets are antigens.
  • Immunoassays make use of the highly specific and sensitive interactions between antigens and their antibodies. Over the last 30 years they have found applications in many diverse areas including clinical chemistry and environmental monitoring. The high specificity and sensitivity arises from the nature of the interactions between the antigens, which are usually the analyte molecules, and their antibodies. With a few exceptions (such as techniques based on surface plasmon resonance, vibrating devices, etc.), a label, reporter or marker must be added if the interactions between the antibodies and the antigens are to be quantified.
  • Several different types of labels have been used in immunoass ys. Popular labels currently used include radioisotopes, fluorescent and chemiluminescent molecules, enzymes, gold particles and coloured latex beads .
  • Lateral flow or immunochromatographic assays are relatively simple immunoassay techniques using coloured latex beads and an example of which is the very successful Clear Blue one-step pregnancy test originally developed by Unilever. It is also an example of a device that with no sample preparation can produce the result of an analysis in a few minutes without the intervention of a skilled operator. There is a requirement for more such devices for use in the field in environmental monitoring or for rapid diagnostics such as point-of-care patient and veterinary testing.
  • simple lateral flow devices are restricted to applications where it is only necessary to determine whether the concentration of the analyte is above or below a threshold value. If more quantitative measurements are required then elaborate measuring equipment has to be used, usually with different markers, and this adds considerably to the complexity of the technique.
  • PMPs are available in a range of different diameters (typically, 0.1 to 20 ⁇ m) from several different suppliers. They have a paramagnetic core (usually iron oxide) with a suitable coating to which capture antibody molecules are attached.
  • a paramagnetic core usually iron oxide
  • an excess of the coated PMPs is introduced into a test solution containing a mixture of the target antigens (analyte) and other species. Eventually, the antigens become attached to the antibodies on the PMPs.
  • an excess of the appropriately labelled secondary antibody (detecting antibody) is introduced into the test solution, which then reacts with the target antigen (analyte) on the PMPs.
  • the PMPs with their attachments can be easily drawn to one side of the reaction vessel and held there by an external magnet whilst the remaining test solution is flushed away and replaced with clean buffer solution.
  • the PMPs are re-suspended in the buffer solution and the quantity of bound label determined using a technique appropriate for the label .
  • the PMPs help to simplify the extraction and washing processes, several steps might still be required, which add to the total time for a complete assay.
  • Another problem is that expensive equipment is usually required to determine the amount of bound label (such as photon- counting equipment for luminescent labels, radiation- counting equipment for radioactive labels, etc.).
  • a magneto-immunoassay system uses the PMPs as the labels, so no additional label is required.
  • a simple magnetometer to determine the quantity of bound PMP labels is described in EP-A-1146347.
  • the immobilization of the magnetic particle is through, on the one hand, the interaction of a binding partner attached to the reactive surface adjacent to a detecting coil and a complementary binding partner (the target analyte) which is introduced into the system in the sample being analysed, and, on the other hand, the target analyte and another complementary binding partner attached to the particle.
  • This assay format is called a sandwich assay and has a dose response with a positive slope.
  • suitably coated paramagnetic particles are bound to a reaction site.
  • paramagnetic particles are "displaced" from the reaction site due to competitive interaction between the target analyte and analyte immobilized on the paramagnetic particles. This will result in a loss on paramagnetic material from the reaction site.
  • This assay format has a dose response with a negative slope. In this mode the particles may be immobilised on the reactive surface prior to sample being added to the liquid or the particles may be added with the sample or following the sample. Alternatively, in a "competitive assay", a. binding partner is attached to the reaction site.
  • a first complementary binding partner (the target analyte) , which is introduced to the system in the sample, competes with a second complementary binding partner (the “competitor") to bind to the reaction site.
  • the second complementary binding partner is attached to the magnetic particle.
  • This form of assay therefore has a dose response with a negative slope.
  • the binding partner may be provided on the magnetic particle and the competitor (the second complementary binding partner) on the reaction site.
  • the target analyte introduced in the sample, competes with the competitor to bind to the magnetic particle.
  • binding partners are attached to the particle and reactive surface through electrostatic and other non-covalent bonds; covalent bonds through reactive groups on the binding partners and particle or reactive surface; through an intermediary biological molecule attached to the surface as above; or through any process of molecular imprinting or melding.
  • a blocking agent is used to bind to vacant binding sites on the reactive surface or particle.
  • Blocking agents used do not bind with either of the binding partners and are typically made from inert proteins or protein hydrolysates, ionic or non-ionic detergents, simple or complex sugars, inert polymers .
  • the reaction takes place in a liquid designed to promote the interaction of the binding partners.
  • This liquid may contain buffer salts, poly-ionic molecules, polymers, simple or complex sugars and proteins.
  • Binding partners may include any substances of biological or chemical origin, such as proteins, nucleic acids, carbohydrates, lipids, drugs, chelating agents, spores, micro-organisms or cells isolated from tissue culture or blood sample or tissue biopsies. "Chemical" substances include non-biological substances such as polymers.
  • binding partners include: any antibody and corresponding antigen, hormone and hormone receptor, hormone and hormone binding protein, drug and drug receptor, enzyme and cofactor, transcription factor and DNA, subunits of a protein complex such as G-proteins, any signalling or transport protein and its control element, lectins and glycoproteins, lectins and carbohydrate moities, receptor protein and lipoprotein, lipid and lipoprotein, DNA and DNA, DNA and RNA, RNA and RNA, PNA and PNA, PNA and DNA, PNA and RNA, cell membrane proteins and virus, cell membrane proteins and spores, cell membrane proteins and bacteria, any cell to cell interaction through any cell surface binding protein such as MHC II and CD4.
  • a protein complex such as G-proteins, any signalling or transport protein and its control element, lectins and glycoproteins, lectins and carbohydrate moities, receptor protein and lipoprotein, lipid and lipoprotein, DNA and DNA, DNA and RNA, RNA and RNA, PNA and PNA, PNA and DNA,
  • a sandwich immunoassay system is used and magnetic labels are immobilized onto a reaction surface and a magnetometer then determines the quantity of immobilised magnetic labels.
  • the basic magnetic particle detection system consists of a coil (inductor) , which forms part of a resonant network, a detector circuit and a test strip or test vessel. During operation the coil is located close to a reaction surface either on the test strip or in the test vessel. Magnetic particles bound on the reaction surface influence the high frequency magnetic field produced by the coil and cause a change in the inductance of the coil .
  • the magnetic particles may contain paramagnetic, ferrimagnetic, anti- erromagnetic or ferromagnetic materials.
  • This change in inductance is a function of the quantity of magnetic particles immobilised on the surface.
  • the change in the inductance of the coil is detected by a change in the resonant frequency of an inductance and capacitance (LC) network, of which the coil forms the inductive component and this is used to determine a measure of the number of particles attached to the reaction surface.
  • LC inductance and capacitance
  • it ' is known to expose the reaction site(s) to a pulsed magnetic field which provides a steep magnetic field gradient so as to attract magnetic particles towards the reaction site or, in the case of a displacement assay, to attract the particles away from the reaction site.
  • a drawback of this approach is the need to provide a magnet system which can generate such a pulsed magnetic field.
  • a method of performing a binding assay comprises supplying a sample including a target analyte to a reaction site having binding partners complementary to the target analyte attached thereto, and a coil and resonant circuit located adjacent the reaction site; providing coated magnetic particles which attach to the target analyte whereby the magnetic particles are caused to bind to the reaction site via the target analyte; monitoring the resonant frequency of, or a change in resonant frequency of, the resonant circuit so as to determine the quantity of magnetic particles bound to the reaction site; and exposing the reaction site to a non-pulsed magnetic field so as to attract magnetic particles towards the reaction site.
  • a method of performing a binding assay comprises supplying a sample including a target analyte to a reaction site having binding partners complementary to the target analyte attached thereto, and a coil and resonant circuit located adjacent the reaction site; providing coated magnetic particles which are initially bound to the reaction site via the binding partners, the target analyte causing the magnetic particles to detach from the reaction site due to preferential binding for the target analytes; monitoring the resonant frequency of, or a change in resonant frequency of, the resonant circuit so as to determine the quantity of magnetic particles bound to the reaction site; and exposing the reaction site to a non- pulsed magnetic field so as to attract magnetic particles away from the reaction site.
  • a method of performing a binding assay comprises supplying a sample including a target analyte to a reaction site having binding partners complementary to the target analyte attached thereto, and a coil and resonant circuit located adjacent the reaction site; providing a competitor to which the binding partners are also complementary and to which coated magnetic particles are attached, whereby the target analyte inhibits binding of the competitor and its attached magnetic particles to the reaction site; monitoring the resonant frequency of, or a change in resonant frequency of, the resonant circuit so as to determine the quantity of magnetic particles bound to the reaction site via the competitor, and exposing the reaction site to a non-pulsed magnetic field so as to attract magnetic particles towards the reaction site.
  • a method of performing a binding assay comprises supplying a sample including a target analyte to a reaction site having a competitor attached thereto, and a coil and resonant circuit located adjacent the reaction site; providing coated magnetic particles having binding partners which are complementary to the target analyte and to the competitor, whereby the target analyte inhibits binding of the magnetic particles to the reaction site via the competitor; monitoring the resonant frequency of, or a change in resonant frequency of, the resonant circuit so as to determine the quantity of magnetic particles bound to the reaction site via the competitor, and exposing the reaction site to a non-pulsed magnetic field so as to attract magnetic particles towards the reaction site.
  • a magnetic particle detector system comprises at least one reaction surface; a corresponding electrical coil located adjacent the or a respective reaction surface; a measuring system for measuring the effect of magnetic particles bound to a reaction surface on the inductance of the associated electrical coil; and means for generating a non-pulsed magnetic field in the vicinity of the reaction site(s) so as to attract magnetic particles either towards the reaction site(s) or away from the reaction site (s) .
  • a pulsed magnetic field it is not necessary to provide a pulsed magnetic field and this allows the use of a magnetic field to be significantly simplified.
  • an electromagnet could be used to provide the non-pulsed or static magnetic field, the invention enables a permanent magnet to be used and therefore avoids the need for any control circuitry.
  • the invention is applicable to a reaction vessel or test strip having a single reaction site but is also applicable to such vessels or strips having a plurality of reaction sites as described and claimed in our co-pending patent application of even date entitled "Magnetic Particle Detector System", GB0410976.5.
  • the sample may comprise any biological or chemical (including non-biological) substances.
  • the sample comprises one of whole blood, plasma, serum and urine.
  • Assays can be performed to detect environmental analytes such as toxins or pollutants in a range of samples such as water samples from rivers, reservoirs, lakes, sewage outlets, farm run-offs, soil extracts and plant extracts.
  • Figure 1 illustrates an arrangement of five coils
  • Figures 2a, 2b and 2c illustrate three examples of a phase locked loop circuit
  • Figure 3 illustrates a typical response of a magnetometer to the presence of magnetic particles
  • Figure 4 illustrates a first example of the circuit of a system according to the invention
  • Figure 5 illustrates a second example of the circuit of a system according to the invention
  • Figure 6 illustrates a third example of the circuit of a system according to the invention
  • Figure 7 is a schematic cross-section through an example of a system according to the invention with magnet omitted
  • Figure 7a depicts an example of a printed circuit board suitable for use in a system according to the invention
  • Figure 8 illustrates the system of Figure 7 in conjunction with an electromagnet
  • Figure 9 illustrates a sandwich assay schematically
  • Figure 10 illustrates a typical response from a sensing coil when a sample is added to the reaction vessel
  • Figure 11 illustrates two dose
  • Magnetic particle detector systems comprise three key elements namely one or more resonant coils, the measuring system or detector circuit, and the test strip or test/reaction vessel. These will be described in turn. Although the invention is applicable to a single reaction site and single detection coil, it will be described with reference to multi-site systems.
  • Resonant Coils A variety of coil designs including helical, conical or flat-plate spiral can be used. Typically, a flat-plate spiral design is used because the electromagnetic field distribution is concentrated close to the surface of the coil so that when the coil is placed adjacent to the reaction surface (s) the electromagnetic field does not penetrate far beyond the reaction surface (s).
  • the coils can be either a single layer flat coil or a multi-layer flat coil. Preferably the coils will be deposited onto a substrate made from an electrically insulating material .
  • Suitable substrate materials include, a polymer, an amorphous material (such as glass, ceramic, etc) , a semiconductor (such as silicon, germanium, gallium arsenide, etc) , a crystalline material (such as mica, graphite, diamond, etc) , or a composite (such as glass or carbon fibre board etc) .
  • the substrate material may be flexible or rigid.
  • the substrate material is ceramic.
  • the coils can be fabricated from any conducting or semiconducting material or mixtures of materials. Examples would be obvious to an expert.
  • gold is used as the major element in the coil fabrication.
  • doped silicon is used as the coil material.
  • the coils can be deposited by a variety of means e.g.
  • gold ink (Agmet ESL part no. 8880-H) is used as the coil material.
  • the diameter of the coils can vary from sub-micrometre sized coils to more than lcm depending on the technology used and the application. The number of turns on the coil determines the inductance of the coil and is chosen to be compatible with the detector circuitry. In a multi-analyte system there is a different binding and affinity partner for each analyte on a reaction surface.
  • each coil has five turns and the track width is lOO ⁇ m with 75 ⁇ m spacing.
  • the thickness of the track is 8 ⁇ m.
  • the inner radius of the coil is 500 ⁇ m.
  • the coils are of identical design, but this is not necessarily the case.
  • the coils on the substrate are arranged relative to the reaction surface, such that the electromagnetic field associated with each coil covers all or a portion of the reaction surface.
  • the coils could be on a support separate from a surface defining the reaction surface and brought adjacent the reaction surface.
  • at least one of the coils will be selected as a reference coil.
  • the reaction surface corresponding to the reference coil is identical to the other surfaces in most respects except that it contains no capture molecules, in which case no magnetic particles can be captured and they might, or might not, contain a pre-determined number of magnetic particles.
  • the reaction surface or surfaces contains capture molecules of a certain type so that a predetermined number of magnetic particles always binds regardless of the concentration of the analyte .
  • the resonant frequency of the reference coil (s) can then be compared with the resonant frequencies of the test coils. This has the benefit of reducing errors in readings caused by factors such as noise, interference and temperature variations .
  • the detector circuit The coils are connected in parallel or in series with one or more capacitors to form a LC resonant network.
  • the capacitor values are chosen in order to create circuits that resonate at frequencies compatible with the measurement circuit .
  • Resonant frequency measurements can be carried out using the LC network to control the output frequency of an oscillator such as a Colpitts, Hartley or Armstrong oscillator.
  • a phase lock loop (PLL) circuit is used to determine the resonant frequency of the LC network.
  • PLL phase lock loop
  • the detector circuitry comprises five main stages.
  • VCO voltage controlled sine-wave oscillator
  • LC network 2 including the coil L and a capacitor C
  • phase detector 3 and a loop filter network 4.
  • the VCO 1 drives the LC network 2.
  • the initial frequency of the VCO 1 should closely match the resonant frequency of the LC network 2.
  • the phase detector 3 senses a phase difference across the resistor R and produces an error signal.
  • the error signal is filtered and supplied to the VCO 1. This changes the oscillator frequency to again match the resonant circuit.
  • a stable resonant circuit is provided.
  • the inductance of the resonant coil L changes when magnetic particles are immobilised on the reaction surface close to the coil. The change is dependent on the quantity of particles present.
  • This inductance change results in a variation in the resonant frequency of the LC network 2. Consequently, as the VCO 1 and the resonant circuit frequencies are no longer identical, a phase difference is detected across the resistor R and an error voltage is supplied to the VCO to alter the output frequency appropriately.
  • the new stable frequency produced by the VCO is the resonant frequency of the network with magnetic particles close to the coil.
  • a typical plot of the number of magnetic particles versus decrease in resonant frequency as detected by a meter 5, is shown in Figure 3.
  • the resonant frequency of a LC network 6 incorporating the coil 7 and a variable capacitor 8 is locked onto the output frequency of a very stable sine wave oscillator 9 (such a temperature regulated crystal- controlled oscillator) .
  • the output from the oscillator 9 is fed to the LC network 6 via a resistor R as before.
  • the values of L and C are chosen so that the resonant frequency of the LC network is close to the output frequency of the oscillator, but in this case, C is a variable voltage- controlled capacitor (varicap or varactor) 8.
  • a phase detector circuit (PSD) 10 senses the phase of the signal across R and, as before, produces a dc-error signal, which in this case is fed via a loop filter 11 to the varactor 8.
  • the error signal adjusts the value of C until the resonant frequency of the LC network matches exactly the output frequency of the stable oscillator.
  • a sample containing magnetic particles brought near to coil causes the inductance of the coil to increase and the PSD circuit 10 responds by changing the error signal so that resonant frequency of the LC network again matches that of the oscillator.
  • the magnitude of the error signal V DC is related to the number of magnetic particles in the sample and is monitored by a meter (not shown) .
  • each coil can be connected to a respective circuit of the type shown in Figure 2a or Figure
  • phase detected (PFD) 110 is phase detected (PFD) 110 to give a DC error and applied as an automatic control signal to the VCO sense tuned circuit variable capacitance diodes. This re-adjusts the resonance of the tuned sense circuit and keeps it locked to the reference signal .
  • the DC error voltage in milli and micro volts is the measure of the sample.
  • Preferred circuits to be used in multi-coil, multi-analyte systems we describe various circuit arrangements we have actually used for multi-coil measurements and describe other possible circuit arrangements.
  • the controller mentioned in all these circuits could be dedicated hardware, such as a group of logic circuits, a microprocessor or a personal computer.
  • one phase locked loop circuit 25 is used to measure the resonant frequencies of the reference LC network and the test LC networks in sequence using a programmable radio-frequency switch 24 ( Figure 4) .
  • each coil 20,21 could be trimmed during manufacture to have identical electrical characteristics and then a single common capacitor (C c ) connected to the pole of the switch 24 would form a resonant circuit with a particular coil when the switch has selected that coil: in this way, the resonant frequency of each coil would be identical prior to adding the test sample.
  • C c common capacitor
  • the controller 22 moves the switch 24 sequentially from one LC network to the next and the resonant frequency of each is measured and stored by the controller.
  • a disadvantage experienced with the circuit shown in Figure 4 is that the PLL circuit can take a long time to settle down after the switch has moved to a new position, which means that data are not collected fast enough to give the reliable kinetic readings using the technique described in the last section.
  • An embodiment that reduces this problem is shown in Figure 5.
  • one of the coils L R and one of the capacitors C R are used as a reference circuit 31.
  • a PLL circuit 30 measures the resonant reference frequency (f R ) of the L R C R circuit 31.
  • the test coils (L sl to L Sn ) are connected in parallel with respective varactor capacitors (C S1 to C Sn ) and the resonant frequency of each LC circuit 32 1 ...32 n determined by individual PLL circuits (PLL to PLL .
  • the resonant frequency of L S1 C S1 at the output of PLL- L is fed into a mixer circuit 33 ! .
  • the reference frequency f R is also fed into the mixer circuit 33 x which produces a beat frequency that is equal to the difference in the resonant frequency of L S1 C S1 network 32 x and f R .
  • a similar arrangement of PPL and mixers 33 2 ...33 n circuits is associated with each of the other test LC circuits.
  • each mixer circuit is connected via a programmable switch 34 to a controller 35, which could be dedicated hardware or a personal computer.
  • the controller 35 adjusts the values of x to C n until there is zero beat frequency from each mixer circuit 33 ! ...33 n .
  • the resonant frequencies of the associated LC circuits decrease, producing beat frequencies in the separate mixer circuits, which are read by the controller 35.
  • An advantage of this circuit is that it is much faster than the one shown in Figure 4.
  • a disadvantage is that it uses a lot of electronic components.
  • a potential problem when using substrates containing many closely-spaced coils is that crosstalk could occur between the coils, leading to faulty operation of the phase detectors.
  • One way of reducing this potential problem is to operate each coil at widely differing frequencies so that pickup by one coil of the electromagnetic radiation from another can be easily filtered out.
  • An alternative approach is to ensure that each coil runs at exactly the same frequency.
  • Figure 6 shows such an arrangement.
  • a crystal-controlled oscillator 40 produces a high-purity sine wave of a fixed, stable, frequency.
  • the oscillator could also be temperature controlled to give greater stability.
  • This waveform is fed to all n of the LC resonant circuits 41 1 ...41 n on the sensing device via suitable feed resistors Rl to Rn as shown.
  • L and C are chosen so that the resonant frequency of each LC network is close to the fixed frequency of the crystal- controlled oscillator 40.
  • Each LC network 41 1 ...41 n has a phase-sensitive detector (PSD1 to PSDn) connected across the feed resistors. The output of each PSD is filtered and then connected via a programmable switch 43 to a controller 42. The output of each PSD is a dc voltage that is read in turn by the controller 42 which also provides a measuring system.
  • PSD1 to PSDn phase-sensitive detector
  • the controller 42 Before samples are added to the reaction vessel, the controller 42 starts by selecting the output from PSD1 and then adjusts C sl , which is a varactor, until the output voltage from PSD1 is 0 : at this point there is no difference in phase in the sinusoidal waveform across Rl and the resonant frequency of the L S1 C S1 network exactly matches the frequency of the sine-wave from the crystal- controlled oscillator.
  • the controller 42 now moves to each position of the switch 43 in turn and repeats the process for each LC network until the resonant frequency of all the LC networks exactly matches the output frequency of the crystal-controlled oscillator.
  • At least one of the coils can be a reference coil.
  • Test strip or test vessel Binding reactions between binding partners where one partner is the analyte in the sample and the other partner is the capture molecule immobilised on the reaction surface take place on a substrate in a test vessel or on a test strip.
  • the immobilised binding partners on the reaction surface will depend on the nature of the analysis and could be a multi-layer arrangement containing linker molecules to fix the capture molecules to a substrate material and blocking molecules to reduce non-specific binding.
  • the number of magnetic particles immobilised on a suitable reaction surface is related to the concentration of the analyte.
  • Suitable substrate materials could be a ceramic, polymer, amorphous material (such as glass) , a semiconductor (such as silicon, germanium, gallium arsenide, etc.), a crystalline material (such as mica, sapphire, diamond, etc.), or a composite (such as glass or carbon fibre board etc.) depending on the nature of the immobilised binding partner in the reaction surface.
  • the substrate material will be thin (for example 1mm) .
  • the reaction surfaces for each analyte may be in separate defined areas on the substrate .
  • the substrate also forms a base or other wall of the test vessel 49.
  • each sensing coil must be close to its respective analyte reaction surface.
  • the coils 50,51... are mounted outside the reaction vessel 49 but positioned close to their respective analyte reaction layers 52,53, which are attached to the substrate 54 forming the wall of the reaction vessel and are inside the vessel.
  • the substrate for the coils and the substrate for the reaction surface (s) may be made from different materials.
  • the reaction surfaces and the coils are both mounted on the same, thin substrate.
  • the reaction surfaces are deposited in defined areas on one side of the substrate and the coils 50,51 are formed on the other side directly opposite their respective reaction surfaces ( Figure 7) .
  • the coils are first deposited on the substrate, covered with a thin insulating layer and then coated with a thin layer of their respective reaction surfaces.
  • the dimensions of the test vessel will be dependent on the volume of the sample and size of the reaction surfaces.
  • the reaction surfaces are coated on one side of a substrate with the coils on the other side.
  • the substrate 54 also forms the base of the test vessel. A typical size for this substrate would be 9mm by 9mm.
  • the walls of the vessel are formed from a hole cut in a sheet of printed circuit board (PCB) 55: any insulating material, such as glass, mica or ceramic, is also effective for this purpose. The depth of the well is determined by the thickness of the PCB and this is typically 1 mm.
  • PCB printed circuit board
  • the coils are deposited on the external side of the substrate. Connections between the coils and the detector circuit can be made by a variety of means including wire bonding, W strip or mechanical clips.
  • the PCB not only acts as the walls of the vessel but also as a connector as tracks are etched onto the board. Capacitors and other components from the detection circuit can be mounted on the substrate 54 and/or the PCB 55.
  • An example of a PCB layout is shown in Figure 7a.
  • the walls of the reaction vessel are formed by a square hole 55' in the PCB 55. Typically, hole 55' is about 9mm by 9mm in size.
  • the tuning capacitors C sl ...C sn ( ⁇ 5% tolerance) could be positioned as close as possible to each coil on the edge of the hole 55 ' .
  • the track lengths could be equalised whilst minimising crosstalk by producing tracks having "meandering" paths.
  • all the leads on the PCB including those connecting the PCB to the controller 35 and those connecting the capacitors C sl ... C sn to the coils) could also be designed in order to match the lengths of the tracks to one another as closely as possible. All the earth connections of the coils could be linked in a star arrangement on the underlying substrate.
  • Typical PCB dimensions may be about 50mm to 24mm, the hole 55' being centred across the width of the PCB 55 adjacent one end of the PCB 55.
  • a test vessel is formed by creating a well in a sheet of material and the coils are deposited on or fabricated in one, or more, of the sides of the vessel.
  • coils are formed on a flexible substrate material, such as a polymer. Using integrated or thick film circuit techniques, it is possible to incorporate some, or all, of the components such as the capacitors, phase-locked loop circuits, programmable switches and mixer circuits into a single device that could be fixed to the substrate along with the coils. Components could then be trimmed in the manufacturing stage and variations in coil, and other component characteristics minimised.
  • a temperature-controlled heater could also be incorporated on the substrate by depositing a metal film onto the substrate.
  • a heater may be necessary in field applications when the ambient temperature is so low that it affects the chemical reactions in the vessel and, or, produces such large temperature drift in the values of the components on the substrate that they are outside their calibration range.
  • Pulsed magnetic fields were used in the earlier experiments, and these were produced by applying a pulsed current to an electromagnet.
  • An intense, localised, magnetic field with a steep field gradient is obtained by placing iron or steel studs 61,62 on the electromagnet and positioning then directly below each sensing coil 50,51 so that the magnetic particles are pulled towards the centre of the coils.
  • the pole pieces of the electromagnet could be shaped accordingly.
  • Figure 9 shows schematically what happens when the external magnetic field pulls a coated magnetic particle, P through the test solution containing the analyte Q towards an immobilised capture molecule, S.
  • the capture molecules on P bind to Q to form a complex, R.
  • Ki P + Q ** R K 2 K x and K 2 are the rate constants as shown. If there is strong affinity between the coated magnetic particle and the analyte molecules, then K 2 may be ignored.
  • the reaction between P and Q is likely to be rapid and if the test solution containing Q is premixed with a solution containing P before the magnetic field is switched on, then the resultant solution will contain mainly the complex R. When the magnetic field is switched on, the complex R is attracted to the immobilized capture molecules, S, and a sandwich complex, T, forms with the magnetic particle immobilised in the reaction surface on the substrate.
  • K 3 and K 4 are the rate constants. Again if there is a strong affinity between the analyte molecule and the immobilised capture molecule in the reaction surface, then K 4 may be ignored.
  • the build up of the sandwich complex [T] t on the substrate with time, t, should follow a first order reaction:
  • the signal recorded by the sensing coil is inversely related to [T] t ; so the rate of fall in resonant frequency of the sensing coil just after the test sample is added to the reaction mixture is a maximum and directly related to the initial concentration [Q] of the analyte molecules.
  • An alternative method is to wait for a certain period, t l7 after adding the solutions when the reactions have settled down and then to measure the gradient of the rate of fall in the resonant frequency over the time period, t 2 - t x .
  • the rate at which the resonant frequency of the coil falls over a particular time period after adding the sample is related to the concentration of the analyte and this is determined by measuring the gradient of line CD.
  • This gradient can be determined in just 3 or 4 cycles of the electromagnet, which is considerably shorter than in previous methods. If this measurement is repeated with a sample containing a different concentration of the analyte, then the gradient of line CD changes accordingly.
  • subtracting the gradient of line AB from line CD reduces errors caused by drift in the resonance coil.
  • one of the coils could be a reference coil and drift in the resonant frequency of this coil could be used to correct for drift in the others.
  • the upper surface was then activated by applying polymerised glutaraldehyde for 30 minutes (prepared by adding 500 ⁇ l of 0.1M sodium hydroxide in 5 ml of 5% glutaraldehyde and neutralized with 500 ⁇ l of 0.1M hydrochloric acid after 30 minutes at room temperature) . Following activation, the surface of the ceramic substrate was washed with methanol for 1 minute.
  • Polymerised glutaraldehyde prepared by adding 500 ⁇ l of 0.1M sodium hydroxide in 5 ml of 5% glutaraldehyde and neutralized with 500 ⁇ l of 0.1M hydrochloric acid after 30 minutes at room temperature
  • Paramagnetic particles were prepared by coating Seradyn protein G coated particles with mouse anti-human Tnl antibody or mouse anti-human CKMB antibody (Randox Laboratories Ltd.) and then diluted 1:1000 times in 0.1 M phosphate buffer (pH 8.1). Ten ⁇ l of paramagnetic particles were washed 3 times in the phosphate buffer and then mixed with 30 ⁇ l of the antibody solution and incubated for 10 minutes at room temperature in a slowly rotating sample mixer (Dynal UK Ltd) .
  • paramagnetic particles were then washed 3 times in phosphate buffer before being re-suspended in 20 nM dimethyl pimelimidate (Sigma-Aldich, UK) and incubated at room temperature with rotational mixing for a further 30 minutes to promote cross linking.
  • the reaction was stopped by suspending the paramagnetic particles 50 mM Tris (hydroxy ethyl) aminoethane buffer (pH 7.3) . Unoccupied sites on the particles were blocked using 1% bovine serum albumin in PBS.
  • a symmetrical pulsed waveform with a frequency of approximately 0.05 Hz (approximately 10s on and 10s off) was used to drive the power supply to the electromagnet causing the magnet field to be alternately on and off.
  • the electromagnet was "on"
  • the paramagnetic particles were pulled on to the reactive surface.
  • the unbound paramagnetic particles were free to move in the buffer.
  • the results were calculated as explained above by plotting the difference in the slope of the recorded signal before the addition of paramagnetic particles and that recorded over the (t 2 - t 2 ) period.
  • k is a constant that depends on the current passing through the coil and the magnetic permeability of the medium surrounding the coil.
  • a spiral, coil consisting of m turns may be approximated to m concentric coils with radii, a x , a 2 , a 3 ...and a m . As the same current passes through each turn of the coil, the total field produced by the spiral coil is:
  • This model is a simplification as it deals only with the magnetic field and neglects such things as the permittivity of the substrate and the buffer solution. It also neglects the electric field associated with the rapidly oscillating magnetic field which will be affected by the high dielectric constant and high conductivity of the of the buffer solution: if these are taken into account then it is likely that the electromagnetic field created by the sensing coil falls even more rapidly than shown in Figure 14.
  • the particles used in these experiments have magnetic domains.
  • the studs on the electromagnet or permanent magnet are positioned below the sensing coil and produce an intense, localised, magnetic field with a rapidly increasing flux density that induces magnetic moments into the particles and attracts them towards the reaction surface ( Figure 15) .
  • Magnetic particles with attached analyte molecules bind quantitatively to the capture molecules on the reaction surface and the particles become immobilised.
  • the magnetic domains in the immobilised particles will attempt to align along the direction of the magnetic field created by the stud.
  • the immobilised particles have a large magnetic moment on the reaction surface which produces a significant perturbation in the intense, high-frequency magnetic field produced by the sensing coil, resulting in an increase in the inductance of the coil and a decrease in the resonant frequency.
  • the unbound magnetic particles are free to move and are subject to Brownian motion.
  • an unbound particle with its magnetic moment aligned along the steady field produced by the stud could be rotated by Brownian motion through 180° so that the moment is now in the wrong direction; the domain could flip its orientation by a process called Neel relaxation, but this is a relatively slow process compared with Brownian motion; so it is likely that the particle will have to wait until the Brownian motion returns the magnetic moment to alignment.
  • Neel relaxation a process called Neel relaxation
  • Unbound particles further away from the reaction surface will also be affected by Brownian motion and, as the field produced by the studs is weaker here than the surface, the magnetic moments of these particles will be more strongly influenced by the Brownian motion.
  • the high-frequency field produced by the sensing coil is also weaker further away from the reaction surface and so these unbound particles produce a negligible response in the sensing coil. In this way, the sensing system is able to distinguish between bound and unbound particles.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Investigating Or Analyzing Materials By The Use Of Magnetic Means (AREA)

Abstract

techniques d'exécution de dosages par liaison. Une première technique consiste: à placer un échantillon comprenant un analyte cible sur un site de réaction présentant des partenaires de liaison complémentaires de l'analyte cible, site adjacent à une bobine et à un circuit résonant; à prendre des particules magnétiques enduites qui se fixent à l'analyte cible, lesdites particules se liant au site de réaction via l'analyte cible; à surveiller la fréquence de résonance, ou bien un changement de fréquence de résonance, du circuit résonant de manière à déterminer la quantité de particules magnétiques qui se sont liées aux site de réaction; et à exposer le site de réaction à un champ magnétique non pulsé afin d'attirer les particules magnétiques vers le site de réaction.
PCT/GB2005/001892 2004-05-17 2005-05-17 Systeme de detection de particules magnetiques et technique d'execution d'une d'un dosage par liaison WO2005111614A1 (fr)

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WO2007122293A1 (fr) 2006-04-21 2007-11-01 Magnasense Oy Dispositif de mesure de particules magnétiques et procédé correspondant
WO2009148668A3 (fr) * 2008-03-07 2010-03-04 California Institute Of Technology Détection de particule magnétique par changement d'inductance efficace
WO2010058059A1 (fr) * 2008-11-18 2010-05-27 University Of Jyväskylä Procédé pour détecter des séquences nucléotidiques spécifiques
US8520211B2 (en) 2008-04-09 2013-08-27 Koninklijke Philips N.V. Carrier for optical detection in small sample volumes
CN103608118A (zh) * 2011-06-21 2014-02-26 西门子公司 用于从包含矿石颗粒磁颗粒凝聚物的悬浮液中获取非磁性矿石的方法
US9157891B2 (en) 2008-10-16 2015-10-13 Koninklijke Philips N.V. Biosensor with quadrupole magnetic actuation system
US9610584B2 (en) 2007-03-21 2017-04-04 University Of The West Of England, Bristol Particle facilitated testing
US9841421B2 (en) 2010-11-30 2017-12-12 Koninklijke Philips N.V. Sensor device for magnetically actuated particles
WO2020081876A1 (fr) * 2018-10-17 2020-04-23 Hemex Health, Inc. Systèmes et procédés de diagnostic
US11053473B2 (en) 2019-06-25 2021-07-06 Hemex Health, Inc. External sonication
CN113109395A (zh) * 2021-04-20 2021-07-13 南昌大学 一种测量磁性复合材料中磁性组元含量的无损测试方法
CN114341641A (zh) * 2020-05-25 2022-04-12 金·保罗·杰里米 磁性纳米颗粒对于分析物的检测和定量的用途

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2016401A1 (fr) * 2006-04-21 2009-01-21 Magnasense OY Dispositif de mesure de particules magnétiques et procédé correspondant
JP2009534641A (ja) * 2006-04-21 2009-09-24 マグナセンセ オユ 磁性粒子測定デバイスおよび方法
US8026716B2 (en) 2006-04-21 2011-09-27 Magnasense Technologies Oy Device for measuring magnetic particles and corresponding method
AU2007242719B2 (en) * 2006-04-21 2012-08-16 Magnasense Technologies Oy Device for measuring magnetic particles and corresponding method
WO2007122293A1 (fr) 2006-04-21 2007-11-01 Magnasense Oy Dispositif de mesure de particules magnétiques et procédé correspondant
EP2016401A4 (fr) * 2006-04-21 2014-09-10 Magnasense Technologies Oy Dispositif de mesure de particules magnétiques et procédé correspondant
US9610584B2 (en) 2007-03-21 2017-04-04 University Of The West Of England, Bristol Particle facilitated testing
WO2009148668A3 (fr) * 2008-03-07 2010-03-04 California Institute Of Technology Détection de particule magnétique par changement d'inductance efficace
US8520211B2 (en) 2008-04-09 2013-08-27 Koninklijke Philips N.V. Carrier for optical detection in small sample volumes
US9157891B2 (en) 2008-10-16 2015-10-13 Koninklijke Philips N.V. Biosensor with quadrupole magnetic actuation system
WO2010058059A1 (fr) * 2008-11-18 2010-05-27 University Of Jyväskylä Procédé pour détecter des séquences nucléotidiques spécifiques
US9841421B2 (en) 2010-11-30 2017-12-12 Koninklijke Philips N.V. Sensor device for magnetically actuated particles
CN103608118A (zh) * 2011-06-21 2014-02-26 西门子公司 用于从包含矿石颗粒磁颗粒凝聚物的悬浮液中获取非磁性矿石的方法
WO2020081876A1 (fr) * 2018-10-17 2020-04-23 Hemex Health, Inc. Systèmes et procédés de diagnostic
EP3867635A4 (fr) * 2018-10-17 2022-07-13 Hemex Health, Inc. Systèmes et procédés de diagnostic
US11053473B2 (en) 2019-06-25 2021-07-06 Hemex Health, Inc. External sonication
CN114341641A (zh) * 2020-05-25 2022-04-12 金·保罗·杰里米 磁性纳米颗粒对于分析物的检测和定量的用途
CN113109395A (zh) * 2021-04-20 2021-07-13 南昌大学 一种测量磁性复合材料中磁性组元含量的无损测试方法

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